Trypsin Inhibitor Research Articles

Quantitative characterization of non-specific interaction of two globular proteins with Dextran T70 in a binary mixture.

In a bid to quantify the contribution of molecular structure to non-specific interactions leading to functionally important structural changes in cellular processes, the self-interaction of dextran-T70 (DT70) and its interaction with each of bovine serum albumin (BSA) and ovomucoid trypsin inhibitor (OVO) were studied at pH 7.4 between 5 and 37°C. The dependences of the apparent molecular weight of each of BSA, OVO and DT70 on the concentration of DT70 were independent of temperature. The activity coefficient of the interaction of each species on DT70 concentration was also independent of temperature. The change in activity coefficient was however dependent on the molecular structure and size of the interacting species. The energy of insertion of each macromolecule in DT70 increased in the order DT70 > BSA > OVO. These findings show that although the enthalpic contribution is negligible, the extent of the entropic contribution to the macromolecular activity coefficient of interaction is chiefly the consequence of the exclusion volume of the interacting macromolecules.

Whole-genome sequencing and assessment of a novel protein- and gossypol-degrading Bacillus subtilis strain isolated from intestinal digesta of Tibetan Pigs

BackgroundWith the rapid development of animal husbandry, the demand for protein feed resources is increasing. Cottonseed meal (CSM) and soybean meal (SBM) are rich sources of protein. However, their application is limited due to the existence of anti-nutrients, which can be harmful to the digestion and absorption. A strain of Bacillus subtilis (Mafic-Y7) was isolated from digesta of intestines of Tibetan pigs. The strain showed high protease activity, which helps in degrading proteinic anti-nutritional factors in grain meal and in vitro degradation of free gossypol. In order to better understand this isolated strain, whole genome of Mafic-Y7 strain was sequenced and analyzed. Different effects on various grain meals were identified.ResultThe GC-depth Poisson distributions showed no bias suggesting high-quality genome assembly of Mafic-Y7. The whole genome sequencing showed that one chromosome with 4,248,845 base pairs(bp)and the genes total length with 3,736,524 bp was predicted in Mafic-Y7. Additionally, Mafic-Y7 possessed 4,254 protein-coding genes, and several protease genes were annotated by aligning them with databases. There are 55 protease genes, one phytase gene and one laccase gene were annotated in the gene sequence of Mafic-Y7. The average nucleotide identity between Mafic-Y7 and the GCA-000009045.1 homologous genome was 0.9938, suggesting a close genetic relationship between them at the species level. Compared with the closest four whole genomes, Mafic-Y7 was annotated the most abundant of protease genes (55 genes). The fermentation supernatant of Mafic-Y7 could increase the content of small peptides, water-soluble proteins, and acid-soluble proteins in vitro by 411%, 281% and 317% in SBM and 420%, 257% and 338% in CSM. After fermentation in grain meal by Mafic-Y7, the degradation rate of anti-nutritional factors in SBM, such as trypsin inhibitor, glycinin, and β-conglycinin was greater than 70%, and lectin was greater than 30%. The degradation rates of anti-nutritional factors in CSM, such as gossypol and phytic acid, were 82% and 26%, respectively.

Unveiling the therapeutic potential of azopyridine derivatives for trypsin inhibition: a DFT and In-Vitro approach

Heterocyclic azo derivatives have emerged as promising scaffolds for drug development. This study focused on the synthesis, computational analysis, and biological evaluation of a series of azopyridine derivatives (1a, 1d, 1 g, 1 h, 1 m, 1p, and 1s) as potential trypsin inhibitors. Density Functional Theory calculations indicated that derivative 1 h exhibited the lowest HOMO-LUMO energy gap 3.167 eV and was characterised as a soft molecule, suggesting strong binding capabilities. Molecular docking studies confirmed that 1 h binds favourably to the active site of trypsin with a glide score of −6.581 kcal/mol and binding energy of −29.95 kcal/mol. Along with docking studies, the stability of the trypsin-1 h complex was further analyzed using molecular dynamic simulations at 200 ns. The results showed that the ligand molecule 1 h bound strongly at the active site of trypsin. In-vitro enzyme assays determined the IC50 value of the molecule as 100 µM, demonstrating enhanced potency. These results indicate that AzPy derivatives, particularly 1 h, hold considerable promise as therapeutic agents for inflammatory disorders and cancer, paving the way for further exploration in drug development and targeted therapies. Further research is warranted to explore 1h’s efficacy, safety, and structure-activity relationships. Highlights: DFT studies were used to classify molecules based on their softness and hardness. Molecular docking, simulation, and in-vitro studies have identified potential anti-trypsin activity of candidate molecules. Experimental and computational calculations were in close agreement.

Acute toxicity of trypsin inhibitor from tamarind seeds in embryo and adult zebrafish (Danio rerio)

The trypsin inhibitor isolated from tamarind seeds (TTI) is being investigated for potential applications in the treatment of noncommunicable diseases (NCD), such as hypertension, obesity, and diabetes. This study aimed to assess TTI embryotoxicity and acute toxicity in adult zebrafish (Danio rerio). TTI was extracted and isolated from tamarind seeds. Embryonic and adult zebrafish were exposed for 96 hours to three concentrations of TTI (12.5, 25, and 50 mg/L). Zebrafish embryos (n=60 per group) were evaluated for survival, hatching, malformations, and potential developmental marker alterations, in addition to cardiotoxicity and neurotoxicity tests. For acute toxicity assessment in adults (n=20 per group), survival and locomotor and anxiety-like behaviors were assessed, along with genotoxicity (micronucleus) evaluation. Embryos exposed to TTI showed no significant adverse effects, presented normal heart rates and positive reflex response in the neurotoxicity tests. In adult fish, TTI did not cause mortality or significant behavioral changes, suggesting no neurotoxicity and no genotoxicity. Histopathological analyses of the whole body showed only changes in the liver and spinal cord, similar to those observed in the control group not exposed to TTI. These findings indicate TTI's biosafety and therapeutic potential in complex organisms. Further research is required to evaluate its long-term effects and efficacy in treating non-communicable diseases.

Engineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

Phenolic and Anti-Nutrients Compositions of Selected Underutilized Seeds: African Oil Bean Seed (Pentaclethra macrophylla), Cashew Nut Seed (Anacardium occidentale), Cucumeropsis Seed (Cucurbita maxima), Groundnut Seed (Arachis hypogeal) and Soya Bean Seed (Glycine max)

The study aimed to assess the levels of anti- nutrients and phenolic compounds in under-utilized seeds such as African oil bean seed (Pentaclethra macrophylla), cashew nut seed (Anacardium occidentale), cucumeropsis seed (Cucurbita maxima), groundnut seed (Arachis hypogeal), and soya bean seed (Glycine max). The goal was to understand the impact of these compounds on the nutritional value of the seeds and to identify potential preventive measures. Neglected and under-utilized crops are plant species that have historically been used for food, fiber, fodder, oil, or medicinal purposes but have diminished in importance over time due to specific supply and utilization constraints. These constraints may include poor shelf life, unrecognized nutritional value, low consumer awareness, and reputational issues (such as being considered famine food or "poor people’s food," often due to changes in agricultural practices). Anti- nutrients are substances commonly present in food that can be harmful to humans and can limit the availability of nutrients to the body. Sun-dried soya bean and groundnut seeds were ground, while African oil bean, cucumeropsis, and cashew nut seeds were mechanically dehulled and ground without heat. Oxalate and phytate levels were determined through titration, and carotenoid levels were determined spectrophotometrically. Various methods were used to determine the presence of tannins, trypsin inhibitors, cyanogenic glycosides, hemagglutinins, saponins, alkaloids, phenols, steroids, and flavonoids. It was found that African oil bean seed had higher concentrations of the analyzed anti-nutrients and phenolic compounds compared to the other seeds studied.

Construction of the technological process and design development of a multifunctional unit for processing vital soybeans with ultrasound. Feasibility of water-crude suspension

Cereals and legumes have a porous structure, being an inert carrier during mass exchange processes; they contain voids in the form of pores and stomata, on the surface of which there are foreign extractable substances in a solid state, in particular, trypsin and urease inhibitor proteins. These substances, due to their harmful effect on the final product, are subject to removal by extraction. One of the methods for inactivating anti-nutritional substances is ultrasonic treatment of soybeans.The article discusses the technology of inactivating inhibitors that worsen food and protein value of vital soybeans. The developed technology was aimed at dissolving a solid substance - soybean inhibitor protein in a liquid, carried out due to molecular diffusion, intensifying the processes of mass transfer and mass return. When creating a suspension by mixing the solid (crushed soybean grain) and liquid (water) phases, water molecules, which are a solvent, penetrate under the action of capillary forces into the pores and stomata of the treated body, begin to penetrate. On this basis, technological directions of research have been established, a sequence has been built and the physical essence of operations has been substantiated, consisting of four main ones - grinding soybean grain, creating a “water-soybean” suspension, ultrasonic (US) treatment of soybeans, drying. As a result of the conducted research, optimal technological parameters have been established for intensifying the process of inactivation of anti-nutritional substances contained in native soybeans: the degree of grinding of soybean grains is 0.5-1.0 mm; the ratio of water-soy suspension (1:1). The efficiency of the developed technological process will be factors affecting the quality of the resulting products, the duration and energy intensity of the process, resource conservation, simplicity of design and low cost of manufacture, the possibility of automation.

The impact of milling degree on physicochemical, nutritional, rheological, textural properties, and in vitro digestibility of rice: From brown rice to white rice

The objective of this study was to characterize the rice milling fractions acquired at each stage of a commercial milling system. This characterization included an analysis of color, ash content, dietary fiber, mineral composition, as well as antinutritional compounds like phytic acid, trypsin inhibitor activity, and saponin. Additionally, we investigated in vitro starch and in vitro protein digestibility, along with pasting, cooking, and textural properties.Our findings revealed that milling improved the visual appeal of rice (e.g., volume expansion, weight gain, whiteness) and notably enhanced its starch digestibility. However, milling reduced dietary fiber, mineral content, antinutrients, cooking time, and texture characteristics such as hardness, chewiness, gumminess, springiness, and cohesiveness to varying degrees. Certain parameters, such as dietary fiber, exhibited a gradual change with the duration of milling, while others, such as mineral content and texture, showed significant variation at the initial stage of milling, particularly at the first mill.

In-situ electrochemically synthesized “artificial” Gly m TI antibody for soybean allergen quantification in complex foods

Biosensors, especially those designed for detecting food allergens like Gly m TI in soybean, play a crucial role in safeguarding individuals who suffer from adverse food allergies, extending to both individual well-being and broader public health considerations. Furthermore, their integration into food production and monitoring processes aids in compliance with regulatory standards, reducing the incidence of allergen-related recalls and protecting vulnerable populations. Technological advancements in biosensor development, such as increased portability, real-time monitoring capabilities, and user-friendly interfaces, have expanded their practical applications, making them indispensable in various settings, including manufacturing plants, food service establishments, and even at-home use by consumers.For the first time, a biosensor targeting the Gly m TI allergen based on molecularly imprinted polymer (MIP) technology was developed to detect/quantify soybean in complex food matrices and effectively address the detection challenges of complex and processed foods. The Gly m TI-MIP underwent a thorough validation process using anti-Gly m TI IgG raised as a polyclonal response to the trypsin inhibitor. Gly m TI-MIP was successfully tested across a range of food matrices, including tree nuts (e.g., peanuts, walnuts, and hazelnuts) and legumes (e.g., lentils, beans, and lupine), presenting minimal cross-reactivity with lupine and walnut. The innovative approach provided a linear response in the 1 ag mL−1 - 10 μg mL−1 range, with a LOD<1 ag mL−1. Applying the Gly m TI-MIP sensor to complex model foods allowed to detect 0.1 mg kg−1 (0.00001 %) of soybean protein isolate in biscuits, ham, and sausages before and after the respective thermal treatments. The innovative biosensor can significantly improve food safety protocols by addressing the complexities of tracing allergens in processed and unprocessed food products. By ensuring rigorous allergen control, these biosensors may support global food trade compliance with international safety standards, boost consumer confidence, and promote transparency in food labeling, ultimately contributing to a safer food supply chain.

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, designated Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch–clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

DbGTi protein attenuates chromium (VI)-induced oxidative stress via activation of the Nrf2/HO-1 signalling pathway in zebrafish (Danio rerio) larval model

Hexavalent chromium (Cr (VI)) contamination poses a significant threat to environmental and human health due to its ability to induce oxidative stress. Conventional strategies to counter Cr (VI)-induced oxidative stress, like antioxidants and chelating agents, face efficacy limitations and adverse effects. The present study is intended to counteract the limitations of conventional strategies by introducing a trypsin inhibitor isolated from Dioscorea bulbifera L. tubers, known as DbGTi protein, against Cr (VI)-induced developmental toxicity and oxidative stress. Through a comprehensive array of biochemical assays, behavioural tests, and gene expression analyses, this study interprets the underlying mechanisms of the DbGTi protein. Results demonstrated that the DbGTi protein effectively restored antioxidant defense systems, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione S-transferase (GST), and glutathione peroxidase (GTPx), thereby mitigating cellular damage, reducing cell death, and enhancing neuro-biomarkers. qRT-PCR analysis of mRNA expression profiling revealed the upregulation of genes associated with antioxidant defense (sod, cat, gpx) and defense pathway (nrf2, hmox-1a), further highlighting the protective effects of DbGTi protein against Cr (VI)-induced oxidative stress.

A Conserved Tryptophan (Trp10) at the Hydrophobic Core Modulates the Stability and Inhibitory Activity of Potato I Type Inhibitors.

Different inhibitor families have their own conserved three-dimensional structures, but how these structures determine whether a protein can become an inhibitor is still unknown. The buckwheat trypsin inhibitor (BTI) pertains to the Potato I type inhibitor family, which is a simple and essential bio-molecule that serves as a model for the investigation of protease-inhibitor interaction. To: study the effects of mutations at Trp10 and Ile25 in the hydrophobic cavity(scaffold) of rBTI on its inhibitory activity and stability. A site-directed mutagenesis and molecular modeling were performed using the sequence of BTI. The hydrogen bonds formed by all amino acids and conformational differences of Trp53 were analyzed in the tertiary structures of rBTI and mutants. Mutant rBTI-W10A almost completely lost its inhibitory activity (retaining 10%), while rBTI-I25A retained about 50% of its inhibitory activity. Both rBTI-W10A and rBTI-I25A could be degraded by trypsin. The hydrogen bond analysis results showed that mutating Trp10 or Ile25 weakened the specific cohesion interactions in the hydrophobic core of rBTI, disrupting the tight hydrogen bond network in the cavity. This further led to difficulty in maintaining the binding loop conformation, ultimately causing the Trp53 to undergo conformational changes. It was also difficult for residues in the mutants to form hydrogen bonds with amino acids in bovine trypsin; thus, the mutants could not stably bind to trypsin. Our findings suggest that the hydrophobic core is also an important factor in the maintenance of inhibitory activity and stability of rBTI.

Comparison of rice bran characteristics fermented by mono- or di-strain probiotics based on omics technology

Rice bran is a premium agricultural byproduct rich in nutrients and bioactive substances. The aim of this study was to compare the effects of fermentation with Bacillus subtilis, Lactobacillus plantarum, and a mixture of both on the bacterial communities and metabolic functions of rice bran based on omics techniques. The results showed that probiotic fermentation decreased crude fiber (CF) content, phytic acid, and trypsin inhibitor activity (TIA), and increased trichloroacetic acid soluble protein (TCA-SP), reducing sugar (RS), and total phenol (TP) in rice bran. Ten dominant genera with relative abundances greater than 1% were successfully identified, and probiotic fermentation significantly reduced the relative abundances of Enterobacter, Kasakonia, and Pantoea. A total of 63 differentially expressed metabolites (DEMs) were identified and further analysis revealed that probiotic fermentation increased essential amino acids, phenolic compounds, and organic acids in rice bran. Additionally, the di-strain probiotic fermentation of Bacillus subtilis and Lactobacillus plantarum facilitated the growth of beneficial microbiota (Limosilactobacillus, Prevotella and Dialister) and the production of bioactive substances (lactic acid, catechol, beta-tyrosine, etc.). In conclusion, co-fermentation of di-strain probiotics is a valuable method to produce functional compounds and enhance the health-promoting properties of rice bran.

Novel Fungicide from Waste Oil Palm Fruit Bunch for Sustainable Management of Tomato Wilt Disease Caused by Fusarium oxysporum - experimental and in silico studies

Tomato wilt, a widespread and persistent disease caused by the fungus Fusarium oxysporum, can be effectively controlled through biological approaches, promoting ecosystem sustainability and enhancing agricultural productivity and quality. This study explored the use of empty oil palm fruit bunch (EOPFB) waste as a natural fungicide to control tomato Fusarium wilt disease through in vitro, pilot scale, and molecular docking experiments. EOPFB aqueous extract showed the highest mycelium growth inhibition of 68.3 %, with the commercial fungicide Mancozeb exhibiting a moderate 42.30 % inhibition, and sterile distilled water having a negligible effect of 0.24 %. The inhibition rate varied with concentration, peaking at 72.63 % at 0.15 g/mL and dropping to 54.80 % at 0.01 g/mL. In terms of wilt disease incidence and severity, EOPFB performed best, recording the lowest incidence of 47.20 % and severity of 15.58 %. Mancozeb followed closely, with an incidence of 46.30 % and severity of 26.23 %. Sterile distilled water, on the other hand, showed the highest incidence of 76.28 % and severity of 56.17 %. Molecular docking simulations corroborated the experimental results, validating the potency of EOPFB as an inhibitor of trypsin from F. oxysporum, and identifying 2-amino-5,6-dimethyl-3 H-pyrimidine-4-one as the primary active compound responsible for the fungicidal activity. This phytochemical, abundant in the crude ethanolic extract of EOPFB, was found to have a higher binding affinity (6.1 kcal/mol) than Mancozeb (4.8 kcal/mol), at the trypsin target. These findings demonstrate the potential of EOPFB, an agricultural waste product, as a sustainable and eco-friendly solution for managing tomato Fusarium wilt disease, contributing to increased tomato production. It also highlights the potential of 2-amino-5,6-dimethyl-3 H-pyrimidine-4-one as a lead compound for the development of novel, natural fungicides, and underscores the value of EOPFB as a rich source of bioactive compounds with agricultural and biotechnological applications.

Exploring the Impact of Solid-State Fermentation on Fava Bean Flour: A Comparative Study of Aspergillus oryzae and Rhizopus oligosporus.

Fava bean (Vicia faba L.) is a protein-rich pulse with high nutritional value, but its functional and sensory characteristics limit its application in foods. Solid-state fermentation (SSF) can modify the composition of plant proteins, modulate its functionality, and enhance the sensory aspects. In this study, fava bean flour (FB) was fermented with Aspergillus oryzae and Rhizopus oligosporus to produce FBA and FBR, respectively, ingredients with distinct nutritional, functional, and aroma characteristics. The protein content increased by 20% in FBA and 8% in FBR, while fat levels rose more significantly in FBR (+40%). The overall content of fermentable oligo-, di-, mono-saccharides, and polyols (FODMAPs) decreased by 47% (FBA) and 57% (FBR), although polyol production by A. oryzae was observed. SSF improved the nutritional profile of FBA and FBR, with a notable increase in the concentration of essential amino acids observed, and a reduction in most antinutrients, with the exception of trypsin inhibitors. SSF resulted in the formation of aggregates, which increased the particle size and reduced protein solubility. Emulsions prepared with the fermented ingredients separated faster, and the foaming capacity of both FBA and FBR was decreased, but an increase in water-holding capacity was observed. SSF resulted in the production of predominantly savoury-associated aroma compounds, with compounds characteristic of metallic and mouldy aromas reduced. These results indicate the potential of SSF to transform FB with enhanced nutritional value and improved sensory and functional properties.

The Effects of Single- or Mixed-Strain Fermentation of Red Bean Sourdough, with or without Wheat Bran, on Bread Making Performance and Its Potential Health Benefits in Mice Model.

The effects of single- (Lactobacillus fermentum) or mixed-strain (Lactobacillus fermentum, Kluyveromyces marxianus) fermentation of red bean with or without wheat bran on sourdough bread quality and nutritional aspects were investigated. The results showed that, compared to unfermented controls, the tannins, phytic acid, and trypsin inhibitor levels were significantly reduced, whereas the phytochemical (TPC, TFC, and gallic acid) and soluble dietary fiber were increased in sourdough. Meanwhile, more outstanding changes were obtained in sourdough following a mixed-strain than single-strain fermentation, which might be associated with its corresponding β-glucosidase, feruloyl esterase, and phytase activities. An increased specific volume, reduced crumb firmness, and greater sensory evaluation of bread was achieved after mixed-strain fermentation. Moreover, diets containing sourdough, especially those prepared with mixed-strain-fermented red bean with wheat bran, significantly decreased serum pro-inflammatory cytokines levels, and improved the lipid profile, HDL/LDL ratio, glucose tolerance, and insulin sensitivity of mice. Moreover, gut microbiota diversity increased towards beneficial genera (e.g., Bifidobacterium), accompanied with a greater increase in short-chain fatty acid production in mice fed on sourdough-based bread diets compared to their controls and white bread. In conclusion, mixed-strain fermentation's synergistic effect on high fiber-legume substrate improved the baking, sensory quality, and prebiotic effect of bread, leading to potential health benefits in mice.

Evaluation of antinutrients in improved and local cultivars of green gram (Vigna radiata (L.) Wilczek)

Besides being an excellent source of high-quality nutrients, legumes are deposits of antinutritional factors (ANFs) that serve as natural defences against herbivory and pathogens. This investigation aimed to study the concentration of different ANFs in 13 green gram (Vigna radiata (L.) Wilczek) cultivars and explore how varietal differences affect their levels. Ten improved, and three local cultivars of green gram were evaluated for antinutritional content using different spectrophotometric assays. Results revealed variability across cultivars with 0.45 to 1.14 g/100 g of phytic acid, 1.11 to 1.99 g/100 g of saponins, 0.91 to 1.78 mg CE/g of tannins, 17.17 to 25.55 mg GAE/g of total phenolic content, 4.41 to 10.44 TUI/mg of trypsin inhibitors, 0.15 to 0.28 g/100 g of raffinose, 0.14 to 0.36 g/100 g stachyose and 1.60 to 2.88 g/100 g of verbascose. These significant variations highlight the wide range of ANF composition found among green gram cultivars. Such data could aid in exploring the nutraceutical and pharmacological potential of these compounds.

Complex of Defense Polypeptides of Wheatgrass (Elytrigia elongata) Associated with Plant Immunity to Biotic and Abiotic Stress Factors.

Plant defense polypeptides play a crucial role in providing plants with constitutive immunity against various biotic and abiotic stressors. In this study, we explored a complex of proteins from wheatgrass (Elytrigia elongata) spikelets to estimate their role in the plant's tolerance to various environmental factors. The current research shows that in vitro protein extracts from E. elongata spikelets possess antifungal activity against certain Fusarium species, which are specific cereal pathogens, at concentrations of 1-2 mg/mL. In this study, we reproduced these antifungal activities using a 4 mg/mL extract in artificial fungal infection experiments on wheat grain (Triticum aestivum) under controlled laboratory conditions. Furthermore, the tested extract demonstrated a protective effect on Saccharomyces cerevisiae exposed to hyper-salinity stress at a concentration of 2 mg/mL. A combined scheme of fractionation and structural identification was applied for the estimation of the diversity of defense polypeptides. Defensins, lipid-transfer proteins, hydrolase inhibitors (cereal bifunctional trypsin/alpha-amylase inhibitors from a Bowman-Birk trypsin inhibitor), and high-molecular-weight disease resistance proteins were isolated from the extract. Thus, wheatgrass spikelets appear to be a reservoir of defense polypeptides. Our findings contribute to a deeper understanding of plant defense proteins and peptides and their involvement in the adaptation to various stress factors, and they reveal the regulatory effect at the ecosystem level.

Development of an effective method for purifying trypsin using a recombinant inhibitor

A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and Km value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30–50 °C and pH range of 4.0–10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.

Quality Assessment and Sensory Evaluation of Complementary Food from Orange Flesh Sweet Potato (OFSP), Soybean, and Tropical Almond Seed Composite Flour

Childhood malnutrition especially vitamin A deficiency and protein-energy malnutrition are widely prevalent in many parts of the world, particularly in developing countries due to the high cost of commercial infant food. Complementary food from OFSP, Soybean and Tropical Almond Seed Flour was produced to improve the nutritional content of locally made infant food. The study evaluated the quality and sensory attributes of complementary food. A commercial sample was used as a reference standard. The sample formulations were in the ratios; 100:0:0, 90:5:5, 80:15:5, 70:25:5. The anti-nutrient composition ranged thus: alkaloid (0.122-0.961%), trypsin inhibitor (0.003-0.455%), tannin (0.036-0.321%), oxalate (0.017-0.049%). The total essential and total non-essential amino acid composition respectively ranged thus in the samples; 14.12-59.55 g/100g of protein and 30.21-41.65 g/100g of protein. Sensory evaluation results showed that all the complementary food samples were generally accepted by the panellists. The composite flour herein produced significantly (p&lt;0.05) increased the nutritional, amino acid, and sensory attributes of the complementary food in contrast to the control sample.