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In the agri-food context, bioprotective cultures represent an interesting alternative to chemical preservatives. The aim of this study was to evaluate the in vitro and in situ antifungal activity of lactic acid bacteria (LAB) and propionibacteria against bakery product spoilage fungi. Firstly, the biodiversity of fungal contaminant in pound cakes and milk bread rolls, as well as the resistance to chemical preservatives of representative isolates (n = 21), were studied. Aspergillus and Eurotium species were the most dominant spoilage fungi and the most resistant towards the tested chemical preservatives. They were followed by Penicillium , Cladosporium and Wallemia spp. Secondly, an in vitro screening showed that the most active isolates against selected fungal targets belonged to the Leuconostoc spp., Lactobacillus reuteri and Lactobacillus buchneri groups among the 270 tested LAB, as well as to the Propionibacterium freudenreichi and Propionibacterium acidipropionici species for the 50 tested propionibacteria. Finally, in situ tests showed that cultures of isolates belonging to the Leuconostoc citreum , Lactobacillus sakei , Lactobacillus plantarum , Lactobacillus spicheri , L. reuteri and Lactobacillus brevis species could delay one or several target fungal growths after bakery product surface spraying. Moreover, different strain cultures led to delayed fungal growths after incorporation in milk bread rolls preparation. The combination of in vitro and in situ approaches allowed for the identification of bacteria exhibiting antifungal activity, providing future prospects for use as bioprotective cultures in bakery products.

Lactobacillus paracasei FT700 was tested for its survival in simulated gastrointestinal conditions ( in vitro ) and for its sensitivity to antibiotics. The strain was used to produce fermented milk and the cell-free supernatant was freeze-dried and applied in cell cultures (monocytes and macrophages) to evaluate its cytotoxicity and immunomodulatory effects. After 180 min at pH 2.0 or in simulated gastric juice, L. paracasei FT700 concentrations were reduced by 4 and 3 log cfu/mL, respectively. Moreover, the strain was susceptible to the majority of antibiotics tested. Freeze dried cell-free fermented milk supernatant (FMS) was toxic to monocytes after 72 h of exposure at 10 mg/mL inducing their apoptosis. However, at concentrations below toxic level, FMS stimulated the differentiation of monocytes into macrophages, as observed by the increased expression of CD71 marker. This immune stimulation was not inflammatory since low production of TNF-α was observed. Overall, L. paracasei FT700 has potential to be used as probiotic in functional fermented milk.

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

Eleven antifungal Lactobacillus strains previously isolated from cow and goat milk were fully characterized using molecular and phenotypic methods. Their antifungal activities were tested in milk and yogurt, against fungal species (Debaryomyces hansenii, Kluyveromyces lactis, Kluyveromyces marxianus, Penicillium brevicompactum, Rhodotorula mucilaginosa and Yarrowia lipolytica) commonly involved in the spoilage of dairy products. The antifungal strains belonged to Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus zeae and Lactobacillus harbinensis species and showed different acidifying and growth capacities in milk. All tested Lactobacillus strains showed an antifungal activity in milk with strain-dependent activity spectra. Lb. harbinensis showed a very strong antifungal effect in yogurt by completely inhibiting all tested fungi as compared to control. The other tested strains were much less effective. It is the first time that the antifungal activity of Lb. harbinensis is described. This strain is a potential candidate for yogurt biopreservation.

Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified asStreptococcus uberis(strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified asWeissella confusaFT424,W. hellenicaFT476,Leuconostoc citreumFT671 andLactobacillus plantarumFT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain wasL. plantarumFT723 that inhibitedPenicillium expansumin modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed againstYarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified asEnterococcus faecalis(strains FT132 and FT522) andLactobacillus paracaseiFT700 were confirmed by SDS–PAGE, as visualized by the digestion of caseins and whey proteins (β-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

International audience; BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that significantly increases morbidity and mortality in nosocomial infections and cystic fibrosis patients. Its pathogenicity especially relies on the production of virulence factors or resistances to many antibiotics. Since multiplication of antibiotic resistance can lead to therapeutic impasses, it becomes necessary to develop new tools for fighting P. aeruginosa infections. The use of probiotics is one of the ways currently being explored. Probiotics are microorganisms that exert a positive effect on the host's health and some of them are known to possess antibacterial activities. Since most of their effects have been shown in the digestive tract, experimental data compatible with the respiratory environment are strongly needed. The main goal of this study was then to test the capacity of lactobacilli to inhibit major virulence factors (elastolytic activity and biofilm formation) associated with P. aeruginosa pathogenicity. RESULTS: Sixty-seven lactobacilli were isolated from the oral cavities of healthy volunteers. These isolates together with 20 lactobacilli isolated from raw milks, were tested for their capacity to decrease biofilm formation and activity of the elastase produced by P. aeruginosa PAO1. Ten isolates, particularly efficient, were accurately identified using a polyphasic approach (API 50 CHL, mass-spectrometry and 16S/rpoA/pheS genes sequencing) and typed by pulsed-field gel electrophoresis (PFGE). The 8 remaining strains belonging to the L. fermentum (6), L. zeae (1) and L. paracasei (1) species were sensitive to all antibiotics tested with the exception of the intrinsic resistance to vancomycin. The strains were all able to grow in artificial saliva. CONCLUSION: Eight strains belonging to L. fermentum, L. zeae and L. paracasei species harbouring anti-elastase and anti-biofilm properties are potential probiotics for fighting P. aeruginosa pulmonary infections. However, further studies are needed in order to test their innocuity and their capacity to behave such as an oropharyngeal barrier against Pseudomonas aeruginosa colonisation in vivo.

International audience; Few antifungal protective cultures adapted to fermented dairy products are commercially available because of the numerous constraints linked to their market implementation. Consumer's demand for naturally preserved food products is growing and the utilization of lactic acid bacteria is a promising way to achieve this goal. In this study, using a 2(5-1) factorial fractional design, we first evaluated the effects of fermentation time, of initial sucrose concentration and of the initial contamination amount of a spoilage yeast, on antifungal activities of single and mixed cultures of Lactobacillus rhamnosus K.C8.3.1I and Lactobacillus harbinensis K.V9.3.1Np in yogurt. L. harbinensis K.V9.3.1Np, the most relevant strain with regard to antifungal activity was then studied to determine its minimal inhibitory inoculation rate, its antifungal stability during storage and its impact on yogurt organoleptic properties. We showed that L. harbinensis K.V9.3.1Np maintained a stable antifungal activity over time, which was not affected by initial sucrose, nor by a reduction of the fermentation time. This inhibitory activity was an all-or-nothing phenomenon. Once L. harbinensis K.V9.3.1Np reached a population of ∼2.5 × 10(6) cfu/g of yogurt at the time of contamination, total inhibition of the yeast was achieved. We also showed that an inoculation rate of 5 × 10(6) cfu/ml in milk had no detrimental effect on yogurt organoleptic properties. In conclusion, L. harbinensis K.V9.3.1Np is a promising antifungal bioprotective strain for yogurt preservation.

Knowledge of fungal diversity in the environment is poor compared with bacterial biodiversity. In this study, we applied the denaturing high-performance liquid chromatography (D-HPLC) technique, combined with the amplification of the ITS1 region from fungal rDNA, for the rapid identification of major fungal species in 9 raw milk samples from cow, ewe and goat, collected at different periods of the year. A total of 27 fungal species were identified. Yeast species belonged to Candida, Cryptococcus, Debaryomyces, Geotrichum, Kluyveromyces, Malassezia, Pichia, Rhodotorula and Trichosporon genera; and mold species belonged to Aspergillus, Chrysosporium, Cladosporium, Engyodontium, Fusarium, Penicillium and Torrubiella genera. Cow milk samples harbored the highest fungal diversity with a maximum of 15 species in a single sample, whereas a maximum of 4 and 6 different species were recovered in goat and ewe milk respectively. Commonly encountered genera in cow and goat milk were Geotrichum candidum, Kluyveromyces marxianus and Candida spp. (C. catenulata and C. inconspicua); whereas Candida parapsilosis was frequently found in ewe milk samples. Most of detected species were previously described in literature data. A few species were uncultured fungi and others (Torrubiella and Malassezia) were described for the first time in milk.

International audience; The genera Propionibacterium and Bifidobacterium are clustered in the class Actinobacteria and form the anaerobic branch of coryneform bacteria. The dairy propionibacteria comprising four species P. freudenreichii, P. acidipropionici, P. jensenii and P. thoenii are industrially important as starter cultures in hard-cheese ripening and recently also as protective bio-preservatives and probiotics. These four species are considered as safe whereas cutaneous Propionibacterium species (also named "acnes group") are pathogens. In contrast, bifidobacteria in fermented dairy products and milk powder are exclusively used as probiotics; selected strains of several species (out of more than thirty) contribute to this task. It has been only rarely found that commensal bifidobacteria have been connected with certain dental and other infections. Consequently, only one single species, Bifidobacterium dentium, is recognized as pathogenic. Genome sequence analysis of Bifidobacterium longum and molecular biological analysis of other probiotic strains confirmed so far the absence of virulence and pathogenecity factors. However, tetracycline resistance genes tet(W), although probably not easy transferable, were found in Bifidobacterium strains, also in Bifidobacterium animalis subsp. lactis, the worldwide most used industrial strain. Conclusively, strains from the Propionibacterium and Bifidobacterium species in dairy food generally represent so far no health hazards.

Background Reuterin produced from glycerol by Lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of Lb. reuteri. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin. The aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols (LMW-SH) such as glutathione, hydrogen peroxide and/or reuterin sensitivity. Reuterin was produced by Lb. reuteri SD2112 in pure glycerol solution, purified and used to test the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). Hydrogen peroxide sensitivity and intracellular LMW-SH concentration were also analysed. Results Our data showed that most tested intestinal bacteria showed MIC below that for a sensitive indicator Escherichia coli (7.5–15 mM). Lactobacilli and Clostridium clostridioforme were more resistant with MIC ranging from 15 to 50 mM. No correlation between bacterial intracellular concentrations of LMW-SH, including glutathione, and reuterin or hydrogen peroxide sensitivities were found. Conclusion Our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of LMW-SH was not directly linked to their capacity to resist reuterin or hydrogen peroxide. This suggests that detoxification by LMW-SH such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide., BMC Microbiology, 7, ISSN:1471-2180