Report. Food Fortification with Micronutrients

Transcription

1 Report Food Fortification with Micronutrients Central Asian Republics, Afghanistan, Pakistan Almaty, 2016 This report is made possible by the generous support of the American people through USAID. The contents are the responsibility of GAIN and do not necessarily reflect the views of USAID or the United States Government. 1

2 Оглавление Background... 4 General Objectives of the Project:... 4 Specific Objectives of the Project:... 4 Support the efforts on harmonization of food fortification standards to improve fortification standards in program supported countries and develop a harmonized standard for wheat flour and edible oil... 5 Regional Expert Group on Harmonization of Wheat Flour Fortification Standards... 5 The first Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards... 5 The second Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards... 6 Support the efforts on the development and adoption of regional fortification regulations... 7 Establishment of an interagency expert group on flour fortification in Kazakhstan... 7 High level advocacy for wheat flour fortification... 8 Support a regulatory capacity assessment and strengthening of the monitoring system and enforcing the fortification law in Kazakhstan... 8 Support a wheat flour cost-benefit analysis in Kazakhstan... 9 Conducting a scientific seminar to present regional and global research findings and evidence on the benefits of wheat flour fortification Summary Annexes Annex 1. First First Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards Annex 2. On the necessity of low extraction wheat flour fortification with vitamin B12 in Central Asia Republics, Afghanistan and Pakistan Annex 3. Analysis and Justification the Possibility of Harmonizing Standards for Refined Wheat Flour Fortification in Central Asia, Afghanistan and Pakistan Annex 4. Analysis and Justification of the Possibility of Harmonizing Standards for Highextraction Wheat Flour Fortification in Afghanistan and Pakistan Annex 5. Draft. National Standard of the Republic of Kazakhstan. Low Extraction Fortified Bakery Wheat Flour. Specification Annex 6. Draft. National Standard of the Republic of Kazakhstan. High Extraction Fortified Bakery Wheat Flour. Specification Annex 7. Draft. External and Internal Quality Control of Iron Fortification of Flour Annex 8. Second Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards Annex 9. Suggestions on rationalization of nutrition for the Kazakhstan population for consideration and decision making at the level of National Coordination Council for Health at the Government of the Republic of Kazakhstan Annex 10. Booklet in Russian. Advantages and benefits of wheat flour fortification Annex 11. Booklet in Kazakh. Advantages and benefits of wheat flour fortification

3 Annex 12. Leaflet in Russian. Advantages and benefits of wheat flour fortification Annex 13. Leaflet in Kazakh. Advantages and benefits of wheat flour fortification Annex 14. Program of Round Table. Advantages and benefits of wheat flour fortification

4 Background In September 2014, the Global Alliance for Improved Nutrition (GAIN) was awarded two-year U.S. Agency for International Development (USAID) funding for the program Regional Fortification in the Central Asia Republics (CAR) and Afghanistan. The program has an emphasis on improving fortification processes, regulations and monitoring in the region, in particular for flour exports from Kazakhstan to Afghanistan and edible oil exports from Pakistan to Afghanistan. One objective is to improve fortification standards in program supported countries and develop a harmonized standard for wheat flour. The USAID-funded Micronutrient Fortification Project in Central Asia, Afghanistan and Pakistan, that works to improve fortification regulations and monitoring in the region, for flour exports from Kazakhstan to Afghanistan and edible oil exports from Pakistan to Afghanistan and on the terms and conditions of this Agreement. The initial year of the program aimed at understanding regional trade of wheat flour and edible oil as well as related legislation and policy. In October 2015, GAIN and USAID co-hosted a regional stakeholder meeting on food fortification that brought together representatives of governments, private sector, academia, civil society, and development partners from Kazakhstan, Afghanistan, Pakistan, Tajikistan, and Uzbekistan. Besides disseminating findings from regional and country assessments, as well as sharing country status on food fortification efforts, the stakeholder meeting also had an objective to form technical working groups. To advance the work on food fortification in the region in October 2015 was signed a grant agreement between GAIN and the Kazakh Academy of Nutrition (KAN). The Kazakh Academy of Nutrition project team, led by KAN's Vice-President, Shamil Tazhibayev, under this agreement, is responsible for: studies on a market availability of fortified flour in Kazakhstan; conducting scientific seminars to support the work on regional harmonization of fortification standards and regulations, and to present results of the studies and other evidence; establishing mechanisms for intersectional cooperation to advance flour fortification work in Kazakhstan; high-level advocacy to help ensure national ownership and support of the fortification program at the highest political level; and assisting the GAIN project team in implementing other work plan activities. General Objectives of the Project: Increase supply of micronutrients to CAR and Afghanistan through fortification of wheat flour and vegetable oil; Strengthen quality control and enforcement of wheat flour and oil fortification with special attention to imported products in all involved countries, particularly Afghanistan and Kazakhstan; Attempt harmonization of standards for fortification in CAR, Afghanistan and Pakistan; and Introduce and test mechanisms to assess extension of use and quality of fortified products at the consumer level in Afghanistan, and in some of the CAR countries. Specific Objectives of the Project: 1. Support the efforts on harmonization of food fortification standards to improve fortification standards in program supported countries and develop a harmonized standard for wheat flour and edible oil. 4

5 2. Support the efforts on the development and adoption of regional fortification regulations for flour and oil inspection and food control including a regional standard for flour based on WHO Guidelines. 3. Establishment of an interagency expert group on flour fortification in Kazakhstan to advance flour fortification work in Kazakhstan. 4. High level advocacy for wheat flour fortification to understand potential stakeholder concerns related to wheat flour in Kazakhstan, to develop evidence, materials and influence methods to address them, and ultimately form national ownership and support of the fortification program at the highest political level. 5. Support a regulatory capacity assessment regulatory framework, personnel capacity, equipment, laboratory capacity, quality testing, surveillance and data analysis) and strengthening of the monitoring system and enforcing the fortification law in Kazakhstan. 6. Support a wheat flour cost-benefit analysis in Kazakhstan, to quantify costs of wheat flour fortification and estimate a monetary value of health and economic benefits over a certain period based on existing evidence in order to support advocacy efforts. 7. Conducting a scientific seminar to present regional and global research findings and evidence on the benefits of wheat flour fortification. Duration of the project: Project start date: October 1, 2015 Project end date: September 30, 2016 Support the efforts on harmonization of food fortification standards to improve fortification standards in program supported countries and develop a harmonized standard for wheat flour and edible oil Regional Expert Group on Harmonization of Wheat Flour Fortification Standards Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan, and Pakistan was formed. It is chaired by the Kazakh Academy of Nutrition and includes specialists representing national standards committees, government regulatory agencies, research institutions, wheat flour producers and respective business associations from six countries - Kazakhstan, Afghanistan, Pakistan, Tajikistan, Kyrgyzstan and Uzbekistan (see the list of members in Annex 1). During November 2015-January 2016, in consultation with international experts, the KAN members of the group developed a technical justification for harmonizing fortification standards for low extraction (refined, white) flour used in all countries of the region, and high extraction (whole) flour mainly used in Afghanistan and Pakistan. These two analytical documents were shared with all the members of expert group for comments and additional input. The documents highlight the need to ensure, at a minimum, adequate and comparable amounts of bioavailable micronutrients in all countries of the region as specified in the WHO recommendations. The first Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards The first Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan was held in Almaty March 14-15, 2016 (Annex 1). All the materials of the Technical Meeting are available at the Web site 5

6 The overall aim of this Technical Meeting was that technical consensus be reached on a unified premix composition to be used in the region. Goals and objectives, the main results and recommendations of the technical meeting are presented as a Summary of Technical Meeting. Technical Meeting was designed for experts representing leading government industry and other stakeholders including fortified wheat flour and oil producers from every project country; policy makers from Ministries of Health, Ministries of Agriculture; representatives of Flour Milling Associations; Food Control Authorities, including representatives of Standardization Committees, government flour and edible oil inspections, research institutions, GAIN and other international organizations (USAID) from project countries. KAN has taken responsibility for all aspects of the workshop organization including identification of participants, preparing invitation letters, logistics, and coordination of presentations. GAIN representatives in Kazakhstan were very helpful in organizing and conducting the Technical Meeting. KAN s staff presented the following presentations at the Technical Meeting (these and other presentations are available on the web site ): Professor Shamil Tazhibayev Analysis and justification of the possibility of harmonizing standards for wheat flour fortification in CAR, Afghanistan and Pakistan. Professor Yuriy Sinyavskiy Regional standards for low- and high- extraction wheat flour fortification - examples for discussion. In accordance with the recommendations of the first Technical Meeting a rationale On the necessity of low extraction wheat flour fortification with vitamin B12 in Central Asia republics, Afghanistan and Pakistan (Annex 2), and Analysis and justification the possibility of harmonizing standards for refined wheat flour fortification in Central Asia, Afghanistan and Pakistan (Annex 3) were developed. In these documents, the level of flour fortification with vitamin B12 reduced from 0,008 ppm to 0,004 ppm, and all of appropriate data were recalculated. The participants of the second Technical Meeting fully endorsed the document Analysis and justification of the possibility of harmonizing standards for high-extraction wheat flour fortification in Afghanistan and Pakistan (Annex 4). On the basis of an updated document Analysis and justification the possibility of harmonizing standards for refined wheat flour fortification in Central Asia, Afghanistan and Pakistan, and in accordance with the recommendations of the Technical Meeting a draft of Standard of the Republic of Kazakhstan Low extraction fortified bakery wheat flour were developed (Annex 5). The draft of the standard was shared with all the members of expert group for use in the development of such national standard in the member countries. In accordance with the recommendations of the Technical Meeting a draft of Standard of the Republic of Kazakhstan High extraction fortified bakery wheat flour were developed as well (Annex 6). The draft of the standard was shared with all the members of expert group for use in the development of such national standard in the member countries. Kazakh Academy of Nutrition, in collaboration with an expert team of Kazakhstan has developed a draft of the Guidelines External and internal quality control of iron fortification of flour (including iron in vitamin-mineral mixtures) (Annex 7). All the Annexes are available at the web site The draft of the document was shared with all the members of expert group for use in the development of such national standard in the member countries. The second Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards The second Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan, Pakistan was held in Almaty September 6, 2016 (Annex 8). All the materials of the second Technical Meeting are available at the Web site Objectives of the second Technical Meeting are the following: 6

7 1) To review and agree on the draft national standards for flour fortification, developed through the use of a single composition of the premix for flour fortification for all countries; 2) To reach agreement and propose a time frame for the adoption of national standards for flour fortification in the participating countries; 3) To review the draft document on quality assurance and quality control (internal and external) of flour fortification, as well as to consider formulation and possibilities for adoption of the document in the participating countries; 4) To present results of the wheat quality analysis in Kazakhstan; 5) To consider the possibility for establishing a system of inter-agency coordination of flour fortification in the participating countries; 6) To discuss the feasibility, timing, and further steps to promote food fortification and to facilitate imports and exports of fortified flour through the harmonization of standards in order to increase the consumption of micronutrients by population in the participating countries. Outcomes of the Second Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan: 1) Consensus on micronutrient composition (unified premix) for flour fortification in all countries reached; 2) The unified premix to serve as a key element of harmonized approach to flour fortification in the regions; 3) Countries agreed to use the draft standards as a template for their national standards to be developed or modified; 4) Countries shared their experiences and underlined the importance of quality assurance and quality control (QA/QC) for harmonization of methodologies and agreed to continue discussions on unified QA/QC approaches; 5) Funds for furtherance of efforts on harmonization are secured and next steps agreed by all countries. Support the efforts on the development and adoption of regional fortification regulations The objective is to develop regional regulations for flour and oil for inspection and food control including a regional standard for flour based on WHO Guidelines. KAN provided the needed technical expertise to assist GAIN in the analysis and development of regional regulations and recommendations for wheat flour and oil inspection and control including a regional standard for wheat flour based on WHO Guidelines. In Kazakhstan and other countriesparticipans were condacted an adaptation of country regulations. Based on regional regulations. KAN organized in Almaty the first (March 14-15, 2016) and the second (September 6, 2016) Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan, described more fully above to facilitate the work. Establishment of an interagency expert group on flour fortification in Kazakhstan The work aimed at improving inter-agency coordination of work on wheat flour fortification. Interagency Coordinating Council to promote flour fortification in Kazakhstan operated in , during implementation of the international program on flour fortification and salt iodization in the framework of the Asian Development Bank (ADB) and then stopped working. Currently, inter-agency coordination activities in the field of Health and Social Welfare organized by the National Coordination Council (NCC) at the Government of the Republic of Kazakhstan under the chairmanship of Deputy Prime Minister of the Republic of Kazakhstan. 7

8 KAN has initiated the establishment of the Interagency Council on the coordination of activities in the field of nutrition and food fortification,, turning such a proposal to the Ministry of Health and Social Protection of the Republic of Kazakhstan (RK MoHSP). MoHSP Kazakhstan proposed to consider issues on nutrition and food fortification at meetings of the NCC. In this regard, KAN has developed a proposal for consideration at meetings of the NCC "Suggestions on rationalization of nutrition for the Kazakhstan population for consideration and decision making at the level of National Coordination Council for Health at the Government of the Republic of Kazakhstan" (Annex 9). The representative of the KAN was included as the member of NCC. NCC meets on average once a quarter. Vice President of KAN Professor Tazhibayev participated at the NCC meeting in May 23, 2016, issued and submitted a proposal to include in the agenda of the next meeting of the NCC the issues on improving nutrition status of population and food fortification with vitamins and trace elements; and the proposal was accepted. After this time, a meeting of the NCC has not yet taken place. High level advocacy for wheat flour fortification The work aimed to understand potential stakeholder concerns related to wheat flour in Kazakhstan, to develop evidence, materials and influence methods to address them, and ultimately form national ownership and support of the fortification program at the highest political level. KAN developed evidence-based materials about the benefits and effectiveness of flour fortification to combat anemia and micronutrient deficiencies (Annexes 10-13) designed for different target groups (e.g., representatives of the Parliament, Ministry of Health and Social Development, Ministry of Agriculture, Ministry of National Economy, Committee for the Protection of Consumer Rights). These materials - that are based on published evidence, global guidance and best practice - used for the formation of stakeholder opinion and in decision-making to support wheat flour fortification at the highest political level. KAN provided the necessary assistance in organizing highlevel advocacy meetings with representatives of the abovementioned ministries and agencies. Professor Shamil Tazhibayev participated in Sixth Annual Central Asian Trade Forum Promoting the expansion of fortified wheat flour trade in Central Asia, Afghanistan and Pakistan (Almaty, 7-8 September 2016, Hotel Rixos) and made a presentation Harmonization of standards for fortified wheat flour in Central Asian Republics, Afghanistan and Pakistan (see attached file 2016-TazhibayevCATF VI-Almaty8-SepEng.pdf). It is a very representative forum, which was attended by the representatives of ministries and agencies, as well as business structures from CARs, Afghanistan and Pakistan. Professor Shamil Tazhibayev made a presentation Wheat Flour Fortification in Kazakhstan: Current Status and Ways to Improve at the Round Table From Evidence to Action: How Wheat Flour Fortification Can Impact Public Health and Economy of the Republic of Kazakhstan, which took place in Astana, 30 September 2016, Hotel Rixos (see attached file 2016-Tazhibayev-FF- AstanaEng30.09). The representatives of many ministries and agencies, as well as business structures in Kazakhstan particitaed in the Round Table. Support a regulatory capacity assessment and strengthening of the monitoring system and enforcing the fortification law in Kazakhstan The objective is to assess wheat flour fortification regulatory capacity (regulatory framework, personnel capacity, equipment, laboratory capacity, quality testing, surveillance and data analysis) to assist the Kazakh government in strengthening its monitoring system and enforcing the fortification law. KAN provided the needed support to the Kazakh government and GAIN in conducting an analysis of the flour fortification regulatory system and its capacity, and in the development of 8

9 guidelines and/or materials to improve the system performance, and in the execution of the law on mandatory flour fortification. In particular, as it mentioned above, Kazakh Academy of Nutrition, in collaboration with an expert team of Kazakhstan has developed a draft of the Guidelines External and internal quality control of iron fortification of flour (including iron in vitamin-mineral mixtures) (Annex 7). Countries in the Region shared their experiences, underlined the importance of quality assurance and quality control (QA/QC) for harmonization of methodologies at the second Regional Meeting, and agreed to continue discussions on unified QA/QC approaches. Support a wheat flour cost-benefit analysis in Kazakhstan The objective is to quantify costs of wheat flour fortification in Kazakhstan and estimate a monetary value of health and economic benefits over a certain period of time based on existing evidence in order to support advocacy efforts. The study will be led by GAIN/HQ and international consultants who are subject matter experts. KAN provided the needed technical assistance to the project team in the development of methodology, identification of partners and organizing their work, reviewing proposed study tools, obtaining some of the data, and reviewing the report. In particular, the KAN has provided the following data for the analysis of costs and benefits of wheat flour fortification in Kazakhstan: Demographic indicators according to the Demographic Yearbook of Kazakhstan for the years of 2014 and Prevalence of anemia, iron deficiency, iron deficiency anemia and folic acid deficiency among the various sex-age groups of population (children aged 6-59 months, 5-11 and years old, pregnant women, women aged years and men aged years), according to the results of a national study conducted by KAN in Kazakhstan in 2008 and Number and percentage (per 1000 births) of neural tube defects, in 2014 and 2015 according to the National Genetic Register (data of the Republican medical-genetic consultation of the Scientific Center for Obstetrics, Gynecology and Perinatology MoHSP RK). KAN s staff participated in meetings on Skype jointly with interested representatives of national and international organizations (USAID, GAIN), devoted to the discussion of costs and benefits of wheat flour fortification, and held on 25 February and 29 April Professor Tazhibayev Shamil participated in an interactive seminar, "The Economic Consequences of nutritional micronutrient deficiencies in Tajikistan", which took place in Dushanbe on March, 2016 and made a presentation on "Regional perspectives on food fortification and its influence in the region". The Majlisi Oli of Republic of Tajikistan, Ministry of Health and Social Protection of Population of Tajikistan, and United States Agency organized the seminar for International Development and the Global Alliance for Improved Nutrition. KAN participated in conducting of the Round Table From Evidence to Action: How Wheat Flour Fortification Can Impact Public Health and Economy of the Republic of Kazakhstan, which took place in Astana, 30 September 2016, Hotel Rixos (Annex 14). As it mentioned above, Professor Shamil Tazhibayev made a presentation Wheat Flour Fortification in Kazakhstan: Current Status and Ways to Improve at the Round Table (see attached file 2016-Tazhibayev-FF-AstanaEng30.09). 9

10 Conducting a scientific seminar to present regional and global research findings and evidence on the benefits of wheat flour fortification The objective is to present findings of the research studies focused on regional evidence, cost and benefits of fortification (e.g., cost benefit analysis, market availability, iron deficiency prevalence) to high-level decision makers and other stakeholders of the region in the domain of health care, production, import and export of wheat flour to support national ownership of the fortification program at the highest political level. KAN took responsibility for all aspects of planning, organizing and conducting the regional seminar, including concept development, identification of participants, securing their participation, development of agenda, preparation of invitation letters, logistics, and coordination of presentations. As stated above, KAN has organized and held two Technical Meetings, as well as participated in conducting of the Round Table: First Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan, March 2016, Almaty, Rixos Hotel. Second Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan, 6 September 2016, Almaty, Rixos Hotel. Round Table From Evidence to Action: How Wheat Flour Fortification Can Impact Public Health and Economy of the Republic of Kazakhstan, Astana, 30 September 2016, Hotel Rixos. Regional and global research findings and evidence on the benefits of wheat flour fortification were presented at these forums. Programs of these forums in which various aspects of wheat flour fortification were presented, are reflected in the relevant sections above. Summary 1) Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan, and Pakistan was formed. 2) Kazakh Academy of Nutrition developed the following materials and documents that are discussed and approved at the Technical meetings of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards: On the necessity of low extraction wheat flour fortification with vitamin B12 in Central Asia republics, Afghanistan and Pakistan. Analysis and justification the possibility of harmonizing standards for refined wheat flour fortification in Central Asia, Afghanistan and Pakistan. Analysis and justification of the possibility of harmonizing standards for high-extraction wheat flour fortification in Afghanistan and Pakistan. Draft of National Standard of the Republic of Kazakhstan Low extraction fortified bakery wheat flour. Draft of National Standard of the Republic of Kazakhstan High extraction fortified bakery wheat flour. 10

11 Draft of the Guidelines External and internal quality control of iron fortification of flour (including iron in vitamin-mineral mixtures). These drafts of Kazakhstan National Standards, guidelines and other normative documents used for the development of similar documents in all countries participating in the project. 3) Two Technical Meetings and one Round Table have ben conducted: The first Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan was held in Almaty March 14-15, The second Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan, Pakistan was held in Almaty September 6, Round Table From Evidence to Action: How Wheat Flour Fortification Can Impact Public Health and Economy of the Republic of Kazakhstan, Astana, 30 September 2016, Hotel Rixos. All the developed materials and documents, as well as global, regional and country aspects of flour fortification have been discussed in these Technical Meetings and Round Table. 4) Kazakh Academy of Nutrition developed "Suggestions on rationalization of nutrition for the Kazakhstan population for consideration and decision making at the level of National Coordination Council for Health at the Government of the Republic of Kazakhstan". These Suggestions are accepted for review and will be considered at the next meeting of the National Coordination Council. 5) Kazakh Academy of Nutrition provided the results of national studies on the prevalence of anemia, iron deficiency, folic acid, vitamin A and iodine deficiency in Kazakhstan, as well as relevant statistical data, which are nesessary for the costs and benefits analysis of wheat flour fortification in Kazakhstan. 6) Evidence-based materials about the benefits and effectiveness of flour fortification to combat anemia and micronutrient deficiencies (leaflet, buklet and video material) designed for different target groups are developed. On an ongoing basis, advocacy for wheat flour fortification, including high-level one is provided. 7) It is recommended to provide further support of countries participating in the project for the adoption and implementation into practice the relevant standards and other regulatory documents, and guidelines for harmonization of standards for flour fortification, as well as quality assurance and quality control of fortified flour. Analysis of the results of activities in these areas could be presented and discussed at the Third Technical Meeting of the Regional Technical Expert Group on harmonization of standards for flour fortification in Central Asian Republics, Afghanistan and Pakistan. 11

12 Annexes Annex 1. First First Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards First Technical Meeting Regional Expert Group on Harmonization of Wheat Flour Fortification Standards Central Asian Republics, Afghanistan, Pakistan March 2016 Almaty, Rixos Hotel Summary This report is made possible by the generous support of the American people through USAID. The contents are the responsibility of GAIN and do not necessarily reflect the views of USAID or the United States Government. 12

13 Background In September 2014, GAIN was awarded two-year USAID funding for the program Regional Fortification in the Central Asia Republics and Afghanistan. The program has an emphasis on improving fortification processes, regulations and monitoring in the region, in particular for flour exports from Kazakhstan to Afghanistan and edible oil exports from Pakistan to Afghanistan. One objective is to improve fortification standards in program supported countries and develop a harmonized standard for wheat flour. The initial year of the program aimed at understanding regional trade of wheat flour and edible oil as well as related legislation and policy. In October 2015, GAIN and USAID co-hosted a regional stakeholder meeting on food fortification that brought together representatives of governments, private sector, academia, civil society, and development partners from Kazakhstan, Afghanistan, Pakistan, Tajikistan, and Uzbekistan. Besides disseminating findings from regional and country assessments, as well as sharing country status on food fortification efforts, the stakeholder meeting also had an objective to form technical working groups. Several joint priorities were identified, including facilitation of cross-border trade through harmonized standards of wheat flour. A Regional Expert Group on Wheat Flour Fortification Standards and Potential for Regional Harmonization was formed. It is chaired by the Kazakh Academy of Nutrition (KAN) and includes specialists representing national standards committees, government regulatory agencies, research institutions, wheat flour producers and respective business associations from six countries - Kazakhstan, Afghanistan, Pakistan, Tajikistan, Kyrgyzstan and Uzbekistan (see the list of members in Annex 1). During November 2015-January 2016, in consultation with international experts, the KAN members of the group developed a technical justification for harmonizing fortification standards for low extraction (refined, white) flour used in all countries of the region, and high extraction (whole) flour mainly used in Afghanistan and Pakistan. These two analytical documents were shared with the expert group for comments and additional input. The documents highlight the need to ensure, at a minimum, adequate and comparable amounts of bioavailable micronutrients in all countries of the region as specified in the WHO recommendations. The overall aim of this Technical Meeting was that technical consensus be reached on a unified premix composition to be used in the region. Agenda is presented in Annex 2. Two options for the micronutrients in premix were proposed: High extraction flour Iron (NaFeEDTA) Zinc (Zinc oxide) Vit B9 (Folic acid) Vit B12 (Cyancobalamin) Low extraction flour Iron (NaFeEDTA and Ferrous sulfate, dried to increase iron content at a reasonable cost) Zinc (Zinc oxide) Vit B9 (Folic acid) Vit B12 (Cyancobalamin) Vit B1 (Thiamine) Vit B2 (Riboflavin) Vit B3 (Niacin) Objectives 1. To review the draft technical justification for harmonization of wheat flour fortification standards in the region and obtain feedback from regional experts; 2. To reach agreement and propose a harmonized standard for wheat flour fortification; and 3. To discuss the feasibility, timeline, and the next steps for development or modification of national wheat flour standards based on the agreements reached at the regional level. 13

14 Participants The invited meeting participants included regional expert group members, international subject matter experts, and selected representatives of the donor and the implementing partner - see the full list in Annex 1. Main Results 1. Technical justification of regional harmonization of wheat flour fortification standards was presented and thoroughly discussed by the experts in the course of the 2-day technical meeting. 2. Experts from each country of the region presented their comments on harmonization of flour fortification standards. 3. Detailed discussion of flour fortification standards harmonization continued in two working groups of experts on fortification of 1) low-extraction; and 2) high-extraction wheat flour. The recommendations developed by these two expert working groups are presented below. 4. Experts arrived at a consensus and agreed on the following premix composition to harmonize standards on low and high extraction wheat flour fortification, which is fully consistent with the recommendations of the World Health Organization 1 : High extraction flour Low extraction flour Micronutrients Levels, ppm Micronutrients Levels, ppm Iron (NaFeEDTA) 15,0 Iron (NaFeEDTA) 15,0 Zinc (Zinc oxide) 30,0 Zinc (Zinc oxide) 30,0 Vit B9 (Folic acid) 1,0 Vit B9 (Folic acid) 1,0 Vit B12 (Cyancobalamin) 0,008 Vit B12 (Cyancobalamin) 0,008 Vit B1 (Thiamine) 2,0 Vit B2 (Riboflavin) 3,0 Vit B3 (Niacin) 10,0 5. Participants discussed and agreed on the following next steps for development or modification of the national flour fortification standards based on the regional consensus: - Kazakh Academy of Nutrition: to design justification for harmonization of low-extraction wheat flour fortification standards without Ferrous Sulfate before May 1, 2016; - Working group members in each of the target countries: a) to coordinate and agree the proposed premix formula with key ministries, government agencies, institutions and organizations before July 1, 2016; b) to develop draft national standards for fortified low and high extraction flour and present these standards at a regional food fortification conference on September 9-10, 2016 (tentatively). 6. Taking into account that the present regional GAIN/USAID project on food fortification in the CAR, Afghanistan and Pakistan ends in October 2016, it was proposed to initiate search for alternative sources of funding for further improvement of flour fortification agenda in the region based on the harmonized standards. 7. Disseminate workshop materials through web-resources and media (KAN s and GAIN s web pages, social networks, etc.) Annex 1 1 WHO, FAO, UNICEF, GAIN, MI, & FFI. Recommendations on wheat and maize flour fortification. Meeting Report: Interim Consensus Statement. Geneva, World Health Organization,

15 The members of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan, Pakistan Afghanistan: 1. Dr. M. Wakil Rahimi - Food and Agricultural Products Standards Department Manager, Afghan National Standards Authority (ANSA). Mobile No: (+93 ) , (+93) wakilrahimi@yahoo.com 2. Dr. Homyoun Lodin, head of Public Nutrition Department, Ministry of Public Health. nutrition.moph@gmail.com 3. Dr. Sedequllah Reshteen, Head of Legal Department, Ministry of Health sedequllahreshteen@gmail.com Kazakhstan: 1. Shamil Tazhibayev Vice President of Kazakh Academy of Nutrition. Address: 66 Klochkov Street, , Almaty, Kazakhstan. Tel. office: +7 (727) Mobile: stazhibayev@kan-kaz.org 2. Yuriy Sinyavskiy Vice President of Kazakh Academy of Nutrition. Address: 66 Klochkov Street, , Almaty, Kazakhstan. Tel. office: +7 (727) Mobile: sinyavskiy@list.ru 3. Evgeniy Gan President of Union of Grain Processors of Kazakhstan. Address: 6 Potanin Street, Astana, Kazakhstan. Tel. office: +7 (7172) , ; , Mobile: ewgan2005@mail.ru admin@kaznan.kz 4. Yelena Kuleshova Head of the Technical Standardizing Department, Institute of Standartization and Certification of Kazakhstan. elena_kuleshova@mail.ru Tel. office: +7 (7172) Mobile: Rakhimzhanova Maral Tleulesovna - Chief Expert for Health Surveillance of the Committee on Consumer Protection of the RK. Tel. Office: +7 (7172) Cell: mrt20@mail.ru Kyrgyz Republic 1. Berdimamat Adanbaev - Director of the Center for Standardization and Metrology of the Ministry of Economy of the Kyrgyz Republic. Tel.: ; b.adanbaev@mail.ru или nism@nism.gov.kg 2. Gulmira Kozhobergenova Civil Alliance on Nutrition and Food Security - gulmiraka@yandex.ru 3. Bazarbaev T.R.- The head of nutrition department, Department of disease prevention and state sanitary supervision, the MOH btoko@mail.ru 4. Bubudzhan Arykbayeva The Department of State Sanitary and Epidemiological Surveillance of the Ministry of Health of the Kyrgyz Republic - abk_cgsn@mail.ru Pakistan: 1. Dr. Shahzad Alam Chairman, Pakistan Council of Scientific and industrial Research (Government). Address: Head office, 1-Constitution Avenue, G-5/2, Islamabad, Pakistan. Office Phone: Cell Phone: chairmanpcsir@gmail.com 2. Dr. Abdul Baseer Khan Director Nutrition/ Secretary National Fortification Alliance, Ministry of National Health Services, Regulation and Coordination. Address: /O National Aids Control Program, National Institute of Health, Chak Shahzad Islamabad Pakistan. Office Phone: Cell Phone: achakzaibk@gmail.com 3. Mr. Barkat Saeed Memon Director General, Pakistan Standards and Quality Control Authority (Government). Address: Director General Pakistan Standards & Quality Control Authority 15

16 Government of Pakistan Block-77, Pakistan Secretariat, Saddar Karachi Pakistan. Office Phone: Cell Phone: Tajikistan: 1. Sultonova Ismonscho - Head of State Supervision Department in the Sphere of Agriculture (work); (cell). info@standard.tj 2. Abduvalieva Adiba- Head of Standardization Department, Tajikstandard (work); (cell). info@standard.tj 3. Dr Vokhidov Abdusalom, the Deputy Director of the Republican Center of the Pediatry and Pediatric Surgery. avokhidov@hotmail.com Uzbekistan: (only contact persons yet, focal persons will be determined soon) 1. Sherzod Atakhanov Food Fortification Program Manager, CPIB 'Health-III', Tashkent, Uzbekistan. Phone: (+99871) ; (Add 104). Fax: (+99871) ; Mobile phone: ( ) satakhanov@jpib.uz 2. Amankul Baikulov Consultant on food fortification in Uzbekistan. Mobile: abaykulov@jpib.uz 3. Diloram Gafurova Director, «Donmahsulatlary IIChM LLC». 16

17 Annex 2 The Program of the First Technical Meeting of the Regional Expert Group for Regional Harmonization of Wheat Flour Fortification Standards Day 1 March 14, :40 9:00 Registration Morning session 1 - the need and potential for regional harmonization of wheat flour fortification standards Moderator: Toregeldy Sharmanov, President, Kazakh Academy of Nutrition (KAN) 9:00 9:20 Opening remarks. Meeting objectives. Rationale for regional harmonization. Toregeldy Sharmanov, President, Kazakh Academy of Nutrition (KAN) Omar Dary, Ph.D., Health Science Specialist (Nutrition), USAID, Bureau of Global Health Yuliya Beloslyudtseva, Kazakhstan Country Coordinator, GAIN 9:20 10:00 The need and scientific approaches for regional harmonization of food fortification standards. Examples of exiting regional standards. Omar Dary, Ph.D., USAID Health Science Specialist (Nutrition), Bureau of Global Health 10:00 10:30 Analysis and justification of the possibility of harmonizing standards for wheat flour fortification in CAR, Afghanistan and Pakistan Shamil Tazhibayev, Vice President, KAN 10:30 10:45 Discussion 10:45 11:00 Coffee Break Morning session 2 - Country comments/feedback on the proposed technical justification for regional harmonization Moderator: Omar Dary, Ph.D., USAID Health Science Specialist (Nutrition), Bureau of Global Health 11:00 11:10 Feedback from Afghanistan, Dr. Homayoun Ludin, Director, Public Nutrition Department, Ministry of Public Health 11:10 11:20 Feedback from Kazakhstan, Evgeniy Gan, President, Union of Grain of Kazakhstan 11:20 11:30 Feedback from the Kyrgyz Republic, Berdimamat Adanbaev, Director of the Center for Standardization and Metrology of the Ministry of Economy 11:30 11:40 Feedback from Pakistan, Dr. Abdul Baseer Khan, Director Nutrition/ Secretary National Fortification Alliance, Ministry of National Health Services, Regulation and Coordination 11:40 11:50 Feedback from Tajikistan, Abdusalom Vokhidov, Deputy Director of the Republican Center of the Pediatrics and Pediatric Surgery 17

18 11:50 12:00 Feedback from Uzbekistan, Amankul Baikulov, Consultant, Food Fortification Program in Uzbekistan 12:00 12:30 Discussion 12:30 13:30 Lunch Afternoon session - Breakout discussions to reach technical consensus on harmonization of standards Moderator: Yuliya Beloslyudtseva, Kazakhstan Country Coordinator, GAIN 13:30 14:00 Regional standards for low- and high- extraction wheat flour fortification - examples for discussion Yuriy Sinyavskiy, Vice President, KAN 14: Discussion :30 (coffee break from 15:20-15:40) Low extraction flour expert sub-group Chair: Shamil Tazhibayev, Vice President, KAN High extraction flour expert sub-group Chair: Quentin Johnson, Technical Adviser, GAIN The discussion will cover the following: - an overview of the proposed justification/standards - clarification of input parameters (e.g., wheat flour consumption levels) - comments and concerns regarding the proposed justification/approach - a detailed discussion of each of the identified issues to reach an acceptable solution - the next steps to finalize the Regional Fortification Standards for low- and high extraction flour - feasibility, procedures and timeline for adoption of the Regional Standard at the country level 17:30-17:40 Day 1 Wrap-up Shamil Tazhibayev, Vice President, KAN Yuliya Beloslyudtseva, Kazakhstan Country Coordinator, GAIN 18:00-20:00 Networking dinner Day 2 March 15, 2016 Morning session 1 - Standards harmonization - presentations, remaining issues, development of solutions Moderator: Quentin Johnson, Technical Adviser, GAIN 9:00-9:20 Group 1 presentation. 9:00-9:20 Group 2 presentation. 9:40-10:40 Discussion. Development of solutions to each of the remaining issues. 18

19 10:40 11:00 Coffee break 11:00-12:30 Discussion (cont.). Conclusions. Next steps. 12:30 14:00 Lunch The time in the afternoon is reserved for additional discussions as necessary to reach regional agreements and commitments or for additional presentations 16:00-16:30 Close out session (summary of the deliberations and next steps) Shamil Tazhibayev, Vice President, KAN Quentin Johnson, Technical Adviser, GAIN Yuliya Beloslyudtseva, Kazakhstan Country Coordinator, GAIN Proposed ideas for other presentations: A comparative analysis of approaches for the development of national fortification standards and related technical protocols in CAR, Afghanistan and Pakistan 19

20 Annex 3 Participants of thetechnical Meeting Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan Almaty, Kazakhstan, March 2016 # Name Position Country 1 M. Wakil Rahimi Manager, Food and Agricultural Products Standards Department, Afghan National Standards AFG Authority (ANSA) 2 Homayoun Ludin Director, Public Nutrition Department, Ministry of Public Health AFG 3 Sedequllah Reshteen Head of Policy Department, Ministry of Health AFG 4 Abdul Baseer Khan Director Nutrition/ Secretary National Fortification Alliance, Ministry of National Health Services, PAK Regulation and Coordination 5 Barkat Saeed Memon Director General, Pakistan Standards and Quality Control Authority PAK 6 Toregeldy Sharmanov President, Kazakh Academy of Nutrition KAZ 7 Shamil Tazhibayev Vice-President, Kazakh Academy of Nutrition KAZ 8 Yuri Sinyavskiy Vice-President, Kazakh Academy of Nutrition KAZ 9 Oksana Dolmatova Chief Researcher, Kazakh Academy of Nutrition KAZ 10 Akkumis Salkhanova Leading Researcher, Kazakh Academy of Nutrition KAZ 11 Baurzhan Amirov Senior Researcher, Kazakh Academy of Nutrition KAZ 12 Evgeniy Gan President, Kazakhstan Union of Grain Processors KAZ 13 Maral Rakhimzhanova Chief Expert for Health Surveillance of the Committee on Consumer Protection of the RK KAZ 14 Yelena Kuleshova Head of the Technical Standardizing Department, KAZ Institute of Standardization and Certification 15 Dauletbek Kogashov Department of health care organizations, Ministry of KAZ Health and Social Development, RK 16 Aigul Nurgabylova UNICEF Kazakhstan KAZ 17 Berdimamat Adanbaev Director of the Center for Standardization and KYR Metrology of the Ministry of Economy 18 Gulmira Civil Alliance on Nutrition and Food Security KYR Kozhobergenova 19 T.R. Bazarbayev Head of Nutrition Division, the Department of State KYR Sanitary and Epidemiological Surveillance of the Ministry of Health of the Kyrgyz Republic 20 Ismonscho Sultonova Head of State Supervision Department in the Sphere TAJ of Agriculture 21 Adiba Abduvalieva Head of Standardization Department, TajikStandard TAJ 22 Abdusalom Vokhidov Deputy Director of the Republican Center of the TAJ Pediatry and Pediatric Surgery 23 Sherzod Atakhanov Head of Flour Fortification Program in Uzbekistan UZB 24 Amankul Baikulov Consultant, Food Fortification Program in UZB Uzbekistan 25 Diloram Gafurova Director, «Donmahsulatlary IIChM LLC» UZB 26 Omar Dary Health Science Specialist (Nutrition), USAID/WDC USAID 27 Yuliya Beloslyudtseva Kazakhstan Country Coordinator GAIN/KAZ 28 Quentin Johnson Technical Adviser GAIN 20

21 Recommendations of the working group on harmonization of low-extraction flour fortification standards Annex 4 1) Regional expert group arrived at a consensus and approved the document developed by the Kazakh Academy of Nutrition ( Analysis and justification of the possibility of harmonizing standards for low-extraction wheat flour fortification in target countries of the region: Afghanistan, Kazakhstan, Kyrgyzstan, Pakistan, Tajikistan, and Uzbekistan ), based on the WHO recommendations. 2) Expert group members will provide updated entry data on wheat flour consumption in each of their countries. 3) FF materials will be posted on the KAN s web-resource and/or Google platform to be shared with all stakeholders. 4) Proposed premix formula should include iron compound in the form of NaFeEDTA (15 ppm as recommended by the WHO) without adding Ferrous Sulfate. 5) As it pertains to vitamin В12 in the premix: KAN will develop a justification to support wheat flour fortification with vitamin B12 for target countries of the region before May 1, ) KAN to develop a justification for low-extraction wheat flour fortification standards without Ferrous Sulfate before May 1, ) Working group members in each of the target countries: a) to coordinate and agree the proposed premix formula with key ministries, government agencies, institutions and organizations before July 1, 2016; b) to develop draft national standards for fortified low-extraction flour before September 1, In this regard, the KAN experts will develop a draft template of national flour fortification standard in line with achieved consensus and share it with all target countries to facilitate development/adjustment of the national flour fortification standards. 8) It was jointly proposed to initiate search for alternative sources of funding for further improvement of flour fortification agenda in the region based on the harmonized standards, including the following actions: a) Organization of a high-level conference for decision-makers on aspects of the state support of flour fortification using the harmonized standards (premix purchase, fortification technology, preferential taxation, etc.); b) Improvement of national FF legislation in the countries of the region (specifically, to ensure and provide legislative framework for mandatory fortification of imported wheat flour). c) Monitoring of fortification, quality assurance and control, as well as coverage and availability of fortified flour to the population. d) Fortification program effectiveness evaluation, including anemia and micronutrient deficiencies prevention. e) Organization of an extended conference on the results of program evaluation with an overall goal to further improve implementation of the program, transfer and dissemination of experience, considering best practices and lessons learned. 21

22 Recommendations of the working group on harmonization of high-extraction flour fortification standards Annex 5 1) More stakeholder involvement at country level (ensuring engagement of flour millers). Creation of enabling environment for production, import and export of flour including legislation and tax exemptions. 2) To fortify high-extraction wheat flour with NaFeEDTA, folic acid, vitamin B12, and zinc. 3) Cost benefit analysis (CBA) in each of target countries in the region based on the current national conditions. Disseminate CBA results and findings among key decision-makers and top-level officials. 4) To study experience and best practices on premix revolving funds (using the example of Uzbekistan where revolving fund for premixes has been functioning for 10 years). 5) Establish Country Working Groups for development of harmonized FF standards. 22

23 Annex 2. On the necessity of low extraction wheat flour fortification with vitamin B12 in Central Asia Republics, Afghanistan and Pakistan Kazakh Academy of Nutrition USAID/GAIN MICRONUTRIENT FORTIFICATION PROJECT IN CENTRAL ASIA, AFGHANISTAN AND PAKISTAN ON THE NECESSITY OF LOW EXTRACTION WHEAT FLOUR FORTIFICATION WITH VITAMIN B12 IN CENTRAL ASIA REPUBLICS, AFGHANISTAN AND PAKISTAN The Document have been developed by Shamil Tazhibayev Professor, Vice President of Kazakh Academy of Nutrition under the edition of: Turegeldy Sharmanov Academician of RAS and NAS of RK, President of Kazakh Academy of Nutrition Omar Dary Professor, Health Science Specialist (Nutrition), USAID, Bureau of Global Health and Quentin Johnson Coordinator, Training and Technical Support Group, the Food Fortification Initiative Almaty

24 Contents 1. Background 2 2. Physiological functions of vitamin B Food sources 3 4. Absorption 3 5. Indicators of deficiency 4 6. The causes of deficiency 4 7. Health consequences and manifestations of deficiency 6 8. Prevalence of deficiency 7 9. Prevention of deficiency Requirements Is it needed to fortify flour with vitamin B12 in Central Asia Republics, Afghanistan and Pakistan? Conclusion Literature Acknowledgement 16 24

25 1. Background In October 2015, GAIN and USAID co-hosted a regional stakeholder meeting on food fortification that brought together representatives of governments, private sector, academia, civil society, and development partners from Kazakhstan, Afghanistan, Pakistan, Tajikistan, and Uzbekistan. Several joint priorities were identified, including facilitation of cross-border trade through harmonized standards of wheat flour. A Regional Expert Group on Wheat Flour Fortification Standards and Potential for Regional Harmonization was formed. It is chaired by the Kazakh Academy of Nutrition (KAN) and includes specialists representing national standards committees, government regulatory agencies, research institutions, wheat flour producers and respective business associations from six countries - Kazakhstan, Afghanistan, Pakistan, Tajikistan, Kyrgyzstan and Uzbekistan. During November 2015-January 2016, in consultation with international experts, the KAN members of the group developed a technical justification for harmonizing fortification standards for low extraction (refined, white) flour used in all countries of the region, and high extraction (whole) flour mainly used in Afghanistan and Pakistan. These two analytical documents were shared with the expert group for comments and additional input. The documents highlight the need to ensure, at a minimum, adequate and comparable amounts of bioavailable micronutrients in all countries of the region as specified in the WHO recommendations. Two options for the micronutrients in premix were proposed: High extraction flour Iron (NaFeEDTA) Zinc (Zinc oxide) Vit B9 (Folic acid) Vit B12 (Cyancobalamin) Low extraction flour Iron (NaFeEDTA and Ferrous sulfate, dried to increase iron content at a reasonable cost) Zinc (Zinc oxide) Vit B9 (Folic acid) Vit B12 (Cyancobalamin) Vit B1 (Thiamine) Vit B2 (Riboflavin) Vit B3 (Niacin) At the Technical Meeting of Regional expert group on harmonization of wheat flour fortification standards in Central Asian Republics, Afghanistan and Pakistan, in Almaty City, March 2016, the experts arrived at a consensus and agreed on the following premix composition to harmonize standards on low and high extraction wheat flour fortification, which is fully consistent with the recommendations of the World Health Organization [1]: High extraction flour Low extraction flour Micronutrients Levels, ppm Micronutrients Levels, ppm Iron (NaFeEDTA) 15,0 Iron (NaFeEDTA) 15,0 Zinc (Zinc oxide) 30,0 Zinc (Zinc oxide) 30,0 Vit B9 (Folic acid) 1,0 Vit B9 (Folic acid) 1,0 Vit B12 (Cyancobalamin) 0,008 Vit B12 (Cyancobalamin) 0,008 Vit B1 (Thiamine) 2,0 Vit B2 (Riboflavin) 3,0 Vit B3 (Niacin) 10,0 25

26 At the same time, Regional Expert Group invited the Kazakh Academy of Nutrition (KAN) to develop a justification to support wheat flour fortification with vitamin B12 for target countries of the region before May 1, Physiological functions of vitamin B12 Vitamin B12 (cobalamin) is a water-soluble vitamin. After the body uses these vitamins, leftover amounts leave the body through the urine. Vitamin B12, like the other B vitamins, is important for protein metabolism. It helps in the formation of red blood cells and in the maintenance of neurological function and the central nervous system. Vitamin B12 is also essential for DNA synthesis and for cellular energy production, the latter mainly from odd-chain fatty acids [2]. In mammalian cells there are only two vitamin B12-dependent enzymes [3]. Vitamin B12 functions as a cofactor for methioine synthase and L-methylmalonyl-CoA mutase [4]. Vitamin B12 (cobalamin) is a cofactor in the synthesis of an amino acid, methionine, and which is the main source of methyl groups for protein, nucleic acids, and neurotransmitter synthesis. Its metabolic role is closely linked to that of folate in that one of the vitamin B12-dependent enzymes, methionine synthase, is vital to the functioning of the methylation cycle in which 5- methyltetrahydrofolate acts as a source of methyl donor groups which are necessary for cell metabolism and survival. Deficiency of this vitamin can thus impair the utilization of folate and causes neurological deterioration, megaloblastic anaemia, elevated plasma homocysteine and possibly, impaired immune function. In infants and young children it can cause severe developmental delays [5]. Thus, the metabolism and the functions of vitamin B12 and folic acid are closely linked. Deficiency of vitamin B12, similar to that of folate, has been associated to the occurrence of neural tube defects [6-10]. 3. Food sources Food sources of vitamin B12 are the only animal products such as meat, eggs, and dairy and fish products [11, 12]. Plants do not contain this vitamin. Most microorganisms, including bacteria and algae, synthesize vitamin B12, and they constitute the only source of the vitamin, that is then transferred to tissues and organs of the animals where those microorganisms live [13]. The vitamin B12 synthesized in microorganisms enters the human food chain through incorporation into food of animal origin. In many animals, gastrointestinal fermentation supports the growth of these vitamin B12 synthesizing microorganisms, and subsequently the vitamin is absorbed and incorporated into the animal tissues. Because plants do not synthesize vitamin B12, individuals who consume diets completely free of animal products (vegan diets) are at risk of vitamin B12 deficiency. 4. Absorption The absorption of vitamin B12 in humans is complex [14]. Vitamin B12 in food is bound to proteins and is only released by the action of a high concentration of hydrochloric acid present in the stomach. This process results in the free form of the vitamin, which is immediately bound to a mixture of glycoproteins secreted by the stomach and salivary glands. These glycoproteins, called R-binders (or haptocorrins), protect vitamin B12 from chemical denaturation in the stomach. The stomach s parietal cells, which secrete hydrochloric acid, also secrete a glycoprotein called intrinsic factor. Intrinsic factor binds vitamin B12 and ultimately enables its active absorption. The vitamin B12 intrinsic factor complex then proceeds to the lower end of the small intestine, where it is absorbed by phagocytosis by specific ileal receptors [13]. Individuals consume about 2.4 μg vitamin B12 per day, of which 50-60% is absorbed [15]. Body s ability to absorb vitamin B12 from dietary supplements is limited by the capacity of intrinsic factor. Only about 10 mcg of a 500 mcg oral supplement is actually absorbed in healthy people [4]. 26

27 Following absorption, vitamin B12 is stored in the liver. A small proportion (1 5%) of free vitamin B12 is absorbed from the intestine by passive diffusion in an intrinsic factor independent process. 5. Indicators of deficiency Vitamin B12 status is usually assessed by measuring concentrations in plasma or serum. Although elevated urinary and plasma methylmalonic acid (MMA) levels are more specific, and often more sensitive, indicators of vitamin B12 deficiency. However, MMA concentrations are more difficult and expensive to measure than those of vitamin B12. Elevated homocysteine is a good predictor of vitamin B12 status (as also for folate status). The following cut-offs are used to define of Vitamin B12 deficiency [16-18]: <150 pmol/l (<203 µg/l) of Vitamin B12 in serum or plasma (reflects both recent intake and body stores) are indicative of deficiency; pmol/l ( µg/l) suggest depletion [19] and some authors have also suggested the values of pmol/l ( µg/l) for identifying marginal values [20]. If values above the cut-off do not necessarily indicate adequate status; if values are marginal, analysis of serum methylmalonic acid is indicated); >271 nmol/l of MMA in serum or plasma (increased when supply of vitamin B12 is low; preferred indicator since increased levels are highly specific to vitamin B12 deficiency); mmol/l ( mg/l) of total homocysteine (free and bound) in plasma (is a good predictor of vitamin B12 status: it is also increased in cases of inadequate folate status, and therefore it is not specific. Homocysteine concentrations also vary by vitamin B2 and B6 status, and they are influenced by gender, race and renal insufficiency. 6. The causes of deficiency The main risk factors for Vitamin B12 deficiency are: low intakes of animal products; malabsorption from food due to gastric atrophy induced by Helicobacter pylori, or bacterial overgrowth; genetic disorder of vitamin B12 metabolism. Deficiency linked to improper absorption rather than low consumption is the main reason of vitamin B12 in developed countries [21, 22]. However, in developing or in transition countries, However, in developing or in transition countries, the primary cause of vitamin B12 deficiency might be insufficient consumption of animal products. An inadequate intake, combined with increased requirement, and disturbed absorption (malabsorption) are exacerbating causes. The presence of internal factors in the digestive tract is an essential condition for absorption of vitamin B12 from food. In this regard, the vitamin B12 deficiency in humans can be developed because of its absorption disorders in the gastrointestinal tract in atrophic gastritis, surgical resection and removing the stomach. This is due to the secretion disorders of "intrinsic factor" which is necessary for absorption of vitamin B12. Normally this factor is released by cells of the gastric mucosa. Anemia caused by a lack of or reduction in the secretion of intrinsic factor is called pernicious anemia [23]. Decreasing diet energy intake to reduce body weight, which leads to a decrease in vitamins and minerals consumption may contribute to the development of vitamin B12 deficiency. Besides deficiency of intrinsic factor, the most common causes of vitamin B12 deficiency are the following: poor nutrition (especially veganism), old age (decreased absorption), increased demand (early childhood, pregnancy and lactation), alcoholism, long-term consumption of hypnotics, stomach injury (including surgical, autoimmune, and infectious and others), pancreas, and liver disease. Since vitamin B12 deposited in the liver, liver disease (especially viral) lead to a loss of vitamin in its depo. Pernicious anemia. 27

28 Malabsorption of vitamin B12 can occur at several points during digestion [14]. By far the most important condition resulting in vitamin B12 malabsorption is the autoimmune disease called pernicious anaemia (PA). In most cases of PA, antibodies are produced against the parietal cells causing them to atrophy, and lose their ability to produce intrinsic factor and secrete hydrochloric acid. In some forms of PA, the parietal cells remain intact but autoantibodies are produced against the intrinsic factor itself and attach to it, thus preventing it from binding vitamin B12. In another less common form of PA, the antibodies allow vitamin B12 to bind to the intrinsic factor but prevent the absorption of the intrinsic factor vitamin B12 complex by the ileal receptors. The incidence of PA increases markedly with age. PA also results in an inability to reabsorb the vitamin B12, which is secreted in the bile. Biliary secretion of vitamin B12 is estimated to be between 0.3 and 0.5mсg/day. Interruption of this so-called enterohepatic circulation of vitamin B12 causes the body to go into a significant negative balance for the vitamin. Once PA has been established, the lack of absorption of new vitamin B12 is compounded by the loss of the vitamin because of negative balance. When the stores have been depleted, the final stages of deficiency are often quite rapid, resulting in death in a period of months if left untreated. Atrophic gastritis. Historically, PA was considered to be the major cause of vitamin B12 deficiency, but it was a fairly rare condition, perhaps affecting between one and a few per cent of elderly populations. More recently, it has been suggested that a far more common problem is that of hypochlorhydria associated with atrophic gastritis, where there is a progressive reduction with age of the ability of the parietal cells to secrete hydrochloric acid [24]. It is claimed that perhaps up to one quarter of elderly subjects could have various degrees of hypochlorhydria as a result of atrophic gastritis. It has also been suggested that bacterial overgrowth in the stomach and intestine in individuals suffering from atrophic gastritis may also reduce vitamin B12 absorption. The absence of acid in the stomach is postulated to prevent the release of protein-bound vitamin B12 contained in food but not to interfere with the absorption of the free vitamin B12 found in fortified foods or supplements. Atrophic gastritis does not prevent the reabsorption of biliary vitamin B12 and therefore does not result in the negative balance seen in individuals with PA. Nonetheless, it is agreed that with time, a reduction in the amount of vitamin B12 absorbed from the diet will eventually deplete vitamin B12 stores, resulting in overt deficiency. When considering recommended nutrient intakes (RNIs) for vitamin B12 for the elderly, it is important to take into account the absorption of vitamin B12 from sources such as fortified foods or supplements as compared with dietary vitamin B12. Absorption of intakes of less than mсg/day is complete - that is, for daily intakes of less than mсg of free vitamin B12, the intrinsic factor-mediated system absorbs that entire amount. It is probable that this is also true of vitamin B12 in fortified foods, although this has not been specifically examined. However, absorption of food-bound vitamin B12 has been reported to vary from 9% to 60% depending on the study and the source of the vitamin, which is perhaps related to its incomplete release from food [16]. This has led many to estimate absorption as being up to 50% to correct for the bioavailability of vitamin B12 from food. Vitamin B12 interaction with folate. One of the vitamin B12-dependent enzymes, methionine synthase, functions in one of the two folate cycles, namely, the methylation cycle. This cycle is necessary to maintain availability of the methyl donor, S-adenosylmethionine. Interruption of the cycle reduces the level of S- adenosylmethionine. This occurs in PA and other causes of vitamin B12 deficiency, producing as a result demyelination of the peripheral nerves and the spinal column, giving rise to the clinical condition called subacute combined degeneration [13,15]. This neuropathy is one of the main presenting conditions in PA. The other principal presenting condition in PA is a megaloblastic anaemia morphologically identical to that seen in folate deficiency. Disruption of the methylation cycle also causes a lack of DNA biosynthesis and anaemia. 28

29 The methyl trap hypothesis is based on the fact that once the cofactor 5,10- methylenetetrahydrofolate is reduced by its reductase to form 5-methyltetrahydrofolate, the reverse reaction cannot occur. This suggests that the only way for the 5-methyltetrahydrofolate to be recycled to tetrahydrofolate, and thus to participate in DNA biosynthesis and cell division, is through the vitamin B12-dependent enzyme methionine synthase. When the activity of this synthase is compromised, as it would be in PA, the cellular folate will become progressively trapped as 5- methyltetrahydrofolate. This will result in a cellular pseudo-folate deficiency where, despite adequate amounts of folate, anaemia will develop, which is identical to that seen in true folate deficiency. Clinical symptoms of PA, therefore, include neuropathy, anaemia, or both. Treatment with vitamin B12, if given intramuscularly, will reactivate methionine synthase, allowing myelination to restart. 7. Health consequences and manifestations of deficiency The main health consequences and manifestations of vitamin B12 deficiency are the following: megaloblastic anaemia (the combination of severe folate deficiency and vitamin B12 deficiency can result in megaloblastic anaemia); severe deficiency can cause developmental delays, poor neurobehavioral performance and growth in infants and children, nerve demyelination and neurological dysfunction. vitamin B12 deficiency can serve as the risk factor for: neural tube defects; elevated plasma homocysteine; impaired cognitive function. The following symptoms develops at the deficiency of vitamin B12: Megaloblastic anemia, fatigue, weakness, constipation, loss of appetite, and weight loss [12]. Vitamin B12 and folate are required for DNA synthesis. A deficiency of any of these vitamins can cause megaloblastic anemia, which is characterized by the production of large and immature red (erythrocytes) and white (leukocytes) blood cells. In addition, vitamin B12 deficiency can cause neuropsychiatric disorders irrespective of the presence of anemia. Neurological changes, such as numbness and tingling in the hands and feet [25]. The development of neuropathy in vitamin B12 deficiency is associated with a defect in the synthesis of myelin required for the formation of the shell of the nerve fibers. The appearance of neurological symptoms prior to the development of anemia may be associated with the introduction of folate without cobalamin. Treatment by folate of the patient with vitamin B12 deficiency can correct the formation and function of red blood cells, but does not prevent the neurological disorders. In this case, the bone marrow function is stimulated; thereby maintaining an adequate erythropoiesis, but neuropathy continues to develop. Difficulty maintaining balance, depression, confusion, dementia, poor memory and soreness of the mouth or tounge [26]. By assessing bioavailable vitamin B-12 status in a large Canadian cohort accrued before and after FA fortification, it was found a 3-fold increase in the risk of neural tube defects (NTDs) in mothers who had vitamin B-12 status in the lower quartile, regardless of folic acid (FA) fortification [27]. Infancy: failure to thrive, movement disorders, developmental delays and megaloblastic anemia [28]. Elderly: vitamin B-12 deficiency is often associated with cognitive deficits. Cognition in the elderly may also be adversely affected at concentrations of vitamin B-12 above the traditional cutoffs for deficiency. By using markers such as holotranscobalamin and methylmalonic acid, it has been found that cognition is associated with vitamin B-12 status across the normal range. Possible mediators of this relation include brain atrophy and white matter damage, both of which are associated with low vitamin B-12 status. It is suggested that the elderly in particular should be encouraged to maintain a good, rather than just an adequate, vitamin B-12 status by dietary means [29]. 8. Prevalence of deficiency n considering the vitamin B-12 fortification of flour, it is important to know who is at risk of vitamin B-12 deficiency and whether those individuals would benefit from flour fortification. The 29

30 recent review by WHO showed that the majority of data on the prevalence of folate and vitamin B12 deficiencies are derived from relatively small, local surveys, but these and national survey data from a few countries suggest that deficiencies of both of these vitamins may be a public health problem that could affect many millions of people throughout the world. Low blood concentrations of the vitamins occur across population groups and in countries in various stages of development [30]. In large surveys in the United States and the United Kingdom, it has been demonstrated that the prevalence of vitamin B12 deficiency varies by age range, affecting at least 3% of those aged years old, 4% of those aged years, and 6% of those 60 years or over. Marginal depletion, defined in this study as a serum cobalamin of pmol/l, affects 15% of those aged years old and more than 20% of those aged 60 years or over [31]. In developing countries, deficiency is much more common, starting in early life and persisting across the life span. Inadequate intake, due to low consumption of animal-source foods, is the main cause of low serum vitamin B-12 in younger adults and likely the main cause in poor populations worldwide. In older persons, food-bound cobalamin malabsorption becomes the predominant cause of deficiency, at least in part due to gastric atrophy, but it is likely that most elderly can absorb the vitamin from fortified food. Fortification of flour with vitamin B-12 is likely to improve the status of most persons with low stores of this vitamin. In countries where vitamin B12 deficiency has been assessed at the national level, low serum vitamin B12 concentrations were prevalent, i.e. in Venezuela (11 12% in preschool and school-aged children), Germany (15% in women of reproductive age), the United Kingdom (31% of the elderly) and New Zealand (12% of the elderly). In smaller studies, a high proportion of low plasma vitamin B12 concentrations were found in Kenya (40% in schoolaged children), Zimbabwe (24% of the elderly), Israel (21% in adults), and India (46% in adults), while in other countries such as Botswana (preschool-aged children), Thailand (school-aged children) and Japan (adults), <1% of plasma vitamin B12 concentrations were low [19, 32-34]. So, there is a limitation connected with a lack of data on vitamin B12 deficiency simply because, to date, little investigation has taken place. This means that the prevalence of many deficiencies suspected of being relatively common (e.g. riboflavin (vitamin B2), vitamin B12, zinc and calcium) is not well known. In some cases, however, evidence of a deficiency in one micronutrient predicts the existence of deficiencies in others. For example, a high prevalence of anaemia and vitamin A deficiency is often accompanied by zinc, vitamin B12 and riboflavin (vitamin B2) deficiencies, because the underlying problem in all cases is an inadequate intake of animal source foods [5]. 9. Prevention of deficiency Prevention of vitamin B12 deficiency include three main approaches: consumption of animal products, which are the only sources of cobalamin; fortification of food with vitamin B12 (usually flour and intermediate cereal products are fortified); vitamin B12 supplementation by multivitamin or multivitamin-mineral preparations. In poor countries, flour fortification would potentially improve vitamin B-12 status in a much larger proportion of the population because of low usual intake of the vitamin in animal-source foods. Here individuals could benefit across the life span, although it could be especially beneficial for pregnant and lactating women, children, and the elderly. Confirming the efficacy of different levels of addition of the vitamin to flour on vitamin B-12 status and functional outcomes in different populations, including the elderly in different stages of gastric atrophy, should be a research priority. In wealthier countries, vitamin B-12 fortification of flour is most likely to lower the prevalence of inadequacy in the elderly, in those who consume low amounts of animal-source foods and fortified cereals, and in nonusers of supplements [35]. The work suggests that vitamin B-12 fortification, analogous to the FA fortification program, may reduce NTDs more than FA fortification alone. A multicenter randomized controlled trial comparing periconceptional vitamin B-12 in combination with FA against FA alone is warranted [27]. 30

31 Deficiencies in at least some of neglected micronutrients (i.e. in zinc, vitamins B2 and B12, niacin, vitamin D and calcium) are likely to be common throughout much of the developing world and among the poorest populations in the industrialized nations. Fortification provides a means of lowering the prevalence of deficiencies in all of these micronutrients, and their inclusion in mass fortification programmes could produce significant public health benefits [5]. This approach is important for prevention of anemia as well, since the presence of vitamins A and B12, folate and riboflavin deficiencies also increases the risk of anaemia, and vitamin B12, as with folate deficiency, the prevalence of neural tube defects [36]. 10. Requirements The daily human requirements for vitamin B12 are minimal, and it is measured in micrograms, depending on age (Table 1). Table 1 Recommended nutrient intakes (RNIs*) for vitamin B12 in different groups of population [18] Population groups RNI (mcg/day) Infants and children 0-6 months 0, months 0,7 1-3 years 0,9 4-6 years 1,2 7-9 years 1,8 Adolescents years 2,4 Adults years 2,4 65+ years 2,4 Pregnant women 2,6 Lactating women 2,8 * RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population. Upper limits. The absorption of vitamin B12 mediated by the glycoprotein, intrinsic factor, is limited to mсg per meal because of the limited capacity of the receptors. In addition, between 1% and 3% of any particular oral administration of vitamin B12 is absorbed by passive diffusion. Thus, if 1000 mсg vitamin B12 (sometimes used to treat those with pernicious anemia - PA) is taken orally, the amount absorbed would be 2.0 mсg by active absorption plus up to about 30 mсg by passive diffusion. Intake of 1000 mсg vitamin B12 has never been reported to have any adverse sideeffects [16]. Similar large amounts have been used in some preparations of nutritional supplements without apparent ill effects. However, there are no established benefits for such amounts. Such high intakes thus represent no benefit outside those with malabsorption and should probably be avoided for the population at large. 11. Is it needed to fortify flour with vitamin B12 in Central Asia Republics, Afghanistan and Pakistan? Unfortunately, no data on the prevalence of vitamin B12 deficiency in Central Asia Republics (CAR), Afghanistan and Pakistan. There are only a nationally representative data on the levels of vitamin B12 intake in the various foodstuffs of animal origin in Kazakhstan in 2008 [37]. These findings will be discussed below and used to justify the need for fortification of flour with vitamin B12 in CAR, Afghanistan and Pakistan. 31

32 The main suppliers of vitamin B12 in the diet of people in Kazakhstan have been exclusively the following animal origin products (Table 2): meat 2.5 mcg/day (87.3%), milk 0.28 mcg/day (9.8%), fish 0.05 mcg/day (1.6%) and eggs 0.04 mcg/day (1.3%). Average daily consumption of cyanocobalamin by males was 3.3 mcg/day and by females mcg/day, average intake by males and females 2.9 mcg/day at the requirement of adolescents and adults for this vitamin equal to 2.4 mcg/day. Table 2 - Average daily consumption of animal origin products, and vitamin B12 intake (mcg/day) be males and females Nutrients and Measurement Males Females Total food items units Mean Median Mean Median average Vitamin В12, total mcg/day 3,3 2,2 2,6 1,7 2,9 Meat and meat g/day products Vitamin B12 in mcg/day 2,9 1,9 2,3 1,5 2,5 the composition of meat Milk and milk g/day products Vitamin B12 in mcg/day 0,32 0,22 0,25 0,17 0,28 the composition of milk Fish and fish g/day products Vitamin B12 in mcg/day 0,05 0,04 0,04 0,03 0,05 the composition of fish Eggs g/day Vitamin B12 in the composition of eggs mcg/day 0,04 0,03 0,03 0,02 0,04 However, even at quite satisfactory average level of cobalamin intake, 33-56% of respondents had been exposed to the risk of vitamin B12 deficiency (Table 3). 32

33 Table 3 - Percentage of people aged years with a high risk of vitamin B12 deficiency Residence The number of people surveyed % of people with vitamin B12 intake level 2/3 of RNI * лет 19 лет males females males females Total Urban Rural * RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population Such a high percentage of people at risk of vitamin B12 deficiency at a reasonable average level of cobalamin intake, may be due to inequalities of consumption of animal products, which are an expensive part of the diet. This is confirmed by statistically significant differences in the levels of vitamin B12 intake, depending on the income level of the population (Table 4). Table 4 - The consumption of vitamin B12 (in mg / day) of men and women years depending on the level of income (KZT/month A ) Пол Р ,43 KZT/month Р20 >3571, KZT/month Percentile, income levels Р < KZT/month Р KZT/month Males 1 st group 5 th group 6 th group 2 nd group В12, mcg/day 2,66 2,91 3,54 3,56 Females B 3 rd group 7 th group 8 th group 4 th group В12, mcg/day 1,89 2,09 3,35 3,49 A - Exchange rate of KZT to dollar averaged 120/1 at the end of July and in August 2008, when the fieldwork for data collection was conducted. B - The difference was statistically significant at t 2,64 (p <0.0042) at using the multiple comparison analysis according to Holm-Bonferroni [38, 39], namely at comparison of Groups 3-4 (t = -2,9); Groups 7-8 (t = -3,0); Groups 7-4 (t = 5,2). It can be expected that the non-uniformity of consumption of animal origin products takes place in other member-countries. This is confirmed by the World Bank data about the differences in gross domestic product per capita and income inequality in member-countries (Table 5). Moreover, the level of GDP in Kazakhstan many times higher than in other member-countries, and Gini indices on income inequality is lower than in Kyrgyzstan, Pakistan, Tajikistan, and Uzbekistan. 33

34 Table 5 - Gross domestic product (GDP) per capita and Gini indices on income inequality in the CARs and Afghanistan and Pakistan Countries GDP [40] Gini indices on income inequality [41] Year Per capita/year, US$ Year Gini index score A Gini index rank B Afghanistan , Kazakhstan , Kyrgyzstan , Pakistan , Tajikistan , Uzbekistan , A 0 = Perfect equality, 100 = perfect inequality. B The countries with a Gini index are ranked from most equal (#1) to most unequal (#145). Moreover, a consumption of animal products, and thus vitamin B12, in other member-states are significantly lower than in Kazakhstan (Table 6). In particular, the intake of vitamin B12 from food in other member-countries are less of RNI for persons 10 years and older (2.4 mcg / day), ranging from 29.6% RNI (Afghanistan) to 91.2% RNI (Kyrgyzstan). Only in Kazakhstan vitamin B12 intake from food is above of RNI and it is equal to 122.3% of RNI. However, even in Kazakhstan, as described above, 33-56% of respondents were exposed to vitamin B12 deficiency due to uneven consumption of dietary sources of cobalamin, which may be caused by income inequality of population. These data indicate the feasibility of wheat flour fortification with vitamin B12 in all participating countries, including Kazakhstan. In this case the level of flour fortification with vitamin B12, equal to 0,004 ppm, can be used as opposed to the recommended by the World Health Organization (WHO) level equal to 0,008 ppm, as there is currently no data on the prevalence of vitamin B12 deficiency in the participating countries. Later, after studying the prevalence of vitamin B12 deficiency in the member-countries, the level of flour fortification with vitamin B12 may be revised in the light of the data received. Table 6 Estimated intake of Vitamin B12 and its food sources in CAR and Afghanistan and Pakistan Country Year Per capita intake: all population A) Per capita intake: adults, years B) kg/year g/day kg/year g/day per 100 g of product Vit B12 intake, mcg C) per capita/day Vit B12 intake levels, % of RNI Afghanistan 2013 Meat 12,33 33,78 14,796 40,536 1,41 0,572 Milk 62,23 170,49 74, ,58 0,06 0,123 8 Fish 0,07 0,19 0,084 0,228 0,3 0,001 Eggs 1,30 3,56 1,56 4,272 0,35 0,015 Total 0,710 29,6 Kazakhstan D) 2008 Meat 66,80 183,00 1,41 2,580 Milk 163,52 448,00 0,06 0,269 Fish 5,84 16,00 0,30 0,048 Eggs 4,02 11,00 0,35 0,039 34

35 Country Year Per capita intake: all population A) Per capita intake: adults, years B) Vit B12 intake, mcg C) Vit B12 intake levels, % of RNI kg/year g/day kg/year g/day per 100 g of product per capita/day Total 2, ,3 Kyrgyzstan 2011 Meat 36,83 100,90 44, ,08 1,41 1,707 Milk 208,22 570,47 249,86 684,56 0,06 0, Fish 1,85 5,07 2,22 6,084 0,3 0,018 Eggs 4,61 12,63 5,532 15,156 0,35 0,053 Total 2,189 91,2 Pakistan 2013 Meat 16,41 44,96 19,692 53,952 1,41 0,761 Milk 183,13 501,73 219,75 602,07 0,06 0, Fish 1,92 5,26 2,304 6,312 0,3 0,019 Eggs 2,95 8,08 3,54 9,696 0,35 0,034 Total 1,175 49,0 Tajikstan 2011 Meat 13,95 38,22 16,74 45,864 1,41 0,647 Milk 52,85 144,79 63,42 173,74 0,06 0,104 8 Fish 0,13 0,36 0,156 0,432 0,3 0,001 Eggs 1,79 4,90 2,148 5,88 0,35 0,021 Total 0,773 32,2 Uzbekistan 2011 Meat 33,89 92,85 40, ,42 1,41 1,571 Milk 133,92 366,90 160,70 440,28 0,06 0,264 4 Fish 0,46 1,26 0,552 1,512 0,3 0,005 Eggs 4,81 13,18 5,772 15,816 0,35 0,055 Total 1,895 79,0 Note: A) FAO stat data B) - Estimated data obtained taking into account that the consumption of food by adults per capita 20% higher than by entire population. The exception is Kazakhstan, where the data of 2008 national studies are used. C) - Estimated data in comparison with appropriate Kazakhstan s national studies data of 2008 D) - The results of National nutrition study in Kazakhstan, 2008 Table 7 summarizes estimated intake levels of vitamin B12 with animal origin food sources and with fortified wheat flour at the levels of fortification 0,004 ppm and 0,008 ppm in CARs and Afghanistan and Pakistan. These data confirm the feasibility of flour fortification with 0,004 ppm of vitamin B12 in CARs and Afghanistan and Pakistan, which could serve as a good means for preventing cobalamin deficiency based on actual consumption of its food sources. 35

36 Table 7 Estimated intake levels of vitamin B12 by animal origin food sources and by fortified wheat flour* in CAR and Afghanistan and Pakistan Countries Vitamin B12 intake levels, % of RNI By animal origin food sources, adults By fortified wheat flour, people years old, at fortification levels: years old 0,004 ppm 0,008 ppm Afghanistan 29,6 47,9-62,2 95,8-124,4 Kazakhstan 122,3 28,2-36,2 56,4-73,3 Kyrgyzstan 91,2 41,2-53,6 82,4-107 Pakistan 49,0 34,0-44,1 67,9-88,2 Tajikistan 32,2 38,2-49,7 76,4-99,3 Uzbekistan 79,0 51,0-66,2 101,9-132,4 * - Fortification level of wheat flour by vitamin B12 is 0,004 ppm 12. Conclusion The above given analysis of the available data confirm the feasibility of flour fortification with 0,004 ppm of vitamin B12 in Central-Asian Republics and Afghanistan and Pakistan. It is proposed to use the following premix composition to harmonize standards on low extraction wheat flour fortification. Low extraction flour Micronutrients Levels, ppm Iron (NaFeEDTA) 15,0 Zinc (Zinc oxide) 30,0 Vit B9 (Folic acid) 1,0 Vit B12 (Cyancobalamin) 0,004 Vit B1 (Thiamine) 2,0 Vit B2 (Riboflavin) 3,0 Vit B3 (Niacin) 10,0 Attachment: The updated version of the document Analysis and justification the possibility of harmonizing standards for refined wheat flour fortification in Central Asia, Afghanistan and Pakistan. In this document, the level of flour fortification with vitamin B12 reduced from 0,008 ppm to 0,004 ppm, and all of appropriate data are recalculated. 36

37 13. Literature 1. WHO, FAO, UNICEF, GAIN, MI, & FFI. Recommendations on wheat and maize flour fortification. Meeting Report: Interim Consensus Statement. Geneva, World Health Organization, Fiona O Leary and Samir Samman. Vitamin B12 in Health and Disease. Nutrients 2010, 2, ; doi: /nu Scott, J.M. & Weir, D.G Folate/vitamin B12 interrelationships. Essays in Biochemistry, p Virendra Goyal. Vitamin B12 A brief overview. IAIM 2015; 2(4): Guidelines on food fortification with micronutrients, edited by Lindsay Allen, Bruno de Benoist, Omar Dary and Richard Hurrell. WHO and FAO, 2006, 376 p. 6. Kirke, P.N.; Molloy, A.M.; Daly, L.E.; Burke, H.; Weir, D.G.; Scott, J.M. Maternal plasma folate and vitamin B12 are independent risk factors for neural tube defects. Q. J. Med. 1993, 86, Suarez, L.; Hendricks, K.; Felkner, M.; Gunter, E. Maternal serum B12 levels and risk for neural tube defects in a Texas-Mexico border population. Ann. Epidemiol. 2003, 13, Gaber, K.R.; Farag, M.K.; Soliman, S.E.; El-Bassyouni, H.T.; El-Kamah, G. Maternal vitamin B12 and the risk of fetal neural tube defects in Egyptian patients. Clin. Lab. 2007, 53, Zhang, T.; Xin, R.; Gu, X.; Wang, F.; Pei, L.; Lin, L.; Chen, G.; Wu, J.; Zheng, X. Maternal serum vitamin B12, folate and homocysteine and the risk of neural tube defects in the offspring in a high-risk area of China. Public Health Nutr. 2009, 12, Molloy, A.M.; Kirke, P.N.; Troendle, J.F.; Burke, H.; Sutton, M.; Brody, L.C.; Scott, J.M.; Mills, J.L. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic Acid fortification. Pediatrics 2009, 123, Antony AC. Vegetarianism and vitamin B-12 (cobalamin) deficiency. Am J Clin Nutr 2003;78: L. R. McDowell. Vitamins in Animal and Human Nutrition, John Wiley & Sons, Weir DG, Scott JM. Vitamin B12. In: Shils ME et al., eds. Modern nutrition in health and disease. Baltimore, MA, Williams & Wilkins, 1999: Weir DG, Scott JM. Cobalamins physiology, dietary sources and requirements. In: Sadler M, Strain JJ, Caballero B, eds. Encyclopedia of human nutrition. Volume 1. San Diego, CA, Academic Press, 1998: Carmel R. How I treat cobalamin (vitamin B12) deficiency. Blood 2008;112: Food and Nutrition Board. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington, DC, National Academy Press, 1998, 591 p Rucker RB et al. Handbook of vitamins. 3rd ed. New York, Marcel Dekker, Vitamin and mineral requirements in human nutrition. Report of a Joint FAO/WHO Expert Consultation on Human Vitamin and Mineral Requirements, Bangkok,Thailand, September nd ed. Geneva,World Health Organization, Allen LH. Folate and vitamin B12 status in the Americas. Nutrition Reviews, 2004, 62 (6 Pt 2):S29 S Selhub J, Morris MS, Jacques PF, Rosenberg IH. Folate vitamin B-12 interaction in relation to cognitive impairment, anemia, and biochemical indicators of vitamin B-12 deficiency. Am J Clin Nutr 2009;89(suppl):702S 6S. 21. M. Kikuchi, S. Kashii, Y. Honda, Y. H Tamura, K. Kaneda, A. Akaike. Protective effects of methylcobalamin, a vitamin B12 analog, against glutamate-induced neurotoxicity in retinal cell culture. Investigative Ophthalmology and Visual Science, 1997; 38(5): Virendra Goyal. Review Article - Vitamin B12. IOSR Journal Of Pharmacy, Volume 5, Issue 3 (March 2015), PP

38 23. Chanarin I. The megaloblastic anaemias, 2nd ed. Oxford, Blackwell Scientific Publications, Carmel R. Prevalence of undiagnosed pernicious anaemia in the elderly. Archives of Internal Medicine, 1996, 156: R. Banerjee and S. W. Ragsdale, The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes, Annual Review of Biochemistry, vol. 72, pp , S. K. Ghosh, N. Rawal, S. K. Syed, W. K. Paik, and S. D. Kim, Enzymic methylation of myelin basic protein in myelin, Biochemical Journal, vol. 275, part 2, pp , Thompson MD, Cole DEC, Ray JG. Vitamin B-12 and neural tube defects: the Canadian experience. Am J Clin Nutr 2009;89(suppl):697S 701S. 28. Pfohl-Leszkowicz, G. Keith, and G. Dirheimer, Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: methylcobalamin can act as a methyl donor, Biochemistry, vol. 30, no. 32, pp , Smith AD, Refsum H. Vitamin B-12 and cognition in the elderly. Am J Clin Nutr 2009;89 (suppl):707s 11S. 30. Conclusions of a WHO Technical Consultation on folate and vitamin B12 deficiencies. Food and Nutrition Bulletin, vol. 29, no. 2 (supplement), S , The United Nations University. 31. How common is vitamin B12 deficiency? Am J Clin Nutr February 2009, vol. 89 no S-696S Refsum H et al. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. American Journal of Clinical Nutrition, 2001, 74: Siekmann JH et al. Kenyan school children have multiple micronutrient deficiencies, but increased plasma vitamin B-12 is the only detectable micronutrient response to meat or milk supplementation. Journal of Nutrition, 2003, 133:3972S 3980S. 34. Review of the magnitude of Folate and Vitamin B12 deficiencies worldwide. McLean E, de Benoist B, Allen LH, Green R. Is it time for vitamin B-12 fortification? What are the questions?. Am J Clin Nutr 2009;89(suppl):712S 6S. 36. Allen LH, Casterline-Sabel JE. Prevalence and causes of nutritional anemias. In: Ramakrishnan U, ed. Nutritional Anemias. Boca Raton, FL, CRC Press, 2000: A study to assess the nutritional status and health of the population in Kazakhstan: final report (in Russian). Kazakh Academy of Nutrition, 2008, 295 p. 38. Holm, S. (1979). "A simple sequentially rejective multiple test procedure". Scandinavian Journal of Statistics 6 (2): Bernard Rosner. Fundamentals of Biostatistics, 4th edit P. 40. World Bank World Bank Acknowledgement: This document was made possible by the generous support of the American people through the United States Agency for International Development (USAID). The contests are the responsibility of the Kazakh Academy of Nutrition (KAN) and GAIN and do not necessarily reflect the views of USAID or the United States Government. 38

39 Annex 3. Analysis and Justification the Possibility of Harmonizing Standards for Refined Wheat Flour Fortification in Central Asia, Afghanistan and Pakistan Kazakh Academy of Nutrition USAID/GAIN MICRONUTRIENT FORTIFICATION PROJECT IN CENTRAL ASIA, AFGHANISTAN AND PAKISTAN ANALYSIS AND JUSTIFICATION THE POSSIBILITY OF HARMONIZING STANDARDS FOR REFINED WHEAT FLOUR FORTIFICATION IN CENTRAL ASIA, AFGHANISTAN AND PAKISTAN The Document have been developed by Shamil Tazhibayev Professor, Vice President of Kazakh Academy of Nutrition under the edition of: Turegeldy Sharmanov Academician of RAS and NAS of RK, President of Kazakh Academy of Nutrition Omar Dary Professor, Health Science Specialist (Nutrition), USAID, Bureau of Global Health and Quentin Johnson Coordinator, Training and Technical Support Group, the Food Fortification Initiative Almaty

40 Contents 1. Comparison of standards for refined wheat flour fortification, operating in the Member Countries with the relevant recommendations of the World Health 3 Organization (WHO) 2. Standards for mandatory and voluntary fortification of wheat flour 5 3. Setting the level of bioavailability of minerals in Afghanistan, Kazakhstan, Kyrgyzstan, Pakistan, Tajikistan and Uzbekistan 6 4. Premix formulation for fortification of refined wheat flour 9 5. Establishment of production and regulatory parameters for fortification of refined wheat flour Daily intake of micronutrients by population groups in the composition of fortified wheat flour, by countries, in % EAR/day Daily intake of micronutrients by population groups in the composition of fortified 18 wheat flour, by countries, in % RNI/day 8. Brief description of the annexes Conclusion 24 Annex 1 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Afghanistan Annex 2 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Kazakhstan Annex 3 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Kyrgyzstan Annex 4 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Pakistan Annex 5 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Tajikistan Annex 6 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Uzbekistan Literature 43 Acknowledgement

41 1. Comparison of standards for refined wheat flour fortification, operating in the Member Countries with the relevant recommendations of the World Health Organization (WHO). According to an interim consensus statement, endorsed by WHO and other institutions about recommendations on wheat and maize flour fortification [1] (reference), the average levels of nutrients that can be added to fortifying wheat flour depending on the: estimated average per capita consumption (g/day) of wheat flour for all the recommended micronutrients; extraction level of flour (low or high) for iron and zinc; fortifying nutrient for all the recommended micronutrients, and others are required by the nutrition situation of the country (Table 1). NaFeEDTA, ferrous sulfate, ferrous fumarate and electrolytic iron may serve as the source of iron. However electrolytic iron cannot be used neither as an iron source in cases where the estimated average per capita consumption of wheat flour is less than 150 g/day nor for high-extraction flour (i.e. whole flour). (high-extraction-rate = 80% wheat flour [2]; so, low-extraction-rate = <80% wheat flour). This is because very high levels of electrolytic iron that are needed could negatively affect sensory properties of fortified flour. The average per capita consumption of wheat flour in the member countries is [3]: 258,7 g/day in Kazakhstan; 311,3 g/day in Pakistan; 350,3 g/day in Tajikistan; 377,9 g/day in Kyrgyzstan; 439,0 g/day in Afghanistan; 467,3 g/day in Uzbekistan. Due to differences in the average per day consumption of wheat flour per person: Kazakhstan assigned to countries where average consumption of wheat flour per capita is g/day; Pakistan, Tajikistan, Kyrgyzstan, Afghanistan and Uzbekistan to countries where average consumption of wheat flour per capita is >300 g/day. Overall, the average levels of per capita per day consumption of flour in the participating countries are high and may contribute to the harmonization of standards for flour fortification. In 4 countries (Kazakhstan, Kyrgyzstan, Tajikistan and Uzbekistan) flour is currently requested to be fortified with 6 micronutrients: vitamins B1, B2, B3 (niacin) and B9 (folate), iron and zinc; in Afghanistan, flour is expected to be fortified by 4 micronutrients (vitamins B9 and B12, iron and zinc); in Pakistan, currently only by 2 micronutrients - vitamin B9 and iron, are required at the national level (Table 2). Here, it is important to point out that in Afghanistan and Pakistan, the consumption of high-extraction flour is common, while in the other countries low extraction flour is more popular. For high-extraction flour, the incorporation of vitamins B1, B2, B3 (niacin) is unnecessary as the intrinsic content of these micronutrients in that type of flour is high The levels of added micronutrients to the flour: in Kazakhstan and Tajikistan slightly higher for supreme grade flour than for the first grade one; in Kyrgyzstan such differences do not exist; in Uzbekistan the data are given for fortification of only the first grade flour; in Afghanistan and Pakistan, data referred to the flour called Atta, which traditionally refers to high-extraction flour. The levels of added micronutrients to the flour in comparison with the WHO-endorsed recommendations are: slightly higher in Uzbekistan and Pakistan for vitamin В9; somewhat lower in Kazakhstan and Pakistan for iron; somewhat lower in Kazakhstan, Tajikistan and Uzbekistan for zinc; 41

42 other parameters in those countries comply with the recommendations of WHO; in Afghanistan and Kyrgyzstan, all the parameters are relevant with the WHO document. Table 1 WHO interim consensus statement about recommendations for fortified flours, 2009 a : Average levels of nutrients to consider adding to fortified wheat flour based on extraction, fortificant compound, and estimated per capita flour availability Nutrient Flour extraction rate Compound Level of nutrient to be added in parts per million (ppm) by estimated average per capita wheat flour availability (g/day) b <75g/day c >300g/day g/day g/day Iron Low NaFeEDTA Ferrous sulphate Ferrous fumarate Electrolytic iron NR d NR d High NaFeEDTA Folic acid Low or Folic acid high Vitamin Low or Cyanocobalamin B12 high Vitamin Low or Vit A palmitate A high Zinc e Low Zinc oxide High Zinc oxide Notes: a WHO, FAO, UNICEF, GAIN, MI, & FFI. Recommendations on wheat and maize flour fortification. Meeting Report: Interim Consensus Statement. Geneva, World Health Organization, b These estimated levels consider only wheat flour as main fortification vehicle in a public health program. If other mass-fortification programs with other food vehicles are implemented effectively, these suggested fortification levels may need to be adjusted downwards as needed. c Estimated per capita consumption of <75 g/day does not allow for addition of sufficient level of fortificant to cover micronutrients needs for women of childbearing age. Fortification of additional food vehicles and other interventions should be considered. d NR = Not Recommended because very high levels of electrolytic iron needed could negatively affect sensory properties of fortified flour. e These amounts of zinc fortification assume 5 mg zinc intake and no additional phytate intake from other dietary sources 42

43 Table 2 Summary data on the levels of micronutrients to be added in parts per million (ppm) to fortified wheat flour according to standards in Central Asian Republics, Afghanistan and Pakistan Items Kazakhstan Kyrgyzstan Tajikistan Uzbekistan Pakistan Afghanistan premium & premium premium & 1st grade 1st grade & 1st grade Atta [8] Atta [9] 1st grade [4] [7] [5] [6] Vitamin 2.0 & 1.6 2,0 2.0 & 1.6 1,6 n/f n/f B1 Vitamin B2 Vitamin B3 Vitamin B9 WHO, 2009 Vitamin B & 2.4 3,0 3.0 & 2.4 2,4 n/f n/f 10.0 & , & 8.0 8,0 n/f n/f 1.5 & 1.2 1,0 1.5 & 1.2 1,2 1,5 1,0 1,3 1,0 1,0 1,0 1,0 1,0 n/f n/f n/f n/f n/f 0,008 WHO, ,01 0,008 0,008 0,008 0,008 0,008 Iron 50.0 & , & ,0 10,0 15,0 WHO, ,0 15,0 40,0 40,0 15,0 15,0 Electrolytic Sodium Sodium Sodium Iron Electrolytic Electrolytic Iron EDTA Iron EDTA EDTA Zinc 22.0 & , & ,6 n/a 30,0 WHO, ,0 30,0 30,0 30,0 30,0 30,0 Notes: n/f not fortified Wheat flour consumption in g/capita/day: < 300 in Kazakhstan > 300 in Afghanistan, Kyrgyzstan, Pakistan, Tajikistan and Uzbekistan 2. Standards for mandatory and voluntary fortification of wheat flour. It seems to be appropriate to develop the following standards for: Mandatory fortification of flour with micronutrients endorsed by the World Health Organization, namely, by vitamins B9 and B12, iron and zinc for all types of flour, plus B1, B2 and B3 for refined (low extraction) flour; As the high-extraction wheat flour contains good amounts of the latter vitamins (B1, B2 and B3); those would not be added to this type of flour; Carry out studies of vitamin A and vitamin D deficiencies in all the countries in order to establish if the addition of these other micronutrients would be appropriate for the whole region. 43

44 3. Setting the level of bioavailability of minerals in Afghanistan, Kazakhstan, Kyrgyzstan, Pakistan, Tajikistan and Uzbekistan. The Food Agriculture Organization of the United Nations/World Health Organization set iron bioavailability at 5% for a strict vegetarian diet, at 10% when some meat and ascorbic acid was added, and at 15% for diets rich in meat and fruits [10]. In our calculations, we used the following assumptions: The diet of the population of Kazakhstan, where the average per capita intake of wheat flour is about 250 g/day, and generous contribution of meat/poultry and vegetables/fruits can be attributed to the group with a high bioavailability (15%) of minerals. Other participating countries where the average per capita intake of low-extraction wheat flour is more than 300 g/day, may be referred to the group with 10% or moderate bioavailability (Kyrgyzstan, Tajikistan and Uzbekistan), and for those consuming high-extraction wheat flour as 5% or low bioavailability (Afghanistan and Pakistan). In the case of zinc, the first country would have good bioavailability (30%), and the rest moderate bioavailability (15%). This subdivision of diet dependent levels of minerals bioavailability we took into account for calculating the levels of micronutrients in fortified flour and comparing them with vitamin and mineral requirements in human nutrition according to WHO/FAO data [11]. Our considerations are based on the average per capita consumption levels of main promoters (meat and meat products calculated per meat, vegetables and fruits) and inhibitors (cereals, which are reach in phytic acids) in the member countries (Table 3). Table 3 Per capita consumption of main dietary promoters and inhibitors of iron absorption in member countries* Promotors Per capita consumption (g/day or ratio**) of main dietary promoters and inhibitors & of iron absorption, by countries inhibitors Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan g/da y rati o g/da y rati o g/da y rati o g/da y rati o g/da y rati o g/da y rati o Promoter s Meat & 34 0, , ,5 45 0,2 38 0,2 93 0,4 meat products (heme iron) Vegetable 79 0, , ,7 72 0, , ,1 s (vitamin C) Fruits 93 0, ,0 85 0,4 80 0,4 78 0, ,8 (vitamin C) Total 0,8 3,0 1,6 0,8 1,3 2,3 Inhibitors Wheat 351, 13,6 25,9 1,0 37,8 1,5 248, 9,6 35,0 1,4 46,7 1,8 flour 2 8 Milk and 171 0, , , , , ,5 dairy products (calcium)? Total 13,8 2,0 2,2 10,3 1,6 2,3 44

45 Promotors & inhibitors Difference (between promoters & inhibitors) Per capita consumption (g/day or ratio**) of main dietary promoters and inhibitors of iron absorption, by countries Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan g/da y rati o g/da y rati o g/da y rati o g/da y rati o g/da y rati o g/da y rati o -13 1,0-0,6-9,5-0,3 0,0 Notes: * - Adapted from reference [12] ** - with respect to indicators of Kazakhstan Based on the data in Table 4 and literature sources [12, 13], it can be recommended to use a high (15%) of bioavailability of minerals in Kazakhstan, a moderate (10%) in Kyrgyzstan, Tajikistan and Uzbekistan, and low (5%) in Afghanistan and Pakistan. There are other dietary promoters and inhibitors of iron absorption (Table 4). However, the data on per capita consumption for some of those promoters and inhibitors are not available. Food and/or food compounds Promoters Acid ascorbic Heme iron Muscle tissue, the digestion products of meat, fish or poultry Fermented or germinated food and condiments Caseinophosphopeptides (CPPs) Polyoxycarbonic acids Inhibitors Phytate or phytic acid Polyphenols Calcium Proteins Table 4 - Dietary promoters and inhibitors of iron absorption. Source: Adapted from reference [14] Comments Present in fruits, juices and vegetables such as green leaves, peppers Present in meat, poultry, fish and seafood (~40% of the total iron) 30g of muscle has the enhancer property as 25mg of ascorbic acid, possible due to the presence of cysteine- containing peptides or a multitude of small peptides Sauerkraut and soy sauce (cooking, fermentation, or germination of food reduces the amount of phytates) The CPPs added to fruit beverage (grape and orange) appears to improve iron bioavailability*. Such as citrate and malate Present in cereal grains, high-extraction flour, legumes, and seeds Foods that contain the most potent inhibitors (e.g. tannins) resistant to the influence of enhancers include tea, coffee, cocoa, herbal infusions (tea) in general, certain spices (e.g. oregano), and some vegetables Particularly from milk and milk products found as calcium phosphate, inhibit absorption of non-heme and heme iron Proteins from products like milk and eggs, and albumin, casein, and soy protein (independent of the phytate content) 45

46 The total iron content of a diet provides little information about its content of bioavailable iron, which is considerably influenced by the foods in the diet and can vary 10-fold from different meals of similar iron content [15]. Although a vegetarian diet is likely to contain iron in amounts equivalent to amounts in a nonvegetarian diet, the iron from a vegetarian diet is likely to be substantially less available for absorption [16] because of differences in the chemical form of iron and the accompanying constituents that enhance or inhibit iron absorption [17]. The chemical form of iron is an important factor affecting the iron availability of vegetarian diets. In the diet inorganic iron-salts (non-heme) are present in plants and animal tissues, and organic iron (heme), which comes from hemoglobin (blood) and myoglobin (red muscle), is present in animal food sources. Heme-iron absorption is less affected by dietary compounds with the exception of calcium compounds [14]. Less than 40% of the iron in meat, poultry, and fish [18] is in the heme form, which is more efficiently absorbed than the remaining nonheme iron present in these and all other foods [19]. Nonvegetarian diets with substantial amounts of red meat supply about 2 mg/d, or 10 15%, of the total iron in the heme form (10). Heme iron is better absorbed (around 15 40%) than nonheme iron (around 1 15%) [20]. Whole cereals contain phytic acids and polyphenols, which decrease iron bioavailability [21]. The phytic acid levels in low extraction wheat flour is low compared to high extraction flour. Also bread in most of the countries in CAR is fermented using yeast. Pakistan and Afghanistan have high consumption of unleavened bread made from high extraction flour. This means the phytic acid is not broken in the latter country by the fermentation step. The fact is that the whole wheat flour (i.e. high extraction flour) is a good source of phytic acid, which is the main inhibitor of non-heme iron absorption from plant products. The phosphate groups of phytic acid are negatively charged under physiologically relevant conditions, resulting in phytate chelation of cations such as iron and zinc, making these minerals less available for absorption [22]. Consuming of phytic acid can substantially reduce iron absorption [23, 24]. Phytate content in refined (white) flour is about 100 mg/100 g, and in wheat and whole-wheat flour it is about 600 mg/100 g [25]. Polyphenols also forms insoluble complexes with iron thereby reducing its bioavailability to the body. In addition, in Kazakhstan the consumption level of meat and meat products is much higher compared to other participating countries. The bioavailability of the heme iron from meat and meat products [26] is significantly higher (15-40%) than the bioavailability of non-heme iron from plant foods [27]. Heme-iron absorption is less affected by dietary compounds with the exception of calcium compounds [28]. The inhibitory effect of calcium on iron absorption was recognized many years ago, and the presence of large amounts of calcium can inhibit the absorption of iron from a fortified food [12]. Different studies have been conducted but they often give conflicting results because several factors influence the interaction between calcium and iron absorption [29]. Milk and dairy products are good sources of calcium. But, absorption of iron and zinc from milk products is higher than from vegetable products, even it is considered that calcium from milk and milk products found as calcium phosphate, inhibit absorption of non-heme and heme iron [14], and again there are conflicting results. For example, goat milk consumption leads to a better recovery of body Fe stores, minimizing Ca-Fe interactions and improving Fe status and its absorption [30]. The absorption of iron from the cereal-based diets was not inhibited by cow s milk [31]. On the other hand, milk contains calcium and caseins, which inhibit absorption of both non-heme and heme iron. It enters into the mucosal cells by different pathways and leave in the same form which implies that calcium inhibit the intracellular transport of iron [32]. To ensure an appropriate iron absorption, the intake of iron should be high enough to improve or maintain iron status. This condition might be attained adding sufficient iron to the fortification vehicle and/or incorporating simultaneously absorption enhancers. In the case of wheat flour fortification, the only practical enhancer to add is EDTA or using as an iron source NaFeEDTA; iron in the form of ferric sodium EDTA is 2 to 3 times more bioavailable than from other mineral sources 46

47 and that it is efficiently incorporated into haemoglobin [33]; iron from ferric sodium EDTA has a high bioavailability despite the presence of inhibitory factors that form insoluble complexes [34]. Vitamin C, which can increase absorption of both native iron and fortification iron due to both its reducing power and chelating actions [35], is destroyed during baking. Bovine hemoglobin is not easily accepted or it is too expensive for being used as a fortificant. More than half of the zinc in US diets is derived from animal foods, and one quarter of the zinc comes from beef [36]. The bioavailability of zinc from vegetarian diets is also to be less than that of nonvegetarian diets. Plant foods rich in zinc such as legumes, whole grains, nuts, and seeds are also high in phytic acid, an inhibitor of zinc bioavailability [37]. Bioavailability of zinc is enhanced by dietary protein [38], but plant sources of protein are also generally high in phytic acid. Because of lower absorption of zinc, those consuming vegetarian diets, especially with phytate-zinc molar ratios > 15, may require as much as 50% more zinc than nonvegetarians [39]. Thus, the iron and zinc from vegetarian diets are generally less bioavailable than from nonvegetarian diets because of reduced meat intake as well as the tendency to consume more phytic acid and other plant-based inhibitors of iron and zinc absorption. Iron bioavailability is estimated to be around 5-12% for vegetarian diets and 14-18% for mixed diets. These values are used to generate dietary reference values for all population groups [40]. Considering all factors that may influence iron bioavailability, the estimated average absorption iron rate for a typical western diet is between 15-18% [41, 42]. 4. Premix formulation for fortification of refined wheat flour. In view of the above WHO recommendations, the average per capita consumption of wheat flour g/day in the participating countries, and the characteristics of diets with low and moderate bioavailability of minerals, premix composition is formulated for mandatory fortification of refined (white) flour, which is presented in Table 5. 47

48 Table 5 The main parameters of the premix for fortification of refined wheat flour. Nutrient Fortificant compound Selected FL (mg/kg flour) Amount of fortificant (mg/kg flour) Fortificant (g/kg premix) Premix Formulation Nutrient (g/kg premix) Cost (US$/kg) Vit. B-1 Thiamin mononitrate 2,0 2,5 9,9 8 $0,25 2,8 (Thiamin) Vit. B-2 Riboflavin 3,0 3,0 12,0 12 $0,72 8,3 (Riboflavin) Vit. B-3 (Niacin) Niacinamide 10,0 10,1 40,4 40 $0,40 4,7 Vit. B-9 (Folate) Folic Acid 1,0 1,1 4,4 4 $0,49 5,6 Vit. B-12 Vit. B % WS 0,004 4,0 16,0 0,02 $0,64 7,4 Iron NaFeEDTA ,4 461,5 60 $3,00 34,5 Zinc Zinc oxide 30 37,5 150,0 120 $0,88 10,1 Filling material (at least 25%) 26,0 305,7 $0,31 3,5 TOTAL 199,6 1000,0 Estimated cost of manufacturing, quality control and delivery (Approx. US$2/kg premix) % Cost $2,00 23,0 Approximate Cost per kg = $8,68 100,0 Estimated cost of micronutrient premix per metric ton of fortified product*: $2,17 0,43 % of the Price Minimum Amount (grams per MT) 200 Maximum Dilution Factor = 1/ 5010 Selected Amount (grams per MT) 250** Selected Dilution Factor = 1/ 4000 Notes: * - The costs of the fortificants are always the largest cost of the fortification process, when it carried out by formal and centralized factories. ** - This value should be larger than the estimated minimum amount per metric ton (above). 48

49 5. Establishment of production and regulatory parameters for fortification of refined wheat flour. Based on the composition of the premix, production and regulatory parameters for fortification of refined (white) flour are established, which are presented in Table 6. 49

50 Table 6 The main production and regulatory parameters for fortification of refined wheat flour Nutrient Fortificant compound Selected FL Production Parameters Regulatory Parameters (mg/kg flour) mfl (1) (mg/kg flour) Average (2) (mg/kg flour) MFL (3) (mg/kg flour) LmL (4) (mg/kg flour) MTL (5) (mg/kg flour) Vit. B-1 Thiamin mononitrate 2,0 1,5 2,8 4,1 1,3 4,1 (Thiamin) Vit. B-2 Riboflavin 3,0 2,0 3,6 5,2 1,8 5,2 (Riboflavin) Vit. B-3 (Niacin) Niacinamide 10,0 11,0 20,0 29,0 9,9 29,0 Vit. B-9 (Folate) Folic Acid 1,0 0,7 1,3 1,9 0,6 1,9 Vit. B-12 Vit. B % WS 0,004 0,002 0,004 0,006 0,002 0,006 Iron NaFeEDTA Zinc Zinc oxide Notes: (1) mfl = Minimum Fortification Level, using equation 2. (2) Average = Selected Fortification Level + Intrinsic content of micronutrient in unfortified food. (3) MFL = Maximum Fortification Level, using equation 3. (4) LmL= Legal Minimum Level, using equation 4. (5) MTL = Maximum Tolerable Level, equivalent to MFL but only for those micronutrients with safety concerns, and rounded. 50

51 6. Daily intake of micronutrients by population groups in the composition of fortified wheat flour, by countries, in % EAR/day. Terms [43]: EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population. All basic calculations in this and other sections are conducted using the Formulator, developed by Omar Dary и Michael Hainsworth [44]. In terms of daily vitamin B1 (thiamin) intake in the composition of fortified wheat flour, in % EAR/day, in all population groups (Table 7) the participating countries are located in the following ascending order: Kazakhstan: 27% to 35% in different age and gender groups Pakistan: 32 to 42% Tajikistan: 36% to 47% Kyrgyzstan: 39% to 51% Afghanistan: 45% to 59% Uzbekistan: 48% to 63%. So, the value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which was the smallest in Kazakhstan and the largest in Uzbekistan. In terms of daily vitamin B2 (riboflavin) intake in the composition of fortified wheat flour, in % EAR/day, in all population groups the participating countries are located in the following ascending order: Kazakhstan: 50% to 66% in different age and gender groups Pakistan: 60% to 78% Tajikistan: 68% to 88% Kyrgyzstan: 73% to 94% Afghanistan: 85% to 110% Uzbekistan: 90% to 117%. The value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which was the smallest in Kazakhstan and the largest in Uzbekistan, as well. In terms of daily vitamin B3 (niacin) intake in the composition of fortified wheat flour, in % EAR/day, in all population groups the participating countries are located in the following ascending order: Kazakhstan: 14% to 18% in different age and gender groups Pakistan: 17% to 22% Tajikistan: 19% to 24% Kyrgyzstan: 20% to 26% Afghanistan: 23% to 30% Uzbekistan: 25% to 32%. Again, the value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which was the smallest in Kazakhstan and the largest in Uzbekistan. That is, that the value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which was the smallest in Kazakhstan and the largest in Uzbekistan. In terms of daily folic acid intake in the composition of fortified wheat flour, in % EAR/day, in all population groups the participating countries are located in the following ascending order: Kazakhstan: 79% to 113% in different age and gender groups Pakistan: 96% to 136% Tajikistan: 108% to 154% Kyrgyzstan: 116% to 166% Afghanistan: 135% to 192% 51

52 Uzbekistan: 240% to 205%. So, the value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which was the smallest in Kazakhstan and the largest in Uzbekistan. In a similar manner the participating countries are arranged by the level of daily intake of added vitamin В12 in % EAR/day: Kazakhstan: 31% to 47% Pakistan: 37% to 57% Tajikistan: 42% to 64% Kyrgyzstan: 45% to 69% Afghanistan: 52% to 80% Uzbekistan: 56% to 85%. Unlike vitamins, by the level of daily iron intake in the composition of fortified wheat flour, in % EAR/day, for all groups of population, the participating countries are located in the following ascending order: Pakistan: 21% to 85% Afghanistan: 30% to 120% Tajikistan: 36% to 143% Kyrgyzstan: 39% to 154% Kazakhstan: 39% to 158% Uzbekistan: 48% to 191%. That is, that the value of this indicator is also correlated with an average per capita consumption of wheat flour in g/day, with the exception of Kazakhstan, where the value of the index was higher than in Pakistan, Afghanistan, Tajikistan and Kyrgyzstan, but lower than in Uzbekistan. This is because the average diet in Kazakhstan attributed, as mentioned above, to the group with a high bioavailability of minerals and the diet of other participating countries - to the group with a moderate (Kyrgyzstan, Tajikistan and Uzbekistan) low (Afghanistan and Pakistan) bioavailability of minerals. In terms of daily zinc intake in the composition of fortified wheat flour, in % EAR/day, in all population groups the participating countries are located in the following ascending order: Pakistan: 50% to 90% Afghanistan: 71% to 127% Kazakhstan: 84% to 150%. Tajikistan: 114% to 203% Kyrgyzstan: 123% to 219% Uzbekistan: 152% to 271% Table 7 Daily intake of micronutrients by population groups in the composition of fortified wheat flour, by countries, in % EAR/day Nutrient Additional daily intake of nutrients by countries population groups, in %EAR/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan Children, 1-3 years Vit. B-1 54,8 32,3 47,2 38,9 43,7 58,4 (Thiamin) Vit. B-2 103,5 61,0 89,1 73,4 82,6 110,2 (Riboflavin) Vit. B-3 29,9 17,6 25,8 21,2 23,9 31,8 (Niacin) Vit. B-9 189,8 111,9 163,4 134,6 151,5 202,1 (Folate) Vit. B-12 79,8 47,0 68,7 56,6 63,7 84,9 Iron (NaFeEDTA) 97,9 129,8 126,4 69,4 117,2 156,3 52

53 Nutrient Additional daily intake of nutrients by countries population groups, in %EAR/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan Zinc 70,5 84,0 122,8 50,0 113,8 151,8 Children, 4-6 years Vit. B-1 58,0 34,2 50,0 41,1 46,3 61,8 (Thiamin) Vit. B-2 109,6 64,6 94,4 77,7 87,5 116,7 (Riboflavin) Vit. B-3 28,5 16,8 24,5 20,2 22,7 30,3 (Niacin) Vit. B-9 180,9 106,6 155,7 128,3 144,3 192,5 (Folate) Vit. B-12 70,2 41,4 60,4 49,8 56,0 74,7 Iron 119,4 158,3 154,1 84,7 142,9 190,6 (NaFeEDTA) Zinc 77,4 91,2 133,2 54,9 123,5 164,7 Children, 7-9 years Vit. B-1 48,6 28,6 41,8 34,4 38,8 51,7 (Thiamin) Vit. B-2 91,7 54,1 79,0 65,0 73,2 97,6 (Riboflavin) Vit. B-3 23,9 14,1 20,5 16,9 19,0 25,4 (Niacin) Vit. B-9 151,4 89,2 130,3 107,3 120,8 161,1 (Folate) Vit. B-12 58,7 34,6 50,6 41,7 46,9 62,5 Iron 106,1 140,6 137,0 75,2 127,0 169,4 (NaFeEDTA) Zinc 83,3 98,1 143,3 59,0 132,9 177,2 Males, years Vit. B-1 54,5 32,1 46,9 38,7 43,5 58,0 (Thiamin) Vit. B-2 95,1 56,0 81,8 67,4 75,9 101,2 (Riboflavin) Vit. B-3 27,9 16,4 24,0 19,8 22,3 29,7 (Niacin) Vit. B-9 177,0 104,3 152,4 125,5 141,2 188,4 (Folate) Vit. B-12 68,7 40,5 59,1 48,7 54,8 73,1 Iron 48,5 64,3 60,8 34,4 56,4 75,2 (NaFeEDTA) Zinc 85,0 99,6 145,5 60,3 134,9 180,0 Males, years Vit. B-1 59,3 34,9 51,0 42,0 47,3 63,1 (Thiamin) Vit. B-2 103,3 60,9 89,0 73,3 82,5 110,0 (Riboflavin) Vit. B-3 30,3 17,9 26,1 21,5 24,2 32,3 (Niacin) Vit. B-9 (Folate) 192,4 113,4 165,6 136,4 153,5 204,8 53

54 Nutrient Additional daily intake of nutrients by countries population groups, in %EAR/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan Vit. B-12 74,7 44,0 64,3 52,9 59,6 79,5 Iron 63,9 84,8 82,5 45,3 76,5 102,1 (NaFeEDTA) Zinc 112,9 133,0 194,3 80,0 180,2 240,3 Males, years Vit. B-1 58,1 34,2 50,0 41,2 46,3 61,8 (Thiamin) Vit. B-2 101,3 59,7 87,2 71,8 80,8 107,8 (Riboflavin) Vit. B-3 29,7 17,5 25,6 21,1 23,7 31,6 (Niacin) Vit. B-9 188,6 111,1 162,3 133,7 150,5 200,7 (Folate) Vit. B-12 73,2 43,1 63,0 51,9 58,4 77,9 Iron 62,6 83,1 80,9 44,4 75,0 100,0 (NaFeEDTA) Zinc 110,6 130,4 190,5 78,4 176,6 235,5 Males, +65 years Vit. B-1 48,6 28,6 41,8 34,5 38,8 51,7 (Thiamin) Vit. B-2 84,7 49,9 72,9 60,1 67,6 90,2 (Riboflavin) Vit. B-3 24,9 14,7 21,4 17,6 19,8 26,5 (Niacin) Vit. B-9 157,8 93,0 135,8 111,9 125,9 167,9 (Folate) Vit. B-12 61,2 36,1 52,7 43,4 48,9 65,2 Iron 52,4 69,5 67,7 37,2 62,7 83,7 (NaFeEDTA) Zinc 92,6 109,1 159,4 65,6 147,7 197,1 Females, years Vit. B-1 49,8 29,3 42,9 35,3 39,7 53,0 (Thiamin) Vit. B-2 94,8 55,9 81,6 67,2 75,7 100,9 (Riboflavin) Vit. B-3 (Niacin) 23,3 13,8 20,1 16,6 18,6 24,9 Vit. B-9 148,2 87,3 127,5 105,1 118,2 157,7 (Folate) Vit. B-12 57,5 33,9 49,5 40,8 45,9 61,2 Iron 29,6 39,3 38,9 21,0 36,0 48,1 (NaFeEDTA) Zinc 84,5 99,6 145,5 59,9 134,9 179,9 Females, years Vit. B-1 51,1 30,1 44,0 36,2 40,8 54,4 (Thiamin) Vit. B-2 (Riboflavin) 96,5 56,9 83,0 68,4 77,0 102,7 54

55 Nutrient Additional daily intake of nutrients by countries population groups, in %EAR/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan Vit. B-3 27,4 16,1 23,6 19,4 21,8 29,1 (Niacin) Vit. B-9 152,0 89,6 130,8 107,8 121,3 161,8 (Folate) Vit. B-12 59,0 34,8 50,8 41,8 47,1 62,8 Iron 39,3 52,1 50,7 27,9 47,0 62,7 (NaFeEDTA) Zinc 127,4 150,2 219,3 90,3 203,3 271,2 Females, years Vit. B-1 51,1 29,7 43,4 35,8 40,2 53,7 (Thiamin) Vit. B-2 96,5 56,1 82,0 67,5 76,0 101,4 (Riboflavin) Vit. B-3 (Niacin) 27,4 15,9 23,3 19,2 21,6 28,8 Vit. B-9 150,1 88,4 129,2 106,4 119,8 159,8 (Folate) Vit. B-12 58,2 34,3 50,1 41,3 46,5 62,0 Iron 100,9 133,8 130,3 71,6 120,8 161,1 (NaFeEDTA) Zinc 125,8 148,3 216,6 89,2 200,7 267,8 Females, +65 years Vit. B-1 45,3 26,7 39,0 32,1 36,1 48,2 (Thiamin) Vit. B-2 85,5 50,4 73,6 60,6 68,2 91,0 (Riboflavin) Vit. B-3 24,3 14,3 20,9 17,2 19,4 25,8 (Niacin) Vit. B-9 134,7 79,4 115,9 95,5 107,5 143,4 (Folate) Vit. B-12 52,3 30,8 45,0 37,1 41,7 55,6 Iron 65,3 86,5 84,3 46,3 78,1 104,2 (NaFeEDTA) Zinc 112,9 133,0 194,3 80,0 180,2 240,3 Note: EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and sex-specific population. 55

56 7. Daily intake of micronutrients by population groups in the composition of fortified wheat flour, by countries, in % RNI/day. In terms of total daily vitamin B1 (thiamin) intake groups in the composition of fortified wheat flour, in % RNI/day, in all population groups (Table 8) the participating countries are located in the following ascending order: Kazakhstan: 22% to 29% in different age and gender groups Pakistan: 28% to 35% Tajikistan: 30% to 39% Kyrgyzstan: 33% to 43% Afghanistan: 38% to 49% Uzbekistan: 40% to 53%. The value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which is the smallest in Kazakhstan and the largest in Uzbekistan. In terms of total daily vitamin B2 (riboflavin) intake groups in the composition of fortified wheat flour, in % RNI/day, in all population groups (Table 8) the participating countries are located in the following ascending order: Kazakhstan: 42% to 52% in different age and gender groups Pakistan: 50% to 62% Tajikistan: 56% to 70% Kyrgyzstan: 61% to 76% Afghanistan: 71% to 88% Uzbekistan: 75% to 93%. The value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which is the smallest in Kazakhstan and the largest in Uzbekistan, as well. The value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which is the smallest in Kazakhstan and the largest in Uzbekistan. In terms of total daily vitamin B3 (niacin) intake groups in the composition of fortified wheat flour, in % RNI/day, in all population groups (Table 8) the participating countries are located in the following ascending order: Kazakhstan: 11% to 14% in different age and gender groups Pakistan: 13% to 17% Tajikistan: 14% to 19% Kyrgyzstan: 16% to 20% Afghanistan: 18% to 23% Uzbekistan: 19% to 25%. The value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which is the smallest in Kazakhstan and the largest in Uzbekistan, as well. In a similar manner, the participating countries are arranged by the levels of total daily intake In terms of total daily folic acid intake groups in the composition of fortified wheat flour, in % RNI/day, in all population groups (Table 8) the participating countries are located in the following ascending order: Kazakhstan: 64% to 91% in different age and gender groups Pakistan: 76% to 109% Tajikistan: 86% to 123% Kyrgyzstan: 93% to 133% Afghanistan: 108% to 156% Uzbekistan: 115% to 164%. That is, that the value of this indicator is correlated with an average per capita consumption of wheat flour in g/day, which is the smallest in Kazakhstan and the largest in Uzbekistan. In a similar manner, the participating countries are arranged by the levels of total daily intake of vitamin В12, in % RNI/day: 56

57 Kazakhstan: 26% to 37% Pakistan: 31% to 44% Tajikistan: 35% to 50% Kyrgyzstan: 38% to 54% Afghanistan: 44% to 62% Uzbekistan: 46% to 66%. Unlike vitamins, by the level of total daily intake of iron, in % RNI/day, for all groups of population, the participating countries are located in the following ascending order: Pakistan: 11% to 35% Afghanistan: 16% to 49% Tajikistan: 19% to 59% Kyrgyzstan: 21% to 63% Kazakhstan: 21% to 65% Uzbekistan: 25% to 78%. That is, that the value of this indicator is also correlated with an average per capita consumption of wheat flour in g/day, with the exception of Kazakhstan, where the value of the index was slightly higher than in Pakistan, Afghanistan, Tajikistan and Kyrgyzstan, but lower than in Uzbekistan. This is because the average diet in Kazakhstan attributed, as mentioned above, to the group with a high bioavailability of minerals and the diet of other participating countries - to the group with a moderate (Kyrgyzstan, Tajikistan and Uzbekistan) and low (Afghanistan and Pakistan) bioavailability of minerals. In terms of total daily zinc intake, in % RNI/day, in all population groups the participating countries are located in the following ascending order: Pakistan: 41% to 75% Afghanistan: 59% to 106% Kazakhstan: 70% to 125%. Tajikistan: 95% to 169% Kyrgyzstan: 102% to 183% Uzbekistan: 127% to 226% Table 8 Daily intake of micronutrients by population groups in the composition of fortified wheat flour, by countries, in % RNI/day Nutrient Total daily intake of nutrients by countries population groups, in % RNI/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan Children, 1-3 years Vit. B-1 43,9 25,8 37,8 31,1 35,0 46,7 (Thiamin) Vit. B-2 82,8 48,8 71,3 58,7 66,1 88,2 (Riboflavin) Vit. B-3 23,0 13,6 19,8 16,3 18,4 24,5 (Niacin) Vit. B-9 151,9 89,5 130,7 107,7 121,2 161,7 (Folate) Vit. B-12 61,4 36,2 52,8 43,5 49,0 65,3 Iron 42,0 55,7 54,2 29,8 50,3 67,1 (NaFeEDTA) Zinc 58,7 70,0 102,3 41,6 94,8 126,5 Children, 4-6 years Vit. B-1 46,4 27,4 40,0 32,9 37,0 49,4 (Thiamin) 57

58 Nutrient Total daily intake of nutrients by countries population groups, in % RNI/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan Vit. B-2 87,7 51,7 75,5 62,2 70,0 93,3 (Riboflavin) Vit. B-3 21,9 12,9 18,9 15,5 17,5 23,3 (Niacin) Vit. B-9 144,7 85,3 124,6 102,6 115,5 154,0 (Folate) Vit. B-12 58,5 34,5 50,3 41,5 46,7 62,3 Iron 49,1 65,1 63,4 34,8 58,8 78,4 (NaFeEDTA) Zinc 64,5 76,0 111,0 45,7 102,9 137,3 Children, 7-9 years Vit. B-1 38,9 22,9 33,4 27,6 31,0 41,4 (Thiamin) Vit. B-2 73,4 43,2 63,2 52,0 58,6 78,1 (Riboflavin) Vit. B-3 18,3 10,8 15,8 13,0 14,6 19,5 (Niacin) Vit. B-9 121,1 71,4 104,2 85,9 96,6 128,9 (Folate) Vit. B-12 48,9 28,9 42,1 34,7 39,1 52,1 Iron 43,7 57,9 56,4 31,0 52,2 69,7 (NaFeEDTA) Zinc 69,4 81,8 119,4 49,2 110,7 147,7 Males, years Vit. B-1 45,4 26,8 39,1 32,2 36,3 48,4 (Thiamin) Vit. B-2 79,2 46,7 68,2 56,2 63,2 84,3 (Riboflavin) Vit. B-3 21,5 12,6 18,5 15,2 17,1 22,8 (Niacin) Vit. B-9 141,6 83,5 121,9 100,4 113,0 150,7 (Folate) Vit. B-12 57,2 33,7 49,3 40,6 45,7 60,9 Iron 34,6 45,9 43,5 24,5 40,3 53,7 (NaFeEDTA) Zinc 70,9 83,0 121,3 50,2 112,4 150,0 Males, years Vit. B-1 49,4 29,1 42,5 35,0 39,4 52,6 (Thiamin) Vit. B-2 86,1 50,7 74,1 61,1 68,7 91,7 (Riboflavin) Vit. B-3 23,3 13,7 20,1 16,5 18,6 24,8 (Niacin) Vit. B-9 153,9 90,7 132,5 109,1 122,8 163,8 (Folate) Vit. B-12 62,2 36,7 53,6 44,1 49,7 66,2 Iron 48,1 63,7 62,1 34,1 57,5 76,7 (NaFeEDTA) Zinc 94,1 110,9 162,0 66,7 150,1 200,3 58

59 Nutrient Total daily intake of nutrients by countries population groups, in % RNI/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan Males, years Vit. B-1 48,4 28,5 41,7 34,3 38,6 51,5 (Thiamin) Vit. B-2 84,4 49,7 72,6 59,8 67,3 89,8 (Riboflavin) Vit. B-3 22,9 13,5 19,7 16,2 18,2 24,3 (Niacin) Vit. B-9 150,8 88,9 129,9 107,0 120,4 160,6 (Folate) Vit. B-12 61,0 35,9 52,5 43,2 48,7 64,9 Iron 47,1 62,5 60,8 33,4 56,4 75,2 (NaFeEDTA) Zinc 92,2 108,7 158,7 65,4 147,1 196,3 Males, +65 years Vit. B-1 40,5 23,9 34,9 28,7 32,3 43,1 (Thiamin) Vit. B-2 70,6 41,6 60,8 50,1 56,3 75,2 (Riboflavin) Vit. B-3 19,1 11,3 16,5 13,6 15,3 20,4 (Niacin) Vit. B-9 126,2 74,4 108,7 89,5 100,7 134,4 (Folate) Vit. B-12 51,0 30,1 43,9 36,2 40,7 54,3 Iron 39,4 52,3 50,9 27,9 47,2 62,9 (NaFeEDTA) Zinc 77,1 90,9 132,8 54,7 123,1 164,2 Females, years Vit. B-1 41,5 24,4 35,7 29,4 33,1 44,2 (Thiamin) Vit. B-2 86,2 50,8 74,2 61,1 68,8 91,8 (Riboflavin) Vit. B-3 (Niacin) 18,0 10,6 15,5 12,7 14,3 19,1 Vit. B-9 118,5 69,8 102,0 84,0 94,6 126,2 (Folate) Vit. B-12 47,9 28,2 41,2 34,0 38,2 51,0 Iron 15,6 20,7 20,5 11,1 19,0 25,3 (NaFeEDTA) Zinc 70,4 83,0 121,2 49,9 112,4 149,9 Females, years Vit. B-1 42,6 25,1 36,6 30,2 34,0 45,3 (Thiamin) Vit. B-2 80,4 47,4 69,2 57,0 64,2 85,6 (Riboflavin) Vit. B-3 (Niacin) 21,1 12,4 18,1 14,9 16,8 22,4 Vit. B-9 121,6 71,7 104,7 86,2 97,0 129,4 (Folate) Vit. B-12 49,2 29,0 42,3 34,9 39,2 52,3 59

60 Nutrient Total daily intake of nutrients by countries population groups, in % RNI/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan Iron 17,7 23,5 22,8 12,5 21,2 28,3 (NaFeEDTA) Zinc 106,2 125,1 182,8 75,3 169,4 226,0 Females, years Vit. B-1 42,6 24,8 36,2 29,8 33,5 44,7 (Thiamin) Vit. B-2 80,4 46,8 68,3 56,3 63,3 84,5 (Riboflavin) Vit. B-3 (Niacin) 21,1 12,3 17,9 14,7 16,6 22,1 Vit. B-9 120,1 70,8 103,4 85,1 95,8 127,8 (Folate) Vit. B-12 48,5 28,6 41,8 34,4 38,7 51,7 Iron 45,5 60,3 58,7 32,2 54,4 72,6 (NaFeEDTA) Zinc 104,8 123,5 180,5 74,3 167,3 223,2 Females, +65 years Vit. B-1 37,7 22,2 32,5 26,7 30,1 40,1 (Thiamin) Vit. B-2 71,2 42,0 61,3 50,5 56,8 75,8 (Riboflavin) Vit. B-3 18,7 11,0 16,1 13,2 14,9 19,9 (Niacin) Vit. B-9 107,7 63,5 92,8 76,4 86,0 114,7 (Folate) Vit. B-12 43,6 25,7 37,5 30,9 34,8 46,4 Iron 40,8 54,1 52,7 28,9 48,8 65,1 (NaFeEDTA) Zinc 94,1 110,9 162,0 66,7 150,1 200,3 Note: RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population. 60

61 8. Brief description of the annexes. Annexes 1-6 provide detailed characteristics of fortified refined (white) wheat flour using a premix, the main parameters of which are shown in Table 5. In particular, the following data are given in these annexes: Addition levels each of seven micronutrients (vitamins B1, B2, B3, B9 and B12, iron as NaFeEDTA, and zinc) to flour, in mg/kg flour Adjusted upper limit for each of the micronutrients (vitamins B1, B2, B3, B9 and B12, iron as NaFeEDTA, and zinc), in mg/kg flour Daily intake of micronutrients in the composition of fortified wheat flour, in: o mg/day o % EAR/day o % RNI/day The mentioned information in each member country are presented for the following age and gender groups of population in accordance with WHO recommendations: 1. Children, 1-3 years 2. Children, 4-6 years 3. Children, 7-9 years 4. Males, years 5. Males, years 6. Males, years 7. Males, + 65 years 8. Females, years 9. Females, years 10. Females, years 11. Females, + 65 years Total 6 annexes are drawn up by the number of countries: Afghanistan Annex 1 Kazakhstan Annex 1 Kyrgyzstan Annex 1 Pakistan Annex 1 Tajikistan Annex 1 Uzbekistan Annex 1 61

62 9. Conclusion. 1. For the mandatory fortification of refined (white) wheat flour the following 7 micronutrients are selected: Vitamin В1 (thiamin) Vitamin В2 (riboflavin) Vitamin В9 (niacin) Vitamin В9 (folate) Vitamin В12 (cyanocobalamin) Iron (NaFeEDTA) Zinc (zinc oxide) 2. The proposed addition levels of micronutrients in refined flour: prepared taking into account the average per capita consumption of flour (in g/day) in the participating countries; fully comply with WHO recommendations 3. The proposed levels of refined flour fortification by zinc and vitamins B1, B2, B3, B9 and B12 ensure the following levels of total daily intake (in % RNI/day) of these micronutrients in the composition of refined fortified wheat flour for all population groups in participating countries: Nutrient Vit. B-3 (Niacin) Vit. B-1 (Thiamin) Vit. B-2 (Ribiflavin) Vit. B-12 Vit. B-9 (Folate) Zinc Total daily intake of nutrients by countries population groups, in % RNI/day Afghanistan Kazakhstan Kyrgyzstan Pakistan Tajikistan Uzbekistan 18% to 11% to 16% to 13% to 14% to 19% to 23% 14% 20% 17% 19% 25% 38% to 22% to 33% to 28% to 30% to 40% to 49% 29% 43% 35% 39% 53% 71% to 42% to 61% to 50% to 56% to 75% to 88% 52% 76% 62% 70% 93% 44% to 26% to 38% to 31% to 35% to 46% to 62% 37% 54% 44% 50% 66% 108% to 64% to 93% to 76% to 86% to 115% to 156% 91% 133% 109% 123% 164% 59% to 70% to 102% to 41% to 95% to 127% to 106% 125% 183% 75% 169% 226% 4. The proposed levels of refined flour fortification by iron don t ensure acceptable levels of total daily intake (in% RNI/day) of iron in females of years old in all participating countries: Pakistan: 11% to 13% Afghanistan: 16% to 18% Tajikistan: 19% to 21% Kyrgyzstan: 21% to 23% Kazakhstan: 21% to 24% Uzbekistan: 25% to 28%. 5. The proposed levels of refined flour fortification by iron ensure more acceptable levels of total daily intake (in % RNI/day) of iron in other age and gender population groups in participating countries: Pakistan: 25% to 35% Afghanistan: 35% to 49% Tajikistan: 40% to 59% Kyrgyzstan: 44% to 63% Kazakhstan: 46% to 65% Uzbekistan: 54% to 78%. 62

63 Annex 1 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Afghanistan Afghanistan: Low diet bioavailability for minerals Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Children, 1-3 years: P-50 Food Intake of target group = 162,4 g/day Vit. B-1 (Thiamin) 2,0 ND 0,219 54,8 43,9 Vit. B-2 (Riboflavin) 3,0 ND 0, ,5 82,8 Vit. B-3 (Niacin) 10,0 10,0 1,381 29,9 23,0 Vit. B-9 (Folate) 1,0 0,3 0, ,8 151,9 Vit. B-12 0,004 N.D. 0, ,75 61,35 Iron (NaFeEDTA) 15,0 4,7 2,436 97,9 42,0 Zinc 30,0 7,0 4,873 70,5 58,7 Children, 4-6 years: P-50 Food Intake of target group = 206,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,279 58,0 46,4 Vit. B-2 (Riboflavin) 3,0 ND 0, ,6 87,7 Vit. B-3 (Niacin) 10,0 15,0 1,754 28,5 21,9 Vit. B-9 (Folate) 1,0 0,4 0, ,9 144,7 Vit. B-12 0,004 N.D. 0, ,15 58,45 Iron (NaFeEDTA) 15,0 7,2 3, ,4 49,1 Zinc 30,0 12,0 6,190 77,4 64,5 Children, 7-9 years: P-50 Food Intake of target group = 259,0g/day Vit. B-1 (Thiamin) 2,0 ND 0,350 48,6 38,9 Vit. B-2 (Riboflavin) 3,0 ND 0,660 91,7 73,4 Vit. B-3 (Niacin) 10,0 17,0 2,202 23,9 18,3 Vit. B-9 (Folate) 1,0 0,5 0, ,4 121,1 Vit. B-12 0,004 N.D. 0,001 58,7 48,9 Iron (NaFeEDTA) 15,0 10,1 3, ,1 43,7 Zinc 30,0 12,0 7,770 83,3 69,4 Males, years: P-50 Food Intake of target group = 403,9 g/day Vit. B-1 (Thiamin) 2,0 ND 0,545 54,5 45,4 Vit. B-2 (Riboflavin) 3,0 ND 1,030 95,1 79,2 Vit. B-3 (Niacin) 10,0 25,0 3,433 27,9 21,5 Vit. B-9 (Folate) 1,0 0,7 0, ,0 141,6 Vit. B-12 0,004 N.D. 0, ,65 57,2 Iron (NaFeEDTA) 15,0 19,8 6,058 48,5 34,6 Zinc 30,0 28,0 12,116 85,0 70,9 Males, years: P-50 Food Intake of target group = 439,0 g/day Vit. B-1 (Thiamin) 2,0 ND 0,593 59,3 49,4 Vit. B-2 (Riboflavin) 3,0 ND 1, ,3 86,1 Vit. B-3 (Niacin) 10,0 35,0 3,732 30,3 23,3 Vit. B-9 (Folate) 1,0 1,0 0, ,4 153,9 Vit. B-12 0,004 N.D. 0, ,65 62,2 Iron (NaFeEDTA) 15,0 28,8 6,585 63,9 48,1 Zinc 30,0 45,0 13, ,9 94,1 Males, years: P-50 Food Intake of target group = 430,2 g/day Vit. B-1 (Thiamin) 2,0 ND 0,581 58,1 48,4 Vit. B-2 (Riboflavin) 3,0 ND 1, ,3 84,4 63

64 Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Vit. B-3 (Niacin) 10,0 35,0 3,657 29,7 22,9 Vit. B-9 (Folate) 1,0 1,0 0, ,6 150,8 Vit. B-12 0,004 N.D. 0, ,15 60,95 Iron (NaFeEDTA) 15,0 27 6,453 62,6 47,1 Zinc 30,0 45,0 12, ,6 92,2 Males, +65 years: P-50 Food Intake of target group = 360,0 g/day Vit. B-1 (Thiamin) 2,0 ND 0,486 48,6 40,5 Vit. B-2 (Riboflavin) 3,0 ND 0,918 84,7 70,6 Vit. B-3 (Niacin) 10,0 35,0 3,060 24,9 19,1 Vit. B-9 (Folate) 1,0 1,0 0, ,8 126,2 Vit. B-12 0,004 N.D. 0,001 61,2 51,0 Iron (NaFeEDTA) 15,0 25,2 5,400 52,4 39,4 Zinc 30,0 45,0 10,799 92,6 77,1 Females, years: P-50 Food Intake of target group = 338,0 g/day Vit. B-1 (Thiamin) 2,0 ND 0,456 49,8 41,5 Vit. B-2 (Riboflavin) 3,0 ND 0,862 94,8 86,2 Vit. B-3 (Niacin) 10,0 25,0 2,873 23,3 18,0 Vit. B-9 (Folate) 1,0 0,7 0, ,2 118,5 Vit. B-12 0,004 N.D. 0,001 57,45 47,9 Iron (NaFeEDTA) 10,0 18 5,070 29,6 15,6 Zinc 25,0 28,0 10,141 84,5 70,4 Females, years: P-50 Food Intake of target group = 346,8 g/day Vit. B-1 (Thiamin) 2,0 ND 0,468 51,1 42,6 Vit. B-2 (Riboflavin) 3,0 ND 0,884 96,5 80,4 Vit. B-3 (Niacin) 10,0 35,0 2,948 27,4 21,1 Vit. B-9 (Folate) 1,0 1,0 0, ,0 121,6 Vit. B-12 0,004 N.D. 0,001 58,95 49,15 Iron (NaFeEDTA) 15,0 23,4 5,202 39,3 17,7 Zinc 30,0 45,0 10, ,4 106,2 Females, years: P-50 Food Intake of target group = 342,4 g/day Vit. B-1 (Thiamin) 2,0 ND 0,468 51,1 42,6 Vit. B-2 (Riboflavin) 3,0 ND 0,884 96,5 80,4 Vit. B-3 (Niacin) 10,0 35,0 2,948 27,4 21,1 Vit. B-9 (Folate) 1,0 1,0 0, ,1 120,1 Vit. B-12 0,004 N.D. 0,001 58,2 48,5 Iron (NaFeEDTA) 15,0 25,2 5, ,9 45,5 Zinc 30,0 45,0 10, ,8 104,8 Females, +65 years: P-50 Food Intake of target group = 307,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,415 45,3 37,7 Vit. B-2 (Riboflavin) 3,0 ND 0,784 85,5 71,2 Vit. B-3 (Niacin) 10,0 35,0 2,612 24,3 18,7 Vit. B-9 (Folate) 1,0 1,0 0, ,7 107,7 Vit. B-12 0,004 N.D. 0,001 52,25 43,55 Iron (NaFeEDTA) 15,0 21,6 4,610 65,3 40,8 Zinc 30,0 45,0 9, ,9 94,1 Notes: 64

65 a - These values are calculated taking in consideration the micronutrient losses during storage and distribution, as well as during cooking. EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population. * N.D. = Not determined 65

66 Annex 2 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Kazakhstan Kazakhstan: High diet bioavailability for minerals Nutrient Addition Adjusted Daily intake of micronutrients in the level to upper limit, composition of fortified wheat flour flour, mg/day mg/day a % EAR/day % RNI/day mg/kg Children, 1-3 years: P-50 Food Intake of target group = 95,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,129 32,3 25,8 Vit. B-2 (Riboflavin) 3,0 ND 0,244 61,0 48,8 Vit. B-3 (Niacin) 10,0 10,0 0,814 17,6 13,6 Vit. B-9 (Folate) 1,0 0,3 0, ,9 89,5 Vit. B-12 0,004 ND 0, ,0 36,15 Iron (NaFeEDTA) 15,0 4,7 1, ,8 55,7 Zinc 30,0 7,0 2,872 84,0 70,0 Children, 4-6 years: P-50 Food Intake of target group = 121,6 g/day Vit. B-1 (Thiamin) 2,0 ND 0,164 34,2 27,4 Vit. B-2 (Riboflavin) 3,0 ND 0,310 64,6 51,7 Vit. B-3 (Niacin) 10,0 15,0 1,034 16,8 12,9 Vit. B-9 (Folate) 1,0 0,4 0, ,6 85,3 Vit. B-12 0,004 ND 0, ,4 34,45 Iron (NaFeEDTA) 15,0 7,2 1, ,3 65,1 Zinc 30,0 12,0 3,648 91,2 76,0 Children, 7-9 years: P-50 Food Intake of target group = 152,6 g/day Vit. B-1 (Thiamin) 2,0 ND 0,206 28,6 22,9 Vit. B-2 (Riboflavin) 3,0 ND 0,389 54,1 43,2 Vit. B-3 (Niacin) 10,0 17,0 1,297 14,1 10,8 Vit. B-9 (Folate) 1,0 0,5 0,126 89,2 71,4 Vit. B-12 0,004 ND 0, ,6 28,85 Iron (NaFeEDTA) 15,0 10,1 2, ,6 57,9 Zinc 30,0 12,0 4,579 98,1 81,8 Males, years: P-50 Food Intake of target group = 238,0 g/day Vit. B-1 (Thiamin) 2,0 ND 0,321 32,1 26,8 Vit. B-2 (Riboflavin) 3,0 ND 0,607 56,0 46,7 Vit. B-3 (Niacin) 10,0 25,0 2,023 16,4 12,6 Vit. B-9 (Folate) 1,0 0,7 0, ,3 83,5 Vit. B-12 0,004 ND 0,001 40,5 33,7 Iron (NaFeEDTA) 15,0 19,8 3,570 64,3 45,9 Zinc 30,0 28,0 7,140 99,6 83,0 Males, years: P-50 Food Intake of target group = 258,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,349 34,9 29,1 Vit. B-2 (Riboflavin) 3,0 ND 0,660 60,9 50,7 Vit. B-3 (Niacin) 10,0 35,0 2,199 17,9 13,7 Vit. B-9 (Folate) 1,0 1,0 0, ,4 90,7 Vit. B-12 0,004 ND 0,001 44,0 36,65 Iron (NaFeEDTA) 15,0 28,8 3,881 84,8 63,7 Zinc 30,0 45,0 7, ,0 110,9 Males, years: P-50 Food Intake of target group = 253,5 g/day Vit. B-1 (Thiamin) 2,0 ND 0,342 34,2 28,5 66

67 Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Vit. B-2 (Riboflavin) 3,0 ND 0,646 59,7 49,7 Vit. B-3 (Niacin) 10,0 35,0 2,155 17,5 13,5 Vit. B-9 (Folate) 1,0 1,0 0, ,1 88,9 Vit. B-12 0,004 ND 0,001 43,1 35,9 Iron (NaFeEDTA) 15,0 27,0 3,803 83,1 62,5 Zinc 30,0 45,0 7, ,4 108,7 Males, +65 years: P-50 Food Intake of target group = 212,1 g/day Vit. B-1 (Thiamin) 2,0 ND 0,286 28,6 23,9 Vit. B-2 (Riboflavin) 3,0 ND 0,541 49,9 41,6 Vit. B-3 (Niacin) 10,0 35,0 1,803 14,7 11,3 Vit. B-9 (Folate) 1,0 1,0 0,175 93,0 74,4 Vit. B-12 0,004 ND 0, ,1 30,05 Iron (NaFeEDTA) 15,0 25,2 3,182 69,5 52,3 Zinc 30,0 45,0 6, ,1 90,9 Females, years: P-50 Food Intake of target group = 199,2 g/day Vit. B-1 (Thiamin) 2,0 ND 0,269 29,3 24,4 Vit. B-2 (Riboflavin) 3,0 ND 0,508 55,9 50,8 Vit. B-3 (Niacin) 10,0 25,0 1,693 13,8 10,6 Vit. B-9 (Folate) 1,0 0,7 0,164 87,3 69,8 Vit. B-12 0,004 ND 0, ,9 28,2 Iron (NaFeEDTA) 15,0 18,0 2,988 39,3 20,7 Zinc 30,0 28,0 5,976 99,6 83,0 Females, years: P-50 Food Intake of target group = 204,4 g/day Vit. B-1 (Thiamin) 2,0 ND 0,276 30,1 25,1 Vit. B-2 (Riboflavin) 3,0 ND 0,521 56,9 47,4 Vit. B-3 (Niacin) 10,0 35,0 1,737 16,1 12,4 Vit. B-9 (Folate) 1,0 1,0 0,169 89,6 71,7 Vit. B-12 0,004 ND 0, ,8 28,95 Iron (NaFeEDTA) 15,0 23,4 3,066 52,1 23,5 Zinc 30,0 45,0 6, ,2 125,1 Females, years: P-50 Food Intake of target group = 201,8 g/day Vit. B-1 (Thiamin) 2,0 ND 0,272 29,7 24,8 Vit. B-2 (Riboflavin) 3,0 ND 0,515 56,1 46,8 Vit. B-3 (Niacin) 10,0 35,0 1,715 15,9 12,3 Vit. B-9 (Folate) 1,0 1,0 0,166 88,4 70,8 Vit. B-12 0,004 ND 0, ,3 28,6 Iron (NaFeEDTA) 15,0 25,2 3, ,8 60,3 Zinc 30,0 45,0 6, ,3 123,5 Females, +65 years: P-50 Food Intake of target group = 181,1 g/day Vit. B-1 (Thiamin) 2,0 ND 0,244 26,7 22,2 Vit. B-2 (Riboflavin) 3,0 ND 0,462 50,4 42,0 Vit. B-3 (Niacin) 10,0 35,0 1,539 14,3 11,0 Vit. B-9 (Folate) 1,0 1,0 0,149 79,4 63,5 Vit. B-12 0,004 ND 0, ,8 25,65 Iron (NaFeEDTA) 15,0 21,6 2,716 86,5 54,1 Zinc 30,0 45,0 5, ,0 110,9 67

68 Notes: a - These values are calculated taking in consideration the micronutrient losses during storage and distribution, as well as during cooking. EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population. * N.D. = Not determined 68

69 Annex 3 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Kyrgyzstan Kyrgyzstan: Moderate diet bioavailability for minerals Nutrient Addition Adjusted Daily intake of micronutrients in the level to upper limit, composition of fortified wheat flour flour, mg/day mg/day a % EAR/day % RNI/day mg/kg Children, 1-3 years: P-50 Food Intake of target group = 139,8 g/day Vit. B-1 (Thiamin) 2,0 ND 0,189 47,2 37,8 Vit. B-2 (Riboflavin) 3,0 ND 0,357 89,1 71,3 Vit. B-3 (Niacin) 10,0 10,0 1,188 25,8 19,8 Vit. B-9 (Folate) 1,0 0,3 0, ,4 130,7 Vit. B-12 0,004 N.D. 0, ,7 52,8 Iron (NaFeEDTA) 15,0 4,7 2, ,4 54,2 Zinc 30,0 7,0 4, ,8 102,3 Children, 4-6 years: P-50 Food Intake of target group = 177,6 g/day Vit. B-1 (Thiamin) 2,0 ND 0,240 50,0 40,0 Vit. B-2 (Riboflavin) 3,0 ND 0,453 94,4 75,5 Vit. B-3 (Niacin) 10,0 15,0 1,510 24,5 18,9 Vit. B-9 (Folate) 1,0 0,4 0, ,7 124,6 Vit. B-12 0,004 N.D. 0, ,4 50,3 Iron (NaFeEDTA) 15,0 7,2 2, ,1 63,4 Zinc 30,0 12,0 5, ,2 111,0 Children, 7-9 years: P-50 Food Intake of target group = 223,0 g/day Vit. B-1 (Thiamin) 2,0 ND 0,301 41,8 33,4 Vit. B-2 (Riboflavin) 3,0 ND 0,569 79,0 63,2 Vit. B-3 (Niacin) 10,0 17,0 1,895 20,5 15,8 Vit. B-9 (Folate) 1,0 0,5 0, ,3 104,2 Vit. B-12 0,004 N.D. 0,001 50,6 42,1 Iron (NaFeEDTA) 15,0 10,1 3, ,0 56,4 Zinc 30,0 12,0 6, ,3 119,4 Males, years: P-50 Food Intake of target group = 347,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,469 46,9 39,1 Vit. B-2 (Riboflavin) 3,0 ND 0,887 81,8 68,2 Vit. B-3 (Niacin) 10,0 25,0 2,955 24,0 18,5 Vit. B-9 (Folate) 1,0 0,7 0, ,4 121,9 Vit. B-12 0,004 N.D. 0,001 59,1 49,25 Iron (NaFeEDTA) 15,0 19,8 5,215 60,8 43,5 Zinc 30,0 28,0 10, ,5 121,3 Males, years: P-50 Food Intake of target group = 377,9 g/day Vit. B-1 (Thiamin) 2,0 ND 0,510 51,0 42,5 Vit. B-2 (Riboflavin) 3,0 ND 0,964 89,0 74,1 Vit. B-3 (Niacin) 10,0 35,0 3,212 26,1 20,1 Vit. B-9 (Folate) 1,0 1,0 0, ,6 132,5 Vit. B-12 0,004 N.D. 0, ,3 53,55 Iron (NaFeEDTA) 15,0 28,8 5,669 82,5 62,1 Zinc 30,0 45,0 11, ,3 162,0 Males, years: P-50 Food Intake of target group = 370,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,500 50,0 41,7 69

70 Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Vit. B-2 (Riboflavin) 3,0 ND 0,944 87,2 72,6 Vit. B-3 (Niacin) 10,0 35,0 3,148 25,6 19,7 Vit. B-9 (Folate) 1,0 1,0 0, ,3 129,9 Vit. B-12 0,004 N.D. 0, ,0 52,45 Iron (NaFeEDTA) 15,0 27 5,555 80,9 60,8 Zinc 30,0 45,0 11, ,5 158,7 Males, +65 years: P-50 Food Intake of target group = 309,9 g/day Vit. B-1 (Thiamin) 2,0 ND 0,418 41,8 34,9 Vit. B-2 (Riboflavin) 3,0 ND 0,790 72,9 60,8 Vit. B-3 (Niacin) 10,0 35,0 2,634 21,4 16,5 Vit. B-9 (Folate) 1,0 1,0 0, ,8 108,7 Vit. B-12 0,004 N.D. 0,001 52,7 43,9 Iron (NaFeEDTA) 15,0 25,2 4,648 67,7 50,9 Zinc 30,0 45,0 9, ,4 132,8 Females, years: P-50 Food Intake of target group = 291,0 g/day Vit. B-1 (Thiamin) 2,0 ND 0,393 42,9 35,7 Vit. B-2 (Riboflavin) 3,0 ND 0,742 81,6 74,2 Vit. B-3 (Niacin) 10,0 25,0 2,473 20,1 15,5 Vit. B-9 (Folate) 1,0 0,7 0, ,5 102,0 Vit. B-12 0,004 N.D. 0,001 49,5 41,2 Iron (NaFeEDTA) 15,0 18 4,365 38,9 20,5 Zinc 30,0 28,0 8, ,5 121,2 Females, years: P-50 Food Intake of target group = 298,5 g/day Vit. B-1 (Thiamin) 2,0 ND 0,403 44,0 36,6 Vit. B-2 (Riboflavin) 3,0 ND 0,761 83,0 69,2 Vit. B-3 (Niacin) 10,0 35,0 2,538 23,6 18,1 Vit. B-9 (Folate) 1,0 1,0 0, ,8 104,7 Vit. B-12 0,004 N.D. 0,001 50,8 42,3 Iron (NaFeEDTA) 15,0 23,4 4,478 50,7 22,8 Zinc 30,0 45,0 8, ,3 182,8 Females, years: P-50 Food Intake of target group = 294,8 g/day Vit. B-1 (Thiamin) 2,0 ND 0,398 43,4 36,2 Vit. B-2 (Riboflavin) 3,0 ND 0,752 82,0 68,3 Vit. B-3 (Niacin) 10,0 35,0 2,505 23,3 17,9 Vit. B-9 (Folate) 1,0 1,0 0, ,2 103,4 Vit. B-12 0,004 N.D. 0,001 50,1 41,75 Iron (NaFeEDTA) 15,0 25,2 4, ,3 58,7 Zinc 30,0 45,0 8, ,6 180,5 Females, +65 years: P-50 Food Intake of target group = 264,5 g/day Vit. B-1 (Thiamin) 2,0 ND 0,357 39,0 32,5 Vit. B-2 (Riboflavin) 3,0 ND 0,675 73,6 61,3 Vit. B-3 (Niacin) 10,0 35,0 2,249 20,9 16,1 Vit. B-9 (Folate) 1,0 1,0 0, ,9 92,8 Vit. B-12 0,004 N.D. 0,001 45,0 37,5 Iron (NaFeEDTA) 15,0 21,6 3,968 84,3 52,7 Zinc 30,0 45,0 7, ,3 162,0 70

71 Notes: a - These values are calculated taking in consideration the micronutrient losses during storage and distribution, as well as during cooking. EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population. * N.D. = Not determined 71

72 Annex 4 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Pakistan Pakistan: Low diet bioavailability for minerals Nutrient Addition Adjusted Daily intake of micronutrients in the level to upper limit, composition of fortified wheat flour flour, mg/day mg/day a % EAR/day % RNI/day mg/kg Children, 1-3 years: P-50 Food Intake of target group = 115,2 g/day Vit. B-1 (Thiamin) 2,0 ND 0,155 38,9 31,1 Vit. B-2 (Riboflavin) 3,0 ND 0,294 73,4 58,7 Vit. B-3 (Niacin) 10,0 10,0 0,979 21,2 16,3 Vit. B-9 (Folate) 1,0 0,3 0, ,6 107,7 Vit. B-12 0,004 N.D. 0, ,6 43,5 Iron (NaFeEDTA) 15,0 4,7 1,728 69,4 29,8 Zinc 30,0 7,0 3,455 50,0 41,6 Children, 4-6 years: P-50 Food Intake of target group = 146,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,198 41,1 32,9 Vit. B-2 (Riboflavin) 3,0 ND 0,373 77,7 62,2 Vit. B-3 (Niacin) 10,0 15,0 1,244 20,2 15,5 Vit. B-9 (Folate) 1,0 0,4 0, ,3 102,6 Vit. B-12 0,004 N.D. 0, ,8 41,45 Iron (NaFeEDTA) 15,0 7,2 2,195 84,7 34,8 Zinc 30,0 12,0 4,389 54,9 45,7 Children, 7-9 years: P-50 Food Intake of target group = 183,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,248 34,4 27,6 Vit. B-2 (Riboflavin) 3,0 ND 0,468 65,0 52,0 Vit. B-3 (Niacin) 10,0 17,0 1,561 16,9 13,0 Vit. B-9 (Folate) 1,0 0,5 0, ,3 85,9 Vit. B-12 0,004 N.D. 0, ,7 34,7 Iron (NaFeEDTA) 15,0 10,1 2,755 75,2 31,0 Zinc 30,0 12,0 5,510 59,0 49,2 Males, years: P-50 Food Intake of target group = 286,4 g/day Vit. B-1 (Thiamin) 2,0 ND 0,387 38,7 32,2 Vit. B-2 (Riboflavin) 3,0 ND 0,730 67,4 56,2 Vit. B-3 (Niacin) 10,0 25,0 2,434 19,8 15,2 Vit. B-9 (Folate) 1,0 0,7 0, ,5 100,4 Vit. B-12 0,004 N.D. 0,001 48,7 40,55 Iron (NaFeEDTA) 15,0 19,8 4,296 34,4 24,5 Zinc 30,0 28,0 8,592 60,3 50,2 Males, years: P-50 Food Intake of target group = 311,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,420 42,0 35,0 Vit. B-2 (Riboflavin) 3,0 ND 0,794 73,3 61,1 Vit. B-3 (Niacin) 10,0 35,0 2,646 21,5 16,5 Vit. B-9 (Folate) 1,0 1,0 0, ,4 109,1 Vit. B-12 0,004 N.D. 0,001 52,9 44,1 Iron (NaFeEDTA) 15,0 28,8 4,670 45,3 34,1 Zinc 30,0 45,0 9,339 80,0 66,7 Males, years: P-50 Food Intake of target group = 305,1 g/day Vit. B-1 (Thiamin) 2,0 ND 0,412 41,2 34,3 72

73 Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Vit. B-2 (Riboflavin) 3,0 ND 0,778 71,8 59,8 Vit. B-3 (Niacin) 10,0 35,0 2,593 21,1 16,2 Vit. B-9 (Folate) 1,0 1,0 0, ,7 107,0 Vit. B-12 0,004 N.D. 0,001 51,9 43,2 Iron (NaFeEDTA) 15,0 27 4,576 44,4 33,4 Zinc 30,0 45,0 9,152 78,4 65,4 Males, +65 years: P-50 Food Intake of target group = 255,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,345 34,5 28,7 Vit. B-2 (Riboflavin) 3,0 ND 0,651 60,1 50,1 Vit. B-3 (Niacin) 10,0 35,0 2,170 17,6 13,6 Vit. B-9 (Folate) 1,0 1,0 0, ,9 89,5 Vit. B-12 0,004 N.D. 0,001 43,4 36,15 Iron (NaFeEDTA) 15,0 25,2 3,829 37,2 27,9 Zinc 30,0 45,0 7,658 65,6 54,7 Females, years: P-50 Food Intake of target group = 239,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,324 35,3 29,4 Vit. B-2 (Riboflavin) 3,0 ND 0,611 67,2 61,1 Vit. B-3 (Niacin) 10,0 25,0 2,037 16,6 12,7 Vit. B-9 (Folate) 1,0 0,7 0, ,1 84,0 Vit. B-12 0,004 N.D. 0,001 40,8 33,95 Iron (NaFeEDTA) 15,0 18 3,596 21,0 11,1 Zinc 30,0 28,0 7,191 59,9 49,9 Females, years: P-50 Food Intake of target group = 245,9 g/day Vit. B-1 (Thiamin) 2,0 ND 0,332 36,2 30,2 Vit. B-2 (Riboflavin) 3,0 ND 0,627 68,4 57,0 Vit. B-3 (Niacin) 10,0 35,0 2,090 19,4 14,9 Vit. B-9 (Folate) 1,0 1,0 0, ,8 86,2 Vit. B-12 0,004 N.D. 0,001 41,8 34,85 Iron (NaFeEDTA) 15,0 23,4 3,689 27,9 12,5 Zinc 30,0 45,0 7,378 90,3 75,3 Females, years: P-50 Food Intake of target group = 242,8 g/day Vit. B-1 (Thiamin) 2,0 ND 0,328 35,8 29,8 Vit. B-2 (Riboflavin) 3,0 ND 0,619 67,5 56,3 Vit. B-3 (Niacin) 10,0 35,0 2,064 19,2 14,7 Vit. B-9 (Folate) 1,0 1,0 0, ,4 85,1 Vit. B-12 0,004 N.D. 0,001 41,3 34,4 Iron (NaFeEDTA) 15,0 25,2 3,642 71,6 32,2 Zinc 30,0 45,0 7,284 89,2 74,3 Females, +65 years: P-50 Food Intake of target group = 217,9 g/day Vit. B-1 (Thiamin) 2,0 ND 0,294 32,1 26,7 Vit. B-2 (Riboflavin) 3,0 ND 0,556 60,6 50,5 Vit. B-3 (Niacin) 10,0 35,0 1,852 17,2 13,2 Vit. B-9 (Folate) 1,0 1,0 0,180 95,5 76,4 Vit. B-12 0,004 N.D. 0, ,1 30,85 Iron (NaFeEDTA) 15,0 21,6 3,269 46,3 28,9 Zinc 30,0 45,0 6,537 80,0 66,7 73

74 Notes: a - These values are calculated taking in consideration the micronutrient losses during storage and distribution, as well as during cooking. EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50 apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5) apparently healthy individuals in an age- and sex-specific population. * N.D. = Not determined 74

75 Annex 5 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Tajikistan Tajikistan: Moderate diet bioavailability for minerals Nutrient Addition Adjusted Daily intake of micronutrients in the level to upper limit, composition of fortified wheat flour flour, mg/day mg/day a EAR/day RNI/day mg/kg Children, 1-3 years: P-50 Food Intake of target group = 129,6 g/day Vit. B-1 (Thiamin) 2,0 ND 0,175 43,7 35,0 Vit. B-2 (Riboflavin) 3,0 ND 0,331 82,6 66,1 Vit. B-3 (Niacin) 10,0 10,0 1,102 23,9 18,4 Vit. B-9 (Folate) 1,0 0,3 0, ,5 121,2 Vit. B-12 0,004 N.D. 0, ,7 48,95 Iron (NaFeEDTA) 15,0 4,7 1, ,2 50,3 Zinc 30,0 7,0 3, ,8 94,8 Children, 4-6 years: P-50 Food Intake of target group = 164,6 g/day Vit. B-1 (Thiamin) 2,0 ND 0,222 46,3 37,0 Vit. B-2 (Riboflavin) 3,0 ND 0,420 87,5 70,0 Vit. B-3 (Niacin) 10,0 15,0 1,399 22,7 17,5 Vit. B-9 (Folate) 1,0 0,4 0, ,3 115,5 Vit. B-12 0,004 N.D. 0, ,0 46,65 Iron (NaFeEDTA) 15,0 7,2 2, ,9 58,8 Zinc 30,0 12,0 4, ,5 102,9 Children, 7-9 years: P-50 Food Intake of target group = 206,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,279 38,8 31,0 Vit. B-2 (Riboflavin) 3,0 ND 0,527 73,2 58,6 Vit. B-3 (Niacin) 10,0 17,0 1,757 19,0 14,6 Vit. B-9 (Folate) 1,0 0,5 0, ,8 96,6 Vit. B-12 0,004 N.D. 0, ,9 39,05 Iron (NaFeEDTA) 15,0 10,1 3, ,0 52,2 Zinc 30,0 12,0 6, ,9 110,7 Males, years: P-50 Food Intake of target group = 322,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,435 43,5 36,3 Vit. B-2 (Riboflavin) 3,0 ND 0,822 75,9 63,2 Vit. B-3 (Niacin) 10,0 25,0 2,739 22,3 17,1 Vit. B-9 (Folate) 1,0 0,7 0, ,2 113,0 Vit. B-12 0,004 N.D. 0,001 54,8 45,65 Iron (NaFeEDTA) 15,0 19,8 4,834 56,4 40,3 Zinc 30,0 28,0 9, ,9 112,4 Males, years: P-50 Food Intake of target group = 350,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,473 47,3 39,4 Vit. B-2 (Riboflavin) 3,0 ND 0,893 82,5 68,7 Vit. B-3 (Niacin) 10,0 35,0 2,978 24,2 18,6 Vit. B-9 (Folate) 1,0 1,0 0, ,5 122,8 Vit. B-12 0,004 N.D. 0,001 59,6 49,65 Iron (NaFeEDTA) 15,0 28,8 5,255 76,5 57,5 Zinc 30,0 45,0 10, ,2 150,1 Males, years: P-50 Food Intake of target group = 343,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,463 46,3 38,6 75

76 Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a EAR/day RNI/day Vit. B-2 (Riboflavin) 3,0 ND 0,875 80,8 67,3 Vit. B-3 (Niacin) 10,0 35,0 2,918 23,7 18,2 Vit. B-9 (Folate) 1,0 1,0 0, ,5 120,4 Vit. B-12 0,004 N.D. 0,001 58,4 48,65 Iron (NaFeEDTA) 15,0 27 5,149 75,0 56,4 Zinc 30,0 45,0 10, ,6 147,1 Males, +65 years: P-50 Food Intake of target group = 287,2 g/day Vit. B-1 (Thiamin) 2,0 ND 0,388 38,8 32,3 Vit. B-2 (Riboflavin) 3,0 ND 0,732 67,6 56,3 Vit. B-3 (Niacin) 10,0 35,0 2,442 19,8 15,3 Vit. B-9 (Folate) 1,0 1,0 0, ,9 100,7 Vit. B-12 0,004 N.D. 0,001 48,9 40,7 Iron (NaFeEDTA) 15,0 25,2 4,309 62,7 47,2 Zinc 30,0 45,0 8, ,7 123,1 Females, years: P-50 Food Intake of target group = 269,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,364 39,7 33,1 Vit. B-2 (Riboflavin) 3,0 ND 0,688 75,7 68,8 Vit. B-3 (Niacin) 10,0 25,0 2,293 18,6 14,3 Vit. B-9 (Folate) 1,0 0,7 0, ,2 94,6 Vit. B-12 0,004 N.D. 0,001 45,9 38,2 Iron (NaFeEDTA) 15,0 18 4,046 36,0 19,0 Zinc 30,0 28,0 8, ,9 112,4 Females, years: P-50 Food Intake of target group = 276,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,374 40,8 34,0 Vit. B-2 (Riboflavin) 3,0 ND 0,706 77,0 64,2 Vit. B-3 (Niacin) 10,0 35,0 2,352 21,8 16,8 Vit. B-9 (Folate) 1,0 1,0 0, ,3 97,0 Vit. B-12 0,004 N.D. 0,001 47,1 39,2 Iron (NaFeEDTA) 15,0 23,4 4,151 47,0 21,2 Zinc 30,0 45,0 8, ,3 169,4 Females, years: P-50 Food Intake of target group = 273,2 g/day Vit. B-1 (Thiamin) 2,0 ND 0,369 40,2 33,5 Vit. B-2 (Riboflavin) 3,0 ND 0,697 76,0 63,3 Vit. B-3 (Niacin) 10,0 35,0 2,322 21,6 16,6 Vit. B-9 (Folate) 1,0 1,0 0, ,8 95,8 Vit. B-12 0,004 N.D. 0,001 46,5 38,7 Iron (NaFeEDTA) 15,0 25,2 4, ,8 54,4 Zinc 30,0 45,0 8, ,7 167,3 Females, +65 years: P-50 Food Intake of target group = 245,2 g/day Vit. B-1 (Thiamin) 2,0 ND 0,331 36,1 30,1 Vit. B-2 (Riboflavin) 3,0 ND 0,625 68,2 56,8 Vit. B-3 (Niacin) 10,0 35,0 2,084 19,4 14,9 Vit. B-9 (Folate) 1,0 1,0 0, ,5 86,0 Vit. B-12 0,004 N.D. 0,001 41,7 34,75 Iron (NaFeEDTA) 15,0 21,6 3,678 78,1 48,8 Zinc 30,0 45,0 7, ,2 150,1 76

77 Notes: a - These values are calculated taking in consideration the micronutrient losses during storage and distribution, as well as during cooking. EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50 apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5) apparently healthy individuals in an age- and sex-specific population. * N.D. = Not determined 77

78 Annex 6 Refined wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Uzbekistan Uzbekistan: Moderate diet bioavailability for minerals Nutrient Addition Adjusted Daily intake of micronutrients in the level to upper limit, composition of fortified wheat flour flour, mg/day mg/day a EAR/day RNI/day mg/kg Children, 1-3 years: P-50 Food Intake of target group = 172,9 g/day Vit. B-1 (Thiamin) 2,0 ND 0,233 58,4 46,7 Vit. B-2 (Riboflavin) 3,0 ND 0, ,2 88,2 Vit. B-3 (Niacin) 10,0 10,0 1,470 31,8 24,5 Vit. B-9 (Folate) 1,0 0,3 0, ,1 161,7 Vit. B-12 0,004 N.D. 0, ,9 65,3 Iron (NaFeEDTA) 15,0 4,7 2, ,3 67,1 Zinc 30,0 7,0 5, ,8 126,5 Children, 4-6 years: P-50 Food Intake of target group = 219,6 g/day Vit. B-1 (Thiamin) 2,0 ND 0,297 61,8 49,4 Vit. B-2 (Riboflavin) 3,0 ND 0, ,7 93,3 Vit. B-3 (Niacin) 10,0 15,0 1,867 30,3 23,3 Vit. B-9 (Folate) 1,0 0,4 0, ,5 154,0 Vit. B-12 0,004 N.D. 0, ,7 62,25 Iron (NaFeEDTA) 15,0 7,2 3, ,6 78,4 Zinc 30,0 12,0 6, ,7 137,3 Children, 7-9 years: P-50 Food Intake of target group = 275,7 g/day Vit. B-1 (Thiamin) 2,0 ND 0,372 51,7 41,4 Vit. B-2 (Riboflavin) 3,0 ND 0,703 97,6 78,1 Vit. B-3 (Niacin) 10,0 17,0 2,344 25,4 19,5 Vit. B-9 (Folate) 1,0 0,5 0, ,1 128,9 Vit. B-12 0,004 N.D. 0,001 62,5 52,1 Iron (NaFeEDTA) 15,0 10,1 4, ,4 69,7 Zinc 30,0 12,0 8, ,2 147,7 Males, years: P-50 Food Intake of target group = 429,9 g/day Vit. B-1 (Thiamin) 2,0 ND 0,580 58,0 48,4 Vit. B-2 (Riboflavin) 3,0 ND 1, ,2 84,3 Vit. B-3 (Niacin) 10,0 25,0 3,654 29,7 22,8 Vit. B-9 (Folate) 1,0 0,7 0, ,4 150,7 Vit. B-12 0,004 N.D. 0, ,1 60,9 Iron (NaFeEDTA) 15,0 19,8 6,449 75,2 53,7 Zinc 30,0 28,0 12, ,0 150,0 Males, years: P-50 Food Intake of target group = 467,3 g/day Vit. B-1 (Thiamin) 2,0 ND 0,631 63,1 52,6 Vit. B-2 (Riboflavin) 3,0 ND 1, ,0 91,7 Vit. B-3 (Niacin) 10,0 35,0 3,972 32,3 24,8 Vit. B-9 (Folate) 1,0 1,0 0, ,8 163,8 Vit. B-12 0,004 N.D. 0, ,5 66,2 Iron (NaFeEDTA) 15,0 28,8 7, ,1 76,7 Zinc 30,0 45,0 14, ,3 200,3 Males, years: P-50 Food Intake of target group = 458,0 g/day Vit. B-1 (Thiamin) 2,0 ND 0,618 61,8 51,5 78

79 Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a EAR/day RNI/day Vit. B-2 (Riboflavin) 3,0 ND 1, ,8 89,8 Vit. B-3 (Niacin) 10,0 35,0 3,893 31,6 24,3 Vit. B-9 (Folate) 1,0 1,0 0, ,7 160,6 Vit. B-12 0,004 N.D. 0, ,9 64,9 Iron (NaFeEDTA) 15,0 27 6, ,0 75,2 Zinc 30,0 45,0 13, ,5 196,3 Males, +65 years: P-50 Food Intake of target group = 383,2 g/day Vit. B-1 (Thiamin) 2,0 ND 0,517 51,7 43,1 Vit. B-2 (Riboflavin) 3,0 ND 0,977 90,2 75,2 Vit. B-3 (Niacin) 10,0 35,0 3,257 26,5 20,4 Vit. B-9 (Folate) 1,0 1,0 0, ,9 134,4 Vit. B-12 0,004 N.D. 0, ,2 54,3 Iron (NaFeEDTA) 15,0 25,2 5,748 83,7 62,9 Zinc 30,0 45,0 11, ,1 164,2 Females, years: P-50 Food Intake of target group = 359,8 g/day Vit. B-1 (Thiamin) 2,0 ND 0,486 53,0 44,2 Vit. B-2 (Riboflavin) 3,0 ND 0, ,9 91,8 Vit. B-3 (Niacin) 10,0 25,0 3,058 24,9 19,1 Vit. B-9 (Folate) 1,0 0,7 0, ,7 126,2 Vit. B-12 0,004 N.D. 0,001 61,2 50,95 Iron (NaFeEDTA) 15,0 18 5,397 48,1 25,3 Zinc 30,0 28,0 10, ,9 149,9 Females, years: P-50 Food Intake of target group = 369,2 g/day Vit. B-1 (Thiamin) 2,0 ND 0,498 54,4 45,3 Vit. B-2 (Riboflavin) 3,0 ND 0, ,7 85,6 Vit. B-3 (Niacin) 10,0 35,0 3,138 29,1 22,4 Vit. B-9 (Folate) 1,0 1,0 0, ,8 129,4 Vit. B-12 0,004 N.D. 0, ,8 52,3 Iron (NaFeEDTA) 15,0 23,4 5,538 62,7 28,3 Zinc 30,0 45,0 11, ,2 226,0 Females, years: P-50 Food Intake of target group = 364,5 g/day Vit. B-1 (Thiamin) 2,0 ND 0,492 53,7 44,7 Vit. B-2 (Riboflavin) 3,0 ND 0, ,4 84,5 Vit. B-3 (Niacin) 10,0 35,0 3,098 28,8 22,1 Vit. B-9 (Folate) 1,0 1,0 0, ,8 127,8 Vit. B-12 0,004 N.D. 0,001 62,0 51,65 Iron (NaFeEDTA) 15,0 25,2 5, ,1 72,6 Zinc 30,0 45,0 10, ,8 223,2 Females, +65 years: P-50 Food Intake of target group = 327,1 g/day Vit. B-1 (Thiamin) 2,0 ND 0,442 48,2 40,1 Vit. B-2 (Riboflavin) 3,0 ND 0,834 91,0 75,8 Vit. B-3 (Niacin) 10,0 35,0 2,780 25,8 19,9 Vit. B-9 (Folate) 1,0 1,0 0, ,4 114,7 Vit. B-12 0,004 N.D. 0,001 55,6 46,35 Iron (NaFeEDTA) 15,0 21,6 4, ,2 65,1 Zinc 30,0 45,0 9, ,3 200,3 79

80 Notes: a - These values are calculated taking in consideration the micronutrient losses during storage and distribution, as well as during cooking. EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50 apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5) apparently healthy individuals in an age- and sex-specific population. * N.D. = Not determined 80

81 Literature 1. WHO, FAO, UNICEF, GAIN, MI, & FFI. Recommendations on wheat and maize flour fortification. Meeting Report: Interim Consensus Statement. Geneva, World Health Organization, Wheat Flour Fortification: Current Knowledge and Practical Applications. Summary report of an international technical workshop. Cuernavaca, Mexico, December 1-3, 2004, 31 p KAP Komplex 1 Specification. Wheat flour fortification. American Ingredients Company, September Приказ Министерства здравоохранения Кыргызской Республики «О внесении изменений в приказ МЗ КР г. N 655». г.бишкек, 12 октября 2013 years N ГОСТ РТ «Мука пшеничная хлебопекарная, обогащенная витаминоминеральной toбавкой (премикс), Таджикистан. 7. Мука пшеничная хлебопекарная первого сорта, обогащенная витаминноминеральной смесью. Технические условия. O z DSt 1104:2011, Узбекистан. 8. Pakistan standard specification for fortified wheat atta. PS: ICS No , 30 p. 9. Fortified wheat flour specification. Draft Afghanistan Standard. Afghanistan National Standards Authority, 13 June 2013, 10 p. 10. Food and Agriculture Organization of the United Nations/World Health Organization. Requirements of vitamin A, iron, folate and vitamin B12. FAO Food and Nutrition Series, No 23. Rome: FAO. 1988; Vitamin and mineral requirements in human nutrition. Second edition. WHO/FAO, 2004, 362 p. 12. WHO/FAO. Vitamin and mineral requirements in human nutrition. 2nd ed. Geneva, Switzerland: World Health Organization and Food and Agriculture Organization of the United Nations, Richard Hurrell and Ines Egli. Iron bioavailability and dietary reference values. Am J Clin Nutr 2010; 91(suppl): 1461S 7S Quintaes KD, Cilla A and Barberá R. Iron Bioavailability from Cereal Foods Fortified with Iron. Austin J Nutr Metab. 2015;2(3): Hallberg L, Hulthen L. Prediction of dietary iron absorption: an algorithm for calculating absorption and bioavailability of dietary iron. Am J Clin Nutr 2000;71: (Published erratum appears in Am J Clin Nutr 2000;72:1242.) 16. Food and Nutrition Board, Institute of Medicine. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press, Hunt JR, Roughead ZK. Nonheme-iron absorption, fecal ferritin excretion, and blood indexes of iron status in women consuming controlled lactoovovegetarian diets for 8 wk. Am J Clin Nutr 1999;69: Monsen ER, Hallberg L, Layrisse M, et al. Estimation of available dietary iron. Am J Clin Nutr 1978;31: Cook JD. Adaptation in iron metabolism. Am J Clin Nutr 1990; 51(2): Hallberg L, Hulten L, Gramatkovski E. Iron absorption from the whole diet in men: how effective is the regulation of iron absorption? Am J Clin Nutr 1997;66: Walter HL, Fanny L, Charles C, Christian R. Minerals and phytic acid interaction: is it a real problem for human nutrition. Int J Food Sc Tech. 2002; 37(7): Bohn, L.; Meyer, A.S.; Rasmussen, S.K. Phytate: Impact on environment and human nutrition. A challenge for molecular breeding. J. Zhejiang Univ. Sci. B 2008, 9, By Ryan Andrews. Phytates and phytic acid. 81

82 24. Schlemmer U, et al. Phytate in foods and significance for humans: Food sources, intake, processing, bioavailability, protective role and analysis. Mol Nutr Food res 2009;53:S330-S Rosalind S. Gibson, Karl B. Bailey, Michelle Gibbs, Elaine L. Ferguson. A review of phytate, iron, zinc, and calcium concentrations in plant-based complementary foods used in lowincome countries and implications for bioavailability. Food and Nutrition Bulletin, vol. 31, no. 2 (supplement), p. S134-S West, A.R.; Oates, P.S. Mechanisms of heme iron absorption: Current questions and controversies. World J. Gastroenterol. 2008, 14, Theil, E.C.; Briat, J.-F. Plant Ferritin and Non-Heme Iron Nutrition in Humans; International Food Policy Research Institute and International Center for Tropical Agriculture: Washington, DC, USA, Cook JD, Dassenko SA, Whittaker P. Calcium supplementation: effect on iron absorption. Am J Clin Nutr. 1991; 53: Allen LH, Ahluwalia N. Improving iron status through diet: the applications of knowledge concerning dietary iron bioavailability in human populations. John Snow Incorporated/OMNI Project, Washington: Díaz-Castro J, Lisbona F, Moreno M, Alférez MJM, Campos M, López-Aliaga. Influence of Goat Milk on Iron Deficiency Anemia Recovery. Int J Dairy Sci Process, 2015, 2(1), p Judith R Turnlund, Radojka G Smith, MaryJKretsch, William R Keyes, and Alka G Shah. Milk s effect on the bioavailability of iron from cereal-based diets in young women by use of in vitro and in vivo methods. Am J C/in Nuir 1990;52: Hallberg L, Rossander-Hulten L, Brune M, Gleerup A. Inhibition of haem-iron absorption in man by calcium. Br J Nutr. 1993; 69(2): Scientific Opinion on the use of ferric sodium EDTA as a source of iron added for nutritional purposes to foods for the general population (including food supplements) and to foods for particular nutritional uses. European Food Safety Authority (EFSA), Parma, Italy. EFSA Journal 2010; 8(1):1414, 32 p Hurrell RF, Reddy MB, Burri J, Cook JD, An evaluation of EDTA compounds for iron fortification of cereal-based foods. Br J Nutr 84, Hurrell R. Preventing iron deficiency through food fortification. Nutr Rev. 1997; 55(6): gh_food_fortification_nutr_rev_55_ Subar AF, Krebs-Smith SM, Cook A, Kahle LL. Dietary sources of nutrients among US adults, 1989 to J Am Diet Assoc 1998;98: Harland BF, Oberleas D. Phytate in foods. World Rev Nutr Diet 1987; 52: Sandström B, Arvidsson B, Cederblad A, Bjorn-Rasmussen E. Zinc absorption from composite meals, I: the significance of wheat extraction rate, zinc, calcium, and protein content in meals based on bread. Am J Clin Nutr 1980;33: Food and Nutrition Board, Institute of Medicine. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press, Hurrell R, Egli I. Iron bioavailability and dietary reference values. Am J Clin Nutr. 2010; 91: 1461S-1467S. 41. Scientific Committee on Food (SCF): Nutrient and Energy Intakes for the European Community. Opinion adopted by the SCF on 11 December In Reports of the SCF Series N.o 31: Luxemburg, European Commission Institute of Medicine (IOM). Food and Nutrition Board. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc. National Academy Press: Washington, D.C

83 43. Vitamin and mineral requirements in human nutrition. Second edition. WHO, FAO, Omar Dary and Michael Hainsworth. The Food Fortification Formulator. Technical Determination of Fortification Levels and Standards for Mass Fortification. USAID, April Acknowledgement: This document was made possible by the generous support of the American people through the United States Agency for International Development (USAID). The contests are the responsibility of the Kazakh Academy of Nutrition (KAN) and GAIN and do not necessarily reflect the views of USAID or the United States Government. 83

84 Annex 4. Analysis and Justification of the Possibility of Harmonizing Standards for Highextraction Wheat Flour Fortification in Afghanistan and Pakistan Kazakh Academy of Nutrition USAID/GAIN MICRONUTRIENT FORTIFICATION PROJECT IN CENTRAL ASIA, AFGHANISTAN AND PAKISTAN ANALYSIS AND JUSTIFICATION OF THE POSSIBILITY OF HARMONIZING STANDARDS FOR HIGH-EXTRACTION WHEAT FLOUR FORTIFICATION IN AFGHANISTAN AND PAKISTAN The Document have been developed by Shamil Tazhibayev Professor, Vice President of Kazakh Academy of Nutrition under the edition of: Turegeldy Sharmanov Academician of RAS and NAS of RK, President of Kazakh Academy of Nutrition Omar Dary Professor, Health Science Specialist (Nutrition), USAID, Bureau of Global Health and Quentin Johnson Coordinator, Training and Technical Support Group, the Food Fortification Initiative Almaty

85 85

86 Contents 1. Comparison of standards for high-extraction wheat flour fortification, operating in Afghanistan and Pakistan with the relevant recommendations of the World Health 2 Organization (WHO) 2. Standards for mandatory and voluntary fortification of wheat flour 4 3. Setting the level of bioavailability of minerals from high-extraction wheat flour in 4 Afghanistan and Pakistan 4. Premix formulation for fortification of high-extraction wheat flour 6 5. Establishment of production and regulatory parameters for fortification of highextraction wheat flour 8 6. Daily intake of micronutrients by population groups in the composition of highextraction fortified wheat flour, in Afghanistan and Pakistan, in % EAR/day and % 10 RNI/day 7. Brief description of the annexes Conclusion 13 Annex 1 High-extraction wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Afghanistan Annex 2 High-extraction wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Pakistan Literature 18 Acknowledgement

87 1. Comparison of standards for high-extraction wheat flour fortification, operating in Afghanistan and Pakistan with the relevant recommendations of the World Health Organization (WHO). According to an interim consensus statement, endorsed by WHO and other institutions about recommendations on wheat and maize flour fortification [1] (reference), the average levels of nutrients that can be added to fortifying wheat flour depending on the: estimated average per capita consumption (g/day) of wheat flour for all the recommended micronutrients; extraction level of flour (low or high) for iron and zinc; fortifying nutrient for all the recommended micronutrients, and others are required by the nutrition situation of the country (Table 1). NaFeEDTA, ferrous sulfate, ferrous fumarate and electrolytic iron may serve as the source of iron. However electrolytic iron cannot be used neither as an iron source in cases where the estimated average per capita consumption of wheat flour is less than 150 g/day nor for high-extraction flour (i.e. whole flour). (high-extraction-rate = 80% wheat flour [2]; so, low-extraction-rate = <80% wheat flour). This is because very high levels of electrolytic iron that are needed could negatively affect sensory properties of fortified flour. The average per capita consumption of wheat flour in the member countries is [3]: 311,3 g/day in Pakistan; 439,0 g/day in Afghanistan. Due to differences in the average per day consumption of wheat flour per person: Pakistan and Afghanistan to countries where average consumption of wheat flour per capita is >300 g/day. Overall, the average levels of per capita per day consumption of flour in the both countries are high and may contribute to the harmonization of standards for flour fortification. In Afghanistan, flour is expected to be fortified by 4 micronutrients (vitamins B9 and B12, iron and zinc); in Pakistan, currently only 2 micronutrients - vitamin B9 and iron, are required at the national level (Table 2). The levels of added micronutrients to the flour: In Afghanistan and Pakistan, data referred to the flour called Atta, which traditionally refers to high-extraction flour. The levels of added micronutrients to the flour in comparison with the WHO-endorsed recommendations are: slightly higher in Pakistan for vitamin В9; somewhat lower in Pakistan for iron; lower in Afghanistan for zinc; other parameters in those countries comply with the recommendations of WHO. 87

88 Table 1 WHO interim consensus statement about recommendations for fortified flours, 2009 a : Average levels of nutrients to consider adding to fortified wheat flour based on extraction, fortificant compound, and estimated per capita flour availability Nutrient Flour extraction rate Compound Level of nutrient to be added in parts per million (ppm) by estimated average per capita wheat flour availability (g/day) b <75g/day c >300g/day g/day g/day Iron Low NaFeEDTA Ferrous sulphate Ferrous fumarate Electrolytic iron NR d NR d High NaFeEDTA Folic acid Low or Folic acid high Vitamin Low or Cyanocobalamin B12 high Vitamin Low or Vit A palmitate A high Zinc e Low Zinc oxide High Zinc oxide Notes: a WHO, FAO, UNICEF, GAIN, MI, & FFI. Recommendations on wheat and maize flour fortification. Meeting Report: Interim Consensus Statement. Geneva, World Health Organization, 2009 ( b These estimated levels consider only wheat flour as main fortification vehicle in a public health program. If other mass-fortification programs with other food vehicles are implemented effectively, these suggested fortification levels may need to be adjusted downwards as needed. c Estimated per capita consumption of <75 g/day does not allow for addition of sufficient level of fortificant to cover micronutrients needs for women of childbearing age. Fortification of additional food vehicles and other interventions should be considered. d NR = Not Recommended because very high levels of electrolytic iron needed could negatively affect sensory properties of fortified flour. e These amounts of zinc fortification assume 5 mg zinc intake and no additional phytate intake from other dietary sources 88

89 Table 2 Summary data on the levels of micronutrients to be added in parts per million (ppm) to fortified wheat flour according to standards in Central Asian Republics, Afghanistan and Pakistan Items Pakistan Afghanistan Atta [4] Atta [5] Vitamin B1 n/f n/f Vitamin B2 n/f n/f Vitamin B3 n/f n/f Vitamin B9 1,5 1,0 WHO, ,0 1,0 Vitamin B12 n/f 0,008 WHO, ,008 0,008 Iron 10,0 15,0 WHO, ,0 15,0 Sodium Iron EDTA Sodium Iron EDTA Zinc n/a 30,0 WHO, ,0 30,0 Notes: n/f not fortified Wheat flour consumption in g/capita/day: > 300 in Afghanistan and Pakistan 2. Standards for mandatory and voluntary fortification of wheat flour. It seems to be appropriate to develop the following standards for: Mandatory fortification of flour with micronutrients recommended by the World Health Organization, namely, by vitamins B9 and B12, iron and zinc for all types of flour, plus B1, B2, and B3 for refined (low extraction) flour; As the high-extraction wheat flour contains good amounts of the latter vitamins; those would not be added to this type of flour. Carry out studies of vitamin A and vitamin D deficiencies in all the countries in order to establish if the addition of these other micronutrients would be appropriate for the whole region. 3. Setting the level of bioavailability of minerals from high-extraction wheat flour in Afghanistan and Pakistan. The Food Agriculture Organization of the United Nations/World Health Organization set iron bioavailability at 5% for a strict vegetarian diet, at 10% when some meat and ascorbic acid was added, and at 15% for diets rich in meat and fruits [6]. The diet of the population of Afghanistan and Pakistan can be referred to the group with 5% (low) bioavailability of minerals, because the average per capita intake is: high (311 g/day in Pakistan and 439 g/day in Afghanistan) for wheat flour, which is mainly high-extracted one (inhibitors of iron absorption); very low (45 g/day in Pakistan and 34 g/day in Afghanistan) for meat and meat products, which contain heme iron (promotor of iron absorption); very low for vegetables (72 g/day in Pakistan and 79 g/day in Afghanistan) and fruits (80 g/day in Pakistan and 93 g/day in Afghanistan), which are the reach sources of vitamin C (promotor of iron absorption). This subdivision of diet dependent levels of minerals bioavailability should be taken into account for calculating the levels of micronutrients in fortified flour and comparing them with vitamin and mineral requirements in human nutrition according to WHO/FAO data [7]. The total iron content of a diet provides little information about its content of bioavailable iron, 89

90 which is considerably influenced by the foods in the diet and can vary 10-fold from different meals of similar iron content [8]. Although a vegetarian diet is likely to contain iron in amounts equivalent to amounts in a nonvegetarian diet, the iron from a vegetarian diet is likely to be substantially less available for absorption [9] because of differences in the chemical form of iron and the accompanying constituents that enhance or inhibit iron absorption [10]. The chemical form of iron is an important factor affecting the iron availability of vegetarian diets. In the diet inorganic iron-salts (non-heme) are present in plants and animal tissues, and organic iron (heme), which comes from hemoglobin (blood) and myoglobin (red muscle), is present in animal food sources. Heme-iron absorption is less affected by dietary compounds with the exception of calcium compounds [11]. Less than 40% of the iron in meat, poultry, and fish [12] is in the heme form, which is more efficiently absorbed than the remaining nonheme iron present in these and all other foods [13]. Nonvegetarian diets with substantial amounts of red meat supply about 2 mg/d, or 10 15%, of the total iron in the heme form [6]. Heme iron is better absorbed (around 15 40%) than nonheme iron (around 1 15%) [14]. Whole cereals contain phytic acids and polyphenols, which decrease iron bioavailability [15]. The phytic acid levels in low extraction wheat flour is low compared to high extraction flour. Also bread in most of the countries in CAR is fermented using yeast. Pakistan and Afghanistan have high consumption of unleavened bread made from high extraction flour. This means the phytic acid is not broken in the latter country by the fermentation step. The fact is that the whole wheat flour (i.e. high extraction flour) is a good source of phytic acid, which is the main inhibitor of non-heme iron absorption from plant products. The phosphate groups of phytic acid are negatively charged under physiologically relevant conditions, resulting in phytate chelation of cations such as iron and zinc, making these minerals less available for absorption [14]. Consuming 5-10 mg of phytic acid can reduce iron absorption by 50% [15, 16]. Phytate content in refined (white) flour is about 100 mg/100 g, and in wheat and whole-wheat flour it is about 600 mg/100 g [17]. Polyphenols also forms insoluble complexes with iron thereby reducing its bioavailability to the body. The bioavailability of the heme iron from meat and meat products [18] is significantly higher (15-40%) than the bioavailability of non-heme iron from plant foods [19]. Heme-iron absorption is less affected by dietary compounds with the exception of calcium compounds [20]. The inhibitory effect of calcium on iron absorption was recognized many years ago, and the presence of large amounts of calcium can inhibit the absorption of iron from a fortified food [21]. Different studies have been conducted but they often give conflicting results because several factors influence the interaction between calcium and iron absorption [11]. Milk and dairy products are good sources of calcium. But, absorption of iron and zinc from milk products is higher than from vegetable products, even it is considered that calcium from milk and milk products found as calcium phosphate, inhibit absorption of non-heme and heme iron [11], and again there are conflicting results. For example, goat milk consumption leads to a better recovery of body Fe stores, minimizing Ca-Fe interactions and improving Fe status and its absorption [22]. The absorption of iron from the cereal-based diets was not inhibited by cow s milk [23]. On the other hand, milk contains calcium and caseins, which inhibit absorption of both non-heme and heme iron. It enters into the mucosal cells by different pathways and leave in the same form which implies that calcium inhibit the intracellular transport of iron [24]. To ensure an appropriate iron absorption, the intake of iron should be high enough to improve or maintain iron status. This condition might be attained adding sufficient iron to the fortification vehicle and/or incorporating simultaneously absorption enhancers. In the case of wheat flour fortification, the only practical enhancer to add is EDTA or using as an iron source NaFeEDTA; iron in the form of ferric sodium EDTA is 2 to 3 times more bioavailable than from other mineral sources and that it is efficiently incorporated into haemoglobin [25]; iron from ferric sodium EDTA has a high bioavailability despite the presence of inhibitory factors that form insoluble complexes [26]. Vitamin C, which can increase absorption of both native iron and fortification iron due to both its 90

91 reducing power and chelating actions [27], is destroyed during baking. Bovine hemoglobin is not easily accepted or it is too expensive for being used as a fortificant. More than half of the zinc in US diets is derived from animal foods, and one quarter of the zinc comes from beef [28]. The bioavailability of zinc from vegetarian diets is also to be less than that of nonvegetarian diets. Plant foods rich in zinc such as legumes, whole grains, nuts, and seeds are also high in phytic acid, an inhibitor of zinc bioavailability [29]. Bioavailability of zinc is enhanced by dietary protein [30], but plant sources of protein are also generally high in phytic acid. Because of lower absorption of zinc, those consuming vegetarian diets, especially with phytate-zinc molar ratios > 15, may require as much as 50% more zinc than nonvegetarians [31, 32]. Thus, the iron and zinc from vegetarian diets are generally less bioavailable than from nonvegetarian diets because of reduced meat intake as well as the tendency to consume more phytic acid and other plant-based inhibitors of iron and zinc absorption. Iron bioavailability is estimated to be around 5-12% for vegetarian diets and 14-18% for mixed diets. These values are used to generate dietary reference values for all population groups. Considering all factors that may influence iron bioavailability, the estimated average absorption iron rate for a typical western diet is between 15-18% [33-36]. 4. Premix formulation for fortification of high-extraction wheat flour. In view of the above WHO recommendations, the average per capita consumption of wheat flour g/day in the participating countries, and the characteristics of diets with low and moderate bioavailability of minerals, premix composition is formulated for mandatory fortification of highextraction flour, which is presented in Table 3. 91

92 Table 3 The main parameters of the premix for fortification of high-extraction wheat flour. Nutrient Fortificant compound Selected FL (mg/kg flour) Amount of fortificant (mg/kg flour) Fortificant (g/kg premix) Premix Formulation Nutrient (g/kg premix) Cost (US$/kg) Vit. B-9 (Folate) Folic Acid 1,0 1,1 4,4 4,0 $0,49 6,5 Vit. B-12 Vit. B % WS 0,008 8,0 32,0 0,03 $1,28 16,9 Iron NaFeEDTA 15,0 115,4 461, $3,00 39,6 Zinc Zinc oxide 30,0 37,5 150,0 120,0 $0,45 5,9 Filling material (at least 25%) 40,5 352,0 $0,35 4,6 TOTAL 202,5 1000,0 Estimated cost of manufacturing, quality control and delivery (Approx. US$2/kg premix) % Cost $2,00 26,4 Approximate Cost per kg = $7,57 100,0 Estimated cost of micronutrient premix per metric ton of fortified product*: $1,89 0,38 % of the Price Minimum Amount (grams per MT) 202 Maximum Dilution Factor = 1/ 4938 Selected Amount (grams per MT) 250** Selected Dilution Factor = 1/ 4000 Notes: * - The costs of the fortificants are always the largest cost of the fortification process, when it carried out by formal and centralized factories. ** - This value should be larger than the estimated minimum amount per metric ton (above). 92

93 5. Establishment of production and regulatory parameters for fortification of highextraction wheat flour. Based on the composition of the premix, production and regulatory parameters for fortification of high-extraction flour are established, which are presented in Table 4. 93

94 Table 4 The main production and regulatory parameters for fortification of high-extraction wheat flour Nutrient Fortificant compound Selected FL Production Parameters Regulatory Parameters (mg/kg flour) mfl (1) (mg/kg flour) Average (2) (mg/kg flour) MFL (3) (mg/kg flour) LmL (4) (mg/kg flour) MTL (5) (mg/kg flour) Vit. B-9 (Folate) Folic Acid 1,0 0,8 1,4 2,0 0,7 2,0 Vit. B-12 Vit. B % WS 0,008 0,004 0,008 0,012 0,004 0,012 Iron NaFeEDTA 15,0 36,0 53,0 70,0 36,0 70,0 Zinc Zinc oxide 30,0 40,0 59,0 78,0 40,0 78,0 Notes: (1) mfl = Minimum Fortification Level, using equation 2. (2) Average = Selected Fortification Level + Intrinsic content of micronutrient in unfortified food. (3) MFL = Maximum Fortification Level, using equation 3. (4) LmL= Legal Minimum Level, using equation 4. (5) MTL = Maximum Tolerable Level, equivalent to MFL but only for those micronutrients with safety concerns, and rounded. 94

95 6. Daily intake of micronutrients by population groups in the composition of highextraction fortified wheat flour, in Afghanistan and Pakistan, in % EAR/day and % RNI/day. Terms [37]: EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population. All basic calculations in this and other sections are conducted using the Formulator, developed by Omar Dary и Michael Hainsworth [38]. In terms of daily intake of nutrients, in % EAR/day and % RNI/day, in different age and gender population groups (Table 5) of two countries are located in the following ascending order: Nutrient/Country % EAR/day % RNI/day Folic acid: Pakistan 96% to 135% 76% to 109% Afghanistan 135% to 192% 108% to 154% Vitamin В12: Pakistan 74% to 113% 62% to 88% Afghanistan 105% to 160% 87% to 124% Iron: Pakistan 21% to 85% 11% to 35% Afghanistan 30% to 119% 16% to 49% Zinc: Pakistan 50% to 90% 42% to 75% Afghanistan 71% to 127% 59% to 106% Table 5 Daily intake of micronutrients by population groups in the composition of high-extraction fortified wheat flour, in Afghanistan and Pakistan, in % EAR/day and % RNI/day Nutrient Additional daily intake of nutrients by population groups, in Afghanistan Pakistan % EAR/day % RNI/day % EAR/day % RNI/day Children, 1-3 years Vitamin B-9 (Folate) 189,8 151,9 134,6 107,7 Vitamin B ,5 122,7 113,1 87,0 Iron (NaFeEDTA) 97,9 42,0 69,4 29,8 Zinc 70,5 58,7 50,0 41,6 Children, 4-6 years Vitamin B-9 (Folate) 180,9 144,7 128,3 102,6 Vitamin B ,3 116,9 99,5 82,9 Iron (NaFeEDTA) 119,4 49,1 84,7 34,8 Zinc 77,4 64,5 54,9 45,7 Children, 7-9 years Vitamin B-9 (Folate) 151,4 121,1 107,3 85,9 Vitamin B ,4 97,8 83,3 69,4 Iron (NaFeEDTA) 106,1 43,7 75,2 31,0 Zinc 83,3 69,4 59,0 49,2 Males, years Vitamin B-9 (Folate) 177,0 141,6 125,5 100,4 Vitamin B ,3 114,4 97,4 81,1 95

96 Nutrient Additional daily intake of nutrients by population groups, in Afghanistan Pakistan % EAR/day % RNI/day % EAR/day % RNI/day Iron (NaFeEDTA) 48,5 34,6 34,4 24,5 Zinc 85,0 70,9 60,3 50,2 Males, years Vitamin B-9 (Folate) 192,4 153,9 136,4 109,1 Vitamin B ,3 124,4 105,8 88,2 Iron (NaFeEDTA) 63,9 48,1 45,3 34,1 Zinc 112,9 94,1 80,0 66,7 Males, years Vitamin B-9 (Folate) 188,6 150,8 136,4 109,1 Vitamin B ,3 121,9 105,8 88,2 Iron (NaFeEDTA) 62,6 47,1 45,3 34,1 Zinc 110,6 92,2 80,0 66,7 Males, +65 years Vitamin B-9 (Folate) 157,8 126,2 111,9 89,5 Vitamin B ,4 102,0 86,8 72,3 Iron (NaFeEDTA) 52,4 39,4 37,2 27,9 Zinc 92,6 77,1 65,6 54,7 Females, years Vitamin B-9 (Folate) 148,2 118,5 105,1 84,0 Vitamin B ,9 95,8 81,5 67,9 Iron (NaFeEDTA) 29,6 15,6 21,0 11,1 Zinc 84,5 70,4 59,9 49,9 Females, years Vitamin B-9 (Folate) 152,0 121,6 107,8 86,2 Vitamin B ,9 98,3 83,6 69,7 Iron (NaFeEDTA) 39,3 17,7 27,9 12,5 Zinc 127,4 106,2 90,3 75,3 Females, years Vitamin B-9 (Folate) 150,1 120,1 106,4 85,1 Vitamin B ,4 97,0 82,6 68,8 Iron (NaFeEDTA) 100,9 45,5 71,6 32,2 Zinc 125,8 104,8 89,2 74,3 Females, +65 years Vitamin B-9 (Folate) 134,7 107,7 95,5 76,4 Vitamin B ,5 87,1 74,1 61,7 Iron (NaFeEDTA) 65,3 40,8 46,3 28,9 Zinc 112,9 94,1 80,0 66,7 Notes: EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and sex-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and sex-specific population. 96

97 8. Brief description of the annexes. Annexes 1 and 2 provide detailed characteristics of fortified high-extraction wheat flour using a premix, the main parameters of which are shown in Table 3. In particular, the following data are given in these annexes: Addition levels each of four micronutrients (vitamins B9 and B12, iron as NaFeEDTA, and zinc) to flour, in mg/kg flour Adjusted upper limit for each of the micronutrients (vitamins B9 and B12, iron as NaFeEDTA, and zinc), in mg/kg flour Daily intake of micronutrients in the composition of fortified wheat flour, in: o mg/day o % EAR/day o % RNI/day The mentioned information in each member country are presented for the following age and gender groups of population in accordance with WHO recommendations: 1. Children, 1-3 years 2. Children, 4-6 years 3. Children, 7-9 years 4. Males, years 5. Males, years 6. Males, years 7. Males, + 65 years 8. Females, years 9. Females, years 10. Females, years 11. Females, + 65 years Total 2 annexes are drawn up by the number of countries: Afghanistan Annex 1 Pakistan Annex 1 97

98 9. Conclusion. 1. For the mandatory fortification of high-extraction wheat flour the following 4 micronutrients are selected: Vitamin В9 (folate) Vitamin В12 (cyanocobalamin) Iron (as NaFeEDTA) Zinc (zinc oxide) 2. The proposed addition levels of micronutrients in high-extraction flour: prepared taking into account the average per capita consumption of flour (in g/day) in the participating countries; fully comply with WHO recommendations 3. The proposed levels of high-extraction flour fortification by zinc and vitamins B9 and B12 ensure acceptable levels of total daily intake (in % RNI/day) of these micronutrients in the composition of fortified wheat flour for all gender and age population groups in both countries: Nutrient Afghanistan Pakistan Vitamin B-9 (Folate) 108% to 154% 76% to 109% Vitamin B-12 87% to 124% 62% to 88% Zinc 59% to 106% 42% to 75% 4. The proposed levels of high-extraction flour fortification by iron don t ensure acceptable levels of total daily intake (in% RNI/day) of iron in females of years old in both countries: Pakistan: 11% to 13% Afghanistan: 16% to 18% 5. Even in the other age and gender population groups the proposed levels of high-extraction flour fortification by iron ensure less than 50% RNI/day levels of total daily intake of iron in both countries: Pakistan: 25% to 34% Afghanistan: 35% to 49% 98

99 Annex 1 High-extraction wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Afghanistan Afghanistan: High-extraction wheat flour fortification Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Children, 1-3 years: P-50 Food Intake of target group = 162,4 g/day Vit. B-9 (Folate) 1,0 0,3 0, ,8 151,9 Vit. B-12 0,008 N.D. 0, ,5 122,7 Iron (NaFeEDTA) 15,0 4,7 2,436 97,9 42,0 Zinc 30,0 7,0 4,873 70,5 58,7 Children, 4-6 years: P-50 Food Intake of target group = 206,3 g/day Vit. B-9 (Folate) 1,0 0,4 0, ,9 144,7 Vit. B-12 0,008 N.D. 0, ,3 116,9 Iron (NaFeEDTA) 15,0 7,2 3, ,4 49,1 Zinc 30,0 12,0 6,190 77,4 64,5 Children, 7-9 years: P-50 Food Intake of target group = 259,0g/day Vit. B-9 (Folate) 1,0 0,5 0, ,4 121,1 Vit. B-12 0,008 N.D. 0, ,4 97,8 Iron (NaFeEDTA) 15,0 10,1 3, ,1 43,7 Zinc 30,0 12,0 7,770 83,3 69,4 Males, years: P-50 Food Intake of target group = 403,9 g/day Vit. B-9 (Folate) 1,0 0,7 0, ,0 141,6 Vit. B-12 0,008 N.D. 0, ,3 114,4 Iron (NaFeEDTA) 15,0 19,8 6,058 48,5 34,6 Zinc 30,0 28,0 12,116 85,0 70,9 Males, years: P-50 Food Intake of target group = 439,0 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,4 153,9 Vit. B-12 0,008 N.D. 0, ,3 124,4 Iron (NaFeEDTA) 15,0 28,8 6,585 63,9 48,1 Zinc 30,0 45,0 13, ,9 94,1 Males, years: P-50 Food Intake of target group = 430,2 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,6 150,8 Vit. B-12 0,008 N.D. 0, ,3 121,9 Iron (NaFeEDTA) 15,0 27 6,453 62,6 47,1 Zinc 30,0 45,0 12, ,6 92,2 Males, +65 years: P-50 Food Intake of target group = 360,0 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,8 126,2 Vit. B-12 0,008 N.D. 0, ,4 102,0 Iron (NaFeEDTA) 15,0 25,2 5,400 52,4 39,4 Zinc 30,0 45,0 10,799 92,6 77,1 Females, years: P-50 Food Intake of target group = 338,0 g/day Vit. B-9 (Folate) 1,0 0,7 0, ,2 118,5 Vit. B-12 0,008 N.D. 0, ,9 95,8 Iron (NaFeEDTA) 15,0 18 5,070 29,6 15,6 Zinc 30,0 28,0 10,141 84,5 70,4 Females, years: P-50 Food Intake of target group = 346,8 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,0 121,6 Vit. B-12 0,008 N.D. 0, ,9 98,3 99

100 Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Iron (NaFeEDTA) 15,0 23,4 5,202 39,3 17,7 Zinc 30,0 45,0 10, ,4 106,2 Females, years: P-50 Food Intake of target group = 342,4 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,1 120,1 Vit. B-12 0,008 N.D. 0, ,4 97,0 Iron (NaFeEDTA) 15,0 25,2 5, ,9 45,5 Zinc 30,0 45,0 10, ,8 104,8 Females, +65 years: P-50 Food Intake of target group = 307,3 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,7 107,7 Vit. B-12 0,008 N.D. 0, ,5 87,1 Iron (NaFeEDTA) 15,0 21,6 4,610 65,3 40,8 Zinc 30,0 45,0 9, ,9 94,1 Notes: a - These values are calculated taking in consideration the micronutrient losses during storage and distribution, as well as during cooking. EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and gender-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and gender-specific population. * N.D. = Not determined 100

101 Annex 2 High-extraction wheat flour fortification characteristics and daily intake of micronutrients by population groups in the composition of fortified wheat flour, in Pakistan Pakistan: High-extraction wheat flour fortification Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Children, 1-3 years: P-50 Food Intake of target group = 115,2 g/day Vit. B-9 (Folate) 1,0 0,3 0, ,6 107,7 Vit. B-12 0,008 N.D. 0, ,1 87,0 Iron (NaFeEDTA) 15,0 4,7 1,728 69,4 29,8 Zinc 30,0 7,0 3,455 50,0 41,6 Children, 4-6 years: P-50 Food Intake of target group = 146,3 g/day Vit. B-9 (Folate) 1,0 0,4 0, ,3 102,6 Vit. B-12 0,008 N.D. 0,001 99,5 82,9 Iron (NaFeEDTA) 15,0 7,2 2,195 84,7 34,8 Zinc 30,0 12,0 4,389 54,9 45,7 Children, 7-9 years: P-50 Food Intake of target group = 183,7 g/day Vit. B-9 (Folate) 1,0 0,5 0, ,3 85,9 Vit. B-12 0,008 N.D. 0,001 83,3 69,4 Iron (NaFeEDTA) 15,0 10,1 2,755 75,2 31,0 Zinc 30,0 12,0 5,510 59,0 49,2 Males, years: P-50 Food Intake of target group = 286,4 g/day Vit. B-9 (Folate) 1,0 0,7 0, ,5 100,4 Vit. B-12 0,008 N.D. 0,002 97,4 81,1 Iron (NaFeEDTA) 15,0 19,8 4,296 34,4 24,5 Zinc 30,0 28,0 8,592 60,3 50,2 Males, years: P-50 Food Intake of target group = 311,3 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,4 109,1 Vit. B-12 0,008 N.D. 0, ,8 88,2 Iron (NaFeEDTA) 15,0 28,8 4,670 45,3 34,1 Zinc 30,0 45,0 9,339 80,0 66,7 Males, years: P-50 Food Intake of target group = 305,1 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,4 109,1 Vit. B-12 0,008 N.D. 0, ,8 88,2 Iron (NaFeEDTA) 15,0 28,8 4,670 45,3 34,1 Zinc 30,0 45,0 9,339 80,0 66,7 Males, +65 years: P-50 Food Intake of target group = 255,3 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,9 89,5 Vit. B-12 0,008 N.D. 0,002 86,8 72,3 Iron (NaFeEDTA) 15,0 25,2 3,829 37,2 27,9 Zinc 30,0 45,0 7,658 65,6 54,7 Females, years: P-50 Food Intake of target group = 239,7 g/day Vit. B-9 (Folate) 1,0 0,7 0, ,1 84,0 Vit. B-12 0,008 N.D. 0,002 81,5 67,9 Iron (NaFeEDTA) 15,0 18 3,596 21,0 11,1 Zinc 30,0 28,0 7,191 59,9 49,9 Females, years: P-50 Food Intake of target group = 245,9 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,8 86,2 Vit. B-12 0,008 N.D. 0,002 83,6 69,7 Iron (NaFeEDTA) 15,0 23,4 3,689 27,9 12,5 101

102 Nutrient Addition level to flour, mg/kg Adjusted upper limit, mg/day Daily intake of micronutrients in the composition of fortified wheat flour mg/day a % EAR/day % RNI/day Zinc 30,0 45,0 7,378 90,3 75,3 Females, years: P-50 Food Intake of target group = 242,8 g/day Vit. B-9 (Folate) 1,0 1,0 0, ,4 85,1 Vit. B-12 0,008 N.D. 0,002 82,6 68,8 Iron (NaFeEDTA) 15,0 25,2 3,642 71,6 32,2 Zinc 30,0 45,0 7,284 89,2 74,3 Females, +65 years: P-50 Food Intake of target group = 217,9 g/day Vit. B-9 (Folate) 1,0 1,0 0,180 95,5 76,4 Vit. B-12 0,008 N.D. 0,001 74,1 61,7 Iron (NaFeEDTA) 15,0 21,6 3,269 46,3 28,9 Zinc 30,0 45,0 6,537 80,0 66,7 Notes: a - These values are calculated taking in consideration the micronutrient losses during storage and distribution, as well as during cooking. EAR = Estimated Average Requirement is the daily intake which meets the nutrient requirements of 50% apparently healthy individuals in an age- and gender-specific population. RNI = Recommended nutrient intake is the daily intake which meets the nutrient requirements of almost all (97.5%) apparently healthy individuals in an age- and gender-specific population. * N.D. = Not determined 102

103 Literature 1. WHO, FAO, UNICEF, GAIN, MI, & FFI. Recommendations on wheat and maize flour fortification. Meeting Report: Interim Consensus Statement. Geneva, World Health Organization, Wheat Flour Fortification: Current Knowledge and Practical Applications. Summary report of an international technical workshop. Cuernavaca, Mexico, December 1-3, 2004, 31 p Pakistan standard specification for fortified wheat atta. PS: ICS No , 30 p. 5. Fortified wheat flour specification. Draft Afghanistan Standard. Afghanistan National Standards Authority, 13 June 2013, 10 p. 6. Food and Agriculture Organization of the United Nations/World Health Organization. Requirements of vitamin A, iron, folate and vitamin B12. FAO Food and Nutrition Series, No 23. Rome: FAO. 1988; Vitamin and mineral requirements in human nutrition. Second edition. WHO/FAO, 2004, 362 p. 8. Hallberg L, Hulthen L. Prediction of dietary iron absorption: an algorithm for calculating absorption and bioavailability of dietary iron. Am J Clin Nutr 2000;71: (Published erratum appears in Am J Clin Nutr 2000;72:1242.) 9. Food and Nutrition Board, Institute of Medicine. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press, Hunt JR, Roughead ZK. Nonheme-iron absorption, fecal ferritin excretion, and blood indexes of iron status in women consuming controlled lactoovovegetarian diets for 8 wk. Am J Clin Nutr 1999;69: Quintaes KD, Cilla A and Barberá R. Iron Bioavailability from Cereal Foods Fortified with Iron. Austin J Nutr Metab. 2015;2(3): Monsen ER, Hallberg L, Layrisse M, et al. Estimation of available dietary iron. Am J Clin Nutr 1978;31: Cook JD. Adaptation in iron metabolism. Am J Clin Nutr 1990; 51(2): Hallberg L, Hulten L, Gramatkovski E. Iron absorption from the whole diet in men: how effective is the regulation of iron absorption? Am J Clin Nutr 1997;66: Bohn, L.; Meyer, A.S.; Rasmussen, S.K. Phytate: Impact on environment and human nutrition. A challenge for molecular breeding. J. Zhejiang Univ. Sci. B 2008, 9, By Ryan Andrews. Phytates and phytic acid Schlemmer U, et al. Phytate in foods and significance for humans: Food sources, intake, processing, bioavailability, protective role and analysis. Mol Nutr Food res 2009;53:S330-S Rosalind S. Gibson, Karl B. Bailey, Michelle Gibbs, Elaine L. Ferguson. A review of phytate, iron, zinc, and calcium concentrations in plant-based complementary foods used in lowincome countries and implications for bioavailability. Food and Nutrition Bulletin, vol. 31, no. 2 (supplement), p. S134-S West, A.R.; Oates, P.S. Mechanisms of heme iron absorption: Current questions and controversies. World J. Gastroenterol. 2008, 14, Theil, E.C.; Briat, J.-F. Plant Ferritin and Non-Heme Iron Nutrition in Humans; International Food Policy Research Institute and International Center for Tropical Agriculture: Washington, DC, USA, Cook JD, Dassenko SA, Whittaker P. Calcium supplementation: effect on iron absorption. Am J Clin Nutr. 1991; 53: Guidelines on food fortification with micronutrients. Edited by Lindsay Allen, Bruno de Benoist, Omar Dary and Richard Hurrell. WHO/FAO, 2006, 376 p. 103

104 23. Allen LH, Ahluwalia N. Improving iron status through diet: the applications of knowledge concerning dietary iron bioavailability in human populations. John Snow Incorporated/OMNI Project, Washington: Díaz-Castro J, Lisbona F, Moreno M, Alférez MJM, Campos M, López-Aliaga. Influence of Goat Milk on Iron Deficiency Anemia Recovery. Int J Dairy Sci Process, 2015, 2(1), p Judith R Turnlund, Radojka G Smith, MaryJKretsch, William R Keyes, and Alka G Shah. Milk s effect on the bioavailability of iron from cereal-based diets in young women by use of in vitro and in vivo methods. Am J C/in Nuir 1990;52: Hallberg L, Rossander-Hulten L, Brune M, Gleerup A. Inhibition of haem-iron absorption in man by calcium. Br J Nutr. 1993; 69(2): Scientific Opinion on the use of ferric sodium EDTA as a source of iron added for nutritional purposes to foods for the general population (including food supplements) and to foods for particular nutritional uses. European Food Safety Authority (EFSA), Parma, Italy. EFSA Journal 2010; 8(1):1414, 32 p Hurrell RF, Reddy MB, Burri J, Cook JD, An evaluation of EDTA compounds for iron fortification of cereal-based foods. Br J Nutr 84, Hurrell R. Preventing iron deficiency through food fortification. Nutr Rev. 1997; 55(6): gh_food_fortification_nutr_rev_55_ Subar AF, Krebs-Smith SM, Cook A, Kahle LL. Dietary sources of nutrients among US adults, 1989 to J Am Diet Assoc 1998;98: Harland BF, Oberleas D. Phytate in foods. World Rev Nutr Diet 1987; 52: Sandström B, Arvidsson B, Cederblad A, Bjorn-Rasmussen E. Zinc absorption from composite meals, I: the significance of wheat extraction rate, zinc, calcium, and protein content in meals based on bread. Am J Clin Nutr 1980;33: Food and Nutrition Board, Institute of Medicine. Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academy Press, Hurrell R, Egli I. Iron bioavailability and dietary reference values. Am J Clin Nutr. 2010; 91: 1461S-1467S. 35. Scientific Committee on Food (SCF): Nutrient and Energy Intakes for the European Community. Opinion adopted by the SCF on 11 December In Reports of the SCF Series N.o 31: Luxemburg, European Commission Institute of Medicine (IOM). Food and Nutrition Board. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc. National Academy Press: Washington, D.C Vitamin and mineral requirements in human nutrition. Second edition. WHO, FAO, Omar Dary and Michael Hainsworth. The Food Fortification Formulator. Technical Determination of Fortification Levels and Standards for Mass Fortification. USAID, April Acknowledgement: This document was made possible by the generous support of the American people through the United States Agency for International Development (USAID). The contests are the responsibility of the Kazakh Academy of Nutrition (KAN) and GAIN and do not necessarily reflect the views of USAID or the United States Government. 104

105 Annex 5. Draft. National Standard of the Republic of Kazakhstan. Low Extraction Fortified Bakery Wheat Flour. Specification. NATIONAL STANDARD OF THE REPUBLIC OF KAZAKHSTAN Draft LOW EXTRACTION FORTIFIED BAKERY WHEAT FLOUR Specification ST RK Committee for technical regulation and metrology Ministry of investment and development of the Republic of Kazakhstan (Gosstandard) Astana

106 Introduction 1 DEVELOPED AND ISSUED by: LLP «PO Kazakh Academy of Nutrition»: Committee of Consumer Protection, Ministry of National Economy, Kazakhstan: Department of medical care organization, Ministry of Health and Social Development, Kazakhstan: Union of Grain Processors of Kazakhstan: RSE "Kazakhstani Institute of Standardization and Certification", Kazakhstan: 2 APPROVED AND EFFECTIVE by: The order 3 This standard implements the provisions of Law of RK: «On technical regulation» dd. November 09, II, Technical Regulations of Customs Union (TR CU): «On safety of food products (TR CU 021/2011), «On safety of grain» (TR CU 015/2011), «On safety of package» (005/2011), «Food products in terms of their labeling» (TR CU 022/2011). 4 TERM OF FIRST INSPECTION 201 INSPECTION FREQUENCY 5 years 5 FIRST ISSUE Information about changes to this standard shall be published in the annually issued information index "Normative documents on standardization", and the text changes and amendments - in a monthly published information signs "National Standards". In case of revision (replacement) or cancellation of the standard will be published a notice in the information index "National Standards" This standard may not be in full or in part reproduced, replicated or distributed as the official edition without consent of the Committee for technical regulation and metrology of the Ministry of investment and development of the Republic of Kazakhstan. 106

107 Content REGIONAL STANDARD... Ошибка! Закладка не определена. 1 Application Regulatory references Technical requirements Acceptance procedure Monitoring methods Transportation and storage Manufacturer s guarantees Attachment А Attachment B Attachment C Attachment D

108 UDK : ICS Keywords: low extraction fortified bakery wheat flour, fortificant, premix, pre-mixture, vitamins, microelements, technical requirements, acceptance regulations, control methods, package, labeling, transportation and storage, manufacturer s guarantee. NATIONAL STANDARD OF THE REPUBLIC OF KAZAKHSTAN LOW EXTRACTION FORTIFIED BAKERY WHEAT FLOUR 1 Application area This standard applies to the fortified bakery wheat flour (hereinafter fortified flour) produced from low extraction flour (refined or fine grinding, 80% of wheat flour) according to ST RK 1482 or GOST 26574, and fortified with vitamin-mineral premix or pre-mixture based on it, and intended for the production of bread, bakery, pastry and culinary products. 2 Regulatory references To apply this standard are required the following regulatory references: ST RK GOST R Food products and food stock. Stripping voltammetry methods for determination of toxic elements (cadmium, lead, copper and zinc). and food stock. Stripping voltammetry methods for determination of mass concentration of arsenic. ST RK Wheat flour. General specification. GOST Sieve cloth of silk and synthetic thread. General specification. GOST Flour and bran. Method of determination of moisture content. GOST State system for ensuring the uniformity of measurements. Requirements to the number of packed goods in packages of any type in their manufacture, packaging, sale and import. GOST Flour, groats and bran. Method of determination of metal magnetic admixture. GOST Verification of purchased products. Organization and methods of control. GOST Flour. Method of determination of whiteness. GOST Bread wheat flour. Specifications. GOST Products of grain processing. Packing, labeling, transportation and storage. GOST Food stock and products. Method of determination of mercury. GOST Food products. Method of determination of iron. GOST Food stock and products. Sample preparation. Mineralization for determination of toxic elements. GOST Food stock and products. Method of determination of arsenic. GOST Food stock and products. Method of determination of lead. GOST Food stock and products. Method of determination of cadmium. GOST Flour and bran. Method of acidity determination from washmill. GOST Flour and bran. Method of ash content determination. GOST Flour. Method of determination of autolytic activity. GOST Flour and bran. Methods of determination of color, odor, taste and crunch. GOST Flour and bran. Method of determination of grain products pests contamination and infestation. GOST Flour and bran. Method of determination of flour grain. 108

109 GOST Flour and bran. Acceptance and sampling methods. GOST Bread wheat flour. Method of test laboratory bread making. GOST Grain and its products. Method of determination of falling number. GOST Wheat flour. Method of determination of quantity and quality of gluten. GOST (ISO ) Wheat flour. Physical properties of dough. Rheometric tests by alveograph GOST (ISO ) Determination of wet gluten content. GOST Bags and bag cloth. General specifications. GOST Food stock and products. Atomic absorption method of determination of toxic elements. GOST Food products. Methods of determination of toxic elements by atomic emission method. arsenic. NOTE: Using this standard it is advisable to check validity of reference standards and classifiers in annually published reference index «Regulatory standardization documents» as of the current year and according to appropriate monthly published reference indexes published in current year. If the reference document is replaced (amended), so using this standard it is advisable to be guided by the replacing (amended) document. If the reference document is cancelled without replacement, so the provision containing the reference on it shall be applied in part not affecting this reference. 3 Technical requirements 3.1 The fortified flour shall meet the requirements of this standard TR TS 021/2011 and be produced according to the recipe and process instruction in compliance with requirements [2] and sanitary norms and regulations approved in established procedure ST RK Characteristics Wheat used for production of fortified flour shall comply with requirements of GOST ГОСТ Wheat for milling shall comply with requirements given in table 1. Table 1 Grain parameter The acceptable level, %, not more than The content of barley, rye grains and sprouted grains of these 5.0 crops and wheat (in aggregate) including: Sprouted grain 3.0 Corn cockle 0.1 Extraneous matters: Ergot 0.05 Creeping bitterling, Sophóra alopecuroídes, 0.1 thermopsis lancet (in aggregate), including creeping bitterling and multicolored coronilla (in aggregate) Coronilla 0.1 Heliotropium dasycarpum 0.1 Trichodesma incanum Shall be absent Smut grain (dirty, blue-eyed mold corn) 10.0 Fusarium grain 1.0 Note: Content of sprouted grain shall be determined on analysis results before treatment. 109

110 3.2.3 Requirements to organoleptic and physical and chemical parameters of fortified flour are shown in table 2. Table 2 Parameter Description and standard Color White or white with cream shade, or white with yellow shade Taste Common to the wheat flour, without other tastes, not sour, not bitter Odor Common to the wheat flour, without other odors, not stale* Moisture content, %, maximum 15.5 Presence of mineral impurities No crunch by chewing Metal magnetic admixture, mg per 1 kg 3.0** of flour; size of individual particles in maximum linear dimension 0.3 mm and/or weight of no more than 0.4 mg, maximum Grain products pests contamination Shall be absent (insects, mites) Grain products pests contamination Shall be absent (insects, mites), total contamination density Contamination of bread with potato Shall be absent disease agents (36 hours after test laboratory baking) Weight ratio of vitamins and minerals, mg/kg: В12 (given background value) В9 (given background value) В1 (given background value) В2 (given background value) В3 (given background value) Mass content of iron (given background value): NaFeEDTA Mass content of zinc (given background value) Qualitative reaction of iron content positive * the fortified flour may have a faint smell of vitamins and additives; Note- Moisture content in fortified flour for long storage shall not exceed 14.5% The fortified flour shall meet the quality requirements specified in the table 3. Table 3 Parameter Ash content on dry matter, %, no more than Whiteness, conventional units of RZ-BPL device, not less than Weight fraction of wet gluten, %, not less than Description and standard 0,55-0,75 36,0-55,0 28,0-30,0 110

111 Parameter Description and standard Wet gluten quality, s.u. of IDK device Not lower than II group Fineness of grind,%, no more than: - Residue on sieve, on GOST 4403, no more than 5 of silk fabric 43 or polyamide fabric 45/50 PA or 2 of silk fabric 35 or polyamide fabric number 36/40 PA - Passage through a sieve on GOST Passage through a sieve on GOST 4403 Falling number, «FN», s, not less than 185 Note: 1. Parameter whiteness replaces the parameter ash content on mills equipped with laboratory devices and machinery according to GOST Parameter Falling number FN is determined according to GOST ГОСТ 27676, upon customer s request. 3. To determine the grain of flour is allowable to use other mesh with equivalent sieving capacity In terms of content of toxic elements, pesticides, mycotoxins and of microbiological parameters the fortified flour shall not exceed levels permitted by requirements [2] Quantity of vitamins and microelements added to the fortified flour shall be monitored in the production process by laying, in accordance with formula regulating the weight of premix or premixture to be added per unit mass of the flour stream to be fortified. 3.3 Requirements to the raw materials The primary products of domestic and foreign production used for manufacturing of fortified flour shall: comply with requirements of regulations in the sphere of technical regulations, sanitary rules, standards, other regulations approved in established order, as well as with purchasing contracts for imported products; be authorized for use by the authorized body in the sphere of sanitary and epidemiological welfare For production of fortified flour use the following primary products: wheat flour of first and supreme grades according to the GOST 26574; vitamin and mineral premixes When manufacturing the fortified flour for export the list of vitamins and minerals for fortification and rates of their application shall be agreed in supply contracts and can be accompanied by indication and name of standard or other regulation of the importing country. 3.4 Package Packaging and materials of domestic and foreign production used for packing the fortified flour shall comply with requirements [1], regulating and technical documents establishing possibility of their contact with food products, provide safety and appearance of products by transportation and storage, and approved for use by authorized body in established order Packing the flour according to GOST Bags according to GOST Deviations of net weight of each packing unit from nominal amount shall not exceed regulations of GOST Transport containers shall be clean, dry, without any odors. 3.5 Labeling 111

112 3.5.1 The bags, packs and labels inserted into package shall be labeled according to the requirements of [3]: products name; name and location of manufacturer (legal address including country, and if not match the legal address address of the mill) and organization in the Republic of Kazakhstan authorized by manufacturer to accept claims of consumers on its territory (if any); trade mark (if any); net weight (g, kg); information of ingredients in decreasing order by weight or percentage; manufacturing date and expiry date; storage conditions; nutrition and energy value; batch numbers (if any); bar code (if any); standard marks; common commercialization mark for countries members of Customs Union Additionally the following labeling is required: - large print the word fortified ; - standard logo of fortified products approved in established order, attachment В [4]; - registered trade name of the premix or pre-mixture (if any) used for flour fortification, and the regulating document according to which they have been produced The multipack shall be labeled with details listed above, and additionally indicate the number of consumer packages Transport labeling according to GOST Information may be placed in one or several point convenient for reading. The information can be applied by any method and shall be clear and easy to read Text and labels shall be applied in state and Russian languages or in customer s language according to the contract. 4 Acceptance procedure 4.1 Acceptance procedure according to GOST The primary products used for production of fortified flour are controlled by incoming inspection according to GOST Organoleptic, and physical and chemical parameters, packing and labeling shall be monitored in each batch. 4.4 The toxic elements, pesticides, mycotoxins and microbiological parameters shall be monitored in accordance with the order established by manufacturer in agreement with the authority of sanitary and epidemiological welfare. 4.5 Control and monitoring frequency for content of all vitamins and minerals to be added to the fortified flour shall be performed one a year in duly accredited test laboratories (centers). For monitoring take an average sample of at least 500 g combining and thoroughly mixing single samples taken each 2 hours within at least one shift. 4.6 In case of unsatisfied results of tests at least by one parameter take a double sampling and perform all tests again. The results of the last test apply to the entire batch and are final. 4.7 Arbitration analysis in the event of differences in quality assessment shall be made in duly accredited centers (test laboratories). 4.8 Confirmation tests of compliance and assessment of compliance confirmation shall be performed in accordance with requirements of the State technical regulation system of the Customs Union. 5 Monitoring methods 5.1 Sampling and sample preparation according to GOST

113 5.2 Determination of organoleptic parameters according to GOST In case of discrepancies in quality assessment of the flour in terms of organoleptic parameters (odor, taste, mineral impurities) they shall be determined by tasting the bread baked from this flour. 5.3 Determination of harmful impurities, sprouted grain and rye and barley impurities in the wheat according to GOST (by acceptance of grain). 5.4 Determination of moisture content in flour according to GOST Determination of flour grain according to GOST Determination of ash w/w in the flour according to GOST Determination of quality and quantity of gluten according to ST RK 1054 and GOST 27839, determination of wet gluten by export according to GOST (ISO 5531). 5.8 Determination of whiteness according to GOST Determination of falling number according to GOST Determination of metal magnetic admixture according to GOST Determination of grain products pests contamination and infestation according to GOST Determination of acidity according to GOST Determination of volume yield of bread, shape stability and organoleptic parameters of bread according to GOST Determination of toxic elements: mercury according to GOST 26927; arsenic according to GOST 26930, GOST R 51766, ST RK GOST R 51962; lead according to GOST 26932, GOST 30178, ST RK GOST R 51301; cadmium according to GOST 26933, GOST 30178, ST RK GOST R 51301; as well as according to GOST or methods approved by authorities in established order Determination of pesticides, mycotoxins according to methods approved by authorities in established order Determination of mass content of fortificants (vitamins, iron and zinc) in fortified flour according to GOST 29138, GOST 29139, GOST 29140, GOST 26928, GOST and methods approved in established order. Quality reaction for presence of iron according to methods specified in attachment B Acidity of flour, autolytic activity and physical properties according to GOST 27493, GOST 27495, GOST Transportation and storage 6.1 Transportation and storage according to GOST The fortified flour may be transported by all means of transport in accordance with freight regulations applicable for relative transport, by presence of duly executed sanitary passport. 6.3 The fortified flour shall be stored in dry place, under temperature not higher than 25 0 С and average humidity not more than 70%, without direct sunlight. 7 Manufacturer s guarantees 7.1 Manufacturer guarantees the compliance of the fortified flour with requirements of this standard, if the consumer observes the storage and transportation requirements. 7.2 Shelf Life 12 months from the date of production. 113

114 Attachment А (reference) Nutrient and energy value of the fortified flour per 100 g of product Proteins, g Fat, g Carbohydrates, g Energy value kcal kj 114

115 A. PRINCIPLE Attachment B QUALITATIVE METHODS OF DETERMINATION OF IRON IN FLOUR Ferric iron in acidic media reacts with potassium thiocyanate solution (KSCN) and forms the insoluble red color. Other types of iron like ferrous and elemental iron can also react similarly with oxidizing to ferric iron by means of hydrogen peroxide. Presence of electrolyte or reduced iron can be determined visually by placing a magnet in the flour sample and drawing of iron particles to it. Ferrous iron can be identified by creating insoluble light-blue color called Turnbull s blue or Berlin blue by reaction with ferrocyanide. Reaction occurs very quickly with ferrous sulfate, but can be slow or even not occur with ferrous fumarate due to low solubility of this salt in water. Reaction can also occur with electrolyte iron after oxidation of iron to Fe 2+, but the rate of reaction is very low. B. MATERIALS - Filter Whatman paper 1 - Hand sieve - Watch glass C. REAGENTS - 2N HCl Hydrochloric acid solution. In 200 ml flask add 100 ml of distilled water. Then slowly add 17 ml of concentrated HCl, then add 83 ml of water. - Hydrochloric acid solution N (HCl). In 1 liter volumetric flask add 600 ml of distilled water, add 1.5 ml of 12N HCl- and make to volume with distilled water. - Potassium thiocyanate -10%. Dissolve 10 g of KSCN in 100 ml of water. Before use mix 10 ml of this solution with 10 ml of 2N HCl. - Hydrogen peroxide (H2O2) - 3% (required if the fortification is being made with elemental iron). In 50 ml flask add 5 ml of concentrated 30% H2O2 and make to volume of 45 ml with distilled water. To prepare daily, discard after finished tests. - Potassium ferricyanide -10%. Dissolve 10 g of K3Fe(CN)6 in 100 ml of water. Before use mix 10 ml of this solution with 10 ml of 0.003N HCl. D. PROCEDURE a. Determination of iron in samples containing elemental iron (electrolyte, reduced iron and other) 1. Take a magnet and put it into 1 kg flour sample. 2. Move the magnet inside the sample, and then remove it. 3. The presence of electrolyte or reduced iron is confirmed by presence of small particles of iron on the magnet. b. Determination of iron in samples containing NaFeEDTA 1. Place the filter paper on a watch glass. 2. Wet the paper surface with potassium tiocyanate solution. The liquid shall infiltrate the paper. 3. By means of hand sieve sift a portion of flour sample so that a thin flour level cover the wet filter paper, remove the excessive flour. 4. Add to the flour surface the acidic solution of potassium tiocyanate, wait a few minutes until reaction. 5. Red spots indicate the presence of iron salt, particularly NaFeEDTA. c. Determination of other iron sources including elemental iron 1. Put the filter paper on the watch glass. 2. Wet the paper surface with potassium tiocyanate solution. The liquid shall infiltrate the paper. 115

116 3. By means of hand sieve sift a portion of flour sample so that a thin flour level cover the wet filter paper, remove the excessive flour. 4. Over the flour add a few of acidic potassium tiocyanate solution, wait for some minutes. 5. Add a small quantity of H2O2 solution, wait a few minutes until reaction (formation of iron (III)). 6. Red spots indicate the presence of added iron. (Note: If the result for electrolyte or reduced iron is negative in tests with magnet, so more likely is added the iron in form of ferrous salt). d. Demonstration of presence of ferrous salt (mainly ferrous sulfate) 1. Place the filter paper on the watch glass. 2. Wet the surface of filter paper with potassium ferricyanide solution-10% and N-HCl. The liquid shall infiltrate the filter paper. 3. By means of hand sieve sift a portion of flour sample so that a thin flour level cover the wet filter paper, remove the excessive flour. 4. Add a few of acidic solution of potassium ferricyanide over the flour. 5. Allow to stay a few minutes until reaction. Rapid reaction with clearly visible spots (during 2 minutes after addition of ferricyanide) indicates the presence of ferrous sulfate. Ferrous fumarate and some types of elemental iron also can have this reaction, but slowly (6-7 minutes and more). 6. Elemental forms can be identified by procedure with magnet. For light brown-green spots the reaction for NaFeEDTA is positive. E. CLARIFICATION Number of spots and their homogenous distribution shows the concentration of iron and homogeneity of sample. For comparative evaluation use samples with known amount of the same type of iron as a control sample. 116

117 Attachment C 117

118 Attachment D (reference) REFERENCE LIST 1. Technical regulation of the Customs Union «On safety of package» (TR CU 005/2011) approved by the Decision of the Customs Union Commission dd. August 16, Technical regulation of the Customs Union «On safety of food production» (TR CU 021/2011) approved by the Decision of the Customs Union Commission dd. December 09, Technical regulation of the Customs Union «Food products in terms of labeling» (TR CU 022/2011) approved by the Decision of the Customs Union Commission dd. December 09, Certificate of trade mark of the Intellectual Property Committee of the Ministry of Justice RK dd for goods of class 30 on ICGS (flour, baked goods, iodine-treated cooking salt). 118

119 Annex 6. Draft. National Standard of the Republic of Kazakhstan. High Extraction Fortified Bakery Wheat Flour. Specification. NATIONAL STANDARD OF THE REPUBLIC OF KAZAKHSTAN Draft HIGH EXTRACTION FORTIFIED BAKERY WHEAT FLOUR Specification ST RK Committee for technical regulation and metrology Ministry of investment and development of the Republic of Kazakhstan (Gosstandart) Astana

120 Introduction 1 DEVELOPED AND ISSUED by: LLP «PO Kazakh Academy of Nutrition»: Committee of Consumer Protection, Ministry of National Economy, Kazakhstan: Department of medical care organization, Ministry of Health and Social Development, Kazakhstan: Union of Grain Processors of Kazakhstan: RSE "Kazakhstani Institute of Standardization and Certification", Kazakhstan: 2 APPROVED AND EFFECTIVE by: The order 3 This standard implements the provisions of Law of RK: «On technical regulation» dd. November 09, II, technical regulations of Customs Union: «On safety of food products (TR CU 021/2011), «On safety of grain» (TR CU 015/2011), «On safety of package» (005/2011), «Food products in terms of its labeling» (TR CU 022/2011). 4 TERM OF FIRST INSPECTION 201 INSPECTION FREQUENCY 5 years 5 FIRST ISSUE Information about changes to this standard shall be published in the annually issued information index "Normative documents on standardization", and the text changes and amendments - in a monthly published information signs "National Standards". In case of revision (replacement) or cancellation of the standard will be published a notice in the information index "National Standards" This standard may not be in full or in part reproduced, replicated or distributed as the official edition without consent of the Committee for technical regulation and metrology of the Ministry of investment and development of the Republic of Kazakhstan. 120

121 Content REGIONAL STANDARD... Ошибка! Закладка не определена. 1 Application Regulatory reference Technical requirements Acceptance procedure Monitoring methods Transportation and storage Manufacturer s guarantees Attachment А Attachment B Attachment C Attachment D

122 UDK : ICS Keywords: bread wheat high extraction fortified flour, fortificant, premix, pre-mixture, vitamins, microelements, technical requirements, acceptance regulations, control methods, package, labeling, transportation and storage, manufacturer s guarantee. NATIONAL STANDARD OF THE REPUBLIC OF KAZAKHSTAN HIGH EXTRACTION FORTIFIED BAKERY WHEAT FLOUR 1 Application This standard applies to the fortified bakery wheat flour (hereinafter fortified flour) produced from high extraction flour (wholegrain or coarse grinding, >80% of wheat flour) according to ST RK 1482 or GOST 26574, and fortified with vitamin-mineral premix or pre-mixture based on it, and intended for the production of bread, bakery, pastry and culinary products. 2 Regulatory references To apply this standard are required the following regulatory references: ST RK GOST R Food products and food stock. Stripping voltammetry methods for determination of toxic elements (cadmium, lead, copper and zinc). for determination of mass concentration of arsenic. ST RK Wheat flour. General specification. GOST Sieve cloth of silk and synthetic thread. General specification. GOST Wheat. Requirements for purchases and deliveries. GOST Flour and bran. Method of determination of moisture content. GOST State system for ensuring the uniformity of measurements. Requirements to the number of packed goods in packages of any type in their manufacture, packaging, sale and import. GOST Flour, groats and bran. Method of determination of metal magnetic admixture. GOST Verification of purchased products. Organization and methods of control. GOST Flour. Method of determination of whiteness. GOST Bread wheat flour. Specifications. GOST Products of grain processing. Packing, labeling, transportation and storage. GOST Food stock and products. Method of determination of mercury. GOST Food products. Method of determination of iron. GOST Food stock and products. Sample preparation. Mineralization for determination of toxic elements. GOST Food stock and products. Method of determination of arsenic. GOST Food stock and products. Method of determination of lead. GOST Food stock and products. Method of determination of cadmium. GOST Flour and bran. Method of acidity determination from washmill. GOST Flour and bran. Method of ash content determination. GOST Flour. Method of determination of autolytic activity. GOST Flour and bran. Methods of determination of color, odor, taste and crunch. GOST Flour and bran. Method of determination of grain products pests contamination and infestation. GOST Flour and bran. Method of determination of flour grain. GOST Flour and bran. Acceptance and sampling methods. GOST Bread wheat flour. Method of test laboratory bread making. GOST Grain and its products. Method of determination of falling number. 122

123 GOST Wheat flour. Method of determination of quantity and quality of gluten. GOST (ISO ) Wheat flour. Physical properties of dough. Rheometric tests by alveograph GOST (ISO ) Determination of wet gluten content. GOST Bags and bag cloth. General specifications. GOST Food stock and products. Atomic absorption method of determination of toxic elements. GOST Food products. Methods of determination of toxic elements by atomic emission method. arsenic. NOTE: Using this standard it is advisable to check validity of reference standards and classifiers in annually published reference index «Regulatory standardization documents» as of the current year and according to appropriate monthly published reference indexes published in current year. If the reference document is replaced (amended), so using this standard it is advisable to be guided by the replacing (amended) document. If the reference document is cancelled without replacement, so the provision containing the reference on it shall be applied in part not affecting this reference. 3 Technical requirements 3.1 The fortified flour shall meet the requirements of this standard and be produced according to the recipe and process instruction in compliance with requirements [2] and sanitary norms and regulations approved in established procedure. 3.2 Characteristics Wheat used for production of fortified flour shall comply with requirements of GOST ГОСТ Wheat for milling shall comply with requirements given in table 1. Table 1 Grain parameter The acceptable level, %, not more than The content of barley, rye grains and sprouted grains of these 5.0 crops and wheat (in aggregate) including: Sprouted grain 3.0 Corn cockle 0.1 Extraneous matters: Ergot 0.05 Creeping bitterling, Sophóra alopecuroídes, thermopsis lancet (in aggregate), including creeping bitterling and multicolored coronilla (in aggregate) Coronilla 0.1 Heliotropium dasycarpum 0.1 Trichodesma incanum 0.1 Shall be absent Smut grain (dirty, blue-eyed mold corn) 10.0 Fusarium grain 1.0 Note: Content of sprouted grain shall be determined on analysis results before treatment. 123

124 3.2.3 Requirements to organoleptic and physical and chemical parameters of fortified flour are shown in table 2. Table 2 Parameter Description and standard Color White with yellow or grey shade Taste Common to the wheat flour, without other tastes, not sour, not bitter Odor Common to the wheat flour, without other odors, not stale* Moisture content, %, maximum 15.0 Presence of mineral impurities No crunch by chewing Metal magnetic admixture, mg per 1 kg of 3.0** flour; size of individual particles in maximum linear dimension 0.3 mm and/or weight of no more than 0.4 mg, maximum Grain products pests contamination Shall be absent (insects, mites) Grain products pests contamination Shall be absent (insects, mites), total contamination density Contamination of bread with potato Shall be absent disease agents (36 hours after test laboratory baking) Weight ratio of vitamins and minerals, mg/kg: В12 (given background value) В9 (given background value) Mass content of iron (given background value): NaFeEDTA Mass content of zinc (given background value) Qualitative reaction of iron content positive * the fortified flour may have a faint smell of vitamins and additives; Note- Moisture content in fortified flour for long storage shall not exceed 14.5% The fortified flour shall meet the quality requirements of the table 3. Table 3 Parameter Description and standard Ash w/w on dry basis, %, maximum 1.25 Whiteness, conventional units of RZ-BPL 12.0 device, not less than Weight fraction of wet gluten, %, not less 25.0 than Wet gluten quality, s.u. of IDK device Not lower than II group Fineness of grind,%, no more than: - Residue on sieve, on GOST 4403, no more than 2 silk 27 or polyamide 120 PA 124

125 Parameter Description and standard - Passage through a sieve on GOST No less than 65.0 of silk 38 or polyamide 41/43 PA Falling number, «FN», s, minimum 160 Note: 1. Parameter whiteness replaces the parameter ash content on mills equipped with laboratory devices and machinery according to GOST Parameter Falling number FN is determined according to GOST ГОСТ 27676, upon customer s request. 3. To determine the grain of flour is allowable to use other mesh with equivalent sieving capacity In terms of content of toxic elements, pesticides, mycotoxins and of microbiological parameters the fortified flour shall not exceed levels permitted by requirements [2] Quantity of vitamins and micro nutrients added to the fortified flour shall be monitored in the production process by laying, in accordance with formula regulating the weight of premix or premixture to be added per unit mass of the flour stream to be fortified. 3.3 Requirements to the raw materials The primary products of domestic and foreign production used for manufacturing of fortified flour shall: comply with requirements of regulations in the sphere of technical regulations, sanitary rules, standards, other regulations approved in established order, as well as with purchasing contracts for imported products; be authorized for use by the authorized body in the sphere of sanitary and epidemiological welfare For production of fortified flour use the following primary products: wheat flour of first grade and prima according to the GOST 26574; vitamin and mineral premixes When manufacturing the fortified flour for export the list of vitamins and minerals for fortification and rates of their application shall be agreed in supply contracts and can be accompanied by indication and name of standard or other regulation of the importing country. 3.4 Package Packaging and materials of domestic and foreign production used for packing the fortified flour shall comply with requirements [1], regulating and technical documents establishing possibility of their contact with food products, provide safety and appearance of products by transportation and storage, and approved for use by authorized body in established order Packing the flour according to GOST Bags according to GOST Deviations of net weight of each packing unit from nominal amount shall not exceed regulations of GOST Transport containers shall be clean, dry, without any odors. 3.5 Labeling The bags, packs and labels inserted into package shall be marked according to the requirements of [3]: products name; name and location of manufacturer (legal address including country, and if not match the legal address address of the mill) and organization in the Republic of Kazakhstan authorized by manufacturer to accept claims of consumers on its territory (if any); 125

126 trade mark (if any); net weight (g, kg); information of ingredients in decreasing order by weight or percentage; manufacturing date and expiry date; storage conditions; nutrition and energy value; batch numbers (if any); bar code (if any); standard marks; common commercialization mark for countries members of Customs Union Additionally the following labeling is required: - large print the word fortified ; - standard logo of fortified products approved in established order, attachment В [4]; - registered trade name of the premix or pre-mixture (if any) used for flour fortification, and the regulating document according to which they have been produced The multipack shall be marked with details listed above, and additionally indicate the number of consumer packages Transport labeling according to GOST Information may be placed in one or several point convenient for reading. The information can be applied by any method and shall be clear and easy to read Text and labels shall be applied in state and Russian languages or in customer s language according to the contract. 4 Acceptance procedure 4.1 Acceptance procedure according to GOST The primary products used for production of fortified flour are controlled by incoming inspection according to GOST Organoleptic and physical and chemical parameters, packing and labeling shall be monitored in each batch. 4.4 The toxic elements, pesticides, mycotoxins and microbiological parameters shall be monitored in accordance with the order established by manufacturer in agreement with the authority of sanitary and epidemiological welfare. 4.5 Control and monitoring frequency for content of all vitamins and minerals to be added to the fortified flour shall be performed one a year in duly accredited test laboratories (centers). For monitoring take an average sample of at least 500 g combining and thoroughly mixing single samples taken each 2 hours within at least one shift. 4.6 In case of unsatisfied results of tests at least by one parameter take a double sampling and perform all tests again. The results of the last test apply to the entire batch and are final. 4.7 Arbitration analysis in the event of differences in quality assessment shall be made in duly accredited centers (test laboratories). 4.8 Confirmation tests of compliance and assessment of compliance confirmation shall be performed in accordance with requirements of the State technical regulation system of the Customs Union. 5 Monitoring methods 5.1 Sampling and sample preparation according to GOST Determination of organoleptic parameters according to GOST In case of discrepancies in quality assessment of the flour in terms of organoleptic parameters (odor, taste, mineral impurities) they shall be determined by tasting the bread baked from this flour. 5.3 Determination of harmful impurities, sprouted grain and rye and barley impurities in the wheat according to GOST (by acceptance of grain). 5.4 Determination of moisture content in flour according to GOST Determination of flour grain according to GOST

127 5.6 Determination of ash w/w in the flour according to GOST Determination of quality and quantity of gluten according to ST RK 1054 and GOST 27839, determination of wet gluten by export according to GOST (ISO 5531). 5.8 Determination of whiteness according to GOST Determination of falling number according to GOST Determination of metal magnetic admixture according to GOST Determination of grain products pests contamination and infestation according to GOST Determination of acidity according to GOST Determination of volume yield of bread, shape stability and organoleptic parameters of bread according to GOST Determination of toxic elements: mercury according to GOST 26927; arsenic according to GOST 26930, GOST R 51766, ST RK GOST R 51962; lead according to GOST 26932, GOST 30178, ST RK GOST R 51301; cadmium according to GOST 26933, GOST 30178, ST RK GOST R 51301; as well as according to GOST or methods approved by authorities in established order Determination of pesticides, mycotoxines according to methods approved by authorities in established order Determination of mass content of fortificants (vitamins, iron and zinc) in fortified flour according to GOST 29138, GOST 29139, GOST 29140, GOST 26928, GOST and methods approved in established order. Quality reaction for presence of iron according to methods specified in attachment B Acidity of flour, autolytic activity and physical properties according to GOST 27493, GOST 27495, GOST Transportation and storage 6.1 Transportation and storage according to GOST The fortified flour may be transported by all means of transport in accordance with freight regulations applicable for relative transport, by presence of duly executed sanitary passport. 6.3 The fortified flour shall be stored in dry place, under temperature not higher than 25 0 С and average humidity not more than 70%, without direct sunlight. 7 Manufacturer s guarantees 7.1 Manufacturer guarantees the compliance of the fortified flour with requirements of this standard, if the consumer observes the storage and transportation requirements. 7.2 Shelf Life 12 months from the date of production. 127

128 Attachment А (reference) Nutrition and energy value of the fortified flour per 100 g of product Proteins, g Fat, g Carbohydrates, g Energy value kcal kj 128

129 Attachment B QUALITATIVE METHODS OF DETERMINATION OF IRON IN FLOUR A. PRINCIPLE Ferric iron in acidic media reacts with potassium thiocyanate solution (KSCN) and forms the insoluble red color. Other types of iron like ferrous and elemental iron can also react similarly with oxidizing to ferric iron by means of hydrogen peroxide. Presence of electrolyte or reduced iron can be determined visually by placing a magnet in the flour sample and drawing of iron particles to it. Ferrous iron can be identified by creating insoluble light-blue color called Turnbull s blue or Berlin blue by reaction with ferrocyanide. Reaction occurs very quickly with ferrous sulfate, but can be slow or even not occur with ferrous fumarate due to low solubility of this salt in water. Reaction can also occur with electrolyte iron after oxidation of iron to Fe 2+, but the rate of reaction is very low. B. MATERIALS - Filter Whatman paper 1 - Hand sieve - Watch glass C. REAGENTS - 2N HCl Hydrochloric acid solution. In 200 ml flask add 100 ml of distilled water. Then slowly add 17 ml of concentrated HCl, then add 83 ml of water. - Hydrochloric acid solution N (HCl). In 1 liter volumetric flask add 600 ml of distilled water, add 1.5 ml of 12N HCl- and make to volume with distilled water. - Potassium tiocyanate-10%. Dissolve 10 g of KSCN in 100 ml of water. Before use mix 10 ml of this solution with 10 ml of 2N HCl. - Hydrogen peroxide (H2O2) - 3% (required if the fortification is being made with elemental iron). In 50 ml flask add 5 ml of concentrated 30% H2O2 and make to volume of 45 ml with distilled water. To prepare daily, discard after finished tests. - Potassium ferricyanide-10%. Dissolve 10 g of K3Fe(CN)6 in 100 ml of water. Before use mix 10 ml of this solution with 10 ml of 0.003N HCl. D. PROCEDURE a. Determination of iron in samples containing elemental iron (electrolyte, reduced iron and other) 1. Take a magnet and put it into 1 kg flour sample. 2. Move the magnet inside the sample, and then remove it. 3. The presence of electrolyte or reduced iron is confirmed by presence of small particles of iron on the magnet. b. Determination of iron in samples containing NaFeEDTA 1. Place the filter paper on a watch glass. 2. Wet the paper surface with potassium tiocyanate solution. The liquid shall infiltrate the paper. 3. By means of hand sieve sift a portion of flour sample so that a thin flour level cover the wet filter paper, remove the excessive flour. 4. Add to the flour surface the acidic solution of potassium tiocyanate, wait a few minutes until reaction. 5. Red spots indicate the presence of iron salt, particularly NaFeEDTA. c. Determination of other iron sources including elemental iron 129

130 1. Put the filter paper on the watch glass. 2. Wet the paper surface with potassium tiocyanate solution. The liquid shall infiltrate the paper. 3. By means of hand sieve sift a portion of flour sample so that a thin flour level cover the wet filter paper, remove the excessive flour. 4. Over the flour add a few of acidic potassium tiocyanate solution, wait for some minutes. 5. Add a small quantity of H2O2 solution, wait a few minutes until reaction (formation of iron (III)). 6. Red spots indicate the presence of added iron. (Note: If the result for electrolyte or reduced iron is negative in tests with magnet, so more likely is added the iron in form of ferrous salt). d. Demonstration of presence of ferrous salt (mainly ferrous sulfate) 1. Place the filter paper on the watch glass. 2. Wet the surface of filter paper with potassium ferricyanide solution-10% and N-HCl. The liquid shall infiltrate the filter paper. 3. By means of hand sieve sift a portion of flour sample so that a thin flour level cover the wet filter paper, remove the excessive flour. 4. Add a few of acidic solution of potassium ferricyanide over the flour. 5. Allow to stay a few minutes until reaction. Rapid reaction with clearly visible spots (during 2 minutes after addition of ferricyanide) indicates the presence of ferrous sulfate. Ferrous fumarate and some types of elemental iron also can have this reaction, but slowly (6-7 minutes and more). 6. Elemental forms can be identified by procedure with magnet. For light brown-green spots the reaction for NaFeEDTA is positive. E. CLARIFICATION Number of spots and their homogenous distribution shows the concentration of iron and homogeneity of sample. For comparative evaluation use samples with known amount of the same type of iron as a control sample. 130

131 Attachment C 131

132 Attachment D (reference) REFERENCE LIST 1. Technical regulation of the Customs Union «On safety of package» (TR CU 005/2011) approved by the Decision of the Customs Union Commission dd. August 16, Technical regulation of the Customs Union «On safety of food production» (TR CU 021/2011) approved by the Decision of the Customs Union Commission dd. December 09, Technical regulation of the Customs Union «Food products in terms of labeling» (TR CU 022/2011) approved by the Decision of the Customs Union Commission dd. December 09, Certificate of trade mark of the Intellectual Property Committee of the Ministry of Justice RK dd for goods of class 30 on ICGS (flour, baked goods, iodine-treated cooking salt). 132

133 Annex 7. Draft. External and Internal Quality Control of Iron Fortification of Flour. Draft Ministry of Health and Social Development of the Republic of Kazakhstan Committee of Consumer Rights Protection of the Ministry of National Economy of the Republic of Kazakhstan Kazakh Academy of Nutrition EXTERNAL AND INTERNAL QUALITY CONTROL OF IRON FORTIFICATION OF FLOUR (including iron in vitamin-mineral mixtures) Guidelines Almaty

134 Guidelines have been developed under the leadership of Sharmanov T.Sh. Academician of RAS and NAS of RK, Professor, President of Kazakh Academy of Nutrition, and Quentin Johnson - Coordinator of the Group of Training and Technical Support, Food Fortification Initiative Authors: Kazakh Academy of Nutrition: Amirov B.B. Senior Researcher; Dolmatova O.V. PhD, DBS, Chief Researcher; SinyavskyYu.A. PhD, DBS, Professor, Vice-President; Tazhibayev Sh.S. PhD, MD, DMS, Professor, Vice-President Committee of Consumer Protection, Ministry of National Economy, Kazakhstan: Rakhimzhanova M. Chief Expert, Department for Sanitary and Hygiene Surveillance Department of medical care organization, Ministry of Health and Social Development, Kazakhstan: Kogashov D. Leading Specialist Union of Grain Processors of Kazakhstan: Gan E. PhD, President RSE "Kazakhstani Institute of Standardization and Certification", Kazakhstan: Kuleshova E. Head of Department on methodology, accounting and registration of documents on standardization and certification These Guidelines are based on the requirements of the Code of the Republic of Kazakhstan, dated of 18 September, 2009, On Health of Population and Health Care System, the Law of the Republic of Kazakhstan On Technical Regulation, dated 9 November, 2004, and the Rules of Fortification of Foods Requiring Sanitary and Epidemiology Surveillance, by the Order of the interim Minister of National Economy of the Republic of Kazakhstan, dated of 24 February, 2015, No. 123 (registered in the Registry of Public Registration of Legal Enactments under the No Annotation These Guidelines External and Internal Quality Control of Iron Fortification of Flour (including iron in vitamin-mineral mixtures) describe the organization and management of external and internal quality control of iron fortification of flour. They are intended for the public services and organizations of sanitary-epidemiology surveillance and managers of quality control of flour fortification at flour mills. These Guidelines are based on appropriate international experience and approaches for ensuring flour fortification and quality and safety of end products. 134

135 CONTENT 1. Annotation 2. List of Abbreviations 3. Explanatory Note 4. Introduction 5. Organization of External and Internal Quality Control 6. Methods of Quality Control 7. Management of Correcting Measures and Recommendations on Following the Standards and Requirements of Consumers 8. References 135

136 2. List of Abbreviations In these Guidelines the terms and definitions derived from the Rules of Fortification of Foods Requiring Sanitary and Epidemiology Surveillance (Rules No.123), by the Order of the interim Minister of National Economy of the Republic of Kazakhstan, dated of 24 February, 2015, No. 123 (registered in the Registry of Public Registration of Legal Enactments under the No ) have been used. ExtC external control VMM vitamin-mineral mix IntC internal control QC quality control QA quality assurance PS production standards 3. Explanatory Note Flour fortification and its quality control in Kazakhstan are regulated by the law on mandatory fortification of high extraction flour, dated of At the same time, there is a need for ensuring a uniform observance of norms and standards, in order to assure universal algorithm of fortification quality control irrelevant of the size, equipment fleet of the mill and other parameters, which might affect the quality of the end product. In this regard, these guidelines focus on uniform understanding and observance of quality control activities and realization of a collective responsibility for following the production standards. The goal of the Guidelines is to render a methodological aid to public bodies, individuals and legal entities regardless of their type of property both on independent and institutional levels, which are in charge of quality control of products, for uniform methods and interpretation of results. The guidelines will promote interaction between the external and internal quality control of fortified products and increase consumer satisfaction, as well as motivation of mills in terms of compliance with appropriate standards. 136

137 4. Introduction Among the CIS countries, laws on mandatory fortification of baker s flour with vitamin-mineral premix have been endorsed in Kazakhstan and Kyrgyzstan (in 2009). In Turkmenistan, the mandatory flour fortification is regulated by the Presidential Decree of Among other CIS countries, the flour fortification programs have been adopted in Uzbekistan (by Presidential Decree), Tajikistan, and by Georgia. At the same time, there is no document justifying the uniform approach to the external and internal quality control. These Guidelines are based on the assessment of quality assurance and quality control of flour fortification with iron in the composition of fortification mixes, according to the Rules No Quality assurance (QA) and Quality control (QC) are the procedures to be established at each mill, with a purpose to ensure both systematic and periodical checks of compliance of the mill with the existing standards of wheat flour fortification with vitamin-mineral mix (VMM), or any other fortificant(s) containing iron, with simultaneous observance of established QA and QC procedures and good manufacturing practices, and eventual goal to maintain processes of quality improvement and standard product quality. Table Summary of Characteristics of QA and QC and Their Interdependence in the Whole Process of Good Manufacturing Issue Quality assurance Quality control Definition QA is a set of activities for ensuring quality in the processes by which products are manufactured. products produced. Focus on Goal How What Responsibility Example QA aims to prevent defects with a focus on the process used to make the product. It is a proactive quality process. The goal of QA is to improve manufacturing processes so that defects do not arise. Establish a quality management system, which defines the procedures for manufacture. Prevention of quality problems through planned and systematic activities, including documentation. Everyone on the team involved in manufacturing the product is responsible for quality assurance. Establishing an approved supplier list is an example of QA. QC is a set of activities for monitoring quality in products. The activities focus on identifying defects in the QC aims to identify (and correct) defects in the finished product. QC, therefore, is a reactive process. The goal of QC is to identify defects during, after a product is manufactured, and before it is released. Finding and eliminating quality problems using tools and equipment, so that customer s requirements are continually. The activities or techniques used to achieve and maintain the product quality, process and service. Quality control is usually the responsibility of a specific team that tests the product for defects. Laboratory testing the moisture content of flour is an example of QC. As a tool QA is a managerial tool. QC is monitoring tool. To ensure the quality of fortified flour, there exist independent systems of external control and quality surveillance responsible for: I) Assessment of producers capacity to conform with production standards; II) Periodical inspections and audits of equipment, premixes, methods and knowledge of staff 137

138 of a mill; III) benchmark testing of final products at established time intervals; IV) control of corrective measures and recommendations on compliance with production standards and consumer expectations; 5. Organization of external and internal quality control Overall goal of the External Control (ExtC) is to check the compliance with the established standards of Good Manufacturing Practices (GMP), with a purpose to protect population and consumers from negative effects of inferior quality products. ExtC must ensure public security, protection of rights and freedoms of consumers. ExtC is executed by authorized public bodies and specialized, accredited private organizations, and controlling bodies according to the established schedule, as well as by the requests of mills, public organizations and within the boundaries of established periodicity of checks. In case of flour fortification, public sanitary and epidemiology services are in charge of producer checks. Such checks provide information and identify potential sources of violation of Internal Control (IntC) at the mills, and allow to give specific recommendations on corrective measures to comply with existing Good Manufacturing Practices (GMP) and requirements of normative documents. ExtC and IntC have a common goal to ensure the required quality level of the final product offered to consumers, and they should collaborate in this direction and ensure mutually additive positive effect. The table below describes main characteristics and differences between ExtC and IntC Issue External control Internal control Definition To be defined in normative To be defined by mill leadership based on requirements (standards) standard recommendations, normative standards and customer requirements. Focus Focus on compliance with Focus on conformity to internal Quality Goal normative requirements Goal of the external control is to ensure conformity of existing standards and normative documents to the requirements of GMP How Periodical checks of documentations and assessment of staff competence What Standards of external control, including proper documentation Responsibi lity Example Tool External organization authorized by competent body Public Health inspection is an example of ExtC. Audit is an example of ExtC ExtC is a tool of assessment of compliance of production processes to rules and recommendations on corrective activities Management System Goal of internal control is to ensure conformity to internal Quality Management System Regular monitoring with corrective and preventative action Regular and scheduled activities or technologies chosen by a mill to achieve and maintain proper quality of products, processes and services Internal responsibility of a mill Regular testing of product samples are an example of IntC IntC is a response (reacting to production defect) and corrective tool to ensure conformity with GMP and standards in regard of final quality of the product Mill auditing bodies should analyze the findings of internal control, in order to spare time and resources and avoid duplication of efforts, and focus on ExtC to identify potentially critical elements. Interaction of ExtC and IntC ensures better effect on end result subject to coordination of checks, exchange of reports, access to operational documentation, joint reports to proper leadership and common documentation patterns. Each of listed responsibilities of ExtC can be specified also as follows: 138

139 1) Assessment of producer s capacity to follow the normative requirements and standards. 2) Availability and knowledge of rules related to flour fortification, in accordance with the following checklist: Appropriate standard of Republic of Kazakhstan, and technological instructions; Copies of effective standards or guides on flour fortification and appropriate requirements of the competent body in charge of sanitary and epidemiological security of population; Specifications of the equipment required for flour fortification; A log of preliminary and operative instructing of staff on safety measures at operating with flour fortification; A log of regular technical checks of fortification equipment; A log for registration of laboratory samples and tests of fortified flour with established time interval and analyses of external control (it is a recommended log, according to effective standards currently flour fortification at a mill is monitored by weight method only. No analyses are made). Logs to every day records of quality control at a mill (see, annex 3). Warehouse logs recoding route of premix within the mill and to technological lines. 3) Regular checks and audits Assessment of regularity of order and delivery of fortificants, suppliers, quality documents certificate of origin, certificate of public registration of premix, etc.); Assessment of premises and conditions of storage, release, record of use of fortificants; Visual assessment of mixing equipment and production process; Sampling, sample control, recording and registration of samples and proper documentation. А. External control by governmental bodies: State audit: there exist two types of audit. One is called technical audit, or the extra-enterprise audit, when the competent body in charge of sanitary and epidemiological security of population examines information provided by a mill or association of mills, if the latter is authorized to collect such information, with a purpose to determine documentary quality of flour fortification. This type of audit can be made once a quarter (see, the example of mill s report). Second type of auditing is the inspection or field examination when the competent body in charge of sanitary and epidemiological security of population inspect a mill with a purpose to assess the conformity of enterprise to flour fortification requirements and double checking of data provided for technical audit. The competent body in charge of sanitary and epidemiological security of population determine frequency of field inspections based on the findings of previous operation of a mill and number of claims or violations identified in regard of the specific enterprise. One of important stages is the check of observance of the procedure of acceptance of a premix, including registration of the following information: 1) Check of package for any damage and recording of such damages in the documentation. It is acceptable to have small damages, but it is inacceptable to have serious damaging by water and ruptures of internal bags; 2) Record of date of acceptance and name of controller; 3) Data on type or name of commodity and number of boxes/bags or total mass, comparison with ordered items; 4) Record of batch numbers; 5) Check the availability and recording in the documentation of a test certificate of the premix and certificate of public registration. It may be put in one of boxes or sent separately by fax or . All this information should be recorded in the Report on Premix Acceptance. 6) Each box must be labeled. The label shall bear: a) name of commodity; b) intended use of commodity; c) name of producer and contact information; d) safety measures; d) date of production and shelf life, sometimes presented in batch number; e) batch number; f) recommended use ration; g) 139

140 net weight; h) composition. Б. Premix producer must provide a Certificate of public registration of premix (or excerpt from the Registry of certificates of public registration), test certificate or test protocol (in case of the Kazakh regulations). For each premix batch and for all micronutrients in the composition of premix, there should be provided a proper document/certificate proving quality of a product. Test certificate (sometimes called Quality certificate ) is a formal document confirming the quality of premix. Test certificates for each batch of premix must be stored and presented in case of control checks. The test certificate shall bear the following information: Results of chemical analysis of the premix batch in regard of each nutrient in its composition. The certificate may provide reference data on minimum and maximum levels of elements in the premix, batch or lot number, date of production and shelf life, if these data are not included in the batch number. Premix suppliers shall provide: information on commodity or a data table. Such document should be stored at a mill and provided upon request to ExtC representatives, production staff and department of quality control. This document shall contain the following information: 1) Premix name; 2) Name and contact information of producer; 3) Intended use of premix; 4) Premix composition as a rule, in descending order; 5) Nutritive status of ingredients (that is, a class according to classification of chemical substances allowed for using in food industry); 6) Recommended proportion of addition of premix to flour and levels of content of micronutrients at such proportion; 7) Standards of minimum composition of a premix, as well as standards of maximum composition (if available); 8) Storage and transportation instructions; 9) Shelf life of a premix; 10) Content of pathogens and other microorganisms of concern 6. Control methods The control is accomplished by using the following assessment methods: А. Assessment of knowledge and skills of laboratory staff; Б. Assessment of availability of equipment and reagents for qualitative and quantitative tests; В. Control sampling and exchange of findings with a mill; Г. Provision of an option of qualification tests. External quantitative analysis. Getting a mixed sample of fortified flour: 1) Mixed samples are made by mixing samples of fortified flour collected at established time intervals during the production shift; 2) Mixed samples consist of 5-10 samples collected from production line, for example, during a 8-hour shift; 3) Samples used for preparation of a mixed sample shall of the same volume and be collected at even intervals during the production shift to be representative for the whole product manufactured during the certain period; 4) The goal of the mixed sample is to assess a mean content of nutrients in a certain batch. Use of mixed samples allows decreasing the cost of analyses of fortification via reducing the number of samples required for getting average value. 5) Use of quantitative analysis for checking one or several cases of too high or too low content of nutrients is senseless. Its main goal is to monitor the evolving trends, and as such, the quantitative analysis is a valuable tool. 7. Ensuring correcting activities and recommendations on observance of normatives 140

141 and consumer demands First, it is necessary to organize a regular reporting on the outcomes of external control activities; Second, it is necessary to hold discussions with a mill on corrective activities and deliver methodological support in the development of recommendations to ensure compliance with Good Manufacturing Practices and to organize, if necessary, (re)training of specialists, taking into consideration employee turnover, replacement of equipment, etc.; Third, to establish submissions of follow-up reports on operation of a mill on ensuring the established level of quality before the controlling bodies. 8. Role of laboratory control As the laboratory methods constitute an important part of both external and internal control of fortification quality, the Annex 1 and Annex 4 provide detailed description of methods commonly used around the world for measuring iron, which are recommended for official use by sanitary, and epidemiology services and mills in Kazakhstan. With evolution of laboratory techniques, currently many started shifting to inductively coupled plasma mass spectrometry (ICP-MS) for determination of iron and other inorganic substances. ICP- MAS is a type of mass spectrometry which is capable of detecting metals and several non-metals at concentrations as low as one part in (part per quadrillion, ppq) on non-interfered low-background isotopes. This is achieved by ionizing the sample with inductively coupled plasma and use of a mass spectrometry to separate and quantify ions. Compared to atomic absorption spectroscopy, ICP-MS has greater speed, precision, and sensitivity. 141

142 Annex 1 Recommended (reference) methods of iron determination in flour in the frame of ExtC (Spectrophotometric methods are given in the Annex 4) HPLC method for determination of iron Iron determination with use of Dionex-500 chromatograph Reagents 1) De-ionized water, nano-purification 18.0 mohm 2) Concentrated HCl 36% 3) Iron standard 100 µg/ml in HN03, commercial standard 4) Post column solvent MetPacDionex Corp. P/N ) Concentrated dilutor MetPacDionex Corp. P/N ) 4-(2-pyridazo) resorsinol, analytical grade 7) Monosodium monohydrate Equipment 1) Analytical balance capable to weigh 0.01 mg 2) Measuring glass, 400 ml and 1,000 ml 3) ColumnandbreakerDionexIonPacCS5 AandIonPacCG5A 4) Alumina crucible, 50 ml 5) Flasks, 100 ml 6) Graduated cylinders, 500 ml and 1,000 ml 7) Gas stove 12"х24", 220 V, with remote control 8) HPLC system (Dionex-500, with AD20 absorption detector, pump GP50, sampler AS40) 9) Magnetic rods for mixer, octogonal 15"х3/8", teflone-coated 10) Muffle furnace, 675 С 11) Sonic bath 12) Mixer 13) Disposable pipette, 5 ml 14) Watch glass, 65 mm 15) Volumetric flask, 250 ml 16) Volumetric pipette, class A:1,3,7,10 and 15 ml Preparation of solution Iron standards Using 1, 3, 7, 10 and 15 pipettes, transfer 100 µg/ml iron standards in labelled volumetric (250 ml) flasks, dilute to volume with deionized water, cap and mix. These solutions are equivalent to mg/pound', mg/pound, mg/pound, mg/pound and mg/pound of iron, respectively. Label the day of preparation. One pound = г Mobile phase Add 800 ml deionized water to 1,000 ml graduated cylinder. Fill to volume with dilutor PDCA. Transfer to the reservoir with mobile phase to deliver to HPLC system. Post-column solvent Weigh ± g of the solvent, transferin 1,000 ml flask with screwcap. Place the flask in sonic bath for five minutes, connect the flask to post-column pump. Preparation of samples 1) Sample 5.00±0.01 g flour and place in alumina crucible. Put the crucible in muffle oven. Record the sample number and its position in oven. Repeat the same with all samples and prepare 142

143 flour. 2) Put muffle temperature for 675 С and leave crucibles overnight. 3) Switch off the oven next morning and carefully open the door to speed up the cooling of crucibles. Take cool crucible from oven and place on gas stove located in fume hood. 4) Turn on the fume hood, and add 2 ml of concentrated HCl in each crucible, around evaporation plate. Close crucible with watch glass. Heat crucible until about 1 ml solution remains. Take crucible off the gas stove and let it cool. Notes Avoid full evaporation of hydrochloric acid! If occurs, add 1 ml of concentrated hydrochloric acid and heat it again. Wash off condensate on watch glass with deionized water in labeled reaction tubes. Move the content of crucible in a flask and collect in it deionized water used for washing the crucible several times. Add deionized water to 100 ml volume, cap the flask and mix by tilting. Fill labelled tube with sample and place in sample conveyor. After charging put the container in automatic sampler AS40 and make sure the black point is on the right side of conveyor at looking on the front side of sampler. Analysis 1) Open the start window PeakNet. To load schedule, click on second icon on the left. Close the schedule developed in step after the click, press the last icon (OK), pump starts operation. 2) Switch on post-column pump and press Start. Let the system work for 30 minutes. 3) Load samples in automatic sampler AS40 as appropriate and press button Start. 4) Dionex software automatically calculates iron in mg/pound and prints out results of each standard and samples. Elements by atomic absorption spectrophotometry AACC method Final approval October 16, 1991; Reapproval November 3, 1999 This method determines calcium, copper, iron, magnesium, manganese, and zinc in grains and cereal products. I. Materials Volumetric flask, 1 L Volumetric flasks, 250 ml Volumetric flasks, 100 ml Volumetric flasks, 25 ml Beakers, 250 ml Manual volumetric pipettes ( ml) Porcelain crucibles Watch glasses Pipette tips Graduated tubes Tips for blue pipettes Test tubes, 10 ml II. Equipment Atomic absorption cereal products spectrophotometer. Several commercial models are available. Since each design is somewhat different, with varying requirements of light source, burner flow rate, and detector sensitivity, only the general outline of operating parameters is given in Table 1 (below). Operator must become familiar with settings and procedures adapted to own apparatus 143

144 and use table only as guide to concentration ranges and flame conditions. Single-slot burner may require that lanthanum be added to standard and sample solutions for all elements. Ashing vessels, 150 ml beaker (Pyrex or Vycor) or 30 ml Vycor crucible. Muffle furnace capable of operating at temperatures up to 525. Vortex mixer Analytical balance Hot plate Eppendorf pipette (100 and 500 ml) Table 1. Operating Parameters Element Wavelength Flame Range (µg/ml) Fe 2483 Rich air-acetylene 2-20 Zn III. Reagents 1) Water, distilled-deionized (greater than 10 megohm resistance). Use throughout procedure in all preparation and dilution of solutions. 2) Stock solutions. See Note 1. a. Iron, 1000 μg Fe/ml. Dissolve g pure Fe wire in about 30 ml 6N HCI with boiling. Dilute to 1 liter. b. Zinc, 1000 μg Zn/ml. Dissolve g pure Zn metal in about 10 ml 6N HCl. Dilute to 1 liter. 3) Lanthanum stock solution, 50 g La/liter ~5% HCl. Dissolve g La2O3 (99.99%, low calcium content) in 250 ml concentrated HCl, adding acid slowly. Dilute to l liter. 4) Working standard solutions. a. Calcium, 0, 5, 10, 15, and 20 μg Ca/ml containing 1% La and ~1% HCl. To 25 ml volumetric flasks, add 0, 5, 10, 15, and 20 ml Ca stock solution (reagent 2a). Add 5 ml La solution and dilute to volume. b. Other standard solutions. Dilute aliquots of solutions 2b, 2c, 2d, 2e, and 2f with 0.5N HCl, to make at least four standard solutions of each element within range of determination. IV. Procedure A. Preparation of sample 1) Accurately weigh 1-10 g ground sample (depending on anticipated concentration of element) into ashing vessel. See Note 2. Begin preparation of reagent blank at this point. 2) Char on hot plate or in muffle, then ash at 500 overnight. If not completely ashed, cool sample and wet with a few drops of concentrated HCl or HNO3, dry at low heat, and re-ash. See Note 3. 3) Break up cake with stirring rod and dissolve in 10 ml concentrated HCl. Boil and evaporate solution nearly to dryness on hot plate. Do not bake residue. 4) Redissolve residue in 20 ml 2N HCl, boiling gently if necessary. 5) Filter through fast paper into 100 ml volumetric flask, washing paper and residue thoroughly with water. Dilute to 100 ml and mix. 6) Measure absorption of solution directly, or dilute with 0.5 N HCl to obtain solutions within ranges of instrument. If Ca is to be determined, add enough La stock solution to make final dilution1% La (i.e., 5 ml La solution to 25 ml flask, 20 ml to 100 ml flask, etc.). See Note 4. B. Method 144

145 1) Set up instrument as in Table I or by previously established optimum settings for apparatus to be used. Secondary or less sensitive lines may be used to reduce necessary dilution if desired. See Ref.2. 2) Read at least 4 standard solutions within analytical range before and after each group of 6 to 12samples. Flush burner with water between samples, and reestablish zero absorption point each time. 3) Prepare calibration curve from average of each standard before and after sample group. See Note 5. 4) Read concentration of samples from plot of absorption against μg/ml. V. Interpretation/Calculations VI. Notes Element (ppm) = (μg/ml x 100) sample weight (g) If original 100 ml volume is diluted, consider this in final calculation. 1) Do not use <2 ml pipettes or <25 ml volumetric flasks in making standards. Prepare working solutions in 0-20 μg/ml range fresh daily. Automatic dilution apparatus may be used. Alternatively, purchased standards may be substituted for stock solutions. 2) Some grains and unfortified products may require ashing a larger sample to increase the concentration of elements to detectable ranges. See Ref. 3. 3) Contamination from labware and reagents can contribute significant amounts of analytical error. Blanks should be run to monitor for contamination. Cleaning protocols including rinses in mineral acids (e.g., nitric and hydrochloric acids) and multiple rinses in distilled/deionized water have proven effective in minimizing metal contamination in labware. If re-ashing is necessary, prepare are-ash blank, using the same amount of concentration HCl or HNO3 as for the samples. 4) Phosphorus interferes in calcium and may interfere in magnesium determination with air-c2h2burners. Eliminate interferences by adding La stock solution to standard and sample solutions so that final dilutions contain 1% La. Phosphorus does not interfere with calcium determination when an N20-C2H2 burner is used. 5) It is recommended that a standard reference material (such as those available from the National Institute of Standards and Technology) be analyzed frequently to ensure accuracy of the determination. Choose reference material that matches as closely as possible the matrix of the sample being analyzed. VII. References 1. AOAC International Official Methods of Analysis of AOAC International. 16th ed. Method The Association, Arlington, VA. 2. Gatehouse, B.M., and Willis, J.B Performance of a simple atomic absorption spectrophotometer. Spectrochim. Acta 17: Zook, E.G., Greene, F.E., and Morris, E.R Nutrient composition of selected wheats and wheat products. VI. Distribution of manganese, copper, nickel, zinc, magnesium, lead, tin, cadmium, chromium, and selenium as determined by atomic absorption spectroscopy and colorimetry. Cereal Chem. 47:

146 Annex 2 - FLOUR FORTIFICATION METHODOLOGY Ensuring standardized monitoring of fortification procedures and records: 1) Store fortification well protected from exposure to light or under the conditions laid down by the manufacturer. It is ideal to keep fortification mixes in their original containers. Once opened, exposure to the light and air should be minimized to prevent product degradation. 2) Obtain and keep on record on record a certificate of compliance for every batch of fortification mix. 3) Employ, and adhere to, strict stock rotation procedures to prevent old stock loosing potency and to comply with the shelf life expiry date. It is recommended to employ the first in, first out (FIFO) system for this purpose. 4) Keep records of grain procurement; 5) Keep records of fortification mix inventory and usage; 6) Keep production records of the amount of fortified bread flour produced; 7) Keep monthly records of the amount of fortification mixes used every month. These records should correspond with the monthly production records; 8) Ensure that all critical stages of the manufacturing process are monitored to ensure the correct dosage levels are maintained through the following measures: 9) Checking of fortification mix feeders to ensure they are delivering the correct dosage levels. This can be done by measuring the weight of fortification mix discharged over a specific time (1 or 2 minutes) and comparing the measurements with the target weight of fortification mix. 10) Performing frequent visual checks to ensure fortification mixes are being used and that no blockages have occurred, and keeping a record of this. 11) Performing regular and random iron spot tests on the bread flour using composite samples of fortified flour. 12) Collect individual samples of fortified flour every 2 hours to form average per shift sample to determine the fortifying iron. Analytical error To minimize analytical and sampling errors, laboratories shall thoroughly mix the samples received from the inspector to form an average shift sample. Then the laboratories of mills or authorized body (in case of external control) shall analyze the sample possibly in duplicate and, more than likely, twice on the same extract rather than twice from the same sample. Total error Laboratory error can be high at 95% confidence level for vitamin analysis in fortified product the result is ±15-20%. Distributing 200 g of pre-mix in 1,000 kg of flour is not easy even with a very good mixer, so we could have a variation >30%. Quality control of the iron fortification at the mill Taking the following actions during the receipt and storage of the iron fortificant will help the mill to assure the quality of the iron in the final product: 1) Request that suppliers include a certificate of compliance with each shipment. A copy of the certificate of compliance should be kept with the permanent records. 2) Maintain a first in, first out policy. Number lots consecutively in order that they are received. For each lot, write the lot number and the date of receipt on the sticker. 3) Arrange storage areas to facilitate the first in, first out policy. Store bags or boxes of the iron fortificant in consecutive order, so that the oldest can be withdrawn first. 4) Store the iron fortificant in conditions that maintain its quality and prevent contamination or deterioration. 5) Record and monitor the movement of the iron fortificant in and out of the warehouse, and control the amounts used in manufacturing. 146

147 6) Maintain records of returned materials and their use and control. 7) Maintain sufficient stocks of iron fortificant. Ask the purchasing department to order a new shipment in sufficient time to avoid running out of iron fortificant. 8) To confirm that the composition of the iron fortificant meets specifications, send samples for laboratory analysis within three months of arrival. Packaging, storage, and handling the premix Premixes are concentrated sources of iron or other micronutrients. Excessive intake of iron in a single dose is poisonous and can be fatal; lower concentrated doses over a prolonged period can also be harmful. For this reason, the boxes must be carefully labelled Not suitable for direct consumption, and workers at the mill must understand and respect this warning. Operators at mill should take precautions such as wearing a dust mask, gloves, etc., to prevent inhalation of and exposure of skin to the premix or iron fortificant. The premix and iron fortificant must be properly handled to minimize degradation of the compound. They must be stored in well-ventilated rooms at low or mild temperatures (preferably not higher than 25 C), and exposure to humidity must be avoided. The amount of commercial premix or iron fortificant needed should be estimated and obtained in quantities small enough so that it does not need to be stored for long periods of time. The production lot number(s) should be properly recorded and the premix used on a first-in/first-out basis. Once a commercial premix or iron fortificant box has been opened, it should be used within the period specified for its shelf life. Fortification of flour in continuous and batch systems Volumeorweightfeeders are used to introduce a premix or mixture in the flow of flour. In a continuous milling system, the iron fortificant or premix is continuously and gradually added as a free-flowing dry powder at a rate dependent on and compatible with the flow of flour along the production conveyor (about 150 g per 1 ton of higher grade flour and 120 g per 1 ton of first grade flour). The homogeneity of micronutrients in the fortified flour is largely dependent on the location of the feeder, and it is very important that good mixing of the micronutrients in flour occurs. The two most common sites for adding micronutrients are 1) before packaging, which facilitates good mixing and 2) where flours from different streams converge, which facilitates excellent mixing. Small- and medium-sized mills may choose to fortify flour using a batch system, where flour is fortified in individual batches rather than in a continuos process. In batch fortification, iron is measured out by weight or volume and added to a batch mixer that is partially or totally filled with flour. The well-mixed, fortified flour is then transferred to pack-out bins and another batch of flour is fortified. The time of satifactory mixing up is determined empirically. Quality control of the iron fortificant or commercial premix A recording and storage system must be in place to log the date the iron is received. The iron or premix must be stored under conditions specified by the manufacturer. Physical characteristics such as color, texture, and odor can be visually examined. The iron or premix should be free-flowing with no lumps or off-odor. The Certificate of Analysis provided by the manufacturer should be reviewed for the grade, particle size, and concentration of iron. The iron should be analyzed using semiquantitative spot tests to ensure the iron level; the analytical methods are presented in Part 3 of this manual. All these observations along with any corrective actions taken when the required specifications were not met must be properly recorded and filed for easy access. Annex 3 gives an example of the type of recording form that can be used. Quality control of iron-flour premix made at the mill The iron-flour premix made at the mill should be analyzed using quantitative methods to verify the concentration of iron in the iron-flour premix. The amount of iron in the premix must be within 10% on either side of the specified mean. The analytical methods to determine the amount of iron in the iron-flour premix and fortified iron are presented in Part 3 of this manual. The mill manager should be responsible for monitoring the quality of iron-flour premix, the date of manufacture, and the addition to bulk flour. The dates and results of all chemical analyses must be 147

148 properly recorded. The mill manager also is responsible for any corrective actions. All records should be filed for easy access. Annex 3 gives an example of the type of recording form that can be used. Quality control of the fortified flour Four methods can be used to supervise and control the addition of iron to flour. Proper recording of all the results of the quality control procedures is essential. An example for recording the quality control results of fortified flour is given in Annex Premix inventory control Comparing the amount of premix used against the flour production records provides a simple way of determining if the correct amount of premix is being used. Another way is to obtain the ratio of the amount of fortified flour produced to the amount of premix used in a predetermined period of time. These methods provide general information on whether the required amount of premix is being added. The mill manager may add this task to his routine quality control activities. 2. Regular weight checks In this case, a plate is put under the feeder to collect the fortificant for usually one to three minutes and the fortificant is weighed to determine if the feeder is dispensing the correct amount of fortificant at that setting; longer collection times are used for lower addition rates. Weight checks should be done every time flour samples are taken for routine quality analysis approximately every two hours. The operator should record the time, weight, and feeder setting. Automated, loss-of-weight feeders are also available, wherein the disappearance of iron fortificant should correspond with calculated usage and any adjustments to the feeder can be made based on these results. The mill manager may add this task to his routine quality control activities. 3. Spot tests Mills can use a simple qualitative spot test for iron to determine if the flour has been fortified or a semi-quantitative spot test that allows the determination of whether the flour is grossly under- or over-fortified. Spot tests are quick and simple, and can be used at various points in the production line using them at the point of pack-out or packed bags will ensure that the flour fortified within a specific range of iron content. The results of qualitative or semi-quantitative tests, however, are not precise enough to make feeder adjustments. It is recommended that semi-quantitative spot tests be conducted every time the mill conducts routine quality control procedures (for example, for moisture or color), approximately once per shift in a continuous fortification process using average shift sample for 12 hours or for every batch of flour produced using the batch fortification process. The laboratory manager or a laboratory technician should be responsible for conducting these tests. 4. Quantitative tests Quantitative tests provide accurate data on the amount of iron in the flour and reflect the efficiency of the entire production process. Ideally, the same flour samples collected for semiquantitative spot tests should be pooled for each production shift, mixed well, and analyzed. However, it is recommended that quantitative tests be conducted as often as routine tests for protein in flour. The tests may need to be performed more frequently when troubleshooting problems in the fortification process. The laboratory manager or a laboratory technician should be responsible for conducting these tests. Sampling the flour for chemical analysis The point at which the flour is sampled, the sampling procedure, and how the sample collected and handled are critical in obtaining reliable and useful results. Samples should be collected at a point in the production line after the addition of the fortificant as well as the blending of fortified flour. Samples must be kept protected from high temperatures and humidity until analyzed. A good sample represents the entire production run averaging out any momentary variation in the fortification process. A composite sample can be obtained by taking a grab of flour at a specified point in the production line every two hours over a shift and mixing them together. This composite sample of fortified flour can then be analyzed for iron content. 148

149 STORAGE OF FLOUR It is important to organize the zone of flour storage operating by the order first in first out, that is to store the bags or package in consecutive order to release first the batches which came to storage are earlier. 149

150 Annex 3 Examples of logs for registration of flour fortification with iron An example of a quality control log for iron fortificant or commercial iron premix Date received Batch # Results of visual examination Color Freeflowing Odor Certificate of analysis check Iron (ppm) (semiquantitati ve test) Observations / corrective actions Mill manager Date An example of a quality control log for iron-flour premix made at the mill Date manufactured Batch number Date tested for iron Iron (ppm) (quantitative test) Date used in bulk flour Observations / corrective actions Mill manager Date An example of a quality control log for fortified flour Date Shi ft and tim e Premix inventory control Tim e (hou rs No. flour sacks produc ed (A) No. fortific ant bags used (B) A B Regular weight checks Colle Weig Feed ction ht er time (mg) setti (min ng & sec) Semiquantitati ve spot test (ppm)1 Quantita tive test (ppm) Observati ons / corrective actions Mill manager Laboratory manager 150

151 Annex 4 Methods of assessment of iron fortification recommended for mills (Internal control) QUALITATIVE METHOD A. Spot test method This method, approved by the AACC, is applicable for qualitative determinations of iron in enriched flour (See Annex 3.3). Principle Ferric iron added to flour reacts with a thiocyanate (KSCN) reagent to form a red colored complex. A higher number of red spots and deeper red color appear with enriched and fortified flour compared with untreated flour. Advantages 1) It is a simple, fast and easy technique requiring no sample treatment. 2) It is inexpensive: only two reagents, KSCN and HCL, are needed. 3) Personnel with minimal training can conduct this assay. 4) It does not require a laboratory; it can be conducted in the flour mill. Limitations It is not quantitative, i.e., it does not determine the amount of iron present in the sample. Notes This method shows only ferric iron. If iron is added in the ferrous form, the sample needs to be oxidized with hydrogen peroxide to convert the ferrous to ferric iron before analysis. Description of the method 1: Iron qualitative method AACC Method First approval May 5, 1960; reapproval November 3, 1999 This method qualitatively determines iron added to flour I. Materials Rectangular glass or rich galvanized iron plate, about 12 x 8 cm Flour trier II. Equipment None III. Reagent Thiocyanate reagent Dissolve 10 g KSCN in 100 ml of water. Mix with equal volume 2N HCL just prior to use. Hydrogen peroxide, 3% IV. Procedure Determination of ferric iron 1) Slick untreated and enriched flour side by side in usual manner (See Method below). 2) Drop approximately 1 ml thiocyanate regent at junction of two flours, in amount sufficient to wet area approximately 1 inch in diameter. 3) Let stand at least 10 minutes. If added ferric compounds are present, deeper red color will be formed than in untreated flour. Small local areas of intense red show up after 20 minutes, indicating location of individual particles of iron compound. (This affords some estimation of uniformity of mixing. 151

152 V. Determination of ferrous iron Follow steps 1 and 2 above. 1) Drop approximately 1 ml of 3% hydrogen peroxide over same area wet by thiocyanate reagent. 2) Repeat step 3 above. The ferrous iron will have been oxidized to ferric state by the hydrogen peroxide. VI. Sampling frequency: Twice per shift Description of the method 2: Method Pekar Color Test (Slick test) Final approval April 13, 1961; Reapproval November 3, 1999 I. Objective Flour color, like ash content, is an indication of milling efficiency. This method is a qualitative test for flour color. The color of flour is visually compared to that of a standard flour. II. Apparatus 1) Rectangular glass or rigid galvanized iron plate, about 12 cm long and 8 cm wide. 2) Flour slick. III. Procedure 1) Place approximately g flour on glass or iron plate. Pack one side in straight line by means of flour slick. Treat same quantity of standard flour used by comparison in same manner, so that straight edges of two flours are adjacent. 2) Carefully move one of the portions so that it will be on contact with the other and slick both with one stroke of flour slick in such a manner that thickness of layer diminishes from about 0.5 cm in middle of plate to thin film at edge. Line of demarcation between two flours should be distinct. Note difference in color. 3) Two emphasize differences in color, cut off edges of layer with flour slick to form rectangle and carefully immerse plate with flour in cold water for 1 minute. 4) Dry at 100 C and note color difference when still moist and when completely dried. Note Since original moisture content of flour has a marked influence on the results of the Pekar test, standard flour and flour tested should be of approximately the same moisture content. IV. Sampling frequency: Twice per shift II. Semi-Quantitative Methods A. Spot test method This method is an adaptation of the AACC s qualitative spot test method. It was developed and is routinely used at INCAP for semi-quantitative determinations of iron in flour. Principle Ferric or ferrous iron added to flour reacts with a thiocyanate (KSCN) reagent, in the presence of hydrogen peroxide, to form a red-colored complex. The number of spots reflects roughly the amount and homogeneity of the iron in the sample. Advantages 1) It is a simple, fast, and easy technique requiring no sample pretreatment. 152

153 2) It is inexpensive. 3) Personnel with minimal training can conduct this assay. Limitations 1) It is semi-quantitative and provides only a rough estimate of the amount of iron in the sample. Notes 1) Iron added to flour in either ferric or ferrous iron can be detected. 2) Flour samples with known concentrations of iron can be tested and the results can be used as a reference for more accurate estimations. Description of the method 1: Semi-quantitative spot test for iron INCAP method IV Adaptation of the AACC qualitative method for iron I. Materials Watch glass Droppers II. Equipment None III. Reagents and Solutions Hydrochloric acid, HCl, 37% Merck 317 Hydrogen peroxide, H2O2, 30% Merck 7209 (it is possible to replace this reagent with commercial oxygenated water). Potassium thiocyanate, KSCN, Merck 5124 or 5125 KSCN 10%: Dissolve 10 g of KSCN in 100 ml distilled water. HCl 2M: To a 500 ml beaker add 100 ml distilled water, then, 17 ml concentrated HCl, and finally 83 ml distilled water. H2O2 3%: Add 9 ml concentrated H2O2 (30%) to 81 ml distilled water. Reagent 1 Immediately before using, mix equal amounts of 10% KSCN and 2M HCl. Mark the levels of 20 and 40 ml on a flask using a pipette. Add 2M HCl up to the first mark and then add 10% KSCN up to the second mark. This is reagent 1. Use within 1 day. Discard the remainder. Reagent 2 3% H2O2. Discard remaining solution at the end of the day. IV. Procedure 1) Take a sample of 10.0 g of flour and place it on the watch glass. With the lower part of another watch glass, press on the flour sample so that it forms a flat surface. 2) Add 5 drops of reagent 1 with the dropper so that it covers an area of 4x4 cm (1.5x1.5 inches). Let stand 15 to 30 seconds 3) Add 5 drops of reagent 2 on the surface covered by reagent 1. Let stand 1 to 2 minutes. V. Interpretation The appearance of red-colored spots indicates the presence of iron. The number of spots is a rough estimate of the amount and homogeneity of iron in the sample. If a more accurate estimation is required, test with known concentrations of iron (30, 60 and 90 ppm) and compare the results with those of the test samples. 153

154 B. Colorimetric method This method was developed by INCAP and is routinely used in its laboratory for the semiquantitative determination of iron in flour (See Annex 3.5). Principle After total combustion of the organic material, by ashing the sample, iron is dissolved in a mildly acidic solution and reduced through the addition of hydroxylamine hydrochloride. Ferrous iron reacts with the chromogen, bathophenanthroline, to form a pink-colored complex. The intensity of the color provides an approximate estimation of the amount of iron in the sample. Advantages 1) It is applicable to various types of food products and ingredients. 2) It is relatively inexpensive. 3) Personnel with minimum training can perform the analyses. Limitations 1) It is semi-quantitative but provides a more accurate estimate than the semi-quantitative spot test. 2) It is a time-consuming procedure involving overnight dry ashing. Samples with a high protein content may take longer to be ashed. 3) It involves handling hot acid solutions; thus, appropriate safety precautions must be taken. Notes 1) The test is based on visual differentiation or categorization and does not require a spectrophotometer. 2) Hydroxylamine hydrochloride reagent must be stored in the refrigerator. 3) It is possible to replace bathophenanthroline reagent with α,α-dipyridyl chromogen. However, slight differences in color become more difficult to distinguish with the dipyridyl reagent. Description of the method 2: Semi-quantitative colorimetric determination of iron in flour INCAP method V INCAP, Chemistry and Biochemistry Laboratory Revision No.2; April, 1997 This method entails total combustion of the organic material by ashing the sample. Then the ash is dissolved in acid and the iron present is reduced through the addition of hydroxylamine hydrochloride reagent. Ferrous iron is determined through the formation of a pink color in the presence of the chromogen bathophenanthroline (4,7-diphenyl-1,10-phenanthroline-disulfonic acid). I. Materials Volumetric flasks 25, 100, and 1000 ml Beakers 500 ml Burette 25 ml Porcelain crucibles Graduated tubes Serological pipettes 5 and 10 ml Volumetric pipettes 1, 2, and 5 ml Test tubes 10 ml II. Equipment Analytical balance (1 mg) Hot plate Muffle furnace 154

155 III. Reagents Sodium acetate (CH3COONa 3H2O), 99%, Fe<200 mg/kg, PM , Merck Art Hydrochloric acid (HCl), 37%, 1.19 g/ml, Fe<28 mg/ml, PM 36.46, Merck Art. 317 Bathophenanthroline, 4,7-diphenyl-1,10-phenanthroline-disulfonic acid (C24H16N2O6S2), PM (free acid, anhydrate) 492.5, Sigma B-1375 Hydroxylamine hydrochloride (NH2OH HCl), PM 69.49, Baker 2196 Iron standard 1 g, Baker DILUT-IT 4777, or any other iron standards, including ferrous ammonium sulfate Deionized water, Fe<1mg/dl A. Preparation of solutions Hydrochloric acid (HCl), 6M: Add 200 ml deionized water to a 500 ml beaker. Slowly add 250 ml concentrated HCl. Let it cool and transfer to a 500 ml volumetric flask and make to volume with deionized water. Transfer to a glass flask and close with a glass stopper. This solution is stable indefinitely. HCl, 0.96M: Add 168 ml deionized water to a 500 ml beaker. Slowly add 32 ml 6M HCl. Transfer to a glass flask and close with a glass stopper. This solution is stable indefinitely. Hydroxylamine hydrochloride, 10%: To a 500 ml beaker add 50 g hydroxylamine hydrochloride and then 400 ml deionized water. Stir with a glass rod. After it is completely dissolved, transfer to a 500 ml volumetric flask and make to volume with deionized water. Transfer to a glass flask and close with a glass stopper. This solution is stable indefinitely. Bathophenanthroline, 0.025% in sodium acetate, 2M (Bathophenanthroline %/CH3COONa-2M): Add to a 500 ml beaker g sodium acetate trihydrate and 0.10 g bathophenanthroline. Add 400 ml deionized water. Dissolve completely with a glass rod using gentle heat if necessary. Make sure that bathophenanthroline is fully dissolved because it is poorly soluble at room temperature. Store in a glass or plastic flask. Discard solution if pink color develops, because this indicates contamination with iron. The solution is stable for 3 to 4 months. B. Preparation of iron standards Standard, 1000 ppm: Dilute reagent DILUT-IT, according to manufacturer s instructions, in a 1 L flask using deionized water. Alternatively, dissolve g Fe(NH4)2(SO4) 6H2O in distilled water, add 2 drops of concentrated HCl, and dilute to 500 ml. Standard, 10 ppm: Add to a 100 ml volumetric flask 1.0 ml (measured with a volumetric pipette) 0f 1000 ppm iron standard. Then add 16 ml of 6V HCl and adjust to volume with deionized water. Standards for visual comparison: Use 25 ml volumetric flasks. To make concentrations of 0.0, 0.8, 1.6, 2.4, and 3.2 ppm, which equivalent to 0, 20, 40, 60, and 80 ppm (mg/kg) of iron in flour, add 2 ml 6V HCl to each flask, then add 2.5 ml 10% hydroxylamine solution, and a corresponding quantity of the 10 ppm iron standard (see table below). Adjust to volume with deionized water. Cover with glass stoppers. These standards are stable for 2 to 4 weeks. Equivalent concentration in mg iron/kg flour IV. Procedure Real concentration in mg iron/l (ppm) A. Ashing the sample Volume of 10 ppm iron standard to be added (ml) 155

156 1) Accurately weigh 2 g of previously homogenized sample. Weigh samples in duplicates. Transfer sample to a porcelain crucible. 2) Ash sample in a muffle furnace at 500 C for 4 hours. The sample is adequately ashed when a white or grey ash is obtained. Cool to room temperature. B. Solubilizing the ash. 1) Add 5 ml of 6M HCl to the porcelain crucible allowing the acid to wash the walls of the crucible and evaporate until dry on the hot plate, taking care that the sample does not splash outside the crucible at any time. 2) Dissolve the residue in exactly 5 ml of 6M HCl and leave for 5 minutes on the hot plate. 3) Filter into a 50 ml flask using a Pasteur pipette. Wash the crucible using several portions of distilled water and quantitatively transfer the contents to the crucible. 4) Add 5 ml of 10% hydroxylamine solution to the flask and mix by gently rotating the flask. Adjust to volume with deionized water. C. Iron determination 1) Label five 10 ml test tubes for 0, 20, 40, 60, and 80 ppm of iron standards and two test tubes for ashed flour samples. 2) Add 2 ml each of iron standard and ashed flour sample solutions using a volumetric pipette. 3) Add 6 ml of bathophenanthroline-0.025%/sodium acetate-2m solution using a burette. Leave for 20 minutes. 4) Compare the color of the sample to that of the standards. V. Interpretation Report results in intervals of 0 to 20, 20 to 40, 40 to 60, or 60 to 80 ppm. If the intensity of the color exceeds that of the 80 ppm standard, dilute 5 ml of the sample solution with 5 ml of 0.96M HCl and mix. Repeat analysis with the diluted sample. If the color of the diluted sample falls in the 20 to 40 ppm interval, the actual concentration is 40 to 60 ppm of iron. VI. Notes The bathophenanthroline reagent can be replaced with α,α-dipyridyl (2,2 -bipyridine) [C10H8N2, PM , Fisher D-95] reagent. In this case, add 4 ml of the chromogen solution instead of 6 ml. the disadvantage to using the dipyridyl reagent is that slight differences in color are more difficult to distinguish. VII. Sampling frequency: once a day III. QUANTITATIVE METHODS A. Spectrophotometric method This method is approved by AACC for quantitative determinations of iron in cereals and cerealbased food products (See Annex 3.6). Principle Organic constituents in a food sample are broken down by dry or wet ashing at a high temperature and the inorganic constituents are dissolved in a mildly acidic solution. Solubilized ferrous iron is then reacted with a chromogenic agent, orthophenanthroline, in the presence of a reducing agent (such as a hydroxylamine hydrochloride), resulting in a pink-colored complex. The concentration of iron is determined by its Spectrophotometric absorbance at 510 nm. Advantages 1) It is applicable to various types of food products and ingredients. 156

157 2) It is a sensitive technique with a detection limit of less than 1 ppm (1 mcg/g or 1 mg/kg) of iron in sample. 3) It is relatively inexpensive compared with atomic absorption spectroscopy (AAS) or emission spectroscopy methods. 4) The ashed solution can be used for the determination of other inorganic elements. Limitations 1) It requires personnel trained to handle corrosive chemicals and to operate the spectrophotometer or colorimeter. 2) It is a time-consuming procedure involving overnight dry ashing. Samples with high protein content may take longer to be ashed. 3) It is relatively expensive involving costs of reagents, muffle furnace (approximately US$1.500), and spectrophotometer (between (US$8.500 and ; Annex 3.1). A fume hood (between US$3.500 and ) is needed for wet ashing of samples. Notes 1) It requires the preparation of an iron standard curve. 2) The reagents must be stored in the refrigerator. 3) Chromogens other than orthophenanthroline, such as α,α-dipyridyl, bathophenanthroline, and ferrozine, also react with ferrous iron and are widely used for iron determinations in various laboratories (See INCAP method VI, Annex 3.7). 4) Although dry ashing is recommended, wet ashing may also be used for Spectrophotometric iron determination. Wet ashing procedures are described in Appendices 3.9 (sample preparation) and Description of the method 1: Iron spectrophotometric method AACC method 40-41B Final approval May 5, 1960; reapproval November 3, 1999 This method determines iron content by reaction with orthophenanthroline and spectrophotometric measurement. It is applicable to cereals and cereal-based products. I. Materials Volumetric flask, 1 L Volumetric flasks, 250 ml Volumetric flasks, 100 ml Volumetric flasks, 25 ml Beakers, ml Manual volumetric pipettes, 1000 ml Watch glasses Pipette tips Graduated tubes Tips for blue pipettes Test tubes, 10 ml II. Equipment Muffle furnace capable of maintaining 550 C Platinum, silica, or porcelain crucible, approximately 60 mm diameter, 35 ml capacity. Porcelain evaporating dishes of about 25 ml capacity are satisfactory. Do not use flat-bottomed dishes of diameter greater than 60 mm. Spectrophotometer or colorimeter Eppendorf pipettes, 100 ml and 500 ml Analytical balance Vortex mixer 157

158 Hot plate III. Reagents 1) Orthophenanthroline solution. Dissolve 0.1 g orthophenanthroline in about 80 ml of water at 80 C, cool, and dilute to 100 ml. store in amber bottle in refrigerator. (Stable for up to several weeks). 2) Iron standard solution, 10 µg Fe/ml. a) Dissolve 0.1 g analytical grade Fe wire in 20 ml HCl and 50 ml water, and dilute to 1 liter. Dilute 100 ml of this solution to 1 liter; or b) Dissolve g Fe(NH4)2(SO4)2 6H2O in water, add 2 drops of HCl, and dilute to 500 ml. dilute 10 ml of this solution to 1 liter. 3) Hydroxylamine hydrochloride solution. Dissolve 10 g NH2OH HCl in water and dilute to 100 ml. store in amber bottle in refrigerator. (This solution is stable for several weeks). 4) Acetate buffer solution. Dissolve 8.3 g anhydrous sodium acetate (previously dried at 100 C) in water, add 12 ml acetic acid, and dilute to 100 ml. (It may be necessary to redistill the acetic acid and purify sodium acetate by recrystallization from water, depending on amount of Fe present). 5) Prepare working standards as follows: Place aliquots of the 10 µg/ml standard solution according to table below into 100 ml volumetric flasks, add 2 ml concentrated HCl to each, and dilute to volume. Aliquot of 10 µg/ml solution taken (ml) Final Fe concentration (ppm) Mix thoroughly by inverting flask times. Using 10 ml of each of these standard solutions, continue under procedure beginning with step 8. 6) Ashing aid a. Magnesium nitrate solution. Dissolve 50 g Mg(NO3)2 6H2O in water and dilute to 100 ml or b. Redistilled HNO3 IV. Procedure 1) Accurately weigh 2-10 g of sample (depending on concentration of iron expected) into clean crucible. (Begin to prepare blank solution at this point in same manner as sample). 2) Char on hot plate or under infrared lamp (optional). 3) Ash overnight in muffle furnace at 550 C. See Notes. 4) Remove crucible from furnace and cool to room temperature. 5) Carefully add 5 ml of concentrated HCl, letting acid rinse upper portion of crucible; evaporate to dryness on steam bath. 6) Dissolve residue by adding 2 ml concentrated HCl, accurately measured; cover with watch glass and heat 5 minutes on steam bath. 7) Rinse watch glass with water, filter quantitatively into 100 ml volumetric flask, dilute to volume, and mix thoroughly. 8) Pipet 10 ml aliquot into 25 ml volumetric flask, and add 1 ml hydroxylamine HCl solution. Mix thoroughly. 9) After 5 minutes, add 5 ml buffer solution and 1 ml orthophenanthroline; dilute to volume. Mix thoroughly. 158

159 10) Let stand 30 minutes, then measure absorbance of samples, standards, and blank solutions in spectrophotometer at 510 nm. Note: a. If color intensity too great, make appropriate dilution of ash solution and continue beginning at step 8. b. Color produced is permanent for several hours. Keep out of direct sunlight. V. Interpretation / Calculations 1) Plot absorbance versus concentration (in ppm) for standard solutions. 2) Obtain concentration of sample solutions from standard curve, subtracting blank value from each. 3) Iron content (mg/100 g) = (C x DF x 10) W where, C = concentration of sample solution (in ppm), DF = dilution factor (if any) from step 10, Note a, W = sample weight in grams. Notes To diminish ashing time or for samples that do not burn practically carbon-free, use one of the following ashing aids: Moisten ash with a) ml magnesium nitrate solution or b) redistilled HNO3. Dry contents and carefully ignite in muffle to prevent spattering. (A white ash with no carbon results in most cases). Do not add these ashing aids to self-rising flour (products containing NaCl) in platinum dish, because of vigorous action. VI. Sampling frequency: once a day Description of the method 2: Spectrophotometric analysis for quantitative determination of iron in foods INCAP method VI INCAP, Chemistry and Biochemistry Laboratory Revision No.3; April, 1997 To analyze inorganic iron in foods, the organic material is first combusted by ashing. The resultant ash is then solubilized and iron reduced to the ferrous form by adding hydroxylamine. The ferrous iron is determined spectrophotometrically after forming a colored complex with any of the following chromogens: α,α-dipyridyl (2,2 -bipyridine), bathophenanthroline (4,7-1,10- phenanthroline-diphenylsulfonic acid), or ferrozine (3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)- 1,2,4-triazine. The absorption maxima of the colored solution is 521 nm for dipyridyl, 535 nm for bathophenanthroline, and 562 nm for ferrozine. The reaction leading to the formation of color is sensitive to ph, thus the ph of the solution is maintained through the addition of 2M sodium acetate buffer. A. Materials Volumetric flask, 1 L Volumetric flasks, 250 ml Volumetric flasks, 100 ml Volumetric flasks, 25 ml Beakers, 250 ml Manual volumetric pipettes ( ml) Porcelain crucibles Watch glasses Pipette tips Graduated tubes Tips for blue pipettes Test tubes, 10 ml 159

160 B. Equipment Vortex mixer Analytical balance Spectrophotometer (521, 535, or 562 nm) Hot plate Muffle furnace Eppendorf pipette (100 and 500 ml) C. Reagents Sodium acetate (CH3COONa 3H2O), 99%, Fe<200 mg/kg, PM , Merck Art Hydrochloric acid (HCl), 37%, 1.19 g/ml, Fe<28 mg/ml, PM 36.46, Merck Art. 317 Α,α-dipyridyl (2,2 -bipyridine) (C10H8N2), PM Fisher D-95 OR Bathophenanthroline, 4,7-diphenyl-1,10-phenanthroline-disulfonic acid (C24H16N2O6S2), PM (free acid, anhydrate) 492.5, Sigma B-1375 Hydroxylamine hydrochloride (NH2OH HCl), PM 69.49, Baker 2196 Iron standards, choose from the following: Electrolytic iron, Merck 3810 or Baker 2234 Ferrous ammonium sulfate, (NH4)2Fe(SO4)2 6H2O, Merck 3792 Iron standard, Merck D. Procedure A. Preparation of solutions Hydrochloric acid (HCl), 6M: Add 200 ml deionized water to a 500 ml beaker. Slowly add 250 ml of concentrated HCl. Let it cool and transfer to a 500 ml volumetric flask and make the to volume with deionized water. Transfer to a glass flask and seal. This solution is stable indefinitely. HCl, 0.96M: Add 168 ml deionized water to a 500 ml beaker. Then slowly add 32 ml 6V HCl. Transfer to a glass flask and seal. This solution is stable indefinitely. Hydroxylamine hydrochloride, 10%: Add to a 500 ml beaker 50 g hydroxylamine hydrochloride, then add 400 ml deionized water. Stir with a glass rod. After it is completely dissolved, transfer to a 500 ml volumetric flask and make to volume with deionized water. Transfer to a glass flask and seal. This solution is stable indefinitely. Dipyridyl, 0.025% in sodium acetate, 2M: Add to a 500 ml beaker g sodium acetate trihydrate and 0.10 g dipyridyl. Add 400 ml deionized water. Dissolve completely using gentle heat if necessary. Make sure that bathophenanthroline is fully dissolved because it is poorly soluble at room temperature. Store in a glass or clear plastic flask. Discard solution if pink color develops as this indicates to contamination with iron. The solution is stable for 3 to 4 months. Bathophenanthroline, 0.025% in sodium acetate, 2M (Bathophenanthroline %/CH3COONa-2M): Add to a 500 ml beaker g sodium acetate trihydrate, and 0.10 g bathophenanthroline. Add 400 ml deionized water. Dissolve completely using gentle heat if necessary. Make sure that bathophenanthroline is fully dissolved because it is poorly soluble at room temperature. Store in a glass or clear plastic flask. Discard solution if pink color develops (indicates to contamination with iron). The solution is stable for 3 to 4 months. B. Preparation of iron standards Standard, 1000 ppm: Dilute reagent DILUT-IT according to manufacturer s instructions, in a 1 L flask, using deionized water. Alternatively, dissolve g Fe(NH4)2(SO4)2 6H2O in distilled water, add 2 drops of concentrated HCl and dilute to 500 ml. Standard, 10 ppm: Add to a 100 ml volumetric flask 1.0 ml (measured with a volumetric pipette) of 1000 ppm iron standard. Then add 16 ml of 6M HCl and make to volume with deionized water. Preparation of standards: Prepare standards in 100 ml volumetric flask. To make concentrations of 0.0, 0.3, 0.6, 1.2, 2.4, and 4.0 ppm, which are equivalent to 0, 7.5, 15, 30, 60, and 160

161 100 ppm (mg/kg) of iron in flour, add 8 ml 6M HCl to each flask, and then add a corresponding quantity of 10 ppm iron standard (see table below). Adjust to volume with deionized water. Store in dark glass flasks and cover with glass stopper. These standards are stable for 2 to 4 weeks. Real concentration in mg iron/l (ppm) Volume of 10 ppm iron standard to be added (ml) C. Ashing the sample 1) Accurately weigh out about 2 g of previously homogenized sample. Weigh samples in duplicates. Transfer sample to a porcelain crucible. 2) Ass sample in s muffle furnace at 500 C for 4 hours. The sample is adequately ashen when a white or grey ash obtained. Cool to room temperature. D. Solubilizing the ash 1) Add 5 ml of 6M HCl to the porcelain crucible allowing the acid to wash walls of the crucible and evaporate until dry on the hot plate, taking care that the sample does not splash outside the crucible at any time. 2) Dissolve the residue in exactly 5 ml of 6M HCl and leave for 5 minutes on the hot plate. 3) Filter into a 50 ml flask. Wash the crucible with several portions of distilled water, and quantitatively transfer the contents of the crucible. E. Iron Determination 1) Label, in duplicate, 10 ml test tubes for standards (0.0, 0.3, 0.6, 1.2, 2.4, 4.0, and up to 10 ppm), control, and samples. 2) Add to each corresponding tube 5 ml of standard, control, or sample. 3) Add 0.5 ml of 10% hydroxylamine solution. Vortex. 4) Add 4 ml of dipyridyl-0.025%/sodium acetate-2m or bathophenanthroline %/sodium acetate-2m solution. Vortex and leave for 20 minutes. 5) Read absorbance of the solution in each tube in a spectrophotometer at 521 nm for dipyridyl or 535 nm for bathophenanthroline. Adjust to zero using distilled water. F. Interpretation / Calculations 1) Plot concentration of iron in ppm (y) versus absorbance (x). 2) The concentration of iron can be calculated directly using a regression equation. To report the concentration of iron in mg of iron per kg of food, multiply the results obtained in ppm (mg/l) as: Iron (mg/kg) = conc.of iron (mg/l) x [(50 x 10-3 L) / (sample in kg)] Iron (mg/kg) = conc. (ppm) / weight (g) x 50 G. Notes 1) Make sure that all glassware are appropriate for mineral analysis. Reagents should of analytical grade with as low concentration of iron as possible. 2) Use only distilled and deionized water with a conductivity less than 2 msi/cm or 10-6 (ohm cm) -1. 3) When using the dipyridyl chromogen, it is critical to maintain the ph of the solution between 5 and 6. If necessary, add sodium acetate buffer solution. 161

162 Micro assay: If manual and Eppendorf pipettes are available, as well as a spectrophotometer capable of reading small cells, this method can be performed in the micro version. The calculations are similar to the macro method. 1) Identify, in duplicate, 10 ml tubes for standards (0.0, 0.3, 0.6, 1.2, 2.4, 4.0, and up to 100 ppm), control, and samples. 2) Add to each corresponding tube 1 ml of standard, control, or sample. 3) Add 0.1 ml 10% hydroxylamine solution and vortex. 4) Add 0.75 ml of dipyridyl-0.025%/sodium acetate-2m or bathophenanthroline %/sodium acetate-2m solution. Vortex and leave for 20 minutes. 5) Read the absorbance of the solution in each tube in a spectrophotometer at 521 nm for dipyridyl or 535 nm for bathophenanthroline. Adjust to zero using distilled water. H. Sampling frequency: Once a day NOTES: Management of a mill should keep in mind, that if one cannot measure it, one cannot control it. 1) Just because you can measure, it does not mean you have to. 2) Make all of such records available for inspection when required by the authorities who are responsible for monitoring the fortification program and in implementing inspection or monitoring for all fortified food products. 3) Codex CAC GL 50 recommends that the inspector samples from the square root of the number of packages, i.e., if a warehouse has 60,000 bags, then the inspector needs to take samples from 245 bags, combine them, mix thoroughly and sub-sample. 4) Taking a package from the packing line is not sampling. 5) Mill will have kept a small sample from each hours production and combined them inspector has the mandate to take a sample from there. References: 1. Кодекс РК «О здоровье народа и системе здравоохранения», Закона Республики Казахстан «О техническом регулировании» от 9 ноября 2004 года 603-II ЗРК. 2. Постановление Правительства Республики Казахстан от 19 января 2008 года 32 «Об утверждении Правил обогащения (фортификации) пищевой продукции, подлежащей санитарно-эпидемиологическому надзору». 3. Schlesinger H.I., and Van Valkenburgh H.B The structure of ferric thiocyanate and the thiocyanate test for iron. J.Am.Chem.Soc., 53: Alcock A.W., and Ediger N.J Influence of flour moisture on the Pekar test. Cereal Chem., 6: AOAC Official Methods of Analysis (1984), No , JAOAC 27: 86, 396, 1944; 28: 77, Andrews J.S., and Felt C. The iron content of cereals. Cereal Chem. 18:8 19.AOAC International Official Methods of Analysis of AOAC International, 16 th ed., 4 th rev. Method The Association. Gaithersburg, MD. 7. Howe M Report of the methods of analysis subcommittee on the determination of iron in cereal products. Cereal Chem, 21: AOAC Official Methods of Analysis (1984). No JAOAC 27: 86, 396, 1944; 28: 77, Measurement of iron status Report of the International Nutritional Anemia Consultative Group (INACG). 162

163 Annex 8. Second Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards Second Technical Meeting Regional Expert Group on Harmonization of Wheat Flour Fortification Standards Central Asian Republics, Afghanistan, Pakistan Agenda 6 September 2016, Almaty, Kazakhstan Rixos Hotel 163

164 Background The USAID/GAIN Afghanistan/Central Asia Regional Food Fortification Program ( ) aims to improve processes, regulations, and monitoring related to wheat flour and edible oil fortification, in order to address ongoing micronutrient deficiency in the region. The Program objectives include increasing supply of micronutrients to the region through fortification of wheat flour and edible oil, strengthening quality control and enforcement of fortification, and facilitation of harmonization of standards for fortification in Central Asia, Afghanistan and Pakistan. In October 2015, GAIN and USAID co-hosted a Regional Stakeholder s Meeting on Food Fortification that brought together representatives of government, private sector, academia, civil society, and development partners from Kazakhstan, Afghanistan, Pakistan, Tajikistan, and Uzbekistan. Several joint priorities were identified at the meeting. A Regional Expert Group on wheat flour fortification standards and potential for regional harmonization, chaired by the Kazakh Academy of Nutrition (KAN), was formed to facilitate regional cross-border trade in fortified wheat flour. The Expert Group included specialists representing national standards committees, government regulatory agencies, research institutions, wheat flour producers and respective business associations from 6 countries: Kazakhstan, Afghanistan, Pakistan, Tajikistan, Kyrgyzstan and Uzbekistan. During November 2015-January 2016, in consultation with international experts, the KAN members of the group developed a technical justification for harmonizing fortification standards for low extraction (refined, white) flour used in all countries of the region, and high extraction (whole) flour mainly used in Afghanistan and Pakistan. These two documents were shared with the Expert Group for comments and additional input. The documents highlight the need to ensure, at a minimum, adequate and comparable amounts of bioavailable micronutrients in all countries of the region as specified in the WHO recommendations. Consensus was reached on a unified premix composition to be used in the region at the Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan, which took place in Almaty, Kazakhstan, March 2016, with participation of all members of the Regional Expert Group and international experts. The following two options of premix have been proposed and adopted by the Technical Expert Group, which provide the following levels of micronutrients in fortified wheat flour (FWF): High extraction flour FWF, ppm Low extraction flour FWF, ppm Iron (NaFeEDTA) 15,0 Iron (NaFeEDTA) 15,0 Zinc (Zinc oxide) 30,0 Zinc (Zinc oxide) 30,0 Vit B9 (Folic acid) 1,0 Vit B9 (Folic acid) 1,0 Vit B12 (Cyancobalamin) 0,008 Vit B12 (Cyancobalamin) 0,004 Vit B1 (Thiamine) 2,0 Vit B2 (Riboflavin) 3,0 Vit B3 (Niacin) 10,0 In accordance with the recommendations of the Technical Meeting, the Kazakh Academy of Nutrition, in collaboration with international experts, prepared a separate scientific justification for the use of vitamin B12 for fortification of low extraction wheat flour in the dose of 0,004 ppm. Taking into account the recommendations of the Technical Meeting, the Kazakh Academy of Nutrition, in collaboration with the Union of Grain Processors of Kazakhstan, drafted the following two standards: 1) Standard of the Republic of Kazakhstan (draft) Low extraction fortified bakery wheat flour 2) Standard of the Republic of Kazakhstan (draft) High extraction fortified bakery wheat flour These documents were sent to all members of the Regional Expert Group on the Harmonization of Standards for use in the development of appropriate national standards in all participating countries. 164

165 At this meeting, it is expected that each country will present the draft national standards for wheat flour fortification, based on previous discussions regarding the use of a single formula of premix. Objectives: 1. To review and agree on the draft national standards for flour fortification, developed through the use of a single composition of the premix for flour fortification for all countries; 2. To reach agreement and propose a time frame for the adoption of national standards for flour fortification in the participating countries; 3. To review the draft document on quality assurance and quality control (internal and external) of flour fortification, as well as to consider formulation and possibilities for adoption of the document in the participating countries; 4. To present results of the wheat quality analysis in Kazakhstan; 5. To consider the possibility for establishing a system of inter-agency coordination of flour fortification in the participating countries; 6. To discuss the feasibility, timing, and further steps to promote food fortification and to facilitate imports and exports of fortified flour through the harmonization of standards in order to increase the consumption of micronutrients by population in the participating countries. Participants: The invited meeting participants include regional expert group members, international subject matter experts, and selected representatives of the donor and the implementing partner see the full list of invited participants in Annex 1. Agenda The Agenda of Technical Meeting is presented in Annex 2. Main Results 1. Experts from the countries-participants and a representative of the Ministry of Health and Medical Industry of Turkmenistan made presentations on the current status of flour fortification in respective countries. 2. Kazakhstan, the Kyrgyz Republic and Uzbekistan have reported on certain progress in the fortification of wheat flour. In Kazakhstan, premix for flour fortification is procured at the expense of mills, in the Kyrgyz Republic with support of the international donor organizations, and in Uzbekistan - through the creation of the revolving fund. In Kazakhstan and the Kyrgyz Republic, production of fortified wheat flour has slightly increased. In Uzbekistan, all flour of the first and supreme grades, produced by "Uzdonmahsulot", is being fortified, which accounts for about 50% of all flour manufactured in the country. 3. In Tajikistan, wheat flour has not being fortified yet, however, the Law of Tajikistan on mandatory fortification of wheat flour has been drafted. It is expected that the law will be adopted by the Parliament of Tajikistan in Turkmenistan premix for wheat flour fortification is supplied at the expense of the state budget, and 100% of the first and supreme grade wheat flour produced in the country are being fortified. 5. Experts from Afghanistan, the Kyrgyz Republic, Tajikistan and Uzbekistan have developed a draft of National standard for low extraction wheat flour fortification on the basis of the draft National standard for low extraction wheat flour fortification of the Republic of Kazakhstan, with the use of unified composition of the premix presented above. These drafts of National standards for low extraction wheat flour fortification will be submitted to the relevant national regulatory authorities 165

166 of countries-participants for consideration and adoption in The use of unified composition of the premix by all member-countries of the project ensures harmonization of standards for low extraction wheat flour fortification in these countries. 6. Experts from the Kyrgyz Republic have proposed not to use vitamin B12 as a part of a premix for low extraction wheat flour fortification. It is possible that the low extraction wheat flour fortification with vitamin B12 in the Kyrgyz Republic will be voluntary. 7. Experts from Afghanistan developed a draft National standard for high extraction wheat flour fortification on the basis of the draft National standard for high extraction wheat flour fortification of the Republic of Kazakhstan, with the use of unified composition of the premix presented above. The drafts of National standards for low and high extraction wheat flour fortification in Afghanistan will be submitted to the relevant national regulatory authorities of the country for consideration and adoption in Experts of the Kazakh Academy of Nutrition presented the draft of Guidelines on external and internal quality control of iron fortification of flour. Regional expert group reached consensus and approved the Draft as a basis for the use by experts from countries-participants to develop similar document in their own countries. 9. The participants discussed and approved the following further steps in promoting the adoption of harmonized standards and food fortification in the Region on the basis of regional consensus: - Adopt National standards for high and low extraction flour fortification by using appropriate unified composition of the premix by the national regulatory authorities of countries-participants. - Carry out a set of actions to ensure legislative grounds in order to guarantee the mandatory fortification of imported wheat flour. - Kazakh Academy of Nutrition: To develop a draft National standard "Vitamin-mineral premix for wheat flour fortification" and "Bread and bakery products fortified with vitamins and mineral supplements. General technical specifications", and to share these drafts with expert groups of member countries, in order to use them for development of relevant documents in all countries. - Develop and adopt in member countries Guidelines on external and internal quality control of iron fortification of flour on the basis of the appropriate document, developed by the Kazakh Academy of Nutrition. - Seek for sources of funding for further improvement of flour fortification agenda in the Region based on the harmonized standards. - Conduct Third Technical Meeting of the Regional expert group in March-April 2016 for reviewing the results of activities, lessons learned and the development of further steps in implementation of harmonized standards for fortification of food products into practice, as well as Guidelines on external and internal quality control of iron fortification of flour in the Central Asian Republics, Afghanistan and Pakistan. 166

167 Agenda: Annex 1 September 6, :40 9:00 Registration Morning session 1 - Country presentations on the current status of wheat flour fortification 9:00 9:10 Opening remarks. Technical workshop objectives. Toregeldy Sharmanov, President, Kazakh Academy of Nutrition (KAN) Moderator: Toregeldy Sharmanov, President, Kazakh Academy of Nutrition (KAN) 9:10 9:20 Welcome Alexander Lane, Kazakhstan Country Director, USAID/CAR Yuri Oksamitniy, UNICEF Representative in Kazakhstan Dora Panagides, Senior Manager, Large Scale Food Fortification, GAIN 9:20 9:30 Representative from Afghanistan Najibullah Safi, General Director, Public health, MOPH, Afghanistan 9:30 9:40 Representative from Kazakhstan Evgeniy Gan, President, Union of Grain of Kazakhstan, Maral Rakhimzhanova, Chief Expert for Health Surveillance of the Committee on Consumer Protection of the Republic of Kazakhstan 9:40 9:50 Representative from the Kyrgyz Republic Mamyrbaeva Tursun, Head of the Scientific Department of the Republic Scientific Medical Library, Ministry of Health, Kyrgyz Republic 9:50 10:00 Representative from Pakistan Khawaja Masood Ahmad, National Coordinator (National Fortification Alliance), Ministry of National Health Services Regulation & Coordination 10:00 10:10 Representative from Tajikistan Abdusalom Vokhidov, Deputy Director of the Republican Center of the Pediatrics and Pediatric Surgery 10:10 10:20 Representative from Uzbekistan Sherzod Atakhanov Head of Flour Fortification Program in Uzbekistan 10:20 10:30 Representative from Turkmenistan Annamurat Nazarov, Head of the Department of Science, Sanitation and Hygiene, Center of Public Health and Nutrition of SSES, Ministry of Health and Medical Industry 10:30 10:40 Discussion 10:40 11:00 Coffee break Morning session 2 Country presentations of draft national standards, developed using a harmonized formula of premix, for low extraction flour fortification Moderator: Quentin Johnson, Technical Adviser, GAIN 167

168 11:00 11:10 Feedback from Afghanistan Dr. Homayoun Ludin, Director, Public Nutrition Department, Ministry of Public Health 11:10-11:20 Feedback from Kazakhstan Yuriy Sinyavskiy, Vice President, KAN 11:20 11:30 Feedback from the Kyrgyz Republic Aytmurzaeva Dinara, Head of Standardization Management, Center for Standardization and Metrology of the Ministry of Economy, Kyrgyz Republic 11:30 11:40 Feedback from Pakistan Khalid Siddique Chaudhary, Director General, Pakistan Standards & Quality Control Authority (PSQCA), Ministry of Science & Technology, Government of Pakistan 11:40 11:50 Feedback from Tajikistan Abdusalom Vokhidov, Deputy Director of the Republican Center of the Pediatrics and Pediatric Surgery 11:50 12:00 Feedback from Uzbekistan Diloram Gafurova, Director, «Donmahsulatlary IIChM LLC» 12:00 12:40 Discussion 12:40 14:00 Lunch Afternoon session 1 Country presentations of draft national standards, developed using a single formula of premix for high extraction flour fortification Moderator: Shamil Tazhibayev, Vice President, KAN 14:00 14:10 Representative from Afghanistan Mohammad Wakil Rahimi, Food Standards Manager, Afghanistan National Standards Authority (ANSA) 14:10-14:20 Representative from Kazakhstan Yuriy Sinyavskiy, Vice President, KAN 14:20 14:30 Representative from Pakistan Khalid Siddique Chaudhry, Director General, Pakistan Standards & Quality Control Authority (PSQCA), Ministry of Science & Technology, Government of Pakistan 14:30 15:00 Discussion Afternoon session 2 Methodological recommendations on external and internal quality assurance and quality control (QA/QC) of flour fortification Moderator: Maral Rakhimzhanova, Chief Expert for Health Surveillance of the Committee on Consumer Protection of the RK 15:00 15:10 Overview of the methodological recommendations on external/internal QA/QC Baurzhan Amirov, Senior Researcher, KAN 168

169 15:10 15:20 Feedback from Uzbekistan Diloram Gafurova, Director, «Donmahsulatlary IIChM LLC» 15:20 15:30 Feedback from the Kyrgyz Republic Arykbaeva Bubudzhan, Head of the Department for prevention of diseases and the State Sanitary and Epidemiological Surveillance, Ministry of Health 15:30 16:00 Feedback from Afghanistan, Pakistan, Tajikistan; discussion Country representatives 16:00 16:20 Discussion 16:20 16:50 Coffee break Afternoon session 3 Review of wheat quality analysis results in Kazakhstan and recommendations on wheat quality assurance Moderator: Quentin Johnson, Technical Adviser, GAIN 16:50 17:00 Reporter Evgeniy Gan, President, Union of Grain of Kazakhstan 17:00 17:15 Discussion Afternoon session 4 Further steps in promoting the adoption of harmonized standards and food fortification in the region. Moderator: Dora Panagides, Senior Manager, Fortification Global Program, GAIN 17:15 17:30 Overview of processes and best practices on standards harmonization Quentin Johnson, Technical Adviser, GAIN Shamil Tazhibayev, Vice President, KAN 17:30-18:10 Discussion Comments and suggestions, representatives of countries and international organizations 18:10-18:20 Wrap-up Shamil Tazhibayev, Vice President, KAN Yuliya Beloslyudtseva, Kazakhstan Country Coordinator, GAIN 18:20 Closeout 19:00-21:00 Networking dinner 169

170 Annex 2 List of Participants for the Second Technical Meeting Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan Almaty, Kazakhstan, 6 September 2016 # Name Position Country 1 Mohammad Wakil Food Standards Manager, Food and Agricultural AFG Rahimi Products Standards Department, Afghanistan National Standards Authority (ANSA) 2 Mohammad Homayoun Director, Public Nutrition Department, Ministry of AFG Ludin Public Health 3 Najibullah Safi General Director Preventive Medicine, Ministry of AFG Public Health 4 Mohibullah Wahdati Country Director, GAIN AFG 5 Zhunus Yergaliyev Economic Advisor to the Ambassador, Kazakh AFG 6 Khawaja Masuood Ahmad 7 Khalid Siddique Chaudhary Embassy to Afghanistan National Coordinator (National Fortification Alliance), Ministry of National Health Services Regulation & Coordination, Pakistan Director General, Pakistan Standards & Quality Control Authority (PSQCA), Ministry of Science & Technology, Government of Pakistan PAK PAK 8 Faiz Rasool Senior Project Manager, Pakistan Regional Food PAK Fortification Project 9 Toregeldy Sharmanov President, Kazakh Academy of Nutrition KAZ 10 Shamil Tazhibayev Vice-President, Kazakh Academy of Nutrition KAZ 11 Yuri Sinyavskiy Vice-President, Kazakh Academy of Nutrition KAZ 12 Oksana Dolmatova Chief Researcher, Kazakh Academy of Nutrition KAZ 13 Artur Iralin Coordinator, Kazakh Academy of Nutrition KAZ 14 Ayan Yergaliyeva Leading Researcher, Kazakh Academy of Nutrition KAZ 15 Baurzhan Amirov Senior Researcher, Kazakh Academy of Nutrition KAZ 16 Evgeniy Gan President, Kazakhstan Union of Grain Processors KAZ 17 Maral Rakhimzhanova Chief Expert for Health Surveillance of the KAZ Committee on Consumer Protection of the RK 18 Yelena Kuleshova Head of Department on methodology, accounting KAZ and registration of documents on standardization and certification, RSE "Kazakhstani Institute of Standardization and Certification", Kazakhstan 19 Baurzhan Bayserkin Director, Department of medical care, Ministry of KAZ Health and Social Development, RK 20 Yuri Oksamitniy UNICEF Representative in Kazakhstan UNICEF/KAZ 21 Arykbaeva Bubudzhan Head of the Department for prevention of diseases KYR and the State Sanitary and Epidemiological Surveillance, Ministry of Health, Kyrgyz Republic 22 Mamyrbaeva Tursun Head of the Scientific Department of the Republic KYR Scientific Medical Library, Ministry of Health, Kyrgyz Republic 23 Aytmurzaeva Dinara Head of Standardization Management, Center for KYR Standardization and Metrology of the Ministry of Economy, Kyrgyz Republic 24 Alexander Shefner Millers Association, Kyrgyz Republic KYR 170

171 25 Damira Abakirova Health and Nutrition Officer, UNICEF, Kyrgyz Republic UNICEF/ KYR 26 Abdusalom Vokhidov Deputy Director of the Republican Center of the TAJ Pediatry and Pediatric Surgery, Tajikistan 27 Azamjon Toshov Senior Specialist, Food and Chemical Laboratory, TAJ State Center for Sanitary and Epidemiological Surveillance, Tajikistan 28 Mutriba Latypova Country Coordinator GAIN/TAJ 29 Sherzod Atakhanov Head of Flour Fortification Program in Uzbekistan UZB 30 Amankul Baikulov Consultant, Food Fortification Program in UZB Uzbekistan 31 Diloram Gafurova Director, «Donmahsulatlary IIChM LLC» UZB 32 John Riordan Director, USAID Uzbekistan Country Office USAID/UZB 33 Bahtiyor Mirzabaev Specialist, USAID Uzbekistan Country Office USAID/UZB 34 Annamurat Nazarov Head of the Department of Science, Sanitation and TUR Hygiene, Center of Public Health and Nutrition of SSES, Ministry of Health and Medical Industry, Turkmenistan 35 Dilara Ayazova Programme Assistant, UNICEF Turkmenistan UNICEF/TUR 36 Amirhossein Yarparvar Regional Health and Nutrition Specialist UNICEF/ Regional CAR 37 Alexander Lane Kazakhstan Country Director USAID/CAR 38 Dora Panagides Senior Manager, Fortification Global Program GAIN 39 Quentin Johnson Technical Adviser GAIN 40 Yuliya Beloslyudtseva Kazakhstan Country Coordinator GAIN/KAZ 41 Aliya Ilyassova Independent Economic Expert GAIN/KAZ 42 Raikhan Nysanbayeva Director, LLC NPO "BioFarmPreparaty" KAZ 171

172 Annex 3 MINUTES of the Second Technical Meeting of the Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan 6 September, 2016, Hotel Rixos, Almaty, Kazakhstan Participants: 42 participants from Central Asian Republics, Afghanistan, Pakistan, and International Organizations Welcome speech, Sharmanov T.Sh., Kaz: It s my privilege to start this meeting. First, I would like to commemorate President of Uzbekistan, Karimov. We have here reps of all experts from all countries in food fortification. USAID priorities: establish a regional group of experts under the leadership of KAN. Group represents associations, producers of wheat flour and experts from all countries. Specialists of KAN developed specifications for high and low extraction flour, according to WHO recommendations. The first meeting in March 14-15, there was reached a consensus on premix composition. Four ingredients for high extraction flour. KAN developed for low extraction flour vitamin content. All recommendation have been shared with all countries, it is suggested that all countries will present their standards at the meeting. Goal: to ensure qualitative fortification with uniform premix for all countries. Reach a consensus on terms of adoption of national standards and develop external and internal control methods and establish intersectoral commissions, and facilitate exports/imports by harmonization of standards. Participants asked to represent themselves and their countries. Let s start our meeting. I welcome all of you, I am very pleased to see high level experts from respective countries. We are experiencing longevity and improvement of quality of live. And we are in charge of the central point in development of our health of population through better nutrition and it is impossible to overestimate your role in this regard. Let s move to program. Lane, USAID/CAR: Welcome to all countries. USAID s important initiative.glad to see multistake group. GAIN and driving force KAN. We will continue to support. Oksamitniy, UNICEF Kaz: thank you. We are friends over 20 years. KAN became an important resource center, including in health of children. Hope this meeting will move ahead the harmonization sustainable development (UN) all countries working on location and adaptation of sustainability goals. And fortification is crucial in this regard. I visit often CAR countries and mother s health is crucial, and aneia and nutrition are very important. UNICEF will continue to collaborate with KAN, GAIN, and USAID. Amir Hussein will help UNICEF. Thank you Dora Panagides, GAIN: pleasure to welcome. Dedication and hard work of KAN would be impossible. Very important documents. Inputs of Quentin, GAIN. Acknowledge USAID and US people. Your participation demonstrates dedication of all countries. Uniform premix for all countries. Harmonization documents on website. Two formulations are agreed upon for two types of flour. Iron content. Back in your countries you should discuss and agree in your countries. We proposed harmonized standard, 50% countries hopefully will accept these standards. Sharmanov, KAN, Kaz: Kazakhstan is behind in fortification, despite Presidential Decree. Under the lead of MOHSD, while Ministry of Agriculture is an interested party. So asked Dr.Bayserkin. He headed SES, and it was transferred to MNE. I ask him to transfer this info to the Minister. Bayserkin, MOHSD, Kaz: I thank Sharmanov for invitation of MOHSD. It is pity that MOHSD is behind, by pushing of ShT this issue was raised. I thank USAID, they help for many years to Kazakhstan in fortification, listed all involved, KAN. Since January Pres endorsed Densaulyk program which includes issues of public health. He stressed the importance of intersectoral collaboration to promote health. One of examples is food fortification which affects health of population, and interests of neighboring countries. We have data on positive effect of fortification. Pres said to decrease maternal and infant mortality to levels of OECD. These goals are not achievable without the fortification programs. I wish good discussions and reach consensus and thank ny teacher ShT. 172

173 Sharmanov, KAN, Kaz: Next time MOHSD will report. Najibula Safi, Afg: GM to all. Important topic. A still continues to import food items. Malnutrition is a major. 40% of children. Iron deficit in 35% in women. Regulation drafted and submitted to Min Justice. Will be approved soon. Iron standard exists. Regional standard to address health. Assure our commitment to joint work. Gan Yevgeny, Kaz: we are getting more attractive, thank you. Fortification program is multifaceted. Important for women and children. And we thankful to USAID and GAIN is establishing a regional team, better understanding and trade. I wish to meet beyond this program as well. We have a lot of friends due to this program. And wish to contribute to furtherance of the program. Mamyrbayeva, Kyrg: Thank you. We also have intersectoral teams and programs and flour fortification is one projects uniting all sectors. Thank you. Presentation look the slides. Premix is procured through MOH and covers all, but the actual fortification is low. Reference data on benefit of fortification. Composition was approved in several stages, including the parliament. Premix is being paid by the WB grant. Premix will be purchased by producers, then the cost of flour may increase. Kazakh flour is cheaper than local. Therefore producers are not willing to fortify. No GOST regarding low/high extraction flour. Regarding WHO attitude to B12 fortification (no evidence). Reference data on B12 slide. We have access to animal products in Kyrgyzstan. Work group recommended to delay B12 fortification, so premix formula will not be changed. Zinc in premix does not affect Zn status, it shall be fortified separately. So, may be we should exclude Zn, while Fe and folic acid are very effective. So, inclusion of additional elements may endanger fortification programs. Sharmanov, KAN, Kaz: Kyrg is ahead of Kaz in fortification, due to more democracy which is very important for the people of Kyrg. Vokhidov, Taj: Thank KAN on development of normative base. Our work group made a good job, in development of laws. Parliament established an initiative work group for drafting Law on fortification. Experience of Taj with WB showed importance of appropriate laws. Listed reps of the work group (MPs). Law drafted and discussed by different groups in June conclusions on a two-month for drafting the Law. Hopefully soon the Law will be adopted. As for GOST, there is a separate presentation. Laws are very important otherwise producers will not follow just recommendations. Food safety coordination council, to be used as a platform of national alliance for food fortification. Atakhanov, Uzb: Thank you for condolences, and organizers of the meeting. Presentation on current situation, GAIN s prant since 2004 to implement National program of flour fortification. Accumulation funds from mills to promote fortification, since Next Law on prevention of micronutrient deficiency in 2010, since 2010 salt and flour must be fortified. In 2013 certain benefits for importation of fortified products, and then tax benefits for producers of fortified product. 50% local producers and 26% importation from Kazakhstan. High grade changed from 3% to 12%, so it is possible to consider fortification of this type in Uzb. Since 2005, flour fortification has increased from 75,000 to 1,516,000 tons in Nazarov, Turkmenistan: No GAIN in the country yet. Presentation. Law on certification 2013; Food safety and quality 2014; Public health care Law 2015; State program Saglyk (Health). Pres decree on Salt iodination and flour fortification with iron 1996; 2006 Folic acid fortification; National program of health nutrition 2013; Technical conditions for iron and folate fortification; External control instructions ; Mandatory fortification of higher and first grade flour; premix content, from Germany (sulfate); Since 2008 with UNICEF memorandum on purchase of premixes (funded by state); awarded for fortification. All four is fortified (20 high grade, 70% first grade). Role of KAN (Tazhibayev, Sharmanov) in establishing the fortification programs in Turkmenistan. Khawaja Ahmed, Pakistan: Since 2001, Nutrition dept under MOH; 2003 National plan to combat micronutrient deficiency; List of initiatives see the slide; NFA goals and objectives; NFA compositions and achievements see the slides; BREAK 173

174 Tazhibayev, KAN, Kaz: two months ago we sent out presentation instructions. However, nobody followed that s why we are late. I liked Uzb that 100% local production are fortified, that is, about 35-40%. Turkmenistan said about 90% are fortified. As for Kyrg, no available data, Kaz, 34% households consume fortified flour, comparable with Uzb. Kyrg: Over 60% imported, as for local only 12% fortified. Because premix is expensive. Quentin, GAIN: Pleasure to be back and meet old and new friends. Session on low extraction flour. Discussions after all presentations. Ludin, Afg: In June 2016 Ministry of PH proposed to assign technical fortification committee to finalize the adaptation of harmonized flour fortification. At second stage 6 members reviewed national standard and compared with KAN proposal. Members agreed upon 7 micronutrients proposed by KAN. B12 changed fro to ppm for low extract flour. Zinc reduced from 50 to 30; Rationale for differences see the slide. Standard will be implemented soon. Sinyavsky, KAN, Kaz: two standards have been developed, with participation KAN, MOHSD, MNE, Standardization committee. Quality requirements to grain for milling (LEF ) see the slide; permissible levels of vitamins and levels see the slide; toxins and pesticides MPC; instructions on organoleptic control; lab control by approved methods; labelling and packing; transportation and storage; nutritive value and then qualitative methods of iron determination; Aytmurzaeva, Kyrg: legislative base two laws; decree of government; state program of food security and nutrition. Standards international 26; 4 Russian Fed standards and 3 national standards of Kyrg; including for flour, premixes; two technical committees involved in development of standards; standards for bakery flour and general purpose flour. Composition and levels of use of premixes are not in standards, to be established by MOH; classification of flour is different, higher, first grade; safety requirements to fortified flour; three forms of assessment of compliance of flour quality: production control, declaration of compliance and state surveillance. Problems: premix production control; no test systems for premix quality control; reconsideration of three standards for introduction of water-soluble iron; no funding for reviewing the standards; Pakistan: challenges and constraints: four problems; national and inter/provincial differences in standards; food depts. In provinces develop their own regulations; since March stakeholders started discussions to harmonize on national and regional levels; NFA and others established core groups and scientific committee; after agreement of all stakeholders in 2016 in next quarter to finalize with KAN and WHO recommendations; by second quarter 2017 consensus will be reached with provinces and other. Vokhidov, Taj: we made the similar work; work groups, academics; draft of first grade flour; developed by state enterprise Galla (grain); with participation of other stakeholders; approved by MOHSD and transferred to the Agency of standardization; table of differences between national and KAN versions of premixes because of differences in nutrition pattern (more animal foods in KAZ/KYR); table of daily intake of micronutrients based on KAN recommendations; draft will be adopted by Tajik standard agency by mid-september. Gafurova, Uzb: Thank the developers of LEF standard. It is convenient with work a template. We have some differences; list of laws on micronutrients and fortification; national standard for bakery flour mandatory; regional draft also includes all these aspects; few differences just due to some technical requirements, requirements to content of metal magnetic admixes asked for comments; requirements to safety in line with national standards; the rest are identical; next table on national standard because of differences between Customs Union and national requirements; agree with micronutrient composition beside ongoing discussion on B12 and increased Zn; comments welcome on levels of vitamins; in conclusion national draft has the same sections as proposed see the slide. Procedures see the slide. Turkmenistan: Standards are identical; they will be reviewed and assessed in terms of further potential implementation Discussion: Afghanistan: few points we are different issue of fortification, safety and quality three things should be reflected; Fe and Zn mandatory, vitamins optional; we are not exporting, we are not changing our national standard; including cost issue producers cannot afford; Uzb exports to Afg; Quentin, GAIN: dealing with exportation, such matters to be discussed directly with Gan and Uzb rather than at the forum; 174

175 Gan, Kaz: to the beginning we discussed what we want to get in the end. I always I said no regional standard is achievable. They are recommendations for harmonization of national standards regardless whatever we may sign here. Because no universal standard can be achieved. But we can make maximally close to each other; Dora, GAIN: safety and quality they should be different level from fortification. Through other mechanisms. Proposed standard has a range, there is not just one figure that makes possible to harmonize. In the end of the day the exporter has to comply with regulations of the importer, including labeling, etc. Tazhibayev, KAN, Kaz: once more we talk about harmonized standard; we want the national standards to use unified premix composition then the national standards will be harmonized. All related to labelling, packages may differ but the unified premix addresses the harmonization. Kyrg raised the issue of B12 and later by Uzb. We analyzed our region, but there are no data available on B12. Based on nutrition pattern in Kazakhstan, because B12 is met only in animal products, and Kyrgyzstan and others the consumption of animal products declines across the region from Kaz to Pak. So proposed to leave B12 for Atta. B12 is used for fortification in a few countries. So, based on Kyrg proposal, we shall decide on B12 either refuse to fortify, or leave ppm for Atta. I do not believe that B12 and Zn fortification does not have any effect. Because many recommendations and meta-analyses do not reject Zn and B12 fortification. So we shall agree on level of B12, and leave Zn in fortification premixes. Turk: Any difference in levels of Zn in foods across the region. Tazhibayev, KAN, Kaz: Zn availability is very low from plant foods. Kyrg: I cited a systematic review which shows that Zn is not beneficial in premix composition. It should administered separately. And we cannot afford introduction of B12. KAZ continues exporting the non-fortified flour. B we cannot identify vitamin in premixes. Quentin, GAIN: I ask Kyrg provide information on Zn to Shamil. UNICEF: exciting debate; important to get evidence; USAID might help in generation of evidence; recommendation on knowledge and evidence generation; think about long-term effects; vitamin D is an example; there is no consent yet on composition of Zn and B12; keep balance between being too scientific and practical. LUNCH BREAK A/n session 1: Tazhibayev, KAN, Kaz: High extraction flour (HEF) never fortified in CAR but this issue is important for Afg and Pak, therefore KAN developed draft standard for HEF fortification Afg: table based on WHO; table comparing Afg and KAN standards; National FF committee agreed on 4 micronutrients; B important for Afg; marking and labeling requirements see the slides; Syniavsky, KAN, Kaz: definition and requirements to HEF; - organoleptic and physicalchemical characteristics; contamination permissible; quality requirements to HEF - see the slides; fortification requirements see the slides; toxins and contaminants for Customs Union countries; assessment of organoleptic properties GOST methods for content of vitamins, iron and zinc); labeling according to specifications; nutritive and energy value see the slide; and lab methods and reagents. Tazhibayev, KAN, Kaz: Afg seems to agree with the proposed. Pak: Our committees agreed B12 and Zn but it is not ratified yet. Hope this month. Dilorom, Uzb: metal magnetic admixture is it possible to use sieve method. Shefner, Kyr: magnetic check should be before the fortification. Just make references to methods used in CIS. Quentin, GAIN: choice of iron on WHO recom EDTA is recommended for HEF. Kind invitation very important on methods of ext/int quality control. Presentation of Amirov - comment of Ludin on importance of QA and QC for regional harmonization Dilorom, Uzb: thanks KAN and based on recommendations of KAN local guidelines developed. Information on ext/int control and regulatory documents and all the aspects, agencies involved; three agencies entitled to use sanctions; internal control by enterprises using spot tests; list of regulatory 175

176 documents see the slide; QC uses qualitative and semi-quantitative methods; we equipped all laboratories via GAIN; HCl availability is a barrier for small labs; training seminars across the country, methods and recording; samples of logs; thresholds of micronutrients for food safety developed in SanPint; external control two labs equipped AASP, HPLC chromatograph. Kyrg: How often ext control and when; total composition of premix or flour Uzb: at certification; not often; premix as entering control; ext/control till 2012 no regular checks; in 2012 we established laboratory monitoring so now quarterly checks; if two checks fail then total analysis at the central lab. Kyrg: quarterly check based on contract or public requirements. What about imported flour. Uzb: public requirement; imported flour is not fortified; that should be indicated in certificate. Gan, Kaz: how many labs in Uzdonmahsulot and about precursors; Dilorom, Uzb: UZDM have license for precursors; small entities do not have; spot test used; standard requires quantitative methods for decision making. Ludin, Afg: problem with using sophisticated equipment and sustainability; no proper training, HPLC expired in Afg. Dilorom, Uzb: we can afford and we have suppliers, state control in all regions total 38 labs across the country, accredited, equipped and have required supplies. Arykbayeva, Kyrg: in the frame of KAN recommendations; we have internal/mills; based on technical regulations: registration, logs, control of premix dose, tests (qualitative) and mills can contract accredited labs for quantitative; single body to control the fortification; external control according to laws on entrepreneurship, besides re-checking; all flour must be fortified higher and first grade; technical audit acceptable as documentary check; number of checks once a year, though it can be individualized depending on risk criteria up to two times; solutions see the slide. Adding: Shefter at production control is very important, fortification since 2005; in addition to logs we developed formula for EDTA a new method; but we need a new mthod, since we have small mills but the main issue use of HCl as precursor; producers are against; to avoid with UNICEF we developed determination in bread; to make a test-system to be used on market; why we started from bread because budget organizations must use fortified bread. We use domestic chemicals instead of HCl, we ask USAID and GAIN to help with development of uniform method for flour. Quentin, GAIN: we can discuss separately the method; Dora, GAIN: unfortified imported flour in both countries Uzb accepts, but Kyrg wants to make mandatory; why unfortified flour enters the country? Kyrg: according to Customs Union, priority to safe Gan, Kaz: Kazakh law says all flour must be fortified but understanding exports requirements if importer demand fortified. Kaz is ready to export all flour fortified if importers ask. Kyrg: who concludes contracts; Gan: between businesses. Damira, UNICEF, Kyrg: it is beneficial to harmonize; Gan: exports fell four-fold to Kyrg in last several years, so the domestic production must be looked at. Uzb: law on micronutrient deficit does not specify iron; but the decree says all flour produced in Uzb must be fortified; about 30-40% of flour is imported. It is impossible to introduce immediately. Tazhibayev, KAN, Kaz: if marketed means the imported too. Uzb: government decided that locally produced shall fall into this category; our technical regulations are close and it is quite acceptable to introduce such restrictions and gradual transition is good. Kyrg: despite two laws in KAZ non-fortified flour exported Ludin, Afg: internal control training provided and GAIN provided support to control unit; and training for millers; external monitoring through support of WFP; collect data from mills; no guidelines and capacity for quality control; working through GAIN support on checklist to start external control; just one main lab at MOH; a few small labs for iron testing spot test; minfin collects samples from suppliers and send to MOH; after regulations approved we will introduce both int/ext control; Pak: with support of GAIN at mills; we have to raise capacity of our external monitoring agencies; capacity building of lab; training for 40 mills; and link them with central lab; Taj: no fortification in Taj; in Nov a law will be adopted; currently a standard of 2004, and all documents at TajStandardr; GAIN helping in establishing ext/int control; Taj learning the experience 176

177 of other countries; currently a consultant is helping with quality control for salt iodination and fortification; Gan, Kaz: my impression int control is insufficient; semi-quant would suffice; only 10% have access to HCl and can use spot methods; others use weight method; we were told the method will be available for electrolyte iron; and I support Shefner s proposal; in external control not much problem; Dora, GAIN: QA/QC is very important; we are interested to work with you; some have systems established, some just starting; some packaging are misleading; not fortified or improperly fortified; Kyrg said about not cheating of consumers; example of VW; we need move forward with agenda. BREAK Gan, Kaz: sampling of grain and flour 5 regions; test protocols from Akmola lab see the slides; all indicators are below the MPCs; the same for flour; recommendations - first virgin land harvest was destroyed due to lack of cleaning and drying facilities; grain humidity now is 8-10%; producers were allowed to increase level of weeds; now it has been decreased; agricultural technology; do not store wet grain over 5 days; forage grain has high microbial contaminated; IV class grain will lead the sales this year. Uzb: do you use pesticides? Gan, Kaz: current technologies require less chemicals; background pollution is les now; Diloram, Uzb: IV class grain should be not less than 25%. Gan, Kaz: something is happening with grain; several years ago 23% was enough; now we use 25%; and we not use nitrogen fertilizers; this year we have higher protein but less gluten; at our mills yield is 73%; Quentin, GAIN: similar information available on Canada and US on web. Dora, GAIN: lot of work done on harmonization; next steps; issues on content of micronutrients; it is not end of journey; quick overview: Quentin, GAIN: best practices in food standards; see the slides; experience for the last 20 years and examples of good successes; elements needed for development of food standards see the slide; objectives of national food laws and standards; based on risk assessment by codex; or on independent, objective and scientific risk assessments; involve all stakeholders; info about codex (FAO/WHO); B12, Zn and others; consensus statements are being reviewed and to be published soon; regulatory based on range an minimum; consistency with codex standard structure; WTO challenging ill based standards. Tazhibayev, KAN, Kaz: rather successful work; teams supported the standards, except Pak (but do not oppose), several proposals from Kyrg about B12 and Zn; Quentin, GAIN and Dora, GAIN said about renewal of WHO recommendations and proposed to link with WHO groups working on B12 and Zn, also we will receive Mrs Mamyrbayeva s information; this work finishes in September, so our work on harmonization further will require funding; in March we raised this issue, and we asked USAID, GAIN and others and there is some hope for continuation till next July. Assuming the funding, first, drafts on LEF and HEF should be adopted till the end of year probably. Second, laws on mandatory fortification of imported flour. It would benefit the importing countries; third, normative documents premix for vitamin premixes; we can develop such document and share with other countries (Nov-March); national standard on bread and bakery products KAN can develop and share with other countries; standards for organization of premixes; then standard for bread and bakery products; again can be shared with other countries; and to hold 3 rd technical meeting, as today, in March-May 2017 and discuss achievements and make plans for future. In Turk in end of September a FortiMass training will be held; for monitoring flour fortification; to propose such methodology for other countries to ensure comparability of monitoring results; and concluding report by the end of July. Dora, GAIN: open floor for discussion; project will be extended through July 2017; third meeting is feasible; you should think what you need for adopting the proposed standards; we are in consensus, except one country; bring one specialist (WHO) on B12 and Zn it is recommended to include; composition-related Zn doubt, but not rule out now; Quentin, GAIN, Shamil may travel, if you feel benefit; UNICEF: consider financial aspects. Dora, GAIN: costing will be brought in the proposals; and its dependence on volumes; Cost considerations should include returns in future. Dora, GAIN: take in account nutrients; 177

178 Kyrg: once more about premix composition; B12 is not included; Zn is in the composition; EDTA iron welcome from exporters; today there is no evidence on benefit of B12, that s why we are raising this issue; B12 we cannot afford; because we cannot hide even single publication; folate must be, because 35% women are anemic; so we want to protect our program because only 12% are fortified. Any modification of regulations for businesses is very complicated. Dora, GAIN: thanks for clarification; please, do not start B12 process; UNICEF: if harmonization happens what next consequences for countries; Quentin, GAIN: outcome of NA agreement (Canada, US and Mexico); covered food fortification; various foods; as a result, much freer trade, lower cost of foods; a lot of flour across the countries; premix price dropped; example of French-speaking Union; increase in trade and reduction in cost; another example WHO Mediterranean region folic acid etc; it was beneficial for regions. Kyrg: what the regional project planning about ext/int control; two issues; Dora, GAIN: QA/QC document for Shamil to comment. Tazhibayev, KAN, Kaz: quality is very important; because small amount shall be distributed in a huge mass of product; to endure the delivery of declared amount of a fortificant; we discussed the proposed document and interesting experiences of countries; to ensure QA/QC equipment, some shortage of reagents, and proposals for a new test without HCl; another issue is eye-check equipment to be addressed somehow by manufacturers; and again the funding rises; because not only regulatory documents but funding too is important for meeting the demands of population. Sharmanov, KAN, Kaz: I ask your excuse for not attending all sessions today; I had a long meeting with deputy ministry of education and science; I am a little bit disappointed by low level of fortification (12%) in Kyrg, and doubts about B12 and Zn, though I always underline the leadership of Kyrgyzstan. I understand your problems, and parliamentarism as an example for us. Today we have discussions about dairy products, we are facing the problem that Russia produces fake dairy products. Info on transfats and other nutrients. About palm oil. Thanks to USAID and GAIN we are succeeding in food fortification. Now I am involved in anti-transfat project, with demonstration of new products without sugar. Dora, GAIN: Prof. Sharmanov you are a real nutrition champion. Tazhibayev, KAN, Kaz: I am glad we are moving ahead; all countries have a consensus on micronutrient composition despite some issues; progress in quality control; all are enthusiasts in fortification and harmonization; many international organizations participated in the meeting; glad to see Turkmen participants a leader in the region first started iodination and fortifies through the state almost 100% of flour (or about 90% of all flour); Uzb have established revolving fund and making great progress; grateful to our consultant; Prof. Omar Dari make a contribution to the program as well; grateful to Dora, GAIN Panagides, we always feel your support and interest in harmonization since the latter is beneficial for trade, flow of commodities. Dora, GAIN: thanks Shamil for support and drafts and incredible work; Yury was also instrumental; Quentin, GAIN, Gan,, also each and everybody. Country teams contributed a lot, in analysis, interest, in regional harmonization and promotion of regional trade, for all your work on presentations. We will work on your suggestions, and proposals and will prioritize and lastly thanks to Yulia for logistics and agenda, for everyday work. Welcome to Thursday session. 178

179 Annex 4 179

180 Annex 9. Suggestions on rationalization of nutrition for the Kazakhstan population for consideration and decision making at the level of National Coordination Council for Health at the Government of the Republic of Kazakhstan Suggestions on rationalization of nutrition for the Kazakhstan population for consideration and decision making at the level of National Coordination Council for Health at the Government of the Republic of Kazakhstan Introduction Nutrition is the key factor determining the public health. According to the World Health Organization (WHO), 60% of all causes of death are directly related to improper nutrition, and 75-80% of harmful environmental factors come to the human body with food products and water. It was calculated that rationalization of diets and nutrition can prevent 30-40% of cancer cases. The major portion of deaths and diseases is associated with such socially important noncommunicable diseases as cardiovascular disease, obesity, cancer, chronic respiratory diseases, diabetes and others, the main casual factors of which, according to unanimous opinion of experts, is the improper diet. Chronic non-communicable diseases take the leading place in the general mortality structure of the Kazakhstan population, and this determines the urgency of the problem. In 2014 in Kazakhstan 53% of adults had overweight or obesity, at this the obesity is spreading among children very quickly. In the Republic are still common the anemia, deficiency in iron and some vitamins, and this causes the significant damage to public health. 1. Prevention of micronutrient deficiency through food fortification in the Republic of Kazakhstan Deficiency of micronutrients (vitamins and minerals) like iron, zinc, folic acid and other B vitamins in diet become the most common and devastating shortage of food in the world. Its devastating effect on survival, development, health, mental capacity and productivity of human labor were widely confirmed. More than three billion people in the world cannot use their full potential as students, parents and workers due to micronutrient deficiencies in their diet. The World Health Organization (WHO) and other international institutions call the micronutrients deficiency the hidden hunger Prevalence of micronutrients deficiency in Kazakhstan The last national representative study [1] carried out by the Kazakh Academy of Nutrition (KAN) on the order of the Ministry of Health of the Republic of Kazakhstan in 2011 found out the high prevalence of deficiency in a number of micronutrients in Kazakhstan (table 1). The most common are the anemia and iron deficiency, and at this the proportion of iron deficiency anemia (IDA) is high, it means that the main anemia ratio falls on IDA. Anemia, including IDA, significantly affects the level of maternal and infant mortality. In addition, the anemia causes delays in physical and mental development and in cognitive skills of children. The reduced performance in school and at work connected with it is associated with losses in national economy of Kazakhstan estimated at more than a billion US Dollars annually [2]. Thus, the result of anemia and vitamin deficiency includes decreased work performance by 17% among manual workers, and by 5% among intellectual workers. 180

181 Table 1 The prevalence of micronutrients deficiency in Kazakhstan among children under 5 years, pregnant and non-pregnant women of childbearing age Index % of persons with micronutrients deficiency Children Pregnant women Non-pregnant women Anemia Iron deficiency Share of iron deficiency anemia Folic acid deficiency Vitamin A deficiency Iodine deficiency Folic acid deficiency (FAD) is the direct cause of congenital deformities, such as congenital anomaly of neural tube, the most severe form of which are anencephaly (no brain) and spina bifida (holorachischisis). Also is expressly indicated the direct connection of FAD and the high risk for atherosclerosis, coronary heart disease, hypertension, prostate cancer, breast and pancreas and large intestine. Folic acid and iron, as well as zinc, vitamins В1, В2 and niacin, are included into premix for wheat flour fortification in Kazakhstan, and mandatory wheat flour fortification could fundamentally solve the problem of vitamin A deficiency. Vitamin A deficiency. Close correlation between the incidence of vitamin A deficiency (VAD) and high infant mortality is well known, as well as those between the degree of predisposition to cancer and the amount of carotenoid (pro-vitamin A) and vitamin A (retinol) deficiency in diet. Incidence of VAD over 20% is considered a serious public health problem and requires urgent intervention to prevent the vitamin deficiency in accordance with recommendations of international organizations (WHO, UNICEF etc.). In Kazakhstan still there is no program for prevention of vitamin A deficiency. Iodine deficiency. In Kazakhstan is implemented the Law On prevention of iodine deficiency disorders of 2003, providing for mandatory salt fortification (iodized salt). In this connection, since 2006 till now over 90% of households currently consume the quality iodized salt, and since 1999 is achieved the three-fold reduction in incidence of iodine deficiency. In 2007 Kazakhstan was certified by international organizations as the country achieved universal salt iodization Salt iodization in Kazakhstan In 1999 the quality iodized salt was obtained by 29% of households, and the iodine deficiency was discovered by 54% of women of reproductive age. In 2002, the Resolution of the Government of the Republic of Kazakhstan On elimination and prevention of iodine deficiency disorders among the population of the Republic of Kazakhstan was issued. In 2003, the Law of RK On prevention of iodine deficiency disorders was issued. In 2006, the national average incidence of iodine deficiency among women of reproductive age was 15.9%. Consequently, achieved the three-fold reduction in iodine deficiency incidence since 1999 due to increased production and availability of iodized salt for the population. In 2007 Kazakhstan was certified as a country achieved the universal salt iodization. Thus, salt iodization is effective and economic efficient mean of preventing the iodine deficiency disorders in Kazakhstan. Costs for salt fortification with iodine are less than 5 KZT per capita per year Flour fortification is an efficient and economic efficient strategy for fight the micronutrients deficiency Why to fortify the flour? 181

182 Vitamins and minerals added to the cereal products, first of all, for prevention of anemia caused by nutrient deficiency and congenital defects of neural tube caused by folic acid deficiency. The nutrition improved by this way also strengthens the immune system and improves performance and cognitive development. Wheat and corn flour and rice fortification is successful because it makes the usual food more nutritious, without changes in eating habits of consumers. There are four main advantages of flour fortification: Prevention of nutritional anemia Prevention of congenital defects of neural tube Increased productivity Economic progress. Kazakhstan has also the following additional benefits connected with wheat flour fortification: Kazakhstan is a large, the world s first exporter of flour, and export of fortified flour is economically more advantageous than of non-fortified. The most significant consumers of Kazakhstan s flour are Afghanistan, Uzbekistan, Tajikistan and Kyrgyzstan. These countries need to purchase the fortified flour. The flour fortification is a prerequisite for humanitarian purchases of Kazakh flour by international organizations. The legislation and implementation of technical standards for mandatory flour fortification do not conflict with international regulations and WTO principles of free trade, provided that this legislation and regulations are applied equally both to local goods and to imported ones. About 70% of national consumption of flour is covered by a relatively centralized production, and plants have the technical capacity to implement the flour fortification process with micronutrients. Flour fortification within the nutrition strategy of countries is supported by global organizations such as WHO, UNICEF, US Center for Disease Control and Prevention (CDC), Global Alliance for Improving Nutrition (GAIN) and Micronutrients Initiative (MI) The effectiveness of flour fortification to fight anemia and micronutrient deficiencies The table 2 shows the information of wheat flour fortification and anemia incidence among the high-risk groups in some countries compared to Kazakhstan, and the table 3 contains information of micronutrients level and sources of iron to be added to fortified flour. 182

183 Table 2 Efficiency of mandatory wheat flour fortification program (WFF) and anemia incidence (in %) among children, pregnant and non-pregnant women of reproductive age, the comparative data for certain countries Country (WFF) Anemia by [3]: NTD, Production, % Consuming, % Children, % Pregnant women,% Nonpregnant women, % per infants USA [4] ,1 12,2 4,61 Canada [5] England [6] Brasilia [7] Argentina [8] Kazakhstan [9-10] ,2 43, Note: NTD Congenital neural tube defects Several reviews of literature [11-13], as well as presented data demonstrate the effectiveness of wheat flour fortification in reducing the anemia incidence, and the success depends on duration of coverage of population with this flour. Moreover, the effect depends on duration of program implementation and consumption of fortified flour. The systematic analysis of results of 185 randomized controlled researches that include fortification of food products and bio-fortification with iron disclose the decrease in anemia incidence by 41% and iron deficiency by 52% [14]. Similar results are found in another large systematic meta-analysis of 201 researches of flour fortification effectiveness: decrease in anemia incidence was found by 45% by children and by 32% by women [15]. The systematic meta-analyze of 18 randomized researches covered 5,142 children under 10 years of age who has received the food products fortified with iron from 6 to 12 months allowed revealing the increase in concentration of hemoglobin in blood by 5.09 g/l [16]. In general, the anemia level decreases slowly enough [17-18]. The flour fortification with iron can improve the iron status of the population subject to three basic conditions [19]: 1) the program is well implemented and monitored; 2) provision and consumption of fortified flour are optimized; 3) the iron compounds are added in recommended concentration. As we can see in the above data, the greatest progress and effect of flour fortification and prevention of micronutrient deficiency are achieved in the United States. Mandatory flour fortification in USA is being performed since 1938, which allowed reducing significantly the iron and folic acid deficiency each year. It prevents more than 5,000 cerebral accidents, 25,000 heart attacks and 100,000 cases of iron deficiency anemia, and this respectively reduces the risk of death and increases the length and quality of life Global progress in flour fortification In the world 85 countries have legislation on mandatory wheat flour fortification, and 14 countries on fortification of corn products, 6 countries on rice fortification. United States of America and Costa Rica are the only countries where all three types of cereals are fortified. Papua New Guinea is the only country fortifying only rice. All 85 countries fortify the wheat flour at least with iron and folic acid, except for Australia that does not include iron, and for Congo, Venezuela, the United Kingdom and Philippines that do not add the folic acid. In 2014 [20] about 30% of world industrial production of wheat flour, 48% of corn flour and 1% of industrial production of milled rice are fortified with at least iron and/or folic acid. 183

184 Flour fortification status in Kazakhstan compared to other countries of Central Asia The wheat flour fortification in Kazakhstan, Kyrgyz Republic, Mongolia, Tajikistan and Uzbekistan started to be implemented successfully in within the international program of Japanese Fund for Poverty Reduction (JFPR), Asian Development Bank (ADB) and UNICEF. The Kazakh Academy of Nutrition (KAN) held scientific support for this program, developed the premix, standards and other regulations for flour fortification that were accepted by all member countries. The legislation on mandatory fortification of wheat flour of first grade and extra currently have all countries of Central Asia except for Tajikistan. Turkmenistan and Uzbekistan have reached almost 100% of fortification of flour of first grade and extra manufactured in these countries. In Kyrgyz Republic more than 50% of flour of first grade and extra is fortified. In Kazakhstan about 25% of flour of first grade and extra being sold in the country is fortified Premix for flour fortification and its procurement in Kazakhstan compared to other countries of Central Asia In Turkmenistan the premix for flour fortification is purchased for many years entirely by the state budget. In Uzbekistan the premix for flour fortification is also purchased entirely by the revolving fund established with participation of the state budget and international projects. In Kyrgyzstan since 2009 as the iron source in premix for flour fortification used NaFeEDTA instead of electrolytic iron. Bioavailability of iron is significantly higher when used a more expensive iron source of form of NaFeEDTA. In Kazakhstan is used the electrolytic iron, which is the cheapest iron source, but availability of the electrolytic iron is significantly lower than from NaFeEDTA. Kazakhstan should consider the possibility to purchase the premix for flour fortification by the state budget, as well as the possibility to replace the iron source in premix to NaFeEDTA Cost and economic efficiency of flour fortification in Kazakhstan Fortification offers an inexpensive and effective way to use the mass food products for impressive improvement of health, and the fortification cost in Kazakhstan is about 2 USD per ton of wheat flour or about 70 KZT/year per capita (at the exchange rate of KZT in February 2016; wheat flour consumption in Kazakhstan is 95 kg/year per capita) [2]. According to forecasts of the World Bank experts, 10-year net profit of Kazakhstan only from wheat flour fortification with iron and folic acid (calculated in 2004) will be 155 million US Dollars due to reducing losses in productivity because of iron and folic acid deficiency, as well as due to prevented deaths from congenital anomalies, reduction in mortality associated with iron and folic acid deficiency [2] Kazakhstan s possibilities in flour fortification The production capacity of mills in Kazakhstan is 9,550,256 МТ of flour per year; at this, they use only 40% of capacities, and the average annual production is about 2 million MT of flour for domestic consumption and about the same for export. In 2014, 100 of 390 mills have fortified the flour (a part of mills operate irregularly), although the flour fortification equipment is installed on most facilities [21]. That is, most of the mills are equipped properly and have experience in production of fortified wheat flour. In the Republic is created the entire necessary legal and regulatory framework for implementation of mandatory flour fortification [22]. Mills potential allows fortification all the flour of first grade and extra both for domestic consumption and export [23]. 184

185 Harmonization of standards for wheat flour fortification in Central Asian Republics, Afghanistan and Pakistan Kazakhstan holds a leading position in the world in the export of wheat flour, which is carried out mainly in the Central Asian Republics (CARs) and Afghanistan. Pakistan also exports flour to Afghanistan. With technical support from the US Agency for International Development (USAID) and the Global Alliance for Improved Nutrition (GAIN a Regional Expert Group on wheat flour fortification standards and potential for regional harmonization, chaired by the Kazakh Academy of Nutrition (KAN), was formed ) in October The Expert Group included specialists representing national standards committees, government regulatory agencies, research institutions, wheat flour producers and respective business associations from 6 countries: Kazakhstan, Afghanistan, Pakistan, Tajikistan, Kyrgyzstan and Uzbekistan. Harmonisation of standards will facilitate regional cross-border trade of fortified wheat flour. In consultation with international experts, the KAN members of the group developed a technical justification for harmonizing fortification standards for low extraction (refined, white) flour used in all countries of the region, and high extraction (whole) flour mainly used in Afghanistan and Pakistan. Consensus was reached on a unified premix composition to be used in the region at the Technical Meeting of Regional Expert Group on Harmonization of Wheat Flour Fortification Standards in Central Asian Republics, Afghanistan and Pakistan, which took place in Almaty, Kazakhstan, March 2016, with participation of all members of the Regional Expert Group and international experts. The following two options of premix have been proposed and adopted by the Technical Expert Group, which provide the following levels of micronutrients in fortified wheat flour (FWF), which comply with international recommendations [24]: High extraction flour FWF, ppm Low extraction flour FWF, ppm Iron (NaFeEDTA) 15,0 Iron (NaFeEDTA) 15,0 Zinc (Zinc oxide) 30,0 Zinc (Zinc oxide) 30,0 Vit B9 (Folic acid) 1,0 Vit B9 (Folic acid) 1,0 Vit B12 (Cyancobalamin) 0,008 Vit B12 (Cyancobalamin) 0,004 Vit B1 (Thiamine) 2,0 Vit B2 (Riboflavin) 3,0 Vit B3 (Niacin) 10,0 Taking into account the recommendations of the Technical Meeting, the Kazakh Academy of Nutrition, in collaboration with the Union of Grain Processors of Kazakhstan, drafted the following two standards: 1) Standard of the Republic of Kazakhstan (draft) Low extraction fortified bakery wheat flour 2) Standard of the Republic of Kazakhstan (draft) High extraction fortified bakery wheat flour On the basis of these documents similar drafts of national standards for all member countries of the program have been developed using a single formula premix for flour fortification, which was discussed and approved at the Second Technical Meeting in Almaty on 5-6 September The proposed Action Plan for the improvement and increase the efficiency of activities on fortification of the first and supreme grades of wheat flour in the Republic of Kazakhstan The proposed Action Plan to support the fortification of food products, for consideration by the Coordination Council under the Government of the Republic of Kazakhstan. 1) Arrange inter-agency coordination coordination and cooperation of all key partners on flour fortification and salt iodization. 2) Develop mechanisms for government subsidies to purchase premix for flour fortification. 185

186 3) Ensure purchasing of premix for flour fortification by state budget, followed by establishment of a revolving fund for centralized purchasing of premix by mills from trusted sources recommended by international organizations. 4) Ensure the state procurement of flour and salt only of fortified flour and iodized salt. 5) Provide baking bread from fortified flour, including by increasing the bakers demand for fortified flour. 6) Achieve the stability of flour fortification program. 7) Realize in full the clauses of the Law of RK On mandatory flour fortification sold in the territory of the Republic of Kazakhstan. 8) Achieve in the coming years 90% fortification of wheat flour of first grade and extra. 9) Include the production volume of fortified food products (fortified flour, iodized salt etc.) to the list of state statistical reporting. 10) Revision and development of the relevant legal acts and food fortification guidelines, as well as harmonization of standards with the countries with which is performed the import/export of fortified food products. 11) Improve the work on quality control and assurance of flour fortification and salt iodization: Cover all mills, bakeries and salt production enterprises. Ensure regularity Arrange the quarterly and annual reporting Distribute reports to all partners and media 12) Strengthen the efforts to raise awareness of population about food products fortification: The importance for prevention of anemia, micronutrients deficiency and iodine deficiency disorders. The importance for normal growth, development, and increase in working capacity, prevention of diseases. When buying the flour to pay attention to the packaging and logo on food fortification. Prefer the fortified wheat flour and iodized salt when buying food products. 13) To conduct the following activities regularly: Monitoring of fortification and availability of fortified foods in the market and at the household level. Bio-monitoring of anemia, deficiency of iron, folic acid, vitamin A and iodine. 3. Reducing the consumption of unhealthy foods and beverages Non-communicable diseases (NCD) represent a leading threat to human health and socioeconomic development they cause more than 80% of deaths in Kazakhstan. Unhealthy food is the most important changeable risk factor of NCD. In our Republic 53% of people aged 15 years and older have overweight (31%) or obesity (22%). Today, all over the world the overweight and obesity are the fifth leading risk of death. Food and drinks rich in calories, simple sugars (sweetness), salt, cholesterol, trans-fat and saturated fatty acids but poor in useful nutrients (vitamins, minerals, dietary fiber, essential fatty acids etc.) play an important role in development and high prevalence of non-communicable diseases (NCD) and obesity. Active marketing of many of these products and especially products having a high content of fat, sugar or salt, challenge efforts to adhere to a healthy diet and maintain healthy weight. In this regard, many countries are moving towards the adoption of various restrictive measures helping to reduce the consumption of unhealthy foods and beverages. Also used incentives aimed at replacing the unhealthy foods and beverages to healthy ones. Proposals to reduce the consumption of unhealthy foods and drinks including acceptance of restrictive measures in the form of excises etc. will be prepared to the second meeting of the Coordination Council. 4. On establishing the Working group for healthy nutrition in the Coordination Council 186

187 Solution of urgent issues concerning rationalization of nutrition aimed at health improvement of population, including in the field of food products fortification (fortification of flour, vegetable oils and other food with micronutrients, salt iodization etc.) is connected with coordination of relevant activities of many ministries, departments and institutions. This coordination could be carried out by Coordination Council by establishing a permanent Working group on healthy nutrition. The Working group could monitor the implementation of Coordination Council decisions on the healthy nutrition issues and prepare appropriate materials for the Coordination Council. List literature 1. Final report of the Kazakh Academy of Nutrition on «Organization of permanent biomonitoring of availability of iodine, iron, folic acid and vitamin A for target groups of population in all regions of the country and cities Astana and Almaty», 2011, 99 p. 2. A preliminary proposal of flour fortification in Kazakhstan: Analyze of economic efficiency and acceptability, strategy and budget for flour fortification in Kazakhstan. Jack Bagryansky, UNICEF Consultant , Decatur, USA 3. The global prevalence of anemia in WHO, 2015, 48 p Final report of the Kazakh Academy of Nutrition on «Organization of permanent biomonitoring of availability of iodine, iron, folic acid and vitamin A for target groups of population in all regions of the country and cities Astana and Almaty», 2011, 99 p Tarun Gera, Harshpal Singh Sachdev, and Erick Boy. Effect of iron fortified foods on hematologic and biological outcomes: systematic review of randomized controlled trials. Am J Clin Nutr August 2012 vol. 96 no Ramesh Athe, M Vishnu Vardhana Rao1, and K Madhavan Nair. Impact of iron-fortified foods on Hb concentration in children (10 years): a systematic review and meta-analysis of randomized controlled trials. Public Health Nutrition: 2013, 17(3), a.pdf&code=5a162a0908a9711f6ca4bcf617ba24df 13. Jai K Das, Rehana A Salam, Rohail Kumar and Zulfiqar A Bhutta. Micronutrient fortification of food and its impact on woman and child health: a systematic review. Systematic Reviews 2013, 2: Tarun Gera, Harshpal Singh Sachdev, and Erick Boy. Effect of iron fortified foods on hematologic and biological outcomes: systematic review of randomized controlled trials. Am J Clin Nutr August 2012 vol. 96 no Jai K Das, Rehana A Salam, Rohail Kumar and Zulfiqar A Bhutta. Micronutrient fortification of food and its impact on woman and child health: a systematic review. Systematic Reviews 2013, 2:67 doi: / Ramesh Athe M Vishnu Vardhana Rao, and K Madhavan Nair. Impact of iron-fortified foods on Hb concentration in children (,10 years): a systematic review and meta-analysis of randomized controlled trials. Review Article. Public Health Nutrition, 2013, 17(3), AnnieRose HarrisonDunn. How effective is fortifying flour at reducing anaemia? Breaking News on Supplements & Nutrition Europe, Helena Pacho n, Rebecca Spohrer, Zuguo Mei, and Mary K. Serdula. Evidence of the effectiveness of flour fortification programs on iron status and anemia: a systematic review. Nutrition ReviewsVR, 2015, Vol. 0(0):

188 19. Iron Fortification Programs And Iron Status. Food Fortification Initiative, FFI-2014-YearInReview16p. #AnnualReport 21. Information of State Sanitary Inspection for Law of RK «The Code of the Republic of Kazakhstan On people s health and healthcare system 193-IV 3RK dd. September 18, Information of the Union of grain processors and millers of Kazakhstan 24. WHO, FAO, UNICEF, GAIN, MI, & FFI. Recommendations on wheat and maize flour fortification. Meeting Report: Interim Consensus Statement. Geneva, World Health Organization,

189 Draft Working Group for healthy nutrition in Coordination Council RK 1. Chairman of the Council Tzoi A.V., Vice-minister of Health and social development 2. Assistant of the Chairman 3. Secretary to the Chairman 4. Vice-chairman 5. Representatives of the Parliament of RK 6. Representatives of the Ministry of Agriculture of RK 7. Representatives of the Ministry of National Economy of RK 8. Representatives of the Ministry of Finance of RK 9. Representatives of the Ministry of Education and Science of RK 10. Representatives of the Ministry of Culture and Sport of RK 11. Representatives of the Ministry of Investments and Development of RK 12. Representatives of the Association of Millers of Kazakhstan 13. Representatives of the Association of Bakers of Kazakhstan 14. Representatives of the Consumer Protection Committee 15. Representatives of the Sanitary and Epidemiological Supervision of RK 16. Representatives of the Chamber of Entrepreneurs 17. Representatives of Kazakh Academy of Nutrition 18. Representatives of NGOs 19. Representatives of international organizations: GAIN, USAID, WHO, UNICEF etc. 189

190 Annex 10. Booklet in Russian. Advantages and benefits of wheat flour fortification. 190

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194 Annex 11. Booklet in Kazakh. Advantages and benefits of wheat flour fortification. 194

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198 Annex 12. Leaflet in Russian. Advantages and benefits of wheat flour fortification. 198

199 Annex 13. Leaflet in Kazakh. Advantages and benefits of wheat flour fortification. 199