Soybean Trypsin Inhibitor Research Articles

Comparison of Conjugates Obtained Using DMSO and DMF as Solvents in the Production of Polyclonal Antibodies and ELISA Development: A Case Study on Bisphenol A

When developing immunochemical test systems, it is necessary to obtain specific antibodies. Their quality depends, among other things, on the immunogen used. When preparing hapten–protein conjugates to obtain antibodies for low-molecular-weight compounds, the key factors are the structure of the hapten itself, the presence of a spacer, the size of the carrier protein and the degree of its modification by hapten molecules. This work shows that one additional factor—the conditions for obtaining the hapten–protein conjugate—is overlooked. In this work, we have synthesized conjugates of bisphenol A derivative 4,4-bis(hydroxyphenyl)valeric acid (BVA), the protein carrier soybean trypsin inhibitor (STI), and bovine serum albumin (BSA) in reaction media combining water with two organic solvents: dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). Namely, BSADMF–BVA, STIDMF–BVA, BSADMSO–BVA and STIDMSO–BVA conjugates were obtained. Rabbit polyclonal antibodies against the BSADMF–BVA conjugate demonstrated basically different interactions in the developed ELISA systems using either STIDMF–BVA or STIDMSO–BVA conjugates. The use of the STIDMF–BVA conjugate demonstrated the absence of competition in combination with antisera obtained from BSADMF–BVA in an ELISA. A competitive interaction was observed only with the use of the STIDMSO–BVA conjugate. Under the selected conditions, the detection limit of bisphenol A was 8.3 ng/mL, and the working range of determined concentrations was 18.5–290.3 ng/mL. The obtained data demonstrate the possibility of achieving sensitive immunoassays by simply varying the reaction media for the hapten–protein conjugation, which could provide an additional tool in the development of immunoassays for other low-molecular-weight compounds.

Credible and Facile Fluorometric Detection of Soybean Trypsin Inhibitor Activity with a Water-Soluble Poly(diphenylacetylene) Derivative

Credible and Facile Fluorometric Detection of Soybean Trypsin Inhibitor Activity with a Water-Soluble Poly(diphenylacetylene) Derivative

Applying bimetallic gold/silver nanoclusters and gold nanoparticles for dairy trypsin and inhibitor analysis via inner-filter approach

Trypsin, a globular protein hydrolase, catalyzes the hydrolysis of peptide bonds at carboxyl groups of arginine and lysine residues, its detection holds paramount significance in the dairy industry, aiding in the diagnosis, management of animal diseases, and preservation of dairy products. This study introduces a highly sensitive fluorometric detection platform utilizing bimetallic gold/silver nanoclusters (Au/Ag NCs) and gold nanoparticles (AuNPs) in an "off-on-off" configuration for precise quantification of dairy trypsin and trypsin inhibitors. This fluorescence variation is leveraged to determine the concentration of trypsin and its inhibitors. Under optimal experimental conditions, the detection of trypsin demonstrates a linear range of 5 ∼ 2000 ng·mL−1 , a lowest detection limit of 1.18 ng·mL−1, and an observed IC50 of 5.94 μg·mL−1 for the soybean trypsin inhibitor (STI). Therefore, it could be inferred that the development of a fluorescence sensing platform presents an important basis for the detection of dairy trypsin.

A sperm-activating trypsin-like protease from the male reproductive tract of Spodoptera litura: Proteomic identification, sequence characterization, gene expression profile, RNAi and the effects of ionizing radiation

Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region.Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage.cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLSP-specific dsRNA into adult males caused a significant reduction after 24 h in tlsp mRNA levels in both locations. The number of eggs laid by females mated to adult males that were given TLSP dsRNA in 10 % honey solution, and the fertility (% hatched) of the eggs were reduced. Injecting pupae with TLSP dsRNA caused the later activation of apyrene sperm motility by adult male prostatic extracts to be significantly reduced compared to controls.Exposure of S. litura pupae to ionizing radiation significantly reduced expression of tlsp mRNA in the prostatic part and accessory gland of irradiated males in both the irradiated generation and also in their (unirradiated) F1 progeny. The implications of these findings for the use of the inherited sterility technique for the control of S. litura and other pest Lepidoptera are discussed.

Enrichment of Trypsin Inhibitor from Soybean Whey Wastewater Using Different Precipitating Agents and Analysis of Their Properties.

Recovering valuable active substances from the by-products of agricultural processing is a crucial concern for scientific researchers. This paper focuses on the enrichment of soybean trypsin inhibitor (STI) from soybean whey wastewater using either ammonium sulfate salting or ethanol precipitation, and discusses their physicochemical properties. The results show that at a 60% ethanol content, the yield of STI was 3.983 mg/mL, whereas the yield was 3.833 mg/mL at 60% ammonium sulfate saturation. The inhibitory activity of STI obtained by ammonium sulfate salting out (A-STI) was higher than that obtained by ethanol precipitation (E-STI). A-STI exhibited better solubility than E-STI at specific temperatures and pH levels, as confirmed by turbidity and surface hydrophobicity measurements. Thermal characterization revealed that both A-STI and E-STI showed thermal transition temperatures above 90 °C. Scanning electron microscopy demonstrated that A-STI had a smooth surface with fewer pores, while E-STI had a rough surface with more pores. In conclusion, there was no significant difference in the yield of A-STI and E-STI (p < 0.05); however, the physicochemical properties of A-STI were superior to those of E-STI, making it more suitable for further processing and utilization. This study provides a theoretical reference for the enrichment of STI from soybean whey wastewater.

Method for Measuring the Mass Fraction of Soybean Trypsin Inhibitor: Features of Development and Certification

Modern industrialization increases the requirements for the accuracy of identifying allergens, especially those that have a negative impact – soy trypsin inhibitor (STI). Correct determination of the presence of STI in food products containing soybeans is key for product safety control and labeling. The authors set a goal to develop and certify a method for measuring the mass fraction of STI using an enzyme-linked immunosorbent assay – a technique that may increase the specificity of the method and avoid false-negative results. The object of research was a method for analyzing food allergens – an enzyme-linked immunosorbent assay carried out with a set of reagents produced by XEMA LLC. In the process of developing the measurement method, the main methodological factors influencing the accuracy of the measurement results were optimized: sample weight, time of its extraction, time and speed of sample centrifugation, ratio of supernatant liquid to ELISA buffer, incubation temperature, interaction time of the allergen-antibody complex with the coloring agent, the wavelength for measuring absorbance, and the maximum time for measuring absorbance after introduction of the stop reagent. The developed method was tested during an interlaboratory experiment with the participation of 5 laboratories. Metrological characteristics were established in accordance with RMG 61–2010. The proposed method was certified in accordance with the requirements of GOST R8.563-2009, No. 102–FZ. The method has a wide range of quantitative determination of the mass fraction of STI from 0.5 to 25.0 μg/kg (ppb) with a detection limit of 0.1 μg/kg (ppb) and a relative error of 40 %. Based on the results of the research, the Federal Information Fund for Ensuring the Uniformity of Measurements (FIF) registered a certified method for identifying and quantifying the content of non-infectious food allergens of plant protein origin in samples of all types of food products and objects related to the requirements for food products, swabs taken from working surfaces during production control using reagent kits for an enzyme-linked immunosorbent assay produced by XEMA LLC No. FR.1.31.2022.43884. The method is intended for use in testing laboratories involved in monitoring the quality and safety of manufactured products; it can be used to confirm product compliance with the mandatory requirements established in the Technical Regulations of the Customs Union TR CU022/2012.

Biomolecular characterization of Antheraea mylitta cocoonase: A secreted protease

Cocoonase is a protease secreted during the emergence of silk moths. In the present study cocoonase of Antheraea mylitta was collected, purified and secondary structure was determined using circular dichroism (CD) spectroscopy which revealed the presence of α-helix 4.3%, β-sheet 55%, turn 8% and random coil 32.7%. The thermal stability of cocoonase was studied using CD spectroscopy while the thermal property was observed using Differential Scanning Calorimetry (DSC). Furthermore, MALDI-TOF peptide mass fingerprinting (PMF) was performed for similar protein identification using the MASCOT server. Using casein as the substrate, the kinetic constants Km and Vmax were 13 × 103 mg/ml and 15.09 × 10−2 μg/mg.s1 respectively. The specific activity of cocoonase was observed to be maximum at temperature 40 °C, pH-8.0. The effect of heavy metals Hg2+, Cd2+, Co2+, Pb2+ showed inhibitory activity at higher concentrations, while few metals like Mn2+, Fe3+ enhanced the activity while the effect of Ca2+ was not much on the activity. Soybean trypsin inhibitor and PMSF showed an inhibitory effect on the activity of cocoonase. Additionally, antioxidant scavenging and fibrinolytic properties were also observed. Furthermore, the imperative information generated through the present study will serve to explore cocoonase for its prospective pharmaceutical applications.

Interpenetrating alginate network as drug delivery matrix: Effects on protein stability and release.

The study investigates the rheological properties and protein release capacity of a uniform hydrogel composed of sodium alginate (SA) and poloxamer (P407). The hydrogel is prepared through the sustained release of calcium ions, resulting in a reinforced and homogeneous interpenetrating networks (IPNs) of SA and P407 polymeric chains. By adjusting the amount of crosslink agent, the hydrogel exhibites an adjustable dissolution ratio and adaptable gelling time. Moreover, the composite showed a well-structured network and superior mechanical strength, enabling the sustained release of both calcium ions and Soybean Trypsin Inhibitor (STI) protein, a model of Bone Morphogenic Protein (BMP). Importantly, the protein release kinetic can be tuned based on the SA content in the polymeric blend, highlighting the versatile nature of this hydrogel for drug delivery purposes.

Electrochemical immunosensor for point-of-care detection of soybean Gly m TI allergen in foods

Soybean is a legume with high technological functionality, commonly used by the food industry as an ingredient in different products. However, soybean is an allergenic food whose undeclared presence in processed foods may represent a public health risk. In this work, it was developed an efficient electrochemical immunosensor, targeting the soybean trypsin inhibitor (Gly m TI) allergen using commercial anti-Gly m TI IgG, aiming at detecting/quantifying minute amounts of soybean in different food formulations. For this purpose, model mixtures of different foods (sausages, cooked-hams, biscuits) were prepared to contain known amounts of soybean protein isolate (100,000–0.1 mg kg−1) and submitted to specific thermal treatments (autoclaving, oven-cooking, baking). The electrochemical immunosensor allowed quantifying down to 0.1 mg kg−1 of soybean in the three food matrices, raw and processed (0.0012 mg of Gly m TI/kg of matrix). Accordingly, the immunosensor is suitable for detecting traces of soybean in raw, processed, and complex foods, thus protecting 99 % of soybean-allergic patients.

Purification of soybean Kunitz trypsin inhibitor and the mechanism of its passivation by lysine and disulfide bond modifications

Soybean trypsin inhibitor（STI）was selectively recovered from soybean whey, a by-product of soybean protein production, by ammonium sulfate salting and the STI was purified up to 5.46-fold (Compared to STI in soybean whey). After purification with a Superdex 75 increase gel filtration preloader column, a 91.5% electrophoretically pure soybean Kunitz trypsin inhibitor（SKTI）was obtained by optical density analysis with a purification ploidy of 33.32-fold and a specific activity of 1072.9 U/mg, and a single band protein with a molecular weight of 20 kDa on SDS-PAGE. SKTI's 85.65% inhibitory activity was maintained after heating at 80 °C for 30 min, and there was no significant change in inhibitory activity in the range of pH 3–10. The secondary structure of SKTI did not change with temperature and pH showing good stability. By chemical bond modification of SKTI, the activity of SKTI was found to have an important connection with lysine and disulfide bond. This study set up a process for the rapid preparation of STI by salinization and the related properties of SKTI was explored to provide more potential for the treatment of soy protein production waste and the application of STI in the food field in the future.

Trypsin from Pyloric Caeca of Asian Seabass: Purification, Characterization, and Its Use in the Hydrolysis of Acid-Soluble Collagen.

The study aimed to purify trypsin from the pyloric caeca of Asian seabass (Lates calcarifer), and investigate its proteolytic capability toward acid-soluble collagen (ASC) in comparison with commercial porcine trypsin (CPT). Trypsin was purified from pyloric caeca, a leftover from the evisceration process, via ammonium sulphate (40-60% saturation) precipitation, and a soybean trypsin inhibitor (SBTI)-Sepharose 4B column. A 18.5-fold purification and a yield of 15.2% were obtained. SDS-PAGE analysis confirmed a single band of trypsin with a molecular weight of 23.5 kDa. Purified trypsin also showed the single band in native-PAGE. The optimal pH and temperature of trypsin for BAPNA (the specific substrate for amidase) hydrolysis were 8.5 and 60 °C, respectively. The trypsin was stable within the pH range of 7.0-9.5 and temperature range of 25-55 °C. Protease inhibition study confirmed that the purified enzyme was trypsin. The purified trypsin had a Michaelis-Menten constant (Km) and catalytic constant (kcat) of 0.078 mM and 5.4 s-1, respectively, when BAPNA was used. For the hydrolysis of TAME (the specific substrate for esterase), the Km and Kcat were 0.09 mM and 4.8 s-1, respectively. Partially purified seabass trypsin (PPST) had a slightly lower hydrolysis capacity toward ASC than CPT, as evidenced by the lower degree of hydrolysis and protein degradation when the former was used. Both the α-chain and β-chain became more degraded as the hydrolysis time increased. Based on MALDI-TOP, peptides with MW of 2992-2970 Da were dominant in the hydrolysates. Therefore, seabass trypsin could be used in the production of hydrolyzed collagen. It could have economic importance to the market, by replacing some commercial proteases, which have religious constraints.

Characterization of digestive proteases in the gut of a basal deuterostome.

Digestive systems are complex organs that allow organisms to absorb energy from their environment to fuel vital processes like growth, development and the maintenance of homeostasis. A comprehensive understanding of digestive physiology is therefore essential to fully understand the energetics of an organism. The digestion of proteins is of particular importance because most heterotrophic organisms are not able to synthesize all essential amino acids. While Echinoderms are basal deuterostomes that share a large genetic similarity with vertebrates their digestion physiology remains largely unexplored. Using a genetic approach, this work demonstrated that several protease genes including an enteropeptidase, aminopeptidase, carboxypeptidase and trypsin involved in mammalian digestive networks are also found in sea urchin larvae. Through characterization including perturbation experiments with different food treatments and pharmacological inhibition of proteases using specific inhibitors, as well as transcriptomic analysis we conclude that the trypsin-2 gene codes for a crucial enzyme for the protein digestion in Strongylocentrotus purpuratus. Measurements of in vivo digestion rates in the transparent sea urchin larva were not altered by pharmacological inhibition of trypsin (Soybean Trypsin Inhibitor) or serine proteases (Aprotinin) suggesting that proteases are not critically involved in the initial step of microalgal breakdown. This work provides new insights into the digestive physiology of a basal deuterostome and allows comparisons from the molecular to the functional level in the digestive systems of vertebrates and mammals. This knowledge will contribute to a better understanding for conserved digestive mechanisms that evolved in close interaction with their biotic and abiotic environment.

Insulin-Loaded Soybean Trypsin Inhibitor-Chitosan Nanoparticles: Preparation, Characterization, and Protective Effect Evaluation.

The aim of this work was to prepare insulin-loaded nanoparticles using soybean trypsin inhibitor (STI) and chitosan (CS) as a potential coating. The nanoparticles were prepared by complex coacervation, and characterized for their particle size, polydispersity index (PDI), and encapsulation efficiency. In addition, the insulin release and enzymatic degradation of nanoparticles in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were evaluated. The results showed that the optimal conditions for preparing insulin-loaded soybean trypsin inhibitor-chitosan (INs-STI-CS) nanoparticles were as follows: CS concentration of 2.0 mg/mL, STI concentration of 1.0 mg/mL, and pH 6.0. The INs-STI-CS nanoparticles prepared at this condition had a high insulin encapsulation efficiency of 85.07%, the particle diameter size was 350 ± 5 nm, and the PDI value was 0.13. The results of the in vitro evaluation of simulated gastrointestinal digestion showed that the prepared nanoparticles could improve the stability of insulin in the gastrointestinal tract. Compared with free insulin, the insulin loaded in INs-STI-CS nanoparticles was retained at 27.71% after 10 h of digestion in the intestinal tract, while free insulin was completely digested. These findings will provide a theoretical basis for improving the stability of oral insulin in the gastrointestinal tract.

Effects of Soybean Trypsin Inhibitor on Pancreatic Oxidative Damage of Mice at Different Growth Periods.

The bioactive components in soybeans have significant physiological functions. However, the intake of soybean trypsin inhibitor (STI) may cause metabolic disorders. To investigate the effect of STI intake on pancreatic injury and its mechanism of action, a five-week animal experiment was conducted, meanwhile, a weekly monitor on the degree of oxidation and antioxidant indexes in the serum and pancreas of the animals was carried out. The results showed that the intake of STI had irreversible damage to the pancreas, according to the analysis of the histological section. Malondialdehyde (MDA) in the pancreatic mitochondria of Group STI increased significantly and reached a maximum (15.7 nmol/mg prot) in the third week. Meanwhile, the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), trypsin (TPS), and somatostatin (SST) were decreased and reached minimum values (10 U/mg prot, 87 U/mg prot, 2.1 U/mg prot, 10 pg/mg prot) compared with the Group Control. The RT-PCR results of the expression of SOD, GSH-Px, TPS, and SST genes were consistent with the above. This study demonstrates that STI causes oxidative structural damage and pancreatic dysfunction by inducing oxidative stress in the pancreas, which could increase with time.

Interactions between Soybean Trypsin Inhibitor and Chitosan in an Aqueous Solution

Supramolecular structures obtained from protein-polysaccharide association may be applied to encapsulate bioactive compounds or to improve the physical stability and texture properties of colloid-based products. In this study, the interaction of 0.1 wt% soybean trypsin inhibitor (STI) with different concentrations of chitosan (CS) in aqueous solutions was investigated under different pH by the analysis of state diagram, turbidity, zeta potential, spectroscopy, and microstructure; the protective effect of STI-CS complex coacervates on STI stability in simulated gastric juice was also discussed. The results suggested that interactions between STI and CS could form soluble/insoluble complexes mainly through hydrophobic interactions (pH 4.0) or electrostatic interactions (pH 6.0). The CD spectra showed that the secondary structure of STI did not change significantly when CS with the same charge was mixed with STI, and the secondary structure of STI was slightly changed when CS with the opposite charge was mixed with STI. Simulated gastric digestion experiments showed that the complex formed by non-covalent bonding had a protective effect on the active protein. This study provides information about the effect of different CS concentrations and pH values on the formation of complexes of CS and STI in an aqueous solution and provides theoretical references for the construction of supramolecular-structured carrier substances based on CS and STI.

Inactivation of soybean trypsin inhibitors under hot alcohol and associated urease activity behavior

大豆は機能性や栄養に富んだ有用な食材であるが, 不快な味や臭い, さらに不活化の困難な栄養阻害因子などを含み, 利用を妨げている. そこで, 栄養阻害因子のトリプシンインヒビター (TI) の不活化にアルコールのタンパク質変性能を利用し, 不活化を検討した. また, 耐熱性のウレアーゼ (UA) とTIの不活化が連動6) するのかについて考察した.

The CASR antagonist NPS2143 induces loss of acrosomal integrity and proteolysis of SPACA1 in boar spermatozoa.

The mechanism by which p32 protein increases during capacitation in boar spermatozoa is unknown. This manuscript shows a new mechanism of induction of p32 in boar spermatozoa: the proteolysis of the phosphorylated and glycosylated form of SPACA1. Protein tyrosine phosphorylation (PY) induction is associated with sperm capacitation. We previously showed that calcium-sensing receptor (CASR) inhibition by NPS2143 induces the 32 kDa tyrosine-phosphorylated protein (p32) in boar spermatozoa. We showed that NPS2143 induced an increase in p32 and loss of acrosomal integrity in live and dead spermatozoa in capacitating conditions (Tyrode's complete medium); the p32 rise occurred in dead spermatozoa, as shown by flow cytometry sorting. EGTA addition blunted the increase in p32, the loss of acrosomal integrity, and the increase in dead spermatozoa induced by NPS2143, indicating that the effects of NPS2143 are calcium-dependent. Mass spectrometry was used to identify which tyrosine-phosphorylated proteins were induced by NPS2143, but only serine/threonine-phosphorylated proteins were found; among these, SPACA1 was identified with different molecular weights (18, 32, and 35-45 kDa). We confirmed tyrosine phosphorylation of SPACA1 at 32 and 35-45 kDa by immunoprecipitation and co-localization of PY and SPACA1 in the presence of NPS2143 by immunofluorescence. The molecular weight of SPACA1 (35-45 kDa) decreased after treatment with peptide-N-glycosidase F, indicating that this protein is N-glycosylated. The soybean trypsin inhibitor (STI), a serine protease inhibitor, suppressed the appearance of p32 and SPACA1 (30 and 32 kDa) induced by NPS2143. Also, 8-Br-cAMP and A23187 treatments induced an increase in p32 and SPACA1 (30-32 kDa) and a parallel induction of the acrosome reaction. These findings suggest that CASR inhibition induces loss of acrosomal integrity and proteolysis of the glycosylated and phosphorylated SPACA1 (35-45 kDa) resulting in a SPACA1 rise at 32 kDa (p32).

Soybean Trypsin Inhibitor(s) Reduce Absorption of Exogenous and Increase Loss of Endogenous Protein in Miniature Pigs

It was the purpose of this study to define whether trypsin inhibitors impair protein digestibility via enhanced loss of exogenous or endogenous protein by quantifying those losses using the homoarginine technique, recently developed in this laboratory. Pigs fitted with permanent ileal T-cannulas were fed test meals containing homoarginine-labeled protein. The meals contained casein and increasing doses of trypsin inhibitors (Experiment 1) or alternatively either heat-treated or raw ground soybeans (Experiment 2). Following a casein meal (425 mmol nitrogen, no trypsin inhibitors), ileal protein was predominantly of endogenous rather than of exogenous origin (105 vs. 9 mmol nitrogen). Addition of isolated trypsin inhibitors (3000 mg) enhanced appearance of both endogenous and exogenous protein at the ileum (by 73 and 9 mmol nitrogen, respectively). Feeding raw instead of heat-treated soybeans in one single test meal caused a significant increase of endogenous protein from 217 +/- 42 to 263 +/- 47 mmol (mean +/- SEM) and of exogenous protein from 16 +/- 3 to 48 +/- 14 mmol. If fed continuously for 1 wk, a raw soybean diet caused endogenous protein loss to rise significantly from 221 +/- 26 to 432 +/- 85 mmol. We conclude that ingestion of food containing trypsin inhibitor affects nitrogen balance more by losses of amino acids of endogenous secreta than by losses of dietary amino acids.

Overexpression of soybean trypsin inhibitor genes decreases defoliation by corn earworm (Helicoverpa zea) in soybean (Glycine max) and Arabidopsis thaliana.

Trypsin inhibitors (TIs) are widely distributed in plants and are known to play a protective role against herbivores. TIs reduce the biological activity of trypsin, an enzyme involved in the breakdown of many different proteins, by inhibiting the activation and catalytic reactions of proteins. Soybean (Glycine max) contains two major TI classes: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Both genes encoding TI inactivate trypsin and chymotrypsin enzymes, which are the main digestive enzymes in the gut fluids of Lepidopteran larvae feeding on soybean. In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. A total of six TIs were tested, including three known soybean trypsin inhibitors (KTI1, KTI2 and KTI3) and three genes encoding novel inhibitors identified in soybean (KTI5, KTI7, and BBI5). Their functional role was further examined by overexpression of the individual TI genes in soybean and Arabidopsis. The endogenous expression patterns of these TI genes varied among soybean tissues, including leaf, stem, seed, and root. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf-punch feeding bioassays detected significant reduction in corn earworm (Helicoverpa zea) larval weight when larvae fed on transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with H. zea on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, bioassays of KTI7 and BBI5 overexpressing lines with soybean cyst nematode (SCN, Heterodera glycines) showed no differences in SCN female index between transgenic and non-transgenic control plants. There were no significant differences in growth and productivity between transgenic and non-transgenic plants grown in the absence of herbivores to full maturity under greenhouse conditions. The present study provides further insight into the potential applications of TI genes for insect resistance improvement in plants.

Inactivation of Soybean Trypsin Inhibitor by Dielectric-Barrier Discharge Plasma and Its Safety Evaluation and Application.

The trypsin inhibitor (TI) is one of the most important anti-nutritive elements in soybeans. As a new nonthermal technology, dielectric-barrier discharge (DBD) cold plasma has attracted increasing attention in food processing. In this research, we investigated the effect of dielectric-barrier discharge (DBD) plasma treatment on soybean trypsin inhibitor content and its structure, evaluated TI toxicity and the safety of its degradation products after treatment with DBD technology in vitro and in vivo, and applied the technology to soybean milk, which was analyzed for quality. Using the statistical analysis of Student's t-test, the results demonstrated that DBD plasma treatment significantly decreased the content of TI (33.8 kV at 1, 3, or 5 min, p &lt; 0.05, p &lt; 0.01, p &lt; 0.001) and destroyed the secondary and tertiary structures of TI. TI was toxic to Caco-2 cells and could inhibit body weight gain, damage liver and kidney functions, and cause moderate or severe lesions in mouse organ tissues, whereas these phenomena were alleviated in mice treated with degradation products of TI after DBD plasma treatment under the optimal condition (33.8 kV at 5 min). The content of TI in DBD-treated soymilk was also significantly reduced (p &lt; 0.001), while the acidity, alkalinity, conductivity, color, and amino acid composition of soymilk were not affected, and there were no statistical differences (p &gt; 0.05). In summary, DBD plasma is a promising non-thermal processing technology used to eliminate TI from soybean products.