The significant involvement of the gut microbiota in the human health suggests that modulation of commensal microbial composition and metabolism through combinations of probiotics and prebiotics could be a dietary strategy to prevent diverse diseases, such as obesity, diabetes, non-alcoholic fatty liver disease, inflammatory bowel disease, and even cancers [4
In the present study, the impact of a synbiotic food supplement on the gut microbiota structure of healthy humans was evaluated by using an integrated molecular approach based on PCR-DGGE and real-time PCR.
DGGE profiles of the predominant fecal microbiota generated complex but overall relatively stable and unique profiles for each individual. Elaboration of DGGE data revealed high SI values between T0 and T1 profiles, and no clustering of banding patterns according to the feeding. These results demonstrated that no significant change in the structure of the gut microbiota of healthy subjects did occur following dietary intervention, confirming previous findings regarding the subject specificity of the predominant fecal communities and their stability over time and resistance to perturbations [9
]. Notably, we cannot exclude an effect of the synbiotic intake on minor bacterial species, an effect that could be investigated using high-throughput sequencing techniques. However, the impact on the dominant colonic microbiota represents the main parameter to evaluate the clinical relevance for the use of a functional food.
Because DGGE can be considered a semiquantitative tool for monitoring the dynamics of the predominant bacterial species of an ecosystem, additional analysis with real-time PCR was performed to obtain a quantitative estimation of the effect of the synbiotic intake on bifidobacteria and lactobacilli populations. In particular, variations in amounts of B. longum
and L. helveticus
were evaluated in order to assess the capability of the probiotic species included in the synbiotic food to pass through the gastrointestinal tract of the human host. Only L. helveticus
concentration increased significantly after the ingestion of the functional food, demonstrating the gut persistence of the probiotic L. helveticus
strain during the feeding period. Since L. helveticus
species is not a natural inhabitant of the human intestine and its presence in feces is diet related [45
], this result was not surprising and suggests that low abundant species could be optimal models for studying the gut colonization of probiotic bacteria. On the other hand, visualization of the gut colonization of a high abundant species, such as B. longum
, is strictly related to its basal concentration. For this reason, we observed the B. longum
increase only in subjects with the lowest concentration of B. longum
species at the time point T0.
The intake of the synbiotic food resulted in significant changes in some gut metabolic activities, as highlighted by the CAP analysis of the fecal metabolic profiles, which pointed out a separation of fecal samples of the subjects on the basis of the synbiotic food intake. Surprisingly little is known about volatile organic compounds formed in the gut. GC-MS/SPME, detecting volatile molecules with high sensitivity, represents a suitable approach to identify microbial metabolites in fecal samples, such as SCFAs, ketones, esters and sulfur compounds [46
Two SCFAs, acetic and valeric acids, were the metabolites showing the highest increase after the synbiotic administration. Although a general increase was observed also for butyric acid, this variation was not statistically significant due to the high variability of the measures. SCFAs are very common in the gut environment, arising from metabolism of undigested carbohydrates, such as dietary fiber and prebiotics, by colonic bacteria. The increase of SCFAs is particularly interesting, as they play a role in regulation of cell proliferation and differentiation of the colonic epithelial cells. Increases in SCFA production have been associated with decreased pH, which may reduce potential pathogenic clostridia, decreased solubility of bile acids, increased absorption of minerals, and reduced ammonia absorption by the protonic dissociation of ammonia and other amines [47
]. Other metabolites whose changes were positively and significantly correlated with the synbiotic intake belonged to the family of methylketones (methyl-5-hepten-2-one, 2-propanone, 2-butanone, 2-pentanone, 2,3-butanedione). In particular, the significant increase of 2-pentanone can be regarded as the most interesting effect associated with the synbiotic food intake. In fact, 2-pentanone, which is a naturally occurring compound in fruits, vegetables and fermented foods, has anti-inflammatory and chemopreventive properties. According to Pettersson et al
], it inhibits the prostaglandin production and COX-2 protein expression in human colon cancer cells. The increase of 2,3-butanedione is interesting since it may have health benefits by impacting on the growth of some bacteria, such as L. delbrueckii
ad Streptococcus thermophilus
]. Furthermore, during glucose catabolism 2,3-butanedione serves as an electron acceptor and can be reduced to 2,3-butanediol via acetoin. This pathway was shown to be important in the removal of toxic amounts of pyruvate and in maintenance of pH homeostasis [49
]. A diverse range of sulfur compounds has been identified in stool samples [41
]. The usual source of sulfur compounds is the microbial breakdown of sulfur containing amino acids and the increase of these compounds suggests an abundance or metabolic activity of bacteria able to breakdown cystein and methionine. In our study, a significant increase of carbon disulfide was observed following the feeding period. Carbon disulfide may be produced by carbonation of hydrogen sulphide as a detoxification mechanism exerted by colonic bacteria. According to Garner et al
], carbon disulfide has been found in 100% of the samples from healthy donors and absent in many samples of patients with Campylobacter jejuni
and Clostridium difficile
. Various esters were detected in all fecal samples. In particular, a significant increase of methyl acetate, ester of methanol and acetic acid, was evident after the synbiotic intake. Methanol is rarely found as free alcohol in the gut, where it is generated from the breakdown of macromolecules including pectins, bran and aspartame. In general, free alcohols and endogenous fatty acids are metabolized into fatty acid esters in liver, pancreas and intestine [50
]. At the intestinal site, esterification of alcohols by colonic bacteria can be regarded as a microbial strategy to remove or trap toxic molecules such as fatty acids and alcohols.
To sum up, the investigation of the fecal volatile metabolites by GC-MS/SPME allowed to correlate the consumption of the synbiotic food with the stimulation of health-promoting metabolic activities of the gut microbiota, such as regulation of the colonic epithelial cell proliferation and differentiation, anti-inflammatory and chemopreventive properties and detoxification processes.