Immunomodulatory properties of non bacterial fraction of kefir: role of organic acids

IRAPORDA C.; RUMBO M; GARROTE G L; ABRAHAM A G

San Miguel de Tucumán

Simposio; IV Simposio Internacional de Bacterias Lácticas (SIBAL); 2013

CERELA

Kefir is obtained by milk fermentation with grains that are formed by a large number of lactic acid bacteria, acetic acid bacteria and yeast in a polysaccharide and protein matrix. It is considered a functional food since it contains bioactive ingredients that offer health beneficial effects and resistance to certain diseases. These beneficial properties could be attributed to the presence of a complex microbiota as well as their metabolic products. The aim of this work is to study the bioactive properties of kefir fermented milk, focusing on the ability to modulate intestinal epithelial innate response. Kefir grains CIDCA AGK1 were used to ferment milk at 20 °C, 24 h. Products were centrifuged, and the supernatants were neutralized and filtered. The effects of supernatants and solutions of lactic and acetic acid (in the same concentration found in the fermented milk) were studied on the reporter system Caco2ccl20:luc. This system consist of intestinal Caco-2 cells stably transfected with a luciferase reporter construction under the control of CCL20 promoter. In response to stimulation with different proinflamatory stimuli (flagellin 1µg/ml, IL-1â 10ng/mL or TNF-á 100ng/mL), the cells induce CCL20 expression. We found that a 30 min pre-incubation of cells, with fermented milk supernatants and also with racemic lactic acid solution (100 mM, pH 7.0) produced a very strong inhibition (>80%) of luciferase activity, independently of the proinflammatory stimuli used, while a solution of acetic acid, neutralized (<10 mM) had no modulatory effect. These treatments did not affect the integrity or viability of the cell culture determined by measuring lactate dehydrogenase activity in cell culture medium and by mitochondrial enzymatic activity. The effect of lactate was also analyzed at transcriptional level using real-time quantitative PCR. It was observed that expression of several chemokines/cytokines (CCL20, CXCL2, CXCL10 and IL-8) involved in the inflammatory response were significantly modulated by this treatment, while it did not affect transcription of genes involved in normal enterocyte function, such as lactate phloryzin hidrolase, intestinal trefoil factor and macrophage migration inhibitory factor. The results obtained in this study showed that the non bacterial fraction obtained from fermented milk with kefir grains has a significant capacity to down-regulate the intestinal epithelial innate response in Vitro. The presence of high levels of lactate in this fraction are at least partly responsible of this property. We are currently dedicated to the study of the mechanisms by which lactate could contribute to the observed effect.