Composition,toxic and antinutritional factors of newly developed cultivars of Brazilian soybean (Glycine max)

Five different recently released Brazilian soybean cultivars (Bays, BR-10, Rio Balsas, Serido and Tropical) were compared for their proximate analyses and presence of antinutritional or toxic factors. As expected, the seeds are rich in proteins, varying from 360·7 to 485·4 g kg⁻¹ flour, and they also have a high amount of fat (from 183·0 to 215·3 g kg⁻¹ flour). Crude extracts from seeds of Bays, BR-10, Serido and Tropical were highly toxic to mice within 1–12 h, depending on the administration route (intraperitoneal, intramuscular or subcutaneous) and dose used while Rio Balsas was not. These acute effects were very similar to those produced by the soytoxin, a neurotoxin that has been recently purified from the commercial soybean sold in Brazil. The amount of trypsin inhibited in the presence of crude extracts ranged from 28·5 to 62·5 g kg⁻¹ flour. Urease was also present and the seed lectin agglutinated preferentially rabbit erythrocytes. A heat treatment at 92°C for 1 min destroyed completely the toxic activity while the haemagglutinating and trypsin inhibitor activities were abolished within 5 min. At these conditions urease was still active. Due to its high protein content, lack of soytoxin, and low levels of trypsin inhibitor, lectin, and urease it is suggested that Rio Balsas could be an alternative for breeding programmes aimed to improve the nutritional quality of soybeans. ©1997 SCI     

Inactivation of trypsin inhibitor,lectin and urease in soybean by hydrothermal treatment

The effects of hydrothermal treating parameters on trypsin inhibitor (TI), lectin and urease activities in whole soybean seeds were investigated by Response Surface Analysis (RSA). Independent variables with equidistant levels examined in this study were the following: treating temperature (levels: 70, 85 and 100°C), treating time (levels: 10, 30 and 50 min), and soaking time (2, 4 and 6 h). The functions between treating parameters and responses values of TI, lectin and urease activities in treated soybean were calculated by multiple, non-linear regression analysis and analysis of variance. Mathematical models were developed in this study to predict TI, lectin and urease activities in soybean during hydrothermal processing and they have been found to be significant (P[%] = 0.1, 1.0, and 0.1, respectively). Differences between the effects of processing parameters on the inactivation of TI, lectin and urease in soybean were observed and they can be seen either from the mathematical models or the typical figures. The modeling of the effects should help in selection of optimal parameters of hydrothermal treatment for destruction of TI. lectin and urease in soybean. Based on the modeling, lectin and urease can be fully inactivated in soybean treated at proper conditions, while remaining TI activity can be expected in soybean treated at any conditions examined in this study.     

Preparation of trypsin‐immobilised chitosan beads and their application to the purification of soybean trypsin inhibitor

BACKGROUND: Trypsin inhibitors are among the most important antinutritional factors in legumes. Recent research has shown that soybean trypsin inhibitor (SBTI) exhibits multiple bioactivities, but very few studies on the purification of SBTI are available. Enzymes are commonly used as biospecific ligands in affinity purification of their substrates or inhibitors. The aim of the present study was to prepare trypsin (EC 3.4.21.4)‐immobilised chitosan beads and use them to purify trypsin inhibitor from soybean whey. RESULTS: Compared with free trypsin, the immobilised trypsin had higher thermal and pH stability. The adsorption ratio of SBTI from crude SBTI aqueous solution by trypsin‐immobilised chitosan beads was 33.3%. The purified SBTI obtained by affinity chromatography was characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis as a single polypeptide band with an M_r of 8.3 kDa belonging to the Bowman–Birk family. CONCLUSION: Trypsin‐immobilised chitosan beads were effectively used in the affinity separation of trypsin inhibitor from soybean seeds, thus indicating that immobilised trypsin may have practical application in the soybean‐processing industry. The results of this study provide a background for further investigation of potential applications of soybean bioactive constituents in the areas of agriculture and food. Copyright © 2008 Society of Chemical Industry     

Identification and characterisation of the major aspartic proteinase activity in Theobroma cacao seeds

Theobroma cacao seeds contain an unusually high level of aspartic proteinase activity. Although this activity is central to the development of high‐quality cocoa flavour, the T cacao polypeptide responsible has not yet been definitively identified. Here we report the identification and characterisation of an active protein complex from T cacao seeds with an apparent molecular weight of approximately 50 kDa. This active complex contains at least two polypeptides: an approximately 30.5 kDa aspartic proteinase, the product of the TcAP2 gene, and an associated polypeptide, the 20.5 kDa trypsin inhibitor protein. The active complex co‐eluted off a size exclusion column with another complex containing the trypsin inhibitor and a putative acid chitinase. The 30.5 kDa TcAP2 proteinase is apparently a monomeric aspartic proteinase with optimal activity between 42 and 47 °C and an optimal pH of 3.0. Significant inactivation of the TcAP2 activity occurs at acid pH around 47–52 °C, a temperature potentially obtained during cocoa bean fermentation. SDS‐PAGE analysis showed that the purified TcAP2 complex efficiently degrades the cacao seed storage protein vicilin into peptides smaller than 10 kDa. In addition, high‐resolution size exclusion chromatography showed that this proteinase is capable of degrading proteins into peptides as small as di‐ and tripeptides, indicating for the first time that the main T cacao seed aspartic proteinase can produce very small peptide products. Our results demonstrate that the aspartic proteinase encoded by the TcAP2 gene plays a critical role in the production of cocoa flavour precursor peptides during cocoa bean fermentation. Copyright © 2005 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF CHYMOTRYPSIN FROM PENAEUS VANNAMEI (CRUSTACEA: DECAPODA)

The purification and characterization of a chymotrypsin from the hepatopan-creas of the white shrimp Penaeus vannamei is described. Only one chymotrypsin was detected in contrast to other shrimp that have two major forms. P. vannamei chymotrypsin has a molecular mass of 33.2 kDa and a pI of 3.1. The molecular mass is high relative to other penaeid chymotrypsins. The proteinase is acid labile and exhibits optimum activity at pH 8. The enzyme is thermostable both at 25 and 37C. It is a serine proteinase. Phenylmethylsulphonyl fluoride and soybean trypsin inhibitor blocked the activity of the enzyme, and it was not affected by chymotrypsin inhibitors such as tosyl-PheCH₂Cl or benzyloxycarbonyl-Phe-CH₂Cl. Protein profiles of the hepatopancreas from two populations varied     

PURIFICATION AND CHARACTERIZATION OF A SERINE PROTEINASE FROM THE TUNA PYLORIC CAECA

A 15.0 kDa serine proteinase with collagenase activity from pyloric caeca of tuna, Thunnus thynnus, was purified in four steps; acetone precipitation, gel filtration chromatography on a Sephadex G‐100, ion‐exchange chromatography on a DEAE‐Sephadex α‐50 and gel filtration chromatography on a Sephadex G‐75 column. The purification and yield were 30.5‐fold and 0.023%, respectively, as compared with those in the starting crude extract. The optimum pH and temperature for the purified collagenolytic enzyme were around pH 7.5 and 55C, respectively. The purified proteinase was strongly inhibited by metal ions (Hg²⁺ and Zn²⁺) and serine proteinase inhibitors (PMSF, TLCK and soybean trypsin inhibitor) suggesting it is a serine protease. The K_m and V_max of the purified enzyme for collagen type I were approximately 3.82 mM and 851.5 U, respectively.     

The influence of tempeh fermentation and conventional cooking on anti‐nutrient level and protein bioavailability (in vitro test) of grass‐pea seeds

BACKGROUND: The solid state fermentation tempeh type is known to result in the decomposition of anti‐nutrients and the improvement of nutritional value of legume seeds. The aim of the research was to study the influence of tempeh fermentation and conventional cooking on (1) levels of 3‐N‐oxalyl‐L ‐2,3‐diaminopropionic acid (ODAP), soluble phenols, trypsin inhibitors and inositol phosphates; and (2) the in vitro bioavailability of proteins in grass‐pea seeds. RESULTS: Tempeh fermentation reduced the level of ODAP, trypsin inhibitors and phytates by 93, 99 and 22%, respectively, and increased protein bioavailability by about 25%. Protein bioavailability from conventionally cooked seeds was higher than from fermented seeds. However, during the in vitro test more soluble protein (approx. 10%) was released from 100 g tempeh product than from cooked seeds. CONCLUSIONS: Solid state fermentation and cooking resulted in seeds of comparable quality but the tempeh contained much less ODAP, which is very promising in the context of popularising grass pea. Copyright © 2008 Society of Chemical Industry     

大豆发芽过程中抗营养因子的变化

大豆蛋白存在着胰蛋白酶抑制因子和凝集素等抗营养因子,影响人类对其的利用。文中采用分光光度法和免疫火箭电泳法测定了发芽过程中东农42、东农823、71434、40567、黑农40五个大豆品种中胰蛋白酶抑制因子和凝集素的含量变化。结果发现:发芽能明显改变大豆中抗营养因子的含量,不同品种的抗营养因子变化程度不同,5种大豆在发芽过程中显示出较为一致的总体趋势。发芽温度对大豆中胰蛋白酶抑制因子的含量有影响。证实在萌发的大豆中存在可失活大豆抗营养因子的内源酶。     

DOUBLE-HEADED TRYPSIN/CHYMOTRYPSIN INHIBITORS FROM HORSE GRAM (DOLICHOS BIFLORUS): PURIFICATION, MOLECULAR AND KINETIC PROPERTIES

Four proteinase inhibitors were purified to homogeneity from horse gram (Dolichos biflorus). These inhibitors are double-headed and inhibit trypsin and chymotrypsin simultaneously and independently. Dissociation constants range between 0.87 and 4.6 × 10^?7 M. Each of the four isoinhibitors possesses a crucial lysine residue at the trypsin reactive site. These inhibitors have molecular masses of 8.5 kDa and isoelectric points of 4.6 to 5.6. They exist mainly as dimers under physiological conditions. Amino acid analysis revealed high levels of half-cystine, serine, aspartate and proline but low levels of methionine and aromatic amino acids. Amino-terminal sequence analysis revealed that each of the four isoinhibitors have a conserved core sequence but are divergent at the N-terminal end. These inhibitors belong to the Bowman-Birk (BBI) family of proteinase inhibitors as reflected by their inhibitory properties, amino acid composition and homology to other BBIs.     

Effects of heating on protein quality of soybean flour devoid of Kunitz inhibitor and lectin

The effects of autoclaving on protein quality of soybean flours prepared from a conventional soybean (CSB) and an isoline lacking Kunitz inhibitor and lectin (KFLF) were studied. The heating was efficient in the urease, trypsin inhibitors and lectin inactivation, being 15 min sufficient to reduce more than 90% of these compounds and provide protein solubility over 80%. The results of PER, NPR and weight gain showed that heating equally improved the nutritional quality of both soybean flours, although higher autoclaving time was required for KFLF. No significant improvement was observed on NPU and in vivo digestibility of the diets containing KFLF at any heating time. As these later results were similar to those obtained with diets containing CSB, they show the importance of the heating to improve the nutritional value and show that other components rather than trypsin inhibitors and lectins impair the nutritive value of raw soybean.     

PURIFICATION AND CHARACTERIZATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF SILVER CARP

Recently, a myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S‐200, High Q ion‐exchange and affinity column of Arginine Sepharose‐4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l ‐3‐carboxy‐trans‐2, 3‐epoxypropionyl‐l ‐leucine‐4‐guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin‐like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.     

Proteinaceous Inhibitors of Trypsin and of Amylases in Developing and Germinating Seeds of Pigeon Pea ( Cajanus cajan(L) Millsp)

Inhibitors of trypsin and amylase in the extracts of developing seeds of 12 pigeon pea cultivars were analysed using a gel-X-ray film contact print technique and an enzyme-inhibitor assay, respectively. The inhibitors of amylase and trypsin in the extracts of germinating seeds of a pigeon pea cultivar (BDN2) were also studied. Nine trypsin inhibitor bands were detected in mature seeds of all the 12 cultivars. Inhibitory activities against amylase and trypsin were not detected in the extracts of seeds collected 11 and 27 days after flowering (DAF) by the enzyme-inhibitor assay. However, up to three trypsin inhibitor bands could be detected in the extracts of seeds collected 27 DAF by the gel-X-ray film contact technique. Two new slow-moving trypsin inhibitor bands were detected in the extracts of germinating seeds of BDN2 cultivar. These bands were prominent in extracts of seeds 10 days after germination (DAG). The amylase inhibitors and trypsin inhibitors in pigeon pea seeds are late synthesised proteins, their highest levels were observed in mature seeds and they were found to be slowly degraded during germination. Significant inhibitor activities were observed even 15 DAG. The amylases in developing seeds are insensitive to endogenous inhibitors.     

Biochemical basis of insect resistance in Vigna unguiculata

The bruchid beetle Callosobruchus maculatus (F.) causes extensive damage to seeds of the cowpea, Vigna unguiculata (L.) (Walp.), when this important tropical foodstuff is stored. A variety of cowpea resistant to attack by this pest has been described. In the present work seeds of a number of cowpea varieties, including the resistant one, were tested for the presence of a physical resistance to C. maculatus, in terms of repulsion of oviposition or of failure of larvae to enter the seeds. No evidence to suggest the presence of a physical resistance was found. When seeds of cowpea varieties were tested for the presence of various antimetabolic secondary compounds, only inhibitory activity against trypsin and, to a much lesser extent, chymotrypsin, could be detected. The resistant variety of cowpea contained a significantly higher level of inhibitors, about twice as much as any other variety. A proteinase inhibitor active against trypsin was purified from cowpea varieties by affinity chromatography on trypsin-Sepharose. The purified inhibitor was shown to inhibit chyraotrypsin also, in such proportions as to account for chymotrypsin inhibition by seed extracts. The inhibitor was shown to consist of a number of isoinhibitors by gel electrophoresis and isoelectric focusing, but no qualitative differences in the inhibitor between varieties could be detected. The antimetabolic nature of the cowpea trypsin inhibitor was confirmed by insect feeding trials in which various protein fractions were added to a basic meal and the effect on larval survival noted. The albumin proteins of cowpea (containing the trypsin inhibitors) at a level of 10% were toxic to larvae of C. maculatus whereas the globulin fractions were not. Further, if cowpea trypsin inhibitor was removed from the albumin proteins they ceased to be toxic. When purified cowpea trypsin inhibitor was added to the basic meal it was shown that a level slightly less than that found in the resistant variety of cowpea caused complete mortality of larvae, whereas lower levels had lesser or no effect. It is concluded that this example of insect resistance in the cowpea is due to an elevated level of trypsin inhibitor.     

ISOLATION AND CHARACTERIZATION OF TRYPSIN INHIBITORS FROM SOME THAI LEGUME SEEDS

Trypsin inhibitors from cultivars of cowpea (Vigna unguiculata (L.) Wasp.), pigeon pea (Cajanus cajan (L.) Millsp.) and bambara groundnuts (Voandzeia subterranea (L.) Thou) grown in Thailand were isolated and characterized. Extraction of seeds with NaCl rendered a higher recovery of trypsin inhibitor than other solvents tested (P<0.05). The extraction time affected the inhibitor recovery (P<0.05). The extraction time of 3 h was optimum for the recovery of trypsin inhibitor from pigeon and bambara groundnuts, whereas 1 h was optimum for cowpea. Based cn inhibitor activity of zones separated by electrophoresis, the molecular mass of the inhibitor from bambara groundnuts was 13 kDa. Two inhibitory bands were observed for cowpea (10 and 18 kDa) and pigeon pea (15 and 25 kDa). Partial purification of inhibitors was achieved by heat‐treatment at 90C for 10 min, followed by ammonium sulfate precipitation with 30–65% saturation. The partially purified inhibitors from four seeds were heat stable up to 30 min at 90C at pH 7.0. The activities were also retained over a wide pH range at 25C but were lost when samples were treated with β‐mercaptoethanol prior to electrophoresis.     

Effect of legume seed extracts on the inhibition of proteolytic activity and muscle degradation of fresh water prawn (Macrobrachiumrosenbergii)

Trypsin inhibitors in the extracts from soybean (Glycine max), adzuki bean (Vigna angularis), bambara groundnut (Vigna subterranea) and red kidney bean (Phaseoulus vulgaris) varied in amount and molecular weight. The soybean extract had the highest level of trypsin inhibitor with molecular weight (MW) of 21 kDa, followed by bambara groundnut extract possessing trypsin inhibitor with MW of 15 kDa. Both extracts showed a more effective inhibition towards crude protease extract (CE) from the hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) than the extracts from adzuki and red kidney beans. Activity staining also reconfirmed the higher inhibitory activity on CE from hepatopancreas by the extracts from both soybean and bambara groundnut. The extracts from all seeds were able to inhibit the degradation of fresh water prawn meat containing CE in a concentration dependent manner. Based on inhibitor study, the extracts from soybean and bambara groundnut can be a potential aid to suppress the muscle softening of fresh water prawn, mediated by trypsin-like proteases released from hepatopancreas, during extended iced storage.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF TROPOMYOSIN-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF YELLOW CROAKER (PSEUDOSCIAENA CROCEA)

A myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of yellow croaker (Pseudosciaena crocea) was isolated from myofibril by heat treatment and chromatographies on Sephacryl S‐200, fast performance liquid chromatography (FPLC) on Mono Q column and high performance liquid chromatography (HPLC) using a Bio‐Sil SEC‐125 column. A single protein band with a molecular weight of 34 kDa was observed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Optimum temperature and pH of the purified protein was 55C and 8.0. Inhibitor susceptibility analysis indicated that the enzymatic activity was effectively suppressed by serine proteinase inhibitors such as Pefabloc 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride and aprotinin, while inhibitors for other proteinases (namely cysteine, asparatic and metallo) did not show any inhibitory effect. At the last purification stage, the electrophoretic study revealed that the proteinase associated with tropomyosin, as the N‐terminal amino acid sequence of the 34 kDa main band protein, exhibited high sequence homology (90%) to α‐tropomyosin from white croaker, human and rat. This result suggested that yellow croaker MBSP is an α‐tropomyosin‐binding proteinase.     

IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARP

Myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55–60C as shown by SDS‐PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as α‐actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril‐bound serine proteinase.     

COMPARATIVE STUDY OF ENZYMATIC CHARACTERISTICS OF TRYPSINS FROM THE PYLORIC CECA OF YELLOW TAIL (SERIOLA QUINQUERADIATA) AND BROWN HAKELING (PHYSICULUS JAPONICUS)

Trypsins from the pyloric ceca of two fish species, yellow tail (Seriola quinqueradiata) and brown hakeling (Physiculus japonicus) were purified by a series of chromatographic separations. Purity increased 62‐ and 106‐fold with approximately 55 and 10% yield for yellow tail trypsin and brown hakeling trypsin, respectively. Final enzyme preparations were homogeneous in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and the molecular weights of both enzymes were estimated to be 24 kDa by SDS‐PAGE. Yellow tail and brown hakeling trypsins had maximal activity at pH 8.0 for hydrolysis of N_α‐p‐tosyl‐L‐arginine methyl ester hydrochloride and was unstable at acidic pH. The optimum temperatures for yellow tail and brown hakeling trypsins were 60 and 50C, respectively. Yellow tail trypsin was stable up to 50C, whereas brown hakeling was stable up to 40C. Both trypsins were stabilized by calcium ions. The activities of both trypsins were strongly inhibited by soybean trypsin inhibitor and N_α‐p‐tosyl‐L‐lysine chloromethyl ketone hydrochloride, and were partially inhibited by ethylenediaminetetraacetic acid. The N‐terminal amino acid sequences of yellow tail trypsin and brown hakeling trypsin were determined as IVGGYECTPYSQPHQVSLNS and IVGGYECPKHSQPHQVSLNS, respectively.     

Biochemical basis of insect resistance in inged bean (Psophocarpus tetragonolobus) seeds

Mature seeds of the inged bean (Psophocarpus tetragonolobus) are toxic to developing larvae of a range of cosmopolitan storage Bruchidae of economic importance, including the copea seed eevil, Callosobruchus maculatus. Insect feeding trials ere carried out in hich protein fractions from seeds of inged bean ere incorporated at a range of concentrations into artificial seeds, and their effects upon development of C maculatus determined. Both albumin and globulin fractions ere toxic to the developing larvae and their toxicity correlated ith their haemagglutinating activity. Assay of Psophocarpin A, B and C fractions demonstrated Psophocarpin B to be the most insecticidal and to contain the highest haemagglutinating activity. Purified basic seed lectin as highly insecticidal to C maculatus larvae, ith an LC₅₀ value of c. 3·5 g kg^?1. The physiological level of this protein in inged bean seeds is sufficient to account for their resistance to attack by C maculatus. inged bean trypsin inhibitor as also purified and tested in artificial seeds against C maculatus. Hoever, even at concentrations in excess of tice the physiological concentration it had no deleterious effects upon development.     

Characterization of proteins from kernel of different soybean varieties

BACKGROUND: Total soybean proteins, storage proteins, glycinin (11S) and β‐conglycinin (7S) fractions and their respective subunits in seven soybean varieties were analyzed. In this work we also present the correlation between concentration and activity of bioactive proteins, lipoxygenase and proteinase inhibitors. RESULTS: Glycinin and β‐conglycinin comprise about 750 g kg^?1 of the bean storage protein and as such account for both quantity and quality of the kernel protein. The 11S concentration of the varieties studied ranged from 503.4 to 602.9 g kg^?1 and those of 7S varied from 178.2 to 230.6 g kg^?1 of total extractable proteins. The ratio of 11S/7S proteins varied from 2.43 to 3.29 among the varieties. A very strong positive correlation was found between the concentration of Kunitz trypsin inhibitor and activity of total trypsin inhibitor (r = 0.96). However, lipoxygenase concentration did not show a strong correlation with lipoxygenase activity. CONCLUSION: It appears that among the seven ZP soybean genotypes there are genotypes with different amounts of subunits that should be bred in the future for a desired level of protein components. Copyright © 2010 Society of Chemical Industry