Case-Control Study of the Association between Single Nucleotide Polymorphisms of Genes Involved in Xenobiotic Detoxification and Antioxidant Protection with the Long-Term Influence of Organochlorine Pesticides on the Population of the Almaty Region.

The association of genetic polymorphisms with the individual sensitivity of humans to the action of pesticide pollution is being actively studied in the world. The aim of this study was a molecular epidemiological analysis of candidate polymorphisms of genes involved in pesticide metabolism, detoxification, and antioxidant protection. Some of the selected polymorphisms also relate to susceptibility to cancer and cardiovascular, respiratory, and immune system diseases in individuals exposed to pesticides for a long time. For a case-control study of a unique cohort of people exposed to organochlorine pesticides for 10 years or more were chosen, a control cohort was selected that matched with the experimental group by the main population characteristics. PCR-PRLF and genome-wide microarray genotyping (GWAS) methods were used. We identified 17 polymorphisms of xenobiotic detoxification genes and 27 polymorphisms of antioxidant defense genes, which had a significantly high statistical association with the negative impact of chronic pesticide intoxication on human health. We also found 17 polymorphisms of xenobiotic detoxification genes and 12 polymorphisms of antioxidant defense genes that have a protective effect. Data obtained added to the list of potential polymorphisms that define a group at high risk or resistant to the negative effects of pesticides.

Individual Risk Assessment for Population Living on the Territories Long-Term Polluted by Organochlorine Pesticides.

The long-term storage of unutilized pesticides raised new problems of long-term environmental contamination. The study presents the results of surveying 151 individuals in 7 villages living close to pesticide-contaminated localities. All individuals have been surveyed concerning their consumption habits and lifestyle characteristics. An assessment of the general exposure risks of the local population was carried out using the analysis of pollutants in food products and the average levels of their consumption in the region. The cohort risk evaluation revealed that the greatest risk was associated with the regular consumption of cucumbers, pears, bell peppers, meat, and milk. The new model to estimate individual risks of long-term pesticide pollution was proposed as a calculation of the combined action of 9 risk factors, including individual genotypes, age, lifestyle, and personal pesticide consumption rates. The analysis of the predictive ability of this model showed that the final score for individual health risks corresponded to the development of chronic diseases. A high level of chromosomal aberrations was evidenced for individual genetic risk manifestations. The combined influence of all risk factors revealed contributions of 24.7% for health status and 14.2% for genetic status, while other impacts go to all unaccounted factors.

The Anxiolytic-like Properties of a Tryptic Hydrolysate of Bovine αs1 Casein Containing α-Casozepine Rely on GABAA Receptor Benzodiazepine Binding Sites but Not the Vagus Nerve.

(1) Background: A tryptic hydrolysate of bovine αs1-casein (CH) exerts anxiolytic-like properties in many species, including humans. This is mainly related to the presence of α-casozepine (α-CZP), which yields these properties in rodents. This study evaluates, in a rat model, the roles of the vagus nerve and the benzodiazepine binding site of GABAA receptors in the mode of action of CH. (2) Methods: The conditioned defensive burying test was used to evaluate anxiety. (3) Results: Participation of the vagus nerve in the mode of action of CH was excluded, as the global anxiety score in vagotomised rats was not significantly different from that of non-vagotomised animals. The blocking of the binding sites of benzodiazepines with flumazenil antagonised CH anxiolytic-like properties. (4) Conclusions: The vagus nerve does not play a role in the anxiolytic-like properties of CH. On the other hand, this anxiolytic-like activity relies on the benzodiazepine binding site of the GABAA receptors. This result is consistent with previous in vitro studies and, more specifically with the discovery of α-CZP, the peptide responsible for the anxiolytic-like properties of CH.

Encapsulation of Salmon Peptides in Marine Liposomes: Physico-Chemical Properties, Antiradical Activities and Biocompatibility Assays.

Salmon byproducts (Salmo salar) generated by the food chain represent a source of long-chain polyunsaturated fatty acids (eicosapentaenoic acid (EPA): 20:5n-3; docosahexaenoic acid (DHA): 22:6n-3) and peptides that can be used as supplements in food for nutraceutical or health applications, such as in the prevention of certain pathologies (e.g., Alzheimer's and cardiovascular diseases). The extraction of polar lipids naturally rich in PUFAs by enzymatic processes without organic solvent (controlled by pH-Stat method), coupled with the production of 1 kDa salmon peptides by membrane filtration, allowed the formulation of nanocarriers. The physicochemical properties of the nanoliposomes (size ranging from 120 to 140 nm, PDI of 0.27, zeta potential between -32 and -46 mV and encapsulation efficiency) were measured, and the bioactivity of salmon hydrolysate peptides was assessed (antioxidant and antiradical activity: ABTS, ORAC, DPPH; iron metal chelation). Salmon peptides exhibited good angiotensin-conversion-enzyme (ACE) inhibition activity, with an IC50 value of 413.43 ± 13.12 µg/mL. Cytotoxicity, metabolic activity and proliferation experiments demonstrated the harmlessness of the nanostructures in these experimental conditions.

Electrically Switchable Nanolever Technology for the Screening of Metal-Chelating Peptides in Hydrolysates.

Metal-chelating peptides (MCP) are considered as indirect antioxidants due to their capacity to inhibit radical chain reaction and oxidation. Here, we propose a new proof of concept for the screening of MCPs present in protein hydrolysates for valorizing their antioxidant properties by using the emerging time-resolved molecular dynamics technology, switchSENSE. This method unveils possible interactions between MCPs and immobilized nickel ions using fluorescence and electro-switchable DNA chips. The switchSENSE method was first set up on synthetic peptides known for their metal-chelating properties. Then, it was applied to soy and tilapia viscera protein hydrolysates. Their Cu2+-chelation capacity was, in addition, determined by UV-visible spectrophotometry as a reference method. The switchSENSE method has displayed a high sensitivity to evidence the presence of MCPs in both hydrolysates. Hence, we demonstrate for the first time that this newly introduced technology is a convenient methodology to screen protein hydrolysates in order to determine the presence of MCPs before launching time-consuming separations.

Food protein-derived anxiolytic peptides: their potential role in anxiety management.

About one in three people are affected by anxiety disorders during their lifetime. Anxiety episodes can be brief due to a stressful event, but anxiety disorders can last at least 6 months. A wide variety of therapeutic drugs are available for the treatment of anxiety disorders, but due to the associated side effects of these anxiolytics, it is interesting to find alternatives. Some food protein hydrolysates or active peptide fragments present in such hydrolysates provide a natural and promising mean for preventing certain forms of anxiety. To date, only a small number of hydrolysates or peptides from food proteins with anxiolytic-like activity have been characterized. Most of these hydrolysates or peptides have displayed potent anxiolytic profiles in animal or clinical studies. The results suggest that these molecules may exert their effects at different levels. This paper reviews the data of the structure/activity relationship, physiological effects displayed in in vitro and in vivo assays, bioavailability, and safety profiles of anxiolytic peptides.

Metabolomics approach based on LC-HRMS for the fast screening of iron(II)-chelating peptides in protein hydrolysates.

Production of iron-chelating peptides from protein hydrolysates requires robust and adequate screening methods to optimize their purification and subsequently valorize their potential antioxidant properties. An original methodology was developed for direct and sensitive screening of iron(II)-chelating peptides based on ion-pair reverse phase liquid chromatography (IP-RPLC) coupled to high-resolution mass spectrometry (HRMS). Peptide mixture was first added to iron(II) solution to form iron(II)-peptide complexes. Then IP-RPLC-HRMS analysis was conducted on this iron-peptide mixture and on the iron-free peptide solution for comparative mass spectra analysis. This protocol, initially applied to a range of low molecular weight standard peptides, allowed detection of [(Peptide-H)+56FeII]+ complex ion for iron(II)-chelating peptides (GGH, EAH, DAH, ßAH, DMH, DTH, DSH). GGH was added in complex peptide mixtures and targeted analysis of [(GGH-H)+56FeII]+ complex showed a limit of detection (LOD) below 0.77 mg L-1 of GGH. This protocol was finally tested in combination with metabolomics software and additional digital processing for non-targeted search for iron(II)-chelating peptides. Applicability of this new screening methodology has been validated by detection of GGH as iron(II)-chelating peptide when added at 0.77 mg L-1 in casein hydrolysate. Graphical abstract.

Quality of Beverage Intake and Cardiometabolic and Kidney Outcomes: Insights From the STANISLAS Cohort.

Background and Aims: Beverages are an important aspect of diet, and their quality can possibly affect health. The Healthy Beverage Index (HBI) has been developed to take into account these effects. This study aimed to highlight the relationships between health and beverage quality by assessing the association of the HBI and its components with kidney and cardiometabolic (CM) outcomes in an initially healthy population-based familial cohort. Methods: This study included 1,271 participants from the STANISLAS cohort. The HBI, which includes 10 components of habitual beverage consumption, was calculated. Associations of the HBI and its components with estimated glomerular filtration rate (eGFR), albuminuria, hypertriglyceridemic waist (HTG waist), metabolic syndrome (MetS), carotid-femoral pulse wave velocity (cfPWV), carotid intima-media thickness (cIMT), and left ventricular mass (LV mass) were analyzed using multivariable linear or logistic regression models. Results: The median HBI score was 89.7 (78.6-95) out of 100 points. While the overall HBI score was not significantly associated with any of the studied outcomes, individual HBI components were found differently associated with the outcomes. cfPWV and cIMT were lower in participants who did not meet the full-fat milk criteria (p = 0.03 and 0.001, respectively). In men, higher cfPWV was observed for the "low Fat milk" (p = 0.06) and "alcohol" (p = 0.03) non-adherence criteria. Odds of HTG waist were higher with the non-adherence to sugar-sweetened beverages criteria (p < 0.001). eGFR was marginally higher with non-adherence to the coffee/tea criteria (p = 0.047). Conclusions: In this initially healthy population, HBI components were differently associated with kidney and cardiometabolic outcomes, despite a good overall HBI score. Our results highlight specific impacts of different beverage types and suggest that beverages could have an impact on kidney and cardiometabolic health.

Affinity of chlordecone and chlordecol for human serum lipoproteins.

Chlordecone (CLD) is a chlorinated persistent organic pollutant (POP) whose presence despite the 1993 ban in agriculture areas has caused numerous public health concerns. CLD accumulates in the liver, and the CLD metabolite, chlordecol (CLD-OH) is found in bile, an important site of excretion for cholesterol transported to the liver via lipoproteins. Here, we studied the real-time molecular interaction between CLD and CLD-OH with human serum lipoproteins, LDL and HDL. While no interaction was detected between CLD and HDL, or between CLD-OH and LDL, relatively high specific affinities were observed between CLD and CLD-OH for LDL and HDL, respectively.

Anxiolytic Activity and Brain Modulation Pattern of the α-Casozepine-Derived Pentapeptide YLGYL in Mice.

α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1­casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α­CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-­CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α­CZP.

Effect of nonenzymatic deamidation on the structure stability of Camelus dromedarius α-lactalbumin.

Camelid α-lactalbumin is the only known protein that can undergo nonenzymatic deamidation on two Asn residues. This leads to the generation of a mixture of unusual isoAsp and d-Asp residues that may impact health. The effect of deamidation on camel α-lactalbumin instability was investigated. Circular dichroism showed that the altered protein acquired secondary structure resulting in an increase in α-helix content. In good agreement, the 3D structure of camel α-lactalbumin determined by X-ray crystallography, displayed a short additional α-helix probably induced by deamidation, compared to the human and bovine counterparts. This α-helix was located in the C-terminal region and included residues 101-106. Differential scanning calorimetry together with the susceptibility to thermolysin showed that the deamidation process reinforced the structural stability of the α-lactalbumin at high temperature and its resistance toward proteolysis.

The X-prolyl dipeptidyl-peptidase PepX of Streptococcus thermophilus initially described as intracellular is also responsible for peptidase extracellular activity.

This study addresses the hypothesis that the extracellular cell-associated X-prolyl dipeptidyl-peptidase activity initially described in Streptococcus thermophilus could be attributable to the intracellular X-prolyl dipeptidyl-peptidase PepX. For this purpose, a PepX-negative mutant of S. thermophilus LMD-9 was constructed by interrupting the pepX gene and named LMD-9-ΔpepX. When cultivated, the S. thermophilus LMD-9 wild type strain grew more rapidly than its ΔpepX mutant counterpart. Thus, the growth rate of the LMD-9-ΔpepX strain was reduced by a factor of 1.5 and 1.6 in milk and LM17 medium (M17 medium supplemented with 2% lactose), respectively. The negative effect of the PepX inactivation on the hydrolysis of ß-casomorphin-7 was also observed. Indeed, when incubated with this peptide, the LMD-9-ΔpepX mutant cells were unable to hydrolyze it, whereas this peptide was completely degraded by the S. thermophilus LMD-9 wild type cells. This hydrolysis was not due to leakage of intracellular PepX, as no peptide hydrolysis was highlighted in cell-free filtrate of wild type strain. Therefore, based on these results, it can be presumed that though lacking an export signal, the intracellular PepX might have accessed the ß-casomorphin-7 externally, perhaps via its galactose-binding domain-like fold, this domain being known to help enzymes bind to several proteins and substrates. Therefore, the identification of novel distinctive features of the proteolytic system of S. thermophilus will further enhance its credibility as a starter in milk fermentation.

New Insights into the Proteolytic System of Streptococcus thermophilus: Use of Isracidin To Characterize Cell-Associated Extracellular Peptidase Activities.

The influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS(+) phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS(-) phenotype) and two mutant strains LMD-9-ΔprtS and LMD-9-ΔprtS-ΔhtrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9. Identification by mass spectrometry of breakdown products of isracidin revealed the existence of novel cell-associated extracellular carboxypeptidase and peptidyl dipeptidase activities in all PrtS(-) strains, besides known cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities. Both aminopeptidase and peptidyl dipeptidase activities were not able to cleave the isracidin at peptide bonds with proline residues. No hydrolysis of isracidin was detected in cell free filtrate for all the strains studied, indicating that no cell lysis had occurred. Taken together, these results suggested the presence of cell-associated extracellular peptidase activities in S. thermophilus strains that could be vital for the growth of PrtS(-) strains.

Probing the catalytic mechanism of bovine CD38/NAD+ glycohydrolase by site directed mutagenesis of key active site residues.

Bovine CD38/NAD(+) glycohydrolase catalyzes the hydrolysis of NAD(+) to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide-ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD(+). To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2'-OH group of NAD(+), was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.

Surface plasmon resonance analysis of the binding mechanism of pharmacological and peptidic inhibitors to human somatic angiotensin I-converting enzyme.

Somatic angiotensin I-converting enzyme (ACE) possesses two catalytic domains and plays a major role in the regulation of blood pressure, thus representing a therapeutic target for the treatment of hypertension. We present a comprehensive surface plasmon resonance (SPR) study of the interaction of human somatic ACE with the pharmacological inhibitors captopril and lisinopril, the bradykinin potentiating peptide BPP-11b, and the food peptidic inhibitors from bovine αs2-casein, F(174)ALPQYLK(181) and F(174)ALPQY(179). SPR binding curves recorded with the high potency inhibitors captopril, lisinopril, and BPP-11b were evaluated both by regression analysis and by kinetic distribution analysis. The results indicated that captopril and lisinopril bound ACE with two K(D)'s differing by a factor 10-20 and >30, respectively (lowest K(D) = 0.1-0.3 nM for both inhibitors). This shows, for the first time in a direct binding assay with the two-domain enzyme, the existence of two binding modes of the pharmacological inhibitors, presumably with the two ACE domains. The BPP-11b-ACE binding curves were complex but showed a predominant interaction with K(D) in the nanomolar range. The caseinopeptides, known to inhibit ACE with an IC50 of 4.3 µM, bound to ACE with K(D) = 3-4 µM. Mapping of the F(174)ALPQY(179) binding site on ACE by sequential binding studies using captopril or BPP-11b indicated that it bound to (or near) the two active sites of ACE, in agreement with the stoichiometry of 2 determined from data fitting. Our results provide a detailed characterization of ACE-inhibitor binding modes and validate SPR for predicting the inhibitory potential of new compounds.

Hydrolysis of milk-derived bioactive peptides by cell-associated extracellular peptidases of Streptococcus thermophilus.

The trend to confer new functional properties to fermented dairy products by supplementation with bioactive peptides is growing in order to encounter the challenge of health-promoting foods. But these functional ingredients have not to be hydrolysed by proteases of bacteria used in the manufacture of these products. One of the two yoghurt bacteria, Streptococcus thermophilus, has long been considered as weakly proteolytic since its only cell wall-associated subtilisin-like protease, called PrtS, is not always present. Nevertheless, a recent study pointed out a possible peptidase activity in certain strains. In this present study, the stability of milk-derived bioactive peptides, e.g. the anxiolytic peptide, αs1-CN-(f91-97), in the presence of two different S. thermophilus strains with PrtS+ or PrtS− phenotype was studied. Both strains appeared to be capable of hydrolysing the αs1-CN-(f91-97) and other bioactive peptides by recurrent removal of N-terminal residues. The hydrolysis was neither due to intracellular peptidases nor to HtrA protease. Results obtained showed that the observed activity originates from the presence at the surface of both strains of an extracellular aminopeptidase activity. Moreover, a cell wall-associated X-prolyl dipeptidyl peptidase activity was also highlighted when ß-casomorphin-7 was used as substrate. All of these findings suggest that, in order to use fermented milks as vector of bioactive peptides, the stability of these bioactive peptides in this kind of products implies to carefully characterize the potential action of the surface proteolytic enzymes of S. thermophilus.

Insights into the mechanism of bovine CD38/NAD+glycohydrolase from the X-ray structures of its Michaelis complex and covalently-trapped intermediates.

Bovine CD38/NAD(+)glycohydrolase (bCD38) catalyses the hydrolysis of NAD(+) into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2'-fluorinated analogs of NAD(+). Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1' of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2'-OH of the substrate NAD(+). Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism.

Binding of divalent metal ions to 1-25 ß-caseinophosphopeptide: an isothermal titration calorimetry study.

To better understand the mechanism of metal ion transport through the gastrointestinal tract to their absorption sites, isothermal titration calorimetry (ITC) was used to investigate the binding of dicationic metals to ß-CN(1-25)4P, a ß-casein tetraphosphorylated peptide. ITC technology was found suitable for studying weak bonds between metal ions and phosphopeptides and provided a direct means of thermodynamic and stoichiometric characterisation of complex formation. Thus, one mole of ß-CN(1-25)4P binds two moles of Ca(2+), Mg(2+) or Zn(2+) under experimental conditions close to those of the ileum (pH 8, 37°C), with rather low binding affinity constants (K=4900-11,200M(-1)). These low affinities should facilitate the release of metal ions during intestinal absorption. By contrast, Cu(2+) did not bind to ß-CN(1-25)4P at pH 8, despite its reported significant affinity towards ß-casein and the 1-25 peptide at near-neutral pH.

Transport across Caco-2 cell monolayer and sensitivity to hydrolysis of two anxiolytic peptides from αs1-casein, α-casozepine, and αs1-casein-f91-97: effect of bile salts.

α-Casozepine and f91-97, peptides from α(s1)-casein, display anxiolytic activity in rats and may have to cross the intestinal epithelium to exert this central effect. We evaluated their resistance to hydrolysis by the peptidases of Caco-2 cells and their ability to cross the cell monolayer. To mimic physiological conditions, two preparations of bile salts were used in noncytotoxic concentrations: porcine bile extract and an equimolar mixture of taurocholate, cholate, and deoxycholate. The presence and composition of bile salts appeared to modulate the peptidase activities of the Caco-2 cells involved (i) in the hydrolysis of α-casozepine, leading to much higher formation of fragments f91-99, f91-98, and f91-97, and (ii) in the hydrolysis of f91-97, leading to lower degradation of this peptide. Transport of α-casozepine across Caco-2 monolayer increased significantly, in the presence of bile extract, and of fragment f91-97, in the presence of bile salts.

In vitro digestibility of α-casozepine, a benzodiazepine-like peptide from bovine casein, and biological activity of its main proteolytic fragment.

α-Casozepine is a peptide, corresponding to the sequence 91-100 of the bovine α(s1)-casein, displaying anxiolytic activity in the rat. The α(s1)-casein tryptic hydrolysate containing this peptide decreases stress effects after oral administration in various species including man. Therefore, the stability of this peptide toward gastric and pancreatic proteases has been assessed by using pepsin, chymotrypsin/trypsin, Corolase PP, pepsin followed by chymotrypsin/trypsin or pepsin followed by Corolase PP. α-Casozepine was slowly degraded by chymotrypsin, much more sensitive to pepsin and Corolase PP but not completely destroyed after 4 h kinetics. The bonds in the region 91 to 95 of the α-casozepine were totally resistant to hydrolysis by all studied proteases. Surprisingly, a fragment, corresponding to the sequence 91-97 and found in all the hydrolysis media in significant amount, possessed an anxiolytic activity in three behavioral tests measuring this parameter. This peptide could participate in the in vivo activity of α-casozepine.