Preliminary Purification and Partial Characterization of a Functional Bacteriocin of Lacticaseibacillus paracasei Zhang and Mining for its Gene Cluster.

Bacteriocins produced by lactic acid bacteria (LAB) have good potential for use as food biopreservatives. Lacticaseibacillus paracasei Zhang (L. paracasei Zhang) is both a food use and a probiotic bacterium. This study aimed to purify and preliminary characterize the active antibacterial metabolite of L. paracasei Zhang. The cell-free supernatant of L. paracasei Zhang was collected and purified by ultrafiltration and gel filtration chromatography. The 1-3 kDa active fraction could inhibit the growth of Staphylococcus aureus but not Escherichia coli. Further antibacterial activity assays revealed its capacity to suppress various foodborne and human opportunistic pathogens (including Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Listeria monocytogenes, and Bacillus cereus), but not fungi. The antibacterial activity showed good tolerance to heat (40 to 100 °C), acid-base (pH 2-3 and pH 6-10), and digestions by a number of industrial and animal/human enzymes (such as trypsin, pepsin, α-amylase, and protease K, except papain); these desired properties make it a suitable biopreservative to be used in harsh and complex industrial production processes. The high papain sensitivity suggested a proteinaceous/peptide nature of the bioactivity. Moreover, our genomic data mining for bacteriocin through BAGEL4 revealed an area of interest encoding a complete set of putative genes required for bacteriocin production. In conclusion, our study showed that L. paracasei Zhang can produce extracellular functional antibacterial metabolite, likely a class II bacteriocin. Our preliminary extraction and characterization of the active metabolite demonstrated that it has good potential to be used as a biopreservative or an agent for suppressing gastrointestinal infections.

Administering Lactiplantibacillus fermentum F6 decreases intestinal Akkermansia muciniphila in a dextran sulfate sodium-induced rat colitis model.

Probiotics are increasingly used to manage gut dysbiosis-related conditions due to their robust ability to manipulate the gut microbial community. However, few studies have reported that probiotics can specifically modulate individual gut microbes. This study demonstrated that administering the probiotic, Lactiplantibacillus fermentum F6, could ameliorate dextran sulfate sodium-induced colitis in a rat model, evidenced by the decreases in the disease activity index score, histopathology grading, and serum pro-inflammatory cytokine levels, as well as the increase in the serum anti-inflammatory cytokine levels. Shotgun metagenomics revealed that the fecal metagenomic of colitis rats receiving the probiotic intervention contained substantially fewer Akkermansia muciniphila than the dextran sulfate sodium group. Thus, the probiotic mechanism might be exerted by reducing specific gut microbial species associated with disease pathogenesis. A new paradigm for designing probiotics that manage diseases through direct and precise manipulation of gut microbes has been provided through this study.

Human Breast Milk: The Role of Its Microbiota and Metabolites in Infant Health.

This review explores the role of microorganisms and metabolites in human breast milk and their impact on neonatal health. Breast milk serves as both a primary source of nutrition for newborns and contributes to the development and maturation of the digestive, immunological, and neurological systems. It has the potential to reduce the risks of infections, allergies, and asthma. As our understanding of the properties of human milk advances, there is growing interest in incorporating its benefits into personalized infant nutrition strategies, particularly in situations in which breastfeeding is not an option. Future infant formula products are expected to emulate the composition and advantages of human milk, aligning with an evolving understanding of infant nutrition. The long-term health implications of human milk are still under investigation.

Postbiotic Administration Ameliorates Colitis and Inflammation in Rats Possibly through Gut Microbiota Modulation.

Postbiotics are preparations of inanimate microorganisms and/or their components that are beneficial to host health. Compared with probiotics, the postbiotic dose required for exerting obvious protective effects is unknown. Thus, we conducted a dose-dependent postbiotic intervention study in dextran sulfate sodium (DSS)-induced colitis rats. The trial included five rat groups, including: control without DSS/postbiotic treatment, group C; 7-day DSS treatment, group D; 14-day low, medium, and high probiotic doses (0.1, 0.2, 0.4 g/kg; groups L, M, H, respectively) after DSS induction. We found that postbiotic intervention effectively mitigated the symptoms and inflammation in colitis rats, evidenced by the improved spleen index, less severe colon tissue damage, and changes in serum cytokine levels (decreases in tumor necrosis factor-α and interleukin-1ß; increase in interleukin-10) in postbiotic groups compared with group D. Moreover, the therapeutic effect was dose-dependent. Fecal metabolomics analysis revealed that the postbiotic recipients had more anti-inflammatory metabolites, namely, salicyloyl phytophingosine, podophylloxin, securinine, baicalein, and diosmetin. Fecal metagenomics analysis revealed that the postbiotic recipients had more beneficial microbes and less pro-inflammatory bacteria. This study confirmed that postbiotics are effective in alleviating colitis in a dose-dependent manner. Our findings are of interest to food scientists, clinicians, and the health food industry.

Dynamic Changes of the Gut Microbiota and Its Functional Metagenomic Potential during the Development of Non-Small Cell Lung Cancer.

The gut microbiota plays a significant role in tumor pathogenesis by regulating the host metabolism and immune response, and there are few studies focused on tracking changes in the gut microbiota from the onset of lung cancer. Therefore, the aim of our study is combining preclinical and clinical research to thoroughly analyze the signatures of fecal microbiota in lung cancer, which will be useful for early diagnosis and predicting the therapeutic efficacy of lung cancer. The first part of this study analyzed the fecal metagenomic differences between patients with non-small cell lung cancer and healthy subjects, and the second part of this work constructed a murine lung cancer model to monitor changes in mouse fecal metagenomics and T cell immunology during lung cancer progression. We found that the fecal microbiota was altered in both humans and mice with lung cancer, characterized by a significantly reduced microbial diversity and number of beneficial microbes, with increases in potential pathogens. The fecal level of Akkermansia muciniphila and the gut metabolic module of the secondary bile acid metabolism were diminished in both humans and mice with lung cancer compared with healthy subjects. Splenomegaly was observed in the lung cancer mice. Flow cytometer analysis of the splenocytes revealed substantial alterations in the proportions of T cell subsets in the lung cancer mice, characterized by significant increases in CD4+Foxp3+CD25+ T regulatory cells (p < 0.05) while significant decreases in CD3+ T cells (p < 0.001), CD4+ T cells (p < 0.001), and the CD4+/CD8+ ratio (p < 0.01). Vertical and longitudinal analyses of the fecal microbiota of the two mouse groups identified some lung cancer biomarkers (including Acutalibacter timonensis, Lachnospiraceae bacterium NSJ-38 sp014337195, etc.). The fecal microbiota of the lung cancer mice had a reduced metagenomic potential for neurotransmitters (melatonin, γ-aminobutyric acid, and histamine) compared with healthy mice. In summary, this study found that the diversity, structure, and composition of gut microbiota vary between cancer and healthy conditions, ultimately leading to changes in the potential for functional metagenomics.

Metagenomic analysis revealed the potential of lactic acid bacteria in improving natural saline-alkali land / El análisis metagenómico reveló el potencial de las bacterias del ácido láctico para mejorar las tierras salino-álcalis naturales

Management and improving saline-alkali land is necessary for sustainable agricultural development. We conducted a field experiment to investigate the effects of spraying lactic acid bacteria (LAB) on the cucumber and tomato plant soils. Three treatments were designed, including spraying of water, viable or sterilized LAB preparations to the soils of cucumber and tomato plants every 20 days. Spraying sterilized or viable LAB could reduce the soil pH, with a more obvious effect by using viable LAB, particularly after multiple applications. Metagenomic sequencing revealed that the soil microbiota in LAB-treated groups had higher alpha-diversity and more nitrogen-fixing bacteria compared with the water-treated groups. Both viable and sterilized LAB, but not water application, increased the complexity of the soil microbiota interactive network. The LAB-treated subgroups were enriched in some KEGG pathways compared with water or sterilized LAB subgroups, such as environmental information processing–related pathways in cucumber plant; and metabolism-related pathways in tomato plant, respectively. Redundancy analysis revealed association between some soil physico-chemical parameters (namely soil pH and total nitrogen) and bacterial biomarkers (namely Rhodocyclaceae, Pseudomonadaceae, Gemmatimonadaceae, and Nitrosomonadales). Our study demonstrated that LAB is a suitable strategy for decreasing soil pH and improving the microbial communities in saline-alkali land.(AU)

Metagenomic analysis revealed the potential of lactic acid bacteria in improving natural saline-alkali land.

Management and improving saline-alkali land is necessary for sustainable agricultural development. We conducted a field experiment to investigate the effects of spraying lactic acid bacteria (LAB) on the cucumber and tomato plant soils. Three treatments were designed, including spraying of water, viable or sterilized LAB preparations to the soils of cucumber and tomato plants every 20 days. Spraying sterilized or viable LAB could reduce the soil pH, with a more obvious effect by using viable LAB, particularly after multiple applications. Metagenomic sequencing revealed that the soil microbiota in LAB-treated groups had higher alpha-diversity and more nitrogen-fixing bacteria compared with the water-treated groups. Both viable and sterilized LAB, but not water application, increased the complexity of the soil microbiota interactive network. The LAB-treated subgroups were enriched in some KEGG pathways compared with water or sterilized LAB subgroups, such as environmental information processing-related pathways in cucumber plant; and metabolism-related pathways in tomato plant, respectively. Redundancy analysis revealed association between some soil physico-chemical parameters (namely soil pH and total nitrogen) and bacterial biomarkers (namely Rhodocyclaceae, Pseudomonadaceae, Gemmatimonadaceae, and Nitrosomonadales). Our study demonstrated that LAB is a suitable strategy for decreasing soil pH and improving the microbial communities in saline-alkali land.

The intricate symbiotic relationship between lactic acid bacterial starters in the milk fermentation ecosystem.

Fermentation is one of the most effective methods of food preservation. Since ancient times, food has been fermented using lactic acid bacteria (LAB). Fermented milk is a very intricate fermentation ecosystem, and the microbial metabolism of fermented milk largely determines its metabolic properties. The two most frequently used dairy starter strains are Streptococcus thermophilus (S. thermophilus) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). To enhance both the culture growth rate and the flavor and quality of the fermented milk, it has long been customary to combine S. thermophilus and L. bulgaricus in milk fermentation due to their mutually beneficial and symbiotic relationship. On the one hand, the symbiotic relationship is reflected by the nutrient co-dependence of the two microbes at the metabolic level. On the other hand, more complex interaction mechanisms, such as quorum sensing between cells, are involved. This review summarizes the application of LAB in fermented dairy products and discusses the symbiotic mechanisms and interactions of milk LAB starter strains from the perspective of nutrient supply and intra- and interspecific quorum sensing. This review provides updated information and knowledge on microbial interactions in a fermented milk ecosystem.

The symbiotic relationship between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is reviewed.Their nutrient co-dependence is discussed.The role of quorum sensing in their interaction is discussed for the first time.This review is of interest to colleagues interested in exploiting LAB starter cultures.

Gut microbiome-targeted therapies for Alzheimer's disease.

The advent of high-throughput 'omics' technologies has improved our knowledge of gut microbiome in human health and disease, including Alzheimer's disease (AD), a neurodegenerative disorder. Frequent bidirectional communications and mutual regulation exist between the gastrointestinal tract and the central nervous system through the gut-brain axis. A large body of research has reported a close association between the gut microbiota and AD development, and restoring a healthy gut microbiota may curb or even improve AD symptoms and progression. Thus, modulation of the gut microbiota has become a novel paradigm for clinical management of AD, and emerging effort has focused on developing potential novel strategies for preventing and/or treating the disease. In this review, we provide an overview of the connection and causal relationship between gut dysbiosis and AD, the mechanisms of gut microbiota in driving AD progression, and the successes and challenges of implementing available gut microbiome-targeted therapies (including probiotics, prebiotics, synbiotics, postbiotics, and fecal microbiota transplantation) in preventive and/or therapeutic preclinical and clinical intervention studies of AD. Finally, we discuss the future directions in this field.

A large-scale comparative genomics study reveals niche-driven and within-sample intra-species functional diversification in Lacticaseibacillus rhamnosus.

Lacticaseibacillus rhamnosus (L. rhamnosus) is widely recognized as a probiotic species, and it exists in a variety of environments including host gut and dairy products. This work aimed at conducting a large-scale comparative genomics analysis of 384 L. rhamnosus genomes (257 whole-sequence or metagenomic-assembled genomes from gut-associated isolates [122 and 135 retrieved from the UHGG and NCBI databases, respectively] and 127 genomes from dairy isolates [34 from the NCBI database; 93 isolated from a cheese sample and sequenced here]). Our results showed that L. rhamnosus had a large and open pan-genome (15,253 pan-genes identified from all 384 genomes; 15,028 pan-genes if the 93 cheese-originated isolates were excluded). The core-gene phylogenetic tree constructed from the 384 L. rhamnosus genomes comprised five phylogenetic branches, with a random distribution of dairy and gut-associated isolates/genomes across the tree. No significant difference was identified in the overall profile of metabolism-related genes between dairy and gut-associated genomes; however, notably, the gut-associated strains/isolates contained more genes coding for specific metabolic pathways and carbohydrate-active enzymes, e.g., lacto-N-biosidase (EC 3.2.1.140; GT20) and lacto-N-biose phosphorylase/galacto-N-biose phosphorylase (EC 2.4.1.211; GH112). Further, we found that there was obvious intra-species diversification of the 93 cheese-originated L. rhamnosus isolates, forming three clades (Clades A, B, and C) in the reconstructed core-gene phylogenetic tree. There were numerous single nucleotide variations (over 10,000) across the three clades. Moreover, significant differences were observed in the content of metabolism-related genes across clades (p < 0.05, Adonis test), characterized by the enrichment in glycoside hydrolases in Clade C and the possession of unique metabolic pathways in each clade. These results implicated genomics/functional diversification of L. rhamnosus in a single food matrix and niche-driven adaptive evolution of isolates from dairy and host gut-associated origins. Our study shed insights into the selection of candidate strains for food industry applications.

Metabolomic Differences between Viable but Nonculturable and Recovered Lacticaseibacillus paracasei Zhang.

The fermentation process can be affected when the starter culture enters the viable but nonculturable (VBNC) state. Therefore, it is of interest to investigate how VBNC cells change physiologically. Lacticaseibacillus (L.) paracasei Zhang is both a probiotic and a starter strain. This study aimed to investigate the metabolomic differences between VBNC and recovered L. paracasei Zhang cells. First, L. paracasei Zhang was induced to enter the VBNC state by keeping the cells in a liquid de Man-Rogosa-Sharpe (MRS) medium at 4 °C for 220 days. Flow cytometry was used to sort the induced VBNC cells, and three different types of culture media (MRS medium, skim milk with 1% yeast extract, and skim milk) were used for cell resuscitation. Cell growth responses in the three types of recovery media suggested that the liquid MRS medium was the most effective in reversing the VBNC state in L. paracasei Zhang. Metabolomics analysis revealed 25 differential metabolites from five main metabolite classes (amino acid, carbohydrate, lipid, vitamin, and purine and pyrimidine). The levels of L-cysteine, L-alanine, L-lysine, and L-arginine notably increased in the revived cells, while cellulose, alginose, and guanine significantly decreased. This study confirmed that VBNC cells had an altered physiology.

Probiotics alleviate constipation and inflammation in late gestating and lactating sows.

Constipation and systemic inflammation are common in late pregnant and lactating sows, which cause health problems like uteritis, mastitis, dystocia, or even stillbirth, further influencing piglets' survival and growth. Probiotic supplementation can improve such issues, but the beneficial mechanism of relieving constipation and enhancing gut motility remains underexplored. This study aimed to investigate the effects and mechanism of probiotic supplementation in drinking water to late pregnant sows on constipation, inflammation, and piglets' growth performance. Seventy-four sows were randomly allocated to probiotic (n = 36) and control (n = 38) groups. Probiotic treatment significantly relieved sow constipation, enhanced serum IL-4 and IL-10 levels while reducing serum IL-1ß, IL-12p40, and TNF-α levels, and increased piglet daily gain and weaning weight. Furthermore, probiotic administration reshaped the sow gut bacteriome and phageome structure/diversity, accompanied by increases in some potentially beneficial bacteria. At 113 days of gestation, the probiotic group was enriched in several gut microbial bioactive metabolites, multiple carbohydrate-active enzymes that degrade pectin and starch, fecal butyrate and acetate, and some serum metabolites involved in vitamin and amino acid metabolism. Our integrated correlation network analysis revealed that the alleviation of constipation and inflammation was associated with changes in the sow gut bacteriome, phageome, bioactive metabolic potential, and metabolism.

Intraspecific microdiversity and ecological drivers of lactic acid bacteria in naturally fermented milk ecosystem.

Traditional fermented milks are produced by inoculating technique, which selects well-adapted microorganisms that have been passed on through generations. Few reports have used naturally fermented milks as model ecosystems to investigate the mechanism of formation of intra-species microbial diversity. Here, we isolated and whole-genome-sequenced a total of 717 lactic acid bacterial isolates obtained from 12 independent naturally fermented milks collect from 12 regions across five countries. We further analyzed the within-sample intra-species phylogenies of 214 Lactobacillus helveticus isolates, 97 Lactococcus lactis subsp. lactis isolates, and 325 Lactobacillus delbrueckii subsp. bulgaricus isolates. We observed a high degree of intra-species genomic and functional gene diversity within-/between-sample(s). Single nucleotide polymorphism-based phylogenetic reconstruction revealed great within-sample intra-species heterogeneity, evolving from multiple lineages. Further phylogenetic reconstruction (presence-absence gene profile) revealed within-sample inter-clade functional diversity (based on carbohydrate-active enzyme- and peptidase-encoding genes) in all three investigated species/subspecies. By identifying and mapping clade-specific genes of intra-sample clades of the three species/subspecies to the respective fermented milk metagenome, we found extensive potential inter-/intra-species horizontal gene transfer events. Finally, the microbial composition of the samples is closely linked to the nucleotide diversity of the respective species/subspecies. Overall, our results contribute to the conservation of lactic acid bacteria resources, providing ecological insights into the microbial ecosystem of naturally fermented dairy products.

Exopolysaccharide-Producing Lacticaseibacillus rhamnosus Space Mutant Improves the Techno-Functional Characteristics of Fermented Cow and Goat Milks.

Lacticaseibacillus rhamnosus Probio-M9 (Probio-M9) is increasingly used as a co-fermentation culture in fermented milk production. Recently, a capsular polysaccharide (CPS)- and exopolysaccharide (EPS)-producing mutant of Probio-M9, HG-R7970-3, was generated by space mutagenesis. This study compared the performance of cow and goat milk fermentation between the non-CPS/-EPS-producing parental strain (Probio-M9) and the CPS/EPS producer (HG-R7970-3), and the stability of products fermented by the two bacteria. Our results showed that using HG-R7970-3 as the fermentative culture could improve the probiotic viable counts, physico-chemical, texture, and rheological properties in both cow and goat milk fermentation. Substantial differences were also observed in the metabolomics profiles between fermented cow and goat milks produced by the two bacteria. Comparing with Probio-M9-fermented cow and goat milks, those fermented by HG-R7970-3 were enriched in a number of flavor compounds and potential functional components, particularly acids, esters, peptides, and intermediate metabolites. Moreover, HG-R7970-3 could improve the post-fermentation flavor retention capacity. These new and added features are of potential to improve the techno-functional qualities of conventional fermented milks produced by Probio-M9, and these differences are likely imparted by the acquired CPS-/EPS-producing ability of the mutant. It merits further investigation into the sensory quality and in vivo function of HG-R7970-3-fermented milks.

Comparative Analysis Using Raman Spectroscopy of the Cellular Constituents of Lacticaseibacillus paracasei Zhang in a Normal and Viable but Nonculturable State.

This study aimed to investigate the molecular composition of a viable but nonculturable (VBNC) state of a probiotic strain, Lacticaseibacillus paracasei Zhang (L. paracasei Zhang), using single-cell Raman spectroscopy (SCRS). Fluorescent microcopy with live/dead cell staining (propidium iodide and SYTO 9), plate counting, and scanning electron microscopy were used in combination to observe bacteria in an induced VBNC state. We induced the VBNC state by incubating the cells in de Man, Rogosa, and Sharpe broth (MRS) at 4 °C. Cells were sampled for subsequent analyses before VBNC induction, during it, and up to 220 days afterwards. We found that, after cold incubation for 220 days, the viable plate count was zero, but active cells could still be observed (as green fluorescent cells) under a fluorescence microscope, indicating that Lacticaseibacillus paracasei Zhang entered the VBNC state under these conditions. Scanning electron microscopy revealed the altered ultra-morphology of the VBNC cells, characterized by a shortened cell length and a wrinkled cell surface. Principal component analysis of the Raman spectra profiles revealed obvious differences in the intracellular biochemical constituents between normal and VBNC cells. Comparative analysis of the Raman spectra identified 12 main differential peaks between normal and VBNC cells, corresponding to carbohydrates, lipids, nucleic acids, and proteins. Our results suggested that there were obvious cellular structural intracellular macromolecular differences between normal and VBNC cells. During the induction of the VBNC state, the relative contents of carbohydrates (such as fructose), saturated fatty acids (such as palmitic acid), nucleic acid constituents, and some amino acids changed obviously, which could constitute a bacterial adaptive mechanism against adverse environmental conditions. Our study provides a theoretical basis for revealing the formation mechanism of a VBNC state in lactic acid bacteria.

Roles of adenine methylation in the physiology of Lacticaseibacillus paracasei.

Lacticaseibacillus paracasei is an economically important bacterial species, used in the food industry and as a probiotic. Here, we investigate the roles of N6-methyladenine (6mA) modification in L. paracasei using multi-omics and high-throughput chromosome conformation capture (Hi-C) analyses. The distribution of 6mA-modified sites varies across the genomes of 28 strains, and appears to be enriched near genes involved in carbohydrate metabolism. A pglX mutant, defective in 6mA modification, shows transcriptomic alterations but only modest changes in growth and genomic spatial organization.

Untargeted mass spectrometry-based metabolomics approach unveils biochemical changes in compound probiotic fermented milk during fermentation.

Probiotic functional products have drawn wide attention because of their increasing popularity. However, few studies have analyzed probiotic-specific metabolism in the fermentation process. This study applied UPLC-QE-MS-based metabolomics to track changes in the milk metabolomes in the course of fermentation by two probiotic strains, Lacticaseibacillus paracasei PC-01 and Bifidobacterium adolescentis B8589. We observed substantial changes in the probiotic fermented milk metabolome between 0 and 36 h of fermentation, and the differences between the milk metabolomes at the interim period (36 h and 60 h) and the ripening stage (60 h and 72 h) were less obvious. A number of time point-specific differential metabolites were identified, mainly belonging to organic acids, amino acids, and fatty acids. Nine of the identified differential metabolites are linked to the tricarboxylic acid cycle, glutamate metabolism, and fatty acid metabolism. The contents of pyruvic acid, γ-aminobutyric acid, and capric acid increased at the end of fermentation, which can contribute to the nutritional quality and functional properties of the probiotic fermented milk. This time-course metabolomics study analyzed probiotic-specific fermentative changes in milk, providing detailed information of probiotic metabolism in a milk matrix and the potential beneficial mechanism of probiotic fermented milk.

Effect of the probiotic strain, Lactiplantibacillus plantarum P9, on chronic constipation: A randomized, double-blind, placebo-controlled study.

Chronic constipation (CC) is a common gastrointestinal condition associated with intestinal inflammation, and the condition considerably impairs patients' quality of life. We conducted a large-scale 42-day randomized, double-blind, placebo-controlled trial to investigate the effect of probiotics in alleviating CC. 163 patients diagnosed with CC (following Rome IV criteria) were randomly divided into probiotic (n = 78; received Lactiplantibacillus plantarum P9 [P9]; 1 ×1011 CFU/day) and placebo (n = 85; received placebo material) groups. Ingesting P9 significantly improved the weekly mean frequency of complete spontaneous bowel movements (CSBMs) and spontaneous bowel movements (SBMs), while significantly reducing the level of worries and concerns (WO; P < 0.05). Comparing with the placebo group, P9 group was significantly enriched in potentially beneficial bacteria (Lactiplantibacillus plantarum and Ruminococcus_B gnavus), while depriving of several bacterial and phage taxa (Oscillospiraceae sp., Lachnospiraceae sp., and Herelleviridae; P < 0.05). Interesting significant correlations were also observed between some clinical parameters and subjects' gut microbiome, including: negative correlation between Oscillospiraceae sp. and SBMs; positive correlation between WO and Oscillospiraceae sp., Lachnospiraceae sp. Additionally, P9 group had significantly (P < 0.05) more predicted gut microbial bioactive potential involved in the metabolism of amino acids (L-asparagine, L-pipecolinic acid), short-/medium-chain fatty acids (valeric acid and caprylic acid). Furthermore, several metabolites (p-cresol, methylamine, trimethylamine) related to the intestinal barrier and transit decreased significantly after P9 administration (P < 0.05). In short, the constipation relief effect of P9 intervention was accompanied by desirable changes in the fecal metagenome and metabolome. Our findings support the notion of applying probiotics in managing CC.