This study evaluated the gastrointestinal implications of low carbohydrate, high fat, Paleolithic style diets through comparison with a cohort of healthy Australians in a cross-sectional study design. Consumption of a long-term Paleolithic diet was associated with markedly higher serum TMAO concentrations, but only in those who adhered to the diet strictly. Romano et al. [42], identified six species of bacteria associated with choline consumption and production of TMA, of which, one was identified in our cohort, Clostridium hathewayi, originating from the genera Hungatella. We did not identify the other species reported by Romano et al. [42], although our methods of short amplicon sequencing are not typically used to identify individual species, but rather provide robust data at the genus level. It is therefore possible the other species were present, but not identified at the species level in our cohort. Nonetheless, the relative abundance of the Hungatella genus was significantly higher in both the SP and PP groups. Our results show that serum TMAO concentrations and Hungatella abundance were inversely associated with total and whole grain consumption, indicating these food groups may downregulate the ability of Hungatella to dominate or interfere with the production of TMA. Notably, TMAO concentrations in the PP group were not statistically different from the controls, despite the increased Hungatella abundance and small to medium effect size noted. The stratification of the Paleolithic group into two groups may have reduced our ability to detect significance of this outcome variable. Furthermore, the lower overall fiber and higher fat content of the PP diet may have influenced the fermentative capacity of the microbiota to produce TMA, given high fat diets may attenuate the fermentation response [43].

Bergeron et al. [44] detected changes in TMAO concentrations after a 2-week dietary intervention with 52 subjects and found low carbohydrate diets, high in RS, were associated with increased plasma TMAO. Conversely, in the current study TMAO was not associated with RS, but inversely associated with grain intake. This may indicate that other components of the grain carbohydrate and fiber are responsible for modulating abundances of Hungatella. Bergeron et al. [44] did not report food group intake for the intervention diets, which limits comparability with our study. In support of the findings presented here is the identification of the genera associated with producing TMA in the fecal microbiota [42], in addition to the statistical association found with red meat intake, a known TMA precursor food [45].

While there were no observed differences in measures of fecal microbiota diversity (α-diversity), a significant group difference (β-diversity) at both the phylum and genus levels were reported. An inverse association was found between the Bacteriodetes:Firmicutes ratio and body fat, supporting previous research showing a reduced ratio was associated with obesity [40, 41, 46]. At the genus level, different community structures were associated with intakes of vegetables, dietary fat, RS, whole grain and dietary fiber. The direction of the shift in microbiota composition was similar for vegetable intake, whole grains, RS and dietary fiber and is likely to be beneficial, given the large body of evidence associated with health benefits from consumption of these food groups [3, 47,48,49,50,51,52,53,54]. Conversely the direction of the shift in microbiota composition associated with fat consumption was in the opposite direction and suggests a more deleterious profile. Reductions to core bacteria including Roseburia, as seen in the current study, have been associated with inflammatory bowel diseases [55]. In animal models, high-fat diets have been shown to drive obesity independently of the composition of the microbiota [56]. There were reductions in the Paleolithic dietary groups to genera Roseburia and Bifidobacterium, known to metabolise carbohydrate and produce butyrate. Moreover, low abundances of known beneficial genera such as Bifidobacterium in the Paleolithic groups support the findings of Brinkworth et al. [13], who reported low abundances of this genera after an 8 week low-carbohydrate diet, comprising 58% fat [13]. A decrease in relative abundance of Bifidobacterium has been previously associated with irritable bowel syndrome, and obesity [57], however, these disorders have also been associated with lower fecal excretion of acetate and butyrate, which was not found in the current cohort. Given that total fat intake was associated with microbiota composition at both the phylum and genus level, and positively correlated with body weight, the differences observed in microbiota composition are unlikely to be beneficial.

Supporting our previous findings from a short-term intervention using the Paleolithic diet [6], the elimination of the grains food group on a Paleolithic diet resulted in a significantly lower intake of RS than the control group, despite no significant differences found in total soluble or insoluble fiber intakes. Intakes of RS in the control group were slightly higher than previous estimates of Australian intakes of 3.4–9.4 g/day [58] and may be due to higher consumption of whole grains than the average Australian intake of 1.5 serves/day [59]. Both groups consumed less than the proposed 20 g RS/day required for bowel health [60] and may be an area for focus in future interventions.

Despite the differences in RS intake, we did not observe any differences between groups in SCFA excretion. While this was an unexpected finding, the high intake of saturated fat in the Paleolithic groups may have influenced the results, given Fava et al. [61], showed fecal SCFA concentrations were higher following a 4-week diet comprising 17% saturated fat in 88 participants at risk of metabolic syndrome. It is not yet understood whether saturated fat leads to changes in fermentation patterns or alterations to colonic uptake of SCFA. Differences in microbiota composition attributed to the intervention diets used by Fava et al. [61] were assessed using fluorescence in situ hybridisation (FISH), which limits comparability with the current study. In addition, the lack of difference we observed for SCFA excretion may have been due to the differences in the sites of fermentation for grains as opposed to vegetables, with the latter likely to be fermented in the distal colon due to the difference in fiber structure, although there is little literature in this area. Even though fecal acetate concentration has been inversely associated with acetate absorption [62] fecal SCFA may not provide a sensitive enough marker to estimate differences in total fermentation along the colon. [63].

Previous short-term interventions using the Paleolithic diet have found increases to HDL cholesterol in diabetics [64], but not in healthy populations [5, 65,66,67], however, the latter also reported reductions to total and LDL cholesterol, in addition to significant weight loss, which may have influenced HDL concentrations. Further, a 2-year intervention showed no significant change in total or HDL cholesterol, despite significant reductions in triglycerides over the intervention period [23]. The current data showed small, but significantly greater HDL concentrations in the Paleolithic groups, which were associated with the reduction in carbohydrate intake, and increase in saturated fat consumption. The findings reported here support previous work showing that saturated fat consumption increases concentrations of calculated HDL cholesterol [68]. The current study also shows a significant relationship between saturated fat consumption and total cholesterol levels. A systematic review and meta-analysis showed that for every 1 mmol/L increase in total cholesterol, the relative risk of CVD was 1.20 for women (95% CI 1.16, 1.24) and 1.24 in men (1.20, 1.28) [69]. Therefore, while there may be conflicting evidence surrounding the specific effect of saturated fat consumption on CVD risk, the positive association noted in the current data between saturated fat and HDL concentrations must be interpreted with caution, as saturated fat intake was also associated with total cholesterol concentrations in the Paleolithic groups and may result in increased CVD risk over a longer-term period. Furthermore, we found a positive association between saturated fat intake, body weight, and BMI, which are known to increase CVD risk [70]. Taken together with the greater observed serum TMAO concentrations, it cannot be concluded that the Paleolithic diet is associated with improved gut health and a reduction in risk of CVD as promoted [71, 72].