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THE RECIPROCAL INTERACTIONS BETWEEN RED RASPBERRY POLYPHENOLS AND GUT MICROBIOME COMPOSITION: PRELIMINARY FINDINGS
Title
THE RECIPROCAL INTERACTIONS BETWEEN RED RASPBERRY POLYPHENOLS AND GUT MICROBIOME COMPOSITION: PRELIMINARY FINDINGS
Creator
Zhang, Xuhuiqun
Date
2017, 2017-07
Description
Red raspberry (Rubus idaeus L.) contains a variety of polyphenols including anthocyanins and ellagitannins. Red raspberry polyphenols absorbed...
Show moreRed raspberry (Rubus idaeus L.) contains a variety of polyphenols including anthocyanins and ellagitannins. Red raspberry polyphenols absorbed in different forms (parent compounds, degradant or microbial metabolite molecules) are subject to xenobiotic metabolism in the intestine, liver, and/or kidney, forming methylate, glucuronide, and sulfate conjugated metabolites. Consistent exposure of certain polyphenols to the gut microbiota may act as prebiotic-like substances feeding the beneficial gut bacteria and changing the gut microbiome composition and function. The dichotomy between the biotransformation of polyphenols into their metabolites by gut microbiota and the modulation of gut microbiome composition by polyphenols is hypothesized to contribute to positive health outcomes. The present study examined the regular consumption of red raspberry purée (RRB) and/or fructo-oligosaccharide (FOS) on gut microbiome composition and subsequent bioavailability of red raspberry polyphenols in healthy volunteers. An 8-week pilot study, including two 4-week chronic treatments and 3 postprandial days, served as a feasibility study and mechanism to collect multiple biological specimens for method development. An ultra high-performance liquid chromatography (HPLC) coupled with electrospray ionization quadrupole time of flight (QTOF) and triple quadrupole (QQQ) mass spectrometer were used to identify and quantify the phenolic compounds in red raspberry purée, plasma and urine samples. Fecal samples were used for the metagenomic study. The sequencing of the 16S ribosomal RNA gene was utilized to study the gut microbiome composition. The red raspberry purée contained 148.55 ± 5.43 mg/100 g fresh weight (FW) polyphenols. Chronic RRB and/or FOS exposure influenced gut microbiome composition: at the phylum level, 4-week FOS (8 g/d), RRB (125 g/d), or FOS plus RRB (8 and 125 g/d, respectively) exposures all decreased Firmicutes and increased Bacteroidetes; at the genus level, 4-week FOS, RRB, or FOS plus RRB exposures all boosted Bacteroides and diminished Blautia; and the increased Akkermansia was only observed after RRB exposure. Chronic RRB and/or FOS exposure also altered the observed RRB polyphenol metabolites: the parent anthocyanins, such as cyanidin 3-O-sophoroside, were lower in plasma and urine after adaptation to RRB, while the production of urolithin A glucuronide (the main microbial-derived metabolite of ellagitannins) increased after FOS, RRB and FOS plus RRB exposure; an effect hypothesized to be related to the altered composition and metabolic activity of the gut microbiota. Overall, these data suggest chronic RRB and/or FOS exposure influenced gut microbiome composition and subsequently increased gut microbial metabolites.
M.S. in Food Safety and Technology, July 2017
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AN IMPROVED VALIDATED METHOD FOR THE DETERMINATION OF SHORT-CHAIN FATTY ACIDS IN HUMAN FECAL SAMPLES BY GC-FID
Title
AN IMPROVED VALIDATED METHOD FOR THE DETERMINATION OF SHORT-CHAIN FATTY ACIDS IN HUMAN FECAL SAMPLES BY GC-FID
Creator
Freeman, Morganne M
Date
2022
Description
Short-chain fatty acids (SCFAs) are metabolites produced by the gut microbiota through the fermentation of non-digestible carbohydrates....
Show moreShort-chain fatty acids (SCFAs) are metabolites produced by the gut microbiota through the fermentation of non-digestible carbohydrates. Recent studies suggest that gut microbiota composition, diet and metabolic status play an important role in the production of SCFAs. Current methods for the analysis of SCFAs are complex and inconsistent between research studies. The primary objective of this study was to develop a simplified method for standardized SCFA analysis in human fecal samples by gas chromatography with flame ionization detection (GC-FID). A secondary objective was to apply the method to fecal samples from a previous randomized, crossover clinical trial comparing participants with pre-diabetes mellitus and insulin resistance (IR-group, n=20) to a metabolically healthy reference group (R-group, n=9) after daily consumption of a red raspberry smoothie (RRB, 1 cup fresh-weight equivalent) with or without fructo-oligosaccharide (RRB + FOS, 1 cup RRB + 8g FOS) over a 4-week intervention period. Extraction parameters, including solvent selection and water content of the sample, were investigated before finalizing the method. Freeze-dried fecal samples (0.5 g) were suspended in 5 mL of milli-Q water, vortexed and centrifuged at 3,214 x g for 10 minutes. The supernatant was transferred to a clean tube, acidified with 5.0 M HCl and centrifuged again at 12,857 x g for 5 minutes. The resulting supernatant was transferred to a GC vial for analysis by GC-FID. Linear regression data for standards at concentrations 5-2000 ppm ranged from 0.99994-0.99998. Limit of detection (LOD) ranged from 0.02-0.23 µg/mL. Limit of quantification (LOQ) ranged from 0.08-0.78 µg/mL. The validated method was then applied to fecal samples collected from a previously conducted study. Nine SCFAs were identified and quantified (acetic, propionic, iso-butyric, butyric, iso-valeric, valeric, 4-methyl valeric, hexanoic and heptanoic acids). Statistical analysis (Student’s t-test, ANCOVA) was performed on PC-SAS 9.4 (SAS Institute). Acetic acid was significantly lower in the IR-group compared to the R-group before starting intervention (baseline, Week 0, IR v R-group, p=0.014). Intervention analysis comparing RRB to RRB + FOS at 4 weeks (WK4) showed a significant difference in 4-methyl valeric acid (p = 0.040) in the R-group. Trends of decreased SCFA content after 4-weeks of RRB and RRB + FOS compared to baseline were observed in both groups, though changes were not significantly different between dietary interventions at 4 weeks (p>0.05). Metabolic status and dietary intervention are discussed in relation to their impact on SCFA content in fecal samples and mechanisms of biological use as a metabolite. Limitations of the study include sample size and using only feces and not other biological samples for SCFAs analysis, which may be considered for future research.
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