DEVELOPMENT AND POPULARIZATION OF VALUE ADDED PRODUCTS FROM SELECTED MEDICINAL MUSHROOMS

Transcription

1 DEVELOPMENT AND POPULARIZATION OF VALUE ADDED PRODUCTS FROM SELECTED MEDICINAL MUSHROOMS a thesis submitted by G. JAWAHAR (08ZN008) In partial fulfillment for the award of the degree of DOCTOR OF PHILOSOPHY under the supervision of DR. R. EMILIN RENITTA DEPARTMENT OF BIOTECHNOLOGY SCHOOL OF BIOTECHNOLOGY AND HEALTH SCIENCES KARUNYA UNIVERSITY (Karunya Institute of Technology and Sciences) (Declared as Deemed-to-be-under Sec-3 of the UGC Act, 1956) Karunya Nagar, Coimbatore INDIA SEPTEMBER 2015

2 ii DECLARATION I, G. JAWAHAR hereby declare that the thesis, entitled Development and popularization of value added products from selected medicinal mushrooms submitted to the Karunya University, in partial fulfillment of the requirements for the award of the Degree of Doctor of Philosophy in Biotechnology is a record of original and independent research work done by me during the period , under the Supervision and guidance of Dr. R. Emilin Renitta, Assistant Professor, Department of Food Processing and Engineering, School of Biotechnology and Health Sciences, Karunya University. The work contained in this thesis has not been previously submitted to meet the requirements for a degree or diploma at this or any other higher education institution. G. Jawahar Signature of Candidate

3 iii BONAFIDE CERTIFICATE Certified that this thesis titled Development and popularization of value added products from selected medicinal mushrooms is the bonafide work of G. JAWAHAR who carried out the research under my supervision. Certified further, that to the best of my knowledge the work reported herein does not form part of any other thesis or dissertation on the basis of which a degree or award was conferred on an earlier occasion on this or any other scholar. Dr. R. Emilin Renitta SUPERVISOR Assistant Professor Department of Food Processing and Engineering School of Biotechnology and Health Sciences Karunya University Countersigned by Dr. J. Jannet Vennila Director School of Biotechnology and Health Sciences Karunya University

4 iv ABSTRACT The purpose of this study focused on the drying characteristics, biochemical, nutritional, antitumor, antioxidant properties, development and popularization of value added products such as biscuits, wafers and chutney powder from Pleurotus ostreatus (Oyster mushroom) and Calocybe indica (Milky mushroom). Drying characteristics and biochemical composition of dried Pleurotus ostreatus and Calocybe indica using Vibrated fluidized dryer and Vacuum shelf dryer were studied. The moisture content and drying rate of Pleurotus ostreatus and Calocybe indica slices varied according to the drying temperatures 50 C, 60 C, 70 C, and 90 C. Rehydration ratio of dried samples were higher at the lower temperature (50 C) in both the samples. Based on drying characteristics, biochemical composition, sensory evaluation and in vitro antioxidant activities, it was found that the optimum temperature for drying both the samples were 70 C without any nutrient loss. The antitumor and in vivo antioxidant activity of Pleurotus ostreatus and Calocybe indica against Dalton's lymphoma Ascites (DLA) induced tumor in Swiss male albino mice results revealed that an ethanolic extract of Pleurotus ostreatus and Calocybe indica (250 mg/kg body weight) could have a direct effect on diminishing the cancer cell development and could have also impacte on their proliferation by enhancing immune responses and antioxidant enzyme

5 v activities in the Swiss male albino mice. After14-days of treatment with Pleurotus ostreatus and Calocybe indica ethanolic extract, it was found that there was a significant decrease in the presence of DLA cells in tumor induced mice. This suggested that the Pleurotus ostreatus and Calocybe indica apparently led to effective reductions in the multiplication of these cancer cells and ultimately, suppressed the malignant course of events. Dietary supplementation of Pleurotus ostreatus and Calocybe indica was evaluated in Swiss male albino mice in comparison with casein control diet. Essential nutritional intake and antioxidant enzyme status were analyzed. The results of the present study indicated that Pleurotus ostreatus and Calocybe indica diets can extend the antioxidant activity and enhance the nutritional value. Dietary supplementation of Pleurotus ostreatus and Calocybe indica mushroom significantly increased the production (Body weight gain), food consumption, assimilation and assimilation efficacy near to casein fed mice. Dieting trials showed that the mice consumed the control diet in more quantities than test diets. There was no non- acceptance by the mice for the control and test diets showed no unhealthy symptoms of deficiency, disease or abnormal toxicities were observed in the mice throughout the experimental period. The shelf life period of value added products such as biscuits, wafers and chutney powder developed from Pleurotus ostreatus and Calocybe indica were

6 vi studied. As months progressed, the composition of protein, carbohydrate, fiber and ash content in Pleurotus ostreatus and Calocybe indica biscuits, wafers and chutney powder were found to be decreased when compared to the initial levels (0 th day storage). A gradual increase in Total Plate Count (TPC) was observed for all the products during storage period. No spoilage occurred during the storage period. This could be the preservative action of ingredients on food products till the 6 th month of storage period. No remarkable changes were observed for organoleptic characteristics of biscuits, wafers and chutney powder prepared from Pleurotus ostreatus and Calocybe indica. The technology transfer programme was conducted for various self-help group people of Mundandurai, Dhaliyur and Semmedu villages of Coimbatore District. This study was undertaken to disseminate the nutritive information, delicacy of mushroom and to creative awareness about the protein rich mushroom to the people. The result of the popularization programme revealed that the scores were found to be high for biscuits compared to wafers and chutney powder given by Mundandurai, Dhaliyur and Semmedu self-help group people of Coimbatore District. The present research revealed that the value added products developed from Pleurotus ostreatus and Calocybe indica had good storage period, nutritive value, antioxidant property, antitumor activity and also received good response

7 vii from self-help group people from various villages of Coimbatore district. Based on observations, it was found that the value added products would be included as a regular diet, since it possess rich nutritive value and medicinal properties. Thus, the present study had proven that the utilization of these edible mushrooms would be increased among people of all age groups. Keywords: Mushrooms, Pleurotus ostreatus, Calocybe indica, cancer, antitumor, antioxidant, dietary supplementation, value addition, biscuits, wafers, chutney powder, popularization.

8 viii ACKNOWLEDGEMENT First of all, from the depth of my heart, I m giving glory, honor and praise to my LORD JESUS CHRIST for enabling me to complete my research work successfully. He alone gave his grace, strength, knowledge and wisdom to accomplish everything ideally in my Ph.D. I am grateful to our beloved founders Late Dr. D.G.S. Dhinakaran and Dr. Paul Dhinakaran, the Chancellor of Karunya University, for their love and prayerful support during the course of my research. I extend my thanks to our Vice-Chancellor, Dr. S. Sundar Manoharan and Registrar, Dr. C. Joseph Kennady for giving me this opportunity to do the research work. Sincere thanks to Dr. A. Hepzibah Christinal, Controller of Examinations, Karunya University for her invaluable support, advice and encouragement to complete the research work successfully. I would like to thank Dr. J. Jannet Vennila, Director, SBT & HS, Karunya University, for motivating me to carry out this research work successfully. I am extremely grateful and deeply indebted to my research supervisor Dr. R. Emilin Renitta, Assistant Professor, Dept., of Food Processing and Engineering, Karunya University for her scholarly guidance, constructive suggestions, technical assistance and continuous support given throughout my research. It is my privilege to thank Dr. M. Lakshmi Prabha, Mrs. Reya Issac, and Dr. C. Guruvayoorappan, Assistant Professors, Dept., of Biotechnology, Karunya University for their valuable suggestions during the course of research work. Sincere thanks to my external Doctoral committee members

9 ix Dr. S. F. Maleeka Begam, and Dr. Jemima Beryl Mohankumar, for their guidance and continuous unrelenting support throughout my research. It is a great pleasure to express my sincere and heartfelt thanks to Mr. J. Jackson Daniel, Dept., of Biotechnology, Karunya University for his care, prayer, support and co-operation. I am grateful to all my friends, Ms. Vijila Moses, Dr. J. Giftson Senapathy, Dr. V. Vinod Prabhu, Dr. S. Ganesh, Mr. P. Lakshmikanthan, Mr. P. Suresh Kumar, Mr. K. Sasikumar, Mr. Paul Venkat and Mr. Mark David Samraj without their active involvement, this thesis would not have seen the light of the day. Sincere thanks to all the prayer warriors from Bethesda International Prayer Centre, Coimbatore for their kind support, valuable prayers and constant encouragement in the paths of my research. I dedicate this dissertation to my loving parents Mr. S. Ganapathy, and Mrs. G. Sulochana Mary for their guidance, love, prayer and sacrifice which made me a blessed man in this earth. I thank all my beloved brothers, sisters and my family members who supported me with their sincere love, care, constant encouragement and financial assistance, which made me to achieve the ambition in my life. Without their active involvement, the thesis would not have been accomplished. Signature of Candidate G. JAWAHAR

10 x TABLE OF CONTENTS TITLE DECLARATION BONAFIDE CERTIFICATE ABSTRACT ACKNOWLEDGEMENT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF SYMBOLS AND ABBREVIATIONS PAGE NO ii iii iv viii x xvii xxi xxv CHAPTER 1 - INTRODUCTION General introduction and need for the study Scope of study Aim and objective of the research Objectives 7 CHAPTER 2 - REVIEW OF LITERATURE History of mushrooms Mushrooms Morphology Taxonomical classification of mushrooms Cultivation Nutritional and medicinal properties of P.ostreatus and 13 C.indica mushrooms

11 xi 2.7 Processing and preservation of mushrooms Drying of mushrooms and its importance in preservation Mushrooms as a source of medicine Value addition of products and popularization 20 CHAPTER 3 DRYING CHARACTERISTICS AND 22 BIOCHMICAL COMPOSTION OF DRIED Pleurotus ostreatus AND Calocybe indica USING VIBRATED FLUIDIZED DRYER AND VACUUM SHELF DRYER 3.1 Introduction Materials and methods Mushroom collection Pretreatment of mushroom Drying methods Rehydration characteristics Estimation of protein Determination of total carbohydrate Determination of total lipid Determination of crude fiber Determination of total ash DPPH free radical scavenging activity Determination of minerals by atomic absorption 31 spectrophotometric method Statistical analysis Results Discussion Conclusion 52

12 xii CHAPTER 4 - INVESTIGATION OF ANTITUMOR AND 53 ANTIOXIDANT ACTIVITY OF Pleurotus ostreatus AND Calocybe indica AGAINST DALTON'S LYMPHOMA ASCITES INDUCED TUMOR IN SWISS MALE ALBINO MICE 4.1 Introduction Materials and methods Preparation of ethanolic extracts of P. ostreatus and 55 C. indica Animal care Culturing and administration of DLA cells Experimental groups Acute toxicity studies Body weight analysis Determination of tumor volume Preparation of blood serum and tissue sample for 57 biochemical studies Enumeration of white blood corpuscles (WBC) Enumeration of red blood corpuscles (RBC) Estimation of haemoglobin (Hb) Estimation of lipid peroxidation (LPO) Superoxide dismutase (SOD) assay Determination of catalase (CAT) Glutathione peroxidase (GPx) assay Glutathione S-transferase (GST) assay Reduced glutathione (GSH) assay 61

13 xiii Ascorbic acid (Vitamin C) assay tocopherol (vitamin E) assay Estimation of protein Histopathological examination Statistical analysis Results Acute toxicity studies of P. ostreatus and C. indica Discussion Conclusion 79 CHAPTER 5 - EVALUATION OF DIETARY 81 SUPPLEMENTATION OF Pleurotus ostreatus AND Calocybe indica IN SWISS MALE ALBINO MICE 5.1 Introduction Materials and methods Animals Grouping of mice Preparation of diets Proximate composition analysis of the diets Feeding trials experiment Enzymatic and non-enzymatic antioxidant assay Statistical analysis Results Discussion Conclusion 99

14 xiv CHAPTER 6 - SHELF LIFE ASSESSMENT OF VALUE 100 ADDED PRODUCTS DEVELOPED FROM Pleurotus ostreatus AND Calocybe indica 6.1 Introduction Materials and methods Formulation ratio of biscuits Preparation of P.ostreatus and C.indica biscuits Physical characteristics of P.ostreatus and C.indica 104 powder incorporated biscuits Formulation ratio of P. ostreatus and C. indica wafers Preparation of P. ostreatus and C. indica wafers Formulation ratio of P. ostreatus and C. indica chutney 105 powder Preparation of P. ostreatus and C. indica chutney powder Estimation of protein, carbohydrate, moisture, ash and 107 crude fiber Free fatty acids (FFA) ph Microbial examinations for total plate count (TPC) Sensory analysis Statistical analysis Results Mushroom biscuits Mushroom wafers Mushroom chutney powder Discussion Conclusion 142

15 xv CHAPTER 7 - POPULARIZATION OF VALUE ADDED 143 PRODUCTS FROM Pleurotus ostreatus AND Calocybe indica THROUGH TECHNOLOGY TRANSFER PROGRAMME 7. 1 Introduction Materials and methods Preparation of biscuits, wafers and chutney powder from 145 P. ostreatus and C. indica Popularization of the products Statistical analysis Results Popularization of value added products prepared from 146 P.ostreatus and C.indica to Mundandurai village (Coimbatore) Popularization of value added products prepared from 146 P.ostreatus and C. indica to Dhaliyur village (Coimbatore) Popularization of value added products prepared from 149 P.ostreatus and C. indica to Semmedu village (Coimbatore) 7. 4 Discussion Conclusion 156 CHAPTER 8 - CONCLUSION AND SUGGESTIONS FOR FURTHER RESEARCH Conclusion 8.2 Suggestions for further research

16 xvi REFERENCES 158 APPENDICES 187 LIST OF PUBLICATIONS 188 CURRICULUM VITAE 189

17 xvii LIST OF TABLES TABLE No TITLE Table 2.1 Table 3.1 (a & b) Table 3.2 (a & b) Table 3.3 (a & b) Table 3.4 (a & b) Table 3.5 (a & b) Table 3.6 Table 4.1 Table 5.1 Table 5.2 Table 6.1 Table 6.2 Table 6.3 Table 6.4 (a & b) Taxonomic Classification of P. ostreatus and C. indica Rehydration ratios of dried Mushrooms under different drying conditions Biochemical composition of P. ostreatus and C. indica dried at 50 C, 70 C and 90 C in Vibrated fluidized dryer Biochemical composition of P. ostreatus and C. indica dried at 50 C, 60 C and 70 C in Vacuum shelf dryer Free radical scavenging activity of P. ostreatus and C. indica using DPPH assay Free radical scavenging activity of P. ostreatus and C. indica using DPPH assay Mineral contents of P. ostreatus and C. indica dried at 70 C temperature using Vibrated fluidized dryer Acute toxicity studies of P. ostreatus and C. indica Composition of control and test diets Effect of normal, control, P. ostreatus and C. indica diets on various parameters in Swiss male albino mice Ingredients for P. ostreatus and C. indica biscuits Ingredients for P. ostreatus and C. indica wafers Ingredients for P. ostreatus and C. indica chutney powder Physical characteristics of P. ostreatus and C. indica mushroom powder incorporated biscuits

18 xviii Table 6.5 (a & b) Table 6.6 (a & b) Table 6.7 (a & b) Table 6.8 (a & b) Table 6.9 (a & b) Table 6.10 (a & b) Table 6.11 (a & b) Table 6.12 (a & b) Table 6.13 (a & b) Changes in the biochemical composition of P. ostreatus biscuits stored in aluminum and laminated packaging material at ambient temperature Changes in the ph, moisture and FFA content of P. ostreatus biscuits stored in aluminum and laminated packaging material at ambient temperature Changes in the Total Plate Count (TPC) of P. ostreatus biscuits stored in aluminum and laminated packaging material at ambient temperature Changes in the organoleptic attributes of biscuits stored in aluminum and laminated packaging material at ambient temperature Changes in the biochemical composition of C. indica biscuits stored in aluminum and laminated packaging material at ambient temperature Changes in the ph, moisture and FFA content of C. indica biscuits stored in aluminum and laminated packaging material at ambient temperature Changes in the Total Plate Count (TPC) of C. indica biscuits stored in aluminum and laminated packaging material at ambient temperature Changes in the organoleptic attributes of C. indica biscuits stored in aluminum and laminated packaging material at ambient temperature Changes in biochemical composition of P. ostreatus wafers stored in aluminum and laminated packaging material at ambient temperature

19 xix Table 6.14 (a & b) Table 6.15 (a & b) Table 6.16 (a & b) Table 6.17 (a & b) Table 6.18 (a & b) Table 6.19 (a & b) Table 6.20 (a & b) Table 6.21 (a & b) Table 6.22 (a & b) Changes in the ph, moisture and FFA content of P. ostreatus wafers stored in aluminum and laminated packaging material at ambient temperature Changes in the Total Plate Count of P. ostreatus wafers stored in aluminum and laminated packaging material at ambient temperature Changes in the organoleptic attributes of P. ostreatus wafers stored in aluminum and laminated packaging material at ambient temperature. Changes in the biochemical composition of C. indica wafers stored in aluminum and laminated packaging material in ambient temperature Changes in the ph, moisture and FFA content of C. indica wafers stored in aluminum and laminated packaging material at ambient temperature Changes in the Total Plate Count (TPC) of C. indica wafers stored in aluminum and laminated packaging material at ambient temperature Changes in the organoleptic attributes of C. indica wafers stored in aluminum and laminated packaging material at ambient temperature Changes in the biochemical composition of P. ostreatus and C. indica chutney powder stored in food grade PET jar at ambient temperature Changes in the ph, moisture and FFA content of P. ostreatus and C. indica chutney powder stored in food grade PET jar at ambient temperature

20 xx Table 6.23 (a & b) Table 6.24 (a & b) Table 7.1 (a & b) Table 7.2 (a & b) Table 7.3 (a & b) Changes in the Total Plate Count of P. ostreatus and C. indica chutney powder stored in food grade PET jar at ambient temperature Changes in the organoleptic attributes of P. ostreatus and C. indica chutney powder stored in food grade PET jar at ambient temperature Sensory scores given by self-help group people of Mundandurai village (Coimbatore) for the value added products developed from P. ostreatus and C. indica Sensory scores given by self-help group people of Dhaliyur village (Coimbatore) for the value added products developed from P. ostreatus and C. indica Sensory scores given by self-help group people of Semmedu village (Coimbatore) for the value added products developed from P. ostreatus and C. indica

21 xxi LIST OF FIGURES Figure 2.1 Figure 2.2 Figure 3.1 Figure 3.2 Pleurotus ostreatus (Oyster Mushroom) Calocybe indica (Milky Mushroom) Drying of P. ostreatus and C. indica in Vibrated fluidized dryer and Vacuum shelf dryer Moisture Content of P. ostreatus dried at 50 C, 70 C and 90 C using Vibrated fluidized Dryer Figure 3.3 Moisture Content of C. indica dried at 50 C, 70 C and 90 C using Vibrated fluidized Dryer Figure 3.4 Drying rate of P. ostreatus dried at 50 C, 70 C and 90 C using Vibrated fluidized Dryer Figure 3.5 Figure 3.6 Drying rate of C. indica at dried 50 C, 70 C and 90 C using Vibrated fluidized Dryer Moisture Content of P. ostreatus dried at 50 C, 60 C and 70 C using Vacuum shelf dryer Figure 3.7 Moisture Content of C. indica dried at 50 C, 60 C and 70 C using Vacuum Shelf Dryer Figure 3.8 Drying rate of P. ostreatus dried at 50 C, 60 C and 70 C using Vacuum Shelf Dryer Figure 3.9 Drying rate of C. indica dried at 50 C, 60 C and 70 C using Vacuum Shelf Dryer

22 xxii Figure Figure Figure Figure Figure Figure Figure Figure Changes in the body weight of Dalton s lymphoma ascites bearing mice after P. ostreatus and C. indica treatment Changes in the tumor fluid volume of Dalton s lymphoma ascites bearing mice after P. ostreatus and C. indica treatment Changes in the total RBC of Dalton s lymphoma ascites bearing mice after P. ostreatus and C. indica treatment Changes in the total WBC count of Dalton s lymphoma ascites bearing mice after P. ostreatus and C. indica treatment Changes in the Haemoglobin (Hb) of Dalton s lymphoma ascites bearing mice after P. ostreatus and C. indica treatment Changes in the Lipid Peroxidation level in the liver of Dalton s lymphoma ascites bearing mice after P. ostreatus and C. indica treatment Changes in the activities of Superoxide Dismutase (SOD) in the liver of Dalton s lymphoma ascites (DLA) bearing mice after P. ostreatus and C. indica treatment Changes in the activities of Catalase (CAT) in the liver of Dalton s lymphoma ascites (DLA) bearing mice after P. ostreatus and C. indica treatment

23 xxiii Figure Figure Figure Figure Figure Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Changes in the activities of Glutathione Peroxidase (GPx) in the liver of Dalton s lymphoma ascites (DLA) bearing mice after P. ostreatus and C. indica treatment Changes in the activities of Glutathione S-Transferase (GST) in the liver of Dalton s lymphoma ascites (DLA) bearing mice after P. ostreatus and C. indica treatment Changes in the activities of Reduced Glutathione (GSH) in the liver of Dalton s lymphoma ascites (DLA) bearing mice after P. ostreatus and C. indica treatment Changes in the activities of Vitamin C in the liver of Dalton s lymphoma ascites (DLA) bearing mice after P. ostreatus and C. indica treatment Changes in the activities of Vitamin E in the liver of Dalton s lymphoma ascites (DLA) bearing mice after P. ostreatus and C. indica treatment Biochemical composition of diet Effect of normal, control, P. ostreatus and C. indica diets on liver Superoxide Dismutase (SOD) activity of Swiss male albino mice Effect of normal, control, P. ostreatus and C. indica diets on liver Catalase (CAT) activity of Swiss male albino mice Effect of normal, control, P. ostreatus and C. indica diets on liver Glutathione Peroxidase (GPx) activity of Swiss male albino mice

24 xxiv Figure 5.5 Figure 5.6 Figure 5.7 Figure 6.1 Figure 6.2 Figure 6.3 Figure 7.1 Figures 7.2 Figures 7.3 Effect of normal, control, P. ostreatus and C. indica diets on liver Reduced Glutathione (GSH) activity of Swiss male albino mice Effect of normal, control, P. ostreatus and C. indica diets on liver Vitamin C activity of Swiss male albino mice Effect of normal, control, P. ostreatus and C. indica diets on liver Vitamin E activity of Swiss male albino mice Biscuits developed from P. ostreatus and C. indica Mushroom wafers developed from P. ostreatus and C. indica Mushroom chutney powder developed from P. ostreatus and C. indica Popularization of value added products prepared from P. ostreatus and C. indica to self-help group people of Mundandurai village Popularization of value added products prepared from P. ostreatus and C. indica to self-help group people of Dhaliyur village Popularization of value added products prepared from P. ostreatus and C. indica to self-help group people of Semmedu village

25 xxv LIST OF SYMBOLS AND ABBREVIATIONS % - Percentage C - Degree centigrade µg - Microgram µl - Microlitre µm - Micromolar ANOVA - Analysis of variance b.wt - Body weight BSA - Bovine serum albumin C. indica - Calocybe indica CAT - Catalase CDNB - 1-Chloro-2,4-dinitrobenzene DLA - Dalton's lymphoma ascites DMRT - Duncan s Multiple Range Test DNA - Deoxyribonucleic acid DPPH - 2, 2-Diphenyl-1-picrylhydrazyl EDTA - Ethylene diamine tetra acetic acid FFA - Free fatty acid GCMS - Gas Chromatography Mass Spectrometry GPx - Glutathione Peroxidase GR - Glutathione Reductase GSH - Reduced glutathione GST - Glutathione S - Transferase H&E - Hematoxylin and Eosin H2O2 - Hydrogen Peroxide Hb - Hemoglobin Hcl - Hydrochloric acid i.p. - Intraperitoneal

26 xxvi Kg - kilograms M - Morality mg - milligrams ml - Milliliter N - Normality Nacl - Sodium Chloride NBT - Nitro-blue tetrazolium NO - Nitric oxide OD - Optical Density OECD - Organisation for Economic Co-operation and Development P. ostreatus - Pleurotus ostreatus PBS - Phosphate buffered saline PMS - Phenazine methosulfate RBC - Red blood corpuscles RNA - Ribonucleic acid RNS - Reactive nitrogen species ROS - Reactive oxygen species S.D. - Standard Deviation SOD - Superoxide dismutase TBA - Thiobarbituric acid TBARS - Thiobarbituric acid reactive substances TCA - Trichloroacetic acid TPC - Total plate count V/V - Volume by volume W/V - Weight by volume WBC - White blood cells WHO - World Health Organization