Antifungal starter culture for packed bread: influence of two storage conditions.

In this study, we analyzed the conservation of a semi-liquid bio-preserver (SL778) developed with Lactobacillus plantarum CRL 778, a lactic acid bacterium (LAB) having antifungal activity. The characteristics of the SL778 starter remained stable during a 14-day storage at 4°C. At -20°C, cell viability and organic acid concentration showed a significant (p<0.05) decrease after 7 days. These differences observed between the storage temperatures tested were reflected in the acidification activity of SL778 during dough fermentation. However, SL778 maintained its antifungal efficacy up to a 14-day storage at both temperatures. Sensory attributes (acidic and spicy tastes and acidic smell) of breads manufactured with starter SL778 (stored at 4 or -20°C) were evaluated. No undesirable difference was detected with respect to bread control without SL778 and bread manufactured with SL778 (stored at 4 or -20°C). In conclusion, the SL778 semi-liquid bio-preserver can be stored at 4 or -20°C without modifying its antifungal activity during 14 days.

Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins.

Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and ß-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly ß-casein, while degradation of αs-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. ß-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.

Genome sequence of the bacteriocin-producing Lactobacillus curvatus strain CRL705.

Lactobacillus curvatus is one of the most prevalent lactic acid bacteria found in fermented meat products. Here, we present the draft genome sequence of Lactobacillus curvatus CRL705, a bacteriocin producer strain isolated from an Argentinean artisanal fermented sausage, which consists of 1,833,251 bp (GC content, 41.9%) and two circular plasmids of 12,342 bp (pRC12; GC, 43.9%) and 18,664 bp (pRC18; GC, 34.4%).

Draft genome sequence of Enterococcus mundtii CRL1656.

We report the draft genome sequence of Enterococcus mundtii CRL1656, which was isolated from the stripping milk of a clinically healthy adult Holstein dairy cow from a dairy farm of the northwestern region of Tucumán (Argentina). The 3.10-Mb genome sequence consists of 450 large contigs and contains 2,741 predicted protein-coding genes.

A combination of two lactic acid bacteria improves the hydrolysis of gliadin during wheat dough fermentation.

The evaluation of gliadin hydrolysis during dough fermentation by using two lactic acid bacteria, Lactobacillus plantarum CRL 775 and Pediococcus pentosaceus CRL 792, as pooled cell suspension (LAB) or cell free extract (CFE) was undertaken. The CFE pool produced a greater (121%) increase in amino acid concentration than the LAB pool (70-80%). These results were correlated with the decrease (76,100 and 64,300 ppm) in the gliadin concentration of doughs supplemented with CFE and LAB, respectively, compared to control doughs. The use of LAB peptidases seemed to be a viable technologic alternative to reduce the gliadin concentration in wheat dough without using living bacteria as starter.

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.

Techno-functional properties of HoPS from lactic acid bacteria of different origins as potential food additives.

Homopolysaccharides (HoPS) produced by lactic acid bacteria (LAB) are highly versatile, biocompatible and safe compounds. In this work, six HoPS from different species of Weisella and Leuconostoc were identified as thermally stable dextrans, with endothermic crystalline deformations between 214 and 239 °C. These dextrans proved to have greater solubility and capacities to retain water and oil than similar polymers in other reports. Furthermore, a surface morphology study presented cubic grumps, stratify mesh with irregular grumps, and highly compact filaments. Assays in vitro revealed moderate antioxidant, browning and foaming activities as well as technological properties, such as anti-syneresis, emulsifying and flocculating activities, even at low concentrations. Taking into account bipolymers' microstructure, functionalities and performance in both, aqueous and hydrophobic matrixes, plus their capacity to maintain themselves at elevated temperatures, we consider these HoPS beneficial and natural food additives.

Inorganic polyphosphate from the immunobiotic Lactobacillus rhamnosus CRL1505 prevents inflammatory response in the respiratory tract.

Lactobacillus (L.) rhamnosus CRL1505 accumulates inorganic polyphosphate (polyP) in its cytoplasm in response to environmental stress. The aim of this study was to evaluate the potential effects of polyP from the immunobiotic CRL1505 on an acute respiratory inflammation murine animal model induced by lipopolysaccharide (LPS). First, the presence of polyP granules in the cytoplasm of CRL1505 strain was evidenced by specific staining. Then, it was demonstrated in the intracellular extracts (ICE) of CRL1505 that polyP chain length is greater than 45 phosphate residues. In addition, the functionality of the genes involved in the polyP metabolism (ppk, ppx1 and ppx2) was corroborated by RT-PCR. Finally, the possible effect of the ICE of CRL1505 strain containing polyP and a synthetic polyP was evaluated in vivo using a murine model of acute lung inflammation. It was observed that the level of cytokines pro-inflammatory (IL-17, IL-6, IL-2, IL-4, INF-γ) in serum was normalized in mice treated with ICE, which would indicate that polyP prevents the local inflammatory response in the respiratory tract. The potential application of ICE from L. rhamnosus CRL1505 as a novel bioproduct for the treatment of respiratory diseases is one of the projections of this work.

Disruption of lipid rafts enhances the effect of lactobacilli on the production of tumor necrosis factor-alpha in mononuclear blood cells.

We investigated, to determine whether disruption of lipid rafts would influence the effect of three selected strains of Lactobacillus on TNF-alpha production by peripheral blood mononuclear cells. Two strains increased TNF-alpha production; and the third one reduced cytokine levels. Disruption of rafts changed the immunomodulatory effect of the three strains. This is the first report on a potential role of rafts in lactobacilli-cell interaction.

Whey fermentation by thermophilic lactic acid bacteria: evolution of carbohydrates and protein content.

Whey, a by-product of the cheese industry usually disposed as waste, is a source of biological and functional valuable proteins. The aim of this work was to evaluate the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Fermentations were performed at 37 and 42 degrees C for 24h in reconstituted whey powder (RW). Carbohydrates, organic acids and amino acids concentrations during fermentation were evaluated by RP-HPLC. Proteolytic activity was measured by the o-phthaldialdehyde test and hydrolysis of whey proteins was analyzed by Tricine SDS-PAGE. The studied strains grew well (2-3log cfu/ml) independently of the temperature used. Streptococcus thermophilus CRL 804 consumed 12% of the initial lactose concentration and produced the highest amount of lactic acid (45 mmol/l) at 24h. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 microg Leu/ml) strain and released the branched chain amino acids Leu and Val. In contrast, Lactobacillus acidophilus CRL 636 and S. thermophilus CRL 804 consumed most of the amino acids present in whey. The studied strains were able to degrade the major whey proteins, alpha-lactalbumin being degraded in a greater extent (2.2-3.4-fold) than beta-lactoglobulin. Two starter cultures were evaluated for their metabolic and proteolytic activities in RW. Both cultures acidified and reduced the lactose content in whey in a greater extent than the strains alone. The amino acid release was higher (86 microg/ml) for the starter SLb (strains CRL 804+CRL 454) than for SLa (strains CRL 804+CRL 636, 37 microg/ml). Regarding alpha-lactalbumin and beta-lactoglobulin degradation, no differences were observed as compared to the values obtained with the single cultures. The starter culture SLb showed high potential to be used for developing fermented whey-based beverages.

New insights into bacterial bile resistance mechanisms: the role of bile salt hydrolase and its impact on human health.

Bile acids (BA), the major components of bile, are biological detergents that facilitate the emulsification and solubilization of dietary lipids and also display potent antimicrobial activity, the bacterial membranes being their main targets. Considering the complicated nature of the stress produced by bile and BA, the microorganism tolerance requires different defence mechanisms including the presence of efflux pumps, bile salt hydrolase (BSH) enzyme, the intrinsic capacity of cells to maintain intracellular homeostasis and modifications in the architecture and composition of the cell membrane. Besides, the expression of proteins involved in carbohydrate and fatty acid metabolism, amino acid and nitrogenous base biosynthesis, and general stress response are commonly affected by the presence of bile. Among the microbial transformations, deconjugation of BA by BSH is the most important. Several studies indicate that BSH activity affects both the host physiology and the microbiota. In fact, it was strongly suggested that BSH could play an important role in the colonization and survival of bacteria in the gut. Also, recent work has shown that BSH and free BA participate in a variety of metabolic processes that include regulation of dietary lipid absorption, cholesterol metabolism, and energy and inflammation homeostasis. In this review we summarize recent advances in the understanding of the mechanisms involved in the tolerance of bacteria to bile, with special emphasis on the contributions of studies applying an omic approach. Besides, the physiological and ecological role of BSH enzyme and its relevance to human health as well as the function of bile acid as metabolic regulator are also discussed.

Inorganic salts and intracellular polyphosphate inclusions play a role in the thermotolerance of the immunobiotic Lactobacillus rhamnosus CRL 1505.

In this work, the thermotolerance of Lactobacillus rhamnosus CRL1505, an immunobiotic strain, was studied as a way to improve the tolerance of the strain to industrial processes involving heat stress. The strain displayed a high intrinsic thermotolerance (55°C, 20 min); however, after 5 min at 60°C in phosphate buffer a two log units decrease in cell viability was observed. Different heat shock media were tested to improve the cell survival. Best results were obtained in the mediumcontaining inorganic salts (KH2PO4, Na2HPO4, MnSO4, and MgSO4) likely as using 10% skim milk. Flow cytometry analysis evinced 25.0% live cells and a large number of injured cells (59.7%) in the inorganic salts medium after heat stress. The morphological changes caused by temperature were visualized by transmission electronic microscopy (TEM). In addition, TEM observations revealed the presence of polyphosphate (polyP) granules in the cells under no-stress conditions. A DAPI-based fluorescence technique, adjusted to Gram-positive bacteria for the first time, was used to determine intracellular polyP levels. Results obtained suggest that the high initial polyP content in L. rhamnosus CRL 1505 together with the presence of inorganic salts in the heat shock medium improve the tolerance of the cells to heat shock. To our knowledge, this is the first report giving evidence of the relationship between polyP and inorganic salts in thermotolerance of lactic acid bacteria.

Effect of a probiotic beverage consumption (Enterococcus faecium CRL 183 and Bifidobacterium longum ATCC 15707) in rats with chemically induced colitis.

BACKGROUND: Some probiotic strains have the potential to assist in relieving the symptoms of inflammatory bowel disease. The impact of daily ingestion of a soy-based product fermented by Enterococcus faecium CRL 183 and Lactobacillus helveticus 416 with the addition of Bifidobacterium longum ATCC 15707 on chemically induced colitis has been investigated thereof within a period of 30 days. METHODS: Colitis was induced by dextran sulfate sodium. The animals were randomly assigned into five groups: Group C: negative control; Group CL: positive control; Group CLF: DSS with the fermented product; Group CLP: DSS with the non-fermented product (placebo); Group CLS: DSS with sulfasalazine. The following parameters were monitored: disease activity index, fecal microbial analyses, gastrointestinal survival of probiotic microorganisms and short-chain fatty acids concentration in the feces. At the end of the protocol the animals' colons were removed so as to conduct a macroscopical and histopathological analysis, cytokines and nitrite quantification. RESULTS: Animals belonging to the CLF group showed fewer symptoms of colitis during the induction period and a lower degree of inflammation and ulceration in their colon compared to the CL, CLS and CLP groups (p<0.05). The colon of the animals in groups CL and CLS presented severe crypt damage, which was absent in CLF and CLP groups. A significant increase in the population of Lactobacillus spp. and Bifidobacterium spp. at the end of the protocol was verified only in the CLF animals (p<0.05). This group also showed an increase in short-chain fatty acids (propionate and acetate). Furthermore, the intestinal survival of E. faecium CRL 183 and B. longum ATCC 15707 in the CLF group has been confirmed by biochemical and molecular analyzes. CONCLUSIONS: The obtained results suggest that a regular intake of the probiotic product, and placebo to a lesser extent, can reduce the severity of DSS-induced colitis on rats.

Effect of bile acid on the cell membrane functionality of lactic acid bacteria for oral administration.

Lactic acid bacteria and other species dwelling in the gut must be tolerant to bile salts. This study sought to determine the effects of the bile salts taurodeoxycholate (TDCA) and deoxycholate (DCA) on Lactobacillus reuteri CRL 1098, a strain of likely probiotic value. When compared to other lactobacilli, L. reuteri showed the highest survival rate but remained sensitive to high (>3 mM) DCA concentrations. DCA produced complete permeabilization of cells, abolished glucose uptake and severely distorted the cell envelope, as shown by electron microscopy. Detailed analytical studies revealed a change in the phospholipid to glycolipid ratio, and also in lipid proportions. The C18:1 W9C form remarkably increased, possibly following a rapid adaptive response during DCA treatment. This study provides the first solid evidence of the mechanism of DCA toxicity in a lactic acid bacterium.

Biopolymers from lactic acid bacteria. Novel applications in foods and beverages.

Lactic acid bacteria (LAB) are microorganisms widely used in the fermented food industry worldwide. Certain LAB are able to produce exopolysaccharides (EPS) either attached to the cell wall (capsular EPS) or released to the extracellular environment (EPS). According to their composition, LAB may synthesize heteropolysaccharides or homopolysaccharides. A wide diversity of EPS are produced by LAB concerning their monomer composition, molecular mass, and structure. Although EPS-producing LAB strains have been traditionally applied in the manufacture of dairy products such as fermented milks and yogurts, their use in the elaboration of low-fat cheeses, diverse type of sourdough breads, and certain beverages are some of the novel applications of these polymers. This work aims to collect the most relevant issues of the former reviews concerning the monomer composition, structure, and yields and biosynthetic enzymes of EPS from LAB; to describe the recently characterized EPS and to present the application of both EPS-producing strains and their polymers in the fermented (specifically beverages and cereal-based) food industry.

Lactobacillus delbrueckii subsp. bulgaricus CRL 454 cleaves allergenic peptides of ß-lactoglobulin.

Whey, a cheese by-product used as a food additive, is produced worldwide at 40.7 million tons per year. ß-Lactoglobulin (BLG), the main whey protein, is poorly digested and is highly allergenic. We aimed to study the contribution of Lactobacillus delbrueckii subsp. bulgaricus CRL 454 to BLG digestion and to analyse its ability to degrade the main allergenic sequences of this protein. Pre-hydrolysis of BLG by L. delbrueckii subsp. bulgaricus CRL 454 increases digestion of BLG assayed by an in vitro simulated gastrointestinal system. Moreover, peptides from hydrolysis of the allergenic sequences V41-K60, Y102-R124, C121-L140 and L149-I162 were found when BLG was hydrolysed by this strain. Interestingly, peptides possessing antioxidant, ACE inhibitory, antimicrobial and immuno-modulating properties were found in BLG degraded by both the Lactobacillus strain and digestive enzymes. To conclude, pre-hydrolysis of BLG by L. delbrueckii subsp. bulgaricus CRL 454 has a positive effect on BLG digestion and could diminish allergenic reactions.

Lactobacilli express cell surface proteins which mediate binding of immobilized collagen and fibronectin.

Binding of immobilized collagen-I (Cn-I) and fibronectin (Fn) by Lactobacillus acidophilus CRL 639 depends on cell-surface proteins. Capsule formation during the stationary growth phase has a negative effect on adherence of Cn-I and Fn. However, cells from the exponential growth phase, which produce no capsule, exhibit maximal binding. Binding is sensitive to trypsin, proteinase K, pronase E, and heat. Gelatin and soluble Cn-I partially inhibit binding of Cn-I although various proteins, sugars and amino acids do not affect binding to Fn. These results indicate that protein-protein interactions mediate adhesion to extracellular matrix proteins. SDS-PAGE and Western blot analyses of surface proteins revealed that several proteins including the major 43-kDa protein of the S-layer are expressed. Monoclonal antibodies showed that Fn binds to a 15-kDa protein, while Cn-I binds to proteins of 45 and 58 kDa.

Efecto antifúngico y antimicotoxigénico de Lactobacillus plantarum CRL 778 a diferentes valores de actividad de agua / Antifungal and antimycotoxigenic effect of Lactobacillus plantarum CRL 778 at different water activity values

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi with high impact Lactic acid bacteria; in food safety due to its toxicity. In the last decade, the presence of OTA was widely reported in different foods. In this study, the ability of Lactobacillus (L.) plantarum CRL 778 to control growth and OTA production by Aspergillus (A.) niger 13D strain, at different water activity (a w) values (0.955, 0.964, 0.971, 0.982, and 0.995) was determined in vitro. Both parame ters were significantly (p<0.05) reduced by the lactobacilli and the effect depended on a w. Greatest growth rate inhibition (46.9%) was obtained at a w = 0.995, which is the most suitable value for growth and production of antifungal metabolites (lactic acid, acetic acid, phenyllac-tic and hydroxyl-phenyllactic acids) by L. plantarum CRL 778. Besides, morphological changes and inhibition of melanin synthesis were observed in colonies of A. niger 13D in presence of L. plantarum CRL 778 at a w ranged between 0.971 and 0.995. In addition, maximum reduction (90%) of OTA production took place at a w = 0.971, while inhibition of fungi growth was more evident at a w =0.995. These findings suggest that L. plantarum CRL 778 could be used for control of ochratoxigenic fungal growth and OTA contamination in different fermented foods with a w values between 0.971 and 0.995.

Ocratoxina A (OTA) es una micotoxina producida por hongos filamentosos con un alto impacto en la seguridad alimentaria debido a su toxicidad. En la última década se ha reportado ampliamente a nivel mundial, la presencia de OTA en diversos alimentos. En este estudio se evaluó in vitro, la capacidad de Lactobacillus (L.) plantarum CRL 778 de controlar el crecimiento y la producción de OTA por Aspergillus (A.) niger 13D, a diferentes valores de actividad de agua (a w): 0.955, 0.964, 0.971,0.982 y 0.995). La cepa láctica redujo significativamente (p <0.05) ambos parámetros, siendo el efecto dependiente del valor de a w. La mayor inhibición del crecimiento (46.9%) se obtuvo a a w =0.995, valor más adecuado para el crecimiento y producción de metabolitos antifúngicos (ácido láctico, ácido acético, ácidos fenil-láctico e hidroxi-fenil láctico) por la cepa láctica. Además, se observaron cambios morfológicos en las colonias de A. niger 13D, crecidas en presencia de L. plantarum CRL 778 a valores de a w de 0.971 y 0.995. El porcentaje máximo de reducción en la producción de OTA (90%) por la cepa láctica se observó a un valor de a w = 0.971, mientras la inhibición del crecimiento fúngico fue mayor cuando a w = 0.995. Estos hallazgos sugieren que L. plantarum CRL 778 podría emplearse para el control de la contaminación por hongos ocratoxigénicos en alimentos con valores de aw comprendidos entre 0.971-0.995.

Genome Sequence of the Cheese-Starter Strain Lactobacillus delbrueckii subsp. lactis CRL 581.

We report the genome sequence of Lactobacillus delbrueckii subsp. lactis CRL 581 (1,911,137 bp, GC 49.7%), a proteolytic strain isolated from a homemade Argentinian hard cheese which has a key role in bacterial nutrition and releases bioactive health-beneficial peptides from milk proteins.

Draft Genome Sequence of Lactobacillus rhamnosus CRL1505, an Immunobiotic Strain Used in Social Food Programs in Argentina.

We report the draft genome sequence of the probiotic Lactobacillus rhamnosus strain CRL1505. This new probiotic strain has been included into official Nutritional Programs in Argentina. The draft genome sequence is composed of 3,417,633 bp with 3,327 coding sequences.