Changes in inhibitory activity and secondary conformation of soybean trypsin inhibitors induced by tea polyphenol complexation

BACKGROUND: Tea polyphenol (TP) is a new food additive for antioxidant application, while soybean is an important resource for food and feed processing. It is therefore of rational and practical significance to investigate the influence of TP on soybean trypsin inhibitors (TIs). The aim of this study was to determine the effects of TP on the inhibitory activity of Kunitz (KTI) and Bowman–Birk (BBTI) TIs and to reveal the relationship between the inhibitory activity and conformation of KTI and BBTI by measurement of circular dichroism (CD) spectra. RESULTS: KTI and BBTI were found to be partially deactivated by TP. BBTI exhibited stronger resistance than KTI to TP deactivation. The unchanged K_M value of trypsin for benzoyl‐DL ‐arginine‐p‐nitroanilide hydrolysis indicated that KTI and BBTI inhibited trypsin in a non‐competitive pattern when complexed with TP. As the TP/TI ratio was increased and the inhibitory activity of KTI and BBTI decreased, the conformation of KTI and BBTI showed relevant changes and the major CD negative bands shifted progressively towards the near‐UV region. CONCLUSION: These results show the deactivation effects of TP on KTI and BBTI and reveal preliminarily the relationship between the inhibitory activity and secondary structure of KTI and BBTI. Copyright © 2009 Society of Chemical Industry     

Changes of trypsin in activity and secondary structure induced by complex with trypsin inhibitors and tea polyphenol

To reveal the relationships between the activity of trypsin and its structural change, changes of trypsin in biological activity induced by complex with Bowman-Birk trypsin inhibitor (BBTI), Kunitz soybean trypsin inhibitor (KSTI, type I-S) and tea polyphenol (TP) were detected and their relationship with the secondary structure changes were studied by far-UV circular dichroism (CD) spectra measurement. BBTI and KSTI were also irradiated by ultrasonic to compare the effects on trypsin. The rank was found as KSTI > BBTI > TP according to their inhibitory activities against trypsin. Yet BBTI exhibited much stronger resistance against ultrasonic irradiation than KSTI. BBTI, KSTI and TP were found inactivate trypsin by modifying the secondary structures and far-UV spectrum of trypsin. Complex of trypsin with ultrasonic-treated BBTI and native BBTI and KSTI exhibited the similar modified effects in secondary structures, decrease of α-helix and β-turn content, increase of β-sheet content and unchanged random coil content basically. But complex of trypsin with ultrasonic-treated KSTI exhibited less modified effects because of inactivation by ultrasonic irradiation. The changes of trypsin in secondary structure induced by complex with TP showed different from those induced by complex with BBTI and KSTI, increase of α-helix content, decrease of random coil content and unchanged β-sheet and β-turn content basically.     

Isolation and characterisation of trypsin from sardinelle (Sardinella aurita) viscera

BACKGROUND: In Tunisia, sardinelle (Sardinella aurita) catches totalled about 13 300 t in 2002. During processing, solid wastes including heads and viscera are generated, representing about 30% of the original raw material. Viscera, one of the most important by‐products of the fishing industry, are recognised as a potential source of digestive enzymes, especially proteases with high activity over a wide range of pH and temperature conditions. This paper describes the purification procedure and some biochemical characterisation of trypsin from S. aurita viscera. RESULTS: Trypsin from the viscera of sardinelle (S. aurita) was purified by fractionation with ammonium sulphate, Sephadex G‐75 gel filtration, Sepharose mono Q anion exchange chromatography, ultrafiltration and a second Sephadex G‐75 gel filtration, resulting in a 5.42‐fold increase in specific activity and 6.1% recovery. The molecular weight of the purified enzyme was estimated to be 24 kDa using size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme showed esterase‐specific activity on N‐α‐benzoyl‐L ‐arginine ethyl ester (BAEE) that was four times greater than its amidase‐specific activity on N‐α‐benzoyl‐DL ‐arginine‐p‐nitroanilide (BAPNA). The optimal pH and temperature for enzyme activity were pH 8 and 55 °C respectively using BAEE as a substrate. The trypsin kinetic constants K_m and k_cat on BAPNA were 1.67 mmol L^?1 and 3.87 s^?1 respectively, while the catalytic efficiency k_cat/K_m was 2.31 s^?1 L mmol^?1. CONCLUSION: Trypsin was purified from sardinelle (S. aurita) viscera. Biochemical characterisation of S. aurita trypsin showed that this enzyme can be used as a possible biotechnological tool in the fish‐processing and food industries. Copyright © 2008 Society of Chemical Industry     

Characterisation of a trypsin/chymotrypsin inhibitor from jack fruit (Artocarpus integrifolia) seeds

Jack fruit seed (Artocarpus integrifolia Hook f) trypsin inhibitor (JSTI) was found to be rich in acidic amino acids and devoid of free thiol groups. The N-terminal and C-terminal amino acids of JSTI were aspartic acid and scrine, respectively. The inhibitor was stable under conditions of extremes of pH (3·0–12·0), at high temperatures and in the presence of denaturing agents. JSTI showed a non-competitive type of inhibition with K_i values of 0·48 ± 0·17 nM and 0·16 ± 0·04 nM for trypsin and chymotrypsin, respectively. The JSTI–trypsin complex exhibited chymotrypsin inhibitory activity suggesting the ‘double-headed’ nature of the inhibitor. Chemical modification of lysine residues resulted in loss of trypsin and chymotrypsin inhibitory activities of JSTI indicating that amino groups are essential for activity.     

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     

Modelling of the inactivation kinetics of the trypsin inhibitors in soy flour

The inactivation kinetics of trypsin inhibitors (TIs) in soy flour was measured over a large range of temperatures (80–134°C) and moisture contents (0.08–0.52 g (g ds)⁻¹). The inactivation of TIs showed a two‐phase inactivation behaviour. The influence of moisture content on the inactivation rate of TIs was large at moisture contents <0.30 g (g ds)⁻¹. Six different inactivation kinetics models were used to describe the decrease of the trypsin inhibitor activity at constant moisture content. The models were compared statistically using a corrected Akaike information criterion. The most parsimonious models at moisture contents ≤0.30 g (g ds)⁻¹ were the model with two first‐order reactions each for a different TI group, and the model with an irreversible inactivation of a native TI to a partially active intermediate TI, followed by a denaturation step. The nth order reaction model was favoured at moisture contents ≥0.40 g (g ds)⁻¹. The kinetics parameters of the model with two firstorder reactions were modelled as a function of moisture content. The overall inactivation model described well the experimental inactivation data of TIs. © 1999 Society of Chemical Industry     

Effects of trypsin inhibitor on plasma antioxidant activity and lipid levels in mice from sweet potato roots

BACKGROUND: Several inflammatory diseases are thought to be related to oxidative injury and reactive oxygen species have been proposed as important causative agents of heart disease and ageing. This study was designed to investigate the effects of sweet potato trypsin inhibitor (SPTI) on antioxidant enzymes, lipid peroxidation and lipid profiles in mice. RESULTS: Twenty mice were randomly divided into four groups and fed with TI (10, 50 and 100 mg kg^?1 BW) as treatment and with saline as a control in addition to regular diets. After 35 days, Trolox equivalent antioxidant capacity (TEAC), triglyceride (TG) and cholesterol levels in plasma and superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), thiobarbituric acid reactive substances (TBARS) in liver were measured. Serum from the group that had received the the highest oral dose of SPTI (100 mg kg^?1 BW) had the highest total antioxidant activity (expressed as 3.59 ± 0.237 mmol L^?1 TEAC). The SOD, catalase and GPx activity of SPTI groups were significantly increased compared with the control group. Malondialdehyde (MDA) was significantly lower in all experimental groups compared with the control one. No significant differences in the concentration of low‐density lipoprotein (LDL)‐cholesterol was found, but high density lipoprotein (HDL)‐cholesterol, triglyceride (TG) and total cholesterol tended to decrease. CONCLUSION: This study showed that the oral intake of SPTI in mice may trigger inflammatory responses which result in an increase in antioxidant enzyme activities, and a decrease in MDA, TG and total cholesterol, which are known risk factors of inflammatory and heart disease. Copyright © 2008 Society of Chemical Industry     

Tolerance of growing pigs to trypsin and chymotrypsin inhibitors in chickpeas (Cicer arietinum) and pigeonpeas (Cajanus cajan)

The threshold level of growing pigs to trypsin and chymotrypsin inhibitors was investigated by adding graded levels of meals rich in these inhibitors to diets and recording responses. Diets were formulated to contain either 250, 500 or 750 g kg^?1 of Opal chickpea, dehulled Tyson chickpea or dehulled pigeonpea meals and pig response compared to that of pigs given a wheat and soya-bean meal control. Trypsin inhibitor levels (mg g^?1) of the diets were, respectively, control, 0.2; chickpea meal 1, 1.2.32; chickpea meal 2, 1.7–4.7; pigeonpea meal, 1.4–3.6. Chymotrypsin inhibitor levels (mg g^?1) of the diets were, respectively, control, 0.2; chickpea meal 1. 0.9–2.2; chickpea meal 2, 1.6–4.5; pigeonpea meal. 0.8–2.1. The diets were offered ad libitum over the 20–50 kg growth phase. Growth responses of the pigs fed the two chickpea meals were similar to those of the pigs fed the control soya-bean meal diet (P>0.05). In contrast, the addition of pigeonpea meal linearly depressed growth rate (P<0.001), feed intake (P<0.05) and increased the feed conversion ratio (P<0.05), inclusion levels of the chickpea meals had no effect on organ weights, whereas the inclusion of pigeonpea meal significantly affected the weights of the liver and pancreas (P<0.05), indicating the presence of other anti-nutritional factors. The results indicate that the growing pig can tolerate dietary levels of at least 4.7 and 4.5 mg g^?1 of trypsin and chymotrypsin inhibitors, respectively. These threshold levels are unlikely to be exceeded in conventional diets containing the majority of grain legumes. The results also indicate that dehulled pigeonpea meal contains an anti-nutritional factor(s) for growing pigs.     

Purification of trypsin/chymotrypsin inhibitor from jack fruit seeds

A trypsin/chymotrypsin inhibitor (JSTI) was isolated from jack fruit seeds (Artocarpus integrifolia Hook f) by ammonium sulphate fractionation and chromatography on DEAE–cellulose and Sephadex G-100. During all stages of purification, the ratio of trypsin and chymotrypsin inhibitory activities remained constant. The purified preparation was found to be homogeneous by gel filtration, polyacrylamide gel electrophoresis (PAGE) and ultra-centrifugation. From the sedimentation coefficient, S _20w value of 3·5 ± 0·15 S. the molecular weight of JSTI was calculated to be 30·00 ± 2·50 kamu. The inhibitor showed a molecular weight of 24·55 kamu on a Sephadex G-75 column when eluted with 6 M guanidine hydrochloride, Under non-denaturing conditions, JSTI exhibited anomalous behaviour on a Sephadex G-200 column. On SDS–PAGE, the inhibitor showed two major bands with molecular weights of 26·30 and 15·00 kamu and two minor bands with molecular weights of 19·50 and 12·00 kamu. The carboxyamidomethylated JSTI showed three trypsin inhibitory activity bands on PAGE, suggesting the presence of isoinhibitors.     

STUDIES ON BLACK GRAM (Phaseolus mungo L.) TRYPSIN INHIBITOR

ABSTRACT A trypsin inhibitor isolated from black gram (Phaseolus mungo L.) had 75 amino acid residues with an estimated molecular weight of 7892. The kinetic constants K_m and V_max as evaluated by the Dixon and Cornish-Bowden plots were 2.7 × 10^?5and 6 × 10^?3M/min, respectively. The dissociation constants of the enzyme-inhibitor complex (Ki) and the enzyme-inhibitor-substrate complex (Ki') were respectively 4 × 10^?7M and 1.9 × 10^?6M. Trypsin inhibition by black gram trypsin inhibitor was of a linear-mixed type. Chemical modification studies suggested the possible involvement of lysine and arginine at the active site of the inhibitor.     

Natural plant enzyme inhibitors. Isolation and characterisation of a trypsin/chymotrypsin inhibitor from indian red wood (Adenanthera pavonia) seeds

A trypsin/chymotrypsin inhibitor was isolated from Indian red wood seeds by extraction with 0.01m HCl, chromatography on diethyl amino ethyl-cellulose, ammonium sulphate fractionation and gel chromatography on Sephadex G-100. The homogeneity of the final product was ascertained by affinity chromatography on trypsin sepharose and chromatography on phenyl sepharose CL-4B columns. During all stages of purification and characterisation the ratio of activities against trypsin and chymotrypsin remained constant at about 1.1:1 indicating that the same factor is responsible for both activities. The size of the inhibitor was found to be 24 000 daltons based on gel chromatographic studies on Sephadex G-100 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molar ratio of interaction between the inhibitor and bovine trypsin for complete inactivation of the enzyme was found to be 1.04:1. Electrophoretic and gel chromatographic studies indicated that the purified inhibitor is capable of undergoing aggregation to form dimers and trimers. Even in the presence of sodium dodecyl sulphate and sodium dodecyl sulphateurea, this phenomenon was discernible. The binding sites on the inhibitor for trypsin and chymotrypsin were not mutually exclusive, based on the data from mixed enzyme studies and on analysis of the inhibitor-enzyme complexes by gel chromatography on Sephadex G-100. Modification of the arginyl residues of the inhibitor resulted in the loss of more of the antitryptic activity than of antichymotryptic activity. Conversely, modification of amino groups resulted in the loss of more of the antichymotryptic activity. The inhibitor was stable to exposure to a wide range of pH (1.0–12.0), but it was completely inactivated on heat-treatment at 100°C for 15 min. The mode of inhibition of trypsin as well as chymotrypsin was non-competitive and K_i values for the inhibitor were 2.92 × 10-¹⁰M and 4.46 × 10-¹⁰M , respectively, for the two enzymes.     

Optimum extraction and recovery of trypsin inhibitor from yellowfin tuna (Thunnus albacores) roe and its biochemical properties

Effect of lipid removal, extraction medium and extraction time on the isolation and recovery of trypsin inhibitor from yellowfin tuna (Thunnus albacores) roe was investigated. Trypsin inhibitor extracted from defatted tuna roe showed the higher specific inhibitory activity than extracted from origin tuna roe. Optimal extraction medium was attained by shaking the defatted yellowfin tuna roe powder in 10 mm Na phosphate buffer (pH 7.0) containing 0.5 m NaCl (P < 0.05). The extraction time affected the inhibitor recovery significantly (P < 0.05). The extraction time of 30 min was optimum for recovery of trypsin inhibitor from yellowfin tuna roe. The biochemical properties of trypsin inhibitor from yellowfin tuna roes were also determined. The inhibitor was stable to heat treatment up to 60C and over a broad pH range (5‐8). Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the trypsin inhibitory activity. However, the activity decreased when trypsin inhibitor was incubated with metal ions at ambient temperature for 30 min.     

Effect of year‐to‐year variation and genotype on trypsin inhibitor level in common bean (Phaseolus vulgaris L) seeds

Changes in trypsin inhibitor (TI) level in common bean (Phaseolus vulgaris L) seeds were investigated in relation to protein content, year‐to‐year variation and genotype. Twenty‐one local populations from the Basilicata region (southern Italy) were tested over 3 years (1995–97). The populations were cultivated in the same environment in which they were traditionally cultivated and are currently grown. A wide variation of TI content, expressed as units of inhibitor per milligram of dry matter (TIU mg^?1 DM), was found within the populations of the collection (x? ± 2σ: 27.67 ± 2.72 in 1995, 25.31 ± 2.82 in 1996, and 23.39 ± 2.14 in 1997). It was found that 16 populations showed a decrease of TI levels from 1995 to 1997; one population showed the opposite trend, in two populations the TI level remained unchanged, and in another two it reached its maximum in the intermediate year. A t‐test showed that only the 1995–97 variation is highly significant (p < 0.02). The increase of TI expression might possibly be related to the drought stress suffered by plants during the vegetative growth stage: rainfall received in 1995 during the growing season was lower (171 mm) than in 1996 (477 mm) and 1997 (388 mm); however, other abiotic or biotic factors cannot be excluded. It is suggested that the extent of TI variation contains a genetic component. Copyright © 2003 Society of Chemical Industry     

Comparative Assessment of Trypsin Inhibitor vis-à-vis Kunitz Trypsin Inhibitor and Bowman-Birk Inhibitor Activities in Soybean

Trypsin inhibitor activity (TIA) in soybean is attributed to two polypeptides, namely, Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Standard spectrophotometric protocol widely followed for estimation of TIA is cumbersome and does not distinguish KTI from BBI. In the present investigation, extraction conditions for KTI were optimized and different forms of this polypeptide were resolved in 180 soybean genotypes of Indian and exotic origin through native PAGE. This led to the identification of three KTI alleles, namely, Ti^a, Ti^b, and Ti^c, with Ti^a occurring in most of the Indian genotypes. Trypsin-KTI complex assay exhibited binding of Ti^a polypeptide with 2.51 fold concentration of trypsin. Subsequently, seeds of selected genotypes were subjected to estimation of KTI and BBI activity through densitometry and enzyme-linked immunosorbent assay (ELISA), respectively; and total TIA through standard spectrophotometric protocol. Summation of KTI and BBI was significantly (P?<?0.05) lower than that of TIA determined through the spectrophotometric method.     

Response of pancreatic secretions to feeding diets with low and high levels of soybean trypsin inhibitors in growing pigs

Studies were carried out to determine the effect of soybean trypsin inhibitors (SBTI) on exocrine pancreatic secretions in growing pigs. Six barrows with an average initial body weight (BW) of 27·1±1·4 kg were fitted with permanent pancreatic re-entrant cannulas and fed two diets according to a crossover design. Two maize starch-based diets were formulated to contain 200 g kg⁻¹ crude protein from either Nutrisoy (food grade defatted soy flour) or autoclaved Nutrisoy. The concentrations of SBTI in Nutrisoy and autoclaved Nutrisoy diets were 13·4 and 3·0 g kg⁻¹, respectively. The experiment consisted of two periods of 9 days each. The average BW at the start of the first and second experimental periods was 33·5±2·7 and 37·2±3·7 kg, respectively. The average BW at the conclusion of the experiment was 41·8±3·9 kg. The volume of pancreatic secretion was higher (P<0·01) when the Nutrisoy, as opposed to the autoclaved Nutrisoy diet was fed (3804 vs 2634 ml (24 h)⁻¹). The concen-tration of nitrogen and protein and specific activities (units litre⁻¹) of amylase, chymotrypsin and trypsin were lower (P<0·05) in pancreatic juice of pigs fed the Nutrisoy diet. There were no differences (P>0·05) in the total secretions of nitrogen (g (24 h)⁻¹) and total activities (units (24 h)⁻¹) of amylase, lipase, chymotrypsin and trypsin in pancreatic juice of pigs fed the Nutrisoy and autoclaved Nutrisoy diets. However, the total secretion of protein was slightly higher (25·7 vs 22·8 g (24 h)⁻¹; P<0·05) in pancreatic juice of pigs fed the autoclaved Nutrisoy diet, which corresponded with the increase in the secretion of protein-bound amino acids. There was also an increase in the total secretion of free amino acids in pancreatic juice. These studies show no effect of SBTI on the total enzyme activities in pancreatic juice of growing pigs. © 1998 SCI.     

PARTIAL CHARACTERIZATION OF TRYPSIN-CHYMOTRYPSIN INHIBITORS FROM BEAN (PHASEOLUS VULGARIS L., VAR. ROSINHA G2): CHEMICAL AND PHYSICAL PROPERTIES

The three trypsin inhibitors A, B and C previously isolated from Brazilian pink bean (Phaseolus vulgaris L. var. Rosinha G2) had molecular weights of 18,200 to 18,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, 20,000 by gel filtration on Sephadex G-100 and 20,400 by sucrose density gradient ultracentrifugation with a Stokes molecular radius of 20 Å, a frictional coefficient of 1.14, a diffusion coefficient of 10.7 × 10^?7 cm²s^?1, a partial specific volume of 0.69 cm³g^?1 and a molar absorptivity of 5.5 × 10³ M^?1 cm^?1 at 280 nm. All three inhibitors bound two moles of trypsin and one mole of chymotrypsin. The K_i values for trypsin were: A, 8.5 × 10^?10 M; B, 1.8 × 10^?10 M and C, 6.8 × 10^?10 M while for chymotrypsin they were: A, 4.4 × 10^?7 M; B, 2.8 × 10^?8 M and C, 3.0 × 10^?8 M. Reductive methylation caused loss of inhibitor activity of all three inhibitors against trypsin without significantly affecting inhibitor activity against chymotrypsin (with exception of inhibitor B), indicating that the inhibitors have lysine in binding site for trypsin. Partial reduction of the disulfide bonds caused loss of inhibitor activity against both trypsin and chymotrypsin with some regain of inhibitor activity following dialysis. Cyanogen bromide cleaved all three inhibitors into two fragments with significant retention of inhibitor activity. Cyanogen bromide-treated inhibitor B had nearly twice the original inhibitor activity against trypsin with no loss of inhibitor activity against chymotrypsin.     

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     

Experimental assessment of toxic phytochemicals in Jatropha curcas: oil,cake, bio‐diesel and glycerol

BACKGROUND: Jatropha curcas seed is a rich source of oil; however, it can not be utilised for nutritional purposes due to presence of toxic and anti‐nutritive compounds. The main objective of the present study was to quantify the toxic phytochemicals present in Indian J. curcas (oil, cake, bio‐diesel and glycerol). RESULTS: The amount of phorbol esters is greater in solvent extracted oil (2.8 g kg^?1) than in expeller oil (2.1 g kg^?1). Liquid chromatography‐mass spectroscopy analysis of the purified compound from an active extract of oil confirmed the presence of phorbol esters. Similarly, the phorbol esters content is greater in solvent extracted cake (1.1 g kg^?1) than in cake after being expelled (0.8 g kg^?1). The phytate and trypsin inhibitory activity of the cake was found to be 98 g kg^?1 and 8347 TIU g^?1 of cake, respectively. Identification of curcin was achieved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and the concentration of curcin was 0.95 g L^?1 of crude concentrate obtained from cake. CONCLUSION: Higher amounts of phorbol esters are present in oil than cake but bio‐diesel and glycerol are free of phorbol esters. The other anti‐nutritional components such as trypsin inhibitors, phytates and curcin are present in cake, so the cake should be detoxified before being used for animal feed. Copyright © 2011 Society of Chemical Industry     

Interaction between four flavonoids and trypsin: effect on the characteristics of trypsin and antioxidant activity of flavonoids

Interaction of flavonoids and enzyme may affect characteristics and physiological activities of both components. In this study, the effects of the interaction between four flavonoids (quercetin, luteolin, kaempferol and apigenin) and trypsin were examined. At the concentration of 2.7 mm , inhibition of trypsin (1.6 U mL^?1) by quercetin, luteolin, kaempferol and apigenin was 46.4%, 32.6%, 26.8% and 17.7%, respectively. In the presence of trypsin, DPPH, ABTS and hydroxyl radical scavenging activities of flavonoids were obviously inhibited. Addition of flavonoids led to fluorescence quenching of trypsin. The decreasing order of binding force between flavonoids and trypsin was quercetin, luteolin, kaempferol and apigenin. It is concluded that the interaction between flavonoids and trypsin depends on the number and position of hydroxyl group of flavonoids.     

Genotype × environment influence on cowpea (Vigna unguiculata (L) walp) antinutritional factors: 1 – trypsin inhibitors,tannins, phytic acid and haemagglutinin

The relative influence of genotype, environment and genotype×environment effects on four antinutritional factors (g kg⁻¹) of importance in cowpea were studied using 15 local and improved cowpea genotypes grown in 12 environments, comprising three locations over four seasons per location. The locations Ago‐Iwoye (6°58′N4°00′E), Mokwa (9°17′N5°04E) and Kano (12°00′N8°31′E) were representatives of the major agroecological zones where cowpeas are produced. Genotypes effects were strongest in controlling trypsin inhibitor content, while the environment was the major source of variation for tannins, haemagglutinin and phytic acid contents. Thus, the variability in the levels of these antinutritional factors in cowpea seeds depends largely on the environment where they are grown. This implies that a cowpea genotype grown and consumed safely in an environment can be poisonous when grown and consumed in another environment. Genotype×environment effects were significant for tannins, haemagglutinins and trypsin inhibitor contents. Correlation coefficients (pooled data) from the three locations indicated that trypsin inhibitor was positively correlated to phytic acid (r=0.59, 0.001<P<0.05) and haemagglutinins (r=0.64, 0.001<P<0.05) but negatively correlated to tannin contents ( r=−0.79, 0.001<P<0.05). © 1999 Society of Chemical Industry