Fecal microbial metabolism of polyphenols and its effects on human gut microbiota

Highlights

• We incubated four dietary polyphenols with human fecal bacteria.

• Fermenta was rich in short-chain fatty acids such as butyrate.

• Fermentation increased bifidobacteria and decreased Firmicutes:Bacteroides ratio.

• Metabolites in the fermenta also specifically increased bifidobacterial growth.

• Metabolites included 3-hydroxyphenylacetic acid and 3-hydroxyphenylpropionic acid.

Abstract

We investigated the biotransformation of four common dietary polyphenols, rutin, quercetin, chlorogenic acid and caffeic acid, in an in vitro mixed culture model of human intestinal microbiota, to determine effects on human gut bacteria. All four compounds were biotransformed rapidly, disappearing from the medium within 0.5 h and later replaced by known phenolic acid breakdown products, at concentrations up to hundreds of micromolar, much higher than in no-polyphenol control experiments. Quantitative PCR was used to measure effects of the polyphenols on the balance between the major groups of intestinal bacteria that are known to influence gut health, i.e., Bifidobacterium spp., Bacteroidetes, and Firmicutes. Fermentation of polyphenols stimulated proliferation of bifidobacteria and decreased the ratio of Firmicutes to Bacteroidetes, relative to controls. Polyphenols also stimulated short chain fatty acid production by the bacteria. Pure bifidobacterial cultures were treated separately with either fermented media isolated from the incubations, the pure test polyphenols, or the biotransformation products detected in the fermentations. Growth stimulation was observed only with fermented polyphenol media and the pure biotransformation products. It appears that dietary polyphenols may have the ability to modify the gut microbial balance, but this effect is indirect, i.e., it is mediated by biotransformation products, rather than the original plant compounds.

Introduction

Polyphenols are important bioactive components in our diet [1]. The two major classes of polyphenols are hydroxycinnamic acids and flavonoids, which account for around half and one quarter of our dietary polyphenol intake respectively [2]. The most abundant hydroxycinnamic acid is caffeic acid, which is widely distributed in fruits [3]. Caffeic acid is commonly esterified with quinic acid to form chlorogenic acid, which is found in apples, berries and kiwifruit, vegetables such as potatoes [1] and in high concentrations in coffee [3]. Of the flavonoids, flavonols are very widespread, with the most common being quercetin [4]. Quercetin is most abundant in onions, curly kale, leeks, broccoli, and blueberries, and is often present as glycosides such as the rutinoside conjugate, rutin [1].

The average dietary intake of polyphenols is about 1 g/day [5]. Once ingested, caffeic acid is much better absorbed than its ester derivative, chlorogenic acid [6]. Chlorogenic acid is not thought to have significant bioavailability until it reaches the colon, where the colonic microbiota rapidly hydrolyse it to liberate caffeic acid [7]. Quercetin aglycone has a very high bioavailability [8], but rutin is absorbed comparatively slowly and most reaches the colon [9]. The colonic microbiota break down rutin to liberate quercetin, some of which is absorbed via the colon and the remainder is biotransformed into simpler phenolic acids [10], [11]. The large proportion of polyphenols that is not absorbed by the small intestine appears to become concentrated in the ileal and colorectal lumen [6].

We have previously demonstrated that polyphenols can influence the viability of gut bacteria and their adhesion to intestinal epithelial cells in vitro [12]. The distal intestine, however, harbours billions of bacteria, with the most populous being anaerobic bacteria from the Bacteroidetes and Firmicutes phyla [13]. Bacteroidetes and Firmicutes are the main groups involved in colonic metabolism of undigested food remnants, including dietary fibre and polyphenols, by a complex metabolic energy-harvesting mechanism based on cross-feeding and co-metabolism [14]. The ratio of Firmicutes to Bacteroides has been found to correlate with body weight [15], with the ratio being higher for obese people. Different polyphenols have been suggested to affect the relative viability of these bacterial groups [16], [17], implying that dietary modulations with polyphenols may play a role in reshaping the gut microbial community and enhancing host microbial interactions to provide beneficial effects such as weight loss [17]. While the individual specific microbiome remains stable lifelong in humans [18], polyphenol-based functional foods may provide opportunities to modulate the microbial balance in the gut.

Another well-studied group in the human gut is the Bifidobacterium spp. (Actinobacteria phylum), which provides distinctive metabolic and probiotic benefits to the host [19]. Bifidobacteria are endowed with an extensive array of carbohydrate utilisation enzymes, and their metabolic potential is further increased by co-operative or mutualistic cross-feeding with major groups such as Bacteroides and butyrate-producing groups of Firmicutes phyla [20]. Bifidobacteria represent only a small proportion (up to 8%) of the total anaerobes in the gastrointestinal tract of adults, and although their numbers can be increased by intake of probiotics, their population decreases again within 14 days, as exogenous bifidobacteria are unable to colonise the gut permanently [21]. Thus, polyphenols could be beneficial if they were to have a prebiotic (growth-promoting) effect on transient diet-derived beneficial bifidobacteria, which would in turn increase the retention of the bifidobacteria in the gut, and could optimise the overall microbial balance.

One of the major modes of energy production in the colon is through the anaerobic microbial metabolism of unassimilated or undigested food to generate short chain fatty acids (SCFAs) including the three major SCFAs - acetate, propionate and butyrate [22], [23]. As well as a cross-feeding resource in the complex food web which comprises the gut microbiota, SCFAs are also beneficial to gut health. Butyrate, in particular, is a major metabolic fuel for colonic cells [22] and protects against colonic disease [23].

Polyphenols are biotransformed by the gut microbiota, initially by deglycosylation, followed by breakdown of flavonoids into relatively simple aromatic carboxylic acids, commonly referred to as phenolic acids [24]. These metabolites may modulate the growth of bacteria in the gut microbial milieu; for example, treatment of human fecal homogenates in vitro with tea polyphenols generally inhibited proliferation, with pathogenic bacteria being inhibited the most [16]. These metabolites are known to mediate many physiological effects; for example, 3,4-dihydroxyphenylacetic acid generated from breakdown of flavonoids can inhibit platelet aggregation [25] and also provide antioxidant protection close to the intestinal wall, which is much more aerobic than the lumen, as it is extensively vascularised with blood capillaries [11].

In this study, we have investigated the metabolism of dietary polyphenols in an in vitro anaerobic model simulating colonic fermentation using isolated total fecal microbiota. We report their effect on the growth of the major bacterial groups and the phenolic acid and SCFA metabolites produced.