Effects of Lacticaseibacillus paracasei 207-27 on intestinal function and its regulatory mechanism in patients with chronic constipation

Effects of Lacticaseibacillus paracasei 207-27 on intestinal function and its regulatory mechanism in patients with chronic constipation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Lacticaseibacillus paracasei 207-27 on intestinal function and its regulatory mechanism in patients with chronic constipation Yanjun An, Yong Jia, Xiaoling zhang, Xiaoqiong Li, Liying Zhu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4108195/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background & Aims: Constipation is a disorder affecting the quality of life of patients, and symptoms following current treatments still need to be improved. Although probiotics treat constipation, the efficacy and safety of this therapy remain controversial. This a randomized, double-blind, placebo-dense controlled trialaimed to investigate the clinical impact of the probiotic bacterium Lacticaseibacillus paracasei 207-27 on chronic constipation. Methods : We recruited 80 patients aged 18–65 with chronic constipation and divided them into probiotic ( Lacticaseibacillus paracasei 207-27) and placebo (maltodextrin) groups. The participants were treated twice daily for 4 weeks, and stool samples and questionnaires were collected before and after treatment. The 16S rRNA sequencing and untargeted metabolomics data were analyzed. Results : Bowel movements, total short-chain fatty acid, and propionic acid were significantly improved in the probiotic group compared with in the placebo (control) group ( p <0.05) following the 4-week intervention. After the intervention, the placebo group had higher Phylum Firmicutes number ( p <0.0001) and abundance of Escherichia-Shigella genus ( p= 0.0257) and Roseburia genus ( p =0.0202) but lower abundances of Actinobacteria phylum ( p =0.0177), Bifidobacterium genus ( p <0.0001), and Roseburia genus ( p =0.0001) than the probiotic group. Probiotic intervention reduced the abundance of the Bacteroides genus ( p =0.0001) and Blautia genus ( p =0.0213) compared with that before intervention (week 0). The analysis of non-target metabolites revealed a significant increase in 3-sulfinoalanine associated with taurine and hypotaurine metabolism in 3-methylxanthine associated with caffeine metabolism and a significant downregulation of 3-methylindolepyruvate associated with tryptophan metabolism after probiotic intervention. Conclusion : Lacticaseibacillusparacasei 207-27 improved constipation symptoms and altered the intestinal microbiota, which could improve patients’ quality of life. Trial registration : chictr.org.cn ChiCTR2200056274, 03/02/2022. Chronic constipation short-chain fatty acids intestinal flora metabolomics association analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. INTRODUCTION Chronic constipation is a functional gastrointestinal tract disease caused by multiple factors, and its incidence is increasing annually ( 1 ), significantly affecting the overall well-being of affected individuals. It may cause intestinal flora disturbance, abnormal intestinal peristalsis, and intestinal water absorption disorder. Based on the variability of traditional drug therapies and dietary controls, nearly half of the patients are unsatisfied with the post-treatment symptoms ( 2 ). Probiotics may improve chronic constipation by controlling the metabolic processes and the composition of the intestinal flora, improving intestinal motility and water absorption ( 3 – 5 ), and are used for treating chronic diseases and are becoming a potential therapy for constipation ( 6 , 7 ); however, the efficacy and safety of probiotic treatment for chronic constipation remain controversial. Recently, untargeted metabolomics has been explored to study the pathogenesis and treatment of chronic constipation ( 3 , 4 ). By comparing the metabolomic data of patients and healthy participants, differences in the metabolite levels in patients with chronic constipation can be detected, providing insights into the pathophysiological mechanisms of chronic constipation. Untargeted metabolomics can be used to diagnose chronic constipation and provide guidance for probiotic therapies. To examine how chitosan affected the abnormalities in plasma metabolomes in mice that had trouble going to the bathroom, Zhang et al. identified metabolic pathways related to sphingolipids, glycerophospholipids, and tryptophan using ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS) ( 5 ). Several metabolomics studies identified specific metabolic abnormalities in patients with chronic constipation, such as disturbances in amino acid metabolism and abnormal bile acid metabolism ( 8 ). These abnormalities may change the composition and function of intestinal bacteria, affecting how the intestine moves and absorbs water. However, the underlying mechanisms of constipation-associated metabolic abnormalities are still largely unknown. This study aimed to explore further the roles and mechanisms of probiotic treatment of chronic constipation, which could help develop new strategies for improving its symptoms. 2. MATERIALS AND METHODS 2.1 Ethical statement This study was registered in the Clinical Trials Database (ID: ChiCTR2200056274), conducted at The Affiliated Hospital of Hangzhou Normal University, and approved by the Ethics Committee. 2.2 Patients This study included 80 patients with constipation. The inclusion criteria were as follows: meeting the Rome IV diagnostic criteria (9), age 18–65 years old, and having a frequency of defecation 1–3 times/week. The exclusion criteria were as follows: individuals diagnosed with gastrointestinal, neurological, cardiovascular, endocrine, renal, or other chronic diseases that may affect bowel movements; those who had used antibiotics within 1 month; those who had used medications to treat constipation or took probiotics within 2 weeks before enrollment; pregnant or lactating women; those who had allergies or were allergic to probiotics. The criteria for dropping out were as follows: poor compliance, inability to complete treatment as required, voluntary withdrawal in the middle of the treatment or loss of visits, and incomplete data. 2.3 Study Design In this randomized, double-blind, placebo-dense controlled trial, the patients were randomized after considering the diary card and the inclusion and exclusion criteria to determine who met the criteria for this study. After preliminary evaluation by the investigator, those who met the inclusion criteria signed an informed consent form and were recruited into a 1-week introductory phase without taking any products to improve bowel movement. These patients were admitted into a 28-day trial of a probiotic or placebo intervention. Patients received one packet twice daily before meals. The participants were instructed to refrain from altering their dietary and lifestyle practices to avoid other probiotics and dietary fibers during the study and to fill in the Bowel Diary Cards every day (day 0–28) to record their bowel symptoms and movements. They were also required to fill in the Quality of Life Scores (PAC-QOL) form and the Constipation Symptom Assessment Form (PAC-SYM ) on days 0, 14, and 28. The Constipation Symptom Assessment Form (PAC-SYM) and stool samples were collected on days 0 and 28 for microbial profiling and metabolomics analysis. 2.4 Preparation of probiotics and placebo Probiotic powder (1 g/bag, 2 billion cfu of La cticaseibacillus paracasei 207-27) was selected as the probiotic (207-27 was originally isolated from healthy infant feces in China and it is now deposited at the Guangdong Microbial Culture Collection Center under the Budapest Treaty, with deposit code GDMCC 60960,which also named Lacticaseibacillus paracasei LPB27 in its commercialized product). The placebo was formulated using maltodextrin (identical in appearance and flavor to the probiotic). Both products were manufactured by the BYHEALTH Co., Ltd. 2.5 Result evaluations Individual stool diary cards were used to compute the number of spontaneous complete bowel movements (SCBM) and Bristol Stool Form Scale (BSFS) per week. The primary outcome was the alteration in the average frequency of SCBM per week during the intervention, and the secondary outcomes were other signs of constipation-related symptoms, such as the amount of improvement in the number of SCBM per week and the scores for the quality of feces every week. The other outcomes were the quality-of-life (PAC-SYM) and constipation symptom assessment scale (PAC-QOL) on days 0, 14, and 28. Stool samples were collected on days 0 and 28 and were stored at -80°C for subsequent microbial profiling and untargeted metabolomics analysis. During the follow-up, safety outcomes, including adverse events, were recorded. 2.6 Analysis of short - chain fatty acids Fecal samples were collected before and after administration, ensuring they were not contaminated with toilet water or urine. Fecal samples (2 g) were weighed, diluted 1:10 in sterile phosphate-buffered saline, and vortexed. The sample was centrifuged at 4°C, 11,000 rpm for 6 min, and the supernatant was collected and transferred to a sterile centrifuge tube. Then, 250 µL of the supernatant was placed into a new, clean container, and 50 µL of the crotonic acid mixture was added with a 1:5 ratio. After filtering through a 0.22 µm membrane, the sample was acidified and stored at -80°C until use. A gas chromatograph (GC-2010 Plus, Shimadzu, Agilent Technologies, Santa Clara, CA, USA) with a 1D mass spectrometer and a DB-FFAP column was used to determine the short chain fatty acid (SCFAs, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) levels. The remaining fecal samples were stored in a -80°C freezer. 2.7 Microbiological analysis DNA was extracted from fecal samples, and 16S rRNA gene amplicons were sequenced using an Illumina PE250 at Minco Biotech (Hangzhou, China). The V4 region of the 16S rRNA gene was amplified using the double barcode primer pair 515F (5'-GTGCCAGCMGCCGCGG-3')-907R (5'-CCGTCAATTCMTTTRAGTTT-3'). The sequencing results were analyzed with QIIME2 software, and the operational taxonomic units (OTUs) were grouped with sequences without repeating at a 97% identity level. These results were examined in detail. To determine the bacterial species, we compared representative sequences from all OTUs to the Silva and RDP databases. Principal component analysis (PCA) was used to examine beta diversity between groups. 2.8 Untargeted metabolomics analysis Nomi performed the LC-MS untargeted metabolomic assays. Data preprocessing was performed by converting raw mass spectrometry downstream files to the mzXML file format using the MSConvert tool in the ProteoWizard software package (v3.0.8789). The R XCMS software package was used to obtain the quantitative list of substances, and the public databases HMDB, mass bank, LipidMaps, McCloud, KEGG, and the self-constructed substance library were used to identify substances. The R package Ropls was used to perform PCA, partial least squares discriminant analysis, and orthogonal partial least squares discriminant analysis downscaling on the sample data. Metabolite molecules were considered statistically significant when the p- value was 1. Pathway analysis, functional pathway enrichment, and topological analysis of the screened differential metabolite molecules were performed using the MetaboAnalyst software package. Using the KEGG Mapper display tool, the pathways identified in the enrichment analysis were used to examined the differential metabolite and pathway maps. 2.9 Statistical analysis Sample size calculations were performed as described previously (10). The primary endpoint was the percentage of patients with SCBM, which was used as an indicator, and a superiority test was used for estimation. If the sample size according to the ratio of those who had four or more bowel movements per week in the intervention group was 80%, and 50% in the control group, with α of 0.05, β of 80%, and an equal proportion of participants in the two groups, the sample size of the intervention group and the control group should be 28, respectively, considering the possibility of intermediate dropouts. Therefore, it was reasonable to include 40 people in each group. The primary and secondary objectives were to assess the population intention-to-treat effect. All data processing and statistical analyses were performed using SPSS v25.0 and GraphPad Prism v.8.0.2 (263). Study data were expressed as mean ± standard deviation (SD) or standard error of the mean (SEM). Two-Way Repeated Measures ANOVA and Bonferroni correction were used for multiple group comparisons. The association was determined using Spearman's linear correlation analysis. Differences were considered statistically significant at p <0.05. 3. RESULTS 3.1 Study parameters In this trial, 80 patients were enrolled and randomized into two groups of 40 patients each. Sixteen patients did not complete the study, ten were lost to follow-up, and six did not comply with the required consecutive intervention. The final analysis included 64 individuals, of which 33 were in the probiotic group and 31 were in the placebo group (Figure 1). 3.2 Baseline characteristics of the participants The baseline characteristics of the participants are presented in Table 1. There were no statistically significant differences in age, sex, body mass index (BMI), or fecal consistency between the groups ( p > 0.05). The population with constipation comprised more women (75%) than men (25%). The participants were considered to have functional constipation according to the Rome IV criteria. Differences in functional constipation parameters PAC-SYM, PAC-QOL, BSFS, and N0. BM/week at baseline were insignificant ( p >0.05). Table . 1 . Demographic and baseline characteristics of participants in the clinical trial. Characteristics Placebo (n=31) Probiotic (n=33) P value Sex (male/female) 6 (0.19)/25 (0.81) 10 (0.30)/23 (0.70) p >0.05 Age (year) (mean ± SD) 36.5 ± 15.53 39.03 ± 15.15 BMI (kg/m 2 ) (mean ± SD) 22.1 ± 3.2 22.5 ± 3.1 PAC-QOL (mean ± SD) 72.94 ± 17.68 70.55 ± 16.73 PAC-SYM (mean ± SD) 1.40 ± 0.53 1.35 ± 0.52 No. of BMs/week (mean ± SD) 2.58 ± 0.56 2.55 ± 0.62 BSFS (mean ± SD) 2.68 ± 0.91 2.52 ± 0.71 Data were presented as mean ± SD. BMI, body mass index; bpm, beats per minute; BSFS, Bristol stool form scale; No. of BMs/week, number of bowel movements per week; PAC-QOL, patient assessment of constipation quality of life; PAC-SYM, patient assessment of constipation symptoms; SD, standard deviation. There were no statistically significant differences across the three groups. 3.3 Clinical efficacy analysis Sixty-four participants were included in the efficacy analysis, with SCBM as the primary efficacy endpoint and the BSFS Stool Scale as the secondary efficacy endpoint. At the end of the 4-week intervention, there was a significant increase in the number of bowel movements (5.12±1.71) in the probiotic group ( p <0.01) compared to the placebo group (3.77±1.56). The Bristol stool form scale of constipated patients in the probiotic group also significantly improved at week 4 (4.20±0.82) compared to week 0 (2.48±0.67). The probiotics showed more effectiveness; this effect started to show at week 2 ( p <0.05) and was more pronounced at week 4 ( p <0.01). This suggests that probiotic treatment is more favorable than placebo to improving symptoms associated with constipation in patients. After 4 weeks of intervention, patients with constipation in both the probiotic and placebo groups experienced symptomatic relief. PAC-SYM scores were significantly lower (0.80±0.13) in the probiotic group compared to baseline (1.35±0.51). Although there was an interaction between time and the group of PAC-SYM scores ( p <0.05), the separate effect of the group was not statistically significant. PAC-QOL scores were substantially lower in both the probiotic and placebo groups compared to the baseline level, but there was no significant difference ( p >0.05), which may be attributed to the placebo effect (Table 2 and Figure 2). Table . 2. Changes from baseline in clinical efficacy parameters of functional constipation in participants receiving placebo or probiotic capsules. Score per week Probiotic (n= 31 ) P lacebo (n= 33 ) P value Mean ± SD Mean ± SD No. of BM/week Week 0 2.42 ± 0.71 2.58 ± 0.56 0.334 Week 1 4.55 ± 2.02 3.84 ± 1.66 0.132 Week 2 4.76 ± 2.05 3.84 ± 1.68 0.055 Week 3 5.03 ± 2.02 4.39 ± 1.58 0.164 Week 4 5.12 ± 1.71 3.77 ± 1.56 0.002 BSFS average Week 0 2.48 ± 0.67 2.64 ± 0.88 0.412 Week 1 3.71 ± 1.12 3.46 ± 1.16 0.390 Week 2 4.13 ± 1.01 3.45 ± 1.03 0.010 Week 3 4.17 ± 0.88 3.63 ± 0.68 0.008 Week 4 4.20 ± 0.82 3.54 ± 1.01 0.005 PAC-SYM score Week 0 1.35 ± 0.51 1.34 ± 0.45 0.957 Week 2 0.86 ± 0.45 0.82 ± 0.46 0.703 Week 4 0.55 ± 0.39 0.62 ± 0.45 0.467 PAC-QOL score Week 0 70.45 ± 16.87 72.13 ± 16.91 0.693 Week 2 52.27 ± 14.66 54.58 ± 15.40 0.541 Week 4 49.48 ± 13.34 48.10 ± 15.51 0.702 BSFS, Bristol stool scale; No. of BMs/week, number of bowel movements per week; PAC-QOL, patient assessment of constipation - quality of life; PAC-SYM, patient assessment of constipation - symptoms; S.D., standard deviation. ANCOVA, Bonferroni correction. 3.4 Effects of probiotics on intestinal flora and their products in patients with chronic constipation 3.4.1 Effects on the main intestinal bacteria We detected Bifidobacterium, Lactobacillus, Enterococcus faecalis , and Faecalibacterium prausnitzii in both groups before and after the intervention by fluorescence quantitative polymerase chain reaction (FQ-PCR) method. The test data showed that the number of Lactobacillus in the probiotic group increased significantly ( p <0.05) with no significant changes in the numbers of Bifidobacterium, Enterococcus faecalis , and Faecalibacterium prausnitzii . The number of Lactobacillus in the probiotic group was significantly different from the placebo group after the intervention( p <0.01), which may be related to the fact that the probiotic ingested was Lacticaseibacillus paracasei (Figure 3). 3.4. 2 Effects on the structure and composition of intestinal flora The α-diversity values of pre-intervention (week 0), post-placebo intervention (post-placebo), and post-probiotic intervention (post-probiotic) were analyzed, and the differences in Chao1, Shannon, and Simpson indices among these three groups were not statistically significant ( p >0.05). Principal component analysis showed no significant clustering in the flora among the three groups (Figures 4–5). Figure 6 shows that, at the phylum level, Firmicutes bacterial gates in both groups increased after the intervention compared with the pre-intervention value, with a more pronounced increase in the placebo group and a significant difference between the two groups (post-placebo vs. post-probiotic, p <0.0001). The Actinomycetes phylum was significantly lower in the placebo group after the intervention (week 0 vs. post-placebo, p =0.0053), with a significant difference between the two groups (post-placebo vs. post-probiotic, p =0.0177). At the genus level, more Bifidobacterium spp. were in the post-intervention probiotic group than in the placebo group (post-placebo vs. post-probiotic, p <0.0001). The post-intervention probiotic group showed reduced Bacteroides spp. (week 0 vs. post-probiotic, p =0.0001) and Blautia spp. Post-intervention probiotic group Bacteroides spp. abundance decreased (week0 vs. post-probiotic, p =0.0001), Blautia spp. abundance decreased (week0 vs. post-probiotic, p =0.0213,) Shigella spp. Escherichia-Shigella decreased in abundance (post-placebo vs. post-probiotic, p =0.0257) and Rochesteria spp. increased in abundance (post-placebo vs. post-probiotic, p =0.0202).At the species level, more Clostridium perfringens were in the post-placebo probiotic group than in the intervention group (post-placebo vs. post-probiotic， p =0.037). 3.4.2 Impact of SCFA Figure 7 shows that probiotic intervention increased total acid production, while the placebo group did not produce the same effect ( p <0.01). Acetic acid content ( p <0.05) and propionic acid content ( p <0.01) increased significantly after intervention with the probiotic, and propionic acid levels were significantly different from the placebo group after the intervention ( p 0.05). All data in the placebo group were not statistically different from pre-intervention ( p >0.05) (Table 3 and Figure 7). Table . 3 . Changes from baseline in SCFA of functional constipation in participants receiving placebo or probiotic capsules. Score Probiotic (n= 33 ) P lacebo (n= 31 ) P value Mean ± SD P value Mean ± SD P value Total acid Pre 7.528 ± 3.083 0.008 7.739 ± 2.315 0.336 0.759 Post 9.564 ± 2.975 8.394 ± 2.898 0.117 AbsΔ 2.036 ± 4.140 0.655 ± 3.730 0.167 Acetic acid Pre 4.409 ± 2.322 0.017 4.612 ± 2.037 0.348 0.713 Post 5.754 ± 2.164 5.163 ± 2.451 0.310 AbsΔ 1.346 ± 3.061 0.552 ± 3.220 0.316 Propionic acid Pre 1.493 ± 1.139 0.029 1.444 ± 0.538 0.714 0.829 Post 2.117 ± 1.142 1.501 ± 0.690 0.012 AbsΔ 0.624 ± 1.571 0.058 ± 0.869 0.082 Butyric acid Pre 1.182 ± 0.939 0.764 1.232 ± 0.663 0.904 0.811 Post 1.246 ± 0.726 1.255 ± 0.734 0.962 AbsΔ 0.064 ± 1.217 0.024 ± 1.083 0.889 Isobutyric acid Pre 0.114 ± 0.063 0.952 1.106 ± 0.066 0.504 0.633 Post 0.114 ± 0.062 0.118 ± 0.080 0.854 AbsΔ 0.001 ± 0.084 0.012 ± 0.098 0.629 Valeric acid Pre 0.148 ± 0.094 0.840 0.158 ± 0.116 0.542 0.688 Post 0.153 ± 0.995 0.175 ± 0.120 0.413 AbsΔ 0.005 ± 0.140 0.0169 ± 0.153 0.745 Isobutyric acid Pre 0.183 ± 0.110 0.876 0.188 ± 0.140 0.842 0.861 Post 0.179 ± 0.094 0.181 ± 0.115 0.934 AbsΔ 0.004 ± 0.135 0.007 ± 0.194 0.936 Student's t test. 3.5 Effect of probiotics on fecal metabolome in patients with chronic constipation We investigated the alterations in fecal metabolites in patients with chronic constipation using a non-targeted LC-MS-based metabolomics method to determine the metabolic pathways and biomarkers most pertinent to understanding and treating disorders associated with constipation. Partial Least Squares Discriminant Analysis (PLS-DA) was used to predict the sample categories. The R2 and Q2 values of the PLSDA permutation test plots were lower than the initial R2 and Q2 values on the upper right, indicating a robust model (Figure 8). 3.5.1 Effects on fecal metabolites Using the PLS-DA multivariate statistical method and setting VIP (Projection of Importance Value of the First Principal Component Variable of PLS-DA) as >1 and p <0.05, 81 secondary differential metabolites were obtained, of which 41 were upregulated and 40 were downregulated (see Appendix). Differential metabolites associated with constipation were as follows: fecal metabolism associated with bile acid metabolism was significantly higher for 3-sulfinoalanine, significantly higher for prostaglandin I2, and lower for 8(S)-HPETE after the probiotic intervention compared to the pre-intervention. In addition, 3-methylindolepyruvate associated with caffeine metabolism, sphingosine associated with sphingolipid metabolism, and O-phosphoethanolamine were elevated, and lanosterin, 24,25-dihydrolanosterol, stigmasterol, and 24-methylenecycloartanol were decreased (Figure 9). 3.5.2 Effects on fecal metabolite enrichment Figure 8A shows the pathway enrichment of the top 20 pathways associated with constipation before and after the probiotic intervention. The pathways closely related to constipation were taurine and hypotaurine metabolism, primary bile acid biosynthesis, caffeine metabolism, and tryptophan metabolism. Other pathways include sphingolipid metabolism, amino acid metabolism, nerve-mediated signaling pathways, protein digestion and absorption, and phenylalanine metabolism. It can be seen that probiotics produced beneficial effects mainly through bile acid metabolism, caffeine metabolism, and tryptophan metabolism pathways. 3.6 Correlation analysis of metabolic differentials with gut differential flora Using Spearman's correlation analysis, we correlated the above differentially enriched metabolites for significant metabolic pathway correlations with the screened genus-level intestinal differential bacterial flora (Figure 8B). A cluster heat map was created using the obtained correlation coefficients. The results of the correlation analysis showed that Bacteroides abundance positively correlated with stigmasterol ( p <0.01), and Blautia abundance positively correlated with 5-aminolevulinic acid ( p <0.05) and negatively correlated with stigmasterol ( p <0.05). The abundance of Roseburia bacteria was positively correlated with O-phosphoethanolamine and 3-methylxanthine ( p <0.05) and negatively correlated with N-acetylputrescine, triethanolamine, and 3-methylindolepyruvate. Escherichia - Shigella abundance was positively correlated with 3-methylindolepyruvate and 2-arachidonoylglycerol ( p <0.05) and negatively correlated with 8(S)-HPETE ( p <0.05). DISCUSSION 4.1 Changes in defecation before and after the probiotic intervention To determine how Lacticaseibacillus paracasei 207-27 affected functional constipation, this study employed a randomized, double-blind, placebo-dense controlled trial. The study's results showed that the probiotic helped relieve the signs of constipation, making bowel movements occur more often and have better physical characteristics. People who were constipated and took Lacticaseibacillus paracasei 207-27 were about 1.5 times more likely to have four or more bowel movements per week than people who received a placebo. The probiotic started to work by the fourth week of treatment ( p <0.01), which suggests that long-term use of Lacticaseibacillus paracasei 207-27 increased the number of bowel movements in individuals with constipation. In the second 2 weeks of the intervention, the probiotic group had significantly better stool characteristics. Their mean BSFS scores were higher than those of the placebo group ( p <0.05), suggesting that Lacticaseibacillus paracasei 207-27 could help people who are constipated pass their stools more quickly through their intestines. Overall, the probiotics' healing effect on the constipation complaints was strongest in the fourth week ( p <0.01). This may indicate that probiotics should be used for a prolonged time to ensure they function and stay effective. The probiotic and placebo groups showed significant improvement in constipation-related symptoms after 4 weeks of treatment based on the PAC-SYM and PAC-QOL questionnaires. Analysis of the simple effect of time on symptom scores showed statistically significant differences in scores at different time points within the group ( p <0.05), but no differences were found in the separate effects of either the symptom scores or the quality-of-life scores. It is similar to a meta-analysis of the placebo effect on chronic idiopathic constipation (11), which, similar to other functional gastrointestinal disorders, had a high placebo effect (4%–44%), possibly explaining the difference between improved fecal frequency under objective measurements and the lack of improvement in overall symptoms under subjective questionnaires, suggesting that psychological interventions could effectively treat constipation. Psychotherapies and antidepressant medications can alleviate the symptoms of functional gastrointestinal disorders in children and adults (12). 4.2 Fecal SCFA and 16S rRNA analysis before and after probiotic intervention At the end of the intervention, we performed SCFAs testing on fecal samples, and the total amount of produced acids was significantly increased in the probiotic group ( p <0.05) compared with that in the placebo group. Probiotics can modulate the microbial community by colonizing the intestines of patients so that more SCFAs are produced by bacteria that ferment undigested carbohydrates in dietary fibers. SCFAs increase propulsive contractile activities in the colon, decrease non-propulsive contractions, restore contraction efficiency, and promote colonic motility (13). Lacticaseibacillus paracasei 207-27 has been shown to improve constipation symptoms by increasing the concentration of short-chain fatty acids in patients with constipation. However, individual SCFAs can induce different changes in motility. For example, butyric acid stimulates colonic Escherichia coli cells to secrete 5-hydroxytryptamine via GPR41/43, promoting colonic motility (14, 15). It also acts as a deacetylase inhibitor and promotes choline acetyltransferase activity (16). This promotes the choline acetyltransferase ChAT gene transcription to produce cholinergic prokinetic effects (16). Propionate increases the secretory activity of the colon and inhibits non-propulsive contractions mediated by PYY or GLP-1 (17). This observation seems to contradict the findings of this study, showing a significant increase in propionate levels before and after the intervention ( p <0.05) and a significant difference compared with the placebo group ( p <0.05), possibly related to the constipation symptoms of the study participants. Studies on ruminants have suggested that methane (CH 4 ) production in the body can be reduced and converted into increased propionic acid production (both of which require H 2 consumption) in the animal's body by the intake of specific diets, thus reducing the amount of excreted methane gas, which is closely associated with constipation. Studies have shown that the degree of respiratory methane production in irritable bowel syndrome is correlated with constipation severity (18, 19). In IBS, individuals with constipation have higher respiratory methane levels (20). Based on an in vitro study of the effects of Bifidobacterium lactis and oligofructose symbiotic bacteria on the intestinal microbiota, the combination of these two types of bacteria reduced CH 4 production in patients with constipation (21). Lactobacillus may also play a role in reducing CH 4 production in individuals with constipation by converting it to propionic acid; however, this hypothesis needs to be confirmed by further studies. The subsequent 16S rRNA analysis showed no significant differences in alpha and beta diversity between the probiotic and placebo groups. Khalif et al. (22) also observed little difference in gut microbiota between healthy individuals and those with constipation unless the transport time was severely prolonged, possibly because the improvement in constipation symptoms was not through mutations at the macro level of the gut flora of the participant but rather through the fine-tuning of parts of the functional gut microbiota. The results of the experiment showed that the number of Lactobacillus in the probiotic group increased significantly and differed from that of the placebo group after 4 weeks of intervention, and there were no significant differences between Bifidobacterium , Faecalibacterium prausnitzii , Clostridium perfringens and Enterococcus faecalis . It has been shown that Lacticaseibacillus paracasei can increase the concentration of acetic acid in the intestinal tract, promote the synthesis of 5-HT in the colon, and reduce water reabsorption in the intestinal tract by enhancing intestinal peristalsis, which in turn promotes an increase in the water content of the faeces(23).On intergroup analysis, we found that the abundance of the Firmicutes phylum was lower in the probiotic group than in the placebo group after the intervention, consistent with previous observations of increased abundance of the thick-walled phylum and decreased abundance of the anaplastic phylum in patients with constipation (24, 25). The Roseburia abundance increased after the probiotic intervention. Roseburia can produce acetic, propionic, and butyric acids, improving intestinal biodiversity (26) and glucose tolerance (27). The abundance of Roseburia -associated bacteria negatively correlates with IBS symptoms (28). The abundance of Roseburia is significantly lower in patients with constipation than in healthy individuals (29). 4.3 Fecal metabolomics analysis before and after probiotic intervention We performed non-targeted metabolite analysis on two groups of fecal samples, and the signaling pathways associated with probiotic intervention for constipation were focused on the following three metabolic pathways: bile acid metabolism, caffeine metabolism, and tryptophan metabolism. Bile acids are synthesized in hepatocytes, excreted in the duodenum with bile via the hepatoenteric cycle, and approximately 95% are reabsorbed from the terminal ileum and transported back to the liver, whereas the remaining 5% of bile acids are converted into various intestinal bile acids in the colon by intestinal commensal bacteria. This metabolic crosstalk between bile acids and intestinal flora is involved in colonic motility, and it has been shown (30) that bile acid metabolism is positively correlated with colonic motility and that abnormal bile acid metabolism is an important feature of intestinal flora and fecal metabolism in patients with slow-transport constipation. In the fecal metabolite enrichment pathway results of the current study, the signaling pathways related to bile acid metabolism were taurine/hypo-taurine metabolism, major bile acid metabolism, and arachidonic acid metabolism, with a significant up-regulation of cysteine sulfinic acid. Taurine/hypo-taurine maintains its normal concentration and function through cysteine sulfinic acid synthesis and metabolism (31), which can be decarboxylated to hypo-taurine and then oxidized to taurine. We believe that the increase in the concentration of cysteine sulfinic acid suggests the activation of this pathway, and more bile acids are amidated by taurine before entry into the duodenum, which improves intestinal motility. However, higher than normal bile acid concentrations in the gut can instead cause diarrhea, and fecal bile acid concentrations have been found to correlate with symptom severity in diarrheal irritable bowel syndrome (32). In an untargeted metabolic analysis of serum from chronically constipated women of childbearing age versus healthy population controls by Liu et al.(8), constipation was associated with caffeine metabolic pathways, which is consistent with our findings. The results also showed that the caffeine metabolism-related metabolite methylxanthine was upregulated after the probiotic intervention, which we believe may be because methylxanthine increases caffeine production, stimulating intestinal motility to improve constipation (33). We showed that 3-methylindolepyruvate was associated with the tryptophan metabolic pathway in the enrichment analyses. Tryptophan metabolism is closely related to gastrointestinal motility (34). It is the precursor of 5-HT, essential for mood regulation, intestinal motility, and secretory activity (35), and has become a key player in the microbiota-gut-brain axis. Kurata et al. found that 3-methylindole causes intestinal epithelial cell dysfunction, closely related to inflammatory bowel disease (IBD) (36). However, the effects of 3-methylindolepyruvate, a 3-methylindole metabolite, on human health remain unknown. However, the microbial production of Aryl hydrocarbon receptor ligands in patients with IBD is reduced compared with that in healthy individuals. Only a few gut microorganisms (e.g., Lactobacillus spp.) have been studied (37). Peptostreptococcus russell can convert tryptophan to the AhR ligand (38), suggesting that Lacticaseibacillus paracasei can promote epithelial cell recovery and maintain barrier integrity by metabolizing tryptophan to produce the AhR ligand. Our analysis also significantly enriched several pathways related to sphingolipid metabolism and neuromodulation, possibly related to the hypolipidemic and weight loss effects of Lacticaseibacillus paracasei (39-42). By analyzing the correlation between different bacteria and metabolites before and after probiotic intervention, we found that Roseburia spp. abundance positively correlated with 3-methylxanthine and negatively correlated with 3-methylindolepyruvate, suggesting that Roseburia spp. was closely associated with caffeine and tryptophan metabolism; further studies on this association should be conducted. Our study also has some limitations. The trial period was short, with probiotic/placebo administration lasting 28 days, and it is still unknown whether the long-term application of probiotics can produce stronger effects. There was no relevant follow-up to examine the change in symptom-relieving effects seen in this study when probiotic intake was discontinued. In summary, we showed that Lacticaseibacillus paracasei 207-27 improved constipation symptoms, increased short-chain fatty acid production, and affected intestinal flora and metabolites. The roles of Lacticaseibacillus paracasei 207-27 in constipation might be related to primary bile acid biosynthesis and the metabolism of taurine, hypotaurine, arachidonic acid, caffeine, tryptophan, and bile acid. 3-Methylxanthine may serve as potential markers for constipation intervention by this probiotic strain. The findings of this study may help develop new treatments for improving chronic constipation. Declarations Ethics approval and consent to participate The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Scientific Research Ethics Committee, Hangzhou Normal University. Written informed consent was obtained from individual or guardian participants. Consent for publication Not applicable. ACKNOWLEDGMENTS We would like to thank The Affiliated Hospital of Hangzhou Normal University，as well as BY-HEALTH Co., Ltd. of China. We are indebted to all the patients taking part in the study. Funding This study was funded by "Pioneer" and "Leading Goose" R&D Program of Zhejiang (Grant No. 2022C03138), "Active Health and Aging Science and Technology Response" of National Key R & Program of China (Grant No. 2022YFC2010101), BY-HEALTH Co., Ltd. of China. Conflict-of-Interest Statement The authors declare no conflict of interest. Author contributions Jinjun Li, Yanjun An, Xiaolei Ze: funding acquisition and project administration. Jinjun Li, Xiaoling Zhang, Yong Jia, Xiaoqiong Li: investigation. Liying Zhu, Xin Wang, Jinjun Li, Yanjun An, Xiaolei Ze: experimentation, data analysis, writing the original draft, reviewing, and editing. Jinjun Li, Yanjun An, Xiaolei Ze: reviewing and editing. Data availability The datasets generated and/or analyzed during the current study are available in the link: ncbi.nlm.nih.gov with accession number PRJNA1092834 References Forootan M, Bagheri N, Darvishi M. Chronic constipation: A review of literature. Medicine (Baltimore) 2018; 97:e10631. Johanson JF, Kralstein J. Chronic constipation: A survey of the patient perspective. Aliment Pharmacol Ther 2007; 25:599-608. Amdanee N, Shao M, Hu X, et al. Serum metabolic profile in schizophrenia patients with antipsychotic-induced constipation and its relationship with gut microbiome. Schizophr Bull 2023; 49:646-658. Wang L, Wang F, Zhang X, et al. Transdermal administration of volatile oil from citrus aurantium-rhizoma atractylodis macrocephalae alleviates constipation in rats by altering host metabolome and intestinal microbiota composition. Oxid Med Cell Longev 2022; 2022:9965334. Zhang X, Hu B, Sun G, et al. Plasma metabolomic pro fi les reveal regulatory effect of chitosan oligosaccharides on loperamide-induced constipation in mice. J Pharm Biomed Anal 2022; 211:114590. van der Schoot A, Helander C, Whelan K, et al. Probiotics and synbiotics in chronic constipation in adults: A systematic review and meta-analysis of randomized controlled trials. Clin Nutr 2022; 41:2759-2777. Yang L, Wang Y, Zhang Y, et al. Gut microbiota: A new avenue to reveal pathological mechanisms of constipation. Appl Microbiol Biotechnol 2022; 106:6899-6913. Liu S, Yang C, Li H, et al. Alteration of serum metabolites in women of reproductive age with chronic constipation. Med Sci Monit 2022; 28:e934117. Mearin F, Lacy BE, Chang L, et al. Bowel disorders. Gastroenterology 2016. Barichella M, Pacchetti C, Bolliri C, et al. Probiotics and prebiotic fiber for constipation associated with parkinson disease: An rct. Neurology 2016; 87:1274-80. Nee J, Sugarman MA, Ballou S, et al. Placebo response in chronic idiopathic constipation: A systematic review and meta-analysis. Am J Gastroenterol 2019; 114:1838-1846. Levy RL, Olden KW, Naliboff BD, et al. Psychosocial aspects of the functional gastrointestinal disorders. Gastroenterology 2006; 130:1447-58. Vincent AD, Wang XY, Parsons SP, et al. Abnormal absorptive colonic motor activity in germ-free mice is rectified by butyrate, an effect possibly mediated by mucosal serotonin. Am J Physiol Gastrointest Liver Physiol 2018; 315:G896-G907. Fukumoto S, Tatewaki M, Yamada T, et al. Short-chain fatty acids stimulate colonic transit via intraluminal 5-ht release in rats. Am J Physiol Regul Integr Comp Physiol 2003; 284:R1269-76. Karaki S, Mitsui R, Hayashi H, et al. Short-chain fatty acid receptor, gpr43, is expressed by enteroendocrine cells and mucosal mast cells in rat intestine. Cell Tissue Res 2006; 324:353-60. Soret R, Chevalier J, De Coppet P, et al. Short-chain fatty acids regulate the enteric neurons and control gastrointestinal motility in rats. Gastroenterology 2010; 138:1772-82. Cherbut C, Ferrier L, Roze C, et al. Short-chain fatty acids modify colonic motility through nerves and polypeptide yy release in the rat. Am J Physiol 1998; 275:G1415-22. Chatterjee S, Park S, Low K, et al. The degree of breath methane production in ibs correlates with the severity of constipation. Am J Gastroenterol 2007; 102:837-41. Teigen L, Mathai PP, Matson M, et al. Methanogen abundance thresholds capable of differentiating in vitro methane production in human stool samples. Dig Dis Sci 2021; 66:3822-3830. Kunkel D, Basseri RJ, Makhani MD, et al. Methane on breath testing is associated with constipation: A systematic review and meta-analysis. Dig Dis Sci 2011; 56:1612-8. Zhang Q, Zhao W, Zhao Y, et al. In vitro study of bifidobacterium lactis bl-99 with fructooligosaccharide synbiotics effected on the intestinal microbiota. Front Nutr 2022; 9:890316. Khalif IL, Quigley EM, Konovitch EA, et al. Alterations in the colonic flora and intestinal permeability and evidence of immune activation in chronic constipation. Dig Liver Dis 2005; 37:838-49. Wang L, Yang S, Mei C, et al. Lactobacillus paracasei relieves constipation by acting on the acetic acid-5-ht-intestinal motility pathway. Foods 2023; 12. Erhardt R, Harnett JE, Steels E, et al. Functional constipation and the effect of prebiotics on the gut microbiota: A review. Br J Nutr 2023; 130:1015-1023. Ohkusa T, Koido S, Nishikawa Y, et al. Gut microbiota and chronic constipation: A review and update. Front Med (Lausanne) 2019; 6:19. Fan P, Nelson CD, Driver JD, et al. Host genetics exerts lifelong effects upon hindgut microbiota and its association with bovine growth and immunity. ISME J 2021; 15:2306-2321. Neyrinck AM, Possemiers S, Verstraete W, et al. Dietary modulation of clostridial cluster xiva gut bacteria (roseburia spp.) by chitin-glucan fiber improves host metabolic alterations induced by high-fat diet in mice. J Nutr Biochem 2012; 23:51-9. Hou Y, Dong L, Lu X, et al. Distinctions between fecal and intestinal mucosal microbiota in subgroups of irritable bowel syndrome. Dig Dis Sci 2022; 67:5580-5592. Sciavilla P, Strati F, Di Paola M, et al. Gut microbiota profiles and characterization of cultivable fungal isolates in ibs patients. Appl Microbiol Biotechnol 2021; 105:3277-3288. Fan Y, Xu C, Xie L, et al. Abnormal bile acid metabolism is an important feature of gut microbiota and fecal metabolites in patients with slow transit constipation. Front Cell Infect Microbiol 2022; 12:956528. Stipanuk MH, Ueki I, Dominy JE, Jr., et al. Cysteine dioxygenase: A robust system for regulation of cellular cysteine levels. Amino Acids 2009; 37:55-63. Wei W, Wang H, Zhang Y, et al. Faecal bile acids and colonic bile acid membrane receptor correlate with symptom severity of diarrhoea-predominant irritable bowel syndrome: A pilot study. Dig Liver Dis 2021; 53:1120-1127. Komada Y, Narisawa H, Ueda F, et al. Relationship between self-reported dietary nutrient intake and self-reported sleep duration among japanese adults. Nutrients 2017; 9. Zheng Z, Tang J, Hu Y, et al. Role of gut microbiota-derived signals in the regulation of gastrointestinal motility. Front Med (Lausanne) 2022; 9:961703. Roth W, Zadeh K, Vekariya R, et al. Tryptophan metabolism and gut-brain homeostasis. Int J Mol Sci 2021; 22. Anonymous. !!! INVALID CITATION !!! (35). Lamas B, Natividad JM, Sokol H. Aryl hydrocarbon receptor and intestinal immunity. Mucosal Immunol 2018; 11:1024-1038. Wlodarska M, Luo C, Kolde R, et al. Indoleacrylic acid produced by commensal peptostreptococcus species suppresses inflammation. Cell Host Microbe 2017; 22:25-37 e6. Kalavathy R, Abdullah N, Jalaludin S, et al. Effects of lactobacillus cultures on growth performance, abdominal fat deposition, serum lipids and weight of organs of broiler chickens. Br Poult Sci 2003; 44:139-44. Wang C, Hu HJ, Dong QQ, et al. Enhancing bile tolerance of lactobacilli is involved in the hypolipidemic effects of liraglutide. Biosci Biotechnol Biochem 2021; 85:1395-1404. Ali SM, Salem FE, Aboulwafa MM, et al. Hypolipidemic activity of lactic acid bacteria: Adjunct therapy for potential probiotics. PLoS One 2022; 17:e0269953. Hamouda RA, Hamza HA, Salem ML, et al. Synergistic hypolipidemic and immunomodulatory activity of lactobacillus and spirulina platensis. Fermentation 2022; 8:220. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4108195","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":300390771,"identity":"f512156b-1f59-4384-b2cd-9c15af95da0f","order_by":0,"name":"Yanjun An","email":"","orcid":"","institution":"The First Hospital of Shanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanjun","middleName":"","lastName":"An","suffix":""},{"id":300390772,"identity":"24a8a450-5237-438a-bab1-20da96e50f94","order_by":1,"name":"Yong Jia","email":"","orcid":"","institution":"Shanxi Traditional Chinese Medicine Institute","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Jia","suffix":""},{"id":300390773,"identity":"dca9cb33-9538-436f-bc29-656a6780b36a","order_by":2,"name":"Xiaoling zhang","email":"","orcid":"","institution":"Shanxi Traditional Chinese Medicine Institute","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"zhang","suffix":""},{"id":300390774,"identity":"a1f818f4-fbdd-446f-806e-246b19e7d17d","order_by":3,"name":"Xiaoqiong Li","email":"","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqiong","middleName":"","lastName":"Li","suffix":""},{"id":300390775,"identity":"8c5289a5-b99e-4d39-853b-726db74e023d","order_by":4,"name":"Liying Zhu","email":"","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Liying","middleName":"","lastName":"Zhu","suffix":""},{"id":300390776,"identity":"baa89aa4-57ee-4d59-a6e3-887f88c18b16","order_by":5,"name":"Xin Wang","email":"","orcid":"","institution":"Shanxi Traditional Chinese Medicine Institute","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Wang","suffix":""},{"id":300390777,"identity":"248a8840-2c9c-4245-8b7f-ceabe657e80b","order_by":6,"name":"Xiaolei Ze","email":"","orcid":"","institution":"BYHEALTH Institute of Nutrition \u0026 Health","correspondingAuthor":false,"prefix":"","firstName":"Xiaolei","middleName":"","lastName":"Ze","suffix":""},{"id":300390778,"identity":"7bbf392f-1db8-4e9d-8730-30494ce3cb37","order_by":7,"name":"Jinjun Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACPmaGhAMMPAwM/ERrYYNpkWwgWguMYXCAaC3sDA8PF8gczjM+f/jhxx8MdvIM7Gfx6wY57PAMnsPFZgeOGUvzMCQbNvDkJRDWwsNzOHHbwQYDaQYG5gQGCR4D4rRsbmb//PMHQz0JWjaw8ZhJ8DAcJlpLeuKMMzxl1jwGxw3beHLwa+HnP5P8mbfHOrG///jmmz8qquX52c/g18LAwJPAwNgD4xggxRRuwH6AgeEHYWWjYBSMglEwggEADms6oHb/sXQAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jinjun","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-03-15 13:29:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4108195/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4108195/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56621651,"identity":"9b81a7fb-db9f-4783-a765-3821d22f2896","added_by":"auto","created_at":"2024-05-16 18:09:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2247980,"visible":true,"origin":"","legend":"\u003cp\u003eEnrollment of participants in the study.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/f6d043425c3aa8a7e5deafae.jpg"},{"id":56621654,"identity":"c801057c-9ecc-4bb5-b7ba-ff9894fa5cee","added_by":"auto","created_at":"2024-05-16 18:09:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2462772,"visible":true,"origin":"","legend":"\u003cp\u003eImprovement in constipation symptoms before and after intervention in the probiotics and placebo groups\u003c/p\u003e\n\u003cp\u003eA, No. of B.M.s/week, number of bowel movements per week. B, BSFS, Bristol stool scale. C, PAC-SYM, patient assessment of constipation-symptoms. D, PAC-QOL, patient assessment of constipation - the quality of life. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, Two-Way Repeated Measures ANCOVA, Bonferroni Correction.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/5e897341cd39c634921c1dbe.jpg"},{"id":56621658,"identity":"5be29ddc-adef-4345-b172-d4ea32a47f0a","added_by":"auto","created_at":"2024-05-16 18:09:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2613457,"visible":true,"origin":"","legend":"\u003cp\u003eFecal microbiology analysis using quantitative PCR\u003c/p\u003e\n\u003cp\u003eA, Bifidobacterium; B, Lactobacillus; C, \u003cem\u003eEnterococcus faecium\u003c/em\u003e; D, \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, were considered with significant differences. Student's t test. PCR, polymerase chain reaction\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/d89f92ccd880de50b3200a9a.jpg"},{"id":56621655,"identity":"25d4bf32-ab31-45d0-a5bd-e2d194a5b45d","added_by":"auto","created_at":"2024-05-16 18:09:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":908441,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha diversity estimates are represented by Chao 1 diversity, Simpson, or Shannon diversity\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/94d1090ed90b11609643d889.jpg"},{"id":56621659,"identity":"5469f16e-a2a2-4627-95a8-ffb36a25e1cb","added_by":"auto","created_at":"2024-05-16 18:09:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":906912,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Component Analysis (PCA) of the gut microbiota\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/e771b6a31a9d9344527f5af7.jpg"},{"id":56621653,"identity":"b991541b-9183-4b9e-89dc-df6a9727d5ca","added_by":"auto","created_at":"2024-05-16 18:09:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1093151,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial microbiota composition. A, Microflora composition at the plylum level. B, Microflora composition at the genus level.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/8f1fd3437b5a12be2c34a2e0.jpg"},{"id":56621656,"identity":"19815411-2e79-4dd6-a45a-3602d1e87e04","added_by":"auto","created_at":"2024-05-16 18:09:25","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2408202,"visible":true,"origin":"","legend":"\u003cp\u003eTotal acid (A), acetic acid (B), propionic acid (C), butyric acid (D), isobutyric acid (E), valeric acid (F), and isovaleric acid (G). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, were considered with significant differences. Student's t test.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/effd8a76461ad09163117940.jpg"},{"id":56621652,"identity":"286965b7-09ca-4014-afc8-cedf8ca33a0f","added_by":"auto","created_at":"2024-05-16 18:09:24","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1838352,"visible":true,"origin":"","legend":"\u003cp\u003eA, PLS-DA Plot (pre-placebo vs post-probiotic). B, Permutation test (pre-probiotic vs post-probiotic).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/8cce1d76295a4f495a162a51.jpg"},{"id":56621657,"identity":"89995176-93ec-4637-bf70-c800d8273798","added_by":"auto","created_at":"2024-05-16 18:09:25","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2569218,"visible":true,"origin":"","legend":"\u003cp\u003eA, KEGG pathway analysis. The X-axis represents the Impact value of enrichment on different metabolic pathways, while the Y-axis represents the metabolic pathways. The numbers represent the corresponding metabolites in the pathway: B, Differentiated microbiota, and differential metabolite correlation analysis heatmap.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/8b68d6e4f4346e3707c9b547.jpg"},{"id":77398210,"identity":"051f88bc-2257-45d1-b0b4-eda10a3f4c1d","added_by":"auto","created_at":"2025-02-28 08:02:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18295542,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4108195/v1/de78f20d-387b-42d6-af8d-83ca34fa866a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Lacticaseibacillus paracasei 207-27 on intestinal function and its regulatory mechanism in patients with chronic constipation","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eChronic constipation is a functional gastrointestinal tract disease caused by multiple factors, and its incidence is increasing annually (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), significantly affecting the overall well-being of affected individuals. It may cause intestinal flora disturbance, abnormal intestinal peristalsis, and intestinal water absorption disorder. Based on the variability of traditional drug therapies and dietary controls, nearly half of the patients are unsatisfied with the post-treatment symptoms (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Probiotics may improve chronic constipation by controlling the metabolic processes and the composition of the intestinal flora, improving intestinal motility and water absorption (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), and are used for treating chronic diseases and are becoming a potential therapy for constipation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e); however, the efficacy and safety of probiotic treatment for chronic constipation remain controversial.\u003c/p\u003e \u003cp\u003eRecently, untargeted metabolomics has been explored to study the pathogenesis and treatment of chronic constipation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). By comparing the metabolomic data of patients and healthy participants, differences in the metabolite levels in patients with chronic constipation can be detected, providing insights into the pathophysiological mechanisms of chronic constipation. Untargeted metabolomics can be used to diagnose chronic constipation and provide guidance for probiotic therapies. To examine how chitosan affected the abnormalities in plasma metabolomes in mice that had trouble going to the bathroom, Zhang et al. identified metabolic pathways related to sphingolipids, glycerophospholipids, and tryptophan using ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Several metabolomics studies identified specific metabolic abnormalities in patients with chronic constipation, such as disturbances in amino acid metabolism and abnormal bile acid metabolism (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). These abnormalities may change the composition and function of intestinal bacteria, affecting how the intestine moves and absorbs water. However, the underlying mechanisms of constipation-associated metabolic abnormalities are still largely unknown.\u003c/p\u003e \u003cp\u003eThis study aimed to explore further the roles and mechanisms of probiotic treatment of chronic constipation, which could help develop new strategies for improving its symptoms.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1 Ethical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was registered in the Clinical Trials Database (ID: ChiCTR2200056274), conducted at The Affiliated Hospital of Hangzhou Normal University, and approved by the Ethics Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included 80 patients with constipation. The inclusion criteria were as follows: meeting the Rome IV diagnostic criteria\u0026nbsp;(9), age 18\u0026ndash;65 years old, and having a frequency of defecation 1\u0026ndash;3 times/week. The exclusion criteria were as follows: individuals diagnosed with gastrointestinal, neurological, cardiovascular, endocrine, renal, or other chronic diseases that may affect bowel movements; those who had used antibiotics within 1 month; those who had used medications to treat constipation or took probiotics within 2 weeks before enrollment; pregnant or lactating women; those who had allergies or were allergic to probiotics. The criteria for dropping out were as follows: poor compliance, inability to complete treatment as required,\u0026nbsp;voluntary withdrawal in the middle of the treatment or loss of visits, and\u0026nbsp;incomplete data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Study Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this\u0026nbsp;randomized, double-blind, placebo-dense controlled trial, the patients were randomized after considering the diary card and the\u0026nbsp;inclusion\u0026nbsp;and\u0026nbsp;exclusion\u0026nbsp;criteria to determine who met the criteria for this study. After preliminary evaluation by the investigator, those who met the inclusion criteria signed an informed consent form and were recruited into a 1-week introductory phase without taking any products to improve bowel movement. These patients were admitted into a 28-day trial of a probiotic or placebo intervention. Patients received one packet twice daily\u0026nbsp;before meals.\u0026nbsp;The participants were instructed to refrain from altering their dietary and lifestyle practices to avoid other probiotics and dietary fibers during the study and to fill in the Bowel Diary Cards every day (day 0\u0026ndash;28) to record their bowel symptoms and movements. They were also required to fill in the Quality of Life Scores (PAC-QOL) form and the Constipation Symptom Assessment Form (PAC-SYM ) on days 0, 14, and 28. The Constipation Symptom Assessment Form (PAC-SYM) and stool samples were collected on days 0 and 28 for microbial profiling and metabolomics analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Preparation of probiotics and placebo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProbiotic powder (1 g/bag, 2 billion cfu of \u003cem\u003eLa\u003c/em\u003e\u003cem\u003ecticaseibacillus paracasei\u003c/em\u003e 207-27) was selected as the probiotic (207-27 was originally isolated from healthy infant feces in China and it is now deposited at the Guangdong Microbial Culture Collection Center \u0026nbsp;under the Budapest Treaty, with deposit code GDMCC 60960,which also named Lacticaseibacillus paracasei LPB27 in its commercialized product). The placebo was formulated using maltodextrin (identical in appearance and flavor to the probiotic). Both products were manufactured by the BYHEALTH Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Result evaluations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndividual stool diary cards were used to compute the number of spontaneous complete bowel movements (SCBM) and Bristol Stool Form Scale (BSFS) per week. The primary outcome was the alteration in the average frequency of SCBM per week during the intervention, and the secondary outcomes were other signs of constipation-related symptoms, such as the amount of improvement in the number of SCBM per week and the scores for the quality of feces every week. The other outcomes were the quality-of-life (PAC-SYM) and constipation symptom assessment scale (PAC-QOL) on\u0026nbsp;days\u0026nbsp;0, 14, and 28. Stool samples were collected on days 0 and 28 and were stored at -80\u0026deg;C for subsequent microbial profiling and untargeted metabolomics\u0026nbsp;analysis. During the follow-up, safety outcomes, including adverse events, were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Analysis of short\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003echain fatty acids\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFecal samples were collected before and after administration, ensuring they were not contaminated with toilet water or urine. Fecal samples (2 g) were weighed, diluted 1:10 in sterile phosphate-buffered saline, and vortexed. The sample was centrifuged at 4\u0026deg;C, 11,000 rpm for 6 min, and the supernatant was collected and transferred to a sterile centrifuge tube. Then, 250 \u0026micro;L of the supernatant was placed into a new, clean container, and 50 \u0026micro;L of the crotonic acid mixture was added with a 1:5 ratio. After filtering through a 0.22 \u0026micro;m membrane, the sample was acidified and stored at -80\u0026deg;C until use. A gas chromatograph (GC-2010 Plus, Shimadzu, Agilent Technologies, Santa Clara, CA, USA) with a 1D mass spectrometer and a DB-FFAP column was used to determine the short chain fatty acid (SCFAs, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid) levels. The remaining fecal samples were stored in a -80\u0026deg;C freezer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Microbiological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from fecal samples, and 16S rRNA gene amplicons were sequenced using an Illumina PE250 at Minco Biotech (Hangzhou, China). The V4 region of the 16S rRNA gene was amplified using the double barcode primer pair 515F (5\u0026apos;-GTGCCAGCMGCCGCGG-3\u0026apos;)-907R (5\u0026apos;-CCGTCAATTCMTTTRAGTTT-3\u0026apos;). The sequencing results were analyzed with QIIME2 software, and the operational taxonomic units (OTUs) were grouped with sequences without repeating at a 97% identity level. These results were examined in detail. To determine the bacterial species, we compared representative sequences from all OTUs to the Silva and RDP databases. Principal component analysis (PCA) was used to examine beta diversity between groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Untargeted metabolomics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNomi performed the LC-MS untargeted metabolomic assays. Data preprocessing was performed by converting raw mass spectrometry downstream files to the mzXML file format using the MSConvert tool in the ProteoWizard software package (v3.0.8789). The R XCMS software package was used to obtain the quantitative list of substances, and the public databases HMDB, mass bank, LipidMaps, McCloud, KEGG, and the self-constructed substance library were used to identify substances. The R package Ropls was used to perform PCA, partial least squares discriminant analysis, and orthogonal partial least squares discriminant analysis downscaling on the sample data. Metabolite molecules were considered statistically significant when the \u003cem\u003ep-\u003c/em\u003evalue was \u0026lt;0.05 and VIP \u0026gt;1. Pathway analysis, functional pathway enrichment, and topological analysis of the screened differential metabolite molecules were performed using the MetaboAnalyst software package. Using the KEGG Mapper display tool, the pathways identified in the enrichment analysis were used to examined the differential metabolite and pathway maps.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSample size calculations were performed as described previously\u0026nbsp;(10). The primary endpoint was the percentage of patients with SCBM, which was used as an indicator, and a superiority test was used for estimation. If the sample size according to the ratio of those who had four or more bowel movements per week in the intervention group was 80%, and 50% in the control group, with \u0026alpha; of 0.05, \u0026beta; of 80%, and an equal proportion of participants in the two groups, the sample size of the intervention group and the control group should be 28, respectively, considering the possibility of intermediate dropouts. Therefore, it was reasonable to include 40 people in each group.\u003c/p\u003e\n\u003cp\u003eThe primary and secondary objectives were to assess the population intention-to-treat effect. All data processing and statistical analyses were performed using SPSS v25.0 and GraphPad Prism v.8.0.2 (263). Study data were expressed as mean \u0026plusmn; standard deviation (SD) or standard error of the mean (SEM). Two-Way Repeated Measures ANOVA and Bonferroni correction were used for multiple group comparisons. The association was determined using Spearman\u0026apos;s linear correlation analysis. Differences were considered statistically significant at \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1 Study parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this trial, 80 patients were enrolled and randomized into two groups of 40 patients each. Sixteen patients did not complete the study, ten were lost to follow-up, and six did not comply with the required consecutive intervention. The final analysis included 64 individuals, of which 33 were in the probiotic group and 31 were in the placebo group (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Baseline characteristics of the participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe baseline characteristics of the participants are presented in Table 1. There were no statistically significant differences in age, sex, body mass index (BMI), or fecal consistency between the groups (\u003cem\u003ep\u003c/em\u003e\u0026gt; 0.05). The population with constipation comprised more women (75%) than men (25%). The participants were considered to have functional constipation according to the Rome IV criteria. Differences in functional constipation parameters PAC-SYM, PAC-QOL, BSFS, and N0. BM/week at baseline were insignificant (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Demographic and baseline characteristics of participants in the clinical trial.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"541\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.00184842883549%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.475046210720887%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlacebo (n=31)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.32347504621072%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProbiotic (n=33)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.199630314232902%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"39.00184842883549%\"\u003e\n \u003cp\u003eSex (male/female)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.475046210720887%\"\u003e\n \u003cp\u003e6 (0.19)/25 (0.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.32347504621072%\"\u003e\n \u003cp\u003e10 (0.30)/23 (0.70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.199630314232902%\" rowspan=\"7\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.421052631578945%\"\u003e\n \u003cp\u003eAge (year) (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.736842105263158%\"\u003e\n \u003cp\u003e36.5 \u0026plusmn; 15.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.842105263157894%\"\u003e\n \u003cp\u003e39.03 \u0026plusmn; 15.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.421052631578945%\"\u003e\n \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e) (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.736842105263158%\"\u003e\n \u003cp\u003e22.1 \u0026plusmn; 3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.842105263157894%\"\u003e\n \u003cp\u003e22.5 \u0026plusmn; 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.421052631578945%\"\u003e\n \u003cp\u003ePAC-QOL (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.736842105263158%\"\u003e\n \u003cp\u003e72.94 \u0026plusmn; 17.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.842105263157894%\"\u003e\n \u003cp\u003e70.55 \u0026plusmn; 16.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.421052631578945%\"\u003e\n \u003cp\u003ePAC-SYM (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.736842105263158%\"\u003e\n \u003cp\u003e1.40 \u0026plusmn; 0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.842105263157894%\"\u003e\n \u003cp\u003e1.35 \u0026plusmn; 0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.421052631578945%\"\u003e\n \u003cp\u003eNo. of BMs/week (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.736842105263158%\"\u003e\n \u003cp\u003e2.58 \u0026plusmn; 0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.842105263157894%\"\u003e\n \u003cp\u003e2.55 \u0026plusmn; 0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"44.421052631578945%\"\u003e\n \u003cp\u003eBSFS (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.736842105263158%\"\u003e\n \u003cp\u003e2.68 \u0026plusmn; 0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.842105263157894%\"\u003e\n \u003cp\u003e2.52 \u0026plusmn; 0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData were presented as mean \u0026plusmn; SD. BMI, body mass index; bpm, beats per minute; BSFS, Bristol stool form scale; No. of BMs/week, number of bowel movements per week; PAC-QOL, patient assessment of constipation quality of life; PAC-SYM, patient assessment of constipation symptoms; SD, standard deviation. There were no statistically significant differences across the three groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Clinical efficacy analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSixty-four participants were included in the efficacy analysis, with SCBM as the primary efficacy endpoint and the BSFS Stool Scale as the secondary efficacy endpoint. At the end of the 4-week intervention, there was a significant increase in the number of bowel movements (5.12\u0026plusmn;1.71) in the probiotic group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01) compared to the placebo group (3.77\u0026plusmn;1.56). The Bristol stool form scale of constipated patients in the probiotic group also significantly improved at week 4 (4.20\u0026plusmn;0.82) compared to week 0 (2.48\u0026plusmn;0.67). The probiotics showed more effectiveness; this effect started to show at week 2 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) and was more pronounced at week 4 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). This suggests that probiotic treatment is more favorable than placebo to improving symptoms associated with constipation in patients.\u003c/p\u003e\n\u003cp\u003eAfter 4 weeks of intervention, patients with constipation in both the probiotic and placebo groups experienced symptomatic relief. PAC-SYM scores were significantly lower (0.80\u0026plusmn;0.13) in the probiotic group compared to baseline (1.35\u0026plusmn;0.51). Although there was an interaction between time and the group of PAC-SYM scores (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), the separate effect of the group was not statistically significant. PAC-QOL scores were substantially lower in both the probiotic and placebo groups compared to the baseline level, but there was no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05), which may be attributed to the placebo effect (Table 2 and Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2.\u003c/strong\u003e Changes from baseline in\u0026nbsp;clinical efficacy parameters of functional constipation in participants receiving placebo or probiotic capsules.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"571\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eScore per week\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e\u003cstrong\u003eProbiotic (n=\u003c/strong\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003elacebo\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(n=\u003c/strong\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"49%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"51%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of BM/week\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e2.42\u0026nbsp;\u0026plusmn; 0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e2.58\u0026nbsp;\u0026plusmn; 0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.334\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e4.55\u0026nbsp;\u0026plusmn;\u0026nbsp;2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e3.84\u0026nbsp;\u0026plusmn; 1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e4.76\u0026nbsp;\u0026plusmn;\u0026nbsp;2.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e3.84\u0026nbsp;\u0026plusmn; 1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.055\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e5.03\u0026nbsp;\u0026plusmn;\u0026nbsp;2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e4.39\u0026nbsp;\u0026plusmn; 1.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.164\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e5.12\u0026nbsp;\u0026plusmn; 1.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e3.77\u0026nbsp;\u0026plusmn; 1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eBSFS average\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e2.48\u0026nbsp;\u0026plusmn; 0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e2.64\u0026nbsp;\u0026plusmn; 0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.412\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e3.71\u0026nbsp;\u0026plusmn;\u0026nbsp;1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e3.46\u0026nbsp;\u0026plusmn;\u0026nbsp;1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.390\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e4.13\u0026nbsp;\u0026plusmn;\u0026nbsp;1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e3.45\u0026nbsp;\u0026plusmn;\u0026nbsp;1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.010\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e4.17\u0026nbsp;\u0026plusmn;\u0026nbsp;0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e3.63\u0026nbsp;\u0026plusmn; 0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e4.20\u0026nbsp;\u0026plusmn;\u0026nbsp;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e3.54\u0026nbsp;\u0026plusmn;\u0026nbsp;1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003ePAC-SYM\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003escore\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e1.35\u0026nbsp;\u0026plusmn; 0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e1.34\u0026nbsp;\u0026plusmn; 0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.957\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e0.86\u0026nbsp;\u0026plusmn; 0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e0.82\u0026nbsp;\u0026plusmn; 0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.703\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e0.55\u0026nbsp;\u0026plusmn; 0.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e0.62\u0026nbsp;\u0026plusmn; 0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.467\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003ePAC-QOL score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e70.45\u0026nbsp;\u0026plusmn; 16.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e72.13\u0026nbsp;\u0026plusmn; 16.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.693\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e52.27\u0026nbsp;\u0026plusmn;\u0026nbsp;14.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e54.58\u0026nbsp;\u0026plusmn;\u0026nbsp;15.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.541\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.394045534150614%\"\u003e\n \u003cp\u003eWeek 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.74430823117338%\"\u003e\n \u003cp\u003e49.48\u0026nbsp;\u0026plusmn; 13.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.79509632224168%\"\u003e\n \u003cp\u003e48.10\u0026nbsp;\u0026plusmn;\u0026nbsp;15.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.066549912434326%\"\u003e\n \u003cp\u003e0.702\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBSFS, Bristol stool scale; No. of BMs/week, number of bowel movements per week; PAC-QOL, patient assessment of constipation - quality of life; PAC-SYM, patient assessment of constipation - symptoms; S.D., standard deviation. ANCOVA, Bonferroni correction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Effects of probiotics on intestinal flora and their products in patients with chronic constipation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEffects on the main intestinal bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe detected Bifidobacterium, Lactobacillus, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, and \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e in both groups before and after the intervention by fluorescence quantitative polymerase chain reaction (FQ-PCR) method. The test data showed that the number of Lactobacillus in the probiotic group increased significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) with no significant changes in the numbers of Bifidobacterium, \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, and \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e . The number of Lactobacillus in the probiotic group was significantly different from the placebo group after the intervention(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), which may be related to the fact that the probiotic ingested was \u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u0026nbsp;\u003c/em\u003e(Figure 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Effects on the structure and composition of intestinal flora\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u0026alpha;-diversity values of pre-intervention (week 0), post-placebo intervention (post-placebo), and post-probiotic intervention (post-probiotic) were analyzed, and the differences in Chao1, Shannon, and Simpson indices among these three groups were not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05). Principal component analysis showed no significant clustering in the flora among the three groups (Figures 4\u0026ndash;5).\u003c/p\u003e\n\u003cp\u003eFigure 6 shows that, at the phylum level, Firmicutes bacterial gates in both groups increased after the intervention compared with the pre-intervention value, with a more pronounced increase in the placebo group and a significant difference between the two groups (post-placebo vs. post-probiotic, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001). The Actinomycetes phylum was significantly lower in the placebo group after the intervention (week 0 vs. post-placebo, \u003cem\u003ep\u003c/em\u003e=0.0053), with a significant difference between the two groups (post-placebo vs. post-probiotic, \u003cem\u003ep\u003c/em\u003e=0.0177). At the genus level, more Bifidobacterium spp. were in the post-intervention probiotic group than in the placebo group (post-placebo vs. post-probiotic, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001). The post-intervention probiotic group showed reduced Bacteroides spp. (week 0 vs. post-probiotic, \u003cem\u003ep\u003c/em\u003e=0.0001) and Blautia spp. Post-intervention probiotic group Bacteroides spp. abundance decreased (week0 vs. post-probiotic, \u003cem\u003ep\u003c/em\u003e=0.0001), Blautia spp. abundance decreased (week0 vs. post-probiotic, \u003cem\u003ep\u003c/em\u003e=0.0213,) Shigella spp. Escherichia-Shigella decreased in abundance (post-placebo vs. post-probiotic, \u003cem\u003ep\u003c/em\u003e=0.0257) and Rochesteria spp. increased in abundance (post-placebo vs. post-probiotic, \u003cem\u003ep\u003c/em\u003e=0.0202).At the species level, more Clostridium perfringens were in the post-placebo probiotic group than in the intervention group\u0026nbsp;(post-placebo\u0026nbsp;vs. post-probiotic，\u003cem\u003ep\u003c/em\u003e=0.037).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2 Impact of SCFA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;7\u0026nbsp;shows that\u0026nbsp;probiotic intervention increased total acid production, while the placebo group did not produce the same effect (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01).\u0026nbsp;Acetic acid content (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) and propionic acid content (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01) increased significantly after intervention with the probiotic, and propionic acid levels were significantly different from the placebo group after the intervention (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u0026nbsp;Butyric acid and valeric acid increased after probiotic intervention, but the difference was insignificant (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05).\u0026nbsp;All data in the placebo group were not statistically different from pre-intervention (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05)\u0026nbsp;(Table\u0026nbsp;3\u0026nbsp;and Figure\u0026nbsp;7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Changes from baseline in\u0026nbsp;SCFA\u0026nbsp;of functional constipation in participants receiving placebo or probiotic capsules.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"545\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eScore\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.211009174311926%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eProbiotic (n=\u003c/strong\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"36.14678899082569%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003elacebo\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(n=\u003c/strong\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.04232804232804%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.841269841269842%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.158730158730158%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.95767195767196%\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003ePre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e7.528\u0026nbsp;\u0026plusmn;\u0026nbsp;3.083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e7.739\u0026nbsp;\u0026plusmn;\u0026nbsp;2.315\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.336\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.759\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.188630490956072%\"\u003e\n \u003cp\u003ePost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.39018087855297%\"\u003e\n \u003cp\u003e9.564\u0026nbsp;\u0026plusmn;\u0026nbsp;2.975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.45736434108527%\"\u003e\n \u003cp\u003e8.394\u0026nbsp;\u0026plusmn;\u0026nbsp;2.898\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.963824289405686%\"\u003e\n \u003cp\u003e0.117\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003eAbs\u0026Delta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e2.036\u0026nbsp;\u0026plusmn;\u0026nbsp;4.140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.655\u0026nbsp;\u0026plusmn;\u0026nbsp;3.730\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.167\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcetic acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003ePre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e4.409\u0026nbsp;\u0026plusmn;\u0026nbsp;2.322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e4.612\u0026nbsp;\u0026plusmn;\u0026nbsp;2.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.348\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.713\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.188630490956072%\"\u003e\n \u003cp\u003ePost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.39018087855297%\"\u003e\n \u003cp\u003e5.754\u0026nbsp;\u0026plusmn;\u0026nbsp;2.164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.45736434108527%\"\u003e\n \u003cp\u003e5.163\u0026nbsp;\u0026plusmn;\u0026nbsp;2.451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.963824289405686%\"\u003e\n \u003cp\u003e0.310\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003eAbs\u0026Delta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e1.346\u0026nbsp;\u0026plusmn;\u0026nbsp;3.061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.552\u0026nbsp;\u0026plusmn;\u0026nbsp;3.220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.316\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003ePropionic acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003ePre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e1.493\u0026nbsp;\u0026plusmn;\u0026nbsp;1.139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e1.444\u0026nbsp;\u0026plusmn; 0.538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.714\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.829\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.188630490956072%\"\u003e\n \u003cp\u003ePost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.39018087855297%\"\u003e\n \u003cp\u003e2.117\u0026nbsp;\u0026plusmn;\u0026nbsp;1.142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.45736434108527%\"\u003e\n \u003cp\u003e1.501\u0026nbsp;\u0026plusmn; 0.690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.963824289405686%\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003eAbs\u0026Delta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e0.624\u0026nbsp;\u0026plusmn;\u0026nbsp;1.571\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.058\u0026nbsp;\u0026plusmn;\u0026nbsp;0.869\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.082\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eButyric acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003ePre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e1.182\u0026nbsp;\u0026plusmn;\u0026nbsp;0.939\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.764\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e1.232\u0026nbsp;\u0026plusmn;\u0026nbsp;0.663\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.904\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.811\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.188630490956072%\"\u003e\n \u003cp\u003ePost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.39018087855297%\"\u003e\n \u003cp\u003e1.246\u0026nbsp;\u0026plusmn;\u0026nbsp;0.726\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.45736434108527%\"\u003e\n \u003cp\u003e1.255\u0026nbsp;\u0026plusmn;\u0026nbsp;0.734\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.963824289405686%\"\u003e\n \u003cp\u003e0.962\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003eAbs\u0026Delta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e0.064\u0026nbsp;\u0026plusmn;\u0026nbsp;1.217\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.024\u0026nbsp;\u0026plusmn;\u0026nbsp;1.083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.889\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsobutyric acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003ePre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e0.114\u0026nbsp;\u0026plusmn; 0.063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e1.106\u0026nbsp;\u0026plusmn; 0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.633\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.188630490956072%\"\u003e\n \u003cp\u003ePost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.39018087855297%\"\u003e\n \u003cp\u003e0.114\u0026nbsp;\u0026plusmn; 0.062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.45736434108527%\"\u003e\n \u003cp\u003e0.118\u0026nbsp;\u0026plusmn; 0.080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.963824289405686%\"\u003e\n \u003cp\u003e0.854\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003eAbs\u0026Delta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e0.001\u0026nbsp;\u0026plusmn;\u0026nbsp;0.084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.012\u0026nbsp;\u0026plusmn;\u0026nbsp;0.098\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.629\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eValeric acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003ePre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e0.148\u0026nbsp;\u0026plusmn;\u0026nbsp;0.094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.840\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.158\u0026nbsp;\u0026plusmn;\u0026nbsp;0.116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.542\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.688\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.188630490956072%\"\u003e\n \u003cp\u003ePost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.39018087855297%\"\u003e\n \u003cp\u003e0.153\u0026nbsp;\u0026plusmn;\u0026nbsp;0.995\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.45736434108527%\"\u003e\n \u003cp\u003e0.175\u0026nbsp;\u0026plusmn;\u0026nbsp;0.120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.963824289405686%\"\u003e\n \u003cp\u003e0.413\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003eAbs\u0026Delta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e0.005\u0026nbsp;\u0026plusmn;\u0026nbsp;0.140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.0169\u0026nbsp;\u0026plusmn;\u0026nbsp;0.153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.745\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsobutyric acid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003ePre\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e0.183\u0026nbsp;\u0026plusmn; 0.110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.188\u0026nbsp;\u0026plusmn; 0.140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\" rowspan=\"2\"\u003e\n \u003cp\u003e0.842\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.861\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.188630490956072%\"\u003e\n \u003cp\u003ePost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.39018087855297%\"\u003e\n \u003cp\u003e0.179\u0026nbsp;\u0026plusmn; 0.094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.45736434108527%\"\u003e\n \u003cp\u003e0.181\u0026nbsp;\u0026plusmn; 0.115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.963824289405686%\"\u003e\n \u003cp\u003e0.934\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.045871559633028%\"\u003e\n \u003cp\u003eAbs\u0026Delta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.44954128440367%\"\u003e\n \u003cp\u003e0.004\u0026nbsp;\u0026plusmn;\u0026nbsp;0.135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.761467889908257%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.91743119266055%\"\u003e\n \u003cp\u003e0.007\u0026nbsp;\u0026plusmn;\u0026nbsp;0.194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.229357798165138%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.596330275229358%\"\u003e\n \u003cp\u003e0.936\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eStudent\u0026apos;s t test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Effect of probiotics on fecal metabolome in patients with chronic constipation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated the alterations in fecal metabolites in patients with chronic constipation using a non-targeted LC-MS-based metabolomics method to determine the metabolic pathways and biomarkers most pertinent to understanding and treating disorders associated with constipation. Partial Least Squares Discriminant Analysis (PLS-DA) was used to predict the sample categories. The R2 and Q2 values of the PLSDA permutation test plots were lower than the initial R2 and Q2 values on the upper right, indicating a robust model (Figure 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.1\u0026nbsp;\u003c/strong\u003eEffects on fecal metabolites\u003c/p\u003e\n\u003cp\u003eUsing the PLS-DA multivariate statistical method and setting VIP (Projection of Importance Value of the First Principal Component Variable of PLS-DA) as \u0026gt;1 and \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, 81 secondary differential metabolites were obtained, of which 41 were upregulated and 40 were downregulated (see Appendix). Differential metabolites associated with constipation were as follows: fecal metabolism associated with bile acid metabolism was significantly higher for 3-sulfinoalanine, significantly higher for prostaglandin I2, and lower for 8(S)-HPETE after the probiotic intervention compared to the pre-intervention. In addition, 3-methylindolepyruvate associated with caffeine metabolism, sphingosine associated with sphingolipid metabolism, and O-phosphoethanolamine were elevated, and lanosterin, 24,25-dihydrolanosterol, stigmasterol, and 24-methylenecycloartanol were decreased (Figure 9).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.2 Effects on fecal metabolite enrichment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 8A shows the pathway enrichment of the top 20 pathways associated with constipation before and after the probiotic intervention. The pathways closely related to constipation were taurine and hypotaurine metabolism, primary bile acid biosynthesis, caffeine metabolism, and tryptophan metabolism. Other pathways include sphingolipid metabolism, amino acid metabolism, nerve-mediated signaling pathways, protein digestion and absorption, and phenylalanine metabolism. It can be seen that probiotics produced beneficial effects mainly through bile acid metabolism, caffeine metabolism, and tryptophan metabolism pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Correlation analysis of metabolic differentials with gut differential flora\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing Spearman\u0026apos;s correlation analysis, we correlated the above differentially enriched metabolites for significant metabolic pathway correlations with the screened genus-level intestinal differential bacterial flora\u0026nbsp;(Figure 8B). A cluster heat map was created using the obtained correlation coefficients. The results of the correlation analysis showed that \u003cem\u003eBacteroides\u0026nbsp;\u003c/em\u003eabundance positively correlated with stigmasterol (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), and \u003cem\u003eBlautia\u003c/em\u003e abundance positively correlated with 5-aminolevulinic acid (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) and negatively correlated with stigmasterol (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). The abundance of \u003cem\u003eRoseburia\u003c/em\u003e bacteria was positively correlated with O-phosphoethanolamine and 3-methylxanthine (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) and negatively correlated with N-acetylputrescine, triethanolamine, and 3-methylindolepyruvate. \u003cem\u003eEscherichia\u003c/em\u003e-\u003cem\u003eShigella\u003c/em\u003e abundance was positively correlated with 3-methylindolepyruvate and 2-arachidonoylglycerol (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) and negatively correlated with 8(S)-HPETE (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003e4.1 Changes in defecation before and after the probiotic intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine how\u0026nbsp;\u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e 207-27 affected functional constipation, this study employed a randomized, double-blind, placebo-dense controlled trial. The study's results showed that the probiotic helped relieve the signs of constipation, making bowel movements occur more often and have better physical characteristics. People who were constipated and took\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u003c/em\u003e 207-27 were about 1.5 times more likely to have four or more bowel movements per week than people who received a placebo. The probiotic started to work by the fourth week of treatment (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), which suggests that long-term use of\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u003c/em\u003e 207-27 increased the number of bowel movements in individuals with constipation. In the second 2 weeks of the intervention, the probiotic group had significantly better stool characteristics. Their mean BSFS scores were higher than those of the placebo group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), suggesting that\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u003c/em\u003e 207-27 could help people who are constipated pass their stools more quickly through their intestines. Overall, the probiotics' healing effect on the constipation complaints was strongest in the fourth week (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). This may indicate that probiotics should be used for a prolonged time to ensure they function and stay effective.\u003c/p\u003e\n\u003cp\u003eThe probiotic and placebo groups showed significant improvement in constipation-related symptoms after 4 weeks of treatment based on the PAC-SYM and PAC-QOL questionnaires. Analysis of the simple effect of time on symptom scores showed statistically significant differences in scores at different time points within the group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), but no differences were found in the separate effects of either the symptom scores or the quality-of-life scores.\u0026nbsp;It is\u0026nbsp;similar to a meta-analysis of the placebo effect on chronic idiopathic constipation\u0026nbsp;(11), which, similar to other functional gastrointestinal disorders, had a high placebo effect (4%–44%), possibly explaining the difference between improved fecal frequency under objective measurements and the lack of improvement in overall symptoms under subjective questionnaires, suggesting that psychological interventions could effectively treat constipation. Psychotherapies and antidepressant medications can alleviate the symptoms of functional gastrointestinal disorders in children and adults\u0026nbsp;(12).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Fecal SCFA and 16S rRNA analysis before and after probiotic intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the intervention, we performed SCFAs\u0026nbsp;testing on fecal samples, and the total amount of produced acids was significantly increased in the probiotic group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) compared with that in the placebo group. Probiotics can modulate the microbial community by colonizing the intestines of patients so that more SCFAs are produced by bacteria that ferment undigested carbohydrates in dietary fibers. SCFAs increase propulsive contractile activities in the colon, decrease non-propulsive contractions, restore contraction efficiency, and promote colonic motility\u0026nbsp;(13).\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u003c/em\u003e 207-27 has been shown to improve constipation symptoms by increasing the concentration of short-chain fatty acids in patients with constipation. However, individual SCFAs can induce different changes in motility. For example, butyric acid stimulates colonic \u003cem\u003eEscherichia coli\u003c/em\u003e cells to secrete 5-hydroxytryptamine via GPR41/43, promoting colonic motility\u0026nbsp;(14, 15). It also acts as a deacetylase inhibitor and promotes choline acetyltransferase activity\u0026nbsp;(16). This promotes the choline acetyltransferase \u003cem\u003eChAT\u003c/em\u003e gene transcription to produce cholinergic prokinetic effects\u0026nbsp;(16). Propionate increases the secretory activity of the colon and inhibits non-propulsive contractions mediated by PYY or GLP-1\u0026nbsp;(17). This observation seems to contradict the findings of this study, showing a significant increase in propionate levels before and after the intervention (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) and a significant difference compared with the placebo group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), possibly related to the constipation symptoms of the study participants. Studies on ruminants have suggested that methane (CH\u003csub\u003e4\u003c/sub\u003e) production in the body can be reduced and converted into increased propionic acid production (both of which require H\u003csub\u003e2\u003c/sub\u003econsumption) in the animal's body by the intake of specific diets, thus reducing the amount of excreted methane gas, which is closely associated with constipation. Studies have shown that the degree of respiratory methane production in irritable bowel syndrome\u0026nbsp;is correlated with constipation severity\u0026nbsp;(18, 19). In IBS, individuals with constipation have higher respiratory methane levels\u0026nbsp;(20). Based on an in vitro study of the effects of \u003cem\u003eBifidobacterium lactis\u003c/em\u003e and oligofructose symbiotic bacteria on the intestinal microbiota, the combination of these two types of bacteria reduced CH\u003csub\u003e4\u003c/sub\u003e production in patients with constipation\u0026nbsp;(21). \u003cem\u003eLactobacillus\u003c/em\u003e may also play a role in reducing CH\u003csub\u003e4\u003c/sub\u003e production in individuals with constipation by converting it to propionic acid; however, this hypothesis needs to be confirmed by further studies.\u003c/p\u003e\n\u003cp\u003eThe subsequent 16S rRNA analysis showed no significant differences in alpha and beta diversity between the\u0026nbsp;probiotic and placebo\u0026nbsp;groups. Khalif et al.\u0026nbsp;(22)\u0026nbsp;also observed little difference in gut microbiota between healthy individuals and those with constipation unless the transport time was severely prolonged, possibly because the improvement in constipation symptoms was not through mutations at the macro level of the gut flora of the participant but rather through the fine-tuning of parts of the functional gut microbiota. The results of the experiment showed that the number of Lactobacillus in the probiotic group increased significantly and differed from that of the placebo group after 4 weeks of intervention, and there were no significant differences between Bifidobacterium\u003cem\u003e,\u0026nbsp;\u003c/em\u003e\u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e, Clostridium perfringens and\u0026nbsp;\u003cem\u003eEnterococcus faecalis\u003c/em\u003e. It has been shown that\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e \u003cem\u003eparacasei\u003c/em\u003e can increase the concentration of acetic acid in the intestinal tract, promote the synthesis of 5-HT in the colon, and reduce water reabsorption in the intestinal tract by enhancing intestinal peristalsis, which in turn promotes an increase in the water content of the faeces(23).On intergroup analysis, we found that the abundance of the Firmicutes phylum was lower in the probiotic group than in the placebo group after the intervention, consistent with previous observations of increased abundance of the thick-walled phylum and decreased abundance of the anaplastic phylum in patients with constipation\u0026nbsp;(24, 25). The \u003cem\u003eRoseburia\u003c/em\u003e abundance increased after the probiotic intervention. \u003cem\u003eRoseburia\u003c/em\u003e can produce acetic, propionic, and butyric acids, improving intestinal biodiversity\u0026nbsp;(26)\u0026nbsp;and glucose tolerance\u0026nbsp;(27). The abundance of \u003cem\u003eRoseburia\u003c/em\u003e-associated bacteria negatively correlates with IBS symptoms\u0026nbsp;(28). The abundance of \u003cem\u003eRoseburia\u003c/em\u003e is significantly lower in patients with constipation than in healthy individuals\u0026nbsp;(29).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Fecal metabolomics analysis before and after probiotic intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed non-targeted metabolite analysis on two groups of fecal samples, and the signaling pathways associated with probiotic intervention for constipation were focused on the following three metabolic pathways: bile acid metabolism, caffeine metabolism, and tryptophan metabolism.\u003c/p\u003e\n\u003cp\u003eBile acids are synthesized in hepatocytes, excreted in the duodenum with bile via the hepatoenteric cycle, and approximately 95% are reabsorbed from the terminal ileum and transported back to the liver, whereas the remaining 5% of bile acids are converted into various intestinal bile acids in the colon by intestinal commensal bacteria. This metabolic crosstalk between bile acids and intestinal flora is involved in colonic motility, and it has been shown\u0026nbsp;(30)\u0026nbsp;that bile acid metabolism is positively correlated with colonic motility and that abnormal bile acid metabolism is an important feature of intestinal flora and fecal metabolism in patients with slow-transport constipation. In the fecal metabolite enrichment pathway results of the current study, the signaling pathways related to bile acid metabolism were taurine/hypo-taurine metabolism, major bile acid metabolism, and arachidonic acid metabolism, with a significant up-regulation of cysteine sulfinic acid. Taurine/hypo-taurine maintains its normal concentration and function through cysteine sulfinic acid synthesis and metabolism\u0026nbsp;(31), which can be decarboxylated to hypo-taurine and then oxidized to taurine. We believe that the increase in the concentration of cysteine sulfinic acid suggests the activation of this pathway, and more bile acids are amidated by taurine before entry into the duodenum, which improves intestinal motility. However, higher than normal bile acid concentrations in the gut can instead cause diarrhea, and fecal bile acid concentrations have been found to correlate with symptom severity in diarrheal irritable bowel syndrome\u0026nbsp;(32).\u003c/p\u003e\n\u003cp\u003eIn an untargeted metabolic analysis of serum from chronically constipated women of childbearing age versus healthy population controls by Liu et al.(8), constipation was associated with caffeine metabolic pathways, which is consistent with our findings. The results also showed that the caffeine metabolism-related metabolite methylxanthine was upregulated after the probiotic intervention, which we believe may be because methylxanthine increases caffeine production, stimulating intestinal motility to improve constipation\u0026nbsp;(33).\u003c/p\u003e\n\u003cp\u003eWe showed that 3-methylindolepyruvate was associated with the tryptophan metabolic pathway in the enrichment analyses. Tryptophan metabolism is closely related to gastrointestinal motility\u0026nbsp;(34). It is the precursor of 5-HT, essential for mood regulation, intestinal motility, and secretory activity\u0026nbsp;(35), and has become a key player in the microbiota-gut-brain axis. Kurata et al. found that 3-methylindole causes intestinal epithelial cell dysfunction, closely related to inflammatory bowel disease (IBD)\u0026nbsp;(36). However, the effects of 3-methylindolepyruvate, a 3-methylindole metabolite, on human health remain unknown. However, the microbial production of Aryl hydrocarbon receptor ligands in patients with IBD is reduced compared with that in healthy individuals. Only a few gut microorganisms (e.g., \u003cem\u003eLactobacillus\u003c/em\u003e spp.) have been studied\u0026nbsp;(37). \u003cem\u003ePeptostreptococcus russell\u003c/em\u003e can convert tryptophan to the AhR ligand\u0026nbsp;(38), suggesting that\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u0026nbsp;\u003c/em\u003ecan promote epithelial cell recovery and maintain barrier integrity by metabolizing tryptophan to produce the AhR ligand. Our analysis also significantly enriched several pathways related to sphingolipid metabolism and neuromodulation, possibly related to the hypolipidemic and weight loss effects of\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u003c/em\u003e (39-42). By analyzing the correlation between different bacteria and metabolites before and after probiotic intervention, we found that \u003cem\u003eRoseburia\u003c/em\u003e spp. abundance positively correlated with 3-methylxanthine and negatively correlated with 3-methylindolepyruvate, suggesting that \u003cem\u003eRoseburia\u0026nbsp;\u003c/em\u003espp. was closely associated with caffeine and tryptophan metabolism; further studies on this association should be conducted.\u003c/p\u003e\n\u003cp\u003eOur study also has some limitations.\u0026nbsp;The trial period was short, with probiotic/placebo administration lasting 28 days, and it is still unknown whether the long-term application of probiotics can produce stronger effects. There was no relevant follow-up to examine the change in symptom-relieving effects seen in this study when probiotic intake was discontinued.\u003c/p\u003e\n\u003cp\u003eIn summary, we showed that\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u003c/em\u003e 207-27 improved constipation symptoms, increased short-chain fatty acid production, and affected intestinal flora and metabolites. The roles of\u0026nbsp;\u003cem\u003eLacticaseibacillus\u003c/em\u003e\u003cem\u003e\u0026nbsp;paracasei\u003c/em\u003e 207-27 in constipation might be related to primary bile acid biosynthesis and the metabolism of taurine, hypotaurine, arachidonic acid, caffeine, tryptophan, and bile acid. 3-Methylxanthine may serve as potential markers for constipation intervention by this probiotic strain. The findings of this study may help develop new treatments for improving chronic constipation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was established, according to the ethical guidelines of the\u003c/p\u003e\n\u003cp\u003eHelsinki Declaration and was approved by the Scientific Research Ethics Committee, Hangzhou Normal University. Written informed consent was obtained from individual or guardian participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank\u0026nbsp;The Affiliated Hospital of Hangzhou Normal University，as well as\u0026nbsp;BY-HEALTH\u0026nbsp;Co.,\u0026nbsp;Ltd.\u0026nbsp;of China. We are indebted to all the patients taking part in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by \"Pioneer\" and \"Leading Goose\" R\u0026amp;D Program of Zhejiang (Grant No. 2022C03138), \"Active Health and Aging Science and Technology Response\" of National Key R \u0026amp; Program of China (Grant No. 2022YFC2010101), BY-HEALTH\u0026nbsp;Co.,\u0026nbsp;Ltd.\u0026nbsp;of China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict-of-Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJinjun Li,\u0026nbsp;Yanjun\u0026nbsp;An,\u0026nbsp;Xiaolei Ze: funding acquisition and project administration. Jinjun Li,\u0026nbsp;Xiaoling Zhang,\u0026nbsp;Yong\u0026nbsp;Jia, Xiaoqiong Li: investigation.\u0026nbsp;Liying Zhu, Xin Wang,\u0026nbsp;Jinjun Li,\u0026nbsp;Yanjun\u0026nbsp;An,\u0026nbsp;Xiaolei Ze: experimentation, data analysis, writing the original draft, reviewing, and editing. Jinjun Li,\u0026nbsp;Yanjun An,\u0026nbsp;Xiaolei Ze: reviewing and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available in the link:\u003c/p\u003e\n\u003cp\u003encbi.nlm.nih.gov with accession number PRJNA1092834\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eForootan M, Bagheri N, Darvishi M. Chronic constipation: A review of literature. Medicine (Baltimore) 2018; 97:e10631.\u003c/li\u003e\n\u003cli\u003eJohanson JF, Kralstein J. Chronic constipation: A survey of the patient perspective. Aliment Pharmacol Ther 2007; 25:599-608.\u003c/li\u003e\n\u003cli\u003eAmdanee N, Shao M, Hu X, et al. Serum metabolic profile in schizophrenia patients with antipsychotic-induced constipation and its relationship with gut microbiome. Schizophr Bull 2023; 49:646-658.\u003c/li\u003e\n\u003cli\u003eWang L, Wang F, Zhang X, et al. Transdermal administration of volatile oil from citrus aurantium-rhizoma atractylodis macrocephalae alleviates constipation in rats by altering host metabolome and intestinal microbiota composition. Oxid Med Cell Longev 2022; 2022:9965334.\u003c/li\u003e\n\u003cli\u003eZhang X, Hu B, Sun G, et al. Plasma metabolomic pro fi les reveal regulatory effect of chitosan oligosaccharides on loperamide-induced constipation in mice. J Pharm Biomed Anal 2022; 211:114590.\u003c/li\u003e\n\u003cli\u003evan der Schoot A, Helander C, Whelan K, et al. Probiotics and synbiotics in chronic constipation in adults: A systematic review and meta-analysis of randomized controlled trials. Clin Nutr 2022; 41:2759-2777.\u003c/li\u003e\n\u003cli\u003eYang L, Wang Y, Zhang Y, et al. Gut microbiota: A new avenue to reveal pathological mechanisms of constipation. Appl Microbiol Biotechnol 2022; 106:6899-6913.\u003c/li\u003e\n\u003cli\u003eLiu S, Yang C, Li H, et al. Alteration of serum metabolites in women of reproductive age with chronic constipation. Med Sci Monit 2022; 28:e934117.\u003c/li\u003e\n\u003cli\u003eMearin F, Lacy BE, Chang L, et al. Bowel disorders. Gastroenterology 2016.\u003c/li\u003e\n\u003cli\u003eBarichella M, Pacchetti C, Bolliri C, et al. Probiotics and prebiotic fiber for constipation associated with parkinson disease: An rct. Neurology 2016; 87:1274-80.\u003c/li\u003e\n\u003cli\u003eNee J, Sugarman MA, Ballou S, et al. Placebo response in chronic idiopathic constipation: A systematic review and meta-analysis. Am J Gastroenterol 2019; 114:1838-1846.\u003c/li\u003e\n\u003cli\u003eLevy RL, Olden KW, Naliboff BD, et al. Psychosocial aspects of the functional gastrointestinal disorders. Gastroenterology 2006; 130:1447-58.\u003c/li\u003e\n\u003cli\u003eVincent AD, Wang XY, Parsons SP, et al. Abnormal absorptive colonic motor activity in germ-free mice is rectified by butyrate, an effect possibly mediated by mucosal serotonin. Am J Physiol Gastrointest Liver Physiol 2018; 315:G896-G907.\u003c/li\u003e\n\u003cli\u003eFukumoto S, Tatewaki M, Yamada T, et al. Short-chain fatty acids stimulate colonic transit via intraluminal 5-ht release in rats. Am J Physiol Regul Integr Comp Physiol 2003; 284:R1269-76.\u003c/li\u003e\n\u003cli\u003eKaraki S, Mitsui R, Hayashi H, et al. Short-chain fatty acid receptor, gpr43, is expressed by enteroendocrine cells and mucosal mast cells in rat intestine. Cell Tissue Res 2006; 324:353-60.\u003c/li\u003e\n\u003cli\u003eSoret R, Chevalier J, De Coppet P, et al. Short-chain fatty acids regulate the enteric neurons and control gastrointestinal motility in rats. Gastroenterology 2010; 138:1772-82.\u003c/li\u003e\n\u003cli\u003eCherbut C, Ferrier L, Roze C, et al. Short-chain fatty acids modify colonic motility through nerves and polypeptide yy release in the rat. Am J Physiol 1998; 275:G1415-22.\u003c/li\u003e\n\u003cli\u003eChatterjee S, Park S, Low K, et al. The degree of breath methane production in ibs correlates with the severity of constipation. Am J Gastroenterol 2007; 102:837-41.\u003c/li\u003e\n\u003cli\u003eTeigen L, Mathai PP, Matson M, et al. Methanogen abundance thresholds capable of differentiating in vitro methane production in human stool samples. Dig Dis Sci 2021; 66:3822-3830.\u003c/li\u003e\n\u003cli\u003eKunkel D, Basseri RJ, Makhani MD, et al. Methane on breath testing is associated with constipation: A systematic review and meta-analysis. Dig Dis Sci 2011; 56:1612-8.\u003c/li\u003e\n\u003cli\u003eZhang Q, Zhao W, Zhao Y, et al. In vitro study of bifidobacterium lactis bl-99 with fructooligosaccharide synbiotics effected on the intestinal microbiota. Front Nutr 2022; 9:890316.\u003c/li\u003e\n\u003cli\u003eKhalif IL, Quigley EM, Konovitch EA, et al. Alterations in the colonic flora and intestinal permeability and evidence of immune activation in chronic constipation. Dig Liver Dis 2005; 37:838-49.\u003c/li\u003e\n\u003cli\u003eWang L, Yang S, Mei C, et al. Lactobacillus paracasei relieves constipation by acting on the acetic acid-5-ht-intestinal motility pathway. Foods 2023; 12.\u003c/li\u003e\n\u003cli\u003eErhardt R, Harnett JE, Steels E, et al. Functional constipation and the effect of prebiotics on the gut microbiota: A review. Br J Nutr 2023; 130:1015-1023.\u003c/li\u003e\n\u003cli\u003eOhkusa T, Koido S, Nishikawa Y, et al. Gut microbiota and chronic constipation: A review and update. Front Med (Lausanne) 2019; 6:19.\u003c/li\u003e\n\u003cli\u003eFan P, Nelson CD, Driver JD, et al. Host genetics exerts lifelong effects upon hindgut microbiota and its association with bovine growth and immunity. ISME J 2021; 15:2306-2321.\u003c/li\u003e\n\u003cli\u003eNeyrinck AM, Possemiers S, Verstraete W, et al. Dietary modulation of clostridial cluster xiva gut bacteria (roseburia spp.) by chitin-glucan fiber improves host metabolic alterations induced by high-fat diet in mice. J Nutr Biochem 2012; 23:51-9.\u003c/li\u003e\n\u003cli\u003eHou Y, Dong L, Lu X, et al. Distinctions between fecal and intestinal mucosal microbiota in subgroups of irritable bowel syndrome. Dig Dis Sci 2022; 67:5580-5592.\u003c/li\u003e\n\u003cli\u003eSciavilla P, Strati F, Di Paola M, et al. Gut microbiota profiles and characterization of cultivable fungal isolates in ibs patients. Appl Microbiol Biotechnol 2021; 105:3277-3288.\u003c/li\u003e\n\u003cli\u003eFan Y, Xu C, Xie L, et al. Abnormal bile acid metabolism is an important feature of gut microbiota and fecal metabolites in patients with slow transit constipation. Front Cell Infect Microbiol 2022; 12:956528.\u003c/li\u003e\n\u003cli\u003eStipanuk MH, Ueki I, Dominy JE, Jr., et al. Cysteine dioxygenase: A robust system for regulation of cellular cysteine levels. Amino Acids 2009; 37:55-63.\u003c/li\u003e\n\u003cli\u003eWei W, Wang H, Zhang Y, et al. Faecal bile acids and colonic bile acid membrane receptor correlate with symptom severity of diarrhoea-predominant irritable bowel syndrome: A pilot study. Dig Liver Dis 2021; 53:1120-1127.\u003c/li\u003e\n\u003cli\u003eKomada Y, Narisawa H, Ueda F, et al. Relationship between self-reported dietary nutrient intake and self-reported sleep duration among japanese adults. Nutrients 2017; 9.\u003c/li\u003e\n\u003cli\u003eZheng Z, Tang J, Hu Y, et al. Role of gut microbiota-derived signals in the regulation of gastrointestinal motility. Front Med (Lausanne) 2022; 9:961703.\u003c/li\u003e\n\u003cli\u003eRoth W, Zadeh K, Vekariya R, et al. Tryptophan metabolism and gut-brain homeostasis. Int J Mol Sci 2021; 22.\u003c/li\u003e\n\u003cli\u003eAnonymous. !!! INVALID CITATION !!! (35).\u003c/li\u003e\n\u003cli\u003eLamas B, Natividad JM, Sokol H. Aryl hydrocarbon receptor and intestinal immunity. Mucosal Immunol 2018; 11:1024-1038.\u003c/li\u003e\n\u003cli\u003eWlodarska M, Luo C, Kolde R, et al. Indoleacrylic acid produced by commensal peptostreptococcus species suppresses inflammation. Cell Host Microbe 2017; 22:25-37 e6.\u003c/li\u003e\n\u003cli\u003eKalavathy R, Abdullah N, Jalaludin S, et al. Effects of lactobacillus cultures on growth performance, abdominal fat deposition, serum lipids and weight of organs of broiler chickens. Br Poult Sci 2003; 44:139-44.\u003c/li\u003e\n\u003cli\u003eWang C, Hu HJ, Dong QQ, et al. Enhancing bile tolerance of lactobacilli is involved in the hypolipidemic effects of liraglutide. Biosci Biotechnol Biochem 2021; 85:1395-1404.\u003c/li\u003e\n\u003cli\u003eAli SM, Salem FE, Aboulwafa MM, et al. Hypolipidemic activity of lactic acid bacteria: Adjunct therapy for potential probiotics. PLoS One 2022; 17:e0269953.\u003c/li\u003e\n\u003cli\u003eHamouda RA, Hamza HA, Salem ML, et al. Synergistic hypolipidemic and immunomodulatory activity of lactobacillus and spirulina platensis. Fermentation 2022; 8:220.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chronic constipation, short-chain fatty acids, intestinal flora, metabolomics, association analysis ","lastPublishedDoi":"10.21203/rs.3.rs-4108195/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4108195/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u0026amp; Aims:\u003c/strong\u003e Constipation is a disorder affecting the quality of life of patients, and symptoms following current treatments still need to be improved. Although probiotics treat constipation, the efficacy and safety of this therapy remain controversial. This a randomized, double-blind, placebo-dense controlled trialaimed to investigate the clinical impact of the probiotic bacterium Lacticaseibacillus\u003cem\u003e paracasei\u003c/em\u003e 207-27 on chronic constipation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We recruited 80 patients aged 18–65 with chronic constipation and divided them into probiotic (\u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e 207-27) and placebo (maltodextrin) groups. The participants were treated twice daily for 4 weeks, and stool samples and questionnaires were collected before and after treatment. The 16S rRNA sequencing and untargeted metabolomics data were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Bowel movements, total short-chain fatty acid, and propionic acid were significantly improved in the probiotic group compared with in the placebo (control) group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) following the 4-week intervention. After the intervention, the placebo group had higher Phylum Firmicutes number (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001) and abundance of \u003cem\u003eEscherichia-Shigella \u003c/em\u003egenus (\u003cem\u003ep=\u003c/em\u003e0.0257) and \u003cem\u003eRoseburia\u003c/em\u003e genus (\u003cem\u003ep\u003c/em\u003e=0.0202) but lower abundances of Actinobacteria phylum (\u003cem\u003ep\u003c/em\u003e=0.0177),\u003cem\u003e Bifidobacterium\u003c/em\u003e genus (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001),\u003cem\u003e \u003c/em\u003eand \u003cem\u003eRoseburia\u003c/em\u003e genus\u003cem\u003e \u003c/em\u003e(\u003cem\u003ep\u003c/em\u003e=0.0001) than the probiotic group. Probiotic intervention reduced the abundance of the \u003cem\u003eBacteroides \u003c/em\u003egenus (\u003cem\u003ep\u003c/em\u003e=0.0001) and\u003cem\u003e Blautia \u003c/em\u003egenus (\u003cem\u003ep\u003c/em\u003e=0.0213) compared with that before intervention (week 0). The analysis of non-target metabolites revealed a significant increase in 3-sulfinoalanine associated with taurine and hypotaurine metabolism in 3-methylxanthine associated with caffeine metabolism and a significant downregulation of 3-methylindolepyruvate associated with tryptophan metabolism after probiotic intervention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: \u003cem\u003eLacticaseibacillusparacasei\u003c/em\u003e 207-27 improved constipation symptoms and altered the intestinal microbiota, which could improve patients’ quality of life.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration\u003c/strong\u003e: chictr.org.cn ChiCTR2200056274, 03/02/2022.\u003c/p\u003e","manuscriptTitle":"Effects of Lacticaseibacillus paracasei 207-27 on intestinal function and its regulatory mechanism in patients with chronic constipation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-16 18:09:15","doi":"10.21203/rs.3.rs-4108195/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"066ece30-d71c-424a-8c5b-d297e0eb2fc7","owner":[],"postedDate":"May 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-28T07:53:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-16 18:09:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4108195","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4108195","identity":"rs-4108195","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}