Estudio de la influencia del aceite de oliva sobre la microbiota del tracto gastrointestinal de roedores

Resumen

A large body of published work indicate the relationship between intestinal microbiota and obesity. There are also many studies about the effects of high fat diets on the gut microbiota, but there are no studies about the effects of extra virgin olive oil, unsaturated fat and important base of Mediterranean diet. During the last few years, increasing evidence of the health benefits of olive oil consumption in the diet has been reported. In this way, we have studied the specific effect on gut bacterial populations of extra virgin olive oil, which is rich in monounsaturated fatty acids and phenolic compounds, in comparison to refined olive oil, which is rich in monounsaturated fatty acids with a low level of phenolic compounds, and to butter, rich in saturated fatty acids and cholesterol. With this purpose, we have designed an experiment based in four groups of mice each one of them fed with a different diet: standard chow diet and three high fat diets, rich in extra virgin olive oil, refined olive oil or butter, during twelve weeks. Using culture-dependent and independent methods, we have been able to study the evolution of symbiont population from faeces. By means of culture-independent methods, the V3 region of 16S rDNA was amplified and separated by denaturing gradient gel electrophoresis (DGGE), followed by sequencing of the most representative bands. When we analysed the results of culture-dependents and independents studies, we observed that different dietary fats had different effects on gut microbial composition. Even more, we detected a different behaviour between extra virgin and refined olive oil and the first one so being most different from butter, which induced changes in the microbial counts similar to those described in obese individuals. In order to see if the type of fat present in the diet determines the characteristics of gut microbiota and this fact can exert a role in the development of metabolic syndrome, we designed another experiment. Twenty six Swiss Webster mice were further studied: those fed with a standard chow diet and with two high fat diets, enriched with extra virgin olive oil and butter. Physiological, hormonal and metabolic parameters were evaluated by the group BIO-221 from the University of Jaén during the experiment and, at final time, DNA was extracted from faeces. Thus the corresponding 16S rDNA was pyrosequenced and the results were analysed. The group fed with butter showed the highest levels in body weight (which correlated positively with Lactobacillus spp. levels) and in blood pressure (correlating with Enterococcus spp.). Moreover, the group fed with extra virgin olive oil presented the lowest levels of triglycerides and the highest relationship between HDL/LDL ratio (showing a direct correlation with Fam. Bacteroidaceae) and also had more percentage of Erysipelotrichaecae and Sutterellaceae (both of them correlated inversely with blood pressure values). When we compared the effects of extra virgin olive oil and butter on some metabolic syndrome parameters, we observed positive effects when extra virgin olive oil was included in the diet in parallel to changes in the percentage of specific OTUs in faeces. This could indicate that this type of fat provides beneficial effects by modulating the intestinal microbiota. On the other hand, when the previous experiment finished and the mice were sacrificed, we extracted the large intestine in order to study and detect methanogenic Archaea, associated with obesity and irritable bowel syndrome. The Archaea, old prokaryot domain present in high levels inside the intestine, cannot be detected by culture-independent methods like DGGE or yrosequencing. In order to detect methanogenic bacteria, we designed an experiment using an interesting feature of this type of bacteria: they show autofluorescence, which can be detected by confocal microscopy. We observed and counted the number of putative methanogenic bacterial cells in each large intestine of each mouse, being the group fed with butter the one that showed a higher number of bacteria per field analysed. Finally, in collaboration with the group BIO-221 from the University of Jaén, the possible correlations between data obtained in the counts of methanogenic bacteria and other physiological variables (food intake, body weight, HDL, LDL and total cholesterol, triglycerides, leptin, ghrelin among others) were studied. Positive significant correlations were found between autofluorescence counts per field and body weight, total cholesterol and insulin in plasma. As we have previously mentioned, a diet rich in fat will condition the populations present in the gut. When we isolated the microbiota contained in faeces and we obtained a collection of Enterococcus (acquired at different times during the experiment), we found similarities between the groups fed with butter and refined olive oil. From the total of strains isolated (91), only thirtyfivewere found to be Enterococcus, but the distribution was not uniform among the different groups of mice, neither over the time. After twelve weeks, the groups fed with butter and refined olive oil showed a similar behaviour between them, while the group fed with extra virgin olive oil did not provided any Enterococcus isolate at final time. These results seem to be similar to the results obtained from culture-independent methods (DGGE). On these same 91 strains isolated, studies were performed to evaluate the antimicrobial properties of the olive oils (extra virgin and refined) and we also studied two of the polyphenols present in the unsaponifiable fraction of extra virgin olive oil. When we analysed in detail the data, we observed an interesting correlation between the number of Enterococcus present in faeces of mice fed with butter and blood pressure value. By massive sequencing results, we could see that the responsible of this fact was Enterococcus faecalis. But, is it the increase in blood pressure due to the presence of E. Faecalis in the intestinal microbiota or vice versa? It was then when we decided to inoculate E. faecalis (orally) in mice in order to answer this question. During the experiment, six weeks, we took measurements of blood pressure. Besides, daily faecal samples were plated in KAA (Kanamycin EsculinAzide Agar). After three weeks of treatment, the group inoculated daily with E. faecalis showed a significant correlation between CFU/g (colony forming units) in KAA and blood pressure values. According to these results we can think that a diet rich in butter may cause the growth of a specific bacterial strain, inducing the correlative increase of blood pressure. A high blood pressure value is one of the main symptoms of metabolic syndrome. Extra virgin olive oil has shown to be beneficial for hypertension and to produce distinct positive effects on intestinal microbiota. With the object to contribute to analyse the relation cause-effects between intestinal microbial changes and blood pressure, we designed an experiment with spontaneous hypertensive rats. These rats were fed with an enriched virgin olive oil diet and standard diet during twelve weeks. Microbial profiles in faeces were studied by culture-independent-culture methods (PCR-DGGE) at the end of the experimental period. In order to quantify selected bacterial groups we used quantitative PCR. The results that we obtained indicated differences in some taxons, which showed inverse correlation with systolic blood pressure values obtained at the end of experimental period.