Gut microbiome-produced metabolites in pigs: a review on their biological functions and the influence of probiotics

SCFAs are fermentation by-products of indigestible polysaccharides, such as fiber and resistant starch, produced by the gut microbiome [38,111–113]. Amino acid fermentation, on the other hand, yields branched-chain fatty acids (BCFAs) [61,114,115]. Acetate, butyrate, and propionate are the major SCFAs produced in the gut, whereas isobutyrate and isovalerate are the main BCFAs in the GIT [61,114]. Lactate, on the other hand, is produced by lactic acid bacteria and utilized by SCFA-producing microbiota via a metabolite cross-feeding mechanism [63,64]. In pigs and other non-ruminant hosts, SCFA and BCFA concentrations along the intestinal tract exhibit heterogeneity, with the highest concentrations produced in the proximal colon [42,64,115,116]. Absorption of these metabolites occurs mainly in the colon [46]. The mechanism by which SCFAs are produced by the gut microbiome, either by anaerobic fermentation or cross-feeding, has been described extensively in previous publications [38,41,63,117,118]. Dietary fibers and gut microbiome composition greatly modulate these mechanisms [6,119]. Because dietary fibers are complex substrates, their complete degradation requires different members of the gut microbiome working in consortia to produce SCFAs [29,33,113]. The phylum Bacillota (Firmicutes) includes many SCFA-producing members, such as Ruminococcaceae and Lachnospiraceae [37,38]. Faecalibacterium, Eubacterium, Roseburia, Blautia, and Ruminococcus can produce SCFAs, particularly butyrate [29,33,35,36]. Clostridium also participates in the production of SCFAs, particularly acetate, in the swine gut [39]. Members of the phylum Actinobacteria, such as Bifidobacterium, and Bacteroidetes, such as Bacteroides fragilis, contribute to the SCFA pool by producing acetate, propionate, and butyrate [40–42]. Although most major SCFA producers in the gut have been characterized, there could be more unknown gut microbial residents involved in SCFA production. On the other hand, lactate is mainly produced by lactic acid bacteria and bifidobacteria from carbohydrate fermentation [120–122]. Meanwhile, in diets low in fiber, the gut microbiome shifts from SCFA to BCFA production, an adaptive mechanism to yield energy [114]. Clostridium, Propionibacterium, Streptococcus, and Bacteroides are the most common producers of BCFAs [61,62].

In recent years, many studies have demonstrated the role of SCFAs, especially butyrate, propionate, and acetate, in maintaining swine gut health [46]. Butyrate, the major energy source for colonocytes, has been extensively studied because of its anti-inflammatory ability and its effects on energy metabolism and homeostasis [44,45,123]. Butyrate also enhances enterocyte proliferation and reduces apoptosis [46]. Zhong et al. reported that enrichment of Lactobacillaceae and Ruminococcaceae also elevates butyrate concentrations, which in turn regulates gut homeostasis by stimulating cell proliferation and suppressing pro-inflammatory cytokines [47]. In addition, Han et al. demonstrated that butyrate reduces pro-inflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)α, IL-8, and IL-12 and protects colon morphology against lipopolysaccharide (LPS)-induced colitis in weaning pigs [48]. Butyrate supplementation also alleviates diarrhea and upregulates tight-junction (TJ) proteins, such as claudin-3, occludin, and ZO-1, in weaning pigs [49]. Meanwhile, propionate has been reported to decrease serum and liver triglyceride levels and improve lipid metabolism in growing pigs [124]. Similar to butyrate, Zhang et al. reported that propionate increased jejunal expression of TJ proteins claudin-1, claudin-4, and occludin in pigs [50]. Propionate also promotes regulatory T cell (Treg) proliferation and regulates their function, resulting in colonic homeostasis [51]. Tregs play a crucial role in suppressing the immune response and their differentiation and potentiation are highly influenced by SCFA activation of G-protein-coupled receptor 43 (GPR43) [125–127]. Acetate, on the other hand, reduces inflammation by suppressing activation of inflammasomes via GPR43 [52]. The beneficial effects of SCFAs on intestinal health may also improve growth performance in pigs. In separate studies, Reyer et al. and McCormack et al. reported that pigs with high feed efficiency ([FE], leaner pigs) are associated with a higher abundance of SCFA-producing microbiota, such as Christensenellaceae, Oscillibacter, Cellulosilyticum, Rothia, Subdoligranulum, and Leeia [53,54]. Infusion of SCFAs in growing pigs resulted in higher feed intake and daily gain, as well as improved carcass and meat quality [55]. Furthermore, Gardiner et al. posited that microbiome-derived SCFAs promote insulin sensitivity and satiety in pigs, through GPR-activated stimulation of glucagon-like peptide-1 (GLP-1) and peptide YY secretion resulting in lower feed intake [6]. Meanwhile, lactate produced by lactic acid bacteria and Bifidobacteria helps to reduce the pH of the GIT, preventing the growth of potentially pathogenic bacteria, such as enterotoxigenic Escherichia coli [120].

BCFAs are produced through the deamination of the branched-chain amino acids valine, leucine, and isoleucine [60,62]. The BCFA concentration serves as a marker for protein fermentation, although it is also affected by the level of dietary carbohydrates [61,62,87,128]. In pigs fed with low dietary fiber, Heo et al. observed a decrease in the levels of BCFAs [129], while the addition of resistant starch also decreased BCFA levels [130]. Similar to butyrate, BCFAs can also be utilized by colonocytes as an energy source, although they are not preferred sources [56]. In previous studies, BCFAs were thought to have a protective function against inflammation in the premature intestine [57,58]. Moreover, BCFAs show a dose-dependent ameliorating effect against the pro-inflammatory cytokines TNFα and interferon (IFN)γ in pigs [59]. However, BCFAs are often associated with other byproducts of protein fermentation, such as ammonia and hydrogen sulfide, and are thus considered potentially harmful metabolites [60,131,132]. BCFAs can impair gut barrier function and enhance the expression of pro-inflammatory cytokines [60]. Nevertheless, additional evidence is required to determine the exact role of BCFAs in intestinal health.

Bile acids are hydroxylated amphipathic steroid acids produced from the liver via cholesterol metabolism [71,73]. In the normal state, bile acid plays an integral role in digestion, lipid absorption, and cholesterol and fat-soluble vitamins uptake [133]. Primary bile acids are produced from the conjugation of bile acids produced in the liver with taurine or glycine (also referred to as conjugated bile acids) [71,133]. The majority (95%) of primary bile acids are reabsorbed in the distal ileum via enterohepatic circulation. However, a small portion of these primary bile acids escapes, flows into the colon, and is metabolized by the microbiome into secondary bile acids [71,73,133]. Gut resident microbiota, such as Lactobacillus, Clostridium, Bacteroides, Bifidobacterium and Enterococcus can deconjugate taurine or glycine from the conjugated bile acids via bile salt hydrolase (BSH) enzyme to produce free primary acids in the colon [74,134]. These primary bile acids are then transformed into secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid (LCA) via 7α-hydroxylation. Members of the Clostridium genus, such as Clostridium hiranonis, C. hylemonae, C. sordellii, and C. scindens, and genus Eubacterium are known to possess the bile acid-inducible (bai) operon, which is responsible for the 7α-hydroxylation activity of these genera [71–74]. There is a bidirectional interaction between bile acids and the gut microbiome – the gut microbiome metabolizing primary bile acids to produce secondary bile acids effectively alters the bile acid pool, while the secondary bile acids regulate the gut microbiome composition through their antimicrobial and cytotoxic activities [28,74,134,135].

According to recent studies, both primary and secondary bile acids interact with nuclear hormone receptors, specifically the Farnesoid X receptor (FXR) or G protein-coupled receptor (TGR5) [65,71]. This interaction is highly dependent on the affinity of each bile acid for the receptors [71]. Bile acid activation of TGR5 plays specific roles in glucose homeostasis (through the production of GLP-1), energy homeostasis, and inhibition of proinflammatory cytokines, whereas FXR activation contributes to the regulation of bile acid synthesis and lipid metabolism [65–67]. However, in dysbiosis, the normal interactions between bile acids and the gut microbiome are altered such that bile acids, especially secondary bile acids, can negatively impact swine intestinal health. Previous in vitro studies in porcine cells have demonstrated that excessive secondary bile acids, particularly LCA and DCA, decreased cell proliferation and gene expression of TJ proteins [68,69]. Moreover, Lin et al. showed that LCA downregulated the gene expression of catalase and superoxide dismutase, which are integral for relieving oxidative stress [69]. Although in vivo experiments focusing on swine bile acid profiles and microbiomes are limited, the results from these studies echo what has been observed in vitro. During a state of undernutrition in piglets, an increase in secondary bile acid concentration was observed, together with a decreased relative abundance of Lactobacillus [68]. Similarly, hyodeoxycholic acid (HDCA) feeding downregulated the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1, markers of epithelial cell proliferation, in the ileum of weaning pigs, while enriching the population of known secondary bile acid producers such as Parabacteroides, Clostridia family XII TCG-001, and Lachnospiraceae [70]. These results suggested that bile acid metabolism in the gut microbiome is a potential target for improving swine health. However, the current data are still limited and thus require further investigation.

Polyamines are low-molecular-weight polycationic molecules produced by the gut microbiome through decarboxylation of aromatic or cationic amino acids [76,136]. Polyamines are important microbiome-derived metabolites, which play crucial roles in gene regulation, signal transduction, DNA and protein synthesis, protection against oxidative stress, and cell proliferation and differentiation [76,77,81,136]. Host colonocytes can produce polyamines (unlike SCFAs); however, the gut microbiome can also significantly contribute to the polyamine profile [87,88]. Putrescine, spermine, and spermidine are the main polyamines produced by the gut microbiome, whereas cadaverine, histamine, tyramine, and 5-aminovalerate are also produced in smaller amounts [77]. In the gut, numerous bacterial groups can metabolize amino acids into polyamines, including Clostridium XIVa group, Roseburia, and Ruminococcus [75]. Bacteroides and Fusobacterium also contribute to polyamine production in the large intestine [76,77]. Enterococcus, Streptococcus, Clostridium, Lactococcus, and Lactobacillus use anaerobic pathways to produce polyamines [77]. Bifidobacterium also has the capacity to produce polyamines [78]. Since these bacteria do not always have a complete set of enzymes to produce polyamines, the colonic microbiome is thought to work collectively to produce polyamines [138]. The polyamine concentration in pig intestines is highly influenced by the dietary protein levels [34,128,139]. Yu et al. reported that extremely low dietary protein levels result in lower tryptamine, cadaverine, and putrescine levels in pigs [140]. Similarly, Chen et al. demonstrated that a reduction in dietary protein from 18% to 12% reduces both ileal and colonic polyamine concentrations in growing pigs [141]. In contrast, a low protein diet enhances polyamine production. For instance, in a low-protein diet with antibiotic treatment, researchers have observed elevated polyamine concentrations in pigs [142,143]. In addition, Peng et al. reported that reduction of dietary crude protein to 15.3% increased total polyamines, especially cadaverine, but decreased upon further dietary protein reduction [144]. These contradictory results may be due to the presence of the amino acid lysine (precursor of cadaverine) even in the low-protein diet, as most researchers have noted in their respective reports.

To date, multiple studies have demonstrated the beneficial effects of polyamines on intestinal health in pigs. Polyamines (mainly putrescine, spermidine, and spermine) regulate swine gestation—they promote placental development, stimulate blastocyst formation, and inhibit apoptosis, potentially improving the survival of the fetus [80,81,145]. Polyamines are also crucial in the growth and maturation of the small intestine in early weaned piglets, as demonstrated by a series of studies [79,82–84]. Fang et al. reported that spermine enhances oxidative stress resistance in piglets [82]. Piglets fed spermine and spermidine-supplemented feed showed increased growth compared to those fed a normal diet, as reported by van Wettere et al. [83]. Lui et al. demonstrated that putrescine alleviates diarrhea and suppresses the expression of TNF-α, IL-6, and IL-8 in weaning piglets [146]. In contrast, Ewtushik et al. observed that polyamines caused detrimental damage to the intestinal morphology of early weaned piglets, which may be due to the concentrations and ratios of polyamines used in the experiment [85].

In addition to BCFAs and polyamines, microbial metabolism of amino acids also produces indolic and phenolic compounds [56,87,147]. These metabolites are produced by fermentation of tryptophan, tyrosine, and phenylalanine. Indole and indoleacetate are produced from the microbial fermentation of tryptophan, whereas tyrosine fermentation yields phenol, p-cresol, and 4-ethylphenol [87,88,94,147]. While phenylalanine fermentation produces phenylacetate and phenylpropionate [94]. High concentrations of indolic and phenolic compounds are produced in the distal colon, where the pH is near neutral and is favorable for the fermentation process to occur [56,87,147]. Amino acid fermentation is highly affected by dietary protein content; as such, elevated concentrations of indole and phenol have been observed in pigs fed a high-protein diet [56,62,132]. Anaerobic bacteria in the colon, such as Bacteroides, Clostridium, Peptostreptococcus, Bifidobacterium, Lactobacillus, and Escherichia are known to be involved in amino acid fermentation [86–88]. For instance, Clostridium sporogenes and Clostridium botulinum can produce indolepropionic acid through tryptophan metabolism [89]. Similarly, Peptostreptococcus contains phenyllactic acid dehydratase, which is responsible for the production of indole-3-acrylate [90]. Lactobacillus produces indole-3-aldehyde and indoleacetic acid from tryptophan [86]. Bifidobacterium can ferment tryptophan into indolelactic acid. Indolic compounds produced by the microbial fermentation of amino acids serve important functions in maintaining colonic health. Indolic compounds are important in stimulating the gut mucosal barrier through the activation of aryl hydrocarbon receptors (AhR) in intestinal epithelial cells [91,92] and upregulating the expression of TJ proteins such as claudin. Indole and its derivatives, such as indolepropionic acid, also reduce inflammation by activating the pregnane X receptor (PXR) [92,148]. In contrast, phenol and its derivatives exert deleterious effects on intestinal homeostasis. Phenol increases gut permeability, whereas p-cresol disrupts colonic cell respiration and ATP synthesis [60,93,94].

Among aromatic amino acids, tryptophan metabolism is perhaps the most studied in relation to pigs because the by-products of tryptophan fermentation, such as indolepropionic acid and indoleacetic acid, confer beneficial health effects [148]. In weaned piglets, increased colonic levels of microbiome-produced indole and indoleacetic acid coincided with the upregulation of AhR and increased TJ proteins ZO-1 and occludin [95]. Liang et al. reported that L-tryptophan supplementation improved the intestinal mucosal barrier of weaned pigs by upregulating ZO-1, ZO-3, and claudin-1, which were linked to the increased population of tryptophan-fermenting commensal bacteria Clostridium and Lactobacillus [150]. Indole and its derivatives also attenuate oxidative stress in diquat-treated pigs, as reported by Fu et al. [96]. On the other hand, skatole, like indole, is also a product of microbial fermentation of tryptophan and is associated with the occurrence of undesirable odor and meat taste called ‘boar-taint’ in barrows [97,98]. In their review, Wesoly and Weiler identified Clostridium and Bacteroides as the main commensal bacteria responsible for skatole formation [97]. Pieper et al. reported that skatole, together with tyrosine-derived products phenol and p-cresol, was elevated by high-protein diets [99]. They also noted that p-cresol negatively affected the growth performance of pigs [99].

Ammonia and hydrogen sulfide are also produced during the microbial fermentation of amino acids in the distal colon [87,103]. Bacterial production of ammonia involves either deamination of amino acids or hydrolysis of urea through ureases [56,88,132]. Several studies have focused on the role of Helicobacter pylori and H. suis in the production of ammonia during glutamine deamination in both humans and pigs [100,101]. Ammonia is considered a toxic fermentation by-product; thus, it is converted back to urea via enterohepatic circulation and excreted in urine [132]. In pigs, as well as in other hosts, a high-protein diet increases the production of ammonia; conversely, decreasing the protein or including fiber in the diet reduces ammonia production [56,103,151,152]. In a high-protein diet, where ammonia production is elevated, ammonia inhibits mitochondrial oxygen consumption and SCFA oxidation [87]. In addition, ammonia impedes the intestinal uptake of butyrate by downregulating monocarboxylate transporter 1 (a transporter protein) [56], which may explain the impaired protective effect of butyrate during increased ammonia levels [60]. However, the deleterious effect of ammonia is dose-dependent and usually requires higher concentrations to cause negative effects, as pointed out by Gilbert et al. [56]. Fermentation of methionine and cysteine, on the other hand, produces hydrogen sulfide [87,88,102]. Fusobacterium, Desulfovibrio, Escherichia, Salmonella, Clostridium, and Enterobacter have been reported to produce hydrogen sulfide from sulfur-containing amino acids [88,102]. These bacteria possess desulfhydrases that utilize these amino acids for energy [87]. Like ammonia, the effect of hydrogen sulfide on the host depends on its concentration. Hydrogen sulfide acts as an energy substrate for colonocytes and a signaling molecule at low micromolar levels; in contrast, at low millimolar levels, it inhibits cytochrome oxidase activity in the mitochondria [102,103]. At high concentrations, hydrogen sulfide can also hinder cell proliferation and induce intestinal inflammation [102]. The effects of gut microbiome-produced ammonia and hydrogen sulfide on the growth performance and intestinal health of pigs are not yet fully understood and require further investigation.

Neurotransmitters are important molecules in the central nervous system as they carry signals that control behavior and cognition [17]. These molecules can be either excitatory (glutamate, dopamine, and acetylcholine) or inhibitory (γ-aminobutyric acid [GABA], norepinephrine, and serotonin). In pigs, environmental stress factors (such as handling, housing, and weaning) have been known to affect the levels of neurotransmitters [153–155]. The gut microbiome regulates the gut–brain axis by producing these neurotransmitters or their precursors [17,25,26,104]. Some gut microorganisms, such as E. coli, Pseudomonas, and other known pathogens, also utilize these neuroactive metabolites as energy sources [104,110]. Strandwitz summarized most neurotransmitter-producing bacteria, such as Bifidobacterium and Lactobacillus (GABA), Bacillus and Escherichia (dopamine), Escherichia, Klebsiella, and Lactobacillus (serotonin) [104]. Biosynthesis of serotonin (5-hydroxytryptamine [5-HT]) by enterochromaffin cells is highly regulated by gut microbiome, specifically spore-forming bacteria [156,157]. Lactic acid bacteria are also known to produce trace amines (neuromodulating metabolites), including tyramine and tryptamine [17,26]. Notably, microbiome-produced neurotransmitters cannot cross the blood–brain barrier; thus, they act locally on the enteric nervous system or vagus nerves (in the case of GABA) or enteric dopaminergic neurons (in the case of dopamine) [17,104,158]. On the other hand, precursors of these neurotransmitters can cross the blood–brain barrier and influence the biosynthesis of neurotransmitters in the brain. The SCFA acetate traverses the blood–brain barrier into the hypothalamus and affects the biosynthesis of glutamate and GABA in the host [17]. Gut microbiome dysbiosis can lead to altered microbial production of neurotransmitters and their precursors, thus affecting behavior and cognition in pigs. In antibiotic-treated pigs, modulation of colonic microbiome leads to a significant reduction in neurotransmitters [159]. There is also significant evidence that altered gut microbiome and tryptophan-serotonin metabolism are associated with tail biting in pigs [105]. In contrast, dietary supplementation with tryptophan has been shown to alter the bacterial production of serotonin [106,107]. These studies emphasize the role of gut microbiome and diet in the biosynthesis of neurotransmitter chemicals, which ultimately affects neurological function in pigs.

Similar to the human gut microbiome, the pig gut microbiome can also synthesize vitamins [6,159,160]. Members of the genus Bacteroides are the main producers of water-soluble vitamin B, including riboflavin, thiamine, biotin, cobalamin, and folates [160,161]. Some species of Lactobacillus also have the capacity to de novo synthesize vitamin B [108,109]. B vitamins serve as coenzymes in neurological processes [110]. Vitamin K, an essential vitamin for proper blood clotting, is produced by the gut microbiome as well [108,110]. These gut microbiome-derived vitamins support the functional metabolism not only of the hosts, but also of other non-vitamin producing bacteria, such as Faecalibacterium, thereby extending their influence on intestinal health [161,162]. Our knowledge of the extent of the effects of gut microbiome-synthesized vitamins on pigs is still scarce and must be investigated in the future.