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.

Enhanced mannitol biosynthesis by the fruit origin strain Fructobacillus tropaeoli CRL 2034.

Mannitol is a natural low-calorie sugar alcohol produced by certain (micro)organisms applicable in foods for diabetics due to its zero glycemic index. In this work, we evaluated mannitol production and yield by the fruit origin strain Fructobacillus tropaeoli CRL 2034 using response surface methodology with central composite design (CCD) as optimization strategy. The effect of the total saccharide (glucose + fructose, 1:2) content (TSC) in the medium (75, 100, 150, 200, and 225 g/l) and stirring (S; 50, 100, 200, 300 and 350 rpm) on mannitol production and yield by this strain was evaluated by using a 22 full-factorial CCD with 4 axial points (α = 1.5) and four replications of the center point, leading to 12 random experimental runs. Fermentations were carried out at 30 °C and pH 5.0 for 24 h. Minitab-15 software was used for experimental design and data analyses. The multiple response prediction analysis established 165 g/l of TSC and 200 rpm of S as optimal culture conditions to reach 85.03 g/l [95% CI (78.68, 91.39)] of mannitol and a yield of 82.02% [95% CI (71.98, 92.06)]. Finally, a validation experiment was conducted at the predicted optimum levels. The results obtained were 81.91 g/l of mannitol with a yield of 77.47% in outstanding agreement with the expected values. The mannitol 2-dehydrogenase enzyme activity was determined with 4.6-4.9 U/mg as the highest value found. To conclude, F. tropaeoli CRL 2034 produced high amounts of high-quality mannitol from fructose, being an excellent candidate for this polyol production.

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.

Physicochemical factors differentially affect the biomass and bacteriocin production by bovine Enterococcus mundtii CRL1656.

Bovine Enterococcus mundtii CRL1656 (Centro de Referencia para Lactobacilos Culture Collection) produces an anti-Listeria and anti-Streptococcus dysgalactiae bacteriocin identified as mundticin CRL1656. The strain and its bacteriocin are candidates to be included in a beneficial product to prevent bovine mastitis as an alternative to antimicrobial agents. To optimize the production of biomass and mundticin CRL1656 by E. mundtii CRL1656, a complete 3 × 2(4) factorial design was applied. The effect of culture medium, initial pH, inoculum size, incubation temperature, and agitation conditions on biomass and bacteriocin production was evaluated simultaneously. Growth parameters were determined using the modified Gompertz model. A nonlinear model was used to estimate the effects of the variables on growth parameters. Bacteriocin production was analyzed using a linear mixed model. Optimal biomass and mundticin CRL1656 production by E. mundtii CRL1656 were obtained in different conditions. Maximal growth was recorded in autolyzed yeast, peptone, tryptone, Tween 80, and glucose or M17 broths, pH 6.5, 5.0% inoculum, 30 °C, with agitation. However, bacteriocin titers were higher in autolyzed yeast, peptone, tryptone, Tween 80, and glucose or de Man-Rogosa-Sharpe (MRS) broths, pH 6.5, 30°C, both with or without agitation. Knowledge of the optimum conditions for growth and bacteriocin production of E. mundtii CRL1656 will allow the obtainment of high levels of biomass and mundticin CRL1656 as bioingredients of potential products to prevent bovine mastitis.

Effect of lyophilization and storage temperature on the activity of salivaricin CRL 1328, a potential bioactive ingredient of a urogenital probiotic product.

Bacteriocins are antimicrobial peptides with potential applications as therapeutic agents for the treatment of microbial infections. The aim of this work was to investigate the effect of different protectors on the activity of salivaricin CRL1328, a bacteriocin produced by Lactobacillus salivarius CRL1328, during the lyophilization process and subsequent storage at different temperatures for 18 months using statistical models. Different protectors such as mannitol, Tween 80, polyethylene glycol 8000 (PEG), monosodium glutamate (MSG), reconstituted skim milk, sucrose and ascorbic acid were used for the lyophilization and storage of salivaricin. The biplot of principal component analysis was used for the interpretation of the interactions between the different factors studied. The antimicrobial activity of salivaricin was dependent mainly on temperature, and also on the time of storage and protector assayed. The stability of salivaricin was higher at -20°C and 4°C than 25°C and decreased during the time of storage; however, salivaricin was active after 18 months of storage at 25°C. Sucrose, mannitol plus sucrose, PEG plus sucrose and MSG were the most effective agents in protecting the bacteriocin during the lyophilization process. Effective maintenance of the activity of the bacteriocin was observed by storage with sucrose and ascorbic acid at -20°C as well as with PEG plus sucrose at 4°C and -20°C. The results obtained suggest that sucrose alone or combined with PEG can effectively maintain the activity of salivaricin during lyophilization and storage. This study provides useful information for the potential application of salivaricin as a bioactive principle for a pharmaceutical formulation.

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, ß-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.

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

PURPOSE OF WORK: To 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 DiSC(3)-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.

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 3(5-1) 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.

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 2(8-2) 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.

Stability of freeze-dried vaginal Lactobacillus strains in the presence of different lyoprotectors.

The industrial use of lactic acid bacteria as probiotic cultures depends on the preservation techniques employed, which are required to guarantee stable cultures in terms of viability and functional activity. The aim of this study was to evaluate the effects of 12% lactose and 12% sucrose suspended in water or reconstituted skim milk on the survival and expression of beneficial characteristics during freeze-drying and subsequent storage of 6 vaginal lactobacilli strains. A cubic polynomial model was also used for the first time to evaluate the effects of different protectors on survival behavior during storage. Different survival patterns were observed among the strains considered. The presence of both lactose and sucrose in water or in 6% skim milk as the suspension medium proved to be effective in maintaining a high degree of survival and expression of potentially probiotic characteristics (production of antimicrobial substances or auto-aggregation capabilities) of most strains after lyophilization and long-term storage. This study constitutes a valuable step to obtain concentrated cultures with the highest stability of microorganisms for pharmaceutical purposes.

Influence of wine-related physicochemical factors on the growth and metabolism of non-Saccharomyces and Saccharomyces yeasts in mixed culture.

The influence of two physicochemical factors involved in winemaking, temperature and SO(2), on the kinetics and metabolic behavior of Kloeckera apiculata and Saccharomyces cerevisiae was examined. Highest biomass was reached at 15 and 25 degrees C for K. apiculata and S. cerevisiae, respectively. Pure cultures of K. apiculata died off early with increasing temperature, but in co-culture with S. cerevisiae it showed higher viability and a change in the death curve from exponential to linear. Statistical analysis revealed that metabolite production was significantly different for the three cultures and also at the different fermentation temperatures. Besides, the interaction between culture type and temperature was significant. At temperatures from 15 to 30 degrees C the mixed culture showed similar ethanol and lower acetic acid production compared with a pure culture of K. apiculata. SO(2) addition slightly increased survival of the non-Saccharomyces species in pure and mixed cultures. Statistical evaluation indicated that culture type and SO(2) addition significantly affected metabolite production, but the interaction between culture and SO(2) was not significant. These results contribute to current knowledge of enological factors and their effect on prevalence and fermentative activities of the composite yeast flora and the statistical significance emphasizes the importance of the combined influence of the culture type and physicochemical factors on the production of fermentation metabolites.

Lactobacillus casei improves resistance to pneumococcal respiratory infection in malnourished mice.

We studied the effect of Lactobacillus casei CRL 431 used as a supplement in a repletion diet on the resistance to Streptococcus pneumoniae respiratory infection in malnourished mice. Weaned mice were malnourished after they consumed a protein-free diet (PFD) for 21 d. Malnourished mice were fed a balanced conventional diet (BCD) with or without supplemental L. casei for 7, 14, or 21 consecutive days, or BCD for 7 d with L. casei supplementation on d 6 and 7 (7dBCD+2dLc). The malnourished control (MNC) group was fed only the PFD, whereas well-nourished control (WNC) mice consumed the BCD ad libitum. Mice were challenged with S. pneumoniae at the end of each dietary treatment. Lung colonization and bacteremia were significantly greater in MNC than in WNC. Normalization of the immune response occurred in malnourished mice fed the BCD for 21 d. L. casei supplementation reduced the time required for a normal response from 21 to 7 d. Mice administered the 7dBCD+2dLc repletion treatment had a more effective pathogen clearance from blood and significantly lower lung damage than MNC. This treatment improved both the number of leukocytes and neutrophils in blood and bronchoalveolar lavages (BAL) and the bactericidal function of phagocytic cells to levels that did not differ from those of WNC. In the 7dBCD+2dLc mice, antipneumococcal IgA in BAL was higher than in WNC, whereas antipneumococcal IgG in serum and BAL did not differ. This study suggests that the addition of L. casei to the repletion diet has a beneficial effect because it accelerates the recovery of the innate immune response and improves the specific immune mechanisms against an S. pneumoniae respiratory infection in malnourished mice.

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.

Evaluation and comparison of lactobacilli characteristics in the mouths of patients with or without cavities.

Lactobacilli were considered the prime cariogenic microorganisms until phylogenetic related bacteria, mutans streptococci, were associated with caries. Today, lactobacilli are still considered a factor in determining the predisposition to develop cavities. As a first step in colonization, microorganisms adhere to oral tissues. Based on this fact, the purpose of our study was to determine if there was a statistical association or difference related to the state of oral health with the surface characteristics of lactobacilli. Patients were classified as caries-free and caries-active. Interviews were performed to establish the nutritional and oral hygiene habits. The previously reported characteristics of isolated lactobacilli's quantification and association to dental tissues were determined. Although the nutritional habits for caries-free and caries-active patients were similar, the patients' oral hygiene and dental care determined differences in risk indices. The number of lactobacilli was statistically lower in saliva of subjects with good oral health. Certain species of lactobacilli could not be associated to specific areas of the mouth, although some species could be localized. Lactobacilli from caries active (CA) subjects showed a greater ability to adhere to hydrophobic substances, had a greater salt agglutination property, and showed lower production of inhibitory substances. Lactobacilli from caries free (CF) subjects were better able to inhibit oral, potentially pathogenic, microorganisms. These studies prove that preliminary differences between oral lactobacilli in CF and CA patients exist. Non-specific and specific adhesion mechanisms in bacteria should be further demonstrated.

Comparison of the growth and hydrogen peroxide production by vaginal probiotic lactobacilli under different culture conditions.

OBJECTIVE: The purpose of this study was to determine the optimal conditions for the growth and hydrogen peroxide production by vaginal lactobacilli. STUDY DESIGN: Two vaginal lactobacillus strains were cultured under different growth conditions (temperature, pH, agitation, and growth media) with a fractional factorial experimental design. RESULTS: The optimal growth conditions of Lactobacillus paracasei CRL (Centro de Referencia para Lactobacilus Culture Collection) 1289 and Lactobacillus crispatus CRL 1266 were temperature 37 degrees C and initial pH 6.5, nonagitated cultures, in autolysat de levure; peptone, tryptone, Tween 80 et glucose (LAPTg) broth for L paracasei or in de Man-Rogosa-Sharpe (MRS) and LAPTg broths for L crispatus. The hydrogen peroxide production, detected only in agitated cultures, was higher at 37 degrees C and pH 6.5. The oxidative metabolite produced the self-inhibition of the lactobacilli growth. In mixed cultures of lactobacilli and Staphylococcus aureus under the optimal conditions of hydrogen peroxide production, the pathogen growth was inhibited. CONCLUSION: The results provided information about the factors that affect the hydrogen peroxide production and about the most favorable conditions with which to obtain the highest biomass in the shortest possible time.

Surface characteristics of lactobacilli isolated from human vagina.

In the present paper, the taxonomic classification of 134 lactobacilli isolates from vaginal samples of 200 women of Tucumán, Argentina, is reported. They were clustered in three metabolic groups of the genus Lactobacillus, most belonging to the obligately homofermentative group (56%), mainly represented by Lactobacillus delbrueckii subsp. delbrueckii and L. acidophilus. In the facultatively heterofermentative group (24%), the dominant species were L. paracasei subsp. paracasei and L. agilis, and in the obligately heterofermentative group (20%), L. brevis was the dominant species. All strains were studied for surface characteristics and adhesion-predicting properties. A correlation between the methods employed for hydrophobicity testing of the different isolates (Microbial Adhesion to Hydrocarbons and Salt Aggregation Test) is reported. Most strains were highly hydrophobic. Their hemagglutination capability with human erythrocytes was also tested, which was positive only for a few strains. Some isolates were self-aggregating. From our results, strains that shared the properties assayed were selected for further testing of some other desirable characteristics, such as antagonistic substance production, adhesion to biological substrates, and appropriate technological properties, to suggest the elaboration of a probiotic for the vaginal tract.