Comparative study of enzymes related to proline metabolism in tepary bean (Phaseolus acutifolius) and common bean (Phaseolus vulgaris) under drought and irrigated conditions,and various urea concentrations

There are several mechanisms used by plants for survival in adverse environments such as drought, high temperature and salinity. The objective of this study was to evaluate the drought tolerance of tepary bean as a function of biochemical processes linked to isozyme synthesis and changes in enzymatic activity related to proline metabolism. Mature seeds of common beans var. flor de mayo, Phaseolus vulgaris and tepary beans Phaseolus acutifolius were grown under two water conditions (irrigation and drought), and four levels of urea. Vertical electrophoresis and spectrophotometric techniques were used to evaluate protein patterns, glutamate dehydrogenase (GDH), proline oxidase (PO) and pyrroline-5-carboxylate reductase (P5C reductase) enzyme activities. These enzymes were studied because they are directly related to protein synthesis. Electrophoretic patterns showed more proteins in tepary beans than in common beans with limited irrigation. GDH showed only one isozyme, with a molecular weight between 240 to 270 kDa. A decrease in PO activity was observed in common beans under drought stress with a value of 237 mol/min, in comparison to irrigation conditions of 580 mol/min. GDH and P5C reductase enzymes have had higher activity in common beans than in tepary beans under water stress. There was a significant difference only in glutamate dehydrogenase enzyme with respect to urea level. The results suggest that drought tolerance of tepary beans is due to biochemical processes related to proline metabolic enzymes.     

Proximate composition and antinutritional factors in rice bean (Vigna umbellata)

Thirteen promising strains of Rice bean (Vigna umbellata) were analysed for their proximate compositions and antinutritional factors. Protein content in these varieties ranged from 17.50 to 23.10 per cent, ash from 3.06 to 4.48 per cent, ether extract from 2.4 to 3.9 per cent and crude fibre from 1.70 to 4.25 per cent. Trypsin inhibitor activity ranged from 112.63 to 163.98 units/g and polyphenols ranged from 0.58 to 1.19 per cent. Phytohemagglutinating activity was present in all the strains, except one, RB-32. Oligosaccharides, viz., raffinose, stachyose and verbascose, ranged from 0.32 to 0.91, 0.95 to 1.98 and 1.40 to 2.58 per cent, respectively. Attempts have been made to compare the results with a standard variety each of cowpea (Vigna unguiculata), moong (Vigna radiata) and mash (Vigna mungo).     

Effects of heat treatments and germination on trypsin inhibitor activity and polyphenols in jack bean (Canavalia ensiformis L. DC)

The application of dry heat to seeds and meal was not effective in inactivating the TI and reducing the polyphenol content. Soaking for 24 h followed by cooking for 20 min, was equally effective in destroying the TI activity. Germination of jack bean seeds for 40 h decreased the levels of TI and polyphenols by 31% and 35%, respectively.     

Effects of heat treatments on trypsin inhibitor and hemagglutinating activities in winged bean

The effect of different heat treatments on inactivation of trypsin inhibitor and hemagglutinin of winged bean was investigated. Trypsin inhibitor extracted from winged bean meal was stable at 60 °C for 60 min. At 80 °C, the activity of the extracted inhibitor decreased by 25% within 5 min, and continued to decline gradually to a loss of 45% of activity after 30 min. When the extracted inhibitor was incubated at 100 °C, it exhibited a triphasic pattern of inactivation. The winged bean extract incubated at 60 °C lost 60% of its hemaggluinating activity within 30 min. At 80 °C, there was a complete loss of activity within 5 min. The microwave treatment to winged bean meal had no effect on trypsin inhibitor or hemagglutinating activities in the meal. However, infrared treatment to winged bean seeds for 60 seconds was effective in destroying most of the trypsin inhibitor and hemagglutinating activities. Autoclave treatment (120 °C at 15 lb pressure) for 10 min inactivated trypsin inhibitor and hemagglutinin in winged bean meal almost completely. Cooking of presoaked beans in boiling water for 30 min was effective in destroying most of the trypsin inhibitor and hemagglutinating activities.     

Effect of domestic processing,cooking and germination on the trypsin inhibitor activity and tannin content of faba bean (Vicia faba)

The effects of domestic processing methods like soaking for 12 h dehulling, ordinary cooking of whole as well as dehulled seeds at 151bs/inch² pressure for 15 and 25 min, and germination for 24, 36 and 48 h were observed on trypsin inhibitor (TIA) and tannin content of two varieties of faba bean viz. VH-131 and WF. A non-significant reduction in TIA was observed on soaking and dehulling, while tannins showed a reduction of 42 and 51% on soaking in VH 131 and WF, respectively. Dehulled seeds showed a decline in tannin content by 70 to 73% in two varieties. There was a significant reduction in TIA (75–76 per cent) and tannin (76–81%) on cooking, while autoclaving for 25 min almost completely eliminated both of these stress factors. Germination of seeds for 48 h led to a reduction of 64–65% in TIA and 90–91% in tannins, which was more than germination for 24 and 36 h. Soaked seeds gave positive results of both these antinutrients.     

Soaking and cooking parameters of tepary beans: effects of cooking time and cooking temperature on hardness and activity of nutritional antagonists

Tepary samples were examined for patterns of hydration, dry matter losses during the processes of soaking and cooking, residual hardness in partially cooked samples and heat lability of endogenous proteinaceous antinutritional factors. At 24 °C, teparies imbibed water equivalent to their weight (100% hydration) in 4 h and continued to absorb water rapidly for an additional 4 h before reaching an equilibrium hydration. During the processes of soaking and cooking, materials leached from raw beans represented 7.3 and 13.5% of their dry weight, 4.3 and 12.4% of their protein content, 7.1 and 12.2% of their stored carbohydrate and 22.4 and 33.4% of their mineral levels, respectively. In samples prepared at different cooking times (60, 90, 120, 150, 180 min) and cooking temperatures (80, 85, 90, 95°C), longer times and higher temperatures resulted in greater reductions in residual bean hardness; interactive effects of time and temperature treatments were significant. Residual activity of trypsin and chymotrypsin inhibitors in partially-cooked samples appeared to be negligible. In addition, at least 80% of the original hemaglutinating activity of lectins in raw beans was lost during partial-cooking of samples under all cooking regimes.     

Effect of alkali treatments on the nutritive value of common bean (Phaseolus vulgaris)

The effect of alkali treatments of common bean seeds with red seed coat on the stability of antinutritional factors such as tannins, phytates and trypsim inhibitors, vitamins such as niacin and riboflavin and on protein quality has been studied. The samples were processed by soaking and soaking and pressure cooking in alkalies such as sodium hydroxide, sodium carbonate and sodium bicarbonate. At low temperature sodium carbonate and at high temperature sodium bicarbonate were found to be most effective in the extraction of tannins. At both low and high temperatures sodium carbonate was found to be more efficient in destruction of phytates. In the case of trypsin inhibitors, extraction at both low and high temperatures with sodium bicarbonate was most effective. Sodium hydroxide treatment was found to be better as far as the retention of niacin and riboflavin was considered.     

Involvement of carboxypeptidase in the degradation of the mung bean (Vigna radiata) trypsin inhibitor during germination and early seedling growth

Examination of the substrate specifity of the carboxypeptidase activity of ungerminated and germinated mung beans (Vigna radiata (L.) Wilczek) reveals the presence of two distinct enzymes. The first of these, carboxypeptidase I, is maximally active against carbobenzyloxy-Ala-Phe. It is present in large amounts in the cotyledons of ungerminated seeds, and declines rapidly during germination. The second, carboxypeptidase II, is most active against carbobenzyloxy-Phe-Ala. This enzyme increases in the cotyledons during germination and seedling growth.The possible involvement of one or both of these enzymes in the degradation of the native mung bean trypsin inhibitor (MBTI - F) and its proteolytically modified forms (MBTI - E, - C, and - E') during germination was also examined. The enzyme catalyzing one step in the conversion of MBTI - E to MBTI - C has been isolated with 557-fold purification from germinated mung beans. The inhibitor-degrading enzyme has a molecular weight of approximately 60,000. It is optimally active against MBTI - E at between pH 4.0 to 4.5. No activity was found with MBTI - F, - E', or - C as substrates. The enzyme has been identified as the carboxypeptidase II on the basis of (1) coelution during chromatography, (2) action on synthetic and natural substrates, (3) sensitivity to enzyme inhibitors, and (4) temporal variation during germination. The MBTI degrading carboxypeptidase removes the two carboxyl-terminal residues from MBTI - E to produce an inhibitor species that co-migrates with MBTI - C on polyacrylamide gel electrophoresis, but has not been subjected to the internal cleavage and proteolysis at the amino-terminus found in MBTI - C.Supported by National Science Foundation Grants PCM 8003854 and PCM 8301202Abbreviations used are: TNBS, 2,4,6-trinitrobenzenesulfonic acid; Cbz-, carbobenzyloxy-; PMSF, phenylmethylsulfonyl fluoride; MBTI, mung bean trypsin inhibitor; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis.     

Trypsin inhibitor in moth bean: thermal stability and changes during germination and cooking

Trypsin inhibitor from moth bean was studied for thermal stability and changes during germination and cooking. The application of dry heat did not inactivate the inhibitor. However, autoclaving at 120°C at 15lbs pressure destroyed inhibitor activity completely. The extracted inhibitor lost 70% activity in 60 min when incubated at 100°C. Soaking of moth bean seeds for 8 h decreased trypsin inhibitor activity by 20%. The germination of seeds for 24 h resulted in 70% reduction in inhibitor activity. No activity was detected in 48 h germinated seeds. Germination (for 24 h) followed by cooking of moth bean seeds destroyed the trypsin inhibitor completely.     

Tepary beans (Phaseolus acutifolius var. latifolius): a potential food source for African and Middle Eastern cultures

Tepary bean (Phaseolus acutifolius var. latifolius), a native North American legume adapted to arid/semiarid lands was partially evaluated as a potential food source for people of African and Middle Eastern regions. To indicate the acceptability of these pulses, traditional Nigerian and Saudi Arabian foodstuffs were formulated with teparies substituted for beans commonly used in these regions. Organoleptic evaluation of these food products by students native to the area of recipe origin indicated the dishes to be moderately to highly acceptable. Chemical analyses of this species revealed tepary beans to be very similar in proximate composition, amino acid profile and content of various minerals to other grain legumes (cowpeas, chickpeas and fava beans) of the African/Middle Eastern region. Protein contents of tepary samples averaged 23.0%, whereas protein quality was found limiting in sulfur amino acids. Laboratory examination of raw tepary samples for antinutritional factors uncovered levels of flatulent oligo saccharides, trypsin inhibitors and phytic acid commonly associated with grain legumes. However, lectin assays revealed greater agglutination associated with tepary composites than in fava, chickpea and cowpea samples. All bean samples were simmered resulting in fully cooked materials which exhibited low levels of nutritional antagonists. Cooked beans were considered safe and nutritionally acceptable.Arizona Agricultural Experiment Station Paper No. 3814.     

The effect of slow-cooking on the trypsin inhibitor and hemagglutinating activities and in vitro digestibility of brown beans (Phaseolus vulgaris,var.Stella) and kidney beans (Phaseolus vulgaris,var.Montcalm)

Brown beans and kidney beans were subjected to two modes of cooking in a household slow cooker: (A), a fixed low setting for 10 h, and (B), a high setting for 2.5 h and a low setting for 7.5 h. Temperature changes in the beans were recorded. With treatment A over 90% of the hemagglutinating and trypsin inhibitor activities occurred after 6 h at which time the temperature had reached 80°C. With treatment B inactivation of these activities was almost complete at the end of 2 h when a maximum temperature of 100°C had been attained. The in vitro digestibility of the bean protein was considerably increased by either treatment. By way of contrast, only 20 min of heating was required to destroy these activities when the beans were brought to a boil in an open vessel.     

Studies on protein quality of green gram (Phaseolus aureus)

Raw green gram contains trypsin inhibitor (5.6 × 10^–3 units/0.1 g) while cooking destroyed the trypsin inhibitor in green gram. No destruction was observed in the case of lysine, methionine, cystine, threonine and tryptophan. Sulphur containing amino acids have been found to be the only limiting amino acids in green gram from studies on blood levels of amino acids and PER experiments. Raw green gram, when fed as a sole source of protein promoted an extremely low growth rate, (13.2 g/4 weeks) and PER (0.77) in rats. Cooking slightly improved the nutritive value of the gram, as indicated by increase in gain in body weight (24 g/4 weeks) as well as PER (1.22). The presence of inhibitors in green gram did not appear to affect some of the essential amino acids as observed from the blood amino acid levels.     

The effect of feeding soya bean trypsin inhibitor and repeated injections of cholecystokinin on rat pancreas

The effects of dietary soya bean trypsin inhibitor (SBTI) and of the daily injection of pure CCK or porcine intedstinal extract have been studied on the rat pancreas. The following parameters were measured: weight, DNA, RNA, trypsin, chymotrypsin, amylase, lipase, insulin and glucagon. The effects of dietary SBTI on the level of gastrin in the antrum have also been studied. Oral SBTI and i.p. CCK caused similar hypertrophy and hyperplasia but not the same activities of the enzymes. It is suggested that SBTI stimulates the secretion of at least two gastro-intestinal hormones: CCK and gastrin.     

Blanching of green bean (Phaseolus vulgaris)

Experiments with one and two steps blanching of green beans have been carried out. Inactivation of the peroxydase requires more heating than inactivation of the enzymes which gives rise to off flavour from aldehydes. When blanching for about one minute to inactivate lipoxygenase, aldehyde formation of flavour ceases. The content of vitamin C decreases during blanching according to a first order reaction. Since considerable loss of vitamin C occurs during blanching, the treatment time should be reduced to a minimum. During preblanching at 65–75 °C and final blanching, chlorophyll is degraded to pheophytin and the surface colour expressed by the Hunter-values (–a/b) increases with time which means that the colour of the beans changes from green to yellow. The firmness of beans, which was measured by use of a tenderometer, decreases during blanching according to a first order reaction with 40 kcal/mole activation energy. Preblanching at 65–75 °C increases the firmness of the beans linearly with treatment time. This increase in firmness is stable after final blanching at 95 °C and even after thawing of frozen beans.     

Influence of spices on protein utilisation of winged bean (Prophocarpus tetragonolobus) and horsegram (Dolichos biflorus)

The influence of a mixture of eleven spices commonly consumed in India on the utilisation of protein from boiled winged bean (Psophocarpus tetragonolobus) and horsegram (Dolichos biflorus) was studied at 10 and 20 percent level of protein intake in experimental rats. Spices used in the mixture include red chillies (Capsicum annum), black pepper (Piper nigrum), coriander (Coriandrum sativum), cumin (Cuminum cyminum), garlic (Allium sativum), ajowan (Carum copticum), turmeric (Curcuma longa), caraway seeds (Carum carui) and fennel seeds (Foeniculum vulgare). Addition of this spice mixture at 1.5% level of the diet decreased the TD of both legumes, significantly only in the case of horsegram. A significant increase was observed in the BV of both the legumes at both levels of protein tested.     

Tannins,Trypsin Inhibitors and Lectin Cytotoxicity in Tepary (<Emphasis Type="Italic">Phaseolus acutifolius</Emphasis>) and Common (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) Beans

This study compared the levels of antinutritional components and cytotoxic effect of extracts, from tepary (Phaseolus acutifolius) and common (Phaseolus vulgaris) beans. Antinutritional factors were evaluated by determining their effect on the viability of epithelial cells isolated from rat small intestine. The protein and carbohydrates content were similar in all the genotypes studied (20 and 60%, respectively). Common beans presented higher content of trypsin inhibitors, tannins and lectins than tepary beans. There was not a significant correlation between tannins and cooking time. However, water absorption and cooking time correlated significantly (p < 0.05). Considerable variation was observed in lectin activity (1302–18161 Ul/mg) of extracts from different beans. Tannins, lectins, trypsin inhibitors and fat content differed between bean varieties whereas protein content was similar. The percent cellularity on rat epithelial cells was significantly different among protein extracts from different bean cultivars and ranged between 53.5% and 87.4% (p < 0.05). These results suggest that the incorporation of tepary beans in the diet would not alter the current nutritional contribution of common beans or introduce adverse toxic effects. The agronomic characteristics of tepary beans make them attractive for cultivation. However, the harder to cook phenomenon may be a limiting factor that needs further consideration.     

Phytic acid in stored common bean seeds (Phaseolus vulgaris L.)

Four varieties of common bean seeds stored at 4°C, 80% relative humidity, for one to eight years showed no differences in proximal chemical composition, Seeds 5–6 years old absorbed more water than 1 to 4 year-old seeds. The cooking time required for five year-old seeds was 6 hours, while the fresh seeds needed 3/4-to-one hour cooking time. The most remarkable difference was in phytic acid content, which decreased 94% to 98% during long storage.     

Unavailable carbohydrates in common bean cotyledon (Phaseolus vulgaris L.) Canario group

Unavailable carbohydrates in cotyledon flour were quantified from four varieties of common bean (Phaseolus vulgaris L) belonging to the Canario group. The pectin content, as percentage of anhydrogalacturonic acid, ranged between 1.57% and 2.3%. Percentages of hemicellulose A and B were similar, with average values of 2.81% and 2.61%, respectively. The polysaccharide extracted with 10% trichloroacetic acid was determined; its yield was 3.17%. This value dropped to 2.35% when the cotyledon flour was previously hydrolyzed with polygalacturonase. The average value of cellulose was 1.36%.     

Comparison of procedures to determine protein content of developing bean seeds (Phaseolus vulgaris)

Micro-Kjeldahl, Lowry and Bradford procedures were compared for determining the protein content ofPhaseolus vulgaris seeds during their development. Micro-Kjeldahl and biuret techniques were also compared with mature seeds of a normal and a genetically-improved bean cultivar. The protein contents of casein and soy protein isolate were as well estimated by these four methods. For many samples of both bean and food protein products large disagreements were found between micro-Kjeldahl and the other three procedures.     

Relationship between content and intake of bean polyphenolics and protein digestibility in humans

The purpose of this study was to determine the effect of bean polyphenolics on bean protein digestibility in humans. Steam pressure cooking decreased tannic acid in beans by: 38.2 (white), 23.9 (red), 38.5 (white:black mixture, 50:50), 20.5 (Ipala black) and 44.4% (Jalpatagua black). The reduction in catechin was: 57.5, 97.6, 83.0, 78.7 and 88.4%, respectively. The content of tannic acid in dried cooked beans was: 284.9 in white, 834.5 in red, 620.2 in the white:black mixture, 1,021 in Ipala black and 1,009 mg/100 g in the Jalpatagua black bean. These percentages correlate significantly with fecal nitrogen (r=0.29, p<0.05). Catechin of the dried cooked beans was 7.5 in white, 27.4 in red, 25.4 in the white:black mixture (50:50), and 60.1 and 55.5 mg/100 g in each of the black cultivars, and correlated significantly with fecal nitrogen (r=0.34, p<0.01), absorbed nitrogen (r=–0.37, p<0.01) and protein digestibility (r=–0.35, p<0.01). It was concluded that one of the factors that reduces bean protein digestibility is the polyphenlic compound.