Date 0-7639 1. 4 u u . is u‘Clefll S [3 cc 5 fl Umvcrsxty f :5: M This is to certify that the thesis entitled PHYSICO-CHEMICAL CONSIDERATIONS IN THE STORAGE 0F PROCESSED COWPEA PRODUCTS presented by MARK E . UKHUN has been accepted towards fulfillment of the requirements for PH.D. FOOD SCIENCE 8 HUMAN (1 gr in e ee NUIRITION may“ Major prft/ lO-th-80 OVERDUE FINES: 25¢ per day per item .- RETURNING LIBRARY MATERIALS: Place In book neturn to regime ‘ ~ _ charge from ctrculatton records ‘ .- PHYSICO-CHEMICAL CONSIDERATIONS IN THE STORAGE OF PROCESSED COWPEA PRODUCTS By Mark E. Ukhun A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition ' 1980 49/ )7 6:74/af ABSTRACT PHYSICO-CHEMICAL CONSIDERATIONS IN THE STORAGE OF PROCESSED COWPEA PRODUCTS By Mark E. Ukhun Raw and drum—dried cooked COWpea powders were pre- pared and stored for six months at water activities (Aw's) of 0.11, 0.33 and 0.75 at a constant temperature of 25° C ' and at 5°, 25° and 40° c at a constant Aw of 0.75. Physico-chemical evaluations of these powders re- vealed that lipid oxidation decreased when the Aw was increased from 0.11 to 0.33 and increased when the Aw was increased from 0.33 to 0.75. Temperature increases had the effect of increasing lipid oxidation. Although the raw samples had lower oxidation status immediately after processing, they had oxidized more than the drum- dried samples at the end of storage. Refrigeration tem- perature effectively minimized lipid oxidation in all the samples. Unsaturated fatty acids decreased in the course of storage. The saturated/unsaturated fatty acid ratio was about unity at the beginning of storage, but this ratio increased at all temperatures, save 5° C, and at all Aw's, as the length of storage increased. The more Mark E. Ukhun highly unsaturated fatty acids oxidized faster than the less unsaturated ones. Lipid free-amino groups decreased in storage with increases in Aw and temperature. Powder whiteness decreased with increased AW and temperature. Losses of reducing sugar and available lysine followed expected trends vis-a—vis browning trends with losses recorded at higher Aw and temperature. The loss of soluble protein in the drum-dried samples followed losses of both reducing sugar and avail- able lysine and therefore appeared to be due to Maillard browning. In the raw samples, the first three months were marked by higher soluble protein contents as the Aw was increased, after which the reverse was true, as would be expected from Maillard browning. Changes in pH, although following color changes, were minimal over a six month storage. Temperature of 40° C appeared to have led to more marked decreases in pH than all the other storage conditions. There was no measurable ascorbic acid in the sam- ples; while riboflavin and thiamine were relatively high in these powders. Increasing Aw led to increasing losses of both vitamins in the drum-dried samples. The Opposite was true in the raw samples. Mark E. Ukhun Examination of the relationships between the various cowPea quality parameters studied indicated that: a. Lipid oxidation may account for some of the losses of riboflavin and thiamine the lipid free—amino groups may have been lost in storage as a result of both oxidation and carbonyl—amine reactions, as in browning the decrease in the pH of the powders during storage may be partly accounted for by the loss of the basic amino groups of the lipid free-amino groups and the lysine the loss of reducing sugar and of the available lysine was, at least, partly due to carbonyl- amine reactions as in browning the browning process was probably responsible for the loss of protein solubility during storage. Drum drying caused minimal losses in the nutritional parameters of these powders but, in storage, drum-drying, in fact,reduced the rates of losses in the nutritional qualities. Proximate analyses of the products revealed high protein content, low total lipid, high carbohydrate and fair amounts of crude fiber and ash contents. Some of the problems observed in storage were cakiness at Aw 0.75 at all temperatures, and mold growth Mark E. Ukhun was noticed after five months Of storage at an Aw of 0.75 at a temperature of 25° C. Based on the results obtained in this work, it is apparent that the optimum storage conditions for these powders are an Aw of 0.11 and temperature of 50 C. DEDICATION The facts and truths of Science testify to the facts and truths of the existence and omnipotence of’God. A belief in Science is not incompatible with a belief in God. To Him and to all the members of my family who rep- resent part of His wonderful creation, this work is dedicated. ii ACKNOWLEDGEMENT I am profoundly appreciative of the persistent com- mitment of my major advisor, Professor L.R. Dugan, Jr. to bringing out the very best in me throughout the period of my academic life at Michigan State University. For willingly and effectively serving on my committee, I am equally indebted to Professors D. R. Dilley, J.I. Gray, W.G. Bergen and M.A. Uebersax. Finally, the moral support and the amicable atmosphere generated by my colleagues at the Food Science Lipid Labor- atory of Midnigan State University is greatly appreciated. I particularly wish to thank Ms. Susan Cuppett for her tech- nical assistance. iii TABLE OF CONTENTS PAGE List of Tables. . . . . . . . . . . . . . . . . . vii List of Figures . . . . . . . . . . . . . . . . . ix INTRODUCTION. . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . 5 Processing of Legumes . . . . . . . . . . . 7 Storage of Legumes. . . . ll Storage and the Protein Quality of Foods. . 14 Lipid Content and Quality of Stored Protein in Foods. . . . . 16 Water Activity and the Quality of Stored. Foods . . . . . . . . . . . . . . . . 25 Stability of Lipids . . . . . . . . . 31 Evaluation of the State of Oxidation. . 33 Ultraviolet (UV) Absorption Methods . . 33 Evaluation of Protein Quality . . . 33 Browning Reactions. . . . . . . . . . . . . 39 Non-Oxidative Browning. . . . . . . . . . . 40 Caramelization. . . . . . . . #1 Sugar Amine or Maillard Browning. . . . . . 42 MATERIALS . . . . . . . . . . . . . . . . . . . . 46 Preparation of Drum-Dried COWpea Powder . . 47 Preparation of Raw COWpea Powders . . . . . 48 Experimental Set-Up . . . . . . . . . . . . #8 METHODS . . . . . . . . . . . . . . . . . . . . . 49 Extraction of Total Lipids. . . . . . . #9 Measurement of Lipid Diene Conjugation. . . 50 Gas-Liquid Chromatography . . . . . . 50 Preparation of Fatty- -Acid Methyl esters . . 5O Gas-Liquid Chromatography of Methyl esters. 51 Extraction of Reducing Sugars . . . 51 iv Determination of Available Lysine. . . . . . . . 52 A. SpectrOphotometric Method . . 52 B. High Performance Liquid Chromatography (HPLC) Method . . . . ........ . . . 53 Measurement of Browning Pigments . . . . . . . . 53 Color Measurement by the Hunter Colorimeter. . . 53 Determination of Soluble Protein . . . . . 54 Determination of pH. . . . . . 56 Determination of Lipid Free-Amino Groups . . 56 Determination of Ascorbic Acid . . . . . . . 57 Fluorometric Determination of Riboflavin . . . . 57 Fluorometric Determination of Thiamine . . . . . 58 Proximate Analysis of Cowpea Powders . . . . . . 58 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 59 Proximate Analyses of Raw and Drum-Dried COWpea Powders . . . . . . . . . 59 Lipid Oxidation As Measured by Diene Conjugation. . . . . . . . . 62 Fatty-Acid Content of. COWpea Powders . . . . . . 7O Lipid Free-Amino Groups. . . . . . . . . . . . . 78 Color Changes and Browning . . . . . . . . . . . 84 Changes in Reducing Sugar. . . . . . 91 Changes in the Available Lysine Content. . . . . 98 Comparison of HPLC and Spectrophotometric Methods in the Determination of Available Lysine Lysine . . . . . . . . . . . . .106 Changes in Soluble Protein . . . . . . . . . . .109 Changes in pH. . . . . . . . . . . . . . . . .117 Ascorbic Acid. . . . . . . . . . . . . . . . . .122 Changes in Thiamine Content. . . . .132 Comparison of Processing and Storage Stabil- ities of Thiamine and Riboflavin . . .139 Some Physical Problems Observed in the COWpea Powders. . . . .139 Relationship Between the Various Quality Para- meters of Stored Cowpea Powders. . . .141 1. Riboflavin Content of Stored Powders as Affected by Lipid Oxidation. . . . .141 2. Effect of Lipid Oxidation on the Lipid Free-Amino Group. . . . . . . . .154 3. Relationship Between Lipid Free-Amino Groups and Browning . . . . . .158 4. Relationship Between Lipid Free Amino Groups and pH . . . . . .164 5. Relationship Between the Available Lysine Content and the pH of Stored Powder. . . . . . . . . . .167 6. Relationship Between the Reducing Sugar and the Available Lysine Con- tents of the Cowpea Powder in Storage .173 Relationship Between the Available Lysine and Solubl Contents. . . . . Relationship Betw and Browning. SUMMARY AND CONCLUSIONS. e Protein een Lipid Oxidation . . 182 SUGGESTIONS FOR FURTHER RESEARCH . BIBLIOGRAPHY . vi 177 . 189 197 200 TABLE PAGE 1. Proximate Analysis of COWpeas. . . . . . . . . 60 2. Effect of Water Activity and Temperature on the Absorbance at 233 hm of Lipid Derived from Raw COWpea Powder . . . . . . . . . . . . 65 3. Estimated Induction Periods (Months) of Stored Cowpea Powders. . . . . . . . . . . . . 68 4. Calculated Rates of Lipid Oxidation in Stored Cowpea Powders. . . . . . . . . . . . 71 5. Fatty Acid Analysis of Lipids Derived from Original Raw and Drum-Dried COWpea Powders . . 71 6. Changes in the S/Uoratios of COWpea Powder Lipids Stored at 25 C . . . . . . . . . . . 73 7. Changes in the S/U Ratios of COWpea Powder Lipids Stored at Aw 0.75 . . . . . . . . . . . 74 8. Calculated Rates of Loss of Percentage Un- saturated Fatty Acids of Stored Cowpea Powder Lipids Over A Six Month Period . . . . . . . . 77 9. Effects of Water Activity and Temperature on the Lipid Free- Amino Group Content (ug/g sample) of Raw Cowpea Powder During Storage. . . . .81 10. Calculated Rates of Loss of Lipid Free-Amino Groups 0 O O D O O O I O O O I I O O O I O O O 83 11. Effects of Water Activity and Tem erature on the Browning Intensity ("L" value? of Raw CowPea Powder During Storage . . . . . . . . . 87 12. Calculated Rates of’Loss of Whiteness (Increase in Browning) in Stored Cowpea Powders. . . . . . . . . . . . . . . . . . . . 89 13. Effects of Water Activity and Temperature on LIST OF TABLES the Percentage Reducing Sugar Content of Raw COWpea Powder During Storage . . . . . . . . . 94 vii TABLE PAGE 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Calculated' Rates of Loss of Reducing Sugar in Stored COWpea Powders. . . . . . . . 97 Effects of Water Activity and Temperature on the Available Lysine Content of Raw COWpea Powder During Storage( (g/lOOg powder) . . .101 Calculated‘ Rates of Loss of Available Lysine (g/lOO of sample) in Stored COWpea Powders. .105 Comparison of Spectrophotometric and HPLC Methods in the Determination of Available Lysine (g/lOOg powder). . . . . . . . . . . .108 Effects of Water Activity and Tem erature ‘ on the Soluble Protein Content (%§ of Raw Cowpea Powder During Storage. . . . . . . . .112 Calculated Rates of Loss of Soluble Protein in Stored Cowpea Powders . . . . . . . . .116 Effects of Water Activity and Temperature on the pH' of Raw COWpea Powder During Storage .120 Effects of Water Activity and Temperature on the Riboflavin Content (mg/100g sample) of Raw Cowpea Powder During Storage. . . . . . .127 'Percentage Losses of Riboflavin in Stored COWpea Powders. . . . . . . . . . . . . . . .129 Effects of Water Activity and Temperature on the Thiamine Content (mg/100g sample) of Raw Cowpea Powders During Storage . . . . . . . .135 ‘Percentage Losses of Thiamine in Stored C6wpea Powders. . . . . . . . . . . . . . . .136 Statistical Analyses of Data. . . . . . . . .142 Correlation Coefficients Between the Various Quality Parameters. . . . . . . . . . . . . .144 Base Values for Raw and Drum-Dried Samples .195 viii LIST OF FIGURES FIGURE PAGE l. 10. ll. 12. Effect of A on Stored Lipid Absorbance at 233 nm in Drum-Dried Cowpea Powders During Storage. . . . . . . . . . . . . . . . 63 Effect of Temperature on Lipid Absorbance at 233 nm in Drum-Dried Cowpea Powders During Storage. . . . . . . . . . . . . . . . . 64 Effect ofAw on.the Lipid Free-Amino Group Content of‘ Drum-Dried CQWpea Powders During Storage 0 o o o o o c c o o o o o o o o o c o o 79 Effect of Temperature on the Lipid Free-Amino Group Content of Drum-Dried COWpea Powders During Storage. . . . . . . . . . . . . . . . . 80 Water Activity.Effect on the "L" Values of Drum-Dried Cowpea Powders During Storage. . . . 85 Temperature Effect on the "L" Values of Drum- Dried Cowpea Powders During Storage . . . . . . 86 Effect of A2 on the Reducing Sugar Content of Drum-Dried Cowpea Powders During Storage, , , 92 Effect of Temperature on the Reducing Sugar Content of Drum-Dried COWpea Powders During Storage . . . . . . . . . . . . . . . . . . . . 93 Water Activity Effect on the Available Lysine Content of Drum-Dried COWpea Powders During Storage . . . . . . . . . . . . . . . . . . . . 99 Temperature Effect on the Available Lysine Content of Drum-Dried COWpea Powders During Storage 0 I I O O O O C O O O O O O O O - O O O .100 Effect of Water Activity on the Soluble Pro- tein Content of Drum-Dried Cowpea Powders During Storage. . . . . . . . . . . . . . .110 Effect of Temperature on the Soluble Protein Content of Drum—Dried Cowpea Powders During Storage 0 O O I O I O O O O I O O I O O O O O 0 111 ix "I FIGURE PAGE 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Effect of Water Activity on the pH of Drum-Dried Cowpea Powders During Storage. . . . . . . . . . . . . . . . . . . 118 Effect of Temperature on the pH of Drum- Dried COWpea Powders During Storage. . . . . 119 Effects of Water Activity on the Ribo- flavin Content of Drum-Dried COWpea Powders During Storage . . . . . . . . . . . . . . . 125 Effect of Temperature on the Riboflavin Content of Drum-Dried COWpea Powders During Storage . . . . . . . . . . . . . . . 126 Effect of Water Activity on the Thiamine Content of Drum-Dried Cowpea Powders During Storage. I O O O O I O O O O O O I I O O O O 133 Effect of Temperature on the Thiamine Con- tent of Drum-Dried COWpea Powders During Storage. . . . . . . . . . . . . . . . . . 134 Effect of Lipid Oxidation on the Riboflavin Content of Drum Dried Cowpea Powder Stored for Six Months at 25°C. . . . . . . . . . . 147 Effect of Lipid Oxidation on the Riboflavin Content of Drum-Dried COWpea Powder Stored for Six Months at Aw 0.75. . . . . . . . . . 148 Effect of lipid oxidation on the Riboflavin Content of Raw Cowpea Powder Stored for Six months at 25 C O O O O O O O O I l I O I O 149 Effect of Lipid Oxidation on the Riboflavin Content of Raw Cowpea Powder Stored for Six Months at Aw 0.75. . . . . . . . . . . . . . 150 Effect of Lipid Oxidation on the Lipid Freed Amino Group Content of Drum-DriedoCowpea Pow- der Stored for Six Months at 25 C. - - - - 155 Effect of Lipid Oxidation on the Lipid Free- Amino Group Content of Raw COWpea Powder Stored for Six Months at 25° . . . . . . . 156 Effect of Browning on the Lipid Free—Amino Group Content of Drum-DriedoCOWpea Powder Stored for Six Months at 25 C . . . . . . . 159 X FIGURES PAGE 26. Effect of Browning on the Lipid Free-Amino Group Content of Drum-Dried Cowpea Powder Stored for Six Months at Aw 0.75. . . . . . . 160 27. Effect of Browning on the Lipid Free-Amino Group Content of RawOCowpea Powder Stored for Six Months at 25 C . . . . . . . . . . . 161 28. Effect of Browning on the Lipid Free—Amino Group Content of Raw Cowpea Powder Stored for Six Months at Aw 0.75 . . . . . . . . . . . . 162 29. Relationship Between the pH and the Lipid Free-Amino Group Content of Drum-DriedOCow- pea Powder Stored for Six Months at 25 C . . 165 30. Relationship Between the pH and the Lipid Free-Amino Group Content of RaW'Cowpea Powders Stored for six Months at 250C - . . . 166 31. Relationship Between Available Lysine Content and pH of Drum-Dgied Cowpea Powders Stored for Six Months at 25 C . . . . . . . . . . . . . 168 32. Relationship Between Available Lysine Content and pH of Drum-Dried Powder Stored for Six Months at Aw 0075 o o o o o o o o o o o o o I 169 33. Relationship Between Available Lysine Content and pH of Raw Cowpea Powders Stored for Six months at 250 C o o o o o o o o o o o o o o o 170 34. Relationship Between Available Lysine and pH of Raw Cowpea Powder Stored for Six Months at Aw 0.75. . . . . . . . . . . . . . . . . . 171 35. Relationship Between Available Lysine and Reducing Sugar Contents of Drum-Dried Cowpea Powder Stored for Six Months at 25° C . . . . 174 36. Relationship Between Available Lysine and Reducing Sugar Contents of aw Cowpea Powder Stored for Six Months at 25 C. . . . . . . . 175 37. Relationship Between the Available Lysine and Soluble Protein Contents of Drum-Dried COWpea Powders Stored for Six Months at 250C . . . . 178 38. Relationship Between the Available Lysine and Soluble Protein Contents of Drum-Dried COWpea Powders Stored for Six Months at Aw 0.75. . . 179 xi FIGURES 39. 40. 41. 42. 43. an. Relationship Between the Available Lysine and Soluble Protein Contents of Raw COWpea Powders Stored for Six Months at 25 C. Relationship Between the Available Lysine and Soluble Protein Contents of Raw COWpea Powders Stored for Six Months at Aw 0.75 Relationship Between Diene Conjugation of Lipid and the Browning Intensity of Drum- Dried Cowpea Powders Stored for Six Months at 250 C Relationship Between Diene Conjugation of Lipid and the Browning Intensity of Drum- Dried COWpea Powder Stored for Six Months at Aw 0.75 Relationship Between Diene Conjugation of Lipid and the Browning Intensity of Raw Cowpea Powder Stored for Six Months at 25 C. Relationship Between Diene Conjugation of Lipid and the Browning Intensity of Raw Cowpea Powder Stored for Six Months at 0.75. xii PAGE 180 181 183 184 185 186 INTRODUCTION Cooked dry bean, pea and lentil powders were de- veloped by Morris (1961) in response to requests from growers and processors for a dried product in conven- ience form. These powders are adaptable for use in a variety of dry mixes, such as soup, bean leaf or patty, and dips for crackers. All of the new products have excellent quality immediately after processing and eval- uations have indicated that they have a satisfactory shelf—life when appropriately packaged (Boggs, 23 a1. 1964). Powders prepared from legume seeds may represent just another convenience food, but it has another po- tential which might even be more significant. It is in- creasingly being realized that the world's protein sup- ply is becoming more and more critical especially in view of the ever increasing world population. In the so-called developing countries of the world where, in fact, populations are rising faster than agricultural out- put and protein-food production, the protein problem is particularly serious. When compared to other plant sources, the legumes as a group are high in protein. This, com- bined with the fact that they are consumed by large sections of people in the third world countries, have 1 2 increased speculations and raised hopes that one possible way to meet the world's protein problem is by supplying cheap, stable protein foods from legumes. A 1966 esti- mate by Altschul puts the annual protein production from legumes at 8.5M tons, while according to Bressani gt g1. (1963), about 20-30% of the protein intake of the econ- omically poor in Latin America comes from legumes. Cowpea (Vigna unguiculata) is an example of a legume with high protein content. Analysis by Bressani gt a1. (1961) puts the value at about 24%. Another analy- sis by Oyenuga (1968) shows the protein to be well bal- anced with respect to the essential amino-acids. It is high in lysine but rather deficient in methionine, as is true of most other legumes (Silbernagel, 1971). Cowpea is a legume crOp which grows prolifically in the warm and humid tropical conditions of Nigeria. It is consumed by a large section of the population-—rich and poor-—in the form of various indigenous Nigerian dishes such as "moin-moin" and "akara". As a member of the so-called developing countries of the world, Nigeria stands to gain by increased utilization of the legume in meeting the protein needs of its population. It is also true, however, that growing incomes and urbanization have created a real potential for convenience foods prepared from the cowpeas. Such convenience foods should grow in importance in a country like Nigeria where eXpectations are running higher everyday while they Should also be 3 readily marketable in a country like the U.S.A. where expectations have always been high. With that in mind, the objective of this work was to prepare COWpea powders by drum-drying and simple milling operations and to study physico-chemical changes in these powders during a six month storage period at water acti- vity of 0.11, 0.33 and 0.75 at a constant temperature of 250 C (room temperature) and at temperatures of 50 C, 25° C and 400 C with the water activity (AW) held con- stant at 0.75. The Aw's of 0.11, 0.33 and 0.75 were chosen to reflect the conditions which might exist in foods stored in the three main climatic belts of Nigeria. These are 0.11 for the Northern Belt, 0.33 for the Middle Belt, and 0.75 for the Southern Belt. Incidentally, the Aw's of 0.11, 0.33 and 0.75 also represent, generally, the three main zones of Aw described by Labuza (1971) at which dif- ferent reactions occur at varying rates in dehydrated foods. 50 C was chosen to represent refrigeration temper- ature, 250 C for ambient temperature and 400 C to repre- sent accelerated storage condition. Moreover, there are some sections of Nigeria (Northern) where 40° C temper- atures occur at certain times of the year. . Drum-drying, rather than freeze-drying or spray drying, was chosen as the dehydration technique purely for reasons of cost. It was thought that the powders 4 should be affordable by the average consumer by lowering production cost which usually translates to lower prices. This would be important particularly in a country like Nigeria. The physico-chemical studies undertaken in the research included: lipid oxidation as measured by changes in diene conjugation and changes in the fatty-acid con- tent, reducing sugar, soluble protein, available lysine, color, pH, and ascorbic acid, riboflavin and thiamine content; These parameters were measured on a monthly .basis over a six month storage period. To obtain a base value against which storage changes were assessed, all the above measurements were made on the fresh products immediately after processing. Observations were also made from time to time to monitor certain physical characteristics while the effects of the type of processing were examined by comparing the ' drum-dried samples with the raw samples. It was hoped that at the end of the studies, it would be possible to arrive at a picture of the type of changes one can eXpect in dried COWpea products and, on the basis of this, be able to recommend optimum storage conditions for them. LITERATURE REVIEW Cowpeas (Vigna unguiculata) have always constituted an important part of the diets of the peoples of West Africa. Lewicki (1974) noted that even as early as the middle ages COWpeas were very important in the foods of the West African peoples. Oyenuga (1968) has described vividly the importance of this legume crop in the diets of modern-day Nigerians. Cowpeas, belong to the family Leguminosae which consists of three subfamilies, Caesalpiniaceae, Papilion- aceae and Mimosaceae. Vigna unguiculata is a warmth- loving herbaceous annual sensitive to cold and severe frost. Chemical analysis by Oyenuga (1968) of cowpeas grown in Nigeria revealed that: a. The seeds are high in protein and soluble carbohydrate, low in crude fiber and oil, and contain a fair amount of minerals. b. They contain all the essential amino-acids in amounts sufficient for growth. c. The amino-acid pattern of cowpeas is similar to that of whole hen's eggs and is of high biological value. 6 d. Cowpeas contain fair amounts of thiamine, niacin and riboflavin and some carotene but are deficient in ascorbic acid. e. Like many legumes, the seeds are rich in phosphorus with only fair amounts of calcium and iron. f. The major and trace elements are well dis- tributed in the cOWpea seed. g. They contain only about 2:5 percent fat with 52 percent of the fatty acids being unsaturated while some 40 percent occurs as the saturated palmitic and stearic acids. Such composition in combination with a high degree of acceptability have made cowpeas highly desirable as ingredients in soups, gravies, stews and other food prod- ucts such as "moin-moin" and "akara".which are very popu- lar in Nigeria. The legumes as a group represent a valueable source of protein for the ever-increasing pOpulation of the world especially in the so-called developing countries of the world. According to Altschul (1966) more than 8.5M tons of protein are obtained from legumes annually. Another estimate by Bressani gt g1. (1963) showed that about 20-30% of the protein intake of the poor in Latin America countries is derived from legumes. However, despite the well documented high protein content of legumes, their nutritional value is limited by their low content of the sulphur amino-acids methionine 7 and cystine. Working with navy beans, Silbernagel (1971) reported that although the average protein content of this legume was about 25%, its real nutritional value was limited to about 8%. He attributed this to the low content of the sulphur amino-acids and suggested supple- mentation with these amino-acids to improve their nutri- tive quality. Another reason why the nutritive value of a legume might be lower than expected would be its content of anti- nutritional factors such as hemagglutinins, saponins and antitrypsin. Ricin, a hemagglutinin was the first toxic plant protein to be isolated (Liener, 1966). It is known that relatively mild heat treatment is sufficient to in- activate these antinutritional factors. Kakade and Evans (1963) destroyed the antitrypsin factors in navy beans by autoclaving for 5 minutes at a temperature of 1210 0. Processing of Legumes One p0pular processing method for cowpeas in Nigeria involves, among other things, cooking the whole legume ’seeds in water, the aim being to detoxify the product, soften the seeds and eliminate the coat. According to Bressani and Elias (1974), a minimum of two hours is re- quired for cooking soaked dry beans at atmospheric press- ure While Dovlo gt g1. (1975) noted that the cooking time varies with the variety of cowpea used, with cooking time 8 at atmospheric pressure ranging from about 1% hours to about 2% hours. Wet and dry methods are available for the dehulling of legume seeds if dehulling prior to cooking is desired. A wet method of dehulling involves soaking the seeds over- night followed by gentle rubbing to separate the seed coats from the cotyledons. Because the seed coats have lower density than the cotyledons, they rise to the sur- face and float when water is added. A dry method de- scribed by Dovlo (1975) involves pounding the dry beans with suitable equipment such as a mortar and pestle fol— lowed by winnowing to remove the seed coats. Attempts have been made at applying intermediate technology to the processing of legumes in Nigeria. Siegel and Fawcett (1976) in an investigative work with three de- hulling units-—an attrition-type mill, a barley pearler and a grain thresher-—have found that the George 0. Hill thresher was effective in mechanically dehulling brown cowpeas. Earlier,Reichert gi‘gl. (1974) had reported that flour obtained from such dehulled cowpea seeds were as good as those dehulled by traditional methods of pounding and winnowing. The processes of soaking and dehulling COWpea seeds have both good and bad effects. Small losses of important nutrients are incurred with the dehulling process while water soluble components may be lost as a result of soaking. 9 For example, Ogunmodede (1972) has reported that the soaking process leads to losses of thiamine and niacin. 0n the other hand, Kakade and Evans (1966) have reported that soaking Phaseolus vulgarig for four days led to a 75% reduction in the activity of hemagglutinin and a 28% reduction in the activity of the trypsin inhibitor. Again, dehulling may also result in improved digestibility of the bean. Because of the long cooking times required for legume seeds, several processes aimed at overcoming this problem have been described in the literature. One such process described by Rockland and Metzler (1967) for lima beans involves soaking the beans in solutions of inorganic salts for about an hour during which the beans are given a vacuum treatment by the Hydravac Process. The beans are then allowed to soak in the salt solution for another six hours after which they are rinsed and dried. The process was thought to yield a quick cooking legume product, first by loosening the seed coats from the cotyledons such that the latter is able to imbibe the sale solution. Once im- bibed, the cotyledon proteins are dispersed and solubilized by the hydrating medium which is comprised of 2.5% sodium chloride, 1.0% sodium tripolyphosphate, 0.75% sodium bi- carbonate and 0.25% sodium carbonate. According to Rock- land and Metzler (1967), the polyphosphates act as metal chelating agents which aid in breaking metal salt-protein bonds. In a later report by Rockland (1974), it was 10 stated that it is possible to reduce the cooking time of dry beans by as much as 80%. Steinkraus gt g1. (1964) have also described a process for the preparation of quick cooking dry beans. Essentially, the method involved soaking the beans in water for about 15 minutes, cooking with steam, dipping in a 20% sucrose solution at 1600 F and finally dehy- drating. Recently, the processing of legumes has taken a new dimension with the introduction of spray and drum- drying techniques. These methods produce instant legume powders or ready-to-eat products which are projected to gain a wider share of the market for legumes in view of the reported decline in the demand for canned whole beans. Morris (1961) was the first to introduce the concept of instant legume powders while Bakker-Arkema gt g1. (1966), developed the idea of spray drying cooked legume powders. Later, in 1967, they also applied the drum-dehydration technique in preparing legume powders. In evaluating both products, Bakker-Arkema gt g1. (1967) pointed out that the drum-dried products had a lower free starch index which translated to better rehydration properties than the spray dried products. Moreover, Bakker-Arkema gt g1. (1967) also indicated that the drum-drying process saved about 2500 BTU per pound of beans when compared to the spray drying tech- nique. The elimination of the pureeing process in the drum-drying operation accounted for the saving in energy. 11 More recently.other workers have applied the drum- drying technique to the production of ready—to-eat legume products. Onayemi and Potter (1976) and Kon gt gt. (1974) have prepared COWpea powders by using the drum-drier. Storage of Legumes Many chemical and physical changes occur in both fresh and dry legume seeds when they are stored. Worthington and Burns (1971) noted that, when fresh cowpea seeds were stored at 1100 F for 12 hours, the firmness of the beans increased significantly. Hoover (1955) indicated that COWpeas are a good source of ascorbic acid only if they are harvested early and are utilized soon after harvest or stored at about 400 F. Temperatures appreciably higher than 400 F or long storage time had the effect of decreasing the ascorbic acid content of fresh COWpeas. According to Burr (1973), prolonged storage of dry beans may lead to undesirable changes in their cooking characteristics which are dependent on the moisture con- tent, time and temperature of storage. In a study by Dexter gt g1. (1955), holding dry beans at temperatures below 38° 0 decreased the water holding capacities of the beans. Bean discoloration and rancidity were associated with high moisture content. _Working with varieties of Phaseolus vulgartg, Morris and Wood (1956) reported that samples with the 12 highest moisture contents also suffered the greatest qual- ity changes as judged by lipid acid values, texture and color. Similarly, Burr gt gt. (1968) observed that high moisture contents were associated with losses in bean quality as measured by the cooking time. Moisture con- tents higher than 10% were observed to increase the cook- ing time of beans. In his work on the quick cooking characteristics of dry lima beans, Rockland (1966) reported increases in cooking times after 3 months storage at 38 and 700 F. Flavor, color and odor problems were also reported. Obviously, the nutritional quality of legumes change during storage. When Mitchell and Beadles (1949) stored soybeans at room temperature, the nitrogen digesti- bility of the beans was reported to decrease from about 85% to about 75%. The corresponding loss in the nutritive value of the protein was about 28%. Supplementation with methionine helped increase the biological value of the beans. Bressani gt_gt. (1961) hinted that the limiting amino-acid in beans is methionine. However, in studies by Molina gt gt. (1975), the methionine and lysine contents of raw beans were reported to increase during storage sug- gesting an increase in the protein quality of the beans during storage. Results from protein.eflficiency ratio (PER) measurements did not indicate this. Earlier, Evans gt gt. (1974) had reported that the PER values correlated well 13 with methionine and lysine availability. It was there- fore reasoned from the results of Molina gt gt. (1975), that processing and storage may adversely affect the availability of lysine and the sulfur amino acids. Indeed, when Ben-Gera and Zimmerman (1964) stored raw defatted soy- bean flakes of 9.1% moisture content at 30° c for 12 weeks, the available lysine content decreased by 18%. This agreed with the later findings of Yannai and Zimmerman (1970) in which the available lysine content of stored soybean was reported to decrease. According to Harrington (1963), dry legume seeds may also lose their ability to germinate if stored under conditions of high relative humidity. Increased respir- ation rates and loss of catalase and phosphatase acti- vities were reported by Morris and Wood (1956) to occur in stored dry legume seeds with high moisture contents. The vitamin content of legume seeds is also af- fected by storage conditions. Although Chitre gt gt. (1955) did not find statistically significant losses of niacin and thiamine in pulses stored for six mohths, Dawson gt gt. (1952) reported that pea beans, stored at 24° C and at relative humidities ranging from 67 to 95%, lost about 20% of thiamine, even at low moisture content. Microbial attack is an important occurence in stored legumes. Heavy attack by Cladosporium of stored dry beans was reported by Swanson gt gt. (1970). Similar- ly, when Watson and Morris (1954) stored beans at a high 14 relative humidity of 98%, mold growth was reported after six weeks of storage. Christenson and Kaufman (1969) have hinted that if the temperature of storage is too high and if the moisture content of stored legume seeds was high enough, storage fungi could attack the product to cause mustiness, germ damage, caking and bin-burning. With the advent of preprocessed legume powders, some work has been reported on the storage stability of these products. The work by Burr 23.§l- (1969) with dry instant legume powders showed that a moisture content of 4%, a temperature of 22° C and a nitrogen atmosphere were effective in maintaining product quality as judged by lipid oxidation and taste panel assessments. They were also able to demonstrate the effectiveness of the anti- oxidant butylatedhydroxytoluene in extending the shelf life of these products. Counter (1969) also reported that a nitrogen atmosphere could be used to extent the shelf-life of instant navy bean powder. Storgge and the Protein ggality of Foods The concept of food protein quality is so important that many works have been published on this topic. Hodson and Miller (1957) reported marked browning in stored milk proteins and that this browning was accom- panied by significant loss of lysine. In milk casein samples stored in air, Hodson and Kruger (1947) reported losses of some amino acids particularly lysine, methionine, 15 arginine and histidine. Nitrogen atmosphere storage helped to reduce these losses. Stevens and McGinnis (1947) also observed decreased lysine availability in studies with stored milk protein. Jones and Gersdorff (1941) indicated that the protein quality factors adversely affected by increasing length of storage of stored wheat flour were digesti- bility, solubility and true protein content. The report of Mitchell and Beadles (1949) also noted that decreased in-vitro protein digestibility associated with decreased protein solubility occurred in stored wheat and corn. Schultz and Thomas (1949) mentioned lysine losses in stored corn. Attempts have been made by various authors to ex- plain the loss of protein quality in stored foods. Ac- cording to Melnick and Oser (1949), the loss of protein quality in stored foods may occur as a result of the formation of new and enzymatically resistant peptide link- ages. They pointed out that the direct destruction of es- sential amino-acids of the proteins was not as important as the formation of these new and resistant linkages. This agrees with the reasoning of Mitchell and Block (1946) who stated that the formation of unusual cross-linkages be- tween proteins and.or peptides may lead to reduced pro- tein digestibility. According to Seegers and Matill (1935), enzyme action on these unusual linkages produce unusual peptides which resist absorption and fecal elimination and 16 which are neither absorbed nor excreted in the urine. Sim- ilarly, Henry and Kon (1948) have stated that browning, due to the long term storage of a food, may act to alter the digestibility of the food protein. Clark and Tannenbaum (1970) did isolate products of the Maillard reaction which have been described by Henry and Kon (1948) to be resis- tant to enzymatic action. In their work, McInroy gt gt. (1945) were able to correlate browning with the formation of enzyme and even acid resistant linkages which corres- pondingly led to decreased protein value. The report of Hill and Patton (1947) showed that apart from lysine, the amount of other essential amino acids of a protein may also be affected by storage browning. The indole group of tryptophan and the imidazole group of histidine, for example, may be involved in these reactions. As shown clearly by Ben-Gera and Zimmerman (1964), storage or heat browning lead to loss of available lysine by a blocking of its epsilon amino group by reaction with reducing sugars or by reaction with the free carboxyl groups of aspartic and glutamic acid. Lipid Content and anlity of Stored Protein in Foods There are many reports which have correlated the protein quality of stored foods with their degrees of lipid oxidation, or with other reactions. Ponting, gt gt. (1964) mentioned lipid oxidation as contributing to the protin quality of stored vegetable l7 foods by reaction between the products of lipid oxidation which supply carbonyl functions and proteins which supply amino functions in the non-enzymatic browning process. Similarly, according to Carpenter gt gt. (1962) in their work with stored fish meal, secondary products of lipid oxidation were able to react with the epsilon amino group of lysine in the non-enzymatic browning process to cause deterioration of the fish meaL protein. By forming resistant complexes with leaf protein concentrates, the products of lipid oxidation helped re— duce the enzyme digestibility of the stored leaf protein (Buchanan, 1969). The greater the storage time and the moisture content of the leaf protein concentrates, the greater was the reported loss in digestibility. When Lea gt gt. (1960) stored herring meal for a year at 20° C and with a moisture content of about 6%, lipid oxidation was demonstrated to lower the nutritive value of the product by damaging the protein constituent of the stored herring meal. Work by Olley and Duncan (1965) with frozen stored fish muscle was able to link the degree of fatty-acid re- lease from these samples with lower protein quality accru- ing from a denaturation phenomenon. According to Koch (1962), lipid oxidation during the freeze-drying of foods causes changes in the rehydra- tion properties, texture and flavor, and thereby limits the shelf-life of these foods, because of the interaction between the products of lipid oxidation and proteins. 18 Solubility changes and the loss of protein amino- acids were reported by Roubal and Tappel (1966a) to be the result of enzyme and protein damage from peroxidizing lipids resulting in polymerization of proteins with poly- unsaturated lipids as oxidation progresses. Radical at- tack rather than aldehyde attack, on protein is mainly re- sponsible for protein damage in a lipid-protein system. (Roubal and Tappel, 1966b). The exact mechanism of the protein-lipid inter- actions in food systems which lead to the deterioration of stored foods have not yet been fully established. Pos- sibly, the binding of lipids to proteins or vice versa may depend on such factors as configuration, multiple attach- ment sites, and/or the matching of polarity between op- posing groups in much the same manner as for the combi- nation of enzyme and substrate. Chapman (1969) referred to metal ions and electro- static and hydrOphobic binding as being important in the interaction between lipids and proteins. In consonance with this, Gurd (1960) suggested that the interactions might be between similar types of functional groups in the two classes of compounds such as between non-polar hydrOphobic residues of the fatty-acid moieties of lipids and the similar residue of certain of the side chain groups of proteins; while other interactions may involve polar or charged groups. Primary covalent linkages such as ester bonds were regarded as being of little importance. 19 In their studies on a gelatin—linoleate system in the dry state, Zirlin and Karel (1960) observed that gel- atin-lipid reactions lead to the scission of protein coup- led with cross-linking. Narayan and Kummerow (1958) have referred to complex formation in the presence of oxidized lipids; with the reaction being environment dependent and different proteins responding differently. Complex forma- tion was attributed to secondary bonds such as hydrogen bonding. The isolation of fluroescent compounds with -C=N- functional groups from an oxidizing system consisting of methyl linoleate and coho salmon myosin and the observa- tion that the epsilon amino groups of myosin were destroyed led Braddock and Dugan (1973) to speculate a cross linking reaction between amino groups and the products of fatty- acid oxidation. Lysine, methionine and histidine were found to be most susceptible to such destruction. Working with carbonyl compunds, Schwenke (1975) stated that blocking of the L-epsilon-amino groups alters the iso-electric point, electrophoretic prOperties, solu- bility and precipitation characteristics of proteins. Ac- cordingly, he speculated that radicals from oxidizing lipids play a role in protein destruction with the possi- bility that carbonyl-amino reactions, as in.Maillard browning, form Schiff bases and secondary products. Jarenback and Liljemark (1975) reported that lino— leic acid hydrOperoxides were much more effective than 20 linoleic acid in reducing the amount of protein in a K01- extract. They therefore suggested more extensive binding between the linoleic acid hydroperoxides and protein than between the linoleic acid and protein. Crawford gt,gt. (1967) have implicated malonalde- hyde in protein destruction in food systems. They stated that malonaldehyde's reactivity is enhanced by resonance stabilization. As such they react, with free amino-acids in proteins by nucleOphilic attack ttg an SN-2 mechanism to produce enamines; the epsilon-NH2 group of lysine and the N—terminal,% -NH2 groups of aspartic acid being in- volved in these reactions. Similarly, Ohio and Tappel (1969) observed that malonaldehyde produced from oxid- izing lipids reacted with enzymes to produce yellow fluore- scent compounds with a consequent loss of enzyme activity. In addition, -SH groups of the proteins, tyrosine, lysine and histidine were destroyed by the malonaldehyde. Then, work by Fletcher and Tappel (1971) using fatty-acids bound to horse serum albumin showed too that polyunsaturated fatty-acids undergoing oxidation reacted with amino-acids to yield Schiff base compounds which fluoresced. 8 Ganage and Matsushita (1973) have, however, hinted that both radical and non-radical products of oxidizing lipids can polymerize with proteins. This accounts for the observation by Roubal (1971) that free radical attack on food proteins rather than aldehyde reactions were mainly responsible for amino-acid losses. 21 Phospholipids are apparently one type of lipid that are involved in these protein destroying reactions. Ac- cording to Bidlack and Tappel (1973a), fluorescent mater- ials were produced in systems composed of microsomal mem- branes and oxidizing lipids. In the course of time, the amounts of extractable phosphatidyl ethanolamine decreased as was the number of reactive amino groups in the phospha— tidyl ethanolamine. Obviously, carbonyl compounds from oxidizing lipids, including malonaldehyde were involved in Schiff base formation reaction with the phospholipid free amine. A similar explanation was advanced for the results of Dillard and Tappel (1973) in which fluorescent products with an excitation at 360 nm and emission at 430-460 nm also led to lower phospholipid free amine. Another work by Reiss and Tappel (1973) using a system composed of deoxyribonucleic acid and polyunsaturated fatty-acid indicated the production of such fluorescent compounds. Again, Bidlack and Tappel (1973b) showed that during the oxidation of phOSphatidyl serine and phospha- tidyl ethanolamine, fluorescent compounds were produced and that the fluorescent intensity increased with in- creasing peroxidation. Along the same lines, Corliss and Dugan (1970) indicated a link between phosphatidyl ethanolamine oxidation and browning. It would appear that the destructive effects to proteins in some protein—lipid food systems is matched by a stabilizing effect on the lipid component of the system. 22 Bishov and Henick (1972) reported an antioxidant effect of autolyzed yeast protein (AYP) and hydrolyzed vegetable protein (HVP) in a freeze-dried model system of lipid, carboxymethyl cellulose, the protein and distilled water. Earlier work by Bishov gt gt. (1967) had indicated im- proved fat stability in dehydrated proteinaceous food mixes of chicken flavored soup and gravy mix. The univer- sality of synergism between protein hydrolyzate and pheno- lic antioxidants in stabilizing lipids was also demon- strated by Bishov and Henick (1975). The ability of protein to impart stability in such a system was also found to vary with the type of protein, method of preparation, pH, and eXperimental condition. For example, in their work reported above, differences in the stabilizing effect between the AYP and HVP were attributed to the milder conditions of autolysis in com- parison to those of acid induced hydrolysis, which gave better stabilizing potential to the former. HVP is ob- tained by hydrolyzing soy protein with an acid followed by alkaline neutralization. The salt which may be thus formed is also thought to lower its antioxidant effective- ness. AYP, on the other hand, is obtained with minimum heat treatment since the yeast cells are self-digesting. In consonance with the above, Hayes gt gt. (1977) has hinted, with respect to soy flour antioxidant, that there is no clear cut, generalized relationship between concentration of added soy flour and the degree of 23 stability obtained. They added that the nature of other product ingredients, the character of the fat or oil in- volved and whether the soy flour is full fat or defatted all probably complicate the outcome. The antioxidant activity of soy flour has been as- cribed to its natural components: such as isoflavones which Walz (1931) isolated. Amino acids and peptides are others. Soybeans normally contain small quantities of peptides and amino acids which are present as a result of incomplete protein synthesis or possibly because of some protein degradation (Smith and Circle, 1972a). Another class of compounds associated with the antioxi- dant activity of soy flour has been the phospholipids and Smith and Circle (1972a) have indicated that only part of the phospholipids are removed in commercial ex- traction of soybean flakes. In their studies with methyl linoleate in freeze- dried model systems, Karel gt gt. (1966) found that cer- tain amino acids including histidine, d.-amino-butyric acid, lysine and cysteine had substantial antioxidant activity and that the nature of this activity was differ- ent from that observed with propyl gallate since the main effect of the amino compounds was to prolong the induction period and to affect the initial rate of oxidation. No effect was present in the more rapid, bimolecular phase of oxidation, whereas prOpyl gallate had an inhibiting effect in the latter stage. 24 As a follow-up to this, Tjho and Karel (1969) re- ported that histidine in low concentration has an anti- oxidant effect in the very early stages of oxidation fol- lowed by a slight pro—oxidant effect in the later stages. Working with herring oil, Marcuse (1960) observed that while histidine had an antioxidant effect, cysteine was pro-oxidant. Marcuse (1961) also reported on a study of the antioxidant effects of several amino acids added to aqueous solutions of linoleate at pH 7.5 and concluded that: 1. Histidine, alanine, methionine, and lysine re- duced oxygan absorption by linoleate by as much as 50-80%. 2. Each amino acid had an Optimum concentration for the antioxidant activity and at high concentrations showed an activity inversion; becoming pro-oxidant rather than antioxidant in its action. 3. The antioxidant activity depended on pH, the presence of other antioxidants or synergists, and the state of oxidation of linoleate. 4. Since antioxidant prOperties were shown in the absence of toc0pherol and other antioxidants, amino acids may act as primary antioxidants. Then in 1962, Marcuse observed that the anti~ oxidant effect is enhanced, and the pro-oxidant effect is lowered by addition of phosphate. Working on the stability of amine salts of lino- leic acid, Dorlores and Lopieks (1971) suggested that 25 the carboxylic acid group of the fatty acid form a salt with one of the amino groups of the basic amino acids, leaving the other amino group available to affect the- double bond area of the fatty acid. If the latter in- teraction is a charge-transfer complex, it is a very weak one involving only partial donation of'electrons and lit- tle bond distortion. It would appear that the protective effect is specifically associated with the solid state. The rigid crystalline lattice of lysinium lino- leate could act as a barrier to the diffusion of oxygen and oxidizer intermediates, and to the prOpagation of chain reaction. On the other hand, studies of the oxida- tion in solution by Koch gt gt. (1971) suggest that the salt complex also prolongs the induction period and re- tards the rate of autoxidation. Therefore, one might speculate that,initially the solid complex physically prevents oxygen from entering the reaction. After the oxygen succeeds in attacking the organic substrate, the oxidation would be terminated by a mechanism in which the basic amino acid acts as an oxygen scavenger or a free-radical chain terminator (Dorlores and Lopieks, 1971). Water Activity ggd the Quality of Stored Foods Water activity is defined by the ratio P/Po where P is the partial pressure of water in a sample and Po is 26 the vapor pressure of pure water at the same temperature (Ayerst, 1965). Water with full activity does not exist except in the pure state. The remaining types of water are present in biological matter and can best be visualized by con- sidering the changes in water activity that occur during a process of drying or freezing (also a drying process with the removed water deposited internally in the form of pure ice-crystals). This approach is greatly facili— tated by considering plots of water activity versus mois- ture content (Fennema, 1976). According to Rockland (1969), when foods are stored under conditions of differing rela- tive humidities they either take in or give up moisture to attain a moisture content which represents the equili- brum moisture content. It is this moisture content that is actually employed in the plots referred to by Fennema (1976) and which is known as a fmoisture sorption iso- therm". Water activity may indicate both the structure of the material and the manner in which the water is found (Heldman, 1972). The first water removed, Type III, has an activity slightly less than that of pure water. This type of water which represents the majority of water in plant and animal food tissues is easily removable and is readily available for growth of micro-organisms and chemi— cal reactions. As it is removed, the remaining water gradually assumes a lower activity. When all of the Type 27 III water has been removed, the moisture content is about 12-25% and the water activity is about 0.8 depending on the type of product and the temperature. Type II water is substantially more difficult to remove than Type III water, and removal of a given increment of Type 11 water results in a much greater reduction of the remaining water's activity than occurs when a like increment of Type 111 water is removed. Partial removal of Type II water eliminates the last possibility of microbial growth and greatly reduces most kinds of chemical reactions. Com- plete or near complete removal of Type 11 water 3-7% mois- ture level, depending on the product and temperature, cor- responds approximately to Optimum stability of dry pro- ducts that contain significant amounts of oxidizable lipids. Partial removal of Type I water can be accomplished by con- ventional dehydration but not by freezing. Type I water is very tightly bound and is what some authors refer to as "true bound water". The degree of binding is such that any reactions depending on solvation are so slow as to be unmeasurable (Fennema, 1976). Other authors have used different terms while agree- ing in their characterization of the three types of water described by Fennema above. Wolf gt gt. (1973) referred to Type I water as constituting the fmonolayer region? in which water is held on polar sites and bound with rela- tively high energy in a single layer distribution over the surfaces of the food. The region, according to them 28 involves water activity up to 0.3. In describing Type II water, Sitt (1958) indicated that it is water found in the 0.3-0.7 Aw region of the isotherm and that here water exists in multiple layers held by forces weaker than those in the monolayer, i.e. Van der Waal forces and similar forces. Brunauer gt gt. (1938) in prOpounding their multi- molecular adsorption theory described the Type III water as that occupying the zone above 0.7 Aw in the sorption isotherm. In this zone, water is thought to exist rela- tively free and macro capillary forces are at work here. Ayerst (1965) hinted that the stability of foods is related to their water activity. Since then, several authors have attempted to relate water activity to the stability of low moisture foods. The descriptions by Labuza (1968), Rockland (1969) and Labuza gt gt. (1970) tell the general story. In the monolayer region, one sees an acceleration of lipid phase reactions such as oxidative rancidity. According to Labuza (1971), the small amount of water that is needed to inhibit oxidative rancidity in the mono- layer region apparently functions by facilitating de- struction of free radicals, hydrogren bonding to hydro- peroxides and slowing the rate of their conversion to other products, and hydrating or reacting with metals and there- by reducing their abilities to catalyze oxidation. As the water content is increased above the optimum level into the multilayer region, the lipid oxidation increases, .29 presumably, because of increased catalyst mobility and because new catalyst surfaces are eXposed as the food matrix swells. Non-enzymatic browning is accelerated if Type II water is present since the reactants are water soluble. At high water activity, however, dilution effect sets in to lower the rate. Because of increased enzymatic activity in this region, enzymatic processes such as enzymatic browning would be expected to increase too. The region above 0.7 Aw in the sorption isotherm is marked by mold, yeast and bacterial activity. It is known that desorption isotherms and resorp- tion isotherms usually do not coincide, i.e. they exhibit hysteresis. At any given moisture content, the Aw during desorption is less than the Aw during resorption and at any given Aw the moisture content during desorption is greater than that during resorption. The cause is appar- ently related to interactions of nonaqueous constituents during desorption so that some adsorption sites are ir- reversibly lost, and differences in water vapor pressure needed to fill and empty capillaries of irregular shape (Rao, 1941). The lower moisture contents of samples dur- ing resorption results in their having greater viscosities than samples of the same Aw during desorption and one con- sequence, according to Labuza gt gt. (1972), is that oxi- dation at a given AW proceeds more slowly in resorption 30 samples. Wolf gt gt. (1972) have related the extent of hysteresis to the quality of foods in storage. Water activity may also be important in the qual- ity of stored foods by the type of interactions that can occur between water and proteins. In most proteins, about 40% of the total amino-acids have non-polar side chains, such as the methyl group of alanine, the benzyl group of phenylalanine, the iSOprOpyl group of valine, the mercap- tomethyl group of cysteine and the secondary butyl and isobutyl groups of leucines. There is good agreement that these non-polar groups have a structure-forming action on adjacent water and that the interaction between water and the non-polar groups has an important influence on the re- activity of the protein and on its native tertiary con- formation (Schackman, 1963). The mechanisms of interaction between water and non-polar groups, however, is a subject of disagreement. According to Kauzmann (1959) and Scherage gt gt. (1962) the mechanism involves hydrophobic association in which, after a protein is placed in an aqueous environment, an alteration in tertiary structure occurs so that hydro- phobic, non-polar groups can associate forming, in essence, an intramolecular micelle and thereby minimizing contact of these groups with water. The net effect is that a pro- tein with a large number of exposed non-polar groups would have the greatest likelihood of aggregation and precipi- tation. 31 Klotz (1958) proposed a different mechanism. He noted that the non—polar side chains of certain amino- acids are analogous to compounds known to form crystalline clathrate hydrates. Since a large number of these groups exist along the protein macromolecule, Klotz has reasoned that they act cooperatively to induce a "stabilized ar- rangement of water in a microsc0pically crystalline array" with a structure similar to clathrate hydrates. Hydro- phobic groups according to this view, would be abundant on the exterior of the molecule. The water structure may also be enhanced by such non-polar compounds as alcohols, fatty acids and free amino-acids. Stability of Lipids Dugan (1976) has described stability as the capa- bility of a fat, oil, or fatty food to maintain a fresh taste and odor during storage and use and that it is re- lated to composition of the lipid moiety, the nature and degree of stress on the system, the presence or absence of prooxidants and antioxidants, and the effectiveness of packaging. He added that fats with substantial unsat- uration in the fatty acids are usually unstable or moder- ately unstable and foods containing them reflect this in- stability; but that vegetable oils usually tend to be more stable than some of the animal fats, such as lard, even 32 though the total unsaturation of the vegetable oils may be greater because natural antioxidants are usually pres- ent in the vegetable oils. Smith and Dunkley (1962) and Waters (1971) have also referred to the promoting effects of light, especially of short wave length, heat and metal catalysts, especially iron and COpper, in the autoxida- tion of the lipid components of foods. The stability of the lipid or fat component of a food is, of course, related and relatable to the level of deterioration of that food. Lipolysis is a term which refers to the hydrolysis of ester linkages of lipids re- sulting from enzymes, from thermal stress, or from chem- ical action. A major cause of food deterioration is also attributable to fat rancidity as a result of oxidation-— "oxidative rancidity". Lipolytic rancidity is thought to pose less of a flavor problem than oxidative rancidity be- cause the former develops off-flavor only in those fats which contain short-chain fatty acids (less than 014). 33 Evaluation of the_§tate of Oxidation Ultraviolet (UV) Absorption Method The ultraviolet spectrum is particularly useful in composition studies on fats and in the analysis and iden- tification of fatty materials. Spectral examination has become indispensable in studying and following chemical reactions of fatty materials, especially cis-trans and position isomerization, oxidation and polymerization. The spectra of fats can indicate whether they have been mis- handled, since oxidation and polymerization products have ' characteristically different spectra from those of fresh materials (Swern, 1964). He has also stated that unsat- urated fatty materials absorb light in the ultraviolet re- gion of the spectrum. This region is between 200-400 nm. In the case of non-conjugated and saturated materials, the absorption is weak and general and cannot be used for ana- lytical purposes. In describing the application of UV absorption to oxidation studies, Mehlenbacher (1960) also indicated that the oxidation of polyunsaturated fatty acids produces per- oxides and the position of the double bond shifts to a con- jugated form. Conjugated linkages give rise to character- istic and intense absorption bands within the spectral range of 200-400 nm, while the absorption of isolated dou- ble bonds within the same region is very weak. This, he 34 added is the basis for the ultraviolet absorption method for determining the state of oxidation. However, many workers including Rusoff 2£.§l-(1945). and Pitt and Morton (1957) have reported ultraviolet spect- ral data on numerous non-conjugated compounds. In addition, when ultraviolet absorption methods indicate the presence of small quantities of conjugated compounds in natural fats, the results must be carefully interpreted because non-conjugated polyunsaturated com- ponents may have undergone conjugation as a result of autoxidation or other mishandling (Swern, 1964). He had earlier reported (1961) that the oxidation of polyun— saturated fatty acids is accompanied by increased ultra- violet absorption and that the magnitude of change is not easily related to the degree of oxidation because the effects upon the various unsaturated fatty acids are different in quality and magnitude as depicted in the analysis of a sample containing dienoic, trienoic, tetra- enoic and pentaenoic groups where the total diene content was due not only to linoleic acid, but also to each of the more unsaturated acids. Therefore, according to Swern, the spectral change for a given substance should be used as a relative measure of oxidation, rather than its meas- urement. In a study on the shelf-life stability of peanut butters during long and short-term storage, Angelo gt gt. (1975) found good correlation between the 35 spectrophotometric determination of conjugated diene hydroperoxides and the peroxide value determinations, over four and twelve week periods of storage. The spec- trophotometric estimation of conjugated hydroperoxides was stated to require smaller samples, to be quicker, more accurate and simpler than the method of peroxide value determination. The non-requirement of additional reagents and non-dependence upon a chemical reaction or color development were also stated as obvious advantages over the method of peroxide value determination. Vitamin A, which has a characteristic maximum at 325 nm can be determined in fats and in Vitamin A concentrates by ab- sorption in the ultraviolet region of the electromagnetic spectrum. ,8 -unsaturated carbonyl compounds can be de— tected in autoxidized fats. Ultraviolet absorption.has been used fairly extensively in studying the autoxidation of drying oils since the conjugation ofpolyunsaturated components parallels oxygen absorption (Swern, 1964). 36 Evaluation of Protein Quality One method for assessing the quality of a protein material involves measurement of protein efficiency ratio (PER). It is a ratio of the weight gained in grams by test animals, usually rats, to the weight in grams of the protein consumed. In short, PER measures protein quality by assessing its ability to support the growth of test animals. The method was develOped by Osborne 2£.§l- (1919). Onayemi and Potter (1976) used the PER procedure to eval- uate the protein quality of drum-dried COWpea powders in storage. They found that the PER of stored powders in- creased from 1.64 without methionine supplementation to 2.65 at the 0.6% addition level. Another method of protein evaluation was developed by Mitchell (1924) which measures the nitrogen retention of test animals over a period of time, When they are fed first a diet devoid of protein and then fed the protein material. Instead of using rats, Elliot (1963) used the mea- dow vole (Mtggotus_pennsylvanicus) over a six-day test period to assess protein quality. Later, Iriarte (1969) .adopted the procedure to measure the PER value of navy bean powder. Results from the study agreed with those obtained later by Miller gt gt. (1973) and Bressani gt gt. (1963)- 37 Methods involving the use of micro-organisms have been developed (Ford, 1960). Protein quality may also be evaluated by methods based on its digestibility by animals as was done by Chen gt gt. (1962). When measuring protein quality using laboratory animals, age, species, strain and general condition of the animals must be taken into account (Ford, 1962). The limitations imposed by age, species, strain and the time required to perform biological evaluations pf protein quality have led to the development of chemical methods. Oser (1951) used essential amino acid index as a measure of protein quality. Moore gt gt. (1958) used essential amino-acid analyses while the chemical score was used by Mitchell and Block (1946). It is known that deficiency of any of the essential amino-acid in a protein will prevent the utilization of the protein, Said gt gt. (1974). So, if direct destruc- tion of essential amino—acids have occurred as might happen in processing and storage the chemical methods referred to above may be a good assessment of protein quality. However, it is known that chemical analyses might reveal the presence of an essential amino-acid in a pro- tein sample even when this amino-acid is not available to the animal. Based on this knowledge, many workers have used in-vitro enzymatic digestibility procedures to meas- ure protein quality (Melnick gt gt., 1946; Eldred and 38 Rodney, 1945; Clandinin, 1949: Clandinin gt gt., 1951; Jones gt gt., 1942; Evans and St. Johns, 1945; Evans and Butts, 1948: Jones and Gersdorff, 1941; and Carpen— ter, 1958). Based on the pepsin digest residue index (PDRI) of Sheffner gt gt. (1956), Akeson and Stahman (1964) used a double enzyme system of pepsin and pancreatin in measuring protein quality. Other workers have attempted to measure protein quality simply by measuring chemically available lysine. Carpenter gt gt. (1957, 1958 and 1960) reported good agreement between results obtained biologically and those obtained by measuring chemically available lysine. Sim- ilarly, Buttersworth and Fox (1963) established a re— lationship between protein solubility and chemically avail- able lysine. Mattu and Mauron (1967) also obtained good agreement between in-vivo and in~vitro evaluations of pro- tein quality based on available lysine measurements. Although chemical assessments of protein quality have the advantages of simplicity, lower cost, and rapid- ity, the specificity of proteases (Byers, 1967) and ionic environment (Saunders, gt gt., 1973) must be taken into account. 39 Browning Reactions "Browning reactions are a part of the natural pro- cesses of decay; e.g. the drying and decomposition of vege- tation of humus, peat, and coal. When ordered, life- supporting substances are deprived of the protection of water, they become oxidized and/or chemically dehydrated to reactive intermediates: these polymerize into highly ordered dull-brown humic substances. The dull-brown color itself indicates a disordered array of red and yellow chromophores and a diffuse light absorption spectrum. Browning, therefore, consists of a series of "downhill" exothermic chemical reactions that proceed spontaneously after the "uphill" activation of energyefilled molecules? (Hodge and Osman 1976). The conventional thing has been to classify browning reactions according to the catalysts involved. Hodge and Osman (1976) however, believe that since the initial re- actions are either oxidative or non-oxidative in which catalysis by enzymes may or may not be involved, browning reactions should be classified as oxidative and non- oxidative,not enzymic or non-enzymic as is now done. When enzymes are involved in oxidative browning, they catalyze only the first step in the process. For example, as a first step only, catecholase or catechol oxi- dase, ascorbate oxidase, or lipoxidase will catalyze the conversion, respectively, of phenol, enediol, and/or 40 conjugated diene functional groups to reactive carbonyl compounds. The reactions that follow this transformation are non-enzymic. Even the initial oxidation reaction can occur, although much more slowly, in the absence of the oxidases. For example, the conversion of ascorbic acid to the vicinal tricarbonyl compound dehydroascorbic acid can occur enzymically or non-enzymically. Once formed, dehydroascorbic acid can react non-enzymically with amino- acids to produce red and brown polymers (Ranganna and Setty, 1974). As indicated by Phillips (1963) and by Weitzel _t gt.. (1957), oxidations and subsequent degradations with browning are induced in carbohydrates by ultra- violet and ionizing radiations. Alditols are oxidized to aldoses, aldoses are oxidized to osones (glycosuloses) and aldonic acids, aldonic acids are oxidized to 2-keto aldonic acids (which enolize to ascrobic acids) and car- bonyl groups are introduced into polysaccharides to in- itiate their degradation ble -elimination reactions. Non-Oxidative Browning Non-oxidative browning may also involve enzymes in the initial step. For example, in the supposedly non- enzymatic browning process in natural products, the first step involving the release of reactive reducing sugars from their conjugates is catalyzed by the glycosyl hydro- lase enzyme. Hodge (1967) indicated that this was an 41 important step in color and flavor development in dates, honey, maple syrup, chocolate, and vanilla. After the sugars have been released enzymatically or non-enzymatically, they go through ring opening, eno— lization, dehydration, and fragmentation reactions to produce more reactive hydroxycarbonyl, cL-dicarbonyl and a1} -unsaturated carbonyl compounds. These carbonylic in- termediates then react to produce brown polymers and flavor compounds, as in caramelization of sugars. Brown- ing may, however, be accelerated greatly by condensation with amines or amino-acids. When this happens, according to Hodge and Osman (1976), nitrogen is found covalently bonded in some of the simple and products such as pyrrole aldehydes, alkyl pyrazines and imidazoles and in the more complex ones like the melanoidin polymers of foods. Caramelization: The essential reactions in the carameli- zation process are (a) inversion of sucrose to D-glucose and D-fructose (b) equilibration of anomeric and ring forms (c) condensation, intermolecular, i.e.: acid- catalyzed reversion of starch sugars to di, tri- and higher oligosaccharides, (d) condensation, intramolecular, i.e.: formation of glycosans and difrustose dianhydrides, (e) isomerization of aldoses to ketoses (f) dehydration reactions and (g) browning (formation of unsaturated polymers) (Hodge and Osman, 1976). 42 Suggrgggtne or Matttgrd Browning: The name flMaillard Browning" derives from the fact that Maillard (1912) first attempted to characterize the sequence of reactions involved in the process by studying the reactions between the carbonyls of reducing sugars and free amino-acids. However, it has to be noted that, as has been demon- strated by many workers, the carbonyl function could come also from lipid oxidation-products rather than from reducing sugars (Jones and Gersdorff, 1941; Tappel, 1955; Lea, 1958; Burton gt gt., 1962; Hoffman, 1962; Koch, 1962; Narayan and Kummerow, 1958; Crawford gt gt., 1967). Hodge (1953) made a review of the chemistry of the Maillard browning but a more recent description by Hodge and Osman (1976) is presented below. A. Initial state (Colorless: no absorption in near-ultraviolet). Reactions involve condensation, enolization, Amadori rearrangement. With proteins, glucose and free amino groups combine in 1:1 ratio. PrOperties include increase in the reducing power in alkaline solution. Storage of a colorless 1:1 glucose-protein product produces browning and insolubility. B. Intermediate stage (buff yellow; strong ab- sorption in near-ultraviolet range). Reactions involve sugar dehydration to 3-deoxy- glucosone and its -3, 4- ene, HMF, and 43 2- (hydroxyacetyl) furan; sugar fragmentation: formation of l-dicarbonyl compounds, reductones, pigments. PrOperties include the fact that ad- dition of sulfite decolorizes: reducing power in acidic solution develops, pH decreases: sugars disappear faster than amino acids. With proteins, acid hydrolysis fails to regenerate the sugar (D-glucose). Positive Elson—Morgan test for amino sugars (Amadori compounds). C. Final stages (red—brown and dark brown color) Reactions involve aldol condensation: poly- merization; Strecker degradation of ct-amino acids to aldehydes and N-heterocyclics at ele- vated temperatures. CO2 evolves. PrOperties include acidity, caramel-like and roasted aromas develop: colloidal and insoluble melan- oidins form; fluorescences: reductone re- ducing power in acid solution; addition of sulfite does not decolorize. cx-Amino acids, peptides, and proteins offer basic amino groups when the pH of the adjacent medium rises above the iso-electric point of the amino compound. An exposed epsilon-amino group of lysine residues in proteins reacts like a separate amine. Some reactive non-protein amino compounds present in foods include 4-amino butyric acid, thiamine, and piperidine-Z-carboxylic acid. If spoilage 44 has begun, ammonia, methylamines, and histamines could also be present to react. . According to Overend, gt gt. (1961) the initial rate of browning of a reducing sugar with an amino com- pound is dependent On the rate at which the sugar's ring Opens to its reducible form. Pentoses and 2-deoxy-D- ribose were reported to undergo browning faster than 2- deoxy hexoses which in turn brown more readily than hexoses. Among the hexoses. D-galactose, D-mannose, and D-glucose show decreasing browning rates (Lea and Liau, 1972). Hodge (1967) referred to the Strecker degradation of amino-acids. When temperatures are high enough Ct-dicar- bonyl compounds such as 3-deoxyglucosone, pyruvaldehyde, glyoxal, or dehydroascorbic acid will cause the degrada- tion of an x-amino acid to the next lower aldehyde. This has been demonstrated by Fujiimaki gt gt. (1969) and Wang ai.§1- (1969). Some melanoidins are soluble in water and aqueous alcohol while some are insoluble. As hinted by Kato gt gt. (1968) and by Kirigaya (1968), they usually exhibit re- ductone reducing power, even after dialysis and antioxi— dant activity toward linoleic acid and in strongly browned foods and dark-beers. The lowering of food quality which normally accom- panies Maillard browning have previously been discussed. In the report of Fragne and Adrian (1971). lysine, methion- ine, and the N—terminal amino-acids of proteins were 45 indicated to react most strongly with pentoses, less strongly with D-galactose and D-mannose, less with D- glucose, still less with D-fructose and lactose, and least with sucrose. Sucrose was reported to react very strongly with lactalbumin, although not with lacto- (globulin. "The losses of lysine and methionine that have been demonstrated in model systems of proteins and sugars were Obtained under rather drastic conditions that do not occur during normal processing of high protein foods. However, losses of these amino-acids in roasted and flaked cereals (e.g.: breakfast foods) may be significant be- cause they are not abundant at the outset. More important are the Off—flavors and dark colors that deveIOp from the Maillard reaction in dehydrated products. These sensory defects may cause consumer rejection of otherwise nutri- tious foods." (Hodge 1976). 46 MATERIALS Cowbea (Vigna unguiculata) was obtained from National Seed Co. in New Orleans. Reagents and Solvents: All reagents and solvents were of analytical grade, except those for HPLC and spectro- photometric measurements which were Of spectro grade. All reagents and solvents when not in use were stored in accordance with the specifications of the manu- facturers. 47 Preparation of Drum—Dried Cowpga Powders The drum-dried COWpea powders were prepared from the COWpea seeds obtained from a seed company in New Orleans as outlined in the flow diagram below. Sorting and Weighing of Raw Seeds t Steam Blanching l Atmospheric CookO in Retort at 212 F i Mashing With Wooden Spoon Stirrer $ Puree Formation in Blender Drum Drying (single) Drum speed = 3 rpm $ Drum clearance =0.0l6" Steam(pressure = 45psi . ' ternal) Manual Crumbllng 1n _ of Flakes NO. of passes — l 5 ‘ Comminution Into NO. 1 Powder Standard Sieve with 1 Tyler Equivalent 16 mesh Packaging. 48 Preparation of Raw Cowpea Powders Raw cowpea powders were prepared from the raw cow- pea seeds by using the mill (Hobart Mfg. CO., Model #3430) located in the Animal Science Department Building at Michigan State University. Powders able to pass through NO. 1 Standard Sieve with Tyler Equivalent of 16 mesh were thus obtained and packaged ready for storage studies. Egperimental Set Up Drum—dried and raw cowpea powders obtained as de- scribed above were employed in the storage studies. 400 g of samples were weighed into 500 ml beakers and stored in desiccators where water-activities of 0.11, 0.33 and 0.75 were obtained according to the method de- scribed by Rockland (1960) and at a uniform temperature of 25° 0. Similar samples were stored at three different temperatures of 5° C, 25° C and 40° 0 all at a uniform water activity of 0.75. Samples were analyzed on a monthly basis for the various physical and chemical parameters of quality. 49 METHODS Extraction of Total Lipids Total lipids were extracted by the method described by Folch gt gt. (1957). This method makes use of a 2:1 v/v chloroform-methanol solvent. 50 grams of sample were homogenized at high speed in a Vortex homogenizer with 300 ml of the 2:1 v/v chloro- form-methanal solvent for three minutes. The homogenized sample was then filtered through a Buchner funnel fitted with a #1 Whatman filter paper after which the resulting filtrate was transferred to a 500 ml separatory funnel. The residual cake was re-extracted with 100 ml of addition- al solvent for one minute. The crude lipid extract so obtained was washed with 0.2 its volume with 0.74% KCl solution and allowed to stand overnight at refrigeration temperature to facili- tate its separation. The chloroform layer was then col- lected by passing the solution through a glass funnel con- taining about 20 grams of anhydrous sodium sulfate into a glass-stoppered round bottomed flask. After this step, the solvent was evaporated at 20° C by using a rotary vacuum evaporator (Rinco Instrument CO.). A stream of nitrogen was used to evaporate traces of chloroform. 50 Lipid so obtained was stored at refrigeration temp- erature when not in immediate use. Measureggnt of Lipid Diene Conjugation Lipid oxidation in the stored samples was followed by taking lipid diene conjugation readings on a monthly basis over a six-month storage period. 10 mg of lipid Obtained as already described was placed into 30 ml test-tube into which 10 ml of iso-octane (2,2,4-trimethylpentane) was poured and mixed thoroughly .in a Fisher mini shaker. The mixture was then filtered through a Whatman NO. 1 filter paper and absorbance values taken in duplicate on a Beckman DU spectrophotometer at 233 nm using iso-octane as blank. Gas-Liquid Chromatogpaphy Fatty acid contents of cowpea lipids were determined by gas-liquid chromatography. Preparation of Fatty:Acid Methyl Esters A rapid method described by Metcalfe gt gt. (1966) for preparing methyl esters from lipids was used to prepare these esters from the lipid extract obtained from stored samples. 51 4 ml of 0.5N methanolic NaOH was added to 150 mg of lipid extract in a 50 ml volumetric flask and the mix- ture heated on a steam bath until the lipid globules were in solution (about 5 minutes). This was then fol— lowed by boiling the soaps with 5 ml Of BFB-methanol for 2 minutes to give a quantitative conversion Of the fatty- acids to methyl esters. Enough of a saturated solution of sodium chloride was then added to the flask to float the methyl esters up into the narrow neck of the flask where they were readily withdrawn with a syringe. Gas-Liguid Chromatogrgphy of Methyl Esterg Gas-liquid chromatography was carried out on a 5830 A Gas Chromatograph (Hewlett-Packard) equipped with a com- puterized integrator for peak area and percentage fatty- acid quantifications. The column was of glass (6' x fit O.d.) packed with 15% DEGS on 80/100 mesh Chromosorb W. The operating con- ditions used were: Injection temperature 2203 C FID Temp. 2500 C Oven Temp. 190 0 Chart speed 1.00 Carrier gas flow rate 27 ml/min Extraction of Reducing Sugars The Official Method of Analysis of the Association of Official Analytical Chemists (AOAC) (1970) was used to 52 extract sugars from the samples and to determine the re— ducing sugar content of the stored powders. Determination of Avatlable Lysine A. §pectrgphotometric Method The reagent, l-fluoro-Z,4-dinitrobenzene (FDNB) has been used by Sanger (1945) and Porter and Sanger (1948) to determine the free epsilon-amino groups of proteins. To study the functional role Of epsilon-amino groups in the biological activity of proteins, Massey and Hartley (1956), Ikenaka (1959) and Blass and Raymond (1960) also used the reagent. Carpenter (1960) developed a method involving the use of FDNB to determine the available lysine content of protein foodstuffs and observed a good correlation be- tween the available lysine content and the biological value of the protein (Boyne 22.21- 1961: Carpenter and March, 1961). Other investigators such as Kakade and Evans (1966) and Boctor and Harper (1968) reached the same conclusion. However, the FDNB method is laborious and time consuming for the routine analysis of proteins. To overcome this limitation, Kakade and Liener (1969) developed a method using the reagent 2,4,6— trinitrobenzesulfonic acid (TNBS) which specifically reacts with primary amino groups. (Okumaya and Satake, 1960: Satake, gt gt., 1960; and Kotaki and Satake, 1964). 53 The method has the added advantage of differentia- ting between the free epsilon (£)-amino and N-terminal amino groups of proteins. The method was used in this project to determine the available lysine content Of the cowpea powder samples. B. High Performancegtiquid Chromatography_(HPLC) Method Peterson and Warthesen (1979) have described a method which employs a high performance liquid chromato- graphy technique. Advantages claimed for this technique include speed of resolution and the avoidance of inter- ference from some products formed during sample hydro- lysis. This method was used in this study to compare it with the spectrOphotometric method described by Kakade and Liener (1969). Measurement Of Browning Pigments The procedure of Choi gt gt. (1949) as modified by Karel and Labuza (1968) was used to measure the intensity of browning in the stored samples. Color Measurements By The Hunter Colorimeter The color of the stored samples were also measured on a monthly basis by using the Hunter Colorimeter (Hunter Associates Laboratory, Inc., VA). 54 Enough amount of sample (40 gms) was added to the colorimeter cup to cover the bottom of the cup such that there were no transparent areas. After standardizing the equipment with a white plate standard supplied with the equipment, "L" values which measure the degree of white- ness of the samples, were determined. Determination of Soluble Protein The first thing that was done in this determina- tion was to extract the soluble protein from the cOWpea samples. 5.0 g of cowpea powder was *weighed accurately into 50 m1 centrifuge tube containing 10 m1 8 M urea: 1 N sodium hydroxide (1:1 v/v). The COWpea powder was allowed to be in contact with the extracting solvent for 40 minutes after which the extracts were clarified by centrifugation at 10,000 g for 25 minutes. Love (1975) used a 15 minute centrifugation period but experience gained in course of this research revealed that at least a 25 minute centrifugation was necessary to Obtain a truly clear extract. This is a crucial step in this determin- ation because cloudly extracts can lead to gross over- estimation of soluble protein content of food stuffs like cowpea powder. A model HR—l, IEC centrifuge was used in this study. The clear aliquots so Obtained were used in the determinatiOn of the soluble protein content according to 55 the procedure described by Leggett-Bailey, (1969) and Gornall, (1949)- First, a standard curve using bovine serum albumin (BSA) was prepared as described below. 5.0 ml of clear BSA solution (10 gms in 100 ml solution were pipetted into a stOppered graduated cylinder, and diluted to 50 ml with 0.9 percent sodium chloride and mixed. In duplicate, a series of nine cuvettes .(Or test- tubes) were prepared. Into them 2.0, 1.85, 1.70, 1.55, 1.40, 1.20, 1.00, 0.80, and 0.60 ($50.9 percent saline were successively pipetted. Then in the same order, 0, 0.15, 0.30, 0.45, 0.60, 0.80, 1.00, 1.20, and 1.40 ml of the diluted serum was added. 8 ml of Biuret reagent (1.50 g cupric sulfate t 6.0 g potassium tartrate dissolved in 500 ml of water. With swirling, 300 ml of 10 percent sodium hydroxide and made to volume with water) were then added, mixed and absorbance read after 30 minutes at 540 mu. Absorbance at this wavelength using a digital spectrOphotometer was plotted against the calculated mg BSA to obtain a stand- ard curve whose linearity extended up to 25 mg BSA. Values for the sample were read off within this range of linearity. The cuvettescontaining 2.0 ml saline i.e. 22.6 percent sodium sulfate solution served as blank. To 2 m1 of the cowpea powder extract, was added 8.0 m1 of the Biuret reagent and mixed and its absorbance read 56 Off at 540 mu after 30 minutes. The corresponding amount of soluble protein was read Off from the standard curve. Determination of pH The pH of the samples were determined by weighing accurately 5.00 g of material into a 100 ml beaker after which 50 ml of deionized distilled water were added. The sample was stirred into the water with a glass-rod and left to stand for 30 minutes. This allowed the aqueous phase to separate from the solid phase, while allowing for acid extraction. pH readings were Obtained on a digital pH meter (Leeds and Northrup Model #7421) by allowing the pH elec- trode to dip down into the sample slurry which was stirred with a glass rod just before electrode immersion. Before use, the pH meter was standardized with buf- fer solutions of pH 4.01 and 7.0. Determination of Lipid Free-Amino Grqus DeKoning (1966), Lea and Rhodes (1955) and Magee gt gt. (1960), all have used the reaction with ninhydrin for the detection and analysis of lipids containing free amino groups. Ninhydrin reagents, however, are difficult to use since reproducible color yields are obtained only with very careful control of all conditions and color yields 57 vary widely with different substances. In contrast, re- actions with trinitrobenzene sulfonic acid (TNBS) yield uniformly reproducible and stable intensities for free amino groups of a variety of substances both lipid and~ non-lipid. The procedure described by Satake (1960) for amino- acids cannot be applied to lipids because of their hydro- phobic nature. Accordingly, Siakotos (1967) has described a rapid method for the determination of lipids containing free amino groups with TNBS. This method was used in this work. Determination Of Ascorbic Acid The titrimetric method described by the Association of Vitamin Chemists (1966) was used to determine the as- corbic acid content of the cowpea powders. Fluorometric Determination Of Riboflavin The Association of Vitamin Chemists (1966) fluoro- metric prodedure was used to determine the riboflavin con- tent of the stored cowpea powder samples. Riboflavin fluoresces in radiation of wavelength 440 to 500 nm with intensity Of fluorescence proportional to the concentration of riboflavin in dilute solutions. The riboflavin is meas- ured in terms of the difference between the fluorescence before and after chemical reduction. 58 Since riboflavin is light-sensitive and is most readily destroyed by light in the blue and violet regions, it was necessary to perform all Operations in this study in the absence of strong light. Fluorometrtg Determination of Thiamine The method described by the Association of Vitamin Chemists (1966) was used to determine the thiamine con- tent of the stored COWpea products. Proximate Analysis of Cowpeg_Powders Proximate analyses of both the drum-dried and raw milled cowpea powders involving moisture, ash, protein, fiber and lipid determinations were made. Except for fiber which was determined according to the procedure described by Whitehouse gt gt. (1945) all other deter- minations were made in accordance with the procedure stipulated in the AOAC (1970). RESULTS AND DISCUSSION Proximate Analyses of Raw and Drum-Dried qupea Powders The proximate analyses for both the raw and drum- dried COWpea powders are shown in Table 1. Both samples are high in crude protein content. This, in combination with the reported good profile of the essential amino- acids particularly lysine (Bressani gt gt., 1961), makes cowpea products a nutritionally valuable source of pro- tein. Drum-drying of the cooked beans did not appreci- ably alter the crude protein content. The slight dea crease in the crude protein content of the drum-dried powder may have been due to losses during the thermal treatments Of blanching, atmospheric cooking and the drum dehydration process itself. 59 60 TABLE 1 PROXIMATE ANALYSIS OF COWPEAS a Drum-Dried Raw Un:Processed Parameter Cooked Sagple - Sample Moisture,, % 3.26 10.17 Crude Fibre, % 2.19 2.32 Total Lipid, % 1.97 2.08 Crude Protein, % 25.22 26.15 Total Ash, 96 3.48 3.44 Carbohydrate, % (by difference) 67.14 66.01 a = (i) values are the means of duplicate readings (ii) values, except for moisture, are based on the % dry matter content. 61 The powders had low lipid contents just like most legumes. Many flavor problems of foods are associated with their lipid content which are subject to hydrolytic or oxidative rancidity. Although it is also true that the lipid content of foods may also contribute positively to their flavor, the low lipid content reported for these ' powders may be important in maintaining their wholesome- ness during storage. The very small difference between the total lipid content of the raw and drum-dried powders may also have been due to losses during the thermal treatments associ- ated with the whole drum-drying Operation. Both powders were low in crude fiber content which agrees with the results of Bressani gt gt. (1961). Drum- drying and cooking had virtually no effect on the crude fiber content. Apparently the conditions employed were not severe enough to cause changes in the amount of crude fiber which normally constitutes one of the more stable components Of plant materials. In addition to this, the dehulling step which the few workers who have prepared drum-dried powders from COWpeas, such as Onayemi and Potter (1976) and Kon gt gt. (1974), have included in their pro- cedure, was omitted in this study. First, the inclusion of the seed hulls did not impart any objectionable color to the product as long as the cooked beans were mashed and blended prOperly and uniformly before the drum-drying 62 step. Second, many food nutrients especially minerals and vitamins are lost when the hulls are removed. Fin- ally, the increasingly documented speculation of the im— portance Of food fibers in promoting gut peristalsis, food energy dilution, and in preventing certain forms of colonic cancer demanded that the seed hulls be retained in the preparation of the COWpea powders used in this study. The low ash contents of both samples also agree with literature values (Oyenuga, 1968) and Bressani, gt gt. (1961). The only marked difference between the raw and drum- dried cooked powders occurred in the moisture content values. As the table shows, the drum-drying process was very effective in reducing the moisture content of the powders. Lipid Oxidation As Measured by Diene Conjugation Figures 1 and 2 and Table 2 show the effects of varying the water activities, temperature, and the stor- age time on lipid oxidation as measured by lipid diene conjugation absorbance at 233 nm., for both the raw and drum-dried COWpea powders. It is seen that an increase of water-activitiy (Aw) from 0.11 to 0.33 led to a decrease in the lipid oxidation of each product when the samples were stored at a constant temperature of 25° 0. However, when the Lipid Absorbance (233 nm) 63 0.140- 0.120- 00 = AW 0.11 4. g. = Aw 0.33 {Iii = Aw 0.75 0.100- 0.080- . A. 0.060r- e o l/gl"“““t 0.040 0.020I- 0.000 ' L H J 4 L 0 1 2 3 4 5 Time in Storage at 25° 0 (Months) FIGURE’l. Effect of A on Lipid Absorbance at 233 nm In Drum-Dried Cowpea Powders During Storage 64 2.400 '- 4: a: -- 5° C .. =25° c _ 0 00 -40 C O 2.000 - o 1.600 - I 1.200 -- ° l E. :1 R I $0.800 - (D O C (U ,Q L: 8 0 400 - '2 o p ' ' ° _— a“ O 000 .~’——— 1 A I 0 l 2 3 4 5 6 Time in Storage At AW 0.75 (Months) Figure 2. Effect of Temperature on Lipid Absorbance at 233 nm.1n Drum—Dried Cowpea Powders During Storage 65 TABLE 2 EFFECT OF WATER ACTIVITY AND TEMPERATURE ON THE ABSORBANCE AT 233 NM* OF LIPID DERIVED FROM RAW COWPEA POWDER *Values are the means Of duplicate readings Aw Temperature 0.11 0.33 0.75 5° C 25° c 40° c _gINITIAL READtNGS 0.030 0.030 0.030 0.030 0.030 0.030 FIRST MONTH'S READINGS 0.038 0.030 0.039 0.032 0.033 0.700 __ SECOND MONTH'S READINGS ___ 0.041 0.036 0.042 0.031 0.036 1.007 THIRD MONTH'S READINGS 0.073 0.038 0.074 0.033 0.075 1.409 FOURTH MONTH'S READINGS 0.138 0.078 0.141 0.032 0.143 1.804 FIFTH MONTH'S READINGS 0.165 0.115 0.148 0.034 0.146 1.843 SIXTH MONTH'S READINGS 0.173 0.137 0.150 0.035 0.152 1.862 66 Aw was raised to 0.75 from 0.33, an increase in lipid oxidation was recorded. This means that there was a multistage trend in the relationship of lipid oxidation to changes in the Aw of the system. This trend is in agreement with many others described in the literature (Ayerst, 1965: Labuza, 1968; Labuza, 1970). In attempting to explain the lowered lipid oxida- tion at higher water activities, Labuza (1971) stated that water at these high water activities may be facil- itating the destruction of free radicals, reducing the rate of hydroperoxide conversion to other products by hydrogen bonding to them and by hydrating or reacting with prooxidant metal catalysts so that they are ren- dered ineffective. The recorded increase in lipid oxi- dation when the Aw was increased from 0.33 to 0.75 may also be explained according to Labuza (1971) who spec- ulated that such high Aw water may be promoting catalyst mobility and or new catalyst surface exposure. The effect of storage was to increase lipid oxida- tion as the length of storage increased. It was estimated that for the raw COWpea powders at 0.11 Aw the induction period for lipid oxidation was about two months. At 0.33 Aw, the induction period increased to 3% months: but at Aw 0.75, the induction period decreased to 2 months again. This is another way of looking at the protective effect of water against lipid oxidation in the stored samples, since shorter induction periods indicate a greater tendency to oxidize. 67 It was not possible to determine precisely the induction periods for the drum-dried samples because of the small monthly changes in absorbance values as de- picted in Figures 1 and 2. In both the raw and drum-dried cowpea powders, increases in temperature from 5° C to 25° C to 40° C led to increases in lipid oxidation. Chemical reactions generally are accelerated when temperatures are increased. This is even more evident for the initiation process of lipid autoxidation. The energy requirement for radical production by rupture of a CH bond is about 80 Kcal which is regarded as excessive. Accordingly, any input of ther- mal energy is likely to increase oxidation as is demon- strated graphically in the Figures and Table. It is also obvious that a temperature Of 5° C was quite effective in protecting the cowpea powders against any measurable in- crease in oxidation over the time span of this study. At this temperature, there was no discernible end of what might be called an induction period for the raw samples. At 25° C it was about 2 months and at 40° C it was less' than 1 month. The drum-dried COWpea powder had no dis- cernible end of an induction period. At 40° C, it was again less than 1 month. These values are shown in Table 3. The potency of temperature as high as 40° C in in- ducing COWpea lipid oxidation is apparent. Apart from supplying greater energy for the oxidation processes, the high storage temperature of 40° C may also have acted 68 TABLE 3 ESTIMATED INDUCTION PERIODS (MONTHS) OF STORED COWPEA POWDERS 25° C Aw 0.11 Aw 0.33 Aw 0.75 Drum Dried Powder - - - Raw Powder 2 3.5 2 Aw 0.75 5_°__Q 25°C 40°C Drum Dried Powder 1 i 1 Raw Powder 3 3 1 No clear cut induction period m: II No discernable end of induction period because of high stability 69 to increase the rate of oxygen diffusion into the system to promote faster lipid oxidation. This would have been particularly important in view of the milling and drum- drying operation which reduced the COWpeaS to powders with more surface area and porosity for oxygen diffusion and oxidation reactions. It also means that, in the warm areas of this country and in all of Nigeria, 10w temper- ature storage (5° C) is advised for these products if lipid oxidation and the consequent flavor problems are to be avoided. Initially, the raw samples had lower oxidation status than the drum-dried samples but, at the end of the six month storage period, the raw samples had oxidized more than the drum-dried samples. The thermal treatment associated with the drum-drying process may have caused some initial oxidation of the drum-dried cowpea powder lipids to account for its higher oxidation status. The higher oxidation status of the raw powders at the end of storage may have been due to the fact that both autoxida- tion andwlipoxygenase;catalyzed oxidation were occuring in these samples. Lipoxygenase, which is principally a plant enzyme, is known to be distributed widely in legumes. For example, Erickson (1967) and Erickson and Suensson (1970) demonstrated lipoxygenase activity in peas, and recommended blanching to inactivate these enzymes. These would have been inactivated in the drum-dried samples and not in the raw powders in this study. 70 Table 4 shows the rate of oxidation (increase in lipid absorbance per month) for the COWpea powders which was calculated from the following devised formula: R = 40X T Where AX: = Total change in value under consideration (lipid oxidation value as measured by diene conjugation absorbance at 233 nm), over a six month period. T = length of storage R = rate of change in oxidation, over the storage period. (increase in lipid conjugated diene absorbance per month). The table reveals that the raw samples had a higher monthly rate of oxidation than the drum-dried samples at all the Aw's and at 5° C and 25° C. It is interesting that at 40° C, the raw samples had a slightly lower value than the drum dried samples probably because exposure to that temperature for as long as six months was sufficient to cause lipoxygenase denaturation and or inactivation. The values tabulated in Table 4 also show the multistage phenom- enon in lipid oxidation rate with changing Aw earlier on referred to. Egtty-Acid Content of Cowpea Powders The fatty-acid composition of the cowpea powders used in this study was analyzed soon after preparation and the results are shown in Table 5. TABLElt COWPEA POWDERS CALCULATED* RATES OF LIPID OXIDATION IN STORED Drum Dried Powders Raw Powders Drum Dried Powders Raw Powders *Increase in lipid absorbance per month 25°C Aw 0.11 Aw 0.33 Aw 0.75 0.007 0.006 0.007 0.024 0.018 0.020 Aw 0.75 O O O 5 C 25 c 40 c 0.001 0.008 0.312 0.001 0.020 0.305 TABLE 5 FATTY ACID ANALYSES OF LIPIDS DERIVED FROM ORIGINAL RAW AND DRUM-DRIED COWPEA POWDERS .% FATTY ggtp FATTY ACID RAW __QBUM-DRIED Oleic Acid 13.20 12.90 Linoleic Acid 27.81 27.41 Linolenic Acid 13.63 13.03 Palmitic Acid 35.14 37.16 Stearic Acid 7.44 7.52 Arachidic Acid 2.78 2.47 72 Palmitic acid was the predominant fatty-acid in both the raw and drum-dried samples followed by linoleic acid, Oleic acid, stearic acid and arachidic acid in the order stated. Takayema EI.§1- (1965) did report the predominance of palmitic acid in the COWpea variety (Vignia sinensis) which he studied but found no arachidic acid as a component fatty-acid Of the bean lipids. Varietal dif- ferences apparently account for this. Although Oyenuga (1968) reported arachidic acid as a component fatty-acid of Vigna unguiggtata lipid, he also reported traces of both behenic and lignoceric acids. The last two fatty- acids were not detected in the samples used in this re- search. It has to be borne in mind, however, that Oyenuga worked with samples obtained in Nigeria where climatic and agronomic practices are not exactly the same as in the United States of America. The calculated ratios of saturated to unsaturated fatty acid (S/U ratios) at various stages of this experi- ment are shown in Tables 6 and 7. The initial S/U ratio represent results obtained by calculation from the fatty- acid profiles Of both the raw and drum-dried cowpea pow— ders immediately after preparation. In effect, they rep- resent base values with which subsequent values in the course of storage were compared. The values indicate that the S/U ratios for both samples were slightly below unity indicating that the unsaturated fatty—acids were present at slightly higher levels than the saturated acids. The 73 TABLE 6 CHANGES IN THE *S/U RgTIOS 0F COWPEA POWDER LIPIDS STORED AT 25 C ** INITIAL READINGS Aw 0.11 Aw 0.33 Aw0'75 Drum Dried Powders 0.875 0.875 0.875 Raw Powders 0.830 0.830 0.830 FIRST MONTH READINGS Drum Dried Powders 0.925 0.882 0.918 Raw Powders 0.933 0.860 0.933 THIRD MONTH READINGS Drum Dried Powders 1.117 0.971 1.120 Raw Powders - 1.171 1.021 1.187 SIXTH MONTH READINGS Drum Dried Powders 1.438 1.060 1.431 Raw Powders 1.620 — 1.204 1.620 *Saturated/Unsaturated fatty-acid ratio **Readings Obtained immediately after drum-drying and milling 74 TABLE 7 CHANGES IN THE *S/U RATIOS 0F COWPEA POWDER LIPIDS STORED AT Aw 0.75 Drum Dried Powders Raw Powders Drum Dried Powders Raw Powders Drum.Dried Powders Raw Powders Drum Dried Powders Raw Powders ** INITIAL READINGS Aw 0.75 5°C 25°C 40°C 0.875 0.875 0.875 0.830 0.830 0.830 FIRST MONTH READINGS 0.875 0.918 1.339 0.830 0.933 1.452 THIRD MONTH READINGS 0.845 1.189 2.149 0.890 0.865 SIXTH MONTH READINGS 1.431 2.394 1.622 3.149 **Readings obtained immediately after drum-drying and milling *Saturated/Unsaturated fatty-acids ratio 75 slightly higher value for the drum-dried sample is to be eXpected from their somewhat higher oxidation status which would have caused a greater loss of the unsaturated fatty-acids vis-a-vis the saturated ones. The Tables under consideration reveal also that at all temperatures except 5° C, and at all Aw‘s, the ratio of saturated to unsaturated fatty-acids increased with increasing length of storage. This probably was due to oxidation. That the samples oxidized at all Aw's and at all temperatures with the exception of 5° C has already been referred to and demonstrated by changes in the lipid diene conjugation. If the unsaturated fatty-acids oxidized more than the saturated fatty—acids, the S/U ratio.would be ex- pected to increase in the course of storage. Moreover, in agreement with oxidation measurement obtained by lipid diene conjugation, the raw samples at all the conditions of storage had a somewhat lower S/U ratio at the beginning of storage. At the end of storage the raw samples at- tained slightly higher values than the drum-dried powders. Again, a combination of lipoxygenase catalyzed autoxida- tion and autoxidation is inferred. The stability Of the S/U ratio at 5° C, is a reflection of the ability of low temperature to retard oxidation of the lipid unsaturated fatty-acids. Using the same formula stated earlier, a calcula- tion was made of the rate of loss, (decrease in percentage 76 fatty acid per month) of the unsaturated fatty-acids in the stored powders. The results are given in Table 8. There is a clear indication that the rate of loss for each fatty-acid tended to be related to its level of unsaturation with the more unsaturated acids showing losses greater than the less unsaturated ones, obviously, due to the greater oxidation rates of the former. The higher values obtained for the raw COWpea powders har- monize with the concept that the raw samples may have eXperienced both autoxidation and lipoxygenase catalyzed oxidation. The effect of increasing the Aw from 0.11 to 0.33 was to decrease the S/U ratio indicating a protection of unsaturated fatty-acids against oxidation by the higher Aw of 0.33. However, the ratio tended to increase when the Aw was increased from 0.33 to 0.75. This is con- sistent with the multistage trend observed in oxidation assessment by lipid diene conjugation. It would appear that an Aw of 0.33 is the critical value for lipid sta- bility in the samples studied here. An Aw of about 0.33. has been found to be the most protective against lipid oxidation for most dehydrated intermediate moisture foods (Labuza, 1971). Increases in temperature from 5° C to 25° C to 40° C led to corresponding increases in the S/U ratio. The results are consistent with the expectation that oxidation would proceed faster at higher temperatures 77 TABLE 8 CALCULATED" RATES OF LOSS OF PERCENTAGE UNSATURATED FATTY ACIDS 0F STORED COWPEA POWDER LIPIDS OVER A SIX MONTH PERIOD RAW POWDERS 25°C AWO . ll AWO . 33 AWO . 75 Oleic Acid 0.597 0.367 0.60 Linoleic Acid 0.882 0.498 0.885 Linolenic Acid 1.267 0.680 1.260 DRUM -DR IED POWDERS Oleic ACid 0.428 0.227 0.437 Linoleic Acid 0.622 0.238 0.600 Linolenic Acid 1.005 0. 335 0. 998 we AW 0 . 75 O O O 5 C 25 C 40 C Oleic Acid 0.027 0.603 1.197 Linoleic Acid 0.040 0.882 1.622 Linolenic ACid 0.101 1.265 2.272 DRUM-DRIED POWDERS Oleic Acid 0.008 0.433 0.932 Linoleic Acid 0.002 0.598 1.285 Linolenic Acid 0.065 1.002 1.763 *Decrease in percentage unsaturated fatty acid per month 78 in such a way that the unsaturated fatty-acids would be oxidized in preference to the saturated acids. Finally, palmitic acid was the main saturated fatty-acid which tended to increase relatively in stor- age as the percentage unsaturated fatty-acid decreased. Stearic and arachidic acids showed smaller increases. Liptd Free—Amino Grggps The effects of changing both the temperature and the Aw on the lipid free-amino groups of the stored cow- pea powders are discernable in Figures 3 and 4 and Table 9. Increases in temperature and Aw lowered the lipid free-amino group concentrations in both the raw and drum- dried cowpea powders. The reason prObably lies in the fact that increasing Aw and increasing temperature pro- moted increased carbonyl-amine reactions. .Indeed, the literature indicates that phospholipids which contain amino groups such as phosphatidyl ethanolamine and phosphatidyl serine are capable of contributing to the browning ob- served in foods. For example, Lea (1956) noted that glu- close in dried egg yolk was able to enter into Maillard type browning reactions with the amino-phospholipids Of the dried eggs. Similarly Folch (1948) reported that phosphatidyl ethanolamine isolated from brain Changed color from white to a tan brown color only after two weeks of 79 ‘BE‘ 0 124.00 — o 0 2120.00 - Q. s C0 C0 3 '00 3 0.. g 00 = AW 0.11 a. <9 . g = AW 0.33 0 3116.00 F *‘V = A 0.75 s w ‘5 CD CD S4 in “U --1 Q u-t .4 112.00 4 , . I ' . O 1 2 3 5 Time in Storage at 25° 0 (Months) FIGURE 3 Effect of A on the Lipid Free-Amino Group Con— tent of Drum-Dried COWpea Powders During Storage ”3.? ,_4 Q. s m U) €119.00. 3” O. 5 O S... c: O c .g - O C t O. ‘25 ° CL. *_‘ =40 C '3 u-i Q. '1 111.00 1 ‘ ‘ L I _1 0 l 2 3 4 5 6 Time in Storage at Aw 0.75 (Months) Figure 4: Effect of Temperature on the Lipid Free- 8O Amino Group Content of Drum-Dried COWpea Powders During Storage. 81 TABLE 9 EFFECTS OF WATER ACTIVITY AND TEMPERATURE ON THE LIPID FREE-AMINO GROUP CONTENT (ug/g sample)* 0F RAW COWPEA POWDER DURING STORAGE .11 Temperature 0.11 0.33 0.75 5° C 25° C 40° C _‘_ INITIAL READINGS 126.72 126.72 126.72 126.72 126.72 126.72 FIRST MONTH'S READINGS 126.23 126.12 126.36 126.67 126.33 125.19 SECOND MONTH'S READINGS 126.30 126.05 126.01 126.55 125.84 124.03 THIRD MONTH'S READINGS 124.07 123.08 122.16 126.02 124.05 122.14 FOURTH MONTH'S READtNGS 122.04 120.04 118.00 125.00 122.14 116.04 FIFTH MONTg;S READINGS 122.05 120.00 116.13 124.66 118.27 110.53 ___ SIXTH MONTH'S READINGS 122.01 120.02 116.04 123.44 117.42 111.00 *Values are the means of duplicate readings 82 storage in a vacuum desiccator implicating Maillard re- actions as the occurring phenomenon in the browning pro- cess. The calculated rate of losses (Decrease in lipid free-amino group (ug/g sample) per month) for the two types of COWpea powders are shown in Table 10. The raw samples again had higher rates of losses. This was pro- bably because the enzyme system was left intact in'the raw COWpea powders so that more oxidation products cap- able of reacting with the amino groups were formed. Even the so—called non-enzymatic Maillard browning may also have been accelerated by these intact enzyme systems such as by glycosyl hydrolase enzymes capable of releasing glu- cose from complex polymers (Hodge, 1967). Increases in temperature and water activity had the effect of increasing this rate of phospholipid amino loss. This would be predictable from increasing brown- ing in both cases. That increasing the Aw from 0.11 to 0.33 did not lead to reduced rate of lipid amino loss would seem to indicate that carbonyl-amine reactions are more important than oxidation in predicting losses in lipid amino groups. The multistage trend in rate which would be linked to lipid oxidation as was discussed earl- ier was not observed in the trend of lipid amino losses with changing Aw. Finally, the efficacy of the refrigeration temper- ature of 5° C in slowing the rate of lipid amino losses 83 TABLE 10 CALCULATED* RATES OF LOSS OF LIPID FREE-AMINO GROUPS Aw 0.75 o o 0 5 C 25 C 40 C Drum Dried Powders 0.038 ~0-800 2.060 Raw Powders 0.538 1.495 2.365 25°C . Aw 0.11 Aw 0.33 Aw 0.75 Drum Dried Powders 0.452 0.700 0-800 Raw Powders 0.703 1.017 1.501 *Decrease in lipid free-amino group (ug/g powder) per month 84 and that of 40° C in accelerating it is quite obvious from Table 8. This again would be predictable from the observed ability of 50 C to slow browning rate and/or oxidation and of 40° C to do just the Opposite. Color Changes and Browning The level of whiteness in both the drum-dried and the raw cowPea powders were assessed by recording the "L" values in.a standardized Hunter Colorimeter as has already been described. The trend in color changes for both samples are shown graphically in Figures 5 and 6 and Table 11. The figures and table show loss of whiteness with increasing Aw and with increasing temperature and storage time. The effectiveness of the refrigeration temperature of 50 C in maintaining whiteness or in inhibiting browning is also apparent. In contrast, a storage temperature of 400 C was quite effective in inducing browning in both types of powders. Changing the Aw from 0.11 to 0.33 and 0.75 did not appear to alter seriously the intensities of browning of the powders, yet the small differences in the levels of coloration were persistent and consistent through- out the period Of storage. Johnson (1966) assessed loss of whiteness in flour by taking reflectance values on a disc reflectance equipment and was able to relate loss of white- ness to decreased reflectance values. 85 7O'OOF CDC) = Aw 0.11 .0 =Aw 0.33 6 . — 900 it =A 0.75 W 67.00- g 0 g \ ' . ° > ”. :q‘ 0 65.004 63.00L. J l L 1 I 0 1 2 3 4 5 Time in Storage at 250 C (Months) FIGURE 5 Water Activity Effect on the TL" Values of Drum- Dried COWpea Powders During Storage 86 85000. O C>C> = 5 C o o = 25° C = 4 O c 44 O 70oOOb o “0 W 50.00- U) (D 3 H C0 >. v—‘| 30.00 - 10 00 ‘ ‘ l . J 0 1 2 3 4 5 Time in Storage at AW 0.75 (Months) FIGURE 6 Temperature Effect on the "L" Values of Drum- Dried Cowpea Powders During Storage 87 TABLE 11 EFFECTS OF WATER ACTIVITY AND TEMPERATURE ON THE BROWNING INTENSITY ("L" VALUE)* OF RAW COWPEA POWDER DURING STORAGE Aw Temperature 0 11 0.33 0.75 5° C 25° C 46° 0 INITIALgfiEADINGS 81.94 81.94 81.94 81.94 81.94 81.94 FIRST MONTH'S READINGS 81.45 81.45 81.20 81.70 81.60 44.60 SECOND MONTH'S READINGS 81.25 81.20 80.60 81.50 81.00 38.70 THIRD MONTH'S READINGS 81.30 81.00 80.20 81.25 80.15 31.10 FOURTH MONTH'S READINGS 81.00 80.90 80.20 81.00 80.10 27.70 FIFTH MONTH'S READINGS 81.00 80.55 80.05 80.60 80.05 23.40 __p SIXTH MONTH'S READINGS 80.65 80.15 79.60 80.20 79.55 15.00 *Values are the means of duplicate readings 88 Counter (1969) did not observe consistent changes in color by using the Hunter Colorimeter, on stored navy bean powders. In anticipation of the type of problem hinted by Counter, a second method of color measurement described by Choi gt a1. (1949) and modified by Karel and Labuza (1968) was used in this work. There was good agreement with the data obtained from the Hunter Colori- meter measurements in this study. Loss of whiteness was obviously due to browning and browning itself may have been caused by the Maillard type reactions between the epsilon-amino group of lysine and other such reactive amino groups in various kinds of compounds such as phospholipids and the carbonyl groups of such compounds as reducing sugars and lipid oxidation products such as malonaldehyde, aldehydes and ketones. The literature cites many such instances (Henry and Kon, 1948; Ben-Gera and Zimmerman, 1964; Hodson and Miller, 1957; Ponting gt g1. 1964; Carpenter, g£,gl. 1962; Brad- dock and Dugan, 1973 and Schwenke, 1975). Browning in stored food may also be due to ascorbic acid browning. However, neither the raw nor the drum-dried COWpea pow- ders contained any measurable ascorbic acid; and as such it cannot be assigned a role in the loss of sample white- ness here. Table 12 shows the rate of browning (decrease in "L" values per month) of the raw cowPea powders to be generally greater than that for the drum-dried samples. 89 TABLE 12 CALCULATED* RATES OF LOSS OF WHITENESS (INCREASE IN BROWNING) IN STORED COWPEA POWDERS Aw 0.75 o o o 5 C 25 C 40 C Drum Dried Powders 0.158 0.208 6.142 Raw Powders 0.280 0.342 4.933 25° 0 AW 0.11 Aw 0-33 Aw 0-75 Drum Dried Powders 0.208 0.0.6? 0,206 Raw Powders 0.133 0.217 0,343 * Decrease in "L" values per month 90 Simply, this may have been due to the activity of poly- phenolase enzymes in the raw bean powder which caused the so-called enzymatic browning. This would be in ad- dition to the so-called non-enzymatic or Maillard browning. In addition, it is speculated here that amylase hydro- lytic enzymes in the raw sample may also have acted to release reactive glucose and or fructose molecules which were subsequently mobilized in Maillard browning reactions. Thirdly, the lipoxygenase enzymes Which may have been pre- sent in the raw COWpea powders may have caused some oxi- dation of the COWpea lipid to yield reactive carbonyl groups to further increase their potential for Maillard browning. Because of the possible thermal inactivation of these enzymes in the drum-dried cooked samples, they would not have contributed to browning. If the initial values of browning are examined in the figures and table in reference which represent the level of whitenss of the powders immediately after drum- drying and milling respectively, it would be seen that the drum-drying process greatly increased browning of the drum-dried powders. The high temperatures of the drum-dryer are enough to induce caramelization reactions described by Hodge and Osman (1976) in addition to some Maillard type reactions between free and reactive amino groups and reactive carbonyl groups. The drum-dehydration 91 process is known to cause pre-storage browning as has been indicated by many workers such as Bakker-Arkema gt a1. (1967), Onayani and Potter (1976), Morris (1961) and Kon gt g1. (1974). The loss of whiteness, if it is not accompanied by serious nutritional losses, should not be a problem in these products. The changes in nutritional parameters brought about by the drum—dehydration applied in this work are discussed later. Loss of whiteness could only be a marketing problem if peOple consciously or uncon~ sciously eXpect these products to be white. Since these are new products, only marketing reSearch can tell what consumers eXpect their color to be. Changes in Reducing Sugar The percentage changes of the reducing sugar in the stored cowpea powders are shown in Figures 7 and 8 and Table 13. Increasing the Aw from 0.11 to 0.33 and to 0.75 led to decreases in the percentage amounts of reducing sugar in the cowpea powders. The effect of increasing the temperature from 5° C to 25° C to 400 C similarly caused decreases in the amount of reducing sugar. An explanation for this loss would appear to be that higher Aw and temperature promoted greater browning reaction involving the carbonyl groups of the reducing sugars and reactive amino groups in the powders. Higher 92 0.4C. 00 = AW 0.11 O. = Aw 0.33 * ¥ 2 AW 0.75 o O o O 0.30' . 3% 0 La CO *8? (a . O 11:0 C. -H O :5 "C! CD °‘0.20"' 0.10 ‘ ' l ‘ ‘ O 1 2 3 4 5 Time in Storage at 250 C (months) FIGURE 7 Effect of A, on the Reducing Sugar Content of Drum Dried CowPea Powders During Storage 93 0.50__ 00 = 5: c .. =25 c ** =40O C 0.40... , o o o 0.30 - g R 300.20 P V) 8° -r-1 0 :5 'C 090.10 - Cr. 0.00 ‘ 14- l s; I l 0 1 2 3 4 5 Time in Storage at Aw 0.75 (months) FIGURE 8 Effect of Temperature on the Reducing Sugar Content of Drum—Dried Cowpea Powders During Storage. 94 EFFECTS OF WATER ACTIVITY AND TEMPERATURE ON TABLE 13 THE PERCENTAGE REDUCING SUGAR CONTENT* OF RAW COWPEA POWDER DURING STORAGE A ML Temperature 0 11 0.33 0.75 5° 0 25° c 40° 0 INITIAL READINGS 0.40 0.40 0.40 ‘0.40 0.40 0.40 FIRST MONTH’S READINGS 0.39 0.37 0.36 0.39 0.36 0.29 SECOND MONTH'S READINGS 0.36 0.35 0.34 0.39 0.33 0.20 THIRD MONTH'S;B§ADING§_ 0.33 0.31 0.28 0.37 0.30 0.13 FOURTH MONTH'S READINGS 0.31 0.27 0.23 0.35 0.27 0.09 FIFTH MONTH'S READINGS 0.28 0.24 0.19 0.36 0.22 0.07 .__ SIXTH MONTH'S READINGS 4__ 0.26 0.24 0.18 0.36 0.20 0.04 *Values are the means of five readingS 95 Aw and temperature have been reported to cause greater browning. The speculation here that Maillard browning may account for the observed trend is consistent with the reports of many workers in the field. Hodge (1953) indicated the involvement of reducing sugar carbonyl groups in such browning reactions. Overend gt g1.(l96l) made mention of the role of the reducing sugars in these browning reactions when he indicated that the initial rate of browning of a reducing sugar with an amino com- pound is dependent on the rate at which the sugar's ring Opens to the reducible form. Fragne and Adrian (1971) listed some reducing sugars such as D—galactose, D-mannose, D-glucose, D-fructose and even sucrose which are capable of reacting with amino acids and N-terminal amino acids of proteins in the Maillard browning reactions. The amount of reducing sugar at 0.11 was higher than that at 0.33 probably because, as Fennema (1976) has hinted, the browning intensity is lower at 0.11 because the degree of water binding at the Aw of 0.11 is greater and thus reactions depending on solution such as Maillard browning would be slower at Aw 0.11 than at Aw 0.33 or at Aw 0.75. Non-enzymatic browning may have been accel- erated at the higher Aw's because the reactants were water soluble. Love (1975) reported a similar loss of reducing sugars as the Aw was increased from 0.11 to 0.33. Increasing storage time led to losses of reducing Sugars in the samples. 96 The calculated rates of loss (decrease in percen- tage reducing sugar per month) of reducing sugars are given in Table 14. The rate of loss was higher in the raw samples than in the drum-dried samples. In the raw cowpea powders, browning phenomenon may have been coupled with other phenomena to account for the higher rate of reducing sugar loss. With the enzyme system intact, mitochondrial linked respiration starting with the glycolytic pathway of sugar metabolism is possible. It might be expected that, since the hydrolytic enzymes responsible for the release of reducing sugars from their complex carbohydrate polymers would be eXpected to be functional in the raw powders, their rate of re- ducing sugars loss should be lower. That would be true if such released sugars were allowed to accumulate. If they were mobilized in browning and in respiration as fast as they were released this speculation would not hold. In fact, it is known that, in contrast to what Operates in potatoes, some commodities notably seeds (peas, corn, beans, among others), synthesis of starch rather than de- gradation may predominate after harvest. (Amir gt g1. 1971). Then Levitt (1972) has noted that most stress conditions such as mechanical damage (milling in the case of this study) and exposure to extreme temperatures may result in a burst of respiratory activity and, as stated by Haard (1976), "the relatively rapid rate of respiration 97 TABLE 14 CALCULATED*'RATES OF LOSS OF REDUCING SUGAR IN STORED COWPEA POWDERS Aw 0.75 o o o 5 C 25 C 40 C Drum Dried Powders 0.002 0.033 0.048 Raw Powders 0.007 0.034 0.060 25°C Aw 0.11 AW 0.33 Aw 0.75 Drum Dried Powders _ 0.015 0.020 0.033 Raw Powders 0.023 0.028 0.037 *Decrease in percentage reducing sugar per month 98 in leguminous seeds and certain vegetables, such as sweet corn and asparagus, appears to relate to starch synthesis.f The refrigeration temperature of 5° C gave a com- paratively minimal rate of loss of reducing sugar indi- cating again that one of the ways to guarantee the whole- someness of this product would be refrigeration. As can be seen from the Figures and Table the drum- dried samples initially, had slightly lower reducing sugar content than the raw powders. The difference does not appear to be considerable. The preparation process of the drum-dried samples used in this work was carefully done to minimize leaching losses during the cooking stage. All the water present in the beans after cooking was used to mash them before drying on the drum-drier. The very small loss of reducing sugar indicated in the results may have occurred by some limited reactions with other components of the beans or by similar losses during the drum-drying operation itself, such as by caramelization. Changes In The Available Lysine Content Figures 9 and 10 and Table 15 show the changes in the available lysine content of the cowpea powders and measured spectrophotometrically according to the proced- ure described by Kakade and Liener (1969). Increasing Aw from 0.11 to 0.33 and to 0.75 led to decreases in the available lysine content of both the raw 99 1'9OOP 1.500 r- 0 o . o o 0 EB <3 $1.00“)- (1) C2 \ a 00 = AW 0.11 q ..'= Aw 0'33 (D g 44 = Aw 0.75 H H m > <2 0.500 I 4111 I 1 11 0 l 2 3 4 5 Time in Storage at 250 C (months) FIGURE 9 Water Activity Effect on the Available Lysine Content of Drum-Dried Cowpea Powders During Storage 100 1.700,. 00 = 50 C .. = 250 0 ‘;H‘ = 400(3 1.500 C C . C O 1.000 .. Available Lysine (g/100 g) 0.500 - 0.300 I l 0 1 2 3 4 5 Time in Storage at Aw 0.75 (months) FIGURE 10 Temperature Effect on the Available Lysine Content of Drum-Dried COWpea Powders During Storage 101 TABLE 15 EFFECTS OF WATER ACTIVITY AND TEMPERATURE ON THE AVAILABLE LYSINE CONTENT OF RAW COWPEA POWDER DURING STORAGE (g/lOOg powder)** Aw Temperature 0.11 0.33 0.75 5° C 25° C 40° C INITIAL READINGS* 1.762 1.762 1.762 1.762 1.762 1.762 FIRST MONTH'S READINGS 1.701 1.692 1.688 1.760 1.684 1.276 .__ SECOND MONTH'S READINGS 1.640 1.613 1.584 1.761 1.585 1.027 THIRD MONTH'S READING§__ 1.600 1.562 1.403 1.758 1.407 0.978 FOURTH MONTH ' S REAQINGS 1.541 1.484 1.306 1.757 1.301 0.768 FIFTH MONTH'S READINGS 1.472 1.395 1.208 1.759 1.204 0.607 __ SIxTH MONTH'S REAglgGS 1.393 1.304 1.200 1.756 1.202 0.402 *Readings obtained immediately after milling **Values are the means of duplicate readings 102 and drum-dried cowpea powders. The inference here is that because Maillard browning also increased with in- creasing Aw as has already been demonstrated here in agreement with the description of Labuza (1970), the available lysine content also decreased in correspondence. This would have been because of the well documented ability of the epsilon-amino group of lysine to enter into Mail- lard browning reactions with the carbonyl groups of such compounds as reducing sugars and carbonyl-amine reactions with oxidation products such as malonaldehyde and similar products. According to Carpenter and Booth (1973), the lysine portion of a protein is able to enter into such reactions because it is the only essential amino acid that still has a free amino group in its condensed form as it exists in the protein molecule. Similarly, Bjarnason and Carpenter (1969) have speculated on the possibility of amide linkage formation between the epsilon-NH2 groups of lysine and carbonyl containing molecules. So also have Eldred and Rodney (1946), Henry e_t_ £11.- (1948) among many others. Increase of temperature from 5° C to 25° C and to 400 C also caused decreases in the available lysine con- tent of the powders. The results here agree with those of many others. Onayemi and Potter (1976) reported that the available lysine content of freshly dried cowpea pow- ders was of the order 97% of total lysine and that this value decreased to 94% at 37° C in 24 weeks. In their 103 work with lysine availability in rice meal, Tsao gt g;. (1978) indicated that elevated temperatures led to de- creased lysine availability as similarly reported for the cowPea powder used in the study. Again, it is obvious that Maillard browning ex- plains the increased loss of lysine availability with in- creasing temperature. It would be recalled that these temperature increases also led to increased browning. In agreement with this, Baldwin 23.31. (1951), working with several protein sources, concluded that, Maillard browning reactions between the free amino groups of the protein lysine and the reducing sugars present were responsible for the reduced protein quality when they were subjected to adverse temperatures. The effect of length of storage time was to de- crease the available lysine content of both powders as the storage time increased. The samples held at 50 C recorded only very small losses when compared to those held at room temperature of 250 C and at 400 C in parti- cular. Of interest too is the way these decreases were recorded. When Wolf gt g1. (1979) subjected soy protein isolates to thermal treatment, they reported that the loss of available lysine was initially very rapid followed by a transition phase which itself was followed by an equilibrium and final phase in which there was no measur- able 1oss of available lysine. The results of this study at all Aw's and at 5° C and 25° C do not appear to follow 104 the sequence observed by Wolf gt a1. The only one that approaches that type of description to any remarkable degree are the samples stored at 400 C. This is not surprising in view of the fact that only the 400 C stor- age temperature could be considered thermal stress on the cowPea powders when compared to 00 C and 250 C. It would even become a more important stress condition the longer the powders were held in storage. Using the formula designated previously, the rates of loss (decrease in available lysine (g/100 g sample) per month) of available lysine at all Aw's and at all temper- atures were calculated and the results are shown in Table 16. The rates increased with increased temperatures and with increased water activities with very negligible rates incurred at 50 C and comparatively remarkable rates re- corded at 400 C. The trend agrees with what was seen in the case of browning and with other measures of Maillard browning such as by the rate of loss of reducing sugar. The table also reveals that the rate of loss of available lysine was definitely higher in the raw powders than in the drum—dried material. This is a plus. It means that once the drum-dehydration process has been op- timized to minimize adverse effects on the quality of the finished product, one can eXpect smaller losses of avail- able lysine during storage of the drum-dried powders than the raw or undehydrated powders. A look at the base values illustrated in Table 2? demonstrates this point. The 105 TABLE 16 CALCULATED* RATES OF LOSS OF AVAILABLE LYSINE (g/100 of sample) IN STORED COWPEA POWDERS Aw 0.75 o o o 5 C 25 C 40 C Drum Dried Powders 0.0005 0.058 0.172 Raw Powders 0.001 0.093 0.227 25° C Aw 0.11 Aw 0.33 Aw 0.75 Drum Dried Powders 0.030 0.051 0.058 Raw Powders 0.062 0.076 0.094 *Decrease in available lysine (g/lOO of sample) per month 106 available lysine content of the drum-dried samples was somewhat higher apparently because Of processing effects, but once in storage, the rate of loss of available lysine of the raw samples was higher than that of the drum-dried. It is likely that over a very long period of storage, the higher rate of loss would have acted to compensate for the initial lower available lysine content of the drum-dried samples. Eventually, they would probably have higher avail- able lysine values than the raw samples. Comparison of HPLC and Spectrophotometric Methods_;n_the Determination of Available Lysine The determination of available lysine of a.protein food stuff is a very important parameter for evaluating its protein quality. Block and Mitchell, (1946) and Harris and Mattil (1940) noted that the discrepancy sometimes ob; served between amino acid analyses and feeding trials in asseSSing protein quality was associated with the amino- acid lysine. Then Finley and Friedman (1973) asserted that, among other things, the availability of essential amino-acids such as lysine affected the nutritive value of a protein food. Moreover, according to Carpenter and Booth (1973), lysine is of great nutritional importance because it is the only essential amino-acid in a protein molecule which still has a reactive amino group in the molecule to engage in various types of reactions of which the most important is probably Maillard browning. Because 107 'of this, many workers have attempted to establish precise procedures to evaluate the lysine availability of protein foods. This is of particular interest in a protein food like cowpea where the high content of lysine is one reason it is seen as a potential for meeting the world's protein needs. A correct estimation of its available lysine con- tent, rather than total lysine, will give a more correct picture of its usefulness as a source of protein. With that in mind, two methods were employed in evaluating the available lysine content of the cowPea pow- ders used in this research: A spectrOphotometric method described by Kakade and Liener (1969) and that by Peterson and Warthesen (1979) which uses high performance liquid chromatography. The aim was to learn whether they showed agreement at critical points in the course of this re- search: immediately after milling and drum-dehydration, mid-way (3 months) in storage and at the end of storage. The results are tabulated in Table 17. The results appear to agree reasonably well, with minor differences here and there. Such differences may be accounted for by the dif- ferent reagents used in this study. The spectrOphotometric method described by Kakade and Liener (1969) uses the reag- ent 2,4,6-trinitrobenzene sulfonic acid which specifically reacts with primary amino groups coupled with the ability of the method to differentiate between free epsilon amino and N-terminal amino groups of proteins. The method is therefore likely to be very precise. 108 can: an posampno mmcacmom * *me.o com. H onwm. o 130MIA, scOH.H .Mmfl.a mnopzom 3mm *mms.o .Hoo. H *Lom. o ..mso. H ammo.H .soH.H moossoe ooHsa soon mozHasmm Amazes :eme smoH.H .mos.H sooH.H .mom.H *smH.H .oom. H mnoosom smm Imemo.H .msH.H mmmH.H .Hmm.H mmmH.H .oom. H meooSoo ooHoa ease mozHasmm maze: aera #man.a .N©N.H *MHM.H .Nwm.a *nan.a .Nmm.a mumpsom 38m .Mmom.a .oam.H MWON.H .omnwd tmom.d .c¢m.H mumpZom powuo Essa II .I n . WM. I I 0 mumHoawm waasz. ms.o as no.0 a. HH.o as ammo. 6 nos. 0 anm. o .mouIH .sson.H .onm.HII mmoszod som ,IhmsH. o .sHm. o IIummIpIIIbeHIIII,_sso~.H .msm.H msoozoa ooHao some mozHo< mo onB CU H CH O ,0 E 0.04 4 0.00‘ ‘ L 1 0.03 0.43 0.83 1.23 1.63 2.08 Lipid Absorbance at 233 nm FIGURE 20 Effect of Lipid Oxidation on the Riboflavin Content of Drum Dried Cowpea Powder Stored for Six Months at Aw 0.75 15 O A 0.11 w Aw 0.33 AW 0.75 sample) 0.0 C)Riboflavin ng/lOOg , - 'l 1 “A 00! J ‘v 0.00 L 0.03 0.07 0.11 0.15 0.19 Lipid Absorbance at 233 nm FIGURE 21 Effect of lipid Oxidation on the Riboflavin Con- tent of Raw COWpea Powder Stored for Six months at 25 0.0. 151 0.16 l 1 - 5°C _ O 2 — 25 C 3 = 40° 0 0.12 b O 3 O C Q. E CG U) 89 0.08 P 0 1—1 \ 8’ 2 C‘. -«—4 > CO H c... O ,0 ~¢-4 m 0.04 r- 3 0.00 I 1 1 _L git ; 0.03 0.43 0.83 1.23 1.63 2.03 Lipid Absorbance at 233 nm FIGURE 22 Effect of Lipid Oxidation on the Riboflavin Con- tent of Raw COWpea Powder Stored for Six Months at Aw 0.75 152 these time periods correspond to the estimated. induction periods for the COWpea lipid seem to indicate that oxida- tion-linked destruction of riboflavin accounts partially for the loss of riboflavin as the Aw was increased from 0.11 to 0.33 and to 0.75. Before the end of the induction period, the photo- destruction of riboflavin may have been the prevailing phenomenon, since the samples were not shielded from light during storage. Figure 21 also reveals the multistage phenomenon in the oxidation of the raw COWpea powder lipid with in- creases in Aw. In accordance with the concept that oxida- tion-linked destruction is one mechanism for the loss of riboflavin in these powders, the higher riboflavin content of samples with an Aw of 0.33 than of those with an Aw of 0.11 is interesting. However, samples stored at an Aw of 0.75 had the highest riboflavin content at the end of stor— age. This does not fit the multistage phenomenon in lipid oxidation depicted in Figure 21. Clearly, other mechanisms are called into play here given the complexity of the sys- tem. Could it be that by some mechanism that the high AW of 0.75 acted to mitigate the destructive effect of light? Increased browning with increased Aw has already been noted in this work. Could it be that the reductones, d.-dicarbonyl compounds and similar antioxidant compounds produced in the course of browning Operated to protect the riboflavin against oxidative destruction? In addition, there may have been a 153 dilution effect of other reactants capable of causing loss of riboflavin in these powders at the high Aw 0f 0-75- Figure 19 also reveals the multistage phenomenon in the oxidation of the drum-dried cowpea lipid. The ef- fect of Aw on the relationship between lipid oxidation and the riboflavin content of the cowpea powder is different in some respect from that observed with the raw powder. First, it has been established that the negative correla— tion that exists between lipid oxidation and the riboflavin contents of these powders suggest a role for lipid oxida- tion in the loss of riboflavin. However, the lack of cor- respondence between the multistage phenomenon in lipid oxidation depicted in Figure 19 and the riboflavin content of the drum dried powder suggest that other mechanisms are involved. It was suggested earlier that increases in Aw in these powders may have acted to accelerate the re- actions involved in the destruction of riboflavin by in- creasing the solubility of the vitamins. Riboflavin loss at low Aw is more directly related to oxidation changes than at higher Aw where more rapid losses of riboflavin occur prior to extensive evidence of lipid oxidation. Temperature also has a strong role since, even at high AW, there is extensive loss of ribo— flavin prior to much evidence of lipid oxidation-this is apparent in both drum-dried and raw powders. The nature of the curves with increases of temper- ature from 5° to 25° and to 400 is equally interesting. In 154 the drum—dried powder stored at 50 C, the riboflavin con- tent was maintained and the degree of lipid oxidation was less than in the raw powders. This is predictable from the ability of low temperature to greatly reduce the rate of lipid oxidation as was earlier discussed and from the ability of peroxidase enzymes in raw powders to accelerate riboflavin destruction through their oxidation products. At 250 C, the relationship between both parameters was essentially linear. However, during the first two or three months of storage, large losses of riboflavin did not relate very strongly with the diene conjugation of lipids. This was apparently because lipid oxidation was still in the induction phase, and that the photo-destruction of riboflavin was the dominant process. At 400 C, losses of riboflayfi1followed diene conjugation of lipids much earlier (within a month in storage) because of the effect of the high temperature in greatly accelerating oxidation. 2. Effect of Lipid Oxidation On The Lipid Free-Amino Group As seen in Figures 23 and 24, the relationship be- tween the lipid free-amino group and the degree of lipid oxidation in the drum—dried powder at all Aw was essen- tially linear and the calculated correlation coefficients were negative indicating that one way by which the lipid free-amino groups were lost in the powder during storage was through lipid oxidation, which provided 126 124 122 Lipid Free—Amino Group (mg/g sample) 118 0 155. 1 = Aw 0.11 2 = Aw 0.33 C) = 3 Aw 0.75 o o .1 2 3 I I I -O4 0.06 0-08 0‘10 ‘Lipid Absorbance at 233 nm FIGURE 23 Effect of Lipid Oxidation on the Lipid Free-Amino Group Content of Drum-Dried Cowpea Powder Stored for Six Months at 250 C 124 122 120 Lipid Free Amino Group (ug/g sample) 118 116 FIGURE 24 156 l = A 0.11 W 2 = Aw 0.33 3 = Aw 0.75 L '0 L (3 -—Cx3 l p 2 b L -1 3 __L 0.03 0.07 0.11 0.15 0.19 Lipid Absorbance at 233 nm Effect of Lipid Oxidation on the Lipid Free AminoeGroup ContentoofWRaw Cowpea Powder Stored For Six Months at25 C l57 carbonyl-containing compounds. The carbonyl groups may then have engaged in carbonyl—amine reactions with the lipid free-amino groups. In the raw powders, the relationship was also es- sentially linear and the calculated correlation coeffic- ients negative. However at Aw 0.11 and 0.33, the last three months were marked by a weak relationship between both parameters. It means that late in storage under the conditions indicated above, loss of lipid free—amino groups may occur by other mechanisms. A mechanism may have been reactions or interactions between the amino group of the phospholipids and the car- bonyl group of reducing sugar. Another indication that other mechanisms are in- volved in the loss of lipid free-amino groups in these powders during storage is that their lipid free-amino group content, at the end of storage, did not correspond to that predictable from the multistage phenomenon in lipid oxidation depicted clearly in Figures 23 and 24. For ex- ample, one would have expected the COWpea powder with an Aw of 0.11 to have the lowest lipid free-amino group con- tent if lipid oxidation was solely responsible for the loss of these groups during storage. The fact that increasing browning with increasing Aw was previously noted in this work suggests that the higher lipid free-amino group con- tent observed in Figures 23 and 24 with increasing Aw was due to browning losses involving reactions between the free-amino groups of the cowpea phospholipid and the 158 carbonyl groups of carbonyl-containing compounds such as reducing sugars and lipid oxidation products. Indeed, Figures 25 and 27 reveal that the higher the Aw the lower was the lipid free—amino group content of the COWpea powders at the end of storage. This was matched by decreasing "L" values (increasing browning) as the Aw was increased from 0.11 to 0.33 and to 0.75. 3. Relationshipretween Lipid Free-Amino Group and Browning Browning as estimated by "L" values were plotted against the lipid free-amino groups of the COWpea powders as shown in Figures 25-28. Decreases in "L" value (in- creased browning) correspond to decreases in the lipid free- amino group. It appears, therefore, that the loss of lipid free-amino group relates in part to carbonyl-amine reactions which lead to browning. The extent of this relationship depended on the con- dition of storage. At 25°C/Aw 0.75, for example, there was virtually no observable relationship between the loss of lipid free-amino group and changes in the browning inten- sity in the raw powder throughout the six months of storage. While the lipid free-amino group was decreasing, the brown- ing intensity remained virtually constant. However, it was shown earlier that after about three months in storage, lipid oxidation was negatively related to the lipid free- amino group content of the raw powder under the same condi- tions of storage. It is suggested, therefore, that the loss 159 126 «D /, O F 1. 125L- 1. 124_ 7.? "S. 123 " 1 0' ‘O/ E U) 3 m 3 v 122 P 8‘ - 8 J.- chL11 0 - o 2 —£HVO.33 c g 121 3 - Aw 0'75 ‘5 CD (1) 5.4 m p '8. 120 «4 ,q 119 I ’1 65 "L" Values 66 FIGURE 25 Effect of Browning on the Lipid Free-Amino Group Content of D um-Dried Conea Powder Stored For Six Months at 25 C 160 126 F. 124 - 73 21 g .122 L- 4‘ U) \ 2 90.) 1'3 :3 120 - O 53 8 l “ 5° c ".4 = 0 E 118 _ 2 25 0 l g 3 = 40° c 5... CL. "C -v-( 3* A 116 1- 114 - 3 112 20 l 1 , 1 1 30 40 5O 6O 70 "L" Values FIGURE 26 Effect of Browning on the Lipid Free-Amino Group Content of Drum-Dried COWpea Powder Stored For Six Months at Aw 0.75 161 129 H II Aw 0.11 Aw 0.33 N II 127 5 \x) ll 125 _ 123 ,. 121 _. 119 _ Lipid Free-Amino Group (ug/g sample) 117 _, a» 4 115 I 1 .1 79 8O 81 82 "L" Values FIGURE 27 Effect of Browning on the Lipid Free~Amino Group Content of Raw COWpea Powder Stored For Six Months at 250 C 127 125 123 121 119 117 Lipid Free-Amino Group (ug/g sample) 115 113 111 FIGURE 28 162 ._ .1 -— 2 1 — 5° 0 2 = 25° 0 3 = 40° c 1 1 1 50 7O , 90 "L" Values Effect of Browning on the Lipid Free-Amino Group Content of Raw CowPea Powder Stored for Six Months At Aw 0.75 163 of the lipid free-amino group in the raw powders stored at 25° C and at Aw 0.75 from about the third to about the sixth month of storage was a function of lipid oxidation rather than of browning. At 400 C, the losses of lipid free-amino groups followed decreases in "L" values (increased browning) dur- ing the first three months. This implies that browning may also lead to losses of lipid free-amino groups, al- though, after this period, the relation between both para- meters was weaker. Data previously referred to reveal that during the first three months of storage at 400 C, lipid oxidation was also contributing to the losses of the lipid free-amino groups. The same picture emerges when other figures relating browning to lipid free-amino groups and those relating oxi- dation to lipid free-amino groups, in the drum-dried powders are examined. It is quite obvious, therefore, that a combination of the browning phenomenon and lipid oxidation account for the loss of the lipid free-amino groups in these powders during storage. In fact, Acosta gt ai. (1966), Roubal (1967) and Mattsson and Swartling (1963) have all reported on the great susceptibility of phosphatidyl ethanolamine and phOSphatidyl serine to oxidation. Malonaldehyde and other substances which can react with 2-thiobarbituric acid, produced during the oxidation of polyunsaturated fatty-acids can react with 164 amino-acids and proteins (Kwon pp ai., 1965). More speci- fically, according to Lea (1956) oxidation products such as aldehydes, epoxides and hydroperoxides could contribute to the loss of the free-amino group of phosphatidyl ethano- lamine. Lea (1956) similarly noted specifically that the decrease in the free—amino group of phosophatidyl ethano- lamine might result from its reaction with carbonyl- containing lipid oxidation products. 4. Relationship Betweenipipid Free:Amino Gppups and_pH As depicted graphically in Figures 29 and 30, de- creases in lipid free-amino groups corresponded to de- creases in the pH of the powders during storage. This is another indication that the free-amino groups of the cowpea lipids were involved in carbonyl-amine reactions as in browning, since losses of basic amino groups should lead to the lowering of pH. The slepes of the curves reflect the buffering capacity of the system. Earlier, it was shown that losses of lipid free-amino groups related in part to browning and that increases in Aw led to increases in browning of the cowpea powders. Figures 29 and 30 show that higher Aw correspond to lower pH and lower lipid free-amino group contents of both the raw and drum-dried COWpea powders. The indication here is clear, therefore, that carbonyl-amine reactions between lipid free-amino groups and carbonyl groups of compounds such as those of lipid oxidation products and reducing sugars 165 126 T. .I .3 O 124 ,_ 75 H Q. E to O U) \ go 0 9 3° 1 Q :5 O is 122 — 8 l = Aw 0.11 g 2 = Aw 0.33 I 8 3 - Aw 0.75 $4 EL. '0 -r-4 D. «4 14 120 118 1 14 6.00 6.50 7.00 P3 FIGURE 29 Relationship Between the pH and the Lipid Free- Amino Group Content of Drum-Daied COWpea Powder Stored for Six Months at 250 166 128 - l = A 0.11 6 w 12 .. _ 2 - AW 0.33 f‘ 3 = A 0.75 m w H 04 E a 124 .- do \ 00 3 3122- 100' 5.4 c: O o C 4-1 ilZOL 2H 41 o p a. 'U .H a. u-i “1118' 3 léb: l 4 l 5.00 6.00 7-00 pH FIGURE 30 Relationship Between the pH and the Lipid Free- Amino Group Content of Raw COWpea Powders Stored for Six Months at 250 C 167 referred to earlier, and which lead to browning, is one way by which lipid free-amino groups are lost. This loss then translates, in part, to lower pH values for these powders because of the decreased basicity and corresponding increased acidity. Apparently, pH values were affected not only by the loss of the basic amino groups of lysine and lipid but by other interactions or reactions. The exact nature of these interactions or reactions is not clear from these studies. The N-terminal amino groups of proteins, peptides, glyco- proteins, lipo-proteins, organic acids produced in the course of lipid oxidation and cell metabolism could be involved in various types of reactions or interactions to influence the pH of these powders. Phenomena such as lipolysis and Strecker degradation have already been referred to as pos— sible mechanisms leading to the lowering of pH. 5. Relationship Between the Available Lysine Contepi ind the pH of Stored Powder. Figure 31—34 depict the relationship between the available lysine content and the pH values of the stored cOWpea powder. In the drum-dried powders, the low cor- relation coefficient between both parameters at 59 C is due to the already discussed inhibition of loss of available lysine at this temperature. In the drum-dried powder stored at 400 C/Aw 0.75, loss of available lysine did not lead to observable changes in the pH of these powders between the 168 7.00.. 1 = Aw 0.11 2 = Aw 0.33 3 = Aw 0.75 6.50- :1: Q. 2 '— 1 6.00 '3' L - . Q: 1.00 1.20 1.40 Available Lysine (g/100 g sample) FIGURE 31 Relationship Between Available Lysine Content and pH of Drum-Dried CowPea Powders Stored For Six Months at 25° C 169 7000 r 1 - 5° 0 2 =25° 0 if 0 1 3 =40 C 6.00.. 2 , ’I.‘ Q. C O 3 5.00 41 —1 J 0.30 0.50 0.70 0.90 1.10 1.30 1.40 Available Lysine (g/100 g sample) FIGURE 32 Relationship Between Available Lysine Content And pH of DrumeDried Powder Stored for Six Months At Aw 0.75 170 7.00" 1 = 2 : 3 = 6.00L. pH is 1.00 1.20 N 1.40 1.60 1.80 Available Lysine (g/100 g sample) FIGURE 33 Relationship Between Available Lysine Content and pH of Raw COWpea Powders Stored for Six Months at 250 C 171 7IOCP l = 5° 0 2 = 25° c w’ 3 = 40° c .“ C 6.0C' 4. 3 i o 1 =5, 5.00.. 2 O 3 4.00l— 1- I _J 0.40 1.00 1.80 Available Lysine (g/100 g sample) FIGURE 34 Relationship Between Available Lysine and pH of Raw COWpea Powder Stored for Six Months at AW 0.75 172 2nd and 6th month of storage. This relationship is dif- ficult to explain. Apart from this apparent discrepancy, the relationship between the available lysine content and the pH of the COWpea powders in storage was as would be expected from the fact that losses of the basic amino groups of lysine in carbonyl-amine reactions would lead to the lowering of pH. The nature of some of the curves with changing Aw's and temperature is also interesting. As shown in Figure 32, at 5° C a vertical curve signifying a weak relationship be- tween the pH and the available lysine content of the drum- dried powder was obtained. At both 25° C and 40° C, how- ever, the relationship was stronger as is obvious from the correlation coefficients shown in Table 26. As depicted in Figure 33, an essentially linear re— lationship exists between both parameters in the raw powder stored at Aw'0.11 and at Aw 0.33- At an Aw of 0.75, however, while the first four months of storage gave an essentially linear relationship between both parameters, the last two months were characterized by a weak relationship between the pH and the available lysine content. This indicates that at this time in storage, decrease in pH occurred by other mechanisms such as by reaction between lipid free- amino group and reducing sugar andfor the products of lipid oxidation. Examination of Figures 29 and 30 indicates a role for the reactions involving lipid free-amino groups in causing pH decreases. In particular, Figure 29 shows that 173 for the drum-dried COWpea powder stored at an Aw of 0.75, the weak relation between available lysine content and pH during the last two months in storage could be explained by the loss of lipid free-amino groups in browning reactions as shown in Figures relating browning to lipid free—amino group content of the powder. Figures 31 and 33 reveal that higher Aw corresponded to lower pH values. This in combination with the observa- tion that high Aw corresponded to higher browning and that strong correlation exists between reducing sugar and avail- able lysine (Table 26) and between lipid oxidation and browning leads to the conclusion that browning reactions ac- count, at least partially, for the decreases in pH and avail- able lysine content in these powders during storage. Figure 34 shows the nature of the curves as a func- tion of temperature. The weak relationship at 5° C has already been discussed here. At 25° C, the first four months were marked by an essentially linear relationship between the pH and the available lysine content of the raw powder. The last two months were characterized by a weak relationship probably because of the same reason already advanced. At 40° C, the relationship between both para- meters is linear for the whole six months of storage. 6. Relationship Between the Reducing Sugar and the Avail- able Lysine Content of the Cowpea Powder in Storagi. Figures 35 and 36 clearly show that essentially lin- ear relation existed between the reducing sugar and the 174 ( 0.33, 0.31_ 0.29... 0.27_ = A 0.11 :3) w 0:; 0.25 _ = AW 0.33 g = Aw 0.75 -.-4 C.) 3 "U (D ‘1‘ 0.23 *- 6° 2 0.21 _ 3 fl ‘ f 0.19 L ‘ 1.00 1.20 1.40 Available Lysine (g/100g sample) FIGURE 35 Relationship Between Available Lysine and Re- ducing Sugar Contentscforum-Dried COWpea Powder Stored for Six Months at 250 C % Reducing Sugar 175 0.40 P 0.36 - 0.32 - 3 0.16 L—L L I I l 1.00 1.20 1.40 1.60 1.80 Available Lysine (g/100 g sample) FIGURE 36 Relationship Between Available Lysine and Reducing Sugar Contents of Raw COWpea Powder Stored for Six Months at 250 C 176 available lysine contents of the cowpea powders in storage. A positive correlation exists between both parameters as shown in Table 26 and implicates both in Maillard browning reactions. The literature in support of this has been re- ferred to earlier in the discussion. However, at Aw 0.75/25° C, and between the 4th and 6th month of storage, decreases in the available lysine content of the drum-dried powder was not matched by cor- responding decreases in the reducing sugar content. Sim- ilarly, at 25° c/AW 0.75 and between the 5th and 6th month of storage, decrease in the reducing sugar content of the drum-dried powder did not lead to any measurable change in the available lysine content. In the first case, re- action between the epsilon amino group of lysine and the carbonyl group of lipid oxidation product may have occurred. This is borne out by later observation that lipid oxida- tion showed a strong relationship with browning. In the second case, the positive correlation between browning and lipid free amino group implies that the loss of reducing sugar was by reaction with lipid free-amino group. Another aspect of Figures 35 and 36 which shows that reducing sugar reaction with lysine is a dominant cause of lysine loss is the amounts of both constituents at the end of storage as influenced by the Aw' The higher the Aw the lower were both the available lysine and the reducing sugar contents of the powders. This observation is predictable from the fact that in the Maillard reaction 177 the epsilon amino group of lysine reacts with the carbonyl group of reducing sugar to produce a Schiff's base compound from which other products of the browning reaction such as the amadori compound and melanoidin pigments are pro- duced successively. 7. Relationship Between the Available Lysine and Soluble Protein Contents Except at 5° C/Aw 0.75 the generally recorded posi- tive correlation between the available lysine and the solu- ble protein contents of the cowpea powders during storage is predictable from the documented effect of Maillard browning and other carbonyl-amine reactions between pro- teins, peptides, amino acids and carbonyl containing com- pounds in lowering protein solubility and digestibility because of the formation of new and resistant linkages. However, in the raw powder, the nature of the s10pes of the curves in Figures 37-40 as the Aw was increased from 0.11 to 0.33 and to 0.75 indicate a weaker relationship between the available lysine and the soluble protein during the first three months in storage. Large decreases in the soluble protein content were accompanied by much smaller decreases in the available lysine content. This agrees with earlier suggestion that during the first three months, other processes other than non-enzymatic browning reaction between the epsilon amino group of lysine and carbonyl group may account for the loss of protein solubility in the raw 178 13.0 11.0 c: -H (D 4.: O 5... CL, ,3 9.0 ,Q '3 H O U) R 7.0 5'01.00 1.10 1f20‘ 1°30 1.40 Available Lysine (g/100 g sample) FIGURE 37 Relationship Between the Available Lysine and Soluble Protein Contents of Drum-Dried Cowpea Powders Stored for Six Months at 25 C 179 14.0_ ‘b 1 = 5° 0 12.0.. 2 = 25° 0 '1 3 = 40° 0 10.0,. C‘. «'4 CD +3 0 d: 8.0,. (D H ,0 3 H O U) , ¥ *6.0- 2 5 4.0- 3 * * ‘ i 2.0; l I 4 l J 1 0.30 0.50 0.70 0.90 1.10 1.30 Available Lysine (g/100 g sample) FIGURE 38 Relationship Between the Available Lysine and Soluble Protein Contents of Drum—Dried COWpea Powders Stored for Six Months At Aw 0.75 180 12.00r_ 11.00,, 1 2 C . .3 9.00 3 4.1 O 5.4 a. (D 3 7.00_ 3 H O U) as 5.00P- ~3 3.004 7,- l J 121.40 1.60 itc— Available Lysine (g/100 g sample) FIGURE 39 Relationship Between the Available Lysine and Soluble Protein Contents of Raw COWpea Powders Stored for Six Months at 250 C 1.00 181 11.00__ ‘0 ; 1 - 5°C ' .l _ O 9.00- 2 ‘ 25 C l 3 = 40‘? c S: -r-l 3 g 7.00- 0... CD H ,0 3 '3 ca 5.00 - was 3.00 - 2 i 3 , Q‘ t? * ’t l.OO 4L 3 _J 0.40 0.80 1.20 1.60 2.00 Available Lysine (g/100 g sample) FIGURE #0 Relationship Between the Available Lysine and Soluble Protein Contents of Raw COWpea Powders Stored for Six Months at Aw 0.75 182 powder. Proteolysis by enzymes and/or configurational changes which eXposed more polar groups was suggested as the dominant mechanism. The effect of increases in temperature in shifting the curves to the left as is apparent in Figures 38 and 40 is also noteworthy. This agrees with the stronger cor- relation between the available lysine and the soluble pro- tein contents of the powders, as depicted in Table 26. At 40° C, however, especially at the last months of storage, great changes in the lysine contents of the powders occurred without corresponding large losses of protein solubility. Obviously protein solubility in these powders is affected by other reactions apart from those involving lysine. 8. Relationship Between Lipid Oxidation and Browning The nature of the relationship between lipid oxida- tion and browning of the COWpea powders as depicted in Figures 41—44 clearly indicates that as speculated earlier in the discussion, lipid oxidation products apparently can take part in the browning reactions. At certain periods of storage, the figures also show that lipid oxidation may not account for browning trends. In these cases, it is interesting to note the strong relationship that exists between reducing sugar and available lysine; whic provides indirect evidence for the involvement of reducing sugar, 183 67.0 P 66.6 m 66.2 m 3 H m > i; = 65.8 Aw 0.11 65.4 3 2 = Aw 0'33 _ l 3 - Aw 0-75 65.0 L. 1 1 L 3 0.0u .0.05 0.06 0.07 0.08 Lipid Absorbance at 233 nm FIGURE 41 Relationship Between Diene Conjugation of Lipid ‘ and the Browning Intensity of Drum-Dried Cowpea Powders Stored for Six Months at 25° C 184 70.0 60.0 50.0 Value "L H 40.0 30.0 1 1 1 0.04 0.44 0.84 1.24 1.64 2.04 20.0 ' Lipid Absorbance at 233 nm FIGURE 42 Relationship Between Diene Conjugation of Lipid and the Browning Intensity of Drum-Dried COWpea Powder Stored for Six Months at Aw 0.75 185 82.0 7 ‘. l = AW 0.11 . 2 = Aw 0.33 : 3 = AW 0.75 'I‘ 81.0 b 0' A. C U) Q) 3 H # .. g l :3 u a 80.0 P ¥ * 3 79 O I I l i __J 0.03 0.07 0.11 0.15 0.19 Lipid Absorbance at 233 nm FIGURE 43 Relationship Between Diene Conjugation of Lipid and the Browning Intensity of Raw COWpea Powder Stored for Six Months at 25° C 186 100.0 " 90.0 70.0 Values "L" 50.0 30.0 10-0 J | 1 1 1 0 - 3 1-23 ' 1-63 2.03 0.03 0:43 Lipid Absorbance at 233 nm FIGURE 44 Relationship Between Diene Conjugation of Lipid and the Browning Intensity of Raw COWpea Powder Stored for Six Months at Aw 0.75 187 rather than lipid oxidation products, in the browning re- action. It is inferred, therefore, that both lipid oxida- tion products and reducing sugars may constitute the sources of the carbonyl groups needed for the browning phenomenon responsible for effects of pH, soluble protein, lipid free-amino groups and available lysine. The effect of Aw on the nature of the curves in the drum-dried powder deserves mention. At Aws of 0.11 and 0.75, the last three months are marked by virtually no lipid oxidation with large increases in browning in- tensities, in contrast to the observation that during the first three or four months, increased lipid oxidation led to increased browning. This means that Aws of 0.11 and 0.75 promoted or allowed for lipid oxidation at the early months of storage and that the products of lipid oxida- tion took part in carbonyl-amine reactions which led to browning. Interestingly, at 0.33 Aw, the first few months in storage showed large losses in the whiteness of the powders which were not accompanied by similar increases in lipid oxidation. This indicates the ability of an Aw of 0.33 to inhibit lipid oxidation in these powders. The similarity in this regard between the Aws of 0.11 and 0.75 fits the multistage nature of lipid oxida- tion noted earlier in the discussion. 188 ' A similar protective effect of an Aw of 0.33 in minimizing lipid oxidation in the raw COWpea powder is seen in Figure 43. ' As shown in Table 26, the effect of increasing temperature from 5° C to 250 C and to'40O C was to in- crease the correlation between browning and lipid oxi- dation in the raw and drum-dried cowpea powders during storage. This apparently is a reflection of increasing lipid oxidation and browning with increasing temperature. Temperature is a greater factor in the relation- ship of lipid oxidation to browning than is Aw which is quite variable. SUMMARY AMQgCONCLUSION The objective in this project was to prepare cow- pea powders by drum-drying and simple milling operations and study physico-chemical changes in the powders during six months' storage at water activities (Aw's) of 0.11, 0.33, and 0.75 with temperatures held constant at 25° c and at different temperatures of 5° C, 250 C and 400 C with Aw held constant at 0.75. The effects of the type of processing on their physico-chemical characteristics were also examined by analyses of the freshly prepared products the values of which also represented base values against which storage changes were evaluated. Physico-chemical characteristics examined and the observed changes were: 1. Lip;d0xidation: This was assessed by absor- bance at 233 nm due to conjugated diene in the system. A multistage trend in lipid oxidation was obtained with lipid oxidation decreasing when Aw was increased from 0.1l to 0-33 and increasing when Aw was increased from 0.33 to 0.75. The classical eXplanation of Labuza (197l) accounts for this trend. With the raw samples showing the greater rate of oxi- dation, increases of temperature from 5° C to 25° C and to 189 190 400 C led to increases in lipid oxidation. The storage temperature of 50 C proved quite effective in mitigating oxidation in both powders. 2. FattyeAcids: Gas liquid chromatography of the total lipid from the powders revealed palmitic acid to be the dominant fatty-acid in these products. The ratio of saturated to unsaturated fatty-acid (S/U ratio) was slight- ly below unity. The unsaturated fatty acids were oleic, linoleic and linolenic. At all Aw's and at all temperatures except 50 C, the S/U ratio increased as the length of storage increased due to preferential loss of unsaturated fatty acids via oxidation. The rate of loss for each fatty acid tended to be related to its level of unsaturation, with the more un- saturated acids having the greater losses. Increase of Aw from 0.11 to 0.33 led to a decrease in the S/U ratio indicating a reduced loss of unsaturated fatty-acids by oxidation at Aw 0.33; but the rate tended to increase slightly when the Aw was increased from 0.33 to 0.75. This was in good agreement with the results ob- tained from oxidation measurements of diene conjugation absorbance at 233 nm. Palmitic acid was the saturated fatty-acid which tended to increase more in a relative sense in storage as the unsaturated fatty-acids decreased. Other saturated l9l fatty acids-stearic and arachidic——showed much smaller increases. 3. Lipid Free-Amino Group: Measurement of lipid free—amino groups by the rapid spectrOphotometric method of Siakotos (1967) showed that increases both in temper- ature and Aw led to lowering of lipid free-amino groups and that the rate of loss was higher for the raw samples than those which had been drum-dried. 4. Qplor Changes and Browning: Color changes were followed by measuring the "L“ values (“whiteness values") on a Hunter Colorimeter and it was observed that loss of whiteness followed increases in Aw and temperature although at 50 C there was virtually no loss of whiteness. With the rate of browning and loss of whiteness higher again in the raw samples than in the drum-dried powders, it is apparent that while browning in the former may have included both enzymic and non-enzymic, browning in the latter probably was non-enzymic. 5. Reducing Sugar: Increases both in Aw and tem- perature meant higher losses of reducing sugars and as the length of storage time increased the loss of reducing sugars in the powders increased} Probably because of respiratory losses and sugar- starch interconversions, in combination with Maillard browning losses, the raw powders registered higher re- ducing sugar losses than the drum-dried powders. 192 6. Available Lysine: The loss of available lysine, as estimated by the spectrophotometric method of Kakade and Liener (1969) and by the high performance liquid chromo- tography method of Peterson and Warthesen (1979), was as would be eXpected from carbonyl-amine reactions: higher losses with increasing Aw and temperature and with increas- ing length of storage. The effectiveness of 50 C in min- imizing losses was again demonstrated. 7. Soluble Protein: Soluble protein was assessed by the Biuret procedure. The soluble protein in the drum-dried powders de- creased as the'Aw and temperature were increased and there- by implicated the browning process. In the raw samples, the effect of increasing Aw for the first three months was to increase the water soluble protein content probably because of hydrolytic actions of proteolytic enzymes and/croonfigurational changes which expose more polar groups. After this three month period, increases in Aw led to decreases in water soluble protein probably because Maillard browning became the dominant process. Protein solubility was virtually unchanged during storage at 50 C. 8. pg: Changes in pH followed browning and the small changes in pH after a six month storage period in- dicate buffering capacity of the system. At 400 C, 193 however, there were more marked and noticeable decreases. in pH than at other storage conditions. The small decreases in pH observed in the powders may have been caused by a combination of loss of basic amino group in carbonyl-amine reactions, Strecker degra- dation phenomenon, especially, at 400 C, deamination phen- omenon in the raw and lipolysis also in the raw. 9. Agcorbic Acid: There was no measurable ascorbic acid in either the raw or drum-dried COWpea powders. lO. Riboflavin: Losses of riboflavin occurred at 250 C and at 400 C at all Aw's as the storage time in— creased, probably by conversion to lumichrome, because of the prevailing pH condition in the powder. 5° C was, however, very effective in minimizing these losses. Higher losses with increasing Aw were noted in the drum-dried powders probably because of greater solubility of the vitamin: but for the raw powders, higher vitamin values with higher Aw's were observed probably because of lower oxidation—linked destruction. ll. Thiamine: The trend was the same as described for riboflavin. Relationship Between the Various Quality Parameters Examination of the relationships between the var- ious cowpea quality parameters indicated that: 194 a. lipid oxidation may account for some of the losses of riboflavin b. the lipid free-amino groups may have been lost in storage as a result of both oxidation and carbonyl-amine reactions: as in browning c. the decrease in the pH of the powders during storage may be partly accounted for by the loss of the basic amino groups of the lipid free-amino groups and the available lysine d. the loss of reducing sugar and of the avail- able lysine was at least partly due to Maillard browning reaction e. the browning process was probably responsible for the loss of protein solubility. Some Effects of Drum Drying Some of the effects of the process of drum-drying, When compared to the milled powders (Table 250 were that there was slight oxidation, slight increase in pH, slight reduction in the percentage reducing sugar content, some increase in the soluble protein content, loss of whiteness, decreased available lysine content, about 25% loss of ribo- flavin, about 17% loss of thiamine, very slight loss of lipid free—amino group and some loss of unsaturated fatty- acid with corresponding increases in the percentage of saturated fatty-acids. 195 TABLE 27 BASE VALUES*FOR RAW AND DRUM-DRIED SAMPLES EgRAMETER ¥_QRUMpQRIED SAMPLES RAW SAMPLE Absorbance at 233 nm of lipid extract 0.041 0.030 pH 6.67 6.56 % Reducing Sugar 0.34 0.40 % Soluble Protein 13.08 . 10.83 Color by "L-value" 66.71 81.94 Available lysine g/lOOg sample 1.346 (1.207)* 1.762 (1.313)* Riboflavin mg/100 g sample 0.122 (25.153% loss) 0.163 Thiamine mg/100 g sample 0.714 (17.263% loss) 0.863 Lipid free—amino group ug/g sample 126-03 126.72 *Obtained by HPLC procedure *Values are the means of at least duplicate readings obtained immediately after processing. 196 Proximate Analyses: Results from the proximate analyses for both powders indicated that the products are high in protein content, low in total lipid, high in carbohydrate content and fair in crude fiber and ash. The drum-drying process was able to reduce moisture content from about 10% to about 4%. Some Physical Problems Observed: Some of the physical problems observed in the course of storage of these powders included cakiness at Aw 0.75 at all temperatures. Cakiness appears to be determined more by Aw than by temperature. Another was mold growth which occurred after five months of storage at Aw 0.75/25O C storage condition. General: These powders will keep well if they are stored under appropriate conditions. The drum-drying process does not appear to cause serious losses of nutrients immediately after processing. In storage, the process is even more beneficial. It will slow most of the deteriorative re- actions in these powders which appear to center around browning and loss of vitamins, if they are stored under improper conditions. The powder if stored at Aw, 0.11 and 50 c, will maintain most of its useful nutritional and physical 197 attributes. At an Aw of 0.11, the only problem might be lipid oxidation but this is only after two or three months in storage. The use of antioxidants should alleviate this problem. Therefore, these powders represent one more po— tential useful convenience food item and should also be of importance in meeting the protein problem of the world. Suggestions For Furthg; Research Every research undertaking raises new and inter- esting issues. This has been the case in this work. Most of the explanations offered for the observa- tions made in this work have been based on hard data: but some were largely speculative as is indeed true of science in its drive to uncover the truth. It would be interesting to monitor changes in lipo- xygenase activity in the raw powders to see if this can account for the higher rates of lipid oxidation observed in the raw powders when compared to the drum-dried pow- ders. It should also be of interest to isolate the phos- pholipids of cowpeas, characterize them and then make oxi- dation studies at the different Aw's and temperatures to see what actual role, if any, phospholipid oxidation plays in the loss of lipid free-amino groups which were reported in this work. 198 It was noted in this work also that for about the first three months increase of water-activity from 0.11 to 0.33 and to 0.75 resulted in higher water soluble pro- tein contents of the powders. It was postulated that pro- teolytic enzymes and/or configurational changes which ex- pose more polar sites may account for this. It would be useful to isolate or at least identify the enzyme involved if it is true that enzymes actually were involved. Once identified, experiments should be conducted to relate the activity of the enzyme to changes in Aw. Only then would it be possible to explain in precise terms what was re- sponsible for the increased water soluble protein content in the first two or three months in the powders. An interesting observation was made with regard to the thiamine and riboflavin contents of the powders as the Aw was increased. In the drum-dried powders, increases in Aw led to increases in loss of these vitamins, but the raw samples recorded lower losSes with increasing Aw' Work should be conducted into the mechanisms of destruction of the vitamins in the cowpea powders. This would be a use- ful step in trying to prevent such losses. Because of the recommendation that the cowpea pow- ders be stabilized against lipid oxidation by uSing BHA, BHT and preferably ascorbic acid which is absent in these powders, research should be conducted into the effective- ness of these and other antioxidants in the conea powder systems. An antioxidant effective in one system might be 199 ineffective in another. Moreover, the optimum level of such antioxidants which protects these products without causing deleterious effects and which meet Federal stand- ards needs to be worked out. The same would be true of the suggested antimycotic and anti-caking agents referred to in the discussion. It might also be useful to investigate the effects of various types of packaging such as air, vacuum and stor- age conditions such as hypobaric on the shelf-life of these products. The effect of using light screening pack- aging materials would be particularly interesting research in terms of riboflavin content. The packaging method eme ployed in this research (beaker exposed to light and at- mosphere) is not the best for maintaining shelf-life of stored foods. 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