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

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

Streptococcus thermophilus, an emerging and promising tool for heterologous expression: Advantages and future trends.

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.

Use of the dynamic gastro-intestinal model TIM to explore the survival of the yogurt bacterium Streptococcus thermophilus and the metabolic activities induced in the simulated human gut.

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.

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

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

The naturally competent strain Streptococcus thermophilus LMD-9 as a new tool to anchor heterologous proteins on the cell surface.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: We demonstrated S. thermophilus LMD-9 could heterologously secrete a high molecular weight protein and probably covalently anchor it to the cell-wall.

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

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

Quantitative and qualitative variability of the caseinolytic potential of different strains of Pseudomonas fluorescens: implications for the stability of casein micelles of UHT milks during their storage.

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.

Variability of hydrolysis of ß-, αs1-, and αs2-caseins by 10 strains of Streptococcus thermophilus and resulting bioactive peptides.

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 with α(s1)-, α(s2)-, or ß-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the ß-casein was preferentially hydrolyzed, followed by α(s2)-casein and then α(s1)-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 α(s1)-casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from ß-casein, 5 from α(s2)-casein, and 2 from α(s1)-casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors.

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

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

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

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

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.

P. fluorescens is responsible for the highest depredation of milk because of its capacity to synthesize extracellular lipase and protease which hydrolyze milk fat and proteins. Several P. fluorescens synthesize an extracellular caseinolytic metalloprotease, called AprX. It is important to rapidly detect the presence of a contamination of raw milk by a strain, especially a P. fluorescens strain, having a high potential of depredation. If standard plate count procedures are often employed, they are time consuming and do not permit to rapidly evaluate the potential of depredation. An alternative method consists to search the aprX gene, but such a method remains of low sensitivity and does not allow evaluating the real potential of depredation of the contaminant. After a milk depredation event, three strains of Pseudomonas spp. (F, 2312 and 2313) have been isolated from a dairy plant. Using molecular and phenotypic approaches, these strains were identified as P. fluorescens strains. Their respective extracellular caseinolytic potential was characterized as well as that of several collection strains of P. fluorescens. It appeared that these strains secreted one protease of about 45 kDa, that their extracellular caseinolytic potential was highly variable for one strain to another and that the one of strain F was the highest. The protease secreted by the strain F was purified and its N-terminal sequence established. It shared 100% identity with the domain 14-34 of extracellular alkaline endoprotease sequences which are called AprX for some of them. Its gene was sequenced as well as that of two collection strains of P. fluorescens having a significant lower extracellular caseinolytic potential. The genomic environment of the aprX gene as well as its expression during the strain growth was investigated. It appears that the difference of extracellular caseinolytic potential which has been observed between the three strains does not mainly result from the AprX sequence/structure but it might rather result from the aprX level of expression.

Genetic instability of whiG gene during the aerial mycelium development of Streptomyces ambofaciens ATCC23877 under different conditions of nitrogen limitations.

In Streptomyces ambofaciens, white papillae that genetic instability events generate during aerial mycelium growth, give rise to Pig-pap mutants which are unable to sporulate and devoid of large genome rearrangement. Knowing that genetic and environmental factors can influence the number of papillae per colony, we investigated the effect of nutrient limitated conditions of growth on the formation of white papillae. We observed that under nitrogen limitation and, most particularly, under amino acid limitation, the number of papillae per colony dramatically increased. Most of the Pig-pap mutants deriving from such papillae displayed a mutation in the whiG gene, which encodes the sigma factor sigma(whiG) which is absolutely required for the sporulation process. In most cases, the mutation led to a loss of function. We showed that the Pig-pap mutants deriving from papillae appearing under usual growth conditions also frequently displayed null mutation of whiG too. As the whiG mutation ratio among the Pig-pap mutants isolated with or without nitrogen limited conditions did not change, the results described in this paper suggest that the production of papillae could constitute a response of S. ambofaciens to an amino acid limitation.

Sigma factor WhiG and its regulation constitute a target of a mutational phenomenon occurring during aerial mycelium growth in Streptomyces ambofaciens ATCC23877.

The genetic instability of Streptomyces ambofaciens affects the pigmentation of colonies and generates a variety of mutants the majority of which display large genome rearrangements. Among them, the Pig-pap mutants, which probably result from a mutational event occurring during aerial mycelium growth, display specific features, since they are unable to sporulate and do not harbor any large detectable genome rearrangements. To identify the mutational event causing their phenotype, three Pig-pap mutants originating from three independent mutational events were characterized. These mutants exhibited a whiG-like phenotype which was suppressed by the introduction of one copy of Streptomyces coelicolor whiG. Their own whiG gene was devoid of mutations and appeared to be transcribed at a level similar to that of the WT. However, whiH, the expression of which depends on sigma(WhiG), was not transcribed in any of the three Pig-pap mutants, suggesting that the sigma(WhiG) was absent or inactive. This suggests that in these Pig-pap mutants, the regulation of sigma(WhiG) might be affected. Finally, the introduction of S. coelicolor whiG in one of these Pig-pap mutants restored not only pigmentation and sporulation, but also the ability to once again form white papillae. Analyses of transgene whiG in these papillae revealed that it constitutes a mutational target during aerial mycelium formation when integrated into the genome of this Pig-pap mutant.

Spontaneous chromosome circularization and amplification of a new amplifiable unit of DNA belonging to the terminal inverted repeats in Streptomyces ambofaciens ATCC 23877.

In Streptomyces, the linear chromosomal DNA is highly unstable and undergoes large rearrangements usually at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, AUD ( amplifiable unit of DNA). In Streptomyces ambofaciens, two amplifiable regions (AUD6 and AUD90), located approximately 600 kb and 1,200 kb from the right chromosomal end respectively, have been characterized. Here, the isolation and molecular characterization of a new S. ambofaciens mutant strain exhibiting a green-pigmented phenotype is described; the wild-type produces a gray pigment. In this mutant, both chromosome ends were deleted, which probably led to circularization of the chromosome. These deletions were associated with amplification of a sequence belonging to the chromosomal terminal inverted repeats (TIRs), which might constitute the new fragment generated by the chromosomal circularization.