Elena Bru

Influence of pH, temperature and culture media on the growth and bacteriocin production by vaginal Lactobacillus salivarius CRL 1328

Aims: To study the influence of pH, temperature and culture medium on the growth and bacteriocin production by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328. 
Methods and Results: The study was performed using a complete factorial experimental design. Lactobacillus salivarius was cultivated in LAPTg and MRS broths, adjusted to specific initial pH, and at different temperatures of incubation. The growth, which was evaluated by the Gompertz model, was higher in MRS broth than in LAPTg broth. The initial pH of the culture medium and the temperature had a dramatic effect on the production of bacteriocin. The optimal conditions for bacteriocin production were different to those for optimal growth. The decrease in the pH of the culture medium was parallel to the growth; pH had similar final values in both the MRS and the LAPTg broths. 
Conclusions: The optimal growth conditions were recorded in MRS broth, with an initial pH of 6·5 and a temperature of 37°C. The maximum bacteriocin activity was obtained in LAPTg after 6 h at 37°C, and at an initial pH of 6·5 or 8·0. 
Significance and Impact of the Study: The application of a complete factorial design, and the evaluation of the growth parameters through the Gompertz model, enabled a rapid and simultaneous exploration of the influence of pH, temperature and growth medium on both growth and bacteriocin production by vaginal Lact. salivarius CRL 1328.

Identification of small peptides arising from hydrolysis of meat proteins in dry fermented sausages.

In this study, proteolysis and low molecular weight (LMW) peptides (<3kDa) from commercial Argentinean fermented sausages were characterized by applying a peptidomic approach. Protein profiles and peptides obtained by Tricine-SDS-PAGE and RP-HPLC-MS, respectively, allowed distinguishing two different types of fermented sausages, although no specific biomarkers relating to commercial brands or quality were recognized. From electrophoresis, α-actin, myoglobin, creatine kinase M-type and L-lactate dehydrogenase were degraded at different intensities. In addition, a partial characterization of fermented sausage peptidome through the identification of 36 peptides, in the range of 1000-2100 Da, arising from sarcoplasmic (28) and myofibrillar (8) proteins was achieved. These peptides had been originated from α-actin, myoglobin, and creatine kinase M-type, but also from the hydrolysis of other proteins not previously reported. Although muscle enzymes exerted a major role on peptidogenesis, microbial contribution cannot be excluded as it was postulated herein. This work represents a first peptidomic approach for fermented sausages, thereby providing a baseline to define key peptides acting as potential biomarkers.

Diversity in growth and protein degradation by dairy relevant lactic acid bacteria species in reconstituted whey

The high nutritional value of whey makes it an interesting substrate for the development of fermented foods. The aim of this work was to evaluate the growth and proteolytic activity of sixty-four strains of lactic acid bacteria in whey to further formulate a starter culture for the development of fermented whey-based beverages. Fermentations were performed at 37 °C for 24 h in 10 and 16% (w/v) reconstituted whey powder. Cultivable populations, pH, and proteolytic activity (o-phthaldialdehyde test) were determined at 6 and 24 h incubation. Hydrolysis of whey proteins was analysed by Tricine SDS-PAGE. A principal component analysis (PCA) was applied to evaluate the behaviour of strains. Forty-six percent of the strains grew between 1 and 2 Δlog CFU/ml while 19% grew less than 0·9 Δlog CFU/ml in both reconstituted whey solutions. Regarding the proteolytic activity, most of the lactobacilli released amino acids and small peptides during the first 6 h incubation while streptococci consumed the amino acids initially present in whey to sustain growth. Whey proteins were degraded by the studied strains although to different extents. Special attention was paid to the main allergenic whey protein, β-lactoglobulin, which was degraded the most by Lactobacillus acidophilus CRL 636 and Lb. delbrueckii subsp. bulgaricus CRL 656. The strain variability observed and the PCA applied in this study allowed selecting appropriate strains able to improve the nutritional characteristics (through amino group release and protein degradation) and storage (decrease in pH) of whey.

Optimization of Low-Cost Culture Media for the Production of Biomass and Bacteriocin by a Urogenital Lactobacillus salivarius Strain

The aim of this work was to formulate a culture medium of lower cost than conventional laboratory media, in order to simultaneously obtain high amounts of both biomass and bacteriocin of vaginal Lactobacillus salivarius CRL 1328. The growth assays under different culture conditions were performed by using a 28−2 central composite experimental design, with a central point and sixteen additional points. The factors taken into consideration were glucose, lactose, yeast extract, tryptone, ammonium citrate, sodium acetate, MgSO4 and MnSO4. The simultaneous presence of a carbon source (mainly glucose), a nitrogen source (mainly yeast extract) and salts (mainly MnSO4, MgSO4 and sodium acetate) allowed the highest cell biomass and bacteriocin levels to be reached in the experimental design. Through the application of the desirability function, several optimal medium compositions to achieve efficient production of biomass and bacteriocin were predicted. The optimized growth media allow a cost reduction of around 25 to 40% compared with conventional broths. The results obtained represent an advance in the search of the most suitable strategies for the production of bioactive compounds for pharmaceutical products to prevent or treat female urogenital infections.

Estimation of combined effects of carbon and nitrogen sources on the growth and bacteriocin production of Lactobacillus salivarius from human source.

Vaginal Lactobacillus salivarius CRL 1328 (Centro de Referencia para Lactobacilos Culture Collection) or the bacteriocin that produces could be included in a urogenital probiotic formula to prevent urogenital infections in women. The objective of this work was to determine the effects of different carbon and nitrogen sources on the growth and bacteriocin production of this microorganism. A fractional factorial design 35–1 was applied to evaluate the effects of five nutrients (glucose, lactose, yeast extract, tryptone and meat peptone), at three different concentrations (0, 1 and 2%). Results were statistically analyzed for linear and quadratic effects of nutrients, along with their interactions. All the nutrients tested stimulated the cell growth and bacteriocin production, but lactose had not a significant influence on the last response. The linear effects of higher magnitude on biomass and bacteriocin production were those of yeast extract and tryptone. The results demonstrated that there were significant interactions between the different nutrients, depending on the response evaluated. Maximum bacteriocin production was reached in different growth media with a lower cost than conventional culture media used in the laboratory. These findings will contribute to the design of a pharmaceutical product for the restoration of ecological balance of urogenital tract. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Application of fluorescent techniques to evaluate the survival of probiotic lactobacilli to bile acid

Purpose of workTo apply a fluorescent dye as an alternative technique to evaluate the survival of potentially probiotic lactobacilli to bile acids (BA) as first step in the design of probiotic functional foods.The use of lactic acid bacteria (LAB) in the functional food design depends on their ability to survive in the gastrointestinal tract where bile is an important natural barrier. Bile is mainly constituted by conjugated BA, which can be hydrolyzed to free BA and taurine or glycine. Changes in the transmembrane electrical potential (ΔΨ) of probiotic LAB strains due to the effect of conjugated and free BA were measured and showed that the majority of the tested LAB strains had greater sensibility to free BA than to their respective conjugated acids. Variations in the ΔΨ of the microorganism correlated well with bacterial viability determined by standard plate count method. We therefore propose the DiSC3-based fluorescent technique as a rapid and effective method to evaluate the resistance of probiotic lactobacilli to bile as first step for strain selection to be included in functional foods.

Biocontrol of Listeria monocytogenes in a meat model using a combination of a bacteriocinogenic strain with curing additives

The aim of this work was to evaluate the effect of meat curing agents on the bioprotective activity of the bacteriocinogenic strain, Enterococcus (E.) mundtii CRL35 against Listeria (L.) monocytogenes during meat fermentation. The ability of E. mundtii CRL35 to grow, acidify and produce bacteriocin in situ was assayed in a meat model system in the presence of curing additives (CA). E. mundtii CRL35 showed optimal growth and acidification rates in the presence of CA. More importantly, the highest bacteriocin titer was achieved in the presence of these food agents. In addition, the CA produced a statistical significant enhancement of the enterocin CRL35 activity. This positive effect was demonstrated in vitro in a meat based culture medium, by time-kill kinetics and finally by using a beaker sausage model with a challenge experiment with the pathogenic L. monocytogenes FBUNT strain. E. mundtii CRL35 was found to be a promising strain of use as a safety adjunct culture in meat industry and a novel functional supplement for sausage fermentation, ensuring hygiene and quality of the final product.

Statistical Models to Optimize Production of Probiotic Characteristics

The use of probiotic microorganisms has been widely promoted in the last 20 years. Probiotics have applied to the gastrointestinal tract as capsules or as fermented milks. The characteristics of the strains proposed as probiotics have been published or patented after elaboration of the product. The first step in designing a probiotic product is to isolate and characterize strains with beneficial properties. Our group has reported the isolation and characterization of 134 strains of lactobacilli from the vagina of women from our city. Surface properties and the production of antagonistic substances has also been published. From these studies, certain strains were selected because they shared probiotic characteristics. The second step in the design of a pharmaceutical product is to determine the optimal conditions for obtaining the highest amount of viable microorganisms, as well as the best conditions to produce antagonistic substances. To perform these types of experiments, laboratory assays can be carried out to determine the optimum conditions in which the microorganisms can be cultured. Growth parameters and optimal growth conditions can be studied by the classical methodology or by the application of alternative methodologies. Alternative methods have been widely used in the food industry, including experimental designs to test multiple conditions at the same time, complemented by the use of statistical models to evaluate and compare all the conditions tested. The object of this chapter is to describe the experimental designs used to study the technological characteristics of vaginal probiotic lactobacilli as well as the application of statistical models for evaluation of the different conditions tested for the production of biomass and antagonistic substances in these bacteria. With this objective in mind, in the present chapter, the following areas are explored: 1. Growth experiments performed to study the effect of temperature, pH, agitation, and culture media on the growth and production of antagonistic substances by vaginal lactobacilli. 2. Fractional and complete factorial experimental designs used to perform the growth experiments. 3. Statistical models applied to evaluate the growth parameters and their modifications under the conditions assayed.

Successful management with fosfomycin + ceftazidime of an infection caused by multiple highly-related subtypes of multidrug-resistant and extensively drug-resistant KPC-producing Serratia marcescens

Fil: Rodriguez, Maria Cecilia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Tucuman. Centro de Referencia para Lactobacilos; Argentina