In vitro enzymatic hydrolysis of protein and protein pattern change of soya and faba beans during germination

In addition to technological processes like heat treatment, germination can be an alternative process for the improvement of protein quality of legumes. This was demonstrated by enzymatic protein hydrolysis of flour of germinated faba and soya beans, using a pepsinpancreatin enzyme system. SDS-PAGE was used to study the changes in protein pattern of these legumes during germination. In addition, the effect of germination on the content of condensed tannins in flour from germinated faba beans and trypsin inhibitors in flour from germinated soya beans were studied. Germination for five days resulted in a maximum increase in enzymatic protein hydrolysis by 21.3% in flour from faba beans and by 25.7% in flour from soya beans after 12 hours of germination. Protein patterns, obtained with SDS-PAGE demonstrated a considerable protein breakdown during germination between day 2 and 3 in faba beans and between day 1 and 2 in soya beans. The tannin content in flour from faba beans decreased by 29.7% after seven days of germination, but the tannin content of the hulls of the faba beans did not change during that period of germination. The trypsin inhibitors in flour from soya beans decreased by 25.5% after seven days of germination. We conclude that the increased enzymatic hydrolysis of protein in both legumes cannot be explained by a decrease of tannins or trypsin inhibitors. The possible explanation is that through degradation of proteins during germination of the legumes, the cleaved protein fragments are more susceptable for hydrolysis by pepsin-pancreatin.     

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.     

Comparative study of the chemical composition of wild and cultivated beans (Phaseolus vulgaris)

Five wildPhaseolus vulgaris beans were compared with five cultivatedPhaseolus vulgaris beans in proximate composition, total (true) protein, amino acid composition, and toxic and antinutritional factors. The wild beans contained more protein (25.5% vs. 21.7%), ash (5.15 vs. 4.15%), crude fiber (7.08% vs. 5.04%) compared to cultivated beans while the former contained less fat (0.56 vs. 0.89%) and carbohydrates (61.64 vs. 68.05%). Sulfur amino acids were found to be limiting in both groups of bean as expected; however, the cultivated beans had a higher content of the limiting amino acids. Therefore, the cultivated beans showed a better amino acid profile than the wild beans. Toxic factors were not found in either type of bean; the determinations included saponins, alkaloids, and cyanogenic glycosides. The antinutritional factors investigated were hemagglutinins (lectins) and trypsin inhibitors. The wild beans presented a higher content of trypsin inhibitors (28 TUI per mg) and lectins (9.6) than the cultivated beans did (21 TUI per mg and 7 respectively). From the chemical point of view, domestication seems to be positive; however, the better protein nutritive quality of the cultivated beans should be further confirmed by biological assays.     

Antinutritional factors in anasazi and other pinto beans (Phaseolus vulgaris L.)

Antinutritional factors of anasazi bean were compared to traditional pinto bean (Phaseolus vulgaris L.). Anasazi beans contained less (p<0.001) soluble and bound condensed tannins compared to pinto beans. No differences (p>0.05) in stachyose and raffinose content were found between the two bean types; verbascose was not detected at all. Significant (p<0.05) differences in lectin content were observed between anasazi and pinto bean. The lectins of anasazi beans were classified as non toxic and those of the pinto beans as toxic types. No differences (p>0.05) in inhibitor activity against human and bovine trypsin and chymotrypsin were found between the two bean types.     

Quick-cooking beans (Phaseolus vulgaris L.): II. Phytates,oligosaccharides, and antienzymes

Effects of the quick-cooking processes on phytate and oligosaccharide levels, and on trypsin and chymotrypsin inhibitors, were investigated in three bean varieties (Phaseolus vulgaris L.: Great Northern, red kidney, and pinto). Beans soaked in distilled water had lower levels of phytate-P than those soaked in a mixed salt solution. Leaching losses of oligosaccharides were nearly the same in different soaking treatments for all the beans except kidney beans. Residual trypsin inhibitor activities (TIA) in cooked quick-cooking beans were about 10% compared with about 20% for chymotrypsin inhibitor activities (CTIA) in the same bean products. -Irradiation was more effective in reducing TIA than CTIA and paralleled destruction by moist heat.Utah Agricultural Experiment Station Journal article no. 2480.     

Studies on α-amylase and trypsin inhibitors in legume seeds using agar diffusion and isoelectric focusing techniques

Amylolytic and tryptic inhibitors of faba bean extracts were determined by an agar diffusion test. The amylolytic inhibitor had protein characters. Furthermore, water-soluble trypsin inhibitors ofCicer arietinum, Lens esculenta, Lupinus termis, Phaseolus vulgaris, Pisum sativum, Trigonella foenum-graecum andVicia faba which were separated by polyacrylamide gel isoelectric focusing (PAGIF) in thin-layers, showed species specific patterns. Negative staining showed 10 bands for French beans, 9 for fenugreek seeds, 8 for lentils and chickpeas, 7 for peas and 6 for faba beans. Lupin seeds were free from trypsin inhibitors. Treatments (soaking, germination and heat processing) of faba beans reduced the number of trypsin inhibitors in PAGIF patterns, less after soaking and germination, but more after roasting and frying. No inhibitors were detected after cooking.     

Influence of heat processing of African yam bean seed (Sphenostylis stenocarpa) flour on the growth and organ weights of rats

The influence of heat processing of African yam bean seed flour on the growth and organ weights of rats was studied. Body weight change, feed utilization and feed conversion ratio were improved by heat processing. All rats significantly (p0.05) gained weight except those fed raw African yam bean and basal diets (diets 3 and 1 respectively). Raw African yam bean diet decreased the growth of rats and had negative effect on the organ weights especially the pancreas which was enlarged. The results indicate that heat processing improved the growth of rats and organ weights due to heat inactivation of toxic factors especially trypsin inhibitors.     

The presence and inactivation of trypsin inhibitors,tannins, lectins and amylase inhibitors in legume seeds during germination. A review

During the germination of legume seeds, enzymes become active in order to degrade starch, storage-protein and proteinaceous antinutritional factors. The degradation of storage-protein is necessary to make peptides and amino acids available in order to stimulate seed growth and early plant growth. Proteinaceous antinutritional factors such as amylase inhibitors, lectins and trypsin inhibitors are present in legume seeds and protect them against predators. However, during germination, they degrade to a lower level by the action of several enzymes. The effect of germination on the content and activity of amylase inhibitors, lectins, tannins and trypsin inhibitors is discussed.     

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.     

The use of enzymic hydrolysis in vitro to study the digestibility of some phaseolus seed proteins

In vitro proteolysis was used to study the digestibility of the major seed protein phaseolin, and the trypsin inhibitor, purified fromPhaseolus vulgaris seeds. Whereas denatured phaseolin was fully digested by the enzymic method used [4], its native form was only partially digested. Trypsin inhibitor, both native and after heat treatment, was poorly digested. It was also found that native trypsin had some resistance to digestion conferred upon it by the presence of the inhibitor.     

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.     

Genetic control of phaseolin protein expression in seeds of common bean,Phaseolus vulgaris L.

Phaseolin, the major globulin seed storage protein of common bean,Phaseolus vulgaris L., accounts for up to 50% of the total seed protein. The rapid accumulation of phaseolin in the maturing seeds begins about 14 days after flowering and continues for some 12–14 days longer. However, the amount and rate of phaseolin accumulation, related to variation in onset, length, termination, and rate of synthesis, have been shown to vary between genotypes.Only three phaseolin electrophoretic types, designated T, S, and C after the cultivars Tendergreen, Sanilac, and Contender, respectively, have been identified among over 100 cultivated accessions. The narrow ranges of molecular weights and isoelectric points of the 14 protein polypeptides of phaseolin, as well as the homology observed from peptide mapping, suggest that the phaseolin polypeptides are similar proteins. Based on the results of crosses among cultivars having the three electrophoretic patterns, the genes controlling the polypeptides of each of the phaseolin types appear to be tightly linked, inherited in a block and the alleles are codominant.Substantial variation in phaseolin content, based on estimations using rocket immunoelectrophoresis, has been found among bean lines. Although most segregating populations show continuous distributions and quantitative inheritance, some inbred backcross lines having enhanced phaseolin accumulation appear to carry a few genes with major effects. A single gene that reduces the amount of phaseolin to less than one-half of the normal levels has been identified recently in an accession of wildP. vulgaris.     

Effect of Cooking Procedures and Storage on Starch Bioavailability in Common Beans (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Common commercial beans were cooked using two procedures: under pressure (autoclaving) and traditional cooking. Total starch extraction was higher in beans cooked with the traditional procedure (41.69–42.81%) than in the autoclaved samples (37.04–38.16%) and did not change during storage at 4 °C. However, available and total resistant starch levels in vitro were not influenced by the cooking procedure or storage. Retrograded resistant starch content was higher in beans cooked with the traditional process (2.65–2.79%) than in autoclaved beans (1.62–1.94%). The initial in vitro -amylolysis rate in freshly cooked beans was higher in the autoclaved preparation than in the beans cooked by the traditional process, but final hydrolysis indices (90 min) were similar for both samples. None of the bean samples showed statistical differences in -amylolysis behavior (=0.05) after storage at 4 °C for 96 hour.     

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.     

Chemical changes during the fermentation of African locust-bean (Parkia filicoidea Welw) seeds for production of ‘Daddawa’

African locust-bean (Parkia filicoidea Welw) seeds were allowed to ferment under natural conditions and the chemical changes occurring during the fermentation were studied. Chemical analysis of fermented and unfermented beans showed a marked decrease in the total sugar content; crude and true protein levels all increased. The results of the chemical analyses indicate that fermentation resulted in protein enrichment of the fermented locust bean (daddaw). Fermentation also imparts a characteristic flavour and odour to the beans.     

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.     

Impact of microwave heating on hemagglutinins,trypsin inhibitors and protein quality of selected legume seeds

Selected legume seeds (dry and soaked) including faba beans, peas, chickpeas, soybeans, lentils and common beans containing 8 and 25% moisture, respectively, were subjected to microwave heating, and to a conventional cooking method to determine the heating effect on toxic compounds and protein quality. Trypsin inhibitors, hemagglutinins and available lysine were analyzed, and laboratory rats were used to determine digestibility and protein efficiency ratio (PER). Results indicated that microwaving destroyed trypsin inhibitors to a similar degree to that observed in beans cooked using the conventional method without affecting PER for raw seeds with low antinutrients content (faba beans, peas, chickpeas and lentils). Microwave-heated soaked soybeans had a higher amount of destroyed trypsin inhibitors, along with a higher PER, compared with microwave-heated dry soybeans. Microwave heating of common beans failed to destroy hemagglutinins and trypsin inhibitors, and consequently their digestibility and PER values were poor. Finally it was concluded that microwave heating constitutes an adequate method for destroying hemagglutinins and trypsin inhibitors without affecting protein quality of most legume seeds, except for common beans that despite of this process retained the antinutritional substances.     

The endogenous endoprotease inhibitors of barley and malt and their roles in malting and brewing

Endoproteases play an important role in barley germination by controlling the hydrolysis of the grain's storage proteins into peptides and amino acids that are needed by the young plant. During malting, the commercial version of this process, many high M_r barley biopolymers are converted into malt nutrients that can be utilized by yeasts during brewing. However, barley and malt both contain endogenous proteins that inhibit the enzymatic activities of these proteases. High levels of these inhibitors can cause brewing problems by preventing the proteases from producing optimal levels of soluble proteins and amino acids. Both high and low M_r inhibitors of cysteine proteases occur in barley and malt. Two of the high M_r inhibitors, lipid transfer protein 1 (LTP1) and LTP2, have been purified and studied. Recently, members of the trypsin/alpha-amylase inhibitor protein family (CM proteins) have been shown to inhibit the activity of SEP-1, a purified serine class barley protease. No inhibitors of aspartic proteases or metalloproteases have yet been purified, but it has been reported that endogenous metalloprotease inhibitors do exist. The inhibitors of the cysteine proteases and metalloproteases are probably the ones most important for brewing, because members of these two protease classes apparently catalyse most of the protein hydrolysis that occurs during malt mashing and, presumably, also during malting. More biochemical studies are needed to clarify how these proteins interact with the proteases to control protein hydrolysis during germination.     

Effect of particle size on kinetics of starch digestion in milled barley and sorghum grains by porcine alpha-amylase

The influence of milled grain particle size on the kinetics of enzymatic starch digestion was examined. Two types of cereals (barley and sorghum) were ground, and the resulting grounds separated by size using sieving, with sizes ranging from 0.1 to 3 mm. In vitro enzymatic digestion was performed, using pancreatic alpha-amylase, amyloglucosidase and protease, to determine fractional-digestion rates over 24 h. The resulting glucose production rate data were well fitted by simple first-order kinetics. For each sieve screen size, the digestion rate of barley was always higher than that of sorghum. The rate coefficients for digestion showed a decrease with increasing size, and could be well fitted by an inverse square relationship. This is consistent with the supposition that starch digestion in these systems is controlled by diffusion of enzyme through the grain fragment. Apparent diffusion coefficients of alpha-amylase obtained by fitting the size dependence were 0.76 (sorghum) and 1.7 (barley) × 10⁻⁷ cm² s⁻¹, 9 (sorghum) and 4 (barley) times slower than predicted for a molecule of the size of alpha-amylase in water.     

Effect of soaking,cooking and germination on the oligosaccharide content of selected Nigerian legume seeds

The identity and quantity of and effect ofprocessing on raffinose oligosaccharides in raw,mature seeds of lima beans (Phaseolus lunatus),pigeon peas (Cajanus cajan), African yam beans(Sphenostylis sternocarpa) and jackbeans (Canavalia ensiformis) were investigated. Sucrose,raffinose, stachyose and verbascose were identified byHPLC in all the legume seeds. The total-galactoside contents of the seeds in decreasing order were African yam beans3.84 mg/100 mg; white lima beans 3.62 mg/100 mg; creampigeon peas 3.51 mg/100 mg; red lima beans3.37 mg/100 mg; jackbeans 2.83 mg/100 mg and brownpigeon peas 2.34 mg/100 mg. The predominantoligosaccharide was verbascose in pigeon peas andstachyose in the other three legumes. Cooking unsoakedseeds brought about a greater reduction in the total-galactoside content than soaking for ninehours. The removal of oligosaccharides was higher inlegumes cooked in alkaline solution than in water.Germination quantitatively reduced raffinose,stachyose and verbascose while sucrose was increasedin all seeds except red lima beans and jackbeans.