Over the past decades, an increasing amount of evidence has been emerging on the importance of the gut microbiota driving many biological functions that are directly associated with host health (1). This has subsequently led to an interest in understanding mechanisms and to potentially modulating the microbiota for health benefits.

Within a healthy individual, the intestinal microbiota is reasonably stable in composition, but there is potential for targeted modification toward a more favorable community composition to optimize health benefits. Human intervention studies have shown a clear link between dietary intake and the gut microbiota, where the species composition will adapt in response to dietary change, determined by competition for substrates and gut conditions (2, 3). Therefore, dietary intake is considered the primary modulator of long-term microbiota composition. This association has been observed in long-term prospective studies. These longitudinal data contribute to the increasing causal evidence for the role of the gut microbiota and its metabolites (including short-chain fatty acids, SCFA) on various clinically important indicators of health, such as immune cell function and gut barrier integrity (4–7). In addition, evidence from human intervention studies targeting modification of the microbiota has shown encouraging effects on systemic inflammation, metabolic health, allergies, and a variety of gastrointestinal diseases (8–15). The most salient evidence for the direct link between the gut microbiota and host health comes from the overwhelming success of invasive fecal microbiota transplants (FMT) in the treatment of recurrent Clostridium difficile infection (16). This approach focuses on correcting the underlying gut microbiota dysbiosis via drastic changes in the microbiota by replacing host microbiota with those of a healthy donor.

For less invasive approaches, specific foods and dietary patterns can also favorably influence the gut microbiota and metabolite production. In specific instances these health effects have been attributed to prebiotics. A prebiotic is defined as a “selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health” (17, 18). Prebiotic supplementation can affect the composition and the activity of the gut microbiota (19, 20). Much of the research on these compounds has focused on dietary fibers (particularly oligosaccharides) but other non-carbohydrate compounds, mainly derived from plant materials as well as polyphenols, have been investigated as naturally-occurring prebiotics (21, 22). An example are cocoa-derived flavanols since they are reported to stimulate lactic acid bacteria in vivo and in vitro in healthy volunteers (n = 22) consuming them daily for 4 weeks (23). Further research has shown that lactic acid bacteria in the human gut can convert phenolic compounds, such as flavonoids to biologically active metabolites, and it is believed that these metabolites confer the health benefits to host health. Consumption of a fermented milk product over a 4-weeks period, increased the production of bacterial metabolites, such as butyrate and other short chain fatty acids (SCFAs) in participants with irritable bowel syndrome (IBS-Constipation type Rome III criteria). Patients (n = 28) also experienced improvements in self-reported symptoms (i.e., abdominal distention, acceleration of colonic transit time), further strengthening the role of this fermented milk in improving gut health (24). The study authors suggest that these findings were compatible with the observation that butyrate (a short chain fatty acid) has previously been shown to also improve intestinal motility and visceral sensitivity in IBS-C (17, 18, 25, 26). Consequently, renewed efforts are investigating fermentation-derived metabolites in foods, assessing their ability to favorably modulate microbiota composition and activity, thereby acting as potential prebiotics.

Fermented foods are produced through controlled microbial growth, and these food and drink products have recently surged in popularity due to their proposed health benefits (27). Yet, there is limited evidence on the real-world effectiveness of most commercially available fermented food and beverages on gastrointestinal health. Several studies have shown that microorganisms from fermented foods can reach the gastrointestinal tract, in the same way as probiotics which are considered “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (28). Studies however indicate that the presence of probiotic species of bacteria appears to be transient when consumption is discontinued in animal and human studies (29, 30).

At present, there is insufficient clinical evidence to make evidence-based recommendations for the consumption of fermented foods to improve the gut microbiota profile. A recent critical review by Stiemsma et al. identified 19 human interventions using fermented foods without probiotic bacterial addition post-fermentation. The heterogeneity in the findings, largely attributed to the variety of foods investigated, highlighted the need to study the bioactive compounds and other beneficial by-products of fermentation individually to ascertain their relative role in mediating health effects (31).

In this study we evaluate a deproteinised whey concentrate, high in L-(+)-Lactic acid, fermented by a member of the genus Lactobacillus, establishing its potential as a putative prebiotic. The main aim was to investigate the effect of daily consumption of the whey product on fecal short chain fatty acid composition, on gut microbiota metabolism and composition, and determine if these could be linked to improvement in glycemic control. We also explored the potential underlying physiological mechanisms via assessment of self-reported intestinal function (regularity and consistency of bowel movements).

We present the results of a diet study investigating the effects of dietary supplementation with a fermented whey product containing bacterially derived L-(+)-Lactic acid on various health parameters and microbial activity. Following daily FWC supplementation in healthy adults, a higher proportion of butyrate and propionate were detected in fecal samples, although total SCFA levels remained unchanged (Figure 5). This may be attributable to the high L-(+)-Lactic acid content (70 g/L) of the FWC influencing the activity of lactate utilizers within the host microbiota. However, we were unable to detect a significant change in the composition of the microbiota following the addition of FWC to the diet. No significant changes were detected in alpha diversity (Figure 6, Shannon-diversity index), bacterial abundance at the phylum level (Figure 7), or in beta diversity (Figure 8, Bray-Curtis dissimilarity) over the course of the supplementation. This may in part be due to the large inter-individual variability in bacterial species composition which our study was not sufficiently powered to overcome. Further investigations would be needed to determine whether oral ingestion of lactate might stimulate cross-feeding of lactate utilizing species in the colon resulting in production of butyrate or propionate in healthy volunteers (48, 49).

Lactate is rarely present in healthy adult feces due to extensive cross-feeding between different bacterial species whereby any lactate produced provides a substrate for lactate-utilizers in the microbiota (1, 43). In this study lactate was not detected in feces, even after supplementation with FWC rich in L-(+)-Lactic acid. This could indicate that the L-(+)-lactic acid was either not able to reach the colon and was absorbed in the upper GI tract, or was completely metabolized by microorganisms (50). There is considerable uncertainty on the biodistribution of orally (exogenous) ingested L-(+)-lactic acid in humans due to the continual flux of naturally occurring lactate from metabolic processes, even at rest (51). Compared to endogenously produced lactic acid, the lactic acid consumed from the FWC will have a substantially different biodistribution, as it is subjected to the multitude of digestive processes all along the human gastrointestinal tract. In particular, the initial stages of exogenous lactate digestion (absorption and distribution) will differ to endogenous lactate, which is directly released from the human cell into circulation (52, 53). In healthy adults, plasma lactate is primarily cleared by the liver and mitochondria-rich tissue, such as skeletal muscle (54, 55).

Exploring the fate of exogenous lactate, produced by bacterial fermentation, within the gastrointestinal tract would improve our understanding of dietary lactic acid intake and the downstream effects on host-microbiota composition, and eventually bioactivity. Commonly consumed fermented foods are naturally rich in L-(+)-Lactic, yogurt can reach up to 2%. This warrants further investigation into the effect of regular ingestion of these levels and whether these contribute to the beneficial health effect associated with regular yogurt consumption (56). The FWC used in our study contains 7% L-(+)-lactic acid (or 70 g/L), meaning that the daily intake of the volunteers corresponded to 2.8 g of L-(+)-lactic acid, comparable with concentrations in a pot of yogurt. It is important to note that this lactic acid was administered independent of any live bacteria or food matrix (in contrast to fermented foods), which may have affected its absorption. It would be interesting to investigate whether regular ingestion of these levels of lactate contribute to the beneficial health effect associated with regular yogurt consumption (56).

A further aspect to consider is that systemic lactate can cross the gut epithelial barrier and thus it is possible that not all FWC lactate directly reached the colon (56). Even if dietary lactate is absorbed into circulation in the upper GIT, at least some of it may reach the colon via indirect mechanisms (systemic circulation and transport back into the colon). A previous study by Scheiman et al., linked the presence of Veillonella bacteria, conversion of circulating lactate to propionate, and enhanced athletic performance (57). The results from our study were in line with these previous findings yet it remains unknown if this mechanism of peripheral lactate transport into the GIT can be observed independent of exercise. Our data shows that consumption of FWC [containing L-(+)-lactic acid] increases relative amounts of butyrate produced in feces, but only in participants who had higher levels of Veillonellaceae (Dialister) species (see Supplementary Table 1A).

Overall, there were no changes in bacterial diversity as a result of FWC supplementation. Both the alpha diversity (the number of different taxa) and the Shannon index (sample richness) indicated similar bacterial abundance levels across the study period. However, when the data were stratified into responders and non-responders based on increased fecal butyrate detection, the Veillonellaceae family were significantly higher in responders (FDR = 0.0047) at both sampling timepoints. This may indicate that those individuals with a higher resident population of bacteria capable of converting lactate to butyrate/propionate, have higher concentrations of these SCFA in feces following lactate consumption. Consequently, the modified SCFA profile indicates that the lactic acid might stimulate microbial metabolic activity without detectable changes in microbial composition. Metatranscriptomic analysis of the microbiota would show which genes were differentially expressed by bacterial species following FWC supplementation and provide detailed insights into changes in metabolic activity. Confirmed prebiotic compounds, such as inulin (a natural soluble fiber) have an effect on microbiota composition as demonstrated in many studies including a study with 12 healthy volunteers investigating the effects of inulin consumption for 21 days on the microbiota where increases in the Bifidobacteria population were observed (58).

There were no changes in fasting insulin levels following the FWC supplementation, but inter-individual differences in response profiles existed. Insulin sensitivity could be more accurately studied with participants at risk of developing diabetes to study any long-term effects on glycaemic control. Previous data indicate that this benefit might be more pronounced in persons with a higher BMI. This was shown in a double-blind, randomized, placebo-controlled crossover study, where a daily dose of 20 g inulin-propionate ester was able to alter insulin sensitivity significantly (59).

There were several limitations associated with our study. There is increasing evidence that gut microbiota richness is linked to stool consistency and water content, which were not assessed in this study (60). This pilot trial was exploratory and thus did not use a control group or a cross over design with placebo treatment. As the study was conducted in a non-blinded manner, we cannot exclude treatment effect at least partially explaining the observed changes in the subjective GI well-being questionnaire. It is important to also bear in mind that the participants were free-living and were able to consume food ad-libitum, and diet is one of the key factors affecting microbiota diversity. Although in this study we collect 3-days food diaries to monitor changes in energy intake, it was not possible to assess changes in food intake patterns and food types consumed but macronutrient intake was fairly consistent at both the timepoints when diaries were completed. Weighed food diaries, as a dietary assessment method, have their limitations, and in our study also do not directly correspond with the timing of stool sample collection which is a further source of uncertainty (57).

We observed increased fecal propionate and butyrate concentrations following FWC consumption. The health effects of fecal butyrate can be attributed to the fact that colonocytes utilize bacterially-produced butyrate as their primary energy source, whereas most other cell types utilize glucose (61). Changes in gut permeability may improve gut health and confer long-term benefit to the host which we were unable to investigate in our short-term study. Changes in the microbiota can be attributed to a huge variety of factors including diet, immune challenges, exercise, variations in GI transit time, intestinal pH, host secretions, regulation of gene expression of the host and/or the microbiota and other environmental factors. Therefore, it is incredibly difficult to definitively attribute any changes in metabolic activity of the microbiota to the FWC supplementation. As highlighted by Stiemsma et al., compositional changes to the gut microbiota in addition to changes to immune or metabolic factors remain the most studied links between fermented food consumption and health outcomes (31). An increased focus on functional analyses, such as SCFA production, and the balance of SCFA production and absorption in humans as a result of fermented food consumption might shed light on their health benefits.

Probiotics and prebiotics are established dietary interventions seeking to correct underlying microbiota dysbiosis and leading ultimately to improved host health. However, a potentially new subtype of gut-directed supplements aiming to correct underlying microbiota dysbiosis in order to influence host health are post-biotics. The term post-biotic refers to “soluble factors (products or metabolic byproducts) secreted by live bacteria or released after bacterial lysis” (62). The key difference is that while probiotics, by definition, need to contain viable bacterial cells, post-biotics do not (28). Post-biotics must originate from bacteria, differentiating them from prebiotics, which tend to be fermentable carbohydrates. At present the few studies which have investigated the effectiveness of post-biotics indicate positive effects on host immune response both in animal and humans (62, 63).

This project was undertaken to evaluate the effect of bacterially derived FWC, with a high L-(+)-Lactic acid content, on microbiota composition and activity and determine potential effects on host health. Based on our data it is likely that the effects of FWC might be linked to the high lactic acid content specifically stimulating species of the resident microbiota. Consequently, we could consider FWC as a putative prebiotic based on the changes in microbial activity, or as a putative post-biotic since the lactic acid component is a product of the Lactobacillus fermentation of the whey, and is safe for human consumption at these concentrations. Confirmation of the FWC in either category would require establishment of a definitive health benefit. Future controlled human studies are required to establish our exploratory data in a larger participant group, with additional biological measures to establish the biodistribution of L-(+)-Lactic acid post-ingestion, and establish a health benefit. Taken together, this will help us understand if microbiota driven effects, such as the observed changes in SCFA profile, are directly beneficial to the host.

The datasets generated for this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: http://www.ebi.ac.uk/ena/data/view/PRJEB30495.

The studies involving human participants were reviewed and approved by Research ethics committee of the Rowett Institute, University of Aberdeen (Reference Nr. 799). The patients/participants provided their written informed consent to participate in this study.

AJ, KS, and AS: conceptualization and funding acquisition. NM and JM: methodology. GH and SS: formal analysis. NS, NM, JM, and CF: investigation and resources by A.Vogel Bioforce AG. GH, SS, and NS: data curation. NS: writing-original draft preparation. AJ, KS, CF, and JM: writing-review and editing. NS and SS: visualization. AJ and KS: supervision. All authors contributed to the article and approved the submitted version.

AS was employed by the company Bioforce AG. A.Vogel Bioforce AG, AJ, and KS have conducted other contract research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.