Trypsin inhibitors and nutritive value in cereals

Chemical assays demonstrated that rye and barley cultivars contained relatively high levels of trypsin inhibitor activity as compared to oat and wheat cultivars, and there was a low degree of stability to prolonged wet heat treatment. In feeding trials with broiler chicks, incorporation of 67% raw barley or 50% raw rye in the rations enhanced feed intake and weight gains, and the marginal increases in pancreas weight were not reversed by feeding autoclaved cereals. Raw rye cultivars fed at the 75% level in mouse diets reduced weight gains, feed efficiency, protein digestibility, protein efficiency ratio and biological value. Autoclaving to inactivate trypsin inhibitors, or ether extraction to remove the resorcinols, failed to improve the nutritive value of rye diets for mice. It appeared that the protease inhibitors in the four cereals were relatively weak inhibitors of trypsin in the digestive system despite stability to dry heat and acid pH.     

Enzyme inhibitors in tuber crops and their thermal stability

Tubers of Cassava (Manihot esculenta), yams (Dioscorea esculenta),aroids (Amorphophallus campanulatus, Colocasia esculenta, Xanthosoma sagittifolium) and Coleus (Solenostemon rotundifolius) were screened for inhibitory activities against amylase, trypsin and chymotrypsin. Coleus tuber possessed the highest anti-amylase activity, whereas Colocasia tuber was the most potent source of anti-tryptic and anti-chymotryptic activity. Xanthosoma tubers exhibited amylase inhibitory activity and Amorphophallus tubers antiprotease activity. Dioscorea esculenta had low levels of amylase and chymotrypsin inhibitors, while Cassava tubers were totally free of inhibitors. When tubers were processed by pressure cooking, there was significant reduction/complete elimination in inhibitory activity. Partial retention of inhibition was observed in the case of amylase inhibitor in Dioscorea, chymotrypsin inhibitor in Colocasia and trypsin inhibitor in Colocasia, Coleus and Amorphophallus. In vitro experiments on heat stability of the different inhibitors revealed almost similar pattern of inactivation.     

Detection of the lunasin peptide in oats (Avena sativa L)

We report the first discovery of lunasin in oats (Avena sativa L). Lunasin is a novel cancer preventive, anti-inflammatory and cholesterol-reducing peptide originally isolated from soy and later found in cereals (barley, rye, wheat, triticale). Lunasin was detected in oats using LC–MS/MS analysis. The chromatograms and mass spectra of lunasin isolated from five oat genotypes were compared with those of the synthetic lunasin peptide. We measured the lunasin content in harvests of two years and found that all tested oat genotypes contained the lunasin peptide. However, we observed genotype-related fluctuations in the lunasin content. Notably, the middle early oat variety ‘Ivory’ contained the highest and the most stable lunasin level at 0.197 ± 0.01 mg per g of grain in year 2010 and 0.195 ± 0.009 mg per g of grain in 2011. We also characterized the selected oat genotypes by measuring the contents of protein, β-glucans, fat, starch and moisture in the grains. However, we did not find correlation between lunasin and protein, and β-glucan content. Lunasin isolated from oat showed similar to the synthetic lunasin antioxidant effects. The detection of lunasin complements a list of bioactive compounds present in oats and strengthens recommendations to use oat products.     

Chemical composition and nutritive value of husked and naked oats grain

11 samples of oats from the 2005 crop of the field experiment carried out in RSD Lipnik were examined. Naked grain oats were represented by a standard Polar cultivar and 5 strains (STH 6102, STH 6856, STH 7146, STH 7256, STH 1692), whereas husked grains were represented by a standard Bohun cultivar and 4 strains (STH 684, STH 688, STH 729, STH 840). The examined naked grain oats differ considerably from husked grain oats in their chemical composition. A statistically significantly higher content of protein (P ≤ 0.05), a higher level of fat (P ≤ 0.01) and a lower value of crude fibre (P ≤ 0.01) were observed in the grains of naked oats compared with the husked grains. The protein of oats is characterised by a favourable amino acid composition of high quality protein which can be confirmed by the EAAI values. The first amino acid limiting the nutritive value of protein was lysine in nearly all oat samples. The characteristic feature of the Polar cultivar was isoleucine deficit. The positive nutritive values for oat grains, in particular those for naked oat grains, make it possible to use them as food for humans and feed for monogastric animals.     

Purification and Characterization of a Glutenin Hydrolysing Proteinase from Wheat Damaged by the New Zealand Wheat Bug, Nysius huttoni

A glutenin hydrolysing enzyme (bug proteinase), present in New Zealand wheat damaged by Nysius huttoni, was purified 50000-fold by anion exchange, hydrophobic interaction, immobilized metal ion affinity and gel filtration chromatography. The enzyme had an apparent M_r of 14·1k as determined by gel filtration chromatography. SDS-PAGE showed a major protein band of M_r 30k and six minor bands of M_r 13·2-28·5k, none of which was a glycoprotein. Isoelectric focusing revealed two major enzyme active bands (pI 9·6 and 9·2) and three minor activity bands (pI 9·9, 8·8 and 8·2). IEF showed no protein contaminants in the most purified sample. The enzymes had optimum activity at pH 8·9 and 45°C. The activity was stable in the pH range 4·5-11 and at 50°C for 20 min at pH 8·9. The bug proteinase was shown to be a serine proteinase by inhibition with phenylmethylsulphonyl fluoride and potato proteinase inhibitors (POT-IC and POT-ID). Thirty other proteinaceous serine proteinase inhibitors did not inhibit the enzyme. Bread baking with partially purified enzyme produced loaves with the poor quality characteristics of loaves made with bug-damage wheat.     

Effect of heat treatment on the flavor of oat flakes

In order to assess the effect of heat treatment procedure on the flavor and volatile compounds of oats, raw oats, kiln and dried oats, dehulled oats and oat flakes were analysed. A sensory profile method was used to monitor changes in flavor. It changed from hay-like for raw oats into nutty, bread-like for oat flakes. Headspace solid phase microextraction (SPME) and solvent assisted flavor evaporation (SAFE) techniques were applied for isolation of aroma compounds. The most abundant compound in headspace was hexanal – its concentration varied from 176 to 1671 μg/kg depending on the processing stage. The key aroma compounds of oat flakes identified using gas chromatography – olfactometry and aroma extract dilution analysis (AEDA) were 2-methyl-3-furanthiol with roast/cooked oatmeal flavor together with methional, dimethyl trisulfide, 1-octen-3-ol, 2-methyl-3,5-diethylpyrazine.     

Interspecific and Intraspecific Variation in Grain and Groat Characteristics of Wild Oat (Avena) Species: Very High Groat (1→3),(1→4)-β- -glucan in an Avena atlantica Genotype

Grain characteristics and groat composition have been evaluated in 35 genotypes from nine taxonomic species of Avena, including three species (A. agadiriana, A. atlantica, A. damascena) for which no previous data are available. There was substantial interspecific and intraspecific variation for all characteristics measured. The proportion of groat in the grain ranged from 32·7–62·1%, and mean groat weight from 2·4–37·4 mg. Groat protein concentrations ranged from 13·9–41·3%, and exceeded 32% in one A. atlantica, two A. damascena and one A. murphyi genotype. Groat β-glucan concentration showed very wide variation (2·2–11·3%) are there were substantial interspecific and intraspecific differences. The highest β-glucan concentrations were found in genotypes of A. atlantica. Although there were interspecific and intraspecific variations in groat oil concentration (4·2–10·6%), and in fatty acid composition, data were within previously reported ranges for A. sativa. Overall these data indicate that some of the genotypes of the wild species studied may be of value for breeding oats with improved levels of β-glucan and protein, and that further studies are warranted into both interspecific and intraspecific variations in grain quality factors in wild oat species.     

CO2-defatted oats: Solubility,emulsification and foaming properties

Functional properties of conventional oat materials are relatively poor with respect to foam and emulsion formation and stabilization. This is largely due to the poor solubility of oat proteins and the presence of lipids in aqueous extracts of oats. In the experimental part of this study, extracts were prepared from different type oat flours (oat endosperm flour, oat fine flour, CO₂-defatted whole oat flour and CO₂-defatted oat flour) with a buffered aqueous extraction procedure at acidic (pH 4.5 and 6.5) and basic (pH 8.5 and 10.5) regions. The solubility of proteins was the highest at pH 10.5 and NaCl concentration of 2%. Among the extracts, CO₂-oat flour showed improved foaming and emulsifying properties at basic pH values. The presence of 0.1% NaCl resulted in the lowest foam volumes, but the emulsion activity and stability values being the highest. Sucrose addition resulted in increased foam and emulsion stability of suspensions. Heat treatment at 80 °C impaired foam properties, whereas the stability of emulsions increased with the increase in temperature from 20 °C to 80 °C. CO₂-extracted oats can be useful raw materials in beverages and other aqueous applications where protein functionality plays an important role.     

Continuous Spectrophotometric Assay and Properties of Ascorbic Acid Oxidising Factors in Wheat

A continuous spectrophotometric assay was developed to measure ascorbic acid oxidation in crude Na₂SO₄ extracts of flour. The rate of ascorbic acid oxidation in flour extracts measured using this method was similar to the rate in flour-water suspensions and 2–4 fold less than the rate in dough measured using an indophenol-xylene extraction method. Flour extracts appeared to contain two ascorbic acid oxidising factors; one with optimal activity at pH 6·3 and 30 °C and the other with optimal activity at pH 10 and 40 °C. The pH 6·3 factor had properties similar to those of ascorbate oxidase (EC 1·10·3·3) in its pH and temperature stability, strong inhibition by NaN₃, KCN and diethyldithiocarbamate, inactivation by proteases, and greater stereospecificity towards -ascorbic acid than -isoascorbic acid. The pH 6·3 factor was most concentrated in the pollard milling fraction of wheat and was lowest in flour. The pH 10 factor had several properties indicating non-enzymic oxidation of ascorbic acid; it was not inactivated by proteases, it was inhibited poorly or not at all by the above ascorbate oxidase inhibitors, and it had low specificity for stereoisomers of ascorbic acid.     

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.     

Composition,functional properties,and biological evaluation of a plastein from cassava leaf protein

Rats lost weight continuously in a feeding trial to evaluate biologically a cassava leaf protein concentrate (LPC). The crude LPC had a high tannin content, 2.2%; 47% protein, and high levels of chymotrypsin and trypsin inhibitors, 27000 and 29500 units of inhibition, respectively. The application of the plastein reaction to the crude cassava LPC yielded a product with 90% protein, brought about a tenfold reduction of the tannin content, and lowered the trypsin and chymostrypsin inhibitors concentration to a non-detectable level. The cassava plastein showed the following value for the biological parameters: PER=2.2, NPR=3.4, TD=82.5%, BV=73.6, and NPU=60.7. The cassava LPC has a deep green colour, is bitter and astringent, and is insoluble in water. The plastein has a bland taste, a pale cream colour, is soluble in water, and has a high capacity of forming emulsions which are stable to thermal processing.     

Production of Proteases by Fusarium Species Grown on Barley Grains and in Media Containing Cereal Proteins

The production of proteases by the cereal plant pathogens Fusarium culmorum, F. graminearum and F. poae was followed through seven days of cultivation. The fungi were grown in mineral and in gluten culture media, and on autoclaved barley grains. The proteolytic activities of each sample were analysed at pH 2·2, 5·0 and 8·0 and the pH optima of the most active proteases were determined. All of the fungi grown in the gluten medium produced proteases that were active at pH levels between 6 and 10 and were most active at about pH 9·0. Fusarium poae also produced acid protease(s) with pH optima between 3.0 and 3.5 when grown in the gluten medium. No protease activity was detected in the cultures that were grown in the mineral medium, except that a small amount was formed after the glucose substrate was depleted. When grown on the barley grain medium the Fusarium species produced protease activities that were similar to the neutral and alkaline ones present in the gluten cultures, but no pH 2·2 protease activity was detected. The alkaline proteases had some characteristics that were similar to those of chymotrypsin.     

The effect of heat treatment on the soluble protein content of oats

The effect of heat treatment on the soluble protein content in oat groats (Kerstin commercial variety) was evaluated using asymmetric flow field-flow fractionation (AF4) in combination with online multiangle light scattering (MALS) and UV detection. The AF4 method was used to separate the monomeric proteins from globulin hexamer and aggregate proteins and β-glucan polysaccharides in the soluble oat protein fraction. The total amount of soluble protein (with respect to total protein) was reduced to 35.7 ± 4.5 wt. % in heat treated oats from 74.6 ± 5.3 wt. % in non-heat treated oats. The ratio of monomeric to globulin hexamer and aggregate proteins was reduced from 1.82 to 1.48 as a result of heat treatment. Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the selective elimination of protein bands associated with the albumin and prolamin protein fractions as a result of heat treatment. These results were supported through amino acid analysis by cation exchange chromatography coupled with UV detection which revealed a reduction in amino acid residues associated with prolamin. The globulin proteins were found to be less sensitive to heat treatment.     

Starch granule morphology in oat endosperm

Mature and developing oat (Avena sativa) grains were sectioned and image analysis methods used to estimate the starch granule-size distribution and morphology in endosperm cells. This showed that oat endosperm cells contain two types of starch granule: compound starch granules such as those seen in rice endosperm and in most other grasses; and simple granules similar to the B-type starch granules seen in the endosperm of Triticeae species such as wheat (Triticum aestivum). The simple granules in oats are similar in size and relative abundance to B-type granules in Triticeae suggesting that they may share a common evolutionary origin. However, there is a fundamental difference between oats and Triticeae in the timing of granule initiation during grain development. In Triticeae, the B-type granules initiate several days after the A-type granules whereas in oats, both the simple and compound granule types initiate at the same time, in early grain development.     

Effects of heat treatment and germination on trypsin and chymotrypsin inhibitory activities in sorghum (Sorghum bicolor (L.) Moench) seeds

There was no appreciable change in proteinase inhibitory activity in sorghum upon dry heat treatment. However, moist heating reduced trypsin and chymotrypsin inhibitors to a greater degree. Germination (5 days) brought about complete reduction in proteinase inhibitory activity.     

Development of standard fingerprints of naked oats using chromatography combined with principal component analysis and cluster analysis

Oat samples of different varieties were collected from various habitats for the determination of avenacoside, β-glucan and fatty contents. The variation coefficients of the three components were 12.13%, 20.79% and 22.46%, respectively. Thus, those three indicators cannot represent the information of all samples, and are not suitable for evaluating the quality of oat raw materials and products. Fatty acid profiles were analyzed using gas chromatography combined with principal component analysis (PCA) and cluster analysis. Fourteen leather oat varieties were distinguished through a PCA scores scatterplot. Forty-six naked oat varieties were selected by cluster analysis and eleven characteristic peaks in these naked oats were identified. Finally, accurate fatty acid standard fingerprints of naked oats were constructed. The results of methodological indicated high precision, reproducibility and stability, in line with fingerprint testing requirements. This study fills the knowledge gap for naked oat fingerprint information, expands the grain fatty acid database, and lays the foundations of a grain nutritional liposome identification technology system.     

Preparation and antinutritional characteristics of dry bean (Phaseolus vulgaris L.) protein concentrates

Protein concentrates and starches were prepared by a wet extraction process from five dry bean (Phaseolus vulgaris L.) cultivars. The protein contents ranged from 69.7–76.4%. Concentrates prepared from dehulled beans under similar conditions had higher protein contents (80.6–87.9%). Each additional washing of the concentrates with distilled water increased their protein content. However, the protein recovery progressively decreased. The yield of starch ranged from 48.0–51.1% of the starting material. The solubility of bean proteins was minimal at pH 4.0, and under alkaline conditions, it was influenced by the tannin contents of the concentrates. Protein concentrates had lower trypsin, chymotrypsin, and amylase inhibitory activities as well as lower phytic acid and tannin contents compared to whole bean flours.     

Variation in Oat Groats Due to Variety, Storage and Heat Treatment. I: Phenolic Compounds

Low molecular weight phenolic compounds present in heat processed oats (Avena sativaL) were analysed. The oat grains were of three varieties (Kapp, Mustang and Svea), stored at different relative humidities (30, 55 or 80%) and periods (3·5 or 15·5 months) and processed with or without hulls. Eleven UV-absorbing compounds detected by High Performance Liquid Chromatography were subjected to univariate and multivariate statistical analysis. The selected compounds included caffeic acid,p-coumaric acid, ferulic acid, vanillic acid,p-hydroxybenzaldehyde, vanillin, coniferyl alcohol, three avenanthramides and one unidentified substance. The levels of vanillic acid, vanillin and, especially,p-coumaric acid,p-hydroxybenzaldehyde and coniferyl alcohol increased significantly in samples processed with hulls, but not in samples processed without hulls. Ferulic acid increased in both processes, while caffeic acid and the avenanthramides were found to decrease during processing. Storage of unprocessed samples for 1 year generally increased the levels of phenolic acids and aldehydes. For the phenolic acids (except ferulic acid), this increase was most pronounced after storage at high relative humidity (80%). The avenanthramides were present at their highest levels in Mustang, caffeic acid in Svea and Mustang, the unidentified compound in Svea, while all the other compounds studied were present predominantly in the variety Kapp.     

Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K⁺ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K⁺ channel blocking activity was also compared.     

Effect of barley and oat flour types and sourdoughs on dough rheology and bread quality of composite wheat bread

The potential of sourdough to improve bread quality of barley and oat enriched wheat breads may depend on the characteristics of the added flour (cereal type, variety, extraction rate). We compared the effect of different barley flours and oat bran (substitution level 40%), unfermented and as sourdoughs (20% of total flour), on composite wheat dough and bread characteristics by combining empirical rheological analyses (DoughLab, SMS/Kieffer Dough and Gluten Extensibility Rig) with small-scale baking of hearth loaves. Whole grain barley flour sourdough increased resistance to extension (Rmax) of the dough and improved the form ratio of hearth loaves compared to unfermented whole grain barley flour. However, sourdough showed little effect on the breads prepared with sifted barley flour or oat bran. The breads made with oat bran showed highest bread volume, lowest crumb firmness and highest β-glucan calcofluor weight average molecular weight (MW). The heat treatment of oat bran inactivated endogenous enzymes resulting in less β-glucan degradation. High MW β-glucans will increase the viscosity of the doughs water phase, which in turn may stabilise gas cells and may therefore be the reason for the higher bread volume of the oat bran breads observed in our study.