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Apples are a rich source of polyphenols and fiber. Proanthocyanidins (PAs), the largest polyphenolic class in apples, can reach the colon almost intact where they interact with the gut microbiota producing simple phenolic acids. These metabolites have the potential to modulate gut microbiota composition and activity and impact on host physiology. A randomized, controlled, crossover, dietary intervention study was performed to determine the broad effects of whole apple intake on fecal gut microbiota composition and activity. Forty heathy mildly hypercholesterolemic volunteers (23 women, 17 men), with a mean BMI (± SD) 25.3 ± 3.7 kg/m2 and age 51 ± 11 years, consumed 2 apples/day (Renetta Canada, rich in PAs), or a sugar matched control apple beverage, for 8 weeks separated by a 4-week washout period in a random order. Fecal and 24-h urine samples were collected before and after each treatment. The broad effects of apple intake on fecal gut microbiota composition were explored by the high throughput sequencing (HTS) of 16S rRNA gene lllumina MiSeq sequencing (V3-V4 region). Sequencing data analysis was performed using the Quantitative Insight Into Microbial Ecology (QIIME) open-source pipeline version 1.9.1. Specific bacterial groups were also enumerated using the quantitative Fluorescence In Situ Hybridization (FISH). Furthermore, the potential formation of microbial polyphenol metabolites, after apple intake, was explored in urine using Liquid Chromatography (LC) High-Resolution Mass Spectrometry (HRMS) metabolomics. Preliminary analysis showed no changes in gut microbiota abundances measured by Illumina MiSeq, after correction for multiple testing. Apple intake significantly decreased Enterobacteriaceae population (P = 0.04) compared to the control beverage, as determined with FISH. Twenty-four polyphenol microbial metabolites were identified in higher concentrations in the apple group (P < 0.05) compared to the control, including valerolactones, valeric and phenolic acids. In conclusion, preliminary data suggest that the daily intake of 2 Renetta Canada apples significantly decreased Enterobacteriaceae population, a family known for its pathogenic members, in healthy mildly hypercholesterolemic subjects. Moreover, several polyphenol microbial metabolites were identified, suggesting that microbial activity is crucial and a prerequisite for the absorption of apple polyphenols, producing active metabolites with potential health benefits.
Conjugated linoleic acids (CLAs) show a number of putative health-promoting activities including anti-carcinogenic, anti-adipogenic, anti-diabetogenic, anti-inflammatory and antioxidant actions. CLAs are naturally produced by ruminal bacteria and several studies demonstrate that various lactobacilli and bifidobacteria are also able to produce CLAs in vitro from linoleic acid (LA). However, the ability of the human gut microbiota to produce CLA is less extensively studied. Our hypothesis is that the human gut microbiota is able to convert LA to CLA, and that the readily fermentable fiber inulin would positively modulate the growth of CLA-producing bacteria and, consequently increase the CLA content in the intestine.
The capability of the faecal microbiota from five healthy donors to produce CLA was tested in anaerobic batch cultures for 48 hours at pH 5.5 and 6.5. Test treatments were linoleic acid (LA; 1 mg/mL) + bovine serum albumin (BSA; 0.2 mg/mL), and LA (1 mg/mL) + BSA (0.2 mg/mL) + inulin (1%, w/v) compared to a control BSA (0.2 mg/mL) fermentation. The microbial composition was analyzed 0, 24 and 48 hours after starting the fermentation by 16S rRNA gene Illumina MiSeq sequencing (V3-V4 region). CLAs were quantified by Ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) and bi-dimensional gas chromatography (GC x GC).
The inclusion of LA + BSA + inulin at pH 5.5 significantly increased the relative abundance of Collinsella aerofaciens (p < 0.05), and tended to increase the relative abundance of bifidobacteria. LA + BSA + inulin at both pH 5.5 and 6.5 reduced the relative abundance of Parabacteroides, Bilophila, Clostridia and Enterobacteriaceae (p < 0.05). The concentration of CLA, in particular the isomer cis9,trans11 C18:2, was significantly higher in the LA + BSA + inulin group at pH 5.5 after 24 and 48 hours fermentation.
The data show that the treatment LA + BSA + inulin at pH 5.5 induce substantial changes in microbiota composition, including bifidogenesis and CLA production in a human intestinal microbiota model. The changes of relative abundance detected are consistent with changes in gut bacteria previously linked to human health. Collinsella aerofaciens has been reported for reducing bloating, in particular in subjects suffering from irritable bowel syndrome, while Clostridia, Bilophila and Enterobacteriaceae causes human infections. In addition, the increase of bifidobacteria and LAB, which have previously been shown in vitro to produce CLA, may also be involved in CLA production under simulated cecal microbiome. These preclinical observations warrant confirmation in suitably designed animal and human mechanistic studies.
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