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Exploring and deciphering the biodiversity of oil bodies (OBs) recovered from oilseeds are of growing interest in the preparation of sustainable, natural and healthy plant-based food products. This study focused on chia (

An in vitro approach is proposed to study the release of an Active Pharmaceutical Ingredient-Ionic Liquid (API-IL) from a natural biopolymer matrix based on zein, a maize storage protein. Zein can be processed in the molten state with 20 w% [Lidocainium][Ibuprofenate] added as API-IL also acting as plasticizer and potentially co-plasticized by glycerol. The thermal stability of the matrix is checked, as well as the in vivo biological activity of the API-IL confirming anesthetic and anti-inflammatory activities. Model tablets are thermomolded at 130 °C (∅20 mm, 0.2 mm thick) and submitted to simulated digestion based on the INFOGEST static protocol of gastrointestinal food digestion at 37 °C (2 h under gastric conditions followed by 2 h under intestinal ones). The release of the API-IL is evaluated by HPLC-UV to dissociate lidocainium, that shows a progressive release (35 % after 2 h and 60 % after 4 h digestion), from ibuprofenate, that is mainly released under intestinal conditions due to low solubility in acidic conditions. The monitoring of the tablets reveals release mechanisms based on diffusion without noticeable erosion of the matrix. These results demonstrate the interest of this thermoplastic material to provide a relevant drug delivery system.

International audience; Hemp seed oil bodies (HSOBs) are of growing interest in response to the demand of consumers for healthy and natural plant-based food formulations. In this study, we used minimal processing including aqueous extraction by grinding and centrifugation to obtain HSOBs. We determined the lipid composition of HSBOs, their micro-structure, and the impact of the homogenization pressure, pH and minerals on their surface properties and the physical stability of the emulsions. HSOBs contain high levels of well-balanced PUFA with LA/ALA =2.9, γ-tocopherol, lutein and phytosterols. The mean diameter of HSOBs was 2.3 ±0.1 μm with an isoelectric point in the range of pH 4.4 to 4.6. Homogenization of hemp seed extracts induced a decrease in the size of HSOBs but did not eliminate the sedimentation of the protein bodies composed of the globulin edestin. By changing the surface properties of HSOBs, pH values below 6 and NaCl induced the aggregation of HSOBs, while CaCl2 induced both aggregation and membrane-fusion mediated coalescence of HSOBs by involving probably the anionic phos-pholipids together with membrane proteins. This study will contribute to extend the range of novel food products and designed emulsions containing hemp seed proteins and oil bodies.

International audience; Titanium dioxide is a food additive that has raised some concerns for humans due to the presence of nanoparticles. We were interested in knowing the fate of TiO2 particles in the gastro-intestinal tract and their potential effect on digestive enzymes. For this purpose, we analysed the behaviour of two different food grade TiO2 samples (E171) and one nano-sized TiO2 sample (P25) through a standardized static in vitro digestion protocol simulating the oral, gastric and intestinal phases with appropriate juices including enzymes. Both E171 and P25 TiO2 particles remained intact in the digestive fluids but formed large agglomerates, and especially in the intestinal fluid where up to 500 mu m sized particles have been identified. The formation of these agglomerates is mediated by the adsorption of mainly alpha-amylase and divalent cations. Pepsin was also identified to adsorb onto TiO2 particles but only in the case of silica-covered E171. In the salivary conditions, TiO2 exerted an inhibitory action on the enzymatic activity of alpha-amylase. The activity was reduced by a factor dependent on enzyme concentrations (up to 34% at 1 mg mL(-1)) but this inhibitory effect was reduced to hardly 10% in the intestinal fluid. In the gastric phase, pepsin was not affected by any form of TiO2. Our results hint that food grade TiO2 has a limited impact on the global digestion of carbohydrates and proteins. However, the reduced activity specifically observed in the oral phase deserves deeper investigation to prevent any adverse health effects related to the slowdown of carbohydrate metabolism.

International audience; A total of 544 isolates of lactic acid bacteria isolated from fermented products: 451 isolates from fermented products from Thailand and 93 isolated from French fermented products were screened for bacteriocin production. 10 isolates showed antimicrobial activities. Identification of the selected strains by random amplification of polymorphic DNA-polymerase change reaction using the primers E1, E2 and M13 showed three different RAPD patterns. One of the isolates from each product was chosen and good antimicrobial activity were displayed by 3 strains including MP6/2, PKS2-1 and OV1-1 producing in MRS broth bacteriocin-like substance (BLIS) inhibiting Candida albicans ATCC 90028, Carnobacterium maltraromaticum NCDO 2760, Lactobacillus sakei subsp. sakei JCM 1157, Listeria innocula CIP 80.11, Listeria ivanovii SLCC 2379, Pseudomonasc aeruginosa ATCC 27853, Staphylococcus aureus CIP 76.25 and Streptococcus mutans used as indicator strains. However, inhibitory activities of MP6/2 and PKS2-1 were related to the production of either organic acid, while OV2-1 was not affected by heating. The inhibitory activities of these three isolates were sensitive to proteolytic enzymes. 16S rDNA gene sequencing allowed to identify the MP6/2 as Pediococcus pentosaceus while PKS2-1 and OV1-1 were identified as Enterococcus faecium. It was concluded that the obtained isolates may be used as bio-preservative cultures to produce fermented foods.

This study investigated the digestibility of proteins in a pea protein-fortified sponge cake, as well as the impact of the degree of structure of the bolus produced by elderly subjects on the digestibility of proteins by combining ex vivo and in vitro approaches via the standardized protocol INFOGEST. The sponge cakes were consumed by a group of 20 elderly subjects with contrasting physiology, their boli were recovered just before swallowing, and their apparent viscosity was measured to delineate the bolus degree of structure. According to this criterion, two pools were formed with boli from subjects selected at the extremes: low viscosity and high viscosity, with apparent viscosity values (at 120 s-1 ) of 124 ± 18 and 208 ± 19 Pa s, respectively. The sponge cakes and the two pools underwent in vitro digestion. Protein hydrolysis kinetics was followed by measuring the released primary amino groups (NH2 ) and by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis at different time points. For all samples, the representative bands of pea proteins disappear gradually during digestion, accompanied by the appearance of bands indicating the presence of proteins with MW

Four strains of lactic acid bacteria (LAB), isolated from various Thai traditional fermented foods, were investigated on free radical scavenging activity of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS). Antioxidant activity by all isolated LAB in intracellular cell free ex- tracts (ICFE) was higher than in intact cells. The LAB strains FF and FB, isolated from fermented foods (fish and sliced beef), their (ICFE) showed the highest radical- scavenging activity for DPPH and ABTS at 65% and 60%, respectively for the former strain. The detection of amino acid decarboxylase of LAB were investigated by growing on decarboxylase medium and quantitatively evaluated by confirmation of amine forming capacity using an HPLC procedure. In this work, all tested LAB strains were tyramine producer; however, the weakest strain was FF followed by strain FB. In contrast, all of them did not produce histamine. Based on 16S rRNA genes, strain FF was identified as Lactobacillus fermentum, while strain FB was Lactobacillus plantarum. Both are potential starter cultures for producing fermented meat sausages.

International audience; Meat is recognized as an important source of protein and amino acids. Before consumption, meat products are transformed, and processing modifies the protein characteristics and impacts their digestibility. The present study aims to evaluate the impact of physicochemical modifications (solubility, oxidation, and denaturation) on the degree of in vitro digestibility and the digestion rate of high-pressure-treated cooked ham during the different process steps (curing, cooking and high-pressure treatment). Samples were homogenized and digested by pepsin and then by trypsin. The curing step increased the degree of proteolysis and the digestion rate compared with those of raw meat. Brining and tumbling caused partial denaturation of the proteins, which provided digestive enzymes better access to their specific cleavage sites. Otherwise, the substantial denaturation and protein oxidation induced by the cooking step decreased the degree of digestibility and significantly increased the digestion rate of cooked ham. Finally, the high-pressure treatment decreased the digestibility of cooked ham and simultaneously increased its digestion rate. The denaturation and oxidation phenomena leading to protein aggregation thus masked the cleavage sites necessary for the digestive enzymes. Therefore, each step of the processing of high-pressure cooked ham had an impact on the protein digestion parameters: the curing step promoted the digestibility and proteolysis rate of pork protein, while cooking and high-pressure treatment reduced the digestibility and proteolysis rate of pork protein.

Resume Introduction Les proteines du lait camelin presentent une faible homologie avec celles des caseines bovines, alors que la β-lactoglobuline, principale proteine allergisante du lait bovin, est completement absente du lait camelin. Cette difference de composition peut induire des reponses antigeniques differentes de celles des proteines du lait bovin. Ce travail consiste en une etude de l’immunoreactivite des proteines du lait camelin contre des serums d’enfants ayant une allergie confirmee aux proteines du lait de vache. Methodes L’etude a ete realisee sur les caseines et les proteines seriques du lait camelin de la race Targui du Sud algerien sur des serums de 10 patients ayant une allergie aux proteines du lait de vache confirmee par des tests cliniques et presentant des manifestations variees. L’immunoreactivite des proteines du lait camelin et bovin a ete etudiee par Elisa indirect contre des serums d’enfants ayant une allergie confirmee aux proteines du lait de vache. Resultats La totalite des serums etudies ont reagi avec les caseines bovines, alors que seulement 40 % des serums ont eu une reponse positive contre les caseines camelines avec une reponse diminuee de 16 a 92 % par rapport a celle des caseines du lait bovin. La totalite des proteines seriques camelines ont reagi avec les serums cependant les reponses sont largement plus faibles que celle des proteines seriques bovines. L’immunoreactivite des proteines seriques camelines varie de 36 a 85 % par rapport a celles des proteines seriques bovines. Discussion Les proteines du lait camelin semblent donc avoir un potentiel antigenique tres faible par comparaison a celle du lait bovin, ceci a ete note par differentes etudes (Restani et al., 2002 ; Vereda, 2006 ; El-Agamy et al., 2007 ; Fiocchi et al., 2010). Conclusion L’utilisation du lait camelin comme substitut du lait bovin comporte un risque evident a cause de son immunoreactivite residuelle. En revanche, il peut etre propose comme formule hypoallergenique preventive pour des enfants a risque vu ces faibles reponses par comparaison a celles des proteines bovines.

The aim of this study was to explore the biochemical and genetic features of the two-peptide bacteriocin produced by a Lactobacillus plantarum strain isolated from Italian type salami produced in Brazil ( Lb. plantarum MBSa4). Identification of bacteriocinogenic Lb. plantarum MBSa4 was performed by 16S rRNA sequencing. Expressed bacteriocin was evaluated for spectrum of activity, heat and pH stability, mechanism of action, and molecular mass. Partial purification was achieved by cation-exchange, and reversed phase - HPLC. Total DNA of Lb. plantarum MBSa4 was extracted and tested for presence of previously described bacteriocin genes. Bacteriocin MBSa4 was heat-stable, unaffected by pH 2.0 to 6.0 and active against all tested Listeria monocytogenes strains and most of tested fungi. Maximal production (1600 AU/ml) in MRS broth occurred after 22 h at 25 °C, presenting bacteriostatic activity as result of combined action of two components. The molecular mass determined by SDS-PAGE was 2.3 kDa. PCR-amplified DNA indicated the same nucleotide sequence of plantaricin W. Results indicate that Lb. plantarum MBSa4 produces plantaricin W, a two-peptide lantibiotic with remarkable anti- Listeria activity.

International audience; The main aim of this study was to develop a continuous microwave treatment system of whey proteins and then apply this process at 37 degrees C, 50 degrees C, 65 degrees C and 70 degrees C to achieve pepsinolysis and produce extensively hydrolysed bovine whey protein hydrolysates with low allergenic properties. The microwave process was compared to a conventional thermal treatment with similar temperature set points. Both processes were deeply analysed in terms of the thermal kinetics and operating conditions. The pepsin hydrolysates obtained by the continuous microwave treatment and conventional heating were characterized by SDS-PAGE and RP-HPLC. The allergenicity of the whey protein hydrolysates was explored using a human IgE sensitized rat basophil leukaemia cell assay. The results indicate that extensively hydrolysed whey protein hydrolysates were obtained by microwave only at 65 degrees C and in a shorter time compared with the conventional thermal treatment. In the same temperature conditions under conventional heating, -lactoglobulin was resistant to pepsinolysis, and 37% of it remained intact. As demonstrated by an in vitro degranulation assay using specific human IgE-sensitized rat basophils, the extensively hydrolysed whey protein obtained by microwave showed maximum degranulation values of 6.53% compared to those of the native whey protein isolate (45.97%) and hence elicited no more allergenic reactions in basophils. This work emphasizes the potential industrial use of microwave heating specific to milk protein processing to reduce their allergenicity and improve their end-use properties.

AIM The objective was to obtain lactic acid bacteria (LAB) capable of hydrolysing immunoreactive proteins in milk, to optimize the hydrolysis, to determine the proteolysis kinetics and to test the safety of the best hydrolytic strain. METHODS AND RESULTS Brazilian cheese was used as source of LAB capable of hydrolysing main milk allergens. Proteolytic isolates were submitted to RAPD-PCR for the characterization of clonal diversity. Optimized hydrolysis was strain and protein fraction dependent. 16S rDNA sequencing identified three proteolytic strains: Enterococcus faecalis VB43, that hydrolysed αS1 -, αS2 - and β-caseins, α-lactalbumin and β-lactoglobulin (partial hydrolysis), and Pediococcus acidilactici VB90 and Weissella viridescens VB111, that caused partial hydrolysis of αS1 - and αS2 -caseins. Enterococcus faecalis VB43 tested negative for virulence genes asa1, agg, efaA, hyl, esp, cylLL and cylLS but positive for genes ace and gelE. Ethylenediamine tetra-acetic acid inhibited the proteolysis, indicating that the main proteases of E. faecalis VB43 are metalloproteases. CONCLUSION Brazilian artisanal cheese is a good source of LAB capable of hydrolysing allergenic proteins in milk. One isolate (E. faecalis VB43) presented outstanding activity against these proteins and lacked most of the tested virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY Enterococcus faecalis VB43 presents good potential for the manufacture of hypoallergenic dairy products.

Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the β-subunit from β-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins. Fil: Biscola, V.. Universidade de Sao Paulo; Brasil. Centre de Recherche de Nantes. Institut National de la Recherche Agronomique; Francia Fil: Rodríguez de Olmos, Antonieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina. Centre de Recherche de Nantes. Institut National de la Recherche Agronomique; Francia Fil: Choiset, Y.. Centre de Recherche de Nantes. Institut National de la Recherche Agronomique; Francia Fil: Rabesona, H.. Centre de Recherche de Nantes. Institut National de la Recherche Agronomique; Francia Fil: Garro, Marisa Selva. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Mozzi, Fernanda Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Chobert, J. M.. Centre de Recherche de Nantes. Institut National de la Recherche Agronomique; Francia Fil: Drouet, M.. Centre hospitalier universitaire d'Angers; Francia Fil: Haertlé, T.. Centre de Recherche de Nantes. Institut National de la Recherche Agronomique; Francia. University Of Tehran; Irán Fil: Franco, B. D. G. M.. Universidade de Sao Paulo; Brasil

Lactococcus lactis KTH0-1S isolated from Thai traditional fermented shrimp (Kung-som) is able to produce heat-stable bacteriocin and inhibits food spoilage bacteria and food-borne pathogens. The inhibitory effect of bacteriocin remained intact after treatment with different pHs and after heating, but was sensitive to some proteolytic enzymes. Addition of bacteriocin KTH0-1S to Staphylococcus aureus cultures decreased viable cell counts by 2.8 log CFU/ml, demonstrating a bactericidal mode of action. Furthermore, the growth of S. aureus decreased significantly after 12-h co-cultivation with bacteriocinogenic strain. The molecular mass of bacteriocin KTH0-1S was found to be 3.346 kDa after ammonium sulfate precipitation, reversed phase (C-8 Sep-Pak), cation-exchange chromatography, RP-HPLC on C-8 column and mass spectrometry (MS/MS) analysis. Bacteriocin KTH0-1S was identified as nisin Z using PCR amplification and sequencing. The majority of tested virulence factors were absent, confirming the safety. Evidenced inhibitory effect of this strain, the absence of virulence factors creates the possibility for its application as protective culture to inhibit pathogenic bacteria in the several fermented seafood products.

International audience; We evaluated the vasorelaxant effect of camel and bovine casein tryptic hydrolysates (CTH and BTH, respectively) and their mechanism of action in aorta and mesenteric artery of Wistar Kyoto rats. CTH produced a more potent relaxation than that induced by BTH. This relaxation was endothelium-dependent and was mediated through the activation of the nitric oxide (NO)/cyclic guanosine 3,5-monophosphate (cGMP) pathway. The intracellular level of cGMP was increased by CTH. Moreover, CTH-induced relaxation was inhibited by HOE 140, a B2 bradykinin receptors antagonist, indicating that B2 bradykinin receptors play a role in the CTH effect. In endothelium-intact mesenteric artery, CTH and BTH produced a similar concentration-dependent relaxation with a lesser maximal effect than in aortic rings. Moreover, this relaxation was inhibited by Nu nitro-L-arginine methyl ester hydrochloride, a NO synthase inhibitor, but the addition of indomethacin, a cyclooxygenase inhibitor, and tetraethylammonium, a K+ channels blocker did not potentiate the inhibition.

International audience; Antifungal activity of lactic acid bacteria (LAB) has been observed in many food fermentation processes. Organic acids produced during the growth of these microorganisms play a major role in antagonism towards molds. The present study characterizes antifungal compounds produced during growth of Lactobacillus harbinensis K.V9.3.1Np that was previously isolated from raw cow milk and found to possess strong antifungal activities. A pH-controlled batch fermentation in enriched milk protein medium with immobilized cells of this bacterium was carried out in the presence of disrupted cells of Debaryomyces hansenii UBOCC-A-211003 and Kluyveromyces lactis UBOCC-A-212021. After seven days of fermentation, cell-free culture supernatants with robust antifungal activities against D. hansenii, Penicillium expansum and Penicillium roqueforti were obtained. These cell-free supernatants contained high amounts of organic acids such as lactic and acetic acids as well as hexanoic acid (9 mM), which the latter revealed to be the most efficient antifungal compound. The antifungal activity of hexanoic acid was influenced by the environmental conditions including the nature of the matrix and the pH. Under the conditions tested, even if organic acids were key antifungal agents, they were only part of a complex mixture of various molecules that acted in a synergistic manner. (C) 2013 Elsevier Ltd. All rights reserved.

The aim of this work was to study the antifungal properties of durancins isolated from Enterococcus durans A5-11 and of their chemically synthesized fragments. Enterococcus durans A5-11 is a lactic acid bacteria strain isolated from traditional Mongolian airag cheese. This strain inhibits the growth of several fungi including Fusarium culmorum, Penicillium roqueforti and Debaryomyces hansenii. It produces two bacteriocins: durancin A5-11a and durancin A5-11b, which have similar antimicrobial properties. The whole durancins A5-11a and A5-11b, as well as their N- and C-terminal fragments were synthesized, and their antifungal properties were studied. C-terminal fragments of both durancins showed stronger antifungal activities than other tested peptides. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l−1 of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra-structure of the yeast cells. Chemically synthesized durancins and their synthetic fragments showed different antimicrobial properties from each other. N-terminal peptides show activities against both bacterial and fungal strains tested. C-terminal peptides have specific activities against tested fungal strain and do not show antibacterial activity. However, the C-terminal fragment enhances the activity of the N-terminal fragment in the whole bacteriocins against bacteria. Significance and Impact of the Study Antifungal properties of durancins isolated from Enterococcus durans A5-11 and of their chemically synthesized fragments were determined. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l−1 of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra-structure of the yeast cells. This work contributes to improve understanding of molecular causes of antimicrobial activities of bacteriocins and their fragments. It may be proposed that the studied peptides affect all the yeast cellular and intramembranes including cytoplasmatic reticulum and nuclear and vacuolar membranes.

International audience; We evaluated on the one hand, the vasorelaxant effect of camel and bovine casein tryptic hydrolysates (CTH and BTH, respectively) and their mechanism of action in thoracic aorta taken from 36 week old male Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) and, on the other hand, the putative antihypertensive effect of CTH and BTH in awake 36 week old male SHR. Relaxation to CTH and BTH was impaired in SHR thoracic aorta compared with that observed in WKY rat thoracic aorta. This relaxation was endothelium-dependent and mediated through the activation of the NO pathway in both rat strains. Oral administration of CTH (800 mg kg(-1) day(-1) per os for 15 days) decreased blood pressure and heart rate only during the first week of treatment in aged SHR. The same dose of BTH did not modify blood pressure and heart rate throughout the 15 days of treatment.

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faccalis (Ent. faccalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (alpha(S1)-, alpha(S2)-, and beta-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Eat. faccalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42 degrees C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecolis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

Lactic acid bacteria (LAB) symbionts of honeybees are certainly playing key roles in their host's colony functioning. The defense against bacterial pathogen invasion by endogenous LAB has been considered as promising and usable phenomenon. This study addresses bacteriocinogeny as one of antibacterial action mode displayed by bacteria. The honeybee endogenous LAB isolated from worker honeybee intestines (61 strains), queen honeybee intestines (16 strains) and beebread (25 strains) were tested for bacteriocin production ability. We checked also well characterized bacteriocin producing LAB strains against bacteria causing American foulbrood (AFB) – Paenibacillus larvae aiming possible use of exogenous LAB for control AFB in honeybees and in the same time to observe the vulnerability of endogenous bacteria exposed to bacteriocin producers. We demonstrated that none of 102 studied LAB strains, isolated from worker honeybee intestines, from queen honeybee intestines and from beebread, produced bacteriocins detectable by the well diffusion method (WDM). All of them failed to inhibit two strains of P. larvae . Three exogenous bacteriocin-producing LAB strains were tested against the same pathogens and against 25 endogenous bacterial isolates representing 11 different LAB species. The screening showed that all the tested exogenous bacteriocin-producing strains inhibited the tested P. larvae strains. The endogenous LAB strains exhibited varied sensitivity profiles when treated with bacteriocin-producing strains. This raises similar challenges to those observed in antibiotic applications leading to dysbacteriosis, even though the efficacy of these bacteriocins against P. larvae in an in vitro system is evident.

The proteolytic activity of thirty-three Lisolates from Mongolian tarag was tested on skimmed milk. The strain displaying the highest proteolytic activity was purified and presented by 16S rDNA sequencing 99.9 % homology with Lactobacillus paracasei 1-4-2A. It was named L. paracasei I-N-10. Proteases of L. paracasei I-N-10 hydrolyze predominately beta-casein and in some level alpha(S2)-casein; hydrolysis of alpha(S1)-casein was not observed. Proteolytic activity was optimal at 42 A degrees C and neutral pH. Proteases of L. paracasei I-N-10 were inhibited by serine- and metalloproteases inhibitors. PCR amplification revealed the presence of prtP gene, which was identical to prtP gene of L. paracasei genus. Mass spectrometry analysis of beta-casein hydrolysate allowed to characterize 7 peptides resulting from proteolysis by L. paracasei I-N-10. The isolated strain was able to cleave beta-casein in different sites including 2 of the major linear epitopes implicated in its allergenicity. Being sensitive to main antibiotics classes, L. paracasei I-N-10 could be considered as safe and used as starter culture with a potential role in decreasing beta-casein immuno-reactivity.

Proteolytic activity of Lactobacillus helveticus A75 and effects of caseins hydrolysis by this strain on their immuno-reactivity were studied. Proteases of L. helveticus A75 hydrolyzed both αS1- and β-caseins and were inhibited by serine- and metallo-proteases inhibitors. Polymerase chain reaction amplification revealed the presence of prtH gene, which was 99% identical to prtH gene of L. helveticus CNRZ 32. Effect of proteolysis of αS1- and β-caseins by L. helveticus A75 on their immunoglobulin E (IgE) binding ability was studied by an enzyme-linked immunosorbent assay with the pool of eight sera from cow milk allergy patients. The IgE binding ability of hydrolyzed proteins was lower than that of intact proteins, which indicates that hydrolysis of αS1- and β-caseins by proteases of L. helveticus A75 decreases their recognition by specific IgE. Half maximal inhibitory concentration values were 2.01 and 2.28 µg/mL for αS1- and β-casein, respectively, and increased until 9.08 and 13.65 µg/mL, respectively, after their hydrolysis by L. helveticus A75. PRACTICAL APPLICATIONS The obtained results yield new information about proteolytic activity and potential application of lactobacilli isolated from traditional Azerbaijani cheeses. Isolated strain could represent new adjunct cultures for the dairy industry.

Effect of combined microwave (MW) and enzymatic hydrolysis on the human immunoglobulin E (IgE)-binding properties of beta-lactoglobulin (beta-lg) and other whey proteins (WP) was investigated. Separated beta-lg and full whey protein isolate (WPI) were hydrolyzed with trypsin, chymotrypsin, mixture of trypsin/chymotrypsin, and pepsin at three microwave power levels: 50 W during 1 and 5 min, 100 and 200 W during 1 and 3 min. The immunoreactivity of the obtained hydrolysates resulting from combined microwave protease treatment was assessed using sera of young patients allergic to bovine whey proteins. The application of microwave treatment at 200 W enhances the hydrolysis of beta-lg by pepsin in 3 min and decreases significantly its immunoreactivity. The extensive hydrolysis of the microwave-treated beta-lg and WPI with trypsin, chymotrypsin, and the mixture of trypsin with chymotrypsin did not have an impact on the IgE binding of the products obtained in all the studied conditions.

The interest of consumers for diverse fermented foods has increased in recent years thanks to the positive perception of their impact on consumer health considered as beneficial. Hence, there is an evident need for search of novel ways and for new food preservation agents of natural origins. In this aspect, lactic acid bacteria are very good candidates. It should be highlighted also that the onset of food allergies is rising significantly in recent years. The reduction of the immunoreactivity of food proteins could be achieved thanks to pre-prandial proteolysis occurring in fermented dairy (and other food) systems changing the allergen presentation or cleaving the allergenic protein epitopes, and produce hypoallergenic products. Fil: El-Ghaish, Shady. Institut National de la Recherche Agronomique; Francia Fil: Ahmadova, Aynur. Institut National de la Recherche Agronomique; Francia Fil: Hadji-Sfaxi, Imen. Institut National de la Recherche Agronomique; Francia Fil: El Merchefi, Kamel Eddine. Institut National de la Recherche Agronomique; Francia. Université d’Oran. Département de biologie. Laboratoire de Physiologie de la Nutrition et sécurité Alimentaire; Argelia Fil: Bazukyan, Inga. Yerevan State University. Faculty of Biology; Armenia Fil: Choiset, Yvan. Institut National de la Recherche Agronomique; Francia Fil: Rabesona, Hanitra. Institut National de la Recherche Agronomique; Francia Fil: Sitohy, Mahmoud. Institut National de la Recherche Agronomique; Francia. Zagazig University. Faculty of Agriculture. Biochemistry Department; Egipto Fil: Popov, Yuri G.. Yerevan State University. Faculty of Biology; Armenia Fil: Kuliev, Akif A.. Baku State University. Biotechnical and Biochemistry Chair; Azerbaiyán Fil: Mozzi, Fernanda Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Chobert, Jean-Marc. Institut National de la Recherche Agronomique; Francia Fil: Haertle, Thomas. Institut National de la Recherche Agronomique; Francia

International audience; In this work, we tried to investigate the impact of treatment with instant controlled pressure drop technology (DIC) on the allergenicity of milk proteins. In DIC treatment, milk proteins are exposed to a high-pressure saturated steam (0.4 MPa, 144 degrees C) during 25 s and ended with an abrupt pressure drop towards a vacuum (5 kPa, 32 degrees C). Immunoglobulin E (IgE)-binding ability is determined with indirect enzyme linked immuno spectrophotometric analysis and western blot test. Results indicate significant changes in the IgE binding of treated proteins with DIC. Treated caseins showed increase in the IgE-binding ability; this indicates that DIC enhance the antigenicity of treated caseins. In the opposite, treated whey proteins (ss-lactoglobulin and alpha-lactalbumin) showed decrease in the IgE binding of these proteins. DIC treatment at 0.4 MPa during 25 s causes a greater reduction of the allergenicity of whey proteins than treatment with 0.6 MPa for 25 s.

Low-density lipoproteins (LDL) are considered to be the main contributors to the exceptional emulsifying activity of hen egg yolk. However, the lack of understanding of the molecular basis for LDL functionality is a significant obstacle for good control of yolk emulsions. Consequently, we have attempted to link the structure and the characteristics of LDL with their emulsifying properties. After purification of LDL, we have determined their protein and lipid compositions, their ultrastructure, and then extracted their apoproteins for physicochemical characterisation. LDL are composed of about 12% of proteins and 87% of lipids and present a spherical shape with a mean diameter of about 35 nm. LDL solubility is high, whatever the medium conditions, because of their low density. LDL contain five major apoproteins out of which the apoprotein of 15 kDa is considered to be the most surface-active. After extraction, this apoprotein showed a high proportion of amphipathic α-helix chains, explaining the high capacity of this apoprotein to adsorb at the oil–water interface.

The 7-kDa lipid transfer proteins, LTP2s, share some amino-acid sequence similarities with the 9-kDa isoforms, LTP1s. Both proteins display an identical cysteine motif and, in this regard, LTP2s have been classified as lipid transfer proteins. However, in contrast with LTP1s, no data are available on their structure, cysteine pairings, lipid transfer and lipid binding properties. We reported on the isolation of two isoforms of 7-kDa lipid transfer protein, LTP2, from wheat seeds and showed for the first time that they indeed display lipid transfer activity. Trypsin and chymotrypsin digestions of the native LTP2 afforded the sequence of both isoforms and assignment of disulfide bonds. The cysteine pairings, Cys10–Cys24, Cys25–Cys60, Cys2–Cys34, Cys36–Cys67, revealed a mismatch at the Cys34-X-Cys36 motif of LTP2 compared to LTP1. Moreover, the secondary structure as determined by circular dichroism suggested an identical proportion of α helices, β sheets and random coils. By analogy with the structure of the LTP1, we discussed what structural changes are required to accommodate the LTP2 disulfide pattern.

Von Ebner gland protein (VEGP) and odorant-binding protein (OBP) were purified from porcine lingual epithelium and nasal mucosa, respectively. Both VEGP and OBP preparations were homogeneous as indicated by SDS–PAGE, isoelectric focusing, gel-filtration and electrospray mass spectrometry. However, high-sensitivity differential scanning calorimetry (HS-DSC) yielded multiphasic denaturation thermograms for both proteins indicating their conformational heterogeneity. The unfolding transition of VEGP is observed at extremely high temperatures (about 110°C), which is unexpected for a protein with significant structural homology to OBP and other lipocalins. Isothermal titration calorimetry (ITC) did not detect the binding of either aspartame or denatonium saccharide to VEGP nor did it detect binding of 2-isobutyl-3-methoxypyrazine (IBMP) to OBP. Extraction of OBP with mixed organic solvents eliminated the conformational heterogeneity and the protein showed a reversible two-state transition in HS-DSC thereafter. ITC also showed that the extracted OBP was able to bind IBMP. These results imply that tightly bound endogenous ligands increase the thermal stability of OBP and block the binding of other ligands. In contrast to OBP, the extraction of VEGP with organic solvents failed to promote binding or to establish thermal homogeneity, most likely because of the irreversible denaturation of VEGP. Thus, the elucidation of the functional behaviour of VEGP is closely related to the exhaustive purging of its endogenous ligands which otherwise very efficiently mask ligand binding sites of this protein.

Aims The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. Methods and Results Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact.sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2 center dot 0 to 6 center dot 0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600AUml(-1)) in MRS broth occurred after 20h at 25 degrees C. The molecular mass of produced bacteriocin was 4303 center dot 3Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-alpha, T-beta, X, P, G and Q). Conclusions In the studied conditions, Lact.sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. Significance and Impact of the Study The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact.sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2 center dot 0-6 center dot 0) and activity against L.monocytogenes food isolates bring up a potential technological application to improve food safety.

International audience; beta-Lactoglobulin (beta-Lg) is the major whey protein -of bovine milk present at a concentration of 2-3 g L-1. Its biological role is still not well-known. However, many studies have suggested that beta-Lg may play either nutritional or specific transporter role. The high affinity of beta-Lg for retinol and other retinoids was reported. The results of interaction studies of beta-Lg with carotenoids, that is, beta-carotene, beta-cryptoxanthin, and alpha-carotene, which display similar structures are reported in this study. The affinities of beta-Lg for binding of retinoids and carotenoids were compared, providing more information about the binding site(s) of these molecules by beta-Lg. Interactions were followed by the measurements of quenching of beta-Lg tryptophan fluorescence and retinol fluorescence. The obtained results indicate that carotenoids are bound by beta-Lg with high affinity of the order of 10(-8) M. Measurement of retinol competition with carotenoids for binding by beta-Lg suggests that the binding of these two ligands occurs at two different sites of beta-Lg.

International audience; The aims of this study were to characterize inhibitory activity spectra, some probiotic properties and safety of Lactobacillus curvatus A61 for its future application in production of fermented foods. The studied strain was isolated from traditional homemade cheese manufactured in Azerbaijan. The cell-free supernatant of culture of Lb. curvatus A61 inhibited the growth of tested LAB, as well as of Listeria monocytogenes and Bacillus cereus strains. The strain presented antifungal activity and inhibited the growth of Cladosporium and Fusarium ssp. during co-cultivation on agar media. PCR amplification with specific primers revealed the presence of curvacin A encoding gene in Lb. curvatus A61. Bacteriocin produced by the studied strain was heat stable and active in a broad pH range, and in the presence of Triton X-20, Triton X-80, Triton X-100, beta-mercaptoethanol, Na-EDTA, SDS and NaCl. The mode of action of bacteriocin against selected indicator strains was found to be bacteriostatic. Lb. curvatus A61 was resistant to physiological concentrations of bile salts and showed high auto-aggregation ability, as well as co-aggregation ability with pathogenic L monocytogenes strains. It was sensitive to chloramphenicol, penicillin, tetracycline, ciprofloxacin and vancomycin, but resistant to ampicillin and gentamicin. (C) 2013 Elsevier Ltd. All rights reserved.

The aims of this study were to characterize bacteriocin activity, some functional and probiotic properties, and to evaluate the safety of Enterococcus faecium AQ71 isolated from Azerbaijani Motal cheese. The studied strain inhibited the growth of selected tested LAB, Listeria monocytogenes and Bacillus cereus strains. PCR amplification with specific primers indicated that E. faecium AQ71 carry genes encoding enterocins P. L50A, L50B and A. Bacteriocin(s) produced by the studied strain was/were heat stable and active in a broad pH range. Triton X-20, Triton X-80, Triton X-100, beta-mercaptoethanol, Na-EDTA, SDS and NaCl did not affect the antimicrobial activity of the strain. The cell free supernatants of the strain caused the lysis of cells of Lactobacillus brevis F145 and inhibited the growth of L. monocytogenes. E. faecium AQ71 was negative for the tested virulence factors and did not present multi-resistance to antibiotics. The strain was resistant to physiological concentrations of bile salts and showed good auto-aggregation ability as well as co-aggregation ability with L. monocytogenes. E. faecium AQ71 exhibited good esterase, esterase lipase, acid phosphatase and aminopeptidase activities. (C) 2012 Elsevier Ltd. All rights reserved.

Five bacterial cocci isolates were selected from a wide pool of 503 isolates collected from traditional Egyptian dairy products on the basis of their inhibitory activities against Lactobacillus brevis F145, Lactobacillus bulgaricus 340, Enterococcus faecium HKLHS, Listeria ivanovii ATCC, Listeria innocua CIP 80.11 and Listeria monocytogenes EGDe 107776. These 5 isolates were identified as E. faecium TX1330 and E. faecium E980 by 16S rDNA amplification and sequencing. The antibacterial activity of the two strains was not affected by treatment of the cell free culture supernatant with catalase but their activities disappeared completely when digested with protease K, α-chymotrypsin and trypsin. The antimicrobial substance was stable over a wide range of pH (2-10) and was active after heating at 100 °C for 10 min. Bacteriocin yield in two strains reached a maximum (1,600 AU/ml) at the end of the exponential phase (6 h) and remained stable until the end of 24 h-incubation period when the medium reached pH 5.5. Maximal production of bacteriocin was obtained when growing the bacterial cells at temperatures ranging between 30 and 37 °C. Bacteriocin production was unaffected when the bacterial cells grew under severe conditions of pH (9.6) and in high salt (6.5% NaCl). Thanks to PCR gene amplification the bacteriocins produced by E. faecium TX1330 could be identified as enterocins A and B structural genes, while the bacteriocins produced by E. faecium E980 could be identified as enterocins P and L50A structural genes, which can be classified into two enterocin subclasses (IIa and IIc), respectively. PCR amplification demonstrated that the two studied strains did not contain virulence factors asal, cyl A and B, ace, efaAfs and espfm. These two strains were sensitive to most of the tested antibiotics but were resistant to tetracycline. E. faecium E980 was also resistant to chloramphenicol.

Proteinase activity ofLactobacillus fermentumIFO3956 cells was higher when they were grown on milk-based media than on 10% reconstituted skim milk. The lowest protease activity was observed when cells were grown on milk-free media. The extraction of milk-induced cell-bound proteases fromLb. fermentumIFO3956 was most efficient using 1% Tween 80 while the use of 1% SDS inhibited all proteolytic activity. Two bands of ∼35 and >100 kDa were observed by zymogram, indicating that proteolytic activity corresponded to the presence of at least two types of enzymes or two molecular forms of one enzyme. Mass spectrometry analyses of αS1-casein hydrolysates detected 24 peptides with sizes ranging from 5 to 36 amino acids, including 9 phosphorylated peptides, resulting from the fermentation ofLb. fermentumIFO3956 of αS1-casein. Most of the identified peptides originated from the N-terminal portion of αS1-casein. The studied bacterial strain could hydrolyze αS1-casein in many sites including the epitopes triggering the allergic reactions against αS1-caseine.g.at the positions 23, 30, 41, 71, 91, 98, 126, 179. After hydrolysis of αS1-casein withLb. fermentumIFO3956 the recognition and the binding of this casein to IgE from the pooled sera of 18 patients with cow's milk allergy was significantly reduced.

The whey protein β-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41-K60; Y102-R124; L149-I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS-PAGE and RP-HPLC. Peptides released were identified by LC-MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8 h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3-5 year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products. Fil: Pescuma, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Rabesona, Hanitra. Institut National de la Recherche Agronomique; Francia Fil: Drouet, Martine. Unité Allergologie Générale et Pneumologie; Francia Fil: Choiset, Yvan. Institut National de la Recherche Agronomique; Francia Fil: Haertlé, Thomas. Institut National de la Recherche Agronomique; Francia Fil: Mozzi, Fernanda Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Font, Graciela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Chobert, Jean-Marc. Institut National de la Recherche Agronomique; Francia

International audience; beta-Lactoglobulin (beta-LG) is one of the cow's major milk proteins and the most abundant whey protein. This globular protein of about 18 kDa is folded, forming a beta-barrel (or calyx) structure. This structure is stabilized by two disulfide bonds and can be altered by heating above 65 degrees C. beta-LG is also one of the major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. During heating in the presence of reducing sugars, beta-LG is also submitted to the Maillard reaction, which at the first stage consists of the covalent fixation of sugars on the epsilon-amino groups of lysyl residues. The following steps are condensation and polymerization reactions leading to the formation of melanoidins (brown pigments). Despite the frequency of use of heating during milk transformation, the effects of heat-induced denaturation and of glycation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) pa! tients are not fully understood. The objectives of our work were to evaluate the effect of heat-induced denaturation of bovine beta-LG on binding of IgE from CMA patients and to determine the effect of moderate glycation on the degree of recognition by IgE. We showed that heat-induced denaturation (loss of tertiary and secondary structures) of beta-LG is associated with weaker binding of IgE from CMA patients. It was also shown that moderate glycation of beta-LG in early stages of Maillard reaction has only a small effect on its recognition by IgE, whereas a high degree of glycation has a clear "masking" effect on the recognition of epitopes. This demonstrates the importance of epsilon-amino groups of lysines in the definition of epitopes recognized by IgE.

International audience; BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.

Le passage de la forme non pathogène de la protéine prion normalement présente chez l’individu sain (PrPC) vers la forme pathogène (PrPSc) se traduit par une augmentation de la proportion de feuillet bêta dans la protéine, favorisant son agrégation, la formation de fibrilles et la résistance à la protéinase K. La structure tridimensionnelle de PrPC, déterminée pour quatre espèces, est extrêmement conservée. Elle comporte un segment désordonné et très flexible à l’extrémité N-terminale et une partie globulaire, constituée de deux brins bêta (S1, S2) et de trois hélices alpha (H1 à H3) associés par des boucles (L1 à L5). Le fragment de la protéine correspondant à l’hélice H1 se structure en hélice de façon autonome. En revanche, le peptide comportant la région H1-L3- S2 (PrPH1-L3-S2) montre, comme la protéine, une capacité à adopter différentes conformations. Ces résultats contribuent à proposer l’hélice H1 comme l’un des motifs structuraux de la protéine capables d’initier la transconformation, c’est-à-dire la transformation de la protéine prion normale en protéine prion pathogène. Le rôle clé de l’hélice H1 dans la transconformation a été étayé par une série d’études physicochimiques, détaillées dans l’article, réalisées à l’aide d’une série de peptides de tailles variées (9 à 33 résidus, séquence ovine) ciblés sur la région [133-165] qui comporte la succession des motifs structuraux L2-H1-L3-S2. Les principaux résultats de cette étude montrent la grande stabilité de l’hélice H1, en particulier en présence de la boucle L2 ou des deux boucles L2 et L3. L’absence de la boucle L2 et la présence du brin bêta S2 sont en revanche des facteurs de déstabilisation de l’hélice H1. La boucle L2 pourrait d’ailleurs jouer un rôle tout particulier comme le suggère l’observation d’une interaction entre cette boucle et la protéine PrPC. Une telle interaction pourrait être mise en jeu dans les mécanismes intervenant dans l’interaction protéine prion saine/protéine prion pathogène impliquée dans la propagation de la maladie. Ces résultats, qui devront être confirmés et développés, conduisent à proposer la boucle L2 et le feuillet S2 comme deux régions assurant la « régulation » de la stabilité de l’hélice H1, qui apparaît comme une région clef dans les processus de conversion pathogène., The conversion of the prion protein from the normalnon-pathogenic cellular form (PrPC), to the pathogenicmisfolded isoform (PrPSc) involves an increase of theamount of beta sheet in the protein structure, whichfavours aggregation, formation of fibrils and resistanceto proteinase K. The three-dimensional structure ofPrPc, which has been determined for four species, isextremely conserved. It contains a flexible segment atthe N-terminus and a globular part composed of twobeta-strands (S1, S2) and three alpha-helices (H1 to H3) associated by loops (L1 to L5). The protein fragmentcorresponding to helix H1 is an autonomousregion that can adopt a helical structure by itself. Bycontrast, the peptide that contains region H1-L3-S2(PrPH1-L3-S2) adopts different conformations, as alsoshown for the protein. These results allow proposinghelix H1 as one of the structural motifs of the prionprotein able to initiate the transconformation, i.e. theconversion of the benign prion protein into its pathogenicisoform. The key role played by helix H1 in thetransconformation was analysed in a physicochemicalstudy using a series of peptides of different sequencesand lengths (9 to 33 residues, sheep sequence) designedin the [133-165] region that contains the structuralmotifs L2-H1-L3-S2. The main results, which aredescribed here, show the great stability of helix H1,particularly in the presence of either loop L2 or loopsL2 and L3. The absence of loop L2 associated to thepresence of beta-strand S2 decreases the stability ofhelix H1. It is possible that loop L2 could play a specialrole, as suggested by the fact that this loop can interactwith PrPC. Such an interaction could contribute tothe mechanisms that are involved in the interactionbenign prion protein / pathogenic prion protein, whichis implicated in the propagation of the disease. Theseresults, which should be confirmed and developped,lead to propose loop L2 and strand S2 as two regionsthat could assume a "regulation" of the stability ofhelix H1, which appears as a key region in the pathogenicconversion process.

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.

Model peptides mimicking a repeating consensus sequence present in gliadin (a wheat storage protein) were used to investigate the structural requirements for tyrosine-tyrosine cross-linking catalyzed by horseradish peroxidase. It was found that the position of tyrosine in the sequence, the vicinity of positive charges, and the length of the peptide are parameters affecting the degree of polymerization. The yields of polytyrosines obtained after oxidation of purified gliadins by two kinds of peroxidases (plant peroxidases and fungus Mn-dependent peroxidase), which differ in their mechanisms, were compared. The efficiency of cross-linking proteins via tyrosine-tyrosine aromatic ring condensation was enhanced when Mn-dependent peroxidase was used, which acts as a distant catalyst as compared to horseradish peroxidase which proceeds by direct contact catalysis. To correlate molecular modifications with certain technological potentials, we investigated the rheological properties of some films obtained from peroxidase-treated gliadins. One effect of enzymatic treatment was to increase the tensile strength of the films.

International audience; The monoclonal antibody, HT13 recognizes human choriogonadotropin (CG) bound to the extracellular domain of its receptor, but not to the full-length receptor. The HT13 epitope is located in the regions of residues 15-17 and 73-75 of the human CG alpha-subunit. Only one synthetic peptide, lutropin (LH)/CG-receptor-(481-497)-peptide (EL2 peptide), which spans the second putative extracellular loop of the LH/CG-receptor endodomain, prevents recognition of human CG by HT13 mAb. EL2 peptide decreases hormone-induced cAMP production, but not high-affinity binding. An anti-EL2 serum also displays the capacity to inhibit human CG-stimulated cAMP production. These results suggest that the second extracellular loop of the receptor is in contact with the HT13 epitope of human CG alpha-subunit and is involved in signal transduction. A relative orientation of the hormone versus the endodomain is proposed.

Pituitary gonadotropin hormones lutropin (LH) and follitropin (FSH) control steroidogenesis and gametogenesis in male and female gonads through interaction with G protein-coupled receptors, LHR and FSHR. In the male, LH acts on Leydig cells and is mostly responsible for the acquisition of puberty and the production of androgens while FSH, together with androgens, regulates spermatogenesis within Sertoli cells. We have engineered filamentous phages displaying mouse LHR and human FSHR decapeptides chosen in hormone binding regions. Peptides from both receptors displayed on phages belong either to the receptor specific exon 1 (amino acids 18–27) or to the homologous exon 4 (amino acids 98–107). Vaccination of prepubertal BALB c male mice with hybrid phages using sub-cutaneous or intraperitoneal injections induced immunity against receptors. Anti-receptor immunization produced agonist or antagonist effects depending only on the circulating levels of the antibodies. Both anti-LHR and anti-FSHR vaccines induced efficient as well as reversible male contraception, through different mechanisms: targeting LH receptors inhibited or hyperstimulated Leydig cell testosterone production while targeting FSH receptors did not affect testosterone levels.

Molecular forms of the porcine LH/CG receptor (pLHR) and complexes between hCG and either the full-length pLHR or its extracellular domain (ectodomain) have been produced in various recombinant systems. In COS cells and in the baculovirus insect cells system, the co-expression of the ecto- and endo-domains reconstituted a functional receptor where the association of the two domains seems to depend upon the presence of disulfide bridges. According to previous observations [39], synthetic peptides mimicking three regions of the ectodomain (21-38, 100-115, 250-272) were found to inhibit hormone binding and stimulation of cAMP production. Antisera raised against these peptides contained anti-peptide antibodies (Ab) able to interfere with hormone signalling. Moreover, the results of peptide mapping indicated that some peptides stretches may be more involved in signalling rather than in binding. Immunochemical mapping based on monoclonal antibodies (mAbs) was used to probe the hCG-ectodomain complex. It appeared that mAbs directed to epitopes present on the 'beta-tip' of hCG (assembled from the beta subunit loops 3 and 1, and previously designated site IIIb) and on the 'alpha-tip' (alpha subunit loops 1 and 3, site IIIa) bound to hCG-receptor complexes, whereas a conformational epitope (defined by the alpha-beta interface between beta seat belt C-terminus and alpha loop 2, site II) was masked. Interestingly, we and others previously reported that, in the hCG-full length receptor complex, site IIIa was shielded to mAb binding. A peptide mimicking the second extracellular loop (EL2) of the receptor endodomain was found to prevent the binding of a mAb directed to site IIIa, suggesting that this region of the endodomain may be interacting with the 'alpha-tip'. In the full-length, membrane anchored pLHR, the EL2 peptide inhibited hCG-induced cAMP production, but not binding. The possibility of inhibiting stimulation without inhibition of binding gives support to the 'negative specificity' hypothesis [6]. Thus, the ectodomain of the glycoprotein hormone receptors might be considered as a screening device preventing access of any glycoprotein hormone to the signalling peptide keys of the endodomain, which otherwise would be sensitive to any alpha subunit stimulation. Finally, antibody binding to site IIIa on the hCG-ectodomain complex was also hindered by an anti-peptide mAb directed against a peptide encoded by the eighth exon (pE x 8) of the LHR. This suggests that pEx8 is vicinal to the alpha-tip of hCG and to EL2 in the hCG-full length receptor complex. Altogether, these observations help to build up a topological model of the hCG-receptor complex.

Background Cow’s milk allergy (CMA) is one of the most widespread human allergies, especially in very young children. One of the main bovine milk allergen is aS1-casein which is considered as intrinsically unfolded protein. Its epitope determinants have been partially characterized using synthetic peptides and crucial amino acid residues for IgE-binding have been identified. However, this very useful approach for epitope characterization neglects local interactions or local structures formed in the fulllength protein and their impact on the accessibility of epitopes for IgE.