Protease Inhibitors of Chickpea (Cicer arietinumL) During Seed Development

Developing seeds of two chickpea varieties were collected on the 20th, 40th and 60th day after flowering (DAF) and analysed for trypsin inhibitors (TI). The varieties differed in their TI profiles and activity units. The TI band and activity were not detectable at 20 DAF in the varieties Annigeri and BDN 9-3. Annigeri showed the highest total and specific TI activity, and five isoinhibitors at 40 DAF, which decreased to four at 60 DAF. The appearance and disappearance of TI bands during seed development indicate their specific post-translational modification and/or the presence of isolated gene groups. © 1997 SCI.     

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.     

Effects of extrusion and conventional processing methods on protein and antinutritional factor contents in pea seeds

The effects of high temperature short time (HTST) treatment compared with other conventional processes on protein, phytic acid, condensed tannins, polyphenols, trypsin, chymotrypsin and α-amylase inhibitor activities and haemagglutinating activities in Renata, Solara and Ballet pea seeds were investigated. Ballet cultivar showed highest protein, phytic acid, tannin, polyphenol contents and trypsin and chymotrypsin inhibitory activities. All pea cultivars contained trypsin- and chymotrypsin-inhibiting activity and lectins but only Solara had α-amylase inhibitory activity. Under extrusion conditions (148°C, 25% moisture and 100 rpm) this thermal processing method was the most effective in condensed tannin, trypsin, chymotrypsin, α-amylase inhibitors and haemagglutinating activity reduction, without modifying protein content as occurs by dehulling, soaking and germination treatments. Trypsin and chymotrypsin inhibitors and haemagglutinating activities in peas were more readily abolished by extrusion treatment than was chymotrypsin inhibitory activity.     

Cultivar differences and effect of pigeon pea seeds boiling on trypsin inhibitor activity and in vitro digestibility of protein and starch

Soaking and ordinary boiling of soaked seeds of five cultivars of pigeon pea (Cajanus cajan L.) significantly (P ≤ 0.05) lowered trypsin inhibitor activity and improved protein and starch digestibility when compared to unprocessed seeds. Cultivar differences were also significant. Boiling of soaked seeds proved most effective in improving nutritional quality.     

Purification and characterisation of protease inhibitors from pigeon pea (cajanus cajan (l) millsp) seeds

Two protease inhibitors from Cajanus cajan seeds have been purified to homogeneity by trichloroacetic acid (TCA) solubilisation, ion-exchange and gel-filtration chromatography followed by preparative polyacrylamide gel electro-phoresis. One of the inhibitors, Cajanus trypsin-chymotrypsin inhibitor (CTCI), inhibits both bovine trypsin and chymotrypsin while the other, Cajanus trypsin inhibitor(CTI), inhibits only bovine trypsin. The two inhibitors contained no carbohydrate and had an isoelectric point of 6. CTCI and CTI had average molecular weights of 15000 and 10500, respectively. The purified inhibitors in solution were stable to heat at 80°C for 15 min and pH 7–10. In the pH range 3–5, 80% of the activity was retained. Autoclaving totally destroyed the inhibitor activity. CTCI had two sites for trypsin binding and one site for chymotrypsin binding while CTI had only one site for trypsin binding. The inhibitors were very specific towards mammalian serine proteases and did not inhibit other proteases or serine proteases of bacterial origin.     

Characterisation of trypsin and α-chymotrypsin inhibitors in Australian wattle seed (Acacia victoriae Bentham)

Crude water extracts from Australian wattle seed (Acacia victoriae Bentham) and their salt (ammonium sulphate)-precipitated fractions were analysed for trypsin and α-chymotrypsin (chymotrypsin) inhibitor activity, using gel electrophoresis and spectrophotometric methods. Three different bands with molecular weight 30.20, 38.03 and 39.81 kDa were active, with the 50% salt-precipitated fraction exhibiting highest activity and number of active bands. The same proteins also appeared to be responsible for both trypsin and chymotrypsin inhibitor activity. To establish conditions for the inactivation of these inhibitors, whole seed and uncoated (dehulled) cotyledon were subjected to different heat treatments. Moist heat treatment at 100 °C for 30 s was sufficient to inactivate both protease inhibitors although the trypsin inhibitor was found to be more heat-resistant than was the chymotrypsin inhibitor. Soaking overnight, before thermal treatment, improved the trypsin inhibitor activity but enhanced the efficiency of thermal inactivation in both inhibitors.     

Chemical and nutritional quality changes in germinating seeds of Cajanus cajan L.

Changes in the chemical constituents and nutritive quality of germinating seeds of pigeon pea, Cajanus cajan L. were studied in seeds that were germinated for 0, 1, 2, 3, 4 and 5 days. The differently germinated seeds were analysed for proximate composition, mineral elements, structural carbohydrates, calorific value, nutritive and non-nutritive matter and certain antinutritional factors. Germination significantly altered the nutrient composition of the seed, causing marked increase in calorific value. Crude protein, soluble carbohydrate, cellular and organic cellular contents, cellulose, lignin, non-nutritive matter, total oxalate and phytic acid contents of the seed were negatively correlated with germination, whereas the reverse was the case with the seed's contents of fat, crude fibre, total ash, soluble ash, acid-insoluble ash, cell wall carbohydrate, hemicellulose, iron, manganese, calcium, magnesium, copper, phosphorus, food energy, digestible energy, tannins, total phenolics and trypsin inhibitory activity. It was concluded that the increased contents of tannins, total phenolics and trypsin inhibitory activity of the seed during the progressive germination might limit its nutritive quality.     

Protease inhibitors in germinating echinocloa (Echinocloa fruneutacea) seeds: Changes in protease inhibitory activities during germination

Antitryptic activity in echinocloa seedlings disappeared completely by the seventh day during germination, whereas antichymotryptic activity remained at a level comparable with that of the dormant seeds. Purified trypsin/chymotrypsin inhibitor from dormant echinocloa seeds was used as a substrate to study the nature of proteases arising during germination. A carboxyl protease localized in the root portion of the seedlings was responsible for the inactivation of the seed inhibitor with reference to the loss of antitryptic activity, and a thiol protease diminished its antichymotryptic activity in vitro. The shoot portion of the seedlings contained both antichymotryptic activity and antitryptic activity. The root extract preferentially destroyed the antitryptic activity of the shoot extract. The data suggest that the trypsin/chymotrypsin inhibitor of echinocloa seed is inactivated and mobilized during plant growth and that a new inhibitor appears in the shoot portion of the plant during growth.     

Antinutritional and/or toxic factors in soybean (Glycine max (L) Merril) seeds: comparison of different cultivars adapted to the southern region of Brazil

Soybean (Glycine max (L) Merril) seeds are known to contain different proteins displaying antinutritional and/or toxic effects, such as soybean agglutinin (an N‐acetylgalactosamine‐specific lectin), proteinase inhibitors (Kunitz‐ and Bowman–Birk‐type trypsin and chymotrypsin inhibitors) and urease (seed and tissue isoforms). Two other toxic proteins were previously isolated from soybeans, soyatoxin (21 kDa) and soybean toxin (18.4 kDa), which are immunologically related to canatoxin, a toxic protein from Canavalia ensiformis (jackbean) seeds. In this work we have screened crude extracts from seeds of six different soybean cultivars, which together represent most of the crop harvested in the southern region of Brazil, for the presence of urease, trypsin inhibitory and haemagglutination activities, intraperitoneal toxicity in mice and immunoreactivity against anti‐canatoxin antibodies. Significant differences were found in the contents of proteinase inhibitors, lectin, urease activity and lethality in mice. The relevance of these findings to the agronomic qualities and to the choice of soybean cultivars to be used as food or feed is discussed. Copyright © 2004 Society of Chemical Industry     

Identification and stability of trypsin inhibitor isoforms in pea (Pisum sativum L.) cultivars grown in New Zealand

Trypsin inhibitor activity (TIA) of 17 spring-sown field pea cultivars grown in New Zealand ranged from 0.33 to 0.75 TIU/mg DM. These values were much lower than those reported for most European pea cultivars. After soaking and cooking, values fell by 42–91%, with an average reduction of 78% (0.07–0.19 TIU/mg DM). After heat treatment, the residual percentage of TIA was negatively correlated to the amount of TIA in the raw seed. Six to ten trypsin isoinhibitors were observed in each cultivar of the raw extracts, with isoelectric points ranging from 4.6 to 7.6. Only three of the isoinhibitors, with isoelectric points of 5.1, 5.9 and 7.6, remained after heat treatment.     

Effect of Germination on Chemical Composition,Biochemical Constituents and Antinutritional Factors of Soya Bean (Glycine max) Seeds

The published scientific data concerning the effects of germination on chemical composition, biochemical constituents and anti-nutritional factors of soya bean are reviewed. The amino acid profile did not change to a great extent; only a noticeable increase in aspartic acid was observed whereas there was a gradual decrease in the available lysine level and lipid content as germination progressed. Both the total protein content and the nonprotein nitrogen increased after 5 days of germination. Dietary fibres are partially degraded in germinated seeds. Germinated soya bean is an excellent source of ascorbic acid and riboflavin. Niacin contents increased distinctly after germination. Germination induced a reduction in lipase inhibitor activity. The galactosyl oligosaccharides drastically decreased in germinated seeds. After 4 days of germination, the activity of certain lectins decreased to 4% of that of ungerminated soya beans. The phytic acid in the seeds was degraded by the phytase activated during germination, thus increasing the availability of the minerals present in the germinated seeds. Germination can degrade both Kunitz soya bean trypsin inhibitor and the major Bowman–Birk soya bean trypsin inhibitor; the degradation is enhanced, if germination process lasts more than 4 days. © 1997 SCI.     

The effect of germination on seed trypsin inhibitors in Vicia faba and Cicer arietinum

During germination, trypsin inhibitor (TI) content increased relative to total protein and dry weight in cotyledons from two varieties of Vicia faba. These data, together with changes in total protein profiles and amounts, suggest that TI proteins are not hydrolysed in parallel with other seed proteins and dry matter. In contrast, TI content in germinating Cicer arietinum seeds remained constant during the germination period; in this case, however, substantial changes in the total protein profile were not observed. TI isoform analyses indicated that compositional changes occurred during germination in both species. Copyright © 2004 Society of Chemical Industry     

光照强度对光敏感型和光不敏感型烟草种子发芽的影响

为研究光照强度对光敏感型和光不敏感型烟草种子发芽的影响,选用19个烟草品种种子分别于光照和黑暗条件下发芽,筛选出光照敏感型品种NC297、光照较敏感型品种NC102和光照不敏感型品种MS云烟87、云烟97等。以光照敏感型品种NC297和光照不敏感型品种MS云烟87种子为材料,研究其在不同光照强度下的发芽情况。结果表明,2个类型的种子发芽势、发芽率以及发芽指数均随着光照强度的增强而提高,当光照强度超过3730 lx后,又逐渐降低,发芽时间则呈相反趋势。研究认为光敏感型和光不敏感型烟草种子发芽的最适光照强度均为3730 lx。     

Immunoaffinity chromatography purification and characterisation of pea trypsin inhibitors

Trypsin inhibitors from pea (Pisum sativum (L) cultivar Progreta grown in Denmark) have been isolated and shown to consist of at least nine pea proteinase inhibitors (PPI) with inhibitor activity toward both trypsin and chymotrypsin. The isoelectric points of the inhibitors were in the range 4.9–7.8. From this PPI mixture at least four inhibitors were isolated by immunoaflinity chromatography on a column containing immobilised monoclonal antibody (mAb) with inhibitor specificity. The PPI isolated by immunoaffinity chromatography were further separated by HPLC, and subsequent SDS-PAGE analysis showed molecular weights for four of the PPI in the range 11.3–14.2 kD. Their pI were determined by isoelectric focusing, mAbs were used for immunochemical characterisation and their amino acid composition showed a high (14.7–21%) content of cysteine. Tryptophan was not present in any of the isolated PPI. The data now obtained support the resemblance of PPI with inhibitors of the Bowman-Birk class and the differences in immunochemical properties of the various PPI indicate that pea has at least two gene loci coding for the inhibitors.     

Detection and partial characterisation of subtilisin inhibitors in legume seeds by isoelectric focusing

Seed extracts of ten legume species were subjected to isoelectric focusing in acrylamide gels and inhibitors of subtilisin, three other bacterial proteases, trypsin and chymotrypsin were detected by a negative-staining technique based on the chromogenic substrate, acetyl-D, L-phenylalanine-2-naphthylester. In addition to complex patterns of trypsin and chymotrypsin inhibitor zones, most legume seeds examined exhibited one major and one minor inhibitor of subtilisin, and the other bacterial proteases. However, in cowpea and black gram only the major, and in lentil and soya bean only two minor subtilisin inhibitor zones were detected. Isoelectric points of the subtilisin inhibitors range from 4.4 to 5.9. The inhibitor patterns obtained by isoelectric focusing of extracts mixed with bacterial protease confirmed that inhibitors of the bacterial proteases represent a new type of inhibitor different from the well characterised trypsin and chymotrypsin inhibitors.     

Studies on the biological responses of rats to seed trypsin inhibitors using near-isogenic lines of Pisum sativum L (pea)

In order to determine the true antinutritional status of pea seed trypsin inhibitor (TI) proteins, pea lines are being produced that are near-isogenic except for the genetic locus, Tri, containing the TI structural genes. These lines are based on selection from the progeny of a cross between lines showing quantitative variation in TI content, as well as TI isoform and gene polymorphisms that serve as markers. Chemical analyses revealed that the composition of seeds from lines of each near-isogenic pair was extremely similar, except for a more than five-fold difference in TI content. Such lines provide material that is superior to the diverse lines previously used for nutritional assessment of pea TI. The specific biological effects of pea TI were studied by including the near-isogenic lines in standardised rat diets. The results indicated that TI content was correlated with a significant negative effect on protein digestibility and biological value. The difference in TI content of the pea seeds was reflected in the relative activity of pancreatic chymotrypsin whereas the activities of trypsin, lipase and amylase were less clearly affected. © 1999 Society of Chemical Industry     

Trypsin inhibitors from Pisum sativum L exhibit identical epitopes

Pea (Pisum sativum L) trypsin inhibitor, known to be a mixture of at least eight different peptides exhibiting different charges as shown by electrophoresis, was subjected to an immunochemical analysis. By PAGE-SDS analysis, only one large diffuse band was detected showing that pea trypsin inhibitor peptides have a molecular weight between 12000 and 15000 amu. After preparative non-denaturing electrophoresis, four major bands, as judged by Coomassie blue staining, were purified and each of them was used to raise specific antibodies in rabbits. By ELISA test, immunoelectrophoresis and absorption on an immunoaffinity column, it was shown that each antiserum directed against any one of the four bands completely cross-reacted against each other. Thus, it can be concluded that each component of the pea trypsin inhibitor should exhibit a very strong sequence homology.     

Further characterisation of protease inhibitors from pigeon pea (cajanus cajan (l) millsp) seeds

Cajanus trypsin inhibitor (CTI) and Cajanus trypsin-chymotrypsin inhibitor (CTCI) previously purified from cv TAT-10 were further characterised. The modification of the inhibitors revealed the presence of lysine at the trypsin reactive site in both CTI and CTCI. Modification of tyrosine at the reactive site of CTCI did not abolish chymotrypsin inhibition suggesting the presence of leucine or phenylalanine as reported in other chymotrypsin inhibitors. CTCI did not contain tryptophan. Dissociation constants for the inhibitors with bovine trypsin were in the region of 0.69 nmol (CTCI) and 0.029 nmol (CTI). Although the protease inhibitors lost their inhibitory activity on exposure to 2-mercaptoethanol they remained attached to the enzyme. The inhibitors were not very effective against the protease from Helicoverpa armigera which is a serious field pest of Cajanus. Dissociation constants for the inhibitors with the larval enzyme were in the region of 100 nmol.     

Biochemical characterisation of α-amylase inhibitors from Achyranthes aspera and their interactions with digestive amylases of coleopteran and lepidopteran insects

BACKGROUND: Starchy seeds are an important food and a source of dietary ingredients in many countries. However, they suffer from extensive predation by bruchids (weevils) and other pests. α‐Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. RESULTS: A proteinaceous α‐amylase inhibitor from the seeds of Achyranthes aspera was identified, purified and characterised. In electrophoretic analysis, two prominent amylase inhibitor activity bands (AI1 and AI2) were detected. The inhibitor was purified 9.99‐fold with 1206.95 total amylase inhibitor units mg^?1 protein. The molecular weight of the purified inhibitor was around 6 kDa. The isolated α‐amylase inhibitor was found to be resistant to heat and proteolysis. Feeding analysis of Callosobruchus maculatus larvae on a diet containing seed powder of A. aspera revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fifth day of feeding. The isolated inhibitor inhibited the majority of amylase isoforms of C. maculatus, Tribolium confusum and Helicoverpa armigera in electrophoretic analysis and solution assays. CONCLUSION: The information obtained in the present investigation could be useful for a genetic engineering approach that would make seeds resistant to storage pest infestations. Copyright © 2011 Society of Chemical Industry     

Effects of Processing on the Reduction of β-ODAP (β-N-Oxalyl-L-2,3-diaminopropionic acid) and Anti-Nutrients of Khesari Dhal,Lathyrus sativus

Effects of different processing techniques on the neurotoxin, β-ODAP (β- N -oxalyl-L-2,3-diaminopropionic acid), and the anti-nutritional compounds (phytate, polyphenols, trypsin and amylase inhibitors, and lectins) within four lines of Lathyrus sativus (high-, medium- and low-ODAP, and so-called ODAP-free) were investigated. Soaking of seeds in various media reduced the contents of these compounds to a varying and significant extent; losses were higher in freshly boiled water, alkaline and tamarind solutions than after soaking in drinking water. The highest losses in boiled water (65–70%) were observed for β-ODAP, followed by trypsin inhibitors (42–48%) and polyphenols (30–37%). Ordinary cooking and pressure cooking of pre-soaked seeds were found to be most effective in reducing the levels of all the natural toxicants examined, whilst fermentation and germination were more effective in destroying both of the enzyme inhibitors (amylase inhibitors by 69–71%; trypsin inhibitors by 65–66%) than either phytates or polyphenols. Lectins were not affected by most of these processes except by pressure cooking and fermentation. Dehusking of pre-soaked seeds significantly reduced β-ODAP levels, but this reduction was lower for the anti-nutrients. These findings and the high water solubility suggest that a simple and effective means of detoxifying Lathyrus by removing this neurotoxic amino acid may be practicable.