A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative.

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

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.

Prebound State Discovered in the Unbinding Pathway of Fluorinated Variants of the Trypsin-BPTI Complex Using Random Acceleration Molecular Dynamics Simulations.

The serine protease trypsin forms a tightly bound inhibitor complex with the bovine pancreatic trypsin inhibitor (BPTI). The complex is stabilized by the P1 residue Lys15, which interacts with negatively charged amino acids at the bottom of the S1 pocket. Truncating the P1 residue of wildtype BPTI to α-aminobutyric acid (Abu) leaves a complex with moderate inhibitor strength, which is held in place by additional hydrogen bonds at the protein-protein interface. Fluorination of the Abu residue partially restores the inhibitor strength. The mechanism with which fluorination can restore the inhibitor strength is unknown, and accurate computational investigation requires knowledge of the binding and unbinding pathways. The preferred unbinding pathway is likely to be complex, as encounter states have been described before, and unrestrained umbrella sampling simulations of these complexes suggest additional energetic minima. Here, we use random acceleration molecular dynamics to find a new metastable state in the unbinding pathway of Abu-BPTI variants and wildtype BPTI from trypsin, which we call the prebound state. The prebound state and the fully bound state differ by a substantial shift in the position, a slight shift in the orientation of the BPTI variants, and changes in the interaction pattern. Particularly important is the breaking of three hydrogen bonds around Arg17. Fluorination of the P1 residue lowers the energy barrier of the transition between the fully bound state and prebound state and also lowers the energy minimum of the prebound state. While the effect of fluorination is in general difficult to quantify, here, it is in part caused by favorable stabilization of a hydrogen bond between Gln194 and Cys14. The interaction pattern of the prebound state offers insights into the inhibitory mechanism of BPTI and might add valuable information for the design of serine protease inhibitors.

Riboflavin-induced photo-ATRP electrochemical strategy for detection of biomarker trypsin.

The detection of trypsin and its inhibitors is important for both clinical diagnosis and disease treatment. Abnormal trypsin activity affects pancreatic function and leads to corresponding pathological changes in the body. Therefore, the study presented a riboflavin-induced photo-ATRP electrochemical assay of trypsin activity and its inhibitor, including detection of trypsin activity in real urine samples. Experiments were performed on indium tin oxide (ITO) electrodes modified with sulfhydryl groups of 3-mercaptopropionic acid, and target trypsin-specific cleavage of BSA-Au nanocluster (BSA-Au NCs) was followed by the modification of Au NCs to the electrodes using Au-S. The Au NCs immobilized monodeoxy-monomercapto-ß-cyclodextrin@adamantan-2-amine (SH-ß-CD@2-NH2-Ada) host-guest inclusion complexes to the electrode surfaces via Au-S. In a two-component photo-initiator system consisting of riboflavin as an initiator and ascorbic acid (AA) as a mild reducing agent under mild blue light radiation, a large number of electroactive substances were grafted onto the electrode surface to generate electrochemical signals. In addition, we have successfully realized the detection of clinical drug inhibitors of trypsin. The detection limit of the system is as low as 0.0024 ng/mL, which much littler than the average standard of trypsin in the patient's urine or serum. It's worth noting that this work will provide researchers with a different route to design electrochemical sensors based on non-covalent recognition strategies.

Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

Decoding the Duality of Antinutrients: Assessing the Impact of Protein Extraction Methods on Plant-Based Protein Sources.

This review aims to provide an updated overview of the effects of protein extraction/recovery on antinutritional factors (ANFs) in plant protein ingredients, such as protein-rich fractions, protein concentrates, and isolates. ANFs mainly include lectins, trypsin inhibitors, phytic acid, phenolic compounds, oxalates, saponins, tannins, and cyanogenic glycosides. The current technologies used to recover proteins (e.g., wet extraction, dry fractionation) and novel technologies (e.g., membrane processing) are included in this review. The mechanisms involved during protein extraction/recovery that may enhance or decrease the ANF content in plant protein ingredients are discussed. However, studies on the effects of protein extraction/recovery on specific ANFs are still scarce, especially for novel technologies such as ultrasound- and microwave-assisted extraction and membrane processing. Although the negative effects of ANFs on protein digestibility and the overall absorption of plant proteins and other nutrients are a health concern, it is also important to highlight the potential positive effects of ANFs. This is particularly relevant given the rise of novel protein ingredients in the market and the potential presence or absence of these factors and their effects on consumers' health.

The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

Effect of different physical pre-treatments on physicochemical and techno-functional properties, and on the antinutritional factors of lentils (Lens culinaris spp).

Lentils have a valuable physicochemical profile, which can be affected by the presence of antinutrients that may impair the benefits arising from their consumption. Different treatments can be used to reduce these undesirable compounds, although they can also affect the general composition and behaviour of the lentils. Thus, the effect of different processing methods on the physicochemical and techno-functional properties, as well as on the antinutritional factors of different lentil varieties was studied. Phytic acid was eliminated during germination, while tannins and trypsin inhibitors are mostly affected by cooking. Functional properties were also altered by processing, these being dependent on the concentration of different nutrients in lentils. All the studied treatments affected the physicochemical profile of lentils and their functional properties. Cooking and germination appear to be the most effective in reducing antinutritional factors and improving the physicochemical profile of the lentils, meeting the current nutritional demands of today's society.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from Bauhinia pulchella (Benth) Seeds.

INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

Exploring Photoswitchable Binding Interactions with Small-Molecule- and Peptide-Based Inhibitors of Trypsin.

The ability to photochemically activate a drug, both when and where needed, requires optimisation of the difference in biological activity between each isomeric state. As a step to this goal, we report small-molecule- and peptide-based inhibitors of the same protease-trypsin-to better understand how photoswitchable drugs interact with their biological target. The best peptidic inhibitor displayed a more than fivefold difference in inhibitory activity between isomeric states, whereas the best small-molecule inhibitor only showed a 3.4-fold difference. Docking and molecular modelling suggest this result is due to a large change in 3D structure in the key binding residues of the peptidic inhibitor upon isomerisation; this is not observed for the small-molecule inhibitor. Hence, we demonstrate that significant structural changes in critical binding motifs upon irradiation are essential for maximising the difference in biological activity between isomeric states. This is an important consideration in the design of future photoswitchable drugs for clinical applications.

Dietary wheat amylase trypsin inhibitors exacerbate CNS inflammation in experimental multiple sclerosis.

OBJECTIVE: Wheat has become a main staple globally. We studied the effect of defined pro-inflammatory dietary proteins, wheat amylase trypsin inhibitors (ATI), activating intestinal myeloid cells via toll-like receptor 4, in experimental autoimmune encephalitis (EAE), a model of multiple sclerosis (MS). DESIGN: EAE was induced in C57BL/6J mice on standardised dietary regimes with defined content of gluten/ATI. Mice received a gluten and ATI-free diet with defined carbohydrate and protein (casein/zein) content, supplemented with: (a) 25% of gluten and 0.75% ATI; (b) 25% gluten and 0.19% ATI or (c) 1.5% purified ATI. The effect of dietary ATI on clinical EAE severity, on intestinal, mesenteric lymph node, splenic and central nervous system (CNS) subsets of myeloid cells and lymphocytes was analysed. Activation of peripheral blood mononuclear cells from patients with MS and healthy controls was compared. RESULTS: Dietary ATI dose-dependently caused significantly higher EAE clinical scores compared with mice on other dietary regimes, including on gluten alone. This was mediated by increased numbers and activation of pro-inflammatory intestinal, lymph node, splenic and CNS myeloid cells and of CNS-infiltrating encephalitogenic T-lymphocytes. Expectedly, ATI activated peripheral blood monocytes from both patients with MS and healthy controls. CONCLUSIONS: Dietary wheat ATI activate murine and human myeloid cells. The amount of ATI present in an average human wheat-based diet caused mild intestinal inflammation, which was propagated to extraintestinal sites, leading to exacerbation of CNS inflammation and worsening of clinical symptoms in EAE. These results support the importance of the gut-brain axis in inflammatory CNS disease.

Development of a rapid process for purification of Bowman-Birk and Kunitz inhibitors from legume seeds, and evaluation of their biophysical, insecticidal, and antimicrobial properties.

Bowman-Birk inhibitor (BBI ~10 kDa) and Kunitz inhibitor (KI ~20 kDa) are serine protease/proteinase inhibitor(s) [PI(s)] ubiquitously found in several Leguminous plant species with insecticidal and therapeutic properties. Due to narrow molecular mass differences, the separation of these inhibitors from a single seed variety is tedious. The present study is aimed to develop a rapid protocol (<24 h) for purifying BBI and KI from legume seeds using mild trichloroacetic acid (TCA) extraction followed by trypsin-affinity chromatography. The mature seeds of Vigna radiata and Cajanus platycarpus are used as a model to purify BBI and KI using this protocol. The BBI and KI purified from the seeds of V. radiata are labeled as VrBBI & VrKI, and C. platycarpus are labeled as CpBBI & CpKI, respectively. These PIs are confirmed by immunodetection and MALDI-TOF studies and further characterized for their structural (CD & fluorescence spectroscopy) and functional properties (temperature & DTT stability). BBI(s) purified using the above process are effective in the management of castor semi-looper 'Achaea janata', while KI(s) are effective in the management of pod borer 'Helicoverpa armigera'. Besides, both BBI(s) and KI(s) have significant potential in controlling the growth of methicillin-sensitive 'Staphylococcus aureus', a gram-positive pathogenic bacterium.

Ultrasound-enhanced interfacial adsorption and inactivation of soy trypsin inhibitors.

In this study, liquid-liquid interfacial protein adsorption was proposed as a means of inactivating soy trypsin inhibitors (TIs, including Kunitz (KTI) and Bowman-Birk inhibitor (BBI)). Hexane-water was first selected as a model system to compare three emulsification methods (hand shaking, rotor-stator and ultrasound mixing). Ultrasound could generate the smallest and least polydisperse emulsion droplets, resulting in highest interfacial adsorption amount of KTI and BBI as well as the highest inactivation percentage of TIs (p < 0.05). Therefore, ultrasound was selected to further explore the effect of the non-aqueous phase on interfacial adsorption and inactivation kinetics of TIs in a food emulsion system containing vegetable oil (VTO). The adsorption amounts of KTI and BBI in the VTO-aqueous emulsion increased by âˆ¼ 25 % compared to the hexane-aqueous emulsion. In addition, the adsorption amounts of KTI and BBI were rapidly increased as a function of sonication time, especially for the hexane-aqueous emulsion system. This result suggests that such inactivation of TIs could be implemented in continuous systems for large-scale processing. Finally, the pathways of interface-induced inactivation of BBI and KTI were investigated based on separate experiments on individual BBI and KTI systems. The results showed that the interface adsorption caused the changes in the secondary and tertiary structure of KTI that led to its activitation. However, BBI was quite stable at the liquid-liquid interface without significant conformational change. Overall, ultrasound-assisted interfacial adsorption can be considered a rapid and highly efficient method to inactivate KTI.

Kinetic and mechanistic study of ultrasonic inactivation of Kunitz (KTI) and Bowman-Birk (BBI) inhibitors in relation to process-relevant parameters.

In this study, quantitative monitoring of low-frequency (20 kHz) and high-frequency (355 kHz) ultrasound-induced inactivation of Kunitz (KTI) and Bowman-Birk inhibitor (BBI) using RP-HPLC was achieved, and its consistency with a traditional TI activity assay was verified. The effect of TI concentration, ultrasonic frequency, power density and pH on inactivation kinetics of KTI and BBI was explored. Results showed that the pseudo-first-order kinetic rate constants of KTI and BBI were decreased by over 60% when the initial TI concentration was increased from 100 mg/L to 1000 mg/L. Also, the amounts of inactivated KTI and BBI were increased by around 4-fold at the higher TI concentration of 1000 mg/L (20 kHz, 1.71 W/mL and pH 4). The colloidal environment and ultrasonic conditions influenced the secondary and tertiary structure and particle size of TIs in LF-induced inactivation. In comparison, the abovementioned conditions affected the oxidation of methionine and the conformational change of TIs in HF-induced inactivation.

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor.

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman-Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P41, with Rwork /Rfree values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme-inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.

In vivo developmental studies of Helicoverpa armigera and in silico molecular interactions with trypsin reveal the bio-insecticidal potential of trypsin inhibitor (SSTI) isolated from Solanum surattense.

We report the impact of gut protease inhibition on the development of Helicoverpa armigera by trypsin inhibitor and the use of molecular modeling to understand the mechanism of trypsin inhibition. Larvae of H. armigera fed on an artificial diet containing 150 and 300 µg/ml SSTI showed a negative impact on the insects' development in terms of mean larval weight, larval fatality, survival rate, and nutritional indices. Prominent physical abnormalities like curled wings, malformed appendages, and small body size were observed during the development. Gene expression studies revealed down regulation in trypsin (HaTry 1, 2, 3, 4, 6, 8) and chymotrypsin (HaChy 1, 2, 3, 4) genes of the larval gut upon treatment of SSTI. Homology modeling has been used to build the three-dimensional structure of SSTI, which showed ß-sheets having a stable canonical inhibitory loop (CIL) with conserved lysine residue. Molecular docking studies showed the strong binding of SSTI at the active site of trypsin. Molecular dynamic (MD) simulation revealed the stable interactions of the rigid CIL of SSTI at the active site of trypsin, leading to its destabilization. Conserved lysine63 of the P1 site in SSTI forms a strong hydrogen bonding network with residues Asp189 and Ser190 of trypsin.

A review of thermosensitive antinutritional factors in plant-based foods.

Legumes and cereals account for the vast proportion of people's daily intake of plant-based foods. Meanwhile, a large number of antinutritional factors in legumes and cereals hinder the body absorption of nutrients and reduce the nutritional value of food. In this paper, the antinutritional effects, determination, and passivation methods of thermosensitive antinutritional factors such as trypsin inhibitors, urease, lipoxygenase, and lectin were reviewed to provide theoretical help to reduce antinutritional factors in food and improve the utilization rate of plant-based food nutrition. Since trypsin inhibitors and lectin have been more extensively studied and reviewed previously, the review mainly focused on urease and lipoxygenase. This review summarized the information of thermosensitive antinutritional factors, trypsin inhibitors, urease, lipoxygenase, and lectin, in cereals and legumes. The antinutritional effects, and physical and chemical properties of trypsin inhibitors, urease, lipoxygenase, and lectin were introduced. At the same time, the research methods for the detection and inactivation of these four antinutritional factors were also summarized in the order of research conducted time. The rapid determination and inactivation of antinutrients will be the focus of attention for the food industry in the future to improve the nutritional value of food. Exploring what structural changes could passivation technologies bring to antinutritional factors will provide a theoretical basis for further understanding the mechanisms of antinutritional factor inactivation. PRACTICAL APPLICATIONS: Antinutritional factors in plant-based foods hinder the absorption of nutrients and reduce the nutritional value of the food. Among them, thermosensitive antinutritional factors, such as trypsin inhibitors, urease, lipoxygenase, and lectins, have a high proportion among the antinutritional factors. In this paper, we investigate thermosensitive antinutritional factors from three perspectives: the antinutritional effect of thermosensitive antinutritional factors, determination, and passivation methods. The current passivation methods for thermosensitive antinutritional factors revolve around biological, physical, and chemical aspects, and their elimination mechanisms still need further research, especially at the protein structure level. Reducing the level of antinutritional factors in the future food industry while controlling the loss of other nutrients in food is a goal that needs to be balanced. This paper reviews the antinutritional effects of thermosensitive antinutritional factors and passivation methods, expecting to provide new research ideas to improve the nutrient utilization of food.

Wheat amylase/trypsin inhibitors (ATIs): occurrence, function and health aspects.

Amylase/trypsin inhibitors (ATIs) are widely consumed in cereal-based foods and have been implicated in adverse reactions to wheat exposure, such as respiratory and food allergy, and intestinal responses associated with coeliac disease and non-coeliac wheat sensitivity. ATIs occur in multiple isoforms which differ in the amounts present in different types of wheat (including ancient and modern ones). Measuring ATIs and their isoforms is an analytical challenge as is their isolation for use in studies addressing their potential effects on the human body. ATI isoforms differ in their spectrum of bioactive effects in the human gastrointestinal (GI), which may include enzyme inhibition, inflammation and immune responses and of which much is not known. Similarly, although modifications during food processing (exposure to heat, moisture, salt, acid, fermentation) may affect their structure and activity as shown in vitro, it is important to relate these changes to effects that may present in the GI tract. Finally, much of our knowledge of their potential biological effects is based on studies in vitro and in animal models. Validation by human studies using processed foods as commonly consumed is warranted. We conclude that more detailed understanding of these factors may allow the effects of ATIs on human health to be better understood and when possible, to be ameliorated, for example by innovative food processing. We therefore review in short our current knowledge of these proteins, focusing on features which relate to their biological activity and identifying gaps in our knowledge and research priorities.