Influence of kefiran on mice intestinal microbiota

HAMET M F, LONDERO A, MEDRANO M, GARROTE G L, PÉREZ P F; ABRAHAM A. G.

San Miguel de Tucumán

Simposio; III Simposio Internacional de Bacterias Lácticas. II Encuentro Red BAL; 2009

Cerela-AAM

During the last years, the application of polysaccharides in functional foods has gained attention. Kefiran, the polysaccharide obtained from kefir grains, is a water-soluble branched glucogalactan with a molecular weight over 4 x 106 Da. On account of its structure it could not be hydrolysed by gastric enzymes son it can reach to small intestine. Furthermore, biological activity of kefiran has been demonstrated recently. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis is recognized as one of the most suitable and widely culture independent technique applied to study complex microbial communities. The aim of this work was to evaluate the effect of kefiran on intestinal microbiota in mice, specially focusing on Bifidobacterium by DGGE analysis. Mice Balb/C were divided into three groups and Kefiran (300 mg/L in water) was administrated ad libitum to during 0, 2 and 7 days. The number of animal in each group was 5 to 7 (N= 15 to 21) and the experiment was performed three times. After treatments, faecal samples were then removed and resuspended in 500 ul of PBS and immediately frozen at - 80 °C. DNA was isolated according to the stool DNA extraction kit (Bioneer, Korea). PCR products were obtained using universal primers for Eubacteria for16S rDNA (518r y GC-338f). Bifidobacterium genus specific PCR was performed using for16S rRNA gen target Bif 164f and GC-Bif 662. Amplification program used for total bacteria was 94°/5 min; 35 cycles of 94°/30 seg, 60°/60 seg, 72°/30 seg and then 72°/5 min. For Bifidobacterium the amplification program was 95°/5 min; 40 cycles of 94°/45 seg, 52°/50 seg, 72°/50 seg and then 72°/7 min. Different denaturing gradients of urea: formamide were assayed  and 40:60 and 45:65 were selected for Eubacteria and bifidobacteria analysis respectively. Electrophoresis was conducted with a constant voltage of 100 V at 60 °C for 16 hours in both cases. SYBR-gold was employed for gel staining and visualized by UV trans-illumination. Differences in the number of bands were analyzed using a Gel-Compare program. The compositions of the faecal microbiota, as reflected by DGGE profiles generated from bacterial DNAs, differed between dietary groups. An increase in the number of amplification products in the prebiotic groups was observed. Those animals that consumed kefiran showed new bands not detected in control mice. Regarding profiles obtained using Bifidobacterium specific DGGE primers an increment in number of bands was observed in treated mice after 2 to 7 days of kefiran administration. These preliminary results show that kefiran have influence in fecal mice microbiota and suggest that consumption of this polysaccharide could improve the establishment of probiotic microorganisms as bifidobacteria in the gut.