Evaluating the influence of diet on human intestinal microflora using molecular techniques

Microbial communities in the gut play an important role in human health. Researchers have begun to characterize this complex ecosystem but many fundamental questions including the influence of diet remain unanswered. Previous studies have relied on conventional microbiological methods but these lack sensitivity that is needed to detect changes in such a complex system. The purpose of this study was twofold: to evaluate the feasibility of using a combination of Polymerase Chain Reaction and Denaturing Gel Gradient Electrophoresis (PCR-DGGE) to monitor changes in intestinal microflora and to investigate the influence of diet on microbial communities in the human intestine. Alterations in microbial communities were investigated based on two types of diet modifications: (a) supplementation with a prebiotic dietary component (i.e., soy isolate enriched with high non-digestible oligosaccharides (NDO)) and (b) a controlled diet made up of a set of foods commonly consumed as part of regular diet with no added prebiotics or probiotics. The effects of the controlled (camp) diet were compared with a free-choice diet (non-controlled, home diet) consumed before and between periods of controlled diet. Thus, in the soy NDO study, we attempted to investigate the prebiotic property of the soy isolate, while in the second study, we aimed to evaluate whether variability in the human intestinal microflora profiles can be reduced when consuming a diet made up of the same foods over an extended period of time. Fecal samples were collected from human subjects at regular intervals throughout the study. Total DNA extracted from the fecal samples was used to amplify the bacterial 16S rRNA gene by PCR using domain specific primers. Intestinal microbial communities were profiled using DGGE separation of the PCR amplicons and Bionumerics software was used to compare DGGE bacterial fingerprint profiles. Bands were selected from the DGGE profiles, excised from the respective gels, sequenced for identification and grouped into a phylogenetic tree using neighbor-joining algorithm. In addition, the archaeal community was also studied using similar techniques. Overall, it was found that human intestinal microbial communities were largely made up of profiles composed of 10 to 20 intense bands. There was considerable inter-subject variability in the bacterial community profiles, but a smaller intra-subject variability. In the soy NDO study, the soy isolate was observed to enhance the growth of Bifidobacterium spp. These results provide evidence of the prebiotic effect of the NDO enriched soy isolate. In the study with regular diet, DGGE fingerprint data indicate that bacterial communities can be modified by changing the diet. Specifically, changes in intestinal microbial community profiles were observed before and after participation in a clinical camp. Data indicate that subjects attained a set of DGGE fingerprint profiles as a response to non-controlled diet at home. Within 2 to 4 days of controlled diet at camp, these profiles changed to another set. The camp fingerprint profiles remained as long as their diet remained unchanged. In addition, each individual assumed their own unique fingerprint profiles even though the same types of foods were given to all subjects during camp. In contrast, intestinal archaeal community had a simpler profile composed of 1 to 2 bands. Within an individual, the presence of these bands varied over time regardless of diet. The findings suggest that unlike bacteria that are consistently found in the gut, Archaea appear intermittently. In summary, data from DGGE and sequencing indicate that the composition of the human intestinal microflora can be modified by diet. Sequences obtained from DGGE data showed that special food supplement (soy isolate enriched with NDO) had prebiotic property, whereby it enhanced the growth of Bifidobacterium spp. when administered at a dose of 5 to 15 g/d for 16 days. Further, DGGE fingerprint profiles indicated that the human intestinal microflora can also be modified by regular food items when they are consumed consistently over a period of time. Overall, these data also imply that with optimized conditions and appropriate analytical software, molecular fingerprinting technique such as PCR-DGGE can be applied in clinical studies as a rapid and reliable method to evaluate microbial community changes in the gut in response to dietary treatment