TECHNICAL REPORT INTERNATIONAL SPONSORED R & D PROJECT IDENTIFYING INCIDENCE, LEVEL AND SOURCE OF AFLATOXIN IN PAKISTANI WHEAT AND WHEAT FLOUR

Transcription

1 TECHNICAL REPORT INTERNATIONAL SPONSORED R & D PROJECT IDENTIFYING INCIDENCE, LEVEL AND SOURCE OF AFLATOXIN IN PAKISTANI WHEAT AND WHEAT FLOUR By DR. IJAZ AHMAD Principal Scientific Officer (PSO)/ Principal Investigator/ Team Leader and DR. QURATUL AIN SYED CSO/ Director General PCSIR/ Deputy Team Leader December 2017 Budget: Rs Millions FINNANCIAL SUPPORT FROM GLOBAL ALLIANCE FOR IMPROVED NUTRITION (GAIN) FOOD AND BIOTECHNOLOGY RESEARCH CENTRE PCSIR LABORATORIES COMPLEX, LAHORE, Pakistan

2 IDENTIFYING INCIDENCE, LEVEL AND SOURCE OF AFLATOXIN IN PAKISTANI WHEAT AND WHEAT FLOUR Written by: DR. IJAZ AHMAD Principal Scientific Officer (PSO)/ Principal Investigator/ Team Leader PCSIR Laboratories Complex, Lahore, Pakistan DR. QURATUL AIN SYED CSO/ Director General PCSIR/ Deputy Team Leader PCSIR Laboratories Complex, Lahore, Pakistan Reviewed by: DAVID MORGAN Senior Technical Specialist - Quality & Safety GAIN - Global Alliance for Improved Nutrition 1st Floor, Churchill House, 142/146 Old Street, London EC1V 9BW CH. FAIZ RASOOL Senior Program Manager Global Alliance for Improved Nutrition (GAIN), Islamabad-Pakistan

3 ACKNOWLEDGEMENTS We are cordially thankful to Pakistan Flour Mill Association, who helped to make this project successful. Moreover, we appreciate the leadership role of Global Alliance for Improved Nutrition (GAIN), who is always in front line to make this type of projects successful. We are also thankful to Food Department for their kind facilitation for sampling. At the end I am cordially grateful to administration of PCSIR who helped to complete this project. DR. IJAZ AHMAD Principal Scientific Officer (PSO) PCSIR Laboratories Complex, Lahore

4 TABLE OF CONTENTS S. No. Contents Page No. Acknowledgement Table of contents List of Tables iii iv v List of Figures 1 Introduction Aflatoxin Testing Services by PCSIR Recognition of PCSIR 3 2 Objectives 3 3 Literature review and technical parameters 4 4 Project team 5 5 Materials and methods Selection of Target area Collection of samples Moisture Content Physical Tests Aflatoxin (B1, B2, G1, G2) Analysis Data ownership by GAIN & PCSIR Statistical analysis 8 6 Results and discussion Storage conditions Moisture contents Physical tests Aflatoxin (B1, B2, G1, G2) Analysis Validation of Aflatoxin study 37 7 Summary 39 8 References 41 9 Annexure Vi

5 LIST OF TABLES S. No. Contents Page No. 1 Summary of collected samples. 7 2 Analysis of wheat grain samples from Godowns of Punjab Analysis of wheat grain and wheat flour samples from Flour Mills of Punjab Moisture contents in wheat flour samples from markets of Punjab Analysis of wheat grain samples from Godowns of KPK Analysis of wheat grain and wheat flour samples from Flour Mills of KPK Moisture contents in wheat flour samples from markets of KPK Analysis of wheat grain samples from Godowns of Baluchistan Analysis of wheat grain and wheat flour samples from Flour Mills of Baluchistan Moisture contents in wheat flour samples from markets of Baluchistan Analysis of wheat grain samples from Sindh Moisture contents in wheat flour samples from Flour Mills of Sindh Moisture contents in wheat flour samples from markets of Sindh Analysis of variance (mean squares) (Punjab, KPK, Baluchistan and Pakistan) Moisture and physical analysis of wheat grains and wheat flours (Pakistan) Comparison of means with respect to different areas and location. x are mean ±SE for Moisture (%). 17 Comparison of means with respect to different areas and location (Punjab and KPK). x are mean ±SE for physical tests of wheat grains. 18 Comparison of means with respect to different areas and location (Baluchistan and Sindh). x are mean ±SE for physical tests of wheat grains

6 LIST OF FIGURES S. No. Contents Page No. 1 a. Moisture (%) Punjab b. Moisture (%) Punjab (Region wise, Box Plot) Moisture (%) KPK Moisture (%) Baluchistan. Moisture (%) Sindh. 5 a. Physical Tests of samples from Punjab (District wise and region wise). b. Physical Tests of samples from Punjab (District wise, Box Plot). c. Physical Tests of samples from Punjab (Region wise), Box Plot. 6 a. Physical Tests of samples from KPK. b. Physical Tests of samples from KPK (Box Plot) Physical Tests of samples from Baluchistan (Box Plot) Physical Tests samples from Sindh (Box Plot) a. Moisture (%) and physical parameters of samples from Pakistan (Province wise). b. Moisture (%) and physical parameters of samples from Pakistan (Province wise, Box Plot)

7 1. INTRODUCTION The quality assurance of wheat grains and wheat flour is of prime importance. It is helpful for the production of good quality wheat flour. The Government of Pakistan has shown a strong commitment to enhance the export of wheat flour. GAIN is also supporting export of wheat flour from Pakistan to Afghanistan. Food and Biotechnology Research Centre (FBRC), PCSIR Laboratories Complex, Lahore is equipped to conduct quality analysis including aflatoxin tests on different foods and food products. This project is undertaken to evaluate the quality of wheat grains and flour in Punjab, Khyber Pakhtunkhwa (KPK), Baluchistan and Sindh. The project addresses a very sensitive issue i.e. mycotoxins which are considered to be one of the most important contaminants in foods and feeds. Mycotoxins are toxic secondary metabolites produced by molds (fungi) that may contaminate agricultural commodities. According to Food and Agriculture Organization (FAO), more than 25% of the world s agricultural production is contaminated with mycotoxins, resulting in economic losses in the grain industry (Cazzaniga et. al., 2001). Mycotoxins are the harmful toxins including Aflatoxin (AF), deoxynivalenol (DON) and fumonisins, which cause illness and eventually death in humans and animals (Dowling 1997). The contaminated food and feedstuffs may be pathogenic for animals and humans. Consequently, guidelines regarding the permissible levels of mycotoxins present in food & feed products, and in raw materials have been established (FAO 1995). However, in cereals particularly wheat and wheat flour aflatoxin (B1, B2, G1, G2) is more common. Commonly used method to analyze mycotoxins is Thin-Layer Chromatography (TLC). Furthermore, there is a concern at national and international levels to prevent and reduce mycotoxin contamination in wheat flour. The study will help better understand the status and magnitude of mycotoxins contamination in wheat and wheat flour in Pakistan. The specific area of attention will be to improve the quality assurance and safety of wheat and wheat flour which will be of great interest for policy makers as well as for public safety.

8 This project will be helpful for quality assurance of wheat grains and wheat flour in Punjab, KPK, Baluchistan and Sindh. It will also support our flour industry to minimize mycotoxins to a permissible level and will be helpful in promoting human/ animal health. Standards keep citizens safe and ensure that the quality of the products they use are of high standards and good for consumers. Pakistan is one of the world's important food exporting countries and due to the strict regulation of importing countries, the best food is exported and sometimes contaminated food is sold domestically. So it is necessary to monitor and control contamination of aflatoxins in wheat and wheat flour in both domestic and international trade (Alim et al., 2014). According to JECFA (1998), in 25% population having Hepatitis B Virus (HBV), with strict aflatoxins standard permissible limit from 20 ppb to 10 ppb, reduced liver cancer by 300 cases/ year/ billion people. 1.1 Aflatoxin Testing Services by PCSIR FBRC, PCSIR Laboratories Complex, Lahore is providing accredited testing services to local manufacturers and exporters for aflatoxin testing and evaluation of compliance with respect to the requirements of various importing countries as well as for the local market. FBRC, PCSIR Laboratories Complex, Lahore is equipped to conduct aflatoxin tests on rice, legumes, spices, nuts etc. FBRC, PCSIR Laboratories Complex, Lahore is also providing technical services for Human Resource Development like internship training to undergraduate students and research work of post graduate students of different universities and other institutions. Recently we have provided training for aflatoxin testing to scientific staff of Central ITD Laboratory, Chaklala (ILO 172/ and ILO 556/ ). Aflatoxin contamination depends upon environmental and storage conditions. A study was conducted in PCSIR Laboratories Complex, Lahore (ILO No. 632, Dated: ), with the collaboration of GAIN. Seventy five (75) samples were analyzed for aflatoxin (ppb) (B 1, B 2, G 1, G 2 ). The samples were collected from 11 flour mills representing 8 cities of Punjab Pakistan. All the samples were free from aflatoxin.

9 During 2016, quality evaluation of wheat flour samples was done for Punjab Food Department (258 samples) and for Police Special Branch (38 samples). Moisture%, ash%, dry gluten% and acidity% were performed for samples collected from Sasta Ramaza Bazars in Pakistan. 1.2 Recognition of PCSIR: PCSIR Laboratory holds the following recognitions: ISO Accreditation by the Pakistan National Accreditation Council (PNAC) for testing of aflatoxin (B1, B2, G1, G2) in cereals, nuts and spices. ISO 9001 Certified by TUV NORD 2. OBJECTIVES The objectives of the project cover support towards reducing contaminated wheat and wheat flour leading to Food Safety & Quality Management (FSQM) and export enhancement program of Government of Pakistan. The proposed project was aimed at Assessment of Pakistani wheat & wheat flour for: a) General Objective of the Project: The General Objective of the Project was to develop an improved understanding of aflatoxin contamination of wheat flour in Pakistan as a baseline and to facilitate a subsequent program to monitor and manage this. b) Specific Objectives of the Project The specific objectives include: i. Assessment of the current quality situation of wheat and wheat flour for assuring food safety and standards. ii. Assessment of the contamination level of aflatoxins in wheat and wheat flour. This project will also enhance the Quality Assurance and Quality Control for Production of Wheat Flour in Punjab, KPK, Baluchistan and Sindh.

10 3. LITERATURE REVIEW AND TECHNICAL PARAMETERS: The most prominent mycotoxins are aflatoxins, zearalenone (ZEA), ochratoxin, patulin, deoxynivalenol (DON) and fumonisin (Gaag et. al., 2003). These compounds cause adverse health effects such as kidney and liver damage (deterioration), mutagenic effects, birth defects, and cancers (specially liver cancer) that result in symptoms ranging from skin irritation to immune suppression, neurotoxicity and death (Bennett and Klich 2003). Aflatoxin B1 (AFB1) and fumonisins are human carcinogens, and patulin is suspected as human carcinogen. DON and other trichothecenes, as well as AFB1 are likely to exert immuno-suppressive effects, and fumonisin B1 (FB1) may contribute to neural tube defects. Renal dysfunction due to OTA (ochratoxin A) exposure is also a potential problem. There is also uncertainty related to the effects of chronic, low level, long-term exposure to single and/ or multiple mycotoxins, which may be the case even for individuals consuming a diverse diet (Lopez-Garcia et. al., 1999). In Pakistan a large number of manufacturers are involved in production of wheat flour. The evaluation of aflatoxin will ultimately enhance the efficiency of locally produced products by providing a competitive environment which will not only protect a common consumer rights but also increase the productivity and streamline the manufacturing processes to produce quality products. Halt (1994) discussed the results of contamination with aflatoxin-producing fungi and aflatoxin B1 (545 samples of wheat grains and 238 samples of flour). Aspergillus (34.87%) dominated in analyzed samples. The presence of Aspergillus flavus, the known aflatoxin producer, was detected in 9.94% of analyzed samples. Aflatoxin B1 was found in 76.8% of samples contaminated with A. flavus. The highest contamination with aflatoxin B1 was detected in wheat grain samples (16.3 micrograms/kg). Contamination was lower in flour samples (4.13 micrograms/kg). FAO and WHO standards says, aflatoxin should not exceed 30 micrograms/kg in food products, only two samples did not meet this criteria. Ghasemi-Kebria et. al. (2013) tested one hundred wheat flour (WF) samples. The mean levels of total aflatoxin and aflatoxin B1 was 1.99 and 0.53 ng g -1, respectively. A positive relationship between aflatoxin (AF) level of WF samples and the risk of esophageal cancer (EC) was observed. It was found that humidity of silos was the most important determinant of AF contamination of WF.

11 4. Project Team. The professional team of PCSIR is involved in Research and Academia. Dr. Ijaz Ahmad (PSO) has completed his Ph. D from NIFSAT, University of Agriculture, Faisalabad in cereal science with special reference to Pakistani wheat varieties. He has completed "Advance Research Study" from SCUT, China. Recently he completed his Post-Doc from Michigan State University, USA in He is HEC (Higher Education Commission) approved Ph. D Supervisor in Pakistan. He has also experience of teaching in University. He has also supervised M. Phil. students. He also provided supervisory guidance for M. Sc and Ph. D students of different universities. He served in Punjab Food Department for 8 years as Storage Office (Food), so he has experience of grain handling and storage. Dr. Quratul Ain Syed (CSO) has also supervised different students. This team is also involved in providing Consultancy/ Advisory Services to different industries. The detail of the professional team of PCSIR Laboratories Complex, Lahore is given below: Sr. No. Team Designation 1 Dr. Ijaz Ahmad, PSO Team Leader 2 Dr. Quratul Ain Syed, CSO Deputy Team Leader 3 Dr. Sakhawat Ali, CSO Member 4 Alim un Nisa, SSO Member 5 Dr. Muhammad Khalid Saeed, SSO Member 6 Dr. Muhammad Ashraf, SSO Member 7 Dr. Shahid Masood, SSO Member 8 Mr. Imran Kaleem, SSO Member 9 Naseem Zehra, SO Member 10 Sajila Hina, SO Member

12 5. MATERIALS AND METHODS 5.1 Selection of Target area The target areas for collecting samples were selected for the following reasons: i. Punjab province, particularly Central and South Punjab is the major wheat producing area. North is the main hub for the export of wheat flour to Afghanistan and other countries. ii. Three provinces (Punjab, KPK and Baluchistan) are the main target areas of GAIN and the most of the export is initiated from these areas. Sindh province was also included to collect national level data in Pakistan. 5.2 Collection of samples PCSIR conducted this study with impartiality. Eight visits were accompanied by representative of GAIN also and samples were taken by one or both parties together. Sampling was done from a number of Government godowns, so that to have representative samples. At the time of sampling, storage conditions of wheat grains at different locations was recorded on sampling form to enhance the data we receive. It allowed a standard set of information to be collected. The following conditions were recorded: time in storage, evidence of any critical condition (evidence of mould, water, infestation), storage condition/ proper cleanliness etc. (Annexure 1). The main market centers with highest estimated market volumes and population density were selected as sample collection sites. The sampling was done from provincial market centers located in the urban centers/ industrial centers. The urban centers (city and towns) have high concentration of population. These are market hubs and through which most of the product volumes are exported and supplied to the population.

13 Table 1. Summary of collected samples. Samples from Punjab Sr. No. City Wheat Grain Sample From Flour From Mill Godown Wheat Flour Sample From Flour From Market Mill Total No. of Samples 1 Islamabad Rawalpindi Gujrat Sialkot Lahore Sheikhupura Gujranwala Sargodha Faisalabad Jhang Toba Tek Singh Pirmahal Kamalia Sahiwal Multan Bahawalpur Muzaffargarh Total Samples from KPK (Khyber Pakhtunkhwa) Sr. No. City Wheat Grain Sample From Flour From Mill Godown Wheat Flour Sample From Flour From Market Mill Total No. of Samples 1 DI Khan Abbotabad Peshawar Total Samples from Baluchistan

14 Sr. No. City Wheat Grain Sample From Flour From Mill Godown Wheat Flour Sample From Flour From Market Mill Total No. of Samples 1 Quetta Pishin Total Samples from Sindh Sr. No. City Wheat Grain Sample From Flour From Mill Godown Wheat Flour Sample From Flour From Market Mill Total No. of Samples 1 Karachi Hyderabad Sukhar Total Total number of samples = Moisture Content Moisture was determined according to the respective method as described in method 44-15A of AACC (2000). The moisture content was determined by drying 2-3 g sample in an air forced draft oven and temperature was maintained at o C. Wheat flour samples were analyzed for moisture contents by Rapid Moisture Tester (MT-C, Brabender, Germany) (9-11 g sample at 130 o C for 30 min). 5.4 Physical Tests Physical tests were performed to determine the level of foreign material in the stored grain. The wheat grains were subjected to physical test according to Federal Grain Inspection Service (FGIS, 1993). Parameters used for physical test were healthy kernel, grain impurity/ foreign matter (FM), shrunken and broken grains, damaged grains (insect, fungus, heat damaged) and live weevils etc. Sieve test was also conducted to check the presence of live weevils in flour. 5.5 Aflatoxin (B1, B2, G1, G2) Analysis In this study thin layer chromatographic (TLC) technique was used for the determination of aflatoxin (B1, B2, G1, G2) in all samples. Estimation of aflatoxins in toxic extracts was done by comparison with standard technique (AOAC, 2005). The determination of aflatoxin by TLC is accredited (ISO Accreditation by the Pakistan National Accreditation Council (PNAC) for testing of aflatoxin (B1, B2, G1, G2) in cereals). Detectable limit of this test by TLC (B1 & G ppb, B2 & G ppb). The concentration (ug/ml) of Standard used (B1: 2.02, B2: 0.500, G1: 2.01: G2: 0.503).

15 Thirty random samples were tested by Eliza for verification of results. Twenty seven random samples were also sent to other Labs for validation. It improved the authenticity of the results. 5.6 Data ownership by GAIN & PCSIR PCSIR and GAIN have ownership for the data obtained by this study. GAIN & PCSIR also have collaborative sharing/ dissemination of data under the Ministry of Science & Technology. 5.7 Statistical analysis The data was statistically evaluated by using the SPSS software. Analysis of variance was carried out following the method described by (Steel et al., 1997). Means were compared by Tukey HSD Test. Box plots and Bar graphs (Mean ±1 SE) were prepared. 6. RESULTS AND DISCUSSION 6.1 Storage conditions The general condition of godowns was observed during sample collection. The most of godowns were of good condition. Only a few godowns were rated as fair condition. The floor surface of only one Government godown was damp. No infection in godowns was observed during sample collection. The samples of season and were collected from government godowns. Type of storage: only two samples were obtained from Ganjis (Temporary open storage/ outdoor storage). All other samples of wheat grains were obtained from godowns. Samples of wheat grains obtained from jute bags were 20% and samples from Polypropylene bags (PP bags) were 80%. 6.2 Moisture contents Moisture contents of wheat grains and wheat flour were analyzed. The results are presented in the Table 2 to 13. Analysis of Variance (ANOVA) results are presented in Table 14. Minimum and maximum values for moisture contents are presented in Table 15. Comparison of means with respect to different areas and location for Moisture (%) are given in Table 16. The moisture contents of wheat grains and wheat flour exhibited highly significant differences with respect to different districts and regions of Punjab. The moisture contents of wheat grains and wheat flour

16 also exhibited significant differences with respect to different districts of KPK and Baluchistan (Table 14). It was observed that moisture contents ranged from 7.12% to 11.49% in wheat grains from Godowns (Table 2) and 7.17% to 10.67% in wheat grains from flour mills of Punjab (Table 3). The moisture contents ranged from 7.8% to 14.30% in wheat flour from flour mills of Punjab (Table 3) and 9.92% to 15.07% in wheat flour from markets of Punjab (Table 4). The moisture (%) for different provinces is also explained in Fig 1 to 4. The data indicated that moisture contents ranged from 6.94% to 11.49% in wheat grains and 7.8% to 15.07% in wheat flour in Pakistan. The mean moisture contents in grains from Punjab exhibited higher values in Central Punjab region and lower values in South Punjab (Fig 1a). It may be due to the effect of warm climate in South Punjab. The Box Plot showed the minimum and maximum values of data. The median values in Box plot for wheat flour from Flour Mills of Punjab indicated that the median values are almost in the middle, indicating almost symmetric data (Fig 1b). The overall data about moisture contents for Pakistan is presented in Fig 9. In KPK province, wheat flour from Peshawar city showed higher moisture contents as compared to other cities (Fig 2). Box Plot indicated that moisture % in flour from Flour Mills of Peshawar also exhibited higher median value than other cities of KPK (Fig 2). Punjab Pure Food Regulations (2017) states that moisture content shall not exceed 12% in wheat grains and not more than 14% in wheat flour. The results of the present study indicates that all the wheat grain samples (from Government godowns and from flour mills) confirms to the standard. However, only 18 wheat flour samples (5.4%) contained higher moisture content. 6.3 Physical tests The parameters used for physical test were healthy kernel, grain impurity/ foreign matter (FM), shrunken and broken grains, damaged grains (insect, fungus, heat damaged) and live weevils etc. Minimum and maximum values for physical parameters are shown in Table 15. Comparison of means with respect to different areas and location for physical tests are presented in Table 17 (Punjab and KPK) and Table 18 (Baluchistan and Pakistan). The Foreign Matter contents of wheat grains exhibited non significant differences with respect to location in Punjab. However all other physical parameters were significantly different in Punjab, KPK and Baluchistan (Table 14).

17 It was observed that healthy kernel ranged (82.6% to 98.67%), grain impurity/ foreign matter (FM) ranged (0.22% to 5.06%), shrunken and broken grains ranged (0.45% to 8.23%), damaged grains (insect, fungus, heat damaged) ranged from (0.47 to 8.11%) in wheat grains from flour mills in Punjab. The physical tests for different provinces are also elaborated in Fig 5 to 8. The data indicated healthy kernel ranged (82.6% to 98.67%), grain impurity/ foreign matter (FM) ranged (0.22% to 6.76%), shrunken and broken grains ranged (0.0% to 11.09%), damaged grains (insect, fungus, heat damaged) ranged from (0.0 to 8.11%) in wheat grains from Pakistan. The overall data about different physical parameters for Pakistan is presented in Fig 9. Wheat grains from godowns of South Punjab exhibited more Foreign Matter and Broken & Shrunken grains than north and central region. However, more damaged grains were present in grains from flour mills of south Punjab (Fig 5 a). The Box Plot (Fig 5 c) indicated that the majority of samples fall in similar range with respect to physical parameters. In KPK province, what grains from godowns of Peshawar city showed higher Broken & Shrunken grains (Fig 6 a). Overall data indicated that wheat grains from godowns of Sindh province contained higher damaged grains (Fig 9 a). Sieve test was also conducted to check the presence of live weevils in flour. It was observed that live weevils were not present in any wheat grain and wheat flour sample. This may be due to reason that the most of samples were obtained from godowns which were already fumigated to avoid possible insect attack etc. The season was also favorable for storage (not hot and humid). Punjab Pure Food Regulations (2017) states that sound grains shall not lower than 93.5%, foreign matter shall not more than 1.5% and damaged grains shall not more than 5%. The present study indicated that 78.76%, 57.52% and 92.92% samples confirms to the standard with respect to healthy kernel, foreign matter and damaged kernels, respectively. The study of physical parameters of wheat samples revealed that 21.24%, 42.48% and 7.08% samples failed to meet standard with respect to healthy kernels, foreign matter and damaged kernels, respectively. This may be due to purchase of wheat as per Fair Average Quality (FAQ). However, FAQ specifications for wheat exports are not possible because importers have their own specifications and minimum parameters of quality and safety indicators, including test weights, protein content and falling numbers etc. 6.4 Aflatoxin (B1, B2, G1, G2) Analysis

18 Aflatoxin (B1, B2, G1, G2) was determined in wheat grain and wheat flour samples. It was observed that no sample was found contaminated with Aflatoxin (B1, B2, G1, G2) in the present study. It may be due to reason that the most of samples were obtained from godowns which were already fumigated to avoid possible insect attack etc. The previous studies also favor this reflection. The effects of phosphine fumigation on survival and growth of storage fungi in wheat was studied by Hocking and Banks (1991). They found that phosphine may be useful in retarding fungal spoilage during short-term storage of high moisture grain (15-19% moisture content in wheat). De Castro et al., (2000) investigated the effect of phosphine on the growth of A. flavus and aflatoxin production in maize stored at different moisture contents. It was observed that aflatoxin production was severely affected by phosphine even at low concentrations. Similarly Habib et al., (2005) studied the effect of Phosphine Fumigation on the Growth of Aspergillus flavus and Aflatoxin Production. The results also revealed significant effect of phosphine (PH3) on Aspergillus flavus growth reduction. The season was also favorable for storage (not hot and humid) which also provided retarding environment for aflatoxin production. Previously it was demonstrated that aflatoxin-producing molds can grow exponentially in conventional multi-month storage as a result of a combination of heat and high humidity (Hell et al., 2010). Fungi produce aflatoxins in the presence of higher moisture, temperature and adequate substratum. That is why warm and wet regions are the most favorable environments for aflatoxins (Ramesh et el., 2013). The detectable limit of Aflatoxin in this analysis by TLC was (B1 & G ppb, B2 & G ppb). The concentration (ug/ml) of Standard used (B1: 2.02, B2: 0.500, G1: 2.01: G2: 0.503). Thirty random samples were also tested with Eliza for verification of results. The analysis of samples by Eliza also showed that Aflatoxin (B1, B2, G1, G2) was Not Detected (ND). Maliha et al., (2010) already reported no detectable levels of AFB1 in wheat grains from Pakistan. The frequency and quantity of AFB1 levels were very low in wheat and barley samples compared to other agricultural commodities (Reddy and Salleh 2010). Food processing can reduce mycotoxin levels by physical removal. Further processing such as milling and extrusion can also reduce mycotoxin content (Karlovsky et al., 2016). According to Official Journal of the European Union (2010), the maximum permissible level of aflatoxin in wheat and wheat flour is 2 ppb (B 1 ) and 4 ppb (total aflatoxin). As no sample was found contaminated with aflatoxin, it indicates that there may be zero or very low incidence of other mycotoxins. Previously it is described that Aspergillus flavus occur in a

19 wide range of temperatures in substrates with more hydrocarbon percentage. However, a lot of non-aflatoxigenic strains act similar, and therefore the fungus existence does not intimate the presence of aflatoxins (Khlangwiset et al., 2011). The contamination of aflatoxin might be due to improper post harvest technology and storage condition (Ramesh et al., 2013). However, Rehan (2017) depicted that Pakistani wheat flour is free from aflatoxin contamination. It might be due to the developed circumstance of post-harvest conditions, and good processing conditions as healthy actions taken for the application of GAP (Good Agricultural Practices). Thus wheat flour from Punjab Province is adequate for human consumption and also for export. The present study also indicated that the Food Safety condition of wheat and wheat flour for both domestic and export market is adequate in Pakistan. However, aflatoxin contamination should be monitored to ensure food quality and safety, and prime attention should be given to wheat, which is staple food of Pakistan and has sufficient potential for export. Table 2. Analysis of wheat grain samples from Godowns of Punjab. Sr. No. City Sample ID Moisture (%) Physical Tests HK FM SBG DG 1 Islamabad 1 G-ISB-GDN G-ISB-GDN G-ISB-GDN G-ISB-GDN Rawalpindi 5 G-RWP-GDN G-RWP-GDN G-RWP-GDN G-RWP-GDN Gujrat 9 G-GUJ-GDN G-GUJ-GDN Sialkot 11 G-SKT-GDN G-SKT-GDN Lahore 13 G-LHR-GDN G-LHR-GDN G-LHR-GDN G-LHR-GDN G-LHR-GDN G-LHR-GDN Sheikhupura 19 G-SKP-GDN G-SKP-GDN Gujranwala 21 G-GRW-GDN G-GRW-GDN Sargodha 23 G-SRG-GDN G-SRG-GDN Faisalabad 25 G-FSB-GDN G-FSB-GDN G-FSB-GDN G--FSB-GDN G--FSB-GDN

20 30 30 G--FSB-GDN Jhang 31 G-JHG-GDN G-JHG-GDN T T Singh 33 G-TTS-GDN G-TTS-GDN G-Rajana-GDN G-Rajana-GDN G-Pirmahal-GDN G- Pirmahal-GDN G-Kamalia-GDN G- Kamalia-GDN Sahiwal 41 G-SHW-GDN G-SHW-GDN Multan 43 G-MLT-GDN G-MLT-GDN Bahawalpur 45 G-BWP-GDN G-BWP-GDN G-BWP-GDN G-BWP-GDN Muzaffargarh 49 G-MZG-GDN G-MZG-GDN HK: Healthy Kernels, FM: Foreign Matter, SBG: Shrunken and Broken Grains, DG: Damaged Grains Table 3. Analysis of wheat grain and wheat flour samples from Flour Mills of Punjab. Sr. No. City Wheat Grain Physical Tests (Grain) Sr. Wheat Flour Sample ID Moistur e (%) HK FM SBG DG No. Sample ID Moist ure % 51 Islamabad 51 G-ISB-FM F-ISB-FM G-ISB-FM F-ISB-FM Rawalpindi 53 G-RWP-FM F-RWP-FM G-RWP-FM F-RWP-FM Gujrat 55 G-GUJ-FM F-GUJ-FM Sialkot 56 G-SKT-FM F-SKT-FM Lahore 57 G-LHR-FM F-LHR-FM G- LHR-FM F- LHR-FM G-LHR-FM F-LHR-FM Sheikhupura 60 G-SKP-FM F-SKP-FM Gujranwala 61 G-GRW-FM F-GRW-FM Sargodha 62 G-SRG-FM F-SRG-FM Faisalabad 63 G-FSB-FM F-FSB-FM G-FSB-FM F-FSB-FM G-FSB-FM F-FSB-FM Jhang 66 G-JHG-FM F-JHG-FM T T Singh 67 G-TTS-FM F-TTS-FM G-Rajana-FM F-Rajana-FM G-Pirmahal-FM F-Pirmahal-FM G-Kamalia-FM F-Kamalia- FM Sahiwal 71 G-SHW-FM F-SHW-FM 7.80

21 72 Multan 72 G-MLT-FM F-MLT-FM Bahawalpur 73 G-BWP-FM F-BWP-FM G-BWP-FM F-BWP-FM Muzaffargarh 75 G-MZG-FM F-MZG-FM HK: Healthy Kernels, FM: Foreign Matter, SBG: Shrunken and Broken Grains, DG: Damaged Grains Table 4. Moisture contents in wheat flour samples from markets of Punjab. Sr. City Sample ID Moisture Sr. City Sample ID Moisture (%) No. (%) No. 101 Islamabad 26 F-ISB-MKT Faisalabad 76 F-FSB-MKT F-ISB-MKT F-FSB-MKT F-ISB-MKT F-FSB-MKT F-ISB-MKT F-FSB-MKT F-ISB-MKT F-FSB-MKT F-ISB-MKT F-FSB-MKT F-ISB-MKT F-FSB-MKT F-ISB-MKT F- FSB-MKT Rawalpindi 34 F-RWP-MKT F- FSB-MKT F-RWP-MKT F- FSB-MKT F-RWP-MKT Jhang 86 F-JHG-MKT F-RWP-MKT F-JHG-MKT F-RWP-MKT F-JHG-MKT F-RWP-MKT F-JHG-MKT F-RWP-MKT T T Singh 90 F-TTS-MKT F-RWP-MKT F-TTS-MKT Gujrat 42 F-GUJ-MKT F-TTS-MKT F-GUJ-MKT F-TTS-MKT F-GUJ-MKT F-Rajana-MKT F-GUJ-MKT F-Rajana-MKT Sialkot 46 F-SKT-MKT F-Rajana-MKT F-SKT-MKT F-Rajana-MKT F-SKT-MKT F-Pirmahal- MKT F-SKT-MKT F-Pirmahal- MKT Lahore 50 F-LHR-MKT F-Pirmahal- MKT F-LHR-MKT F-Pirmahal- MKT F-LHR-MKT F-Kamalia- MKT F-LHR-MKT F-Kamalia- MKT F-LHR-MKT F-Kamalia- MKT F-LHR-MKT F-Kamalia- MKT F-LHR-MKT Sahiwal 106 F-SHW-MKT 12.13

22 F-LHR-MKT F-SHW-MKT F-LHR-MKT F-SHW-MKT F-LHR-MKT F-SHW-MKT F-LHR-MKT Multan 110 F-MLT-MKT F-LHR-MKT F-MLT-MKT Sheikhupura 62 F-SKP-MKT F-MLT-MKT F-SKP-MKT F-MLT-MKT F-SKP-MKT Bahawalpur 114 F-BWP-MKT F-SKP-MKT F-BWP-MKT Gujranwala 66 F-GRW-MKT F-BWP-MKT F-GRW-MKT F-BWP-MKT F-GRW-MKT F-BWP-MKT F-GRW-MKT F-BWP-MKT Sargodha 70 F-SRG-MKT F-BWP-MKT F-SRG-MKT F-BWP-MKT F-SRG-MKT Muzaffargarh 122 F-MZG-MKT F-SRG-MKT F-MZG-MKT Faisalabad 74 F-FSB-MKT F-MZG-MKT F-FSB-MKT F-MZG-MKT Table 5. Analysis of wheat grain samples from Godowns of KPK (Khyber Pakhtunkhwa). Sr. No. City Sample ID Moisture (%) Physical Tests HK FM SBG DG 201 DI Khan 1 G-DIK-GDN G-DIK-GDN G-DIK-GDN G-DIK-GDN Abbotabad 5 G-ABT-GDN Peshawar 6 G-PWR-GDN G-PWR-GDN G-PWR-GDN G-PWR-GDN G-PWR-GDN G-PWR-GDN HK: Healthy Kernels, FM: Foreign Matter, SBG: Shrunken and Broken Grains, DG: Damaged Grains Table 6. Analysis of wheat grain and wheat flour samples from Flour Mills of KPK. Sr. Wheat Grain Physical Tests (Grain) Sr. Wheat Flour No. City Sample ID Moisture HK FM SBG DG No. Sample (%) ID 212 DI Khan 12 G-DIK-FM F-DIK-FM G-DIK-FM F-DIK-FM Abbotabad 14 G-ABT-FM F-ABT-FM Peshawar 15 G-PWR-FM F-PWR-FM Moisture %

23 G-PWR-FM F-PWR-FM G-PWR-FM F-PWR-FM HK: Healthy Kernels, FM: Foreign Matter, SBG: Shrunken and Broken Grains, DG: Damaged Grains, KPK: Khyber Pakhtunkhwa Table 7. Moisture contents in wheat flour samples from markets of KPK (Khyber Pakhtunkhwa). Sr. No. City Sample ID Moisture (%) Sr. No. City Sample ID Moisture (%) 224 DI Khan 7 F-DIK-MKT Abbotabad 20 F-ABT-MKT F-DIK-MKT Peshawar 21 F-PWR-MKT F-DIK-MKT F-PWR -MKT F-DIK-MKT F-PWR -MKT F-DIK-MKT F-PWR-MKT F-DIK-MKT F-PWR -MKT F-DIK-MKT F-PWR-MKT F-DIK-MKT F-PWR-MKT Abbotabad 15 F-ABT-MKT F-PWR-MKT F-ABT-MKT F-PWR-MKT F-ABT-MKT F-PWR-MKT F-ABT-MKT F-PWR-MKT F-ABT -MKT F-PWR-MKT Table 8. Analysis of wheat grain samples from Godowns of Baluchistan. Sr. City Sample ID Moisture Physical Tests No. (%) HK FM SBG DG 250 Quetta 1 G-QTA-GDN G-QTA-GDN G-QTA-GDN G-QTA-GDN G-QTA GDN G-QTA-GDN Pishin 7 G-PSH-GDN G-PSH-GDN G-PSH-GDN G-PSH-GDN G-PSH-GDN Table 9. Analysis of wheat grain and wheat flour samples from Flour Mills of Baluchistan. Sr. No. City Wheat Grain Physical Tests (Grain) Sr. Wheat Flour Sample ID HK FM SBG DG No. Sample ID Moisture Moisture (%) 261 Quetta 12 G-QTA-FM F-QTA-FM G-QTA-FM F-QTA-FM G-QTA-FM F-QTA-FM Pishin 15 G-PSH-FM F-PSH-FM 10 %

24 G-PSH-FM F-PSH-FM F-PSH-FM 9 Table 10. Moisture contents in wheat flour samples from markets of Baluchistan. Sr. No. City Sample ID Moisture (%) Sr. No. City Sample ID Moisture (%) 272 Quetta 7 F-QTA-MKT Pishin 19 F-PSH-MKT F-QTA-MKT F-PSH-MKT F-QTA-MKT F-PSH-MKT F-QTA-MKT F-PSH -MKT F-QTA-MKT F-PSH -MKT F-QTA-MKT F-PSH-MKT F-QTA-MKT F-PSH -MKT F-QTA-MKT F-PSH-MKT F-QTA-MKT F-PSH-MKT F-QTA-MKT F-PSH-MKT F-QTA-MKT F-PSH-MKT F-QTA-MKT F-PSH-MKT F-PSH-MKT Table 11. Analysis of wheat grain samples from Sindh. Sr. City Sample ID Moisture Physical Tests No. (%) HK FM SBG DG Godowns of Sindh 297 Sukhar 1 G-SKR-GDN G-SKR-GDN Flour Mills of Sindh 299 Hyderabad 3 G-HDR-FM G-HDR-FM Sukhar 5 G-SKR-FM HK: Healthy Kernels, FM: Foreign Matter, SBG: Shrunken and Broken Grains, DG: Damaged Grains Table 12. Moisture contents in wheat flour samples from Flour Mills of Sindh. Sr. No. City Wheat Flour Sample ID Moisture % 302 Karachi 1 F-KHI-FM F-KHI-FM F-KHI-FM F-HDR-FM F-HDR-FM F-SKR-FM 11.83

25 Table 13. Moisture contents in wheat flour samples from markets of Sindh. Sr. No. City Sample ID Moisture (%) Sr. No. City Sample ID Moisture (%) 308 Karachi 7 F-KHI-MKT Hyderabad 20 F-HDR-MKT F-KHI-MKT F-HDR-MKT F-KHI-MKT F-HDR -MKT F-KHI-MKT F-HDR -MKT F-KHI-MKT F-HDR-MKT F-KHI-MKT F-HDR -MKT F-KHI-MKT F-HDR-MKT F-KHI-MKT Sukhar 27 F-SKR-MKT F-KHI-MKT F-SKR-MKT F-KHI-MKT F-SKR-MKT F-KHI-MKT F-SKR-MKT F-KHI-MKT F-SKR-MKT Hyderabad 19-F-HDR-MKT F-SKR-MKT 7.91 Table 14. Analysis of variance (mean squares) (Punjab, KPK, Baluchistan and Pakistan). Source variation District Area x District Error Total Source variation Region Area x Region Error Total Source variation District Area x District Error of of of Degrees of freedom Degrees of freedom Degrees of freedom Wheat flour Moisture (%) ** ** ** Punjab (district wise). Moisture (%) ** ** ** Foreign matter (%) NS ** ** Wheat Grains SBG (%) ** ** ** Damage grains (%) ** ** ** Punjab (Region wise: North, Central, South). Wheat flour Wheat Grains Moisture Moisture Foreign SBG (%) Damage (%) (%) matter (%) grains (%) ** ** ** ** ** * ** ** ** ** ** ** ** ** ** KPK (Khyber Pakhtunkhwa) (district wise). Wheat flour Wheat Grains Moisture Moisture Foreign SBG (%) Damage (%) (%) matter (%) grains (%) * ** ** * ** NS ** ** ** ** ** ** ** ** ** Healthy grains (%) ** ** ** Healthy grains (%) * ** ** Healthy grains (%) ** ** **

26 Total 17 Source of variation District Area x District Error Total Source variation of Province Area x Province Error Total Degrees of freedom Degrees of freedom Wheat flour Moisture (%) ** ** ** Baluchistan (district wise). Wheat Grains Moisture (%) * ** ** Foreign matter (%) ** ** ** SBG (%) ** ** ** Pakistan (Province wise) Wheat flour Wheat Grains Moisture Moisture Foreign SBG (%) (%) (%) matter (%) NS ** * NS ** NS NS NS NS NS ** NS Damage grains (%) ** ** ** Damage grains (%) NS ** * Healthy grains (%) ** ** ** NS = Non-significant (P>0.05); * = Significant (P<0.05); ** = Highly significant (P<0.01). SBG: Shrunken & broken Grains Healthy grains (%) NS ** NS Table 15. Moisture and physical analysis of wheat grains and wheat flours (Pakistan). Moisture (%) Grains (Godown) Moisture (%) Grains (Flour Mill) Minimum Maximum Mean SD SE Minimum Maximum Mean SD SE Punjab KPK Baluchistan Sindh Moisture (%) flour (Mill) Moisture (%) flour (Market) Minimum Maximum Mean SD SE Minimum Maximum Mean SD SE Punjab KPK Baluchistan Sindh Foreign Matter (%) Grains (Godown) Foreign Matter (%) Grains (Flour Mill) Minimum Maximum Mean SD SE Minimum Maximum Mean SD SE Punjab KPK Baluchistan Sindh Shrunken and broken grains (%) Godown Shrunken and broken grains (%) (Flour Mill) Minimum Maximum Mean SD SE Minimum Maximum Mean SD SE Punjab

27 KPK Baluchistan Sindh Damaged Grains (%) (Godown) Damaged Grains (%) (Flour Mill) Minimum Maximum Mean SD SE Minimum Maximum Mean SD SE Punjab KPK Baluchistan Sindh Healthy Grains (%) (Godown) Healthy Grain (%) (Flour Mill) Minimum Maximum Mean SD SE Minimum Maximum Mean SD SE Punjab KPK Baluchistan Sindh KPK (Khyber Pakhtunkhwa) Table 16. Comparison of means with respect to different areas and location. x are mean ±SE for Moisture (%). Punjab Wheat Grains Wheat Flour Area Mean (Punjab) Mean Godown Flour Mills Market Flour Mills Central 8.44±0.03b 8.64±0.03a 8.54±0.05A 12.85±0.04a 11.95±0.05d 12.40±0.20A North 7.98±0.01d 8.45±0.04ab 8.22±0.11B 12.28±0.01c 12.17±0.01c 12.23±0.03B South 7.91±0.08d 8.20±0.02c 8.05±0.07C 12.15±0.03c 12.69±0.03b 12.42±0.12A Mean 8.11±0.09B 8.43±0.07A 12.43±0.11A 12.27±0.11B KPK (Khyber Pakhtunkhwa) Wheat Grains Wheat Flour Area Mean (Punjab) Mean Godown Flour Mills Market Flour Mills Central 9.12±0.05a 8.53±0.23b 8.83±0.17A 11.30±0.03c 10.72±0.04d 11.01±0.13A North 7.87±0.01c 7.82±0.16c 7.84±0.07B 10.39±0.01e 10.29±0.01e 10.34±0.02C South 7.99±0.01bc 7.84±0.03c 7.91±0.03B 12.33±0.01b 12.83±0.03a 12.58±0.11A Mean 8.32±0.20A 8.06±0.14B 11.34±0.28A 11.28±0.39B Area (Baluchistan) Baluchistan Wheat Grains Wheat Flour Mean Godown Flour Mills Market Flour Mills Mean

28 Quetta 8.15±0.02a 7.39±0.21b 7.77±0.19A 11.20±0.007c 12.10±0.043a 11.65±0.20A Pishin 7.24±0.01b 7.47±0.04b 7.35±0.05B 11.72±0.052b 10.36±0.027d 11.04±0.30B Mean 7.70±0.20A 7.43±0.10B 11.46±0.119A 11.23±0.390B Pakistan Area Wheat Grains Wheat Flour (Pakistan) Mean Mean Godown Flour Mills Market Flour Mills Punjab 8.32± ± ±0.12B 11.34±0.28bc 11.28±0.39bc 11.31±0.23B KPK 8.33± ± ±0.08AB 12.66±0.16a 12.18±0.26ab 12.42±0.15A Baluchistan 7.70± ± ±0.12C 11.46±0.12bc 11.23±0.39bc 11.34±0.20B Sindh 8.49± ± ±0.28A 10.22±0.34c 11.95±0.11ab 11.08±0.27B Mean 8.28±0.08A 8.45±0.10A 12.07±0.16A 11.95±0.18A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). Small letters represent comparison among interaction means and capital letters are used for overall mean. Table 17. Comparison of means with respect to different areas and location (Punjab and KPK). x are mean ±SE for physical tests of wheat grains. Foreign Matter Shrunken and broken grains Area Mean (Punjab) Mean Godown Flour Mills Godown Flour Mills Central 1.900±0.011b 1.817±0.007c 1.859±0.019A 4.62±0.02a 3.86±0.01c 4.24±0.17A North 1.619±0.007d 1.388±0.010e 1.503±0.052B 3.73±0.03d 4.21±0.01b 3.97±0.11C South 2.481±0.012a 1.195±0.010f 1.838±0.288A 4.72±0.01a 3.47±0.05e 4.10±0.28B Mean 2.000±0.127A 1.467±0.092B 4.36±0.16A 3.85±0.11B Damaged grains Healthy grains Area Mean (Punjab) Mean Godown Flour Mills Godown Flour Mills Central 1.278±0.008d 2.136±0.003c 1.707±0.192C 92.31±0.03c 92.26±0.04c 92.28±0.02B North 1.161±0.014e 2.403±0.016b 1.782±0.278B 93.52±0.02a 92.09±0.03cd 92.81±0.32A South 1.175±0.015e 2.914±0.005a 2.044±0.389A 91.85±0.04d 92.95±0.14b 92.40±0.25B Mean 1.204±0.019B 2.484±0.114A 92.56±0.25A 92.43±0.14B Foreign Matter Shrunken and broken grains Area Mean (KPK) Mean Godown Flour Mills Godown Flour Mills Abbotabad 2.273±0.041a 1.327±0.052c 1.800±0.214A 2.233±0.04c 1.670±0.044e 1.952±0.129C

29 DI Khan 1.419±0.005c 2.252±0.003a 1.835±0.186A 1.809±0.02de 2.562±0.049b 2.186±0.170B Peshawar 1.584±0.016b 1.285±0.018c 1.435±0.068B 6.682±0.03a 1.841±0.014d 4.262±1.082A Mean 1.759±0.131A 1.621±0.159B 3.575±0.78A 2.024±0.138B Damaged grains Healthy grains Area Mean (KPK) Mean Godown Flour Mills Godown Flour Mills Abbotabad 0.00±0.000f 1.050±0.029b 0.525±0.235B 95.47±0.08b 96.33±0.24a 95.90±0.22A DI Khan 0.43±0.003e 0.620±0.005d 0.527±0.042B 96.44±0.07a 95.57±0.09b 96.00±0.20A Peshawar 1.28±0.006a 0.751±0.004c 1.017±0.119A 90.52±0.04c 96.19±0.08a 93.36±1.27B Mean 0.57±0.188B 0.807±0.064A 94.14±0.92B 96.03±0.14A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05).. KPK (Khyber Pakhtunkhwa). Small letters represent comparison among interaction means and capital letters are used for overall mean Table 18. Comparison of means with respect to different areas and location (Baluchistan and Pakistan). x are mean ±SE for physical tests of wheat grains. Foreign Matter Shrunken and broken grains Area Mean (Baluchistan) Mean Godown Flour Mills Godown Flour Mills Quetta 1.10±0.01c 1.39±0.031b 1.25±0.067B 1.83±0.005c 2.72±0.032b 2.27±0.199A Pishin 1.77±0.03a 1.14±0.016c 1.46±0.141A 0.44±0.009d 3.10±0.018a 1.77±0.593B Mean 1.44±0.15A 1.27±0.058B 1.14±0.310B 2.91±0.086A Damaged grains Healthy grains Area Mean (Baluchistan) Mean Godown Flour Mills Godown Flour Mills Quetta 0.93±0.010c 0.84±0.004d 0.89±0.02B 96.18±0.02b 95.25±0.038c 95.72±0.21A Pishin 1.02±0.016b 1.60±0.038a 1.31±0.13A 96.74±0.03a 94.16±0.002d 95.45±0.58B Mean 0.97±0.022B 1.22±0.169A 96.46±0.13A 94.71±0.245B Foreign Matter Shrunken and broken grains Area Mean Pakistan Mean Godown Flour Mills Godown Flour Mills Punjab 1.88± ± ±0.11A 4.62± ± ±0.23A KPK 1.76± ± ±0.10A 3.57± ± ±0.43B

30 Baluchistan 1.44± ± ±0.08A 1.14± ± ±0.31B Sindh 1.74± ± ±0.82A 1.38± ± ±0.06B Mean 1.82±0.09A 1.92±0.17A 3.98±0.29A 3.45±0.25A Damaged grains Healthy grains Area Mean Pakistan Mean Godown Flour Mills Godown Flour Mills Punjab 1.32±0.11c 2.41±0.28b 1.87±0.16B 92.31± ± ±0.31B KPK 0.57±0.19c 0.81±0.06c 0.69±0.10C 94.14± ± ±0.50A Baluchistan 0.97±0.02c 1.22±0.17c 1.10±0.09BC 96.46± ± ±0.29A Sindh 4.57±0.01a 3.16±0.76ab 3.63±0.55A 92.30± ± ±0.31B Mean 1.34±0.13A 2.15±0.22A 92.97±0.30A 92.72±0.41A Means sharing similar letter in a row or in a column are statistically non-significant (P>0.05). KPK (Khyber Pakhtunkhwa) Small letters represent comparison among interaction means and capital letters are used for overall mean Wheat Grain Wheat Flour

31 Grains (godowns of Punjab) Grains (flour mills of Punjab) Flour (flour mills of Punjab) Flour (markets of Punjab) Fig 1 a. Moisture (%) Punjab Grains (godowns of Punjab) Grains (flour mills of Punjab)

32 Flour (flour mills of Punjab) Flour (market of Punjab) Fig 1 b. Moisture (%), Punjab (Region wise, Box Plot). MOISTURE

33 GRAINS Grains (godowns of KPK) Grains (flour mills of KPK) FLOUR Flour (flour mills of KPK) Flour (market of KPK) Fig 2. Moisture (%) KPK (Khyber Pakhtunkhwa). Grains (GDN Baluchistan) Moisture (%) grains (flour mills Baluchistan)

34 Flour (flour mills of Baluchistan) Fig 3. Moisture (%) Baluchistan. Flour (markets of Baluchistan) Moisture (%) grains (GDN Sindh) Moisture (%) grains (flour mills Sindh) Flour (flour mills of Sindh) Fig 4. Moisture (%) Sindh. Flour (markets of Sindh)

35 Fig. 5 a. Physical Tests of samples from Punjab (District wise and region wise). Foreign Matter in grain (godowns of Punjab) Foreign Matter in grain (flour mills of Punjab)

36 Shrunken & Broken grain (godowns of Punjab) Shrunken & Broken grain (flour mills of Punjab) Damaged grain (godowns of Punjab) Damaged grain (flour mills of Punjab) Healthy grain (godowns of Punjab) Healthy grain (flour mills of Punjab) Fig. 5 b. Physical Tests of samples from Punjab (District wise, Box Plot).

37 Foreign Mater in grain (godowns of Punjab) Foreign Matter in grain (flour mills of Punjab) Shrunken & Broken grain (godowns of Punjab) Shrunken & Broken grain (flour mills of Punjab) Damaged grain (godowns of Punjab) Damaged grain (flour mills of Punjab) Healthy grain (godowns of Punjab) Healthy grain (flour mills of Punjab) Fig. 5 c. Physical Tests of samples from Punjab (Region wise, Box Plot).

38 Fig. 6 a. Physical Tests of samples from KPK (Khyber Pakhtunkhwa).

39 FM grain (godowns of KPK) FM grain (flour mills of KPK) Tab19 SB grain (godowns of KPK) Tab20 SB grain (flour mills of KPK) Damaged grain (godowns of KPK) Damaged grain (flour mills of KPK) Healthy grain (godowns of KPK) Healthy grain (flour mills of KPK) Fig. 6 b. Physical Tests of samples from KPK (Khyber Pakhtunkhwa) (Box Plot).

40 Foreign Matter in grains (GDN Baluchistan) Foreign Matter in grains (Flour mills of Baluchistan) SB grains (GDN of Baluchistan) SB grains (Flour mills of Baluchistan) Damaged grains (GDN of Baluchistan) Damaged grains (Flour mills of Baluchistan) Healthy grains (godowns of Baluchistan) Healthy grains (Flour Mills of Baluchistan) Fig. 7. Physical Tests of samples from Baluchistan (Box Plot). GDN (Godown), SB Grains (Shrunken & Broken Grains)

41 Foreign matter in grains (Godown of Sindh) Foreign matter in grains (flour mills Sindh) Shrunken & Broken grains (godowns of Sindh) Shrunken & Broken grains (Flour mills of Sindh) Damaged grains (Godowns of Sindh) Damaged grains (Flour Mills of Sindh) Healthy grains (godowns of Sindh) Healthy grains (Flour Mills of Sindh) Fig. 8. Physical Tests samples from Sindh (Box Plot).