Compositional and functional changes in Acacia tenuissima seeds due to processing.

The chemical, functional, anti-nutritional, and antioxidant composition of raw and roasted Acacia tenuissima seeds were determined to evaluate the impact of heat processing. The raw seeds were relatively high in protein (25.2 %), fat (10.3 %) and crude fiber (13.2 %), potassium (760 mg/100 g), magnesium (111 mg/100 g) and calcium (66.5 mg/100 g). Linolenic (34 %), margaric acid (24 %) and oleic acid (16 %) were the notable fatty acids present in the raw seeds. The proximate and fatty acid composition of A. tenuissima were not significantly impacted by roasting. Acacia tenuissima flour had excellent foaming capacity, foaming stability and water solubility making it a potential functional ingredient in the baking industry. Anti-nutrients were present in the raw seeds but at low quantities (trypsin inhibitor activity: 0.22 TIU/g, saponin: 2.29 %) while the antioxidant activity was high at 94 % compared to other Acacia species. Only the water solubility and trypsin inhibitor activity of A. tenuissima decreased significantly after roasting. Roasting had no significant impact on the seed constituents and their properties except for water solubility, and trypsin inhibitor activity which decreased, making it an ideal ingredient to be used in baked products.

A fast and cost-effective procedure for reliable measurement of trypsin inhibitor activity in soy and soy products.

Rapid and accurate measurement of trypsin inhibitor is critical for soy processors to assess the quality of soy meal. Currently, trypsin inhibitor activity is measured using the American Oil Chemists' Society (AOCS) and the American Association of Cereal Chemists International (AACCI) approved method. We have modified and improved the AACCI/AOCS approved method resulting in the elimination of several time-consuming steps and drastically reducing the assay volume. By employing our simplified procedure, we have measured trypsin inhibitor activity of several soy and soy products. A side-by side comparison of our simplified procedure with AOCS approved method revealed strikingly similar results indicating that several time-consuming and tedious steps associated with AACCI/AOCS approved methods can be eliminated without sacrificing the accuracy of the assay. Moreover, we demonstrate that our assay can also be carried out in 96-well microplates which will enable high-throughput screening of large number of soy meal samples.

Chia (Salvia hispanica L.) Seeds Contain a Highly Stable Trypsin Inhibitor with Potential for Bacterial Management Alone or in Drug Combination Therapy with Oxacillin.

The emergence of antibiotic resistance poses a serious and challenging threat to healthcare systems, making it imperative to discover novel therapeutic options. This work reports the isolation and characterization of a thermostable trypsin inhibitor from chia (Salvia hispanica L.) seeds, with antibacterial activity against Staphylococcus aureus sensitive and resistant to methicillin. The trypsin inhibitor ShTI was purified from chia seeds through crude extract heat treatment, followed by affinity and reversed-phase chromatography. Tricine-SDS-PAGE revealed a single glycoprotein band of ~ 11 kDa under nonreducing conditions, confirmed by mass spectrometry analysis (11.558 kDa). ShTI was remarkably stable under high temperatures (100 °C; 120 min) and a broad pH range (2-10; 30 min). Upon exposure to DTT (0.1 M; 120 min), ShTI antitrypsin activity was partially lost (~ 38%), indicating the participation of disulfide bridges in its structure. ShTI is a competitive inhibitor (Ki = 1.79 × 10-8 M; IC50 = 1.74 × 10-8 M) that forms a 1:1 stoichiometry ratio for the ShTI:trypsin complex. ShTI displayed antibacterial activity alone (MICs range from 15.83 to 19.03 µM) and in combination with oxacillin (FICI range from 0.20 to 0.33) against strains of S. aureus, including methicillin-resistant strains. Overproduction of reactive oxygen species and plasma membrane pore formation are involved in the antibacterial action mode of ShTI. Overall, ShTI represents a novel candidate for use as a therapeutic agent for the bacterial management of S. aureus infections.

Chemical Composition and in vitro Anti-inflammatory Activity of Wheat Germ Oil Depending on the Extraction Procedure.

This study aimed to examine the chemical composition of wheat germ oil extracted by three different methods, and to evaluate its inhibitory effect on the cyclooxygenase and proteinase activities. The results showed that the contents of policosanols, tocopherols and phytosterols were affected by the extraction procedure. However, the fatty acid composition of the different oil extracts was nearly the same. Among the tested oils samples, cold pressed oil exhibited the strongest inhibitory activity against proteinase (93.4%, IC50 =195.7 µg/mL) and cyclooxygenase 1 (80.5%, IC50 =58.6 µg/mL). Furthermore, the cold pressed oil had the highest content of octacosanol, ß-sitosterol and α-linolenic acid, suggesting that those bioactive compounds could be essential for the potent ani-cyclooxygenase activity. The present data revealed that wheat germ oil contained cyclooxygenase and trypsin inhibitors, which are the promising therapeutic target for the treatment of various inflammatory diseases. Thus, wheat germ oil might be used to develop functional foods and pharmaceutic products for the human health.

Detoxification of jatropha kernel meal to utilize it as aqua-feed.

BACKGROUND: Jatropha is an oilseed crop with high kernel oil (55-58%) and protein (26-29%) contents, which makes it a good source of biodiesel and animal/aqua-feed. However, the presence of anti-nutritional toxins, such as phorbol esters, lectins, trypsin inhibitor, phytate, and saponins, restricts its use as feed. This paper describes chemical, ultraviolet (UV) radiation, and biological treatments for detoxification of jatropha kernel meal. Raw, defatted, and one-time and two-times mechanically expressed oil samples were analyzed for toxins. Chemical treatment involved heating with 90% methanol and 4% sodium hydroxide. UV treatment was carried out at UV light intensity of 53.4 mW cm-2 for 30 min. For biological treatment, cell-free extract from Pseudomonas aeruginosa (strain PAO1) was mixed with kernel meal for detoxification. RESULTS: Among treatments, chemical treatment was most effective in reducing all toxins, with phorbol esters in the range 0.034-0.052 mg g-1 , lectin 0.082-10.766 mg g-1 , trypsin inhibitor 10.499-11.350 mg g-1 , phytate 2.475-5.769 mg g-1 , and saponins 0.044-0.098 mg g-1 . Biological treatment reduced all toxins except phytate, whereas UV treatment could not reduce any of toxins and, hence, cannot be used for aqua-feed preparation. Pellets prepared from chemically detoxified kernel meal with the least oil content (defatted) resulted in the highest strength (70.93 N). CONCLUSION: Chemically treated jatropha kernel meal can be used for aqua-feed pellet preparation because of its low toxin content. The highest compressive strength was obtained for pellets with the least oil content (defatted). Biological treatment time must have been extended for many hours instead of 24 h. Jatropha kernel meal treated chemically can be recommended for aqua-feed manufacturing. © 2021 Society of Chemical Industry.

Trypsin inhibitor content and activity of soaking water whey as waste in soy milk processing.

Soybean soaking water whey (SWW) is obtained as the waste of soy milk production and mostly represents an environmental problem. The aim of this study was to assess the content of proteins and content and activity of trypsin inhibitors of fresh SWW, obtained during soy milk production. Two zones of Bowman-Birk trypsin inhibitors (BBI) were detected. One was identified as a monomeric form of BBI (0.61-2.93%) and the other one was identified as a polymeric form of BBI (0.45-3.33%). The degree of BBI extraction (1.88-5.49%) was influenced by the soybean genotype and the grain size, i.e. it increased with increasing grain size. Kunitz trypsin inhibitor was not detected. Total proteins were found in traces in SWW (0.03-0.06%). Low residual trypsin inhibitor activity (0.32-0.55%) suggested that SWW can potentially be applied for preparing food or feed. In that case it will not be waste but a cheap functional supplement with BBI as a biologically active component.

Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars.

Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC-MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.

Exploring the Diversity of Cysteine-Rich Natural Product Peptides via MS/MS Fingerprint Ions.

Natural product extracts present inherently complex matrices in which the identification of novel bioactive peptide species is challenged by low-abundance masses and significant structural and sequence diversity. Additionally, discovery efforts often result in the re-identification of known compounds, where modifications derived in vivo or during sample handling may obscure true sequence identity. Herein, we identify mass spectral (MS2) "fingerprint" ions characteristic of cyclotides, a diverse and biologically active family of botanical cysteine-rich peptides, based on regions of high sequence homology. We couple mass shift analysis with MS2 spectral fingerprint ions cross referenced with CyBase-a cyclotide database-to discern unique mass species in Viola communis extracts from mass species that are likely already characterized and those with common modifications. The approach is extended to a related class of cysteine-rich peptides, the trypsin inhibitors, using the characterized botanical species Lagenaria siceraria. Coupling the observation of highly abundant MS2 ions with mass shift analysis, we identify a new set of small, highly disulfide-bound cysteine-rich L. siceraria peptides.

Comparative quantitative LC-MS/MS analysis of 13 amylase/trypsin inhibitors in ancient and modern Triticum species.

Amylase/trypsin inhibitors (ATIs) are major wheat allergens and they are also implicated in causing non-celiac gluten sensitivity and worsening other inflammatory conditions. With only few studies on ATI contents in different Triticum species available so far, we developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method based on stable isotope dilution assays to quantitate the 13 most important ATIs in a well-defined sample set of eight cultivars of common wheat and durum wheat (modern species), as well as spelt, emmer and einkorn (ancient species) grown at three locations in Germany, respectively. Only few ATIs with low contents were detected in einkorn. In contrast, spelt had the highest total ATI contents. Emmer and common wheat had similar total ATI contents, with durum wheat having lower contents than common wheat. Due to the lack of correlation, it was not possible to estimate ATI contents based on crude protein contents. The wheat species had a higher influence on ATI contents than the growing location and the heritability of this trait was high. Despite comparatively low intra-species variability, some cultivars were identified that may be promising candidates for breeding for naturally low ATI contents.

Identification and Quantitation of Amylase Trypsin Inhibitors Across Cultivars Representing the Diversity of Bread Wheat.

α-Amylase/trypsin inhibitors (ATIs) may have a role in nonceliac wheat sensitivity (NCWS) and celiac disease (CD), but the ATI content and diversity across a range of wheat cultivars are not well characterized. Discovery proteomics was used to detect ATIs across two wheat cultivars: Chara and Magenta. Comprehensive mapping of detected ATIs with the ATIs from the recently published wheat genome RefSeq v1.0 shows the presence of three major subclasses: monomeric (9%), dimeric (61%), and chloroform-methanol (CM) type (30%). Subsequently, the level of 18 ATI isoforms (63 peptides) grouped into four subtypes was monitored across 15 commercial wheat cultivars and the eight parental lines from a multiparent advanced-generation intercross (MAGIC) population using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS). The ATI content of wheat cultivars Janz, Sunvale, Diamond Bird, and Longreach Scout was significantly lower than that of other wheat cultivars. The MAGIC parental cultivars Baxter and Xiaoyan 54 contain higher levels (∼115% relative to the average wheat ATI content), whereas cultivar Pastor contained the lowest levels (∼87%). Comprehensive sequence analysis, annotation, chromosomal locations, and epitope mapping enabled us to build an LC-MRM-MS method to monitor and quantify the immunostimulatory ATI proteins potentially related to NCWS, autoimmune diseases, and metabolic disorders. This provides an opportunity to select wheat cultivars with significantly lower levels of ATIs.

Novel gluten-free formulations from lentil flours and nutritional yeast: Evaluation of extrusion effect on phytochemicals and non-nutritional factors.

The food industry is increasingly innovating and applying new processing technologies and ingredients to develop novel food products that meet the consumers' demand. In this study, the effect of extrusion (at 140 °C and 160 °C) was evaluated in different lentil flours formulations enriched with nutritional yeast, in terms of α-galactosides (raffinose, stachyose, verbascose), inositol phosphates (IPs), trypsin inhibitors and lectins content. The content of α-galactosides and IPs was determined by high performance liquid chromatography. Trypsin inhibitor activity (TIA) was evaluated using a small-scale quantitative assay. The lectin content was analyzed using a haemagglutination assay and a Competitive Indirect Enzyme-linked immunosorbent assay. Extrusion promoted a significant increase, up to 85% in total α-galactosides content. After extrusion, IPs content was significantly decreased and TIA as well as lectins content had a reduction higher than 90%. Extrusion demonstrated to have a beneficial effect by increasing desirable prebiotic compounds and decreasing non-nutritional factors.

Trypsin inhibitor, antioxidant and antimicrobial activities as well as chemical composition of potato sprouts originating from yellow- and colored-fleshed varieties.

Potato sprouts could be a valuable resource of phytochemicals such as secondary plant metabolites, potential antioxidants and nutritive compounds. In this work, potato sprout extracts of five varieties were examined; they differed in major glycoalkaloid content, trypsin inhibitor activity, total polyphenol content and antioxidant activity, as well as in antimicrobial activity against Gram + and G - bacteria, and yeast. Sprouts of colored-fleshed tubers were characterized by higher trypsin inhibitor activity than sprouts of yellow potatoes. The strongest microorganism growth inhibition effect was observed for macerate with sprouts from the purple-fleshed Blaue Annelise variety against B. subtilis, whereas C. albicans yeasts were sensitive to macerates with sprouts from purple-fleshed Blue Congo and yellow-fleshed Vineta potato varieties.

Targeted proteomics to monitor the extraction efficiency and levels of barley α-amylase trypsin inhibitors that are implicated in non-coeliac gluten sensitivity.

Plant defense protein α-amylase trypsin inhibitors (ATIs) have been proposed as one of the triggers of non-coeliac gluten sensitivity, however there have been no focused studies on their optimal extraction and quantitation from cereal grains. The efficiency of extraction is of utmost interest for the downstream detection and characterisation. In the present study, three extraction buffers and two modified protocols were investigated using LC-MRM-MS in order to examine their ability to efficiently and repeatably extract ATIs from selected barley cultivars. Initially, three extraction buffers IPA/DTT, urea and Tris-HCl were used to extract ATIs from two selected barley cultivars, Commander and Hindmarsh. The results obtained from the preliminary study showed that IPA/DTT and urea-based buffer extraction could yield ∼70% and ∼45% more ATIs, respectively than a buffer based on Tris-HCl extraction, with all methods showing high repeatability (CV < 15%). A multi-step protocol, employing IPA/DTT and urea improved the extraction efficiency in comparison to the single buffer extraction protocols (p<0.0001). When solutions from parallel extractions using IPA/DTT and urea were combined, the results were comparable (p = 0.99) with a sequential multi-step IPA/DTT-urea protocol. However, the repeatability of the combined process was compromised, as discerned by greater variation (CV>30%). The optimised sequential two-step extraction protocol was successfully used to extract and quantify ATIs from 12 barley cultivars. LC-MS analysis revealed that cv Yagan and cv Scope contain the higher levels (∼143% relative to the average barley ATI content), whereas cultivars Fleet (61%), Baudin (77%) and Commander (79%) contained the lowest levels. The libraries of ATIs identified and the quantitative methods described here provide a foundation for the future application of MS-based quantitative methodologies to detect and quantify ATIs in barley varieties and in food products.

A ratiometric fluorescence sensor for ultra-sensitive detection of trypsin inhibitor in soybean flour using gold nanocluster@carbon nitride quantum dots.

Gold nanocluster@carbon nitride quantum dot nanocomposites protected by bovine serum albumin (BSA-AuNC@CNQDs) were designed as a ratiometric fluorescence nanosensor for ultra-sensitive detection of trypsin inhibitor (TI). CNQDs were prepared via thermal treatment of carbon nitride powder. BSA-CNQDs acted as templates to synthesize BSA-AuNC@CNQDs with dual-emission peaks at 450 and 650 nm. Trypsin can catalyze the hydrolysis of BSA and decompose BSA-AuNC@CNQDs resulting in fluorescence quenching. The fluorescence quenching at 650 nm was prevented by the addition of TI to inhibit the activity of trypsin. The nanosensor-trypsin system showed a satisfactory ability toward TI detection. The ratiometric responses (the ratio of intensity at 650 to 450 nm, I650/I450) had an excellent linearity (R2 = 0.981) with logarithmic values of TI concentrations in the broad range of 1-10,000 ng/mL. The limit of detection (LOD, 0.089 ng/mL) indicates ultra-sensitive detection of TI can be achieved. Additionally, TI in soybean flour was detected by the proposed ratiometric method with satisfactory recoveries (98.15-105.52%) and less than 6% of coefficient of variation. This study reveals that BSA-AuNC@CNQDs have potential applications in detection of TI in real samples.

Chickens' growth performance and pancreas development exposed to soy cake varying in trypsin inhibitor activity, heat-degraded lysine concentration, and protein solubility in potassium hydroxide.

This study focused on the effect of varying trypsin inhibitor activity (TIA), heat-degraded lysine concentration and protein solubility in potassium hydroxide on broiler performance and pancreas weight. Two soybean breeds were subject to varying thermal, hydrothermal, pressure, and kilning processing. This resulted in a total of 34 soy cake variants, widely varying in TIA (0.25 to 23.6 mg/g), heat-degraded lysine (1.40 to 8.60 g/kg), and potassium hydroxide (65.5 to 97.6%), respectively. These soy cake variants as well as a commercial soybean meal extract were included into a common grower and finisher diet for broiler chicks at fixed amounts (grower: 35%; finisher: 25%) and tested in a 35 d fattening experiment with 1680 broiler chicks (grower phase: day 11 to 24; finisher phase day 25 to 35). TIA was the dominant factor affecting zootechnical performance and pancreas weight at slaughter (day 35), depressing liveweight at day 24 (P < 0.006), and day 35 (0.026), weight gain (grower: P < 0.006) and feed: gain ratio during grower phase (P < 0.005) and increasing pancreas weight (P < 0.010) at the time of slaughter. Negative effects of TIA were also visible in soy cake variants below recommended thresholds. This highlights the necessity of complete elimination of TIA in broiler diets as far as technically possible.

Quality control of hospital preparations: Establishment of a simple and rapid method for quantifying ulinastatin in vaginal suppositories.

Ulinastatin vaginal suppositories, used to prevent threatened premature delivery, are frequently used in hospitals. However, there is no established method for quantifying ulinastatin contained in suppositories. Therefore, we investigated a simple and efficient method for quantifying ulinastatin contained in suppositories. Our analytical method involved removal of the base; optimising the enzyme inhibition reaction time and enzyme reaction time; and measuring the absorbance. The modified method was reproducible, operation time was significantly shortened, and cost was reduced to approximately 1/17 of that of the previously reported method. This simple and rapid quantitative method could contribute to the improvement of quality control of ulinastatin vaginal suppositories as an extemporaneous hospital preparation.

Detoxification of Jatropha curcas seed cake by a new soil-borne Enterobacter cloacae strain.

Jatropha curcas seed cake is a by-product generated after oil extraction from J. curcas seeds. Although the protein content is high, the cake contains phorbol esters and antinutritional factors such as phytates, trypsin inhibitors, lectins and tannins. Therefore, it cannot be directly used in food or feed. In this study, the toxic compounds and antinutrients present in J. curcas seed cake were detoxified by fermentation with Enterobacter Z11, a soil-borne isolate. Solid-state fermentation was undertaken under optimized conditions: deoiled cake, 5·0 g; initial moisture content, 50%; temperature, 30°C; and inoculum, 2 × 106 cells per gram of cake. Postfermentation, bacterial growth, pH and the amount of antinutrients were studied. Fermentation reduced the content of phorbol esters, phytates, lectins, tannins and trypsin inhibitors by 51·6, 82·6, 88·9, 37·8 and 90·5%, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain of Enterobacter cloacae Z11 was originally isolated from the soil. To the best of our knowledge, E. cloacae has never been used to remove toxins and antinutritional factors in Jatropha curcas seed cake (JSC). Under the optimized condition, fermentation with the Enterobacter strain decreased the phorbol esters content in JSC by 51·6%, and phytates, tannins, lectins and trypsin inhibitors contents by 83, 38, 89 and 90%, respectively. This study provided a new method with potential to render the seed cake suitable for use in feed. Further study is needed to focus on remaining toxicity and nutritional value post-treatment.

Amylase-Trypsin Inhibitors in Wheat and Other Cereals as Potential Activators of the Effects of Nonceliac Gluten Sensitivity.

Nonceliac gluten sensitivity (NCGS) is a gluten-related gastrointestinal disorder distinct from celiac disease (CD) and gluten allergy that is not easy to diagnose due to the lack of biomarkers. It is characterized by intestinal symptoms and extraintestinal manifestations with the consumption of gluten-containing foods. In contrast to CD, NCGS patients do not present a genetic predisposition or intestinal villi atrophy. Recent studies question the proinflammatory triggering activity of α-gliadin fraction contained in wheat, since it has been demonstrated that the amylase-trypsin inhibitors (ATIs) exert a strong activating effect on the innate immune response. We aimed to analyze the role of ATIs in the activation of innate immunity and in the development of the symptoms characteristic of NCGS. A systematic literature search was made using databases such as MEDLINE, SciELO, Science Direct, and Scopus, with focus on key words such as "amylase-trypsin inhibitors," "wheat," "gluten," and "celiac." Many studies are available on the structure, inhibition mechanism, and immune system effects of ATIs, mainly focused on IgE-mediated reactions. Recently, with the increase of NCGS interest, has increased the literature on the capacity of ATIs contained in wheat to activate the innate immune system. Literature published to date questions the relationship between activation of the innate immune system and gluten in NCGS. ATIs may have acted as interfering contaminant of gluten and appear as potential activator of innate immunity in NCGS patients. In view of their potential impact, more interventional studies are needed to demonstrate the proinflammatory effect of ATIs.

Total proteolytic activity and concentration of alpha-1 antitrypsin in meconium for assessment of the protease/antiprotease balance.

BACKGROUND: During intrauterine life, various proteolytic enzymes and their main inhibitor, alpha-1 antitrypsin, accumulate naturally in meconium. A protease/antiprotease balance is required to maintain the biological stability of the environment in which the fetus develops. METHODS: The pool of active proteases was determined using the EnzChek Protease Assay Kit. The concentration of alpha-1 antitrypsin in meconium was measured by enzyme-linked immunosorbent assay. Serial portions of meconium (n=80) were collected from healthy full-term neonates (n=19). RESULTS: Mean concentrations of active proteases and alpha-1 antitrypsin were 1.55 [standard deviation (SD) 1.3]mgg-1 (range 0.15-6.17) and 3.72 (SD 1.78)mgg-1 (range 0.76-8.55), respectively, with significant correlation (Rs=0.32, p=0.004). A significant increase in the concentration of active proteases was found between the first and last meconium portions (p<0.05). The proteases in the last meconium portions had a higher reaction velocity and affinity for the substrate than the proteases in the first meconium portions. The active protease:alpha-1 antitrypsin ratio was <0.5 in all first meconium portions, but was higher in the last meconium portions. CONCLUSIONS: Strong correlation between the concentrations of active proteases and alpha-1 antitrypsin in meconium may indicate their mutual interaction in the intrauterine environment. Alpha-1 antitrypsin maintains the protease/antiprotease balance during fetal development.

Inactivation Methods of Trypsin Inhibitor in Legumes: A Review.

Seed legumes have played a major role as a crop worldwide, being cultivated on about 12% to 15% of Earth's arable land; nevertheless, their use is limited by, among other things, the presence of several antinutritional factors (ANFs - naturally occurring metabolites that the plant produces to protect itself from pest attacks.) Trypsin inhibitors (TIs) are one of the most relevant ANFs because they reduce digestion and absorption of dietary proteins. Several methods have been developed in order to inactivate TIs, and of these, thermal treatments are the most commonly used. They cause loss of nutrients, affect functional properties, and require high amounts of energy. Given the above, new processes have emerged to improve the nutritional quality of legumes while trying to solve the problems caused by the use of thermal treatments. This review examines and discusses the methods developed by researchers to inactivate TI present in legumes and their effects over nutritional and functional properties.