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Professeur des Universités
Faculté des Sciences et Technologies - Nancy
Université de Lorraine
+33 (0)3 72 74 51 93 | annie.dary@univ-lorraine.fr
Molecules, 29 (7), 1552.
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
In addition to traditional use in fermented dairy products, S. thermophilus also exhibits anti-inflammatory properties both in live and heat-inactivated form. Recent studies have highlighted that some hydrolysates from surface proteins of S. thermophilus could be responsible partially for overall anti-inflammatory activity of this bacterium. It was hypothesized that anti-inflammatory activity could also be attributed to peptides resulting from the digestion of intracellular proteins of S. thermophilus. Therefore, total intracellular proteins (TIP) from two phenotypically different strains, LMD-9 and CNRZ-21N, were recovered by sonication followed by ammonium sulphate precipitation. The molecular masses of the TIP of both strains were very close to each other as observed by SDS-PAGE. The TIP were fractionated by size exclusion fast protein liquid chromatography to obtain a 3–10 kDa intracellular protein (IP) fraction, which was then hydrolysed with pancreatic enzyme preparation, Corolase PP. The hydrolysed IP fraction from each strain exhibited anti-inflammatory activity by modulating pro-inflammatory mediators, particularly IL-1β in LPS-stimulated THP-1 macrophages. However, a decrease in IL-8 secretion was only observed with hydrolysed IP fraction from CNRZ-21N, indicating that strain could be an important parameter in obtaining active hydrolysates. Results showed that peptides from the 3–10 kDa IP fraction of S. thermophilus could therefore be considered as postbiotics with potential beneficial effects on human health. Thus, it can be used as a promising bioactive ingredient for the development of functional foods to prevent low-grade inflammation.
Probiotics and Antimicrobial Proteins, 15 (2), pp. 387-399.
Hadef, S., Idoui, T., Sifour, M., Genay, M., Dary-Mourot, A.
Twenty-five lactic acid bacterial (LAB) strains have been isolated from traditional goat butter and three types of cheese (dry Klila, frech Klila, and Bouhezza) and evaluated for technological abilities, probiotic properties, and potentials as starter cultures. The twenty-five LAB strains comprised eight strains belonging to Lactobacillus, four strains belonging to Lactococcus, eleven strains belonging to Enterococcus, and two strains belonging to Leuconostoc. A non-hierarchical cluster analysis was performed in order to select the performing strains. After carrying out the preliminary phenotypic characterizations and the probiotic potential, three strains designated as BM10, B15, and C30 belonging to the genus Lactobacillus and Enterococcus with good tolerance to acidity were selected. The strains showed a significant resistance to 0.5% bile salts and 0.4% phenol. Hemolytic activity was not detected; in addition, good hydrophobicity and autoaggregation was obtained. A significant antimicrobial activity was exhibited by all selected strains against Listeria innocua. Genotypic identification by 16S rRNA allowed the identification of B15, BM10, and C30 as Lactobacillus plantarum, Lactobacillus casei, and Enterococcus durans, respectively. The results of the current study suggest that the strains isolated from Algerian fermented dairy products have high potential as probiotic starter cultures in the goat butter and cheese industry.
Nutrients, 14 (22), pp. 4777.
Allouche, R., Genay, M., Dary-Mourot, A., Hafeez, Z., Miclo, L.
Streptococcus thermophilus, a food grade bacterium, is extensively used in the manufacture of fermented products such as yogurt and cheeses. It has been shown that S. thermophilus strains exhibited varying anti-inflammatory activities in vitro. Our previous study displayed that this activity could be partially due to peptide(s) generated by trypsin hydrolysis of the surface proteins of S. thermophilus LMD-9. Surface protease PrtS could be the source of these peptides during gastrointestinal digestion. Therefore, peptide hydrolysates were obtained by shaving two phenotypically distinct strains of S. thermophilus (LMD-9 PrtS+ and CNRZ-21N PrtS−) with pepsin, a gastric protease, followed or not by trypsinolysis. The peptide hydrolysates of both strains exhibited anti-inflammatory action through the modulation of pro-inflammatory mediators in LPS-stimulated THP-1 macrophages (COX-2, Pro-IL-1β, IL-1β, and IL-8) and LPS-stimulated HT-29 cells (IL-8). Therefore, peptides released from either PrtS+ or PrtS− strains in the gastrointestinal tract during digestion of a product containing this bacterium may display anti-inflammatory effects and reduce the risk of inflammation-related chronic diseases.
Foods, 11 (8), pp. 1157.
Allouche, R., Hafeez, Z., Papier, F., Dary-Mourot, A., Genay, M., Miclo, L.
Nutrients, 14 (24), pp. 5338.
Pinchaud, K., Hafeez, Z., Auger, S., Chatel, J.-M., Chadi, S., Langella, P., Paoli, J., Dary-Mourot, A., Maguin-Gaté, K., Olivier, J.-L.
Although arachidonic acid (ARA) is the precursor of the majority of eicosanoids, its influence as a food component on health is not well known. Therefore, we investigated its impact on the gut microbiota and gut–brain axis. Groups of male BALB/c mice were fed either a standard diet containing 5% lipids (Std-ARA) or 15%-lipid diets without ARA (HL-ARA) or with 1% ARA (HL + ARA) for 9 weeks. Fatty acid profiles of all three diets were the same. The HL-ARA diet favored the growth of Bifidobacterium pseudolongum contrary to the HL + ARA diet that favored the pro-inflammatory Escherichia–Shigella genus in fecal microbiota. Dietary ARA intake induced 4- and 15-fold colic overexpression of the pro-inflammatory markers IL-1β and CD40, respectively, without affecting those of TNFα and adiponectin. In the brain, dietary ARA intake led to moderate overexpression of GFAP in the hippocampus and cortex. Both the hyperlipidic diets reduced IL-6 and IL-12 in the brain. For the first time, it was shown that dietary ARA altered the gut microbiota, led to low-grade colic inflammation, and induced astrogliosis in the brain. Further work is necessary to determine the involved mechanisms.
Microorganisms, 9 (11), pp. 2380.
Awussi, A. A., Roux, É., Humeau, C., Hafeez, Z., Maigret, B., Chang, O.K., Lecomte, X., Humbert, G., Miclo, L., Genay, M., Perrin, C., Dary-Mourot, A.
Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.
Food & Function, 12, pp. 1415-1431.
Hafeez, Z., Benoit, S., Cakir-Kiefer, C., Dary, A., Miclo, L.
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 is 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 few numbers 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 data of the structure/activity relationship of anxiolytic peptides, their physiological effects displayed in in vitro and in vivo assays, bioavailability, and safety profiles.
Microorganisms, 9 (6), pp. 1113-1113.
Uriot, O., Kebouchi, M., Lorson-Dalibard, É., Galia, W., Denis, S., Chalançon, S., Hafeez, Z., Roux, É., Genay, M., Blanquet-Diot, S., Dary-Mourot, A.
Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic
acid bacterium widely used in dairy industry, requires further documentation of its physiological
status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo
technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under
simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library
were designed to positively select activated genes. Second, recombinant clones were introduced
into complementary models mimicking the human gut, the Netherlands Organization for Applied
Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine,
the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human
feces as a colon model. All inserts of activated clones displayed a promoter activity that differed
from one digestive condition to another. Our results also showed that S. thermophilus adapted its
metabolism to stressful conditions found in the gastric and colonic competitive environment and
modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated
in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses,
a first stage in the identification of gut resistance markers and a key step in probiotic selection.
Food Research International, 131 (x), pp. 108906-108906.
Kebouchi, M., Hafeez, Z., Le Roux, Y., Dary, A., Genay, M.
The mucus, mainly composed of the glycoproteins mucins, is a rheological substance that covers the intestinal epithelium and acts as a protective barrier against a variety of harmful molecules, microbial infection and varying lumen environment conditions. Alterations in the composition or structure of the mucus could lead to various diseases such as inflammatory bowel disease or colorectal cancer. Recent studies revealed that an exogenous intake of probiotic bacteria or other dietary components (such as bioactive peptides and probiotics) derived from food influence mucus layer properties as well as modulate gene expression and secretion of mucins. Therefore, the use of such components for designing new functional ingredients and then foods, could constitute a novel approach to preserve the properties of mucus. After presenting some aspects of the mucus and mucins in the gastrointestinal tract as well as mucus role in the gut health, this review will address role of dietary ingredients in improving mucus/mucin production and provides new suggestions for further investigations of how dietary ingredients/probiotics based functional foods can be developed to maintain or improve the gut health.
Journal of Dairy Science, 102 (1), pp. 113-123.
Hafeez, Z., Cakir-Kiefer, C., Lecomte, X., Miclo, L., Dary, A.
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-delta-pepX. When cultivated, the S. thermophilus LMD-9 wild type strain grew more rapidly than its delta-pepX mutant counterpart. Thus, the growth rate of the LMD-9-delta-pepX 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 β-casomorphin-7 was also observed. Indeed, when incubated with this peptide, the LMD-9-delta-pepX 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 high-lighted 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 β-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.
Journal of Functional Foods, 37, pp. 74-89.
Uriot, O., Denis, S., Junjua, M., Roussel, Y., Dary, A., Blanquet-Diot, S.
Probiotics are defined as live microorganisms that when administered in adequate amount confer a health benefit to the host. To be considered as a probiotic, a bacterial strain must not only be safe but should also survive in the human gastrointestinal tract and exert health benefits on its host. Streptococcus thermophilus is a Gram positive bacterium widely used in dairy fermentations for the production of yogurt and cheese. In contrast with other lactic acid bacteria, the probiotic status of S. thermophilus remains still questioned. This review gives an update of the human trials, in vivo assays in animal models and in vitro experiments, which have assessed the resistance of S. thermophilus to gastrointestinal stresses and have investigated its positive health effects. The underlying mechanisms of action are also described and the probiotic status of the bacterium is debated with respect to the available literature.
Dairy Science and Technology, 96, pp. 623-636.
Galia, W., Jameh, N., Perrin, C., Genay, M., Dary, A.
The acquisition of prtS by Streptococcus thermophilus strains allowed hydrolysis
of caseins into peptides and then to increase their growth in milk. This leads to faster
milk acidification, which is important in dairy industry. However, some strains harboring
the same allele of prtS present different acidification rates, which could be explained by a
difference in the regulation of prtS expression.We chose two strains with the same allele
of prtS (including the same promoter region): one, PB302, is with high acidification rate
while the other, PB18O, is without. They exhibited similar growth in M17, but not in
milk, where PB302 showed better growth. The expression of prtS and activity of PrtS
were lower in PB18O, in the two media tested.We demonstrated that other genes known
to be involved in carbon and nitrogen metabolism were overexpressed in PB302.
Interestingly, these genes were overexpressed in milk compared to M17. Nearly all these
genes possessed a putative CodY-box in their promoter region. Taken together, difference
of gene expression detected in PB302 between milk (low-peptide medium) and
M17 (rich-peptide medium) and presence of a putative CodY-box is a feature of the
transcriptional pattern of CodY-regulated genes. Altogether, our results propose that
acquisition of prtS is not enough in certain strains to achieve rapid milk acidification.
High transcriptional level of dtpT, amiF, ilvC, ilvB, bcaT, livJ, ackA, codY, and prtS in fast
acidifying strain suggests that this transcriptional pattern could be required for fast milk
acidification in Streptococcus thermophilus.
Food Research International, 86, pp. 34-45.
Jameh, N., Galia, W., Awussi, A. A., Roux, É., Genay, M., Perrin, C., Dary, A.
In silico analysis of the genome of Streptococcus thermophilus LMD-9 revealed that this strain has a potential new peptide/nickel ABC transporter. We named this system OTS for Oligopeptide Transporter of S. thermophilus. It is composed of a peptide/nickel binding protein OtsA, two permeases OtsB and OtsC and a double ATPase OtsD. This system was presumably acquired by horizontal transfer from Actinobacteria or distant species like Lactococcus raffinolactis or Enterococcus asini may be via an intermediate like Lactococcus lactis or its ancestor. RT-PCR experiments proved that OTS gene cluster is transcribed and that at least the otsB, otsC, and otsD genes constitute an operon. A mutant LMD-9?ots, partially deleted for the otsA and otsB genes was constructed. Growth of LMD-9 and LMD-9?ots strains was monitored in the presence of different nitrogen sources and in the presence of urea and nickel. Results revealed that OTS is not implicated in nickel transport, but constitutes a new characterized transporter of peptides of small size, possibly di- and tripeptides in S. thermophilus.
LWT - Food Science and Technology, 70 (1), pp. 78-87.
Junjua, M., Kechaou, N., Chain, F., Awussi, A. A., Roussel, Y., Perrin, C., Roux, É., Langella, P., Bermúdez-Humarán, L.G., Le Roux, Y., Chatel, J.-M., Dary, A.
In spite of its contribution to health benefits of yogurt, probiotic properties of Streptococcus thermophilus remain less explored. Hence, we evaluated the capacities of 30 strains of different origins, to resist the stresses prevailing in digestive tracts, of adhering to the mucus producing HT29-MTX cells, as well as their anti-inflammatory properties. First, on the basis of results obtained by multilocus sequence typing, two very closely related groups were distinguished phylogenetically. However, it appeared that in spite of this phylogenetic proximity, resistance to low pH, bile salts and H2O2 and their capacities of adhesion highly varied from one strain to another. Furthermore, most of the strains reduced the production of the pro-inflammatory interleukin IL-8 after co-incubation with HT-29 cells, while they induced production of the anti-inflammatory interleukin IL-10, when incubated with Peripheral Blood Mononuclear Cells. On the basis of ratio of synthesis of IL-10 and of IL-12, currently used to evaluate the anti-inflammatory potential of a probiotic bacterium, three strains appeared to display a strong and promising in vitro anti-inflammatory potential, suggesting that they could be appropriate for elaborating anti-inflammatory functional fermented foods. Finally, the Principal Component Analysis method enabled us to cluster strains into 6 classes displaying distinct phenotypic properties.
Applied Microbiology and Biotechnology, 100 (8), pp. 3667-3679.
Kebouchi, M., Galia, W., Genay, M., Soligot-Hognon, C., Lecomte, X., Awussi, A. A., Perrin, C., Roux, É., Dary, A., Le Roux, Y.
Streptococcus thermophilus (ST) is a lactic acid
bacterium widely used in dairy industry and displays several
properties which could be beneficial for host. The objective of
this study was to investigate, in vitro, the implication of
sortase A (SrtA) and sortase-dependent proteins (SDPs) in
the adhesion of ST LMD-9 strain to intestinal epithelial cells
(IECs) and resistance to bile salt mixture (BSM;
taurocholoate, deoxycholate, and cholate). The effect of mutations
in prtS (protease), mucBP (MUCin-Binding Protein),
and srtA genes in ST LMD-9 in these mechanisms were examined.
The HT29-MTX, HT29-CL.16E, and Caco-2 TC7
cell lines were used. HT29-MTX and HT29-CL.16E cells
express different mucins found in the gastro intestinal tract;
whereas, Caco-2 TC7 express cell surface proteins found in
the small intestine. All mutants showed different adhesion
profiles depending on cell lines. The mutation in genes srtA
and mucBP leads to a significant decrease in LMD-9 adhesion
capacity to Caco-2 TC7 cells. A mutation in mucBP gene has
also shown a significant decrease inLMD-9 adhesion capacity
to HT29-CL.16E cells. However, no difference was observed
using HT29-MTX cells. Furthermore, ST LMD-9 and srtA
mutant were resistant to BSM up to 3 mM. Contrariwise, no
viable bacteria were detected for prtS and mucBP mutants at
this concentration. Two conclusions could be drawn. First,
SDPs could be involved in the LMD-9 adhesion depending
on the cell lines indicating the importance of eukaryotic-cell
surface components in adherence. Second, SDPs could contribute
to resistance to bile salts probably by maintaining the
cell membrane integrity.
Food Microbiology, 53 (A), pp. 2-9.
Lecomte, X., Gagnaire, V., Lortal, S., Dary, A., Genay, M.
Streptococcus thermophilus is the second most used bacterium in dairy industry. It is daily consumed by millions of people through the worldwide consumption of yogurts, cheeses and fermented milks. S. thermophilus presents many features that make it a good candidate for the production of heterologous proteins. First, its ability to be naturally transformable allows obtaining swiftly and easily recombinant strains using various genetic tools available. Second, its Generally Recognised As Safe status and its ability to produce beneficial molecules or to liberate bioactive peptides from milk proteins open up the way for the development of new functional foods to maintain health and well-being of consumers. Finally, its ability to survive the intestinal passage and to be metabolically active in gastrointestinal tract allows considering S. thermophilus as a potential tool for delivering various biological molecules to the gastrointestinal tract. The aim of this review is therefore to take stock of various genetic tools which can be employed in S. thermophilus to produce heterologous proteins and to highlight the advantages and future trends of use of this bacterium as a heterologous expression host.
Food Microbiology, 53 (A), pp. 18-29.
Uriot, O., Galia, W., Awussi, A. A., Perrin, C., Denis, S., Chalancon, S., Lorson, É., Poirson, C., Junjua, M., Le Roux, Y., Alric, M., Dary, A., Blanquet-Diot, S., Roussel, Y.
Streptococcus thermophilus, a lactic acid bacterium used to produce yogurts and cheeses is more and more considered for its potential probiotic properties. This implies that additional information should be obtained regarding its survival and metabolic activity in the human Gastro-Intestinal Tract (GIT). In this study, we screened 30 S. thermophilus strains for urease, small heat shock protein, and amino-acid decarboxylase functions which may play a role in survival in the upper part of the GIT. The survival kinetics of 4 strains was investigated using the TIM, a physiologically relevant in vitro dynamic gastric and small intestinal model. The three strains LMD9, PB18O and EBLST20 showed significantly higher survival than CNRZ21 in all digestive compartments of the TIM, which may be related to the presence of urease and heat shock protein functions. When LMD9 bacterial cells were delivered in a fermented milk formula, a significant improvement of survival in the TIM was observed compared to non-fermented milk. With the RIVET (Recombinase In Vivo Expression Technology) method applied to the LMD9 strain, a promoter located upstream of hisS, responsible for the histidyl-transfer RNA synthesis, was found to be specifically activated in the artificial stomach. The data generated on S. thermophilus survival and its adaptation capacities to the digestive tract are essential to establish a list of biomarkers useful for the selection of probiotic strains.
Journal of Agricultural and Food Chemistry, 63 (34), pp. 7522-7531.
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Lecomte, X., Paris, C., Galia, W., Dary, A., Miclo, L.
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-?prtS and LMD-9-?prtS-?htrA 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.
International Dairy Journal, 49, pp. 78-88.
Matéos, A., Guyard-Nicodème, M., Baglinière, F., Jardin, J., Gaucheron, F., Dary, A., Humbert, G., Gaillard, J.-L.
UHT milk made from milk contaminated by Pseudomonas LBSA1 destabilised during storage. Sedimentation of UHT milk was observed; zeta potential of casein micelles decreased, while contents of noncasein nitrogen and non-protein nitrogen increased. Pseudomonas LBSA1 produced an extracellular
protease that hydrolysed caseins but not whey proteins; this was identified as AprX, a thermoresistant
protease belonging to the serralysin family. This protease showed a broad range of pH activity (pH 6 to
pH 10) and an optimal temperature of activity of 40°C. Peptides released from purified alpha-s1-, beta- and kappa-caseins were determined by tandem mass spectrometry. The identified cleavage sites did not reveal a strong specificity of the extracellular protease. However, the presence of basic or aromatic amino acid residues in the P1 position had a positive influence on cleavage in comparison with acidic amino acid residues or proline.
International Dairy Journal, 38 (2), pp. 104-115.
Chang, O.-K., Roux, É., Awussi, A. A., Miclo, L., Jardin, J., Jameh, N., Dary, A., Humbert, G., Perrin, C.
Bioactive peptides can be produced from milk proteins in fermented products by proteases of lactic acid
bacteria. The cell envelope protease (PrtS) of Streptococcus thermophilus is anchored at the cellwall, but we
recently discovered that the 4F44 strain produces a soluble form that can be recovered in medium supernatant.
This workwas aimed at optimising the production of bioactive peptides from bovine caseins. By
growing S. thermophilus 4F44 in the newly designed YLUNi medium, a high quantity of the soluble form of
PrtS could be produced that could be directly used as the proteolytic agent on sodium caseinate. Peptide
production was monitored by reverse phase-high performance liquid chromatography and tandem mass
spectrometry; of 247 peptides identified, 143 were derived from beta-casein. Twenty-two peptides, already
reported in the literature as bioactive, include ACE-inhibitory, antioxidant, immunomodulating, or antibacterial
peptides; addition of such peptides could improve the health benefits of dairy products.
Food Research International, 63 (A), pp. 71-80.
Hafeez, Z., Cakir-Kiefer, C., Roux, É., Perrin, C., Miclo, L., Dary, A.
Besides their basic nutritional role, dietary proteins contain bioactive peptides which are encrypted in their sequence and may modulate different body functions such as digestive, cardiovascular, immune and nervous systems, and therefore contribute in maintaining consumer health. Currently, milk proteins are considered to be the major source of bioactive peptides. The occurrence of these peptides has already been reported in fermented milk products such as yogurt, sour milk or kefir and some of them have been shown to confer health benefits. This review focuses on different strategies that could be employed to enhance the production of bioactive peptides from the milk proteins that will be consequently used to functionalize the fermented milk products. Three types of strategies are developed. The first exploits the proteolytic system of lactic acid bacteria (LAB) or food grade enzymes or combination of both to release the functional peptides from the milk proteins directly in the fermented milk products. The second concerns the supplementation of the fermented milk products with the bioactive peptides obtained outside of the product through the hydrolysis of the purified proteins by the same enzyme sources. Finally, the last consists in the production of the bioactive peptides, initially identified from the milk-proteins, by microorganisms using recombinant DNA technology.
Journal of Applied Microbiology, 116 (3), pp. 620-631.
Junjua, M., Galia, W., Gaci, N., Uriot, O., Genay, M., Bachmann, H., Kleerebezem, M., Dary, A., Roussel, Y.
Objectifs
Construire et valider l'outil R-IVET chez Streptococcus thermophilus (ST)
Matériel et méthodes et Résultats
Le système R-IVET que nous avons construit dans la souche LMD-9 comprend le plasmide pULNcreB qui permet la fusion transcriptionnelle avec le gène codant le recombinase site-spécifique Cre et la cassette chromosomique contenant un gène de résistance à la spectinomycine flanqué par 2 sites loxP. Sur milieu M17, les promoteurs des gènes codant la protéase PrtS, la protéine de choc thermique Hsp16 et l'opéron lactose ont conduit à la délétion de la cassette, indiquant une activité promotrice dans ces conditions. Le promoteur de l'opéron lactose était également activé durant le transit dans le tractus gastrointestinal d'une souris.
Conclusion
Le système R-IVET développé chez ST est relativement stable, fonctionnel, très sensible et peut être utilisé pour mesurer l'activité des promoteurs qui sont spécifiquement actifs in vivo.
Impact de l'étude
Cette première adaptation de l'outil R-IVET chez ST fournit un outil très précieux permettant une exploration de l'état physiologique de ST dans le TGI de mammifères, lors du processus de fermentation ou dans les produits laitiers.
To construct and validate the recombinase-based in vivo expression technology (R-IVET) tool in Streptococcus thermophilus (ST).
The R-IVET system we constructed in the LMD-9 strain includes the plasmid pULNcreB allowing transcriptional fusion with the gene of the site-specific recombinase Cre and the chromosomal cassette containing a spectinomycin resistance gene flanked by two loxP sites. When tested in M17 medium, promoters of the genes encoding the protease PrtS, the heat-shock protein Hsp16 and of the lactose operon triggered deletion of the cassette, indicating promoter activity in these conditions. The lactose operon promoter was also found to be activated during the transit in the murine gastrointestinal tract.
The R-IVET system developed in ST is relatively stable, functional, very sensitive and can be used to assay activity of promoters, which are specifically active in in vivo conditions.
This first adaptation of R-IVET to ST provides a highly valuable tool allowing an exploration of the physiological state of ST in the GIT of mammals, fermentation processes or dairy products.
Microbial Cell Factories, 13 (1), pp. 82-82.
Lecomte, X., Gagnaire, V., Briard-Bion, V., Jardin, J., Lortal, S., Dary, A., Genay, M.
CONTEXTE:
Des études fondamentales aux procédés industriels, la synthèse de protéines hétérologues par des micro-organismes est largement utilisée. La sécrétion de protéines hétérologues solubles dans le milieu extracellulaire facilite leur récupération, tandis que leur fixation à la surface de la cellule permet l'utilisation de cellules hôtes recombinantes comme supports de protéines ou de peptides. Un des points clés pour mener à bien l'expression hétérologue est de choisir l'hôte approprié. Nous proposons d'élargir le panel des hôtes de sécrétion hétérologues en utilisant Streptococcus thermophilus LMD-9. Cette bactérie lactique a un statut GRAS, est largement utilisée dans la fabrication de yaourts, laits fermentés et fromages, et est facilement transformable par compétence naturelle. Cette étude démontre la faisabilité de la sécrétion d'une protéine hétérologue ancrée à la surface de S. thermophilus. Pour cela, nous avons utilisé la cell envelope proteinase (CEP) PrtH de Lactobacillus helveticus CNRZ32 CIRM-BIA 103.
RÉSULTATS:
En utilisant S. thermophilus LMD-9 comme souche de base, trois souches recombinantes ont été construites: i) un contrôle négatif correspondant à S. thermophilus PrtS-mutant dans lequel le gène codant pour la CEP PrtS a été partiellement délété; ii) un mutant PrtH+ exprimant la pro-protéine PrtH de L. helveticus avec son propre motif d'attachement à la paroi cellulaire (de type S-layer) et iii) un mutant PrtH+WANS exprimant la pro-protéine PrtH avec le motif d'ancrage LPXTG de PrtS. Les niveaux d'expression des gènes prtH+ et prtH+WANS ont été mesurés par RT-PCR quantitative dans les mutants correspondants comparés à celui du gène prtS dans la souche sauvage LMD-9. Les niveaux d'expression des deux gènes recombinants, quel que soit le motif d'ancrage, ont atteint jusqu'à 76% du niveau d'expression de prtS. Les CEP ont été recherchées et identifiées sur la surface de la souche sauvage LMD-9 et des 2 mutants PrtH+ et PrtH+WANS en utilisant la technique du shaving suivie par l'identification des peptides par spectrométrie de masse, ce qui démontre que la sécrétion hétérologue et l'ancrage d'une protéine de plus de 200 kDa était efficace. L'ancrage à la paroi cellulaire semble être plus efficace lorsque le motif LPXTG de PrtS a été utilisé à la place du motif S-layer de PrtH.
CONCLUSIONS:
Nous avons démontré que S. thermophilus LMD-9 était capable de sécréter une protéine hétérologue de poids moléculaire élevé, et probablement de l'ancrer de manière covalente à la paroi cellulaire.
From fundamental studies to industrial processes, synthesis of heterologous protein by micro-organisms is widely employed. The secretion of soluble heterologous proteins in the extracellular medium facilitates their recovery, while their attachment to the cell surface permits the use of the recombinant host cells as protein or peptide supports. One of the key points to carry out heterologous expression is to choose the appropriate host. We propose to enlarge the panel of heterologous secretion hosts by using Streptococcus thermophilus LMD-9. This lactic acid bacterium has a generally recognised as safe status, is widely used in the manufacture of yogurts, fermented milks and cheeses, and is easy to transform by natural competence. This study demonstrates the feasibility of secretion of a heterologous protein anchored to the cell surface by S. thermophilus. For this, we used the cell envelope proteinase (CEP) PrtH of Lactobacillus helveticus CNRZ32 CIRM-BIA 103.
Using S. thermophilus LMD-9 as the background host, three recombinant strains were constructed: i) a negative control corresponding to S. thermophilus PrtS- mutant where the prtS gene encoding its CEP was partially deleted; ii) a PrtH+ mutant expressing the L. helveticus PrtH pro-protein with its own motif (S-layer type) of cell-wall attachment and iii) a PrtH+WANS mutant expressing PrtH pro-protein with the LPXTG anchoring motif from PrtS. The PrtH+ and PrtH+WANS genes expression levels were measured by RT-qPCR in the corresponding mutants and compared to that of prtS gene in the strain LMD-9. The expression levels of both fused prtH CEPs genes, regardless of the anchoring motif, reached up-to more than 76% of the wild-type prtS expression level. CEPs were sought and identified on the cell surface of LMD-9 wild-type strain, PrtH+ and PrtH+WANS mutants using shaving technique followed by peptide identification with tandem mass spectrometry, demonstrating that the heterologous secretion and anchoring of a protein of more than 200 kDa was efficient. The anchoring to the cell-wall seems to be more efficient when the LPXTG motif of PrtS was used instead of the S-layer motif of PrtH.
We demonstrated S. thermophilus LMD-9 could heterologously secrete a high molecular weight protein and probably covalently anchor it to the cell-wall.
Emirates Journal of Food and Agriculture, 26 (4), pp. 309-316.
El Hatmi, H., Jrad, Z., Khorchani, T., Dary, A., Girardet, J.-M.
The aim of this study was to investigate the radical-scavenging properties towards a stable radical cation, ABTS, of Camelus dromedarius whey proteins (CWP) separated onto a cation-exchanger by fast protein liquid chromatography. The highest activities were found for CWP and fraction F1 mainly composed of a-lactalbumin. Fractions F2, F3 and F4 contained a mixture of lactoferrin, immunoglobulins G and probably camel whey basic protein (CWBP). These three fractions displayed low radical-scavenging activities. Lactoferrin was eluted almost pure in the last fraction (F5) but did not possess detectable radical-scavenging activity. The present results suggested that the cation-exchange chromatography is of great interest to yield, in a single step, whey protein fractions with various biological activities, i.e. a highly-enriched α-lactalbumin fraction displaying efficient antioxidant activity, a fraction (pool of F2-F4) mainly composed of heavy-chain immunoglobulins potentially interesting for human therapy and a fraction of pure lactoferrin having numerous biological activities such as antimicrobial and immunomodulating properties.
International Dairy Journal, 31 (2), pp. 55-61.
Baglinière, F., Matéos, A., Tanguy, G., Jardin, J., Briard-bion, V., Rousseau, F., Robert, B., Beaucher, E., Gaillard, J.-L., Amiel, C., Humbert, G., Dary, A., Gaucheron, F.
Destabilisation of ultra high temperature (UHT) treated milk has been linked to residual proteolytic activity after UHT treatment. To understand the physico-chemical modifications of casein micelles by the protease AprX, produced by Pseudomonas fluorescens F, this enzyme was purified and added to raw milkbefore UHT treatment. Destabilisation of the UHT milk, over three months of storage, was investigated at macroscopic, colloidal and molecular scales. A visual destabilisation appeared progressively over time. At colloidal scale, aggregates were formed and a parallel decrease in zeta potential and hydration of casein micelles was observed. At molecular scale, peptides were released from casein micelles and identified by reversed-phase liquid chromatography coupled with tandem mass spectrometry. The alpha-S1-, alpha-S2-, beta- and kappa-caseins were hydrolysed, with a preference for beta-casein. The results were consistent with the proposition that proteolysis by Ps. fluorescens leading to the destabilisation of milk was due to the activity of AprX.
Applied Microbiology and Biotechnology, 97 (22), pp. 9787-9799.
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Jardin, J., Perrin, C., Dary, A., Miclo, L.
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 β-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.
Food Chemistry, 135 (4), pp. 2593-2603.
Baglinière, F., Tanguy, G., Jardin, J., Matéos, A., Briard, V., Rousseau, F., Robert, B., Beaucher, E., Humbert, G., Dary, A., Gaillard, J.-L., Amiel, C., Gaucheron, F.
Pseudomonas fluorescens grows at low temperature and produces thermo-resistant protease(s) that can destabilize UHT (Ultra High Temperature) milk during its storage. The consequences of contamination of microfiltered milk with 9 strains of P. fluorescens on the stability of the corresponding UHT milk during storage had been investigated in this study.
The strains were classified in two groups according to their ability to destabilize UHT milk. For the group of highly destabilizing strains, sedimentations of UHT milks, low values to phosphate test and the presence of aggregates were observed. Zeta potential and hydration of casein micelles decreased, whereas non casein nitrogen (NCN) and non protein nitrogen (NPN) contents increased. The analyses of NCN fraction by liquid chromatography coupled to mass spectrometry indicated that the different casein molecules were hydrolyzed in a similar way for the destabilizing strains suggesting that the same enzyme was implicated. For the group of slightly or not destabilizing strains no visual and biochemical alteration were found. This study showed that destabilization of UHT milk by P. fluorescens was highly variable and strain-dependent.
International Dairy Journal, 23 (2), pp. 91-98.
Chang, O.K., Perrin, C., Galia, W., Saulnier, F., Miclo, L., Roux, E., Driou, A., Humbert, G., Dary, A.
PrtS is the sole cell envelope protease (CEP) characterized in Streptococcus thermophilus. It is believed that it is anchored to the cell wall by sortase A (SrtA) through the LPXTG motif present at its C-terminus. Two soluble proteases corresponding to PrtS in its proenzyme and mature form were detected in the supernatant of S. thermophilus strain 4F44. In this strain, 60% of the PrtS molecules are anchored to the cell wall and 40% released in the medium. Such a release might result from a partial deficiency in the strain 4F44 of SrtA, even if its sequence slightly differs from that of S. thermophilus strain LMD-9, in which PrtS is anchored. Indeed, the presence of an intact LPXTG motif at the C-terminus of the released proteases showed that the linking process driven by SrtA did not occur and these proteases were not released by proteolysis after their anchoring.
Journal of Agricultural and Food Chemistry, 60 (2), pp 554-565.
Miclo, L., Roux, E., Genay, M., Brusseaux, E., Poirson, C., Jameh, N., Perrin, C., Dary, A.
Milk proteins contain numerous potential bioactive peptides, which may be released by digestive proteases or by the proteolytic system of lactic acid bacteria during food processing. The capacity of Streptococcus thermophilus to generate peptides, especially bioactive peptides, from bovine caseins was investigated. Strains expressing various levels of the Cell Envelope Proteinase, PrtS, were incubated either with αs1-, αs2- or β-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the β-casein was preferentially hydrolyzed first, followed by αs2-casein and then αs1-casein. Numbers and types of peptides released were strain-dependent. Hydrolysis appeared to be linked with the accessibility of different casein regions by protease. Analysis of bonds hydrolyzed in the region 1-23 of αs1-casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from β-casein, 5 from αs2-casein and 2 from αs1-casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors.
Journal of Agricultural and Food Chemistry, 59 (9), pp. 4464-4472.
Cakir-Kiefer, C., Le Roux, Y., Balandras, F., Trabalon, M., Dary, A., Laurent, F., Gaillard, J.-L., Miclo, L.
α-Casozepine is a peptide, corresponding to the sequence 91−100 of the bovine αs1-casein, displaying anxiolytic activity in the rat. The αs1-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.
Journal of Agricultural and Food Chemistry, 59 (22), pp. 11956–11965.
Cakir-Kiefer, C., Miclo, L., Balandras, F., Dary, A., Soligot, C., Le Roux, Y.
α-Casozepine and f91–97, peptides from αs1-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.
Journal of Dairy Science, 94 (6), pp. 2779-2793.
Dufour, D., Germon, P., Brusseaux, E., Le Roux, Y., Dary, A.
Mastitis pathogens belonging to Escherichia coli species are often considered as environmental opportunistic pathogens that invade the udder and are rapidly killed by the immune system of cows. However, several studies have reported that some of these strains are able to persist in the udder for prolonged periods or to adhere and invade mammary epithelial cells, suggesting that they might possess some specific properties or genes that could be involved in their capacity to provoke mastitis. The aim of this work was to search for such specific genes in the E. coli strain P4, which was isolated from a case of severe mastitis and is often used to induce experimental mastitis. We established that this strain belongs to phylogenetic group A of the E. coli species, and that its core genome is very similar to that of the commensal nonpathogenic strain E. coli K-12 MG1655. Seventeen transfer RNA loci, known to be frequently associated with genomic islands, were screened and an altered structure was detected for 7 of them. The partial characterization of 5 of these loci (asnT, leuX, pheV, serU, and thrW) and the complete characterization of 1 (argW) revealed the presence of genomic islands that differ from those already described in pathogenic or nonpathogenic E. coli strains.
Can the mammopathogenic Escherichia coli P4 strain have a direct role on the caseinolysis of milk observed during bovine mastitis?
Journal of Dairy Science, 92 (4), pp. 1398-1403.
Dufour, D., Jameh, N., Dary, A., Le Roux, Y.
During bacterial bovine mastitis, the quality of milk is altered because of caseinolysis. Endogenous potential actors in milk responsible for this caseinolysis have been well studied, unlike the exogenous bacterial ones. The aim of this study was to evaluate the direct role in caseinolysis of a mammopathogenic strain, Escherichia coli P4. Secretion of at least 4 extracellular bacterial caseinolytic enzymes was highlighted by zymography, in 3 different growth media, and at each bacterial growth state, suggesting that their expression was constitutive. Different experimental conditions to evaluate caseinolytic potential did not show any significant caseinolytic activity of E. coli P4 and of the 4 extracellular proteases detected, suggesting that the high caseinolysis observed during E. coli bovine mastitis does result from endogenous milk actors.
Variability and molecular typing of Streptococcus thermophilus strains displaying different proteolytic and acidifying properties
International Dairy Journal, 19 (2), pp. 89-95.
Galia, W., Perrin, C., Genay, M., Dary, A.
Proteolytic and acidifying properties of Streptococcus thermophilus strains isolated from yoghurt or cheeses were evaluated. Among 30 strains tested, 12 exhibited cell envelope-associated proteinase activity (PrtS+), three displayed a slight PrtS activity (PrtS+/−) and 15 were PrtS−, despite the presence of the corresponding gene (prtS) in eight of them. Sequencing of the prtS gene in four PrtS− and one PrtS+ strains revealed that the absence of PrtS activity in the PrtS− strain probably results from an alteration of the prtS regulation. The strains displaying the highest acidifying capacities were all PrtS+. All but one PrtS+ strains were phylogenetically close, as shown by the sequencing of their rDNA internal transcribed spacer (ITS) 16S-23S. More specifically, the high proteolytic and acidifying capacities are associated with the presence of a type II-ITS.
Two-dimensional cartography of equine beta-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon
Journal of Dairy Science, 92 (6) pp. 2389-2399.
Matéos, A., Girardet, J.-M., Mollé, D., Dary, A., Miclo, L., Gaillard, J.-L.
Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine β-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. β-Casein prepared from Haflinger mare’s milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that β-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the β-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10°C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of β-casein was achieved by mixing each phosphorylation isoform in its native state with the whole β-casein fraction.
Equine alpha-s1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels of multiple isoforms
Journal of Dairy Science, 92 (8), pp. 3604-3615.
Matéos, A., Miclo, L., Mollé, D., Dary, A., Girardet, J.-M., Gaillard, J.-L.
Alpha-s1-casein was isolated from Haflinger mare’s milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare’s alpha-s1-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of alpha-s1-casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine alpha-s1-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped.
Molecular typing of industrial strains of Pseudomonas spp. isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them
International Journal of Food Microbiology, 125 (2), pp. 188-196.
Dufour, D., Nicodème, M., Perrin, C., Driou, A., Brusseaux, E., Humbert, G., Gaillard, J.-L., Dary, A.
Implication of stringent response in the increase of mutability of the whiG and whiH genes during Streptomyces coelicolor development
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 624 (1-2), pp. 49-60.
Genay, M., Decaris, B., Dary, A.
Genetic instability of whiG gene during the aerial mycelium development of Streptomyces ambofaciens ATCC23877 under different conditions of nitrogen limitations
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 595 (1-2), pp. 80-90.
Genay, M., Catakli, S., Kleinclauss, A., Andrieux, A., Decaris, B., Dary, A.
Sigma factor WhiG and its regulation constitute a target of a mutational phenomenon occurring during aerial mycelium growth in Streptomyces ambofaciens ATCC23877
Research in Microbiology, 156 (3), pp. 328-340.
Catakli, S., Andrieux, A., Decaris, B., Dary, A.
Spontaneous chromosome circularization and amplification of a new amplifiable unit of DNA belonging to the terminal inverted repeats in Streptomyces ambofaciens ATCC 23877
Archives of Microbiology, 179 (6), pp. 387-393.
Catakli, S., Andrieux, A., Leblond, P., Decaris, B., Dary, A.
DNA rearrangements at the extremities of the Streptomyces ambofaciens linear chromosome: Evidence for developmental control
Biochimie, 82 (1), pp. 29-34.
Dary, A., Martin, P., Wenner, T., Decaris, B., Leblond, P.
Identification and typing of Streptomyces strains: Evaluation of interspecific, intraspecific and intraclonal differences by RAPD fingerprinting
Research in Microbiology, 151 (10), pp. 853-864.
Martin, P., Dary, A., Andre, A., Decaris, B.
Evolution of the linear chromosomal DNA in Streptomyces: Is genomic variability developmentally modulated?
Research in Microbiology, 150 (7), pp. 439-445.
Dary, A., Martin, P., Wenner, T., Leblond, P., Decaris, B.
Intraclonal polymorphism in the bacterium Streptomyces ambofaciens ATCC23877: Evidence for a high degree of heterogeneity of the wild type clones
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 430 (1), pp. 75-85.
Martin, P., Dary, A., Andre, A., Fischer, G., Leblond, P., Decaris, B.
Modulation of lipid metabolism and spiramycin biosynthesis in Streptomyces ambofaciens unstable mutants
Applied and Environmental Microbiology, 65 (6), pp. 2730-2737.
Schauner, C., Dary, A., Lebrihi, A., Leblond, P., Decaris, B., Germain, P.
Generation of a genetic polymorphism in clonal populations of the bacterium Streptomyces ambofaciens: Characterization of different mutator states
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 421 (1), pp. 73-82.
Martin, P., Dary, A., Decaris, B.
An amplifiable and deletable locus of Streptomyces ambofaciens RP181110 contains a very large gene homologous to polyketide synthase genes
Microbiology, 142 (10), pp. 2815-2824.
Aigle, B., Schneider, D., Morilhat, C., Vandewiele, D., Dary, A., Holl, A.-C., Simonet, J.-M., Decaris, B.
Large genomic rearrangements of the unstable region in Streptomyces ambofaciens are associated with major changes in global gene expression
Molecular Microbiology, 10 (4), pp. 759-769.
Dary, A., Kaiser, P., Bourget, N., Thompson, C.J., Simonet, J.-M., Decaris, B.
Amplification of a particular DNA sequence in Streptomyces ambofaciens RP181110 reversibly prevents spiramycin production
Research in Microbiology, 143 (1), pp. 99-112.
Dary, A., Bourget, N., Girard, N., Simonet, J.-M., Decaris, B.
Genetic instability in Streptomyces ambofaciens: inducibility and associated genome plasticity
Gene, 115 (1-2), pp. 49-54
Simonet, J.-M., Schneider, D., Volff, J.-N., Dary, A., Decaris, B.
Characterization of a family of multimeric ccc molecules of amplified chromosomal DNA in Streptomyces ambofaciens DSM 40697
FEMS Microbiology Letters, 78 (1), pp. 25-32.
Simonet, B., Dary, A., Decaris, B., Simonet, J.M.
Molecular cloning of a nosiheptide resistance gene from Streptomyces actuosus [CLONAGE D'UN GENE DE RESISTANCE AU NOSIHEPTIDE DE STREPTOMYCES ACTUOSUS]
Comptes Rendus de l'Academie des Sciences - Serie III, 308 (2), pp. 35-41.
Dary, A., Simonet, B., Simonet, J.-M., Decaris, B.
Brevet américain US 2021/0268040 publié le 02/09/2021
Dardevet, D., Auzeloux, I., Jarzaguet, M., David, J., Chatel, J.-M., Dary-Mourot, A., Cherbuy, C., Langella, P.
Brevet international WO 2020/020540 publié le 30/01/2020
Dardevet, D., Auzeloux, I., Jarzaguet, M., David, J., Chatel, J.-M., Dary-Mourot, A., Cherbuy, C., Langella, P.
Brevet français 3084255 déposé le 26/07/2018
Dardevet, D., Auzeloux, I., Jarzaguet, M., David, J., Chatel, J.-M., Dary-Mourot, A., Cherbuy, C., Langella, P.
FEBS 2023 - the 47th FEBS Congress, 08-12 juillet, Tours, France
Allouche, R., Hafeez, Z., Dary, A., Genay, M., Miclo, L.
Streptococcus thermophilus is a dairy starter granted “Generally Recognized as Safe” by the FDA and “Qualified Presumption of Safety” by EFSA. A significant part of the world's population ingests this bacterium when consuming fermented products. Some strains of S. thermophilus, either in the live or heat-inactivated state, and peptides released after shaving and hydrolysis of the surface proteins of some strains of this bacterium displayed anti-inflammatory activity in vitro (Allouche et al.,2022). S. thermophilus cells could undergo lysis during their passage through the digestive tract. Consequently, its intracellular proteins could be hydrolysed by endogenous proteases leading to the release of peptides. We hypothesized that peptides generated from digestion of intracellular protein of S. thermophilus might also contribute to its overall anti-inflammatory effect. Therefore, intracellular proteins from S. thermophilus CNRZ-21N strain were recovered after sonication. After fractionation by size exclusion chromatography, the resulting 3-10 kDa protein fraction was hydrolysed by Corolase PP, a mixture of pancreatic proteases. MS-MS analysis showed that most of the identified peptides belonged to the ribosomal proteins. The hydrolysed fraction showed anti-inflammatory activity on macrophagelike THP-1 cells inflamed by LPS since their secretion of IL-8 and IL-1β cytokines and expression level of Pro-IL-1β were reduced. The results suggest that the peptides released from a fraction of intracellular proteins of S. thermophilus after digestion by Corolase PP may contribute to the anti-inflammatory activity of this bacterium and could be used as a functional ingredient to prevent lowgrade inflammation.
5th Edition of Innovations in Food Science and Human Nutrition (IFHN-2022), 20-21 septembre, Barcelone, Espagne
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
23ème Colloque du Club des Bactéries Lactiques, 08-10 juin, Rennes, France
Allouche, R., Hafeez, Z., Papier, F., Dary-Mourot, A., Genay, M., Miclo, L.
Journée doctorale virtuelle franco-allemande et transfrontalière : Biotechnologies et Sciences de la Vie, 10 novembre, dématérialisée
Allouche, R., Hafeez, Z., Dary-Mourot, A., Genay, M., Miclo, L.
Séminaire de l'École Doctorale SIReNa, 13 février, Nancy, France
Allouche, R., Hafeez, Z., Dary, A., Genay, M., Miclo, L.
Streptococcus thermophilus is widely used as a starter culture in the dairy industry and has been awarded generally recognized as safe status (GRAS) by the American Food and Drug Administration. Some strains of S. thermophilus display an anti-inflammatory activity in vitro (Junjua et al., 2016). Inflammation is a part of the regular host reaction to injury or infection caused by pathogens, damaged cells, irritants and allergens. However, the mechanism of action by which this bacterium modulates inflammatory response remains unclear. It has been shown that the hydrolysis of food proteins or endogenous proteins by some digestive proteases releases peptides with various biological activities. Such peptides can also be generated by the surface proteolytic system of Lactic Acid Bacteria (Hafeez et al., 2014) as S. thermophilus, which produces bioactive peptides from bovine caseins (Miclo et al., 2012). These peptides are inactive within the sequence of the parent protein and display their activity after a hydrolysis step. Thus, the assumption that peptides generated in the gastro-intestinal tract from hydrolysis of S. thermophilus surface or intracellular proteins could display an anti?inflammatory activity and contribute to the overall anti-inflammatory effect of the bacterium can be made. Therefore, it is interesting to explore the role of such peptides in the modulation of inflammation. In a first approach, this study aims to investigate the anti-inflammatory properties of hydrolysates genrated after hydrolysis by gastrointestinal enzymes of surface proteins of S. thermophilus. The method involves the recovery of bacterial surface polypeptides by shaving with pepsin. Supernatant obtained after shaving was analysed by RP-HPLC and showed the release of peptides. The next challenge constitutes evaluation in in vitro cell model of anti-inflammatory activity of the peptides obtained and the characterisation of these peptides by mass spectrometry. This study will lead to novel insights into the modulation of host inflammatory response through probable action of peptides obtained from S. thermophilus.
43ème Colloque de la Société de Neuroendocrinologie, 2-4 octobre, Tours, France
Pinchaud, K., Hafeez, Z., Chatel, J.-M., Chadi, S., Auger, S., Dary, A., Maguin Gaté, K., Olivier, J.-L.
L’acide arachidonique (ARA) est le second acide gras polyinsaturé dans le cerveau. L’apport d’ARA est associé à la consommation d’aliments d’origine animale qui semble sous-estimée dans les régimes occidentaux. Une précédente étude de notre laboratoire a montré qu’un régime enrichi en ARA à hauteur de 1% augmente la sensibilité des souris BalB/C males à la neurotoxicité des oligomères de peptides β-amyloïdes, considérés comme étant un des principaux acteurs de la maladie d’Alzheimer. L’objectif de cette étude est d’évaluer l’impact d’un régime enrichi en ARA sur les fonctions cérébrales au travers de modifications du microbiote intestinal et d’altération des communications intestin-cerveau. Pour cela, deux groupes de souris BalB/C ont été nourries avec un régime moyennement hyperlipidique HL-ARA (15% de lipides non enrichi en ARA) ou HL+ARA (15% de lipides et enrichi en ARA à hauteur de 1%) ou encore Std-ARA 5% de lipides non enrichi en ARA) durant 8 semaines complètes. Notre étude n’a montré aucune différence significative concernant le poids des animaux durant l’expérimentation dans chacun des groupes, excepté une augmentation du poids du tissu adipeux mésentérique pour le groupe HL-ARA. Une augmentation du groupe de Bifidobacteriaceae (anti-inflammatoires) dans le microbiote intestinal des souris nourries avec le régime HL-ARA a été mise en évidence comparé au groupe Std-ARA. Cette augmentation de Bifidobacteriaceae est corrélée avec une plus forte présence de lipides dans le régime mais cet effet est renversé par l’ajout d’ARA dans le régime. Aucune modification des marqueurs de l’inflammation n’a été détecté dans le plasma et les fèces de chacun des groupes de souris, mais une augmentation du marqueur des astrocytes GFAP a été observée dans l’hippocampe des souris nourries avec le régime HL+ARA. Il serait intéressant d’étudier les altérations de communication entre l’intestin et cerveau incluant les signaux neuroendocriniens afin de comprendre la cascade impliquée dans la modulation des fonctions cérébrales par l’intestin.
Arachidonic acid (ARA) is the second polyunsaturated fatty acid in the brain. ARA intake is associated with consumption of animal origin products and seems to be underestimated in western diet. A previous study in our laboratory showed that a diet containing 1% ARA increased the sensitivity of male Balb/C mice to the neurotoxicity of the amyloid-β peptide oligomers, considered as the main Alzheimer’s disease agents. The objective of this study was to evaluate the impact of dietary ARA intake on brain functions through gut microbiota modifications and alteration of gut-brain communications. For this, two groups of male BalB/C mice were orally fed with moderately high fat diet, i.e., HL-ARA (15% lipid without ARA) and HL+ARA (15% lipid with 1% ARA) and the third group was fed on standard diet (Std-ARA, 5% lipids without ARA) during 9 weeks. No significant difference was observed in weight gain among the 3 groups except an increase in mesenteric fat tissue in HL-ARA diet group. An increase in Bifidobacteriaceae group (potentially anti-inflammatory) in gut microbiota of HL-ARA diet group was noted compared to standard diet group. This increase in Bifidobacteriaceae was correlated to high lipid contents in diet but this effect was reversed in HL+ARA diet group. No modifications in inflammatory markers were highlighted in plasma and feces samples of the three groups. Contrariwise, higher expression levels of the Glial fibrillary acidic protein were observed in hippocampus of HL+ARA group. It could be interesting to further investigate alterations of the gut-brain communications including neuroendocrine signals.
22nd International Conference of Functional Food Center (FFC) - 10th International Symposium of Academic Society for Functional Foods and Bioactive Compounds (ASFFBC) at Harvard Medical School, 22-23 septembre, Boston, États-Unis
Hafeez, Z., Perrin, C., Dary, A., Chevalot, I., Kapel, R., Chatel, J.M., Miclo, L.
Background:
Inflammation, a basic host defensive response, is crucial for resistance to injury, infectious agents or other noxious stimuli. Nevertheless, excessive and persistent inflammation often leads to various chronic diseases such as cardiovascular, osteoporosis, diabetes, obesity and gastro-intestinal inflammatory diseases. According to WHO, chronic diseases are the leading cause of morbidity and mortality both in developed and developing countries, and represent 60% of all deaths in the world. This figure rises to 87% in Europe and it is expected that more people will be affected by chronic diseases over the next few decades. Increased treatment-related health care costs have made chronic diseases a real health problem to the societies and the concern to prevent or treat these illnesses through diet has increased.
Daily diet is comprised of variety of nutrients including proteins. Bioactive peptides derived from dietary proteins particularly milk may modulate different body functions both at intestinal and systemic levels and ultimately contribute in maintaining consumer health. It has been shown that casein hydrolysates or peptides present in them exhibit anti-inflammatory activity by inhibiting and/or reducing the expression of inflammatory markers and/or by modulating their activity. For example, besides the fragment 106-169 of the bovine κ-casein, anti-inflammatory activity was demonstrated for 84VPP86 and 74IPP76 peptides from the bovine β-casein which reduce in vivo the mRNA expression of inflammatory cytokines (IL-6 and IL-1β). Three ways for releasing peptides from native proteins can be: (i) in vitro enzymatic hydrolysis, (ii) during gastrointestinal digestion or (iii) during fermentation by lactic acid bacteria.
Streptococcus thermophilus, a lactic acid bacterium previously known for conferring organoleptic properties to dairy products, has also been shown to generate bioactive peptides from milk proteins through its cell envelope proteinase (PrtS), which is anchored to cell wall by the transpeptidase sortase SrtA. However, during fermentation the pH decreases due to production of lactic acid and consequently the activity of PrtS is reduced or inhibited (optimal activity at pH 7.5), resulting in lower peptide contents. Therefore, to overcome this problem, S. thermophilus LMD-9-delta-srtA mutant strain was constructed to release in growth medium PrtS, which after purification was used to hydrolyze a caseinate to produce a hydrolysate with higher peptide content, which was afterward fractionated.
Objectives:
The main goal of the study was to evaluate the immunomodulatory potential of the peptide fractions obtained after fractionation by ultrafiltration and diafiltration of a hydrolysate resulting from the hydrolysis of a caseinate by PrtS purified from the growth culture of S. thermophilus LMD-9-delta-srtA strain, using peripheral blood mononuclear cells (PBMC) from 4 human donors.
Materials and Methods:
S. thermophilus LMD-9-delta-srtA strain was cultured for 8 h in yeast-lactose (YL) medium to obtain PrtS-rich supernatant. Followed by batch chromatography using diethylaminoethyl (DEAE) cellulose DE23 (Pharmacia, Uppsala, Sweden) resin and discontinuous gradient of NaCl, PrtS-rich fraction was concentrated by ultrafiltration using Amicon® Ultrafiltration system (cutoff threshold 50 kDa, Milipore, Jaffery, USA). The concentrated PrtS-rich fraction was then used to hydrolyze an industrial caseinate comprising of 87% proteins (92% casein and 8% whey proteins). This initial hydrolyzate (F1) was further fractionated by ultrafiltration using a 3 kDa cut-off membrane to isolate non-hydrolysed proteins in the form of retentate (F2) and peptide fraction as permeate (F3). The F3 fraction was then concentrated by ultrafiltration and diafiltration using a 1 kDa cutoff membrane to obtain retentate (F4) and permeate (F5). The peptide fractions were co-incubated in vitro with PBMCs (Clinisciences, Nanterre, France) and secretion of the IL-10 anti-inflammatory cytokine and of the IL-12 pro-inflammatory cytokine was measured in the cellular medium. The IL-10/IL-12 ratio makes it possible to evaluate the immunomodulatory potential of the peptide fractions.
Results:
Using DEAE cellulose DE23 batch chromatography with NaCl gradient, the PrtS was predominantly recovered in the fraction eluted by 0.4 M NaCl since about 90% of the initial activity was found in this fraction. Hydrolysis of caseinate by PrtS was not total since size-exclusion chromatography showed that a significant proportion of proteins remained unhydrolyzed under the conditions used. The protein concentration of the hydrolyzate was about 5 g/L with about 1.6 g/L of peptides. F4 fraction containing peptides whose molecular mass was between 1000 and 3000 Da was free of any salts contrary to F3 and F5 fractions. Anti-inflammatory activity of the five peptide fractions was evaluated by quantifying IL-10 and IL-12 production in vitro after co-incubating individually with PBMCs obtained from four healthy donors who did not use anti-inflammatory drugs for a significant period of time. All the fractions tested at concentrations of 0.2 and 1.0 mg of protein matter / mL led to a secretion of IL-10 by PBMCs except F3 and F5 where the secretion of IL-10 was observed only for concentration of 0.2 mg/mL. The absence of secretion with these fractions tested at 1.0 mg/mL was probably due to an excess of salt content. However, F4 fraction induced higher level of IL-10 production (between 70 and 400 pg/mL according to the PBMC donor) followed by F2 fraction regardless of concentration used. No IL-10 was detected when PBS alone was applied. Similarly, none of the peptide fraction favored the release of the pro-inflammatory cytokine IL-12. Results obtained with the cells of all the donors gave the same conclusions.
Conclusion:
PBMC treated with F4 fraction, among all peptide fractions, secreted higher levels of IL-10 in vitro and, therefore, this fraction displayed potential anti-inflammatory activity, as it did not trigger any secretion of IL-12. This peptide fraction is rich in peptides with molecular mass ranging between 1 and 3 kDa. Hence, milk proteins represent a promising source of peptides with potential anti-inflammatory activity and these peptides could be released directly by S. thermophilus or by its protease used as a biotechnological tool. It could be interesting to identify the peptide sequences of the F4 fraction in order to characterize it with the aim of using it in the development of functional fermented milk products.
21ème Colloque du Club des Bactéries Lactiques, 14-15 juin, Lille, France
Kebouchi, M., Genay, M., Uriot, O., Blanquet-Diot, S.-T., Lorson-Dalibard, É., Soligot-Hognon, C., Le Roux, Y., Dary, A.
Streptococcus thermophilus est largement utilisée dans l’industrie laitière pour fabriquer des yaourts et des fromages. Afin de définir son potentiel probiotique, nous avons (i) déterminé la capacité de S. thermophilus LMD-9 à adhérer à des lignées cellulaires (HT29-MTX, HT29-CL.16E et Caco-2) exprimant différentes mucines, (ii) identifié des gènes spécifiquement induits durant l’adhésion aux cellules Caco-2 en utilisant l’approche R-IVET (Recombinase-based In Vivo Expression Technology).
Nous avons montré que la capacité d’adhésion de LMD-9 variait suivant la lignée cellulaire utilisée. Elle adhérait plus aux cellules HT29-CL.16E qui sécrètent majoritairement la mucine intestinale MUC2, suggérant qu’elle pourrait coloniser les intestins in vivo et exercer des effets bénéfiques. Le rôle dans l’adhésion des gènes ctsR, srtA, prtS et mucBP, dont deux codent des protéines de surface pariétales (prtS, mucBP), a également été étudié. Le profil d’adhésion des mutants était différent selon les lignées cellulaires, montrant que l’implication des protéines CtsR, SrtA, PrtS et MucBP dans l’adhésion dépendrait des propriétés des cellules eucaryotes (mucus, type de mucine…).
L’analyse transcriptomique a révélé que le processus d’adhésion ne dépendait pas uniquement des protéines de surface mais d’autres voies métaboliques joueraient un rôle important. Nous avons identifié 31 gènes spécifiquement induits lors de l’adhésion : 4 codant des protéines de surface, 8 des transporteurs de nutriments, 3 des protéines de réponse au stress, 2 des protéines de compétence, 1 régulateur, 5 protéines hypothétiques et 9 autres.
Le processus d’adhésion est donc un processus complexe qui dépend à la fois des propriétés de surface bactériennes et eucaryotes.
Streptococcus thermophilus is widely used in the dairy industry to make yogurts and cheeses. In order to define its probiotic potential, we have (i) determined the ability of S. thermophilus LMD-9 to adhere to cell lines (HT29-MTX, HT29-CL.16E and Caco-2) expressing different mucins, (ii) identified genes specifically induced during adhesion to Caco-2 cells using the R-IVET approach (Recombinase-based In Vivo Expression Technology).
We have shown that the adhesion capacity of LMD-9 varies according to the cell line used. It adhered more to the HT29-CL.16E cells, which mainly secrete intestinal mucin MUC2, suggesting that it could colonize the intestines in vivo and have beneficial effects. The role in adhesion of ctsR, srtA, prtS and mucBP genes, two of which encode parietal surface proteins (prtS, mucBP), was also studied. The adhesion pattern of the mutants was different according to the cell lines, showing that the involvement of the proteins CtsR, SrtA, PrtS and MucBP in the adhesion would depend on the properties of the eukaryotic cells (mucus, mucin type ...).
Transcriptomic analysis revealed that the adhesion process did not depend solely on surface proteins, but other metabolic pathways would play an important role. We identified 31 genes specifically induced during adhesion: 4 encoding surface proteins, 8 nutrient transporters, 3 stress response proteins, 2 competency proteins, 1 regulator, 5 hypothetical proteins and 9 others.
The adhesion process is therefore a complex process that depends on both bacterial and eukaryotic surface properties.
Club des Bactéries Lactiques, 17-19 juin, Lille, France
Galia, W., Roussel, Y., Uriot, O., Junjua, M., Dary, A.
La bactérie Streptococcus thermophilus, largement utilisée comme levain lactique pour la fabrication de yaourt et de certains fromages, est maintenant considérée pour ces propriétés probiotiques potentielles. Afin d’envisager son utilisation comme vecteur probiotique, il est nécessaire de connaitre son état physiologique lors du passage dans le tractus gastro-intestinal (TGI). Pour cela, la technologie RIVET (Recombinase in vivo Expression Technology) a été développée pour S. thermophilus LMD9. C’est un outil de transcriptomique ciblée qui permet de détecter des promoteurs activés chez une bactérie se trouvant dans un environnement complexe. La méthodologie utilise une fusion transcriptionnelle au gène de la recombinase Cre dépourvu de son propre promoteur (cre’) en amont duquel sont insérées des séquences d’ADN de la souche à analyser.
L’outil RIVET que nous avons construit chez la souche LMD9 est constitué de deux composants, le plasmide pULNcreB portant la fusion transcriptionnelle et une cassette chromosomique contenant un gène de résistance à la spectinomycine bordé par deux sites lox, cibles de la recombinase Cre. Une banque a été construite dans pULNcreB avec des fragments d’ADN génomique de la souche LMD9 obtenus par digestion partielle et insérés dans le site de clonage situé en amont du gène cre’. Les plasmides recombinants ont été introduits dans la souche porteuse de la cassette chromosomique donnant 44 000 clones avec une taille moyenne d’insert de 0,5kb, ce qui permet d’estimer la représentativité de la banque à plus de 99% du génome.
Les clones de la banque RIVET ont été administrés à des souris Swiss par voie orale puis récupérés dans les selles après un transit d’une durée de 3 à 6 heures. Tous les clones devenus sensibles à la spectinomycine par recombinaison de la cassette RIVET ont été analysés puis la séquence de l’insert de leurs plasmides pULNcreB déterminée. Quinze séquences à l’activité promotrice induite lors du passage dans le TGI de la souris ont ainsi été identifiées.
3rd International congress of Translational Research in Human Nutrition, 26-27 juin, Clermont-Ferrand, France
Uriot, O., Junjua, M., Kechaou, N., Chain, F., Bermudez-Humaran, L., Langella, P., Alric, M., Dary, A., Chatel, J.-M., Blanquet-Diot, S.
The regular consumption of yogurt, which is the product of milk fermentation by the two lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii sp. bulgaricus, may have benefits for human health. Yogurt has been recently accredited by the European Food Safety Authority (EFSA) with the health claim of lactose digestion improvement. One of the two yogurt bacteria, S. thermophilus, has shown promising probiotic properties by preventing and/or improving gastro-intestinal diseases like colitis or infant diarrhea or by regulating immune responses. Up to date, most of the immunomodulatory properties of S. thermophilus was described in vitro in cell line models, using a limited number of strains. In addition, very little is known about the survival capacity of S. thermophilus throughout the human digestive tract. The aim of the present work was to better document the probiotic potential of S. thermophilus by exploring in cellular models the immunomodulatory properties of a large number of strains and its survival capacity in simulated human digestive conditions.
First, we screened thirty S. thermophilus strains from dairy products to assess their resistance to bile salts and acid pH and their anti/pro-inflammatory activity. The production of both anti-inflammatory and pro-inflammatory interleukins (IL-10, IL-8 and IL-12) was monitored after co-incubation of the strains with two different cellular models: HT29 intestinal cells and Peripheral Blood Mononuclear Cells (PBMC). Large variations in resistance to gastrointestinal stresses were observed. Some of the strains showed very interesting anti-inflammatory properties. Second, the survival of four selected strains was evaluated in the human stomach and small intestine using the dynamic TNO gastro-intestinal model (TIM). We showed that the survival of S. thermophilus was strain-dependent and can be improved if the bacteria were administered as fermented milk and not only inoculated in milk.
Thus, the present study shows that S. thermophilus is able to survive to gastro-intestinal transit in human. In vivo experiments are ongoing to investigate the anti-inflammatory potential of S. thermophilus during experimental colitis in mice. If the in vivo results confirm in vitro data on cellular models, it will give key arguments for a potential probiotic allegation for the selected strains and could lead to the development of fermented milk as functional food.
Printemps de la Cardiologie, 24-25 avril, Strasbourg, France
Zeder-Lutz, G., Zidane, F., Legrani, K., Dary, A., Miclo, L., Altschuh, D., Cakir-Kiefer, C.
Angiotensin I-converting enzyme (ACE), which is a key enzyme of the renin-angiotensin system, is one of target for antihypertensive molecules. Indeed, ACE is well known for its involvement in hypertension which is the main risk factor involved in the development of cardiovascular and kidney diseases and is a major cause of morbidity and mortality. Currently, many pharmacological ACE inhibitors (captopril, lisinopril…) are used for hypertension treatment, but their administration over a long period is associated with some undesirable side effects. Furthermore, there are many publications dealing with antihypertensive peptides from food proteins. ACE-inhibitory peptides, generated by hydrolysis of food proteins, may be a natural alternative to prevent hypertension appearance. However, among the bioactive peptides published in the literature as ACE inhibitors, a very small number really displays an antihypertensive activity in vivo in animals. Moreover, their mechanism of action at the molecular level is still misunderstood.
The objective of our work was to characterize the molecular interactions between ACE and some peptides described as ACE-inhibitors, which have or not a true antihypertensive activity in vivo.
For this purpose, a methodology already developed in our teams was employed (Zidane et al., 2013). It is based on the use, for the first time, of Biacore® technology (SPR). This real time technology provides some important molecular information such as the rate constants of association and dissociation, the stoichiometry and the site of the interaction on ACE. In our study, the direct interaction between ACE and inhibitors (without substrate or ligand) showed dissociation constants (KD) of the same order of magnitude as IC50. Moreover, the formed ACE-inhibitor complexes are unstable. Given these results, it is difficult to attribute significant antihypertensive effect demonstrated in vivo for these peptides (for example IPP, VPP) to the only ACE inhibition.
Young Researchers in Life Sciences, 26-28 mai, Paris, France
Kebouchi, M., Galia, W., Genay, M., Soligot-Hognon, C., Dary, A., Le Roux, Y.
International Pharmabiotics Conference, 29-30 octobre, Paris, France
Uriot, O., Roussel, Y., Denis, S., Chalancon, S., Alric, M., Dary, A., Blanquet-Diot, S.
Yogurt, which is the product of milk fermentation by the two lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii sp. Bulgaricus, has been accredited by the European Food Safety Authority (EFSA) with the health claim of lactose digestion improvement. The very long history of S. thermophilus use in food industry led to the GRAS (Generally Recognized As Safe) status assignment by the American Food and Drug Administration and the QPS (Qualified Presumption of Safety) status by EFSA. Recent in vitro and in vivo studies in animals have shown that S. thermophilus may have beneficial effects on human health. Nevertheless, in contrast with other lactic acid bacteria, the probiotic status of S. thermophilus is still questioned. In order to rule on this issue, it is important to investigate the fate and physiologic status of the bacteria when they are in transit through the human gastro-intestinal tract (GIT).
In the present study, we first investigated the survival capacities of four S. thermophilus strains through the upper human GIT using the dynamic TNO gastro Intestinal Model (TIM). In addition, as it is more and more accepted that the way bacteria are administered is decisive for their survival, we assessed the effect of food matrix on S. thermophilus survival. The RIVET (Recombinase-based in vivo Expression Technology) strategy was then used to explore the physiologic status of S. thermophilus and identify which genes are specifically expressed in the human GIT. These data are essential for an effective selection of strains used in functional food product development and for a potential probiotic allegation for S. thermophilus.
3rd International Conference on Food Digestion, 11-13 mars, Wageningen, Pays-Bas
Uriot, O., Roussel, Y., Denis, S., Galia, W., Alric, M., Dary, A., Blanquet-Diot, S.
Regular consumption of yogurt containing living bacteria provides some health benefits such as improvement of lactose digestibility. Streptococcus thermophilus is one of the two bacteria used to produce yogurt and is also commonly used in cheese making. In order to better understand the interactions between S. thermophilus and the consumer, it is important to investigate the fate and physiologic status of the bacteria when they are in transit through the human gastro-intestinal tract (GIT).
In this study, we first analyzed the survival capacities through the upper human GIT of three S. thermophilus strains using a dynamic gastrointestinal model, the TIM (TNO gastro Intestinal Model) system. The S. thermophilus cells inoculated in a milky liquid matrix were found to be sensitive to the low gastric pH and to the bile salts of the small intestine. When the bacterial cells were delivered in fermented clotted milk, a significant improvement in the survival rate was observed.
The RIVET (Recombinase-based in vivo Expression Technology) strategy was then used to explore the physiologic status of S. thermophilus in the human GIT. This approach enables the identification of genes which are specifically expressed in a complex environment. When applied to the gastric compartment of the TIM, two genes were detected: STER_1905 (hisS) responsible for the histidyl-transfer RNA synthesis and STER_1406 encoding the component of an ABC-type oligopeptide/nickel transport system. Analyses of these two genes and other RIVET functions activated in the stomach and small intestine will provide valuable information on bacterial survival factors. These data are essential for an effective selection of strains used in functional food product development.
19e Colloque du Club des Bactéries Lactiques, 16-18 octobre, Bordeaux, France
Genay, M., Lecomte, X., Briard-Bion, V., Jardin, J., Lortal, S., Gagnaire, V., Dary, A.
La croissance en lait de S. thermophilus et Lb. helveticus dépend de leur système protéolytique, notamment de CEP (Cell Envelope Proteinase) qui hydrolysent les caséines en peptides. Lb. helveticus et S. thermophilus présentent respectivement de 0 à 4 CEP (PrtH à PrtH4) et 0 à 1 CEP (PrtS). Connaître leur activité et spécificité de substrat indépendamment permettra d’orienter la protéolyse dans les produits fermentés selon les souches utilisées. La réalisation de mutants chez Lb. helveticus étant difficile, nous avons choisi d’exprimer de façon hétérologue le gène prtH dans une bactérie lactique naturellement transformable, protéolytique, et qui croît dans les mêmes conditions, S. thermophilus LMD-9. Trois mutants ont été obtenus par remplacement de prtS de LMD-9 : i) PrtS- (prtS remplacé par ery), ii) PrtH+ (exprimant PrtH avec son propre domaine d’ancrage de type S-Layer) et iii) PrtH+WANS (exprimant PrtH avec l’ancrage de PrtS (motif LPXTG)). En lait, les gènes introduits présentent un niveau d’expression similaire à celui de prtS (RT-PCRq). Un shaving de la surface des cellules de S. thermophilus et de l’HPLC-MSMS a permis de détecter 18 protéines extracellulaires, dont HtrA et PrtS chez LMD-9 et PrtH chez les mutants PrtH+ et PrtH+WANS. La présence de formes ancrées immatures et matures de PrtS suggère une automaturation alors que seule la forme immature de PrtH a été détectée, suggérant cette fois-ci la nécessité d’une maturase, comme c’est le cas pour d’autres protéases. Ces résultats montrent que S. thermophilus est un bon outil pour exprimer et ancrer à la paroi des protéines hétérologues d’intérêt. Nos perspectives concernent maintenant la caractérisation de l’activité et de la spécificité des différentes CEP de Lb. helveticus.
The growth in milk of S. thermophilus and Lb. helveticus depends on their proteolytic system, including CEP (Cell Envelope Proteinase) that hydrolyze caseins in peptides. Lb. helveticus and S. thermophilus harbour respectively 0 to 4 CEP (PrtH to PrtH4) and 0 to 1 CEP (PrtS). Better understand their activity and substrate specificity independently will permit to control proteolysis in fermented products according to the strain used. Construction of mutants of Lb. helveticus is not easy, so we chose to express heterologously the prtH gene in a lactic acid bacterium naturally transformable, proteolytic, and growing under the same conditions: S. thermophilus LMD-9. Three mutants were obtained by replacing prtS in LMD-9: i) PrtS- (prtS replaced by ery), ii) PrtH+ (expressing PrtH with its own anchor domain (S-layer) and iii) PrtH+WANS (expressing PrtH with PrtS anchor (LPXTG motif)). In milk, the introduced genes have a similar level of expression to that of prtS (RT-qPCR). A shaving of the cell surface of S. thermophilus and HPLC-MSMS analyses have permit to detect 18 extracellular proteins, for example HtrA and PrtS in LMD-9 and PrtH in PrtH+ and PrtH+WANS mutants. The presence of immature and mature forms of anchored PrtS suggests an autoprocessing maturation while only the immature form of PrtH was detected, suggesting the need for a maturase, as it is the case for other proteases. These results show that S. thermophilus is a good tool to express and anchor to the wall heterologous proteins of interest. Our prospects concern the characterization of the activity and specificity of the different CEP of Lb. helveticus.
8th NIZO dairy conference, 11-13 Septembre, Papendal, Pays-Bas.
Roux, É., Miclo, L., Chang, O.-K., Humbert, G., Dary, A., Perrin, C.
EuroFoodChem XVII, 07-10 mai, Istanbul, Turquie
Zidane, F., Zeder-Lutz, G., Altschuh, D., Dary, A., Miclo, L., Cakir-Kiefer, C.
19e Colloque du Club des Bactéries Lactiques, 16-18 octobre, Bordeaux, France
Roussel, Y., Blanquet-Diot, S., Alric, M., Langella, P., Le Roux, Y., Chatel, J., Dary, A.
Streptococcus thermophilus est massivement consommée au travers des aliments laitiers fermentés tels que les yaourts et les fromages. Afin de mettre en évidence les interactions existant avec l’hôte, nous avons analysé l’état physiologique de cette bactérie dans des modèles complémentaires du tractus gastro-intestinal (TGI) (1) dans des conditions in vitro reproduisant certains stress rencontrés dans le TGI ; (2) dans le système TIM-1 simulant de manière dynamique l’estomac et l’intestin grêle humain et (3) dans le TGI de souris. Un criblage d’analyse de croissance réalisé sur 30 souches nous a permis de montrer une grande variabilité de résistance aux pH acides et aux sels biliaires. La capacité d'adhésion de ces souches mesurée sur les cellules HT29-MTX productrice de mucus a également montré une grande variabilité. Le dosage des cytokines IL-10 et IL-12 produites par des cellules mononuclées du sang périphérique (PBMC) a permis d’identifier des souches au potentiel anti-inflammatoire élevé. La capacité de survie de 3 souches a été testée dans le système TIM-1 où nous avons observé une réduction du nombre de bactéries de l’ordre de 2 à 3 Log, résultant probablement du faible pH stomacal et de la forte concentration de sels biliaires dans l’intestin grêle. La méthodologie RIVET (Recombinase In Vivo expression Technology) appliquée à la souche LMD-9 a permis d’identifier 16 gènes activés lors du transit dans le GIT de souris et 2 gènes activés dans le compartiment gastrique du TIM-1. L’analyse des fonctions bactériennes activées dans le TGI permettra de mieux comprendre la physiologie de S. thermophilus dans l’environnement digestif humain et d’identifier les gènes impliqués dans sa survie et son interaction avec l’hôte.
19e Colloque du Club des Bactéries Lactiques, 16-18 Octobre, Bordeaux, France.
Awussi, A. A., Roux, É., Chang, O.-K., Humbert, G., Dary, A., Perrin, C.
8th NIZO dairy conference, 11-13 septembre, Papendal, Pays-Bas
Lecomte, X., Genay, M., Briard-Bion, V., Jardin, J., Lortal, S., Gagnaire, V., Dary, A.
Streptococcus thermophilus et Lactobacillus helveticus sont des bactéries lactiques utilisées comme co-starters dans la fabrication de produits laitiers fermentés. Leur croissance est dépendante d'un apport en acides aminés par le système protéolytique, composé de protéases pariétales (CEP pour Cell Envelope Proteinase) qui hydrolysent les caséines du lait en peptides, de transporteurs de peptides et de peptidases intracellulaires clivant les peptides en acides aminés libres. Alors que la plupart des bactéries lactiques possède une CEP, les souches de L. helveticus peuvent présenter de 1 à 4 gènes de CEP. Par exemple, chez L. helveticus CNRZ32, les quatre protéases PrtH, PrtH2, PrtH3 et PrtH4 sont potentiellement présentes sur la paroi cellulaire et pourraient participer à la génération de peptides techno- et bio-fonctionnels. À ce jour, l'activité et la spécificité de chaque CEP ne sont toujours pas clairement connues même si elles constituent un défi industriel pour la sélection de souches. Les stratégies biochimiques et génétiques se sont révélées inefficaces pour étudier chaque CEP séparément, nous avons donc décidé d'exprimer les CEP à la surface d'une autre bactérie lactique. Notre stratégie a été d'exprimer PrtH sur la paroi cellulaire de S. thermophilus LMD9. Cette souche a été choisie parce qu'elle pousse dans les mêmes conditions de culture que L. helveticus, elle exprime une unique CEP, PrtS, et est naturellement transformable. Trois mutants ont été obtenus par remplacement allélique de PrtS : i) un contrôle négatif PrtS- (gène prtS délété), ii) un mutant PrtH+ exprimant PrtH avec son motif d'ancrage de type S-layer et iii) un mutant PrtH+WANS exprimant PrtH fusionnée au domaine d'ancrage de PrtS. Tous les gènes sont exprimés comme indiqué par RT-qPCR. Cependant, seule la protéase PrtH+WANS a été détectée par shaving des protéines de la paroi cellulaire et identification de ces peptides par HPLC-MS/MS. Ces premiers résultats montrent que S. thermophilus est un bon outil pour sécréter et ancrer des protéines hétérologues. L'étape suivante consiste à exprimer les trois autres protéases de L. helveticus et de déterminer leur activité catalytique sur les protéines du lait.
Streptococcus thermophilus and Lactobacillus helveticus are lactic acid bacteria (LAB) used in fermented dairy products as co-starters. Their growth depends on the supply of amino acids by a proteolytic system, which contains cell envelope proteinases (CEP) hydrolyzing milk proteins in peptides, peptide transporters and intracellular peptidases hydrolyzing peptides into amino acids. Whereas most LAB possess one CEP, L. helveticus strains can display one to four CEP genes. In L. helveticus CNRZ32, the four PrtH, PrtH2, PrtH3 and PrtH4 are potentially present on the cell?wall and participate to the generation of techno- and bio-functional peptides. To date, activity and specificity of each CEP is still not established although it is an industrial challenge for strain selections. As biochemical and genetical strategies have been ineffective to study each CEP, we decided to express CEP on another LAB surface. Our strategy was to express PrtH on the cell?wall of S. thermophilus LMD9. This strain was chosen because it grows under the same culture conditions as L. helveticus, expresses a unique CEP, PrtS, and is naturally transformable. Three mutants were obtained by allelic replacement of prtS: i) a negative control PrtS- with prtS deleted, ii) a PrtH+ mutant expressing PrtH, using the lactobacilli S-Layer motif as anchoring domain and iii) a PrtH+WANS mutant expressing PrtH fused with PrtS cell-wall spacer and anchor domains. All genes were expressed as shown by RT-qPCR. However, only PrtH+WANS proteinase was detected by shaving of the cell-wall proteins and peptide identification by chromatography coupled on line with ESI orbitrap tandem mass spectrometry. These first results show that S. thermophilus is a good tool to secrete and anchor heterologous proteins on the cell wall. Next step is to express the three others L. helveticus proteinases and to determine their catalytic activity on milk proteins.
18e Colloque du Club des Bactéries Lactiques, 22-24 mai, Clermont-Ferrand, France
Chang, O.-K., Perrin, C., Roux, É., Miclo, L., Humbert, G., Dary, A.
Colloque Adebiotech-SFGP "Peptides issus des procédés d'hydrolyse - Filières Industrielles", 2-3 octobre, Romainville, France
El Hatmi, H., Cakir-Kiefer, C., Miclo, L., Dary, A., Girardet, J.-M.
16th World Congress of Food Science and Technology (IUFoST) & XVII Latin American Seminar of Food Science and Technology (ALACCTA), 5-9 août 2012, Foz do Iguaçu, Brésil
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Dary, A., Miclo, L.
Traditional foods can be functionalized by bioactive peptides either by natural fermentation or ripening and/or by their addition in the form of food ingredients. Milk proteins had been shown as a main source of bioactive peptides. Alpha-casozepine, alpha-s1-CN(f91-100), is a bioactive peptide released by tryptic hydrolysis of bovine alpha-s1-casein that exhibits in vivo anxiolytic activity in three different behavioral tests in rats. The hydrolysate containing this peptide proved effective in clinical studies. In vitro hydrolysis of alpha-casozepine with proteolytic enzymes such as pepsin and chymotrypsin released N-terminal shorter fragment alpha-s1-CN-(f91-97) named heptapeptide that also possesses the same activity. Currently, it has been shown that various Streptococcus thermophilus strains were able to hydrolyze alpha-s1-, alpha-s2- and beta-casein with different efficiency and to release many bioactive peptides but neither alpha-casozepine nor heptapeptide was liberated by the 30 strains of our collection tested. Therefore, to develop a functional food containing such peptides, they could be added directly in dairy products (e.g. fermented milks) and should be resistant to degradation by the proteolytic system of S. thermophilus. The experiments revealed that many bioactive peptides including the heptapeptide were degraded by different S. thermophilus strains even when the strains were lacking the cell surface-associated protease PrtS. Indeed, cell surface-associated aminopeptidase and X-prolyl-dipeptidyl aminopeptidase activities were detected. Therefore, to overcome this problem, we are developing a strategy in which we clone a multicopy of anxiolytic heptapeptide in a plasmid and introduce it into bacteria to produce this peptide as a multimer directly in food product.
18e Colloque du Club des Bactéries Lactiques, 22-24 mai, Clermont-Ferrand, France
Hafeez, Z., Cakir-Kiefer, C., Girardet, J.-M., Dary, A., Miclo, L.
In the last few decades, increased consumers awareness about the health benefits of food products has encouraged the development of various health promoting functional foods. These foods not only contribute to the nutritional requirements but also prevent certain diseases, in order to improve the physical and mental well-being of consumers. In some cases, a bioactive peptide of food origin constitutes the active component of these products. Milk proteins are known to be a major source of bioactive peptides that may impart extra-nutritional benefits. These peptides are encrypted in the sequence of caseins and are released only after protein proteolysis in vitro or by gastric or pancreatic enzymes during the digestion of proteins in vivo or by the enzymes of lactic acid bacteria during manufacture of fermented dairy products. S. thermophilus, that possesses its own proteolytic system, is one of the most widely used lactic acid bacteria in the manufacture of fermented dairy products (yoghurt, cheese). The proteolytic system of S. thermophilus is comprised of cell surface proteinase, peptide transport system and a pool of intracellular peptidases. Thus, the question arises whether bioactive peptides added in the fermented dairy products could remain intact or not in presence of this bacterium. To answer this question, two S. thermophilus strains differing in their cell surface proteolytic activity were evaluated: strain LMD9 which possesses cell envelope proteinase activity (PrtS+) and strain CNRZ1066 without this activity (PrtS-). We studied the resistance of some milk protein derived peptides with different biological activities to S. thermophilus cell wall proteolytic system. We then incubated the peptides, namely anxiolytic [YLGYLEQ - alpha-s1-CN-(f91-97)], antihypertensive [TTMPLW - alpha-s1-CN-(f194-199), FALPQYLK - alpha-s2-CN-(f174-181)] and opioid peptide (YPFPGPI - beta-casomorphin-7, RYLGYLE - alpha-s1-CN-(f90-96)] with the non proliferating cells of LMD-9 and CNRZ1066 strains in phosphate / acetate buffer (12.5 mM, pH 6.5). Surprisingly, two different cell wall-anchored peptidase activities were highlighted. The peptides YLGYLEQ, TTMPLW, RYLGYLE, and FALPQYLK were hydrolyzed by both the strains in a similar way. Liquid chromatography coupled to mass spectrometry analysis revealed that breakdown fragments were generated after successive cleavages of amino acids from the N-terminal of these peptides which provides an evidence of an aminopeptidase activity. However, in case of beta-casomorphin-7, an X-prolyl dipeptidyl aminopeptidase activity was observed which is involved in removal of N-terminal dipeptides containing N-terminal prolyl residue. All the peptides remained stable upon incubation in cell free filtrate of these strains which showed that no intracellular peptidases were released and thus involved in the proteolysis process. Therefore, these peptidase activities may help in optimal bacterial growth which consequently increases the milk fermentation rate but at the same time may also cause degradation of bioactive peptides present or added in the fermented milk products. Hence, screening a strain of S. thermophilus having reduced cell wall proteolytic activity would be required for the production of functional fermented dairy products.
18e Colloque du Club des Bactéries Lactiques, 22-24 mai, Clermont-Ferrand, France
Junjua, M., Chain, F., Kechoua, N., Roussel, Y., Le Roux, Y., Langella, P., Bermúdez-Humarán, L., Chatel, J.-M., Dary, A.
18e Colloque du Club des Bactéries Lactiques, 22-24 mai, Clermont-Ferrand, France
Lecomte, X., Genay, M., Gagnaire, V., Lortal, S., Dary, A.
Actes du séminaire de l'École Doctorale RP2E, 19 janvier, Nancy, France
Hafeez, Z., Miclo, L., Girardet, J.-M., Dary, A., Cakir-Kiefer, C.
Actes du séminaire de l'École Doctorale RP2E, 19 janvier, Nancy, France
Hafeez, Z., Miclo, L., Girardet, J.-M., Dary, A., Cakir-Kiefer, C.
Bénéfices nutrition santé des micro-organismes d'intérêt laitier. Travaux de la Mission Scientifique Syndifrais, 28 mai, Paris, France
Dary, A.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Dary, A., Miclo, L., Cakir-Kiefer, C., Roux, E., Humbert, G., Driou, A., Perrin, C.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Hafeez, Z., Cakir-Kiefer, C., Perrin, C., Dary, A., Miclo, L.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Jameh, N., Perrin, C., Carré, J., Galia, W., Roux, E., Dary, A.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Junjua, M., Roussel, Y., Dary, A.
17e Colloque du Club des Bactéries Lactiques (CBL), 27-29 octobre, Nancy, France
Roux, E., Miclo, L., Brusseaux, E., Poirson, C., Perrin, C., Dary, A.
16e Colloque national de la recherche dans les IUT, 9-11 juin, Angers, France
Junjua M., Genay M., Roussel Y., Dary A.
Actes du séminaire de l'école doctorale RP2E, 28 janvier, Nancy, France.
Jameh, N., Perrin, C., Genay, M., Galia, W., Dary, A.
16e Colloque du Club des Bactéries Lactiques, 27-29 mai, Toulouse, France
Chang O.K., Perrin C., Saulnier F., Driou A., Galia W., Humbert G., Miclo L., Dary A.
Titre : Existence d'une forme extracellulaire de PrtS chez Streptococcus
thermophilus.
Auteur(s) : O. K. Chang, C. Perrin, F. Saulnier, A. Driou, W. Galia, G.
Humbert,
L. Miclo et A. Dary
16e Colloque du Club des Bactéries Lactiques, 27-29 mai 2009, Toulouse, France.
4th IDF Dairy Science and Technology Week, 20-24 avril, Rennes, France
Galia, W., Perrin, C., Jameh, N., Genay, M., Dary, A.
Cloning of a resolvase gene from Lactococcus lactis and potential use in molecular genetics
16e Colloque du Club des Bactéries Lactiques, 27-29 mai, Toulouse, France
Roussel, Y., Genay, M., Junjua, M., Shearman, C., Gasson, M., Dary, A.
Actes du séminaire de l'école doctorale RP2E, 15 janvier, Nancy, France
Chang, O.K., Perrin, C., Driou, A., Galia, W., Humbert, G., Miclo, L., Dary, A.
Recherche de facteurs de virulence chez Escherichia coli P4O32, bactérie inductrice de mammites bovines.
Séminaire de l'École Doctorale RP2E, 26 janvier, Nancy, France
Dufour, D., Le Roux, Y., Dary, A.
Instabilité génétique des gènes whiG et whiH lors du développement de Streptomyces ambofaciens et S. coelicolor : implication de la réponse stringente.
Rencontre du Club des Actinomycètes, juin, Lyon, France
Genay, M., Decaris, B., Dary, A.
Modification de l’instabilité génétique au cours du développement de Streptomyces ambofaciens et Streptomyces coelicolor en fonction de la quantité de nutriments du milieu
Rencontre du Club des Actinomycètes, 6-7 mai, Liège, Belgique
Genay, M., Catakli, S., Dary, A., Decaris, B.
Rapport de contrat avec le Centre Interprofessionnel de l’Économie Laitière (CNIEL), 55 p.
Miclo, L., Brusseaux, E., Poirson, C., Perrin, C., Dary, A.
