The heat-killed microbial cells had not been included into postbiotics until 2021, when the International Scientific Association of Probiotics and Prebiotics (ISAPP) incorporated the deliberately inanimate microbial cells, mostly heat-killed probiotic bacteria cells, into the definition of postbiotics (2, 39–41). To address the biological role of microbial cells, microorganisms were further inanimate by various methods like heating, chemical (e.g., formalin), gamma or ultraviolet rays, and sonication treatments (42–44). Lactobacillus and Bifidobacterium species, the mostly studied probiotics, were found to have beneficial functions in heat-killed formations (45–51). For instance, heat-killed Lactobacillus salivarius subsp. salicinius AP-32, L. rhamnosus CT-53, L. paracasei ET-66 could significantly inhibit the invasion of oral pathogens to improve oral health (45). Besides, heat-killed Lactobacillus and Bifidobacterium species are found to exhibit immunomodulatory effect. Heat-killed Bifidobacterium breve M-16V cells suppressed proinflammatory cytokine production in spleen cells and affected intestinal metabolism (46). Oral administration of heat-killed L. plantarum L-137 could enhance protection against IFV infection together with increased IFN-β production in the serum of infected mice at an early stage of infection (47). Lactobacillus rhamnosus GG (LGG) could ameliorate intestinal inflammation, and promote cytoprotective responses in the developing murine gut, thus playing a role in inflammatory bowel disease treatment (16, 48, 49). Li et al. found the heat-killed LGG could also decrease lipopolysaccharide (LPS)-induced proinflammatory mediators and increase anti-inflammatory mediators (50). Ueno et al. proved that administration of heat-killed Lactobacillus brevis SBC8803 could successfully maintain intestinal homeostasis and cure intestinal inflammation, which manifesting the potential for CRC treatment (51). Recently, Akkermansia muciniphila was found to be beneficial in the prevention of various diseases through maintaining the integrity of gut barrier (52). Implantation of live Akkermansia muciniphila exerted protective role against liver injury, colitis and CRC as well (53–55). Interestingly, Wang et al. showed that the pasteurized bacterium of Akkermansia muciniphila strikingly inhibit colitis associated tumorigenesis in mice, indicating the beneficial role of Akkermansia muciniphila derived components against CRC progression (56). Meanwhile, Kim et al. reported that heat-killed B. bifidum MG731, L. reuteri MG5346, and L. casei MG4584 significantly delayed tumor growth in colorectal carcinoma, which give direct evidence of heat-killed bacterial cells for CRC (57).

Cell-free supernatant refers to the biologically active metabolites secreted by microorganisms. Here we mainly focus on the cell-free supernatant of bacteria, which are prepared after the bacterial cells were incubated, centrifuged, and removed (34). CFS has been reported to show antimicrobial, antioxidant, and antitumor activity (16, 58–63).

The antimicrobial activity refers to both antibacterial and antifungal activity (58, 59). The Saccharomyces cerevisiae CFS showed anti-biofilm activity against Staphylococcus aureus, which is the widely reported pathogen in both food and clinical environment (58, 64). The anti-biofilm property of S. cerevisiae CFS can be thus utilized to reduce the risk of infection by S. aureus in human (65). In addition, the cell-free supernatants derived from Lactobacillus paracasei subsp. Paracasei SM20 and Propionibacterium jensenii SM11 coculture all showed high antifungal activity against Candida pulcherrim and Rhodotorula mucilaginosa (59). Interestingly, CFS of B. animalis subsp. Lactis DSMZ 23032, L. acidophilus DSMZ 23033, L. rhamnosus SD11, and Lactobacillus brevis DSMZ 23034 exhibited strong antioxidant activity through DPPH radical scavenging and oxidative stress hypersensitivity in vivo (60). Pourramezan et al. found that there was a relationship between the antibacterial activity and antioxidant activity of CFS of Lactobacillus (61).

For Lactobacillus and Bifidobacterium species, their CFS were found to inhibit cancer progression (62). For example, L. casei and LGG cultures could prevent the invasion of colon cancer cells (62). In recent years, Lactobacillus cell-free supernatant (LCFS) has attracted more and more attention to its potential benefits on CRC prevention (63). The anti-cancer effects of L. fermentum CFS has been tested which proved that it could induce apoptotic cell death in three dimensional (3D) spheroids of colorectal cancer cells in vitro (63). Besides, the mRNA levels of apoptosis markers were dramatically induced after treating with LCFS in 3D system (63). Pahumunto et al. showed that CFS of Lacticaseibacillus paracasei SD1, Lacticaseibacillus rhamnosus SD4, Lacticaseibacillus rhamnosus SD11, and Lacticaseibacillus rhamnosus GG could inhibit pro-inflammatory cytokine expression in Caco-2 cells and inhibit the growth of Caco-2 cells, supporting its role in preventing CRC (16).

According to the more recent definition of postbiotics by the ISSAP, cell components can be regarded as another major type of postbiotics. Two categories of cellular components were intensively investigated by far. Teichoic acids (TAs) are major constituent of bacterial cell walls, including both wall teichoic acids (WTAs) covalently linked to peptidoglycan and lipoteichoic acids (LTAs) anchored to the cytoplasmic membrane (66, 67). WTA was reported to be effective in modulating bacterial colonization (29), while LTA was discovered to exert immune-modulatory activity by recognizing Toll-like receptors (68). LTA could interact with TLR2 and TLR6 to active host immune response (69). In particular, LTA of Lactobacillus paracasei D3-5 could enhance mucin (Muc2) expression by modulating TLR-2/p38-MAPK/NF-kB pathway, which in turn reduces inflammation (70). In addition, LTA of Lactobacillus plantarum inhibited the phosphorylation of ERK and p38 kinase as well as the activation of NF-κB, resulting in decreased IL-8 production and inflammation suppression in porcine intestinal epithelial cells (71).

Peptidoglycan (PGN) is the conserved structure of Gram-negative bacterial which could be recognized by nucleotide-binding oligomerization domain-1 (NOD1) (72). Once activated by PGN, NOD1 could further activate the downstream NF-κB (72) and MAPK (73) pathway through interacting with a caspase activated and recruitment domain (CARD) and its adaptor protein, the receptor-interacting protein 2 (RIP2) (74–76). Besides, the PGN/Nod1/RIP2 pathway could induce autophagy and inflammatory signaling in response to bacterial infection (77). PGN of the Lactobacillus paracasei subsp. paracasei M5 strain exerted cytotoxic effects on HT-29 cells and induced a mitochondria-mediated apoptosis in colon cancer (78).

Exopolysaccharides (EPS) is a subclass of polysaccharides that constitute the cell wall structure of bacteria and is characterized by less attachment to the cell surface compared with capsular polysaccharides (79). EPS can be structurally divided into two categories: homopolysaccharide (HoPS) and heteropolysaccharide (HePS). HoPS was composed of a single type of monosaccharides, such as D-glucose or D-fructose. Crosslinking of D-glucose through α- or β-glycosidic linkage resulted in α- or β- glucans, respectively, while connection of D-fructose through β-glycosidic linkage forms β-fructans (80). In contrast, HePS are composed of different types of monosaccharides, such as D-glucose, D-galactose, and L-Rhamnose (80). Besides, N-acetylation is also found to be presented on HePS, which further diversified their molecular structures (81).

Bacterial EPS has been evaluated as industrially important biopolymers with significant commercial potential (82). The effects of EPS on human health (and in particular, CRC) have also been recognized (80). EPS can be synthesized by a wide range of bacterial species such as Bifidobacteria sp. and Lactobacillus sp., which benefits the gut homeostasis (83). The protective role of EPS in CRC has been demonstrated previously. The EPS extracted from Lactobacillus acidophilus was found to inhibit the growth of Caco-2 cells in a dose-dependent manner (84). Similarly, Lactobacillus-derived EPS was found to suppress HT-29 cell growth by inducing the G0/G1 cell cycle arrest and apoptosis (85). In line with that, EPS produced by microalgae Chlorella pyrenoidosa, Scenedesmus sp., and Chlorococcum sp. was found to induce anti-tumor effect on both HCT116 and HCT8 cells (86), similar to EPS produced from Chlorella zofingiensis and Chlorella vulgaris (87). Taken together, these data suggest a universal protective effect of EPS against CRC independent of its sources. Nevertheless, the in vivo activity of EPS in CRC protection has not been fully clarified yet. It was revealed that EPS from Lactobacillus rhamnosus is critical in providing the “shield” for bacteria itself against the host immune defense, which is beneficial for the maintenance of gut microbiota homeostasis (88). Apart from that, EPS from Bifidobacterium longum subsp. Longum significantly alleviated DSS-induced colitis by maintaining the integrity of gut mucosal barrier (89), which was supported by the significantly altered level of EPS from Rhizopus nigricans in CRC mouse model (90). Taken together, these studies support the beneficial role of EPS against CRC.

Short chain fatty acids (SCFAs) are a subset of saturated fatty acids with six or less carbon molecules including acetate, propionate, butyrate, pentatonic (valeric) acid, and hexanoic (caproic) acid (91). SCFAs are the main metabolic products of indigestible saccharides by anaerobic bacterial fermentation in human gut (91). Deficiency of SCFAs production can lead to the pathogenesis of many human diseases, such as autoimmune syndromes from allergies to asthma, metabolic diseases, neurological diseases, and cancers (92).

The protective effects of SCFAs against colorectal cancer were first discovered in the early 1980s. Whitehead et al. found that butyrate stimulation significantly inhibited the proliferation rate of human colorectal cancer cell line LIM1215 (93), accompanied by a similar finding that both propionate and butyrate suppressed the growth of human colorectal cancer HT-29 cells (94). In an independent study, it was shown that SCFA treatment, either alone or in combination, significantly increased the proliferation of colon crypts, suggesting a symbiotic effect on colorectal health, which eventually prevents the formation or progression of colorectal cancer (95). The anti-cancer effect of SCFAs is also supported by epidemiological studies revealing an inverse correlation between the level of dietary fibers and the incidence of human colorectal cancer (96). In line with this, the proportions of major SCFAs in enema samples of patients with colon polyps or colorectal cancer were strikingly different from healthy subjects, indicating the niche-dependent activity of SCFAs in response to CRC (97). Moreover, Gibson et al. investigated the fermentative production of butyrate in vivo as well as its protective effect against colorectal cancer in a rat model (98). It was found that the intake of natural fiber was associated with high concentration of butyrate in colon tissue and reduction of colorectal tumor formation, which highlighted the protective effect of butyrate in colorectal cancer. Propionate and valerate are also members of SCFAs effective in arresting cell growth or suppressing differentiation of human colon carcinoma cells (99). Although the in vivo activity of SCFAs in the context of CRC is still under investigation, the anti-inflammatory effects of SCFAs have been well-described and are essential for the inhibition of colitis and inflammatory bowel disease (IBD), which are both considered as the major risks for the development of CRC (100, 101). Of note, recent studies uncovered the physiological role of SCFA in preventing colitis-induced CRC in mice, as administration of SCFA mix strongly suppressed tumor burden in mice treated with Azoxymethane (AOM)/dextran sodium sulfate (DSS) which were commonly used for the induction of colitis-associated colorectal cancer (100). Colon inflammation as well as pathological score index was also reduced, consistent with the decreased secretion of pro-inflammatory cytokines including interlukin-17 (IL-17), IL-6, and tumor necrosis factor-α (TNF-α) (100, 102). Overall, these results highlighted the clinical potential of SCFAs in protection against CRC.

Enzymes are recognized as key regulators driving the metabolism of all organisms, with no exception for gut microbiome (103). In fact, enzymes encoded by microbes play essential roles in host-microbe interactions (103). Early studies on the epidemiology of CRC revealed that there was a negative correlation between the abundance of lactic acid-producing bacteria Bifidobacterium in human gut and the incidence of colorectal cancer (104). Lactic acid bacteria (LAB) are active in fermentation via synthesizing various enzymes, and the protective effects on colorectal cancer by dietary supplementation of different lactic acid-producing bacterial species have been well-established in murine studies (105–108). Therefore, the potential role of bacterial enzymes in inhibiting CRC development has also been indicated. In fact, it was shown that administration of Bifidobacterium longum and Lactobacillus acidophilus generated better outcome of AOM-induced colorectal cancer in rats, and was accompanied by increased β-glucuronidase and nitro-reductase activity in fecal samples (109). It was also demonstrated that the inhibitory effect of Bifidobacterium longum on colon carcinogenesis was induced by 2-Amino-3-methylimidazo [4,5-f] quinoline (IQ), a food mutagen (106). Consistently, supplementation of coconut cake was found to remarkably reduce the incidence of 1,2-dimethyl hydrazine (DMH)-induced colorectal cancer occurrence in a rat model, which was associated with the upregulation of β-glucuronidase and mucinase (107). In addition, oral administration of a catalase-producing Lactococcus lactis can prevent DMH-induced colorectal cancer in mice (108).

Other types of metabolites produced by gut microbes are also involved in regulating CRC progression, which include the microbially modified secondary bile acids (secondary BAs). Primary bile acids (primary BAs) are cholesterol derivatives that synthesized in the liver and then secreted into intestine for lipid absorption. Once utilized, most bile acids are recycled to liver via enterohepatic or systemic circulation. Meanwhile, a proportion of primary BAs is transported to colon where they are metabolized by gut microbiome to produce secondary BAs. The detailed signal transduction pathways of BA metabolism have been comprehensively reviewed (110). The relationship of diet, BAs, gut microbiota and CRC has been revealed in recent studies (110, 111). It was reported that the consumption of “unhealthy” western diet is associated with the increase of secondary BAs, especially deoxycholic acid (DCA) and lithocholic acid (LCA) in the gut, while the increased level of bile acids is tightly correlated with high CRC incidence (112–115). Therefore, secondary BAs are primarily considered as carcinogens in human gastrointestinal cancers (116). In addition, high-fat diets promote the synthesis and colonic delivery of bile acids, which stimulate the growth and activity of 7α-dehydroxylating bacteria to convert the primary BAs into secondary BAs (117). Animal studies also showed the effects of high fact diet in modulating the composition and activity of gut microbiota, which lead to increased BA secretion and colon tumorigenesis (118–120). Diet intervention has been implicated to be critical in preventing CRC through the regulation of BA levels in gut (121). These results indicate the biological role of gut microbial secreted BA in regulating the development of CRC. On the other side, ursodeoxycholic acid (UDCA), another member of secondary BAs (122), was effective in inducing apoptosis of colon cells in vitro (123), which is distinct from DCA (124). Consistently, another study uncovered the role of UDCA in inhibiting the proliferation of colon cancer cells by regulating oxidative stress and cancer stem-like cell growth (125). Mechanistically, it was revealed that UDCA suppressed the malignant progression of colorectal cancer by inhibiting the activation of Hippo/YAP pathway (126). Animal studies demonstrated the protective role of UDCA in chemical induced colon tumorigenesis (127, 128). According to a cross-sectional study, by analyzing the relationship between ursodiol use and colonic dysplasia (the precursor to colon cancer) in patients with ulcerative colitis and primary sclerosing cholangitis, it was revealed that the application of UCDA is associated with lower prevalence of colonic neoplasia (129). Although UDCA has been approved for the treatment of primary biliary cirrhosis by FDA (130), the mechanism of action of UCDA, the relationship of UCDA with gut microbiota, as well as the clinical use of UCDA in CRC treatment remains undetermined and therefore warrants further investigation.

Tryptophan is one of the essential amino acids supplied by the intake of dietary proteins (131). Despite the absorption of the majority of ingested proteins in small intestine, a small fraction of amino acids can reach colon where they can be absorbed by local microbes (132). Tryptophan metabolism is complicated and dependent on two different pathways: the kynurenine (Kyn) pathway and the indolic pathway (133). The significance of microbial tryptophan metabolites in human health has been investigated (134). In the context of CRC, it has been shown that Tryptophan metabolites derived from gut microbes regulate the homeostasis of gut immunity. The deficiency of Tryptophan secretion due to gut dysbiosis may cause exacerbated inflammatory response which eventually led to colorectal tumorigenesis (135). In fact, alteration of fecal tryptophan metabolism was correlated with shifted microbiota, which may be involved in the pathogenesis of colorectal cancer (136).

Bacteriocins are defined as ribosomally synthesized antimicrobial proteins or peptides. It could be divided into two categories: class I bacteriocins are modified by post-translational modifications; class II bacteriocins remain unaltered (137, 138). Given the fact that bacteriocins have antimicrobial activity, bacteriocins targeting CRC associated bacterial pathogens may have potential benefits against CRC. For example, Streptococcus salivarius DPC6993 which could secrete salivaricin against F. nucleatum in an ex vivo model of human colon, was shown to be effective in reducing the risk of CRC (139). Probiotics-derived bacteriocins, including plantaricin JLA-9, plantaricin W, lactococcin A, and lactococcin MMFII directly interact with CRC-promoting protein COX2 and modulate inflammasome complex NLRP3 and NF-kB pathways to reduce CRC-associated inflammation. They also have the potential of activating autophagy and apoptosis by regulating PI3K/AKT and caspase pathways in CRC (140).

In addition to the above-mentioned molecules, several other types of gut microbial derived molecules were also beneficial to human health. For instance, vitamins secreted by gut microbes, including B-group vitamins and vitamin K, were shown to be effective in preventing CRC (141, 142). Neurotransmitter substances synthesized by gut microbes, such as γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NE), brain-derived neurotrophic factor (BDNF), and acetyl choline (29), were critical in regulating gut-brain axis (143, 144). However, their roles in CRC still awaits further investigation. In conclusion, a variety of probiotic bacteria-derived molecules have been identified, and the potential of these bacterial derived “postbiotics” in preventing/treating CRC have also been highlighted.

Yeast is one kind of highly adaptable microorganisms with proved benefits to human health. Yeast cell wall, particularly the mannan-oligosaccharides, were reported to be able to bind to fimbriated bacteria in the gut and prevent their attachment to gut mucosa which eventually prevents the proliferation and colonization of those bacterial pathogens (145). Furthermore, mannan-oligosaccharides (MOS) in the YCW have a strong binding affinity with type I-fimbriae in the gut (146, 147). The probiotic yeast S. boulardii, widely incorporated in dairy food, grains, fruit juices, chocolate, coffee, and tea, was demonstrated to ameliorate diarrheal conditions. Cell wall extracts of S. cerevisiae and S. boulardii could adsorb aflatoxin B1 (148), cholera toxin (149), and pathogenic E. coli (150) which are possibly by cell wall polysaccharides such as β-glucan, mannoprotein and chitin (148, 151). Furthermore, by adsorbing these, the cell wall extracts of S. boulardii could enhance immune responses in the intestinal mucosa (152). As for the relationship between YCW and CRC, the β-glucan of S. boulardii cell wall extract has been proven to prevent colon carcinogenesis in vitro (151). In addition, EPS was also found in probiotic yeast species like Kluyveromyces marxianus and Pichia kudriavzevii, indicating the complicated natural source of EPS (153). Similarly, EPS derived from yeast species Kluyveromyces marxianus and Pichia kudriavzevii showed a comparable anti-colorectal cancer activity (153). Collectively, these results highlight the potential of YCW as postbiotics against CRC.

The anaerobic gut fungal commensals (such as Anaeromyces robustus, Caecomyces churrovis, Neocallimastix californiae, and Piromyces finnis) are reported to encode diverse biosynthetic enzymes for natural products and antimicrobial peptides (AMP), including polyketide synthases (PKS), non-ribosomal peptide synthases (NRPS), etc., (154). A significant proportion of fungal products were detected in the fecal samples of gnotobiotic mice, which may affect the growth and differentiation of host epithelial and immune cells (155).

A postbiotic yeast cell wall-based blend containing hydrolyzed yeast cell wall of Saccharomyces cerevisiae, organic acids (n-butyric acid), vitamins (ascorbic acid), and essential oils can partially improve the health of pigs which were treated with multiple mycotoxins including aflatoxin B1 and deoxynivalenol (156). The potential of using S. boulardii-derived postbiotics (for instance, polyamines, organic acids, enzymes, etc.) as dietary supplements has also been suggested (157). However, the benefits of non-YCW fungal products in CRC remain unclear which awaits further investigation in the future.

The current hallmarks of cancer embody eight hallmark capabilities and two enabling characteristics, include sustaining proliferative signaling, evading growth suppressors, avoiding immune destruction, enabling replicative immortality, tumor-promoting inflammation, activating invasion and metastasis, inducing or accessing vasculature, genome instability and mutation, resisting cell death and deregulating cellular metabolism (165, 166). With the growing evidence and our increasing understanding of cancer biology, emerging hallmarks and enabling characteristics are now introduced into the hallmarks of cancer, involving “unlocking phenotypic plasticity,” “non-mutational epigenetic reprogramming,” “polymorphic microbiomes,” and “senescent cells” (167). Among these, excessive cell proliferation mainly resulted from three types of mutations: mitogen or its signal transducing molecules to convey mitogen information, mutation of late-G1 cell-cycle checkpoints regulated by pRB, and uncontrolled expression of Myc (168–170). Therefore, one of the anti-cancer strategies is to block cancer cell proliferation, and that is how most postbiotics works against cancer (171). It was observed that the ratio of G0/G1 cells increased when treated with gemcitabine together with butyrate, a common postbiotic from intestinal bacteria. The percentage of cells in S-phase significantly decreased when treated with butyrate in PANC-1 cell, indicating that butyrate could inhibit the proliferation of human pancreatic cancer cells (172). Postbiotic metabolites produced by certain strains of L. plantarum exhibited selective cytotoxic activity via antiproliferation of human breast cancer cells, suggesting their potential against breast cancers (173). In addition, Lazarova et al. proposed that butyrate can induce cell cycle arrest which was dependent on the modulation of Wnt signal activity in CRC (174). When combined with chemotherapeutic agents like irinotecan, butyrate significantly inhibited cell proliferation which promoted the efficacy of chemotherapies for the treatment of colorectal cancer (175).

In addition to butyrate, varies of postbiotics derived from Lactobacillus also exhibited anti-proliferative activities on colorectal cancer cells. For instance, the cell-free culture supernatant of Lactobacillus has inhibitory effect on the proliferation of colorectal cancer cells (176). Another interesting study reported that cell free cultural supernatants of Lactobacillus conjugated with various linoleic acids significantly reduced the transcriptional level of genes required for colon cancer cell growth and proliferation, such as CDK1/2/6, PLK1, and SKP2, indicating the potential benefits of those metabolites in treating CRC (177).

Cell apoptosis is a programmed cell death with distinct morphological characteristics, including cell shrinkage, pyknosis, membrane blebbing, chromatin condensation and formation of cytoplasmic blebs (178, 179). Many diseases are related with apoptosis, such as AIDS (Acquired Immuno-Deficiency Syndrome), neurodegenerative diseases, autoimmune disorders, infectious disease, and cancer (180, 181). All somatic cells including cancer cells need to suppress apoptosis mainly by input of survival and trophic signals to survive (171). Besides, apoptosis also contributes to chemotherapy resistance (182). It’s a potential therapy to target the lesions that suppress apoptosis in tumor cells for cancer apart from inhibiting cancer cell proliferation (183–185). In recent years, postbiotics have been investigated to induce apoptosis in order to treat cancer (172, 173, 186). Postbiotic metabolites produced by six strains of L. plantarum induced apoptosis of MCF-7 breast cancer cells (173). Similarly, Kim et al. reported that heat-killed B. bifidum MG731, L. reuteri MG5346, and L. rhamnosus MG5200 induced apoptosis in human gastric cancer MKN1 cell to block tumor cell growth significantly. SCFAs, as one of the intensively investigated postbiotics, were also found to induce apoptosis in cancer treatment (186). SCFAs activated the mitochondrial apoptosis pathway and Fas death receptor apoptosis pathway in the breast cancer cell line 4T1 in vitro (186). Combining butyrate together with gemcitabine were found to decrease the percentage of live cells while increasing that of apoptotic cells in human pancreatic cancer cell lines (172). The underlying mechanism of butyrate’s anticarcinogenic effect is to modulate gene expression of key regulators in apoptosis and cell cycle such as cell cycle inhibitor p21 and proapoptotic protein BAK (99, 187–190).

Therefore, it is a potential way to harness postbiotics to treat CRC by inducing colorectal cancer cell apoptosis (183–185). The cultural supernatant of L. gallinarum significantly promoted apoptosis in CRC cell lines, HCT116 and LoVo, without impacting the cell growth of normal colonic epithelial cell line (NCM460), showing its specific beneficial potential against CRC (191). Consistently, Streptococcus thermophilus could secrete β-Galactosidase promoting CRC cells apoptosis and suppress the growth of CRC xenograft (192). Ma et al. discovered the induction of tumor apoptosis through PI3K/Akt signaling and caspase-3 activation in mice by the increasing production of beneficial metabolites including SCFAs when administrating sitosterol (193). Clostridium butyricum can suppress intestinal tumor development by modulating Wnt signaling and gut microbiota, including increasing the abundance of SCFAs-producing bacteria, especially butyrate-producing bacteria, thus suggest their potential efficacy against CRC (194). The in vitro activity of postbiotics on colorectal cancer cells is illustrated in Figure 2. On one side, postbiotics can efficiently inhibit colorectal cancer proliferation by disrupting the normal cell cycle of cancer cells; on the other side, postbiotics are effective in inducting cancer cell death through apoptotic pathways.

The research on the effect of postbiotics on gut microbiota had been emerging in the past few years. It was found that postbiotics could modulate the composition and abundance of gut microbiota by promoting the growth of probiotics in the gastrointestinal tract of both healthy people and patients with different kinds of diseases (32). Terada et al. studied the effects of the consumption of heat-killed Enterococcus faecalis EC-12 on microbiota in healthy adults, and found that the levels of bifidobacteria and lactobacilli were significantly increased, while the abundance of lecithinase-positive clostridia including Clostridium perfringens, and Enterobacteriaceae were significantly decreased (195).

Postbiotics are reported to affect the gut microbiota of individuals with GI disorders, including inflammatory bowel disease (IBD). Sassone-Corsi et al. reported that microcins produced by Escherichia coli Nissle 1917 (EcN) could mediate inter and intra-species competition among the Enterobacteriaceae in the inflamed gut (196). The two major categories of IBD are Crohn’s disease (CD) and ulcerative colitis (197). In IBD patients, the pattern of gut microbiota dysbiosis is primarily characterized by a reduction in the abundance of bacterial species within the phylum of Firmicutes and Bacteroidetes and a relative increase of bacterial species belonging to the Enterobacteriaceae family, within the Phylum of Proteobacteria (198). Sokol et al. found that Faecalibacterium prausnitzii supernatant can effectively treat the intestinal microbiota disorders caused by CD and inhibit intestinal inflammation (197). Microbially modified bile acids were correlated with bacterial growth, activation of inflammation, and gut epithelium damage, suggesting an important role as components of intestinal antimicrobial defense (199, 200). It was revealed that cholic acid (primary bile acid) treatment significantly altered the composition of gut microbiota and promoted intestinal carcinogenesis, which is tightly correlated with the transformation of cholic acid to deoxycholic acid via bacterial 7α-dehydroxylation reaction, indicating the biological role of deoxycholic acid in gut microbiota homeostasis and CRC progression (201). Additionally, UCDA, which is mentioned earlier, was shown beneficial in improving the outcome of colitis via gut miciobiota modulation (202). It was also found that postbiotics could influence behavior in a mouse model of Citrobacter-induced colitis. A postbiotic derived from Limosilactobacillus fermentum and Lactobacillus delbrueckii shortened the small intestine and increased colon crypt depth in the mouse model, showing the protective effect of postbiotics on colitis (203).

The bacteriocin preparation fermented by lactic acid bacteria (LAB), lacidophilin tablets, has been extensively studied (204). Continuous intervention of lacidophilin tablets can reduce harmful bacteria and increase beneficial bacteria and normal bacteria. Lacidophilin tablets could adjust the composition and diversity of intestinal microbiota quickly in antibiotic-associated diarrhea (AAD) mice, and reconstruct the microbial communities which were mainly beneficial bacteria (205, 206). Specifically, lacidophilin-treated mice dramatically increased the abundance of Lactobacillus and decreased that of potential pathogens, such as Bacteroides, Parabacteroides, and Parasutterella, to nearly normal level (206). The interaction between postbiotics and anxiety and depressive disorder has also been studied. It showed that ADR-159 treatment, a heat-killed fermentate generated by two Lactobacillus strains, led to subtle but distinct changes in murine gut microbiota together with increased sociability and lower baseline corticosterone levels (stress hormone), supporting a beneficial effect on anxiety disorder and depressive disorder (207).

The in vivo function of postbiotics in regulating gut microbial composition in the context of CRC has not been fully elucidated. However, accumulating evidence showing the influence of gut microbial composition on CRC progression. Kimoto-Nira et al. investigated influence of long-term consumption of a Lactococcus lactis strain G50 on the intestinal flora of the senescence-accelerated mouse, showing that G50 could suppress the intestinal growth of H₂S-producing bacteria (208). The H₂S-producing intestinal bacteria include Salmonella sp., Shigella sp., and Citrobacter sp., all of which are associated with CRC (209). Gut microbiota dysbiosis was discovered in CRC patients. Clostridium butyricum supernatant was reported to decrease the relative abundance of pathogenic bacteria, such as Desulfovibrio, Odoribacter, and Helicobacter, which are related to CRC (194). Collectively, these results highlight the potential role of postbiotics in the prevention of CRC.

The first line of host defense against both native gut commensals and external pathogens is the intestinal mucosal barrier, which consists of tightly connected epithelial cells and is protected by two host-secreted mucous layers, inner mucus layer and outer mucus layer (210, 211). The intestinal mucus layer was composed of secreted mucin glycoproteins and other substances, which were secreted by goblet cell (212). It is found that mucin can not only provide adhesion sites for intestinal symbiotic bacteria, but can also separate the pathogens from Caco-2 cells (213). However, several intestinal pathogens could secrete enzymes to disrupt the mucous barrier or promote their invasion capability to facilitate their colonization and successful infection (214). Postbiotics could modulate gut commensalism by affecting the intestinal mucosal barrier. Bacterial SCFAs and other metabolites can induce mucus biosynthesis in germ-free mice (215, 216). For example, butyrate-treated human colorectal cells LS174T cells increased mucin protein expression, which enhanced adherence of probiotics Lactobacillus and Bifidobacterium and eventually inhibited the pathogenic Escherichia coli (E. coli) attachment (217, 218). Other probiotics supernatant, such as Lactobacillus rhamnosus GG culture supernatant, have been proved to promote mucin production to help the gut colonization of symbiotic bacteria and exert a preventive effect on most gut-derived pathogenic infections including E. coli K1 (218).

Adhesion is an important step for gut commensalism. If postbiotics provide adhesins such as fimbriae and lectins, they can compete with resident microorganisms for adhesion sites (219, 220). The isolated lectin domains of Llp1 and Llp2 exhibit significant inhibitory activity against biofilm formation of various pathogens, including clinical Salmonella species and pathogenic E. coli (220). EPS are extracellular polysaccharides attached to the bacterial cell wall which can affect adhesion by shielding cell surface adhesins or acting as ligands (221). It was found that EPS produced by LAB could block the adhesion of harmful bacteria to intestinal epithelial cells by inhibiting binding of adhesion factors to bacterial cell surface (222). Besides, different kinds of EPS could also modulate LAB’s efficiency of adhesion to intestinal epithelial cells in vitro (223). In addition to EPS, it was also found that heat-treated Lactobacillus acidophilus strain LB could inhibit the adhesion of entero-invasive pathogens to human intestinal Caco-2 cells (224). Thus, postbiotics may exert benefit in treating CRC by modulating gut commensalism through intestinal barrier remodulation (225).

Postbiotics can modulate gut pathogenesis not only through competitive inhibition of receptor adhesion but also by changing the intestine barrier function or expression of specific regulatory genes. Tsilingiri et al. first confirmed the anti-inflammatory activity of the Lactobacillus paracasei supernatant (SN) after Salmonella infection of healthy tissue (226). They showed that the secretion of TNF-α, which is a proinflammatory cytokine, was increased after Salmonella infection. Proinflammatory cytokines are positive mediators of inflammation (227). But this effect was eliminated when SN was added together with Salmonella, manifesting an inflammatory potential of Salmonella (226). Mechanistically, SN can stimulate the epithelium cells resistant to Salmonella invasion rather than affecting Salmonella proliferation (226). Similarly, acetate was discovered to significantly increase resistance to enterohaemorrhagic E. coli O157:H7 infection in a mouse model (228). The could be attributed to the sealing properties of acetate on the intestinal barrier, eventually preventing lethal toxins from entering the general circulation (228). Ramakrishna et al. showed that some probiotics and their metabolites prevent pathogen invasion not only by blocking adherence sites but also by upregulating gene expression of MUC2 and antimicrobial peptides (229). Consistent with this is that Mack et al. showed that part of the beneficial effect of L. ramnosus and its supernatant was mediated by induction of mucin genes in intestinal epithelial cells (230).

The gut microbiota dysbiosis could promote the progression of CRC probably through pro-inflammatory responses (231–233). For example, the next generation sequencing analysis revealed that enrichment in Fusobacterium could promote inflammation in CRC (234). Inflammatory responses play an important role in different stages including initiation, malignant conversion, invasion, and metastasis of tumor such as CRC development (235). Some anti-inflammatory drugs like aspirin and sulindac were proved to prevent CRC and decrease side effects of inflammatory symptoms, hence bring new insights of CRC prevention and treatment (236, 237). Thus, the immunomodulatory activity of postbiotics can be harnessed in CRC prevention and treatment. For example, bile acids were reported to modulate gastrointestinal inflammation and play a role in CRC prevention (110). Niacin, which is also called nicotinic acid or vitamin B3, can be regarded as another kind of postbiotic because it could also be produced by intestinal microbes and exerts benefits to human health (238). It was reported that niacin can modulate immune responses through G protein-coupled receptor 109a (GPCR109a) to prevent colitis and colon cancer (239, 240). Apart from the anti-inflammation activity, postbiotics can also ameliorate CRC progression by inhibiting the enzymatic activity of pathogenic bacteria and reducing the amount and activity of virulence factors to attenuate gut pathogenesis (241, 242). However, certain microorganisms like Fusobacterium nucleatum, Escherichia coli NC101 and Bacteroides fragilis not only induce inflammation and ROS-mediated genotoxicity but also secrete toxins which induce DNA damage responses to promote CRC (243–245).

Gut microbiota-host interaction is critical for human health. Host inflammatory response is one of the most important signals modulating the homeostasis between gut microbiota and host. It was reported that inflammatory cell infiltration in adipose tissues, liver, and pancreatic islets is associated with an increase in LPS plasma levels, possibly derived from gut bacteria (246, 247). Moreover, the gut microbiota can regulate host inflammation by affecting cytokine secretion as well as the differentiation of inflammatory cell types, demonstrating the dynamic interplay between gut microbiota and the host immune (248, 249).

For instance, F. nucleatum promoted carcinogenesis by stimulating Wnt signaling pathway in CRC (250, 251). And the adhesion molecule FadA from F. nucleatum can bind to E cadherin, leading to activation of β-catenin and tumor development (252). Bacteroides fragilis was also known as the risk factor of CRC. Bacteroides fragilis toxins can bind to specific colonic epithelial receptor to activate Wnt and NF-κB signaling cascades, leading to an increased proinflammatory reactions including T_H17 response and induction of DNA damage response, aggravating the colorectal tumorigenesis (253, 254). All the discussed in vivo biological roles of postbiotics in preventing CRC are illustrated in Figure 3. The beneficial roles of postbiotics in vivo are achieved via the following ways: postbiotics are effective in modulating gut microbial composition and the abundance of certain microbial species, which lead to homeostasis of gut microbiota; postbiotics can also benefit gut commensalism by promoting the integrity of intestinal mucosa tissue; postbiotics can specifically inhibit the invasion of pathogenic microbes in the gut; postbiotics are involved in the interaction between gut microbiota and host cells.