University of Ghana http://ugspace.ug.edu.gh COLLEGE OF BASIC AND APPLIED SCIENCES SCHOOL OF BIOLOGICAL SCIENCES MICROBIAL INACTIVATION BY GAMMA IRRADIATION OF POWDERED SUN-DRIED LEGON-18 PEPPER (CAPSICUM ANNUUM L.) AND ITS IMPACT ON PRODUCT QUALITY BY BERNARD TAWIAH ODAI THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF DOCTOR OF PHILOSOPHY DEGREE IN FOOD SCIENCE DEPARTMENT OF NUTRITION AND FOOD SCIENCE July, 2019 University of Ghana http://ugspace.ug.edu.gh DECLARATION i University of Ghana http://ugspace.ug.edu.gh DEDICATION This thesis is dedicated to the Glory of God and to the memory of my late daddy, Julius Nii-Odai who was a source of motivation to me in my academic endeavour. ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGMENTS My sincerest and profound gratitude to the Almighty God for bringing me this far in my career. I am grateful to all my supervisors for their support throughout this study. Grateful for their inputs. Thanks to the Ghana Atomic Energy Commission especially, Prof. B. J. B. Nyarko, Prof. K. E. Danso and Mr. A. Adu-Gyamfi for the permission granted me to pursue the programme. I am highly indebted to Mr. M. L. Akye and Mr. C. Bonsu of Noguchi Memorial Institute for Medical Research (NMIMR) for the various assistance offered during the period of working in the Bacteriology Department of the Institute. I am grateful for the assistance offered to me by the staff of the Central Laboratory at KNUST, Dr. D. Azanu, Mr. W. Appaw and colleagues, and Mr. Solomon Dowuona of Food Research Institute. Finally, I so much appreciate the supportive role played by family, especially my mother and siblings. iii University of Ghana http://ugspace.ug.edu.gh ABSTRACT Pepper powder (being a spice) has been known to be contaminated with several pathogenic microorganisms. These organisms tend to make red pepper powder a source of potential health hazard. The FAO/WHO preliminary report indicated a global concern on the management of these pathogens in foods and the need to reduce or eliminate the health hazards associated with them. Some of these pathogens have been identified with both dried and powdered pepper samples in Ghana. This warranted a study to investigate the use of gamma irradiation on these pathogens in Legon-18 (Capsicum annuum L.) pepper powder and also to determine the impact of the gamma radiation treatment on the quality parameters of the samples stored at two different temperatures. Samples of powdered Legon-18 pepper were obtained from a local farmer. Known weights of the samples were sterilised by gamma irradiation at 20 kGy, and a cocktail of Listeria monocytogenes, Bacillus cereus, Salmonella Typhimurium, Staphylococcus aureus and Escherichia coli of pre-determined cell count (colony forming unit/millilitre), were inoculated into them. The samples were irradiated at 1, 2, 4, and 5 kGy with 0 kGy as control, to determine an effective dose of gamma irradiation that could lead to complete inactivation of the pathogens inoculated in the samples. The samples were stored at 4 oC and 28±2 oC. Enumeration of the different pathogens was carried out on days 0, 2, 5, 12, 21, 30, 45 and 60 in storage. The effects of gamma irradiation and storage on the quality parameters of unsterile samples were determined. These were irradiated at 1, 2, 4 and 5 kGy. Unirradiated samples served as control (0 kGy). The CIELAB colour components were determined using the Minolta Chroma-meter. Carotenoids and capsaicinoids in the samples were quantified using high performance liquid chromatography. The results suggest that gamma irradiation treatment completely inactivated L. monocytogenes and S. Typhimurium only at day 60 at 2 and 4 kGy. E. coli could not thrive in the samples after 30 days of storage when not exposed to gamma irradiation. S. aureus could be completely inactivated at 4 kGy only after 45 days (no detection of S. aureus in the samples after). All iv University of Ghana http://ugspace.ug.edu.gh the pathogens could be completely inactivated at 2, 4 and 5 kGy. The optimum dose for complete inactivation of the pathogens excluding B. cereus was 2 kGy which is subject to a storage period of over 45 days. All pathogens used in the study were completely inactivated at 5 kGy even on day zero. Gamma irradiation treatments and storage significantly (p<0.05) affected the quality parameters of the samples. Losses of colour parameters, carotenoids and capsaicinoids were more pronounced in the samples that were stored at 28±2 oC as compared with the samples that were stored at 4 oC. Percentage losses for the samples stored at 4 oC were in the range of 83.32 and 83.81%, 50.00% to 53.72%, 33.53% to 37.80%, 54.52% to 58.60%, 40.25% and 56.00%, 78.35 to 81.71% and percentage increase in the range of 372.36% to 429.14% for lightness, redness, yellowness, browning index, chroma, hue and total colour difference respectively. The total colour difference, hue, chroma, browning index, yellowness, redness and lightness of the samples that were stored at 28±2 oC were in the range of 410.72% to 417.50%, 80.13% to 84.75%, 39.55% to 57.00 %, 50.69% to 54.02%, 32.98% to 37.37%, 51.86 to 55.06, 72.77 to 76.98% respectively. Moisture content, total titratable acidity and pH of the samples were stable. Capsaicinoid content ranged from 118 in the unirradiated samples to 221.00 (mg/100g) in the irradiated samples. At the end of the storage period there was a loss of 22.46%, 9.95%, 11.78%, 9.86% and 9.53% in the samples that were irradiated at 0, 1, 2, 4and 5 kGy respectively and stored at 4 oC and for the unirradiated samples, 16.43%, 11.11%, 10.31% and 10.67% for the samples that were irradiated at 1, 2, 4 and 5 kGy respectively and stored at 28±2 oC. Gamma irradiation caused an increase of 6.33, 17.68, 18.79 % and 20.95% in the samples irradiated at 1, 2, 4 and 5 kGy respectively. Beta cryptoxanthin, beta carotene and capsanthin ranged from 1.04 to 2.11, 5.36 to 10.27 and 1.12 to 1.48 (mg/100 g) in the irradiated samples, respectively. Gamma irradiation and storage caused some reductions in the contents of all the pigments analysed (p<0.05). v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ................................................................................................................. i DEDICATION .................................................................................................................... ii ACKNOWLEDGMENTS ................................................................................................. iii ABSTRACT ...................................................................................................................... iv TABLE OF CONTENTS .................................................................................................. vi LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES ......................................................................................................... xiii LIST OF ABREVIATIONS ............................................................................................. xv CHAPTER ONE ................................................................................................................. 1 1.0 GENERAL INTRODUCTION .................................................................................... 1 1.1 Background ............................................................................................................... 1 1.2 Rationale ................................................................................................................... 2 1.3 Objectives .................................................................................................................. 3 1.3.1 Main Objective ................................................................................................ 3 1.3.2 Specific objectives .......................................................................................... 4 CHAPTER TWO ................................................................................................................ 5 2. 0 LITERATURE REVIEW ............................................................................................ 5 2.1 Spices ........................................................................................................................ 5 2.2 Peppers: origin, botany and production .................................................................... 6 2.3 Nutritional Composition ........................................................................................... 7 2.4 World production and economic importance .......................................................... 10 2.5 Characteristics and quality indices of chili pepper and pepper products ................ 11 2.5.1 Colour ............................................................................................................ 11 2.5.2 Pungency ....................................................................................................... 12 2.6 Uses of chili pepper and pepper products ............................................................... 14 2.7 Pepper production in Ghana .................................................................................... 14 2.8 Microbial ecology of chili pepper powder .............................................................. 15 2.9 Processing ............................................................................................................... 15 2.9.1 Pepper powder ............................................................................................... 16 2.9.2 Harvesting of pepper fruits ............................................................................ 16 2.9.3 Pre-treatment ................................................................................................. 17 2.9.4 Drying of the raw material ............................................................................ 17 2.9.4.1 Sun drying .................................................................................................. 17 2.9.4.2 Solar drying ............................................................................................... 18 2.9.4.3 Super-heated steam (SHS) ......................................................................... 18 vi University of Ghana http://ugspace.ug.edu.gh 2.10. Cleaning ............................................................................................................... 18 2.11 Milling (Particle size reduction) ........................................................................... 19 2.12 Packaging and storage ........................................................................................... 19 2.13 Microbial decontamination of chili pepper powder .............................................. 19 2.13.1 Super-heated steam ..................................................................................... 19 2.13.2 Radio frequency heating ............................................................................. 20 2.13.3 Food Irradiation (gamma rays, electron beams and X-rays) ....................... 20 2.13.4 General classification of food irradiation doses .......................................... 21 2.14 Use of Gamma rays in microbial decontamination of foods .......................... 22 2.15 Regulation of food irradiation ........................................................................ 23 CHAPTER THREE .......................................................................................................... 24 3.0 MATERIALS AND METHODS ............................................................................... 24 3.1 Study design ............................................................................................................ 24 3.1.1 First phase ..................................................................................................... 24 3.1.2 Second phase ................................................................................................. 24 3.2 Pepper powder samples and microbial culture ....................................................... 25 3.2.1 Pepper sample ............................................................................................... 25 3.2.1.1 State of pepper samples .............................................................................. 26 3.2.2 Microbial pathogens ...................................................................................... 26 3.2.3 Sample preparation ........................................................................................ 26 3.3 Microbial inactivation of inoculated pathogens in pepper samples ........................ 27 3.3.1 Inoculum preparation .................................................................................... 27 3.3.1.1 Salmonella Typhimurium, Escherichia coli .............................................. 27 3.3.1.2 Listeria monocytogenes ............................................................................. 27 3.3.1.3 Bacillus cereus ........................................................................................... 28 3.3.1.4 Staphylococcus aureus ............................................................................... 28 3.3.2 Inoculum formulation and inoculation of pepper samples ............................ 29 3.3.3 Irradiation of powdered pepper samples ....................................................... 29 3.3.4 Microbial enumeration using viable counts .................................................. 30 3.4 Determination of the effect of gamma irradiation on quality and physicochemical parameters of pepper powder ........................................................................................ 31 3.4.1 Irradiation of Samples ................................................................................... 31 3.4.2 Determination of colour ................................................................................ 32 3.4.2.1 Hue, Chroma, colour difference and browning index ............................... 32 3.4.3 Moisture content ............................................................................................ 33 3.4.4 pH .................................................................................................................. 33 3.4.5 Total titratable acidity ................................................................................... 34 vii University of Ghana http://ugspace.ug.edu.gh 3.4.6 Carotenoids ................................................................................................... 34 3.4.6.1 Source of standards and reagents ............................................................... 34 3.4.6.2 Extraction ................................................................................................... 34 3.4.6.3 Quantification ............................................................................................ 35 3.4.7 Capsaicinoids ................................................................................................ 35 3.4.7.1 Extraction ................................................................................................... 35 3.4.7.2 Quantification ............................................................................................ 36 3.4.7.3 Scoville heat units ...................................................................................... 36 3.5 Data analysis ........................................................................................................... 36 CHAPTER FOUR ............................................................................................................ 37 4.0 RESULTS AND DISCUSSION ................................................................................. 37 4.1 Characteristics of pepper samples used .................................................................. 37 4.1.1 Sample sterilization and inoculation with cocktail of pathogens. ................. 37 4.2 Microbial inactivation of inoculated pathogens in sun-dried Legon-18 pepper powder ........................................................................................................................... 37 4.2.1 Effect of gamma irradiation, storage time and temperature on the inactivation of Salmonella enterica Typhimurium ................................................... 38 4.2.2 Effect of gamma irradiation, storage time and temperature on the inactivation of Escherichia coli ................................................................................. 40 4.2.3 Effect of gamma irradiation, storage time and temperature on the inactivation of Bacillus cereus .................................................................................. 42 4.2.4 Effect of gamma irradiation, storage time and temperature on the inactivation of Listeria monocytogenes ..................................................................... 43 4.2.5 Effect of gamma irradiation, storage time and temperature on the inactivation of Staphylococcus aureus ...................................................................... 44 4.2.6 Effect of gamma irradiation on all the organisms at 4 oC and 28±2 oC. ........ 46 4.3 The effect of gamma irradiation on the quality parameters of pepper powder ....... 49 4.3.1 Effect on colour ............................................................................................. 49 4.3.1.1 Effect on L* values .................................................................................... 49 4.3.1.2 Effect on a* values ..................................................................................... 51 4.3.1.3 Effect on b* values .................................................................................... 53 4.3.1.4 Effect on chroma ........................................................................................ 54 4.3.1.5 Effect on browning index .......................................................................... 56 4.3.1.6 Effect on colour difference ........................................................................ 58 4.3.1.7 Effect on hue .............................................................................................. 60 4.3.2 Effect on physiochemical properties ............................................................. 62 4.3.2.1 Effect on pH and Titratable acidity (TTA) ................................................ 62 viii University of Ghana http://ugspace.ug.edu.gh 4.3.2.2 Effect on moisture content ......................................................................... 64 4.3.3 Effect of gamma irradiation and storage on the capsaicinoids and SHU ...... 65 4.3.3.1 Effect on capsaicin ..................................................................................... 65 4.3.3.2 Effect on dihydrocapsaicin ........................................................................ 67 4.3.3.3 Effect on total capsaicinoids ...................................................................... 69 4.3.3.4 Effect on Scoville Heat Units (hotness index) ........................................... 71 4.3.4 Impact of gamma irradiation and storage weeks on the carotenoids content 73 4.3.4.1 Effect on beta cryptoxanthin ...................................................................... 73 4.3.4.2 Effect on beta carotene .............................................................................. 75 4.3.4.3 Effect on capsanthin .................................................................................. 76 4.4 Optimization of microbial inactivation by gamma irradiation and storage days on S. Typhimurium, E. coli, B. cereus, L. monocytogenes and S. aureus using response surface methodology (Central Composite Design) ....................................................... 78 4.4.1 Salmonella Typhimurium .............................................................................. 78 4.4.2 Escherichia coli ............................................................................................. 80 4.4.3 Bacillus cereus .............................................................................................. 82 4.4.4 Listeria monocytogenes ................................................................................ 83 4.4.5 Staphylococcus aureus .................................................................................. 86 4.5 Optimization of the effect of gamma irradiation and storage on the quality and components of legon-18 pepper samples (Central Composite Design) ........................ 87 4.5.1 L* values ....................................................................................................... 87 4.5.2 a* values ....................................................................................................... 89 4.5.3 b* values ....................................................................................................... 91 4.5.4 Chroma ......................................................................................................... 93 4.5.5 Browning index ............................................................................................ 94 4.5.6 Total colour difference ................................................................................. 96 4.5.7 Hue ................................................................................................................ 97 4.5.8 pH ................................................................................................................. 99 4.5.9 Moisture content ......................................................................................... 101 4.5.10 Titratable acidity ....................................................................................... 102 4.5.11 Capsaicin ................................................................................................... 104 4.5.12 Dihydrocapsaicin ...................................................................................... 105 4.5.13 Total capsaicin .......................................................................................... 107 4.5.14 Scoville Heat Units (SHU) ....................................................................... 108 4.5.15 Beta carotene ............................................................................................ 110 4.5.16 Beta cryptoxanthin .................................................................................... 112 4.5.17 Capsanthin ................................................................................................ 113 ix University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE ............................................................................................................ 115 5.0 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS ............................ 115 5.1 Summary ............................................................................................................... 115 5.2 Conclusions ........................................................................................................... 117 5.3 Recommendations ................................................................................................. 117 REFERENCES ............................................................................................................... 118 APPENDICES ................................................................................................................ 139 x University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 2.1. Spice classification based on plant part ............................................................. 5 Table 2.2. Classification of spices based on flavour .......................................................... 5 Table 2.3. Proximate composition of dried chili pepper powder ....................................... 8 Table 2.4. General Elemental composition of dried chili pepper powder .......................... 8 Table 2.5. General nutrient composition of dried chili pepper powder .............................. 9 Table 3.1. Central composite rotational design matrix (template) for two independent variables for inactivation of pathogens at both temperatures ...................... 25 Table 3.2. Central composite rotational design matrix (template) for two independent variables on quality parameters at both temperatures .................................. 25 Table 4.1. Effect of gamma irradiation and storage on the survival of S. Typhimurium at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) ................... 39 Table 4.2. Effect of gamma irradiation and storage on the survival of E. coli at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) .................................. 41 Table 4.3. Effect of gamma irradiation and storage on the inactivation of B. cereus at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) ...................... 42 Table 4.4. Effect of gamma irradiation and storage on the survival of L. monocytogenes at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) ................ 44 Table 4.5. Effect of gamma irradiation and storage on the survival of S. aureus at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) ........................... 45 Table 4.6. Effect of gamma radiation on the survival of microorganisms in powdered Legon-18 pepper (C. annuum) at 4 oC. ........................................................ 47 Table 4.7. Effect of gamma radiation on the survival of microorganisms in powdered Legon-18 pepper (C. annuum) at 28±2 oC ................................................... 48 Table 4.8. L* values of Legon-18 pepper powder after gamma irradiation, and during storage at different storage conditions. ........................................................ 50 Table 4.9. The a* values of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ................................................................. 52 Table 4.10. B*values of Legon-18 pepper powder after gamma irradiation, during storage at different temperatures ................................................................. 54 Table 4.11. Chroma of Legon-18 pepper powder after gamma irradiation, during storage at different temperatures .............................................................................. 55 xi University of Ghana http://ugspace.ug.edu.gh Table 4.12. Browning index of Legon-18 pepper powder after gamma irradiation, during storage at different temperatures ................................................................. 57 Table 4.13. Colour difference of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ...................................................... 59 Table 4.14. Hue of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures .............................................................................. 61 Table 4.15. The pH of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ................................................................. 62 Table 4.16. Total titratable acidity of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ............................................... 63 Table 4.17. Moisture content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ...................................................... 64 Table 4.18. Capsaicin content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ...................................................... 66 Table 4.19. Dihydrocapsaicin content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ............................. 69 Table 4.20. Total capsaicinoids of Legon-18 pepper powder after gamma irradiation and during storage at different temperatures ...................................................... 70 Table 4.21. Scoville heat unit of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ...................................................... 72 Table 4.22. Beta cryptoxanthin content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ............................. 74 Table 4.23. Beta carotene content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures ............................................... 76 Table 4.24. Capsanthin content of Legon-18 pepper powder pepper powder after gamma irradiation, and during storage at different temperatures ............................. 77 xii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Fig. 2.1. Flow chart for chili (red) pepper powder production (Source: Schweiggert et al., 2007). .............................................................................................................. 16 Fig. 3.1. Bulked Samples in pouches for irradiation ........................................................ 29 Fig. 3.2. Samples on palette in irradiation chamber (area) at the Gamma Irradiation Facility. ........................................................................................................... 30 Fig. 3.3. Packed Legon-18 pepper samples for gamma irradiation .................................. 31 Fig. 3.4. Pepper samples on palette in irradiation chamber (area) at the Gamma Irradiation Facility .......................................................................................... 32 Fig. 4.1. Effect of gamma irradiation and storage on the survival of S. Typhimurium at 4oC. ................................................................................................................. 78 Fig. 4.2. Effect of gamma irradiation and storage on the survival of S. Typhimurium at 28±2 oC ........................................................................................................... 79 Fig. 4.3. Effect of gamma irradiation and storage on the survival of E. coli at 4oC. ........ 80 Fig. 4.4. Effect of gamma irradiation and storage on the survival of E. coli at 28±2 oC. . 81 Fig. 4.5. Effect of gamma irradiation and storage on the survival of B. cereus at 4oC. ... 82 Fig. 4.6. Effect of gamma irradiation and storage on the survival of B. cereus at 28±2 oC. ........................................................................................................................ 83 Fig. 4.7. Effect of gamma irradiation and storage on the survival of L. monocytogenes at 4oC. ................................................................................................................. 84 Fig. 4.8. Effect of gamma irradiation and storage on the survival of L. monocytogenes at 28±2 oC. .......................................................................................................... 85 Fig. 4.9. Effect of gamma irradiation and storage on the survival of S. aureus at 4oC. ... 86 Fig. 4.10. Effect of gamma irradiation and storage on the survival of S. aureus at 28±2 oC ........................................................................................................................ 87 Fig. 4.11. L* values after gamma irradiation and during storage stored at 4 oC .............. 88 Fig. 4.12. L* values after gamma irradiation and during storage at 28±2 oC ................... 89 Fig. 4.13. a* values after gamma irradiation and during storage at 4 oC. ......................... 90 Fig. 4.14. a* values after gamma irradiation and during storage at 28±2 oC .................... 91 Fig. 4.15. b* values after gamma irradiation and during storage at 4 oC .......................... 92 Fig. 4.16. b* values after gamma irradiation and during storage at 28±2 oC ................... 92 Fig. 4.17. Chroma after gamma irradiation and during storage at 4 oC ............................ 93 Fig. 4.18. Chroma after gamma irradiation and during storage at 28±2 oC ...................... 94 Fig. 4.19. Browning index after gamma irradiation and during storage at 4 oC ............... 95 Fig. 4.20. Browning index after gamma irradiation and during storage at 28±2 oC ......... 96 xiii University of Ghana http://ugspace.ug.edu.gh Fig. 4. 21. Total Colour Difference after gamma irradiation and during storage at 4 oC . 97 Fig. 4.22. Total Colour Difference after gamma irradiation and during storage at 28±2 o ........................................................................................................................ 97 Fig. 4.23. Hue after gamma irradiation and storage at 4 oC ............................................. 98 Fig. 4.24. Hue after gamma irradiation and storage at 28±2 oC ....................................... 99 Fig. 4.25. pH after gamma irradiation and during storage at 28±2 oC. ........................... 100 Fig. 4.26. pH after gamma irradiation and during storage at 4 oC. ................................. 100 Fig. 4.27. Moisture content (%) after gamma irradiation and during storage at 4 oC ..... 101 Fig. 4.28. Moisture content (%) after gamma irradiation and during storage at 28±2 oC. ...................................................................................................................... 102 Fig. 4.29. Titratable acidity after gamma irradiation and during storage at 4 oC ........... 103 Fig. 4.30. Titratable acidity after gamma irradiation and during storage 28±2 oC ......... 103 Fig. 4.31. Capsaicin content after gamma irradiation and during storage at 4 oC .......... 104 Fig. 4.32. Capsaicin content after gamma irradiation and during storage at 28±2 oC .... 105 Fig. 4.33. Dihydrocapsaicin content after gamma irradiation and during storage at 4 oC ...................................................................................................................... 106 Fig. 4.34. Dihydrocapsaicin content after gamma irradiation and during storage at 28±2 oC .................................................................................................................. 106 Fig. 4.35. Total capsaicinoids content after gamma irradiation and during storage at 4 o ...................................................................................................................... 107 Fig. 4.36. Total capsaicinoids content after gamma irradiation and during storage at 28±2 oC .................................................................................................................. 108 Fig. 4.37. SHU after gamma irradiation and during storage at 4 oC ............................... 109 Fig. 4.38. SHU after gamma irradiation and during storage at 28±2 oC ......................... 109 Fig. 4.39. Beta carotene content after gamma irradiation and during storage at 4 oC .... 111 Fig. 4.40. Beta carotene content after gamma irradiation and during storage at 28±2 oC. ...................................................................................................................... 111 Fig. 4.41. Beta cryptoxanthin content after gamma irradiation and during storage at 4 oC. ...................................................................................................................... 112 Fig. 4.42. Beta cryptoxanthin content after gamma irradiation and during storage at 28±2 oC. ................................................................................................................. 113 Fig. 4.43. Capsanthin content after gamma irradiation and during storage at 4 oC. ....... 114 Fig. 4.44. Capsanthin content of gamma irradiation and during storage in Legon-18 stored at 28±2 oC. .......................................................................................... 114 xiv University of Ghana http://ugspace.ug.edu.gh LIST OF ABREVIATIONS ANOVA Analysis of variance AOAC Association of Official and Applied Chemists AU Australia BD Becton Dickinson BHI Brain heart infusion BI Browning index BPA Baird Parker Agar BPW Buffered peptone water CDC Centre for Disease Control and Prevention CFU Colony forming unit CT-SMAC Cefixime tellurite sorbitol MacConkey DOH Department of Health EU European Union FAO Food and Agriculture Organization FSAI Food Safety Authority of Ireland HPLC High performance liquid chromatography IAEA International Atomic Energy Agency MOFA Ministry of Food and Agriculture MYP Mannitol egg yolk polymyxin NMIMR Noguchi Memorial Institute for Medical Research PBS Phosphate Buffer Saline PLS Palcam Listeria selective SHU Scoville Heat Units xv University of Ghana http://ugspace.ug.edu.gh TSB Tryptic soy broth TTA Total titratable acidity USDA United States Department of Agriculture WHO World Health Organization XLD Xylose lysine deoxycholate YE Yeast extract xvi University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 GENERAL INTRODUCTION 1.1 Background Red pepper (Capsicum annuum), commonly known as chili, has been identified as one of the widely used spices in the world (Lee et al., 2004). It is considered as the second most important vegetable after tomato (Liu et al., 2013; Ali, 2006; Ochoa-Alejo and Ramirez- Malagon, 2001; Yoon, 1989). Chili is produced in several countries (Pinto et al., 2016; Rufino and Penteado, 2006). The ripened matured fruits (red in colour) are harvested and processed into fine powder. The powder is used as a spice, food colourant, a flavouring (Jung et al., 2015) and for the preparation of a special sauce known as ‘shito’ in Ghana (Doku, 2015). The commercial quality indices of red pepper powder include the pungency and colour (Jung et al., 2015). The pungency (hotness) of red pepper powder is due to the presence of capsaicinoids. Dihydrocapsaicin and capsaicin are the main capsaicinoids responsible for the hotness of pepper (Jung et al., 2015; Orellana-Escobedo et al., 2013; Lee et al., 2004). Capsanthin is the main carotenoid that gives pepper its red colour (Jung et al., 2015; Giuffrida et al., 2013). Processed spices, including pepper powder, have been known to be reservoirs of microorganisms (Greig, 2015; Jung et al., 2015; Witkowska et al., 2011; DOH/VICTORIA/AU, 2010; Kahraman and Ozmen, 2009; Shamsuddeen, 2009; Bhunia, 2008; Koch et al., 2005; Kaul and Taneja, 1989; Kovács-Domján; 1988; Christensen et al., 1967) owing to their agricultural origin and processing methods (Jung et al., 2015; Buckenhuskes and Rendlen, 2004; Oularbi and Mansouri, 1996). Piggott and Othman 1 University of Ghana http://ugspace.ug.edu.gh (1993) and Boer et al. (1985) indicated that pepper can have high microbial contamination of viable counts exceeding 10 7 colony forming unit per gram with most of these being spore formers. Pathogenic bacteria such as Salmonella Montevideo have been associated with pepper in the United States of America (CDC, 2010). Other pathogenic bacteria associated with pepper include Staphylococcus aureus, Escherichia coli, spore formers such as Bacillus subtillis, B atrophaeus, B. cereus and fungi such as Aspergillus flavus, A. parasiticus, A. nomius, A. pseudotarii (Koohy‐Kamaly-Dehkordy et al., 2013; Oh et al., 2012; Higa, 2011; Aydin et al., 2007; Buckenhuskes and Rendlen, 2004; Gustavsen and Breen, 1984). Listeria monocytogenes, a soil borne pathogen, has also been associated with pepper (Kara et al., 2015). 1.2 Rationale Microbial decontamination methods that have been used in spice such as pepper, include the use of fumigants (ethylene oxide, gaseous ozone) and irradiation techniques such as ultra-violet decontamination, radio frequency heating, cold plasma treatment, gamma irradiation, electron beam and x-rays sterilization (Jung et al., 2015; Cheon et al., 2015; Kim et al., 2013; Witkowska et al., 2011; Rico et al., 2010; Cember and Johnson, 2009; Schweiggert et al., 2007; Gregoire et al., 2003; Oslon, 1998 ). Some of these methods tend to have adverse effects on the quality (both chemical and sensorial) parameters of the spices. The use of ethylene oxide has been banned by the European Union because it is now classified as a carcinogen (Lilie et al., 2007; Shweggert et al., 2007; Almela et al., 2002; Tainter and Grenis, 2001). The Joint Food and Agriculture Organization (FAO), the International Atomic Energy Agency (IAEA) and the World Health Organization (WHO) have approved the use of gamma irradiation for the decontamination of foods and it is used in over 51 countries (IAEA, 2008; WHO, 1981). 2 University of Ghana http://ugspace.ug.edu.gh Pathogenic microorganisms have been associated with powdered pepper in Ghana (Saba and Gonzalez, 2012). The occurrence of pathogens in powdered pepper may be due to handling and processing conditions (Yankey, 2014). The FAO and WHO expert consultation panel on ranking of low moisture foods lists Listeria monocytogenes, Bacillus cereus, Salmonella spp, Staphylococcus aureus, and pathogenic E. coli as some of the microbial hazards associated with low moisture foods which include powdered products (WHO/FAO, 2014). Effective strategies for the reduction or complete elimination of these health hazards in low moisture foods are urgently required. This study therefore is in response to such a research gap to deliver powdered pepper with low microbial loads. The goal was towards reducing the food safety risks associated with powdered pepper using gamma radiation. This is in line with suggestion by other authors (Farkas and Andrassy, 1998; Byun et al., 1996; Cho et al., 1986) that gamma irradiation is more effective for the decontamination of spices than other methods such as ozone treatment, steam-heat treatment and ethylene oxide without any adverse effects. 1.3 Objectives 1.3.1 Main Objective The main objectives of the study were to investigate the microbial decontamination of pepper (Capsicum annuum L.) powder using gamma irradiation and to determine the effects of irradiation on the quality parameters of the pepper powder stored at different temperatures. 3 University of Ghana http://ugspace.ug.edu.gh 1.3.2 Specific objectives The specific objectives of the study were to: 1. investigate inactivation effects of gamma irradiation on pathogenic microorganisms in powdered pepper 2. determine the effect of irradiation treatment for microbial inactivation on the quality of the powder 3. determine the optimum irradiation conditions (dosage and storage time) for the effective reduction of pathogens in powdered pepper 4 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2. 0 LITERATURE REVIEW 2.1 Spices These are plant parts that are used based on their flavor and other properties (Embuscado, 2015; Herman, 2015). They can be classified into various groups such as their taxonomy, taste or flavor, or the part of the plant from which they were obtained (El-Sayed and Youssef, 2019; Embuscado, 2015; Herman, 2015). Tables 2.1 and 2.2 indicate the various classification of spices. Table 2.1. Spice classification based on plant part Plant part Spice Aril Mace Bulbs Onion, garlic, leek Root Ginger, turmeric Seed Dill, mustard, fennel, nut meg, fenugreek Leaves Basil, oregano, baby leaf, thyme Fruits Chili, allspice, clove, black pepper Pistil, flower, bud Saffron, clove Bark Cassia, cinnamon Source: El-Sayed and Youssef, 2019; Embuscado, 2015; Herman, 2015. Table 2.2. Classification of spices based on flavour Flavour Spice Hot chilies, cayenne peppers, white and black peppers, mustard Mild coriander, paprika Aromatic cinnamon, clove, nutmeg, dill, funnel Vegetables onion, garlic, shallot Source: El-Sayed and Youssef, 2019; Embuscado, 2015; Herman, 2015. Other classification based on taxonomy indicates that spices in general are flowering plants or Angiospermae (Embuscado, 2015). 5 University of Ghana http://ugspace.ug.edu.gh 2.2 Peppers: origin, botany and production Peppers (Capsicum spp) belong to the family Solanacea (do Rego et al., 2016). Other crops in the Solanaceae include tomatoes, potatoes, tobacco (Ryan and Pearce, 2003; Lovell, 1993). Capsicum species are known to have originated from the South Americas, and have been domesticated for several millennia in the American continent, notably Latin America. This was introduced to Europe in the 15th century from where its worldwide distribution began (Liu et al., 2013; Perry et al., 2007; Andrews, 1995a) and subsequently introduced to Africa and Asia through the spice trade (Anon, 2014; Kumar et al., 2006). According to Pickersgill (1969), it is one of the most cultivated crops in the Americas before the advent of agriculture. Capsicum is made up of thirty species of which twenty-five are wild and only five have been domesticated (Bosland and Votava, 2000). Costa et al. (2009) indicated that Brazil became the centre of diversity after the cultivation of pepper in the humid and tropical parts of both South and Central America for over seven millennia. The species of pepper domesticated include Capsicum annuum comprises of chili (dried for chili powder and paprika) as well as hot peppers, sweet pepper from Mexico (Kraft et al., 2014); C. chinense also known as aromatic hot pepper from the Amazonia Region; C. frutescens (Bird eye) from the coastal regions of the southern part of tropical South America. C. frutescence and C. chinense in tropical Africa are treated as a single species as C. annuum (Grubben et al., 2004; Purseglove et al., 1981). Among the species of Capsicum, C. annuum is the most widely cultivated worldwide, mostly used in commercial cultivar breeding programmes, making it the most economically important (Bosland and Votava, 2000). FAOSTAT (2007) indicated that pepper production is done in about seventy-seven countries worldwide. It is estimated that pepper cultivation is done on over three million 6 University of Ghana http://ugspace.ug.edu.gh hectares of land yearly (Doku, 2015). In terms of production, over 89% of pepper production is done in Asia, 7% in the Americas and 4% in Africa, Europe and the Middle East (Rufino and Penteado 2006). According to Liu et al. (2013), there are two main types of pepper in terms of their botany. Those of the wild (Capsicum) which are perennials whiles the ones commercially cultivated are annuals. Peppers in general are herbaceous plants that turn woody with age, some of which can grow as high as 6 feet (Doku, 2015). The capsicum plant produces flowers that have five petals, sepals, pistils and stamens. There are a range of colour for the stamens, petals and pistils. These can be greenish-white, greenish yellow, white or purple which is variety and species dependent. The identification of the various domesticated species can be attributed to the differences in the colour of the filaments, seed, corolla, and flower patterns per node (Liu et al., 2013; Walsh and Hoot, 2001; DeWitt and Bosland, 1993). Pepper is a self-pollinated crop which does not show any inbreeding depression. They can be cross-pollinated by insects, with an outcrossing rate ranging from 70-90% been cultivar-dependent (Liu et al., 2013; Andersson et al., 2007). The fruits have different shapes and colour, some constituents including vitamin C, carotenoids, capsaicinoids. Capsicum spp can be cultivated in the tropics from sea level to elevations of over 6,000 feet. They require a temperature range of 21 and 30 °C. 2.3 Nutritional Composition Peppers have high nutritional value which varies from pepper species, variety, fruit age and condition of postharvest treatments. They are a good source of vitamin K and vitamin 7 University of Ghana http://ugspace.ug.edu.gh B6, riboflavin, niacin, alpha tocopherol, vitamin A and vitamin C, (Liu et al., 2013; Lin and Saltveit, 2012; Nadeem et al., 2011), etc. Table 2.1-2.3 indicates the various composition of dried chili pepper. Table 2.3. Proximate composition of dried chili pepper powder Proximate component g/100 g Water 10.75 Protein 13.46 Total lipid (fat) 14.28 Ash 11.81 Carbohydrate (by difference) 49.7 kJ/100g Energy 1179 Source: USDA National Nutrient Database for Standard Reference 28 slightly revised May 2016. Table 2.4. General Elemental composition of dried chili pepper powder Minerals (Element) mg /100 g Calcium, Ca 330 Iron, Fe 17.3 Magnesium, Mg 149 Phosphorus, P 300 Potassium, K 1950 Sodium, Na 2867 Zinc, Zn 4.3 Copper, Cu 1 Manganese, Mn 1.7 µg/100g Selenium, Se 20.4 Source: USDA National Nutrient Database for Standard Reference 28 slightly revised May 2016. 8 University of Ghana http://ugspace.ug.edu.gh Table 2.5. General nutrient composition of dried chili pepper powder Nutrient Unit Value per 100 g Vitamins Vitamin C, total ascorbic acid Mg 0.7 Thiamin Mg 0.25 Riboflavin Mg 0.94 Niacin Mg 11.6 Pantothenic acid Mg 0.888 Vitamin B-6 Mg 2.094 Folate, total µg 28 Folate, food µg 28 Folate, DFE µg 28 Choline, total Mg 66.5 Betaine Mg 2.7 Vitamin A, RAE µg 1483 Carotene, beta µg 15000 Carotene, alpha µg 2090 Cryptoxanthin, beta µg 3490 Vitamin A, IU IU 29650 Lycopene µg 21 Lutein + zeaxanthin µg 310 Vitamin E (alpha-tocopherol) Mg 38.14 Tocopherol, beta Mg 0.24 Tocopherol, gamma Mg 3.41 Vitamin K (phylloquinone) µg 105.7 Lipids Value per 100 g Fatty acids, total saturated G 2.462 Fatty acids, total monounsaturated G 3.211 Fatty acids, total polyunsaturated G 8.006 Phytosterols Mg 83 Carbohydrates Fibre, total dietary 34.8 Sugars, total 7.19 Sucrose 0.76 Glucose (dextrose) 2.14 Fructose 4.29 Source: USDA National Nutrient Database for Standard Reference 28 slightly revised May 2016 Software v.3.8.6.5. 9 University of Ghana http://ugspace.ug.edu.gh Table 2.5. General nutrient composition of dried chili pepper powder (continued) Amino Acids g/100 g Tryptophan 0.07 Threonine 0.27 Isoleucine 0.39 Leucine 0.63 Lysine 0.36 Methionine 0.13 Cystine 0.18 Phenylalanine 0.37 Tyrosine 0.19 Valine 0.54 Arginine 0.49 Histidine 0.18 Alanine 0.45 Aspartic acid 1.69 Glutamic acid 1.59 Glycine 0.6 Proline 1.25 Serine 0.23 Source: USDA National Nutrient Database for Standard Reference 28 slightly revised May 2016 Software v.3.8.6.5. 2.4 World production and economic importance Pinto et al. (2016) indicated that 89% of the total production area for peppers world-wide is in the Asian continent comprising of countries such as India, China, Vietnam, Thailand, Sri Lanka, and Indonesia. The second largest region of production are United States of America and Mexico which accounts for 7% of pepper production globally. The least areas of pepper production (total worldwide production) can be located in Africa, Europe and the Middle East. These account for 4% (Rufino and Penteado 2006). Pepper export and imports yielded over $96,397 billion, being the second largest earner in terms of vegetable export in 2011 (FAOSTAT, 2011). 10 University of Ghana http://ugspace.ug.edu.gh 2.5 Characteristics and quality indices of chili pepper and pepper products 2.5.1 Colour The colour of pepper (as well as its products such as powder, flakes, prickles and paste) is dependent on the presence of carotenoids available in the tissue of the pepper fruit. These carotenoids have been identified to be red, orange and yellow pigments which are found in plants. According to Biacs et al. (1989), Griesbach and Stommel (2008c), Gnayfeed et al. (2001) and Deli and Molnar (2002), pepper fruits have been identified to contain over 30 different carotenoids. These carotenoids include β carotene and wide range of xanthophylls such as zeaxanthin, β cryptoxanthin, lutein and a host of others. Of all the carotenoids indicated to occur in pepper fruits, the only carotenoids that possess provitamin A activity are β carotene and β cryptoxanthin. The oxidative cleavages of β carotene and cryptoxanthin yield retinal which is needed by humans. Wall et al. (2011) indicated that, the amount of β carotene in pepper both dried and fresh is sufficient to meet the total amount needed by an adult (man) on daily basis. Beta (β) carotene content of chili pepper had been indicated in literature. Topuz and Odzemir (2003) and Topuz et al. (2009) indicated that the β carotene content in some pepper varieties were in the range of 69.5±8.4 to 120.3±11.0 (mg/kg) and Kim et al. (2004) reported a range of 80.74±13.50 to 129.40±3.73 (mg/100g) in Korean chili pepper. The quality of chili pepper in terms of colour is attributed to the presence of carotenoids (which is variety–based), stage of maturity, growing conditions, ripeness. The red colour of pepper fruits is derived from the xanthophylls such as capsanthin and capsorubin. According to Deli and Molnar (2002), Maoka et al. (2001), Ha et al. (2007), Topuz and Ozdemir, (2007), the red colour of pepper fruits is mainly due to the pigment capsanthin; since it contributes the most red colour in a percentage range of 37 to 80 (as the total carotenoids in pepper). Topuz and Odzemir (2003), Kim et al. (2004), Topuz and Odzemir (2007), Jung et al. (2015), reported that the 11 University of Ghana http://ugspace.ug.edu.gh capsanthin content in red pepper was 550.77±20.61 to 951.38 (mg/kg), 15.41±1.28 to 27.11±6.97 (mg/100g), 769.0±71.3 to 1270±89 (mg/kg) and 5.07±0.57 to 6.00±0.07 (µg/100mg), in dried and irradiated pepper, respectively. Topuz and Odzemir (2003) reported a beta cryptoxanthin content of red pepper (paprika) as 113.5±11.0 to 165.5±12.3 (mg/kg) and Kim et al. (2004) also reported that cryptoxanthin in Korean red pepper was l22.06±1.42 to 45.39 (mg/100 g). 2.5.2 Pungency The hotness of pepper and pepper products is attributed to the availability of capsaicinoids which are a group of related alkaloids formed due to the condensation of vanillylamine with a medium chain branched fatty acid (Luo et al., 2011). There are over 20 capsaicinoids which can be differentiated from each other based on the fatty acid structure. The most abundant capsaicinoids available in pungent or hot peppers are capsaicin and dihydrocapsaicin (Davis et al., 2007; Materska and Perucka, 2005). Others include nornornorcapsaicin, nordihydrocapsaicin, nornorcapsaicin, norcapsaicin, homodihydrocapsaicin, nonivamide and homocapsaicin, (Manirakiza et al. 2003; Pruthi 2003b; Fujinari, 1997). Giuffrida et al. (2013), indicated the proportion of capsaicin and dihydrocapsaicin as 92.5% of the total capsaicinoids found in some varieties of capsicum. Capsaicin can be found in the stem end of the pod (fruit) which is in the range of 0.1% to 1.0% of the total fruit (pod) weight (Al Othman et al., 2011; Tucker, 2001). The pericarp tissues contain the capsaicinoids. The production site for capsaicinoids in red peppers are the glands on the placenta of the pepper, thus making the white ribs and the glands the hottest part of red pepper (Dong, 2000; Bosland, 1992). The amount of capsaicinoids in capsicum fruits varies widely based on the variety, maturity level of the plant, fruit, time of harvest, storage conditions (which are influenced by humidity, temperature, exposure 12 University of Ghana http://ugspace.ug.edu.gh to light, water activity, and contact with oxygen), environmental conditions during cultivation as well as edaphic factor (Al Othman et al., 2011; Rhim and Hong, 2011; Menozzi-Smarrito et al., 2009; Antonious et al., 2006; Tucker, 2001). Capsaicin and dihydrocapsain dominates the flavour of C. annuum. The capsaicin content of 28.18- 32.35 (mg/100g) was observed by Lee et al. (2004); Topuz et al. (2009) observed an amount of 499-657 (mg/kg); 0.52-0.56 (mg/g) was observed by Rico et al. (2010); 707 to 38871(µg/g) in 12 pepper varieties investigated by Giuffrida et al. (2013); a content ranging from 1407.98 to 1859.42 (µg/g) by Giuffrida et al. (2014) was reported in red pepper powder and 23.44 to 36.87 (mg/100g) by Jung et al. (2015) and Nagy et al. (2015) reported range of 197 and 440.8(ug/g); and Jung et al. (2015) recorded a capsaicin content of 7.03-7.40 (µg/100g) in both irradiated and unirradiated samples. Dihydrocapsaicin content of some pepper varieties had been reported in literature. Topuz and Odzemir (2004) reported a range of 121±4.53 to 134±4.26 (mg/kg); a range of 308±21 to 14132±1323 (µ/g) by Giuffrida et al. (2013); Giuffrida et al. (2014) reported a range of 811.32±24.66 to 1077.20±54.55 (µg/g); Nagy et al. (2015) reported 88.9 to 780 (µg/g) in some Hungarian varieties, 4.14±0.27 (mg/100g) in some Korean red pepper by Cheon et al. (2015) and a range of 3.23±0.12 to 3.4±0.1 (µg/100g) by Jung et al. (2015). The hotness of pepper and pepper products (as well as its products such as powder, flakes, prickles and paste) is measured in Scoville Heat Units (SHU) which was first developed in 1912 by Wilbur Scoville (Giuffrida et al., 2013; Al Othman et al., 2011; Scoville, 1912). Five classifications of the pungency of pepper has been identified based on the Scoville heat units (Weiss, 2002). These are non-pungent (0–700 SHU), mildly pungent (700–3,000 SHU), moderately pungent (3,000–25,000 SHU), highly pungent (25,000–70,000 SHU) and very highly pungent (>80,000 SHU). The SHU of some pepper varieties indicated by 13 University of Ghana http://ugspace.ug.edu.gh Lee et al. (2004) and Orellana-Escobedo et al. (2013) are 717±28 to 750±50 (x1000 in mg/100g) and 961.13± 7.91 to 211247.65±656.47 (x10000 in mg/kg). 2.6 Uses of chili pepper and pepper products Reilly et al. (2001) and Bosland and Votava (2000), indicated that Capsicum and Capsicum derived components and ingredients have diverse applications, including as food additives, in traditional medicines, drugs, health-promoting products, pests control in agricultural fields, cosmetics and use as self-defence in pepper sprays. In the food industry, oleoresins which is an extract of carotenoids from pepper is used as a food colorant. The powder of chili pepper is used in various ethnic foods worldwide as components of drinks, soups, stews, sauces, and other fermented foods such as kimchi, gochujang, sauerkraut (Raju et al., 2010); in the cosmetic as well as the pharmaceutical industry for many products. 2.7 Pepper production in Ghana Pepper is not native to Ghana. It was introduced into Ghana in the 15th Century (La Anyane, 1963). Chili peppers are produced in Ghana for both the local and the international markets. The main varieties cultivated in Ghana are the Bird’s eye (which are mostly cultivated in Thailand and India) and the Legon-18 which was bred by Crop Scientists from the University of Ghana (MOFA, 2007). The peppers are cultivated in the Volta, Central, Greater Accra, Eastern, Northern and other Regions of Ghana. About two- thirds of the overall chili pepper production in Ghana is done in the Northern Savannah belt for both the local market and the international market. Over 198,000 households are into chilli production in the Northern Savanah belt of Ghana. Irrigation is used to serve as water supply for dry season production. Chilli peppers from Ghana are exported to the 14 University of Ghana http://ugspace.ug.edu.gh European Union, most of which come from farms in the southern part of Ghana under rain fed conditions (Asase, 2014; Sualihu, 2012; Schipmann, 2006). 2.8 Microbial ecology of chili pepper powder Chili pepper powder (which is a product of chili pepper fruits) has been identified to be a reservoir of many pathogenic microorganisms (FSAI, 2005). This is due to its cultivation (chili pepper crop), production, processing and storage conditions, insanitary drying conditions, improper sanitation procedures, failure on the part of processors to validate and verify antimicrobial intervention treatment which will facilitate an effective level of the inactivation of pathogens, cross contamination of food materials etc. (Bakobie et al., 2017; Jung et al., 2015; Gurtler et al., 2014; Rico et al., 2010; Moreira, 2009; Threlfall, 2009; Antai, 1998). Notable organisms implicated in the contamination of chili pepper include Bacillus cereus, Salmonella species, Listeria monocytogenes, Staphylococcus aureus, Aspergillus species, E. coli, Cladosporium species, Penicillium species, Clostridium perfringens (Bakobie et al., 2017; Nokwanda and Ijabadeniyi, 2013; Van et al., 2013; Sualihu, 2012; CDC, 2010; Hampikyan et al., 2009; Vij et al., 2006; Banerjee and Sakar, 2003). 2.9 Processing Chili pepper fruits are processed into pastes, flakes, powder and pickles (Kim et al., 2017; Jung et al., 2015). 15 University of Ghana http://ugspace.ug.edu.gh 2.9.1 Pepper powder The process of red pepper powder production varies from one place to the other. The general procedure for the production of red pepper powder is depicted in Figure 2.1. Freshly harvested raw material Pre-treatments Drying Cleaning Packaging/storage Grinding/milling Microbial d econtamination Fig. 2.1. Flow chart for chili (red) pepper powder production (Source: Schweiggert et al., 2007). 2.9.2 Harvesting of pepper fruits Harvesting of fruits for spice production are either manually or mechanically done (Pruthi et al., 2003b). In Ghana, harvesting of pepper is mostly done manually (Asase, 2014; MOFA, 2007). 16 University of Ghana http://ugspace.ug.edu.gh 2.9.3 Pre-treatment Pre-treatments of pepper fruits is done before drying (Fig 2.1). Sorting of fruits is done and other undesirable components of the fruits are removed. Processing may involve blanching or boiling of the fruits before drying, however, not all processors boil (Owusu- Kwarteng, 2017; Obeng-Ofori et al, 2007). Blanching before drying aids in obtaining pepper powder with best organoleptic and nutritional qualities (Doymaz and Pala, 2010; Tunde-Akintunde, 2010; Wiriya et al., 2009) In Ghana, harvested, cleaned fruits are blanched or boiled before drying (Owusu-Kwarteng et al., 2017). 2.9.4 Drying of the raw material There are various drying methods employed in pepper powder production. These include solar drying, sun or open air drying and super-heated steam drying. 2.9.4.1 Sun drying This is also known as open air drying. It is the commonest drying method employed for chili pepper. Drying period can last for several days which may be due to environmental conditions during the drying period. Generally, the moisture content of the dried produce may be in the range of 4% to 12 % (Jung et al., 2015; Montoya-Ballesteros et al., 2014; Topuz et al., 2011; Schweiggert et al., 2007; Topuz et al., 2004). Sun drying is the main mode of drying pepper in Africa, Central and South America and Asia. In Ghana, pepper is dried either on a tarpaulin or on a raised platform (Sualihu, 2012; Schipmann, 2006). Variations in the moisture content of the final product, inability to control temperature of drying (due to variations in the climatic conditions such as solar radiation, humidity, wind speed and current), variations in colour (a quality parameter of red pepper powder) and poor microbial quality are the disadvantages associated with this method of drying 17 University of Ghana http://ugspace.ug.edu.gh (Montoya-Ballesteros et al., 2014; Phomkong et al., 2010; Topuz and Odzemir, 2004; KPIC, 2001). 2.9.4.2 Solar drying This involves the use of solar energy devices (equipment or materials) that are able to harness (concentrate) solar energy with or without natural airflow inside the dryer. Several types exist which are used in vegetable drying. In Ghana, they are also used in the drying of chili pepper (Owusu-Kwarteng et al., 2017; Schipmann, 2006). 2.9.4.3 Super-heated steam (SHS) This is a drying method that makes use of steam. It is done by exposing the material to be dried to high steam temperature, leading to loss of moisture from the surface of the material. There is no resistance to moisture diffusion in the process and so the rate of drying is controlled by heat transfer only (Devahastin and Suvarnakuta 2008; Mujumdar and Huang, 2007; Kudra and Mujumdar,2002). Kiang and Jon (2015) described the system for superheat steam as operating in a closed cycle and it is made up of a heat chamber, compressor, heat exchanger and a blower to blow the steam on the material. Some advantages of using the SHS is its higher energy efficiency in terms of drying due to higher drying rates. Other advantages include its environmental friendliness (Devahastin and Suvarnakuta, 2008). 2.10 Cleaning Dried pepper samples are examined and cleaned where appropriate before milling (Schweiggert et al., 2007). 18 University of Ghana http://ugspace.ug.edu.gh 2.11 Milling (Particle size reduction) Dried pepper samples are well examined and cleaned before milling. Grinding (milling) is done using equipment such as hammer, electric and cryogenic mills (Schipmann, 2006; Minguez-Mosquera et al., 2000). 2.12 Packaging and storage At the end of processing, powdered pepper is packed and stored in appropriate materials (Schweiggert et al., 2007). During storage, temperature and humidity are critical since these tend to have effect on the quality parameters of chili powder (Rico et al., 2010; Wang et al., 2009; Almela et al., 2008). 2.13 Microbial decontamination of chili pepper powder There are several processes used in microbial decontamination to enhance food safety and food preservation (Farkas, 2007). These include the following; 2.13.1 Super-heated steam This is a process of applying high steam temperature to a material leading to the evaporation of water from the material. The treatment aids in the decontamination of vegetative microorganisms and inactivation of spores, resulting from the high-temperature and the absence of oxygen from the chamber during the process. The method has been used in the decontamination of pepper (Rico et al., 2010; Devahastin and Suvarnakuta, 2008; Schweiggert et al., 2007; Tainter and Grenis, 2001). 19 University of Ghana http://ugspace.ug.edu.gh 2.13.2 Radio frequency heating Radio-frequency heating is the method of using the band of electromagnetic spectrum which covers a frequency range of 1 to 300MHz’ (Ryynanen, 1995). This method of heating, also called dielectric heating, allows quick and uniform heating of food materials due to the application of high-voltage electric current to the electrodes. According to Tang et al. (2005), it can be used for the heating of various food stuffs to different temperatures. Radio-frequency heating has been used for the inactivation of Escherichia coli O157:H7 and Salmonella Typhimurium on black and red pepper (Kim, 2012). 2.13.3 Food Irradiation (gamma rays, electron beams and X-rays) Food irradiation is a process whereby foods are exposed to ionizing radiations such as gamma rays from radio nuclides like Co60 and Ce137; X-rays and electrons generated from machine sources operated at or a below an energy level of 5 million electron volts and at or below an energy level of 10 MeV to meet certain technical objectives respectively (O’Hara, 2013; Arvanitoyannis and Tserkezou, 2010; Cleland, 2010; Ignacio, 2008; Confederation of British Industry, 2007; Codex, 2003; Andress et al., 1998, Olso, 1998; Loaharanu, 1996). Some of the uses of gamma irradiation in the food industry include in sprout inhibition, radiation quarantine treatment, microbial decontamination and delay ripening in fruits and vegetables. X-rays has been used in pepper decontamination (Jung et al., 2015). Gamma, X-rays and Electron beams are three main sources of ionizing radiations used in the food industry (Riganakos, 2010). Gamma irradiations are produced by radio nuclides such as cobalt-60 (being the commonest) with energy levels of 1.17 and 1.33 MeV; and caesium-135 which has an energy level of 0.662 MeV; X-rays or decelerating rays are produced from accelerators 20 University of Ghana http://ugspace.ug.edu.gh with a maximum quantum energy of electrons not exceeding 5 MeV and Electron beams produced from linear accelerators such as the Van De Graaf generator with a quantum energy not exceeding 10 MeV. 2.13.4 General classification of food irradiation doses Food irradiation doses have classified into various ranges of doses in order to meet specific targets ranging from low dose to high dose irradiations. 1. Low doses These are doses of gamma irradiation that are less than 1 kGy. These doses are used mainly for phytosanitary purposes, sprout inhibitions in roots, tubers and bulbs, ripening delay in fruits and vegetables, inactivation of parasites and egg treatment. Other uses include, the treatment of fruits, insect disinfestation and quarantine purposes (IFIS, 2020; Carbo Verde, 2018; Hallman and Loaharanu, 2016; Kalyani and Manjula, 2014; IAEA, 2004; ICGFI, 1991). The application of these low doses to food is also known as radurization. 2. Medium doses This process is also known as radicidation which involves the use of doses ranging from 1 kGy to 10 kGy. These doses are used for the control of foodborne pathogens as well as the extension of shelf life of food materials such as fresh produce, meat and meat products, poultry and poultry products, spices as well as dried foods (Miller, 2015; Hallman, 2011). 21 University of Ghana http://ugspace.ug.edu.gh 3. High doses This involves the use of doses in the range of 10 kGy to 100 kGy. These doses are meant for shelf-stable meats, foods meant for astronauts, sterility of foods meant for immunocompromised individuals, microbial decontamination of some spices, muscle food sterilization purposes and some Korean foods (Song et al., 2018; USFDA, 2016; Miller, 2015; Kalyani and Manjula, 2014; IAEA, 2002). This process of irradiation is also known as radappertization (IAEA, 2002). 2.14 Use of Gamma rays in microbial decontamination of foods Gamma rays of specific energies originate from the spontaneous disintegration of either artificial or natural radionuclides (radioisotopes). They disintegrate spontaneously or decay to a stable state due to their instability. The most commonly used radioisotope for food irradiation by gamma rays is the 60Co. 60Co is produced by the neutron bombardment in a nuclear reactor of 59Co which is then doubly encapsulated in a stainless steel material known as pencils to prevent the leakage into the environment during usage (Riganakos, 2010). The basics for the use of the gamma rays in microbial decontamination is the ability of the rays to pick electrons from a material leading to free electrons and thus take part in a chemical reaction. This will lead to the disruption or destruction of the DNA, thus inactivating the organism. The advantages with the use of 60Co is its ability to deeply penetrate the food material. It is also considered to pose a low risk to the environment and further has a uniform distribution in terms of its dose in the food material (Riganakos, 2010). Gamma irradiation has been used in the decontamination of several foods (Song et al., 2009; Stewart et al., 2001; CFR, 1986). Some of these include, the decontamination of Korean medicinal herbs (Kim et al., 2000); inactivation of psychrotrophic bacteria in squid 22 University of Ghana http://ugspace.ug.edu.gh rings at 4.8 kGy (Tomac et al., 2013); inactivation of Escherichia coli O157:H7 and Salmonella Typhimurium in black and red pepper at 5 kGy (Song et al., 2014); improving the hygienic quality of red pepper powder in Korea (Jung et al., 2015); reduction of mesophilic bacteria and fungi in Korean red pepper powder (Lee et al., 2004); and inactivation of Salmonella Typhimurium in pea nut butter (Ban and Kang, 2014). Rico et al. (2010) indicated the effect of the use of gamma irradiation and steam on the physiochemical and microbiological properties of dried red pepper. Gamma rays have been used on spices and other food commodities to determine their impact on the quality parameters of such commodities (Waje et al., 2008; Ray and Bhunia, 2007; Farkas, 2006; Clavero et al., 1994). Some of these include the effect of gamma rays on the quality parameters of Korean pepper with reference to the colour, pungency and volatiles (Lee, 2004). Jung et al. (2015) investigated the effect of gamma irradiation on the physicochemical qualities of red pepper powder; Topuz and Odzemir (2004) investigated the effect of gamma irradiation, storage and drying method on the quality parameters of some spices, Rico et al. (2010) investigated the impact of gamma irradiation and steam on the physicochemical properties of dried red pepper. 2.15 Regulation of food irradiation The World Trade Organization, the World Health organization and the Food and agriculture Organization and International Atomic Energy Agencies are some of the notable regulatory agencies for food irradiation. The General Standard for the Irradiation of Food by Codex in 1980, indicated that foods irradiated up to doses of 10 kGy are safe. Notwithstanding, foods that are irradiated above 10 kGy are also safe for the consumer (Roberts, 2016; Loaharanu, 2003). 23 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Study design The study was conducted in two phases. 3.1.1 First phase Firstly, a microbial inactivation test on powdered pepper samples was done. Six kilograms of pepper powder was acquired and divided into 120 sub-samples of 10 g weight each. Each sub sample was pre-contaminated with a cock tail of pathogens (ranging from 6.00±0.08 to 6.49±0.08 cfu/g) containing Bacillus cereus, Salmonella Typhimurium, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus. The sub samples were further divided into five subsets and irradiated at 0, 1, 2 4and 5 kGy of gamma radiation for similar durations to de-contaminate them. Viable counts were then done on the irradiated samples using standard microbiological procedures (Jeong et al., 2010; Cheon et al.,2015; Tango et al., 2016; Deng et al., 2015; Luo et al., 2016). The 0 kGy served as control. 3.1.2 Second phase In the second phase, pepper powder samples that were not pre-inoculated with pathogens were irradiated at same dosage of 0, 1, 2, 4 and 5 kGy and stored at different temperatures. They were then analysed to determine the effect of the irradiation on the quality parameters of powdered pepper. A central composite rotational design for k = 2 (Cochran and Cox, 1957) yielded 13 experimental runs for the study, including 4 centre points. The Central composite design matrix for the two independent variables for both the pathogens and quality parameters are indicated in tables 3.1 and 3.2, respectively. 24 University of Ghana http://ugspace.ug.edu.gh Table 3.1. Central composite rotational design matrix (template) for two independent variables for inactivation of pathogens at both temperatures Run order Doses (kGy) Days Survival of organism (log cfu/g) 1 2 60 2 2 30 3 0 0 4 4 0 5 2 0 6 2 30 7 4 30 8 0 60 9 0 30 10 4 60 11 2 30 12 2 30 13 2 30 Table 3.2. Central composite rotational design matrix (template) for two independent variables on quality parameters at both temperatures Run order Doses (kGy) Storage weeks Quality parameter 1 2 4 2 2 4 3 4 4 4 0 0 5 2 4 6 4 0 7 0 4 8 4 8 9 2 4 10 0 8 11 2 4 12 2 8 13 2 0 3.2 Pepper powder samples and microbial culture 3.2.1 Pepper sample The variety of chilli pepper used was Legon-18, and the pepper powder used for the study was purchased from a local farmer. 25 University of Ghana http://ugspace.ug.edu.gh 3.2.1.1 State of pepper samples According to the suppliers, they were processed from sun-dried, fully ripened Legon-18 pepper fruits. In order to obtain the powder, the fruits were sorted from unwholesome fruits and the fruit stalks were broken; blanched and sun-dried by open-air drying on raised platforms, cleaned, milled and packaged. 3.2.2 Microbial pathogens Pure strains of Listeria monocytogenes (NCTC 7973/ATCC 35152) and Bacillus cereus (NCTC 7464/ATCC 10876) were obtained (purchased) from Becton Dickinson in the United States of America; Salmonella Typhimurium, Esherichia coli (25922) and Staphylococcus aureus (25923) strains were obtained from the Microbiological Collection Centre of the Noguchi Memorial Institute for Medical Research of the University of Ghana. 3.2.3 Sample preparation Pepper powder samples were sterilized by irradiation and its sterility tested. Samples were contained in sealed pouches and irradiated at the Gamma Irradiation Facility of the Radiation Technology Centre under the Biotechnology and Nuclear Agriculture Research Institute of the Ghana Atomic Energy Commission, Kwabenya. Irradiation was done from a cobalt – sixty (60Co) source (which is a category IV wet storage gamma irradiator) to remove background microflora according to the method employed by Deng et al. (2015). Sterility tests were conducted to confirm the inactivation of the background microflora. Selective media for Bacillus cereus, Salmonella Typhimurium, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus were prepared and poured under sterile conditions and used for the sterility tests according to the procedures of Luo et al. (2016), 26 University of Ghana http://ugspace.ug.edu.gh Tango et al. (2016), Ducic et al. (2016), Deng et al. (2015), Cheon et al. (2015) and Jeong et al. (2010). The procedures of Deng et al. (2015) and Jeong et al. (2010) were used with some modifications. Ten grams of the pepper samples from the pouches were weighed into sterile stomacher (Seward, UK) pouches, and 90 ml of sterile buffered saline water added. The samples were homogenised and serially diluted and spread-plated. 3.3 Microbial inactivation of inoculated pathogens in pepper samples 3.3.1 Inoculum preparation 3.3.1.1 Salmonella Typhimurium, Escherichia coli Inoculum preparation and inoculation were done according to the procedures of Cheon et al. (2015), Jeong et al. (2010) and Deng et al. (2015) with some modifications. The stock culture which was in storage at -80 oC in 0.7ml of Tryptic Soy Broth (TSB; Disco) and 0.3ml of 50% glycerol was streaked onto Tryptic Soy Agar (TSA; Difco), incubated at 37 oC for 24 h and then stored at 4 oC. The cells were harvested by centrifugation at 4000 g for 20 minutes at 4 oC and washed three times with phosphate buffered saline (PBS). The final pellets were re-suspended in 10 ml of PBS which corresponded to approximately 107- 108 CFU/ml. 3.3.1.2 Listeria monocytogenes Samples of pepper were inoculated by the method of Luo et al. (2016). The inoculum was prepared by transferring 0.1 ml of the stock cultures into 10 ml of tryptic soy broth (TSB; Becton Dickinson Diagnostic Systems, Sparks, Maryland; BD). This was incubated at 37 oC for 24 h. Cells were centrifuged at 3000 g for 10 min at 4 oC and harvested. The pellets were washed and re-suspended in 10 ml of 0.1% sterile PBS and adjusted to make a final cell concentration of approximately 109 CFU/ml. 27 University of Ghana http://ugspace.ug.edu.gh 3.3.1.3 Bacillus cereus The pepper samples were inoculated according to the method of Luo et al. (2016). The B. cereus strain used was maintained at -80 oC in tryptic soy broth (TSB, Difco, Sparks, MD, USA) with 0.6% yeast extract (YE, Difco) and 20% glycerol until use. The inoculum was prepared by transferring frozen suspension of B. cereus in TSB for overnight incubation at 35 oC with continuous shaking. The cultured strain was streaked onto mannitol egg yolk polymyxin agar (MYP, BD) with egg yolk enrichment 50% and antimicrobic vial P (BD), and incubated at 37 oC for 24 h. After that, single colonies from the incubated plate was transferred to tubes filled with 10 mL TSB and was incubated at 35 oC for 24 h. They were centrifuged at 4000 g for 10 min at 4 oC, and the supernatants decanted. The cell pellets were washed twice with 0.1% sterile PBS and resuspended in 10 mL of the same solution to obtain final cell concentration of approximately 8.0 log CFU/mL. 3.3.1.4 Staphylococcus aureus The samples were inoculated according to the method of Tango et al. (2016). The strain used for the inoculation was in storage at -70 oC in tryptic soy broth (TSB, Difco) containing 25% glycerol and 0.6% yeast extract (YE, Difco) was transferred onto Baird Parker Agar (BPA, BD) plates and incubated at 37 oC for 24 h. A single colony was used for inoculating into10 mL of BHI broth followed by incubation overnight at 37 oC. Following the broth culture, 10 mL of the culture was centrifuged (3000 x g for 10 min at 4 oC). The harvested cells were washed twice in 10 mL of 0.1% buffered peptone water (pH 7.2) (BPW, Difco) to obtain a final cell concentration of approximately 8 log CFU/mL. 28 University of Ghana http://ugspace.ug.edu.gh 3.3.2 Inoculum formulation and inoculation of pepper samples A cocktail (approximate equal proportions) of the organisms was prepared and used in the study. One millilitre of the suspension was added to 10 g of the powdered pepper samples in sterile pouches. They were thoroughly mixed and dried in a biosafety hood for 1 h. 3.3.3 Irradiation of powdered pepper samples Inoculated pepper (10 g) was placed in pouches (dimensions of 0.118 m in width and 0.170 m in length) (Figure 3.1 and 3.2) and irradiated at 1, 2, 4 and 5 kGy at a dose rate of 2.01 kGy/h. Unirradiated samples were used as control (0 kGy). Fig. 3.1. Bulked Samples in pouches for irradiation 29 University of Ghana http://ugspace.ug.edu.gh Fig. 3.2. Samples on palette in irradiation chamber (area) at the Gamma Irradiation Facility 3.3.4 Microbial enumeration using viable counts Enumeration for the various organisms was done according to the procedures of Luo et al. (2016), Tango et al. (2016), Ducic et al. (2016), Deng et al. (2015), Cheon et al. (2015) and Jeong et al. (2010) with modifications. Ninety millilitres of PBS was poured into 10 grams of the inoculated pepper in sterile pouches (stomacher bags) and homogenized in a stomacher. The samples were serially diluted onto the various media for the various organisms used in the study and spread plated under sterile conditions onto the various media. XLD agar (BD) was used for S. Typhimurium, L. monocytogenes on PLS agar (BD), E. coli on CT-SMAC (BD), B. cereus on MYP agar (BD) and BP (BD) agar for S. aureus. All the organisms were incubated at 37 oC for 24 h except L. monocytogenes which was incubated for 48 h for days 0, 2, 5, 12, 21, 30, 45 and 60 and colony counted on a colony counter. 30 University of Ghana http://ugspace.ug.edu.gh 3.4 Determination of the effect of gamma irradiation on quality and physicochemical parameters of pepper powder 3.4.1 Irradiation of Samples Uninoculated pepper powder samples in pouches (dimensions of 0.118 m in width and 0.170 m in length) were irradiated at 1, 2, 4 and 5 kGy at a dose rate of 1.55 kGy/h. The control samples were labelled as 0 kGy (unirradiated inoculated sample). Fig. 3.3. Packed Legon-18 pepper samples for gamma irradiation 31 University of Ghana http://ugspace.ug.edu.gh Fig. 3.4. Pepper samples on palette in irradiation chamber (area) at the Gamma Irradiation Facility 3.4.2 Determination of colour The surface colour of the samples was determined according to the procedure of Lee et al. (2004). Six grams of the sample was put into a petri dish and the colour measured using the Huntler L (lightness), a (±, redness/ greenness) and b (±, yellowness/blueness) with a CR 410 MINOLTA Chroma-meter (Konica Minolta, Japan). 3.4.2.1 Hue, Chroma, colour difference and browning index The hue, chroma and colour difference were determined using the procedure of Koide and Shi, 2007. The hue was calculated using the mathematical formula below. -1 𝒃 ∗ Hue = tan ( ) 𝒂∗ and the chroma as Chroma = (𝑎∗2 + 𝑏∗2)1/2 32 University of Ghana http://ugspace.ug.edu.gh The colour difference (Jung et al., 2015) was calculated using the formula ∆𝑬*= [(∆𝐿 ∗2) + (∆𝑎∗2) + (∆𝑏∗2)]1/2 The Browning Index (BI) using the L, a* and b* according to Mohammadi et al. (2008). [100(𝑥−0.31)] BI= 0.17 (𝑎∗+1.75𝐿∗) Where x= (5.645𝐿∗+𝑎∗−3.012𝑏∗) 3.4.3 Moisture content The moisture content of the pepper samples (both irradiated and unirradiated) were determined for the period of the study according to the method of AOAC (2000). Two grams of the powder were weighed into petri plates in triplicates. The samples were dried in the oven for 2 hours at 130 o C in an oven (Gallenkamp, United Kingdom). The plates were covered while in the oven, transferred into a desiccator whiles hot to cool to room temperature before they were weighed. The percentage moisture content of the samples was calculated using the formula proposed by Nielson (2017). 𝑊2−𝑊3 % Moisture Content = X 100 𝑊2−𝑊1 Where W1 is the weight of the empty petri plate, W2 the weight of petri plates and wet sample and W3 is the weight of the petri dish and dried sample (Nielson, 2017). 3.4.4 pH The pH of the pepper samples were determined according to the procedure of AOAC (2000). Ten grams of the samples (unirradiated and irradiated samples) were weighed and mixed with 100 ml of distilled water and filtered. The pH of the filtrate was measured using a standard pH meter (WTW Wissenschaftlich-Technishe Werskstatten, Gerrmany) after calibration. 33 University of Ghana http://ugspace.ug.edu.gh 3.4.5 Total titratable acidity The total titratable acidity (TTA) of the pepper powder (both irradiated and unirradiated) were determined using the AOAC (2000) method. Ten grams of the samples was mixed with 100 ml of distilled water and filtered. Ten millilitres of the filtrate were pippeted into a 25 ml conical flask. The aliquot was diluted with 50 ml distilled water to minimize the interference from the colour of the pepper samples. Three drops of phenolphthalein (1%) was added and titrated against 0.1 M NaOH TTA was determined in triplicates, expressed as % citric acid (Nielson, 2017) and computed as follows 𝑇𝑖𝑡𝑟𝑒 𝑣𝑎𝑙𝑢𝑒 𝑋 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑋 𝑚𝑖𝑙𝑙𝑖 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑎𝑐𝑖𝑑 𝑋 100 % acid= 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 3.4.6 Carotenoids The main carotenoids analysed in the pepper samples are beta carotene, beta cryptoxanthin, capsanthin and zeaxanthin using reverse phase HPLC according the procedures of Giuffrida et al. (2014) and Topuz et al. (2003). 3.4.6.1 Source of standards and reagents Standards of the various carotenoids of interest in the study were purchased from Extrasynthese (Lyon, France). 3.4.6.2 Extraction One gram of the pepper samples were weighed and contents extracted using accelerated solvent extractor equipped with 100-ml stainless steel extraction cells. The cells were loaded with the pepper sample mixed with inert sea sand which had been homogenised. The cells were filled with 90 % ethanol to a pressure of 1500 psi. Heat was applied for the 34 University of Ghana http://ugspace.ug.edu.gh initial period of heat-up time, after which static extraction took place after all the valves were closed. The cells were rinsed with the extraction solvent and purged with N2 gas for 2 minutes. The extracts were collected from the cells with 20 ml falcon tubes after the system was depressurised. 3.4.6.3 Quantification The extracts were filtered into a 2 ml glass vial by using a 0.45 µm membrane filter (Millipore), and then used for HPLC injection. The chromatographic separation was performed on a reversed-phase column AQUA 5u C18 125A (150 x4.60, 5um). The binary gradient (acetone:water at the beginning 75:25). Calibration curves of the various carotenoids (standards) were drawn using Microsoft Excel, 2010 (Appendix XCX-XCII). 3.4.7 Capsaicinoids The capsaicinoids analysed in the samples include capsaicin and dihydrocapsaicin. Pressurized liquid (Fluid Management Systems, USA) extractor was used to extract the capsaicinoids in the pepper samples and quantified using an HPLC. Standards of capsaicin and dihydrocapsaicin were purchased from Extrasynthese (Lyon, France). 3.4.7.1 Extraction A gram (1.0 g) of the pepper samples were weighed and contents extracted using accelerated solvent extractor equipped with 100-ml stainless steel extraction cells. The cells were loaded with the pepper sample mixed with inert sea sand which had been homogenised. The cells were filled with 90 % ethanol to a pressure of 1500 psi. Heat was applied for the initial period of heat-up time, after which static extraction took place after all the valves were closed. The cells were rinsed with the extraction solvent and purged 35 University of Ghana http://ugspace.ug.edu.gh with N2 gas for 2 minutes. The extracts were collected from the cells after the system was depressurised (Mustafa and Turner, 2011; Barbero et al., 2006). 3.4.7.2 Quantification The extracts were filtered into a 2 ml glass vial by using a 0.45 µm membrane filter (Millipore), and then used for HPLC injection. Qualitative and quantitative analysis of the capsaicinoids profile was carried out by the HPLC PDA detector (PerkinElmer Flexar, UK) equipped with a reversed phase column SunFire TM C18 (5um, 4.6 x150 mm from Waters) thermostated at 30 oC. Separation of the compounds was performed by using an isocratic mixture of water: acetonitrile 55:45 v/v. The detection wavelength was set at 280nm (Giuffrida et al., 2013). Calibration curves of the various capsaicinoids (standards) were drawn using Microsoft Excel, 2010 (Appendix XCXIII-XCIV). 3.4.7.3 Scoville heat units The Scoville heat units and the total capsaicinoids were calculated using the methods of Kim et al. (2004) and Orellana-Escobedo et al. (2013). Total capsaicinoids= capsaicin + dihydrocapsaicin SHU= [(% capsaicin x 16.1) + (% dihydrocapsaicin x 16.1)] x 10000. 3.5 Data analysis Data was analysed with StatGraphics Centurion XV.I and Minitab (version 14) statistical soft wares. Means were separated using Least Significant Difference (p<0.05). Response surface regression procedures were used to determine the optimum dose and storage time for effective reduction of pathogens in powdered pepper. 36 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS AND DISCUSSION 4.1 Characteristics of pepper samples used The powdered pepper samples, weighing 6 kg, were packed in food grade polyethylene bags when procured. Microbial analysis done on the samples detected Staphylococcus aureus. The presence of Staphylococcus aureus in powdered pepper had been indicated in a previous study by Sualihu (2012). Parameters such as pH (5.00±0.01 and 5.12±0.02 of the unirradiated samples and the sterile samples respectively), moisture content (10.03±0.6 and 10.04±0.28 of the unirradiated samples and the sterile samples respectively) and titratable acidity (0.264±0.001 and 0.268±0.018 the unirradiated samples and the sterile samples respectively) were not affected (p>0.05) by gamma irradiation after sterilization in comparison with the unirradiated samples which are similar to the observations of Atuobi-Yeboah et al. (2016). 4.1.1 Sample sterilization and inoculation with cocktail of pathogens. Considering that Staphylococcus aureus was detected in the pepper powder obtained from the farmer, the samples were sterilized by irradiation at 20 kGy before it was used for further experimentations. The microbial analysis of samples after the sterilization process showed no detection of pathogens. 4.2. Microbial inactivation of inoculated pathogens in sun-dried Legon-18 pepper powder The effect of gamma irradiation on the pathogens in the samples have been indicated in Tables 4.1 to 4.7. The microbial count (log cfug-1) of the various pathogens ranged from no detection (0) to 6.69 log cfug-1throughout the study period. Gamma irradiation caused 37 University of Ghana http://ugspace.ug.edu.gh inactivation of the organisms in the samples that were irradiated at 1, 2, 4 and 5 kGy. Dose- dependent effects were observed. As doses of gamma irradiation increased, the log cfug-1 of the organisms were reduced. A general reduction in the microbial count of all the organisms was observed throughout the period of study. 4.2.1 Effect of gamma irradiation, storage time and temperature on the inactivation of Salmonella enterica Typhimurium The log cfug-1 of S. Typhimurium inoculated in the samples and irradiated ranged from 6.49±0.08 to the point where no detection was observed (Table 4.1) at both storage temperatures (Table 4.1; appendix XCXV; XCXVI). This pathogen was inactivated at the various doses of gamma irradiation irrespective of the storage temperature (appendices CV and CVI), which was dose–dependent (p<0.05). The least inactivation of S. Typhimurium was observed in the samples irradiated at 1 kGy (showing a higher survival of the pathogen as in Table 4.1). The observed dose-dependent effect on the inactivation of S. Typhimurium in all the samples might be attributed to the levels of injuries caused to the cells at the various doses of irradiation as explained by Wu (2008). Byun et al. (2001), however, indicated that the lethality of gamma irradiation on microorganisms was due to the inability of the organisms to adapt to their surroundings (food material), signifying a post-irradiation effect. Deng et al. (2015) and Song et al. (2014) also observed the inactivation of Salmonella Typhimurium in red pepper powder) at gamma irradiations of 1, 2, 3 and 5 kGy, which was also dose-dependent. 38 University of Ghana http://ugspace.ug.edu.gh Table 4.1. Effect of gamma irradiation and storage on the survival of S. Typhimurium at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) STORAGE Microbial count (log cfug-1) DAYS TEMP 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 6.49±0.08Aa 5.57±0.11Ba 5.37±0.07Ca 4.40±0.09Da ND 2 6.38±0.08Aa 5.55±0.11Ba 5.18±0.05Cb 3.89±0.08Db ND 5 5.72±0.11Ab 5.32±0.11Bb 5.00±0.11Cc 3.65±0.08Dc ND 12 o 5.62±0.11 Abc 5.10±0.11Bc 4.80±0.09Cd 3.44±0.08Dd ND 4 C 21 5.52±0.11Ac 4.90±0.09Bd 4.60±0.09Ce 3.02±0.09De ND 30 5.12±0.11Ad 4.60±0.09Be 3.65±0.08Cf 2.72±0.11Df ND 45 4.99±0.11Ad 3.79±0.08Bf 2.88±0.11Cg 1.46±0.13Dg ND 60 4.65±0.09e ND ND ND ND 0 6.49±0.08Aa 5.21±0.13Ba 4.83±0.11Ca 4.04±0.11Da ND 2 6.02±0.08Ab 5.19±0.13Ba 4.62±0.11Cb 3.52±0.09Db ND 5 5.66±0.13Ac 4.96±0.13Bb 4.49±0.11Cbc 3.33±0.09Dbc ND 12 28±2 5.55±0.13Ac 4.69±0.11Bc 4.33±0.11Cc 3.24±0.09Dd ND 21 oC 5.36±0.13Ad 4.67±0.11Bc 3.99±11Cd 2.88±0.11De ND 30 4.59±0.11Ae 4.09±0.11Bd 3.14±0.09Ce 2.21±0.13Df ND 45 3.83±0.09Af 3.32±0.09Be 2.42±0.13Cf 1.25±0.09Dg ND 60 3.83±0.09Af ND ND ND ND Least Significant Difference: Means with the same letters (upper cases, doses within a particular temperature regeme) in the same row are not significantly (P>0.05) different from each other and means with the same letters in the same column (lower case, doses per day within the same temperature regeme) are not significantly different (P>0.05) from each other. Key: ND= not detected. TEMP.= Temperature There were significant decreases (p<0.05) in the log cfu/g of the pathogen during storage at the different temperatures in all samples irradiated. Similarly, Song et al. (2006) reported a consistent decrease in the count of microorganisms exposed to gamma irradiation in storage. The S. Typhimurium count during storage was higher in the control (unirradiated) samples stored at 4 oC than samples stored at ambient 28±2 oC (4.65±0.09 and 3.38±0.09 log cfu/g, respectively). At both storage temperatures, as days of storage increased inactivation of the pathogen increased in all samples until no detection occurred on the 60th day. 39 University of Ghana http://ugspace.ug.edu.gh Inactivations during storage as indicated by Ban and Kang (2014) decreases in microbial counts were significant (p<0.05) statistically (Table 4.1; appendix XCXV and XCXVI). 4.2.2 Effect of gamma irradiation, storage time and temperature on the inactivation of Escherichia coli Table 4.2 shows the data on the effect of the gamma irradiation on E. coli in the pepper samples during storage. Appendices XCXVII and XCXVIII shows the pattern of the counts. Generally, there was a significant reduction in the colony counts (p<0.05) with the irradiation treatment. The extent of reduction increased with increasing dosage of the gamma radiation, varying from about 1.5 log cfug-1 with the 1kGy treatment to no detection with 5 kGy. Beyond the irradiation treatment (Day 0), however reduction in counts continued but was similar in all samples, including the control and at the two different storage temperatures. Length of storage also had a significant effect (p<0.05) on the inactivation of the pathogen. In previous studies, Deng et al. (2015) and Song et al. (2014) observed a dose-dependent inactivation of E. coli by gamma irradiation in pepper samples which is similar to this study. 40 University of Ghana http://ugspace.ug.edu.gh Table 4.2. Effect of gamma irradiation and storage on the survival of E. coli at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) STORAGE Microbial count (log cfug-1) DAYS TEMP. 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 6.69±0.08Aa 5.17±0.11Ba 4.59±0.11Ca 3.51±0.11Da ND 2 6.43±0.09Ab 4.84±0.11Bb 4.24±0.11Cb 3.11±0.11Db ND 5 6.24±0.08Ac 4.44±0.11Bc 3.94±0.11Cc 2.81±0.15Dc ND 12 5.67±0.11Ad 4.24±0.11Bd 3.66±0.19Cd Dd 4 o 2.41±0.15 ND C 21 4.85±0.09Ae 4.07±0.11Bd 3.03±0.09Ce 2.11±0.15De ND 30 3.53±0.09Af 3.43±0.09Ae 2.87±0.13Be 1.78±0.12Cf ND 45 ND ND ND ND ND 60 ND ND ND ND ND 0 6.40±0.09Aa 4.12±0.09Ba 3.84±0.08Ba 2.57±0.11Ca ND 2 6.14±0.08Ab 3.85±0.08Bba 3.49±0.08Bb 2.17±0.11Cb ND 5 5.94±0.76Ac 3.49±0.08Bba 3.19±0.21Bc 2.05±0.2Cb ND 12 28±2 5.37±0.11Ad 3.29±0.72Bba 3.06±0.08Bc 1.40±0.09Cc ND 21 oC 4.55±0.08Ae 3.14±0.68Bb 2.32±0.11Cd 1.10±0.09Dd ND 30 3.14±0.08Af 2.67±0.58ABc 2.15±0.11Be 1.06±0.03Cd ND 45 ND ND ND ND ND 60 ND ND ND ND ND Least Significant Difference: Means with the same letters (upper cases, doses within a particular temperature regeme) in the same row are not significantly (P>0.05) different from each other and means with the same letters in the same column (lower case, doses per day within the same temperature regeme) are not significantly different (P>0.05) from each other. Key: ND= not detected. The inactivation and dose-dependent effect can be attributed to the impact of the damage caused to the cells by the gamma rays. Ban and Kang (2014), indicated that the inactivation of an organism is dose-dependent (inactivation of an organisms exposed to gamma irradiation increases with an increasing dose), which was also observed in this study. Higher survival rates (least inactivation) were observed in the samples stored at 4 oC than the samples that were stored at 28±2 oC for the study period. Thayer and Boyd (1993), indicated that the resistance of bacteria to gamma irradiation during post-irradiation storage is increased at lower temperatures than at higher temperatures. This is in agreement with the effect of gamma irradiation on the activation of pathogens in this study. 41 University of Ghana http://ugspace.ug.edu.gh 4.2.3 Effect of gamma irradiation, storage time and temperature on the inactivation of Bacillus cereus The enumeration of Bacillus cereus after the irradiation treatment gave about 6 log cfu/g for the samples with no irradiation and about 5.0 log cfu/g to no detection for samples irradiated (Table 4.3; appendix XCXIX and C). Table 4.3. Effect of gamma irradiation and storage on the inactivation of B. cereus at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) STORAGE Microbial count (log cfug-1) DAYS TEMP. 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 6.15±0.08Ac 5.62±0.11Bc 5.15±0.11Ca 4.97±0.11Ca ND 2 6.17±0.08Ac 5.62±0.11Bb 5.15±0.11Ca 4.55±0.09Db ND 5 6.09±0.08Ac 5.96±0.08Ab 4.78±0.09Bb 4.23±0.09Cc ND 12 o 6.32±0.08 Ab 5.85±0.11Bb 4.55±0.09Cc 3.79±0.08Dd ND 4 C 21 6.48±0.08Aa 6.15±0.08Ba 4.80±0.09Cb 4.30±0.09Dc ND 30 6.17±0.08Ac 5.67±0.11Bc 4.45±0.09Cc 3.74±0.08Dd ND 45 5.87±0.11Ad 5.17±0.11Bd 3.97±0.09Cd 3.54±0.08De ND 60 5.07±0.11Ae 3.95±0.09Be 3.02±0.17Ce 2.65±0.04Df ND 0 6.00±0.08Ac 5.41±0.13Bc 4.93±0.09Ca 4.74±0.11Ca ND 2 6.02±0.08Ac 5.64±0.13Bb 4.87±0.14Ca 4.34±0.11Db ND 5 5.94±0.08Ac 5.78±0.13Ab 4.55±0.11Bb 4.02±0.11Bc ND 12 28±2 6.17±0.08Ab 5.75±0.13Bb 4.24±0.11Cc 3.48±0.09Dd ND 21 oC 6.33±0.08Aa 5.87±0.13Ba 4.49±0.11Cb 3.43±0.09Dc ND 30 6.02±0.08Ac 5.36±0.13Bc 4.14±0.11Cc 3.23±0.09Dd ND 45 5.72±0.13Ad 4.90±0.09Bd 3.30±0.09Cd 2.98±0.09De ND 60 4.89±0.11Ae 3.64±0.11Be 2.47±0.13Be 2.33±0.13Bf ND Least Significant Difference: Means with the same letters (upper cases, doses within a particular temperature regeme) in the same row are not significantly (P>0.05) different from each other and means with the same letters in the same column (lower case, doses per day within the same temperature regeme) are not significantly different (P>0.05) from each other. Key: ND= not detected. Generally, there were significant (p<0.05) reductions in the colony counts during storage, however the effects were more severe in samples treated with higher doses. The trends were similar in the samples at the two different storage temperatures, and the longer the storage period, especially beyond 45 days, the more reduction in the counts. Dose- dependent effect of gamma irradiation on the samples were observed as inactivation 42 University of Ghana http://ugspace.ug.edu.gh increased with increasing dose of gamma irradiation (Jung et al., 2015; Carcel et al., 2015; Song et al., 2014; Ban and Kang, 2014; Rico et al., 2010; Stewart, 2001; Simic, 1983). At the end of the storage period, 81.50% and 82.44% of the organisms were counted in control samples and 0% in the samples that were irradiated 5 kGy at 28±2 oC and 4 oC respectively. 4.2.4 Effect of gamma irradiation, storage time and temperature on the inactivation of Listeria monocytogenes Presented in Table 4.4 and appendices CI and CII are the data on the survival of L. monocytogenes after the gamma irradiation treatment and during storage at the different temperatures. Inactivation of L. monocytogenes by gamma irradiation was observed with all the irradiation doses applied. There was a dose-dependent effect (p<0.05) in all cases, with complete inactivation in the 2 and 4 kGy samples by the 60th day (Table 4.4, appendices CXIX and CXX), compared to the high survival in the control (unirradiated samples). The progress of inactivation in the 1 kGy sample stored at 4℃ was not consistent with time, as shown in Table 4.4, as the observed differences in the survival data were not statistically significant (p > 0.05). Percentage of survival at the 60th day were 77.48 and 76.34at 4℃ and 28±2 oC, respectively for the control (unirradiated) samples; 35.46% and 31.71% at 4℃ and 28±2 oC for the 1 kGy samples and no detection in the 2, 4 kGy, and 5 kGy samples at both temperatures. 43 University of Ghana http://ugspace.ug.edu.gh Table 4.4. Effect of gamma irradiation and storage on the survival of L. monocytogenes at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) STORAGE Microbial count (log cfug-1) DAYS TEMP. 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 6.26±0.08Aa 5.87±0.11Ba 4.97±0.11Ca 4.83±0.12Da ND 2 6.15±0.08Aa 5.42±0.11Bb 4.46±0.09Cb 3.81±0.11Db ND 5 6.04±0.08Aa 5.28±0.11Bb 4.26±0.09Cc 3.12±0.11Dc ND 12 o 5.78±0.11 Ab 5.02±0.11Bc 4.10±0.09Cc 2.86±0.11Dd ND 4 C 21 5.42±0.11Ac 4.90±0.09Bc 3.84±0.09Cd 2.66±0.13Dde ND 30 5.18±0.11Ad 4.30±0.09Bd 3.15±0.08Ce 2.46±0.13De ND 45 5.07±0.11Ad 3.19±0.08Be 2.47±0.11Cf 1.96±0.13Df ND 60 4.85±0.09Ae 2.22±0.11Bf ND ND ND 0 5.96±0.08Aa 5.37±0.11Ba 4.20±0.09Ca 4.55±0.09Da ND 2 5.88±0.08Aa 5.11±0.11Bb 4.16±0.09Cb 3.53±0.11Db ND 5 5.77±0.08Aa 4.97±0.11Bb 3.96±0.09Cc 2.87±0.11Dc ND 12 28±2 5.48±0.11Ab 4.70±0.11Bc 3.79±0.09Cc 2.56±0.11Dd ND 21 oC 5.12±0.11Ac 4.60±0.09Bc 3.54±0.08Cd 2.37±0.13De ND 30 4.86±0.11Ad 4.00±0.09Bd 2.88±0.08Ce 2.17±0.13Df ND 45 4.75±0.11Ad 2.92±0.08Be 2.16±0.11Cf 1.65±0.13Dg ND 60 4.55±0.09Ae 1.89±0.11Bf ND ND ND Least Significant Difference::Means with the same letters (upper cases, doses within a particular temperature regeme) in the same row are not significantly (P>0.05) different from each other and means with the same letters in the same column (lower case, doses per day within the same temperature regeme) are not significantly different (P>0.05) from each other. Key: ND= not detected The trends of inactivation of L. monocytogenes in this study were similar to trends reported in literature (Jeong and Kang, 2017; Mukhopadhyay et al., 2013; Rico et al., 2010). The mechanisms of inactivation are expected to be similar to that earlier described for the other pathogens. 4.2.5 Effect of gamma irradiation, storage time and temperature on the inactivation of Staphylococcus aureus Data on the survival of S. aureus after exposure to gamma irradiation treatment during storage at different temperatures are depicted in Table 4.5. 44 University of Ghana http://ugspace.ug.edu.gh Table 4.5. Effect of gamma irradiation and storage on the survival of S. aureus at 4 oC and 28±2 oC in powdered Legon-18 pepper (C. annuum) STORAGE Microbial count (log cfug-1) DAYS TEMP. 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 6.43±0.08Aa 5.56±0.13Ba 4.79±0.11Ca 2.86±0.13Da ND 2 6.01±0.09Ab 5.36±0.13Bb 4.64±0.11Cb 2.71±0.13Da ND 5 5.81±0.13Ab 4.96±0.13Bb 4.50±0.11Cc 1.96±0.13Db ND 12 o 5.41±0.13 Ac 4.74±0.13Bc 4.14±0.11Cd 1.91±0.13Db ND 4 C 21 5.21±0.13Acd 4.59±0.11Bc 3.83±0.09Ce 1.52±0.13Dc ND 30 5.06±0.13Ad 4.42±0.11Bd 3.13±0.09Cf 1.26±0.13Dd ND 45 4.69±0.11Ae 3.78±0.09Be 3.08±0.09Cg ND ND 60 4.37±0.11f ND ND ND ND 0 5.94±0.08Aa 5.27±0.11Ba 4.55±0.09Ca 2.57±0.11Da ND 2 5.56±0.08Ab 4.75±0.09Bb 4.30±0.09Cb 2.17±0.11Db ND 5 5.32±0.11Ac 4.60±0.09Bb 4.10±0.09Cc 1.82±0.11Dc ND 12 4.80±0.11Ad 4.40±0.09Bc 3.79±0.11Dd o 1.42±0.11 Dd ND 28±2 C 21 4.65±0.09Ad 4.30±0.09Bc 3.49±0.08Ce 1.22±0.11De ND 30 4.45±0.09Ae 4.13±0.11Bd 2.63±0.11Cf 1.17±0.11Df ND 45 4.10±0.09Af 3.09±0.08Be ND ND ND 60 ND ND ND ND ND Least Significant Difference:: Means with the same letters (upper cases, doses within a particular temperature regeme) in the same row are not significantly different (p>0.05) from each other and means with the same letters in the same column (lower case, doses per day within the same temperature regeme) are not significantly different (P>0.05) from each other. Key: ND= not detected. Inactivation (p<0.05) of S. aureus was observed at 1, 2 and 4 kGy (Table 4.5, appendices CIII, CIV, CXVI and CXVII) which was dose-dependent and a complete inactivation in samples irradiated at 1, 2 and 4 kGy. Inactivation trends observed in S. aureus is typical in literature (Jung et al., 2015; Ban and Kang, 2014; Song et al., 2014; Arzina et al., 2012; Rico et al., 2010). This has been attributed to the increase in the lethality of the corresponding dose that the organisms were exposed to due to more production of radiolytic compounds that affected the cellular content of the organism leading to a corresponding effect. 45 University of Ghana http://ugspace.ug.edu.gh 4.2.6 Effect of gamma irradiation on all the organisms at 4 oC and 28±2 oC. Presented in Tables 4.6 and 4.7 are the comparative of the effect of gamma irradiation and storage period on the survival of E. coli, S. aureus, S. Typhimurium, B. cereus and L. monocytogenes (appendix CXXI). Generally, microbial counts reduced with increasing storage time and increasing doses of gamma irradiation used. The observed reductions were statistically significant (p<0.05). Generally, the inactivation was more pronounced in E. coli after day 30 and less in B. cereus. Song et al. (2014) observed E. coli to be more sensitive when E. coli and S Typhimurium were irradiated together in both black and red pepper powder samples. Deng et al. (2015) also made a similar observation when E. coli, S Typhimurium and Aspergillus niger were inoculated in pepper and irradiated. Jeong and Kang (2017) and Clavero et al. (1994) reported that the most resistant gram-negative organism to gamma irradiation is Salmonella, as observed in this study (Table 4.6). L. monocytogenes which is Gram-positive, was more resistant to gamma irradiation than E. coli and S. Typhimurium, which is similar to the observations of Jeong and Kang (2017). Zahran et al. (2008) indicated that B. cereus was more resistant to gamma irradiation than L. monocytogenes and this was also observed during this study. In this study, the most resistant of the organisms was B. cereus it was the only organism detected throughout the 60 days of storage at all the doses irrespective of the storage temperatures. A dose- dependent effect was however observed. 46 University of Ghana http://ugspace.ug.edu.gh Table 4.6. Effect of gamma radiation on the survival of microorganisms in powdered Legon-18 pepper (C. annuum) at 4 oC. STORAGE Microbial count (log cfug-1) ORGANISMS 5 DAYS 0 kGy 1 kGy 2 kGy 4 kGy kGy E. coli 6.69±0.08A 5.17±0.11C 4.59±0.11D 3.51±0.11C ND S. aureus 6.43±0.08B 5.56±0.13B 4.79±0.11C 2.86±0.13D ND 0 L. monocytogenes 6.26±0.08C 5.87±0.11A 4.97±0.11BC 4.83±0.12A ND S. Typhimurium 6.49±0.08B 5.57±0.11B 5.37±0.07A 4.40±0.09B ND B. cereus 6.15±0.08C 5.62±0.11B 5.15±0.11B 4.97±0.11A ND E. coli 6.43±0.09A 4.84±0.11C 4.24±0.11D 3.11±0.11C ND S. aureus 6.01±0.09C 5.36±0.13B 4.64±0.11B 2.71±0.13D ND 2 L. monocytogenes 6.15±0.08BC 5.42±0.11B 4.46±0.09C 3.81±0.11B ND S. Typhimurium 6.38±0.08A 5.55±0.11B 5.18±0.05A 3.89±0.08B ND B. cereus 6.17±0.08B 5.62±0.11A 5.15±0.11A 4.55±0.09A ND E. coli 6.24±0.08A 4.44±0.11D 3.94±0.11E 2.81±0.15D ND S. aureus 5.81±0.13C 4.96±0.13C 4.50±0.11C 1.96±0.13E ND 5 L. monocytogenes 6.04±0.08B 5.28±0.11B 4.26±0.09D 3.12±0.11C ND S. Typhimurium 5.72±0.11C 5.32±0.11B 5.00±0.11A 3.65±0.08B ND B. cereus 6.09±0.08AB 5.96±0.08A 4.78±0.09B 4.23±0.09A ND E. coli 5.67±0.11B 4.24±0.11D 3.66±0.19D 2.41±0.15D ND S. aureus 5.41±0.13C 4.74±0.13C 4.14±0.11C 1.91±0.13E ND 12 L. monocytogenes 5.78±0.11B 5.02±0.11B 4.10±0.09C 2.86±0.11C ND S. Typhimurium 5.62±0.11B 5.10±0.11B 4.80±0.09A 3.44±0.08B ND B. cereus 6.32±0.08A 5.85±0.11A 4.55±0.09B 3.79±0.08A ND E. coli 4.85±0.09D 4.07±0.11E 3.03±0.09D 2.11±0.15D ND S. aureus 5.21±0.13C 4.59±0.11D 3.83±0.09C 1.52±0.13E ND 21 L. monocytogenes 5.42±0.11B 4.90±0.09B 3.84±0.09C 2.66±0.13C ND S. Typhimurium 5.52±0.11B 4.90±0.09B 4.60±0.09B 3.02±0.09B ND B. cereus 6.48±0.08A 6.15±0.08A 4.80±0.09A 4.30±0.09A ND E. coli 3.53±0.09C 3.43±0.09D 2.87±0.13D 1.78±0.12D ND S. aureus 5.06±0.13B 4.42±0.11C 3.13±0.09C 1.26±0.13E ND 30 L. monocytogenes 5.18±0.11B 4.30±0.09C 3.15±0.08C 2.46±0.13C ND S. Typhimurium 5.12±0.11B 4.60±0.09B 3.65±0.08B 2.72±0.11B ND B. cereus 6.17±0.08A 5.67±0.11A 4.45±0.09A 3.74±0.08A ND E. coli ND ND ND ND ND S. aureus 4.69±0.11C 3.78±0.09B 3.08±0.09B ND ND 45 L. monocytogenes 5.07±0.11B 3.19±0.08C 2.47±0.11D 1.96±0.13B ND S. Typhimurium 4.99±0.11B 3.79±0.08B 2.88±0.11C 1.46±0.13C ND B. cereus 5.87±0.11A 5.17±0.11A 3.97±0.09A 3.54±0.08A ND E. coli ND ND ND ND ND S. aureus 4.37±0.11C ND ND ND ND 60 L. monocytogenes 4.85±0.09B ND ND ND ND S. Typhimurium ND ND ND ND ND B. cereus 5.07±0.11A 3.95±0.09 3.02±0.17 2.65±0.04 ND Least significance difference. Means with the same letters (upper cases, doses within a particular a day) in the same column are not significantly (P>0.05) different from each other for all the orgnisms. 47 University of Ghana http://ugspace.ug.edu.gh Table 4.7. Effect of gamma radiation on the survival of microorganisms in powdered Legon-18 pepper (C. annuum) at 28±2 oC STORAGE Microbial count (log cfug-1) ORGANISMS 5 DAYS 0 kGy 1 kGy 2 kGy 4 kGy kGy E. coli 6.40±0.09A 4.12±0.09B 3.84±0.08D 2.57±0.11D ND S. aureus 5.94±0.08B 5.27±0.11A 4.55±0.09B 2.57±0.11D ND 0 L. monocytogenes 5.96±0.08B 5.37±0.11A 4.20±0.09C 4.55±0.09B ND S. Typhimurium 6.49±0.08A 5.21±0.13A 4.83±0.11A 4.04±0.11C ND B. cereus 6.00±0.08B 5.41±0.13A 4.93±0.09A 4.74±0.11A ND E. coli 6.14±0.08A 3.85±0.08C 3.49±0.08D 2.17±0.11C ND S. aureus 5.56±0.08C 4.75±0.09B 4.30±0.09C 2.17±0.11C ND 2 L. monocytogenes 5.88±0.08B 5.11±0.11AB 4.16±0.09C 3.53±0.11B ND S. Typhimurium 6.02±0.08AB 5.19±0.13AB 4.62±0.11B 3.52±0.09B ND B. cereus 6.02±0.08AB 5.64±0.13A 4.87±0.14A 4.34±0.11A ND E. coli 5.94±0.76A 3.49±0.08C 3.19±0.21C 2.05±0.20D ND S. aureus 5.32±0.11C 4.60±0.09B 4.10±0.09B 1.82±0.11D ND 5 L. monocytogenes 5.77±0.08AB 4.97±0.11B 3.96±0.09B 2.87±0.11C ND S. Typhimurium 5.66±0.13B 4.96±0.13B 4.49±0.11A 3.33±0.09B ND B. cereus 5.94±0.08A 5.78±0.13A 4.55±0.11A 4.02±0.11A ND E. coli 5.37±0.11B 3.29±0.72C 3.06±0.08C 1.40±0.09D ND S. aureus 4.80±0.11C 4.40±0.09B 3.79±0.11B 1.42±0.11D ND 12 L. monocytogenes 5.48±0.11B 4.70±0.11B 3.79±0.09B 2.56±0.11C ND S. Typhimurium 5.55±0.13B 4.69±0.11B 4.33±0.11A 3.24±0.09B ND B. cereus 6.17±0.08A 5.75±0.13A 4.24±0.11A 3.48±0.09A ND E. coli 4.55±0.08D 3.14±0.68C 2.32±0.11D 1.10±0.09D ND S. aureus 4.65±0.09D 4.30±0.09B 3.49±0.08C 1.22±0.11D ND 21 L. monocytogenes 5.12±0.11C 4.60±0.09B 3.54±0.08C 2.37±0.13C ND S. Typhimurium 5.36±0.13B 4.67±0.11B 3.99±11B 2.88±0.11B ND B. cereus 6.33±0.08A 5.87±0.13A 4.49±0.11A 3.43±0.09A ND E. coli 3.14±0.08D 2.67±0.58C 2.15±0.11E 1.06±0.03C ND S. aureus 4.45±0.09C 4.13±0.11B 2.63±0.11D 1.17±0.11C ND 30 L. monocytogenes 4.86±0.11B 4.00±0.09B 2.88±0.08C 2.17±0.13B ND S. Typhimurium 4.59±0.11C 4.09±0.11B 3.14±0.09B 2.21±0.13B ND B. cereus 6.02±0.08A 5.36±0.13A 4.14±0.11A 3.23±0.09A ND E. coli ND ND ND ND ND S. aureus 4.10±0.09C 3.09±0.08C ND ND ND 45 L. monocytogenes 4.75±0.11B 2.92±0.08C 2.16±0.11C 1.65±0.13B ND S. Typhimurium 3.83±0.09D 3.32±0.09B 2.42±0.13B 1.25±0.09C ND B. cereus 5.72±0.13A 4.90±0.09A 3.30±0.09A 2.98±0.09A ND E. coli ND ND ND ND ND S. aureus ND ND ND ND ND 60 L. monocytogenes 4.55±0.09B 1.89±0.11B ND ND ND S. Typhimurium 3.18±0.09C ND ND ND ND B. cereus 4.89±0.11A 3.64±0.11A 2.47±0.13A 2.33±0.13A ND Least significance difference: Means with the same letters (upper cases, doses within a particular a day) in the same column are not significantly (P>0.05) different from each other for all the orgnisms. 48 University of Ghana http://ugspace.ug.edu.gh After 60 days of storage, the microbial counts of 4.37±0.11 (67.96%), 4.85±0.09 (77.48%) and 5.07±0.11 (82.44%) for S. aureus, L. monocytogenes and B. cereus respectively at 4 oC were recorded. Percentage microbial count in the samples stored at 28±2 oC were 76.34%, 49.00% and 77.25% of L. monocytogenes, S. Typhimurium and B. cereus respectively were detected. 4.3 The effect of gamma irradiation on the quality parameters of pepper powder 4.3.1 Effect on colour 4.3.1.1 Effect on L* values Table 4.8 depicts the lightness (L* values), that is the brightness or whiteness of the samples irradiated at different doses and stored at different temperatures for different periods. Gamma irradiation had a statistically significant (p<0.05) impact on the L* values of the pepper samples. There were marginal increases in the values of the samples immediately after the treatment, especially as the irradiation doses increased. During storage however, there were some discoloration (loss of brightness) in the samples. This effect is observed through to the fourth week of storage, after which there were some marginal stability. The decrease in L* values during storage was more pronounced in the samples stored at 28±2 oC. At the end of the storage period there was less than 20% loss in the L*values of all the samples irrespective of the doses applied as well as the storage condition. The L* values of both irradiated and unirradiated pepper samples have been indicated in literature. Lee et al. (2004) indicated that the L* values of some Korean pepper samples were in the range about 30.77 in both irradiated and unirradiated samples; Topuz et al. (2009) reported a range of 40.50 to 49.18 in Anaheim variety and 40.47 to 48.11 in Jalapeno; while Rico et al. (2010) recorded about 51 and about 52 in unirradiated and irradiated samples, respectively; Jung et al. (2015) reported about 46 in some Korean 49 University of Ghana http://ugspace.ug.edu.gh varieties and Song et al. (2014) also indicated values of about 27 in unirradiated samples and about 34 in irradiated samples which were lower than the L* values of Legon-18 pepper powder. The reduction in the lightness of the colour of the pepper samples can be attributed to the deterioration caused by oxidation of the carotenoids in the pepper samples (Schweiggert et al., 2005). Table 4.8. L* values of Legon-18 pepper powder after gamma irradiation, and during storage at different storage conditions. S S L-values W C 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 50.93±0.01Ha 50.96±0.01Hab 51.09±0.14Gb 51.42±0.06Hc 51.85±0.00Hd 1 49.81±0.01Ga 49.88±0.01Ga 50.21±0.06Fb 50.42±0.15Gc 50.96±0.09Gd 2 48.60±0.01Fa 48.93±0.04Fb 49.16±0.01Ec 49.47±0.02Fd 50.20±0.05Fe 3 44.46±0.01Ea 45.65±0.01Eb 46.33±0.01Dc 47.35±0.03Ed 48.73±0.06Ee 4 4 oC 42.84±0.00Ba 43.02±0.00Db 43.55±0.09Cc 43.91±0.03Dd 44.59±0.07De 5 42.86±0.00Da 42.88±0.00Ba 42.96±0.01Bc 42.88±0.01Aa 42.96±0.01Cc 6 42.85±0.00Ca 42.94±0.02Cb 42.99±0.03Bc 43.27±0.02Bd 43.93±0.07Bd 7 42.60±0.00Ba 43.01±0.01Db 43.58±0.12Cc 43.73±0.01Cd 43.89±0.0.4Be 8 42.51±0.00Aa 42.69±0.01Ab 42.82±0.03Ac 42.97±0.01Ad 43.20±0.07Ae 0 50.49±0.04Ia 50.63±0.02Ib 50.76±0.02Ic 51.05±0.09Id 51.66±0.02Ie 1 49.48±0.03Ha 49.80±0.02Hb 49.88±0.18Hb 50.12±0.13Hc 50.62±0.10Hd 2 47.10±0.10Ga 47.37±0.13Gb 47.70±0.10Gc 48.24±0.04Gd 49.83±0.04Ge 3 43.53±0.06Fa 44.58±0.30Fb 45.29±0.06Fc 46.22±0.06Fd 47.26±0.12Fe 28±2 4 41.29±0.06Ea 42.27±0.11Eb 43.18±0.06Eco 43.55±0.28 Ed 44.22±0.06Ee C 5 40.22±0.12Da 40.78±0.10Db 41.25±0.11Dc 41.91±0.06Dd 42.25±0.10De 6 38.98±0.08Ca 40.28±0.11Cb 40.98±0.02Cc 41.51±0.14Cd 43.00±0.05Ce 7 38.28±0.06Ba 39.00±0.08Bb 40.20±0.05Bc 40.75±0.11Bd 41.18±0.08Be 8 36.74±0.06Aa 37.18±0.12Ab 38.12±0.11Ac 39.30±0.13Ad 40.65±0.10Ae Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORGE WEEKS; SC-STORAGE CONDITION The observed pattern in the differences recorded in L*values of the pepper samples stored at both temperatures are similar to the observations of Liu et al. (2010). They indicated that temperature during storage led to the reduction of L* values in tomato powder. Rhim and Hong (2011) indicated that storage temperature leads to the reduction in L* values in dried pepper products during storage. This observation may be attributed to the rate of 50 University of Ghana http://ugspace.ug.edu.gh non-enzymatic browning and package-free space of the packaging material (Rhim and Hong, 2011). 4.3.1.2 Effect on a* values The a* values which depict the redness of the pepper powder are indicated in Table 4.9. The value was about 21.5 for the control sample at day 0 but increased to about 24.5 after the irradiation treatment at day 0. The increase with irradiation was dose-dependent. During the storage periods, the a* values significantly decreased. The patterns of decrease were similar at the different storage temperatures. The a* value of pepper powder has been variously reported in literature. Lee et al. (2004) reported the redness of unirradiated Korean red pepper powder to be 26.21, Topuz et al. (2009) measured the redness of some Turkish pepper varieties to be 29.56, Rico et al. (2010) reported a value of 20.78 in samples analysed, Arslan and Ozcan (2011) reported a* values of some pepper products to be over 30 and Jung et al. (2015) reported 17.5 for red peeper powder analysed. 51 University of Ghana http://ugspace.ug.edu.gh Table 4.9. The a* values of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures S a* values SC W 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 21.48±0.02Ia 21.87±0.01Hb 22.60±0.02Ic 23.55±0.05Id 24.50±0.13Ie 1 19.07±0.02Ha 19.93±0.07Gb 20.61±0.06Hc 21.32±0.06Hd 22.62±0.05He 2 18.00±0.06Ga 18.54±0.17Fb 19.34±0.03Gc 20.42±0.21Gd 21.17±0.05Ge 3 17.38±0.06Fa 18.24±0.10Eb 18.75±0.08Fc 19.48±0.00Fd 20.67±0.02Fe o 4 4 C 15.30±0.05Ea 16.11±0.16Db 16.87±0.02Ec 17.46±0.09Ed 18.88±0.02Ee 5 14.77±0.03Da 16.24±0.03Db 16.66±0.03Dc 17.63±0.01Dd 18.27±0.06De 6 14.21±0.01Ca 14.84±0.01Cb 15.21±0.05Cc 15.59±0.01Cd 17.91±0.01Ce 7 10.08±0.00Ba 10.58±0.00Bb 11.24±0.31Bc 11.32±0.06Bc 12.80±0.13Bd 8 9.94±0.07Aa 10.34±0.03Ab 10.98±0.01Ac 11.09±0.11Ac 12.25±0.18Ad 0 21.45±0.01Ia 21.86±0.02Hb 22.56±0.02Ic 23.52±0.08Id 24.41±0.01Ie 1 18.64±0.02Ha 19.22±0.06Gb 19.83±0.07Hc 20.25±0.1Hd 21.48±0.04He 2 17.19±0.01Ga 18.22±0.06Fb 19.46±0.02Gc 19.87±0.02Gd 20.35±0.01Ge 3 17.04±0.01 Fa 18.18±0.02Fb 18.68±0.04Fc 19.30±0.05Fd 20.00±0.05Fe 28±2 4 o 15.03±0.01 Ea 15.84±0.05Eb 16.36±0.02Ec 16.88±0.01Ed 17.45±0.06Ee C 5 11.03±0.02Da 11.45±0.01Db 12.69±0.30Dc 13.69±0.11Dd 16.31±0.12De 6 10.85±0.01Ca 10.93±0.01Ca 11.48±0.14Cb 12.29±0.12Cc 15.64±0.17Cd 7 9.83±0.0.5Ba 10.14±0.03Bb 10.77±0.09Bc 11.13±0.12Bd 12.00±0.01Be 8 9.64±0.01Aa 9.99±0.02Ab 10.32±0.06Ac 10.77±0.09Ad 11.75±0.01Ae Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. The differences in the values reported can be attributed to the varietal differences, edaphic factors, methods of drying employed, as well as the storage conditions of the samples (Cheon et al., 2015; Kim et al., 2012; Rico et al., 2010). Almela et al., (2002), indicated that higher temperatures tend to promote the degradation of colour in paprika. In this study, higher redness (a*) of the samples were observed in the samples that were stored at refrigeration temperature (4 oC) than the samples that were stored at 28±2 oC. This observation is similar to the findings of Almela et al. (2002). At the end of the 8 weeks storage period, there was over 50% loss in the a* values for both storage conditions. 52 University of Ghana http://ugspace.ug.edu.gh 4.3.1.3 Effect on b* values The results of the colour parameter b* (yellowness) are shown in Table 4.10. The b* value for the control sample (unirradiated) was about 22 at day 0 but increased to about 24 in sample irradiated with 5 kGy gamma rays. The increase was dose-dependent which was statistically significant (p<0.05). The b* values decreased with storage. The b* values reported by Lee et al. (2004) was less than 17 in both irradiated and unirradiated Korean pepper samples, Rico et al. (2010) recorded less than 27 in both irradiated and unirradiated samples; less than 16 in other varieties (Song et al., 2014) and Jung et al. (2015) reported values less than 11 in both irradiated and unirradiated samples. Legon-18 values were higher than some of the b* values reported in literature, except during storage. The observed differences may be due to the differences in terms of variety, edaphic factors, stage of maturity during harvest, post-harvest treatments as well as drying methods (Won et al., 2015; Rhim and Hong, 2011; Topuz et al., 2009, Kim et al., 2004). The general reduction observed in the b* values of the samples irrespective of the storage condition and the doses of gamma irradiation that the samples were exposed to, might be due to the breakdown of the pigments that are responsible for the yellowness of the pepper samples which is similar in previous report (Kim et al., 2005). Rico et al. (2010), observed that red pepper samples that were stored at room temperature (20±2 oC) had higher losses of colour as compared with the samples that were stored at 4 oC. In this study, the samples that were stored at 4 oC had higher b* values as compared with the samples that were stored at 28±2 oC. This may be attributed to the higher rate of colour degradation of colour in the samples at the higher temperature (Rico et al., 2010). 53 University of Ghana http://ugspace.ug.edu.gh Table 4.10. B*values of Legon-18 pepper powder after gamma irradiation, during storage at different temperatures b* values SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 22.10±0.01Ia 22.82±b0.06Hb 23.48±0.03Ic 23.98±0.01Id 24.59±0.01Ie 1 17.28±0.01Ha 18.05±0.03Gb 18.82±0.06Hc 19.17±0.06Hd 20.03±0.08He 2 15.77±0.01Ga 16.43±0.08Fb 17.22±0.06Gc 18.08±0.06Gd 19.08±0.06Ge 3 15.38±0.04Fa 16.18±0.06Eb 16.95±0.05Fc 17.42±0.04Fd 18.18±0.11 Fe 4 4 o 13.05±0.02 Ea 13.89±0.02Db 14.43±0.07Ec 15.25±0.07Ed 17.70±0.01Ee C 5 12.64±0.04Da 13.17±0.03Cb 13.89±0.02Dc 14.28±0.01Dd 15.37±0.03De 6 12.56±0.00Ca 12.95±0.05Bb 13.21±0.06Cc 13.95±0.01Cd 14.89±0.01Ce 7 7.64±0.01Ba 7.94±0.02Ab 8.22±0.03Bc 8.89±0.13Bd 9.68±0.04Be 8 7.41±0.02Aa 7.87±0.03Ab 8.01±0.01Ac 8.41±0.03Ad 9.19±0.03Ae 0 21.10±0.01Ia 22.92±0.12Ib 23.19±0.13Hc 23.98±0.01Id 24.23±0.01He 1 16.55±0.02Ha 17.23±0.07Hb 17.96±0.02Gc 18.46±0.02Hd 19.60±0.04Ge 2 13.82±0.01Ga 14.24±0.03Gb 14.91±0.03Fc 15.52±0.06Gd 16.38±0.06Fe 3 28 11.98±0.04Fa 12.58±0.08Fb 13.27±0.02Ec 13.97±0.02Fd 15.24±0.11Ee 4 ±2 8.71±0.01Ea 9.19±0.03Eb 9.92±0.06Dc 10.52±0.06Ed 11.38±0.01De 5 oC 8.35±0.1Da 8.92±0.06Db 9.27±0.33Cc 9.88±0.02Dd 10.40±0.03Ce 6 7.78±0.01Ca 8.40±0.00Cb 9.00±0.10Bc 9.63±0.05Cd 10.52±0.01Ce 7 7.53±0.01Ba 8.13±0.03Bb 8.83±0.05Bc 9.20±0.13Bd 10.11±0.03Be 8 7.46±0.01Aa 7.56±0.01Ab 7.85±0.06Ac 8.48±0.05Ad 9.16±0.03Ae Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION The loss of the yellowness (b* values) of the samples during storage was remarkable in all the samples. At the end of the study, there was loss of over 60% of the b* values for all the doses applied and at the different storage temperatures. 4.3.1.4 Effect on chroma Table 4.11 indicates the chroma values (which depicts the colourfulness quantitatively) of the samples. The chroma of the samples were in the range of 12.19±0.01 and 34.71±0.10. There was a dose-dependent effect on the chroma of the pepper from week 0 to week 8 irrespective of the doses applied as well as the temperature of storage. The chroma values at both temperatures reduced as the weeks progressed. Higher values of chroma were 54 University of Ghana http://ugspace.ug.edu.gh observed in the samples that were irradiated as compared with the samples that were not irradiated irrespective of the storage condition. Table 4.11. Chroma of Legon-18 pepper powder after gamma irradiation, during storage at different temperatures Chroma SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 30.81±0.01Ia 31.61±0.05Ib 32.58±0.03Ic 33.61±0.03d 34.71±0.10Ie 1 25.73±0.03Ha 26.89±0.05Hb 27.91±0.04Hc 28.67±0.01d 30.22±0.02He 2 23.93±0.04Ga 24.78±0.09Gb 25.89±0.03Gc 27.27±0.19d 28.50±0.05Ge 3 23.21±0.05Fa 24.38±0.05Eb 25.28±0.08Fc 26.14±0.03d 27.53±0.07 Fe 4 4 20.11±0.06Ea 21.27±0.12Eb 22.20±0.03Eco 23.18±0.07 d 25.87±0.02Ee C 5 19.44±0.02Da 20.91±0.02Db 21.69±0.02Dc 22.68±0.004d 23.88±0.04De 6 18.97±0.004Ca 19.70±0.07Cb 20.15±0.03Cc 20.92±0.01d 23.29±0.00Ce 7 12.65±0.003Ba 13.23±0.01Bb 13.92±0.24Bc 14.39±0.12d 16.05±0.08Be 8 12.40±0.04Aa 12.99±0.04Ab 13.59±0.01Ac 13.92±0.09d 15.31±0.16Ae 0 30.08±0.01Ia 31.68±0.01Ib 32.35±0.08Ic 33.59±0.05Id 34.39±0.02Ie 1 24.93±0.01Ha 25.81±0.09Hb 26.75±0.04Hc 27.40±0.08Hd 29.08±0.18He 2 22.05±0.02Ga 23.12±0.06Gb 24.51±0.03Gc 25.21±0.05Gd 26.13±0.06Ge 3 28 20.83±0.03Fa 22.11±0.05Fb 22.92±0.03Fc 23.82±0.04Fd 25.14±0.08Fe 4 ±2 17.37±0.01Ea 18.31±0.03Eb 19.13±0.03Ec 19.89±0.04Ed 20.84±0.04Ee 5 oC 13.83±0.04Da 14.52±0.04Db 15.72±0.03Dc 16.88±0.06Dd 19.34±0.15De 6 13.35±0.01Ca 13.79±0.01Cb 14.59±0.05Cc 15.61±0.08Cd 18.84±0.13Ce 7 12.38±0.04Ba 13.00±0.04Bb 13.93±0.10Bc 14.44±0.14Bd 15.69±0.04Be 8 12.19±0.01Aa 12.53±0.01Ab 12.96±0.03Ac 13.71±0.09Ad 14.90±0.1Ae Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION The colourfulness of the pepper samples was less than what was observed in the samples that were studied by Topuz et al. (2009). They observed that the colourfulness of Jalapeno red pepper powder was in the range of 33.57 to 35.53 over a period of 90 days of storage at -18 oC. The chroma of Legon-18 was higher than the chroma of the Anaheim samples. Since the values of a* and b* reduced with time due to the oxidation of the carotenoids in the samples irrespective of the storage condition (Schweiggert et al., 2005), the observed 55 University of Ghana http://ugspace.ug.edu.gh phenomenon in terms of the colourfulness can be attributed to the behaviour of the a* and b* values which reduced gradually in the samples as a result of the breakdown of the pigments (carotenoids) in the samples (Schweiggert et al., 2005). Chroma values in the samples were generally higher in the ones that were stored at 4 oC as compared with the samples that were stored at 28±2 oC is consistent with literature (Sirisoontaralak and Noomhorm, 2006). 4.3.1.5 Effect on browning index The values for browning index (which ascertains the rate of decolouration) are shown Table 4.12. The browning index of the samples reduced with time. Gamma irradiation had effect on browning index of the pepper samples at both storage temperatures (p<0.05). Due to the effect of gamma irradiation (bleaching effect on the colour), the browning index of the irradiated pepper samples were higher than the samples that were not irradiated- giving an indication of the higher likelihood of becoming browner as compared with the samples that were not irradiated. Differences observed in the browning index of the samples were storage-temperature dependent. The higher values of browning index obtained in the samples at the beginning of the study was higher than what was observed in mushroom samples irradiated and stored for twelve months period (Kortei et al., 2015). 56 University of Ghana http://ugspace.ug.edu.gh Table 4.12. Browning index of Legon-18 pepper powder after gamma irradiation, during storage at different temperatures Browning index SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 86.03±0.04Ia 88.91±0.24Ib 91.85±0.2Hc 94.10±0.07Hd 96.46±0.18Ie 1 69.43±0.18Ha 72.85±0.11Hb 75.60±0.23Gc 77.30±0.39Gd 80.76±0.20He 2 65.20±0.14Ga 67.50±0.14Gb 70.65±0.17Fc 74.31±0.44Fd 77.21±0.27Ge 3 69.84±0.13Fa 71.71±0.13Fb 73.82±0.26 Ec 74.60±0.14Fd 76.25±0.34Fe 4 4 o 61.41±0.21 Ea 65.25±0.23Eb 67.39±0.08Dc 70.49±0.28 Ed 80.01±0.13Ee C 5 59.17±0.03Da 63.26±0.08Db 66.21±0.03Cc 69.22±0.01Dd 73.94±0.11De 6 58.05±0.01Ca 60.20±0.23Cb 61.60±0.19Bc 64.22±0.05Cd 69.90±0.14Ce 7 36.41±0.02Ba 37.74±0.0B4 39.04±0.35Ac 40.97±0.41Bd 45.43±0.13Be 8 35.62±0.05Aa 37.43±0.14Ab 38.75±0.02Ac 39.96±0.2Ad 43.87±0.30Ae 0 83.54±0.09Ia 89.94±0.41Hb 91.53±0.16Hc 94.87±0.14Id 95.51±0.11He 1 67.14±0.02Ha 69.49±0.29Gb 72.43±0.29Gc 74.16±0.19Hd 78.53±0.26Ge 2 60.33±0.10Ga 62.69±0.12Fb 65.98±0.08Fc 67.59±0.15Gd 68.42±0.42Fe 3 59.55±0.10Fa 61.60±0.58Eb 63.46±0.12Ec 65.12±0.09Fd28± 68.46±0.70 Fe 4 2 49.06±0.05Ea 50.61±0.10Db 52.50±0.12Dc 54.69±0.54Ed 57.30±0.09Ee o 5 C 42.58±0.41Da 44.47±0.31Cb 47.06±1.42Cc 49.81±0.22Dd 55.18±0.40De 6 41.82±0.08Ca 42.50±0.10Bb 44.52±0.09Bc 47.22±0.15Cd 53.48±0.30 Ce 7 39.97±0.17Ba 41.71±0.23Ab 43.71±0.26Bc 44.85±0.62Bd 48.73±0.10Be 8 41.19±0.07Aa 41.65±0.15Aab 42.10±0.28Ab 43.62±0.44Ac 45.87±0.08Ae Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. Reduction in the browning index with time may be attributed to the reduction in the pigments of the pepper samples used in the study (Schweiggert et al., 2005). At the end of the study, less than 50% of the browning index was observed in all the samples irrespective of the doses applied as well as the storage condition (temperature). The effect of temperature on the colour degradation (L*, a*, b*) played a role in the differences thus observed in the samples stored at 4oC as compared with the samples stored at 28±2 oC which is similar to literature (Rico et al., 2010; Liu et al., 2010; Sirisoontaralak and Noomhorm, 2006; Almela et al., 2002). 57 University of Ghana http://ugspace.ug.edu.gh 4.3.1.6 Effect on colour difference The colour difference (which is an indication of the magnitude of the change in colour between the samples stored and the day 0 samples (control or irradiated) (Martins and Silva, 2002; Patras et al., 2011)) are indicated in Table 4.13. The differences ranged from 5.14±0.07 and 23.62±0.03. A general rise in the colour difference of the samples of the storage weeks for all the storage conditions was observed. There was a general dose- dependent effect on the colour difference of all the samples for the period of the study. There were no significant differences (p>0.05) in the samples during weeks seven and eight for all the samples stored at 4 oC. There was significant (p<0.05) differences in all the samples that were stored at 28±2 oC during the period of the study. Colour difference in the samples was due to the change in the a*, b* and L*-values of the samples (as there was the breakdown of the pigments in the pepper samples) as well as the effect of storage (Topuz et al., 2011; Topuz, et al., 2009; Schweiggert et al., 2005). The total colour difference of some irradiated pepper samples ranged from 0.24 to 1.12 (Nieto- Sandoval et al., 1999), 0.19 to 0.33 in samples stored at 4 oC and 1.05 to 1.37 (Rico et al., 2010) and 0.6 to 0.9 in red Korean pepper samples (Jung et al., 2015) which were lower than the total colour difference of Legon-18 the pepper samples used in this study. The colour difference in the samples in this study was greater than 3 and this indicates ‘very distinctive’ colour difference of the samples from the onset of the study to the end of the study irrespective of the storage temperature (Adekunte et al., 2010). The samples that were stored at 28±2 oC had a higher total colour difference (Table 4.13) as compared with the ones that were stored at 4 oC. 58 University of Ghana http://ugspace.ug.edu.gh Table 4.13. Colour difference of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures Total Colour Change SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 1 5.50±0.06Ad 5.26±0.03Ac 5.14±0.07Ab 5.50±0.06Ad 5.01±0.07Aa 2 7.59±0.03Bc 7.48±0.10Bc 7.32±0.06Bb 7.59±0.03Bc 6.64±0.03Ba 3 10.18±0.04Cd 9.24±0.03Cc 8.94±0.12Cb 10.18±0.04Cd 8.10±0.11Ca 4 4 13.62±0.06Dd 13.27±0.06Dc 13.09±0.07Db 13.62±0.06Dd 11.49±0.12Da o 5 C 14.13±0.02Ec 13.78±0.03Ea 13.90±0.08Eb 14.13±0.02Ec 14.25±0.08Ea 6 20.21±0.02Fa 20.30±0.05Fa 20.45±0.11Fb 20.21±0.02Fb 20.56±0.06Fc 7 20.48±0.03Ga 20.61±0.08Ga 21.03±0.07Gb 20.48±0.03Gc 21.50±0.23Gc 8 20.48±0.03Ga 20.61±0.08Gb 21.03±0.07Gc 20.48±0.03Ga 21.50±0.23Gb 1 5.43±0.01Aa 6.33±0.16Ac 5.97±0.13Ab 5.43±0.01Ac 5.57±0.15Aa 2 9.09±0.06Ba 9.97±0.05Bd 9.36±0.05Bb 9.09±0.06Bc 9.02±0.15Ba 3 12.28±0.04Cc 12.54±0.22Cd 11.98±0.07Cb 12.28±0.04Cb 10.93±0.13Ca 28± 4 16.71±0.05Dc 17.17±0.04Db 16.49±0.17Da 16.71±0.05 Db 16.39±0.45Da 2 5 oC 19.40±0.06 Eb 20.03±0.09Ec 19.53±0.34Eb 19.40±0.06Eb 18.59±0.16Ea 6 20.54±0.05Fb 20.92±0.04Fc 20.49±0.08Fb 20.54±0.05Fb 18.44±0.04Ea 7 21.63±0.04Gb 22.17±0.10Gd 21.37±0.18Ga 21.63±0.04Gc 21.52±0.07Gab 8 22.67±0.06Hb 23.62±0.03Ge 23.34±0.04Hd 22.67±0.06Hc 22.55±0.05Ga Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION. Rico et al. (2010), Liu et al. (2010) and Almela et al. (2002) had indicated the effect of temperature (higher temperatures tend to promote ‘increased’ colour change) on the colour of particular food products. The temperature-dependent total colour differences observed in the samples is according to literature, thus the rate of colour degradation on the samples that were stored at 4 oC was lower due to a slower degradation rate than the samples that were stored at 28±2 oC (Liu et al., 2010; Rico et al., 2010; Almela et al., 2002). There were percentage increases of 372.36, 374.73, 382.36, 372.36 and 390.91 for the samples at 0, 1, 2, 4 and 5 kGy respectively in the samples that were stored at 4 oC. In the samples that were stored 28±2 oC had differences up to 417.50%, 434.99%, 429.83%, 428.18% and 415.29% for the samples irradiated at 0, 1, 2, 4 kGy and 5 kGy respectively. 59 University of Ghana http://ugspace.ug.edu.gh 4.3.1.7 Effect on hue The results of the hue angle which depicts the qualitative attribute (in terms of angle) of the colour of a commodity (Pathare et al., 2012) are shown in Table 4.14. The hue angle of the samples ranged from 30.08±0.14 to 46.35±0.13. There was a general reduction in the hue of the samples at both temperatures of storage. Generally, there was a dose- dependent effect on the hue of the pepper samples that were stored for the period at both storage temperatures which was statistically significant (p<0.05). Storage temperature had an effect on the hue of the samples (p<0.05). A previous study conducted by Topuz et al. (2009), showed that the hue of pepper powders from two varieties stored at -18 oC was in the range of 31.35 to 41.18, however in the samples used in this study, the hue ranged from 30.08±0.14 to 46.35±0.13 which is an indication of varietal and different storage conditions. The observed pattern in the hue of the pepper samples which is in the range of redness, may be attributed to the effect of gamma irradiation on the a* and b* of the pepper samples. This can be attributed to the effect of the breakdown of the pigments of the pepper samples (Schweiggert et al., 2005) irrespective of the storage conditions. 60 University of Ghana http://ugspace.ug.edu.gh Table 4.14. Hue of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures Hue SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 45.82±0.01Fd Fa 46.22±0.06Hd 46.09±0.01Gc 45.52±0.07Fb 45.11±0.15 1 42.19±0.12Ebc 42.17±0.13Gbc 42.39±0.17Fc 41.96±0.17Eb 41.53±0.12 Ea 2 41.22±0.10Da 41.55±0.37Fab 41.68±0.13Dbc 41.53±0.20Eab 42.03±0.08 Dc 3 Da Fab EFc Eb 41.33±0.18Ca 41.51±0.09 41.59±0.24 42.11±0.10 41.81±0.06 4 4 40.45±0.02Ca 40.76±0.30Dao 40.54±0.17 Ca 41.13±0.23Db 43.15±0.04 Cb C 5 40.55±0.10Cd 39.04±0.08Ca 39.82±0.06Bb 39.01±0.02Ca Bc 40.06±0.11 6 Dc Eb Cb Dd 39.73±0.04Ba41.47±0.01 41.10±0.02 40.98±0.20 41.81±0.01 7 Ab37.15±0.02Bb 36.90±0.05Aab 36.20±0.84Aa 38.16±0.25Bc 37.10±0.38 8 36.72±0.28Ab 37.28±0.03Bd 36.11±0.05Aa 37.19±0.30Acd 36.86±0.30 Acb 0 44.53±0.003Ia 46.35±0.13Hd 45.80±0.03Hc Ib 45.56±0.10Fc 44.78±0.17 1 41.60±0.06Ha 41.87±0.03Gb 42.17±0.13Gc Hc 42.36±0.11Ec 42.38±0.24 2 38.80±0.004Gc 38.01±0.04Eb 37.46±0.04Da 38.00±0.09Cb 38.84±0.43 Gc 3 Fd28 35.12±0.07 Bb 34.67±0.15Ba 35.38±0.06Bb 35.89±0.09Bc 37.32±0.40 4 ±2 30.08±0.14Aa 30.11±0.15Aa 31.22±0.18Ab 31.93±0.15Ac 33.11±0.13 Ed o 5 C Da37.13±0.37Ec 37.92±0.20Ed 36.13±0.33Cb 35.81±0.12Bb 32.51±0.29 6 35.63±0.04Cb 37.53±0.03Dc Ec Ca 38.11±0.65 38.08±0.37Cc 33.92±0.37 7 37.45±0.09Da 38.73±0.03Fb 39.35±0.08Fc 39.60±0.42Dc 40.12±0.14 Bd 8 37.74±0.05Fb 37.14±0.06Ca Abc 37.25±0.35Da 38.23±0.11Cc 37.95±0.05 Least Significant Difference: Means with the same letters (upper case, with the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION The lower values that were observed in the samples stored at 4 oC can be attributed to the higher values in the L*, a*, b* values of the samples which had lower degradation due to the lower effect of rate of degradation in relation to lower temperature as compared with the samples that were stored at 28±2 oC (Rico et al., 2010; Liu et al., 2010; Sirisoontaralak and Noomhorm, 2006; Almela et al., 2002). At the end of the storage period, losses of 19.55%, 18.83%, 21.19%, 18.64%, 19.86% were recorded in the hue of the samples that were irradiated at 5 kGy, 4 kGy, 2 kGy, 1 kGy and 0 kGy respectively and were stored at 4 oC. A loss of 15.25%, 16.60%, 16.35%, 14.15% and 14.78% for the samples that were irradiated at 0, 1, 2 4 kGy and 5 kGy respectively and stored at 28±2 oC. Comparing the 61 University of Ghana http://ugspace.ug.edu.gh storage temperatures, the samples that were stored at 4 oC had lower hue values as compared the ones stored at 28±2 oC. 4.3.2 Effect on physiochemical properties 4.3.2.1 Effect on pH and Titratable acidity (TTA) Tables 4.15 and 4.16 show the pH and TTA of the samples. pH and TTA of the samples were not affected by gamma irradiation. Rico et al. (2010) observed that gamma irradiation and storage did not have effect (p>0.05) on the pH and TTA of dried red pepper samples at 4 oC and 20±2 oC. Similar occurrence was also observed by Liu et al. (2010) for tomato samples stored below 37 oC in powdered tomato samples. The observed pattern in both pH and TTA may be due to the stability of the organic acids in the samples. Table 4.15. The pH of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures pH SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 5.00±0.00Aa 5.05±0.15Aa 5.06±0.01Aa 5.06±0.05Aa 5.09±0.01Aa 1 5.11±0.05Ba 5.15±0.03ABCa 5.12±0.01Ba 5.15±0.01Ba 5.14±0.00Ba 2 5.13±0.03Bb 5.08±0.01Aba 5.12±0.02Bab 5.09±0.01ABab 5.11±0.02Aa 3 5.13±0.01Ba 5.13±0.01Aba 5.14±0.01Ba 5.15±0.01Ba 5.14±0.02 Ba 4 4 o 5.14±0.02 Ba 5.15±0.01ABCa 5.21±0.04Ca 5.16±0.06BCa 5.19±0.01Ca C 5 5.24±0.02Ca 5.29±0.01Ea 5.25±0.01CDa 5.24±0.05CDb 5.24±0.01Db 6 5.21±0.01Ca 5.19±0.04BCDEa 5.22±0.01Ca 5.33±0.03EFa 5.33±0.01Ea 7 5.29±0.02Da 5.27±0.03DEa 5.28±0.01Da 5.26±0.03DEa 5.28±0.01Fa 8 5.40±0.11Eab 5.43±0.01Fb 5.41±0.02Eab 5.36±0.04Fab 5.40±0.01Gab 0 5.09±0.05Aa 5.06±0.00Aa 5.10±0.02Aa 5.07±0.04Aa 5.16±0.04Aa 1 5.14±0.01Aa 5.17±0.01Ba 5.15±0.01Aba 5.16±0.03ABa 5.18±0.01Aa 2 5.14±0.01Aa 5.15±0.0Ba 5.16±0.02Bb 5.15±0.01Bb 5.16±0.03Aa 3 28± 5.16±0.03 Aa 5.15±0.02Ba 5.23±0.01Bb 5.24±0.14Bb 5.24±0.01Bb 4 2 5.25±0.01Ba 5.29±0.01Cb 5.25±0.03Bab 5.26±0.13CDab 5.24±0.00Ba o 5 C 5.24±0.00Bab 5.27±0.01Cab 5.25±0.01Bb 5.27±0.13Db 5.27±0.02Ba 6 5.26±0.00Ba 5.28±0.01Ca 5.27±0Ba 5.28±0.26Da 5.25±0.01Ba 7 5.38±0.08Cab 5.46±0.01Db 5.44±0.06Cab 5.44±0.26Eab 5.48±0.01Ca 8 5.40±0.01Ca 5.46±0.04Da 5.42±0.03Ca 5.41±0.13Ea 5.37±0.03Da Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION 62 University of Ghana http://ugspace.ug.edu.gh Table 4.16. Total titratable acidity of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures Titratable acidity (%) SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 0.224±0.009Aa 0.199±0.009Aa 0.218±0.018Aa 0.224±0.027Aa 0.218±0.00Aa 1 0.263±0.009Bab 0.263±0.009Bab 0.243±0.000Aa 0.275±0.009Bb 0.275±0.00Bb 2 0.263±0.009Ba 0.275±0.009BCa 0.275±0.009Ba 0.269±0.000Ba 0.275±0.00Ba 3 0.275±0.009BCa 0.288±0.009Ca 0.275±0.009Ba 0.282±0.018Ba 0.288±0.00Ba 4 4 oC 0.275±0.009BCa 0.269±0.000Ba 0.275±0.009Ba 0.269±0.000Ba 0.275±0.00Ba 5 0.275±0.009BCa 0.275±0.009BCa 0.275±0.009Ba 0.275±0.009Ba 0.275±0.00Ba 6 0.295±0.001CDa 0.275±0.009BCa 0.288±0.009Ba 0.288±0.027Ba 0.282±0.00Ba 7 0.295±0.018CDa 0.269±0.000Ba 0.282±0.018Ba 0.301±0.009Ba 0.288±0.00Ba 8 0.301±0.009Da 0.275±0.009Ba 0.282±0.018Ba 0.282±0.000Ba 0.288±0.00Ba 0 0.224±0.009Aa 0.224±0.027Aa 0.224±0.009Aa 0.211±0.009Aa 0.218±0.00Aa 1 0.231±0.000ABa 0.231±0.018ABa 0.211±0.027Aa 0.231±0.000Aa 0.231±0.00Aa 2 0.231±0.000ABab 0.243±0.000ABCb 0.224±0.009Aa 0.237±0.009ABab 0.263±0.01CDc 3 0.250±0.009BCb 0.263±0.009BCEab 0.263±0.009Bc 0.263±0.009BCab 0.256±0.0 oCDa 4 28±2 oC 0.263±0.009CDa 0.250±0.009ABCDa 0.269±0.000Ba 0.263±0.009BCa 0.243±0.00BCa 5 0.269±0.000CDa 0.263±0.027BCDEa 0.275±0.009Ba 0.275±0.009CDa 0.275±0.01Da 6 0.275±0.009Da 0.275±0.009CDEa 0.269±0.000Ba 0.263±0.027BCa 0.275±0.01DEa 7 0.282±0.018DEa 0.288±0.009Ea 0.275±0.009Ba 0.288±0.009CDa 0.288±0.01EFa 8 0.301±0.009Ea 0.282±0.018DEa 0.275±0.009Ba 0.295±0.000Da 0.301±0.01Fa Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION. 63 University of Ghana http://ugspace.ug.edu.gh 4.3.2.2 Effect on moisture content The moisture content of the samples are shown in Table 4.17. There was no apparent effect of gamma irradiation on the moisture content of the samples. The moisture content seemed to vary slightly during long storage, however the changes were generally not significant. Table 4.17. Moisture content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures S Moisture Content (%) SW C 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 10.20±0.57Da 10.40±0.00Ca 11.00±0.28Ea 10.60±0.28BCa 10.9±0.14Da 1 10.10±0.07BDa 10.30±0.14BCa 10.90±0.14BCEa 10.40±0.28Ba 10.40±028BCa 2 9.60±0.28BCDa 9.80±0.00BCa 10.10±0.71BCa 10.50±0.42Ba 10.70±0.14CDa 3 9.10±0.14ABCa 9.00±0.28Ba 9.80±0.57ABCEa 9.80±0.00Aba4 9.40±0.00 Aa 4 o 9.30±0.14ABCa 9.50±0.14ABCab 9.90±0.42ABCEab 10.40±0.57Bb 10.20±0.00Bb 5 C 9.70±0.14CDab 8.80±0.14Aba 10.60±0.85BCAab 12.00±0.13Cb 10.30±0.14BCab 6 9.30±0.14Aba 9.40±0.00ABCa 10.30±0.70BCa 10.20±0.57ABa 10.70±0.14CDa 7 8.90±0.14Aba 9.00±0.00ABab 9.70±0.42ABab 10.60±0.13BCb 9.30±0.42Aab 8 8.80±0.00Aa 8.60±0.28Aa 8.90±0.14Aa 8.90±0.14Aa 9.20±0.00Ab 0 9.70±0.14BCa 10.20±0.00BCa 10.30±0.71Ba 10.40±0.57Da 10.70±0.14Eb 1 9.60±0.28Ca 9.90±0.14ABCa 9.90±0.99Ba 10.20±0.00CDa 10.70±0.14Eb 2 2 10.40±0.28Ea 10.10±0.42BCa 9.90±0.14Ba 10.00±0.57CDa 10.60±0.00DEa 3 8 9.20±0.28Aa 9.50±0.14Aba 8.80±0.85Aba 9.20±0.00Ba 9.30±0.14Aa ± 4 9.70±0.28 BCa 10.20±0BCa 9.20±0.57Aba 9.60±0.57BCa 9.90±0.14BCa 2 5 9.80±0.28BCDa 10.70±0.14Cb 9.70±0.14Ba 10.70±0.14Db 10.30±0.42CDEao 6 DEa Aba Ba BCa CDaC 10.30±0.42 9.40±0.85 10.10±0.71 9.50±0.14 10.10±0.42 7 9.00±0.28Aa 9.00±0.85Aa 9.00±0.13Aba 9.10±0.14Aa 9.40±0.28Aa 8 9.20±0.00ABc 9.40±0.00ABd 8.10±0.14Aa 8.40±0.00Ab 9.50±0.14Ad Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION The marginal reduction in moisture content of the samples might be due to the probable permeability of the packaging material to moisture, thereby leading to a loss in the moisture content over the period as well as changes pertaining to the relative humidity in the storage environment (Hussain et al., 2011; Hossain and Gottschalk, 2009; Latapi and Barret, 2006). The observed marginal reduction in the moisture content (Tables 4.17) of 64 University of Ghana http://ugspace.ug.edu.gh the samples was similar to the observations made by Rico et al. (2010) when dried red pepper samples were stored at 4 oC and 20±2 oC. The moisture content of the samples reduced with storage. Das et al. (1994) indicated an increase in the moisture content of tea and they attributed to the activities of microorganisms in the unirradiated samples. In another study, Thomas et al. (2008) also made a similar observation as they found that the moisture content in tea increased in the unirradiated samples and was fairly stable in the irradiated samples. In another study, moisture content of some irradiated tomato powder (freeze dried and solar dried samples) increased with storage in a two-month study (Atuobi-Yeboah et al., 2016). 4.3.3 Effect of gamma irradiation and storage on the capsaicinoids and SHU 4.3.3.1 Effect on capsaicin The capsaicin content of the samples is indicated in Table 4.18. The values were in the range of 118 in the unirradiated samples to 221.00 (mg/100g) in the irradiated samples. Gamma irradiation ‘caused’ significant increases (p<0.05) in the capsaicin content of the samples and was dose-dependent. The values however significantly (p<0.05) reduced during storage with time at both storage temperatures, although it was more pronounced in the samples stored at 28±2 oC. The capsaicin content of Legon-18 was higher than some of the samples indicated in previous studies (Jung et al., 2015; Nagy et al., 2015; Giuffrida et al., 2014; Giuffrida et al., 2013; Rico et al., 2010; Topuz et al., 2009; Lee et al., 2004). Topuz and Odzemir (2004), Wang et al. (2009) and Giuffrida et al. (2014) indicated that gamma irradiation and storage had effect on the capsaicin content of some pepper varieties leading to the reduction of capsaicin in those pepper samples. 65 University of Ghana http://ugspace.ug.edu.gh Table 4.18. Capsaicin content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures Capsaicin (mg/100g) SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 178.74±1.30Ia 192.70±1.90Hb 217.38±6.07Fc 216.06±1.43Hc 221.00±2.29Hc 1 177.18±0.28Ha 188.20±7.43Gb 216.88±0.07Fc 216.96±0.12Hc 217.35±0.37Hc 2 173.25±0.00Ga 182.55±0.85Fb 211.09±0.05Ec 214.39±0.14Gd 216.88±0.11Ge 3 165.73±0.77Fa 176.18±1.05Eb 207.59±0.02Ec 211.19±0.56Fd 213.72±0.17Fe 4 4 oC 161.51±0.15Ea 169.32±0.89Db 201.67±3.64Dc 206.52±0.75Ed 210.53±0.00Ee 5 154.40±1.22Da 165.05±0.52CDb 194.03±0.41Cc 199.61±1.41Dd 207.31±0.39De 6 146.93±0.00Ca 161.40±0.25Cb 182.88±3.85Bc 195.78±1.11Cd 204.44±0.73Ce 7 144.74±0.00Ba 156.52±0.02Bb 178.59±0.00Bc 186.72±1.01Bd 198.95±0.09Be 8 140.34±0.00Aa 150.39±0.04Ab 171.87±0.00Ac 184.10±0.16Ad 190.57±0.11Ae 0 180.33±3.35Ha 190.65±4.97Ha 218.40±15.38Gb 213.23±0.33Ib 219.66±1.48Hb 1 170.81±0.37Ha 184.56±0.41Gb 204.78±0.75Fc 210.52±0.00BHd 218.42±0.34He 2 167.91±0.76Ga 180.38±0.41Fb 194.09±2.97Ec 207.67±0.02Gd 214.85±0.01Ge 3 154.98±1.15Fa 169.74±4.57Eb 182.47±8.76Dc 197.66±0.45Fd 206.04±0.77Fe 28± 4 148.71±1.47Ea 156.72±0.46Db 165.37±0.16Cc 190.81±2.61Ed 198.30±0.11Ee 2 oC 5 144.46±0.39Da 150.59±0.00Cb 156.87±0.89BCc 179.92±0.11Dd 188.55±0.56De 6 135.41±4.13Ca 144.71±0.01Bb 147.81±0.07Abb 170.75±0.33Cc 183.17±0.55Cd 7 123.54±1.21Ba 144.24±0.42Bb 145.36±0.51Ab 164.77±0.50Bc 177.06±0.42Bd 8 118.04±0.49Aa 137.35±0.12Ab 144.50±0.39Ac 159.24±0.33Ad 169.78±1.04Ae Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC- STORAGE CONDITION However, Byun et al. (1996) and Lee et al. (2004) also indicated that capsaicin is stable after gamma irradiation with doses less than 15 kGy. The dose-dependent effect on the capsaicin content of the pepper samples from 0 kGy to 4 kGy was similar to the observations of Giuffrida et al. (2014), Wang et al. (2009) and Topuz and Odzemir (2004). The reduction in the content of capsaicin in the pepper samples may be attributed to the presence of some residual enzymatic induced oxidation as well as the effect of the milling process (Wang et al., 2009). The higher content of capsaicin in the irradiated samples may also be attributed to the changes in the conformation of the molecules as well as the accompanying compounds that affected the extraction of the capsaicin in the pepper samples (Topuz et al., 2004; Subbulakshmi et al., 1991). Rico et al. (2010) reported that 66 University of Ghana http://ugspace.ug.edu.gh the temperature of storage of irradiated red pepper did not affect the capsaicin content in red pepper samples. Wang et al. (2009) and Giuffrida et al. (2014) indicated the effect of storage temperature on the capsaicinoids in red chilli pepper powder as temperature- dependent. The effect of temperature on the kinetics of degradation might have played a role in the higher content of capsaicin in the samples stored at 4 oC as compared to samples stored at 28±2 oC, which is similar to what has been reported in literature (Giuffrida et al., 2014; Wang et al., 2009). 4.3.3.2 Effect on dihydrocapsaicin The dihydrocapsaicin content of the pepper powdered samples are presented in Table 4.19. Dihydrocapsaicin content was significantly affected by gamma irradiation (p<0.05). A dose-dependent effect was observed. A reduction in the dihydrocapsaicin content was observed for all samples during storage at the different temperatures. The dihydrocapsaicin content of Legon-18 was in the range of dihydrocapsaicin content of some pepper varieties in literature (Nagy et al., 2015; Cheon et al., 2015; Jung et al., 2015; Giuffrida et al., 2014; Giuffrida et al., 2013; Topuz and Odzemir, 2004). The observed dose-dependent effect of gamma irradiation on the increment of dihydrocapsaicin in the pepper samples was similar to the observation of Giuffrida et al. (2014), Wang et al. (2009) and Topuz and Odzemir (2004). The general increase in the dihydrocapsaicin content of the samples due to dose- dependence might have resulted in a change in the conformation and the other accompanying compounds that were in the samples and had affected the dihydrocapsaicin content on the extraction (Subbulakshmi et al., 1991) irrespective of the storage temperatures at which the samples were stored. Lee et al. (2000), Lee et al. (2004) and Byun et al. (1996), indicated the stability of dihydrocapsaicin after exposure to gamma irradiation, however, this is in contrast to the current study. Griesbach (2003) indicated 67 University of Ghana http://ugspace.ug.edu.gh that constituents of different varieties of the same crop may behave differently after exposure to gamma irradiation which was observed in this study in terms of the effect of gamma irradiation on the content of dihydrocapsaicin in the samples used in this study. The general reduction in the dihydrocapsaicin content of the pepper samples with time which has been stated elsewhere in literature (Giuffrida et al., 2014; Wang et al., 2009; Topuz and Odzemir 2004,). This may be attributed to the oxidative effect of some enzymes noted for the degradation of dihydrocapsaicin as well as effect of processing (Wang et al., 2009). Samples stored at 4 oC had higher dihydrocapsaicin content than the samples that were stored at 28±2 oC. At the end of the storage period there was a loss of 22.46%, 9.95%, 11.78%, 9.86% and 9.53% in the samples that were irradiated at 0, 1, 2, 4 and 5 kGy respectively and stored at 4 oC and for the unirradiated samples, 16.43%, 11.11%, 10.31% and 10.67% for the samples that were irradiated at 1, 2, 4 and 5 kGy respectively and stored at 28±2 oC. 68 University of Ghana http://ugspace.ug.edu.gh Table 4.19. Dihydrocapsaicin content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures Dihydrocapsaicin (mg/100g) SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 77.12±1.84Ga 79.37±0.02Ib 83.73±0.47Hc 87.91±0.59Hd 88.48±1.11Hd 1 75.48±0.02Fa 79.01±0.03Hb 83.45±0.17Hc 87.71±0.08Hd 88.76±0.04He 2 74.29±0.05Ea 78.48±0.43Gb 82.79±0.11Gc 85.90±0.38Gd 87.21±0.00Ge 3 73.35±0.12Ea 77.49±0.13Fb 80.94±0.13Fc 85.11±0.23Fd 86.38±0.37Fe 4 4 oC 66.15±0.00Da 76.87±0.07Eb 80.08±0.26Ec 83.80±0.45Ed 85.32±0.15Ee 5 64.46±0.63Ca 76.35±0.04Db 79.51±0.04Dc 82.66±0.00Dd 83.56±0.51De 6 63.82±0.58Ca 75.71±0.15Cb 78.66±0.34Cc 81.93±0.04Cd 82.62±0.39Cd 7 61.97±0.00Ba 74.96±0.03Bb 77.13±0.37Bc 80.53±0.05Bd 80.84±0.10Bd 8 59.80±0.15Aa 71.47±0.20Ab 73.87±0.15Ac 79.24±0.32Ad 80.05±0.18Ae 0 76.42±1.36Ga 79.35±0.15Ib 83.68±0.47Hc 87.85±0.59Hd 89.25±0.02He 1 72.94±0.13Fa 78.70±0.03Hb 83.12±0.17Hc 86.24±0.08Hd 87.02±0.08Ge 2 72.10±0.23Ea 77.38±0.43Gb 80.95±0.11Gc 85.17±0.38Gd 86.46±0.33Fe 3 60.38±0.34Ea 75.52±0.13Fb 79.27±0.13Fc 84.15±0.23Fd 85.51±0.23Ee 28± 4 o 59.78±0.06 Da 74.83±0.07Eb 78.67±0.26Ec 82.74±0.45Ed 85.24±0.01Ee 2 C 5 52.04±0.79Ca 74.07±0.04Db 77.91±0.04Dc 81.99±0.00Dd 84.00±0.55De 6 47.94±0.76Ca 72.74±0.15Cb 77.01±0.34Cc 81.14±0.04Cd 82.61±0.07Ce 7 40.90±0.4Ba 68.89±0.03Bb 75.43±0.37Bc 79.32±0.05Bd 81.69±0.41Be 8 37.18±0.4Aa 66.31±0.20Ab 73.88±0.14Ac 78.79±0.32Ad 79.73±0.54Ae Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION. 4.3.3.3 Effect on total capsaicinoids The total capsaicinoids in the samples are indicated Table 4.20. Generally, the samples that were irradiated had higher content of total capsaicinoids as compared to the unirradiated samples. Gamma irradiation caused an increase of 6.33, 17.68, 18.79 % and 20.95% in the samples irradiated at 1, 2, 4 and 5 kGy respectively. The total capsaicinoids reduced with storage just as there was a reduction in the capsaicin and dihydrocapsaicin content of the pepper samples for all the temperature conditions as well as the doses that the samples were exposed to. The total capsaicinoids content of the samples that were stored at 28±2 oC were reduced by percentages of 39.54 for the unirradiated, 24.57 for samples at 1 kGy, 27.7 at 2 kGy, 20.94 for samples at 4 kGy and 0.94% at 5 kGy. 69 University of Ghana http://ugspace.ug.edu.gh Table 4.20. Total capsaicinoids of Legon-18 pepper powder after gamma irradiation and during storage at different temperatures Total capsaicinoids (mg/100 g) SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 255.87±2.57Ia 272.07±1.95Hb 301.11±5.93Hc 303.96±0.89Hcd 309.47±3.32Id 1 252.65±0.39Ha 267.21±7.43Gb 300.33±0.11Hc 304.67±0.18Hc 306.12±0.36Hc 2 247.54±0.30Ga 261.04±1.17Fb 293.88±0.13Gc 300.28±0.46Gd 304.09±0.11Ge 3 239.08±1.06Fa 253.68±1.00Eb 288.53±E0.15Fc 296.29±0.51Fd 300.10±0.47Fe 4 4 oC 227.66±0.21Ea 246.20±0.90Db 281.74±3.90Ec 290.32±1.19Ed 295.85±0.15Ee 5 218.86±1.14Da 241.40±0.54Cb 273.54±0.43Dc 282.27±1.40Dd 290.86±0.20De 6 210.75±0.76Ca 237.10±0.37Cb 261.54±3.71Cc 277.71±1.09Cd 287.06±0.34Ce 7 206.70±0.05Ba 231.49±0.04Bb 255.72±0.37Bc 267.25±1.09Bd 279.79±0.17Be 8 200.14±0.42Aa 221.86±0.17Ab 245.74±0.15Ac 263.35±0.19Ad 270.61±0.14Ae 0 256.75±3.23Ia 270.00±4.90Ia 302.07±15.53Gb 301.08±0.38Ib 308.91±1.49Ib 1 243.74±0.37Ha 263.27±0.41Hb 287.90±0.49Fc 296.77±0.09Hd 305.44±0.35He 2 240.01±0.42Ga 257.76±0.42Gb 275.04±3.17Ec 292.84±0.33Gd 301.31±0.35Ge 3 215.36±1.25Fa 245.26±4.57Fb 261.74±8.49Dc 281.81±0.43Fd 291.55±0.53Fe 28± 4 208.50±1.38Ea 231.56±0.45Eb 244.04±0.28Cc o 273.54±2.64 Ed 283.53±0.10Ee 2 C 5 196.50±0.45Da 224.66±0.08Db 234.78±0.96BCc 261.91±0.23Dd 272.55±0.61De 6 183.35±4.06Ca 217.46±1.15Cb 224.82±0.25ABc 251.90±0.38Cd 265.78±0.62Ce 7 164.44±1.33Ba 213.13±1.11Bb 220.79±0.35Ac 244.09±0.44Bd 309.47±3.32Id 8 155.22±0.50Aa 203.66±0.11Ab 218.39±0.26Ac 238.02±0.19Ad 306.12±0.36Hc Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION The higher total capsaicinoids recorded in the irradiated samples is similar to previous studies (Giuffrida et al., 2014; Wang et al., 2009; Topuz and Odzemir 2004). but contrasts the findings of Lee et al. (2000), Lee et al. (2004) and Byun et al. (1996). The general reduction of total capsaicinoids of the samples may be due to the oxidative effect of some enzymes noted for the degradation of capsaicin and dihydrocapsaicin as well as effect of processing (Wang et al., 2009) and varietal differences (Griesbach, 2003). Since the total capsaicinoids is a computation of the addition of the capsaicin and dihydrocapsaicin (Jung et al., 2015; Orellana-Escobedo et al., 2013; Lee et al., 2004), the effect of temperature on the capsaicin and dihydrocapsaicin obviously will have an effect on the total capsaicin content of the pepper samples. The higher total capsaicin content that were detected in the 70 University of Ghana http://ugspace.ug.edu.gh samples that were stored at 4 oC than the samples that were stored at 28±2 oC may be due to the slower rate of degradation of the both capsaicin and dihydrocapsaicin in the samples that were stored at 4 oC, which is similar to literature (Giuffrida et al., 2014; Wang et al., 2009). 4.3.3.4 Effect on Scoville Heat Units (hotness index) The Scoville Heat Units (SHU) of the pepper samples used in the study are depicted in Table 4.21. The SHU of Legon-18 pepper samples were in the range of 4119.46 (x1000 in mg/100g) in the unirradiated samples and 4893(x1000 in mg/100g). Gamma irradiation affected the SHU of the samples. The hotness of the samples increased with dose from the unirradiated to the irradiated samples which was statistically significant (p<0.05). There was a general reduction in the SHU for all the samples from the beginning to the end of the storage period irrespective of the storage temperatures, however, it was more pronounced in the samples that were stored at 28±2 oC. The SHU of Legon-18 pepper samples is less than that reported by Lee et al. (2004) and Orellana-Escobedo et al. (2013). 71 University of Ghana http://ugspace.ug.edu.gh Table 4.21. Scoville heat unit of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures SCOVILLE HEAT UNIT (x10000 mg/100 g) SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 4119.46±41.33Ia 4380.27±31.39Gb 4847.90±96.17Hc 4893.82±14.41Idc 4982.51±53.49Id 1 4067.74±62.26Ha 4302.05±119.55Fb 4835.38±1.82Hc 4905.17±2.96Hc 4928.46±57.45Hc 2 3985.45±36.96Ga 4202.69±18.90Eb 4731.43±0.21Gc 4834.56±7.47Gd 4865.9118.19Ge 3 3849.15±17.01Fa 4084.19±16.03Db 4645.32±2.41Fc 4770.33±8.15Fd 4831.62±76.45Fe 4 4 oC 3665.39±33.07Ea 3963.75±14.53Cb 4536.07±62.74Ec 4674.10±19.17Ed 4763.26±23.72Ee 5 3523.64±18.43Da 3886.48±8.68Bb 4403.97±6.90Dc 4544.47±22.62Dd 4682.88±32.45De 6 3393.05±12.30Ca 3817.36±5.94Bb 4210.79±59.66Cc 4471.08±17.61Cd 4621.74±54.33Ce 7 3327.91±0.78Ba 3726.92±0.63Ab 4117.08±5.98Bc 4302.80±17.54Bd 4504.64±27.11Be 8 3222.23±67.96Aa 3571.93±2.81Ab 3956.39±2.24Ac 4239.86±3.04Ad 4356.87±22.17Ae 0 4133.65±51.94Ia 4347.07±78.87Ia 4863.39±249.98Gb 4847.44±6.07Ib 4973.43±24.03Ib 1 3924.25±5.95Ha 4238.58±6.53Hb 4635.19±7.85Fc 4777.94±1.49Hd 4917.57±55.95He 2 3864.16±6.88Ga 4149.98±6.83Gb 4428.13±50.99Ec 4714.67±5.36Gd 4851.09±55.64Ge 3 3467.35±20.18Fa 3948.62±73.55Fb 4214.01±136.64Dc 4537.16±6.91Fd 4693.95±85.88Fe 4 28±2 oC 3356.81±22.20Ea 3728.10±7.62Eb 3929.08±4.57Cc 4404.05±4.26Ed 4564.90±16.83Ee 5 3163.62±7.25Da 3617.00±1.27Db 3779.97±15.52BCc 4216.75±3.69Dd 4388.04±97.97De 6 2951.97±65.44Ca 3501.04±18.55Cb 3619.61±3.99ABc 4055.53±6.06Cd 4279.13±10.53Ce 7 2647.55±21.36Ba 3431.42±17.89Bb 3554.76±5.68Ac 3929.79±7.14Bd 4165.96±10.53Be 8 2499.05±7.97Aa 3278.95±1.78Ab 3516.03±4.14Ac 3832.14±3.12Ad 4017.12±11.00Ae Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION 72 University of Ghana http://ugspace.ug.edu.gh The observed reduction in SHU in the samples may be due to the effect of some residual enzymatic action which led to the oxidation of the capsaicinoids, the effect of milling as well as change in the conformation of the molecules and the accompanying compounds that affected the extraction of the capsaicinoids (Giuffrida et al., 2014; Wang et al., 2009; Topuz and Odzemir, 2004; Subbulakshmi et al., 1991). At the end of the storage period, the hotness of the pepper samples irrespective of the storage condition and doses applied were less than the samples that were analysed at the beginning of the study. Capsaicin and dihydrocapsaicin were more stable in the samples stored at 4 oC than the samples stored at 28±2 oC (Wang et al., 2009; Giuffrida et al., 2014) hence the SHU of the samples were higher in the samples that were stored at 4 oC than the samples that were stored at 28±2 oC. Samples that were stored at 28±2 oC were reduced by over 10.00 %. Gamma irradiation caused an increment of over 6.3% in the SHU of the samples used in the study. 4.3.4 Impact of gamma irradiation and storage weeks on the carotenoids content The carotenoids analysed and quantified in this study were beta carotene, beta cryptoxanthin, capsanthin and zeaxanthin which are known to be the main carotenoids in red chili peppers (Giuffrida et al., 2013; Kim et al., 2004). Except for zeaxanthin, beta carotene, beta cryptoxanthin and capsanthin were detected during the HPLC analysis. 4.3.4.1 Effect on beta cryptoxanthin The beta cryptoxanthin content of the samples are shown in Table 4.22. The values were in the range of 1.04 to 1.87 in the irradiated samples to 1.29 to 2.11 (mg/100 g) in the unirradiated samples. Gamma irradiation had effect on the values which was dose- dependent (p<0.05). There was a general reduction in the values during storage 73 University of Ghana http://ugspace.ug.edu.gh irrespective of the storage temperatures the samples were stored at, but more pronounced in the samples that were stored at 28±2 oC. Table 4.22. Beta cryptoxanthin content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures S Beta cryptoxanthin (mg/100 g) SC W 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 Da2.11±0.02He 1.87±0.02Id 1.71±0.00Hc 1.67±0.04Ib 1.55±0.01 1 2.05±0.00Ge 1.84±0.00Hd 1.71±0.00Hc 1.63±0.01Hb 1.59±0.00 Da 2 2.03±0.02Ge 1.75±0.00Gd 1.68±0.00Gc 1.59±0.01Gb 1.56±0.03 Da 3 Ca1.95±0.00Fe 1.70±0.01Fd 1.62±0.02Fc 1.53±0.01Fb 1.48±0.01 4 4 oC BCa1.87±0.00Ee 1.64±0.00Ed 1.57±0.01Ec 1.51±0.01Eb 1.43±0.02 5 De Dd Dc Db 1.40±0.00Ba1.80±0.01 1.55±0.01 1.48±0.00 1.44±0.00 6 1.71±0.00Ce 1.49±0.01Cd 1.41±0.00Cc 1.37±0.00Cb 1.30±0.00 Aa 7 1.66±0.00Be 1.42±0.01Bd 1.36±0.01Bc 1.27±0.00Bb 1.24±0.00 Aa 8 1.50±0.03Ae 1.36±0.00Ad 1.29±0.01Ac 1.23±0.00Ab 1.24±0.11 Aa 0 2.10±0.01Ie 1.87±0.02Id 1.74±0.00Ic 1.67±0.03Ib 1.55±0.00 Ia 1 2.06±0.01He 1.79±0.00Hd 1.68±0.01Hc 1.59±0.01Hb 1.52±0.00 Ha 2 Ga2.00±0.00Ge 1.73±0.02Gd 1.63±0.00Gc 1.53±0.02Gb 1.44±0.00 3 1.91±0.00Fe 1.66±0.00Fd Fa 1.56±0.01Fc 1.45±0.01Fb 1.41±0.02 28±2 4 1.76±0.02Ee 1.59±0.01Ed 1.46±0.01Eco 1.38±0.02 Eb 1.34±0.01 Ea C 5 Da1.65±0.02De 1.44±0.00Dd 1.39±0.01Dc 1.30±0.00Db 1.24±0.00 6 1.54±0.01Ce Ca 1.41±0.01Cd 1.33±0.01Cc 1.22±0.00Cb 1.16±0.01 7 Ba1.43±0.01Be 1.34±0.02Bd 1.26±0.01Bc 1.18±0.00Bb 1.09±0.01 8 1.29±0.01Ae Aa 1.26±0.00Ad 1.15±0.01Ac 1.08±0.01Ab 1.04±0.02 Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION The beta-cryptoxanthin content of the pepper samples of Legon-18 pepper samples was less than the content of a Korean variety found by Kim et al. (2004) and higher than the other variety observed by Topuz and Odzemir (2003). This may be due to varietal differences (Wang et al., 2009). Topuz and Odzemir (2003) and Kim et al. (2004) indicated that radiolytic products formed and absorbed energy during gamma irradiation led to reduced content of carotenoids. The observed reduction in the values due to increasing 74 University of Ghana http://ugspace.ug.edu.gh doses of gamma irradiation is similar to previous studies (Kim et al., 2004; Topuz and Odzemir, 2003). The observed reduction during storage might be attributed to secondary effects of gamma irradiation, storage temperature, oxidation of components, form of pepper samples and the structure of beta cryptoxanthin (Guadarrama-Lezama et al., 2014; Giuffrida et al., 2014; Kim et al., 2006; Perez-Galvez and Minguez-Mosquera, 2001; Tang and Chen, 1999; Goda et al., 1995). 4.3.4.2 Effect on beta carotene Beta carotene content of the pepper samples are indicated in Table 4.23. The beta carotene content of the samples ranged from 5.36 to 10.25 in the irradiated 7.17 to 10.27 (mg/100g) in the unirradiated samples. The content was affected by gamma irradiation which was dose-dependent (p<0.05). There was a marginal reduction in the beta carotene content from unirradiated through to the irradiated samples immediately after gamma irradiation (p<0.05) and also due to storage at the different temperatures. The observed phenomenon in the values are similar as to the observations recorded in beta cryptoxanthin. 75 University of Ghana http://ugspace.ug.edu.gh Table 4.23. Beta carotene content of Legon-18 pepper powder after gamma irradiation, and during storage at different temperatures Beta carotene (mg/100 g) SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 10.27±0.00Id 10.25±0.02Id 9.86±0.02Hc 9.54±0.00Ib 9.38±0.00Ia 1 10.04±0.00Hd 9.92±0.00Hc 9.43±0.00Gb 9.24±0.00Ha 9.24±0.00Ha 2 9.92±0.00Ge 9.66±0.04Gd 9.42±0.06Gc 9.01±0.00Gb 8.78±0.00Ga 3 9.43±0.00Fe 9.57±0.02Fd 8.95±0.06Fc 8.75±0.03Fb 8.48±0.00Fa 4 4 oC 9.20±0.00Ee 9.42±0.02Ed 8.82±0.01Ec 8.55±0.00Eb 8.23±0.02Ea 5 8.91±0.02De 9.26±0.02Dd 8.57±0.02Dc 8.44±0.00Db 8.21±0.00Da 6 8.69±0.07Ce 9.04±0.04Cd 8.36±0.00Cc 8.20±0.02Cb 7.91±0.00Ca 7 8.48±0.00Be 8.89±0.00Bd 7.64±0.00Bc 7.53±0.00Bb 7.26±0.00Ba 8 8.17±0.00Ae 8.51±0.07Ad 7.21±0.02Ac 6.35±0.00Ab 6.01±0.00Aa 0 10.29±0.02Ie 10.25±0.01Id 9.89±0.01Ic 9.53±0.01Ib 9.38±0.00Ia 1 9.96±0.01He 9.77±0.00Hd 9.55±0.02Hc 9.27±0.00Hb 9.20±0.01Ha 2 9.77±0.00Ge 9.43±0.04Gd 8.96±0.02Gc 8.89±0.00Gb 8.67±0.00Ga 3 9.28±0.00 Fe 8.88±0.02Fd 8.69±0.01Fc 8.51±0.00Fb 8.21±0.00Fa 28±2 4 o 8.86±0.00 Ee 8.33±0.01Ed 8.21±0.00Ec 7.87±0.00Eb 7.72±0.00Ea C 5 8.38±0.02De 7.87±0.00Dd 7.54±0.02Dc 7.29±0.04Db 7.06±0.18Da 6 7.91±000Cd 7.29±0.02Cc 7.19±0.01Cc 6.66±0.05Cb 6.27±0.00Ca 7 7.54±0.03Bc 7.17±0.02Bb 6.70±0.19Bc 6.16±0.00Bb 5.57±0.02Ba 8 7.17±0.00Ae 6.68±0.02Ad 6.15±0.02Ac 5.80±0.02Ab 5.36±0.00Aa Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION 4.3.4.3 Effect on capsanthin Table 4.24 shows the capsanthin content of the samples. Capsanthin content of the irradiated were in the range of 1.14 to 1.44 and 1.12 to 1.48 (mg/100 g) in the unirradiated samples. Lower values were recorded in the samples that were irradiated which was statistically significant (p<0.05). There was a dose-dependent effect (resulting in a reduction in the capsanthin content as doses increased) on the content of capsanthin in the samples during storage with some few stabilities in the capsanthin content of the samples. The observed phenomenon is similar to the other pigments discussed earlier. The few stabilities observed in the values is similar to what has been reported in literature (Jung et al., 2015; Rico et al., 2010; Lee et al., 2004). 76 University of Ghana http://ugspace.ug.edu.gh Table 4.24. Capsanthin content of Legon-18 pepper powder pepper powder after gamma irradiation, and during storage at different temperatures Capsanthin (mg/100 g) SW SC 0 kGy 1 kGy 2 kGy 4 kGy 5 kGy 0 1.48±0.00Fe 1.44±0.00Fd 1.41±0.00Ec 1.38±0.01Gb 1.36±0.02Ga 1 1.46±0.01Ee 1.437±0.00Ed 1.40±0.00Dec 1.37±0.00Gb 1.35±0.01Ga 2 1.42±0.00Dc 1.43±0.00Dd 1.39±0.02Dc 1.37±0.00Gb 1.33±0.00GFa 3 1.41±0.00Dd 1.42±0.00Ccd 1.39±0.02Dc 1.34±0.00Fb 1.30±0.00EFa 4 4 oC 1.41±0.00De 1.42±0.00Cd 1.33±0.00Cc 1.32±0.01Eb 1.27±0.00DEa 5 1.41±0.00Dc 1.40±0.00Bd 1.30±0.00Bc 1.30±0.00Db 1.26±0.00DCa 6 1.38±0.02Cd 1.40±0.00Bd 1.29±0.01Bc 1.27±0.00Cb 1.23±0.00Ca 7 1.35±0.02Bc 1.37±0.00Ab 1.26±0.01Aa 1.25±0.01Bb 1.20±0.00Ba 8 1.32±0.02Ae 1.37±0.00Ad 1.25±0.00Ac 1.20±0.00Ab 1.14±0.04Aa 0 1.47±0.00Ie 1.45±0.01Id 1.41±0.00Ic 1.39±0.01Ib 1.35±0.02Ha 1 1.46±0.00He 1.44±0.00Hd 1.40±0.00Hc 1.38±0.00Hb 1.32±0.01Ga 2 1.43±0.00Ge 1.40±0.00Gd 1.37±0.00Gc 1.35±0.00Gb 1.29±0.00Fa 3 1.41±0.00Fe 1.37±0.00Fd 1.36±0.00Fc 1.32±0.01Fb 1.30±0.00Fa 28± 4 o 1.40±0.00 Ed 1.33±0.00Ec 1.29±0.00Eb 1.29±0.01Eb 1.26±0.01Ea 2 C 5 1.36±0.01Dd 1.29±0.01Dc 1.25±0.01Db 1.25±0.00Db 1.23±0.00Da 6 1.32±0.00Cd 1.25±0.01Cc 1.22±0.00Cb 1.21±0.00Cb 1.18±0.00Ca 7 1.28±0.00Be 1.22±0.01Bd 1.18±0.00Bc 1.17±0.00Bb 1.15±0.01Ba 8 1.22±0.01Ae 1.18±0.00Ad 1.16±0.00Ac 1.14±0.00Ab 1.12±0.01Aa Least Significant Difference: Means with the same letters (upper case, within the same column for a particular temperature regeme) are not significantly (P>0.05) different from each other and means with the same letters in the same row (lower case, doses week within the same temperature regeme) are not significantly different (P>0.05) from each other. KEY: SW-STORAGE WEEKS; SC-STORAGE CONDITION. Giuffrida et al. (2014), Guadarrama-Lezama et al. (2014), Kim et al. (2006) and Tang and Chen (1999) indicated that higher temperatures led to lower carotenoid content of agricultural produce. Beta carotene, beta cryptoxanthin and capsanthin content in the samples that were stored at 4 oC were higher than the ones that were stored at 28±2 oC. The observed reduction in the carotenoids investigated in the study might be due to the rate of degradation of the carotenoids at such temperatures (Guadarrama-Lezama et al., 2014; Giuffrida et al., 2014; Kim et al., 2006; Tang and Chen, 1999). 77 University of Ghana http://ugspace.ug.edu.gh 4.4 Optimization of microbial inactivation by gamma irradiation and storage days on S. Typhimurium, E. coli, B. cereus, L. monocytogenes and S. aureus using response surface methodology (Central Composite Design) 4.4.1 Salmonella Typhimurium The estimated regression coefficients from the central composite design (appendix I) for the inactivation (survival) of S. Typhimurium and days of storage in the samples that were stored at 4oC were all not statistically significant (p>0.05) except the constant of regression. A general reduction in the log cfu/g of S. Typhimurium was observed for all doses (Fig.4.1 and appendix XXI). The regression analysis fitted the model at 92.50%. Fig. 4.1. Effect of gamma irradiation and storage on the survival of S. Typhimurium at 4oC The observed inactivation might be due to the effect levels of injuries caused to the cells, the inability of the cells to repair their DNA as well as the post-irradiation effect on the cells (Lucht et al., 1998; Byun et al., 2001; Song et al., 2006; Song et al., 2007; Wu, 2008). 78 University of Ghana http://ugspace.ug.edu.gh Figure 4.2 and appendices III and XXII indicate the inactivation of the pathogen in the samples that were stored at 28±2 oC. The regression coefficients were all statistically significant (p<0.05). The model fitted the inactivation (R2) at 98.80%. A general reduction in the log cfu/g of the organisms was observed. Complete inactivation of the organisms was only during day 60 at all the doses including the control. The gradual inactivation in the irradiated samples over time may be attributed to the post irradiation effect as well as the lethality of the doses applied (Cárcel et al., 2015; Song et al., 2007, Song et al., 2006; Byun et al., 2001; Lucht et al., 1998). Fig. 4.2. Effect of gamma irradiation and storage on the survival of S. Typhimurium at 28±2 oC 79 University of Ghana http://ugspace.ug.edu.gh 4.4.2 Escherichia coli The estimated regression coefficients are indicated in appendix V for the samples stored at 4 oC. There was a general reduction in the count of organisms with time (Fig. 4.3 and appendix V). The model fitted the inactivation (R2) at 99.40%. A gradual inactivation of the pathogen irrespective of the doses, however the highest inactivation (lowest survival rate) was observed in the samples at the various doses (Fig. 4.3 and appendix III). The observed inactivation might be due to other factors like the composition of the material and probably nutrient for growth (Ban and Kang, 2014; Song et al., 2014). Fig. 4.2. Effect of gamma irradiation and storage on the survival of E. coli at 4oC The gradual inactivation especially among the irradiated samples may be attributed to the lethality of the doses as well as the effect of post irradiation effect (Lucht et al., 1998; Byun et al., 2001; Song et al., 2006, Song et al., 2007, Cárcel et al., 2015). 80 University of Ghana http://ugspace.ug.edu.gh Statistically significant (p<0.05) coefficients of regression in terms of doses of gamma irradiation, days of storage, constant of regression was observed for the samples that were stored at 28±2 oC (appendix VII and VIII). The R2 for the model fit was 99.20%. A general reduction in the survival (increase in the inactivation) of E. coli was observed (Fig. 4.4 and appendix XXIV). Fig. 4.3. Effect of gamma irradiation and storage on the survival of E. coli at 28±2 oC Higher inactivation (lower survivability) from day 0 was observed for all the irradiated samples (Fig 3.6 and 3.7). The impact of lethality of the gamma irradiated samples was gradual (Byun et al., 2001; Lucht et al., 1998) thereby leading to a complete inactivation at day 60 (Fig.3.6). These doses applied indicates that complete decontamination of the samples can be achieved with storage. 81 University of Ghana http://ugspace.ug.edu.gh 4.4.3 Bacillus cereus The regression coefficients for the only spore former B. cereus are indicated in appendices appendix IX, X) at storage 4 oC. A general reduction in the survival of the organisms was conspicuous (Fig. 4.5 and appendix XXV). Samples that were stored at 28±2oC had regression coefficients indicated in appendix XI, XII. The experiment fitted the model at an R2 of 97.30%. The highest inactivation during the study was observed in the samples irradiated at 4 kGy (Fig 3.6 and appendix XXVI), which could not achieve complete inactivation of this pathogen except beyond 60 days of storage. Fig. 4.4. Effect of gamma irradiation and storage on the survival of B. cereus at 4oC The gradual inactivation of the pathogen with storage had been reported in literature (Ban and Kang, 2014). 82 University of Ghana http://ugspace.ug.edu.gh Fig. 4.5. Effect of gamma irradiation and storage on the survival of B. cereus at 28±2 oC 4.4.4. Listeria monocytogenes Regression coefficients for the samples that were stored at 4 oC had can be found appendix XIII and XIV. The R2 value was 96.10% for the model fit. A general reduction in the count of the organisms was observed (Fig. 4.7 and appendix XXVII). The complete inactivation of the sample was achieved at both doses 2 and 4 kGy only after a long storage period of 60 days (Fig. 4.7 and appendix XXVII) which is an indication of the effect of lower lethality from the lower doses that were used in the study. The post irradiation effect and the injury caused to the organisms could not inactivate them completely even at day 0 (Byun et al., 2001; Lucht et al., 1998). 83 University of Ghana http://ugspace.ug.edu.gh Fig. 4.6. Effect of gamma irradiation and storage on the survival of L. monocytogenes at 4oC. The complete inactivation of the sample was achieved at both doses 2 and 4 kGy only after a long storage period of 60 days (Fig. 4.7 and appendix XXVII) which is an indication of the effect of lower lethality from the lower doses that were used in the study. The post irradiation effect and the injury caused to the organisms could not inactivate them completely even at day 0 (Byun et al., 2001; Lucht et al., 1998). Inactivation of the organisms was dose dependent which is typical of literature (Torgby- Tetteh et al., 2014; Ban and Kang, 2014; Rico et al., 2010). Higher doses from 5 kGy would have inactivated all the organisms even at day zero (a preliminary observation during the study) which is typical of literature (Song et al., 2014). 84 University of Ghana http://ugspace.ug.edu.gh The coefficient of the regression parameters for the samples irradiated at 28±2 oC are displayed in appendix XV and XVI. The R2 value for the model was 96.80% (appendix XV). A general reduction in the count of the organisms was observed (Fig. 4.8 and appendix XXVIII). Fig. 4.7. Effect of gamma irradiation and storage on the survival of L. monocytogenes at 28±2 oC. Post irradiation effect on the L. monocytogenes was obvious as depicted in Fig. 4.8 due to the gradual inactivation of the pathogen (Cárcel et al., 2015; Song et al, 2007; Song et al, 2006, Byun et al., 2001; Lucht et al., 1998). The injuries that were caused to the organisms could not inactivate them immediately or render the samples microbiologically safe for use until day 60. Higher doses (Table 4.4) could have led to the inactivation even during day zero (Oularbi and Mansouri, 1996). 85 University of Ghana http://ugspace.ug.edu.gh 4.4.5 Staphylococcus aureus The effect of the gamma irradiation on S. aureus were fitted to model at an r2 of 97.60% (appendix XVII). The coefficients of regression are shown in appendix XVII and XVIII. Fig. 4.9 and appendix XXIX depict the effect of days and doses on the survival of the organisms. The most effective dose for the inactivation of S. aureus in this current study was 4 kGy, however its effect was with time which is typical in literature (Cárcel et al., 2015; Song et al., 2007; Song et al., 2006, Byun et al., 2001; Lucht et al., 1998). Fig. 4.8. Effect of gamma irradiation and storage on the survival of S. aureus at 4oC. The coefficient of the regression for the samples that were stored at 28±2 oC were -1.33263, -0.03545 and -0.00254 for the doses, days and interaction between the days and the doses respectively (XIX and XX). The r2 for the model was 94.10%. Interactive effects and effects of the main factors are displayed in Fig. 4.10 and appendix XXX. Inactivation of the pathogen at this storage temperature was gradual due to the doses applied as these doses had a lower lethality on the cells as well as a lower post irradiation effect (Song et al., 2007; Song et al., 2006; Byun et al., 2001; Lucht et al., 1998). The injuries that were 86 University of Ghana http://ugspace.ug.edu.gh caused to the organisms could not inactivate them completely within the first day of (day zero) of the study (Lucht et al., 1998). Fig. 4.9. Effect of gamma irradiation and storage on the survival of S. aureus at 28±2 oC 4.5 Optimization of the effect of gamma irradiation and storage on the quality and components of legon-18 pepper samples (Central Composite Design) 4.5.1 L* values From the estimated regression coefficient (Appendix LXII and LXIII, and Fig. 4.4) for the lightness (brightness) of the pepper samples, the effect of the doses of gamma irradiation applied to the samples was not significant (p>0.05). The estimated regression coefficient for the samples stored at 4 oC (Appendices LXIV, LXV), indicated that the impact of the doses on the lightness or brightness of the colour of the pepper samples was not significant (p>0.05). Effect of storage weeks was significant (p<0.05). The observed pattern might be due to the effect of oxidation of the carotenoids in the samples (Schweiggert et al., 2005). Longer storage days would lead to further ‘darkening’ of the samples (Kim et al., 2004). 87 University of Ghana http://ugspace.ug.edu.gh The impact of the storage weeks on the lightness of the pepper samples were significant (p<0.05). Increasing the storage days thus will have more impact on the brightness of the colour of the pepper than looking at the doses on the pepper samples (Kim et al., 2004). This indicates the impact of the storage weeks on the samples on the samples that were stored at 28±2 oC. The regression fitted the model at 98.6%. Fig. 4.10. L* values after gamma irradiation and during storage stored at 4 oC 88 University of Ghana http://ugspace.ug.edu.gh Fig. 4.11. L* values after gamma irradiation and during storage at 28±2 oC 4.5.2 a* values The coefficient of regression (appendices LXVI and LXVII, and Fig.4.18) for the doses was significant (p<0.05) indicting bleaching effect of the gamma irradiation on the redness of the samples at 28±2 oC indicating that the higher the doses applied the higher the redness of the samples. Though storage weeks significantly (p<0.05) affected the redness of the samples, the coefficient of the regression indicates a ‘reduction’ effect on the redness of the samples. This implies that the longer the samples are stored, the darker (redness of the samples reduces) the samples will become (Almela et al., 2002). There was a dose-week interaction effect (p<0.05), which implies that the redness of the samples of Legon-18 is affected by both the doses and weeks combined which is similar to literature (Cheon et al., 2015; Kim, 2012). The regression coefficients, analysis of variance (ANOVA) for the redness of the pepper samples are displayed in appendix XLVI, XLVII and interactive effect from Fig. 4.13 for 89 University of Ghana http://ugspace.ug.edu.gh the samples that were stored at 4 oC. There was a significant (p<0.05) effect of the doses on the colour from the regression analysis. The dose-dependent effect was conspicuous, however, the regression coefficient for the impact of the weeks of storage was negative and significant (p<0.05). The observed pattern may be attributed to the effect of gamma irradiation on the pigments responsible for the redness (a* values) as well as the effect of oxidation of those pigments (Guadarrama-Lezama et al., 2014; Giuffrida et al., 2014; Kim et al., 2006; Perez-Galvez and Minguez-Mosquera, 2001; Tang and Chen, 1999). Fig. 4.12. a* values after gamma irradiation and during storage at 4 oC 90 University of Ghana http://ugspace.ug.edu.gh Fig. 4.13. a* values after gamma irradiation and during storage at 28±2 oC 4.5.3 b* values The effect of gamma irradiation doses, weeks and the interactive effect of these factors are displayed at appendix LXVIII, LXIX and Fig. 4.14 for the samples that were stored at 28±2 oC. There was a significant effect (p<0.05) of gamma irradiation doses, weeks and the dose-weeks interactions. The yellowness of the samples stored at 4 oC recorded regression coefficients and interactive effects that are displayed in appendix LXX, LXXI and Fig. 4.15. There was a significant effect (p<0.05) of the doses on the effect of the yellowness of the samples, an indication of the dose-dependent effect on the yellowness of the samples. The negative value recorded in the weeks, though significant (p<0.05) indicates that storage weeks led to the reduction in the yellowness of the samples. The observed effects of the doses, gamma irradiation and weeks of storage may be due to the breakdown of the pigments responsible for the yellowness of the samples as well as the effect of storage temperature on the rate of deterioration (Kim et al., 2005; Rico et al., 2010) 91 University of Ghana http://ugspace.ug.edu.gh Fig. 4.14. b* values after gamma irradiation and during storage at 4 oC Fig. 4.15. b* values after gamma irradiation and during storage at 28±2 oC 92 University of Ghana http://ugspace.ug.edu.gh 4.5.4 Chroma The estimated coefficients of the regression for the chroma of the samples, ANOVA for the chroma as well as the interactive effect of the doses and weeks are displayed in appendix XXXV, XXXVI and Fig. 4.18 and appendix XXXVII, XXXVIII and Fig. 4.17 for the samples that were stored at 28±2 oC and 4 oC respectively. The effect of doses, weeks and interactive effect on the chroma of the samples were significant (p<0.05) for the samples at both temperatures. The positive regression coefficient for the doses indicates the effect of ‘bleaching’. Thus, increasing the doses would lead to higher chroma values in the samples, however, the negative coefficient of regression for the weeks indicates that increasing the number of weeks will lead to lower chroma values. The interactive effect of the dose and weeks was also negative depicting a reducing effect on the chroma of the pepper samples with time for samples stored at both temperatures (Schweiggert et al., 2005). Fig. 4.16. Chroma after gamma irradiation and during storage at 4 oC 93 University of Ghana http://ugspace.ug.edu.gh Fig. 4.17. Chroma after gamma irradiation and during storage at 28±2 oC 4.5.5 Browning index The regression coefficients, the ANOVA as well as the interactive effect of both doses applied and the weeks at both storage conditions are depicted in appendices XXXI to XXXIV and Fig. 4.19 and 4.20. The impact of the doses on the browning index of the pepper samples stored at 28±2 oC was not significant (p>0.05) indicating that in optimization of the impact of gamma irradiation, the browning index is not necessarily dependent on the doses applied, however the browning index is dependent on the storage weeks (p<0.05) which led to the reduction in the browning index of the pepper samples. The effect of the doses and weeks (interactive effect) of the pepper samples was not significant (p>0.05) but had a reducing effect on the browning index. The effect of the gamma irradiation and the doses led to the reduction in the browning index of the pepper samples. 94 University of Ghana http://ugspace.ug.edu.gh Fig. 4.18. Browning index after gamma irradiation and during storage at 4 oC Samples that were stored at 4 oC were affected by the doses of gamma irradiation applied (p<0.05). The combined effect of the doses and storage weeks on the browning led to a reduction in the browning index of the pepper samples which was statistically (p>0.05). The effect of the storage weeks led to a reduction (p<0.05) in the browning index of the samples. The dose-dependent effect caused a higher browning index in the samples that were stored at this temperature. This indicates that the longer the storage period the less the browning index of the pepper samples which may be due higher degradation or oxidation of the pigments of the samples (Schweiggert et al., 2005). The browning index of the samples depended on the storage weeks. Increasing the weeks of storage or reducing the weeks of storage will affect the browning index of the samples (Schweiggert et al., 2005). 95 University of Ghana http://ugspace.ug.edu.gh Fig. 4.19. Browning index after gamma irradiation and during storage at 28±2 oC 4.5.6 Total colour difference The total colour difference of the samples which indicates the change in the colour of the samples from the onset to the end of the experiment was optimized. The estimated regression coefficient, the ANOVA and the interactive effect of the doses of gamma irradiation are indicated in appendices XLIII to XLVI and Fig. 4.21 and 4.22. The parameters that affected the total colour differences at both temperatures were not statistically significant (p>0.05) except the weeks of storage (p<0.05). Total colour difference increase with weeks of storage may be attributed to the oxidation and degradation of the pigments responsible for the colour of the samples (Schweiggert et al., 2005; Topuz, et al., 2009). 96 University of Ghana http://ugspace.ug.edu.gh Fig. 4. 20. Total Colour Difference after gamma irradiation and during storage at 4 oC Fig. 4.21. Total Colour Difference after gamma irradiation and during storage at 28±2 oC 4.5.7 Hue The optimization of the process parameters that affected the hue of the pepper samples is indicated in appendices XXXIX to XLIII as well as the interactive effect in Fig. 4.23 and 97 University of Ghana http://ugspace.ug.edu.gh 4.24stored at 28±2 oC and 4 oC. Weeks of storage had a significant (p<0.05) effect on the hue of the samples that were stored at both temperatures, however with a reducing effect. The lower values recorded may be attributed to the breakdown of the pigments responsible for the colour of the samples (Schweiggert et al., 2005). In view of this observation, longer weeks of storage may lead to lower values at both storage temperatures. Fig. 4. 22. Hue after gamma irradiation and storage at 4 oC 98 University of Ghana http://ugspace.ug.edu.gh Fig. 4.23. Hue after gamma irradiation and storage at 28±2 oC 4.5.8 pH The regression coefficient of the pH of the samples are depicted in appendices XC to XCIII as well as the interactive effect of doses and storage weeks at Fig. 4.25 and 4.26. The weeks of storage had a significant effect on the pH of the samples at both storage temperatures (4 oC and 28±2 oC). The values recorded due to storage weeks might be due to the breakdown of organic acids (Atuobi-Yeboah et al., 2016). The pH of the samples stored at different temperatures could only be reduced with storage weeks. The samples may become less acidic during a longer storage time. 99 University of Ghana http://ugspace.ug.edu.gh Fig. 4.24. pH after gamma irradiation and during storage at 28±2 oC Fig. 4.25. pH after gamma irradiation and during storage at 4 oC 100 University of Ghana http://ugspace.ug.edu.gh 4.5.9 Moisture content The regression coefficients for the moisture content of the samples that were stored at both temperatures are depicted in appendices XCVI to XCIX and figures 4.27 and 4.28. There was a significant effect (p<0.05) of the doses on the moisture content for the samples that were stored 28±2 oC. There was no significant effect (p>0.05) of the interaction of the weeks and doses as well as the weeks for the samples stored at 28±2 oC and 4 oC. The effect of the interaction of doses and weeks at both storage conditions led to a general reduction in the moisture content of the samples (p>0.05) which had been indicated previously. The observed effect of irradiation, and irradiation and storage weeks, and doses and on the moisture content may be due to depolymerization of some structural components of samples stored at both temperatures (Rico et al., 2010). Fig. 4.26. Moisture content (%) after gamma irradiation and during storage at 4 oC 101 University of Ghana http://ugspace.ug.edu.gh Fig. 4.27. Moisture content (%) after gamma irradiation and during storage at 28±2 oC 4.5.10 Titratable acidity Appendices XCIV, XCV, LXXXVIII and LXXXIX, Fig. 4.29 and 4.30 indicates the results of the central composite design for the titratable acidity of the samples. The regression coefficients for the TTA of the samples stored at both temperatures were significant only in terms of the storage weeks (p<0.05). The observed phenomenon may be due to the conversion of organic acids to sugars (Liu et al., 2010; Rico et al., 2010). Longer weeks of storage weeks will lead to lower values. 102 University of Ghana http://ugspace.ug.edu.gh Fig. 4.28. Titratable acidity after gamma irradiation and during storage at 4 oC Fig. 4.29. Titratable acidity after gamma irradiation and during storage 28±2 oC 103 University of Ghana http://ugspace.ug.edu.gh 4.5.11 Capsaicin The regression coefficients as well as the ANOVA of all the samples are depicted in appendices LVII to LXI and Fig. 4.31 and 4.32. The doses of gamma irradiation applied and the storage weeks had significant effect (p<0.05) on the capsaicin content of the pepper samples at both temperatures. The observed reduction in the capsaicin content may be due to the oxidation of the capsaicin caused by residual enzymatic activity and the milling process (Wang et al., 2009). The negative values obtained for both the weeks of storage and the interactive effect is an indication of the impact of gamma irradiation and storage weeks that will lead to a reduction in the capsaicin content of the pepper samples with time. Hence, the longer the storage time the less the content of the capsaicin in the pepper samples irrespective of the storage temperature employed. Fig. 4.30. Capsaicin content after gamma irradiation and during storage at 4 oC 104 University of Ghana http://ugspace.ug.edu.gh Fig. 4.31. Capsaicin content after gamma irradiation and during storage at 28±2 oC 4.5.12 Dihydrocapsaicin The regression coefficients, the ANOVA and the interactive effect of doses and weeks for the impact of gamma on the dihydrocapsaicin are displayed in appendices LXXVI to LXXIX and Fig. 4.33 and 4.34. The effect of gamma irradiation doses on the dihydrocapsaicin content of the pepper samples for both temperatures (28±2 oC and 4 oC) was significant (<0.05). However, there was a negative effect (p<0.05) of weeks or storage for both temperature regimes was observed. There was a significant interactive effect of gamma irradiation doses and storage weeks for both temperatures of storage (p<0.05). The observed pattern is similar as stated in capsaicin (Wang et al., 2009, Giuffrida et al., 2014). Higher irradiation doses may lead to higher dihydrocapsaicin content but a reduced content with longer storage periods for both storage temperatures. 105 University of Ghana http://ugspace.ug.edu.gh Fig. 4.32. Dihydrocapsaicin content after gamma irradiation and during storage at 4 oC Fig. 4.33. Dihydrocapsaicin content after gamma irradiation and during storage at 28±2 oC 106 University of Ghana http://ugspace.ug.edu.gh 4.5.13 Total capsaicin The total capsaicinoid content of the samples was based on the addition of the dihydrocapsaicin and capsaicin (Kim et al, 2004). Regression coefficients, ANOVA, the interactive effect of the samples can be found in appendices LXXX to LXXXIII. The doses of gamma irradiation had a significant effect on the total capsaicinoid content of the samples (p<0.05). This indicates that the content increased (p<0.05) with the doses applied irrespective of the temperature and weeks of storage (p>0.05). The weeks of storage led to a reduction in the total capsaicinoid content of the samples but not significant (p>0.05). The observed pattern may be due to the behaviour of the capsaicinoids investigated (Wang et al., 2009, Giuffrida et al., 2014). Fig. 4.34. Total capsaicinoids content after gamma irradiation and during storage at 4 oC 107 University of Ghana http://ugspace.ug.edu.gh Fig. 4.35. Total capsaicinoids content after gamma irradiation and during storage at 28±2 oC 4.5.14 Scoville Heat Units (SHU) The regression coefficients, ANOVA and interactive effect of the doses and weeks are indicated in appendices LXXXIV to LXXXVII and Fig. 4.37 and 4.38 for the sample stored at both temperature regimes (28±2 oC and 4 oC). There was no significant impact (p>0.05) of the doses and storage weeks on the SHU for the samples that were stored at 28±2 oC for effect of doses of irradiation and the weeks of storage as well as the interactive effect of the doses and weeks. However, a reduction of the SHU in the samples was observed irrespective of the storage temperature. A significant effect of gamma irradiation (doses) as well as the weeks of storage on the SHU was observed in the samples that were stored at 4 oC. 108 University of Ghana http://ugspace.ug.edu.gh Fig. 4.36. SHU after gamma irradiation and during storage at 4 oC Fig. 4.37. SHU after gamma irradiation and during storage at 28±2 oC The higher the dose, the higher the SHU (a dose dependent effect on the SHU) of the samples. However, there was a reduction in the SHU per storage weeks. The observed 109 University of Ghana http://ugspace.ug.edu.gh pattern is similar to what was observed in total capsaicinoids (Giuffrida et al., 2014; Wang et al., 2009). 4.5.15 Beta carotene The carotenoids investigated in this samples were beta carotene, beta cryptoxanthin and capsanthin. The regression coefficients, ANOVA and interactive effect of the doses and weeks of storage for the beta carotene content of the samples can be found in appendices LI to LIII, Fig. 4.39 and 4.40 for the samples stored at 28±2 oC and 4 oC. Effect of gamma irradiation was significant (p<0.05) on the beta carotene content in the samples stored at 28±2 oC but not in the samples that were stored at 4 oC. The effect of the weeks of storage was significant (p<0.05) for all the samples that were stored at the various temperatures. Beta carotene content reduced with storage. The interactive effect of doses and weeks led to reduction in the content of beta carotene in the samples irrespective of the storage temperatures. These observations may be attributed to the effects of storage temperature, oxidation of the components of the pigments and its structure and the secondary effects of gamma irradiation (Guadarrama-Lezama et al., 2014; Giuffrida et al., 2014; Kim et al., 2006; Perez-Galvez and Minguez-Mosquera, 2001; Tang and Chen, 1999; Goda et al., 1995). 110 University of Ghana http://ugspace.ug.edu.gh Fig. 4.38. Beta carotene content after gamma irradiation and during storage at 4 oC Fig. 4.39. Beta carotene content after gamma irradiation and during storage at 28±2 oC 111 University of Ghana http://ugspace.ug.edu.gh 4.5.16 Beta cryptoxanthin Beta cryptoxanthin content in the samples were affected significantly (p<0.05) by the doses of gamma irradiation applied to the samples, the weeks of storage and the interactive effect of the doses and weeks of storage irrespective of the temperatures of storage employed (appendices LXXII to LXXV, Fig. 4.41 and 4.42). The doses led to the general reduction in the beta cryptoxanthin content of the samples as well as the storage weeks. These observations in beta cryptoxanthin are similar to beta carotene (Guadarrama- Lezama et al., 2014; Giuffrida et al., 2014; Kim et al., 2006; Perez-Galvez and Minguez- Mosquera, 2001; Goda et al., 1995; Tang and Chen, 1999). Fig. 4.40. Beta cryptoxanthin content after gamma irradiation and during storage at 4 oC 112 University of Ghana http://ugspace.ug.edu.gh Fig. 4.41. Beta cryptoxanthin content after gamma irradiation and during storage at 28±2 oC. 4.5.17 Capsanthin The capsanthin content of the samples were affected (p<0.05) by the doses and the weeks of storage and (appendices XLVII to L). These led to the general reduction of the content of capsanthin in the samples irrespective of the temperature of storage. However, there was no significant (p>0.05) effect of the interaction of the doses and the weeks (appendices XLVII to L; Fig. 4.43 to 4.44). The observed phenomenon in capsanthin is similar to beta carotene (Guadarrama-Lezama et al., 2014; Giuffrida et al., 2014; Kim et al., 2006; Perez- Galvez and Minguez-Mosquera, 2001 Tang and Chen, 1999; Goda et al., 1995). 113 University of Ghana http://ugspace.ug.edu.gh Fig. 4.42. Capsanthin content after gamma irradiation and during storage at 4 oC. Fig. 4.43. Capsanthin content of gamma irradiation and during storage in Legon-18 stored at 28±2 oC. 114 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS 5.1 Summary The study was conducted to determine the inactivation effect of gamma irradiation on selected foodborne pathogens associated with low moisture foods in Capsicum annuum (Legon-18 pepper) powder and its effect on the quality parameters of the pepper powder. Gamma irradiation, storage temperature and period had significant (p<0.05) effects on the inactivation of all the pathogens. Inactivation of the pathogens was dose-dependent. As gamma irradiation dosage increased inactivation rates of microorganisms on the powdered pepper also increased. This was probably due to the level of cellular injuries caused by the increasing doses of gamma irradiation, inability of injured microorganisms to adapt to their surroundings. Post-irradiation radiolysis in the matrix and the effect of storage temperature all may have contributed to the increased rate of microbial inactivation. It was observed that inactivation was more pronounced in the gram-negative microorganisms as compared with the gram-positives probably due to differences in the structural components of gram negatives. Gram-positives have double-stranded DNA which makes them resistant to gamma irradiation as compared with the gram-negatives. Inactivation also increased with increasing storage time. This increased inactivation with progression of storage suggests that lower doses of gamma irradiation can be used for the inactivation of the pathogens if the product will be stored for a longer period. The progression of inactivation with storage could be due to the inability of the pathogens to repair injuries which occurred due to the effects of gamma irradiation and its post effects such as increased radiolysis. On the quality of the pepper samples the doses of gamma irradiation, the storage temperature and the period of storage significantly (p<0.05) affected the colour 115 University of Ghana http://ugspace.ug.edu.gh components of the pepper samples. The dose-dependent effect on the carotenoids (beta carotene, beta cryptoxanthin and capsanthin) determined had a corresponding effect on the colour components of the samples. The red (a* values) components of the samples reduced with storage. The carotenoids (beta carotene and beta cryptoxanthin) responsible for the yellowness of the samples also reduced with storage due to both primary and secondary effect of gamma irradiation, oxidation and temperature. The capsanthin content reduced during storage which had a corresponding effect on the b* values too. Hue, chroma, total colour change (or difference) and browning index of the samples were all affected by the gamma irradiation, storage and temperature. Due to the degradation of the pigments in the samples responsible for the redness, yellowness and brightness of the colour components, a corresponding effect on the hue, chroma, total colour change and browning index during storage was observed. The hotness of the pepper samples was based on the presence of dihydrocapsaicin and capsaicin. The hotness of the samples increased due to the effect of gamma irradiation. A dose-dependent effect was observed (p<0.05) which led to increased hotness of the pepper samples (Capsaicin and dihydrocapsaicin, total capsaicinoids and Scoville Heat Units) after irradiation. There was a reduction in the two capsaicinoids content of the samples which had a corresponding effect on the total capsaicinoids as well as the Scoville Heat Units. The carotenoids in the samples were also affected by the irradiation and the length of storage period. There was a decreasing effect which was dose and length of storage- dependent. 116 University of Ghana http://ugspace.ug.edu.gh 5.2 Conclusions The results of the study suggest that gamma irradiation could be used to decontaminate microbial pathogens in pepper powder. The effects vary with the dosage of the irradiation. The impact of irradiation on the inactivation progresses during storage of the pepper samples. Inactivation by gamma irradiation is more pronounced in gram-negative microorganisms than in the gram-positives organisms. Gamma irradiation had effects on the quality parameters of pepper powder, specifically on the color components and hotness index. The color parameters of L*, a* and b* values increased with increasing doses of gamma irradiation immediately after irradiation, however, these values reduced during storage which was more pronounced at 28 oC. A corresponding effect of gamma irradiation and storage on L*, a* and b* values were observed in the hue, chroma, browning index and total colour change (difference) of the samples. Capsaicinoids, total capsaicinoids and SHU values varied with irradiation doses which reduced during storage. The carotenoids analyzed reduced with increasing doses of gamma irradiation as well as increasing time of storage. All the quality parameters measured reduced with storage. 5.3 Recommendations 1. Gamma irradiation at 5 kGy can be used as an immediate decontaminating dose for Legon-18 pepper. However, 2 kGy can be used for long storage period for less effects of gamma irradiation on the quality parameters. 2. A study should be conducted on the effect of gamma irradiation on the biochemical pathways of macromolecules of the pepper samples. 3. 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Federal Register, 1 April. 136 University of Ghana http://ugspace.ug.edu.gh Van D, J. M., Neil, K. P., Parish, M., Gieraltowski, L., Gould, L. H. and Gombas, K. L. (2013). Foodborne illness outbreaks from microbial contaminants in spices, 1973- 2010. Food Microbiology. 36(2):456–64. Vij, V., E. Ailes, C. Wolyniak, F.J. Angulo, and K.C. Klontz. 2006. Recalls of spices due to bacterial contamination monitored by the U.S. Food and Drug Administration: The predominance of salmonellae. J. Food Prot. 69:233- 237. Waje, C. K., Kim, H. K., Kim, K. S., Todoriki, S., & Kwon, J. H. (2008). Physicochemical and microbiological qualities of steamed and irradiated ground black pepper (Piper nigrum L.). Journal of Agricultural and Food Chemistry. 56(12): 4592–4596. Wall, M. M., Wadell, C. A. and Bosland, P. W. (2011). Variation in Beta carotene and total carotenoid content in fruits of Capsicum. Hort. Science. 36(4):746-749 Walsh, B., and Hoot, S. (2001). Phylogenetic relationships of Capsicum (Solanaceae) using DNA sequences from two noncoding regions: the chloroplast atpb-rbcl spacer region and nuclear waxy introns. International Journal of Plant Science. 162(6): 1409-1418. Wang, Y., Xia, Y, Wang, J., Luo, F. and Huang, Y. (2009). Carotenoids in chili pepper (Capsicum annuum, L.) powder as affected by heating and storage methods. American Society of Agricultural and Biological Engineers. 52(6): 2007-2010. Weiss, E.A. (2002). Spice Crops. CABI Publishing International: New York, NY, USA. pp. 411. WHO (1981) Wholesomeness of irradiated food. Report of a Joint FAO/IAEA/WHO Expert Committee, Technical Report Series 659. p. 33. World Health Organization, Geneva, Switzerland. Wiriya, P. T. Paiboon, and Somchart, S. (2009). Effect of drying air temperature and chemical pretreatments on quality of dried chilli,” International Food Research Journal. 16 (3): 441–454. Witkowska, A.M., Hickey, D.K., Alonso-Gomez, M. & Wilkinson, M.G. (2011). The microbiological quality of commercial herb and spice preparations used in the formulation of a chicken supreme ready meal and microbial survival following a simulated industrial heating process. Food Control. 22(3): 616-625. Won, Y-C., Min, C. S. & Lee, -D-U. (2015). Accelerated Drying and improved Colour Properties of Red Pepper by Pre-treatment of Pulsed Electric Fields. Drying Technology. 33:926-92 137 University of Ghana http://ugspace.ug.edu.gh Wu, V. C. H. (2008). A review of microbial injury and recovery methods in food. Food Microbiology. 25: 735-744. Yankey, J. (2014). Assessment of microbiological contamination of some indigenous spices sold in selected markets in the Kumasi Metropolis. An MPhil. Thesis submitted to the Kwame Nkrumah University of Science and Technology. Yoon, J.Y., Green, S.K., Tschanz, A.T., Tsou, S. C. S. & Chang, L.C. (1989). Pepper Zahran DA, Hendy B. A. and El-Hifnawi, H. N. (2008). Incidence and radiation sensitivity of Bacillus cereus, Listeria monocytogenes and their toxins in some chicken products. World applied Sciences Journal. 5(2):182-188. 138 University of Ghana http://ugspace.ug.edu.gh APPENDICES Appendix I: Estimated Regression Coefficients for Salmonella Typhimurium at 4 oC Term Coefficient SE Coefficient T P-value Constant 6.49945 0.592225 10.975 0* Doses -1.01628 0.454897 -2.234 0.061** Days -0.01864 0.030326 -0.615 0.558** Dose *Doses 0.15147 0.100218 1.511 0.174** Days*Days -0.00045 0.000445 -1.008 0.347** Doses* Days -0.01021 0.005552 -1.839 0.109** S=0.6662 R2=92.50% R2 (adjusted)= 87.20% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. log cfu/g = 6.49945 -1.01628x -0.01864y + 0.15147x2-0.00045y2-0.01021xy Where x=doses of gamma irradiation, y=days of storage Appendix II: Analysis of Variance for Salmonella Typhimurium at 4 oC Source DF Seq SS Adj. SS Adj. MS F ratio P-value Regression 5 38.4606 38.46057 7.69211 17.33 0.001* Linear 2 35.8491 2.64949 1.32475 2.98 0.116** Square 2 1.1109 1.11088 0.55544 1.25 0.343** interaction 1 1.5006 1.50062 1.50062 3.38 0.109** Residual error 7 3.1068 3.10683 0.44383 Lack of fit 3 3.0786 3.07855 1.02618 145.15 0* Pure Error 4 0.0283 0.02828 0.00707 Total 12 41.5674 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix III: Estimated Regression Coefficients for Salmonella Typhimurium at 28±2 oC Term Coefficient SE Coefficient T P value Constant 6.69101 0.25276 26.472 0* Doses -0.98093 0.194149 -5.052 0.001* Days -0.05778 0.012943 -4.464 0.003* Dose *Doses 0.06763 0.042773 1.581 0.158 Days*Days -0.00082 0.00019 -4.293 0.004* Doses* Days 0.01021 0.002369 4.308 0.004* S= 0.2843 R2=98.80% R2 (adjusted)= 98.00% Values marked *are statistically significant (p<0.05). log cfu/g = 6.69101-0.98093x -0.05778y + 0.06763x2-0.00082y2+0.01021xy Where x=doses of gamma irradiation, y=days of storage 139 University of Ghana http://ugspace.ug.edu.gh Appendix IV: Analysis of Variance for Salmonella Typhimurium at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F ratio P value Regression 5 47.1599 47.15987 9.43197 116.66 0* Linear 2 44.169 4.37994 2.18997 27.09 0.001* Square 2 1.4902 1.49023 0.74511 9.22 0.011* interaction 1 1.5006 1.50063 1.50063 18.56 0.004* Residual error 7 0.5659 0.56593 0.08085 Lack of fit 3 0.5305 0.53053 0.17684 19.98 0.007* Pure Error 4 0.0354 0.0354 0.00885 Total 12 47.7258 Values marked *are statistically significant (p<0.05). DF-degree of freedom, Adj. SS- Adjusted Sum of Squares, Adj. MSS-Adjusted mean sum of squares. Appendix V: Estimated Regression Coefficients for Escherichia coli at 4 oC Term Coefficient SE Coefficient T P value Constant 6.62348 0.175951 37.644 0* Doses -0.86001 0.135151 -6.363 0* Days -0.08678 0.00901 -9.631 0* Dose *Doses 0.01448 0.029775 0.486 0.642** Days*Days -0.00038 0.000132 -2.872 0.024* Doses* Days 0.01313 0.001649 7.957 0* S=0.1979 R2=99.40% R2 (adjusted)= 98.90% Values marked *are statistically significant (p<0.05). log cfu/g = 6.62348-0.86001x -0.08678y + 0.01448x2-0.00038y2-0.01313xy Where x=doses of gamma irradiation, y=days of storage 140 University of Ghana http://ugspace.ug.edu.gh Appendix VI: Analysis of Variance for Escherichia coli at 4 oC Source DF Seq SS Adj. SS Adj MS F ratio P value Regression 5 44.3224 44.32239 8.86448 226.27 0* Linear 2 41.5017 6.15866 3.07933 78.6 0* Square 2 0.3401 0.3401 0.17005 4.34 0.059** interaction 1 2.4806 2.48063 2.48063 63.32 0* Residual error 7 0.2742 0.27424 0.03918 Lack of fit 3 0.21 0.20996 0.06999 4.36 0.095** Pure Error 4 0.0643 0.06428 0.01607 Total 12 44.5966 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix VII: Estimated Regression Coefficients for Escherichia coli at 28±2 oC Term Coefficient SE Coefficient T P value Constant 6.26468 0.18587 33.705 0* Doses -1.04197 0.142769 -7.298 0* Days -0.10613 0.009518 -11.151 0* Dose *Doses 0.02039 0.031453 0.648 0.538** Days*Days 0.00004 0.00014 0.251 0.809** Doses* Days 0.01604 0.001742 9.207 0* S=0.2091 R2=99.20% R2 (adjusted)= 98.60% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). log cfu/g = 6.26468-1.04197x -0.10613y + 0.02039x2 +0.00004y2-0.01604xy Where x=doses of gamma irradiation, y=days of storage Appendix VIII: Analysis of Variance for Escherichia coli at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F ratio P value Regression 5 37.1975 37.1975 7.4395 170.17 0* Linear 2 33.4608 9.15531 4.57765 104.71 0* Square 2 0.031 0.03104 0.01552 0.35 0.713** interaction 1 3.7056 3.70563 3.70563 84.76 0* Residual error 7 0.306 0.30603 0.04372 Lack of fit 3 0.2679 0.26791 0.0893 9.37 0.028* Pure Error 4 0.0381 0.03812 0.00953 Total 12 37.5035 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix IX: Estimated Regression Coefficients for Bacillus cereus at 4 oC Term Coefficient SE Coefficient T P value 141 University of Ghana http://ugspace.ug.edu.gh Constant 5.88842 0.295015 19.96 0* Doses -0.45068 0.226606 -1.989 0.087** Days -0.0166 0.015107 -1.099 0.308** Dose *Doses 0.05944 0.049923 1.191 0.273** Days*Days -0.00007 0.000222 -0.312 0.764** Doses* Days -0.00537 0.002766 -1.944 0.093** S= 0.3319 R2=92.30% R2 (adjusted)= 86.90% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). log cfu/g = 5.88842-0.45068x -0.0166y +0.05944x2-0.00007y2-0.00537xy Where x=doses of gamma irradiation, y=days of storage Appendix X: Analysis of Variance for Bacillus cereus at 4 oC Source DF Seq SS Adj. SS Adj. MS F ratio P value Regression 5 9.2929 9.29291 1.858583 16.88 0.001* Linear 2 8.7182 0.66389 0.331943 3.01 0.114** Square 2 0.1587 0.15872 0.079361 0.72 0.519** interaction 1 0.416 0.41602 0.416025 3.78 0.093** Residual error 7 0.771 0.77096 0.110138 Lack of fit 3 0.7516 0.75164 0.250548 51.87 0.001** Pure Error 4 0.0193 0.01932 0.00483 Total 12 10.0639 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XI: Estimated Regression Coefficients for Bacillus cereus at 28±2 oC Term Coefficient SE Coefficient T P value Constant 6.20129 0.212514 29.181 0* Doses -0.92846 0.163235 -5.688 0.001* Days 0.01171 0.010882 1.076 0.317** Dose *Doses 0.14534 0.035962 4.042 0.005* Days*Days -0.00057 0.00016 -3.571 0.009* Doses* Days -0.00571 0.001992 -2.865 0.024* S=0.2391 R2 = 97.30% R2 (adjusted)= 95.40% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). log cfu/g = 6.20129-0.92846x+ 0.01171y +0.14534x2-0.00057y2-0.00571xy Where x=doses of gamma irradiation, y=days of storage Appendix XII: Analysis of Variance for Bacillus cereus at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F ratio P value Regression 5 14.3486 14.34858 2.869715 50.21 0* 142 University of Ghana http://ugspace.ug.edu.gh Linear 2 12.6701 1.85037 0.925187 16.19 0.002* Square 2 1.2093 1.20927 0.604633 10.58 0.008* interaction 1 0.4692 0.46923 0.469225 8.21 0.024* Residual error 7 0.4001 0.40006 0.057151 Lack of fit 3 0.3801 0.38014 0.126712 25.44 0.005* Pure Error 4 0.0199 0.01992 0.00498 Total 12 14.7486 Values marked *are statistically significant (p<0.05). DF-degree of freedom, Adj. SS- Adjusted Sum of Squares, Adj. MSS-Adjusted mean sum of squares. Appendix XIII: Estimated Regression Coefficients for Listeria monocytogenes at 4 oC Term Coefficient SE Coefficient T P value Constant 6.40977 0.436974 14.669 0* Doses -1.42181 0.335646 -4.236 0.004* Days -0.01718 0.022376 -0.768 0.468** Dose *Doses 0.27608 0.073946 3.734 0.007* Days*Days -0.00028 0.000329 -0.848 0.425** Doses* Days -0.0145 0.004096 -3.54 0.009* S=0.4916 R2= 96.10% R2 (adjusted)= 93.30% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05 log cfu/g = 6.40977-1.42181x-0.01718y+0.27608x2-0.00028y2-0.0145xy Where x=doses of gamma irradiation, y=days of storage Appendix XIV: Analysis of Variance Listeria monocytogenes at 4 oC Source DF Seq SS Adj. SS Adj.MS F ratio P value Regression 5 0.009 41.43633 8.28727 34.3 0* Linear 2 34.9472 4.8597 2.42985 10.06 0.009* Square 2 3.4615 3.46152 1.73076 7.16 0.02* interaction 1 3.0276 3.0276 3.0276 12.53 0.009* Residual error 7 1.6914 1.69144 0.24163 Lack of fit 3 1.6632 1.66316 0.55439 78.41 0.001* Pure Error 4 0.0283 0.02828 0.00707 Total 12 43.1278 Values marked *are statistically significant (p<0.05). DF-degree of freedom, Adj. SS- Adjusted Sum of Squares, Adj. MSS-Adjusted mean sum of squares. Appendix XV: Estimated Regression Coefficients for Listeria monocytogenes at 28±2 oC Term Coefficient SE Coefficient T P value Constant 6.01029 0.37126 16.189 0* Doses -1.51086 0.28517 -5.298 0.001* Days -0.01395 0.019011 -0.734 0.487** 143 University of Ghana http://ugspace.ug.edu.gh Dose *Doses 0.29522 0.062826 4.699 0.002* Days*Days -0.00028 0.000279 -0.991 0.355** Doses* Days -0.01325 0.00348 -3.807 0.007* S=0.4176 R2=96.80% R2(adjusted)= 94.50% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). log cfu/g = 6.01029-1.51086x-0.01395y+0.29522x2-0.00028y2-0.01325xy Where x=doses of gamma irradiation, y=days of storage Appendix XVI: Analysis of Variance for Listeria monocytogenes at 28±2 oC Source DF Seq SS Adj. SS Adj.MS F ratio P value Regression 5 36.7905 36.79052 7.3581 42.19 0* Linear 2 30.281 5.34927 2.67463 15.33 0.003* Square 2 3.9815 3.98145 1.99073 11.41 0.006* interaction 1 2.5281 2.5281 2.5281 14.49 0.007* Residual error 7 1.221 1.22096 0.17442 Lack of fit 3 1.1848 1.18476 0.39492 43.64 0.002* Pure Error 4 0.0362 0.0362 0.00905 Total 12 Values marked *are statistically significant (p<0.05). DF-degree of freedom, Adj. SS- Adjusted Sum of Squares, Adj. MS-Adjusted mean squares. Appendix XVII: Estimated Regression Coefficients for Staphylococcus aureus at 4 oC Term Coefficient SE Coefficient T P value Constant 6.22641 0.326483 19.071 0* Doses -0.9313 0.250776 -3.714 0.008* Days -0.06736 0.016718 -4.029 0.005* Dose *Doses -0.00207 0.055248 -0.037 0.971** Days*Days -0.00054 0.000246 -2.187 0.065** Doses* Days 0.01354 0.003061 4.425 0.003* S R2=97.60% R2 (adjusted)= 95.90% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). log cfu/g = 6.22641-0.9313x-0.06736y-0.00207x2-0.00054y2+0.01354xy Where x=doses of gamma irradiation, y=days of storage Appendix XVIII: Analysis of Variance for Staphylococcus aureus at 4 oC Source DF Seq SS Adj. SS Adj. MS F ratio P value Regression 5 38.6157 38.61567 7.72313 57.26 0* Linear 2 35.2104 4.82983 2.41491 17.9 0.002* Square 2 0.7646 0.76463 0.38232 2.83 0.125** 144 University of Ghana http://ugspace.ug.edu.gh interaction 1 2.6406 2.64063 2.64063 19.58 0.003* Residual error 7 0.9442 0.9442 0.13489 Lack of fit 3 0.9049 0.90488 0.30163 30.68 0.003* Pure Error 4 0.0393 0.03932 0.00983 Total 12 39.5599 Values marked *are statistically significant (p<0.05). DF-degree of freedom, Adj. SS- Adjusted Sum of Squares, Adj. MS-Adjusted mean squares. Appendix XIX: Estimated Regression Coefficients for Staphylococcus aureus at 28±2 oC Term Coefficient SE Coefficient T P value Constant 6.65463 0.525041 12.674 0* Doses -1.33263 0.403292 -3.304 0.013* Days -0.03545 0.026886 -1.319 0.229** Dose *Doses 0.11409 0.088849 1.284 0.24** Days*Days -0.00025 0.000395 -0.643 0.541** Doses* Days -0.00254 0.004922 -0.516 0.621** S=0.5906 R2=94.10% R2 (adjusted)= 90.00% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). log cfu/g = 6.65463-1.33263x-0.03545y+0.11409x2-0.00025y2+-0.00254xy Where x=doses of gamma irradiation, y=days of storage 145 University of Ghana http://ugspace.ug.edu.gh Appendix XX: Analysis of Variance for Staphylococcus aureus at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F ratio P value Regression 5 39.2524 39.25239 7.85048 22.5 0* Linear 2 38.5744 5.03934 2.51967 7.22 0.02* Square 2 0.5849 0.58494 0.29247 0.84 0.472** interaction 1 0.093 0.09302 0.09302 0.27 0.621** Residual error 7 2.4419 2.44192 0.34885 Lack of fit 3 2.4221 2.42212 0.80737 163.11 0* Pure Error 4 0.0198 0.0198 0.00495 Total Surf1a2 ce P41l.o69t4 3o f S ATCOLD vs Day s, Dos es (kGy) Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 6 4 log cfu/g 2 60 0 40 Days 0.0 20 1.5 3.0 0 4.5 Doses (kGy) Appendix XXI: Effect of gamma irradiation and storage on the inactivation of S. Typhimurium at 4 oC 146 University of Ghana http://ugspace.ug.edu.gh Surface Plot of SATAMB vs Days, Doses (kGy) 6 4 log cfu/g 2 60 0 40 20 Days0.0 1.5 3.0 0 4.5 Doses (kGy) AppendixS XuXrIfIa: Ecfefe cPt loof tg aomfm Ea CirrCadOiaLtioDn avnsd sDtoaraygse ,o nD thoes ienasc t(ivkatGioyn )of S. typhirium at 28±2 oC 6 4 log cfu/g 2 60 0 40 Days 0.0 20 1.5 3.0 0 4.5 Doses (kGy) Appendix XXIII: Response surface for the inactivation E. coli at 4 oC 147 University of Ghana http://ugspace.ug.edu.gh Surface Plot of ECAMB vs Days, Doses (kGy) 6 4 log cfu/g 2 60 0 40 Days 0.0 20 1.5 3.0 0 4.5 Doses (kGy) Appendix XXIV: Effect of gamma irradiation and storage on the inactivation of E. coli Surface Plot of BCCOLD vs Days, Doses (kGy) at 28±2 oC 6 5 log cfu/g 4 3 60 40 20 Days0.0 1.5 3.0 0 4.5 Doses (kGy) Appendix XXV: Effect of gamma irradiation and storage on the inactivation of B. cereus at 4 oC 148 University of Ghana http://ugspace.ug.edu.gh Surface Plot of BCAMB vs Days, Doses (kGy) 6 log cfu/g 4 60 2 40 0.0 20 Days 1.5 3.0 0 4.5 Doses (kGy) Appendix XXVI: Effect of gamma irradiation and storage on the inactivation of B. cereus at 28±2 oC Surface Plot of LMCOLD vs Days, Doses (kGy) 6 4 log cfu/g 2 60 0 40 20 Days0.0 1.5 3.0 0 4.5 Doses (kGy) Appendix XXVII: Effect of gamma irradiation and storage on the inactivation of L. monocytogenes at 4 oC 149 University of Ghana http://ugspace.ug.edu.gh Surface Plot of LMAMB vs Days, Doses (kGy) 6 4 log cfu/g 2 60 0 40 20 Days0.0 1.5 3.0 0 4.5 Doses (kGy) Appendix XSXVuIrIfI:a Ecfefe cPt olfo gta momf aS irTraAdiAatMionB a nvd sst oDraagey osn, thDe oinsacetisv a(tikonG oyf L). monocytogenes at 28±2 oC 6 4 log cfu/g 2 60 0 40 0.0 20 Days 1.5 3.0 0 4.5 Doses (kGy) Appendix XXIX: Effect of gamma irradiation and storage on the inactivation of S. aureus 28±2 oC 150 University of Ghana http://ugspace.ug.edu.gh Surface Plot of STACOLD vs Days, Doses (kGy) 6 4 log cfu/g 2 0 60 40 20 Days0.0 1.5 3.0 0 4.5 Doses (kGy) Appendix XXX: Effect of gamma irradiation and storage on the inactivation of S. aureus at 4 oC Appendix XXXI. Estimated Regression Coefficient for browning index in Legon-18 irradiated at 28±2 oC Term Coefficient SE Coefficient T P Constant 84.598 2.17134 38.961 0* Doses 2.1451 1.66784 1.286 0.239** Weeks -12.9643 0.83392 -15.546 0* Dose *Doses 0.152 0.36744 0.414 0.692** weeks*weeks 0.9451 0.09186 10.289 0* Doses* weeks -0.2613 0.15266 -1.712 0.131** S R2 =99.00% R2 (adjusted) 98.30% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Browning index= 84.598+ 2.1451x-12.9643y+0.152x2-0.9451y2-0.2613xy Where x=doses of gamma irradiation, y=weeks of storage 151 University of Ghana http://ugspace.ug.edu.gh Appendix XXXII. Analysis of variance for browning index of Legon-18 in irradiated samples at 28±2 oC Source DF Seq. SS Adj. SS Adj. MS F P Regression 5 4221.26 4221.26 844.252 141.50 0.000* Linear 2 3441.18 1451.21 725.603 121.62 0.000* Square 2 762.60 762.60 381.299 63.91 0.000* Interaction 1 17.48 17.48 17.477 2.93 0.131* Residual error 7 41.76 41.76 5.966 Lack of fit 3 6.20 6.20 2.066 0.23 0.870** Pure error 4 35.57 35.57 8.892 Total 12 4263.02 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XXXIII. Estimated Regression Coefficients for browning index in Legon-18 irradiated pepper samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 85.6683 0.73843 116.015 0.000* Doses 3.6037 0.56720 6.353 0.000* Weeks -5.4006 0.28360 -19.043 0.000* Doses*Doses -0.3242 0.12496 -2.594 0.036* Weeks*Weeks -0.1180 0.03124 -3.777 0.007* Doses*Weeks -0.1158 0.05192 -2.230 0.061** S = 0.8307 R2 = 99.9% R2 (adj) = 99.8% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Browning index= 85.6683+ 3.6037x-5.4006y-0.3242x2-0.1180y2-0.1158xy Where x=doses of gamma irradiation, y=weeks of storage Appendix XXXIV. Analysis of Variance for browning index in Legon-18 irradiated pepper samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 4259.45 4259.446 851.889 1234.58 0.000* Linear 2 4233.04 257.816 128.908 186.82 0.000* Square 2 22.97 22.973 11.487 16.65 0.002* Interaction 1 3.43 3.432 3.432 4.97 0.061** Residual Error 7 4.83 4.830 0.690 Lack-of-Fit 3 3.73 3.732 1.244 4.53 0.089** Pure Error 4 1.10 1.098 0.274 Total 12 4264.28 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 152 University of Ghana http://ugspace.ug.edu.gh Appendix XXXV. Estimated Regression Coefficient for Chroma of Legon-18 irradiated pepper samples 28±2 oC stored Term Coefficient SE Coefficient T P Constant 30.1161 0.129921 231.803 0.000* Doses 1.2196 0.099794 12.221 0.000* Weeks -4.0645 0.049897 -81.457 0.000* Doses*Doses -0.0871 0.021986 -3.963 0.005* Weeks*Weeks 0.2258 0.005496 41.089 0.000* Doses*Weeks -0.0583 0.009134 -6.387 0.000* R2 = 100.0% R2 (adj) = 100.0% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Chroma= 30.1161+1.2196x-4.0645y-0.0871x2+0.2258y2-0.0583xy Where x=doses of gamma irradiation, y=weeks of storage Appendix XXXVI. Analysis of Variance for Chroma of Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 591.341 591.341 118.2681 5536.88 0.000* Linear 2 550.993 141.749 70.8746 3318.08 0.000* Square 2 39.476 39.476 19.7382 924.07 0.000* interaction 1 0.871 0.871 0.8714 40.8 0* Residual error 7 0.15 0.15 0.0214 0* Lack of fit 3 0.147 0.147 0.049 75.23 0.001* *Pure Error 4 0.003 0.003 0.0007 Total 12 591.49 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XXXVII. Estimated Regression Coefficient for Chroma Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 30.6021 0.211195 144.9 0* Doses 1.2544 0.162222 7.733 0* Weeks -2.7764 0.081111 -34.224 0* Dose *Doses -0.1192 0.035739 -3.336 0.012* weeks*weeks 0.059 0.008935 6.604 0* Doses* weeks -0.0374 0.014849 -2.519 0.04* S= 0.2376 R2=99.90% R2 (adjusted)= 99.90% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). 153 University of Ghana http://ugspace.ug.edu.gh Chroma= 30.6021+1.2544x-2.7764y-0.1192x2+0.059y2-0.0374xy Where x=doses of gamma irradiation, y=weeks of storage Appendix XXXVIII. Analysis of Variance for Chroma of Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 555.726 555.7260 111.1452 1969.15 0.000* Linear 2 552.862 66.4109 33.2055 588.30 0.000* Square 2 2.506 2.5059 1.2529 22.20 0.001* Interaction 1 0.358 0.3582 0.3582 6.35 0.040* Residual Error 7 0.395 0.3951 0.0564 Lack-of-Fit 3 0.392 0.3920 0.1307 166.05 0.000* Pure Error 4 0.003 0.0031 0.0008 Total 12 556.121 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XXXIX. Estimated Regression Coefficient for hue of Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 43.8198 3.6064 12.151 0.000* Doses 2.4231 2.7701 0.875 0.411** Weeks -5.1340 1.3851 -3.707 0.008* Doses*Doses -0.5143 0.6103 -0.843 0.427** Weeks*Weeks 0.5318 0.1526 3.486 0.010* Doses*Weeks -0.0202 0.2536 -0.079 0.939** S = 4.057 R2 =71.4% R2 (adj) = 51.0% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Hue= 43.8198+2.4231x-5.1340-0.5143x2+0.5318y2-0.0202xy Where x=doses of gamma irradiation, y=weeks of storage 154 University of Ghana http://ugspace.ug.edu.gh Appendix XL. Analysis of Variance for hue of Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 287.797 287.797 57.559 3.50 0.067** Linear 2 83.212 227.415 113.708 6.91 0.022* Square 2 204.481 204.481 102.240 6.21 0.028* Interaction 1 0.104 0.104 0.104 0.01 0.939** Residual Error 7 115.210 115.210 16.459 Lack-of-Fit 3 10.835 10.835 3.612 0.14 0.932* Pure Error 4 104.375 104.375 26.094 Total 12 403.007 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XLI. Estimated Regression Coefficient for hue of Legon-18 samples irradiated and stored at 4 oC Term Coefficient SE Coefficient T P Constant 45.9633 0.32947 139.505 0.000* Doses -0.3098 0.25307 -1.224 0.260** Weeks -1.4909 0.12654 -11.782 0.000* Doses*Doses 0.0723 0.05575 1.297 0.236** Weeks*Weeks 0.0412 0.01394 2.958 0.021* Doses*Weeks 0.0189 0.02316 0.815 0.442** S=0.3706 R2 =99.2% R2 (adj)= 98.7% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Hue= 45.9633-0.3098x-1.4909y+0.0723x2+0.0412y2+0.0189xy Where x=doses of gamma irradiation, y=weeks of storage Appendix XLII. Analysis of Variance for hue for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 123.432 123.4323 24.6865 179.71 0.000* Linear 2 121.195 20.4519 10.2260 74.44 0.000* Square 2 2.146 2.1464 1.0732 7.81 0.016* Interaction 1 0.091 0.0912 0.0912 0.66 0.442** Residual Error 7 0.962 0.9616 0.1374 Lack-of-Fit 3 0.872 0.8719 0.2906 12.96 0.016* Pure Error 4 0.090 0.0897 0.0224 Total 12 124.394 155 University of Ghana http://ugspace.ug.edu.gh Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XLIII. Estimated Regression Coefficient for total colour difference for Legon-18 samples irradiated and stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 5.54376 0.235670 23.523 0.000* Doses 0.09114 0.181022 0.503 0.630** Weeks 3.22515 0.090511 35.633 0.000* Doses*Doses 0.02994 0.039881 0.751 0.477** Weeks*Weeks -0.13183 0.009970 -13.222 0.000* Doses*Weeks -0.01563 0.016569 -0.943 0.377** S=0.2651 R2 = 99.9% R2 (adj) = 99.8% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Total colour difference= 5.54376+0.09114x+3.22515y+0.02994x2-0.13183y2-0.01563xy Where x=doses of gamma irradiation, y=weeks of storage Appendix XLIV. Analysis of Variance for total colour difference for Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 453.739 453.7394 90.7479 1291.16 0.000* Linear 2 439.879 92.0750 46.0375 655.02 0.000* Square 2 13.798 13.7976 6.8988 98.16 0.000* Interaction 1 0.063 0.0625 0.0625 0.89 0.377** Residual Error 7 0.492 0.4920 0.0703 Lack-of-Fit 3 0.400 0.3998 0.1333 5.78 0.062** Pure Error 4 0.092 0.0922 0.0230 Total 12 454.231 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 156 University of Ghana http://ugspace.ug.edu.gh Appendix XLV. Estimated Regression Coefficient for total colour difference for Legon- 18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 5.70076 0.24785 23.001 0.000* Doses -0.36858 0.19038 -1.936 0.094** Weeks 1.91146 0.09519 20.081 0.000* Doses*Doses 0.05726 0.04194 1.365 0.214** Weeks*Weeks -0.00350 0.01049 -0.334 0.748** Doses*Weeks 0.03872 0.01743 2.222 0.062** S = 0.2788 R2= 99.9% R2 (adj) = 99.7% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Total colour difference= 5.70076-0.36858x+1.91146y+0.05726x2-0.00350y2+0.03872xy Where x=doses of gamma irradiation, y=weeks of storage Appendix XLVI. Analysis of Variance for total colour difference for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 369.676 369.6761 73.9352 951.10 0.000* Linear 2 369.144 31.4838 15.7419 202.50 0.000* Square 2 0.148 0.1480 0.0740 0.95 0.431** Interaction 1 0.384 0.3838 0.3838 4.94 0.062** Residual Error 7 0.544 0.5442 0.0777 Lack-of-Fit 3 0.534 0.5344 0.1781 72.92 0.001* Pure Error 4 0.010 0.0098 0.0024 Total 12 370.220 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XLVII. Estimated Regression Coefficient for capsanthin for Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 1.48425 0.011196 132.567 0.000* Doses -0.05904 0.008600 -6.865 0.000* Weeks -0.02035 0.004300 -4.734 0.002* Doses*Doses 0.00887 0.001895 4.684 0.002* Weeks*Weeks -0.00153 0.000474 -3.233 0.014* Doses*Weeks 0.00041 0.000787 0.516 0.622** S = 0.01259 R2 = 99.0% R2 (adj) = 98.3% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Capsanthin content (mg/100g)= 1.48425-0.05904x-0.02035y+0.00887x2- 0.00153y2+0.00041xy Where x=doses of gamma irradiation, y=weeks of storage 157 University of Ghana http://ugspace.ug.edu.gh Appendix XLVIII. Analysis of Variance for capsanthin for Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.112469 0.112469 0.022494 141.80 0.000* Linear 2 0.108556 0.013040 0.006520 41.10 0.000* Square 2 0.003870 0.003870 0.001935 12.20 0.005** Interaction 1 0.000042 0.000042 0.000042 0.27 0.622** Residual Error 7 0.001110 0.001110 0.000159 * Lack-of-Fit 3 0.001090 0.001090 0.000363 72.69 0.001* Pure Error 4 0.000020 0.000020 0.000005 Total 12 0.113579 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XLIX. Estimated Regression Coefficient for Capsanthin for Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 1.47634 0.008836 167.079 0.000* Doses -0.04407 0.006787 -6.493 0.000* Weeks -0.01633 0.003394 -4.811 0.002* Doses*Doses 0.00557 0.001495 3.724 0.007* Weeks*Weeks -0.00058 0.000374 -1.542 0.167** Doses*Weeks -0.00034 0.000621 -0.553 0.597** S = 0.009940 R2 = 98.8% R2 (adj) = 98.0% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Capsanthin content (mg/100g)= 1.47634-0.04407x-0.01633y+0.00557x2-0.00058y2- 0.00034xy Where x=doses of gamma irradiation, y=weeks of storage 158 University of Ghana http://ugspace.ug.edu.gh Appendix L. Analysis of Variance for Capsanthin in Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.059177 0.059177 0.011835 119.78 0.000* Linear 2 0.057774 0.007651 0.003825 38.72 0.000* Square 2 0.001372 0.001372 0.000686 6.94 0.022* Interaction 1 0.000030 0.000030 0.000030 0.31 0.597** Residual Error 7 0.000692 0.000692 0.000099 Lack-of-Fit 3 0.000688 0.000688 0.000229 286.85 0.000* Pure Error 4 0.000003 0.000003 0.000001 Total 12 0.059868 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LI. Estimated Regression Coefficient for beta carotene for Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 10.3257 0.070274 146.936 0.000* Doses -0.3569 0.053978 -6.612 0.000* Weeks -0.3122 0.026989 -11.568 0.000* Doses*Doses 0.0442 0.011892 3.714 0.008** Weeks*Weeks -0.0113 0.002973 -3.791 0.007** Doses*Weeks -0.0195 0.004941 -3.947 0.006** S = 0.07905 R2 = 99.8% R2 (adj) = 99.6% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Beta carotene content (mg/100g) = 10.3257-0.3569x-0.3122y+0.0442x2-0.01132- 0.0195xy Where x=doses of gamma irradiation, y=weeks of storage 159 University of Ghana http://ugspace.ug.edu.gh Appendix LII. Analysis of Variance for beta carotene in irradiated Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 20.5274 20.52740 4.105480 656.95 0.000* Linear 2 20.3026 1.29817 0.649087 103.87 0.000* Square 2 0.1275 0.12748 0.063739 10.20 0.008* Interaction 1 0.0973 0.09734 0.097344 15.58 0.006* Residual Error 7 0.0437 0.04374 0.006249 Lack-of-Fit 3 0.0437 0.04374 0.014581 18225.74 0.000* Pure Error 4 0.0000 0.00000 0.000001 Total 12 20.5711 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LIII. Estimated Regression Coefficient for beta carotene for Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 10.1730 0.122230 83.228 0.000* Doses -0.1829 0.093887 -1.948 0.092** Weeks -0.1203 0.046943 -2.562 0.037* Doses*Doses 0.0135 0.020684 0.652 0.535** Weeks*Weeks -0.0180 0.005171 -3.476 0.010* Doses*Weeks -0.0342 0.008594 -3.985 0.005* S = 0.1375 R2 = 99.0% R2 (adj) = 98.3% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Beta carotene content (mg/100g) = 10.1730-0.1829x-0.1203y+0.0135x2-0.0180y2- 0.0342xy Where x=doses of gamma irradiation, y=weeks of storage 160 University of Ghana http://ugspace.ug.edu.gh Appendix LIV. Analysis of Variance for beta carotene Source DF Seq SS Adj. SS Adj. MS F P Regression 5 12.8529 12.852901 2.570580 135.97 0.000* Linear 2 12.3142 0.232467 0.116233 6.15 0.029* Square 2 0.2384 0.238393 0.119196 6.30 0.027* Interaction 1 0.3003 0.300304 0.300304 15.88 0.005* Residual Error 7 0.1323 0.132342 0.018906 Lack-of-Fit 3 0.1322 0.132240 0.044080 1728.63 0.000* Pure Error 4 0.0001 0.000102 0.000026 Total 12 12.9852 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LIV. Estimated Regression Coefficient for beta cryptoxanthin in Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 2.08787 0.023685 88.153 0.000* Doses -0.21145 0.018193 -11.623 0.000* Weeks -0.08044 0.009096 -8.843 0.000* Doses*Doses 0.02591 0.004008 6.463 0.000* Weeks*Weeks -0.00212 0.001002 -2.113 0.072** Doses*Weeks 0.00650 0.001665 3.903 0.006* S = 0.02664 R2 = 99.4% R2 (adj) = 99.0% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Beta cryptoxanthin content (mg/100g) = 2.08787-0.21145x-0.08044y+0.02591x2- 0.00212y2+0.00650xy Where x=doses of gamma irradiation, y=weeks of storage 161 University of Ghana http://ugspace.ug.edu.gh Appendix LVI. Analysis of Variance for beta cryptoxanthin for Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.884731 0.884731 0.176946 249.26 0.000* Linear 2 0.844158 0.179721 0.089861 126.59 0.000* Square 2 0.029756 0.029756 0.014878 20.96 0.001* Interaction 1 0.010816 0.010816 0.010816 15.24 0.006* Residual Error 7 0.004969 0.004969 0.000710 Lack-of-Fit 3 0.004574 0.004574 0.001525 15.45 0.012* Pure Error 4 0.000395 0.000395 0.000099 Total 12 0.889700 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LVIII. Estimated Regression Coefficient for capsaicin in Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 167.560 7.5796 22.107 0.000* Doses 15.149 5.8220 2.602 0.035* Weeks -7.081 2.9110 -2.433 0.045* Doses*Doses -0.291 1.2826 -0.227 0.827** Weeks*Weeks 0.919 0.3207 2.866 0.024* Doses*Weeks -2.061 0.5329 -3.867 0.006* S = 8.527 R2 = 88.6% R2 (adj) = 80.5% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Capsaicin content (mg/100g) = 167.560+15.149x-7.081y-0.291x2+0.919y2-2.061xy Where x=doses of gamma irradiation, y=weeks of storage 162 University of Ghana http://ugspace.ug.edu.gh Appendix LIX. Analysis of Variance for capsaicin in Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 3963.02 3963.02 792.60 10.90 0.003* Linear 2 2214.98 794.33 397.16 5.46 0.037* Square 2 660.79 660.79 330.39 4.54 0.054** Interaction 1 1087.25 1087.25 1087.25 14.96 0.006* Residual Error 7 508.91 508.91 72.70 Lack-of-Fit 3 508.85 508.85 169.62 10711.54 0.000* Pure Error 4 0.06 0.06 0.02 Total 12 4471.93 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LX. Estimated Regression Coefficient for capsaicin in Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 178.959 2.8531 62.725 0.000* Doses 23.610 2.1915 10.774 0.000* Weeks -1.811 1.0958 -1.653 0.142** Doses*Doses -3.498 0.4828 -7.246 0.000* Weeks*Weeks -0.413 0.1207 -3.420 0.011* Doses*Weeks 0.220 0.2006 1.096 0.309** S = 3.210 R2 = 98.8% R2 (adj) = 97.9% V alues marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Appendix LXI. Analysis of Variance for capsaicin in Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 5754.75 5754.75 1150.95 111.73 0.000* Linear 2 4741.43 1195.72 597.86 58.04 0.000* Square 2 1000.95 1000.95 500.47 48.59 0.000* Interaction 1 12.38 12.38 12.38 1.20 0.309** Residual Error 7 72.11 72.11 10.30 Lack-of-Fit 3 31.58 31.58 10.53 1.04 0.466** Pure Error 4 40.53 40.53 10.13 Total 12 5826.86 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 163 University of Ghana http://ugspace.ug.edu.gh Appendix LXII. Estimated Regression Coefficient for l* values in Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 50.2698 0.63236 79.496 0.000* Doses 0.4747 0.48572 0.977 0.361** Weeks -2.6318 0.24286 -10.837 0.000* Doses*Doses -0.0739 0.10701 -0.690 0.512** Weeks*Weeks 0.1159 0.02675 4.333 0.003* Doses*Weeks 0.0569 0.04446 1.279 0.242** S = 0.7114 R2 = 98.6% R2 (adj) = 97.7% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). L*-values = 50.2698+0.4747x-2.6318y-0.0739x2+0.1159y2+0.0569xy Where x=doses of gamma irradiation, y=weeks of storage Appendix LXIII. Analysis of Variance for l* values for Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 257.793 257.7933 51.5587 101.89 0.000* Linear 2 246.921 59.7378 29.8689 59.03 0.000* Square 2 10.044 10.0441 5.0221 9.92 0.009* Interaction 1 0.828 0.8281 0.8281 1.64 0.242** Residual Error 7 3.542 3.5422 0.5060 Lack-of-Fit 3 0.254 0.2542 0.0847 0.10 0.954** Pure Error 4 3.288 3.2880 0.8220 Total 12 261.336 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 164 University of Ghana http://ugspace.ug.edu.gh Appendix LXIV. Estimated Regression Coefficient for l* values in Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 50.7642 0.149170 340.312 0.000* Doses 0.2187 0.114579 1.909 0.098** Weeks -2.7785 0.057290 -48.500 0.000* Doses*Doses -0.0162 0.025243 -0.644 0.540** Weeks*Weeks 0.2169 0.006311 34.366 0.000* Doses*Weeks 0.0009 0.010488 0.089 0.931** S = 0.1678 R2 = 99.9% R2 (adj) = 99.8% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). L*-values = 50.7642+0.2187x-2.7785y-0.0162x2+0.2169y2+0.0009xy Where x=doses of gamma irradiation, y=weeks of storage Appendix LXV. Analysis of Variance for l* values for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 143.122 143.1221 28.6244 1016.55 0.000* Linear 2 104.762 67.2545 33.6273 1194.22 0.000* Square 2 38.360 38.3598 19.1799 681.15 0.000* Interaction 1 0.000 0.0002 0.0002 0.01 0.931** Residual Error 7 0.197 0.1971 0.0282 Lack-of-Fit 3 0.174 0.1741 0.0580 10.09 0.025* Pure Error 4 0.023 0.0230 0.0057 Total 12 143.319 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 165 University of Ghana http://ugspace.ug.edu.gh Appendix LXVI. Estimated Regression Coefficient for a* values for Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 21.3658 0.111355 191.871 0.000* Doses 0.7704 0.085534 9.007 0.000* Weeks -1.5885 0.042767 -37.144 0.000* Doses*Doses -0.0600 0.018844 -3.184 0.015* Weeks*Weeks 0.0134 0.004711 2.852 0.025* Doses*Weeks -0.0259 0.007829 -3.313 0.013* S = 0.1253 R2 = 100.0% R2 (adj) = 99.9% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). a* values = 21.3658+0.7704x-1.5885y-0.0600x2+0.0134y2-0.0259xy Where x=doses of gamma irradiation, y=weeks of storage Appendix LXVII. Analysis of Variance for a* values for Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 230.334 230.3337 46.0667 2935.75 0.000* Linear 2 229.953 21.7945 10.8972 694.46 0.000* Square 2 0.208 0.2084 0.1042 6.64 0.024* Interaction 1 0.172 0.1722 0.1722 10.98 0.013* Residual Error 7 0.110 0.1098 0.0157 Lack-of-Fit 3 0.108 0.1084 0.0361 103.28 0.000* Pure Error 4 0.001 0.0014 0.0003 Total 12 230.444 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 166 University of Ghana http://ugspace.ug.edu.gh Appendix LXVI. Estimated Regression Coefficient for a* values in Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 19.9285 1.40078 14.227 0.000* Doses 2.7600 1.07596 2.565 0.037* Weeks -2.0608 0.53798 -3.831 0.006* Doses*Doses -0.4393 0.23704 -1.853 0.106** Weeks*Weeks 0.0783 0.05926 1.321 0.228** Doses*Weeks -0.0259 0.09849 -0.263 0.800** S = 1.576 R2 = 93.3% R2 (adj) = 88.5% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). L*-values = 19.9285+2.7600x-1.5885y-0.0600x2+0.0134y2-0.0259xy Where x=doses of gamma irradiation, y=weeks of storage Appendix LXVII. Analysis of Variance for a* values in for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 241.261 241.2615 48.2523 19.43 0.001* Linear 2 231.462 46.0868 23.0434 9.28 0.011* Square 2 9.627 9.6271 4.8135 1.94 0.214* Interaction 1 0.172 0.1722 0.1722 0.07 0.800** Residual Error 7 17.381 17.3814 2.4831 Lack-of-Fit 3 17.380 17.3798 5.7933 14483.20 0.000* Pure Error 4 0.002 0.0016 0.0004 Total 12 258.643 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXVIII. Estimated Regression Coefficient for b* values in for Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 21.2059 0.129684 163.519 0.000* Doses 0.9602 0.099612 9.640 0.000* Weeks -4.5270 0.049806 -90.891 0.000* Doses*Doses -0.0625 0.021946 -2.846 0.025* Weeks*Weeks 0.3497 0.005486 63.739 0.000* Doses*Weeks -0.0566 0.009118 -6.204 0.000* S = 0.1459 R2 = 100.0% R2 (adj) = 99.9% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). 167 University of Ghana http://ugspace.ug.edu.gh b* values = 21.2059+0.9602x-4.5270y-0.0625x2+0.3497y2-0.0566xy Where x=doses of gamma irradiation, y=weeks of storage Appendix LXIX. Analysis of Variance for b* values in for Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 430.250 430.250 86.0499 4043.23 0.000* Linear 2 331.527 176.381 88.1903 4143.80 0.000* Square 2 97.903 97.903 48.9516 2300.09 0.000* Interaction 1 0.819 0.819 0.8190 38.48 0.000* Residual Error 7 0.149 0.149 0.0213 Lack-of-Fit 3 0.136 0.136 0.0454 14.28 0.013* Pure Error 4 0.013 0.013 0.0032 Total 12 430.399 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXX. Estimated Regression Coefficient for b* values in for Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 22.0177 0.176663 124.631 0.000* Doses 0.7916 0.135697 5.833 0.001* Weeks -2.5201 0.067849 -37.142 0.000* Doses*Doses -0.0636 0.029895 -2.128 0.071** Weeks*Weeks 0.0841 0.007474 11.252 0.000* Doses*Weeks -0.0272 0.012421 -2.189 0.065** S = 0.1987 R2 = 99.9% R2 (adj) = 99.9% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). b* values = 22.0177+0.7916x-2.5201y-0.0636x2+0.0841y2-0.0272xy Where x=doses of gamma irradiation, y=weeks of storage 168 University of Ghana http://ugspace.ug.edu.gh Appendix LXXI. Analysis of Variance for b* values for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 356.976 356.9762 71.3952 1807.72 0.000* Linear 2 351.572 54.4858 27.2429 689.79 0.000* Square 2 5.215 5.2154 2.6077 66.03 0.000* Interaction 1 0.189 0.1892 0.1892 4.79 0.065** Residual Error 7 0.276 0.2765 0.0395 Lack-of-Fit 3 0.259 0.2593 0.0864 20.10 0.007* Pure Error 4 0.017 0.0172 0.0043 Total 12 357.253 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXXII. Estimated Regression Coefficient for beta cryptoxanthin in for Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 2.08787 0.023685 88.153 0.000* Doses -0.21145 0.018193 -11.623 0.000* Weeks -0.08044 0.009096 -8.843 0.000* Doses*Doses 0.02591 0.004008 6.463 0.000* Weeks*Weeks -0.00212 0.001002 -2.113 0.072** Doses*Weeks 0.00650 0.001665 3.903 0.006* S = 0.02664 R2 = 99.4% R2 (adj) = 99.0% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXXIII. Analysis of Variance for beta cryptoxanthin in Legon-18 samples irradiated and stored 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.884731 0.884731 0.176946 249.26 0.000* Linear 2 0.844158 0.179721 0.089861 126.59 0.000* Square 2 0.029756 0.029756 0.014878 20.96 0.001* Interaction 1 0.010816 0.010816 0.010816 15.24 0.006* Residual Error 7 0.004969 0.004969 0.000710 Lack-of-Fit 3 0.004574 0.004574 0.001525 15.45 0.012* Pure Error 4 0.000395 0.000395 0.000099 Total 12 0.889700 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 169 University of Ghana http://ugspace.ug.edu.gh Appendix LXXIV. Estimated Regression Coefficient for beta cryptoxanthin for Legon- 18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 2.11232 0.019755 106.926 0.000* Doses -0.24443 0.015174 -16.109 0.000* Weeks -0.04159 0.007587 -5.482 0.001* Doses*Doses 0.03068 0.003343 9.178 0.000* Weeks*Weeks -0.00449 0.000836 -5.368 0.001* Doses*Weeks 0.00784 0.001389 5.647 0.001* S = 0.02222 R2 = 99.4% R2 (adj) = 99.1% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Beta cryptoxanthin (mg/100g) = 2.11232-0.24443x-0.04159y+0.03068x2- 0.00449y2+0.00784xy Where x=doses of gamma irradiation, y=weeks of storage Appendix LXXV. Analysis of Variance for beta cryptoxanthin for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.621762 0.621762 0.124352 251.80 0.000* Linear 2 0.562391 0.161323 0.080661 163.33 0.000* Square 2 0.043621 0.043621 0.021810 44.16 0.000* Interaction 1 0.015750 0.015750 0.015750 31.89 0.001* Residual Error 7 0.003457 0.003457 0.000494 Lack-of-Fit 3 0.003310 0.003310 0.001103 29.98 0.003* Pure Error 4 0.000147 0.000147 0.000037 Total 12 0.625219 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. 170 University of Ghana http://ugspace.ug.edu.gh Appendix LXXVI. Estimated Regression Coefficient for dihydrocapsaicin for Legon-18 samples stored at 28±2oC Term Coefficient SE Coefficient T P Constant 75.1353 3.1039 24.206 0.000* Doses 10.2371 2.3842 4.294 0.004* Weeks -4.2696 1.1921 -3.582 0.009* Doses*Doses -1.9507 0.5253 -3.714 0.008* Weeks*Weeks -0.0077 0.1313 -0.059 0.955** Doses*Weeks 0.9569 0.2182 4.385 0.003* S = 3.492 R2 = 95.8% R2 (adj) = 92.8% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Dihydrocapsaicin (mg/100g) = 75.1353+10.2371x-4.2696y-1.9507x2-0.0077y2+0.9569xy Where x=doses of gamma irradiation, y=weeks of storage Appendix LXXVII. Analysis of Variance for dihydrocapsaicin in Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 1935.51 1935.51 387.101 31.75 0.000* Linear 2 1501.98 329.12 164.559 13.50 0.004* Square 2 199.12 199.12 99.561 8.17 0.015* Interaction 1 234.40 234.40 234.399 19.23 0.003* Residual Error 7 85.34 85.34 12.192 Lack-of-Fit 3 72.73 72.73 24.242 7.68 0.039* Pure Error 4 12.62 12.62 3.154 Total 12 2020.85 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXXVIII. Estimated Regression Coefficient for dihydrocapsaicin in for Legon-18 samples stored at 4 oC. Term Coefficient SE Coefficient T P Constant 77.1818 1.45233 53.144 0.000* Doses 5.9961 1.11555 5.375 0.001* Weeks -2.3500 0.55778 -4.213 0.004* Doses*Doses -0.8681 0.24577 -3.532 0.010* Weeks*Weeks 0.0187 0.06144 0.305 0.769* Doses*Weeks 0.3195 0.10211 3.129 0.017* S = 1.634 R2 = 97.2% R2 (adj) = 95.2% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). 171 University of Ghana http://ugspace.ug.edu.gh Dihydrocapsaicin (mg/100g) = 77.1818+5.9961x-2.3500y-0.8681x2+0.0187y2+0.3195xy Where x=doses of gamma irradiation, y=weeks of storage Appendix LXXIX. Analysis of Variance for dihydrocapsaicin in for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 643.651 643.651 128.730 48.23 0.000* Linear 2 580.835 107.756 53.878 20.19 0.001* Square 2 36.686 36.686 18.343 6.87 0.022* Interaction 1 26.129 26.129 26.129 9.79 0.017* Residual Error 7 18.684 18.684 2.669 Lack-of-Fit 3 10.636 10.636 3.545 1.76 0.293* Pure Error 4 8.048 8.048 2.012 Total 12 662.335 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXXX. Estimated Regression Coefficient for total capsaicinoids in for Legon-18 samples stored at 28±2oC Term Coefficient SE Coefficient T P Constant 253.819 14.8591 17.082 0.000* Doses 27.972 11.4135 2.451 0.044* Weeks -11.842 5.7067 -2.075 0.077** Doses*Doses -4.107 2.5145 -1.633 0.146** Weeks*Weeks -0.019 0.6286 -0.030 0.977** Doses*Weeks 1.107 1.0447 1.059 0.325** S = 16.72 R2 = 89.4% R2 (adj) = 81.8% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Total capsaicinoids (mg/100g) = 253.819+27.972x-11.842y-4.107x2-0.019y2+1.107xy Where x=doses of gamma irradiation, y=weeks of storage 172 University of Ghana http://ugspace.ug.edu.gh Appendix LXXXI. Analysis of Variance for total capsaicinoids in Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 16500.3 16500.30 3300.06 11.81 0.003* Linear 2 15302.4 2486.60 1243.30 4.45 0.057** Square 2 884.4 884.43 442.21 1.58 0.271** Interaction 1 313.5 313.52 313.52 1.12 0.325** Residual Error 7 1955.8 1955.81 279.40 Lack-of-Fit 3 30.5 30.51 10.17 0.02 0.995* Pure Error 4 1925.3 1925.31 481.33 Total 12 18456.1 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXXXII. Estimated Regression Coefficient for total capsaicinoids in Legon- 18 pepper samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 256.030 3.5836 71.445 0.000* Doses 29.938 2.7526 10.876 0.000* Weeks -3.996 1.3763 -2.903 0.023* Doses*Doses -4.449 0.6064 -7.337 0.000* Weeks*Weeks -0.415 0.1516 -2.735 0.029* Doses*Weeks 0.539 0.2520 2.141 0.070** S = 4.031 R2 = 98.9% R2 (adj) = 98.1% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Total capsaicinoids (mg/100g) = 256.030+29.938x-3.996y-4.449x2-0.415y2+0.539xy Where x=doses of gamma irradiation, y=weeks of storage 173 University of Ghana http://ugspace.ug.edu.gh Appendix LXXXIII. Analysis of Variance for total capsaicinoids in for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 10169.3 10169.28 2033.86 125.15 0.000* Linear 2 8638.7 1944.16 972.08 59.82 0.000* Square 2 1456.1 1456.14 728.07 44.80 0.000* Interaction 1 74.5 74.47 74.47 4.58 0.070** Residual Error 7 113.8 113.76 16.25 Lack-of-Fit 3 67.2 67.20 22.40 1.92 0.267** Pure Error 4 46.6 46.56 11.64 Total 12 10283.0 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXXXIV. Estimated Regression Coefficient for SHU for Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 4340.98 217.382 19.969 0.000* Doses 105.47 166.975 0.632 0.548** Weeks -155.02 83.487 -1.857 0.106** Doses*Doses 2.54 36.786 0.069 0.947** Weeks*Weeks -5.51 9.197 -0.599 0.568** Doses*Weeks 19.41 15.284 1.270 0.245** S = 244.5 R2 = 89.3% R2 (adj) = 81.6% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). SHU = 4340.98+105.47x-155.02y+2.54x2-5.51y2+19.41xy Where x=doses of gamma irradiation, y=weeks of storage 174 University of Ghana http://ugspace.ug.edu.gh Appendix LXXXV. Analysis of Variance for SHU for Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 3481352 3481352 696270 11.64 0.003* Linear 2 3361738 212899 106450 1.78 0.237** Square 2 23215 23215 11607 0.19 0.828** Interaction 1 96399 96399 96399 1.61 0.245** Residual Error 7 418594 418594 59799 Lack-of-Fit 3 418545 418545 139515 11327.64 0.000* Pure Error 4 49 49 12 Total 12 3899946 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXXXVI. Estimated Regression Coefficient for SHU in for Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 4122.08 57.699 71.441 0.000* Doses 482.00 44.320 10.876 0.000* Weeks -64.33 22.160 -2.903 0.023* Doses*Doses -71.63 9.764 -7.336 0.000* Weeks*Weeks -6.68 2.441 -2.735 0.029* Doses*Weeks 8.68 4.057 2.141 0.070** S = 64.91 R2 = 98.9% R2 (adj) = 98.1% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. SHU = 4122.08+482.00x-64.33y-71.63x2-6.68y2+8.68xy Where x=doses of gamma irradiation, y=weeks of storage 175 University of Ghana http://ugspace.ug.edu.gh Appendix LXXXVII. Analysis of Variance for SHU in for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 2635999 2635999 527200 125.14 0.000* Linear 2 2239230 503956 251978 59.81 0.000* Square 2 377466 377466 188733 44.80 0.000* Interaction 1 19304 19304 19304 4.58 0.070** Residual Error 7 29491 29491 4213 Lack-of-Fit 3 17421 17421 5807 1.92 0.267** Pure Error 4 12069 12069 3017 Total 12 2665490 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix LXXXVIII. Estimated Regression Coefficient for total titratable acidity in Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 0.213326 0.008200 26.016 0.000* Doses -0.003313 0.006298 -0.526 0.615** Weeks 0.021291 0.003149 6.761 0.000* Doses*Doses 0.002296 0.001388 1.655 0.142** Weeks*Weeks -0.001427 0.000347 -4.115 0.004* Doses*Weeks -0.001201 0.000577 -2.083 0.076** S = 0.009224 R2 = 92.4% R2 (adj) = 86.9% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Total titratable acidity (%) = 0.213326-0.003313x+0.021291y+0.002296x2-0.001427y2- 0.001201xy Where x=doses of gamma irradiation, y=weeks of storage 176 University of Ghana http://ugspace.ug.edu.gh Appendix LXXXIX. Analysis of Variance for titratable acidity in Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.007196 0.007196 0.001439 16.92 0.001* Linear 2 0.005386 0.003917 0.001959 23.02 0.001* Square 2 0.001441 0.001441 0.000721 8.47 0.014* Interaction 1 0.000369 0.000369 0.000369 4.34 0.076** Residual Error 7 0.000596 0.000596 0.000085 Lack-of-Fit 3 0.000479 0.000479 0.000160 5.46 0.067** Pure Error 4 0.000117 0.000117 0.000029 Total 12 0.007792 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares Appendix XC. Estimated Regression Coefficient for pH in for Legon-18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 4.97457 0.035591 139.770 0.000* Doses 0.06088 0.027338 2.227 0.061** Weeks 0.03585 0.013669 2.623 0.034* Doses*Doses -0.01095 0.006023 -1.818 0.112** Weeks*Weeks 0.00195 0.001506 1.295 0.236** Doses*Weeks -0.00344 0.002502 -1.374 0.212* S = 0.04004 R2 = 94.7% R2 (adj) = 90.9% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares pH = 4.97457+0.06088x+0.03585y-0.01095x2+0.00195y2-0.00344xy Where x=doses of gamma irradiation, y=weeks of storage 177 University of Ghana http://ugspace.ug.edu.gh Appendix XCI. Analysis of Variance for pH in Legon-18 pepper samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.200087 0.200087 0.040017 24.96 0.000* Linear 2 0.191083 0.022599 0.011300 7.05 0.021* Square 2 0.005978 0.005978 0.002989 1.86 0.224** Interaction 1 0.003025 0.003025 0.003025 1.89 0.212** Residual Error 7 0.011221 0.011221 0.001603 Lack-of-Fit 3 0.007901 0.007901 0.002634 3.17 0.147** Pure Error 4 0.003320 0.003320 0.000830 Total 12 0.211308 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares Appendix XCII. Estimated Regression Coefficient for pH of Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 5.10463 0.019366 263.590 0.000* Doses 0.00070 0.014875 0.047 0.964** Weeks 0.02869 0.007438 3.857 0.006* Doses*Doses -0.00091 0.003277 -0.276 0.790** Weeks*Weeks 0.00102 0.000819 1.250 0.252** Doses*Weeks 0.00094 0.001362 0.689 0.513** S = 0.02179 R2 = 97.8% R2 (adj) = 96.2% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares pH = 5.10463+0.00070x+0.02869y-0.00091x2+0.00102y2+0.00094xy Where x=doses of gamma irradiation, y=weeks of storage 178 University of Ghana http://ugspace.ug.edu.gh Appendix XCIII. Analysis of Variance for pH of Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.145155 0.145155 0.029031 61.17 0.000* Linear 2 0.144167 0.007279 0.003640 7.67 0.017* Square 2 0.000763 0.000763 0.000382 0.80 0.485** Interaction 1 0.000225 0.000225 0.000225 0.47 0.513** Residual Error 7 0.003322 0.003322 0.000475 Lack-of-Fit 3 0.002122 0.002122 0.000707 2.36 0.213* Pure Error 4 0.001200 0.001200 0.000300 Total 12 0.148477 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares Appendix XCIV. Estimated Regression Coefficient for titratable acidity in Legon-18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 0.220313 0.008866 24.850 0.000* Doses -0.005062 0.006810 -0.743 0.481** Weeks 0.017215 0.003405 5.056 0.001* Doses*Doses 0.001132 0.001500 0.755 0.475** Weeks*Weeks -0.000918 0.000375 -2.447 0.044* Doses*Weeks -0.000400 0.000623 -0.642 0.541** S = 0.009973 R2 = 92.6% R2 (adj) = 87.2% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). Total titratable acidity (%) =0.220313-0.005062x+0.017215y+0.001132x2-0.000918y2- 0.000400xy Where x=doses of gamma irradiation, y=weeks of storage 179 University of Ghana http://ugspace.ug.edu.gh Appendix XCV. Analysis of Variance for total titratable acidity in Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 0.008651 0.008651 0.001730 17.40 0.001* Linear 2 0.008011 0.002543 0.001272 12.78 0.005* Square 2 0.000599 0.000599 0.000300 3.01 0.114** Interaction 1 0.000041 0.000041 0.000041 0.41 0.541** Residual Error 7 0.000696 0.000696 0.000099 Lack-of-Fit 3 0.000695 0.000695 0.000232 946.04 0.000* Pure Error 4 0.000001 0.000001 0.000000 Total 12 0.009347 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares Appendix XCVI. Estimated Regression Coefficient for moisture content in for Legon- 18 samples stored at 4 oC Term Coefficient SE Coefficient T P Constant 9.92989 0.46088 21.545 0.000* Doses -0.05632 0.35401 -0.159 0.878** Weeks 0.03017 0.17701 0.170 0.869** Doses*Doses 0.04741 0.07799 0.608 0.562** Weeks*Weeks -0.01940 0.01950 -0.995 0.353** Doses*Weeks -0.03750 0.03240 -1.157 0.285** S = 0.5185 R2 = 70.5% R2 (adj) = 49.4% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares Moisture content (%) = 9.92989-0.05632x + 0.03017y + 0.04741x2-0.01940y2-0.03750xy Where x=doses of gamma irradiation, y=weeks of storage 180 University of Ghana http://ugspace.ug.edu.gh Appendix XCVII. Analysis of Variance for moisture content in for Legon-18 samples stored at 4 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 4.4892 4.48916 0.897832 3.34 0.074** Linear 2 3.8467 0.01258 0.006292 0.02 0.977** Square 2 0.2825 0.28249 0.141247 0.53 0.613** Interaction 1 0.3600 0.36000 0.360000 1.34 0.285** Residual Error 7 1.8816 1.88161 0.268801 Lack-of-Fit 3 1.3496 1.34961 0.449870 3.38 0.135** Pure Error 4 0.5320 0.53200 0.133000 Total 12 6.3708 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XCVIII. Estimated Regression Coefficient for moisture content in for Legon- 18 samples stored at 28±2 oC Term Coefficient SE Coefficient T P Constant 9.86839 0.232475 42.449 0.000* Doses 0.50316 0.178567 2.818 0.026* Weeks -0.07342 0.089284 -0.822 0.438** Doses*Doses -0.06121 0.039340 -1.556 0.164** Weeks*Weeks -0.00905 0.009835 -0.920 0.388** Doses*Weeks -0.03125 0.016345 -1.912 0.097** S=0.2615 R2 = 91.6% R2 = 85.5% Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Moisture content (%) = 9.86839+0.50316x -0.07342y-0.06121x2-0.00905y2-0.03125xy Where x=doses of gamma irradiation, y=weeks of storage 181 University of Ghana http://ugspace.ug.edu.gh Appendix XCIX. Analysis of Variance for moisture content in for Legon-18 samples stored at 28±2 oC Source DF Seq SS Adj. SS Adj. MS F P Regression 5 5.19203 5.19203 1.03841 15.18 0.001* Linear 2 4.59333 0.55244 0.27622 4.04 0.068** Square 2 0.34870 0.34870 0.17435 2.55 0.147** Interaction 1 0.25000 0.25000 0.25000 3.66 0.097** Residual Error 7 0.47874 0.47874 0.06839 Lack-of-Fit 3 0.08674 0.08674 0.02891 0.30 0.828** Pure Error 4 0.39200 0.39200 0.09800 Total 12 5.67077 Values marked *are statistically significant (p<0.05). Values marked ** are statistically not (p>0.05). DF-degree of freedom, Adj. SS-Adjusted Sum of Squares, Adj. MS- Adjusted mean squares. Appendix XCX. Calibration curve for beta cryptoxanthin. 182 University of Ghana http://ugspace.ug.edu.gh Appendix XCXI. Calibration curve for capsanthin 183 University of Ghana http://ugspace.ug.edu.gh Appendix XCXII. Calibration curve for beta-carotene Appendix XCXIII. Calibration curve for capsaicin 184 University of Ghana http://ugspace.ug.edu.gh Appendix XCXIV. Calibration curve for dihydrocapsaicin Appendix XCXV: Effect of gamma irradiation and storage time (days) on the inactivation of S. Typhimurium at 4 oC. 185 University of Ghana http://ugspace.ug.edu.gh Appendix XCXVI: Effect of gamma irradiation and storage time (days) on the inactivation of S. Typhimurium at 28±2 oC. Appendix XCXVII: Effect of gamma irradiation and storage time (days) on the inactivation of E. coli at 4 oC. 186 University of Ghana http://ugspace.ug.edu.gh Appendix XCXVIII: Effect of gamma irradiation and storage time (days) on the inactivation of E. coli at. 28±2 oC. Appendix XCXIX: Effect of gamma irradiation and storage time (days) on the inactivation of B. cereus at 4 oC. 187 University of Ghana http://ugspace.ug.edu.gh Appendix C: Effect of gamma irradiation and storage time (days) on the inactivation of B. cereus at 28±2 oC. Appendix CI: Effect of gamma irradiation and storage time (days) on the inactivation of L. monocytogenes at 4 oC. 188 University of Ghana http://ugspace.ug.edu.gh Appendix CII: Effect of gamma irradiation and storage time (days) on the inactivation of L. monocytogenes at 28±2 oC. Appendix CIII: Effect of gamma irradiation and storage time (days) on the inactivation of S. aureus 4 oC. 189 University of Ghana http://ugspace.ug.edu.gh Appendix CIV: Effect of gamma irradiation and storage time (days) on the inactivation of S. aureus at 28±2 oC. Appendix CV. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of S. Typhimurium at 4 oC. Source Of Variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 123.5615 17.65164 2651.17 <.001* Doses (B) 4 418.141 104.5353 15700.57 <.001* AB 28 60.69558 2.167699 325.58 <.001* Residual 79 0.525986 0.006658 Total 118 602.9241 Values designated as * are significant (p<0.05) Appendix CVI. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of S. Typhimurium at 28±2 oC. Source of variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 137.967 19.70958 2323.2 <.001* Doses (B) 4 354.2124 88.5531 10437.91 <.001* AB 28 45.0775 1.609911 189.76 <.001* Residual 80 0.678704 0.008484 Total 119 537.9356 Values designated as * are significant (p<0.05) 190 University of Ghana http://ugspace.ug.edu.gh Appendix CVII. ANOVA summary table for the effect of gamma irradiation, temperature and storage time (days) on the inactivation of S. Typhimurium Source Of Variation D.F. S.S. M.S. V.R. F Pr. Storage days (A) 7 2.66E+02 3.80E+01 5017.56 <.001* Temperature (B) 1 4.83E+00 4.83E+00 638.13 <.001* Doses (C) 4 7.77E+02 1.94E+02 25661.91 <.001* AB 7 6.92E-01 9.89E-02 13.07 <.001* AC 28 1.01E+02 3.62E+00 477.95 <.001* BC 4 1.66E+00 4.14E-01 54.7 <.001* ABC 28 4.20E+00 1.50E-01 19.84 <.001* Residual 160 1.21E+00 7.57E-03 Storage days (A) 239 1.16E+03 Total 239 1.16E+03 Values designated as * are significant (p<0.05) Appendix CVIII. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of E. coli at 4 oC. Source Of Variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 178.3944 25.48492 282.82 <.001 Doses (B) 4 198.9963 49.74906 552.09 <.001 AB 28 80.92326 2.89012 32.07 <.001 Residual 79 7.11878 0.09011 Total 118 465.4327 Values designated as * are significant (p<0.05) Appendix CIX. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of E. coli 28±2 oC. Source of variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 181.6851 25.95501 4113.22 <.001* Doses (B) 4 198.4249 49.60623 7861.35 <.001* AB 28 79.05667 2.823453 447.45 <.001* Residual 80 0.504811 0.00631 Total 119 459.6715 Values designated as * are significant (p<0.05) 191 University of Ghana http://ugspace.ug.edu.gh Appendix CX. ANOVA summary table for the effect of gamma irradiation, temperature and storage time (days) on the inactivation of E. coli Source Of Variation D.F. S.S. M.S. V.R. F Pr. Storage days (A) 7 369.77332 52.82476 1108.18 <.001* Temperature (B) 1 0.12732 0.12732 2.67 0.104* Doses (C) 4 405.86058 101.46515 2128.57 <.001* AB 7 0.10083 0.0144 0.3 0.952** AC 28 160.60926 5.73604 120.33 <.001* BC 4 0.41564 0.10391 2.18 0.074** ABC 28 0.57381 0.02049 0.43 0.995** Residual 160 7.62691 0.04767 Storage days (A) 239 Total 239 Values designated as * are significant (p<0.05) Appendix CXI. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of B. cereus at 4 oC. Source Of Variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 23.4511 3.350157 495.62 <.001* Doses (B) 4 542.1026 135.5257 20049.51 <.001* AB 28 10.56801 0.377429 55.84 <.001* Residual 79 0.534004 0.00676 Total 118 576.6557 Values designated as * are significant (p<0.05) Appendix CXII. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of B. cereus at 28±2 oC. Source Of Variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 26.56107 3.794439 443.25 <.001* Doses (B) 4 508.1883 127.0471 14840.98 <.001* AB 28 14.34506 0.512324 59.85 <.001* Residual 80 0.684845 0.008561 Total 119 549.7793 Values designated as * are significant (p<0.05) 192 University of Ghana http://ugspace.ug.edu.gh Appendix CXIII. ANOVA summary table for the effect of gamma irradiation, temperature and storage time (days) on the inactivation of B. cereus Source Of Variation D.F. S.S. M.S. V.R. F Pr. Storage days (A) 7 5.10E+01 7.28E+00 947.54 <.001* Temperature (B) 1 2.91E+00 2.91E+00 379.06 <.001* Doses (C) 4 1.05E+03 2.63E+02 34243.14 <.001* AB 7 2.87E-01 4.10E-02 5.33 <.001* AC 28 2.39E+01 8.54E-01 111.21 <.001* BC 4 1.10E+00 2.76E-01 35.92 <.001* ABC 28 8.49E-01 3.03E-02 3.95 <.001* Residual 160 1.23E+00 7.68E-03 Total 239 1.13E+03 Values designated as * are significant (p<0.05) Appendix CXVI. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of S. aureus 4 oC. Source Of Variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 157.1885 22.4555 2547.36 <.001* Doses (B) 4 376.4077 94.10193 10674.96 <.001* AB 28 64.84396 2.315856 262.71 <.001* Residual 79 0.696401 0.008815 Total 118 599.1366 Values designated as * are significant (p<0.05) Appendix CXVII. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of S. aureus 28±2 oC. Source of variation D.F. S.S. M.S. V.R. F PR. Days (A) 7 155.308 22.18685 3896.01 <.001* Doses (B) 4 317.4543 79.36357 13936.24 <.001* AB 28 71.39257 2.549735 447.73 <.001* Residual 80 0.455581 0.005695 Total 119 544.6104 Values designated as * are significant (p<0.05) 193 University of Ghana http://ugspace.ug.edu.gh Appendix CXVIII. ANOVA summary table for the effect of gamma irradiation, temperature and storage time (days) on the inactivation of S. aureus Source Of Variation D.F. S.S. M.S. V.R. F Pr. Storage days (A) 7 3.16E+02 4.51E+01 6221.86 <.001* Temperature (B) 1 6.65E+00 6.65E+00 916.32 <.001* Doses (C) 4 6.98E+02 1.75E+02 24063.12 <.001* AB 7 3.28E+00 4.68E-01 64.55 <.001* AC 28 1.28E+02 4.56E+00 628.75 <.001* BC 4 2.78E+00 6.96E-01 95.97 <.001* ABC 28 8.64E+00 3.09E-01 42.53 <.001* Residual 160 1.16E+00 7.25E-03 Total 239 1.16E+03 Values designated as * are significant (p<0.05) Appendix CXIX. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of L. monocytogenes 4 oC. Source Of Variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 85.70755 12.24394 1599.23 <.001* Doses (B) 4 427.6144 106.9036 13963.15 <.001* AB 28 41.2907 1.474668 192.61 <.001* Residual 79 0.604834 0.007656 Total 118 555.2175 Values designated as * are significant (p<0.05) Appendix CXX. ANOVA summary table for the effect of gamma irradiation, doses and days on the inactivation of inactivation of L. monocytogenes at 28±2 oC. Source of variation D.F. S.S. M.S. V.R. F Pr. Days (A) 7 81.31138 11.61591 1542.26 <.001* Doses (B) 4 384.1245 96.03113 12750.22 <.001* AB 28 36.04856 1.287448 170.94 <.001* Residual 80 0.602538 0.007532 Total 119 502.087 Values designated as * are significant (p<0.05) 194 University of Ghana http://ugspace.ug.edu.gh Appendix CXXI. ANOVA summary table for the effect of gamma irradiation, temperature and storage time (days) on the inactivation of L. monocytogenes Source Of Variation D.F. S.S. M.S. V.R. F Pr. Storage days (A) 7 1.72E+02 2.46E+01 3246.17 <.001* Temperature (B) 1 3.42E+00 3.42E+00 451.34 <.001* Doses (C) 4 8.15E+02 2.04E+02 26871.05 <.001* AB 7 2.27E-01 3.25E-02 4.28 <.001* AC 28 7.68E+01 2.74E+00 361.58 <.001* BC 4 8.98E-01 2.24E-01 29.6 <.001* ABC 28 4.04E-01 1.44E-02 1.9 <0.007* Residual 160 1.21E+00 7.58E-03 Storage days (A) 239 1.07E+03 Total 239 1.07E+03 Values designated as * are significant (p<0.05) Appendix CXXII. ANOVA summary table for the effect of gamma irradiation, temperature and the type of organism on the log cfu/g in pepper powder Source of variation D.F. S.S. M.S. F PR. Microorganisms (A) 4 477.5767 119.3942 <.001* Storage temperature (B) 1 15.09803 15.09803 <.001* Storage days (C) 7 1043.47 149.0671 <.001* Doses (D) 4 3584.89 896.2224 <.001* AB 4 2.83753 0.70938 <.001* AC 28 131.1655 4.68448 <.001* BC 7 1.30289 0.18613 <.001* AD 16 163.0295 10.18934 <.001* BD 4 4.60666 1.15166 <.001* CD 28 298.3691 10.65604 <.001* ABC 28 3.28127 0.11719 <.001* ABD 16 2.25005 0.14063 <.001* ACD 112 191.8624 1.71306 <.001* BCD 28 2.8387 0.10138 <.001* ABCD 112 11.8287 0.10561 <.001* Residual 800 12.44034 0.01555 Total 1199 5946.847 Values designated as * are significant (p<0.05). 195