Safety, environmental and technological characterization of beneficial autochthonous lactic bacteria, and their vaginal administration to pregnant cows for the design of homologous multi-strain probiotic formulas.

In recent years, veterinary probiotic formulations constitute an interesting alternative to the use of antibiotics in animals for human consumption, but beneficial microorganisms must meet certain requirements to be included in these products. The objective of this work was to evaluate the safety and innocuity of beneficial autochthonous lactic bacteria (BALB) as well as to determine their beneficial, environmental, and technological characterization. Antibiotic resistance was assayed using phenotypic and genotypic methodology. A bovine vaginal fluid simulated medium (MSBVF) was designed where growth, pH changes, and expression of beneficial characteristics of lactic bacteria were evaluated; additionally, the optimal culture conditions in commercial media were determined in order to obtain the highest biomass production of the strains. Finally, the best strains were lyophilized and administered intravaginally to pregnant cows and their permanence in the vagina and adverse effects were evaluated. The results show that most of the strains were resistant to vancomycin, tetracycline, and streptomycin, with a high sensitivity to ampicillin, gentamicin, and clindamycin. The strains evaluated did not show gelatinase or hyaluronidase activity; however, 11 strains produced α-type hemolysis. The optimal growth of the microorganism was obtained in MRS broth, under slight agitation and without pH control. The strains grown in the MSBVF grew well and maintained the probiotic properties. Animals treated with probiotics bacteria did not show systemic or local inflammation. These strains can be included in a probiotic veterinary product to be applied to different bovine mucosa.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented chia sourdough.

Chia, is a gluten-free, rich in proteins, oilseed that is "on trend" as an alternative ingredient in food production, adding nutritional value. As a reservoir of natural biodiversity, lactic acid bacteria development, during spontaneous chia flour fermentation (sourdough) for 10 days, were investigated by culturing and high throughput sequencing (HTS). Culture-dependent analysis showed a rapid increase in total LAB numbers from the second day of sourdough refreshment. Taxonomical identification of LAB isolates by rep-PCR and further 16S rRNA sequencing was performed. Besides Among identified LAB by culture-dependent approach, species from genus Enterococcus were the most abundant; Lactococcus (Lc. lactis), Lactobacillus (L. rhamnosus) and Weissella (W. cibaria) species were also isolated. By HTS, twelve OTUs belonging to LAB genera were identified during chia sourdough fermentation with an increased Lactobacillus diversity. Enterococcus (E.) faecium, E. mundtii, W. cibaria and L. rhamnosus were detected as dominant species in the final propagation stages while Bacillus and Clostridium were mostly present during first fermentation stages. The investigation of biotechnological and safety traits (acidification ability, protein hydrolysis, exopolysaccharides production, antimicrobial activity and antibiotic resistance) of 15 representative LAB strains was performed. Strains characterization led to the selection of Lc. lactis CH179, L. rhamnosus CH34 and W. cibaria CH28 as candidates to be used as novel functional starter culture for gluten-free chia fermented products. As far as we know, this is the first study providing information on the molecular inventory of LAB population during spontaneous fermentation of chia sourdough.

Safety and Growth Optimization of Lactic Acid Bacteria Isolated From Feedlot Cattle for Probiotic Formula Design.

In order to eliminate the widespread use of antibiotics in livestock production, the research for alternatives has increased lately. This study examined the safety of 40 lactic acid bacteria (LAB) isolated from bovine feedlot environment and previously selected as potential probiotics. A high sensitivity prevalence to ampicillin (AMP, 100%), gentamicin (GEN, 96.3%), kanamycin (KAN, 96.3%), clindamycin (CLI, 85.2%), chloramphenicol (CHL, 92.6%) and streptomycin (STR, 88.9%) while moderate and high resistance against erythromycin (ERY, 48%) and tetracycline (TET, 79%) respectively, were determined. Feedlot enterococci and pediococci displayed high resistance to CLI, ERY, GEN and TET (73, 100, 54.5, and 73%, respectively). Among fifteen resistance genes investigated, seven were identified in lactobacilli; their presence not always was correlated with phenotypic resistance. STR resistance genes, aadA and ant(6) were observed in 7.4 and 3.7% of isolates, respectively; genes responsible for aminoglycosides resistance, such as bla (7.4%), and aph(3")-III (3.7%) were also recognized. In addition, resistance cat and tetS genes (3.7 and 7.4%, respectively) were harbored by feedlot lactobacilli strains. The presence of ermB gene in 22.3% of isolates, including two of the six strains phenotypically resistant to ERY, exhibited the highest prevalence among the assessed antibiotics. None of the feedlot lactobacilli harbored virulence factors genes, while positive PCR amplification for ace, agg, fsrA, and atpA genes was found for enterococci. With the objective of producing large cell biomass for probiotic delivery, growth media without peptone but containing glucose and skim milk powder (Mgl and Mlac) were selected as optimal. Lactobacillus acidophilus CRL2074, L. amylovorus CRL2115, L. mucosae CRL2069, and L. rhamnosus CRL2084 were strains selected as free of antibiotic resistance and virulence determinants, able to reach high cell numbers in non-expensive culture media and being compatible among them.