Biochemical Characterization of the Seed Quality of a Collection of White Lupin Landraces from Southern Italy.

Lupin species provide essential nutrients and bioactive compounds. Within pulses, they have one of the highest contents of proteins and fibers and are among the poorest in carbohydrates. The Mediterranean region is an important cradle area of the origin and domestication of cultivated white lupin (Lupinus albus L.). In this work, we present the characterization of 19 white lupin landraces collected from several sites in southern Italy, characterized by different pedoclimatic conditions. The protein contents and electrophoretic patterns, total polyphenols, phytic acid, lipids and phosphorous content, and reducing and anti-tryptic activities have been determined for each landrace. The relationships of the compositional characteristics, the area of origin of landraces and between compositional characteristics and thermo-pluviometric trends that occurred in the genotype comparison field during the two-year period between 2019 and 2020 are compared and discussed. From a nutritional point of view, some of the analyzed landraces differ from the commercial reference. The panel of molecular analyses performed can help in building an identity card for the grain to rapidly identify those varieties with the desired characteristics.

A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium.

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

Binary Alginate-Whey Protein Hydrogels for Antioxidant Encapsulation.

Encapsulation of antioxidants in hydrogels, i.e., three-dimensional networks that retain a significant fraction of water, is a strategy to increase their stability and bioaccessibility. In fact, low oxygen diffusivity in the viscous gelled phase decreases the rate of oxidation. Moreover, some hydrocolloids such as alginate and whey proteins provide a pH-dependent dissolution mechanism, allowing the retention of encapsulated compounds in the gastric environment and their release in the intestine, where they can be absorbed. This paper reviews the information on alginate-whey protein interactions and on the strategies to use binary mixtures of these polymers for antioxidant encapsulation. Results showed that alginate and whey proteins strongly interact, forming hydrogels that can be modulated by alginate molecular mass, mannuronic acid: guluronic acid ratio, pH, Ca2+ or transglutaminase addition. Hydrogels of alginate and whey proteins, in the forms of beads, microparticles, microcapsules, and nanocapsules, generally provide better encapsulation efficiency and release properties for antioxidants with respect to the hydrogel of alginate alone. The main challenges for future studies are to extend knowledge on the interactions among three components, namely alginate, whey proteins, and the encapsulated bioactive compounds, and to investigate the stability of these structures under food processing conditions. This knowledge will represent the rationale basis for the development of structures that can be tailored to specific food applications.

Digestive and gastroprotective effects of Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (syn. A. moschata Wulfen) (Asteraceae): From traditional uses to preclinical studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (syn. A. moschata Wulfen) (Asteraceae) is an alpine endemic plant whose aerial parts are harvested by the locals mainly for the digestive properties. Despite its widespread use, few studies have been conducted to date to verify its bioactivity. AIM OF THE STUDY: The purpose of the work was to meet the tradition confirming with experimental data the popular belief that the consumption of this species offers beneficial effects to the gastrointestinal system. MATERIALS AND METHODS: Using Soxhlet apparatus, the dried aerial parts of A. erba-rotta subsp. moschata were successively extracted with petroleum ether (PET), dichloromethane (DCM) and methanol (MeOH). The essential oil (EO) was obtained by hydrodistillation using a Clevenger apparatus while infusion (AE) was prepared following the traditional local recipe. Their chemical characterization was performed by various techniques including SPME-GC/MS, GC/MS and HPLC/MS-MS. An in vitro biological screening was carried out. The influence of AE on lipid digestion was monitored by titration of free fatty acids (FFA) during pancreatic lipase activity with the pH-stat method. For all extracts and EO, the anti-Helicobacter pylori activity was assessed by the broth microdilution method, the influence on cell viability was evaluated against NCI-N87, OE21 and Caco-2 cell lines and a preliminary toxicity evaluation was done using Brine Shrimp lethality (BSL) assay. The anti-inflammatory potential was evidenced by interleukin IL-1- induced IL8 expression on Caco-2 cells. RESULTS: AE increased by 15% the FFA releasing compared to the pancreatic lipase alone. PET, DCM and MeOH extracts as well as AE and EO were considered active against the growth of both antimicrobial susceptible and resistant strains of H. pylori with MIC values starting from 16 µg/mL. PET and DCM (IC50 = 89 µg/mL and 96 µg/mL, respectively, against Caco-2 cell line) extracts showed the high effect on cell viability while the EO reduced in 50% of cell viability at 1.48 µL/mL (NCI-N87 cells), 1.42 µL/mL (OE21 cells), and 3.44 µL/mL (Caco-2 cells) corroborating the BSL results. In different degrees, all extracts and EO inhibited the IL-1ß-stimulated IL-8 production in Caco-2 cells. CONCLUSIONS: The obtained data are encouraging and provide a scientific basis for the traditional use of A. erba-rotta subsp. moschata as a digestive agent although they need to be further corroborated by studies involving the investigation of both the in vivo activities and the role of the compounds detected in the extracts.

Thermal Shift Assay as a Tool to Evaluate the Release of Breakdown Peptides from Cowpea ß-Vignin during Seed Germination.

The present work aimed to characterize the molecular relationships between structure and function of the seed storage protein ß-vignin, the vicilin storage protein of cowpea (Vigna unguiculata, l. Walp) seeds. The molecular characterization of ß-vignin was carried out firstly by assessing its thermal stability, under different conditions of pH and ionic strength, by thermal shift assay (TSA) using SYPRO Orange fluorescent dye. Secondly, its aggregation propensity was evaluated using a combination of chromatographic and electrophoretic techniques. Two forms of ß-vignin were considered: the native form purified from mature quiescent seeds, and a stable breakdown intermediate of 27 kDa produced while seeds germinate. TSA is a useful tool for determining and following over time the structural changes that occur to the protein during germination. The main result was the molecular characterization of the 27 kDa intermediate breakdown polypeptide, which, to the best of our knowledge, has never been described before. ß-vignin seems to retain its trimeric conformation despite the evident degradation of its polypeptides.

Polyphenol bioactivity evolution during the spontaneous fermentation of vegetal by-products.

Food industry by-products such as grape pomace (GP), tomato pomace (TP), and spent coffee grounds (SCG) are rich in polyphenols (PP) but are easily biodegradable. The aim of this study is to test Spontaneous Fermentation (SF) as treatment to modify PP profile and bioactivity. The results highlighted that the by-products' organic matter and the microbial populations drove the SF evolution; heterolactic, alcoholic, and their mixtures were the predominant metabolisms of TP, GP, and SCG + GP co-fermentation. Increases in the extractable amounts and antiradical activity occurred for all the biomasses. Regarding the aglycate-PPs (APP), i.e. the most bioreactive PPs, significant changes occurred for TP and GP but did not influence the anti-inflammatory bioactivity. The co-fermentation increased significantly chlorogenic acid and consumed most of the APPs, acting as a purification system to obtain a highly concentrated APP fraction, so that the extract might be employed for a specific purpose.

Bioactivities of Pseudocereal Fractionated Seed Proteins and Derived Peptides Relevant for Maintaining Human Well-Being.

Food proteins and peptides are able to exert a variety of well-known bioactivities, some of which are related to well-being and disease prevention in humans and animals. Currently, an active trend in research focuses on chronic inflammation and oxidative stress, delineating their major pathogenetic role in age-related diseases and in some forms of cancer. The present study aims to investigate the potential effects of pseudocereal proteins and their derived peptides on chronic inflammation and oxidative stress. After purification and attribution to protein classes according to classic Osborne's classification, the immune-modulating, antioxidant, and trypsin inhibitor activities of proteins from quinoa (Chenopodium quinoa Willd.), amaranth (Amaranthus retroflexus L.), and buckwheat (Fagopyrum esculentum Moench) seeds have been assessed in vitro. The peptides generated by simulated gastro-intestinal digestion of each fraction have been also investigated for the selected bioactivities. None of the proteins or peptides elicited inflammation in Caco-2 cells; furthermore, all protein fractions showed different degrees of protection of cells from IL-1ß-induced inflammation. Immune-modulating and antioxidant activities were, in general, higher for the albumin fraction. Overall, seed proteins can express these bioactivities mainly after hydrolysis. On the contrary, higher trypsin inhibitor activity was expressed by globulins in their intact form. These findings lay the foundations for the exploitation of these pseudocereal seeds as source of anti-inflammatory molecules.

Chromatography-Independent Fractionation and Newly Identified Molecular Features of the Adzuki Bean (Vigna angularis Willd.) ß-vignin Protein.

Adzuki seed ß-vignin, a vicilin-like globulin, has proven to exert various health-promoting biological activities, notably in cardiovascular health. A simple scalable enrichment procedure of this protein for further nutritional and functional studies is crucial. In this study, a simplified chromatography-independent protein fractionation procedure has been optimized and described. The electrophoretic analysis showed a high degree of homogeneity of ß-vignin isolate. Furthermore, the molecular features of the purified protein were investigated. The adzuki bean ß-vignin was found to have a native size of 146 kDa, and the molecular weight determined was consistent with a trimeric structure. These were identified in two main polypeptide chains (masses of 56-54 kDa) that are glycosylated polypeptides with metal binding capacity, and one minor polypeptide chain with a mass 37 kDa, wherein these features are absent. The in vitro analysis showed a high degree of digestibility of the protein (92%) and potential anti-inflammatory capacity. The results lay the basis not only for further investigation of the health-promoting properties of the adzuki bean ß-vignin protein, but also for a possible application as nutraceutical molecule.

Antioxidant and Anti-Inflammatory Activities of the Crude Extracts of Raw and Fermented Tomato Pomace and Their Correlations with Aglycate-Polyphenols.

Two tomato pomace (TP) were studied as feedstocks to obtain extracts that are rich in polyphenols. TPs prompt degradation impairs biomass safety, thus naturally present microflora were tested to perform conservation, and own lactic bacteria became predominant after 60 days of treatment. The extracts of TPs and TPs fermented (TPF) were chemically characterized and tested for antioxidant and anti-inflammatory activities. Flavonoids and phenolic acids were classed as aglycone-polyphenols (A-PP), the most bioactive polyphenol fraction. Fermentation led to a reduction of the A-PP amount, but no significant change in composition. Antioxidant power increased, despite the A-PP reduction, for the presence of fermentation metabolites having aromatic-substituent. TP and TPF both have anti-inflammatory properties that were strictly dependent upon the A-PP content. Fermentation preserved the anti-inflammatory activity and the Partial Least Square (PLS) identified as the most active molecules naringenin chalcone, kaempferol, gallic acid, and cinnamic acid, together with the definition of the active dose.

Bio-Functional and Structural Properties of Pasta Enriched with a Debranning Fraction from Purple Wheat.

A colored and fiber-rich fraction from the debranning of purple wheat was incorporated at 25% into semolina- and flour-based pasta produced on a pilot-plant scale, with the aim of increasing anthocyanin and total phenolic content with respect to pasta obtained from whole pigmented grains. The debranning fraction impaired the formation of disulfide-stabilized protein networks in semolina-based systems. Recovery of phenolics was impaired by the pasta making process, and cooking decreased the phenolic content in both enriched samples. Cooking-related losses in anthocyanins and total phenolics were similar, but anthocyanins in the cooked semolina-based pasta were around 20% of what was expected from the formulation. HPLC (High Performance Liquid Chromatography) profiling of phenolics was carried out on extracts from either type of enriched pasta both before and after cooking and indicate possible preferential retention of specific compounds in each type of enriched pasta. Extracts from cooked samples of either enriched pasta were tested as inhibitors of enzymes involved in glucose metabolism and uptake, as well as for their capacity of suppressing the response to inflammatory stimuli. Results of both biological tests indicate that the phenolics in extracts from both cooked pasta samples had inhibitory capacities higher than extracts of the original debranning fraction at identical concentrations of total bioactives.

The Bio-Functional Properties of Pigmented Cereals may Involve Synergies among Different Bioactive Species.

This study was aimed at characterizing the anthocyanins and phenolics profile in different varieties of pigmented corn and wheat and in some of their milling fractions. Acid/ethanol extracts were used to assess total anthocyanins, overall antioxidant activity, the overall polyphenol profile, and for evaluating the inhibition of pancreatic α-amylase and of intestinal α-glucosidase. Both enzymes were inhibited in a dose-dependent manner by all extracts, but individual extracts had specific effects on each enzyme. Anti-inflammatory response was evaluated by using acid-free extracts and Caco-2 cells transiently transfected with a luciferase reporter gene responding to cytokine stimulation. The immune response of interleukin-stimulated cells decreased significantly in a dose-dependent manner in the presence of 20-50 µM/l anthocyanins from all grains extracts, again with a different efficiency. The inhibitory ability and the anti-inflammatory capability of these extracts are in most cases higher than in similar extracts from other sources, suggesting that activities in each extract may imply specific synergies between anthocyanins and other phenolics.

New molecular features of cowpea bean (Vigna unguiculata, l. Walp) ß-vignin.

Cowpea seed ß-vignin, a vicilin-like globulin, proved to exert various health favourable effects, including blood cholesterol reduction in animal models. The need of a simple scalable enrichment procedure for further studies for tailored applications of this seed protein is crucial. A chromatography-independent fractionation method allowing to obtain a protein preparation with a high degree of homogeneity was used. Further purification was pursued to deep the molecular characterisation of ß-vignin. The results showed: (i) differing glycosylation patterns of the two constituent polypeptides, in agreement with amino acid sequence features; (ii) the seed accumulation of a gene product never identified before; (iii) metal binding capacity of native protein, a property observed only in few other legume seed vicilins.

Proteolytic cleavage at twin arginine residues affects structural and functional transitions of lupin seed 11S storage globulin.

The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.

TgaA, a VirB1-like component belonging to a putative type IV secretion system of Bifidobacterium bifidum MIMBb75.

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.

Murein lytic enzyme TgaA of Bifidobacterium bifidum MIMBb75 modulates dendritic cell maturation through its cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) amidase domain.

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system.

Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells.

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

Identification in lupin seed of a serine-endopeptidase activity cleaving between twin arginine pairs and causing limited proteolysis of seed storage proteins.

The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses. These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF), leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), and ethylenediaminetetraacetic acid (EDTA), thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Susceptibility of lupin gamma-conglutin, the plasma glucose-lowering protein of lupin seeds, to proteolytic enzymes.

Lupin seed gamma-conglutin, orally administered to animal models, has been shown to display glucose-controlling properties. Therefore, we have addressed the study of gamma-conglutin susceptibility to proteolytic enzymes in vitro as the basis to unveil its metabolic fate in the body. Pepsin treatment at pH 2.0 and 3.0 caused extensive proteolytic breakdown, while at pH 4.0, where pepsin is minimally active, gamma-conglutin was unaffected. Aliquots of the pepsin-treated protein were further incubated with pancreatin at neutral pH. If the protein backbone was already cleaved by pepsin action, then the breakdown by pancreatin was almost complete; alternatively, pancreatin did not affect at all gamma-conglutin polypeptide chain. This was not due to an inhibitory activity of gamma-conglutin, because co-incubation with casein showed complete breakdown of the milk protein. Furthermore, gamma-conglutin was incubated with bromelain, a proteinase effective between pH 4.0 and 7.0. A sharp transition from the uncleavable to the fully cleavable form of gamma-conglutin was observed below pH 4.25. Therefore, it was concluded that (i) gamma-conglutin is resistant to proteolysis at pH greater than 4.0, likely because of a compact native conformation, (ii) an acidic pH renders the protein susceptible to proteases, suggesting the occurrence of a trans conformation, which has also been observed by circular dichroism spectral analysis, and (iii) the protein undergoes an "all or none" degradation pathway, regardless of the enzyme used.

Implication of an outer surface lipoprotein in adhesion of Bifidobacterium bifidum to Caco-2 cells.

We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.

Identification and characterization of a Bowman-Birk inhibitor active towards trypsin but not chymotrypsin in Lupinus albus seeds.

The paper describes the purification, structural characterization and inhibitory properties of a trypsin inhibitor from Lupinus albus L., a leguminous plant believed to be devoid of any protease inhibitor. The protein has been isolated by a newly set-up procedure and characterized by direct amino acid sequencing, MALDI-TOF mass spectroscopy and circular dichroism. Inhibitory properties toward bovine trypsin and chymotrypsin, as well as its thermal and pH stabilities, have been also assessed. The inhibitor is 63 amino acid long (Mr 6858; pI 8.22) and it is capable to inhibit two trypsin molecules simultaneously, with a Kd of 4.2+/-0.4 nM, but not chymotrypsin. BLAST search against UniProtKB/TrEMBL database indicates that the inhibitor belongs to the Bowman-Birk inhibitor (BBI) family. The interest in these serine-protease inhibitors arises from the ability to prevent or suppress carcinogen-induced transformation, as shown in various in vitro and in vivo model systems.