Development of innovative fermented products by exploiting the diversity of immunomodulatory properties and fermentative activity of lactic and propionic acid bacteria.

Many consumers nowadays demand plant-based milk analogs for reasons related to lifestyle, health, diet and sustainability. This has led to the increasing development of new products, fermented or not. The objective of the present study was to develop a plant-based fermented product (based on soy milk analog or on hemp milk analog), as well as mixes, using lactic acid bacteria (LAB) and propionic acid bacteria (PAB) strains, as well as consortia thereof. We screened a collection of 104 strains, from nine LAB species and two PAB species, based on their ability to ferment plant or milk carbohydrates, to acidify goat milk, soy milk analog and hemp milk analog, as well as to hydrolyze proteins isolated from these three products. Strains were also screened for their immunomodulatory ability to induce secretion of two interleukins, i.e., IL-10 and IL-12, in human Peripheral Blood Mononuclear Cells. We selected five strains: Lactobacillus delbrueckii subsp. lactis Bioprox1585, Lactobacillus acidophilus Bioprox6307, Lactococcus lactis Bioprox7116, Streptococcus thermophilus CIRM-BIA251, and Acidipropionibacterium acidipropionici CIRM-BIA2003. We then assembled them in 26 different bacterial consortia. Goat milk and soy milk analog fermented by each of the five strains or by the 26 consortia were tested in vitro, for their ability to modulate inflammation in cultured Human Epithelial Intestinal Cells (HEIC) stimulated by pro-inflammatory Lipopolysaccharides (LPS) from Escherichia coli. Plant-based milk analogs, fermented by one consortium composed of L.delbrueckii subsp. lactis Bioprox1585, Lc.lactis Bioprox7116, and A.acidipropionici CIRM-BIA2003, reduced the secretion of the proinflammatory cytokine IL-8 in HIECs. Such innovative fermented vegetable products thus open perspectives as functional foods targeting gut inflammation.

Dairy starters and fermented dairy products modulate gut mucosal immunity.

The gut microbiota plays a crucial role in the regulation of mucosal immunity and of the function of the intestinal barrier. Dysbiosis is accordingly associated with rupture of mucosal immune homeostasis, leading to inflammatory intestinal diseases. In this context, probiotic bacteria, including a new generation of intestinal probiotics, can maintain intestinal homeostasis and promote health. Surprisingly, little is known about the impact of fermented dairy products in this context, while they represent our main source of live and active bacteria. Indeed, they provide, through our daily diet, a high number of bacteria whose effect on mucosal immunity deserves attention. Among bacteria ingested in fermented dairy products, Streptococcus thermophilus, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii are on top, as they are ingested in high concentrations (close to 109 per gram of product) in fermented milks or cheeses. This review gives an overview of the potential immunomodulatory effects of these main dairy starters. It further explores studies dealing with fermented dairy products containing theses starters, in a context of inflammation.

Data from a proteomic comparative analysis highlight differential adaptation of Lactobacillus delbrueckii subsp. bulgaricus to cow milk versus to soy milk environments.

The article presents a proteomic dataset generated by a comparative analysis, using gel-free nanoLC-MS/MS, of the cellular proteome of Lactobacillus delbrueckii subsp. bulgaricus, a yogurt starter, when cultivated in soy milk versus in cow milk. The CIRM-BIA1592 strain was cultivated in the aqueous phase of soy milk, or of cow milk. Whole-cell proteins were extracted, trypsinolyzed and analyzed by nano LC-MS/MS, prior to identification and to classification by function using the X!Tandem pipeline software and the proteomic data from NCBI.nlm.nigh.gov. Quantification of the proteins was moreover performed to evidence changes in their expression, depending on the culture medium. Data are available via ProteomeXchange with the identifier PXD033905 (http://www.proteomexchange.org/). This article is related to the research article entitled "The stressing life of Lactobacillus delbrueckii subsp. bulgaricus in soy milk", by G.Jan et al. in Food Microbiology, 2022. This proteomic differential analysis indeed revealed major modulation of the stress proteome, with many stress proteins upregulated in the soy environment.

Little Impact of NaCl Reduction in Swiss-Type Cheese.

Reducing salt intake can mitigate the prevalence of metabolic disorders. In fermented foods such as cheeses, however, salt can impact the activity of desirable and undesirable microorganisms and thus affect their properties. This study aimed to investigate the effect of salt level on Swiss-type cheese ripening. Since proteolysis is a major event in cheese ripening, three strains of Lactobacillus helveticus were selected on the cell-envelope proteinase (CEP) they harbor. Their proteolytic activity on caseins was studied at six salt levels (0-4.5%) at pH 7.5 and 5.2. Swiss-type cheeses were manufactured at regular, increased, and decreased salt concentrations, and characterized for their composition and techno-functional properties. L. helveticus strains possessed and expressed the expected CEPs, as shown by PCR and shaving experiments. The two strains of L. helveticus that possessed at least the CEP PrtH3 showed the greatest proteolytic activity. Casein hydrolysis in vitro was similar or higher at pH 5.2, i.e., cheese pH, compared to pH 7.5, and slightly decreased at the highest salt concentrations (3.0 and 4.4%). Similarly, in ripened cheeses, these L. helveticus strains showed 1.5-2.4 more proteolysis, compared to the cheeses manufactured without L. helveticus. Regarding the salt effect, the 30% salt-reduced cheeses showed the same proteolysis as regular cheeses, while the upper-salted cheeses showed a slight decrease (-14%) of the non-protein fraction. The microbial and biochemical composition remained unchanged in the 30%-reduced cheeses. In contrast, Propionibacterium freudenreichii, used as ripening bacteria in Swiss cheese, grew more slowly in upper-salted (1.14%, w/w) cheeses, which induced concomitant changes in the metabolites they consumed (-40% lactic acid) or produced (fivefold decrease in propionic acid). Some cheese techno-functional properties were slightly decreased by salt reduction, as extrusion (-17%) and oiling off (-4%) compared to regular cheeses. Overall, this study showed that a 30% salt reduction has little impact in the properties of Swiss-type cheeses, and that starters and ripening cultures strains could be chosen to compensate changes induced by salt modifications in Swiss-type and other hard cheeses.

The stressing life of Lactobacillus delbrueckii subsp. bulgaricus in soy milk.

Lactobacillus delbrueckii subsp. bulgaricus is a beneficial lactic acid bacterium and constitutes one of the most used, and thus consumed, dairy starters, worldwide. This homofermentative bacterium was the first lactobacillus described and is involved in the fermentation of yogurt and of diverse other fermented products, including cheeses. It has a long history of safe use, as well as documented probiotic lato sensu effects, including alleviation of lactose intolerance. Plant-based fermented products presently experience a considerable development, as a result of evolution of consumers' habits, in a general context of food transition. This requires research and development, and thus scientific knowledge, to allow such transition, including the development of fermented soy milks. These last indeed offer an alternative source of live and active bacteria. The yogurt starters L. delbrueckii subsp. bulgaricus, together with Streptococcus thermophilus, have been implemented to generate yogurt-type fermented soy milks worldwide. While the adaptation of these starters to the dairy environment has been extensively studied, little is known about L. delbrueckii adaptation to the soy environment. We therefore investigated its adaptation to soy milk and compared it to cow's milk. Surprisingly, it did not grow in soy milk, neither alone, nor in co-culture with S. thermophilus. Acidification of soy milk was however faster in the presence of both species. In order to deepen such adaptation, we then compared L. delbrueckii growth and survival in soy milk ultrafiltrate (SUF, the aqueous phase of soy milk) and compared it to cow's milk ultrafiltrate (MUF, the aqueous phase of cow milk). This comparison revealed major differences in terms of cell morphology and proteome composition. Lactobacilli appeared deformed and segmented in soy. Major differences in both the surface and the cellular proteome indicated upregulation of stress proteins, yet downregulation of cell cycle and division machinery. Altogether, these results suggest that soy milk may be a stressing environment for the yogurt starter L. delbrueckii subsp. bulgaricus.

Mixed dairy and plant-based yogurt alternatives: Improving their physical and sensorial properties through formulation and lactic acid bacteria cocultures.

Food transition requires incorporating more plant-based ingredients in our diet, thus leading to the development of new plant-based products, such as yogurt alternatives (YAs). This study aimed at evaluating the impact of lactic acid bacteria (LAB) cocultures and formulation on the physico-chemical and sensory properties of YAs. YAs were made by emulsifying anhydrous milk fat (AMF) or coconut oil in milk and lupin protein suspensions. The starters used, in mono- and cocultures, were the strains Lactococcus lactis NCDO2125, Enteroccocus faecalis CIRM-BIA2412 and Lactiplantibacillus plantarum CIRM-BIA1524. Textural properties and metabolites of YAs were evaluated and their sensory properties compared using a sorting task. Some cocultures led to higher firmness, viscosity, and water holding capacity of YAs, compared to monocultures. AMF and a milk:lupin protein ratio of 67:33 gave firmer and more viscous YAs. YAs were sensorially discriminated on the basis of protein ratio and fat type, but not of starters. The cocultures exhibited more diverse functional outputs, such as texturing, production of flavour compounds, proteolysis, when the strains associated in coculture had distinct capacities. Appropriate associations of LAB and formulation offer interesting solutions to improve the perception of YAs, and ultimately, encourage their consumption.

Positive Interactions between Lactic Acid Bacteria Promoted by Nitrogen-Based Nutritional Dependencies.

Nutritional dependencies, especially those regarding nitrogen sources, govern numerous microbial positive interactions. As for lactic acid bacteria (LAB), responsible for the sanitary, organoleptic, and health properties of most fermented products, such positive interactions have previously been studied between yogurt bacteria. However, they have never been exploited to create artificial cocultures of LAB that would not necessarily coexist naturally, i.e., from different origins. The objective of this study was to promote LAB positive interactions, based on nitrogen dependencies in cocultures, and to investigate how these interactions affect some functional outputs, e.g., acidification rates, carbohydrate consumption, and volatile-compound production. The strategy was to exploit both proteolytic activities and amino acid auxotrophies of LAB. A chemically defined medium was thus developed to specifically allow the growth of six strains used, three proteolytic and three nonproteolytic. Each of the proteolytic strains, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125, and CIRM-BIA244, was cocultured with each one of the nonproteolytic LAB strains, L. lactis NCDO2111 and Lactiplantibacillus plantarum CIRM-BIA465 and CIRM-BIA1524. Bacterial growth was monitored using compartmented chambers to compare growth in mono- and cocultures. Acidification, carbohydrate consumption, and volatile-compound production were evaluated in direct cocultures. Each proteolytic strain induced different types of interactions: strongly positive interactions, weakly positive interactions, and no interactions were seen with E. faecalis CIRM-BIA2412, L. lactis NCDO2125, and L. lactis CIRM-BIA244, respectively. Strong interactions were associated with higher concentrations of tryptophan, valine, phenylalanine, leucine, isoleucine, and peptides. They led to higher acidification rates, lower pH, higher raffinose utilization, and higher concentrations of five volatile compounds. IMPORTANCE Interactions of lactic acid bacteria (LAB) are often studied in association with yeasts or propionibacteria in various fermented food products, and the mechanisms underlying their interactions are being quite well characterized. Concerning interactions between LAB, they have mainly been investigated to test antagonistic interactions. Understanding how they can positively interact could be useful in multiple food-related fields: production of fermented food products with enhanced functional properties or fermentation of new food matrices. This study investigated the exploitation of the proteolytic activity of LAB strains to promote positive interactions between proteolytic and nonproteolytic strains. The results suggest that proteolytic LAB do not equally stimulate nonproteolytic LAB and that the stronger the interactions between LAB are, the more functional outputs we can expect. Thus, this study gives insight into how to create new associations of LAB strains and to guarantee their positive interactions.

Positive Interactions Between Lactic Acid Bacteria Could Be Mediated by Peptides Containing Branched-Chain Amino Acids.

Lactic acid bacteria (LAB) are responsible for the sanitary, organoleptic, and health properties of most fermented products. Positive interactions between pairs of LAB strains, based on nitrogen dependencies, were previously demonstrated. In a chemically defined medium, using milk and lupin proteins as sole nitrogen source, two proteolytic strains were able to sustain the growth of non-proteolytic strains, but one did not. The objective of the present study was, thus, to determine which specific peptides were implicated in the positive interactions observed. Peptides produced and involved in the bacterial interactions were quantified using tandem mass spectrometry (LC-MS/MS). About 2,000 different oligopeptides ranging from 6 to more than 50 amino acids in length were identified during the time-course of the experiment. We performed a clustering approach to decipher the differences in peptide production during fermentation by the three proteolytic strains tested. We also performed sequence alignments on parental proteins and identified the cleavage site profiles of the three bacterial strains. Then, we characterized the peptides that were used by the non-proteolytic strains in monocultures. Hydrophobic and branched-chain amino acids within peptides were identified as essential in the interactions. Ultimately, better understanding how LAB can positively interact could be useful in multiple food-related fields, e.g., production of fermented food products with enhanced functional properties, or fermentation of new food matrices.

Function-Driven Design of Lactic Acid Bacteria Co-cultures to Produce New Fermented Food Associating Milk and Lupin.

Designing bacterial co-cultures adapted to ferment mixes of vegetal and animal resources for food diversification and sustainability is becoming a challenge. Among bacteria used in food fermentation, lactic acid bacteria (LAB) are good candidates, as they are used as starter or adjunct in numerous fermented foods, where they allow preservation, enhanced digestibility, and improved flavor. We developed here a strategy to design LAB co-cultures able to ferment a new food made of bovine milk and lupin flour, consisting in: (i) in silico preselection of LAB species for targeted carbohydrate degradation; (ii) in vitro screening of 97 strains of the selected species for their ability to ferment carbohydrates and hydrolyze proteins from milk and lupin and clustering strains that displayed similar phenotypes; and (iii) assembling strains randomly sampled from clusters that showed complementary phenotypes. The designed co-cultures successfully expressed the targeted traits i.e., hydrolyzed proteins and degraded raffinose family oligosaccharides of lupin and lactose of milk in a large range of concentrations. They also reduced an off-flavor-generating volatile, hexanal, and produced various desirable flavor compounds. Most of the strains in co-cultures achieved higher cell counts than in monoculture, suggesting positive interactions. This work opens new avenues for the development of innovative fermented food products based on functionally complementary strains in the world-wide context of diet diversification.

Differential Adaptation of Propionibacterium freudenreichii CIRM-BIA129 to Cow's Milk Versus Soymilk Environments Modulates Its Stress Tolerance and Proteome.

Propionibacterium freudenreichii is a beneficial bacterium that modulates the gut microbiota, motility and inflammation. It is traditionally consumed within various fermented dairy products. Changes to consumer habits in the context of food transition are, however, driving the demand for non-dairy fermented foods, resulting in a considerable development of plant-based fermented products that require greater scientific knowledge. Fermented soymilks, in particular, offer an alternative source of live probiotics. While the adaptation of lactic acid bacteria (LAB) to such vegetable substrates is well documented, little is known about that of propionibacteria. We therefore investigated the adaptation of Propionibacterium freudenreichii to soymilk by comparison to cow's milk. P. freudenreichii grew in cow's milk but not in soymilk, but it did grow in soymilk when co-cultured with the lactic acid bacterium Lactobacillus plantarum. When grown in soymilk ultrafiltrate (SUF, the aqueous phase of soymilk), P. freudenreichii cells appeared thinner and rectangular-shaped, while they were thicker and more rounded in cow's milk utltrafiltrate (MUF, the aqueous phase of cow milk). The amount of extractable surface proteins (SlpA, SlpB, SlpD, SlpE) was furthermore reduced in SUF, when compared to MUF. This included the SlpB protein, previously shown to modulate adhesion and immunomodulation in P. freudenreichii. Tolerance toward an acid and toward a bile salts challenge were enhanced in SUF. By contrast, tolerance toward an oxidative and a thermal challenge were enhanced in MUF. A whole-cell proteomic approach further identified differential expression of 35 proteins involved in amino acid transport and metabolism (including amino acid dehydrogenase, amino acid transporter), 32 proteins involved in carbohydrate transport and metabolism (including glycosyltransferase, PTS), indicating metabolic adaptation to the substrate. The culture medium also modulated the amount of stress proteins involved in stress remediation: GroEL, OpuCA, CysK, DnaJ, GrpE, in line with the modulation of stress tolerance. Changing the fermented substrate may thus significantly affect the fermentative and probiotic properties of dairy propionibacteria. This needs to be considered when developing new fermented functional foods.

Understanding the Mechanisms of Positive Microbial Interactions That Benefit Lactic Acid Bacteria Co-cultures.

Microorganisms grow in concert, both in natural communities and in artificial or synthetic co-cultures. Positive interactions between associated microbes are paramount to achieve improved substrate conversion and process performance in biotransformation and fermented food production. The mechanisms underlying such positive interactions have been the focus of numerous studies in recent decades and are now starting to be well characterized. Lactic acid bacteria (LAB) contribute to the final organoleptic, nutritional, and health properties of fermented food products. However, interactions in LAB co-cultures have been little studied, apart from the well-characterized LAB co-culture used for yogurt manufacture. LAB are, however, multifunctional microorganisms that display considerable potential to create positive interactions between them. This review describes why LAB co-cultures are of such interest, particularly in foods, and how their extensive nutritional requirements can be used to favor positive interactions. In that respect, our review highlights the benefits of co-cultures in different areas of application, details the mechanisms underlying positive interactions and aims to show how mechanisms based on nutritional interactions can be exploited to create efficient LAB co-cultures.

Taking Advantage of Bacterial Adaptation in Order to Optimize Industrial Production of Dry Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a beneficial bacterium, used both as a probiotic and as a cheese starter. Large-scale production of P. freudenreichii is required to meet growing consumers' demand. Production, drying and storage must be optimized, in order to guarantee high P. freudenreichii viability within powders. Compared to freeze-drying, spray drying constitutes the most productive and efficient, yet the most stressful process, imposing severe oxidative and thermal constraints. The aim of our study was to provide the tools in order to optimize the industrial production of dry P. freudenreichii. Bacterial adaptation is a well-known protective mechanism and may be used to improve bacterial tolerance towards technological stresses. However, the choice of bacterial adaptation type must consider industrial constraints. In this study, we combined (i) modulation of the growth medium composition, (ii) heat-adaptation, and (iii) osmoadaptation, in order to increase P. freudenreichii tolerance towards technological stresses, including thermal and oxidative constraints, using an experimental design. We further investigated optimal growth and adaptation conditions, by monitoring intracellular compatible solutes accumulation. Glucose addition, coupled to heat-adaptation, triggered accumulation of trehalose and of glycine betaine, which further provided high tolerance towards spray drying and storage. This work opens new perspectives for high quality and fast production of live propionibacteria at the industrial scale.

Probiotic Propionibacterium freudenreichii requires SlpB protein to mitigate mucositis induced by chemotherapy.

Propionibacterium freudenreichii CIRM-BIA 129 (P. freudenreichii wild type, WT) is a probiotic bacterium, which exerts immunomodulatory effects. This strain possesses extractable surface proteins, including SlpB, which are involved in anti-inflammatory effect and in adhesion to epithelial cells. We decided to investigate the impact of slpB gene mutation on immunomodulation in vitro and in vivo. In an in vitro assay, P. freudenreichii WT reduced expression of IL-8 (p<0.0001) and TNF-α (p<0.0001) cytokines in LPS-stimulated HT-29 cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. Subsequently, both strains were investigated in vivo in a 5-FU-induced mucositis mice model. Mucositis is a common side effect of cytotoxic chemotherapy with 5-FU, characterized by mucosal injury, inflammation, diarrhea, and weight loss. The WT strain prevented weight loss, reduced inflammation and consequently histopathological scores. Furthermore, it regulated key markers, including Claudin-1 (cld1, p<0.0005) and IL-17a (Il17a, p<0.0001) genes, as well as IL-12 (p<0.0001) and IL-1ß (p<0.0429) cytokines levels. Mutant strain displayed opposite regulatory effect on cld1 expression and on IL-12 levels. This work emphasizes the importance of SlpB in P. freudenreichii ability to reduce mucositis inflammation. It opens perspectives for the development of probiotic products to decrease side effects of chemotherapy using GRAS bacteria with immunomodulatory surface protein properties.

Spatial Distribution of Lactococcus lactis Colonies Modulates the Production of Major Metabolites during the Ripening of a Model Cheese.

In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and number of colonies for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, can influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain with two different spatial distributions, big and small colonies, to monitor the production of the major ripening metabolites, including sugars, organic acids, peptides, free amino acids, and volatile metabolites, over 1 month of ripening. The monitored metabolites were qualitatively the same for both cheeses, but many of them were more abundant in the small-colony cheeses than in the big-colony cheeses over 1 month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis modulated the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work further explores the immobilization of bacteria as colonies within cheese and highlights the consequences of this immobilization on cheese ripening.

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.

Bacterial Colonies in Solid Media and Foods: A Review on Their Growth and Interactions with the Micro-Environment.

Bacteria, either indigenous or added, are immobilized in solid foods where they grow as colonies. Since the 80's, relatively few research groups have explored the implications of bacteria growing as colonies and mostly focused on pathogens in large colonies on agar/gelatine media. It is only recently that high resolution imaging techniques and biophysical characterization techniques increased the understanding of the growth of bacterial colonies, for different sizes of colonies, at the microscopic level and even down to the molecular level. This review covers the studies on bacterial colony growth in agar or gelatine media mimicking the food environment and in model cheese. The following conclusions have been brought to light. Firstly, under unfavorable conditions, mimicking food conditions, the immobilization of bacteria always constrains their growth in comparison with planktonic growth and increases the sensibility of bacteria to environmental stresses. Secondly, the spatial distribution describes both the distance between colonies and the size of the colonies as a function of the initial level of population. By studying the literature, we concluded that there systematically exists a threshold that distinguishes micro-colonies (radius < 100-200 µm) from macro-colonies (radius >200 µm). Micro-colonies growth resembles planktonic growth and no pH microgradients could be observed. Macro-colonies growth is slower than planktonic growth and pH microgradients could be observed in and around them due to diffusion limitations which occur around, but also inside the macro-colonies. Diffusion limitations of milk proteins have been demonstrated in a model cheese around and in the bacterial colonies. In conclusion, the impact of immobilization is predominant for macro-colonies in comparison with micro-colonies. However, the interaction between the colonies and the food matrix itself remains to be further investigated at the microscopic scale.

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments.

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.

Emmental Cheese Environment Enhances Propionibacterium freudenreichii Stress Tolerance.

Dairy propionibacteria are actinomycetales found in various fermented food products. The main species, Propionibacterium freudenreichii, is generally recognized as safe and used both as probiotic and as cheese starter. Its probiotic efficacy tightly depends on its tolerance towards digestive stresses, which can be largely modulated by the ingested delivery vehicle. Indeed, tolerance of this bacterium is enhanced when it is consumed within a fermented dairy product, compared to a dried probiotic preparation. We investigated both stress tolerance and protein neosynthesis upon growth in i) chemically defined or ii) aqueous phase of Emmental cheeses. Although the same final population level was reached in both media, a slower growth and an enhanced survival of CIRM BIA 1 strain of P. freudenreichii subsp. shermanii was observed in Emmental juice, compared to chemically defined medium. This was accompanied by differences in substrates used and products released as well as overexpression of various early stress adaptation proteins in Emmental juice, compared to chemically defined medium, implied in protein folding, in aspartate catabolism, in biosynthesis of valine, leucine and isoleucine, in pyruvate metabolism in citrate cycle, in the propionate metabolism, as well as in oxidoreductases. All these changes led to a higher digestive stress tolerance after growth in Emmental juice. Mechanisms of stress adaptation were induced in this environment, in accordance with enhanced survival. This opens perspectives for the use of hard and semi-hard cheeses as delivery vehicle for probiotics with enhanced efficacy.

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.

Quantitative proteomic analysis of bacterial enzymes released in cheese during ripening.

Due to increasingly available bacterial genomes in databases, proteomic tools have recently been used to screen proteins expressed by micro-organisms in food in order to better understand their metabolism in situ. While the main objective is the systematic identification of proteins, the next step will be to bridge the gap between identification and quantification of these proteins. For that purpose, a new mass spectrometry-based approach was applied, using isobaric tagging reagent for quantitative proteomic analysis (iTRAQ), which are amine specific and yield labelled peptides identical in mass. Experimental Swiss-type cheeses were manufactured from microfiltered milk using Streptococcus thermophilus ITG ST20 and Lactobacillus helveticus ITG LH1 as lactic acid starters. At three ripening times (7, 20 and 69 days), cheese aqueous phases were extracted and enriched in bacterial proteins by fractionation. Each sample, standardised in protein amount prior to proteomic analyses, was: i) analysed by 2D-electrophoresis for qualitative analysis and ii) submitted to trypsinolysis, and labelled with specific iTRAQ tag, one per ripening time. The three labelled samples were mixed together and analysed by nano-LC coupled on-line with ESI-QTOF mass spectrometer. Thirty proteins, both from bacterial or bovine origin, were identified and efficiently quantified. The free bacterial proteins detected were enzymes from the central carbon metabolism as well as stress proteins. Depending on the protein considered, the quantity of these proteins in the cheese aqueous extract increased from 2.5 to 20 fold in concentration from day 7 to day 69 of ripening.