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TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE " lL__ [__ L41— l: _J: mi MSU I. An Affirmative Action/Equal Opportunity Inuitution emu!!!” ‘ L] MECHANISMS OF DRY BEAN (Ehgggglng xnlggzig) HARDENING IN STORAGE: ROLE OF PECTIN METHYLESTERASE, PHYTASE AND LIGNIN IN DECORTICATED HARD AND SOFT MALAWIAN BEAN LANDRACES BY Mercy Mnyembezi flatuleka 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 1990 é¢7~ /9Z§ ABSTRACT MECHANISMS OF DRY BEAN (Ehgfigglnfi ynlggrifi) HARDENING IN STORAGE: ROLE OF PECTIN METHYLESTERASE, PHYTASE AND LIGNIN IN DECORTICATED HARD AND SOFT HALAWIAN BEAN LANDRACES BY Mercy Mnyembezi Mafuleka The phytic acid degradation and lignin formation mechanisms in the hard-to-cook phenomenon of the hard (red) and soft (white) decorticated Malawian beans were examined. Samples stored under varying temperatures, humidities and time periods (60°F or 15.6°C, 95°F or 35°C: sstnn, 85% RH; four and eight months, respectively) were compared to the, control group (35°F or 2°C, 30% RH, zero months). Phytase activities (inorganic phosphate), phytic acid, total pectic and water soluble pectic substances, calcium and magnesium ions, and lignin levels were determined spectrophotome- trically. Titrimetric, micro-kjeldahl and Kramer Shear Press procedures were used to determine total pectic substances' degree of esterification [pectin methylesterase (PME) activities], protein contents and cooked bean firmness, respectively. Increased cooked hardness, elevated phytase and PME activities and slight increases in lignin levels existed in both the hard and soft beans stored under adverse conditions for the extended period (eight months). Positive correlations between cooked bean hardness and phytase activities (r = 0.83), among legume seed hardness and lignin levels (r = 0.71), and between the hardiness of seeds stored for eight months and PME activities (r = 0.67) were found. The phytic acid degradation mechanism appeared to be the dominant system directing the hard-to-cook defect of the beans under adverse conditions over the eight months storage period. DEDICATION To my mother (Mrs. Faidasi Mafuleka) and father (Mr. Macksaida W.T. Mafuleka) ACKNOWLEDGEMENTS The author is deeply greatful to Dr. 0.8. Ott, major professor, for her intelligent and sincere academic guidance, encouragement, understanding and patience during the program of study, research and writing of this dissertation. Dr. Ott’s personal interest in the author's well-being and academic work made her stay a memorable one. The author is greatly indebted to Drs. M.W. Adams, J. Cash, G.L. Hosfield, P.Markakis and M.A. Uebersax for their suggestions, advice and willingness to serve on the author's graduate committee. Special thanks go to Drs. C. Cress for his untiring help and guidance in the statistical data analyses, R. Hammerschmidt for help with lignin determination procedures and G. Acquah for help with SOS-PAGE protein analysis. The author wishes to sincerely thank the Malawi/M80 Bean/Cow Pea Collaborative Research Project for financial, material and moral support during her training program, and the Michigan Agricultural Experiment Station for its financial support of the research. The author would also like to thank all people and friends that helped in one way or the other throughout her program of study. Finally, the author wishes to express her deep appreciation to Mr. M.W.T and Mrs. P. Mafuleka (her parents), her brothers and sisters in Malawi for their spiritual and moral support throughout her study. vi TABLE OF CONTENTS Page LIST OF TABLES ..................................... ix LIST OF FIGURES .................................... xiii INTRODUCTION ....................................... 1 LITERATURE REVIEW .................................. 8 Phytase ........................................ ll Pectin Methylesterase .......................... l4 Lignin ......................................... 17 List Of reference .0.000000000000000000.0.000... 23 CHAPTER 1: DRY BEAN (W W) HARDENING DURING STORAGE: ROLE or PHYTASE AND LIGNIN IN HARD AND SOFT NALAWIAN BEAN LANDRACES . 26 Abstract ....................................... 27 Introduction ................................... 28 Materials and Methods .......................... 33 Results and Discussion ......................... 51 Conclusion ..................................... 70 References ..................................... 71 CHAPTER 2: DRY BEAN (Rngggglnfi ynlggzifi) HARDENING AND THE CONSEQUENCES OF PECTIN METHYLESTERASE ACTIVITY IN STORAGE ....... 83 Abstract ....................................... 84 Introduction ................................... 85 Materials andnethOds OOOOOOOCOOOOOOOOOOOOOOOOO. 88 Results and DiscuSSion OOOOOOOOOOOOOOOOOOOOOOOO. 92 vii conCIuSions O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 References 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 CONCLUSIONS AND FUTURE RESEARCH RECOMMENDATIONS . . . . . APPENDIX 000......O..0.......0......000000000.0...... viii Page 101 102 109 111 CHAPTER 1 Table 1: Table 2: Table 3: CHAPTER 2 Table 4: LIST OF TABLES Page Experimental storage treatment arrangement of decorticated beans ....... 75 Degree of freedom and mean square values for hard-to-cook defect indicators (moisture, Pi, phytic acid, calcium, and phytic acid/calcium ratios) of stored decorticated beans for storage time, temperature, humidity and variety parameters ............................. 76 Degrees of freedom and mean square values for hard-to-cook defect indicators (magnesium, total pectic and water soluble pectic substances, lignin and protein) of stored decorticated beans for storage time, temperature, humidity and variety parameters ...................... 77 Degrees of freedom and mean square values for hard-to-cook defect indicators (total pectic substances' degree of esterifica- tion and cooked bean texture) of stored ix APPENDIX Table Table Table Table Table 5: decorticated beans for storage time, temperature, humidity and variety parameters 0.0.0....OOOOOOOOOOOOOOOOOOOO Moisture content (t) of stored decorticated beans by storage time, temperature, humidity and variety parameters ............................. Inorganic phosphorus content (ug/ml) of stored decorticated beans by storage time, temperature, humidity and variety parameters 000......OOOOOOOOOOOOOOOOOOOOO Phytic acid content (mg/ml) of stored decorticated beans by storage time, temperature, humidity and variety parameters .............................. Cell wall calcium content (ug/ml) of stored decorticated beans by storage time, temperature, humidity and variety parameters .............................. Phytic acid/calcium ratios of stored decorticated beans by storage time, temperature, humidity and variety parameters 0....O...OOOOOOOOOOOOOOOOOOOOO Page 105 112 113 114 115 116 Page Table 10: Cell wall magnesium content (ug/ml) of stored decorticated beans by storage time, temperature, humidity and variety parameters .............................. 117 Table 11: Total pectic substances (mg/ml) of stored decorticated beans by storage time, temperature, humidity and variety parameters .............................. 118 Table 12: Water soluble pectic substances (mg/ml) of stored decorticated beans by storage time, temperature, humidity and variety parameters ........................ ...... 119 Table 13: Lignin content (ug/ml) of stored decorticated beans by storage time, temperature, humidity and variety parameters .............................. 120 Table 14: Protein content (t) of stored decorticated beans by storage time, temperature, humidity and variety parameters .............................. 121 Table 15: Degree of esterification (t) of total pectic substances from stored decorticated beans by storage time, temperature, humidity and variety parameters .0.0.....OOOOOOOOOOOCOOOOOOOOO 122 xi Table 16: Page Cooked bean texture (g force/g) of stored decorticated beans by storage time, temperature, humidity and variety parameters OOOOOOOOOOOCOOOOOOO0.0.00.0... 123 xii CHAPTER 1 Figure Figure Figure Figure Figure 1: LIST OF FIGURES Page Moisture contents of stored decorticated beans as affected by storage time, temperature and humidity parameters ... 78 Inorganic phosphate contents of stored decorticated white beans as affected by storage time, temperature and humidity parameters ............................ 79 Inorganic phosphate contents of stored decorticated red beans as affected by storage time, temperature and humidity parameters ............................ 80 Lignin contents of stored decorticated white beans as affected by storage time, temperature and humidity parameters ... 81 Lignin contents of stored decorticated red beans as affected by storage time, temperature and humidity parameters ... 82 xiii CHAPTER 2 Figure 6: Figure 7: Figure 8: APPENDIX Figure 9: Figure 10: Figure 11: Total pectic substances' degree of esterification of decorticated beans as affected by storage time and relative humidity parameters.................. Cooked texture of stored decorticated white bean landrace as affected by storage time, temperature and humidity parameters .........OOOOOOOOOOOIOOOOO Cooked texture of stored decorticated red bean landrace as affected by storage time, temperature and humidity parameters 0............OOOOOOOOOOOO Hoisture-sorption isotherm for decorticated white beans (Bngggglufi ynlggrifi) at 60°F (lS.6°C)........... Hoisture-sorption isotherm for decorticated white beans (fihgfigglnfi xnlggzifi) at 95°F (35°C) ............ Hoisture-sorption isotherm for decorticated red beans (zhgggglng xnlgnzifi) at 60°F (15.6°C) .......... xiv Page 106 107 108 124 125 126 Page Figure 12: Hoisture-sorption isotherm for decorticated red beans (Ehasgglus yulggzifi at 95°F (35°C) ............. 127 Figure 13: One-dimensional SOS/PAGE of phaseolin of red (hard) and white (soft) Malawian beans stored at 95°F (35°C) and 85% RH for eight months and control conditions (35°F or 2°C, 30% RH, zeromonthS) ......OOOOOOOOOOOOOOOOO 128 INTRODUCTION Common dry beans (Ehasgglus ynlgaris) are abundant and inexpensive protein sources to aid in meeting the dietary protein needs of populations living in developing societies (Bressani et a1, 1961: Jones and Boulter, 1983). However, legume seeds become hard when stored under high temperatures (z 70°F or 21°C) and high relative humidities (RH) (2 70% RH) (Moscoso et al, 1984: Ron and Sanshuck, 1981). Hardened beans have exhibited longer cooking times _ (hence high bean processing energy expenditures) and decreased protein digestibilities (Burr et a1, 1968; Sievwright and Shipe, 1986). These problems associated with the hard-to-cook defect are important in tropical geographic areas, such as Malawi, where temperatures and humidities are high (Moscoso et al, 1984; Jones and Boulter, 1983: Burr et al, 1968). Investigators examining the hard-to-cook defect on a cellular level have proposed two mechanisms. The first hard-to-cook defect proposal has linked phytic acid degradation by the phytase enzyme and pectin desolubilization, through the formation of calcium and magnesium pectates in the middle lamella, to the hard-to- cook phenomenon (Ron and Sanshuck, 1981: Jones and Boulter, 1983: Mattson et al, 1951: Moscoso et a1, 1984: Vindiola 2 et al, 1986). Justification for the phytic acid degradation pathway is substantial. Kon (1979) proved that when beans were exposed to high temperatures (40°C or 104°F to 60°C or 140°F) during soaking, phytase activities were increased. Consequently, within bean tissues, inorganic phosphate (Pi) calcium and magnesium ions accumulated, with a concomitant decrease in phytic acid concentrations. An increase in legume seed cooking time was also noted which was indicative of the hard-to-cook defect. Furthermore, Ron and Sanshuck (1981) demonstrated that by steeping hard-to-cook beans in ethylene-diaminetetraacetic acid (EDTA) and phytic acid, (metal chelators), bean cooking times were lowered through greater cell separations during heat treatment. The amelioration in cell separation implied modifications in the legume seed middle lamella pectins by calcium removal from calcium pectates (Ron and Sanshuck, 1981). Jones and Boulter (1983) added support for the production of water insoluble calcium pectates in the middle lamella of hard-to-cook beans. Mard-to-cook black beans, stored at 34°C (93.2°F) and 70% to 75% RH for up to six months, exhibited reduced pectin solubilities and esterification, and phytic acid levels with accompanying increases in calcium and magnesium pectates and cell wall calcium and magnesium ion concentrations. Jones and Boulter (1983) postulated that lower pectin solubilities were due to phytic acid degradation by phytase which 3 subsequently released Pi, calcium and magnesium ions. The divalent ions were then available to form cation bridges within the pectinaceous middle lamella, hence desolubilize pectins. Pectin desolubilization was thought to be facilitated by pectin de-esterification which created more free carboxyl groups. Whether or not pectin de- esterification by pectin methylesterace (PME) leads to the development of hardened bean texture remains to be determined (Vindiola et al, 1986). To reiterate, the phytic acid hard-to-cook defect proposal involves the following. Several researchers have hypothesized that within the bean cotyledon cells, phytase hydrolyzes phytic- acid, thus releasing Pi, calcium and magnesium ions. Outside the cells, in the middle lamella, PME hydrolyzes pectin to pectinic acid and methanol. The calcium and magnesium ions are thought to migrate from the cell to the middle lamella, therein producing insoluble calcium and magnesium pectinates that cement the cells together. All of these reactions occur at high temperatures (a 70°F or 21°C) and high relative humidities (z 70% RH) (Ron and Sanshuck, 1981: Jones and Boulter, 1983: Vindiola et al, 1986). Unfortunately the phytic acid degradation pathway only partially explains the legume seed hard-to-cook phenomenon, since steeping hardened beans in solutions containing metal chelators (EDTA and phytic acid) does not completely reverse the hardened state (Ron and Sanshuck, 1981). The 4 second hard-to-cook defect proposal suggests lignin formation. Justification for the ligninfication-like mechanism has been documented. Hincks and Stanley (1986) found a reduction in extractable phenols of hardened beans as compared to controls. The decrease in extractable phenols signified increased phenol polymerization which implied that a lignification-like mechanism functioned to restrict cell separation upon cooking of the hardened seeds. Extending these data, Hincks and Stanley (1987) found a positive reaction for lignin in the cell wall corners joining the middle lamella of hardened legume seeds. Lignin concentrations in hard-to-cook beans remain to be quantified (Hincks and Stanley, 1987). Support for the existence of the lignification-like mechanism in the hard-to-cook phenomenon was supplemented by legume seed protein studies by Hohlberg and Stanley (1987). Phaseolin proteins were decreased and small polypeptides were significantly increased in hardened beans in contrast to control (soft) legume seeds. These workers proposed that protein degradation plus free aromatic amino acid accumulations and peroxidase activities increased within the stored bean cotyledons. These reactions represented common initial steps for plant lignification. Holberg and Stanley (1987) hypothesized that small polypeptides and free aromatic amino acids were hydrolyzed leading to polyphenol synthesis. The phenolic substances 5 then migrated from the cotyledon cells to the middle lamella under high temperature and high humidity storage conditions where the phenolic compounds were lignified by the action of peroxidase. Consequently two mechanisms have been offered to explain the development of the hard-to-cook defect of stored legume seeds at z 70% RH and z 70°F (21°C). The phytic acid degradation theory seems to predominate during early storage periods (two to four months). In contrast, the lignin formation appears to prevail during later storage periods (> eight months). Both mechanisms are apparently operating between four and eight months storage. (Mincks and Stanley, 1986). Numerous queries can be raised regarding the two postulated hard-to-cook defect mechanisms. Several problems have been addressed in this dissertation. In order to eliminate hard shell effect (due to water impermeable seed coats), soft and hard beans used in this project were decorticated. Earlier studies concerning the hard-to-cook condition have been conducted predominantly with whole beans (Hincks and Stanley, 1986, 1987: Hohlberg and Stanley, 1987: Ron and Sanshuck, 1981: Vindiola et al, 1986: Moscoso et al, 1984) from a single cultivar/variety. Minimal data have been reported comparing physical (hardness) and chemical changes between soft and hard beans stored under high humidity and high temperature environments, thus both types of legume seeds were used 6 throughout the storage investigations and physical and chemical parameters measured. Furthermore, the involvement of PME, and the extent to which lignin deposition (quantitatively), and phytase activities directed the legume hard-to-cook condition development in decorticated beans over an eight month storage period was unclear. This . project was directed towards elucidating the role of PME, phytase and lignin in the hard-to-cook defect of decorticated hard and soft Malawian beans (Ehgggglgg ynlggzig) over the eight month storage period. The approach followed in the enzyme assay of phytase and pectin methylesterase in the current research was to determine enzyme activities by measuring products formed and/or substrates remaining in decorticated beans following seed exposure to specific temperatures and humidities over specific storage time periods (Schwimmer, 1981). This research study addressed the following hypotheses. l. Decorticated beans stored at high temperature (95°F or 35°C) and high humidity (85% RH) conditions over an eight month storage period would have increased lignin deposition in the bean cotyledon, as compared to the control seeds stored at 35°F (2°C) and 30% RH. Consequently, legume seed hardening would be greater in cooked beans stored under the high temperature and humidity conditions than in the control storage environment. 2. Decorticated beans held under high temperature (95°F 7 or 35°C) and high relative humidity (35% an) environments over eight months would have higher PME and phytase activities in the bean cotyledons, as compared to the legume seed controls stored at 35°F (2°C) and 30% RH. There would be a decrease in water soluble pectic substances with a concomitant increase in calcium and magnesium ion concentrations, as well as an increase in cooked bean hardness in seeds stored under the high temperature and humidity conditions, as opposed to the control storage environment. Both hard and soft beans would exhibit similarities in relation to hardening alterations when held under the' same storage conditions. The legume seed hard-to-cook defect would be directed predominantly by the phytate mechanism as opposed to the lignification-like mechanism in decorticated beans stored under high temperature (95°F or 35°C) and high humidity (85% RH) conditions, over the eight months period. LITERATURE REVIEW Numerous researchers have demonstrated that stored legume seeds under high temperatures (2 70°F or 21°C) and high humidities (2 70% RH) become hard in texture (Burr et al, 1968: Ron and Sanshuck, 1981: Moscoso et al, 1984). Jones and Boulter (1983) considered beans to be hard when the legume seeds failed to soften adequately during cooking procedures (at boiling water temperatures, 99°C or 210.2°F for 1 hour). Failure of legume seeds to soften sufficiently during home food preparation/processing practices has been attributed to two major factors. Vindiola et al (1986) stated that beans resisted heat treatment, because of a hard shell condition. In this situation the bean seed coat was impermeable to water. The hardshell state was promoted by low humidities (g 10% RH) and high temperatures (2 75°F or 23.9°C). Hardening was reversed by hydrothermal treatment or scarification (abrasive treatment). Secondly, these workers disclosed that legume seeds resisted cooking temperatures due to the hard-to-cook phenomenon. In this situation, the bean cotyledons did not soften during boiling, even though the seeds imbibed water. The hard-to-cook state was thought to be promoted by high humidities (z 70% RH) and high temperatures (2 70°F or 21°C). This type of legume seed hardening was irreversible. Two theories have been proposed regarding the hard- to-cook defect. The phytic acid degradation proposal involves the following. Within the bean cotyledon cells, phytase hydrolyzes phytic acid thereby releasing Pi, calcium and magnesium ions. Outside the cells, within the middle lamella, PME hydrolyzes pectin to pectinic acid and methanol. The calcium and magnesium ions are considered to migrate from the cotyledon to the middle lamella, therein producing insoluble calcium and magnesium pectinates that bind the cells together, thus rendering the bean hard (Ron and Sanshuck, 1981: Jones and Boulter, 1983: Vindiola et al. 1986). The second hard-to-cook defect theory suggests lignin involvement. Several researchers have hypothesized- that small polypeptides and free aromatic amino acids were hydrolyzed, leading to polyphenol synthesis. The phenolic substances traverse from the cotyledon to the middle lamella where the phenolic compounds are lignified by peroxidase, causing the bean to harden (Hincks and Stanley, 1986: Hohlberg and Stanley, 1987). This literature review focus upon the previous research support for these two hard-to-cook defect theories. Interest in elucidating the legume seed hard- to-cook defect, and preventing this phenomenon, has been expressed by numerous groups including Food Scientists, Seed Scientists as well as Nutritionist, because hardened beans exhibit longer cooking times, and lower protein digestibilities as demonstrated by the following researchers. 10 Sievwright and Shipe (1986) stored black beans at several temperatures and humidities (2°C or 35.6°F, 50% RH: 5°C or 41°F, 50% RH: 30°C or 86°F, 30% RH: and 40°C or 104°F, 80% RH) for a maximum of six months. Decreases in in-vitro protein digestibilities and phytic acid concentrations with an accompanying increase in seed firmness were found, as storage times and temperatures increased. Tannin contents fluctuated over time, first increasing up to three months, and then decreasing thereafter. The lower in-vitro protein digestibilities were speculated to be the result of proteins complexing with phytic acid and tannin components under high temperature and high humidity conditions, since there was a high negative correlation between in-vitro protein digestibilities and loss of phytic acid (r = -0.90), as well as a decrease in tannin concentrations from protein diafiltrates. A high correlation (r = 0.90) was also noted between lower protein digestibilities and increasing legume seed firmness (Sievwright and Shipe, 1986). Thus, under adverse storage conditions (30°C or 86°F, 80% RH: 40°C or 104°F, 80% RH), black bean firmness increased and protein nutritive values deteriorated. These effects were counteracted by storage at 5°C (41°F) and 50% RH, and soaking the seeds in salt solutions. Salt solutions were .reported to disrupt hydrogen bonding between proteins and magnesium, between phytic acid and proteins, and between 11 minerals (magnesium, calcium) and pectins (Sievwright and Shipe, 1986). Legume seeds comprising high moisture (16%) contents and held under high temperatures (2 70°F or 21°C), or legumes maintained under high humidities z 70% RH) and high temperatures (z 70°F or 21°C) had extended cooking times to reach a softened state (Ron and Sanschuck, 1981: Burt et al, 1968). Ron and Sanshuck (1981) stored California small white beans containing either 16% moisture at 32°C (89.6°F) or 10.5% moisture at 22°C (71.6°F), for a maximum of ten months. Seeds stored at 32°C (89.6°F) had an extended cooking time (300 min) as compared to the cooking time (30‘ min) of beans maintained at 22°C (71.6°F). Lower phytic acid concentrations were observed in beans stored at high temperatures. Enzymatic involvement in the development of the hard-to-cook legume seed defect has been reported by several investigators (Hincks and Stanley, 1986: Vindiola et al, 1986). Phytase Phytase (myo-inositol-hexakisphosphate phospho- hydrolase, EC 3.1.3) is an enzyme that hydrolyzes phytic acid (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to myo-inositol and free orthophosphate ion and/or phosphoric acid (Henderson and Ankrah, 1985: Lolas and Markakis, 1977: Peers, 1953). Optimum phytase activities occur at pH and temperature ranges of 5.0-5.5 and 50°C (122°?) to 60°C 140°F, respectively (Peers, 1953). 12 Phytase inhibition by phytic acid (1.5 mM) and low (0.32- 0.64 mM) Pi concentrations has also been cited (Chang and Schwimmer, 1977). An early investigation by Mattson et al (1951) related legume seed cookability to storage conditions and phytic acid hydrolysis. Peas were stored at 20°C (68°F), 66-95% . RH for a maximum of 12 months.- As storage time advanced, legume seed cookability and phytic acid concentrations declined. To develop methods to reduce heat treatment times of hardened legume seeds, Ron and Sanshuck (1981) explored the cause of dry bean hardening. California small white beans containing two moisture levels (16% and 10.5%) were stored for ten months at 32°C (89.6°F) and 22°C (72.6°F), respectively. As storage times advanced, increases in calcium and magnesium ion concentrations and extended cooking times were found. A negative correlation (r - -0.71) between phytic acid/calcium ratios and length of cooking was also established. Cooking times were reduced by soaking hardened beans in phytic acid and/or EDTA for several hours (14 hours). Photomicrographs of the hardened, phytic acid and EDTA steeped beans revealed enhancement in cell separation. Cell separation of the soaked seeds suggested modifications in the middle lamella pectins when the ratio of phytic acid to calcium was adequate (pH 6.5) (Ron and Sanshuck, 1981). These researchers also postulated that both phytic acid and EDTA l3 removed calcium from calcium pectate during cooking which caused a reduction in heating time. EDTA was a more effective agent than phytic acid in softening the hardened legume seeds, because of its stronger affinity for calcium. The phytic acid and/or EDTA soak did not completely reverse the seed hardened state. Because of the incomplete reversal of the hard-to-cook phenomenon, Ron and Sanshuck (1981) concluded that phytate degradation and subsequent calcium accumulation were not the only factors involved in bean hardening, since firm beans were not completely softened to the level of the unhardened (soft) controls when soaked in EDTA and/or phytic acid. The chemical compositions of several beans and peas (navy, black, kidney, lima, faba, black eyed) revealed that those legume seeds having low calcium and high phytic acid concentrations cooked faster than seeds with high calcium and low phytic acid concentrations (Kon, 1979: Ron and Sanshuck, 1981). These results agree with data reported by Moscoso et al (1984). Legume seeds stored longer than three months (32°C or 89.6°F and 17.9% moisture) were lower in phytic acid and water soluble pectic substance concentrations, higher in calcium and magnesium ion contents, and had reduced cookability as compared to controls (2°C or 35.6°F and 12.5% moisture). All of these findings lend support to the phytic acid degradation mechanism as directing the legume seed hard-to- cook phenomenon (Mattson et al, 1951: Ron, 1979: Ron and l4 Sanshuck, 1981: Moscoso at al, 1984). However, the role of PME in the postulated phytic acid degradation mechanism remains to be elucidated. Pectin.Methylesterase Pectic substances are common plant high molecular weight acid polysaccharides. Pectin (a pectic substance) is composed of D-galacturonic acid and methyl-D- galacturonic units. PME (pectin pectylhydrolase EC 3.1.1.11) is a pectin de-esterifying enzyme that hydrolyzes the methyl group from the methyl-D-galacturonate molecule to produce D-galacturonic acid and methanol (Richard and Noat, 1986: Delincee and Radola, 1970: Versteeg et al, 1978). The cell wall bound enzyme has optimum activities between pH 7.0 and pH 8.5 (Moustacas et al, 1986: Markovic et al, 1983). PME activities have also been found to increase at temperatures ranging from 20°C (68°F) to 35 °C (95°F) (Markovic et al, 1983: Dahodwala et al, 1974). Research relating legume seed hardening during storage to pectic substances and PME enzyme activities are limited and inconclusive (Vindiola et al, 1986). Jones and Boulter (1983), in one part of the study, investigated several physical and chemical properties of beans that hardened due to suboptimal storage conditions. Black beans (Rhgggglng ynlggzig var S-19-N) containing 10% moisture were stored post harvest at 4°C (39.2°F) and moderate relative humidity (65% RH) (Jones and Boulter, 1983). One-half of the legume seeds were hardened at 34°C 15 (93.2°F), 70-75% RH for six months. Hardened beans had lower concentrations of water soluble pectins, phytins, and pectin esterification, as well as reduced imbibition values. Increased calcium and magnesium ion contents and cotyledon electrolyte leakage were measured. The other part of the Jones and Boulter (1983) experiment examined legume seed hardness changes by incubating soft beans (4°C or 39.2°F, 65% RH: controls) with either 0.03M CaClz or CaClz with PME: or CaClz with PME, plus electrolyte induced leakage (with ethanol extraction) for 18 hours at pH 7.0. Bean samples that were incubated with only CaC12 had a 60% increase in cooking times as compared to the untreated controls. PME additions further lengthened (by four min) heat treatment by the CaClz treated group. The slight increase in Cooking time of seeds to which PME was added over those incubated only in CaClz, was considered too small to account for the contribution of the great decrease in pectin esterification (51% to 15%) observed in the six months storage hardened beans (Jones and Boulter, 1983). PME activities were not determined in the incubated samples of the Jones and Boulter (1983) study, rather the indirect effect of the enzyme on bean texture. Beans incubated with CaClz and EMS, plus electrolyte induced leakage failed to soften in texture. Cellular electrolyte leakage of hard-to-cook legume seeds stored at 41°C (105.8°F) and 75% RH for 55 days was first observed by Varriano-Marston and Jackson 16 (1981), thus supporting the Jones and Boulter (1983) data where beans failed to soften when electrolyte leakage was induced. The Jones and Boulter (1983) studies lend further support to the role of the phytic acid degradation mechanism in the hard-to-cook defect. Pectin de- esterification seems to facilitate this process. However, to clearly define the role of PME in bean hardening during storage, both PME activities as well as legume seed texture (hardness) of similarly treated samples should be assessed. The phytic acid degradation proposal only partially explains the legume seed hard-to-cook defect, because this‘ phenomenon was incompletely reversed when seeds were soaked in solutions containing metal chelators (EDTA and phytic acid) (Ron and Sanshuck, 1981). Thus, the involvement of lignin in the hard-to-cook phenomena was suggested by Hincks and Stanley (1986). These workers studied enzymatic (phytase), chemical (phytic acid and phenols) and physical (legume seed structure and hardness) parameters in black beans stored at 30°C (86°F), 85% RH or 15°C (59°F), 35% RH for a maximum of ten months. Generally, beans held at elevated temperatures and humidities developed the hard-to- cook defect. The hard-to-cook legume seeds had reduced cell separations, lower phytic acid and extractable phenol contents and higher phytase activities. The investigators proposed that the decrease in extractable phenols (tannins) was partially due to increased phenol polymerization which l7 implied that a lignification-like mechanism was functioning to restrict cell separation upon cooking. Hincks and Stanley (1986) concluded that during the initial months of storage (two to four months), legume seed hardening changes were dominated by phytic acid degradation reactions, while in later storage periods (2 eight months) the lignin- formation pathway prevailed as the major contributor to bean hardness. Four to eight months storage was advocated as the transition period for the two hard-to-cook defect theories. All of these data imply that legume seed hardening during storage involves multiple routes including phytic acid degradation and lignification-like mechanisms.‘ Lignin Lignin (a collective term) refers to closely related polymeric molecules derived from the phenylpropanoid compounds, coniferyl alcohol and related alcohols (Schubert, 1965: Freudenberg and Neish, 1968). The involvement of lignin in the hard-to-cook defect has been explored further by Stanley's group and other investigators. Freshly harvested black beans stored at 15°C (59°F), 35% RH and 30°C (86°F), 85% RH for a maximum of 10 months were tested for hardness, microstructural changes and occurrence of lignin in the cell wall (Hincks and Stanley, 1987). Seeds stored at elevated temperatures and humidities developed the hard-to-cook defect. The hardened beans had heavy staining in the cell wall corners, 18 secondary cell walls and middle lamella, reflecting high concentrations of lignin in these locations. A lignification-like mechanism was deemed responsible, at least in part, for the hard-to-cook defect in beans. It was postulated that the sequence for the development of the legume seed hard-to-cook phenomenon involved a . phytate/phytase mechanism which was overriden by a lignification mechanism as storage time advanced (Hincks and Stanley, 1987). Although the microstructural and histological evidence identified the presence of lignin in cell walls of hardened beans, lignin remained to be chemically verified (Hincks and Stanley, 1987). Srisuma et al (1989) quantified lignin formation. These workers stored navy beans (Ehgsgglng yfilggris, var. Seafarer) for up to nine months at several temperatures and relative humidities (5°C or 4°F, 40% RH, control: 20°C or 68°F, 73% an: and 35°C or 95°F, 80% RH). Legume seed samples were monitored for hardness: hydroxy-cinnamic acids (ferulic, sinapic and p-Coumaric) and lignin concentrations. The hard-to-cook defect developed in beans stored within the highest temperature (35°C or 95°F) and relative humidity (80% RH) environment. The hardened bean cotyledons and their seed coats were not significantly different in lignin contents (P 5 0.05) from the control legumes. However, seed coats were significantly (P): 0.05) higher in lignin concentrations than cotyledons. Hardened legume seeds were also found to be high in ferulic l9 acids (hydroxycinnamic acids) as compared to the controls. Srisuma et al (1989) concluded that enhanced legume seed lignification was not a major cause of the hard-to-cook. defect in beans. These findings contradict those reported by Molina et al (1976). Lignin protein concentrations were evaluated in hardened black beans stored at 25°C (77°F), 70% RH for a maximum of nine months (Molina et al, 1976). As legume seed hardness increased, lignified cotyledon proteins increased (r - 0.91). The contradictory results reported by Srisuma et al (1989) and Molina et al (1976) may be attributed to differences between lignin extraction procedures. Protein- lignin cross-linking bonds have been reported (Whitmore, 1978). Bond-forming reactions of some carbohydrates and proteins toward coniferyl alcohol dehydrogenation polymer (DHP) (lignin) revealed strong and acidic stable linkages between DHP and proteins (containing hydroxyproline). Hydroxyproline is an important constituent of plant cell proteins. Whitmore (1978) proposed that cell wall proteins containing hydroxyproline cross-linked with DHP through oxidative reactions (involving peroxidase) before massive lignification took place. Protein-lignin cross-linking would reduce extractable lignin, unless the protein-lignin bonds were disrupted prior to/or during the lignin extraction procedure. Molina et al (1976) used a gravimetric method to determine lignin concentrations, while Srisuma et al (1989) followed a spectrophotometric 20 procedure employing numerous lignin extractions, to purify the polymers, followed by lignin derivatization. A more sensitive and simple method is required to minimize or eliminate lignin losses during purification steps. Hohlberg and Stanley (1987) studied the fate of black bean intracellular starches and proteins in relation to bean textural changes during various storage situations (30°C or 85°F, 85% RH: 25°C or 77°F, 55% RH: 15°C or 59°F, 35% RH) for a maximum of ten months. Similarities were found in moisture content, water absorption and hardness of soaked beans following four months of storage among the three environments. No hardshell defect was apparent in any storage situation. However, hardness of cooked legume seeds was significantly different (p 5 0.05) for the storage conditions. Beans maintained at 30°C (86°F), 85% RH and at 24°C (77°F), 65% were reported to have promoted the hard-to-cook defect following three months of storage. There was a significant decrease (P): 0.05) in phaseolin proteins, and a significant increase in small polypeptides (P 5 0.05) with advanced storage time. Hohlberg and Stanley (1987) proposed that phaseolin subunits were possibly hydrolyzed either enzymatically or non- enzymatically after prolonged storage under unfavorable environmental conditions. Protease activities were observed in bean cotyledon extracts. Peroxidase activities were similar among samples stored under the three storage conditions whereas no polyphenol oxidase (catecholase) 21 activities were apparent in any of the protein extracts. However, there was a significant increase (P 5 0.05) in the percentage of free aromatic amino acids as storage time lengthened. Within the bean cotyledon (during storage) protein hydrolysis in conjunction with the presence of aromatic free amino acids and peroxidase activities, represented common steps for polymerization and/or lignification reactions in plants (Hohlberg and Stanley, 1987). The data reviewed signify the fact that the legume seed hard-to-cook defect is controlled by multi-mechanisms, directed by numerous enzyme reactions (phytase, proteases, PME, and peroxidases) with several substrates (Phytic acid, pectic substances, phenolic compounds) and products (minerals, aromatic amino acids, and other lignin monomers including coniferyl alcohol and related alcohols) (Hon, 1979: Ron and Sanshuck, 1981: Jones and Boulter, 1983: Moscoso et al, 1984: Hincks and Stanley, 1986/1987: Hohlberg and Stanley, 1987: Whitmore, 1978). These series of reactions leading to the hard-to-cook defect occur in the middle lamella and cotyledon cells of beans and other legume seeds stored under high temperatures (z 70°F or 21°C) and high humidities (z 70% RH). The research reported in this dissertation was initiated to provide more data to evaluate the phytic acid degradation and lignin formation mechanisms (Ron and Sanshuck, 1981: Jones and Boulter, 1983: Vindiola et al, 1986: Hohlberg and Stanley, 22 1987), as directing the legume seed hard-to-cook defect of hard and soft Malawian beans under tropical storage environments. 23 REFERENCES Bressani, R., Elias, L.G. and Navarrete, D.A. 1961. Nutritive value of Central American beans. IV. The essential amino acid content of samples of black beans, red beans, rice beans, and cow peas of Guatemala. J. Food Sci. 26: 525-528. Burr, H. R., Ron, S. and MOrris, H.J. 1968. Cooking rates of dry beans as influenced by moisture content, temperature and time of storage. Food Technology. 22:336-338. Chang, R. and Schwimmer, S. 1977. Characterization of phytase of beans (Bhaseelus xulgaris)- J- Food Biochem. 1: 45-56. Dahodwala, 8., Humphrey, A. and Weibel, H. 1974. Pectic enzymes: Individual and concerted kinetic behavior of pectinesterase and pectinase. J. Food Sci 39: 920-926. Delincee, H. and Radola, B.J. 1970. Some size and change properties of tomato pectin methylesterase. Biochem. Biophys. Acta. 214: 178-189. Freudenberg, K. and Neish, A.C. 1968. ”Constitution and Biosynthesis of Lignin in Molecular Biology, Biochemistry and Biophysics. Vol. 2,” Springer-Verlag New York Inc., New York, NY. Henderson, H.H. and Ankrah, S.A. 1985. The relationship of endogenous phytase, phytic acid and moisture uptake with cooking time in yigia fab; mingr cv. Aladin. Food Chem. 17:1-11. Hincks, H.J. and Stanley, D.W. 1986. Multiple mechanisms of bean hardening. J. Food Technology 21: 731-750. Hinks, H.J. and Stanley, D.W. 1987. Lignification: Evidence for a role in hard-to-cook beans. J. Food Biochem. 11:41-58. Hohlberg, A.I. and Stanley, D.W. 1987. Hard-to-cook defect in black beans. Protein and starch considerations. J.Agric. Food Chem. 35:571-576. Jones, P.M.B. and Boulter, D. 1983. The cause of reduced cooking rate in Enasgglns ynlgaris following adverse storage conditions. J. Food Sci. 48: 623-626, 649. 24 Ron, 8. 1979. Effect of soaking temperature on cooking and nutritional quality of beans. J. Food Sci. 44: 1329- 1334, 1340. Ron, 8. and Sanshuck, D.W. 1981. Phytate content and its effect on cooking quality of beans. J. Food Process. and Preserv. 5:169-178. Lolas, G.H. and Markakis, P. 1977. The phytase of navy beans (Ehasgglns ynlgarig). J. Food Sci. 42: 1094- 1097, 1106. Markovic, 0., Machova, E. and Slezarik, A. 1983. The ' action of tomato and pectinesterases on oligomeric substrates esterified with diazomethane. Carbohydrate Research. 116: 105- 111. Mattson, S., Akerberg, E., Eriksson, E., Koutler-Anderson, E. and Vahtras, K. 1951. Factors determining the composition and cookability of peas. Acta Agric. Scand. 11: 40-61. Molina, M.R., Baten, M.A., Gomez-Brenes, R.A., King, K.W. and Bressani, R. 1976. Heat treatment: A process to control the development of the hard-to-cook phenomenon in black beans (Ehgsgglug ynlgaris). J. Food Sci. 41: 661-666. Moscoso, W., Bourne, M.C. and Hood, L.F. 1984. Relationships between the hard-to-cook phenomenon in red kidney beans and water absorption, puncture force, pectin, phytic acid and minerals. J. Food Sci. 49: 1577-1583. Moustacas, A.M., Nari, J. Diamantidis, G., Noat, G., Crasnier, H., Borel, H. and Ricard, J. 1986. Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells 2. The role of pectin methylesterase in the modulation of electrostatic effects in soybean cell walls. Eur. J. Biochem. 155: 191-197. Peers, F.G. 1953. The phytase of wheat. Biochem J. 53: 102- 110. Richard, J. and Noat, G. 1986. Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells 1. A theory of the ionic control of a complex multi-enzyme system. Eur. J. Biochem. 155: 183-190. Schubert, W.S. 1965. ”Lignin Biochemistry,” Academic Press, New York, NY. 25 Schwimmer, S. 1981. "Source book of food enzymology." The AVI Publishing Company, Inc. , Westport, CT. Sievwright, C.A. and Shipe, W.F. 1986. Effect of storage conditions and chemical treatments on firmness, invitro protein digestibility, condensed tannins, phytic acid and divalent cations of cooked black beans (Ehfififiglflfi xylgaris). J. Food Sci. 51: 982-987. Srisuma, N., Hammerschmidt, R., Uebersax, M.A., Ruengsakulrah, s., Bennink, M.R., and Hosfield, G.L. 1989. Storage induced changes of phenolic acids and the development of hard-to-cook in dry beans (Ehafigglng ynlgarifi, var. seafarer). J. Food Sci. 54: 311-314, 318. Varriano-Harston, E.V. and Jackson, G.H. 1981. Hard-to-cook phenomenon in beans: Structural changes during storage and imbibition. J. Food Sci. 46: 1379-1385. Versteeg, C., Rombouts, F.M. and Pilnik, W. 1978. Purification and some characteristics of two pectinesterase isoenzymes from orange. Lebensm.-Wiss. U. - Technol. 11: 267-274. Vindiola, O.L., Seib, P.A. and Hoseney, R.C. 1986. Accelerated development of the hard-to-cook state in beans. Cereal Chem. 31: 538-552. Whitmore, F.W. 1978. Lignin-protein complex catalyzed by peroxidase. Plant Science Letters. 13: 241-245. CHAPTER 1 DRY BEAN (Hussein: 303195115) HARDENING DURING STORAGE: ROLE OF PHYTASE AND LIGNIN IN HARD AND SOFT MALAWIAN BEAN LANDRACES 26 ABSTRACT DRY BEAN (W W) HARDENING DURING STORAGE: ROLE OF PHYTASE AND LIGNIN IN HARD AND SOFT HALAWIAN BEAN LANDRACES Phytic acid degradation and lignin formation mechanisms in the hard-to-cook phenomenon of decorticated Malawian red (hard) and white (soft) beans (Ehgsgglng ynlggris) were evaluated. Samples were stored under various temperatures (60°F or 15.6°C, 95°F or 35°C), humidities (55% RH, 85% RH) and time periods (four and eight months) and compared to controls (35°F or 2°C, 30% RH, zero months). Phytase activities (Pi), phytic acid, calcium and magnesium ions, and lignin concentrations were determined spectrophotometrically. Microkjeldahl procedures were used for total protein determinations. Elevated phytase activities and slight increases in lignin levels were produced in both bean landraces held under adverse storage conditions for extended time periods. Positive correlations between cooked bean hardness and phytase activities (r - 0.83), and among legume hardness and lignin levels (r - 0.71) were found. The phytic acid degradation mechanism appeared to be the dominant system directing the hard-to-cook defect in the legume varieties. This is evidenced by increased activities of phytase which were positively correlated to cooked bean texture. 27 28 INTRODUCTION Prior studies have established that legume seeds stored at elevated humidities (z 70% RH) or containing high moisture contents (2 14%) and high temperatures (z 70°F or 21°C) become hard in texture resulting in the hard-to-cook defect (Ron and Sanshuck, 1981: Moscoso et al, 1984). Hard-to-cook has been defined as an irreversible condition where bean cotyledons fail to soften sufficiently during typical food preparation procedures (heating at 99°C or 210.2°F for 60 min) even though the seeds have imbibed water (Jones and Boulter, 1983a: Vindiola et al, 1986). Hardened beans have exhibited extended cooking times, hence increased processing energy expenditures, and reduced protein digestibilities (Burr et al, 1968: Ron and Sanshuck, 1981: Jones and Boulter, 1983a: Sievwright and Shipe, 1986). Tropical geographic areas, such as Malawi, have encountered the hard-to-cook condition, since environmental temperatures and humidities are elevated (z 21°C or 70°F and z 70% RH) (Burr et al, 1968: Malawi Heteology Dept, 1986). Two hard-to-cook mechanisms have been postulated. Supporting literature is reviewed. A proposed phytic acid degradation mechanism involves the hydrolysis of phytic acid by phytase (myo-inositol- hexakisphosphate phospho-hydrolase, EC 3.13), thus releasing Pi, magnesium and calcium ions within cotyledon cells. Concomitantly, within the middle lamella, pectin methylesterase (PME) hydrolyzes pectin to pectinic acid and 29 methanol. The calcium and magnesium ions are considered to migrate from the cotyledon to the middle lamella to produce calcium and magnesium pectates that subsequently bind the legume seed cells. The role of PME in the postulated phytic acid degradation mechanism remains to be determined (Mattson et al, 1951: Lolas and Markakis, 1977: Ron, 1979: Ron and Sanshuck, 1981: Jones and Boulter, 1983a: Hoscoco et al, 1984: Hincks and Stanley, 1986: Vindiola et al, 1985). As storage time advanced, lower phytic acid (Mattson et al, 1951: Moscoso et al, 1984) and water soluble pectic substance levels (Moscoso et al, 1984), elevated calcium and magnesium concentrations (Ron and Sanshuck, 1981: Moscoso et al, 1984), reduced legume seed cookability (Mattson et al, 1951: Ron and Sanshuck, 1981) and firmer bean textures (Moscoso et al, 1984) were found in several legume varieties held under high temperature and humdity conditions. A negative correlation between phytic acid/calcium ratios and length of cooking was established (Ron and Sanshuck, 1981). Cooking times have been reduced by steeping hard-to-cook legume seeds in phytic acid and/or EDTA, but legume seed firmness was not reversed (Ron and Sanshuck, 1981). The steeped seeds demonstrated enhancement in cell separations which implied modifications in the middle lamella pectins when phytic acid/calcium ratios were sufficient (Ron and Sanshuck, 1981). 30 Ron and Sanshuck (1981) deduced that another system must exist besides the postulated phytic acid degradation hard-to-cook mechanism, because of the incomplete reversal of the hard-to-cook defect when legumes were soaked in solutions containing metal chelators. Therefore, a lignin formation mechanism was postulated. Stanley's group has . hypothesized that small polypepetides and free aromatic amino acids were hydrolyzed from large proteins leading to polyphenol synthesis. The phenolic substances traverse from the cotyledon to the middle lamella where the phenolic compounds were lignified by peroxidase (Hincks and Stanley, 1986: Hohlberg and Stanley, 1987). Hard-to-cook black beans have shown reduced cell separations (Hincks and Stanley, 1986), high lignin concentrations in the cell wall corners, secondary cell walls and middle lamella (Hincks and Stanley, 1987), increased lignified cotyledon proteins (Molina et al, 1976), reduced phaseolin protein levels (Hohlberg and Stanley, 1987), raised small polypeptide and free aromatic amino acid concentrations (Hohlberg and Stanley, 1987), decreased phytic acid and extractable phenol contents (Hincks and Stanley, 1986), and increased phytase activities (Hincks and Stanley, 1986). The reduction in extractable phenols may be partially due to increased phenol polymerization (Hincks and Stanley, 186). Within the bean cotyledon during suboptimal storage conditions, protein hydrolysis products in the presence of free 31 aromatic amino acids and enzymatic activities, may represent polymerization and/or lignification reactions in plant tissues (Hohlberg and Stanley, 1987). Srisuma et al (1989) found no differences in navy bean cotyledon and seed coat lignin levels, and concluded that enhanced legume seed lignification was not a major determinant of the hard-to- cook defect. The phytic acid degradation theory seems to predominate during early storage periods (two to four months). In contrast, the lignin formation mechanism appears to prevail during later storage periods (> eight months). Four to eight months storage was advocated as the’ transition period for the two hard-to-cook defect theories (Hincks and Stanley, 1986, 1987). The current study examined several indicators in the proposed phytic acid degradation and lignin formation hard- to-cook defect theories, in order to elucidate the contributions of these indicators in the development of legume seed hardening. The parameters investigated included: phytase activities, lignin, phytic acid, pectic substances, total protein, calcium and magnesium ion concentrations. Changes in these indicators were monitored in soft and hard decorticated Malawian common dry bean landraces (Ehgsgglg§,ynlg§11§) over an eight month storage period. Several hypotheses were addressed. Decorticated beans maintained under high temperature (95°F or 35°C) and high relative humidity (85% RH) environments over eight 32 months would have higher cotyledon phytase activities and lignin concentrations, as compared to the legume seed controls stored at 35°F (2°C) and 30% RH. There would also be a decrease in cotyledon water soluble pectic substances with an accompanying increase in calcium and magnesium ion levels, resulting in increased legume seed hardness. It was also hypothesized that both hard and soft Malawian beans would exhibit similarities in hardening alterations when held under the same storage conditions. Finally, it was hypothesized that the development of the hard-to-cook defect under suboptimal storage conditions would be 7 directed predominantly by the phytic acid degradation mechanism, as opposed to the lignin formation mechanism over the eight month storage period. This is due to activities of phytase which would increase during the first eight months of storage under suboptimal conditions. These phytase activities would be positively correlated to cooked bean texture. 33 IHKTERIALS.AND1HETEODS Environmental Conditions Controlled environmental cubicles located within the Department of Food Science and Human Nutrition at Michigan State University were temperature adjusted to 35°F (2°C), 50°F (15.5°C) and 95°F (35°C) 1 2° prior to initiation of the legume seed storage study. High density polyethylene five gallon containers, equipped with tight fitting lids (Cole-Parmer Instrument Co., Chicago, IL) were placed in the temperature controlled cubicles. Humidities within each container were regulated by the use of saturated salt solutions, except for the control samples. Preliminary work showed the relative humidities within the polyethylene containers held in the refrigerated (35°F or 2°C). Environmental cubicles, average 30% RH i 5%, which translated to 10% moisture content of beans. Saturated salt solutions of potassium chloride and magnesium nitrate (certified ACS, Fisher Scientific Co., Livonia, M1) were used to control and maintain relative humidities at 85% and 55% i 5%, respectively. The saturated salt solutions were prepared according to the Labuza (1984) method. Salt solution choices were based on moisture-sorption isotherm (see Appendix) pilot study results for decorticated bean samples, and equilibrium relative humidity saturated salt solution data reported by Labuza (1984). A digital hygrometer-thermometer (Fisher Scientific Co., Livonia, MI - model No. 11-661-71) was used '34 to measure relative humidities and temperatures within the containers weekly for the first month, two times per month the second and third months, and one time per month during the remaining storage period following initial sample equilibrium. After two months of storage, one time per month humidity and temperature recordings were considered sufficient to monitor and minimize fluctuations in the environmental conditions. The bean storage container lids were modified so as to allow the hygrometer-thermometer sensing probe to be inserted through the top of the lid. This opening was sealed with a rubber stopper when measurements were completed. The storage conditions chosen were based on work by Burr et al (1968), Moscoso et al (1984), Varriano-Marston and Jackson (1981) and Ron and Sanshuck (1981). These researchers demonstrated that beans stored at high temperatures (>70°F) and moderate to high relative humidities (65%-70%) exhibited the hard-to-cook phenomena. In addition, the storage conditions were chosen as relevant and applicable to weather conditions in Malawi where temperatures and relative humidities range from 55°F-82°F (13°C-28°C) and 48%-92%, respectively (Malawi Meteorology Dept., 1985). Sample Preparation Two Malawian bean landraces (varieties), one a small red bean (Acc:6-5) with a thick seed coat (hard1 bean), and the other a large white bean (Acc:2-10) with a thin seed 35 coat (soft1 bean), were generously provided by the Malawi (Africa) Bean/Cow Pea Collaborative Research Project at Michigan State University, East Lansing, MI. A single layer of beans (500 g) was placed in steel wire net baskets constructed in our laboratory. The baskets were suspended approximately four inches above 500 ml deionized water and placed in a desiccator. Beans were allowed to humidity for 3 to 4 days until seed coats loosened. Following humidification, the seeds were sprinkled with ambient temperature deionized water (50 ml H20/500 g seed) to enhance loosening of the seed coat. Seeds were decorticated by hand with the aid of a surgical blade. Upon decortication, seeds were air dried at room temperature for three days. A pilot study demonstrated that previously humidified decorticated beans were similar in moisture content (9.5% - 10%) to decorticated beans (not humidified) following three days of air drying at room temperature. The dried seeds were kept in the lidded polyethylene containers under refrigeration (35°F or 2°C) 1Classification based on responses from Malawian women through interviews conducted by the Malawi Bean/Cow Pea CRSP Project (Personal Communication). These classifications were confirmed by the preliminary data on cooked bean texture in our laboratory which revealed the Malawian hard bean to be about 30% harder than the soft seed (based on "Kramer Shear Press” forces). 36 until initiation of the storage study. All legume seed samples were decorticated prior to storage to eliminate hard shell effects. Experimental Design The experiment was a four-factor factorial model in a completely randomized design where time x temperature x ' humidity x variety were fixed.. All experimental treatments within the experimental design are shown in Table 1. Note that the control was not part of the factorial arrangement. The control was compared to means within the factorial arrangement using the least significant difference (LSD) test (Little and Hills, 1978: Steel and Torrie, 1980). The nine treatment combinations for each landrace were replicated twice. Thirty-six (400 g) decorticated dry bean samples (18 red and 18 white landraces for each replicate) were weighed to the nearest 0.01 g and put into low density polyethylene zip-loc coded bags (6 1/2” x 5 1/2", 1.15 ml thick). Legume samples within each replicate for each variety were randomly assigned to the nine treatment combinations. After randomization, all bagged legume seed samples were treated with approximately 5 g Captan TM dust to control/inhibit mold growth prior to initiation of the storage study. All bean samples were analyzed for the legume seed hard-to-cook indicators following the experimental storage treatments. 37 Legume Flour Preparation All hard-to-cook indicators, except cooked bean texture, were determined using bean flour samples. Legume flour was obtained by grinding a 50 g dry bean sample for five min (80 mesh) using a Braun mill (Model No. KSM2, Lynnfield, MA). Phytic Acid Chemicals and supplies used for the phytic acid determination included: trichloroacetic acid (ACS certified, Baker Chem. Co., Phillipsburg, NJ): ferric chloride, sodium sulfate, sodium acetate (ACS certified, Mallinckrodt Inc, Paris, KY): hydroxylamine hydrochloride (ACS certified, Aldrich Chem. Co., Milwaukee, WI): NaOH (1N Soln. Mallinckrodt Inc, Paris, Ky): HCl (ACS Certified, Fisher Scientific, Livonia, MI): Orthophenanthroline (ACS Certified, Sigma Chem. Co., St. Louis, MO): ferrous ammonium sulfate and ferric nitrate (Pfs, Sigma Chem, Co., St. Louis, MO). Phytic acid and derivatives (phytin) were extracted following the methods of Wheeler and Ferrel (1971) as modified by Lolas and Markakis (1975). Phytic acid concentrations were determined spectrophotometrically at 510 nm, using a UV/visible spectrophotometer (Model 4050, LKB Biotechnology Inc, Gaithersburg, MD). A ferrous ammonium sulfate standard curve was developed relating its concentration to absorbance at 510 nm. A 4:6 Fe/phosphorous atomic ratio was used to 38 calculate bean phytic acid content (Wheeler and Ferrel, 1971). Extraction and Chemical Analysis Finely ground (1 g) legume seed flour was thoroughly mixed with 20 ml 3% trichloroacetic acid (TCA) in a stoppered erlenmeyer flask (250 mL). The slurry was extracted at 60°C (140°F) for 45 min with constant stirring followed by centrifugation at 13,000 RPM (20,202 x G) for ten min (Sorvall centrifuge model RC2-B, Du Pont Co., Hoffman Estates, IL). A ten ml aliquot of supernatant was transferred into a conical centrifuge tube (40 mL) and 5 ml FeCl3 solution (2 mg ferric iron per ml in 3% TCA) was added. The sample was heated for 60 min at 95 - 100°C (203-212°F) in a boiling water bath, with periodic swirling. After 30 min, 3% sodium sulfate in 3% TCA (1-2 drops) were added whenever the supernatant was cloudy. Again, the mixture was centrifuged (10 min, 13,000 RPM), and all of the clear supernatant fraction was decanted. The ferric phytate precipitate was washed (3 times) with 20 ml 3% TCA, followed by a deionized water (once) washing step. In between each washing, the samples were heated for 5 min in a boiling water bath followed by another centrifugation step at 13,000 RPM for 10 min (Wheeler and Ferrel, 1971). Five ml NaOH (0.6N) was added. To coagulate Fe(OH)3, the mixture was heated for 45 min in a boiling water bath, followed by centrifugation (13,000 RPM for 10 min). This precipitate was dissolved in 5 ml HCl 39 (0.5N) while heating in a boiling water bath (10-15 min). The dissolved Fe(OH)3 was transfered to a volumetric flask, (100 ml), and brought up to volume with HCl (0.1N). The solution (1 ml) was transferred to a 25 ml volumetric flask, then one ml hydroxylamine hydrochloride (10%) solution was added, mixed thoroughly and allowed to stand at room temperature (5 min). The mixture was brought to volume using sodium acetate (9.5 ml, 2M), 1 ml orthophenanthroline (0.1%) and HCl (0.1N). The solution was mixed and allowed to stand (5 min) before reading the absorbance at 510 nm. HCl (0.1N) was used for the sample blank (Lolas and Markakis, 1975). Phytase Assay Phytase activities were measured indirectly by determining bean cotyledon Pi ion concentrations following the methods of Watanabe and Olsen (1965), Murphy and Riley (1962), and Pons and Guthrie (1946). Inorganic Phosphate Chemicals, supplies and equipment for the inorganic phosphate method included: ammonium molybdate (ACS certi- fied, Mallinckrodt, Inc, Paris, KY). Antimony potassium tartrate, ascorbic acid (USP, Mallinckrodt, Inc, Paris, KY): H2804 (ACS certified, EM Industries, Inc, Gibbstown, NJ): trichloroacetic acid (ACS certified, Baker, Inc, Phillipsburg, NJ): phosphorus (Standard solution, 20 Mg/ml, Sigma Chem. Co., St. Louis, MO): and a spectronic 40 Spectrophotometer (model 21D, Milton Roy Co., Rochester, NY). Inorganic phosphate was extracted from legume seed flour following the methods of Watanabe and Olsen (1965), Murphy and Riley (1962), and Pons et al (1946). Absorbance was measured spectrophotometrically at 730 nm. An inorganic phosphate standard curve was produced relating concentration to absorbance at 730 nm. TCA (0.75N) served as the sample blank (Pons and Guthrie, 1946). Extraction and Chemical Analysis Finely ground (0.5 g) legume seed flour was mixed thoroughly using 25 ml TCA (0.75N) at room temperature (25°. or 77°F for 60 min) under continuous stirring. The mixture was then centrifuged at 13000 RPM for 10 min and the supernatant (2 ml) was placed in a 25 ml volumetric flask. Five ml of a reagent composed of ammonium molybdate, antimony potassium tartrate, H2804, and ascorbic acid was added to the supernatant fraction. The solution was mixed, brought up to volume with deionized water, and allowed to stand for 10 min at room temperature. Absorbance was read at 730 nm (Pons and Guthrie, 1946: Watanabe and Olsen, 1965: Murphy and Riley, 1962). Total Pectic Substances Extraction Procedure Chemicals and supplies for the extraction of legume seed total pectic substances included: Calgon (Beechum Products, Pittsburgh, PA): HCl (ACS certified), and Celite 41 (analytical filter aid), (Fisher Scientific Co., Livonia, MI): ground pulp (Scheicher and Schuell, Inc, Keen, NH), and macroporous filter paper (pore size: 10-2000 um, Spectrum Medical Industries, Inc., Los Angeles, CA). A digital pH meter (Corning model No. 610A, Corning Co., Medfield, MA) was used to measure sample pH. Total pectic substances were extracted from legume seed flour following the method of Owen et al (1952). Ground seed flour (1 g) was thoroughly mixed with deionized water (40 mL) in a stoppered erlnemeyer flask (250 mL). Calgon (0.12 g) was added followed by pH adjustment to 4.5 with 1.3 ml HCl (0.25N) and NaOH (0.1N) as necessary. The mixture was heated slowly to 203°F (95°C) and maintained at this temperature for 1 hour. Celite filter aid (0.4 g) and ground paper pulp (0.4 g) were added. The mixture was then rapidly filtered by suction through macroporous (2-2000 mm pore size) filter paper (Owen et al, 1952). Chemical Analysis Chemicals and supplies for the determination of legume seed total pectic substances included: NaOH (1N standard solution, Mallinkrodt, Inc, Paris, KY): H2804 (ACS certified, EM Industries, Inc, Gibbstown, NJ): ethanol (Absolute, USP, AAPER Alcohol and Chem. Co., Shelbyville, KY): and carbazole (Pfs, Sigma Chem. Co., St Louis, MO). Following extraction, total pectic substances were analyzed chemically following the method of Owen et al (1952). Total pectic substance concentrations were 42 measured spectrophometrically at 520 nm (Model 4050, LKB Biotechnology, Inc: Gaithersburg, MD). A standard curve was produced relating galacturonic acid hydrate (MW - 212) concentrations to absorbances at 520 nm. NaOH (0.05N) served as the sample blank. The filtrate (1 ml) was added to 9 ml NaOH (0.05N) in a 50 ml volumetric flask, mixed, . and allowed to stand for 30 min at room temperature (25°C or 77°F), then diluted to volume with NaOH (0.05N). The diluted sample (2 ml) was added to ice cold concentrated H2804 (12 ml) in a test tube (25 x 180 mm), vortexed, and heated for 10 min in boiling water bath. The sample was cooled to 68°F (20°C) using an ice bath. Ethanol (1 ml) containing carbazole (1.5 mg) was added, the solution vortexed and allowed to stand (15 min) at room temperature. Color intensity was measured spectrophotometrically at 520 nm (Owen et al, 1952). Water Soluble Pectic Substances Legume seed water soluble pectic substances were determined from the total pectic substances spectro- photometrically at 520 nm by the method of Owen et al (1952). Additional chemicals and supplies for the seed water soluble pectic substance method included: H2804 (ACS certified, EM Industries, Inc, Gibbstown, NJ): Sodium tetraborate (CAS, electrophoresis grade, Fisher Biotech, Fisher Scientific Co., Fair Lawn, NJ): and meta- hydroxydiphenyl (Lot No. C17A, Eastman Kodak Co., Rochester, NY). A water soluble pectic substance 43 galacturonic acid hydrate, (MW - 212) standard curve, relating absorbance at 520 nm to concentration was determined (Owen et al, 1952). NaOH (0.5%) was used for the blank sample. Chemical Analysis The legume seed total pectic substance extraction procedure has been previously described under "Total pectic substances extraction procedure" in this chapter. A sodium tetraborate solution (0.0125M in conc H2804) was added to the total pectic substance filtrate (1 ml) in a stoppered test tube. The mixture was cooled for 10 min in crushed ice, vortexed, heated in a boiling water bath for 5 min at 95-100°C (203-212°F), and again cooled in an ice bath for 20 min (20°C or 58°F). Meta-hydroxydiphenyl (100 microliters) was then added, the sample vortexed and absorbance was read within 5 min at 520 nm (Owen et al, 1952). Calcium and Magnesium ions Chemicals and supplies for the legume seed calcium and magnesium ion determination included: HCl and mannitol (ACS certified, Fisher Scientific Co., Livonia, MI): lanthanum chloride (ACS certified, sigma Chem. Co., St. Louis, MO): calcium (Atomic absorption standard solution: 1000 mg/ml 1% HCl) and magnesium (Atomic absorption standard solution: 1015 mg/ml 1% HNO3 ) (Pfs, Sigma Chem. Co., St. Louis, MO). Equipment for calcium and magnesium determination included: 44 a spectrophotometer (Atomic absorption, model 2380, Perkin-Elmer Co., Norwalk, CT): centrifuge (Model EN 811, International Equipment Co. , Needhamhts, MA): a vacuum drying oven (model No. FF3174X, Lablime, Inc, Chicago, IL): and a muffle furnace (Model FF470/471, Thermolyne Corporation, Dubuque, Iowa). Legume seed calcium and magnesium ions were extracted from seed flour (0.2759) following the method of Jones and Boulter (1983a). The minerals were determined spectrophotometrically at 422.7 nm and 285.2 nm for calcium and magnesium, ions respectively. Standard curves relating legume seed calcium and magnesium ion absorbances to ion concentrations were determined. Deionized water served as the sample blanks. Calcium and Magnesium Ion Extraction and Chemical Analysis Finely ground legume seed flour samples (0.275 g), were thoroughly mixed with 20 ml mannitol (0.33M), centrifuged at 2,000 RPM for four min and the supernatant decanted. The precipitate was washed repeatedly with several solutions (mannitol, deionized water and acetone) to obtain cell wall and starch residue (Jones and Boulter, 1983a). The residue was vacuum oven dried at 70°C (158°F) overnight, ashed at 550°C (1022°F) for 72 hours, and cooled. Ten ml HCl (3N) was added to the ashed samples. Solutions were transferred to erlenemeyer stoppered flask (25 ml), boiled gently for 10 min and cooled to room temperature (25°C or 77°F). Mixtures were then filtered 45 (No. 1 Whatman filter paper) into 100 ml volumetric flasks. Twenty ml lanthanum chloride (1%) was added, and the samples were diluted to volume with deionized water. Calcium and magnesium ions concentrations were determined using atomic absorption spectroscopy at 422.7 nm and 285.2 nm, respectively. Lignin Chemicals, supplies and equipment for the legume seed lignin determination included: methanol (Absolute, ACS certified, Baker, Inc, Phillipsburg, NJ): NaOH (1N standard solution, Mallinckrodt, Inc, Paris, KY): HCl (ASC certified, Fisher Scientific Co., Livonia, MI): thioglycolic acid (70%, Grade IV, Sigma Chem. Co., St. Louis, MO): and the following equipment: a centrifuge (Sorvall superspeed RC2-B automatic refrigerated, Du Pont Co., Hoffman estates, IL) and Spectrophotometer (UV/Visible, Model 4050, LKB Biotechnology, Inc, Gaithersburg, MD). Bean lignin was extracted from seed flour and measured spectrophotometrically at 280 nm following the method of Hammerschmidt (1984). A lignin standard curve was developed relating relative lignin absorbance at 280 nm (in potato suberin) to its concentration. NaOH (0.5N) served as the sample blank. 46 Extraction and Chemical Analysis Finely ground legume seed flour (2 g) was steeped in 10 ml absolute methanol in a stoppered erlenemeyer flask (25ml) and then placed in a shaker water bath at 22°C (71.6°F) for 48 hours. The methanol was decanted and replaced every 12 hours during soaking. Samples were air dried overnight. NaOH (0.5N) was added to the mixture followed by incubation at 22°C (71.6°F) for 16 hours in a shaker water bath to hydrolyze cell wall bound phenolic acids. After hydrolysis, 5 ml HCl (2N) was added to neutralize the slurry. The slurry was filtered by suction through a glass filter (medium porosity), and the residue was washed (four times) with 20 ml deionized water. A final wash was conducted with 20 ml methanol followed by air drying the sample. The dried residue was finely ground, using mortar and pestle, and then mixed with 4.2 ml thioglycolic acid (10%) plus 34 ml HCl (2N) in a stoppered conical centrifuge tube. The sample was heated at 95°C (203°F) for four hours using a water bath and then set aside to cool to ambient temperature. The cooled mixture was centrifuged (13,000 RPM for 10 min). The supernatant was discarded and the residue was washed one time with 34 ml deionized water before recentrifugation (13,000 RPM, 10 min). The washed precipitate was incubated in 5 ml NaOH (0.5N) for 16-18 hours to solubilize lignin-thioglycolic acid (LTGA). Again, the mixture was centrifuged (13,000 RPM, 10 min), the supernatant was collected, and the 47 precipitate was washed with deionized water (2 ml) and recentrifuged (13000 RPM), 10 min). The supernatant was collected and added to that previously assembled. The wash was repeated once with deionized water (2 ml). The pooled supernatants (NaOH extract and water) were collected and placed in a conical centrifuge tube, conc HCl ( 1 ml) was added and the acidified extract was allowed to precipitate under refrigeration at 4°C (39.2°F) for four hours. The refrigerated samples were recentrifuged (13,000 rpm, 10 min) and the supernatant discarded. The pellet was suspended in 2 ml HCL (0.1N), centrifuged (13,000 rpm, 10 min) and the supernantant was eliminated. Washing of the pellet in HCl (0.1N) was repeated again. The pellet was then dissolved in 5 ml NaOH (0.5N) and centrifuged (13,000 rpm, 3 min) to remove insoluble materials (non-LTGA). An aliquot of dissolved LTGA (0.16 ml) was diluted with NaOH (0.5N) to a 4 ml volume and its absorbance was measured spectrophotometrically at 280nm. Proteins Chemicals, supplies and equipment for the legume seed proteins included: Kjeltabs (Pot Sulf/se), boric acid, NaOH pellets, electrolyte (K+ and Cl', 100 mEq/liter) (ACS certified, Fisher Scientific Co., Livonia, MI): H2804 (ACS certified, EM Industries, Inc, Gibbstown, NJ). Equipment included the use of a digestor (Tecator digestion system 40 1016 digester, Fisher scientific Co., Livonia, MI): a Kjeldahl distillation and titration unit (Buch 322/342, 48 Brinkmann Instrument Co., Westbury, NY) a multi-Dosimat recorder (model 655, Metrohm AG CH-9100, Herisau, Switzerland) and a Mettler analytical balance 10.01 mg (Model 100, Fisher Scientific Co., Livonia, MI). Legume seed proteins were determined from seed flour following the Suhre et al (1982) microkjeldahl methods (AOAC actions 24.B01 - 24.803). Preliminary data using these methods revealed linearity with 6 to 8 ml of HCl (0.0987N). Digestion, Distillation and Titration Finely ground bean flour samples (0.3 g) were weighed into kjeldahl flasks, then one kjeltab and 5 ml conc H2804 were added into each sample. Seed four samples were predigested until the solutions were orange in color by increasing the temperature every 15 min. Then the solutions were heated at the highest digestor temperature for two hours or until clear, followed by sample cooling to ambient temperature. The distillation/titration unit reservoirs were filled with appropriate solutions which included electrolyte solution (15 ml), deionized water, NaOH (30%) and boric acid (4%). The control unit was then preheated for two min. The pH of a 4% boric acid solution was measured in receiving vessel (60 ml) to mark the titration end point. Distillation and titration proceeded. The amount of HCl (0.0987N) used for sample titration was displayed and recorded at the titration and point. Legume seed protein contents were determined as follows: 49 % Protein - (VA'Va) x 1.4007 x N x 6.25 where VA and Va - Vol. Std HCl required for sample and blank, respectively: 1.4007 - millieq. wt of nitrogen x 100(%) N - Normality of Std HCl - 0.0987 6.25 - Protein factor (16% Nitrogen) Kjeltab plus conc H2804 (5 ml) were used as sample blanks. Moisture Supplies and equipment for the legume seed moisture content determination included: desicator, aluminum drying trays (4.5 inch dia) (Fisher Scientific Co., Livonia, MI) and vacuum drying oven (Model FF3173X, Hot pack Co., Philadelphia, PA). Moisture contents were indirectly measured using decorticated beans (2g) following the A.A.C.C (1989), Jones and Boulter (1983b), and Hincks and Stanley (1986) methods. In preliminary work, legume seed moisture contents were shown to be constant after vacuum oven drying for 48 hours at 98°C (208.4 °F). Decorticated bean samples were weighed (29) into pre- weighed aluminum drying trays and placed in a desicator until oven dried (A.A.C.C., 1989). The samples were dried in a vacuum (25 - 28 mm Hg) oven at 98°C (208.4°F) for 48 hours. Dried samples were placed in a desiccator, and allowed to cool to room temperature (A.A.C.C., 1989) before weight losses were measured. Moisture contents were 50 calculated as follows: Fresh wt - Dry wt % moisture - ----------------- x 100 Dry wt Statistical Analyses The effects of time, temperature, humidity and variety upon phytase activities (Pi of cotyledon), moisture, phytic acid, calcium, magnesium, water soluble and total pectic substances, protein and lignin contents and phytic acid/calcium ratios of beans were analyzed statistically using analysis of variance (ANOVA) via the MSTAT statistical software package (Freed et al, 1985). The Least Significant Difference (LSD) test was used for multiple comparisons among means (Little and Hills, 1978: Steel and Torrie, 1980). Where significance was indicated, the P 5 0.05 level was meant, unless otherwise stated. Linear correlation coefficients were calculated to determine the relationships between cooked bean texture (hardness) and phytase activities, and between cooked legume seed texture and lignin contents (Little and Hills, 1978: Steel and Torrie, 1980). 51 RESULTS AND DISCUSSION Moisture Moisture content (dry wt basis) results (Table 2) demonstrated that legume seed moisture was significantly (P 5_0.01) influenced by storage conditions (time, temperature, RH). In addition, there were significant time x RH and temperature x RH interactions. The significant interactions implied that the effects of storage time and temperature on seed moisture contents were dependent upon the level of relative humidity, and those of relative humidity were dependent upon the level of temperature and length of storage. Furthermore, bean storage conditions (time, temperature, relative humidity) determined the moisture level within the legume seed. The relationship between storage conditions and legume seed moisture contents could not be attributed to either, time, temperature or relative humidity in isolation. Similar data were reported by Sefa-Dedeh et a1 (1979). These workers found a general increase in moisture contents of cowpeas (yigna nngnignlata) stored for a maximum of 12 months under higher temperature (29°C or 84.2°F) and humidity (85% RH) as compared to cowpeas stored at lower temperatures and humidities (21°C or 69.8°F) and 35% RH. The relationship between storage time, temperature and relative humidity factors and legume seed moisture contents is shown (Fig. 1). Beans stored at 85% RH for four and eight months exhibited significantly higher (P 5 0.05) 52 moisture contents as temperature was raised from 60°F (15.5°C) to 95°F (35°C). These highly humidified (85% RH) seeds also contained significantly (P 5 0.05) greater moisture levels than the control seeds stored at 35°F (2°C) and 30% RH. In addition, beans stored at 95°F (35°C) for eight months contained significantly (P‘s 0.05) higher levels of moisture than beans stored at the same temperature for four months. These data agree with results of Hincks and Stanley (1986) where black bean moisture contents were raised to 16.5% (dry wt basis) when stored at 30°C (86°F) and 85% RH for ten months as compared to control legume seeds (9% moisture) held at 15°C (59°F) and 35% RH. Higher moisture contents of stored beans were achieved at higher temperatures and longer storage periods. These data were anticipated since RH is the ratio between the water vapor mole fraction of a moist air sample, and the water vapor mole fraction of a saturated air sample at the same temperature and pressure (Singh and Heldman, 1984). Moisture-sorption isotherm results (Appendix) previously obtained in our laboratory substantiated the bean moisture content findings since legume seed moisture contents advanced as relative humidities were raised (see Appendix). Phytase Activity (Pi) Biochemical reactions that result in Pi production within bean cotyledons include some of the following: 53 a) Phytic acid ___________ Phytase > Inositol + Pi + Ca, Mg (Hincks and Stanley, 1986: Ron, 1979: Lolas and Markakis, 1975/77: Peers, 1953). b) Respiration reactions such as: ATP --------- > ADP + Pi + Energy (Smith et al, 1983). In dry seeds, respiration reactions are minimal. Following moisture imbibition (during germination), both phytase activities and respiration rates rise (Bonner and Varner, 1965: Lolas and Markakis, 1977: Peers, 1953). storage of beans under high temperature (z 70°F or 21°C) and humidity (z 70% RH) conditions for extended periods has been reported to lead to increased phytase activities (Hincks and Stanley, 1986). Respiration reaction rates in stored legume seeds would be expected to rise slightly when held at high temperature and humidity environments, due to the high moisture content resulting from the high humidity. Pi ANOVA data from stored decorticated beans are shown (Table 2). Pi was significantly influenced by storage time, temperature and variety factors (P 5_0.01). Furthermore, there were significant (P): 0.05) time x temperature x variety and humidity x variety interactions, indicating that the effect of storage time on Pi from legume seeds was dependent upon temperature levels and bean variety, that of temperature was contingent upon length of 54 storage and legume variety, and that of variety was dependent upon temperature levels, humidity and storage time. The variations in Pi levels for beans held at different environmental conditions in the current research were expected to be primarily due to differences in phytase activity levels, since phytic acid phosphorus constitutes . about 70% of total dry bean seed phosphorus (Lolas and Markakis, 1975/77). The relationship between phytase activities (as Pi) and storage conditions cannot be credited to either time, temperature, or RH in isolation. The variations in Pi concentrations due to variety may not reflect differences in phytase activity rates per se, but probably reflect dissimilarities in initial phytase substrate (phytic acid) concentrations. Red bean phytic acid concentrations were lower (P 5 0.01) than in the white bean landrace. Varietal phytic acid content differences were also observed in faba beans by Hussein et al (1989). The relationships between several storage parameters, and Pi levels of white (soft) and red (hard) decorticated beans are illustrated (Figs. 2 and 3). White beans (Fig. 2) stored for four and eight months at two relative humidities (55% and 85% RH) demonstrated significantly (P g 0.05) greater Pi accumulation as storage temperatures increased from 60°F (15.6°C) to 95°F (35°C). These results coincided with those of Ron (1979). Increased phytase activities were demonstrated in black beans steeped (16 hours) at high temperatures (40-60°C or 104-140°F) as 55 compared to control seeds steeped at 20°C (68°F) (Hon, 1979). Legume seeds stored at 95°F (35°C), 85% RH for four or eight months exhibited significantly (P45 0.05) higher Pi levels than those samples stored at the same temperature, but lower RH 55% RH, and the control seeds maintained at 35°F (2°C) and 30% RH (Fig. 2). The highest Pi levels and consequently the greatest phytase activities were produced in beans stored under the maximum time, temperature and RH conditions. Similar data were obtained by Hincks and Stanley (1986) who reported highest phytase activities in beans stored for a maximum of ten months at 30°C (86°F), 85% RH as compared to legumes held at 15°C (59°F) and 35% RH. Red beans (Fig. 3) maintained for four and eight months at 55% and 85% relative humidities exhibited significantly (P 5_0.05) higher Pi contents as temperatures rose from 60°F (15.6°C) to 95°F (35°C). Legume seeds had significantly (P z 0.05) greater Pi when stored for four and eight months at 95°F (35°C) and either 55% or 85% RH as compared to control seeds (35°F or 2°C, 30% RH). Greater Pi levels were evident for red beans held under the maximum time, temperature and both relative humidity (55%, 85% RH) conditions as opposed to the shorter storage period (four months). After four months storage at 55% RH and 95°F (35°C), significantly (P z 0.05) greater amounts of Pi were measured as compared to beans stored at the same temperature, but higher RH (85%). These results were not 56 anticipated, although a similar, but non-significant trend was apparent after eight months storage. No literature could be found to support or refute these findings. Phytase activities were expected to be greater at higher RH than at lower RH. Generally, most biological reactions accelerate as water activities increase (Reed, 1975). Greatest phytase activities in the red bean samples were found in legume seeds stored at the highest temperature (95°F or 35°C), and under both humidities (55% and 85% RH) as compared to beans held at the other storage environments (50°F or 15.5°C) for the 55% and 85% RH: 35°F (2°C), 30% RH - control. It can be deduced that the higher phytase activities in decorticated white beans maintained under high temperature and high humidity storage for eight months contributed greatly to the bean hard-to-cook defect, since these legumes also exhibited increased cooked hardness (data are in Chapter 2 of this dissertation). The same was true for red beans under the same temperature and time period for both the 55% and 85% relative humdity conditions, but the impact was not as apparent. Phytic Acid Phytic acid levels were significantly (P z 0.01) influenced by the variety of beans (Table 2). White beans had greater phytic acid levels than the red bean landrace. Therefore, phytic acid concentration differences among the two bean landraces may explain the significant influence of variety on phytase activities as measured by the amount of 57 Pi within the bean cotyledons (Fig. 2 and 3). Varietal phytic acid content differences have been reported to exist in faba beans (Hussein et al, 1989). Calcium Cell wall calcium levels in stored decorticated red and white beans were significantly (P 5 0.05) influenced by storage temperatures and relative humidities (Table 2). Variety did not influence cell wall calcium concentrations although red beans tended to be slightly higher in calcium than the white bean landrace. Legume seeds stored at 95°F (35°C), 85% RH were greater (P g 0.05) in cell wall calcium contents than beans held at 60°F (16.6°C) and 55% RH. In contrast, seeds stored under the highest temperature and relative humidity conditions (95°F or 35°C, 85% RH) were not significantly (P s 0.05) different in cell wall Calcium levels from the control group (35°F or 2°C, 30% RH). These data concur with others (Ron and Sanshuck, 1981: Moscoso et al (1984). Moscoso et al (1984) found that bean calcium contents were slightly higher (0.49 mg/g) in legume seeds stored for nine months at 32°C (89.6°F), 17.9% moisture than controls (0.47 mg/g dry marc) held at 2°C (35.6°F) and 12.5% moisture. Phytic Acid/Calcium Ratios Phytic acid/calcium ratios of stored decorticated beans were significantly influenced by storage temperature (P z 0.01), relative humidity (P 5 0.05) and variety 58 (P 5 0.05) (Table 2). Legume seeds stored at the highest temperature and humidity levels (95°F or 35°C, 85% RH) exhibited significantly (P g 0.05) lower phytic acid/calcium ratios as compared to the other two storage conditions (50°F or 15.5°C, 55% RH: 35°F or 2°C 30% RH). These ratio data lend support to the Pi results in that a decrease in phytic acid/calcium ratios within each landrace under adverse storage conditions implies advanced phytic acid degradation with concomitant elevation of calcium. Phytase degrades phytic acid to release Pi, calcium and magnesium (Henderson and Ankrah, 1985: Lolas and Markakis, 1977: Peers, 1953: Moscoso et al, 1984: Ron and Sanshuck, 1981). A decline in phytic acid/calcium ratios in beans stored under high temperature (32°C or 89.6°F) and moisture (16%) over 10 months was reported by Ron and Sanshuck (1981). Ron and Sanshuck (1981) determined that low phytic acid/calcium ratios were a characteristic of the hard-to- cook defect in stored legumes. Varietal differences in phytic acid/calcium ratios, observed in the current research, may not reflect differences in phytase activity levels per se, but most likely reflect dissimilarities in initial phytic acid/calcium ratios. Bean varietal differences in phytic acid/calcium ratios have been noted by Ron and Sanshuck (1981). Despite the origin of reduced phytic acid/calcium ratios, under high temperature and relative humidity storage parameters, low ratios have been associated with 59 increased cooked bean hardness (Ron and Sanshuck, 1981: Hussein et al, 1989). The phytic acid/calcium ratio data reported here support the cooked white and red legume seed texture (hardness) data reported in Chapter 2 (Cooked Bean Texture section). A high negative correlation (r - -0.93) was found between white bean hardness and concomitant phytic acid/calcium ratios under low and high temperatures (50°F or 15.5°C, 95°F or 35°C) and humidities (55%, 85% RH) for the four to eight months storage periods. Likewise, a moderately high negative correlation (r - -0.61) was observed between red bean hardness (texture) and the phytic acid/calcium ratios under the same storage conditions. These negative correlations support the phytic acid degradation mechanism. The decrease in legume seed phytic acid/calcium ratios under high temperature and humidity storage conditions has been explained by the increase in phytase activity levels where Pi and calcium and magnesium ions are released (Ron and Sanshuck, 1981). The cations are believed to migrate from inside the cell to middle lamella and desolubilize pectins. Pectin desolubilization in the middle lamella has been shown to limit cell separation of cooked legume seeds, thus causing the hardened bean texture (Jones and Boulter, 1983a/b). iMagnesium Cell wall magnesium data (Table 3) from decorticated beans revealed that cell wall magnesium levels were significantly (P 5 0.01) influenced by bean variety. The red bean 60 landrace had greater magnesium concentrations than the white bean landrace. Varietal differences in legume seed magnesium contents (0.133 - 0.168%) have been demonstrated by Ron and Sanshuck (1981), although their data were not A significant. Storage conditions did not affect bean magnesium levels (Table 2). No literature was found to support or refute these data. Correlation of Cooked Bean Texture and Phytase Activity Phytase activities (cotyledon Pi levels) were positively correlated (r - 0.92) with white cooked bean texture (hardness) for beans stored under two storage temperatures (60°F or 15.6°C, 95°F or 35°C) and relative humidities (55% RH, 85% RH) for four and eight months. A moderately high correlation (r = 0.73) was found between hardness and Pi (phytase activities) of red beans. Similar results were reported by Ron (1979) who found a high positive correlation (r - 0.77) between length of cooking and phytase activities as steeping temperature increased from 40°C (104°F) to 70°C (158°F). Total Pectic Substances Total pectic substances were not significantly influenced by storage conditions (time, temperature, humidity) nor by bean variety (Table 3). These results were expected since the total pectic substances procedure (Owen et al, 1952) was capable of extracting both water soluble and water insoluble pectic substances from legume 61 seed samples. These data are consistent with the total pectic substances results of Moscoso et al (1984). These workers found comparable cotyledon total pectic substance levels in red kidney beans stored for a maximum of nine months under three environments (2°C or 35.6°F, 12.5%HZO: 32°C or 89.5°F, 14.9% H20: 32°C or 89.5°F, 17.9% H20). Water soluble Pectic Substances Water soluble pectic substance (pectin) levels in beans were significantly influenced by storage time (P g 0.05), temperature (P 5 0.05) and variety (P 5 0.01) parameters (Table 3). Thus, storage conditions (time, temperature) and variety will influence the quantity of water soluble pectic substances in decorticated legume seed cotyledons. Lower (P g 0.05) water soluble pectic substances concentrations were measured from beans held at the highest temperature (95°F or 35°C) for the longest storage time period (eight months) in contrast to legumes stored for less time and at lower temperatures (60°F for four months: 35°F or 2°C). These results substantiate data of Jones and Boulter (1983a) and Moscoso et al (1984). Jones and Boulter (1983a) postulated that the reduction in water soluble pectic substance levels was due to phytic acid degradation by phytase which released calcium and magnesium ions. Subsequently, cation bridges were formed within the pectinaceous middle lamella, rendering the pectin insoluble. Pectin desolubilization was thought to be facilitated from pectin de-esterification by the action 62 of PME which increased the availability of free carboxyl sites (Jones and Boulter, 1983a). In support of this theory, relationships were found between decreased phytic acid/calcium ratios and decreased water soluble pectic substance concentrations for white (r - 0.86: r - 0.96) and red beans (r - 0.90: r - 0.62) stored for four and eight months, respectively, at both the low and high temperature and humidity levels (60°F or 15.6°C, 95°F or 35°C and 55% RH, 85% RH). These relationships implied that the depression of water soluble pectic substances from stored seeds (four and eight months) was associated with reduced phytic acid levels with a concomitant elevation in calcium ion concentrations. The decline in water soluble pectic substances due to accumulation of calcium and magnesium cations in the middle lamella has been suggested to be accompanied by a rise in insoluble pectin concentrations and restricted cell separation resulting in hardened beans (Ron and Sanshuck, 1981). The reduction in cell separation of the hard-to- cook legume seeds implied that there were modifications in the middle lamella pectins. Support to this theory was revealed by the negative correlations between cooked legume seed texture (hardness) reported in chapter two (Cooked Bean Texture section) and bean water soluble pectic substance concentrations for white (r - -0.93) and red (r - -0.86) beans for both the four and eight months storage, under both low and high temperatures (60°F or 63 15.5°C, 95°F or 35°C), and low and high humidities (55% RH, 85% RH) . Varietal differences (P): 0.01) among bean landraces in terms of their contents of water soluble pectic substances could be accounted for by the high and low phytic acid/calcium ratios (white and red beans, respectively), rather than differences in phytase activities. Legume seed Pi and water soluble pectic substance contents, phytic acid/calcium ratios and cooked bean texture (hardness) data lend support to the phytate mechanism as directing the subsequent hardening (hard-to- cook defect) of the white and red bean landraces during storage. The effect on legume seed hardness by the phytate system was greater in the white bean landrace (soft bean) which had higher initial phytic acid contents as compared to the red bean landrace (hard bean). Despite the greater contribution of the phytate mechanism towards the development of the hard-to-cook defect in white beans, the hard red beans remained harder following heat treatment (P 5 0.05) than the soft white beans. Lignin ANOVA (Table 3) revealed that lignin concentrations in decorticated legume seeds were significantly influenced by storage time (P 5_0.01), temperature (P): 0.05) and variety (P‘s 0.01). In addition, there were significant temperature x humidity (P g 0.05) and time x temperature x humidity (P 5 0.05) interactions. These interactions 64 implied that the lignin effects due to storage time were dependent on temperature and humidity levels, and those of temperature on storage time lengths and humidity levels. Therefore, bean lignin content changes cannot be ascribed to one factor in isolation from the others. Figures 4 and 5 illustrate the relationship between storage time, temperature, relative humidity and variety factors and bean lignin contents. White beans (Fig. 4) stored for four months at 55% RH exhibited significant lignin concentration increases (P 5 0.05) as temperature increased from 50°F (15.5°C) to 95°F (35°C). Legume seed lignin contents were also raised at 60°F (15.6°C) as humidity increased from 55% RH to 85% RH following storage at four months. The decline in white bean lignin content after four months storage, at 85% RH, as temperature increased from 60°F (15.6°C) to 95°F (35°C) was not expected. There might have been other chemical reactions (protein-lignin cross linking) (Whitmore, 1978), under adverse conditions, that caused the reduction in extractable cotyledon lignin levels which our study did not measure. There was a general, but non-significant decline in lignin levels of decorticated white beans as storage time, temperature and humidity increased (Fig. 4). The decrease in lignin levels from white decorticated beans stored at high temperatures and humidities for eight months was not expected and contradicts the qualitative studies by Hincks and Stanley (1987), demonstrating increased cell 65 wall and middle lamella lignin deposition in hard-to-cook beans stored at 30°C (86°F), 85% RH for a maximum of ten months. Data from the current research are not sufficient to explain causes of the lignin decreasing trend as storage time, temperature and humidity increased. However, it might have been that after eight months storage, there was lignin-protein cross-linking (Whitmore, 1978) causing lignin not to be quantitatively measured following the thioglycolic acid lignin determination procedures used in this study. The qualitative lignin studies (Hincks and Stanley, 1987) did not involve extraction of lignin, rather the staining of lignin in intact cell walls and middle lamella. Increases (P z 0.05) in red bean lignin concentrations were apparent as the relative humidity was raised from 55% RH to 85% RH while maintaining 59°F (15.5°C) temperature following four months storage (Fig. 5). However, like the white bean landrace, red bean lignin levels declined after four months storage, at 85% RH, as temperature increased from 60°F (15.6°C) to 95°F (35°C). These results were not expected. After eight months storage, beans maintained at 55% RH and 85% RH showed significant (P g 0.05) increases in extractable bean lignin as temperatures increased from 60°F (15.6°C) to 95°F (35°C). However, similar to the white bean landrace, there was a general, but non significant decline in lignin levels as storage time increased. 66 Extractable lignin contents from both the red and white bean landraces following eight months storage were similar to the legume seed control group stored at 35°F (2°C), 30% RH. These results agree with data reported by Srisuma et al (1989) who found no significant differences in lignin levels among beans stored under three conditions (5°C or 41°F, 40% RH: 20°C or 58°F, 73% RH: 35°C or 95°F, 80% RH) for nine months. Lignin concentration varietal differences were apparent (Figs. 4 and 5). The red bean landrace exhibited significantly (P s 0.01) higher lignin contents as compared to the white bean variety. These data imply that lignin contributed to the initial hard texture of the hard (red) bean (Figs. 7 and 8, chapter 2). Correlation of Cooked Bean Texture and Lignin There were relationships between cooked bean texture (hardness) as shown in Chapter 2 (Cooked Bean Texture section) and lignin concentrations for both the red (r = 0.78) and white (r - 0.63) bean landraces following eight months storage. No correlations were found after four months storage between white bean texture (hardness) and lignin concentrations (r - 0.22), and between red bean hardness and lignin contents (r - 0.15). After eight months storage, greatest lignin levels were obtained from both legume varieties stored under the highest temperature and relative humidity conditions (95°F or 35°C, 85% RH), although the concentrations were lower than those following 67 four months storage under similar temperature and humidity conditions. These findings (after eight months storage), although not significant, support the qualitative data reported by Hincks and Stanley (1987) that the lignification-like mechanism contributed to the development of the legume hard-to-cook defect in later storage (z eight months) periods . Red beans had a greater magnitude of change in lignin concentrations than white beans after eight months storage at 55% RH and 85% RH as temperature increased from 60°F (15.6°C) to 95°F (35°C) (Figs. 4 and 5). Although not significant, data for red beans coupled with the high positive correlation (r - 0.78) between red bean cooked texture (hardness) and lignin content, as compared to the white beans (r - 0.63), implied that the lignification mechanism might have some contribution to the hard-to-cook defect of the red (hard) bean landrace in contrast to the white (soft) bean variety. Proteins Protein contents were significantly influenced by storage time (P 5 0.05) and variety (P‘s 0.01) (Table 3). Protein concentrations of legume seeds stored for eight months were significantly (P): 0.05) lower than after four months storage as compared to the control seeds. Studies examining the effects of the legume hard-to-cook defect on bean protein levels have reported decreased protein digestibilities (Sievwright and Shipe, 1986) and lower 68 phaseolin protein contents with concomitant increases in small polypeptides (Hohlberg and Stanley, 1987). No studies were found in the literature that investigated the effect of the hard-to-cook defect on total legume seed proteins. The reduction in protein contents after prolonged storage (eight months) could possibly be attributed to a progression in protein-lignin cross- linking. A moderate relationship was apparent between white bean proteins and lignin concentrations (r - -0.52), between the red bean proteins and lignin concentrations (r - -0.47) after eight months storage time. Preliminary work revealed heavy and light phaseolin protein staining on . SDS-Polyacrylamide gel electrophoresis (PAGE) map for the white and red beans, respectively, stored under high temperature (95°F or 35°C) and humidity (85% RH) for the eight months period, as compared to control legume seeds held at 35°F (2°C), 30% RH (see Appendix). These findings lend support to the protein data in the current research and the findings of Hincks and Stanley (1987) in revealing that legume seed protein changes did occur during storage under adverse conditions. Strong negative correlations were evident between cooked bean texture (hardness) shown in chapter two (Cooked Bean Texture section) of white (r - -0.92) and red ’ (r - -0.77) beans, and protein contents following the eight months storage period. Decreases in bean proteins with increases in storage time may have some role in legume seed 69 hardening. Hohlberg and Stanley (1987) proposed that proteins might be involved in the development of the legume seed hard-to-cook defect by providing aromatic amino acids through protein hydrolysis by proteases for the lignification reaction. 70 OONCUUSIONS The legume seed hard-to-cook defect developed in both decorticated Malawaian white (soft) and red (hard) beans stored under the most severe environmental conditions and longest time periods used in this study. In both bean landraces the phytic acid degradation mechanism appeared to ' be the major system directing the hard-to-cook defect. However, the phytate mechanism may be only partially responsible for the hard-to-cook defect in the red (hard) bean landrace. Other contributors may include initial water soluble pectic substances levels in relation to the total pectic substances contents, phytic acid and calcium concentrations. There was a trend that the lignin formation mechanism affected the hardness of the red (hard) more than the white (soft) legume seeds despite the minimal contributions of the lignification mechanism to the hard- to-cook phenomena. Lignin concentrations may have a major impact on the original hardness of the red (firm) beans as opposed to the hardness developed during adverse storage conditions for the period between four to eight months. Further studies are needed in this area, such as quantifying lignin levels in several legume landraces using protein free samples stored under severe conditions to produce the hard-to-cook defect. Also changes in the types of been proteins in relation to lignin formation needs to be addressed. 71 REFERENCES American Associations of Cereal Chemists. 1989. "Approved Methods of the American of Cereal Chemists," Vol. 2: 44-31 - 44-40. Approved Methods Committee, American Association of Cereal Chemists, Inc. St. Paul, MN. Bonner, J. and Varner, J.E. 1965. ”Plant biochemistry." Academic Press, New York, NY. Burr. H.K., Ron, 3. and Morris, H.S. 1968. Cooking rates of dry beans as influenced by moisture content, temperature and time of storage. Food Technology 22: 336-338. Freed, R., Eisensmith, S.P., Goetz, 8., Reicosky, D., Smail, V.W. and Wolberg, P. 1985. "MSTAT: A microcomputer program for the design, management, and analysis of agronomic research experiments," Version 4.0. Michigan State University, East Lansing, MI. Hammerschmidt, R. 1984. Rapid deposition of lignin in potato tuber tissue as a response to fungi non- pathogenic on potato. Physiological Plant Pathology. 24: 33-42. Hincks, M.J. and Stanley, D.W. 1986. Multiple mechanisms of bean hardening. J. Food Technology. 21: 731-750. Hincks. M.J. and Stanley, D.W. 1987. Lignification: Evidence for a role in hard-to-cook beans. J. Food Biochem. 11: 41-58. Hohlberg, A.I. and Stanley, D.W. 1987. Hard-to-cook defect in black beans. Protein and Starch considerations. J. Agric. Food Chem. 35: 571-576. Hussein, L., Ghanern, R., Khalil, S., Nassib, A. and Ezilarab, A. 1989. The effect of phytate and fiber content on cooking quality in faba bean. Journal of Food Quality. 12: 331-340. Jones, P.M.B. and Boulter, D. 1983a. The cause of reduced cooking rate in Phaseglug ynlgazis following adverse storage conditions. J. Food Sci. 48: 623-626, 649. Jones, P.M.B. and Boulter, D. 1983b. The analysis of development of hardbean during storage of black beans (Phasggln§,ynlgazis L). Qual. Plant Plant Foods Hum. Nutr. 33: 77-85. 72 Ron, S. 1979. Effect of soaking temperature on cooking and nutritional quality of beans. J. Food Sci. 44: 1329- 1334, 1340. Ron, 8. and Sanshuck, D.W. 1981. Phytate content and its effect on cooking quality of beans. J. Food Process. and Preserv. 5: 169-178. Little, T.M. and Hills, F.S. 1978. ”Agricultural experimentation: Design and Analysis,” John Wiley and Sons, New York, NY. Lolas, G.M. and Markakis, P. 1975. Phytic acid and other phosphorus compounds of beans (Phasgglng ynlgazis L.). J. Agr. Food Chem. 23: 13-15. Lolas, G.M. and Markakis, P. 1977. The phytase of navy beans (Ehgsgglng ynlggzig). J. Food Sci. 42: 1094- 1097, 1106. Malawi Meteology Dept. 1986. "Malawi climatological summary,” Malawi Meteology Department, Lilongwe, malawi. Mattson, S., Akerberg, E., Eriksson, E., Koutler-Anderson, E. and Vahtras, K. 1951. Factors determining the composition and cookability of peas. Acta Agric. Scand. 11:40-61. Molina, M.R., Baton, M.A., Gomez-Brenes, R.A., King, K.W. and Bressani, R. 1976. Heat treatment: A process to control the development of the hard-to-cook phenomenon in black beans (fingggglng ynlgaris). J. Food Sci. 41:661-666. Moscoso, W., Bourne, M.C. and Hood, L.F. 1984. Relationships between the hard-to-cook phenomenon in red kidney beans and water absorption, puncture force, pectin, phytic acid and minerals. J. Food Sci. 49: 1755-1583. Murphy, J. and Riley, J.P. 1962. A modified single solution of phosphate in natural waters. Anal. Chim. Acta. Owen, H.S., McCready, R.M., Sheperd, A.D., Shultz, T.H., Pippen, E.L., Swensen, H.A., Miers, J.C., Erlandsen, R.F. and Maclay, W.D. 1952. Methods used at Western Regional Research Laboratory for extraction and analysis of pectic materials. AIC-340 U.S. Dept. Agri., Bureau Agr. Ind. Chem., Washington, D.C. Peers, F.G. 1953. The phytase of wheat. Biochem. J. 53: 102-110. 73 Pons, W.A. and Guthrie, J.D. 1946. Determination of inorganic phosphorus in plant materials. Ana. Chem. 18: 184-186. Reed, G. 1975. Enzymes in food processing. In ”Food Science and Technology - A Series of Monographs," Second Edition. p. 573. Academic Press, New York, NY. Sefa-Dedeh 8. Stanley, D.W. and voisey, P.W. 1979. Effect of storage time and conditions on the hard-to-cook defect in cowpeas (yigna,nngnigglg§a). J. Food Sci. 44: 790-796. Sievwright, C.A. and Shipe, W.F. 1986. Effect of storage conditions and chemical treatments on firmness, invitro protein digestibility, condensed tannins, phytic acid and divalent cations of cooked black (Ehafigglns xnlgaris). J. Food Sci. 51: 982-987. Singh, R.P. and Heldman, D.R. 1984. ”Introduction to Food Engineering", Academic Press, Inc. New York, NY. Smith, E.L., Hill, R.L., Lehman, I.R., Lefkowitz, R.S., Handler, P., and White, A. 1983. "Principles of Biochemistry: General Aspects." Seventh Edition. McGraw-Hill Book Company, New York, NY. Srisuma, N., Hammerschmidt, R., Uebersax, M.A., Ruengsakulrah, S., Bennink, M.R. and Hosfield, G.L. 1989. Storage induced changes of phenolic acids and the development of hard-to-cook in dry beans (Phaggglng ynlgaris, var. Seafarer). J. Food Sci. 54: 311-314, 318. Steel, R.G.D. and Torrie, J.H. 1980. "Principles and procedures of statistics: A biometrical approach," Second Edition. McGraw-Hill Book Company, New York, NY. Suhre, F.B., Corrao, P.A., Glover, A. and Malanoski, A.J. 1982. Comparison of three methods for determination of crude protein in meat: Collaborative study. J. Assoc. Off. Anal. Chem. 65: 1339-1345. Vindiola, O.L., Seib, P.A. and Hoseney, R.C. 1986. Accelerated development of the hard-to-cook state in beans. Cereal Chem. 31: 538-552. Watanabe, F.S. and Olsen, S.R. 1965. Test of an ascorbic acid method for determining phosphorus in water and NaHCO extracts from soil. Soil Sci. Soc. Am. Proc. 29: 6 7-678. 74 Wheeler, E.L. and Ferrel, K.E. 1971. A method for phytic acid determination in wheat and wheat fractions. Cereal Chem. 48:312-320. Whitmore, F.W. 1978. Lignin-protein complex catalyzed by peroxidase. Plant Sci. Letters. 13: 241-245. 75 firi...‘ “ca-Lyle. ... Ev. 3!. i‘.:.". .unou Amway oosouommwo ussoauqswun unsoa one made: usoaomcsuus Heauouosu one C.“ ECHO. 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CHAPTER 2 DRY BEAN (Ehgfigglnfi xnlggzifi) HARDENING AND THE CONSEQUENCES OF PECTIN HETHYLESTERASE ACTIVITY IN STORAGE 83 84 ABSTRACT DRY BEAN (Phaseglns ynlgazis) HARDENING AND THE CONSEQUENCES OF PECTIN METHYLESTERASE ACTIVITY IN STORAGE Pectin methylesterase (PME) activities and hardness of two decorticated Malawi bean landraces (Ehgggglus ynlgaris) were evaluated over an eight month storage period at 55% a._ _* and 85% relative humidities, and 60°F (16°C) and 95°F (35°C). PME activities were indirectly measured by determining the degree of esterification of total pectic substances. Cooked bean hardness was determined using a i Kramer Shear Press. Legume seeds stored for four and eight months under these conditions exhibited significantly A (P g 0.05) higher PME activities as compared to legume control seeds stored at 35°F (2°C), 30% RH. Beans stored for eight months had significantly (P): 0.05) higher enzyme activities than seeds stored for four months irrespective of RH. Bean hardening advanced with increased storage time, temperature and relative humidity. Legume varieties demonstrated significant (P g 0.01) textural differences. A positive correlation between the hardness of beans stored for eight months and PME activities was also found. 'I 85 INTRODUCTION Failure of stored common dry beans (Phaseglns ynlggzis) to soften sufficiently during cooking (210.2°F or 99°C for one hour) has been related to the hard-to-cook defect, which is a failure of the cotyledons to soften adequately during cooking procedures even after imbibing water (Vindiola et al., 1986). Several factors have been reported to be involved in the development of the hard-to- cook defect including high temperatures (z 70°F or 21°C), high (3 70%) relative humidities (RH), and phytase activities (Vindiola et al., 1986: Hincks and Stanley, 1986: Burr et al., 1968). The involvement of the enzyme pectin methylesterase (PME) in the development of the hard-to-cook bean defect has been suggested. PME (pectin pectylhydrolase, EC 3.1.1.11) is an enzyme that hydrolyzes the methyl group from the pectin molecule to produce methanol and pectinic acid and/or low methoxyl pectin (Delincee and Radola, 1970: Versteeg et al., 1978). Research that has related PME activities to the hard- to-cook defect of seeds is limited and inconclusive. Jones and Boulter (1983) in the first part of their study incubated fresh black beans (4°C or 39.2°F and 10% moisture: controls) with 0.03M CaC12 or 0.03M CaC12 plus PME at pH 7.0 for 18 hours. Bean samples incubated with only CaC12 had a 60% increase in cooking times as compared to the untreated controls. PME addition further lengthened 86 processing time by four minutes as compared to the CaClz treated group. PME activities were not determined in the incubated samples of the Jones and Boulter (1983) investigation, rather the indirect effect of the enzyme on bean texture. A second component of the Jones and Boulter (1983) experiment measured PME activities in beans stored at 93.2°F (34°C) and 70-75% RH for six months. PME activities were measured by determining the degree of esterification (DE) of the pectin molecules. These workers demonstrated a decrease in pectin DE from 51% to 15%. This implied that PME was active in legume seeds during storage. Bean texture (hardness) was not determined in the. second part (the six months storage study) of the Jones and Boulter (1983) investigation. To clearly define the role of PME in bean hardening during storage, both the PME enzyme activities as well as legume seed texture should be assessed. The need to understand the hard-to-cook mechanism during storage is necessary in order to alleviate problems associated with this defect. Problems encountered include increased cooking times, hence energy consumption increases, and lowered legume seed protein digestibilities (Ron and Sanshuck, 1981: Sievwright and Shipe, 1986). The purpose of this investigation was to evaluate PME activities and textural (hardness) changes in decorticated common dry beans (Rhgggglng yylggxig), over an eight month storage period. This project was initiated to enhance the research 87 data base and to evaluate the proposed mechansim by Vindiola et al. (1986) and Jones and Boulter (1983) to determine whether or not PME is involved in hardening of beans stored under high temperature (2 70°F or 21°C) and high humidities (z 70% RH). The following hypotheses were addressed. Decorticated bean seeds stored in a suboptimal environment (95°F or 35°C: 85% RH) would have increased PME E activities as storage time advanced (0-8 months). Elevated : PME activities would lead to decreased DE of total pectic substances as compared to the control group (35°F or 2°C: 30% RH). Cooked bean hardness would be promoted by E elevated PME activities and a lowering of the water soluble‘ pectic substances with an accompanying rise in insoluble calcium and magnesium pectates. IHATERIALS AND METHODS Please refer to Chapter 1 of this dissertation (Materials and Methods section) for the description of storage environmental conditions, sample preparation procedures and the projects experimental design. _ Pectin Methylesterase Assay _ $ Chemicals and supplies used for the DE determination of total pectic substances included: phenol red indicator '. ‘ J;_‘Itl.“h.‘l l K .' (Sigma Chemical Co., Louis, MO): HCL and NaCl (ACS certified, Baker Chem. Co., Phillipsburg, NJ: NaOH (1N- Mallinckrodt, Inc., Paris, KY) and ethyl alcohol (absolute- , USP, Alcohol and Chemical Co., Shelbyville, KY). Legume seed PME enzyme activities were indirectly measured by determining the DE of total pectic substances extracted from bean flour (1 9), prepared as cited in Chapter 1 (”Legume Flour Preparation” section). Total Pectic Substance Extraction and Titration Total pectic substance extraction and pectic acid titration for the DE determination were carried out according to the method of Owen et al (1952). The bean total pectic substance extraction procedure has been described in Chapter 1 (Total Pectic Substance section). The total pectic substance filtrate (0.5 g) was weighed into an erlenemeyer flask (250 ml) with subsequent addition of ethanol (5 ml), NaCl (1 g), boiled (carbon dioxide-free) deionized water (100 ml) and phenol red 89 indicator (six drops). The mixture was slowly titrated with NaOH (0.1N) to its end point (pink - pH 7.5) to avoid pectin de-esterification. Twenty five ml of NaOH (0.25N) was then added, and the solution was thoroughly mixed. The sample was allowed to de-esterify for 30 minutes at room temperature (25°C or 77°F). Following de-esterification, 25 ml of HCl (0.25N) was added, the sample was titrated with NaOH (0.1N) to the pH 7.5 (pink) and point, and the amount used for titration was recorded. The calculation for the degree of esterification was: N(NaOH) x Vol. of NaOH(ml) x 3.1 wt of sample (9) Accuracy and reproducibility of the total pectic substance extraction/titration method was measured using apple pomace pectin (DE - 65-85%) (de Vries et al, 1986). During preliminary work, mean DE value of 64% was found for total pectic substances from seven apple pomace samples. Bean Processing/Texture since decorticated beans were used in this investigation, conventional canning procedures could not be employed since cooking times were lowered substantially. The following legume seed processing method was determined after several pilot studies. Decorticated dry bean samples (100 g) were placed into pyrex glass canning jars (264 ml), followed by the addition of deionized water (Voisey and Larmond, 1971). Samples were placed into a boiling water 90 bath for 30 min, drained, rinsed and exhausted for 5 min. The jars were then sealed, and the samples pressure cooked at 5 psig (228°F or 109°C) for five min in a pressure cooker (6 qt). Cookability was determined by the method of Jones and Boulter (1983). Upon cooling, the cooked beans were refrigerated for two weeks at 39°F (4°C) for moisture equilibration. The equilibrated samples were drained, ‘enflr rinsed and bean samples (100 g) were used for the texture I determination. Legume seed firmness (hardness) was measured using a Kramer Shear Press (Food Technology Corp., Reston, VA, model No. T-2100-C) with a standard shear- compression cell (model No. CS-1) at 1/3 range setting and 3000 lb transducer force. The amount of force to shear 100 g beans was calculated following the method of Binder and Rockland (1964). The greater the shear force of a sample compared to the control, the harder the texture. Statistical Analyses The effects of time, temperature, humidity and variety upon PME activities (the DE of total pectic substances) and cooked bean texture were analyzed statistically using analysis of variance (ANOVA) via the MSTAT statistical software package (Freed et al, 1985). The Least Significance Difference (LSD) test was used for multiple comparisons among means (Little and Hills, 1978: Steel and Torrie, 1980). When signficance was indicated, the P 5 0.05 level was meant, unless otherwise stated. Linear correlation coefficients were calculated to determine the 91 relationship between bean texture (hardness) and PME activities (Little and Hills, 1978: Steel and Torrie, 1980). 92 RESULTS AND DISCUSSION Pectin Methylesterase Activity PME activities in the middle lamella of plant tissues generally involve the following reactions: Pectin-cooch + H O ---------- > Pec ine-Coo' 3 2 P“3 + Hs + CH3OH (Hagerman and Austin, 1986). Pectin de-esterification can also be initiated non-enzymatically under very acidic (about pH 1.0), or slightly alkaline (2 pH 8.0), or‘ above 90°C (2 194°F) temperature conditions (Deuel and Stutz, 1958: Kohn et al, 1982: Markovic et al, 1983). Pectin esterification in legume seeds used in the current research was expected to be primarily affected by PME activities, since beans are low acid foods with a pH of about 6.0 (Banwart, 1981: Lopez, 1987: Paul and Palmer, 1972). Preliminary data revealed the Malawian hard and soft beans to have an average pH of 6.0. Bean samples for the current study were held at a maximum temperature of 95°F (35°C). ANOVA (Table 4) for the DE data of total pectic. substances (PME activities) from stored decorticated beans revealed that the DE was significantly (P 5 0.01) influenced by storage time. In addition, there was 8’ significant (P 5 0.05) time x humidity interaction which means that the effect of storage time on the DE of total pectic substances from stored legume seeds depended on the 93 level of relative humidity. These results implied that the bean storage conditions (time and relative humidity together) had some effect on the DE of total pectic substances, hence PME activities in the legume seed cotyledons. Higher enzyme activities (by DE) would be expected as storage time increased, since conventionally, one unit of PME equals amount of enzyme that would liberate ' $19!; . . 1L“ 7 ' .I one micromole of carboxyl groups per minute (Versteeg et al, 1978: Markovic et a1, 1983: Kohn et al, 1983: Dahodwala et al, 1974). However to relate storage conditions (time * and relative humidity) to PME activities, changes in the EJ latter cannot be attributed to either time or relative humidity in isolation. Both factors (storage time and humidity) must be considered in interpreting and using these data. Figure 6 illustrates that for the four month storage period, PME activities were significantly greater (P g 0.05) at 55% RH. These results were not expected. Enzyme activities were expected to be higher at 85% RH than 1 at 55% RH. In general, most enzymatic and biological reactions increase with an increase in water activity (Reed, 1975). The PME enzyme activities trend at 55% RH and 85% RH for beans stored for eight months (even though not significantly different) was expected. Considering storage time, beans stored for four and eight months were significantly higher (P 5 0.05) in PME activities than in the control seeds (Fig. 6). In 94 addition, legume seeds stored for eight months were significantly higher (P‘s 0.05) in PME activities than those stored for four months, irrespective of the relative humidities at which they were stored. These results agreed with those found by Jones and Boulter (1983) who reported a decrease in DE of bean pectin from 51% to 15% when stored at 93.2°F (34°C), 70%-75% RH for six months (hence an increase in PME activities), as compared to the control. Unless inhibited prior to initiation of the storage period, PME remains active in beans stored at z 55% RH for four months or longer when storage temperatures are 2 60°F. The impact of this enzyme was greater the longer the legumes were stored. Cooked Bean Texture Texture (hardness) of cooked beans was significantly (P g 0.01) influenced by storage time, temperature, relative humidity and variety (Table 4). In addition, the following interactions were significant: time x temperature (P 5_0.05), time x relative humidity (P g 0.01), temperature x relative humidity (P 5 0.01), and time x temperature x relative humidity (P 5 0.01). These interactions implied that the effects of storage time on legume seed texture (hardness) were dependent on levels of temperature and humidity: those of temperature on time and humidity: and those of relative humidity on time and temperature. When relating storage conditions (time, temperature, relative humidity) to cooked bean texture, 95 the changes in hardness cannot be attributed to one factor in isolation. Burr et al. (1968) measured changes in bean hardness during storage by determining cooking time. These investigators reported that cooking times of pinto beans increased as storage time lengthened. In addition, the researchers also found that the impact of storage time (24 months) and high moisture (16%) content was mainly observed at high temperature (70°F). These results were supported by those of Ron and Sanshuck (1981). These workers found longer cooking times (300 min) for beans containing 16% moisture that were stored under high temperature (89.6°F or 32°C) for ten months, as compared to the control (30 min) which contained 10.5% moisture, and which was stored at 71.5°F (22°C). Figures 7 and 8 depict the relationships between storage time, temperature, relative humidity and variety as these parameters influenced cooked bean texture. White beans (Fig. 7) stored for four months exhibited significant (P 5 0.05) increases in hardness at both the 55% and 85% RH levels as storage temperature increased from 60°F (16°C) to 95°F (35°C). The same trend was observed for the eight month storage period. However, at eight months, the magnitude of change in legume seed hardness at 85% RH was significantly higher (P 5 0.05) between the low (60°F or 16°C) and high (95° or 35°C) temperatures than for the 55% RH stored samples. 96 similar results were found for texture (hardness) of the red legume landrace (Fig. 8). These similarities in changes of seed hardness for the red and white bean varieties during storage suggest that similar mechanisms may operate during the development of the hard-to-cook defect in a hard bean (Malawian red bean) and a soft bean (Malawian white bean). However, changes in legume seed phytic acid, phytic acid/calcium ratios (Kon and Sanshuck, 1981): water soluble pectic substances and pectin DE (Jones and Boulter, 1983): and phenolic components (Stanley and Plhak, 1989: Srisuma et al., 1989) need to be thoroughly examined before a definitive statement can be made regarding the similarities in the mechanisms involved in the development of the hard-to-cook defect in these two bean landraces during storage. Results from our laboratory, (Chapter 1, refer to section on "Phytic Acid Ratios”) revealed that white bean phytic acid/calcium ratios were significantly (P 5 0.05) lower when seeds were stored at a high temperature (95°F or 35°C) and a high humidity (85%) as compared to the white beans control samples (35°F or 2°C, and 30% RH, and those held at 50°F (15.5°C), 55% RH (P 5 0.01). In contrast, the red bean landrace exhibited no significant differences in phytic acid/calcium ratios when stored at 95°F (35°C), 85% RH as compared to stored controls (35°F or 2°C at 35% RH), even though a trend for decreasing phytic acid/calcium ratios was apparent. 97 Dissimilarities between varieties were evident. The red bean landrace had significantly lower (P 5 0.05) phytic acid/calcium ratios than the white bean landrace. These results most likely suggest that phytic acid degradation in the white bean (which had a high initial phytic acid content) may have a greater impact on texture (hardness) of seeds stored at a high temperature (95°F or 35°C) and high humidity (85% RH), as opposed to those legume seeds with a low initial phytic acid content. However, an initial low phytic acid/calcium ratio (as in the red bean) would serve the same purpose with respect to pectin desolubilization leading to harder bean texture, as the reduced ratio achieved under high temperature and humidity (as in the white bean). The findings on the relationship between time, temperature, and relative humidity of the present study support those reported by Burr et al. (1968) and Kon and Sanshuck (1981) who stated that the greatest impact of high humidty and increased storage time on bean hardness occurred at high temperatures. In fact, Jones and Boulter (1983) reported that there was a synergistic relationship between high moisture and high storage temperature on bean hardness. The results reported here also support the fact that problems with the hard-to-cook defect are more prevalent in tropical environments where storage temperatures and humidities are normally high (Bur et al. 1968). In our 98 investigation, beans stored for both four and eight months at high temperature (95°F or 35°C) and high relative humidity (85%) were significantly harder (P < 0.05) than controls stored at 35°F (2°C) and 30% RH for both red and white bean landraces. When comparing the texture of the white and red bean landraces, the latter beans were significantly harder (P 5 0.05) than the former. The red bean variety is classified as a hard bean by plant breeders (Vindiola et al., 1986), a classification based on the seed coat characteristics and perceptions of Malawian women following cooking which was confirmed in our laboratory when bean hardness was measured- using Kramer Shear Force. However, the results of our study suggest that there are more factors contributing to the hardness of the hard bean variety than simply the seed coat characteristics as suggested by Vindiola et al. (1986), since the seed samples in the current research were decorticated before storage. In support of the above suggestion, Hussein et al. (1989) found a significant correlation between phytic acid content of different varieties of faba bean cotyledons with cooking time. Correlation of Cooked Bean Texture and Pectin Methylesterase Activity PME activities were negatively correlated (r - -0.64) with white bean landrace texture at four months, and positively correlated (r - 0.67) at eight months as estimated by DE of total pectic substances. The negative 99 correlation between PME activities and cooked bean texture after four months storage was due to the PME activity increase at the lower humidity level (55% RH) and a decrease at the higher humidity (85% RH). These data were not expected, because generally most biological and enzymatic reactions increase with increase in water activity (Reed, 1975). In contrast, PME activities in the red bean were not correlated (r - 0.29) to been hardness following four months of storage. However, after eight months storage, PME activities were positively correlated (r - 0.66) to bean hardness. These correlations between PME activities and cooked bean texture following four and eight months storage at both the 55% RH and 85% RH conditions provide preliminary, but valuable, information regarding the enzyme's role in the legume seed hard-to-cook phenomenon which was not previously available in the literature. More data are needed, however, to verify the current findings using direct methods in PME assay. The positive correlation between PME activities and bean hardness after eight months storage for both the white and red bean landraces lends support to the hard-to-cook hypothesis proposed by Jones and Boulter (1983). These researchers postulated that during storage, pectin desolubilization in the middle lamella (which occured as a result of calcium and magnesium salt bridge formation with pectin) was facilitated by pectin de-esterification which created more free carboxyl sites. The resulting 100 de-esterified pectins located in the middle lamella were available to react with divalent cations including calcium and magnesium to form insoluble salts that cause the legume seed to harden. Results shown in Chapter 1 (water Soluble Pectic Substances section) revealed a significant decrease in water soluble pectic substances of stored beans with an increase in storage time and temperature. Jones and Boulter (1983) and Ken and Sanshuck (1981) suggested that the source of the divalent cations was mainly from phytic acid degradation. Our laboratory has also shown decreased phytic acid/calcium ratios as storage temperature and humidity increased (Chapter 1, Phytic Acid/calcium Ratio section), lending further support to the Jones and Boulter (1983) hard-to-cook hypothesis. 101 CONCLUSIONS The legume seed hard-to-cook defect was produced in both decorticated Malawian red (hard) and white (soft) bean landraces stored in suboptimal high temperature and humidity conditions over an extended storage time. The hardened beans exhibited increased PME activities. Similarities in legume seed hardness were found between the red (hard) and white (soft) bean landraces. However, the magnitude of change in seed hardness was greatest in the white (soft) as opposed to the red (hard) beans. Therefore, besides PME activities, other factors seem to be influencing hardness changes. Development of the hard-to- cook defect was greater in beans which contained higher .water soluble pectic substances and phytic acid concenrations and higher phytic acid/calcium ratios prior to the initiation of the hard-to-cook phenomena under adverse storage environments. Consequently, varietal differences regarding the initial concentrations of these two compounds, the ratio of phytic acid to calcium, and their effect(s) on the legume hard-to-cook defect remain to be clarified. 102 REFERENCES Banwart, G.J. 1981. ”Basic food microbiology.” The AVI Publishing Company, Inc., Westport, CT. Binder, L.S. and Rockland, L.B. 1964. Use of the automatic recording shear press in cooking studies of large dry lima beans (Ehafiglng lungtgfil. Food Technology. 18: 127-130. Burr, H.K., Kon, s. and Morris, H.J. 1968. Cooking rates dry beans as influenced by moisture content, temperature and time of storage. Food Technology. 22: 336-338. Dahodwala, S., Humphrey, A. and Weibel, M. 1974. Pectic enzymes: Individual and concerted kinetic behavior of pectinesterase and pectinase. J. Food Sci. 39:920-926. Delincee, H. and Radola, B.J. 1970. Some size and change properties of tomato pectin methylesterase. Biochem. Biophys. Acta. 214: 178-189. Deuel, H. and Stutz, E. 1958. Pectic substances and pectic enzymes. Advances in Enzymology. 20:341-381. de Vries, J.A., Hansen, M., Soderberg, J., Glahn, P.E. and Pedersen, J.H. 1986. Distribution of methyloxyl groups in pectins. Carbohydrate Polymers. 6: 165-176. Freed, R., Eisensmith, S.P., Goetz, S., Reicosky, D., Smail, V.W. and Wolberg, P. 1985. "MSTAT: A microcomputer program for the design, management, and analysis of agronomic research experiments" Version 4.0. Michigan State University, East Lansing, Michigan. Hagerman, A.E. and Austin, P.J. 1986. Continuous spectrophotometric assay for plant pectin methylesterase. J. Agric. Food Chem. 34(3):440-444. Hincks, M.J. and Stanley, D.W. 1986. Multiple mechanisms of bean hardening. 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"Principles and procedures of statistics: A biometrical approach," Second Edition. McGraw-Hill Book Company, New York, New York. Variano-Marston, E.V. and Jackson, G.M. 1981. Hard-to- cook phenomenon in beans: Structural changes during storage and imbibition. J. Food Science. 46:1379- 1385. Versteeg, C., Rombouts, F.M. and Pilnik, W. 1978. Purification and some characteristics of two pectinesterase isoenzymes from storage. Lebensm. - Wiss. U.-Techno1. 11: 267-274. Vindiola, O.L., Seib, P.A. and Hoseney, R.C. 1986. Accelerated development of the hard-to-cook state in beans. Cereal Chem. 31: 538-552. Voisey, P.W. and Larmond, E. 1971. Texture of baked beans: A composition of several methods of measurement. J. 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Ln fin 1fijfil‘lfii‘ 0. O. 0. O. 0. O. O. o (D V' N C no (0 V‘ N 1— v- ‘— 1— 1M Mp 6 OOL/JQZWM 6 Figure 9: Hoisture-sorption isotherm for decorticated white beans (Ehgggglnfi xglgazig) at 60°F (15.6°C). 125 90 7'0 I iy (7o) Relative Hum b 1 4 8.04 6.0- <5. <- 16 O 14.0: 12 O- 10.0; 2.04 0.0 4M 10p 6 OOL/J910M 6 Figure 10: Moisture-sorption isoterm for decorticated white beans (zhgggglgg xnlggxig) at 95°F (35°C). 126 JD no _ID [x >\ .38 DA 'IN (”v _:n> ms: 2 ’ (D Er: JD ‘- In LN) fi1fifij1fi1'fi‘l‘lf o 0. O. 0. C2 O. 0. C2 0. QCDd'NOQCDfl'N s—v-s—I—v— 1M Mp EOQL/Jemmb Figure 11: Moisture-sorption isotherm for decorticated red beans (Enaseglus xulsaris) at 60°F (15-6°C)- 127 _u1 no _u3 [\ :>\ L .— -Ln E ‘0 DA . :E:g<3 mv .0 > “3 1:3 2 ' a) (1: _u3 ‘- u) ‘N) 1ESC) 1‘11)- C'z'é. 00 “3 122(3- 1(3JD‘ 44134 121)- ()1) 4M Mp 6 COL/1910M 6 Figure 12: Moisture-sorption isotherm for decorticated red beans (Bhesselns xnlsnris) at 95°F (35°C). Figure 13. 128 One-dimensional SOS/GAGE of phaseolin of red (hard) and white (soft) Malawian beans stored at 95°F (35°C) and 85% RH for eight months and control conditions (35°F or 2°C, 30% RH, zero months). 1 Control red beans 2 = Red beans under adverse conditions 3 = Control-white beans White beans under adverse conditions .5 ll