Probiotic technological and functional characteristics of Lactobacillus strains isolated from chicken gut / Características tecnológicas e funcionais de linhagens de Lactobacillus isoladas do trato gastrointestinal de 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.

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.

Molecular identification of Lactobacillus spp. associated with puba, a Brazilian fermented cassava food.

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.

Molecular identification of Lactobacillus spp. associated with puba, a Brazilian fermented cassava food

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.

Characteristics of Lactobacillus and Gardnerella vaginalis from women with or without bacterial vaginosis and their relationships in gnotobiotic mice.

The objectives of the present study were to evaluate in vitro the production of antagonistic compounds against Gardnerella vaginalis by Lactobacillus strains isolated from women with or without bacterial vaginosis (BV), and to select one of the better Lactobacillus producers of such a substance to be tested in vivo using a gnotobiotic animal model challenged with one of the more sensitive G. vaginalis isolates. A total of 24 isolates from women with and without BV were identified as G. vaginalis. A higher frequency (P<0.05) of this bacterium was observed in women with BV (56.7%) when compared to healthy women (17.6%). A total of 86 strains of Lactobacillus were obtained from healthy women and women with BV. Lactobacillus strains were more frequently present (P<0.05) in healthy women (97.5%) than in women with BV (76.7%). Lactobacillus crispatus was the predominating strain in both healthy women and women with BV. Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus gasseri and Lactobacillus vaginalis were isolated with an intermediate frequency in the two groups. In vitro antagonism assays were performed using as indicators 17 reference strains and the G. vaginalis strains isolated from women with BV and from healthy women. Lactobacillus isolated from healthy women showed the higher antagonistic activity against all the indicator strains when compared with isolates from women with BV. Concerning the indicator strains, G. vaginalis found in women with BV was more resistant to the antagonism, particularly when Lactobacillus isolates from women with BV were used as producer strains. A high vaginal population level of G. vaginalis was obtained by intravaginal inoculation of germ-free mice, and this colonization was accompanied by vaginal histopathological lesions. A tenfold decrease in vaginal population level of G. vaginalis and a reduction of histological lesions were observed when the pathogenic challenge was performed in mice previously monoassociated with an L. johnsonii strain. Concluding, results of the present study suggest that progression of G. vaginalis-associated BV depends in part on a simultaneous presence of Lactobacillus populations with a low antagonistic capacity and of a G. vaginalis strain with a high resistance to this antagonism. The results could also explain why G. vaginalis is frequently found in the vaginal ecosystem of healthy women.

Queijo de minas artesanal da Serra da Canastra: influência da altitude das queijarias nas populações de bactérias acidolácticas / Influence of altitute on lactic acid bacteria population of minas artisanal cheese from Serra da Canastra

Amostras de queijo de minas artesanal foram coletadas em 18 queijarias localizadas em propriedades rurais da região da Serra da Canastra, Minas Gerais, com o objetivo de avaliar a influência da altitude sobre a população de bactérias acidolácticas. As queijarias estavam distribuídas nas altitudes de 600 a 900m, 900 a 1000m e mais de 1000m. Observaram-se populações mais elevadas de bactérias acidolácticas nas amostras de queijo da altitude de 600 a 900m. Lactobacillus rhamnosus, Lactobacillus casei e Lactobacillus plantarum foram os principais microrganismos isolados e identificados por PCR ARDRA 16S-23S rDNA, além de Enterococcus spp., Lactococcus spp. e outras espécies de Lactobacillus. Sugere-se que estas espécies estejam adaptadas ao ambiente de produção do queijo de minas artesanal produzido na região, o que resultaria em características sensoriais próprias do produto.

Samples of minas artisanal cheese were collected in 18 small-scale producer properties located in the rural region of Serra da Canastra, Minas Gerais state, aiming to evaluate the influence of three altitudes, from 600 to 900m, 900 to 1000m, and higher than 1000m, on the lactic acid bacteria (LAB) population. High populations of LAB were observed in the cheese samples, mainly in the lowest altitude. Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus plantarum were the major LAB isolated from the cheese samples and identified according to PCR ARDRA 16S-23S rDNA. Enterococcus spp., Lactococcus spp., and other species of Lactobacillus genus were also found. It is suggested that these microorganisms are adapted to the production environment of the minas artisanal cheese which result in the unique sensorial properties of the product.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis

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.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis.

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.