Occurrence of bacteria with technological and probiotic potential in Argentinian human breast-milk.

Breast milk can be a source of potential probiotic bacteria, but the technological capacity of isolates obtained from this source is not always guaranteed. We aimed at isolating lactobacilli from breast milk samples collected in Argentina, focusing on isolates with functional and technological potential as probiotics. Fourteen Lactobacillus and one Bifidobacterium isolates were obtained from 164 samples donated by 104 mothers. The isolates preliminarily identified by MALDI-TOF, and then the identity was confirmed by partial 16S rRNA gene sequencing. Hydrophobicity was determined (hexadecane and xylene partition). The strains were also co-cultured with murine RAW 264.7 macrophages for screening the capacity to induce the anti-inflammatory cytokine interleukin (IL)-10. Hydrophobicity ranged from 7.4 and 95.9%. The strains Lactobacillus gasseri (70a and 70c) and Lactobacillus plantarum (73a and 73b) were the strains with a higher capacity to induce IL-10 production by macrophages. The technological application was evaluated by freezing dried in 10% lactose or 10% polydextrose. The survival was assessed after accelerated (37 °C, 4 weeks) or long-term (5 and 25 °C, 12 months) storage. Except for Lactobacillus gallinarum 94d, strains lost less than 1 Log10 order cfu/g after long-term (12 months) storage at 5 °C in lactose and polydextrose as protectants. A low correlation between survival to accelerated and long-term storage tests was observed. L. gasseri (70a and 70c) and L. plantarum (73a and 73b) deserve further studies as potential probiotics due to their capacity to induce IL-10 from murine macrophages and their hydrophobicity. In special, L. plantarum 73a was able to confer enhanced protection against Salmonella infection by promoting the immunity of the small intestine.

Postbiotics produced at laboratory and industrial level as potential functional food ingredients with the capacity to protect mice against Salmonella infection.

AIM: To determine the protective capacity against Salmonella infection in mice of the cell-free fraction (postbiotic) of fermented milk, produced at laboratory and industrial level. METHODS AND RESULTS: The proteolytic activity (PA) of 5 commercial cultures and 11 autochthonous Lactobacillus strains was evaluated. The DSM-100H culture displayed the highest PA and it was selected for further studies. The capacity of the postbiotics produced by pH-controlled fermentation to stimulate the production of secretory IgA in faeces and to protect mice against Salmonella infection was evaluated. A significant increase in secretory IgA in faeces of mice fed 14 days the postbiotic obtained at the laboratory (F36) was detected compared to control animals. A significantly higher survival was observed in mice fed the F36 and the FiSD (industrial product) compared to controls. CONCLUSION: The postbiotics obtained showed immunomodulatory and protective capacity against Salmonella infection in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: The pH-controlled milk fermentation by the proteolytic DSM-100H culture could be a suitable strategy to obtain a food ingredient to be added to a given food matrix, not adequate to host viable cells of probiotics, to confer it enhanced functionality and thus expand the functional food market.

Yeasts from autochthonal cheese starters: technological and functional properties.

AIMS: The aim of this work was to identify 20 yeasts isolated from autochthonal cheese starters and evaluate their technological and functional properties. METHODS AND RESULTS: The capacities of the yeasts to grow at different temperatures, pH, NaCl and lactic acid concentrations as well as the proteolytic and lipolytic activities were studied. Moreover, survival to simulated gastrointestinal digestion, hydrophobicity, antimicrobial activity against pathogens and auto- and co-aggregation abilities were evaluated. The sequentiation of a fragment from the 26S rDNA gene indicated that Kluyveromyces marxianus was the predominant species, followed by Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces lactis and Galactomyces geotrichum. RAPD with primer M13 allowed a good differentiation among strains from the same species. All strains normally grew at pH 4.7-5.5 and temperatures between 15 and 35°C. Most of them tolerated 10% NaCl and 3% lactic acid. Some strains showed proteolytic (eight isolates) and/or lipolytic (four isolates) capacities. All strains evidenced high gastrointestinal resistance, moderate hydrophobicity, intermediate auto-aggregation and variable co-aggregation abilities. No strains inhibited the growth of the pathogens assayed. CONCLUSIONS: Some strains from dairy sources showed interesting functional and technological properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has been the first contribution to the identification and characterization of yeasts isolated from autochthonal cheese starters in Argentina. Many strains could be proposed as potential candidates to be used as probiotics and/or as co-starters in cheese productions.

A comprehensive approach to determine the probiotic potential of human-derived Lactobacillus for industrial use.

Specific strains should only be regarded as probiotics if they fulfill certain safety, technological and functional criteria. The aim of this work was to study, from a comprehensive point of view (in vitro and in vivo tests), three Lactobacillus strains (Lactobacillus paracasei JP1, Lactobacillus rhamnosus 64 and Lactobacillus gasseri 37) isolated from feces of local newborns, determining some parameters of technological, biological and functional relevance. All strains were able to adequately grow in different economic culture media (cheese whey, buttermilk and milk), which were also suitable as cryoprotectants. As selective media, LP-MRS was more effective than B-MRS for the enumeration of all strains. The strains were resistant to different technological (frozen storage, high salt content) and biological (simulated gastrointestinal digestion after refrigerated storage in acidified milk, bile exposure) challenges. L. rhamnosus 64 and L. gasseri 37, in particular, were sensible to chloramphenicol, erythromycin, streptomycin, tetracycline and vancomycin, increased the phagocytic activity of peritoneal macrophage and induced the proliferation of IgA producing cells in small intestine when administered to mice. Even when clinical trails are still needed, both strains fulfilled the main criteria proposed by FAO/WHO to consider them as potential probiotics for the formulation of new foods.

Cell viability and immunostimulating and protective capacities of Bifidobacterium longum 51A are differentially affected by technological variables in fermented milks.

AIM: To investigate the cell viability of Bifidobacterium longum 5(1A) in fermented milks and to study its immunostimulating and protective capacity against Salmonella enterica ssp. enterica serovar Typhimurium infection in mice. METHODS AND RESULTS: Bifidobacterium longum 5(1A) was added to milk fermented with different yoghurt starter cultures, before or after fermentation, and viability was monitored during storage (5°C, 28 days). Resistance to simulated gastric acid digestion was assessed. Fermented milks were orally administered to mice for 10 days followed by oral infection with Salmonella Typhimurium. The number of IgA+ cells in the small and large intestine was determined before infection. Survival to infection was monitored for 20 days. Bifidobacterium longum 5(1A) lost viability during storage, but the product containing it was effective for the induction of IgA+ cells proliferation in the gut and for the protection of mice against Salm. Typhimurium infection. CONCLUSIONS: Cell viability of Bif. longum 5(1A) in fermented milks along storage did not condition the capacity of the strain to enhance the number of IgA+ cells in the gut and to protect mice against Salmonella infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The uncoupling of cell viability and functionality demonstrated that, in certain cases, nonviable cells can also exert positive effects.

Diversity among Lactobacillus paracasei phages isolated from a probiotic dairy product plant.

AIMS: To evaluate the phage diversity in the environment of a dairy industry which manufactures a product fermented with a probiotic strain of Lactobacillus paracasei. METHODS AND RESULTS: Twenty-two Lact. paracasei phages were isolated from an industrial plant that manufactures a probiotic dairy product. Among them, six phages were selected based on restriction profiles, and two phages because of their notable thermal resistance during sample processing. Their morphology, host range, calcium dependency and thermal resistance were investigated. All phages belonged to the Siphoviridae family (B1 morphotype), were specific for Lact. casei and paracasei strains showing identical host spectrum, and only one phage was independent of calcium for completing its lytic cycle. Some of the phages showed an extraordinary thermal resistance and were protected by a commercial medium and milk. CONCLUSIONS: Phage diversity in a probiotic product manufacture was generated to a similar or greater extent than during traditional yogurt or cheese making. SIGNIFICANCE AND IMPACT OF THE STUDY: This work emphasizes probiotic phage infections as a new ecological situation beyond yogurt or cheese manufactures, where the balanced coexistence between phages and strains should be directed toward a favourable state, thus achieving a successful fermentation.

Multiplex PCR for the detection and identification of dairy bacteriophages in milk.

Bacteriophage infections of starter lactic acid bacteria are a serious risk in the dairy industry. Phage infection can lead to slow lactic acid production or even the total failure of fermentation. The associated economic losses can be substantial. Rapid and sensitive methods are therefore required to detect and identify phages at all stages of the manufacture of fermented dairy products. This study describes a simple and rapid multiplex PCR method that, in a single reaction, detects the presence of bacteriophages infecting Streptococcus thermophilus and Lactobacillus delbrueckii, plus three genetically distinct 'species' of Lactococcus lactis phages commonly found in dairy plants (P335, 936 and c2). Available bacteriophage genome sequences were examined and the conserved regions used to design five pairs of primers, one for each of the above bacteriophage species. These primers were designed to generate specific fragments of different size depending on the species. Since this method can detect the above phages in untreated milk and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms or for use in those that involve phage-deactivating conditions.

Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption.

AIMS: Three indigenous Lactobacillus delbrueckii subsp. bulgaricus bacteriophages and their adsorption process were characterized. METHODS AND RESULTS: Phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed low burst size and short latent periods. A remarkably high sensitivity to pH was also demonstrated. Indigenous phage genomes were linear and double-stranded DNA molecules of approx. 31-34 kbp, with distinctive restriction patterns. Only one phage genome appeared to contain cohesive ends. Calcium ions did not influence phage adsorption, but it was necessary to accelerate cell lysis and improve plaque formation. The adsorption kinetics were similar on viable and nonviable cells, and the adsorption rates were high between 0 and 50 degrees C. SDS and proteinase K treatments did not influence the phage adsorption but mutanolysin and TCA reduced it appreciably. No significant inhibitory effect on phage adsorption was observed for the saccharides tested. This study also revealed the irreversibility of phage adsorption to their hosts. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on phages of thermophilic lactic acid bacteria.

Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries.

Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml. The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant.

Thermal and chemical inactivation of indigenous Streptococcus thermophilus bacteriophages isolated from Argentinian dairy plants.

Thermal and chemical resistance of five autochthonal bacteriophages of Streptococcus thermophilus, isolated from Cuartirolo cheese wheys and yogurt, was investigated. Times to obtain 99% inactivation of phages (T99) at 63 degrees C and 72 degrees C in three suspension media (enriched tryptic soy broth, reconstituted commercial nonfat skim milk, and tris magnesium gelatin buffer) were determined. The thermal resistance was dependent on the phages studied but not detectable counts (<10 PFU/ml) were only achieved by heating at 90 degrees C during 5 min. The data obtained for the three assayed media did not permit verifying significant differences among them. Sodium hypochlorite (100 ppm) provided a fast inactivation of bacteriophage particles (<10 PFU/ml after 5 min). Ethanol, at concentrations of 75% and 100%, was also effective for phage destruction. Isopropanol was slightly less effective than ethanol at the same concentrations. Peracetic acid (0.15%) was also a very effective agent for phage inactivation. The results showed that these autochthonal bacteriophages were not completely inactivated neither by normal pasteurization treatments nor by some biocides commonly used in disinfection, except sodium hypochlorite and peracetic acid. The practical implications of these findings have pointed out the necessity of recognizing the importance of establishing adequate conditions to assure effective thermal and chemical treatments in dairy plants and laboratory environments.

Natural milk cultures for the production of Argentinian cheeses.

Samples (32) of natural milk cultures used in the Santa Fe, Argentina, area for soft and semihard cheese production were examined. The microbial composition (including lactic acid microflora characterization) and technological parameters (acidifying and proteolytic activities) were evaluated. The cultures contained mainly thermophilic lactic acid bacteria, identified as Streptococcus salivarius subsp. thermophilus (96.8% of the total strains) and Enterococcus spp. The strains showed a low proteolytic activity. The isolates of S. salivarius subsp. thermophilus exhibited a widespread phage resistance. The nonlactic microflora comprised coliforms, yeasts, spore-forming bacteria and lactate fermentative bacteria. The samples showed an acidity level from 0.38 to 0.69% lactic acid (pH from 4.25 to 5.75). The acidifying activity was optimal at 45 degrees C. The advantages and disadvantages of the employment of natural milk starters are discussed.