ABSTRACT
With the increase of antimicrobial resistances due to the widespread use of antibiotics, the search of new probiotics to control aquaculture diseases has a growing public interest. The aim of this study was to isolate bacteria with antimicrobial effect from the gut of marine healthy fishes and select lactic acid bacteria (LAB) as potential probiotics, being strains considered as generally regarded as safe (GRAS) by the European Food Safety Agency (EFSA). Of a total of 45 Gram-positive strains with antimicrobial activity found in a screening of the gut microbiota of 13 marine fishes, nine were identified as LAB by 16S rRNA gene sequencing. LAB strains (five Lactococcus lactis subsp. lactis, two Enterococcus spp., one Lactobacillus plantarum, and one Leuconostoc mesenteroides subsp. mesenteroides) also showed a broad-spectrum antibacterial activity against aquaculture pathogens such as Vibrio harveyi, V. splendidus, and Photobacterium damselae and survived in experimental gastrointestinal conditions when grown in culture media modified with different values of pH and bile salts. These results showed the potential of LAB obtained from the indigenous microbiota of wild marine fishes for use as probiotics in aquaculture.
Subject(s)
Aquaculture , Fishes/microbiology , Gastrointestinal Microbiome , Lactobacillales/isolation & purification , Probiotics/isolation & purification , Animals , Anti-Infective Agents/pharmacology , Bile Acids and Salts/pharmacology , Hydrogen-Ion Concentration , Lactobacillales/genetics , Marine BiologyABSTRACT
The present work describes a new analytical method for direct immunoaffinity column clean-up of ochratoxin A (OTA) in milk samples followed by determination of the toxin using high-performance liquid chromatography with fluorescence detection (HPLC-FD). Two different immunoaffinity cartridges (IAC) were investigated, and Ochraprep columns were chosen because they showed the best results. An average recovery of 89.8% and a mean RSD of 5.8% for artificially contaminated cow's milk in the range of 5-100 ng/L were attained. The calculated limit of detection (LOD) and limit of quantitation (LOQ) were as low as 0.5 and 5 ng/L, respectively. This new easy and fast method avoids a previous liquid-liquid extraction step and therefore the use of toxic chlorinated solvents. Chromatograms of the final extracts were clean and OTA could be easily detected at a retention time of 8.4 min without interferences. To assess the presence of the toxin in cow's milk eight samples of skimmed and four samples of whole milk were analysed and OTA was not detected over the established detection limit.
Subject(s)
Carcinogens/analysis , Chromatography, Affinity/methods , Chromatography, High Pressure Liquid/methods , Food Contamination/analysis , Milk/chemistry , Ochratoxins/analysis , Analytic Sample Preparation Methods , Animals , Aspergillus/chemistry , Cattle , Chemical Fractionation/methods , Food Analysis/methods , Penicillium/chemistry , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, FluorescenceABSTRACT
Lactococcus lactis subsp. lactis IPLA972 is a wild lactococcal strain suitable as a single starter in the manufacture of dairy products. This strain synthesizes lactococcin 972 (Lcn972), a unique bacteriocin that blocks septum formation. In this work, we report on the conditions to optimize biomass and Lcn972 production. In batch cultures, pH 6.8 was found to be optimum for bacteriocin synthesis and both glucose and lactose supported Lcn972 production. The inhibitory activity improved up to eight-fold with increasing carbohydrate concentration. In chemostat cultures, steady states were achieved even at dilution rates higher than mu(max), due to the strong wall growth. Lcn972 behaved as a true primary metabolite, as it was maximally produced when the cells were actively growing. Bacteriocin yields were improved up to ten-fold in chemostat cultures compared with those achieved in batch.
