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Consumption of fumonisin-contaminated foods has a negative influence on the health of humans (carcinogen; oesophageal cancer in Eastern Cape in South Africa). Lactic acid bacteria (LAB) have emerged as a promising natural detoxification agent against mycotoxins. The aim of this study was to visualise the interaction between fumonisins (FB1 and FB2) and LAB: Lactobacillus plantarum FS2, L. delbrueckii subsp. delbrueckii CIP 57.8T and Pediococcus pentosaceus D39, isolated from traditional fermented maize-based products (ogi and mahewu) using confocal laser scanning microscopy (CLSM) and to then quantify the LAB-bound fumonisin using high performance liquid chromatography (HPLC). The objective was to obtain a physically visible and quantifiable binding interaction between fumonisins and LAB strains with the aim of utilising LAB as a possible detoxifying agent. Fumonisins were derivatised using naphthalene-2,3-dicarboxaldehyde (NDA) and then combined with non-fluorescent LAB cells (viable and non-viable). For the quantification of bound fumonisins, viable and non-viable cells were incubated in the presence of predetermined concentrations of fumonisins and the level of fumonisin in the suspension was determined. CLSM showed the derivatised green fluorescent fumonisins binding to the surface of each of the LAB cells. For viable cells, L. plantarum FS2 bound FB1 most effectively while P. pentosaceus D39 bound the least level of FB1. The highest levels of FB2 were bound by L. plantarum R 1096 and the least by L. delbrueckii CIP 57.8 T. For non-viable cells, L. plantarum FS2 was also the most effective for binding both fumonisins with P. pentosaceus D39 and L. delbrueckii CIP 57.8 T being the least effective for FB1 and FB2, respectively. To our knowledge, this is the first study to visualise the interaction between LAB and fumonisins. We demonstrate that LAB isolates from indigenous fermented maize-based beverages bind fumonisins and thus present a potential strategy for their reduction in these traditional foods.

Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans’ color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSM-AAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)5-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters.

International audience; Lactococcus lactis ssp. cremoris and Lactobacillus acidophilus NCFM were cultured in growth media supplemented with CaCl2 to induce intracellular accumulation of Ca2+. Both strains grown with 10 mM Ca2+ demonstrated enhanced thermal stability, with increased survival after spray drying from 49.9% to 64.9% for the coccus and from 35.5% to 43.3% for the rod. The increased intracellular Ca2+ level was confirmed with murexide assay. Adding Ca2+ in the reconstitution solution could not improve the viability of spray dried cells at extended storage stages. We hypothesized that the over-presence of intracellular Ca2+ might stimulate the activity of heat shock proteins, increasing cell survival towards heat stress. The positive effect of calcium partly explained the protective mechanism of some calcium-containing protectants. The reported approach, which improves the stress tolerance of cells, can be combined with the optimization of spray drying conditions and protectant formulation for developing a consolidated powder production scheme.

The organoleptic properties of traditional dairy beverages done with non-conventional dairy species (horse, camel), popular in Central Asia, were rarely described in the literature. To characterize the Volatile Organic Compounds (VOC) profile of fermented mare milk, 12 samples of cow milk, used as matrix were inoculated by different strains from two types of bacteria (bacilli and cocci) isolated from natural fermented mare milk. The analysis performed by Gas Chromatography coupled with Solid-Phase Micro-Extraction allowed identification of 160 different compounds from the 12 strains, and 90 from natural fermented mare milk. After cluster analysis, 3 types of profiles were observed. Those profiles were distinct by the amount of acid compounds (low, medium, high), negatively related to aromatic and aliphatic hydrocarbons. The analysis of the mean volatile compound profile of each type of bacteria (bacilli and cocci) by factorial discriminant analysis showed that 3 molecules (oxime-methoxy-phenyl, propanedioic acid propyl and 2-propanamine) allowed to well class 100% of the samples. Further researches on bacterial identification and experiments with different fermentation matrices from other dairy species will be conducted.

Fermented cereals are part of the main traditional diets of many people in Africa, usually obtained from artisanal production. The intensification of their manufacturing, responding to the consumers demand, requires a better control to ensure their sanitary, nutritional, and taste qualities, hence, the need of selecting accurate and safe starter cultures. In the present study, 48 lactic acid bacteria (LAB) strains, previously isolated from Algerian fermented wheat lemzeiet, were analyzed for different technological properties. 14 LAB strains, belonging to Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Lactobacillus brevis, and Leuconostoc mesenteroides species, decreased rapidly the pH of the flour extract broth close to 4 or below. 91% of strains showed extracellular protease activity, but only 12% were amylolytics. 18 LAB strains inhibited or postponed the growth of three fungal targets Rhodotorula mucilaginosa UBOCC-A-216004, Penicillium verrucosum UBOCC-A-109221, and Aspergillus flavus UBOCC-A-106028. The strains belonging to Lactobacillus spp., Leuconostoc fallax, L. mesenteroides, and Weissella paramesenteroides were the most antifungal ones. Multiplex PCR for biogenic amines' production did not reveal any of the genes involved in the production of putrescine, histamine, and tyramine for 17 of the 48 strains. The obtained results provided several candidates for use as starter culture in the future production of lemzeiet.

Lactobacillus, Pediococcus, Oenococcus and Leuconostoc are the main Lactic Acid Bacteria (LAB) genera present in cider as they are able to survive this hostile environment. LAB play a significant role in cider quality, for example in the process of malolactic fermentation, even though they can also be involved in spoilage of cider (production of biogenic amines, exopolysaccharides, off-flavours…). In this context a better monitoring of the fermentation process is a matter of interest to guarantee cider quality. In the present study, we designed a genus-specific multiplex PCR for a rapid and simultaneous detection of the four main LAB genera involved in cider production. This multiplex PCR worked equally with purified genomic DNA of bacterial isolates and with colonies directly picked from agar plates. This new PCR method was also successfully extended to wine and dairy isolates, and thus constitutes an effective tool to quickly identify LAB associated with fermented foods. Moreover, many biodiversity studies would also benefit from this fast, cheap and reliable identification method.

In the context of a demand for “preservative-free” food products, biopreservation appears as a promising alternative to either replace or reduce the use of chemical preservatives. The purpose of this study was to evaluate the antifungal activity of a collection of lactic acid bacteria (n = 194), and then to evaluate the applicability and efficacy of selected ones used as bioprotective cultures against mold spoilers in dairy and bakery products. First, lactic acid bacteria were isolated from various Algerian raw milk samples and Amoredj, a traditional fermented product. Secondly, in vitro screening tests against Mucor racemosus UBOCC-A-109155, Penicillium commune UBOCC-A-116003, Yarrowia lipolytica UBOCC-A-216006, Aspergillus tubingensis AN, Aspergillus flavus T5 and Paecilomyces formosus AT allowed for the selection of 3 active strains, namely Lactobacillus plantarum CH1, Lactobacillus paracasei B20 and Leuconostoc mesenteroides L1. In situ tests were then performed to validate their activity in actual products (sour cream and sourdough bread) challenged with fungal spoilers. These tests showed that antifungal LAB could slow the fungal target growth and could be candidates of interest for industrial applications. Finally, organic acids and various antifungal compounds produced in sour cream and sourdough bread by the selected LAB, and thus potentially supporting the observed antifungal activity, were identified and quantified by HPLC and LC-QTOF.

Natural sourdoughs are commonly used in bread-making processes, especially for organic bread. Despite its role in bread flavor and dough rise, the stability of the sourdough microbial community during and between bread-making processes is debated. We investigated the dynamics of lactic acid bacteria (LAB) and yeast communities in traditional organic sourdoughs of five French bakeries during the bread-making process and several months apart using classical and molecular microbiology techniques. Sourdoughs were sampled at four steps of the bread-making process with repetition. The analysis of microbial density over 68 sourdough/dough samples revealed that both LAB and yeast counts changed along the bread-making process and between bread-making runs. The species composition was less variable. A total of six LAB and nine yeast species was identified from 520 and 1675 isolates, respectively. The dominant LAB species was Lactobacillus sanfranciscensis, found for all bakeries and each bread-making run. The dominant yeast species changed only once between bread-making processes but differed between bakeries. They mostly belonged to the Kazachstania clade. Overall, this study highlights the change of population density within the bread-making process and between bread-making runs and the relative stability of the sourdough species community during bread-making process.

International audience; Fungi are commonly identified as the cause for dairy food spoilage. This can lead to substantial economic losses for the dairy industry as well as consumer dissatisfaction. In this context, biopreservation of fermented dairy products using lactic acid bacteria, propionibacteria and fungi capable of producing a large range of antifungal metabolites is of major interest. In a previous study, extensive screening was performed in vitro and in situ to select 3 dairy fermentates (derived from Acidipropionibacterium jensenii CIRM-BIA1774, Lactobacillus rhamnosus CIRM-BIA1952 and Mucor lanceolatus UBOCC-A-109193, respectively) with antifungal activity. The aim of the present study was to determine the main compounds responsible for this antifungal activity. Fifty-six known antifungal compounds as well as volatiles were targeted using different analytical methods (conventional LC and GC, GC–MS, LC-QToF). The most abundant antifungal compounds in P. jensenii-, L. rhamnosus- and M. lanceolatus-derived fermentates corresponded to propionic and acetic acids, lactic and acetic acids, and butyric acid,respectively. Many other antifungal compounds (organic acids, free fatty acids, volatile compounds) were identified but at lower levels. In addition, an untargeted approach using nano LC-MS/MS identified a 9-amino acid peptide derived from αs2-casein in the L. rhamnosus-derived fermentate. This peptide inhibited M. racemosus and R. mucilaginosa in vitro. This study provides new insights on the molecules involved in antifungal activities of food-grade microorganism fermentates which could be used as antifungal ingredients in the dairy industry.

Propionibacterium freudenreichii is a beneficial bacterium traditionally used as a cheese-ripening starter and more recently for its probiotic abilities based on the release of beneficial metabolites. In addition to these metabolites (short-chain fatty acids, vitamins, and bifidogenic factor), P. freudenreichii revealed an immunomodulatory effect confirmed in vivo by the ability to protect mice from induced acute colitis. This effect is, however, highly strain-dependent. Local action of metabolites and of immunomodulatory molecules is favoured by the ability of probiotics to adhere to the host cells. This property depends on key surface compounds, still poorly characterized in propionibacteria. We thus investigated the involvement of a specific propionibacterial surface protein in adhesion of the probiotic Propionibacterium freudenreichii to host cells. We compared several strains of Propionibacterium freudenreichii, with respect to adhesion to cultured human intestinal epithelial cells, using a well-established model. We inactivated genes using a previously developed method, based on the integration of a non-replicative plasmid, harbouring a portion of the target gene. We analysed adhesion of the wild type and the mutant strains using fluorescent-assisted flow cytometry and confocal microscopy. We also sought inhibition of adhesion using antibodies directed against surface layer proteins. In the present study, we showed different adhesion rates to cultured human intestinal cells, among strains of P. freudenreichii. The most adhesive one was P. freudenreichii CIRM-BIA 129, which is known to expose surface-layer proteins. We evidenced here the involvement of these proteins in adhesion to cultured human colon cells. We then aimed at deciphering the mechanisms involved in adhesion. Adhesion was inhibited by antibodies raised against SlpB, one of the surface-layer proteins in P. freudenreichii CIRM-BIA 129. Inactivation of the corresponding gene suppressed adhesion, further evidencing the key role of slpB product in cell adhesion. This work confirms the various functions fulfilled by surface-layer proteins, including probiotic/host interactions. It opens new perspectives for the understanding of probiotic determinants in propionibacteria, and for the selection of the most efficient strains within the P. freudenreichii species.

This research investigated the influence of lactic acid bacteria (LAB) strains on ester levels in Bordeaux red wines. These wines were made in five Bordeaux areas in two vintages, using three yeast strains. Malolactic fermentation (MLF) was carried out using industrial starters or indigenous strains, each in triplicate. Ester concentrations were determined by liquid-liquid-extraction- or HS-SPME-GC/MS at various stages in the winemaking process. The levels of most compounds were slightly impacted by LAB, depending on grape variety. Nevertheless, branched hydroxylated esters, such as ethyl 2-hydroxy-3-methylbutanoate and ethyl 2-hydroxy-4-methylpentanoate were the only compounds to be strongly influenced by the bacteria strain, regardless of matrix composition or the yeasts used for alcoholic fermentation. Moreover, the effect observed after MLF persisted over time, for at least 12 months. These esters are apparently important markers of LAB esterase activity. To our knowledge, this was the first time they had been identified in this role.

Probiotics have been adopted to treat and prevent various diseases in humans and animals. They were notably shown to be a promising alternative to prevent mastitis in dairy cattle. This inflammation of the mammary gland is generally of infectious origin and generates extensive economic losses worldwide. In a previous study, we found that Lactobacillus casei BL23 was able to inhibit the internalisation of Staphylococcus aureus, one of the major pathogens involved in mastitis, into bovine mammary epithelial cells (bMEC). In this study, we further explored the capacity of this strain to modulate the innate immune response of bovine mammary epithelial cells during S. aureus infection. L. casei BL23 was able to decrease the expression of several pro-inflammatory cytokines, including interleukins 6, 8, 1α and 1β and tumour necrosis factor alpha, in S. aureus-stimulated bMEC, 8 h post-infection. On the other hand, L. casei did not impair the induction of defensins, such as lingual antimicrobial peptide and defensin β1 in the presence of S. aureus, and even slightly increased the induction of tracheal antimicrobial peptide during S. aureus infection. Finally, this strain did not alter the expression of the pattern recognition receptor nucleotide-binding oligomerisation domain proteins (NOD2). This study demonstrates that L. casei BL23 displayed anti-inflammatory properties on S. aureus-stimulated bMEC. These results open the way to further characterisation of the BL23 probiotic potential in a bovine mammary gland context and to a better understanding of how all these beneficial properties combine in vivo to combat mastitis pathogens.

Fermented milk is a common beverage in many countries, but its flavor is highly variable from one region to another. The fermented mare milk, common beverage in Central Asia, has a typical flavor of koumiss, but those flavors were never described using aroma compound determination. In the present study, six lactic acid bacteria were identified in local koumiss. Due to the apparent technological advantage of Lactobacillus casei and Streptococcus thermophilus (notably their acidification speed), the main objective of this study was to compare their volatile compound profiles. 35 aromatic compounds were detected. To compare the aroma profiles of each strain, the ratio proportion strain/control was calculated for each strain and for each aroma showing significantly different profiles with predominance of 2-methyl-1-propanol and 2-undecanol in milk fermented with L. casei, and of 2,3-pentanedione in milk fermented with S. thermophilus. L. casei strain produced more aroma and its profile was significantly (P>0.03) different from S. thermophilus profile.

Biopreservation represents a complementary approach to traditional hurdle technologies for reducing microbial contaminants (pathogens and spoilers) in food. In the dairy industry that is concerned by fungal spoilage, biopreservation can also be an alternative to preservatives currently used (e.g. natamycin, potassium sorbate). The aim of this study was to develop antifungal fermentates derived from two dairy substrates using a sequential approach including an in vitro screening followed by an in situ validation. The in vitro screening of the antifungal activity of fermentates derivating from 430 lactic acid bacteria (LAB) (23 species), 70 propionibacteria (4 species) and 198 fungi (87 species) was performed against four major spoilage fungi (Penicillium commune, Mucor racemosus, Galactomyces geotrichum and Yarrowia lipolytica) using a cheese-mimicking model. The most active fermentates were obtained from Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei/paracasei and Lactobacillus plantarum among the tested LAB, Propionibacterium jensenii among propionibacteria, and Mucor lanceolatus among the tested fungi. Then, for the 11 most active fermentates, culture conditions were optimized by varying incubation time and temperature in order to enhance their antifungal activity. Finally, the antifungal activity of 3 fermentates of interest obtained from Lactobacillus rhamnosus CIRM-BIA1952, P. jensenii CIRM-BIA1774 and M. lanceolatus UBOCC-A-109193 were evaluated in real dairy products (sour cream and semi-hard cheese) at a pilot-scale using challenge and durability tests. In parallel, the impact of these ingredients on organoleptic properties of the obtained products was also assessed. In semi-hard cheese, application of the selected fermentates on the cheese surface delayed the growth of spoilage molds for up to 21 days, without any effect on organoleptic properties, P. jensenii CIRM-BIA1774 fermentate being the most active. In sour cream, incorporation of the latter fermentate at 2 or 5% yielded a high antifungal activity but was detrimental to the product organoleptic properties. Determination of the concentration limit, compatible with product acceptability, showed that incorporation of this fermentate at 0.4% prevented growth of fungal contaminants in durability tests but had a more limited effect against M. racemosus and P. commune in challenge tests. To our knowledge, this is the first time that the workflow followed in this study, from in vitro screening using dairy matrix to scale-up in cheese and sour cream, is applied for production of natural ingredients relying on a large microbial diversity in terms of species and strains. This approach allowed obtaining several antifungal fermentates which are promising candidates for dairy products biopreservation.

Algerian couscous named lemzeiet is manufactured from fermented wheat. Historically performed in underground silos called matmor, the fermentation of wheat is now generally carried out in plastic jerrycans with or without addition of vinegar at the beginning of the fermentation. Culture-dependent and culture-independent methods (PCR-TTGE) were used to characterize lactic acid bacteria and to determine their dynamic and diversity over a two-year period, with and without the addition of vinegar. Fungi, physicochemical characteristics, and volatile compound profiles were also monitored. The isolates obtained from different stages of fermentation and from both processes were characterized by coupling different molecular methods (16SrRNA/pheS/rpoA gene sequencing, species-specific PCR, RAPD and PFGE). PCR-TTGE revealed very similar profiles for both processes. Sixty-nine isolates were identified as belonging to six genera of 16 species (Enterococcus, Lactobacillus, Leuconostoc, Pediococcus, Weissella, and Streptococcus). The profiles of volatile aroma compounds showed a marked effect of the fermentation process, compared to non-fermented wheat, with 35 of the 40 volatiles detected at amounts 20- to 30-fold higher in fermented wheat samples. This study gives the first insight into lactic acid bacteria population diversity and activity in fermented wheat and will contribute to a better control of the fermentation process.

This chapter reviews scientific research showing that wood offers greater or at least not lower than cleaning guarantees, plastic and stainless steel. Wood in contact with raw milk and cheeses is rapidly covered by a microbial biofilm. In several studies, the researchers showed a high biodiversity of microorganisms present in biofilms not only between the different types of cheeses studied, but also within the same cheese variety, between different wooden vats (tina or gerle) used in different cheese plants. The biodiversity confirms and demonstrates the strong characterisation of the territorial origin of the products. The correct maintenance of wooden vats promotes the selection of a microbial flora able to play an active role in the achievement of the food safety objectives through the biocompetitive activity of lactic acid bacteria (LAB) and the inhibitory activity against pathogenic bacteria, particularly Listeria monocytogenes.

Khliaa Ezir is a traditional and popular meat product, which is produced from whole beef, camel, goat or lamb meat. It is an Algerian and a ready to eat meat product that is marinated, cooked and ripened. The product is for numerous months preserved in an earthenware jar at room temperature. Microbiological investigation on Khliaa Ezir during preparation is a prerequisite and to our knowledge, this is the first study, which provide its preliminary microbiological characterization. Thus, the aim of the present work is to study the evolution of the microflora and microbiological safety of Khliaa Ezir during the main traditional preparation steps. The microbiological counts indicated that Lactic Acid Bacteria (LAB) are the most abundant in the product, namely during the repining and storage period. However, total Coliforms were very low in fresh beef, being eliminated after 3 days of curing. Yeasts and Molds were the highest in fresh beef, and then disappeared after cooking and during the ripening and storage step. None of the pathogenic flora during the whole preparation steps were detected. We think that the cooking temperature applied during thermal treatment (80°C) contributed to the high hygienic quality of Khliaa Ezir. On another hand, a significant increase in pH was observed during the storage period to achieve a final pH of 6.19 ± 0.01 at 30 days of storage.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2017.02.013; The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensisK.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifyingtheir organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the actionmechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability,membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10 days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3 days incubation in control supernatant,while itwas prevented for up to 10 days by the active supernatant. Interestingly, between 66 and 99% of yeast cellswere under a viable but non-cultivable (VNC) state despite an absence ofmembrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.

Understanding the mechanisms behind the typicity of regional wines inevitably brings attention to microorganisms associated with their production. Oenococcus oeni is the main bacterial species involved in wine and cider making. It develops after the yeast-driven alcoholic fermentation and performs the malolactic fermentation, which improves the taste and aromatic complexity of most wines. Here, we have evaluated the diversity and specificity of O. oeni strains in six regions. A total of 235 wines and ciders were collected during spontaneous malolactic fermentations and used to isolate 3,212 bacterial colonies. They were typed by multilocus variable analysis, which disclosed a total of 514 O. oeni strains. Their phylogenetic relationships were evaluated by a second typing method based on single nucleotide polymorphism (SNP) analysis. Taken together, the results indicate that each region holds a high diversity of strains that constitute a unique population. However, strains present in each region belong to diverse phylogenetic groups, and the same groups can be detected in different regions, indicating that strains are not genetically adapted to regions. In contrast, greater strain identity was seen for cider, white wine, or red wine of Burgundy, suggesting that genetic adaptation to these products occurred. IMPORTANCE This study reports the isolation, genotyping, and geographic distribution analysis of the largest collection of O. oeni strains performed to date. It reveals that there is very high diversity of strains in each region, the majority of them being detected in a single region. The study also reports the development of an SNP genotyping method that is useful for analyzing the distribution of O. oeni phylogroups. The results show that strains are not genetically adapted to regions but to specific types of wines. They reveal new phylogroups of strains, particularly two phylogroups associated with white wines and red wines of Burgundy. Taken together, the results shed light on the diversity and specificity of wild strains of O. oeni , which is crucial for understanding their real contribution to the unique properties of wines.

In the present work, the influence of hydraulic retention time (HRT) on dark fermentation metabolism was evaluated through the operation and analysis of a series of four continuous stirred tank reactors (CSTR) at four HRT ranging from 6 h to 24 h. A maximum volumetric hydrogen production rate (VHPR) of 2000 ± 149 mL/L-d corresponding to an H2 yield of 0.86 molH2/mollactose was observed at 6 h HRT. In depth analysis of metabolite profiles and microbial communities showed that low values of HRT favored the emergence of a community dominated by Clostridiaceae-Lachnospiraceae-Enterobacteriaceae, which performed metabolic pathways co-producing hydrogen. In contrast, long HRT led to the establishment of Sporolactobacillaceae-Streptococcaceae microbial community that outcompeted hydrogen producing bacteria and was responsible of lactate production. Results suggested that these two communities mutually excluded themselves and HRT can act as an operational parameter to control the microbial communities and consequently the related metabolic pathways.

Therapeutic DNA vaccines are mostly plasmidic constructs containing a strong promoter that allows in situ transcription and translation of one or many encoded proteins/antigens to induce protective cellular and humoral immune responses against different pathogenic organisms [1–5]. Currently, at least 114 open clinical studies are recruiting patients for distinct clinical phases using a DNA vaccine approach.

A total of 230 isolates of lactic acid bacteria were isolated from gastrointestinal tracts (GIT) of indigenous chickens purchased from local market in Bang Rakam district, Phitsanulok, Thailand. Among 230 isolates, only 7 isolates, named CM6CR07, CM6CR11, CF1GI15, CF1GI14, CF1GI17-1, CF1GI17-2 and CF1GI 19 showed an inhibition zone against indicator strains. Bacteriocin activity was completely inactivated when Neutralized Cell Free Supernatants (NCFS) of the isolates were treated with proteases, confirming the proteinaceous nature of the bacteriocin component. NCFS of all selected bacteriocin producing lactic acid bacteria (SBP-LAB) retained their bacteriocin activity after heating at 100 °C for 30 min. All of SBP-LAB were identified as Enterococcus faecalis. These Ent. faecalis isolates harbored the same virulence genes. All strains were positive for gelE (gelatinase), efaAfs (cell wall adhesins), hyl (hyaluronidase), ace (adhesin of collagen protein), cylLL (cytolysin structural subunit) and cylLS (cytolysin structural subunit) and were negative for asa1 (aggregation substance), agg (aggregation protein involved in adherence to eukaryotic cells), and esp (enterococcal surface protein). These Ent. faecalis showed a wide range of inhibitory spectrum against food-spoilage and food-borne pathogens when studied by agar spot test. The CFS and NCFS also inhibited some of food-spoilage and food-borne pathogenic indicator strains when checked by agar well diffusion assay. All of these Ent. faecalis survived simulated stomach conditions, whereas all of them could survive in conditions of simulated intestinal juice.

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.

Gowe is a traditional Beninese fermented red sorghum beverage made from a blend of malted and precooked nonmalted flour. The main objective of this work was to evaluate the degradation of starch during the production of gowe and to characterise the organic acid and sugar profiles of the product obtained using starter cultures. Inoculation was performed with strains of Lactobacillus fermentum or Weissella confusa and of Kluyveromyces marxianus or Pichia anomala used singly or in combination. Size exclusion chromatography revealed that starch was hydrolysed into high molecular weight dextrins (DP over 35), oligosaccharides and glucose, mainly due to the malting and precooking steps. Glucose was the main free sugar, and lactic acid was the main organic acid. The choice of the inoculation strain directly influenced the acidity but also indirectly the sugar content of the resulting gowe and will thus affect consumer acceptability. (Resume d'auteur)

In this study, we present the draft genome sequence of Carnobacterium divergens V41. This strain was previously reported as producing divercin V41, a bacteriocin of interest for food biopreservation. Its genome revealed also the presence of a gene cluster putatively involved in polyketide production, which is unique in lactic acid bacteria.

Propionibacterium freudenreichii, a dairy starter, can reach a population of almost 109 propionibacteria per gram in Swiss-type cheese at the time of consumption. Also consumed as a probiotic, it displays strain-dependent anti-inflammatory properties mediated by surface proteins that induce IL-10 in leukocytes. We selected 23 strains with varied anti-inflammatory potentials in order to identify the protein(s) involved. After comparative genomic analysis, 12 of these strains were further analysed by surface proteomics, eight of them being further submitted to transcriptomics. The omics data were then correlated to the anti-inflammatory potential evaluated by IL-10 induction. This comparative omics strategy highlighted candidate genes that were further subjected to gene-inactivation validation. This validation confirmed the contribution of surface proteins, including SlpB and SlpE, two proteins with SLH domains known to mediate non-covalent anchorage to the cell-wall. Interestingly, HsdM3, predicted as cytoplasmic and involved in DNA modification, was shown to contribute to anti-inflammatory activity. Finally, we demonstrated that a single protein cannot explain the anti-inflammatory properties of a strain. These properties therefore result from different combinations of surface and cytoplasmic proteins, depending on the strain. Our enhanced understanding of the molecular bases for immunomodulation will enable the relevant screening for bacterial resources with anti-inflammatory properties.

Traditional fermented foods are not only the staple food for most of developing countries but also the key healthy food for developed countries. As the healthy functions of these foods are gradually discovered, more high throughput biotechnologies are being used to promote the fermented food industries. As a result, the microorganisms, process biochemistry, manufacturing, and down-streaming processing, as well as the bioactive metabolites released by the fermenting organisms and, above all, the healthy functions of these foods were extensively researched. The application and progress of biotechnology and biochemistry of traditional fermented food systems are different from each other, as the microorganisms and the food matrices vary widely. Part I (Biochemistry and Biotechnology) of this book (Fermented Foods) discusses the general aspects of biochemistry and biotechnological application of fermented foods involving acetic acid bacteria, lactic acid bacteria, ethanolic yeasts, and fungi in accelerating the many and variable functional factors in the fermented foods as well as metagenomics of fermented foods. The detailed technological interventions involved in different categories of fermented foods such as fermented cereals (bread and sourdough), fermented milk products (yogurt, cheese), fermented sausages, fermented vegetables (kimchi, sauerkraut), fermented legumes (tempeh, natto) and coffee and cocoa fermentations, and fermented beverages (animal- and plant-based) with their potential and actual health benefits, are discussed in Part II (Fermented Foods: Technological Interventions). (Resume d'auteur)

International audience; Mastitis is a mammary gland inflammatory disease often due to bacterial infections. Like many other infections, it used to be considered as a host-pathogen interaction driven by host and bacterial determinants. Until now, the involvement of the bovine mammary gland microbiota in the host-pathogen interaction has been poorly investigated, and mainly during the infectious episode. In this study, the bovine teat microbiome was investigated in 31 quarters corresponding to 27 animals, which were all free of inflammation at sampling time but which had different histories regarding mastitis: From no episode of mastitis on all the previous lactations (Healthy quarter, Hq) to one or several clinical mastitis events (Mastitic quarter, Mq). Several quarters whose status was unclear (possible history of subclinical mastitis) were classified as NDq. Total bacterial DNA was extracted from foremilk samples and swab samples of the teat canal. Taxonomic profiles were determined by pyrosequencing on 16s amplicons of the V3-4 region. Hq quarters showed a higher diversity compared to Mq ones (Shannon index: ~8 and 6, respectively). Clustering of the quarters based on their bacterial composition made it possible to separate Mq and Hq quarters into two separate clusters (C1 and C2, respectively). Discriminant analysis of taxonomic profiles between these clusters revealed several differences and allowed the identification of taxonomic markers in relation to mastitis history. C2 quarters were associated with a higher proportion of the Clostridia class (including genera such as Ruminococcus, Oscillospira, Roseburia, Dorea, etc.), the Bacteroidetes phylum (Prevotella, Bacteroides, Paludibacter, etc.), and the Bifidobacteriales order (Bifidobacterium), whereas C1 quarters showed a higher proportion of the Bacilli class (Staphylococcus) and Chlamydiia class. These results indicate that microbiota is altered in udders which have already developed mastitis, even far from the infectious episode. Microbiome alteration may have resulted from the infection itself and or the associated antibiotic treatment. Alternatively, differences in microbiome composition in udders with a history of mastitis may have occurred prior to the infection and even contributed to infection development. Further investigations on the dynamics of mammary gland microbiota will help to elucidate the contribution of this endogenous microbiota to the mammary gland health.

In this study, Lactococcus lactis was engineered to express mutated internalin A on its surface and to secrete large amounts of listeriolysin O (LLO) in order to improve its potential as a vehicle for DNA vaccination. Western blotting experiments demonstrated that the bacterium expressed LLO in both the cytoplasmic and extracellular compartments, with higher quantities found in the culture supernatants. A hemolytic assay showed that the recombinant strain secreted 250 ng active LLO/mg total protein. This mInlA/LLO-producing strain of L. lactis may be used as an alternative tool in DNA vaccination against a number of infectious diseases or in cancer therapy. Fil: de Azevedo, M. S. P.. Universidade Federal de Minas Gerais; Brasil Fil: Santos Rocha, C.. Universidade Federal de Minas Gerais; Brasil Fil: Pereira, V. B.. Universidade Federal de Minas Gerais; Brasil Fil: de Oliveira Junior, A. .. Universidade Federal de Minas Gerais; Brasil Fil: de Sousa, C. S.. Universidade Federal de Minas Gerais; Brasil Fil: Azevedo, V.. Universidade Federal de Minas Gerais; Brasil Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Chatel, J. M.. Institut National de la Recherche Agronomique; Francia Fil: Miyoshi, A.. Universidade Federal de Minas Gerais; Brasil

International audience; In order to contribute to the description of sourdough LAB composition, MiSeq sequencing and qPCR methods were performed in association with cultural methods. A panel of 16 French organic bakers and farmer-bakers were selected for this work. The lactic acid bacteria (LAB) diversity of their organic sourdoughs was investigated quantitatively and qualitatively combining (i) Lactobacillus sanfranciscensis-specific qPCR, (ii) global sequencing with MiSeq Illumina technology and (iii) molecular isolates identification. In addition, LAB and yeast enumera-tion, pH, Total Titratable Acidity, organic acids and bread specific volume were analyzed. Microbial and physico-chemical data were statistically treated by Principal Component Analysis (PCA) and Hierarchical Ascen-dant Classification (HAC). Total yeast counts were 6 log 10 to 7.6 log 10 CFU/g while LAB counts varied from 7.2 log 10 to 9.6 log 10 CFU/g. Values obtained by L. sanfranciscensis-specific qPCR were estimated between 7.2 and 10.3 log 10 CFU/g, except for one sample at 4.4 log 10 CFU/g. HAC and PCA clustered the sixteen sourdoughs into three classes described by their variables but without links to bakers' practices. L. sanfranciscensis was the dominant species in 13 of the 16 sourdoughs analyzed by Next Generation Sequencing (NGS), by the culture dependent method this species was dominant only in only 10 samples. Based on isolates identification, LAB diversity was higher for 7 sourdoughs with the recovery of L. curvatus, L. brevis, L. heilongjiangensis, L. xiangfangensis, L. koreensis, L. pontis, Weissella sp. and Pediococcus pentosaceus, as the most representative species. L. koreensis, L. heilongjiangensis and L. xiangfangensis were identified in traditional Asian food and here for the first time as dominant in organic sourdough. This study highlighted that L. sanfranciscensis was not the major species in 6/16 sour-dough samples and that a relatively high LAB diversity can be observed in French organic sourdough.

Colloque organisé par : Pr. Catherine Béal - UFR Technologie et Procédés Alimentaires - AgroParisTech Dr. Luis Bermudez - MICALIS - Institut National de la Recherche Agronomique Dr. Marie-Christine Champomier-Verges - MICALIS - Institut National de la Recherche Agronomique, Jouy en Josas Dr. Céline Delbès - Unité de Recherches Fromagères - Institut National de la Recherche Agronomique, Aurillac Dr. Stéphanie-Marie Deutsch - STLO - Institut National de la Recherche Agronomique, Rennes Dr. Véronique Monnet - Institut National de la Recherche Agronomique, Jouy en Josas Dr. Monique Zagorec - Unité SECALIM - Institut National de la Recherche Agronomique, ONIRIS - NantesColloque organisé par :Pr. Catherine Béal - UFR Technologie et Procédés Alimentaires - AgroParisTechDr. Luis Bermudez - MICALIS - Institut National de la Recherche AgronomiqueDr. Marie-Christine Champomier-Verges - MICALIS - Institut National de la Recherche Agronomique, Jouy en JosasDr. Céline Delbès - Unité de Recherches Fromagères - Institut National de la Recherche Agronomique, AurillacDr. Stéphanie-Marie Deutsch - STLO - Institut National de la Recherche Agronomique, RennesDr. Véronique Monnet - Institut National de la Recherche Agronomique, Jouy en JosasDr. Monique Zagorec - Unité SECALIM - Institut National de la Recherche Agronomique, ONIRIS - Nantes; Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. Twenty-one strains were sequenced. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose were only used by some strains. The presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes and was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were to a greater or lesser extent (quantitative phenotype) degraded by some strains. For these sugars, the phenotypes correlated with the premature appearance of a stop codon interrupting protein synthesis, preventing the catabolism of corresponding sugar. These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations.

Dairy propionibacteria (PAB) are used as a ripening starter in combination with Lactic acid bacteria (LAB) for dairy products such as Swiss-type cheese. LAB and PAB have also been studied for their probiotic properties but little is still known about their individual and/or synergistic beneficial effects within dairy matrices. In the context of a rising incidence of Inflammatory Bowel Diseases, it has become crucial to evaluate the immunomodulatory potential of bacteria ingested in large numbers via dairy products. We therefore selected different strains and combinations of technological LAB and PAB. We determined their immunomodulatory potential by IL-10 and IL-12 induction, in human peripheral blood mononuclear cells, on either single or mixed cultures, grown on laboratory medium or directly in milk. Milk was fermented with selected anti-inflammatory strains of LAB or PAB/LAB mixed cultures and the resulting bacterial fractions were also evaluated for these properties, together with starter viability and optimum technological aspects. The most promising fermented milks were evaluated in the context of TNBS- or DSS-induced colitis in mice. The improvement in inflammatory parameters evidenced an alleviation of colitis symptoms as a result of fermented milk consumption. This effect was clearly strain-dependent and modulated by growth within a fermented dairy product. These findings offer new tools and perspectives for the development of immunomodulatory fermented dairy products for targeted populations.

The aim of this study is to highlight the changes in the physiological cellular state of Oenococcus oeni during malolactic fermentation (MLF), and to use its cellular parameters to improve existing knowledge of O. oeni behaviour and to more effectively control the performance of the bacteria during MLF in wine. To do this, measurements of intracellular pH, transmembrane potential and vitality were performed using flow cytometry with different fluorescent probes: CFDA-SE and CDCF, DiBAC and CFDA, respectively. The kinetics of the cellular changes in these parameters were determined during MLF in FT80 synthetic medium and in white wine, as were the kinetics of malic acid consumption. pH(in) measurement throughout the entire growth shows that the pH was equal to the pH of the culture medium during the early stage, increased to pH 6 in the exponential phase, and then decreased to equilibrate with the pH of the medium in the late stationary phase. Membrane potential increased in early MLF and then decreased. The decrease in pHin and membrane potential occurred when all of the malic acid was consumed. Finally, we showed that the higher the Delta pH (pH(in) - pH(eX)) in O. oeni cells was, the shorter the lag phase of the MLF was. To better manage the initiation of MLF in wines, the physiological state of O. oeni cells must be taken into account. These results allow us to understand the sometimes random initiation of MLF in wines inoculated with O. oeni and to suggest ways to improve this control.

Consumption of fermented camel milk, named shubat, is very popular in Central Asia and especially in Kazakhstan where it is known for its medicinal and dietary properties. To identify lactic acid bacteria (LAB) camel milk and shubat were sampled from 4 regions of Kazakhstan with important camel's population. In total, 26 dairy samples from 13 selected farms representing the variability of the farming system in the country were collected. Isolated strains were identified by genotypic approach including PCR using three different pairs of primers (338f/518r; W001/23S1; Lac1/Lac2/Lac3) and 16S rDNA gene sequencing. Three genus were in majority: Lactococcus, Lactobacillus and Enterococcus. The following microorganisms were identified: Enterococcus durans ; Enterococcus faecalis; Enterococcus faecium; Lactobacillus casei; Lactobacillus casei subsp. casei; Lactobacillus curvatus; Lactobacillus kefiri; Lactobacillus paracasei; Lactobacillus sakei; Lactococcus lactis subsp. lactis; Leuconostoc mesenteroides. Identification of camel milk and shubat microflora provides a theoretical foundation for developing starter cultures by using local LAB strains for industrial production of traditional fermented milk products. (Resume d'auteur)

New approaches, mainly based on mass spectrometry techniques, are being developed and appear as a must in the modern food science and microbiology research to investigate food quality and safety. To date, the investigation of cheese ripening mechanisms has mostly used targeted approaches. The aims of the present project were to assess the use of untargeted metabolomics as an approach to investigate the influence of altering one ripening parameter to generate fine differences in the microbial metabolism within cheese. Two cheeses were made which varied with respect to the spatial distribution of bacterial colonies, leading to cheeses with only big or only small colonies. Liquid chromatography high resolution mass spectrometry metabolic fingerprints were acquired on cheese extracts collected after 2, 13 and 27 days of ripening using two different extraction methods (water or acetonitrile) and analyzed using two different simultaneous ionization modes (positive and negative electrospray). Data processing involving XCMS and multivariate statistical analysis highlighted significant discriminant profiles of the cheese metabolomes according to the two different spatial distributions compared. The different fractions investigated (water and acetonitrile extractions in two ionization modes) were complementary and resulted in a view as global as possible of the cheese metabolome which had been modulated by the spatial distribution of bacterial colonies. Some of the metabolites were then identified using an in-house database. These results show the relevance of cheese LC–HRMS fingerprinting to understand the influence of a ripening parameter generating fine differences on microbial metabolism within cheese.

Article in press; [i]Lactobacillus sakei[/i], a lactic acid bacterium naturally found in fresh meat and sea products, isconsidered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbonsources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of 80 (B1) and 70 kDa (B2), except for strain BMG.167which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (Nacetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledgeof their role in fermentation processes where they represent the dominant species.

This study aimed to evaluate molecular microbial markers associated with cocoa fermentation and drying steps. The dynamics of microbial populations were analyzed during postharvest processing (fermentation and sun drying) of cocoa from Ivory Coast. The variations in bacterial and yeast communities structure were monitored by a molecular technique (PCR-DGGE) using 16S rRNA and 26S rRNA profiles. Dominant microbial species were determined by sequencing. Our results showed that treatments of cocoa generated great variations in the microbial ecology in terms of number of species detected. The bacterial ecology quickly underwent dramatical changes during the fermentation step after two days. The dominance of Enterobacteria was first observed and then accompanied by Lactic acid bacteria and at the latest stages by acetic acid bacteria. Furthermore, Hanseniaspora opuntiae yeast species predominated during cocoa bean fermentation and drying processes. In addition, two bacterial species were only identified during the drying step of fermented cocoa beans and are potential candidates for biological markers of this process. (Resume d'auteur)

This chapter reviews recent data upon the potential of lactic acid bacteria (LAB) to improve animal productions, and animal health and well-being. The most important expected benefits of probiotic LAB are growth-promoting effects through better feed utilization and digestion, and biological control of pathogen colonization. They are also used to enhance milk and egg production. According to their functions and properties, additives for animal feed are classified in one or several categories including technological, sensorial, nutritional, or zootechnical additives. In Europe, the authorization of microorganisms used as zootechnical additives for feed is subjected to strict regulation. The chapter addresses the main animal production sectors, their specificities in terms of the production types (meat, milk, egg), the ecosystems to be considered (gut, vaginal, and/or mammary) and presence of LAB therein, the main diseases encountered, and the use of probiotics to prevent these diseases or enhance productivity.

International audience; Genomes can be markedly heterogeneous in conspecific bacterial strains. Genome sequences can be used to analyze genome plasticity via a PCR(2) (plasticity of chromosome revealed by PCR) approach. Small-sized chromosomes can indeed be fully amplified by long-range PCR with a set of primers designed using a reference strain and then applied to several other strains. Analysis of the resulting patterns can reveal genome plasticity. GenoFrag, a software package for the design of primers optimized for PCR(2) [N. Ben Zakour, M. Gautier, R. Andonov, D. Lavenier, M.F. Cochet, P. Veber, A. Sorokin, Y. Le Loir, GenoFrag: Software to design primers optimized for whole genome scanning by long-range PCR amplification, Nucleic Acids Res. 32 (2004) 17-24] was developed for the analysis of bacterial genome plasticity by whole genome amplification in approximately 10-kb-long fragments. By applying GenoFrag, we provide herewith evidence that genome plasticity can be analyzed in lactic acid bacteria using a PCR(2) approach. The genome sequences of Lactococcus lactis IL1403, Lactobacillus plantarum WCFS1, Lactobacillus bulgaricus ATCC11842 and Bifidobacterium longum NCC2705 were used to design four sets of primers. Each set was evaluated in silico to check that it ensured optimum coverage of the bacterial chromosome. To validate the primers generated by GenoFrag, a subset of primers was successfully used in LR-PCR experiments on genomic DNA from four L. bulgaricus strains.

Two starter cultures (A and B) and seven sodium lactate concentrations were evaluated for chicken raw dry-fermented sausage processing. Starter culture B contained more lactic acid bacteria and less staphylococci than starter A. Their effects on acidification and inhibition of pathogens (Listeria monocytogenes and Salmonella spp.) were monitored. Starter culture B grew faster and was less inhibited by sodium lactate, thus inducting a faster and more important pH drop into the sausages. With lower pH, sausages processed with B starter were less contaminated by Listeria monocytogenes. The type of starter was found to influence the end-product pH, lactic acid bacteria content and extent of Listeria monocytogenes contamination. A 30-member panel did not note differences between sausages processed with the different starter cultures when lactate was added. Adding sodium lactate to the sausage mix reduced the pH drop in the dry sausage product. This acidification inhibiting effect of sodium lactate was greater for A. Sodium lactate significantly inhibited lactic acid bacteria development but did not reduce Listeria monocytogenes contamination frequency of the batches, unlike in many literature data. Sodium lactate may however control the acidification of the sausage processed with starter B, in order to obtain moderately acidified fermented sausages. A simple kinetic model was applied to our data. The sodium lactate content and especially the type of starter culture often had a significant effect on the four parameters of this empirical model (lag time, acidification time, initial and final pH).

A new ATP bioluminescence-based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris. The principle of the method is to quantify the release of adenylic-nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin–luciferase. Nisin-induced leakage of AN included ATP from a sensitive L. cremoris to the external medium immediately after the contact with the bacteria. The growth of L. cremoris was correlated with the extracellular AN content. The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration. For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quantification of ATP. The effectiveness threshold, corresponding to a 100% inhibition of L. cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e. for a ANtot:ANext ratio = 1. For an initial concentration of 1.4 × 107 bacteria/mL, the nisin effectiveness threshold is 3.4 ± 0.01 mg nisin/L. It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells. Copyright © 2003 John Wiley & Sons, Ltd.

The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts.

Lactic acid bacteria (LAB) are Gram positive bacteria widely used in the production of fermented food in particular cheese and yoghurts. Bacteriophage infections during fermentation processes have been for many years a major industrial concern and have stimulated numerous research efforts. Better understanding of the molecular mechanisms of bacteriophage interactions with their host bacteria is required for the development of efficient strategies to fight against infections. The bacterial cell wall plays key roles in these interactions. First, bacteriophages must adsorb at the bacterial surface through specific interactions with receptors that are cell wall components. At next step, phages must overcome the barrier constituted by cell wall peptidoglycan to inject DNA inside bacterial cell. Also at the end of the infection cycle, phages synthesize endolysins able to hydrolyze peptidoglycan and lyse bacterial cells to release phage progeny. In the last decade, concomitant development of genomics and structural analysis of cell wall components allowed considerable advances in the knowledge of their structure and function in several model LAB. Here, we describe the present knowledge on the structure of the cell wall glycopolymers of the best characterized LAB emphasizing their structural variations and we present the available data regarding their role in bacteria-phage specific interactions at the different steps of the infection cycle.

Lactic acid (LA) fermentation is considered a simple and useful form of biotechnology to keep and/or enhance the safety, nutritional, sensory and shelf life properties of vegetables and fruits (Demir et al. 2006). As shown in the data from literature of the last decade, the combination of this ancient method of bio-preservation with the current biotechnology tools should allow controlled fermentation processes and the selection of starter cultures to increase the consumption of fresh-like vegetables and fruits (McFeeters 2004, Di Cagno et al. 2013). Lactic acid bacteria (LAB) convert the carbohydrate contents of the vegetables and fruits into LA, which decreases the pH of the fermented products to around 4.0 ensuring stability. Lower pH value restricts the growth of spoilage fl ora and pathogenic bacteria. These bacteria improve the human intestinal microbial balance and enhance health by inhibiting the growth of pathogens such as Escherichia coli, Salmonella and Staphylococcus (Ohmomo et al. 2000, Ross et al. 2002). They are often considered as probiotic, benefi cial for human health and active in lowering the serum cholesterol level (Kaur et al. 2002). They also stimulate immune responses and prevent tumour formation by inhibiting1 CIRAD-PERSYST, UMR Qualisud, TA B95/16, 34398 Montpellier Cedex 5, France. Email: didier.montet@cirad.fr, nadine.zakhia-rozis@cirad.fr 2 Central Tuber Crops Research Institute (Regional Centre), Bhubaneswar 751 019, India. Email: rc_rayctcri@rediffmail.com * Corresponding authorcarcinogenic compounds in the gastro-intestinal tract through reducing fecal bacteria enzyme activity (Nakphaichit et al. 2011) or breaking down certain enterotoxins (Bernardeau et al. 2006).

Aims : Nutritional requirements of #Lactobacillus fermentum$ Ogi E1 were studied in order to define a simplified fermentation medium. Methods and Results : When grown with MRS-medium in 21 bioreactors, a biphasic pattern of growth and metabolite production was observed. Study of nutritional requirements resulted in a simplified medium (SYAM) that allowed, under anaerobiosis, similar results to be obtained as in MRS medium, but without biphasic fermentation kinetics. The best substrates for both growth and amylase production were starch and maltose. Although melibiose, raffinose, fructose, sucrose and glucose also supported growth, lower amylase activity was observed. Conclusions : The physiology of the strain can be investigated with SYAM medium, using either starch or maltose as substrate. The strain also presented potential for alpha-galactoside fermentation. Significance and Impact of the Study : #Lactobacillus fermentum$ was one of the dominant bacteria of African maize dough fermentations. Amylolytic strains with activity against other compounds (i.e. raffinose) suggested a potential to be used as starter for cereal fermentation. (Résumé d'auteur)

The microbial community developing during the spontaneous fermentation of sour cassava starch was investigated by cultivation-independent methods. Denaturing gradient gel electrophoresis (DGGE) of partially amplified 16S rDNA followed by sequencing of the most intense bands showed that the dominant organisms were all lactic acid bacteria (LAB), mainly close relatives of #Bifidobacterium minimum$, #Lactococcus lactis$, #Streptoccocus$ sp., #Enteroccucus saccharolyticus$ and #Lactobacillus plantarum$. Close relatives of #Lb. panis$, #Leuconostoc mesenterides$ and #Ln. citreum$ were also found. A complementary analysis using hybridization of 16S rRNA with phylogenetic probes was necessary to detect the presence of the recently discovered species #Lb. manihotivorans$. Although it represented up to 13% of the total lactic acid bacteria of sour cassava starch, this species could not be detected by DGGE as the PCR product migrated to the same position as #Lc. lactis$. In addition, it was shown that a strong pH decrease in the time course of fermentation was most probably responsible for the competitive selection of acid-resistant LAB vs. both homo and heterofermentative acid-sensitive LAB. (Résumé d'auteur)

Lactobacillus helveticus and Propionibacterium freudenreichii are essential starters in Swiss cheesemaking and the release of their intracellular enzymes through autolysis could significantly influence ripening. To provide evidence of this lysis, cheese made from microfiltered thermized milk inoculated with Lb. helveticus ITGLH77, Prop. freudenreichii ITGP23 and a commercial Streptococcus thermophilus was assayed. Starter viability was determined and lysis was monitored during ripening by protein analysis with SDS-PAGE of aqueous cheese extracts and by immunoblot detection of intracellular proteins: dipeptidase (PepD) for Lb. helveticus and methylmalonyl coenzyme A mutase for Prop. freudenreichii. We verified that the species specificity of these lysis markers was towards the cytoplasms of all the species currently used in Swiss cheese. Lb. helveticus exhibited an almost complete loss of viability (99·9%) from the beginning of ripening in the cold room; concomitantly PepD appeared in the cheese extracts and was detected until the end of ripening. Damaged Lb. helveticus cells were also visualized by scanning electron microscopy. In addition, free PepD was also successfully detected in commercial Swiss-related cheeses. All these results clearly demonstrated the autolysis of Lb. helveticus in Swiss cheese. Prop. freudenreichii ITGP23 grew during warm room ripening and no loss of viability was detected after maximal growth (109 cfu/g cheese). Free methylmalonyl-coenzyme A mutase was detected at the end of ripening during cold storage, when the cheese extracts were concentrated 20-fold, demonstrating that the autolysis of Prop. freudenreichii was tardy and limited.

The most frequently used technique for dehydration of dairy bacteria is freeze drying. Of the other possible preservation techniques used in the dairy industry to produce large amounts of dairy ingredients at commercially viable processing costs, spray drying is one of the main processing tools and the cost is 10 times lower than that of freeze drying. In this work, some examples are presented for different species of dairy bacteria with respect to spray-drying processes (as an alternative approach to freeze drying) and storage conditions for production of powdered bacterial cultures with high viability (100%) and count (1010 g−1). The advantages and disadvantages of this new approach (viability, contamination, yield during process and storage) are discussed.

International audience; Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.