To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192 T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principalcomponent analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.is a nondigestible oligosaccharide that is widely distributed in many plants, such as sugar beet, cane, cabbage, potato, grape, wheat, barley, corn, and the seeds of many legumes (20,21). In Hokkaido, Japan, raffinose is an important agricultural product that is extracted from sugar beets as a by-product of sugar processing. Several prebiotic effects in humans have been reported for this oligosaccharide, including reduction of fecal ammonia and indole (18), improvement of defecation frequency (18), and increased cell numbers among indigenous bifidobacteria (5). Increases in bifidobacterial populations in rats (8) and the suppression of T helper 2 cell-mediated immune responses in mice (19) have also been reported.To obtain a scientific basis for the use of raffinose as a prebiotic, a precise determination of its effects on microbiotic composition in the human intestine, especially on the bifidobacterial population at the species level, is required. Because the majority of microbiota in the huma...

The aims of this study were to screen and characterize lactic acid bacteria (LAB) isolated from gastrointestinal (GI) tract of Cemani chicken, one of Indonesian local chicken and to investigate their potential use as probiotics. LAB were isolated from GI tract using MRSA and GYPA media and incubated anaerobically. Selected LAB were determined their probiotic properties with several assays. Identification of selected LAB was based on 16S rDNA sequences, morphological and biochemical characteristics. Ninety five bacteria were isolated and characterized as lactic acid bacteria (Gram positive, catalase negative, non sporeforming and acid producing). Twenty four isolates of LAB demonstrated antimicrobial activity against Escherichia coli JCM 1649 and Salmonella enteritidis B2586, and three selected isolates, i.e. CCM011, CSP004, and CVM002 showed the highest inhibition activity. The isolates had characters of high cell surface hydrophobicity and inter-isolate coaggregation ability of LAB, high survival at low pH, high phytase and protease activity (but no amylase and lipase activity), weak coaggregation with pathogen and no resistance to the examined antibiotics. The isolates were identified based on sequence analysis of 16S rRNA gene as Lactobacillus salivarius, however, each isolate had different profiles of sugar fermentation. Therefore the three LAB isolates had potential application as probiotics for chicken.Key words: Cemani chicken, gastrointestinal tract, lactic acid bacteria, probiotic ABSTRAK Tujuan penelitian ini adalah untuk menyeleksi dan mengarakterisasi bakteri asam laktat (BAL) yang diisolasi dari saluran pencernaan ayam Cemani, salah satu ayam asli Indonesia dan untuk mengetahui potensi penggunaannya sebagai probiotik. BAL diisolasi dari saluran pencernaan dengan menggunakan medium MRSA dan GYPA dan diinkubasi secara anaerobik. BAL terpilih kemudian ditentukan karakter probiotiknya melalui serangkaian percobaan. Identifikasi isolat BAL terpilih berdasarkan pada sekuen 16S rDNA, karakter morfologi dan biokimianya. Sembilan puluh lima isolat diisolasi dan dikarakterisasi sebagai bakteri asam laktat (Gram positif, katalase negatif, tidak membentuk endospora dan menghasilkan asam). Dua puluh empat isolat BAL memperlihatkan aktivitas antimikrob terhadap E. coli JCM 1649 dan S. enteritidis B2586, dan tiga isolat terpilih, yaitu CCM011, CSP004, dan CVM002 menunjukkan aktivitas penghambatan yang tertinggi. Isolat-isolat tersebut mempunyai karakter pelekatan terhadap permukaan sel yang tinggi, mempunyai kemampuan koagregasi antar BAL, daya tahan yang tinggi pada pH rendah, menunjukkan aktivitas enzim fitase dan protease yang tinggi (tetapi tidak mempunyai aktivitas amilase dan lipase), koagregasi yang lemah terhadap bakteri patogen dan tidak resisten terhadap antibiotik uji. Isolat-isolat tersebut diidentifikasi berdasarkan analisis sekuen gen 16S rRNA sebagai Lactobacillus salivarius, tetapi tiap isolat mempunyai profil fermentasi terhadap gula yang berbeda. Ketiga isolat BAL tersebut berpotensi seba...

We evaluated the effects of Bifidobacterium breve JCM1192(T )and/or raffinose on epithelial proliferation in the rat small and large intestines. WKAH/Hkm Slc rats (4 wk old) were fed a control diet, a diet supplemented with either encapsulated B. breve (30 g/kg diet, 1.5 x 10(7) colony-forming unit/g capsule) or raffinose (30 g/kg diet), or a diet supplemented with both encapsulated B. breve and raffinose, for 3 wk. Epithelial proliferation in the small intestine, as assessed by bromodeoxyuridine immunohistochemistry, was increased only in the B. breve plus raffinose-fed group. We determined the number of bifidobacteria in cecal contents using fluorescence in situ hybridization and confirmed the presence of ingested B. breve only in the B. breve plus raffinose-fed group. This suggests that the ingested B. breve cells used raffinose and were activated in the small intestine, where they subsequently influenced epithelial proliferation. In conclusion, we found a prominent synbiotic effect of encapsulated B. breve in combination with raffinose on epithelial proliferation in rat small intestine but not in large intestine. To our knowledge, this is the first report of a synbiotic that affects epithelial proliferation.

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