At baseline and at the end of 2 weeks of intervention period, body weight, body mass index and composition were determined. Body composition was measured using the Tanita-BC418 bio-electrical impedance analyzer . Height was measured without shoes using a stadiometer and recorded to the nearest 0.1 cm. In the meantime, subjects reported on their overall well-being at baseline and at the end of two weeks by completing the SF-36 general wellness questionnaire. Aliquot of stool samples was diluted, acidified, and filtered, and SCFAs were quantified by gas chromatography flame ionization detection, as previously described. SCFA standard mix was purchased from Sigma-Aldrich . Urinary free rosmarinic acid as a biochemical marker of adherence to spice supplementation was measured by high-performance liquid chromatography according to published methods. Measures of beta-diversity were computed using the weighted and unweighted UniFrac distance metric using phyloseq in R. Differences in whole communities across groups were determined by Permutational Multivariate Analysis of Variance using the Adonis command provided by Vegan in R.
The results were visualized via Principal Coordinate Analysis ordination . OTUs differentially abundant between follow up and baseline visits in subjects receiving spice and placebo interventions were identified using DESeq2. This algorithm performs normalization using size factors estimated by the median-of-ratios method,blueberry packing boxes employs an empirical Bayesian approach to shrink dispersion, and fits the data to negative binomial models. Differential abundance was determined using the Wald test with automatic filtering of low abundance OTUs and automatic calculation of adjusted p-values , and the enriched OTUs were visualized using the ggplot2 package in R. As Bonferroni correction is often considered overly conservative, an extended set of OTUs significant at p < 0.2 was listed. Differentially abundant OTUs between spice and placebo groups were also detected by DESeq2. Null and test models were constructed for each OTU using DESeq. Each model includes variables “time” and “intervention” . Differentiating the two models was an interaction between “time” and “intervention”, which was only present in the test model. To identify OTUs with abundance changes that occurred specifically with spice but not placebo intervention, we used a likelihood ratio test to test for a significant additional contribution of the interaction between time and spice vs. placebo exposure on OTU abundance in the test model when compared to the null model. Here, we report that daily intake of 5 g of mixed spices for 2 weeks in healthy subjects resulted in a significant reduction in the relative abundance of the phylum Firmicutes , and a trend of increasing in phylum Bacteroidetes as compared with a matched control group.
Most healthy adult micro-biota are dominated by these two bacterial phyla, which together make up about 95% of gut micro-biota. A significantly higher abundance of Firmicutes and a higher Firmicutes/Bacteroidetes ratio has been frequently demonstrated in obese individuals, whose gut micro-biome is characterized by an increased capacity for energy harvest, inflammation, and gut barrier disruption. In addition to the shifts observed in Firmicutes and Bacteroidetes abundance, a number of OTUs in the family of Bifidobateriaceae and Bacteroidaceae, as well as the family of Streptococcaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae, and Erispelotrichaceae, were significantly altered between groups . Spice intervention significantly enhanced two Bifidobacterium OTUs of the Bifidobacteriaceae family—B. animalis and B. pseudolongum—as well as one Lactobacillus OTU compared with control group. Bifidobacterium has been shown to associate with the production of a number of potentially health promoting metabolites, including short chain fatty acids, conjugated linoleic acid, and bacteriocins. The abundance of B. animalis was reported to negatively associate with the body mass index. Results from the present study are consistent with published reports in that consuming polyphenol rich foods increases the relative abundance of Bifidobacterium and Lactobacillus and reduces pathogenic Clostridium species. Bifidobacterium and Labtobacillus are considered “healthy bacteria”, and members of the Bifidobacterium and Lactobacillus, as well as Streptococcus, are frequently used as probiotic strains with evident health benefits such as immune-modulation, cancer prevention, inflammation management, and the control of diverse bacterial consortia infections.
We noted that the abundance of all three Ruminococcus spp. were increased. In humans, Ruminococcus spp. were found as abundant members of a “core gut micro-biome” in a majority of humans. Some Ruminococcus spp. in our gut micro-biomes play an important role in helping us degrade and convert complex polysaccharides into a variety of nutrients for their hosts. The slow digestion of these special carbohydrates has been associated with numerous health benefits such as reversing infectious diarrhea and reducing the risk of diabetes and colon cancer. Interestingly, the Bacteroides spp., such as B. fragilis, also have the ability to recognize and metabolize plant- and host-derived polysaccharides, and to produce polysaccharides as well. Polysaccharide A, e.g., synthesized by B. fragilis, can promote immunological tolerance to pathogenic species such as Helicobacter hepaticus and protect the host from inflammation and associated colorectal cancer.SCFAs constitute approximately 10% of the energy source in healthy people. These microbial-derived products are utilized by the host and exert a range of health-promoting functions. Butyrate is used preferentially as an energy source by the gut mucosa, is anti-inflammatory, and protects against colorectal cancer, whereas propionate is largely taken up by the liver and is a good precursor for gluconeogenesis, and promotes satiety and reduction in cholesterol liponeogenesis and protein synthesis. Acetate is the most predominant gut-produced SCFA in peripheral blood and plays a role in prevention of weight gain through an anorectic effect, inflammation, and metabolic dysregulation. Valerate is present in substantially low amount, and research on this SCFA is very limited. A recent study, however, showed that valerate significantly inhibited the growth of C. difficile in vitro and in vivo, suggesting valerate can potentially be used as a safe, microorganism-free method to treat C. difficile infection. In the present work, the level of all four SCFAs was trending higher after spice intervention but the difference did not attain statistical significance due perhaps to the large interpersonal variations and small size of the study population. Nonetheless, our results indicated that spice supplementation can change SCFA production. Human studies investigating the effects of spice supplementation on gut micro-biota composition are very limited. Kang et al., reported that intake of capsaicin powder in healthy subjects increased the Firmicutes/Bacteroidetes ratio, accompanied with increased plasma levels of glucagon-like peptide 1 and gastric inhibitory polypeptide and decreased plasma ghrelin level. In animal models, Van Hul et al., reported that cinnamon bark extract lowered fat mass gain and adipose tissue inflammation in mice fed a high-fat diet leading to reduced liver steatosis and lower plasma nonesterified fatty acid levels that were associated with change on the microbial composition. Spices have been shown to alter serum biochemical parameters related to inflammation or low-grade inflammation induced by high-fat diet and have protective effects against chronic diseases. This evidence indicates that spices may play important role in modulating the growth of gut micro-biota and promoting human health. The observed lack of significant difference in microbial richness is largely attributed to the brevity of the intervention,package of blueberries which is also believed to account to for the lack of change in species evenness as well as of overall microbial composition. Another limitation of the study is that to avoid the sustained effects of spices on micro-biota, our study design was a randomized placebo-controlled and not a crossover clinical trial, therefore, there are large interpersonal variations in gut micro-biota composition and metabolite formation.
Lastly, since this is not a controlled feeding study, dietary recall data analyzed by a dietitian was the only method used to assess participants’ compliance with the beige diet. Analysis of dietary data may have an impact on interpretation of the results. The human gut micro-biome mediates many key biological functions and its imbalance, termed dysbiosis, is associated with a number of inflammatory and metabolic diseases from inflammatory bowel disease to asthma to obesity and insulin resistance . How to effectively shift the micro-biome and restore balance is a key question for disease prevention and treatment. The gut micro-biome is influenced by a number of factors including the nature of the initial colonization at birth , host genotype, age, and diet. As diet is a readily modifiable factor, it is an obvious target for interventions. Several studies have confirmed high inter-individual variability in the bacterial composition of the gut micro-biome in healthy individuals . Despite this high variability at the species level, enterotypes, or distinct clusters at the genus level, were described as core micro-biomes that are independent of age, gender, nationality, or BMI . Although the concept of enterotypes is itself controversial, diet has been shown to play a key role in determining enterotype . Although the core micro-biota within each person are stable over longer time scales , community composition is highly dynamic on shorter time scales . In fact, major shifts occur within 1 day of a significant dietary change . “Blooms” in specific bacterial groups were observed in response to controlled feeding of different fermentable fibers . Dietary changes affect both the structure and function of the gut micro-biome in animals , and humans under controlled feeding conditions . Rapid shifts in micro-biome composition are observed in response to change from a vegetarian to an animal based diet . An ecological perspective helps to delineate the complexity and multi-layered nature of the relationships between the micro-biota, the human host, and both the nutritive and non-nutritive compounds we ingest . The concept of the human gut micro-biome as a distinct ecosystem or collection of micro-ecosystems allows us to identify and characterize the components of the system, including its inputs and outputs. In this case, the inputs of the system include all of the various ingested compounds that can either serve as food substrates or that can be metabolized by or that affect the metabolism of the micro-biota . Some of these inputs, such as probiotics have been studied extensively. It has been well documented that certain sugars such as galactooligosaccharides, fructooligosaccharides, and oligosaccharides found in milk act as prebiotics that support the establishment and growth of certain commensal microbial species . Research has also documented the effects of antibiotics, and pathogens on the micro-biota composition, its recovery or lack of recovery to baseline following resolution, and the various immunological and physiological effects of these perturbations . Yet, little is known about the effects of ingested microorganisms on gut micro-biota composition or function, and even the basic questions of which microbes, how many of them, and how much they vary from diet to diet and meal to meal, have not been answered. We do know about the microbial ecology of various specialty foods where fermentation, colonization, ripening, and/or aging are part of the preparation of these foods, for example pancetta and of course cheese . The microbial ecology of the surfaces of raw plant-derived foods such as fruits and vegetables has also been characterized . Furthermore, it is known that the microbial ecology of endemic microbes found on food surfaces can affect mechanisms by which pathogens colonize these foods . A recent article showed that certain ingested microbes found in foods such as cheese and deli meats were detected in the stool of individuals who consumed them, and that furthermore they were culturable and thus survived transit through the upper intestinal tract . However, the microbial ecology or microbial assemblages of different meals and diets, as well as the total number of live microorganisms ingested in these meals and diets are largely unknown. In fact, studies of the effects of diets and foods on the gut micro-biota rely on dietary recalls and other dietary reporting instruments that were not designed to capture the potential variability in aspects of foods other than their basic macronutrient and micronutrient content. Specifically, current instruments for collecting individual dietary data do not capture the provenance of foods or their preparation, both of which would likely influence certain compositional aspects of the foods, especially the microbes on those foods. We performed a preliminary study designed to generate hypotheses about the microbes we eat, and how they vary in terms of total abundance and relative composition in different meals and dietary patterns typical of American dietary intakes.