The Step of Incorporation of Bacillus coagulans GBI-30 6086 Into "requeijão cremoso" Processed Cheese Does Not Affect Metabolic Homeostasis of Rats.

Dairy product consumption is a common habit in Brazil. These products present a good matrix for probiotic incorporation. Thus, in this study the feasibility of producing a probiotic "requeijão cremoso" incorporated with Bacillus coagulans GBI-30 6086 in three different steps and its metabolic effect in an animal model for 2 weeks has been evaluated. Wistar adult health rats were randomized into one to five groups (n = 8 for each group): Control (C); "requeijão cremoso" without probiotic (RC); probiotic inoculated in the milk before pasteurization at 65°C/30 min (RPP); "requeijão cremoso" inoculated before the fusion step and consequently exposed to 90°C/5 min (RPF); and "requeijão cremoso" inoculated after fusion step, i.e., once the product temperature reached 50°C (RPAF). At the end of treatment, analysis of molecular markers of proteins of stress and antioxidant system, HSP 25, 60, 70 and 90, SOD and catalase were performed in the animals' muscles by Western Blot technique. The HSP25, HSP90 and catalase levels of C, RPP, RPF, and RPAF were similar, indicating that the homeostasis remained unchanged. The incorporation of B. coagulans GBI-30 6086 in the "requeijão cremoso" was shown to be stable and the microorganism remained viable in all steps tested. The incorporation of the probiotic strain in the fusion stage facilitated the technological process, since it allowed a better homogenization of the product and did not affect the maintenance of the metabolic homeostasis of rats.

The resistance of Bacillus, Bifidobacterium, and Lactobacillus strains with claimed probiotic properties in different food matrices exposed to simulated gastrointestinal tract conditions.

The resistance of Bifidobacterium, Lactobacillus, and Bacillus strains with claimed probiotic properties in different food matrices was evaluated. Lactobacillus paracasei PXN 37, Lactobacillus acidophilus La-5, Bifidobacterium animalis subsp. lactis Bb-12, Bifidobacterium breve PXN 25, Bacillus subtilis PXN 21, Bacillus coagulans GBI30 6086 and Bacillus coagulans MTCC 5856 strains were inoculated in "requeijão cremoso" cheese, pasteurized orange juice, and bread. Further, the counts of the strains with claimed probiotic properties were determined throughout the products' shelf-life. Additionally, the survival (%), at the beginning and at the end of their shelf-life, of each strain with claimed probiotic properties inoculated in the three foods was estimated by using a static in vitro system simulating the gastric (pH 2), enteric I (pH 5) and enteric II (pH 7) phases of gastrointestinal tract (GIT). Overall, it has been found that the Bacillus strains with claimed probiotic properties showed greater viability than probiotic Bifidobacterium and Lactobacillus strains no matter the food studied. The percentage of survival of the Bacillus strains with claimed probiotic properties were always above 83%. The Bacillus strains with claimed probiotic properties were able to survive well in all the food matrices tested. Therefore, this study shows that these strains of Bacillus may comprise a feasible strategy for expanding the range of "probiotic food" choices given their high resistance to the composition of foods, manufacturing steps, and resistance to simulated GIT conditions.

Behavior of different Bacillus strains with claimed probiotic properties throughout processed cheese ("requeijão cremoso") manufacturing and storage.

Even though spore-forming bacteria have been mainly linked to spoilage or foodborne pathogens vehiculated through foods, some strains of Bacillus can potentially present probiotic properties. The advantage of incorporating probiotic Bacillus strains in foods relies mainly on the fact that these microorganisms present high resistance to harsh processing conditions. "Requeijão cremoso" is a type of processed cheese highly appreciated in Brazil. During processing, this product is submitted to several harsh conditions (heating at 90 °C, for instance), leading to the inactivation of probiotic bacteria belonging to Lactobacillus and Bifidobacterium genera. That fact has precluded the development of probiotic "requeijão cremoso" products; however, probiotic Bacillus strains may comprise a promising alternative to overcome the low resistance of traditional probiotics to food processing. The objective of this study was to evaluate the behavior of different Bacillus strains with claimed probiotic properties throughout processed cheese ("requeijão cremoso") manufacturing. A total of five different Bacillus strains with claimed probiotic properties (B. coagulans MTCC 5856, B. coagulans GBI-30 6086, B. subtilis PXN 21, B. subtilis PB6, and B. flexus HK1) were individually inoculated at different stages of manufacture - curd pasteurization, coagulation, and fusion - of "requeijão cremoso" and their survival in each of these stages was determined. The survival of B. coagulans GBI-30 6086 was further assessed throughout "requeijão cremoso" production and shelf life (45 days at 6 °C). Besides, the chemical composition, level of proteolysis, and fatty acid profile of the treatments during shelf life were evaluated. The fusion stage was found as the most appropriate for the addition of B. coagulans GBI-30 6086, which allows the production of probiotic "requeijão cremoso" and facilitates the technological process while preventing the occurrence of final product recontamination.

Effect of galactooligosaccharides and Bifidobacterium animalis Bb-12 on growth of Lactobacillus amylovorus DSM 16698, microbial community structure, and metabolite production in an in vitro colonic model set up with human or pig microbiota.

A validated in vitro model of the large intestine (TIM-2), set up with human or pig faeces, was used to evaluate the impact of potentially probiotic Lactobacillus amylovorus DSM 16698, administered alone (i), in the presence of prebiotic galactooligosaccharides (GOS) (ii), and co-administered with probiotic Bifidobacterium animalis ssp. lactis Bb-12 (Bb-12) (iii) on GOS degradation, microbial growth (L. amylovorus, lactobacilli, bifidobacteria and total bacteria) and metabolite production. High performance anion exchange chromatography revealed that GOS degradation was more pronounced in TIM-2 inoculated with pig faeces than with human faeces. Denaturing gradient gel electrophoresis profiling of PCR-amplified 16S rRNA genes detected a more complex Lactobacillus spp. community in pig faecal material than in human faecal inoculum. According to 16S rRNA gene-targeted qPCR, GOS stimulated the growth of lactobacilli and bifidobacteria in faecal material from both materials. The cumulative production of short chain fatty acids and ammonia was higher (P < 0.05) for pig than for human faeces. However, lactate accumulation was higher (P < 0.05) in the human model and increased after co-administration with GOS and Bb-12. This study reinforced the notion that differences in microbiota composition between target host organisms need to be considered when animal data are extrapolated to human, as is often done with pre- and probiotic intervention studies.

In vitro evaluation of gastrointestinal survival of Lactobacillus amylovorus DSM 16698 alone and combined with galactooligosaccharides, milk and/or Bifidobacterium animalis subsp. lactis Bb-12.

Probiotic properties of Lactobacillus amylovorus DSM 16698 were previously demonstrated in piglets. Here, its potential as a human probiotic was studied in vitro, using the TIM-1 system, which is fully validated to simulate the human upper gastrointestinal tract. To evaluate the effect of the food matrix composition on the survival of L. amylovorus DSM 16698 in TIM-1, the microorganism was inoculated alone or with prebiotic galactooligosaccharides (GOS), partially skimmed milk (PSM) and/or commercial probiotic Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12). Samples were collected from TIM-1 for six hours, at one-hour intervals and L. amylovorus populations were enumerated on MRS agar plates with confirmation of identity of selected isolates by randomly amplified polymorphic DNA (RAPD) fingerprinting. The cumulative survival for L. amylovorus alone (control) was 30% at the end of the experiment (t=6h). Co-administration of L. amylovorus with GOS, PSM and/or Bb-12 increased its survival in comparison with the control significantly from the 4th hour after ingestion onwards (P<0.05). Furthermore, by the use of High Performance Anion Exchange Chromatography, both L. amylovorus and Bb-12 were observed to promptly degrade GOS compounds in samples collected from TIM-1, as assessed at t=2h. Hence, food matrix composition interfered with survival and growth of L. amylovorus during passage through TIM-1, providing leads towards optimization of probiotic properties in vivo.

Vaginal lactobacilli as potential probiotics against Candida spp

Urogenital infections affect millions of people every year worldwide. The treatment of these diseases usually requires the use of antimicrobial agents, and more recently, the use of probiotic lactic acid bacteria (LAB) cultures for the management of vaginal infections has been extensively studied. In this work, 11 vaginal lactobacilli isolates, previously obtained from healthy patients, were studied to screen microorganisms with probiotic properties against Candida spp. The LAB were tested for their ability of auto-aggregation, coaggregation with C. albicans, C. glabrata, C. krusei, and C. tropicalis, adhesion to Caco-2 epithelial cells and production of lactic acid and hydrogen peroxide (H2O2). All lactobacilli isolates tested were able to auto-aggregate (ranging from 25.3 percent to 75.4 percent assessed at 4 hours of incubation) and to co-aggregate with the four Candida species into different degrees; among them L. crispatus showed the highest scores of coaggregation. The highest amount of lactic acid was produced by L. salivarius (13.9 g/l), followed by L. johnsonii (6.5 g/l), L. acidophilus (5.5 g/l), and L. jensenii (5.4 g/l). All isolates produced H2O2 , but the highest levels (3 -10 mg/l) were observed for L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, and L. vaginalis. Only L. agilis, L. jensenii, L. johnsonii and L. ruminus were able to adhere to epithelial Caco-2 cells. Among the isolates evaluated, L agilis, L. jensenii, L. johnsonii, and L. ruminus exhibited simultaneously several desirable properties as potential probiotic strains justifying future studies to evaluate their technological properties in different pharmaceutical preparations for human use.

Vaginal lactobacilli as potential probiotics against Candida SPP.

Urogenital infections affect millions of people every year worldwide. The treatment of these diseases usually requires the use of antimicrobial agents, and more recently, the use of probiotic lactic acid bacteria (LAB) cultures for the management of vaginal infections has been extensively studied. In this work, 11 vaginal lactobacilli isolates, previously obtained from healthy patients, were studied to screen microorganisms with probiotic properties against Candida spp. The LAB were tested for their ability of auto-aggregation, co-aggregation with C. albicans, C. glabrata, C. krusei, and C. tropicalis, adhesion to Caco-2 epithelial cells and production of lactic acid and hydrogen peroxide (H2O2). All lactobacilli isolates tested were able to auto-aggregate (ranging from 25.3% to 75.4% assessed at 4 hours of incubation) and to co-aggregate with the four Candida species into different degrees; among them L. crispatus showed the highest scores of co-aggregation. The highest amount of lactic acid was produced by L. salivarius (13.9 g/l), followed by L. johnsonii (6.5 g/l), L. acidophilus (5.5 g/l), and L. jensenii (5.4 g/l). All isolates produced H2O2, but the highest levels (3 - 10 mg/l) were observed for L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, and L. vaginalis. Only L. agilis, L. jensenii, L. johnsonii and L. ruminus were able to adhere to epithelial Caco-2 cells. Among the isolates evaluated, L agilis, L. jensenii, L. johnsonii, and L. ruminus exhibited simultaneously several desirable properties as potential probiotic strains justifying future studies to evaluate their technological properties in different pharmaceutical preparations for human use.

Effect of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the ability of Candida albicans to infect cells and induce inflammation.

Vulvovaginal candidiasis, a high prevailing infection worldwide, is mainly caused by Candida albicans. Probiotic Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 have been previously shown to be useful as adjuvants in the treatment of women with VVC. In order to demonstrate and better understand the anti-Candida activity of the probiotic microorganisms in an in vitro model simulating vaginal candidiasis, a human vaginal epithelial cell line (VK2/E6E7) was infected with C.albicans 3153a and then challenged with probiotic L. rhamnosus GR-1 and/or L. reuteri RC-14 or their respective CFS (alone or in combination). At each time point (0, 6, 12 and 24 hr), numbers of yeast, lactobacilli and viable VK2/E6E7 cells were determined and, at 0, 6 and 12 hr, the supernatants were measured for cytokine levels. We found that C. albicans induced a significant increase in IL-1alpha and IL-8 production by VK2/E6E7 cells. After lactobacilli challenge, epithelial cells did not alter IL-6, IL-1alpha, RANTES and VEGF levels. However, CFS from the probiotic microorganisms up-regulated IL-8 and IP-10 levels secreted by VK2/E6E7 cells infected with C. albicans. At 24 hr of co-incubation, L. reuteri RC-14 alone and in combination with L. rhamnosus GR-1 decreased the yeast population recoverable from the cells. In conclusion, L. reuteri RC-14 alone and together with L. rhamnosus GR-1 have the potential to inhibit the yeast growth and their CFS may up-regulate IL-8 and IP-10 secretion by VK2/E6E7 cells, which could possibly have played an important role in helping to clear VVC in vivo.

Improved cure of bacterial vaginosis with single dose of tinidazole (2 g), Lactobacillus rhamnosus GR-1, and Lactobacillus reuteri RC-14: a randomized, double-blind, placebo-controlled trial.

Bacterial vaginosis (BV) is the most prevalent vaginal infection worldwide and is characterized by depletion of the indigenous lactobacilli. Antimicrobial therapy is often ineffective. We hypothesized that probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 might provide an adjunct to antimicrobial treatment and improve cure rates. Sixty-four Brazilian women diagnosed with BV were randomly assigned to receive a single dose of tinidazole (2 g) supplemented with either 2 placebo capsules or 2 capsules containing L. rhamnosus GR-1 and L. reuteri RC-14 every morning for the following 4 weeks. At the end of treatment (day 28), the probiotic group had a significantly higher cure rate of BV (87.5%) than the placebo group (50.0%) (p = 0.001). In addition, according to the Gram-stain Nugent score, more women were assessed with "normal" vaginal microbiota in the probiotic group (75.0% vs. 34.4% in the placebo group; p = 0.011). This study shows that probiotic lactobacilli can provide benefits to women being treated with antibiotics for an infectious condition.

Analysis of vaginal lactobacilli from healthy and infected Brazilian women.

Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H(2)O(2)-producing lactobacilli were not associated with protection against VVC.

Evaluation of bacteriocin-producing Lactobacillus sakei 1 against Listeria monocytogenes 1/2a growth and haemolytic activity

Lactobacillus sakei 1 produtor de bacteriocina foi cultivado em caldo Infusão Cérebro-Coração por 24h a 25ºC. O sobrenadante da cultura foi neutralizado, esterilizado por filtração e usado para testar a atividade da bacteriocina frente a Listeria monocytogenes 1/2a, a 8ºC e 15ºC. Lactobacillus sakei ATCC 15521 não bacteriocinogênico, foi utilizado como controle negativo. L. monocytogenes 1/2a foi inoculada no sobrenadante da cultura de L.sakei 1 e L. sakei ATCC 15521 e as populações listeriais foram determinadas após 0, 5 e 10 dias. A produção de bacteriocina foi quantificada como unidades arbitrárias por mL (UA/mL), utilizando-se o teste de antagonismo em ágar. Adicionalmente, para investigar se o padrão de virulência de L. monocytogenes poderia ter mudado após exposição à bacteriocina, foi avaliada a habilidade de L. monocytogenes em causar hemólise em hemáceas de carneiro, antes e após exposição à bacteriocina a 8ºC. Na presença do peptídeo antimicrobiano, a 8ºC, a população de L. monocytogenes foi reduzida, mas o desenvolvimento de células resistentes foi observado. A 15ºC, não houve diferença entre os grupos controle e teste. Além disso, a atividade hemolítica de L. monocytogenes 1/2a não foi alterada pela exposição à bacteriocina de L. sakei, 1/2a não foi alterada pela exposição à bacteriocina de L. sakei, o que sugere que não houve mudança em seu padrão de virulência.