RESUMO O queijo minas artesanal é produzido por culturas starters endógenas, incluindo bactérias ácido-láticas (BAL). Algumas BAL podem possuir potencial probiótico. Com isso, este estudo teve como objetivo avaliar as propriedades probióticas in vitro de lactobacilos isolados de queijo minas artesanal produzido no estado de Minas Gerais. Dez amostras de lactobacilos, previamente isoladas desses queijos, foram submetidas aos seguintes testes: susceptibilidade aos antimicrobianos, tolerância ao suco gástrico artificial e aos sais biliares, produção de peróxido de hidrogênio e antagonismo contra micro-organismos patogênicos e não patogênicos. Apenas L. plantarum (C0) foi sensível a todos os antimicrobianos testados, enquanto as outras amostras de BAL foram resistentes a, pelo menos, uma droga testada. Seis amostras foram tolerantes ao suco gástrico artificial, e L. brevis (A6) apresentou crescimento nesse meio. Três amostras foram tolerantes aos sais biliares. Apenas L. brevis (E35) produziu peróxido de hidrogênio. Diferença (P<0,05) foi observada entre as médias dos halos de inibição de lactobacilos contra Enterococcus faecalis ATCC 19433 e Lactobacillus plantarum C24 no teste do spot-on-the-lawn. Todas as amostras de lactobacilos inibiram Escherichia coli ATCC 25922, Salmonella enterica var. Typhimurium ATCC 14028 no teste de antagonismo em cocultura (P<0,0001). A maioria das amostras de lactobacilos apresentou potencial probiótico in vitro. Com base nas amostras testadas, L. brevis (A6) apresentou os melhores resultados, considerando-se todos os testes probióticos in vitro.

ABSTRACT Minas artisanal cheese is made from endogenous starter cultures, including lactic acid bacteria (LAB). Some LAB may possess probiotic potential. Thus, this study aimed to evaluate the in vitro probiotic properties of lactobacilli isolated from Minas artisanal cheeses produced in Minas Gerais. Ten samples of lactobacilli, formerly isolated from those cheeses, were submitted to the following assays: antimicrobial susceptibility, tolerance to artificial gastric juice and biliary salts, production of hydrogen peroxide and antagonism against pathogenic and non-pathogenic micro-organisms. Only L. plantarum (C0) was sensitive to all tested antimicrobials, while the other LAB samples were resistant to at least one drug. Six samples were tolerant to artificial gastric juice, and L. brevis (A6) even grew in that medium. Three samples were tolerant to biliary salts. Only L. brevis (E35) produced hydrogen peroxide. Difference (P< 0.05) was observed among the means of inhibition haloes of lactobacilli against Enterococcus faecalis ATCC 19433 and Lactobacillus plantarum C24 in spot-on-the-lawn assay. All samples of lactobacilli inhibited Escherichia coli ATCC 25922, Salmonella enterica var. Typhimurium ATCC 14028 in co-culture antagonism test (P< 0.0001). Most lactobacilli samples showed in vitro probiotic potential. From the tested samples, L. brevis (A6) presented the best results considering all in vitro probiotic tests.

A habilidade de uma cepa de Lactobacillus rhamnosus isolada de um recém-nascido saudável de reduzir as consequências patológicas para o hospedeiro após infecção experimental por Salmonella enterica subsp. enterica sorov. Typhimurium foi avaliada em camundongos gnotobióticos e convencionais. Os camundongos convencionais receberam 0,1mL de leite probiótico por dia (0,8 log unidade formadora de colônia), 10 dias antes do desafio oral com S. Typhimurium (5,0 log unidade formadora de colônia), e continuaram recebendo probiótico até o término do experimento. O tratamento com probiótico nos camundongos gnotobióticos consistiu em uma única dose de leite probiótico no início do experimento e desafio oral após 10 dias (3,0 log unidade formadora de colônia). Em termos histopatológicos e morfométricos, a proteção foi observada no fígado e nos intestinos nos animais gnotobióticos e convencionais, respectivamente. No entanto, a população de S. Typhimurium foi similar em ambos os grupos tratado e controle de animais gnotobióticos. Desta forma, conclui-se que a proteção conferida pela cepa de L. rhamnosus contra o desafio experimental S. Typhimurium foi observada em diferentes sítios anatômicos nos animais convencionais e gnotobióticos e que essa proteção não foi devido à redução da população de S. Typhimurium nos intestinos.

The ability of a Lactobacillus rhamnosus strain isolated from a healthy breast-fed human newborn to reduce the pathological consequences for the host due to an experimental oral infection with Salmonella enterica subsp. enterica serov. Typhimurium in vivo was determined using gnotobiotic and conventional mice. Conventional mice received 0.1mL probiotic milk (8.0 log colony-forming unit) daily for 10 days before the oral pathogenic challenge (5.0 log colony-forming unit). Then probiotic treatment was continued until the end of the experiment. Probiotic treatment in germ-free mice consisted of a single dose of the probiotic milk at the beginning of the experiment and a challenge with S. Typhimurium 10 days later (3.0 log colony-forming unit). A protective effect was observed in both gnotobiotic and conventional animals in terms of histopathologic and morphometric data, but in different anatomical sites. This protection was observed in liver and intestines, respectively, for gnotobiotic and conventional mice. However, S. Typhimurium populations were similar in the feces of both treated and control gnotobiotic mice. We conclude that a protective effect of L. rhamnosus against experimental S. Typhimurium was observed. This protection was not due to the reduction of the population of pathogenic bacteria in the intestine.

Lactobacillus spp. isolados de diferentes porções do trato gastrintestinal de frangos foram testados quanto à capacidade de se manterem viáveis em uma emulsão água/óleo (A/O) contendo óleos de gergelim e de girassol. Após sessenta dias de estocagem sob refrigeração, três de cinco linhagens testadas sobreviveram em concentração igual ou superior a 10 6 UFC/g. Lactobacillus reuteri 2M14C, que apresentou maior capacidade de sobrevivência na emulsão desenvolvida (10 7 UFC/g), foi testado quanto à sua capacidade de sobreviver às condições do trato gastrintestinal in vivo em camundongos gnotobióticos. Após dez dias de monoassociação com L. reuteri 2M14C, foi testada também a capacidade de estimulação da atividade fagocítica de macrófagos peritoneais. A linhagem permaneceu em número superior a 10 9 UFC/g de conteúdo fecal durante os dez dias de monoassociação, e os camundongos monoassociados apresentaram atividade fagocítica maior (P<0,05) que a do grupo controle isento de germe. Os resultados sugerem que a formulação proposta é capaz de manter a viabilidade de células de lactobacilos para adição em ração animal, necessitando, no entanto, de um acompanhamento dessa viabilidade por tempo maior de estocagem. Além disso, os resultados demonstram que Lactobacillus reuteri 2M14C é um forte candidato a ser adicionado em formulações probióticas para uso em frangos.

Lactobacillus spp. isolated from different portions of chickens' gastrointestinal tract were evaluated concerning their ability to survive in a water-in-oil (W/0) emulsion containing sesame and sunflower oil. After sixty days of emulsion storage under refrigeration, three of five strains tested survived in number equal to or higher than 10(6)cfu/g. Lactobacillus reuteri 2M14C, which presented the highest survival in W/O emulsion (10(7)cfu/g), was tested for its capacity to resist throughout the passage through gnotobiotic mice gastrointestinal tract and for the ability to stimulate murine peritoneal macrophages phagocytosis. This strain remained at a number above 10(9)cfu/g feces during ten days of monoassociation, and monoassociated mice showed phagocytic activity significantly greater than the germ-free controls (P<0.05). The results suggest that the formulation can be used to incorporate viable Lactobacillus spp. cells in animal feed. Moreover, the results suggest that L. reuteri 2M14C is a strong candidate to be incorporated in probiotic formulations for use in chicken.

Puba or carimã is a Brazilian staple food obtained by spontaneous submerged fermentation of cassava roots. A total of 116 lactobacilli and three cocci isolates from 20 commercial puba samples were recovered on de Man, Rogosa and Sharpe agar (MRS); they were characterized for their antagonistic activity against foodborne pathogens and identified taxonomically by classical and molecular methods. In all samples, lactic acid bacteria were recovered as the dominant microbiota (7.86 ± 0.41 log10 CFU/g). 16S-23S rRNA ARDRA pattern assigned 116 isolates to the Lactobacillus genus, represented by the species Lactobacillus fermentum (59 isolates), Lactobacillus delbrueckii (18 isolates), Lactobacillus casei (9 isolates), Lactobacillus reuteri (6 isolates), Lactobacillus brevis (3 isolates), Lactobacillus gasseri (2 isolates), Lactobacillus nagelii (1 isolate), and Lactobacillus plantarum group (18 isolates). recA gene-multiplex PCR analysis revealed that L. plantarum group isolates belonged to Lactobacillus plantarum (15 isolates) and Lactobacillus paraplantarum (3 isolates). Genomic diversity was investigated by molecular typing with rep (repetitive sequence)-based PCR using the primer ERIC2 (enterobacterial repetitive intergenic consensus). The Lactobacillus isolates exhibited genetic heterogeneity and species-specific fingerprint patterns. All the isolates showed antagonistic activity against the foodborne pathogenic bacteria tested. This antibacterial effect was attributed to acid production, except in the cases of three isolates that apparently produced bacteriocin-like inhibitory substances. This study provides the first insight into the genetic diversity of Lactobacillus spp. of puba.

Staphylococcus aureus foi inoculado em queijos produzidos de forma estéril em laboratório, juntamente com Lactobacillus rhamnosus e Lactococcus lactis, isolados de queijo de coalho artesanal e identificados por PCR-ARDRA16S-23S. L. lactis foi capaz de reduzir a contagem de S. aureus no primeiro dia após produção (P<0,05) dos queijos de 3,3x10(7)UFC/g para 1,0x10(7)UFC/g. L. rhamnosus não impediu o crescimento de S. aureus. A presença das cepas acidoláticas, principalmente L. lactis, mostrou ainda potencial de inibição da produção de enterotoxina estafilocócica do tipo B, sendo que a concentração de enterotoxinas no 15° dia foi inferior ao limite de detecção pelo kit comercial utilizado. Concluiu-se que a presença das bactérias acidoláticas estudadas pode contribuir para a melhoria da qualidade sanitária de queijos artesanais.

Staphylococcus aureus was inoculated in cheese elaborated with Lactobacillus rhamnosus and Lactococcus lactis, produced in a sterile lab, previously isolated from Brazilian coalho artisanal cheese and identified by PCR-ARDRA16S-23S. L. lactis was able to reduce the staphylococcal count on the first day after cheese production (P<0.05) from 3.33x10(7)CFU/g to 1.0x10(7)CFU/g. L. rhamnosus did not inhibit S. aureus growth. The presence of lactic acid bacteria (LAB), mainly L. lactis, also showed evidences of inhibition of the enterotoxin B production, and the staphylococcal enterotoxins concentration on the 15th day was lower than the commercial kit threshold. Hence, the studied LAB may contribute to the sanitary quality of artisanal cheeses.

Avaliaram-se iogurtes de leite de cabra contendo ou não Bifidobacterium longum, B. breve, B. pseudolongum ou B. bifidum, adicionados ou não de aroma de morango. Os dados obtidos nas análises higiênico-sanitárias e físico-químicas foram dentro dos valores exigidos pela legislação brasileira; apenas o valor encontrado para lactose esteve abaixo do recomendado. Quanto às bactérias do iogurte, não houve diferença na contagem de Streptococcus salivarius subsp. thermophilus durante a estocagem, e não foi detectado Lactobacillus delbrueckii subsp. bulgaricus nas diluições utilizadas. A enumeração de Bifidobacterium spp. manteve-se entre 10(6) e 10(8)UFC/mL e não diferiu entre as espécies ao longo do tempo. Considerando-se a adição ou não de aroma, a análise das variáveis tempo e aroma não mostrou diferença estatística. A contagem entre os Bifidobacterium spp. demonstrou que nenhum microrganismo apresentou um comportamento superior a outro. Na análise sensorial, as amostras de iogurtes adicionados ou não de Bifidobacterium spp. e adicionados de aroma de morango não apresentaram diferenças entre si. O estudo mostrou ser possível a elaboração de iogurte de leite de cabra adicionado de Bifidobacterium spp. e de aroma de morango com qualidade assegurada, potencial para uso probiótico e boa aceitação pelo consumidor.

Goat milk yogurt with or without Bifidobacterium longum, B. breve, B. pseudolongum, or B. bifidum, and supplemented or not with strawberry aroma were evaluated. Data obtained from hygienic-sanitary and physicochemical analyses were within the values recommended by the Brazilian legislation, except for lactose contents, which were lower than the recommended. In relation to the yogurt bacteria, there was no difference in the counts of Streptococcus salivarius subsp. thermophilus during the storage, and Lactobacillus delbrueckii subsp. bulgaricus was not detected. The counts of Bifidobacterium spp.ranged from 10(6) to 10(8)CFU/mL, without difference between the species along the time. Considering the addition of flavor or not, the analysis of aroma and time variables showed no statistical difference. The counts of Bifidobacterium spp. demonstrated that none showed a superior value. For the hedonic scale test, there was no difference between yogurts supplemented or not with Bifidobacterium spp., and added or not with strawberry aroma. The study showed to be possible the elaboration of goat milk yogurt supplemented with Bifidobacterium spp. and strawberry aroma, presenting safety, potential for probiotic use, and good acceptability by consumers.

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

Lactobacillus delbrueckii UFV-H2b20 has been shown to increase clearance of bacteria injected into the blood of germ-free mice. Moreover, it induces the production of type 1 cytokines by human peripheral mononuclear cells. The objective of the present study was to investigate the production of inflammatory cytokines [interleukin-12 (IL-12 p40), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)] triggered in vitro by live, heat-killed or lysozyme-treated L. delbrueckii UFV-H2b20 and in vivo by a live preparation. Germ-free, L. delbrueckii-monoassociated and lipopolysaccharide (LPS)-resistant C3H/HeJ mice were used as experimental models. UFV-H2b20 induced the production of IL-12 p40 and TNF-α by peritoneal cells and IFN-γ by spleen cells from germ-free or monoassociated Swiss/NIH mice and LPS-hyporesponsive mice (around 40 ng/mL for IL-12 p40, 200 pg/mL for TNF-α and 10 ng/mL for IFN-γ). Heat treatment of L. delbrueckii did not affect the production of these cytokines. Lysozyme treatment decreased IL-12 p40 production by peritoneal cells from C3H/HeJ mice, but did not affect TNF-α production by these cells or IFN-γ production by spleen cells from the same mouse strain. TNF-α production by peritoneal cells from Swiss/NIH L. delbrueckii-monoassociated mice was inhibited by lysozyme treatment. When testing IL-12 p40 and IFN-γ levels in sera from germ-free or monoassociated Swiss/NIH mice systemically challenged with Escherichia coli we observed that IL-12 p40 was produced at marginally higher levels by monoassociated mice than by germ-free mice (40 vs 60 ng/mL), but IFN-γ was produced earlier and at higher levels by monoassociated mice (monoassociated 4 and 14 ng/mL 4 and 8 h after infection, germfree 0 and 7.5 ng/mL at the same times). These results show that L. delbrueckii UFV-H2b20 stimulates the production of type 1 cytokines in vitro and in vivo, therefore suggesting that L. delbrueckii might have adjuvant properties in infection in which these cytokines play a major role.

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Several studies have reported a decrease in serum cholesterol during the consumption of large doses of fermented dairy products or lactobacillus strains. The proposed mechanism for this effect is the removal or assimilation of intestinal cholesterol by the bacteria, reducing cholesterol absorption. Although this effect was demonstrated in vitro, its relevance in vivo is still controversial. Furthermore, few studies have investigated the role of lactobacilli in atherogenesis. The aim of the present study was to determine the effect of Lactobacillus delbrueckii on cholesterol metabolism in germ-free mice and the possible hypocholesterolemic and antiatherogenic action of these bacteria using atherosclerosis-prone apolipoprotein E (apo E) knock-out (KO) mice. For this purpose, Swiss/NIH germ-free mice were monoassociated with L. delbrueckii and fed a hypercholesterolemic diet for four weeks. In addition, apo E KO mice were fed a normal chow diet and treated with L. delbrueckii for 6 weeks. There was a reduction in cholesterol excretion in germ-free mice, which was not associated with changes in blood or liver cholesterol concentration. In apo E KO mice, no effect of L. delbrueckii was detected in blood, liver or fecal cholesterol. The atherosclerotic lesion in the aorta was also similar in mice receiving or not these bacteria. In conclusion, these results suggest that, although L. delbrueckii treatment was able to reduce cholesterol excretion in germ-free mice, no hypocholesterolemic or antiatherogenic effect was observed in apo E KO mice.

Quatro cepas de Lactobacillus spp. e duas cepas de Lactococcus spp. isoladas de queijos de coalho artesanal e industrial foram testadas quanto às suas atividades antimicrobianas. Observou-se atividade antagonista dessas bactérias ácido-lácticas frente a elas, a outras bactérias ácido-lácticas isoladas de queijo de coalho, aos patógenos isolados dos mesmos queijos e a cepas de patógenos de referência. Verificou-se diferença (P<0,05) entre as atividades antagonistas, exceto quando as próprias bactérias ácido-lácticas foram utilizadas como reveladoras. Lactobacillus spp. apresentaram as atividades antagonistas mais potentes.

Four strains of Lactobacillus spp. and two strains of Lactococcus spp. were isolated from artisanal and industrial "coalho" cheese and tested for inhibitory activity. It was observed antagonistic activity of the lactic acid bacteria against themselves and other lactic acid bacteria and pathogens isolated from the same cheese samples, as well as against pathogenic strains from other sources. Significant difference (P<0.05) was observed among the antagonistic activities, except when lactic acid bacteria were used as indicator. From the tested bacteria, Lactobacillus spp. showed the strongest antagonistic activity.

An experimental infection with Salmonella enterica subsp. enterica serovar Typhimurium was evaluated in gnotobiotic mice previously exposed to a plasmid-free non-pathogenic Escherichia coli (EMO strain). Mice were exposed to EMO (experimental) or not (control) 10 days before challenge with Salmonella Typhimurium (10² colony forming units (CFU)/mouse). Survival after challenge was higher (P < 0.05) in the experimental group (16%) than in the control animals (0%). Histopathological examination of the colon and ileum mucosa of the experimental group showed less extensive lesions such as edema, cell inflammatory infiltration and hyperemia. The epithelial cells of the mucosal surface and the production of the mucous layer were also better preserved in the experimental group. The population levels of Salmonella Typhimurium in the feces were initially 10-fold lower (P < 0.05) in the experimental groups. However, 3 days after challenge both experimental and control groups showed similar population levels ranging from 10(8) to()10(9) CFU/g of feces. The intestinal contents of total and anti-Salmonella Typhimurium sIgA were higher in the experimental groups 10 days after inoculation of E. coli EMO strain. Translocation of Salmonella Typhimurium to the spleen was 10-fold lower (P < 0.05) in the experimental group only on day 3 after infection. This was not related to an increase in the bacterial blood clearance of the animals, as shown by experimental venous challenge with E. coli B41. In conclusion, treatment of mice with E. coli EMO strain promoted a relative protection against experimental infection with Salmonella Typhimurium. This protection was not due to the reduction of the population of pathogens in the intestine but was probably related to stimulation of the immune response.

Probiotics are formulations containing live microorganisms or microbial stimulants that have some beneficial influence on the maintenance of a balanced intestinal microbiota and on the resistance to infections. The search for probiotics to be used in prevention or treatment of enteric infections, as an alternative to antibiotic therapy, has gained significant impulse in the last few years. Several studies have demonstrated the beneficial effects of lactic acid bacteria in controlling infection by intestinal pathogens and in boosting the host's nonspecific immune response. Here, we studied the use of Lactobacillus acidophilus UFV-H2b20, a lactic acid bacterium isolated from a human newborn from Viçosa, Minas Gerais, Brazil, as a probiotic. A suspension containing 108 cells of Lactobacillus acidophilus UFV-H2b20 was inoculated into groups of at least five conventional and germfree Swiss mice to determine its capacity to stimulate the host mononuclear phagocytic activity. We demonstrate that this strain can survive the stressing conditions of the intestinal tract in vivo. Moreover, the monoassociation of germfree mice with this strain for seven days improved the host's macrophage phagocytic capacity, as demonstrated by the clearance of a Gram-negative bacterium inoculated intravenously. Monoassociated mice showed an undetectable number of circulating E. coli, while 0.1% of the original inoculum was still present in germfree animals. Mice treated with viable or heat-killed Lactobacillus acidophilus UFV-H2b20 presented similarly improved clearance capacity when compared with germfree controls. In addition, monoassociated mice had twice the amount of Kupffer cells, which are responsible for the clearance of circulating bacteria, compared to germfree controls. These results suggest that the L. acidophilus strain used here stimulates a nonspecific immune response and is a strong candidate to be used as a probiotic.

The association of vertebrate hosts with the indigenous microbiota and its effect on the response to infections has long been a subject of scientific curiosity. From the first theory supported by Louis Pasteur that life would be impossible in the absence of associated microorganisms to the development of germfree mammals for research, a lot was learned about how the normal microbiota influences the environment in which pathogens may find themselves. In the present review, we attempt to summarize the more recent results from our group and others on the influence of the normal microbiota on the outcome of parasitic infections. Our results and those of others point to a complex relationship between the mammalian system and its indigenous microbiota, leading to greater resistance to some infections and enhanced susceptibility to others