Microbial ecology involved in the ripening of naturally fermented llama meat sausages. A focus on lactobacilli diversity.

Llama represents for the Andean regions a valid alternative to bovine and pork meat and thanks to the high proteins and low fat content; it can constitute a good product for the novel food market. In this study, culture-dependent and independent methods were applied to investigate the microbial ecology of naturally fermented llama sausages produced in Northwest Argentina. Two different production technologies of llama sausage were investigated: a pilot-plant scale (P) and an artisanal one (A). Results obtained by High-Throughput Sequencing (HTS) of 16S rRNA amplicons showed that the production technologies influenced the development of microbial communities with a different composition throughout the entire fermentation process. Both sequencing and microbiological counts demonstrated that Lactic Acid Bacteria (LAB) contributed largely to the dominant microbiota. When a total of 230 isolates were approached by RAPD-PCR, presumptive LAB strains from P production exhibited an initial variability in RAPD fingerprints switching to a single profile at the final of ripening, while A production revealed a more heterogeneous RAPD pattern during the whole fermentation process. The constant presence of Lactobacillus sakei along the fermentation in both productions was revealed by HTS and confirmed by species-specific PCR from isolated strains. The technological characterization of Lb. sakei isolates evidenced their ability to grow at 15°C, pH4.5 and 5% NaCl (95%). Most strains hydrolyzed myofibrillar and sarcoplasmic proteins. Bacteriocins encoding genes and antimicrobial resistance were found in 35% and 42.5% of the strains, respectively. An appropriate choice of a combination of autochthonous strains in a starter formulation is fundamental to improve and standardize llama sausages safety and quality.

Lactic acid bacteria isolated from young calves--characterization and potential as probiotics.

Lactic acid bacteria (LAB) are widely used as probiotics in humans and animals to restore the ecological balance of different mucosa. They help in the physiological functions of newborn calves that are susceptible to a variety of syndromes. The criteria for the selection of strains for the design of probiotic products are not available. Based in the host-specificity of the indigenous microbiota, 96 LAB isolates from faeces and oral cavity of calves were obtained. The surface properties were screened showing a small number of highly hydrophobic or autoagglutinating isolates. Also, a group produced H(2)O(2) and were able to inhibit pathogens, and two strains were bacteriocin-producers. Some grew at very low pH and high bile concentrations. The strains sharing some of the specific properties evaluated were identified genetically, assayed their compatibility and exopolysaccharide production. The results allow going further in the establishment of criteria to select strains to be included in a multi-strain-probiotic-product to be further assayed in animals.

Screening of surface properties and antagonistic substances production by lactic acid bacteria isolated from the mammary gland of healthy and mastitic cows.

Bovine mastitis (BM) is a costly disease in dairy cattle production. The prevention and treatment of mastitis is performed by applying antimicrobial products that negatively affect milk quality. In the last years, the use of probiotic microorganisms to prevent infections in humans and animals has being aggressively studied. Samples from teat canal and milk (foremilk and stripping) were taken from healthy and mastitic mammary quarters. A screening of the surface properties and antagonistic substances production of lactic acid bacteria (LAB) isolated from the mammary gland was performed to select potential probiotic strains to prevent mastitis. Somatic cell count, physico-chemical and microbiological studies were carried out. Pre-selected microorganisms were genetically identified. Compared with stripping milk, foremilk showed lower levels of fat and higher levels of pH, density, microorganism numbers, lower percentage of strains with mean and high hydrophobicity and mean autoaggregation and higher number of strains able to produce hydrogen peroxide and bacteriocins. The other parameters analyzed were not statistically significant. One hundred and two LAB strains were isolated. Most of them had low degrees of hydrophobicity and autoaggregation. No correlation between these properties was found. Antagonistic metabolites were mainly produced by strains isolated from healthy quarters. Most of the pre-selected strains were identified as Streptococcus bovis and Weissella paramesenteroides. Three bacteriocin-producers were found and their products partially characterized. The results of this work are the basis for the further design of a specie-specific probiotic product able to prevent BM.

Inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus gasseri isolated from the vaginal tract of cattle.

Infectious diseases are the major reproductive complication during postpartum. The bacteria that colonize the postpartum uterus of cattle are specific as well as opportunistic pathogens that include Staphylococcus aureus. The antibiotics and antiseptic agents used in the treatment of postpartum acute infections induce residues in foods, spread of bacterial resistance, increase in financial costs and failures in defense mechanisms of the host. Preventive treatment with probiotic products could decrease the use of antibiotics in dairy farming systems. Lactobacilli are present in the vaginal microflora of healthy cows. They can prevent pathogen colonization by mechanisms such as the production of antagonistic substances as lactic acid, H2O2, or bacteriocins. The aim of the present study was the selection of H2O2 generating lactobacilli from a group of 72 strains isolated from the vagina of cattle. Both Lactobacillus gasseri CRL1421 and Lactobacillus gasseri CRL1412, which share some probiotic properties, produce H2O2, detected by the plate colorimetric method. They were chosen to study the kinetics of H2O2 production under different culture conditions. Both microorganisms produced greater amounts of H2O2 in aerated than in static cultures. As L. gasseri CRL1421 had a greater capacity to generate H2O2, associative cultures with this strain and S. aureus were conducted. A significant decrease in the growth of the pathogen was detected after culture for 6h, this effect being greater under aerated conditions. The addition of catalase to mixed cultures partially abolished the inhibition, an effect that could be attributed to the combined action of H2O2 and other antagonistic metabolites. The simultaneous addition of catalase and NaOH to these cultures restored S. aureus growth. This observation suggests that the inhibition was produced by the combination of H2O2 and lactic acid, both released by the lactobacilli. Electron microphotographs showed the damage caused by the lactobacilli supernatant on the pathogenic cells. The treatment of S. aureus with lactic acid and hydrogen peroxide evidenced that each metabolite produced a different type of morphological damage. The number of viable cells obtained agrees with the electron microscopy observations. The results support the idea that L. gasseri CRL1421 could be successfully included in a probiotic product to prevent S. aureus infection in cows.

Bacterial surface characteristics applied to selection of probiotic microorganisms.

For the study of probiotic microorganisms, the in vitro selection tests need to be based on a solid scientific foundation. Surface characteristics, one of the in vitro properties are used to evaluate the potentially probiotic strains of lactobacilli. Bacterial surface properties have been associated with attachment to a variety of substrata. Bacterial adhesion to tissues is considered the first step, and such adhesion can also determine the colonization capability of a microorganism. Through adhesion ability and colonization of tissues, probiotic microorganisms can prevent pathogen access by steric interactions or specific blockage on cell receptors. One of the main characteristics studied is the hydrophobic nature of the bacterial cell surface. To test this property, Rosenberg and Doyle divided microbial cell hydrophobicity assays into two categories. The first includes contact angle measurements (CAMs), partitioning of cells into one or another liquid phase (TTP), and adsorption of individual hydrophobic molecular probes at the cell surface. The second category includes microbial adhesion to hydrocarbons (MATH), hydrophobic interaction chromatography (HIC), and adhesion to polystyrene and other hydrophobic solid surfaces. The tests included in the first category measure hydrophobic properties of the outer cell surface as a whole; those in the second measure hydrophobicity in terms of adhesion. Finally, those bacterium classified as hydrophobic can be considered as able to mediate adhesion. The objective of this chapter is to describe three different methods applied in our laboratory for the study of bacterial surface properties. They can be used to screen characteristics of lactobacillus strains for probiotic purposes. They are: Microbial adhesion to hydrocarbons (MATH); Salt aggregation test (SAT); Hemagglutination (HA) reaction.