Skin-associated lactic acid bacteria from North American bullfrogs as potential control agents of Batrachochytrium dendrobatidis.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis and has been a key driver in the catastrophic decline of amphibians globally. While many strategies have been proposed to mitigate Bd outbreaks, few have been successful. In recent years, the use of probiotic formulations that protect an amphibian host by killing or inhibiting Bd have shown promise as an effective chytridiomycosis control strategy. The North American bullfrog (Lithobates catesbeianus) is a common carrier of Bd and harbours a diverse skin microbiota that includes lactic acid bacteria (LAB), a microbial group containing species classified as safe and conferring host benefits. We investigated beneficial/probiotic properties: anti-Bd activity, and adhesion and colonisation characteristics (hydrophobicity, biofilm formation and exopolysaccharide-EPS production) in two confirmed LAB (cLAB-Enterococcus gallinarum CRL 1826, Lactococcus garvieae CRL 1828) and 60 presumptive LAB (pLAB) [together named as LABs] isolated from bullfrog skin.We challenged LABs against eight genetically diverse Bd isolates and found that 32% of the LABs inhibited at least one Bd isolate with varying rates of inhibition. Thus, we established a score of sensitivity from highest (BdGPL AVS7) to lowest (BdGPL C2A) for the studied Bd isolates. We further reveal key factors underlying host adhesion and colonisation of LABs. Specifically, 90.3% of LABs exhibited hydrophilic properties that may promote adhesion to the cutaneous mucus, with the remaining isolates (9.7%) being hydrophobic in nature with a surface polarity compatible with colonisation of acidic, basic or both substrate types. We also found that 59.7% of LABs showed EPS synthesis and 66.1% produced biofilm at different levels: 21% weak, 29% moderate, and 16.1% strong. Together all these properties enhance colonisation of the host surface (mucus or epithelial cells) and may confer protective benefits against Bd through competitive exclusion. Correspondence analysis indicated that biofilm synthesis was LABs specific with high aggregating bacteria correlating with strong biofilm producers, and EPS producers being correlated to negative biofilm producing LABs. We performed Random Amplified Polymorphic DNA (RAPD)-PCR analysis and demonstrated a higher degree of genetic diversity among rod-shaped pLAB than cocci. Based on the LAB genetic analysis and specific probiotic selection criteria that involve beneficial properties, we sequenced 16 pLAB which were identified as Pediococcus pentosaceus, Enterococcus thailandicus, Lactobacillus pentosus/L. plantarum, L. brevis, and L. curvatus. Compatibility assays performed with cLAB and the 16 species described above indicate that all tested LAB can be included in a mixed probiotic formula. Based on our analyses, we suggest that E. gallinarum CRL 1826, L. garvieae CRL 1828, and P. pentosaceus 15 and 18B represent optimal probiotic candidates for Bd control and mitigation.

Effect of physicochemical factors on glycerol production by simultaneous cultures of wine micro-organisms using the response surface method.

AIM: To evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc2, Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). METHODS AND RESULTS: Multifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6 days yeasts prevailed, especially S. cerevisiae (10(9) CFU ml(-1)), while O. oeni reached 10(7) CFU ml(-1). At initial fixed pH 5·5, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26°C, 50 mg l(-1) SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26°C, 60·24 mg l(-1) SO2 and pH 5·5 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. CONCLUSIONS: We propose a predictive condition to improve the performance of mixed cultures for must fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: To optimize the culture conditions to design mixed starters containing autochthonous yeasts and O. oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics.

Survival and beneficial properties of lactic acid bacteria from raniculture subjected to freeze-drying and storage.

AIM: To evaluate the effect of freeze-drying and storage conditions on the viability and beneficial properties of lactic acid bacteria (LAB) for raniculture. METHODS AND RESULTS: Lactococcus lactis CRL 1584, L. lactis CRL 1827, Lactococcus garvieae CRL 1828 and Lactobacillus plantarum CRL 1606 viability under different conditions was studied. 10% lactose and 5% skim milk + 5% lactose were excellent lyoprotectants, but 5% skim milk + 5% lactose and whey protein concentrated (WPC) or WPC + sugars were the lower cost lyoprotective options. The effect of temperature depended on both lyoprotectants and storage time. Thus, for Lactococcus, skim milk, skim milk + sucrose and WPC + sucrose were selected for lyophilization and storage at 4°C and skim milk + lactose for 25°C. For Lact. plantarum CRL 1606, the best lyoprotectants for lyophilization and storage at 4°C were milk + sugars and WPS + sucrose and, at 25°C, skim milk + sucrose. CONCLUSIONS: Lactic acid bacteria viability after freeze-drying was strain-specific and depended on the lyoprotectant used. Highest viability was obtained when stored at 4°C, and the beneficial properties remained stable for 18 months independently of storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: The studies reported for the first time in this work are of primary interest to obtain dried bacteria to be included in beneficial products for raniculture.

Production and stability of pediocin N5p in grape juice medium.

The production and stability of pediocin N5p from Pediococcus pentosaceus, isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml-1) were observed at a initial pH of 7.5 and 30 degrees C. The activity of the inhibitory substance was stable between pH values from 2.0 to 5.0 at 4 degrees and 30 degrees C. At pH 10.0 it was completely inactivated. When submitted to 30 min at 80 degrees, 100 degrees and 115 degrees C, maximal stability was observed at pH 2.0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40-80 mg 1-1 SO2, independently or combined with different ethanol concentrations, affect inhibitory activity.