Characterization of volatile compounds produced by Lactobacillus helveticus strains in a hard cheese model.

Starter cultures of Lactobacillus helveticus used in hard cooked cheeses play an important role in flavor development. In this work, we studied the capacity of three strains of L. helveticus, two autochthonous (Lh138 and Lh209) and one commercial (LhB02), to grow and to produce volatile compounds in a hard cheese extract. Bacterial counts, pH, profiles of organic acids, carbohydrates, and volatile compounds were analyzed during incubation of extracts for 14 days at 37 ℃. Lactobacilli populations were maintained at 106 CFU ml-1 for Lh138, while decreases of approx. 2 log orders were found for LhB02 and Lh209. Both Lh209 and LhB02 slightly increased the acetic acid content whereas mild increase in lactic acid was produced by Lh138. The patterns of volatiles were dependent on the strain which reflect their distinct enzymatic machineries: LhB02 and Lh209 produced a greater diversity of compounds, while Lh138 was the least producer strain. Extracts inoculated with LhB02 and Lh 209 were characterized by ketones, esters, alcohols, aldehydes, and acids, whereas in the extracts with Lh138 the main compounds belonged to aromatic, aldehydes, and ketones groups. Therefore, Lh209 and LhB02 could represent the best cheese starters to improve and intensify the flavor, and even a starter composed by combinations of LhB02 or Lh209 with Lh138 could also be a strategy to diversify cheese flavor.

Microencapsulation of Lactobacillus helveticus and Lactobacillus delbrueckii using alginate and gellan gum.

Sodium alginate (SA) at 2% (w/v) and low acylated gellan gum (LAG) at 0.2% (w/v) were used to microencapsulate Lactobacillus helveticus and Lactobacillus delbrueckii spp lactis by employing the internal ionic gelation technique through water-oil emulsions at three different stirring rates: 480, 800 and 1200 rpm. The flow behavior of the biopolymer dispersions, the activation energy of the emulsion, the microencapsulation efficiency, the size distribution, the microcapsules morphology and the effect of the stirring rate on the culture viability were analyzed. All of the dispersions exhibited a non-Newtonian shear-thinning flow behavior because the apparent viscosity decreased in value when the shear rate was increased. The activation energy was calculated using the Arrhenius-like equation; the value obtained for the emulsion was 32.59 kJ/mol. It was observed that at 400 rpm, the microencapsulation efficiency was 92.83%, whereas at 800 and 1200 rpm, the stirring rates reduced the efficiency to 15.83% and 4.56%, respectively, evidencing the sensitivity of the microorganisms to the shear rate (13.36 and 20.05 s(-1)). Both optical and scanning electron microscopy (SEM) showed spherical microcapsules with irregular topography due to the presence of holes on its surface. The obtained size distribution range was modified when the stirring rate was increased. At 400 rpm, bimodal behavior was observed in the range of 20-420 µm; at 800 and 1200 rpm, the behavior became unimodal and the range was from 20 to 200 µm and 20 to 160 µm, respectively.

Milk fermentation products of L. helveticus R389 activate calcineurin as a signal to promote gut mucosal immunity.

BACKGROUND: Fermented milks containing probiotic bacteria are a way of delivering bioactive constituents to targets in the gastrointestinal tract. We reported previously that the fermentation of milk at constant pH 6 by L. helveticus R389 increased its content of peptide fractions, and the oral administration of the non-bacterial fraction (FMSpH6) to mice increased total secretory IgA in the intestinal lumen and enhanced the number of IgA and various cytokines producing cells as well as the secretion of IL-6 by small intestine epithelial cells. We also demonstrated that this FMSpH6 was effective for the prevention of Salmonella typhimurium infection in mice. In this work, we studied in mice the impact of the oral administration of the supernatant of milk fermented by L. helveticus R389 on the gut physiology by measuring parameters such as calcium channels and E-cadherin expression, the activation of the biological signal calcineurin and mast and goblet cells, as a way to determine some mechanisms involved in the immunomodulating effects of the milk fermentation products, observed in previous studies. We analyzed the impact of the supernatant of milk fermented by L. helveticus R389 at pH6-controlled on the expression of calcineurin and on the reinforcement of the ephitelial barrier, measuring parameters such as calcium channels and E-cadherin expression and in the reinforcement of the non-specific immunity determining mast cells and goblet cells associated to the gut. RESULTS: We observed an enhanced expression of TRPV6 channels in the duodenum, indicating an improved capacity for dietary Ca2+ uptake. We demonstrated an enhanced expression of calcineurin in the small intestine, able to upregulate immune parameters such as IL-2 and TNF production, with an increase in the number of these cytokines secreting cells. We determined an increase in the number of mucosal mast cells and goblet cells, which would mean an improved state of mucosal surveillance at sites of infection. CONCLUSION: The oral administration of the supernatant of milk fermented by L. helveticus R389 enhanced the gut mucosal immunity by improving the mechanisms that reinforce the epithelial and non-specific barriers and the gut functioning at sites of infection, with an improvement in the expression of the enzyme calcineurin, an important signal in the network that activates the gut immune system. The results of this work contribute to revealing the mechanisms underlying the immunomodulation of the gut immune function by fermented milks with probiotic bacteria.

Rapid restoration of colonic goblet cells induced by a hydrolyzed diet containing probiotics in experimental malnutrition.

PURPOSE: To investigate the effects of the addition of probiotic bacteria to a hydrolyzed diet on the recovery of goblet cells during renutrition in an animal model of malnutrition. METHODS: Twenty-six male Wistar rats (200-250g) were included in the study. Six were kept under normal conditions (sham group) while twenty received an aproteic diet for 15 days, and were randomized thereafter to receive a hydrolyzed diet containing (n=6; probiotic group) or not (n=6; hydrolyzed group) probiotics (10(6) cfu/g of Streptococcus thermophilus e Lactobacillus helveticus); or immediately killed (n=8; aproteic group). Histological slides containing cecal and sigmoid biopsies were used to counting the number of goblet cells and the goblet cells/colonocytes ratio. RESULTS: Malnutrition diminished the population of goblet cells in all sites. Goblet cells/colonocytes ratio of the probiotic group was significantly greater than hydrolyzed group at the ceccum (0.39 +/- 0.03 vs. 0.34 +/-0.02; p=0.02). Only rats fed with probiotics showed complete restoration of the normal goblet cells/colonocytes ratio at the sigmoid (0.37 +/- 0,02 vs. 0.22 +/- 0,03; p<0,001). CONCLUSION: Streptococcus thermophilus and Lactobacillus helveticus added to a renutrition diet enhance the recovery of mucosal atrophy induced by malnutrition and especially induce a rapid restoration of goblet cells population in the malnourished colonic mucosa.

Milk fermented by Lactobacillus helveticus R389 and its non-bacterial fraction confer enhanced protection against Salmonella enteritidis serovar Typhimurium infection in mice.

Bacterial infections in the gastrointestinal tract represent a major global health problem, even in the presence of normally effective mucosal immune mechanisms. Milk fermented by Lactobacillus helveticus R389 (FM) or its non-bacterial fraction obtained by milk fermentation at controlled pH 6 (NBF) are able to activate the small intestine mucosal immune response according to previous studies. In this work we aimed at comparing their protection capacity against an infection by Salmonella enteritidis serovar Typhimurium and at studying the mechanisms involved. In a completely randomized design, BALB/c mice received FM or NBF for 2, 5 or 7 consecutive days, followed by a single oral challenge with S. Typhimurium (10(7) cells/mouse). The increase in the number of IgA+ cells in the lamina propria of the small intestine, after the feeding periods, was accompanied by an increase in the luminal content of total S-IgA. However, no antibodies were produced against the NBF. In mice given the FM or the NBF for 7 consecutive days, lower levels of liver colonization on day 7 post-challenge with S. Typhimurium, higher luminal contents of specific anti-Salmonella S-IgA, higher percentages of survival to infection and lower numbers of MIP-1alpha+ cells in the lamina propria were observed. In this work we observed that in both the FM or the NBF there are active principles that confer enhanced protection against S. Typhimurium infection. However, the mechanisms underlying mucosal immunomodulation and protection are different. In those mechanisms, the mucosal immune response would seem to be more involved than the competitive or exclusion mechanisms between L. helveticus R389 and S. enteritidis serovar Typhimurium.

Rapid restoration of colonic goblet cells induced by a hydrolyzed diet containing probiotics in experimental malnutrition / Restauração rápida de células caliciformes induzida por uma dieta hidrolisada contendo probióticos em desnutrição experimental

PURPOSE: To investigate the effects of the addition of probiotic bacteria to a hydrolyzed diet on the recovery of goblet cells during renutrition in an animal model of malnutrition. METHODS: Twenty-six male Wistar rats (200-250g) were included in the study. Six were kept under normal conditions (sham group) while twenty received an aproteic diet for 15 days, and were randomized thereafter to receive a hydrolyzed diet containing (n=6; probiotic group) or not (n=6; hydrolyzed group) probiotics (10(6) cfu/g of Streptococcus thermophilus e Lactobacillus helveticus); or immediately killed (n=8; aproteic group). Histological slides containing cecal and sigmoid biopsies were used to counting the number of goblet cells and the goblet cells/colonocytes ratio. RESULTS: Malnutrition diminished the population of goblet cells in all sites. Goblet cells/colonocytes ratio of the probiotic group was significantly greater than hydrolyzed group at the ceccum (0.39 ± 0.03 vs. 0.34 ± 0.02; p=0.02). Only rats fed with probiotics showed complete restoration of the normal goblet cells/colonocytes ratio at the sigmoid (0.37 ± 0,02 vs. 0.22 ± 0,03; p<0,001). CONCLUSION: Streptococcus thermophilus and Lactobacillus helveticus added to a renutrition diet enhance the recovery of mucocal atrophy induced by malnutrition and especially induce a rapid restoration of goblet cells population in the malnourished colonic mucosa.(AU)

OBJETIVO: Investigar os efeitos da adição de probióticos em uma dieta hidrolisada na recuperação de células caliciformes durante a renutriçao em um modelo animal de desnutrição. MÉTODOS: Vinte e seis ratos Wistar (200-250g) foram incluídos no estudo. Seis foram mantidos em condições normais (grupo sham) enquanto que 20 receberam uma dieta aproteica por 15 dias, e foram randomizados para receber uma dieta hidrolisada com (n=6; grupo probiótico) ou sem (n=6; grupo hidrolisado) probióticos (10(6) cfu/g of Streptococcus thermophilus e Lactobacillus helveticus); ou foram sacrificados imediatamente (n=8; grupo aproteico). Cortes histológicos contendo biopsias do ceco e sigmoide foram examinados e o número de células caliciformas e a razão caliciformes/colonócitos foram contados. RESULTADOS: A desnutrição diminuiu o número de células caliciformes em todo o cólon. A razão células caliciformes/colonócitos do grupo probiótico foi significantemente maior que o do grupo hidrolizado no ceco (0.39 ± 0.03 vs. 0.34 ± 0.02; p=0.02). Somente os ratos alimentados com probióticos mostrou restauração completa da relação células caliciformes/colonócitos no sigmóide (0.37 ± 0,02 vs. 0.22 ± 0,03; p<0,001). CONCLUSÃO: Streptococcus thermophilus and Lactobacillus helveticus adicionados a uma dieta de renutrição melhora a recuperação da atrofia mucosa induzida pela desnutrição e especialmente induzem a uma rápida restauração da população de células caliciformes na mucosa colônica desnutrida.(AU)

Rapid restoration of colonic goblet cells induced by a hydrolyzed diet containing probiotics in experimental malnutrition

PURPOSE: To investigate the effects of the addition of probiotic bacteria to a hydrolyzed diet on the recovery of goblet cells during renutrition in an animal model of malnutrition. METHODS: Twenty-six male Wistar rats (200-250g) were included in the study. Six were kept under normal conditions (sham group) while twenty received an aproteic diet for 15 days, and were randomized thereafter to receive a hydrolyzed diet containing (n=6; probiotic group) or not (n=6; hydrolyzed group) probiotics (10(6) cfu/g of Streptococcus thermophilus e Lactobacillus helveticus); or immediately killed (n=8; aproteic group). Histological slides containing cecal and sigmoid biopsies were used to counting the number of goblet cells and the goblet cells/colonocytes ratio. RESULTS: Malnutrition diminished the population of goblet cells in all sites. Goblet cells/colonocytes ratio of the probiotic group was significantly greater than hydrolyzed group at the ceccum (0.39 ± 0.03 vs. 0.34 ± 0.02; p=0.02). Only rats fed with probiotics showed complete restoration of the normal goblet cells/colonocytes ratio at the sigmoid (0.37 ± 0,02 vs. 0.22 ± 0,03; p<0,001). CONCLUSION: Streptococcus thermophilus and Lactobacillus helveticus added to a renutrition diet enhance the recovery of mucocal atrophy induced by malnutrition and especially induce a rapid restoration of goblet cells population in the malnourished colonic mucosa.

OBJETIVO: Investigar os efeitos da adição de probióticos em uma dieta hidrolisada na recuperação de células caliciformes durante a renutriçao em um modelo animal de desnutrição. MÉTODOS: Vinte e seis ratos Wistar (200-250g) foram incluídos no estudo. Seis foram mantidos em condições normais (grupo sham) enquanto que 20 receberam uma dieta aproteica por 15 dias, e foram randomizados para receber uma dieta hidrolisada com (n=6; grupo probiótico) ou sem (n=6; grupo hidrolisado) probióticos (10(6) cfu/g of Streptococcus thermophilus e Lactobacillus helveticus); ou foram sacrificados imediatamente (n=8; grupo aproteico). Cortes histológicos contendo biopsias do ceco e sigmoide foram examinados e o número de células caliciformas e a razão caliciformes/colonócitos foram contados. RESULTADOS: A desnutrição diminuiu o número de células caliciformes em todo o cólon. A razão células caliciformes/colonócitos do grupo probiótico foi significantemente maior que o do grupo hidrolizado no ceco (0.39 ± 0.03 vs. 0.34 ± 0.02; p=0.02). Somente os ratos alimentados com probióticos mostrou restauração completa da relação células caliciformes/colonócitos no sigmóide (0.37 ± 0,02 vs. 0.22 ± 0,03; p<0,001). CONCLUSÃO: Streptococcus thermophilus and Lactobacillus helveticus adicionados a uma dieta de renutrição melhora a recuperação da atrofia mucosa induzida pela desnutrição e especialmente induzem a uma rápida restauração da população de células caliciformes na mucosa colônica desnutrida.