During the last decades, a significant number of studies have explored the role of gut microbiota in several pathophysiological conditions including obesity, cancer, inflammatory bowel diseases, metabolic syndrome and neurological diseases [1, 2]. Diet components as well as dietary habits have been implicated in the modulation of the gut microbiota composition and functionality [3, 4]. Fermented foods and especially dairy fermented products containing high populations of live microorganisms have been tested for their efficacy in the modulation of the gut microbiota composition [5, 6].

Yogurt, a well-known fermented dairy product, is included in almost all the heart-healthy dietary patterns, while it should be noted that the benefits of low-fat and fat-free compared to the full-fat dairy products are still under discussion [7, 8]. Reviews and meta-analyses have concluded that yogurt consumption has an inverse association with the risk of type 2 diabetes (T2D), whilst no significant association seems to exist between yogurt consumption and the risk of stroke, hypertension, coronary artery disease (CAD) and cardiovascular diseases (CVDs). Meanwhile, yogurt consumption and risk of all-cause and CVD mortality has been inversely associated [9, 10, 11]. The results from a recent meta-analysis suggest that dairy intake might improve inflammatory biomarkers but without taking into account the yogurt intake per se as the real cause of the observed changes [12].

Yogurt is a fermented food containing 10${}^7$ colony-forming unit (CFU)/g viable cells from Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus bacteria [13]. The frequent yogurt consumption impacts significantly the composition of the gut microbiota resulting in increased bifidobacterial and lactobacilli populations [14, 15]. Yogurt can also be used as a food matrix for other functional ingredients such as other bacterial cultures, flavoring ingredients, nutritive carbohydrate sweeteners, vitamins etc. [16], while any unwelcome organoleptic properties of the active ingredients can be concealed when it is flavored [17].

We have previously demonstrated that the daily consumption for 8 weeks of a yogurt, fortified with an olive oil pomace extract (OOPLE), attenuated ex vivo platelet-rich plasma (PRP) aggregation induced by PAF, decreased IL-6 and IL-10 levels, and also favorably modulated Platelet-activating factor (PAF) metabolic enzymes, in healthy mainly overweight adults [18, 19]. It has been demonstrated that the OOPLE enrichment contains PAF receptor (PAF-R) inhibitors [20] and has the capacity to prevent the development of atherosclerotic plaques in rabbits fed a high cholesterol diet and also to regress the formed plaques in the same animal model [21]. PAF, chemically identified as 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine [22] is a potent endogenous lipid mediator that induces platelet activation, aggregation and secretion as well as mobilization of intracellular calcium, while its intravenous administration in experimental animals leads to anaphylactic shock and even in death in higher concentrations [23]. PAF is implicated in a variety of pathological conditions where inflammation, thrombosis and immune activation has a recognized role [24, 25, 26, 27]. More specifically, PAF binding to its receptor (PAF-R), a G-protein-coupled receptor expressed in many cell types, triggers signaling pathways that result in the activation of a variety of kinases, in the production of nitric oxide and in the production of arachidonic acid metabolites through the activation of cytoplasmic phospholipase A${}_2$ [28]. Additionally, the role of PAF in inflammatory bowel diseases has been described [29, 30, 31, 32]. PAF concentration in plasma, cells and tissues is mainly controlled via its biosynthetic and catabolic pathways. Two distinct pathways are responsible for PAF biosynthesis, the de novo and the remodeling pathway. The de novo pathway utilizes alkylacetylglycerols as substrates and with the action of 1-alkyl-2-acetyl-sn-glycerol choline phosphotransferase (PAF-CPT) is producing the elementary PAF levels [33], while the remodeling pathway uses Lyso-PAF as substrate that is subsequently acetylated by the action of the acetyl-CoA:lyso–platelet-activating factor acetyltransferase (Lyso-PAF AT), is mainly activated under inflammatory conditions [34]. PAF biological action is contributed to the presence of the acetyl group in the sn-2 position of the glycerol backbone and thus is inactivated when this group is hydrolyzed by intracellular PAF-specific acetylhydrolase (PAF-AH) and its plasma isoform lipoprotein-associated phospholipase A${}_2$ (LpPLA${}_2$) [35].

The role of PAF in the development of inflammatory bowel diseases has been well-established in necrotizing enterocolitis (NEC) where it has been reported that PAF-AH reduces the incidence of NEC [36]. In addition, PAF-R is constitutively expressed in human intestinal epithelium [37]. This data has led the authors to suggest that PAF is produced by the human intestinal epithelium and exerts autocrine and/or paracrine action [37]. Additionally, PAF upregulates both Toll-Like Receptors 4 (TLR4) mRNA and protein expression in intestinal epithelial cell lines and the PAF-PAF-R complex interacts with and subsequently activates TLR4 [38, 39]. These receptors recognize specific repetitive patterns associated with bacterial products such as lipopolysaccharide (LPS) and mediate cellular responses that lead also to inflammation [40]. It has also been reported that PAF-R also acts as a recognition receptor for the phosphorylcholine group of Gram-positive lipoteichoic acid (LTA) as well as for the Gram-negative LPS [41, 42].

According to the above, the aim of the present study was to investigate the impact of the daily consumption of a low-fat yogurt fortified with PAF-R antagonists, or the plain one, on gut microbiota and faecal metabolites. Additionally, potential associations between inflammatory indices, hemostatic markers and PAF metabolic enzymes with gut microbiota characteristics and faecal metabolites in humans were investigated. For this purpose, a randomized, three-arm, double-blind, placebo-controlled, parallel-group trial was performed in apparently healthy mainly overweight adults.

The present clinical trial aimed to investigate whether the intake of a yogurt on a daily basis, fortified with a polar lipid extract of olive-oil by-products that contains PAF-R antagonists, could affect gut microbiota and faecal metabolites in apparently healthy, mainly overweight, participants. According to the results, the consumption of the enriched yogurt resulted in a significant increase in the levels of Bifidobacterium spp., C. perfringens group and F/B ratio. On the other hand, a significant increase in the levels of Lactobacillus and C. perfringens group was observed after the intake of the plain yogurt. Additionally, a higher % increase in molar ratio of BSCFAs was detected for both yogurt groups after the 8 week-intervention compared to control. The consumption of the enriched yogurt also resulted in a significant drop in faecal caproic levels and a trend for lower ratio of butyrate to total VFAs compared to baseline levels.

Previous intervention trials in adults have reported increased levels of bifidobacteria and in some cases lactobacilli populations after daily yogurt intake [15, 48, 49] even though no effect in faecal bifidobacteria has also been stated [50, 51]. No differences were detected in the faecal levels of clostridia and E. coli in previous studies [48, 51], while in a study that included over 65 years old Irish subjects, the presence of C. perfringens was negatively correlated with the number of Bifidobacterium spp. recovered [52]. Similar bacterial changes, with a concomitant decrease in Bacteroides vulgatus levels, after yogurt consumption have been previously attributed to the normalization of the bacterial density groups, probably reflecting a more balanced gut microbiota profile [53, 54]. Furthermore, it should be noted that despite the increase of C. perfringens group levels after yogurt consumption in our study, no diarrheic episodes or severe gastrointestinal side effects were reported during the intervention period. The increase in the levels of Bifidobacterium spp. observed after the intake of the enriched yogurt may be also due to the existence of olive pomace carbohydrates and polysaccharides since it has been documented that fructo-oligosaccharides promote the growth of endogenous beneficial organisms such as bifidobacteria [55].

It should note that the OOPLE extract contained 30% w/w maltodextrin (MDX) as a protective colloid for emulsions as well as an emulsion polymerization material, resulting in an intake of approximately 0.23 g MDX/day. It has been reported that MDX dosages ranging between 0.5 and 15 g/d for 1–16 weeks resulted in a significant increase of various Firmicutes members, in increased acetate and propionate levels, in decreased BSCFAs while the effect of MDX on butyrate levels varied between studies [56, 57]. The significant increase in F/B ratio after the consumption of the enriched yogurt was probably due to the raise of baseline Firmicutes levels and could be merely attributed to the presence of MDX in the food matrix, though daily intake of MDX in our study was much lower compared to the previous reported. Furthermore, elevated F/B ratio may be resulted by components present in the OOPLE extract of the yogurt, since a similar rise in F/B ratio has already been documented in mice fed with a virgin olive oil enriched diet [58].

In our study, SCFAs analysis indicated a significant drop in faecal caproic levels and a trend for lower ratio of butyrate to total VFAs after the consumption of the enriched yogurt. Caproic acid, also characterized as medium chain fatty acid (MCFA), seems to exert pro-inflammatory properties through the activation of p38 MAPK signaling and also its serum concentration is increased in multiple sclerosis patients, although its role in other pathological conditions is still under consideration [59, 60]. It has been suggested that caproic acid is produced from the elongation pathway of acetyl-CoA, -the product of lactate oxidation-, resulting in the formation of butyryl-CoA and hexanoyl-CoA [61]. The decrease of both butyrate and caproic acid detected after the intake of the enriched yogurt is consistent with the above biosynthetic pathway. Additionally, a significant higher % change in molar ratio of BSCFAs was detected for both enriched and plain yogurt group after the 8 week-intervention compared to control. Even though, no significant differences were detected between yogurt groups in the % changes of the gut microbiota and faecal metabolites, plain yogurt had a trend for higher % change in iso-butyrate concentration whereas enriched yogurt group had a trend for higher % increase in iso-valerate concentration. The elevated BSCFAs and specifically the increase in iso-butyrate and iso-valerate levels after yogurt consumption was somewhat expected since it has been documented that they are mainly produced during protein fermentation and especially from valine and leucine degradation by the intestinal microbiota carried out mainly by genera Bacteroides and Clostridium [62, 63, 64].

The relation between gut microbiota and PAF metabolism has not been studied with the exception of one study that revealed no connection between Akkermansia muciniphila and LpPLA${}_2$ activity [65]. Regarding PAF action, it has been reported that L. acidophilus, as a probiotic, counteracts PAF-induced inflammation in cell line derived from normal human colon and Caco-2 cells by reducing nuclear factor kappa B activation and IL-8 production [66]. Lyso-PAF that is the precursor molecule for the remodeling PAF biosynthesis, as well as the product of PAF-acetylhydrolases action, has been detected in faecal samples of healthy donors and also PAF and lyso-PAF levels have been augmented in ileoanal anastomosis and pouchitis [67]. As far as yogurt intake is considered, the microbial species present in yogurts and especially S. thermophilus and L. acidophilus have the metabolic capacity to synthesize phospholipids among them glyceryl-ether analogs [66] and lyso-PAF plasma levels were directly associated with full-fat dairy intake including yogurt [68]. Additionally, the existence of PAF inhibitors in the lipid fraction of yogurts has been documented [66, 69, 70, 71]. In the plain yogurt group, the increase in C. perfringens seems to reduce both PAF catabolism through LpPLA${}_2$ action and the calcium-independent PAF remodeling biosynthetic pathway, while the increase in Lactobacillus group appears also to reduce intracellular PAF catabolism (PAF-AH activity), indicating that PAF levels may be increased. Similar to our results, phospholipase C from C. perfringens had been documented to rise PAF levels by stimulating PAF remodeling biosynthetic pathway in cultured intestinal epithelial cells, but no data presented whether this stimulation was observed in the presence or absence of calcium [72]. In the same group, Bifidobacterium spp. reinforced PAF action in platelets as lower concentration of PAF was needed to induce ex vivo PRP aggregation implying the secretion of a PAF analog that is in accordance with previous data [73]. Interestingly, in the enriched yogurt group, the increase of C. perfringens group was also inversely correlated with the plasma catabolic enzyme, LpPLA${}_2$, but the increased F/B ratio was positively correlated with the LpPLA${}_2$ activity taking under consideration the LDL levels, the main carrier of LpPLA${}_2$. In addition, we have previously reported that the intake of the enriched yogurt resulted in lower LpPLA${}_2$-to-LDL ratio at 8 weeks compared to the plain yogurt [19]. Moreover, C. perfringens group seems to inhibit PAF action as a positively correlation with PAF EC${}_{50}$ values, lower response of platelets against PAF, was detected. Since, this correlation was not observed in the plain yogurt group, the implication of PAF-R antagonists present in the OOPLE extract cannot be excluded and the reduced PAF action has already been documented in this group [18]. Additionally, it was recently reported that PAF is a weak activating agonist and an allosteric inducer of FFAR2 (free fatty acid receptor 2) also known as GPR43, a G protein-coupled receptor that responds to short-chain fatty acids, especially acetate and propionate, produced by gut microbiota fermentation, through a receptor cross-talk mechanism and the PAF induced transactivation of FFAR2 was inhibited by the PAFR specific antagonist WEB in human neutrophils [74]. Since the OOPLE extract contains PAF-R antagonists, it may also be implicated in the inhibition of the PAF induced transactivation of FFAR2.

Daily low-fat yogurt consumption for 8 weeks led to a significant increase in the levels of C. perfringens group that was inversely associated with the plasma catabolic enzyme of PAF, namely LpPLA${}_2$, that is considered a cardiovascular risk marker and has been associated with inflammation and atherosclerosis. Moreover, in the enriched with PAF-R antagonists yogurt group, the increased levels of C. perfringens group were also associated with lower PAF action. An increase in the levels of Bifidobacterium spp. and a significant drop in faecal caproic levels was also detected in the enriched yogurt group indicating a favorably effect of PAF antagonists on gut microbiota in healthy overweight subjects although further exploration is needed to elucidate the mechanism.

The participants were apparently healthy overweight adults of Greek ethnicity with medium adherence to Mediterranean diet, thus the findings may not apply to different population groups. Even though the study protocol included 92 participants, stool samples were provided before and after the intervention from only 51 adults (58% participation rate). Additionally, the participants of Group B that provided stool samples presented a metabolically slightly less healthy profile that may have influenced their response to the intervention. PAF levels were not evaluated that would provide a direct information regarding its relation with specific microbial species. Gut microbiota diversity could be further analyzed in-depth by e.g., 16S rRNA sequencing. Also, we did not measure other microbial-derived metabolites such as trimethylamine, a precursor of trimethylamine N-oxide that has been associated with platelet activity. On the other hand, the study design (i.e., randomized, double-blind) eliminated any potential sources of bias (i.e., selection, performance and detection bias). Lastly, subjective tools and methods, i.e., self-administered records, phone calls were used to evaluate the compliance of participants to the study protocol.

To our knowledge, this is the first study that has investigated the impact of two yogurts, a plain one and an one enriched with PAF inhibitors on gut microbiota and faecal metabolites in healthy overweight subjects and report significant associations between gut microbiota and PAF metabolism and action.

Data are available from the corresponding author upon reasonable request.

SA designed the research study, supervised its implementation and drafted the manuscript; EKM performed the evaluation of stool characteristics, gut microbiota qPCR-based analysis and SCFAs, performed the statistical analyses and critically revised the manuscript; AK designed and supervised the gut microbiota analysis and SCFAs and critically revised the manuscript; EF supervised the biochemical analyses, performed the statistical analyses and critically revised the manuscript; MD performed the biochemical analyses. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All participants were informed about the objectives and procedures of the study and provided their written consent before enrollment. The study took place at the Department of Nutrition and Dietetics of Harokopio University in Athens, Greece, from October 2014 to June 2016. The trial adhered to the guidelines of the Declaration of Helsinki (1989) of the World Medical Association, was approved by the Bioethics Committee of Harokopio University (40/30-10-2013) and was registered in ClinicalTrials.gov (NCT02259205).

The authors would like to thank the participants of this study for their involvement, as well as all the personnel and investigators for their contribution in the enrollment, randomization and dietary evaluation of the participants and in conducting the biochemical analysis of blood samples: Christos Kokkalis, M.D., Mary Yannakoulia, Costas Anastasiou, George Milias, Tzortzis Nomikos, Antigoni Tsiafitsa, Margarita Christea, Ioanna Vlachogianni, Marianna Xanthopoulou, Chrysa Argyrou and Stella Zouloumi.

The European Regional Development Fund of the EU and the Greek Ministry of Education and Religious Affairs, Sport and Culture/GGET – ЕΥDЕ-ЕΤАΚ have cofinanced this work, through the Operational Program Competitiveness and Entrepreneurship (OPC II), NSRF 2007–2013, Action “SYNERGASIA 2011” Project: 11SYN_2_652, entitled “Cardioprotective properties of yogurt enriched with bioactive lipids from oil production by-products”.

Smaragdi Antonopoulou states that given her role as Guest Editor, she had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Dr. Amedeo Amedei and Dr. Eugene Rosenberg. Smaragdi Antonopoulou has a relevant patent (Hellenic Industrial Property Organisation, 1008550—25/08/2015 issued). The other authors declare no conflict of interest.