The role of the gut microbiome in health may be enacted by the action of the bioactive molecules they release in the human colon. Important substrates for the gut microbiota include plant polyphenols and dietary fibre and the main bioactive molecules released include phenolic acids (PA) and short-chain fatty acids (SCFAs), respectively. Common dietary polyphenols include rutin and quercetin, which are found in fruits and vegetables such as tomatoes, onions, kale, tea, some berries, citrus fruits, and apricots [1
]. These and other flavonoids have been associated with reduced risk of cardiovascular disease and other health benefits. In a meta-analysis of 18 randomised clinical trials [3
], flavonol intake, mainly quercetin, reduced total cholesterol, triacylglyceride (TAG), low-density lipoprotein (LDL), systolic blood pressure (SBP), diastolic blood pressure (DBP) and increased high-density lipoprotein (HDL) levels. A meta-analysis of seven case-control studies showed an inverse association between flavonol and procyanidin intake and development of colorectal cancer [4
Bioavailability is a key factor influencing the health promoting potential of plant (poly)phenolics. The majority of polyphenols are not absorbed (only 7.2% of ingested polyphenols are found in blood and tissues) [5
]. Thus, most ingested polyphenols reach the colon, where they are catabolised to PA by the colonic microbiota [6
], although inter-individual variations do exist [11
]. These catabolites may be responsible for some of the health benefits attributed to polyphenol-rich foods [7
Colonic metabolism of rutin and quercetin, studied in vivo and in vitro, leads to the formation of PA, mainly 3,4-dihydroxyphenylaceticacid (3,4-diOHPAA), 3-hydroxyphenylaceticacid (3-OHPAA), 4-hydroxybenzoicacid (4-HBA), 3,4-dihydroxybenzoicacid (3,4-diOHBA), and 3-hydroxyphenylpropionicacid (3-OHPPA) [1
]. The recovery of quercetin in the ileal fluid of ileostomy patients was 83–86% [13
] when ingested in its glycosidic form (rutin) and ~77% when ingested as quercetin aglycones [13
The bioavailability of polyphenols is also dependent on their interaction with other substrates in the presence of the gut microbiota. Polyphenols are, most often, ingested alongside dietary fibres, sometimes in the same foods, and may be found together in plant cell walls. Therefore, they enter the colon at similar time.
Both fibre and polyphenols have been proposed to alter the growth and diversity of the gut microbiota, by which they are both catabolised. Most dietary fibres are fermented to SCFAs resulting in a fall in colonic pH. Some fibres selectively increase the populations of specific groups of bacteria, and therefore, act as prebiotics [16
]. This, along with lower pH may alter the metabolism of some compounds. Both antibacterial and prebiotic effects have also been described for (poly)phenol compounds [18
] and PA have also demonstrated antimicrobial impact especially towards pathogenic bacteria, with less impact on the commensal bacteria [24
]. Aglycones such as quercetin have been shown to have stronger antibacterial properties than their glycosidic forms such as rutin.
Thus, polyphenols and fibres are converted to bioactive molecules by the gut bacteria and can also influence the composition of the microbiota. There is very little evidence, however, of any interactions between the polyphenols and fibre, and the resultant release of PA and SCFAs. Inclusion of a fermentable carbohydrate (glucose) in the fermentation media of faecal batch cultures, accelerated the degradation of rutin, releasing quercetin and further metabolites [1
]. However, there is a lack of evidence on the interactions between dietary fibres, polyphenols, and the gut microbiota.
In this study, we investigated such interactions in vitro using a range of dietary fibres. Raftiline, ispaghula (psyllium), and pectin were chosen for their varying viscosity and fermentation rates as some of the potential inhibitory effect of fibre on polyphenol bioavailability could be linked to their viscosity and their fermentability [25
]. Rutin and quercetin were chosen as they are very abundant in the diet alongside dietary fibre.
We aimed to determine whether the three fermentable fibres influenced the colonic microbiota metabolism of rutin and the release of PA, using an in vitro fermentation model. The impact of quercetin, rutin, and their colonic metabolites on the production of SCFAs from the fermentation of soluble fibres by the gut microbiota was also evaluated.
This study has shown clear interactions between the inclusion of fibres and polyphenols in cultures of gut microbiota, which resulted in a change in the release of bioactive catabolites. In vitro fermentation of the polyphenolic compound quercetin-3-O-rutinoside led to the production of seven PA, as previously described [1
]. The most abundant PA was PAA, followed by 3,4-diOHPAA, 3-OHPAA, 4-OHPAA, 3-OHPPA, 4-OHPPA, and 4-OHBA. Phenolic acid from the background diet, determined based on their presence in the faecal sample alone incubations, included PAA, 3-OHPAA (in one participant only), 4-OHPAA, 3-OHPPA, and 4-OHPPA. The differences detected for rutin metabolism between participants were transient production of 4-OHBA in 2/10 participants and 3-OHPAA in 9/10 participants (Table 2
). It has been proposed that there are different metabotypes where individual microbiomes produce different patterns of metabolites, including PA from the same polyphenol source [34
]. We did not detect any other difference in metabotypes from rutin metabolism between the participants (Table S1
). A larger study may be required to further investigate differences in metabotypes. The paired study model allowed the use of FS-alone incubation as a control, despite the presence of PA from the background diet.
The dietary fibres raftiline, ispaghula, and pectin were chosen due to their varying viscosity and rates and patterns of fermentation. We did not measure viscosity in our study. However, it was clear on visual assessment of the fermentation vessels that raftline was not viscous and remained liquid throughout the fermentation. Pectin and ispaghula were viscous, with ispaghula displaying higher viscosity and forming a thick gel. Pectin and Ispaghula maintained their viscosity throughout the fermentation.
Raftiline as a fibre with higher fermentability (raftiline) had greater inhibitory impact on the PA production from rutin catabolism than ispaghula, which had the highest viscosity and lower fermentability. We did not detect an impact of rutin or quercetin at a concentration of 28 µmol/L on the pH or SCFA and gas production from these fibres. These results are in line with an in vitro fermentation study by Aura, O’leary [31
] which also demonstrated no impact from rutin and its metabolites on the pH of fermentation vessels.
The production of 3,4-diOHPAA in the current study and all previous studies demonstrates it as a dominant PA from rutin/quercetin colonic degradation, followed by 3-OHPAA. Previous studies did not detect PAA in high concentrations as was found in faecal incubations of this study. This may be attributed to LCMS analysis in previous studies compared to GCMS analysis used in this study. Phenyl acetic acid is non-polar in its non-conjugated form and has a low molecular weight [35
]; therefore, it is better detected by the GCMS due to poor ionisation in the LCMS. An improved sensitivity of GCMS for the detection of PAA and PBA compared to low sensitivity of LCMS has been demonstrated in other studies [36
]. Furthermore, FS-alone incubations resulted in 4.3 ± 1.6 µg/mL compared to 5.5 ± 3.3 µg/mL of PAA production from rutin incubations. Thus, the majority (77.4%) of PAA detected in our study was from the background diet.
Contrary to previous reports by Jaganath, Mullen [1
] and Aura, O’leary [31
], we did not detect 3,4-DiOHBA or 3,4-DiOHPPA. This may be due to rapid conversion of 3,4-DiOHPPA to 4-OHPPA and 3,4-DiOHPPA to 3-OHPPA, which were both identified in our samples. Our findings support the findings of Aura et al. [31
], that 3-OHPAA increases at 6 h concomitantly with the plateauing of 3,4-diOHPAA production. This suggests that 3-OHPAA is produced as a result of further degradation of 3,4-diOHPAA (Figure 2
In this study, 3,4-diOHPAA, PAA and total PA were inhibited by raftiline (81.6%, 92.5% and 85.5% reduction, respectively), pectin (66.8%, 95%, 78.1% reduction, respectively) and ispaghula (27.6%, 47.2%, 42.3% reduction, respectively). While 3-OHPAA and 4-OHPAA were inhibited by only raftiline (98.4%, 76% reduction, respectively) and pectin (89.6%, 74.6% reduction, respectively); 3-OHPPA and 4-OHPPA were not inhibited by any of the fibres. Only 2/10 participants produced 4-OHBA in rutin incubation and 1/10 in raftiline + rutin incubations. Pectin and raftiline completely inhibited the production of 4-OHBA (Table 1
The highly fermentable fibres such as raftiline exhibited an inhibition of PA production as early as 2 h post fermentation; ispaghula having a slower fermentation rate demonstrated an impact only at the 24 h sampling point and not across time, with the exception of its impact on PAA which was seen across time.
Raftiline and pectin had a higher inhibition percentage on total PA production than ispaghula. No difference was seen between raftiline (85.5% reduction) and pectin (78.1% reduction). Raftiline also inhibited PA production from polyphenolics present in the background diet, which was shown in higher PA concentration in FS-only incubations compared to raftiline only incubations.
These results are novel and demonstrate the importance of considering the impact of fibre viscosity and fermentability when studying their impact on the gut microbiota and their influence on PA production. There is insufficient evidence on the impact of carbohydrates on colonic metabolism of rutin.
In this study, the impact of fermentable fibres with low and high viscosity and fermentation rates on the degradation of rutin by colonic bacteria were considered. This enabled better understanding of the role of viscosity or fermentability on the bioavailability of polyphenol metabolite.
Most studies have been designed to investigate the antibacterial properties of polyphenols [18
] and not their impact on the colonic metabolite production of the microbiota. Such studies can only provide an assumption that the observed antibacterial activity of polyphenols will alter colonic metabolite production, such as SCFA production from fibre. Our study did not show any impact of polyphenolics on SCFA production from fibre catabolism. Additionally, PA production had no effect on incubation media pH or gas production.
Dietary fibres and dietary polyphenols may compete for catabolism by gut bacteria and their catabolism may affect the other [9
]. The consideration of the prebiotic potential of fibres in conjunction to the antibacterial properties of the polyphenols is of importance. The potential impact of rutin and quercetin on the microbiota may have been ameliorated by the prebiotic properties of raftiline, pectin, and ispaghula. The findings of this study are limited by the absence of microbial assessment. Focusing on microbial assessment in association with their catabolic by-products and measurement of fibre viscosity in future studies would provide further insight to the impact of fibres and polyphenols on the microbiota.
Most studies [20
] investigating the effects of polyphenols on gut microbiota have used high concentrations of polyphenols over long periods of time predominantly for their potential application in the food and pharmacology industry. They have not taken into consideration the bioavailability of these polyphenols. The concentration of rutin and quercetin (17.0 µg/mL rutin and 8.46 µg/mL quercetin) used in this study are based on their physiological bioavailability as recovered in the ileal fluid of patients 0–24 h after consuming tomato juice supplemented with 175 µmol/L rutin [43
]. This was much lower than the lowest MIC found for these compounds in vitro. We did not measure the changes in bacterial populations in the study.
Some PA were found in cultures of faecal samples alone. The reason for this is unclear. Compliance to the low polyphenol diet was shown by the dietary record of participants analysed using the Phenol Explorer database [44
It is important to note that our findings did not confirm whether the impact of fibres on PA production was inhibitory or just retardation. The total sum of PA showed a linear increase suggesting that the final total amount could have been much higher if more time were available. As demonstrated by Pérez-Jiménez, Serrano [45
], the release of antioxidants associated with dietary fibre was delayed.
The role of the microbiota in health and disease is currently a subject of considerable interest [46
]. Previous studies reporting health benefits from consumption of polyphenols or fibre did not take into consideration the interaction of these two components, which are most often found in combination in foods. The health claims for fibre has led the food industry to incorporate fibre into many products which are naturally high in polyphenols. This may ameliorate the potential health benefits of polyphenols, and is therefore of relevance to the food industry. This study shows that the impact of fibres on bioavailability of food polyphenols and subsequent benefits on health needs to be considered in this regard, with careful evaluation of all parameters that may influence health outcomes, from the perspective of the individual, the food chosen, and the study methods.