The dynamic revolution of intestinal flora and bile acids profiles revealed the hypolipidemic effect of lotus seed resistant starch

The dynamic revolution of intestinal flora and bile acids profiles revealed the hypolipidemic effect of lotus seed resistant starch | 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 The dynamic revolution of intestinal flora and bile acids profiles revealed the hypolipidemic effect of lotus seed resistant starch Suzhen Lei, Yijun Jiang, Xiaoliang Cai, Zhixiong Lin, Yi Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4210834/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 Our research group had shown that lotus seed resistant starch (LRS) had hypolipidemic effect, but its mechanism is still being studied. Bile acids are important metabolic pathway of cholesterol, accelerating the conversion of cholesterol into bile acids and excreting them in the fecal may be one of the effective ways to reduce cholesterol levels in the body. This study aimed to reveal the lipid-lowering effect of LRS from the perspectives of fecal microbiota and bile acids. Herein, a rat model of hyperlipidemia was established and intervened with LRS. Fecal samples from different periods were collected to study the changes in microbiota and bile acids, and the correlation network diagram was established to reveal the lipid-lowering mechanism of LRS. The results showed that LRS inhibited the growth of Prevotella and Allobaculum in hyperlipidemic rats. Meanwhile LRS promoted the excretion of cholic acid (CA), chenodeoxycholic acid (CDCA), alpha-muricholic acid (α-MCA), ursodeoxycholic acid (UDCA), ursocholic acid (UCA), 7-ketodeoxycholic acid (7-keto-DCA) in hyperlipidemic rats. Furthermore, total cholesterol (TCHO), low-density lipoprotein cholesterol (LDL-C) were negatively correlated with CA, CDCA, UDCA and UCA, and TCHO was positively correlated with Prevotella . Triglycerides (TG) was negatively correlated with CA, CDCA, 7-keto-DCA and UCA, while high-density lipoprotein cholesterol (HDL-C) was positively correlated with α-MCA. Regulating the gut microbiota such as Prevotella and accelerating the transformation of liver cholesterol into primary bile acids (CA, CDCA) for excretion from the body was one of the effective means for LRS to ameliorate blood lipid levels in hyperlipidemic rats. Lotus seed resistant starch Fecal flora Bile acids Hypolipidemic effect Correlation network Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Primary bile acids are synthesized from cholesterol in the liver through the classical or alternative bile acid synthesis pathway, and then enter the intestine. Some of the primary bile acids form secondary bile acids by a series of microbial enzymes secreted by the gut flora, and excreted from the body with food debris [ 1 – 3 ]. Hyperlipidemia is a kind of metabolic disease caused by lipid metabolism disorders, and usually high cholesterol is one of its main characteristics [ 4 , 5 ]. Bile acids are important metabolic pathway of cholesterol, accelerating the conversion of cholesterol into bile acids and excreting them in the fecal may be one of the effective ways to reduce cholesterol levels in the body [ 1 , 6 ]. Tang et al. [ 7 ] suggested that one of the main reasons that consumption of fragrant rapeseed oil could improve the blood lipid level in hyperlipidemic rats was that it promoted the excretion of bile acids from the body. Wang et al. [ 8 ] also suggested that hyperoside lowered cholesterol through bile acids excretion. The high concentration of secondary bile acids is toxic to intestinal wall cells, and the production of secondary bile acids is closely related to the composition of intestinal flora [ 9 ]. A previous study had shown that the carbohydrate available to bacteria, such as resistant starch, changed the type and content of bile acids by regulating the structure and composition of gut flora [ 10 ]. Lei et al. [ 11 ] showed that resistant starch could regulate the microbial community mediating the conversion of sodium taurocholate. LRS is a type 3 resistant starch formed by gelatinization and regeneration of lotus seed starch with high amylose content [ 12 ]. Our previous studies had shown that LRS could reduce serum cholesterol and improve dyslipidemia in hyperlipidemic mice and rats. Meanwhile, LRS regulated intestinal flora and reduced the conversion of primary bile acids to secondary bile acids in rats [ 13 – 15 ]. At present, the research on the relationship between resistant starch, intestinal flora and bile acids is one of the hot spots in contemporary food science, but many researchers tend to focus on the result of lowering blood lipid, while this paper pays more attention to the relationship between bacterial flora and bile acids in the process of lowering blood lipid, so as to achieve the goal of lowering blood lipid. Therefore, in this paper, 16S rRNA technology and bile acids targeted metabolomics technology were used to determine the changes of fecal flora and bile acids profiles in normal rats and hyperlipidemic rats at different periods, and then the correlation heat map of fecal bacteria and bile acids was constructed to reveal the hypolipidemic effect of LRS. This result is expected to provide a theoretical basis for the hypolipidemic mechanism of LRS. 2. Materials and methods 2.1 Reagents and Materials Fresh lotus seeds obtained from Green Field Fujian Food Co., Ltd. (Sanming, China) and LRS were prepared in the laboratory according to previous studies [ 11 , 16 ]. Fresh lotus seeds undergo thawing, juice extraction, filtration, precipitation, washing, and drying to obtain lotus seed starch. LRS was obtained through steps such as high-temperature gelatinization, retrogradation, purification, and drying of lotus seed starch. A total of 46 bile acids standards were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. (Shanghai, China), and the information is shown in Table 1 . Chromatographic grade methanol and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, USA), and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Table 1 Bile acids standards information No. Full name Abbreviation CAS No. Full name Abbreviation CAS 1 glycocholic acid GCA 475-31-0 24 taurodeoxycholate acid TDCA 1180-95-6 2 glycochenodeoxycholic acid GCDCA 16564-43-5 25 lithocholic acid LCA 434-13-9 3 taurocholic acid TCA 145-42-6 26 cholic acid CA 81-25-4 4 taurochenodeoxycholic acid TCDCA 516-35-8 27 chenodeoxycholic acid CDCA 474-25-9 5 glycoursodeoxycholic acid GUDCA 64480-66-6 28 hyocholic acid HCA 547-75-1 6 allocholic acid Allo-CA 2464-18-8 29 23-norcholic acid 23-nor-CA 60696-62-0 7 ursodeoxycholic acid UDCA 128-13-2 30 glycohyocholic acid GHCA 32747-08-3 8 deoxycholic acid DCA 83-44-3 31 23-nordeoxycholic acid 23-nor-DCA 53608-86-9 9 tauroursodeoxycholic acid TUDCA 14605-22-2 32 isolithocholic acid isoLCA 1534-35-6 10 hyodeoxycholic acid HDCA 83-49-8 33 12-ketolithocholic acid 12-keto-LCA 5130-29-0 11 apocholic acid ACA 641-81-6 34 dehydrolithocholic acid Dehydro-LCA 1553-56-6 12 glycodeoxycholic acid GDCA 16409-34-0 35 lithocholic acid 3-sulfate LCA-3-S 34669-57-3 13 glycolithocholic acid GLCA 474-74-8 36 chenodeoxycholic acid-3-β-D-glucuronide CDCA-3-β-D-G 58814-71-4 14 alpha-muricholic acid α-MCA 2393-58-0 37 3β-ursodeoxycholic acid 3β-UDCA 78919-26-3 15 beta-muricholic acid β-MCA 2393-59-1 38 3-dehydrocholic acid 3-Dehydro-CA 2304-89-4 16 7-ketolithocholic acid 7-keto-LCA 4651-67-6 39 chenodeoxycholic acid 24 - acyl - β-D-glucuronide CDCA-24-Acyl-β-D-G 208038-27-1 17 tauro-α-muricholic acid T-α-MCA 25613-05-2 40 12-ketochenodeoxycholic acid 12-keto-CDCA 2458-08-4 18 tauro-β-muricholic acid T-β-MCA 145022-92-0 41 7,12-diketolithocholic acid 7,12-Diketo-LCA 517-33-9 19 omega-murichoclic acid ω-MCA 6830-03-1 42 dehydrocholic acid Dehydro-CA 81-23-2 20 murideoxycholic acid MDCA 668-49-5 43 ursocholic acid UCA 2955-27-3 21 taurohyodeoxycholic acid THDCA 38411-85-7 44 7-ketodeoxycholic acid 7-keto-DCA 911-40-0 22 taurohyocholic acid THCA N/A 45 isodeoxycholic acid isoDCA 566-17-6 23 taurolithocholic acid TLCA 6042-32-6 46 3β-cholic acid 3β-CA 3338-16-7 2.2 Animal experiment design and fecal collection All animal experimental procedures were approved by the Animal Ethics Committee of Fujian Academy of Chinese Medicine (Approval number: FJATCM-IAEC2021002). The feces were sourced from specific pathogen free grade Sprague Dawley male rats purchased from Slack Laboratory Animals Co., Ltd. (Shanghai, China, permit number: SCXK (Hu) 2017-0005). The experimental methods refer to previous study [ 17 ], and the process is shown in Fig. 1 . After 1 week of adaptive feeding, a total of 24 rats were randomly divided into two groups with 12 rats in each group. One group was fed basic diet and the other group was fed high-fat diet. The high-fat feed was purchased from Fuzhou Wu's Animal Testing Co., Ltd. (Fuzhou, China), and formula was as follows: basic feed 89.25%, egg yolk powder 5%, lard 5%, cholesterol 0.5% and porcine bile salt 0.25%. After 2 weeks of feeding, blood was collected from the fundus venous plexus of rats from the two groups. The levels of TG, TCHO, HDL-C and LDL-C were measured by a fully automatic biochemical analyzer to determine the success of a rat model with hyperlipidemia. The hyperlipidemic rats were randomly divided into two groups with 6 rats in each group. Meanwhile the rats fed basic diet were also randomly divided into two groups with 6 rats in each group. LRS intervention was performed for 4 weeks, and the gavage dose of LRS was 0.25 g /100 g · day according to the Dietary Guidelines for U.S. Residents. Animal group information was as follows: rats fed a basal diet (NC group), rats fed a basal diet and gavage LRS (NC_LRS group), rats fed a high-fat diet (MC group), and rats fed a high-fat diet and gavage LRS (MC_LRS group). Physiological indexes of rats and the construction of hyperlipidemia models can be referred to previous study [ 13 ]. Feces from each group of rats were collected during three stages: early modeling (detection point 1), end of modeling (detection point 2), and end of LRS intervention (detection point 3). The feces were collected in sterile freeze-storage tubes at 8 am, and stored in a -80℃ refrigerator for use. The naming of the samples is shown in Table 2 . The feces of three rats with no significant difference in physiological indicators between each group were used for the determination of microbial communities and bile acids. Table 2 Name information of each sample Group Stage NC NC_LRS MC MC_LRS detection point 1 NC_1 NC_LRS_1 MC_1 MC_LRS_1 detection point 2 NC_2 NC_LRS_2 MC_2 MC_LRS_2 detection point 3 NC_3 NC_LRS_3 MC_3 MC_LRS_3 2.3 Fecal flora determination Illumina next generation 16S rRNA gene amplicon sequencing technology was used to sequence fecal microflora of rats in each group. The method can be referred to previous studies [ 18 , 19 ]. First, the total DNA of each sample was extracted, and its purity, concentration and integrity were tested. Then the 16S V3-V4 region was amplified by PCR, and the PCR products were recovered and purified. Finally, PCR products were quantified and sequenced in Miseq PE300 platform after library construction. After data optimization of the original sequence obtained by sequencing, the amplicon sequence variant (ASV) representative sequence and abundance information can be obtained. Based on the representative sequence and abundance information of ASVs, a series of statistical analyses were conducted to analyze the community diversity, composition and species differences. 2.4 Determination of fecal bile acids Fecal bile acids were measured using targeted metabolomics techniques, which can refer to previous study with appropriate modifications [ 20 ]. First of all, metabolite extraction of feces was carried out: 50 mg of feces was weighed and added with 400 µL extraction solution (methanol: water = 4:1), then ground by freezing grinder for 6 min (-10°C, 50 Hz), ultrasounded at low temperature for 30 min (5°C, 40 KHz), and stood at -20°C for 30 min. 200 µL supernatant was centrifugally taken for on-machine detection. Secondly, standard bile acids solutions of different concentrations were provided: 1 mg of each of 46 bile acids standard products was weighed, dissolved with methanol and fixed to 1 mL, then vortically mixed to obtain standard reserve solution. Finally, Liquid chromatography-mass spectrometry was used to determine the standard solution and the sample to be tested under the same conditions. Specific parameters were as follows: Chromatographic conditions: ExionLC AD system, Waters BEH C18 (150 * 2.1 mm, 1.7 µm) liquid chromatographic column, column temperature 40°C, injection volume 1 µL. Mobile phase A (0.1% formic acid-water solution), mobile phase B (0.1% formic acid-acetonitrile). Mass spectrometry conditions: AB SCIEX QTRAP 6500+, using negative mode detection. Curtain gas 35, collision gas medium, ionspray voltage − 4500. Temperature 500, ion source gas 1, 40, ion source gas 2, 50. 2.5 Data analysis and statistics Experimental data were represented as the mean ± standard deviation, and SPSS25.0 software was used for significance analysis and multiple comparisons. One-way analysis of variance was analyzed using Duncan's multiple range tests. Drawings were generated using OriginPro8.5 and GraphPad Prism8.0 software. The Pearson correlation coefficient was calculated, and correlation heatmaps or relationship network diagrams were drawn using R language and Cytoscape software. 3. Results and analysis 3.1 Analysis of the overall difference of fecal flora The overall difference of fecal flora in normal rats and hyperlipidemic rats were analyzed, as shown in Fig. 2 . In normal rats, fecal flora of NC_1, NC_2, NC_LRS_1 and NC_LRS_2 groups were mainly in the first and fourth quadrants, while that of NC_3 and NC_LRS_3 groups were mainly in the second and third quadrants (Fig. 2 (a)). This result indicated that at detection point 3, the fecal flora structure of rats had been greatly changed. The structure of gut microbiota is related to age, and as the feeding time increases, the fecal flora of rats gradually changed [ 21 ]. Further, according to non-metric multidimensional scale (NMDS) and sample clustering analysis (Fig. 2 (b) (c)), NC_1, NC_2, NC_LRS_1 and NC_LRS_2 groups had high similarity in fecal flora, while NC_3 and NC_LRS_3 groups had high similarity in fecal flora. The results were consistent with those of principal co-ordinates analysis (PCoA). Previous study had also shown that gut flora changed dynamically as they grow [ 22 ]. As shown in Fig. 2 (d) (e) (f), fecal flora structure of rats in MC_1 and MC_2 groups was different, and that of rats in MC_LRS_1 and MC_LRS_2 groups was also different to some extent, indicating that high-fat diet changed the fecal flora structure of normal rats. The alteration of fecal flora in rats by high-fat diet had been reported in previous study [ 23 ]. In hyperlipidemic rats, the fecal flora structure of rats in MC_3 and MC_LRS_3 groups was different from that in other groups, indicating that the fecal flora structure of rats with hyperlipidemia had a great change at detection point 3. The similarity in fecal flora structure between MC_3 group and MC_LRS_3 group rats was high. In terms of the overall difference analysis, LRS had no significant effect on fecal flora structure of hyperlipidemic rats during the feeding time of detection point 2 and detection point 3, and the fecal flora composition of rats in each group was further analyzed. 3.2 Community composition of fecal flora at different classification levels Fecal flora community composition of rats in each group at different periods is shown in Fig. 3 . According to Fig. 3 (a), Firmicutes and Bacteroidota were the main microorganisms in rat feces at the phylum level, accounting for more than 80% of the total number of microorganisms. It was followed by a small amount of Actinobacteriota, Spirochaetota, Desulfobacterota and Proteobacteria. The results were consistent with previous research [ 24 ]. In normal rats, the relative abundance of Firmicutes in feces of NC_2 group was higher than that of NC_1 group, while the relative abundance of Bacteroidota, Actinobacteriota and Spirochaetota was lower than that of NC_1 group. Compared with NC_2 group, the relative abundance of Bacteroidota increased, while the relative abundance of Firmicute, Actinobacteriota and Spirochaetota decreased in NC_3 group. The trend of relative abundance of Firmicute, Bacteroidota and Actinobacteriota in the feces of NC_LRS group was the same as that of NC group. The relative abundance of Firmicute in NC_LRS_3 group was lower than that in NC_3 group, while the relative abundance of Bacteroidota was higher. The impact of LRS on the composition of fecal flora should be further analyzed. In addition, Compared to NC_2 group, the relative abundance of Firmicute in MC_2 group increased, while the relative abundance of Actinobacteriota decreased. In hyperlipidemic rats, the relative abundance of Firmicute in feces of MC_2 group was higher than that of MC_1 group, while the relative abundance of Bacteroidota and Actinobacteriota was lower than that of MC_1 group. Yan et al. [ 25 ] also found that the relative abundance of Firmicute increased while Bacteroidota decreased in rats with high fat diet when studying the lowering of blood lipid of rice buckwheat. Compared with MC_2 group, the relative abundance of Bacteroidota increased while the relative abundance of Firmicute and Actinobacteriota decreased in MC_3 group. In the MC_LRS group, the relative abundance of Firmicute in feces of MC_LRS_3 group was the highest, followed by MC_LRS_2 group, and the relative abundance of Firmicute in feces of MC_LRS_1 group was the lowest. On the contrary, MC_LRS_1 group had the highest relative abundance of Bacteroidota, followed by MC_LRS_2 and MC_LRS_3 groups. Compared with MC_3 group, the relative abundance of Firmicute and Actinobacteriota increased, while the relative abundance of Bacteroidota decreased in MC_LRS_3 group. After the intervention of LRS, the composition of fecal flora of rats in MC group and MC_LRS group was different, mainly Firmicute and Bacteroidota. The relative abundance of Firmicute increased and the relative abundance of Bacteroidota decreased after LRS administration in hyperlipidemic rats. Zhang et al. [ 17 ] also found that the relative abundance of Bacteroidota decreased in hyperlipidemic mice treated with resistant starch when studying specific gut microbiota properties related to anti-hyperlipidemic action of resistant starch. As shown in Fig. 3 (b), at the genus level, the main microorganisms in rat feces including Lactobacillus , norank_f__Muribaculaceae , Prevotella , unclassified_f__Lachnospiraceae , Romboutsia , Turicibacter , Alloprevotella , Bacillus , Lachnospiraceae_NK4A136_group and Bifidobacterium , accounted for more than 50% of the total number of microorganisms. Among them, the Lactobacillus , unclassified_f__Lachnospiraceae , Romboutsia , Turicibacter , Bacillus , Lachnospiraceae_NK4A136_group belong to Firmicute, norank_f__Muribaculaceae , Prevotella and Alloprevotella belong to Bacteroidota, while Bifidobacterium belong to Actinobacteriota. The results were consistent with previous research [ 24 ]. In normal rats, the relative abundance of Lactobacillus , Prevotella , Lachnospiraceae_NK4A136_group and Bifidobacterium in NC_2 group were lower than that in NC_1 group, while the relative abundance of norank_f__Muribaculaceae was higher than that of NC_1 group. Compared with NC_2 group, the relative abundance of Lactobacillus , norank_f__Muribaculaceae and Prevotella in feces in NC_3 group increased. The trend of relative abundance of Lactobacillus and Prevotella in feces in NC_LRS group was the same as that in NC group. In addition, compared to NC_2 group, the relative abundance of norank_f__Muribaculaceae and unclassified_f__Lachnospiraceae decreased, while the relative abundance of Prevotella and Romboutsia increased in MC_2 group. In hyperlipidemic rats, the relative abundance of Lactobacillus and norank_f__Muribaculaceae in feces of MC_2 group were lower than that of MC_1 group and MC_3 group, while the relative abundance of Prevotella and Romboutsia were higher than those of MC_1 and MC_3 groups. The trend of relative abundance of norank_f__Muribaculaceae , Prevotella and unclassified_f__Lachnospiraceae in the feces of MC_LRS group and MC group was different. This result indicated that LRS had regulatory effect on the relative abundance of norank_f__Muribaculaceae , Prevotella and unclassified_f__Lachnospiraceae in hyperlipidemic rats. Previous study also had shown that resistant starch had a certain regulatory effect on intestinal flora in hyperlipidemic rats [ 26 ]. 3.3 Analysis of species difference of fecal flora Further, differences in fecal flora of rats in each group were analyzed at phylum and genus levels, as shown in Fig. 4 , 5 . As can be seen from Fig. 4 , the main species of fecal flora in rats at the phylum level were Firmicute and Bacteroidota, followed by Actinobacteriota. Compared with detection points 1 and 2, the relative abundance of Firmicute, Actinobacteriota, Spirochaetota and Desulfobacterota in rat feces of NC_3 group were low, and the relative abundance of Bacteroidota was high (Fig. 4 (a)). Compared with detection point 2, the relative abundance of Firmicute, Actinobacteriota, Spirochaetota and Desulfobacterota in the feces of rats in NC_LRS_3 group were decreased and the relative abundance of Bacteroidota was increased (Fig. 4 (b)). The variation trend of fecal flora in NC group and NC_LRS group was similar at the phylum level, which should be further discussed. In the rats with hyperlipidemia, compared with detection point 2, the relative abundance of Firmicute and Actinobacteriota decreased, while the relative abundance of Bacteroidota increased in feces of MC_3 group (Fig. 4 (c)). After the intervention of LRS, the relative abundance of Firmicute and Actinobacteriota increased while the relative abundance of Bacteroidota decreased (Fig. 4 (d)). Compared with normal rats, LRS had a more obvious effect on fecal bacteria community of hyperlipidemic rats, mainly reflected in Firmicute, Bacteroidota and Actinobacteriota. Zheng et al. [ 27 ] also showed that resistant starch could ameliorate gut microbiota disorder in hyperlipidemic rats. At the genus level, the differences among the top 15 species in the relative abundance of fecal flora of rats in each group were analyzed, as shown in Fig. 5 . The main species included Lactobacillus , norank_f__Muribaculaceae , Prevotella and unclassified_f__Lachnospiraceae in feces of normal rats. Compared with NC_2 group, the relative abundance of Lactobacillus , norank_f__Muribaculaceae , Prevotella , Romboutsi a, Ruminococcus and Prevotellaceae_NK3B31_group increased, while the relative abundance of unclassified_f__Lachnospiraceae , Lachnospiraceae_NK4A136_group , Bifidobacterium and Bacillus reduced in NC_3 group (Fig. 5 (a)). Compared with NC_LRS_2 group, the relative abundance of norank_f__Muribaculaceae , Lactobacillus , Prevotella and Bacillus increased while the relative abundance unclassified_f__Lachnospiraceae , Alloprevotella , Turicibacter , Lachnospiraceae_NK4A136_group , Romboutsia , Ruminococcus and Clostridium_sensu_stricto_1 decreased (Fig. 5 (b)). Between detection point 2 and detection point 3, the trends of relative abundance of Bacillus , Alloprevotella , Turicibacter , Romboutsia , Ruminococcus and norank_f__norank_o__Clostridia_UCG-014 in NC group and NC_LRS group were different. The results showed that LRS could promote Bacillus and norank_f__norank_o__Clostridia_UCG-014 proliferation in normal rats. Resistant starch was a kind of polysaccharide [ 28 ], and polysaccharide promoted intestinal norank_f__norank_o__Clostridia_UCG-014 proliferation had been reported in previous study [ 29 ]. In hyperlipidemic rats, the relative abundance of Lactobacillus , norank_f__Muribaculaceae , unclassified_f__Lachnospiraceae , Allobaculum , Prevotellaceae_NK3B31_group in MC_3 group were higher than that of MC_2 group, while the relative abundance of Prevotella , Romboutsia , Turicibacter , Alloprevotella , Staphylococcus , Bacillus , Roseburia and Bifidobacterium were lower than that of MC_2 group (Fig. 5 (c)). After LRS intervention, the relative abundance of Lactobacillus , norank_f__Muribaculaceae , Allobaculum , Alloprevotella , Blautia and Ruminococcus increased, while the relative abundance of Prevotella , unclassified_f__Lachnospiraceae , Romboutsia , Bacillus , Bacteroides and Staphylococcus reduced in MC_LRS_3 group (Fig. 5 (d)). During the period of intervention, the change trends of relative abundance of Blautia , Ruminococcus , unclassified_f__Lachnospiraceae , Alloprevotella and Bacteroides in MC group and MC_LRS group were different. The results showed that intragastric administration of LRS to hyperlipidemic rats, the relative abundance of Blautia , Ruminococcus , Alloprevotella increased, and the relative abundance of unclassified_f__Lachnospiraceae and Bacteroides decreased in feces. Previous studies had also shown that resistant starch could promote proliferation of Blautia and Alloprevotella [ 17 , 30 ]. In addition, compared with the MC group, the variation amplitude of the relative abundance of Prevotella at detection point 3 and 2 in the MC_LRS group were higher than that of the MC group, while the variation amplitude of the relative abundance of Bacillus , Romboutsia and Allobaculum at detection point 3 and 2 were lower than that of the MC group. These results indicated that LRS promoted the proliferation of Bacillus and Romboutsia , and inhibited the growth of Prevotella and Allobaculum in hyperlipidemic rats. Liang et al. [ 31 ] also found that the relative abundance of Allobaculum decreased in the high-resistant starch group when studying the lowering of blood lipid by resistant starch. In short, the regulation effects of LRS on microflora of normal rats and hyperlipidemic rats were different. 3.4 Effect of LRS on the overall distribution of fecal bile acids Targeted metabolomics technology was used to measure fecal bile acids at detection points 1, 2 and 3 of rats in each group, and 46 kinds of bile acids were obtained. They were classified into primary bile acids and secondary bile acids, and total bile acids were the sum of primary bile acids and secondary bile acids, as shown in Fig. 6 . The proportion changes of primary or secondary bile acids in the feces of rats in different groups at different stages can be found in Figure S1 , S2. As can be seen from Fig. 6 (a), the contents of secondary bile acids in rat feces were high. In normal rats, compared with NC_1 group, the contents of total bile acids and secondary bile acids in feces of NC_2 group were increased, while the content of primary bile acids was decreased. The contents of total bile acids, secondary bile acids and primary bile acids in feces in NC_3 group were lower than those in NC_2 group. The change trend of fecal bile acids in NC_LRS group was the same as that in NC group. The results indicated that LRS had no significant effect on the overall distribution of fecal bile acids in normal rats, which should be further analyzed. In addition, the contents of total bile acids, primary bile acids and secondary bile acids in feces of MC_2 group were increased compared with those of MC_1 group. Tang et al. [ 7 ] also found that the content of total bile acids in feces of rats with high fat diet increased significantly when studying the lowering of blood lipid by rapeseed oil. In hyperlipidemic rats, compared with MC_2 group, the contents of total bile acids and secondary bile acids in feces of rats in MC_3 group were increased. After LRS intervention, the variation trend of fecal bile acids in MC_LRS group was different from that in MC group. Compared with MC_LRS_2 group, total bile acids content in feces of rats in MC_LRS_3 group had no significant change, while primary bile acids content was increased and secondary bile acids content was decreased. These results indicated that LRS could promote the excretion of primary bile acids and reduce the conversion of primary bile acids to secondary bile acids in hyperlipidemic rats. Secondary bile acids are produced by a series of microbial enzymes secreted by intestinal flora acting on primary bile acids. The types and contents of bile acids in feces of rats in different groups were related to the feces flora [ 2 , 3 ]. The types of primary bile acids and secondary bile acids in feces of rats in each group were further analyzed, as shown in Fig. 6 (b) (c). As shown in Fig. 6 (b), the primary bile acids in rat feces were mainly free primary bile acids, including α-MCA, β-MCA, CA and CDCA. In normal rats, compared with NC_2 group, the total amount of primary bile acids in feces of NC_3 group decreased, mainly α-MCA and β-MCA contents decreased. The change trends of α-MCA and β-MCA contents in feces of rats in NC_LRS group were the same as those in NC group. In addition, compared with MC_1 group, the total amount of primary bile acids in feces of MC_2 group was increased, including β-MCA, CA and CDCA contents. Previous studies had shown that a high-fat diet increased the secretion of 12α-hydroxyl bile acids and promoted fecal bile acids excretion [ 32 , 33 ]. In hyperlipidemic rats, the total amount of primary bile acids in feces of rats in MC_2 and MC_3 groups had no significant change. After LRS intervention, the variation trend of fecal primary bile acids in MC_LRS group was different from that in MC group. Compared with MC_LRS_2 group, the content of primary bile acids in feces of rats in MC_LRS_3 group was increased, mainly including β-MCA and CA. Intragastric administration of LRS in hyperlipidemic rats could promote the excretion of primary bile acids (β-MCA, CA). As a new type of dietary fiber, resistant starch had a certain effect on bile acids profiles [ 15 ]. Previous study had also shown that dietary fiber promoted CA excretion in feces of rats on a high fat diet [ 34 ]. As shown in Fig. 6 (c), the types of secondary bile acids in normal rat feces were mainly free secondary bile acids, including DCA, HDCA, ω-MCA, LCA, MDCA, Dehydro-LCA, isoLCA, etc. Compared with NC_2 group, the total content of secondary bile acids in feces of rats in NC_3 group was decreased, mainly including DCA, ω-MCA and LCA. Between detection point 2 and detection point 3, the variation trends of ω-MCA and LCA in feces of rats in NC_LRS group were the same as those in NC group, while the variation trend of Dehydro-LCA was different from that in NC group. In hyperlipidemic rats, the types of secondary bile acids were mainly free secondary bile acids, including UDCA, DCA, HDCA, ω-MCA, MDCA, LCA, HCA, etc. The results were consistent with previous research [ 33 ]. Compared with the MC_1 group, the total amount of secondary bile acids in feces of rats in MC_2 group was increased, including the contents of DCA, HDCA and LCA, and the high concentration of secondary bile acids in feces was associated with cholesterol cholelithiasis and other diseases [ 35 ]. Compared with MC_2 group, the total amount of secondary bile acids in feces of rats in MC_3 group increased, mainly including the contents of HDCA and ω-MCA. After LRS intervention, the total amount of secondary bile acids in feces of rats in MC_LRS_3 group was lower than that in MC_LRS_2 group, mainly including the reduction of HDCA, MDCA and LCA. The excretion of HDCA, MDCA and LCA in feces of hyperlipidemic rats was reduced by intragastric administration of LRS. As a new type of dietary fiber, LRS could promote the direct excretion of primary bile acids, and reduce the conversion of primary bile acids to secondary bile acids in hyperlipidemic rats [ 11 , 36 ]. 3.5 Effect of LRS on fecal primary bile acids profiles Further, the influence of LRS on the primary bile acids profiles of normal rats and hyperlipidemic rats were explored, and the bile acids changes at detection points 2 and 3 were discussed. As shown in Fig. 7 , it is the difference of rat feces primary bile acids at detection point 3 and point 2. The difference above the X-axis indicates the increase of bile acids content; otherwise, it indicates the decrease of bile acids content. In normal rats, compared with NC_2 group, the contents of α-MCA, β-MCA, CA and CDCA in feces of NC_3 group were decreased, while the contents of TCA and T-β-MCA were increased, with significant changes in α-MCA and β-MCA (Fig. 7 (a)). Compared with NC_LRS_2 group, the contents of α-MCA, β-MCA, CA and CDCA in feces of rats in NC_LRS_3 group were decreased, while the contents of TCA and T-β-MCA were increased (Fig. 7 (b)). The change trend of fecal primary bile acids profiles in NC_LRS group was the same as that in NC group. However, the amplitude of change was different, mainly reflected in α-MCA, CA and CDCA, suggesting that the excretion of fecal CA and CDCA could be promoted by gavage of LRS in normal rats. Previous study had also shown that the bile acid profile was closely related to dietary fiber in the diet [ 37 ]. In hyperlipidemic rats, compared with MC_2 group, the contents of TCA, β-MCA, T-β-MCA and CA in feces of rats in MC_3 group were increased, while the content of CDCA was decreased, with great changes in CDCA and CA (Fig. 7 (c)). The contents of α-MCA, β-MCA and CA in feces of rats in MC_LRS_3 group were increased, while the content of CDCA was decreased compared with those in MC_LRS_2 group (Fig. 7 (d)). The variation trend or amplitude of fecal primary bile acids profiles of rats in MC_LRS group were different from those in MC group, mainly manifested in α-MCA, β-MCA, T-β-MCA, CA and CDCA. These results indicated that the intragastric administration of LRS could promote the excretion of free primary bile acids (α-MCA, β-MCA, CA, and CDCA) and reduce the excretion of combined primary bile acids (TCA, T-β-MCA) in hyperlipidemic rats. Promoting the excretion of free primary bile acids may be one of the effective ways for LRS to reduce cholesterol in hyperlipidemic rats [ 1 , 13 ]. In addition, LRS could promote the excretion of free primary bile acids (CA and CDCA) in rats, which may be because resistant starch had certain adsorption effect on bile acids and could accelerate their excretion in the body [ 38 , 39 ]. 3.6 Effect of LRS on fecal secondary bile acids profiles In order to explore the effect of LRS on the secondary bile acids profiles of normal rats and hyperlipidemic rats, the top 10 secondary bile acids in two kinds of rat feces were analyzed according to their contents. As shown in Fig. 8 , it is the difference of secondary bile acids in rat feces at detection point 3 and point 2. The difference above the X-axis indicates the increase of bile acids content; otherwise, it indicates the decrease of bile acids content. In normal rats, compared with NC_2 group, the contents of DCA, HDCA, ω-MCA, MDCA, LCA and isoLCA in feces of NC_3 group were decreased, while the content of Dehydro-LCA was increased, and ω-MCA and LCA were significantly changed (Fig. 8 (a)). Although the variation trends of DCA, HDCA, ω-MCA, MDCA and LCA in feces of rats in NC_LRS group were the same as those in NC group, the variation amplitude of secondary bile acids profiles was different. They were mainly reflected in DCA, HDCA, ω-MCA, LCA and isoLCA. Meanwhile, the trend of Dehydro-LCA in feces of rats in NC_LRS group was different from that in NC group (Fig. 8 (b)). The results showed that the excretion of DCA, HDCA, LCA and isoLCA could be promoted and the contents of ω-MCA and Dehydro-LCA could be reduced in normal rats by gavage of LRS. Secondary bile acids are closely related to gut microbiota, and previous study had shown that a diet rich in resistant starch resulted in altered microbiota-dependent secondary bile acids pool size and composition [ 40 ]. In hyperlipidemic rats, compared with MC_2 group, the contents of DCA, HDCA, ω-MCA, MDCA, HCA and 12-keto-CDCA in feces of MC_3 group were increased, while the contents of UDCA, LCA, UCA and 7-keto-DCA were decreased, among which HDCA and ω-MCA had great changes (Fig. 8 (c)). Compared with MC_LRS_2 group, the contents of HDCA, ω-MCA, MDCA, LCA and HCA were decreased, while the contents of UDCA, UCA and 7-keto-DCA were increased in feces of rats in MC_LRS_3 group (Fig. 8 (d)). There were differences in the change trend of secondary bile acids profiles between MC_LRS group and MC group, mainly UDCA, HDCA, ω-MCA, MDCA, LCA, HCA, UCA and 7-keto-DCA. Intragastric administration of LRS in hyperlipidemic rats could promote the excretion of UDCA, UCA and 7-keto-DCA, and reduce the contents of HDCA, ω-MCA, MDCA, LCA and HCA in feces. The effects of LRS on secondary bile acids profiles in feces of normal rats and hyperlipidemic rats were different, which might be because resistant starch had different regulation effects on the bacterial community of the two kinds of rats, and different bacterial community structure would affect the type and content of secondary bile acids [ 41 – 43 ]. 3.7 Correlation analysis between fecal flora and bile acids According to the composition of fecal flora and bile acids profiles, LRS had different effects on fecal flora and bile acids profiles of normal rats and hyperlipidemic rats. Bile acids are closely related to intestinal flora, so pearson correlation coefficient was further used to analyze the correlation among fecal flora, primary bile acids and secondary bile acids of rats. As shown in Fig. 9 , it is the correlation heat map between the top 15 bacteria genera with the relative abundance of normal rat fecal flora and the primary and secondary bile acids, among which the secondary bile acids are the top 10 according to the content in normal rat feces. In normal rats (Fig. 9 (a)), free primary bile acids were mainly positively correlated with Bifidobacterium and Turicibacter , and negatively correlated with norank_f__norank_o__Clostridia_UCG-014 . Among them, β-MCA had significant positive correlation with Bifidobacterium and Turicibacter (p < 0.05), α-MCA and CDCA were positively correlated with Bifidobacterium (0.001 < p < 0.01), while β-MCA and α-MCA were negatively correlated with norank_f__norank_o__Clostridia_UCG-014 (0.001 < p < 0.01). Further, as can be seen from Fig. 9 (b), HDCA was significantly positively correlated with norank_f__norank_o__Clostridia_UCG-014 (0.001 < p < 0.01). Meanwhile HDCA was positively correlated with Ruminococcus (p < 0.05), and had a significant negative correlation with Bifidobacterium (p < 0.05). The results indicated that the relative abundance of norank_f__norank_o__Clostridia_UCG-014 in normal rat feces was related to the type and content of bile acids [ 42 , 44 ]. After the intervention of LRS, the relative abundance of norank_f__norank_o__Clostridia_UCG-014 in normal rats could be adjusted. The regulation of intestinal flora by resistant starch had been previously reported [ 17 , 27 ]. As can be seen form Fig. 10 (a), free primary bile acids (CDCA and CA) were significantly correlated with fecal flora in hyperlipidemic rats. Among them, CDCA was positively correlated with Romboutsia , Bacillus , Turicibacter and Staphylococcus . CA was positively correlated with Blautia and Allobaculum , while had a significant negative correlated with Bifidobacterium (p < 0.05). In addition, α-MCA was positively correlated with Ruminococcus (p < 0.05), and was significantly negative correlated with Prevotella (p < 0.05). Furthermore, in hyperlipidemic rats (Fig. 10 (b)), UDCA were significantly positive correlated with Romboutsia , Turicibacter and Staphylococcus (0.001 < p < 0.01), and were positively correlated with Roseburia (p < 0.05). There was a significant negative correlation between UCA and Prevotella , and a significant positive correlation between UCA and Romboutsia (0.001 < p < 0.01). In the feeding process, Romboutsia , Bacillus , Blautia and Prevotella in feces of hyperlipidemic rats were closely related to bile acids. This result was consistent with previous studies that Blautia and Prevotella are bile acids metabolism related bacteria [ 42 , 45 ]. 3.8 Correlation analysis of blood lipid indicators, fecal flora and bile acids Further, correlation analysis was carried out on the blood lipid indicators, fecal flora and bile acids of rats, as shown in Fig. 11 . The blood lipid indicators (TCHO, TG, LDL-C, HDL-C) were obtained by measuring the abdominal arterial blood of rats in each group after the end of the experimental period. The data can be viewed from previous study, which showed that oral administration of LRS in hyperlipidemic rats could ameliorate their blood lipid levels [ 13 ]. TCHO, LDL-C were negatively correlated with CA, CDCA, UDCA and UCA, and TCHO was positively correlated with Prevotella . At the same time, TG was negatively correlated with CA, CDCA, 7-keto-DCA and UCA, while HDL-C was positively correlated with α-MCA. There was a certain correlation between fecal bile acids and bacteria. For example, CDCA was negatively correlated with unclassified_f__Lachnospiraceae , UDCA was negatively correlated with Bifidobacterium , and 7-keto-DCA was positively correlated with Ruminococcus . One of the effective methods for LRS to ameliorated blood lipids in hyperlipidemic rats may be to regulate the relative abundance of Prevotella , unclassified_f__Lachnospiraceae , Ruminococcus , and promote the direct excretion of primary bile acids (CA, CDCA, α-MCA) and secondary bile acids such as UDCA and UCA, and then accelerate the transformation of liver cholesterol into primary bile acids. Reducing blood lipids by accelerating the production and expulsion of bile acids from the liver had been previously reported [ 7 , 8 ]. 4. Conclusion In this study, 16S rRNA technology and bile acids targeted metabolomics technology were used to determine the changes of fecal flora and bile acids profiles in normal rats and hyperlipidemic rats at different periods, aiming to reveal the hypolipidemic effect of LRS. Our findings suggested that LRS regulated the relative abundance of Prevotella , unclassified_f__Lachnospiraceae , Ruminococcus in hyperlipidemic rats, and promoted the direct excretion of primary bile acids (CA, CDCA, α-MCA) and secondary bile acids such as UDCA and UCA. Regulating the gut microbiota and accelerating the transformation of liver cholesterol into primary bile acids for excretion from the body was one of the effective means for LRS to reduce blood lipid levels in hyperlipidemic rats. The present study provides a theoretical basis for the hypolipidemic mechanism of LRS. Abbreviations ACA, apocholic acid; Allo-CA, allocholic acid； α-MCA, alpha-muricholic acid; β-MCA, beta-muricholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; CDCA-3β-D-G, chenodeoxycholic acid-3β-D-glucuronide; CDCA-24-Acyl-β-D-G, chenodeoxycholic acid 24-acyl-β-D-glucuronide; DCA, deoxycholic acid; Dehydro-LCA, dehydrolithocholic acid; Dehydro-CA, dehydrocholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GDCA, glycodeoxycholic acid; GHCA, glycohyocholic acid; GLCA, glycolithocholic acid; GUDCA, glycoursodeoxycholic acid; HCA, hyocholic acid; HDCA, hyodeoxycholic acid; HDL-C, high density lipoprotein cholesterol; isoDCA, isodeoxycholic acid; isoLCA, isolithocholic acid; LCA, lithocholic acid; LCA-3-S, lithocholic acid-3-sulfate; LDL-C, low density lipoprotein cholesterol; LRS, lotus seed resistant starch; MDCA, murideoxycholic acid; ω-MCA, omega-murichoclic acid; T-α-MCA, tauro-α-muricholic acid; T-β-MCA, tauro-β-muricholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TCHO, total cholesterol; TDCA, taurodeoxycholate acid; TG, triglycerides; THCA, taurohyocholic acid; THDCA, taurohyodeoxycholic acid; TLCA, taurolithocholic acid; TUDCA, tauroursodeoxycholic acid; UCA, ursocholic acid; UDCA, ursodeoxycholic acid; 3-Dehydro-CA, 3-dehydrocholic acid; 3β-CA, 3β-cholic acid; 3β-UDCA, 3β-ursodeoxycholic acid; 7-keto-DCA, 7-ketodeoxycholic acid; 7-keto-LCA, 7-ketolithocholic acid; 7,12-Diketo-LCA, 7,12-diketolithocholic acid; 12-keto-CDCA, 12-ketochenodeoxycholicacid; 12-keto-LCA, 12-ketolithocholic acid; 23-nor-CA, 23-norcholic acid; 23-nor-DCA, 23-nordeoxycholic acid. Declarations Author Contributions Suzhen Lei: Conceptualization, formal analysis, Writing - Original Draft. Yijun Jiang : Software, Methodology. Xiaoliang Cai : Investigation, Validation. Zhixiong Lin : Formal analysis. Yi Zhang: Project administration, supervision. Hongliang Zeng: Conceptualization, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. Ethic al Approval All animal studies were performed in compliance with the Guidelines for the Care and Use of Laboratory Animals (NIH Publication 85-23, 1996) published by the U.S. National Institutes of Health. Meanwhile all animal experimental procedures were approved by the Animal Ethics Committee of Fujian Academy of Chinese Medicine (Approval number: FJATCM-IAEC2021002). Consent to Participate Informed consent was obtained from all individual participants included in the study. Consent to Publish Not applicable. 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Jiang B, Yuan G, Wu J, Wu Q, Li L, Jiang P (2022) Prevotella copri ameliorates cholestasis and liver fibrosis in primary sclerosing cholangitis by enhancing the FXR signalling pathway. Biochim. Biophys. Acta, Mol. Basis Dis. 1868:166320. https://doi.org/10.1016/j.bbadis.2021.166320. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx 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. 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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-4210834","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":287990661,"identity":"2f4f633a-4cee-4c99-97ad-ef5b4cd1d24f","order_by":0,"name":"Suzhen Lei","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Suzhen","middleName":"","lastName":"Lei","suffix":""},{"id":287990662,"identity":"22b25fd3-62e5-41d2-b684-5888a2c12607","order_by":1,"name":"Yijun Jiang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yijun","middleName":"","lastName":"Jiang","suffix":""},{"id":287990663,"identity":"a4ac8015-9884-4edf-a6e8-4ef34837278a","order_by":2,"name":"Xiaoliang Cai","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoliang","middleName":"","lastName":"Cai","suffix":""},{"id":287990665,"identity":"ae1dab42-420d-42aa-89b6-df94b12b4671","order_by":3,"name":"Zhixiong Lin","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Zhixiong","middleName":"","lastName":"Lin","suffix":""},{"id":287990668,"identity":"c97e4b14-033b-4bb1-b333-bc201d6e36f3","order_by":4,"name":"Yi Zhang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":287990671,"identity":"62d76d4a-566b-40a4-9c76-ab9e729fa9d9","order_by":5,"name":"Hongliang Zeng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIie3OOwrCQBCA4VkWUq2kHZvkCmuteJYNgdhapZRdArHJAQLxEt4gIZAytkIEBS+QMhaCm9LCPDqL/buB+ZgBMJn+MaSyBdjYQHM9WZMIUSmIYCktMY+UM4ibKaVe3QXtmHFowxLsTA4TcipUxESDWDFO0roEvOXDhKJ3fIJoDnBPOF3EJXAUw8RCT6lO1OjqK/Q9hTBNJBM58p6QKQQ1iVjg46qy9kVS7xheR4ib+oXqNlt0Knp+dOHasdMR8lXefzpj32QymUy/+gCCgUGrMGQkcgAAAABJRU5ErkJggg==","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Hongliang","middleName":"","lastName":"Zeng","suffix":""}],"badges":[],"createdAt":"2024-04-03 07:44:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4210834/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4210834/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54425508,"identity":"a7ceedf1-8434-443a-9f2f-e20d34a23315","added_by":"auto","created_at":"2024-04-10 09:17:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":438468,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of animal experiment process.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/7ef7e52fe0cba8961e15e709.png"},{"id":54425519,"identity":"1b5b0b6c-3d4d-4f95-872b-d1ee9591d086","added_by":"auto","created_at":"2024-04-10 09:17:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":708264,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the overall difference of fecal flora in rats: (a) principal co-ordinates analysis (PCoA); (b) non-metric multidimensional scale (NMDS); (c) sample level clustering analysis; (d) PCoA; (e) NMDS; (f) sample level clustering analysis. Note: The overall difference analysis of normal rats was (a) (b) (c); and (d), (e), (f) were the overall differences among hyperlipidemic rats.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/a69957780faa9b981f15a177.png"},{"id":54425515,"identity":"3a9f758d-561f-4493-a104-e283e3b98217","added_by":"auto","created_at":"2024-04-10 09:17:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":635422,"visible":true,"origin":"","legend":"\u003cp\u003eClassification of taxa in feces microbial communities of rats: (a) phylum level, (b) genus level.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/a0e746ffb548e490f9f0647c.png"},{"id":54425501,"identity":"f1aba392-ac76-45de-b8d6-69bae0f5f4e0","added_by":"auto","created_at":"2024-04-10 09:17:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":289276,"visible":true,"origin":"","legend":"\u003cp\u003eDifference of rat fecal flora in each group at phylum level in different periods: (a) NC group; (b) NC_LRS group; (c) MC group; (d) MC_LRS group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/869d8d6c9fcd28efcfe905a9.png"},{"id":54425520,"identity":"c35db3f0-1b97-4a2c-88ac-dd3d51a3931c","added_by":"auto","created_at":"2024-04-10 09:17:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":553994,"visible":true,"origin":"","legend":"\u003cp\u003eDifference of rat fecal flora in each group at genus level in different periods: (a) NC group; (b) NC_LRS group; (c) MC group; (d) MC_LRS group.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/cf56e26f333189a1538a55c7.png"},{"id":54426121,"identity":"8d41b667-acef-4914-8cfb-dc55ae0ad30b","added_by":"auto","created_at":"2024-04-10 09:25:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1780916,"visible":true,"origin":"","legend":"\u003cp\u003eOverall distribution of fecal bile acids in each group at different periods: (a) total bile acids; (b) primary bile acids; (c) secondary bile acids.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/d2db57eb1f9a17bc1c0ab6a4.png"},{"id":54425506,"identity":"e1666e0c-4736-4125-8180-7ef776a930c7","added_by":"auto","created_at":"2024-04-10 09:17:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":975009,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in fecal primary bile acid content of rats in each group at detection points 3 and 2. Note: Primary bile acids content change value = primary bile acids content at detection point 3 - primary bile acids content at detection point 2\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/2f21f7927bb2968f2b43412b.png"},{"id":54425509,"identity":"16fae135-dfed-48cf-89ba-ccefe56e975d","added_by":"auto","created_at":"2024-04-10 09:17:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1027375,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in fecal secondary bile acids content of rats in each group at detection points 3 and 2. Note: Secondary bile acids content change value = secondary bile acids content at detection point 3 - secondary bile acids content at detection point 2.\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/5416f1ac24655fcce93e51ea.png"},{"id":54425513,"identity":"5fb55cb2-fdba-4964-882e-4a8ae7d49610","added_by":"auto","created_at":"2024-04-10 09:17:54","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":505266,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of correlation between fecal flora and bile acids in normal rats: (a) primary bile acids; (b) secondary bile acids.\u003c/p\u003e","description":"","filename":"floatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/d3843b7100d27f6efbf96bfa.png"},{"id":54425512,"identity":"d8d36336-793c-4577-937e-30d48daeaa6b","added_by":"auto","created_at":"2024-04-10 09:17:54","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":471693,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of correlation between fecal flora and bile acids in hyperlipidemic rats: (a) primary bile acids; (b) secondary bile acids.\u003c/p\u003e","description":"","filename":"floatimage19.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/5bc80b2a2d8256fc3729bb6c.png"},{"id":54425511,"identity":"d32d4a10-7643-424d-a2e1-955ed554bab7","added_by":"auto","created_at":"2024-04-10 09:17:54","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1912181,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation network of blood lipid index, fecal flora and bile acids\u003c/p\u003e","description":"","filename":"floatimage21.png","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/051ad8f43ae9c0bb2876bd81.png"},{"id":54522944,"identity":"ac5342d4-db8f-4207-8693-6ca4a32f7d37","added_by":"auto","created_at":"2024-04-11 18:38:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5553136,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/da8d193b-3dff-4c1c-a33e-14e362b628e8.pdf"},{"id":54426120,"identity":"51637981-40f2-4f97-adb7-97d1f25e43a6","added_by":"auto","created_at":"2024-04-10 09:25:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1540052,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4210834/v1/133289c95f2596db30ec5b18.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The dynamic revolution of intestinal flora and bile acids profiles revealed the hypolipidemic effect of lotus seed resistant starch","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePrimary bile acids are synthesized from cholesterol in the liver through the classical or alternative bile acid synthesis pathway, and then enter the intestine. Some of the primary bile acids form secondary bile acids by a series of microbial enzymes secreted by the gut flora, and excreted from the body with food debris [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hyperlipidemia is a kind of metabolic disease caused by lipid metabolism disorders, and usually high cholesterol is one of its main characteristics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Bile acids are important metabolic pathway of cholesterol, accelerating the conversion of cholesterol into bile acids and excreting them in the fecal may be one of the effective ways to reduce cholesterol levels in the body [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Tang et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] suggested that one of the main reasons that consumption of fragrant rapeseed oil could improve the blood lipid level in hyperlipidemic rats was that it promoted the excretion of bile acids from the body. Wang et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] also suggested that hyperoside lowered cholesterol through bile acids excretion.\u003c/p\u003e \u003cp\u003eThe high concentration of secondary bile acids is toxic to intestinal wall cells, and the production of secondary bile acids is closely related to the composition of intestinal flora [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A previous study had shown that the carbohydrate available to bacteria, such as resistant starch, changed the type and content of bile acids by regulating the structure and composition of gut flora [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Lei et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] showed that resistant starch could regulate the microbial community mediating the conversion of sodium taurocholate. LRS is a type 3 resistant starch formed by gelatinization and regeneration of lotus seed starch with high amylose content [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our previous studies had shown that LRS could reduce serum cholesterol and improve dyslipidemia in hyperlipidemic mice and rats. Meanwhile, LRS regulated intestinal flora and reduced the conversion of primary bile acids to secondary bile acids in rats [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. At present, the research on the relationship between resistant starch, intestinal flora and bile acids is one of the hot spots in contemporary food science, but many researchers tend to focus on the result of lowering blood lipid, while this paper pays more attention to the relationship between bacterial flora and bile acids in the process of lowering blood lipid, so as to achieve the goal of lowering blood lipid.\u003c/p\u003e \u003cp\u003eTherefore, in this paper, 16S rRNA technology and bile acids targeted metabolomics technology were used to determine the changes of fecal flora and bile acids profiles in normal rats and hyperlipidemic rats at different periods, and then the correlation heat map of fecal bacteria and bile acids was constructed to reveal the hypolipidemic effect of LRS. This result is expected to provide a theoretical basis for the hypolipidemic mechanism of LRS.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents and Materials\u003c/h2\u003e \u003cp\u003eFresh lotus seeds obtained from Green Field Fujian Food Co., Ltd. (Sanming, China) and LRS were prepared in the laboratory according to previous studies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Fresh lotus seeds undergo thawing, juice extraction, filtration, precipitation, washing, and drying to obtain lotus seed starch. LRS was obtained through steps such as high-temperature gelatinization, retrogradation, purification, and drying of lotus seed starch. A total of 46 bile acids standards were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd. (Shanghai, China), and the information is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Chromatographic grade methanol and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, USA), and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBile acids standards information\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv 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\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eglycochenodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16564-43-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003elithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e434-13-9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etaurocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e145-42-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echolic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e81-25-4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etaurochenodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e516-35-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echenodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e474-25-9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eglycoursodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGUDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64480-66-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ehyocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e547-75-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eallocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAllo-CA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2464-18-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23-norcholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23-nor-CA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e60696-62-0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eursodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e128-13-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eglycohyocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGHCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e32747-08-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83-44-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23-nordeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e23-nor-DCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e53608-86-9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etauroursodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTUDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14605-22-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eisolithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eisoLCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e1534-35-6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehyodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83-49-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12-ketolithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12-keto-LCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e5130-29-0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eapocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e641-81-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003edehydrolithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDehydro-LCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e1553-56-6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eglycodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16409-34-0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003elithocholic acid 3-sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLCA-3-S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e34669-57-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eglycolithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGLCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e474-74-8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echenodeoxycholic acid-3-β-D-glucuronide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCDCA-3-β-D-G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e58814-71-4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ealpha-muricholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eα-MCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2393-58-0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3β-ursodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3β-UDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e78919-26-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ebeta-muricholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ-MCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2393-59-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3-dehydrocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3-Dehydro-CA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e2304-89-4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7-ketolithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7-keto-LCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4651-67-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003echenodeoxycholic acid 24 - acyl - β-D-glucuronide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCDCA-24-Acyl-β-D-G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e208038-27-1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etauro-α-muricholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT-α-MCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25613-05-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12-ketochenodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12-keto-CDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e2458-08-4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etauro-β-muricholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT-β-MCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e145022-92-0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7,12-diketolithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7,12-Diketo-LCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e517-33-9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eomega-murichoclic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eω-MCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6830-03-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003edehydrocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDehydro-CA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e81-23-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emurideoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e668-49-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eursocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eUCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e2955-27-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etaurohyodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTHDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38411-85-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7-ketodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7-keto-DCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e911-40-0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etaurohyocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTHCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eisodeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eisoDCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e566-17-6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etaurolithocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTLCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6042-32-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3β-cholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3β-CA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c8\"\u003e \u003cp\u003e3338-16-7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animal experiment design and fecal collection\u003c/h2\u003e \u003cp\u003e All animal experimental procedures were approved by the Animal Ethics Committee of Fujian Academy of Chinese Medicine (Approval number: FJATCM-IAEC2021002). The feces were sourced from specific pathogen free grade Sprague Dawley male rats purchased from Slack Laboratory Animals Co., Ltd. (Shanghai, China, permit number: SCXK (Hu) 2017-0005). The experimental methods refer to previous study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and the process is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. After 1 week of adaptive feeding, a total of 24 rats were randomly divided into two groups with 12 rats in each group. One group was fed basic diet and the other group was fed high-fat diet. The high-fat feed was purchased from Fuzhou Wu's Animal Testing Co., Ltd. (Fuzhou, China), and formula was as follows: basic feed 89.25%, egg yolk powder 5%, lard 5%, cholesterol 0.5% and porcine bile salt 0.25%. After 2 weeks of feeding, blood was collected from the fundus venous plexus of rats from the two groups. The levels of TG, TCHO, HDL-C and LDL-C were measured by a fully automatic biochemical analyzer to determine the success of a rat model with hyperlipidemia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe hyperlipidemic rats were randomly divided into two groups with 6 rats in each group. Meanwhile the rats fed basic diet were also randomly divided into two groups with 6 rats in each group. LRS intervention was performed for 4 weeks, and the gavage dose of LRS was 0.25 g /100 g \u0026middot; day according to the Dietary Guidelines for U.S. Residents. Animal group information was as follows: rats fed a basal diet (NC group), rats fed a basal diet and gavage LRS (NC_LRS group), rats fed a high-fat diet (MC group), and rats fed a high-fat diet and gavage LRS (MC_LRS group). Physiological indexes of rats and the construction of hyperlipidemia models can be referred to previous study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Feces from each group of rats were collected during three stages: early modeling (detection point 1), end of modeling (detection point 2), and end of LRS intervention (detection point 3). The feces were collected in sterile freeze-storage tubes at 8 am, and stored in a -80℃ refrigerator for use. The naming of the samples is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The feces of three rats with no significant difference in physiological indicators between each group were used for the determination of microbial communities and bile acids.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eName information of each sample\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC_LRS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMC_LRS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003edetection point 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC_LRS_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMC_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMC_LRS_1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003edetection point 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC_LRS_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMC_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMC_LRS_2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003edetection point 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC_LRS_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMC_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMC_LRS_3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fecal flora determination\u003c/h2\u003e \u003cp\u003eIllumina next generation 16S rRNA gene amplicon sequencing technology was used to sequence fecal microflora of rats in each group. The method can be referred to previous studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. First, the total DNA of each sample was extracted, and its purity, concentration and integrity were tested. Then the 16S V3-V4 region was amplified by PCR, and the PCR products were recovered and purified. Finally, PCR products were quantified and sequenced in Miseq PE300 platform after library construction. After data optimization of the original sequence obtained by sequencing, the amplicon sequence variant (ASV) representative sequence and abundance information can be obtained. Based on the representative sequence and abundance information of ASVs, a series of statistical analyses were conducted to analyze the community diversity, composition and species differences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Determination of fecal bile acids\u003c/h2\u003e \u003cp\u003eFecal bile acids were measured using targeted metabolomics techniques, which can refer to previous study with appropriate modifications [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. First of all, metabolite extraction of feces was carried out: 50 mg of feces was weighed and added with 400 \u0026micro;L extraction solution (methanol: water\u0026thinsp;=\u0026thinsp;4:1), then ground by freezing grinder for 6 min (-10\u0026deg;C, 50 Hz), ultrasounded at low temperature for 30 min (5\u0026deg;C, 40 KHz), and stood at -20\u0026deg;C for 30 min. 200 \u0026micro;L supernatant was centrifugally taken for on-machine detection. Secondly, standard bile acids solutions of different concentrations were provided: 1 mg of each of 46 bile acids standard products was weighed, dissolved with methanol and fixed to 1 mL, then vortically mixed to obtain standard reserve solution. Finally, Liquid chromatography-mass spectrometry was used to determine the standard solution and the sample to be tested under the same conditions. Specific parameters were as follows:\u003c/p\u003e \u003cp\u003eChromatographic conditions: ExionLC AD system, Waters BEH C18 (150 * 2.1 mm, 1.7 \u0026micro;m) liquid chromatographic column, column temperature 40\u0026deg;C, injection volume 1 \u0026micro;L. Mobile phase A (0.1% formic acid-water solution), mobile phase B (0.1% formic acid-acetonitrile).\u003c/p\u003e \u003cp\u003eMass spectrometry conditions: AB SCIEX QTRAP 6500+, using negative mode detection. Curtain gas 35, collision gas medium, ionspray voltage \u0026minus;\u0026thinsp;4500. Temperature 500, ion source gas 1, 40, ion source gas 2, 50.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data analysis and statistics\u003c/h2\u003e \u003cp\u003eExperimental data were represented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and SPSS25.0 software was used for significance analysis and multiple comparisons. One-way analysis of variance was analyzed using Duncan's multiple range tests. Drawings were generated using OriginPro8.5 and GraphPad Prism8.0 software. The Pearson correlation coefficient was calculated, and correlation heatmaps or relationship network diagrams were drawn using R language and Cytoscape software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and analysis","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Analysis of the overall difference of fecal flora\u003c/h2\u003e \u003cp\u003eThe overall difference of fecal flora in normal rats and hyperlipidemic rats were analyzed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In normal rats, fecal flora of NC_1, NC_2, NC_LRS_1 and NC_LRS_2 groups were mainly in the first and fourth quadrants, while that of NC_3 and NC_LRS_3 groups were mainly in the second and third quadrants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a)). This result indicated that at detection point 3, the fecal flora structure of rats had been greatly changed. The structure of gut microbiota is related to age, and as the feeding time increases, the fecal flora of rats gradually changed [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Further, according to non-metric multidimensional scale (NMDS) and sample clustering analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b) (c)), NC_1, NC_2, NC_LRS_1 and NC_LRS_2 groups had high similarity in fecal flora, while NC_3 and NC_LRS_3 groups had high similarity in fecal flora. The results were consistent with those of principal co-ordinates analysis (PCoA). Previous study had also shown that gut flora changed dynamically as they grow [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (d) (e) (f), fecal flora structure of rats in MC_1 and MC_2 groups was different, and that of rats in MC_LRS_1 and MC_LRS_2 groups was also different to some extent, indicating that high-fat diet changed the fecal flora structure of normal rats. The alteration of fecal flora in rats by high-fat diet had been reported in previous study [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In hyperlipidemic rats, the fecal flora structure of rats in MC_3 and MC_LRS_3 groups was different from that in other groups, indicating that the fecal flora structure of rats with hyperlipidemia had a great change at detection point 3. The similarity in fecal flora structure between MC_3 group and MC_LRS_3 group rats was high. In terms of the overall difference analysis, LRS had no significant effect on fecal flora structure of hyperlipidemic rats during the feeding time of detection point 2 and detection point 3, and the fecal flora composition of rats in each group was further analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Community composition of fecal flora at different classification levels\u003c/h2\u003e \u003cp\u003eFecal flora community composition of rats in each group at different periods is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (a), Firmicutes and Bacteroidota were the main microorganisms in rat feces at the phylum level, accounting for more than 80% of the total number of microorganisms. It was followed by a small amount of Actinobacteriota, Spirochaetota, Desulfobacterota and Proteobacteria. The results were consistent with previous research [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In normal rats, the relative abundance of Firmicutes in feces of NC_2 group was higher than that of NC_1 group, while the relative abundance of Bacteroidota, Actinobacteriota and Spirochaetota was lower than that of NC_1 group. Compared with NC_2 group, the relative abundance of Bacteroidota increased, while the relative abundance of Firmicute, Actinobacteriota and Spirochaetota decreased in NC_3 group. The trend of relative abundance of Firmicute, Bacteroidota and Actinobacteriota in the feces of NC_LRS group was the same as that of NC group. The relative abundance of Firmicute in NC_LRS_3 group was lower than that in NC_3 group, while the relative abundance of Bacteroidota was higher. The impact of LRS on the composition of fecal flora should be further analyzed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, Compared to NC_2 group, the relative abundance of Firmicute in MC_2 group increased, while the relative abundance of Actinobacteriota decreased. In hyperlipidemic rats, the relative abundance of Firmicute in feces of MC_2 group was higher than that of MC_1 group, while the relative abundance of Bacteroidota and Actinobacteriota was lower than that of MC_1 group. Yan et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] also found that the relative abundance of Firmicute increased while Bacteroidota decreased in rats with high fat diet when studying the lowering of blood lipid of rice buckwheat. Compared with MC_2 group, the relative abundance of Bacteroidota increased while the relative abundance of Firmicute and Actinobacteriota decreased in MC_3 group. In the MC_LRS group, the relative abundance of Firmicute in feces of MC_LRS_3 group was the highest, followed by MC_LRS_2 group, and the relative abundance of Firmicute in feces of MC_LRS_1 group was the lowest. On the contrary, MC_LRS_1 group had the highest relative abundance of Bacteroidota, followed by MC_LRS_2 and MC_LRS_3 groups. Compared with MC_3 group, the relative abundance of Firmicute and Actinobacteriota increased, while the relative abundance of Bacteroidota decreased in MC_LRS_3 group. After the intervention of LRS, the composition of fecal flora of rats in MC group and MC_LRS group was different, mainly Firmicute and Bacteroidota. The relative abundance of Firmicute increased and the relative abundance of Bacteroidota decreased after LRS administration in hyperlipidemic rats. Zhang et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] also found that the relative abundance of Bacteroidota decreased in hyperlipidemic mice treated with resistant starch when studying specific gut microbiota properties related to anti-hyperlipidemic action of resistant starch.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (b), at the genus level, the main microorganisms in rat feces including \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e, accounted for more than 50% of the total number of microorganisms. Among them, the \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e belong to Firmicute, \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eAlloprevotella\u003c/em\u003e belong to Bacteroidota, while \u003cem\u003eBifidobacterium\u003c/em\u003e belong to Actinobacteriota. The results were consistent with previous research [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In normal rats, the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e in NC_2 group were lower than that in NC_1 group, while the relative abundance of \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e was higher than that of NC_1 group. Compared with NC_2 group, the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e in feces in NC_3 group increased. The trend of relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e in feces in NC_LRS group was the same as that in NC group.\u003c/p\u003e \u003cp\u003eIn addition, compared to NC_2 group, the relative abundance of \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e and \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e decreased, while the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eRomboutsia\u003c/em\u003e increased in MC_2 group. In hyperlipidemic rats, the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e in feces of MC_2 group were lower than that of MC_1 group and MC_3 group, while the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eRomboutsia\u003c/em\u003e were higher than those of MC_1 and MC_3 groups. The trend of relative abundance of \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e in the feces of MC_LRS group and MC group was different. This result indicated that LRS had regulatory effect on the relative abundance of \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e in hyperlipidemic rats. Previous study also had shown that resistant starch had a certain regulatory effect on intestinal flora in hyperlipidemic rats [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Analysis of species difference of fecal flora\u003c/h2\u003e \u003cp\u003eFurther, differences in fecal flora of rats in each group were analyzed at phylum and genus levels, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the main species of fecal flora in rats at the phylum level were Firmicute and Bacteroidota, followed by Actinobacteriota. Compared with detection points 1 and 2, the relative abundance of Firmicute, Actinobacteriota, Spirochaetota and Desulfobacterota in rat feces of NC_3 group were low, and the relative abundance of Bacteroidota was high (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a)). Compared with detection point 2, the relative abundance of Firmicute, Actinobacteriota, Spirochaetota and Desulfobacterota in the feces of rats in NC_LRS_3 group were decreased and the relative abundance of Bacteroidota was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (b)). The variation trend of fecal flora in NC group and NC_LRS group was similar at the phylum level, which should be further discussed. In the rats with hyperlipidemia, compared with detection point 2, the relative abundance of Firmicute and Actinobacteriota decreased, while the relative abundance of Bacteroidota increased in feces of MC_3 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (c)). After the intervention of LRS, the relative abundance of Firmicute and Actinobacteriota increased while the relative abundance of Bacteroidota decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (d)). Compared with normal rats, LRS had a more obvious effect on fecal bacteria community of hyperlipidemic rats, mainly reflected in Firmicute, Bacteroidota and Actinobacteriota. Zheng et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] also showed that resistant starch could ameliorate gut microbiota disorder in hyperlipidemic rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the genus level, the differences among the top 15 species in the relative abundance of fecal flora of rats in each group were analyzed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The main species included \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e in feces of normal rats. Compared with NC_2 group, the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eRomboutsi\u003c/em\u003ea, \u003cem\u003eRuminococcus\u003c/em\u003e and \u003cem\u003ePrevotellaceae_NK3B31_group\u003c/em\u003e increased, while the relative abundance of \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e reduced in NC_3 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (a)). Compared with NC_LRS_2 group, the relative abundance of \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e increased while the relative abundance \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e and \u003cem\u003eClostridium_sensu_stricto_1\u003c/em\u003e decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (b)). Between detection point 2 and detection point 3, the trends of relative abundance of \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e and \u003cem\u003enorank_f__norank_o__Clostridia_UCG-014\u003c/em\u003e in NC group and NC_LRS group were different. The results showed that LRS could promote \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003enorank_f__norank_o__Clostridia_UCG-014\u003c/em\u003e proliferation in normal rats. Resistant starch was a kind of polysaccharide [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and polysaccharide promoted intestinal \u003cem\u003enorank_f__norank_o__Clostridia_UCG-014\u003c/em\u003e proliferation had been reported in previous study [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn hyperlipidemic rats, the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eAllobaculum\u003c/em\u003e, \u003cem\u003ePrevotellaceae_NK3B31_group\u003c/em\u003e in MC_3 group were higher than that of MC_2 group, while the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eRoseburia\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e were lower than that of MC_2 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (c)). After LRS intervention, the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003enorank_f__Muribaculaceae\u003c/em\u003e, \u003cem\u003eAllobaculum\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eRuminococcus\u003c/em\u003e increased, while the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eStaphylococcus\u003c/em\u003e reduced in MC_LRS_3 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (d)). During the period of intervention, the change trends of relative abundance of \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e, \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e and \u003cem\u003eBacteroides\u003c/em\u003e in MC group and MC_LRS group were different. The results showed that intragastric administration of LRS to hyperlipidemic rats, the relative abundance of \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e increased, and the relative abundance of \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e and \u003cem\u003eBacteroides\u003c/em\u003e decreased in feces. Previous studies had also shown that resistant starch could promote proliferation of \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eAlloprevotella\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, compared with the MC group, the variation amplitude of the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e at detection point 3 and 2 in the MC_LRS group were higher than that of the MC group, while the variation amplitude of the relative abundance of \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eRomboutsia\u003c/em\u003e and \u003cem\u003eAllobaculum\u003c/em\u003e at detection point 3 and 2 were lower than that of the MC group. These results indicated that LRS promoted the proliferation of \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eRomboutsia\u003c/em\u003e, and inhibited the growth of \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eAllobaculum\u003c/em\u003e in hyperlipidemic rats. Liang et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] also found that the relative abundance of \u003cem\u003eAllobaculum\u003c/em\u003e decreased in the high-resistant starch group when studying the lowering of blood lipid by resistant starch. In short, the regulation effects of LRS on microflora of normal rats and hyperlipidemic rats were different.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of LRS on the overall distribution of fecal bile acids\u003c/h2\u003e \u003cp\u003eTargeted metabolomics technology was used to measure fecal bile acids at detection points 1, 2 and 3 of rats in each group, and 46 kinds of bile acids were obtained. They were classified into primary bile acids and secondary bile acids, and total bile acids were the sum of primary bile acids and secondary bile acids, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The proportion changes of primary or secondary bile acids in the feces of rats in different groups at different stages can be found in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (a), the contents of secondary bile acids in rat feces were high. In normal rats, compared with NC_1 group, the contents of total bile acids and secondary bile acids in feces of NC_2 group were increased, while the content of primary bile acids was decreased. The contents of total bile acids, secondary bile acids and primary bile acids in feces in NC_3 group were lower than those in NC_2 group. The change trend of fecal bile acids in NC_LRS group was the same as that in NC group. The results indicated that LRS had no significant effect on the overall distribution of fecal bile acids in normal rats, which should be further analyzed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the contents of total bile acids, primary bile acids and secondary bile acids in feces of MC_2 group were increased compared with those of MC_1 group. Tang et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] also found that the content of total bile acids in feces of rats with high fat diet increased significantly when studying the lowering of blood lipid by rapeseed oil. In hyperlipidemic rats, compared with MC_2 group, the contents of total bile acids and secondary bile acids in feces of rats in MC_3 group were increased. After LRS intervention, the variation trend of fecal bile acids in MC_LRS group was different from that in MC group. Compared with MC_LRS_2 group, total bile acids content in feces of rats in MC_LRS_3 group had no significant change, while primary bile acids content was increased and secondary bile acids content was decreased. These results indicated that LRS could promote the excretion of primary bile acids and reduce the conversion of primary bile acids to secondary bile acids in hyperlipidemic rats. Secondary bile acids are produced by a series of microbial enzymes secreted by intestinal flora acting on primary bile acids. The types and contents of bile acids in feces of rats in different groups were related to the feces flora [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe types of primary bile acids and secondary bile acids in feces of rats in each group were further analyzed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (b) (c). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (b), the primary bile acids in rat feces were mainly free primary bile acids, including α-MCA, β-MCA, CA and CDCA. In normal rats, compared with NC_2 group, the total amount of primary bile acids in feces of NC_3 group decreased, mainly α-MCA and β-MCA contents decreased. The change trends of α-MCA and β-MCA contents in feces of rats in NC_LRS group were the same as those in NC group. In addition, compared with MC_1 group, the total amount of primary bile acids in feces of MC_2 group was increased, including β-MCA, CA and CDCA contents. Previous studies had shown that a high-fat diet increased the secretion of 12α-hydroxyl bile acids and promoted fecal bile acids excretion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In hyperlipidemic rats, the total amount of primary bile acids in feces of rats in MC_2 and MC_3 groups had no significant change. After LRS intervention, the variation trend of fecal primary bile acids in MC_LRS group was different from that in MC group. Compared with MC_LRS_2 group, the content of primary bile acids in feces of rats in MC_LRS_3 group was increased, mainly including β-MCA and CA. Intragastric administration of LRS in hyperlipidemic rats could promote the excretion of primary bile acids (β-MCA, CA). As a new type of dietary fiber, resistant starch had a certain effect on bile acids profiles [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous study had also shown that dietary fiber promoted CA excretion in feces of rats on a high fat diet [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (c), the types of secondary bile acids in normal rat feces were mainly free secondary bile acids, including DCA, HDCA, ω-MCA, LCA, MDCA, Dehydro-LCA, isoLCA, etc. Compared with NC_2 group, the total content of secondary bile acids in feces of rats in NC_3 group was decreased, mainly including DCA, ω-MCA and LCA. Between detection point 2 and detection point 3, the variation trends of ω-MCA and LCA in feces of rats in NC_LRS group were the same as those in NC group, while the variation trend of Dehydro-LCA was different from that in NC group. In hyperlipidemic rats, the types of secondary bile acids were mainly free secondary bile acids, including UDCA, DCA, HDCA, ω-MCA, MDCA, LCA, HCA, etc. The results were consistent with previous research [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Compared with the MC_1 group, the total amount of secondary bile acids in feces of rats in MC_2 group was increased, including the contents of DCA, HDCA and LCA, and the high concentration of secondary bile acids in feces was associated with cholesterol cholelithiasis and other diseases [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Compared with MC_2 group, the total amount of secondary bile acids in feces of rats in MC_3 group increased, mainly including the contents of HDCA and ω-MCA. After LRS intervention, the total amount of secondary bile acids in feces of rats in MC_LRS_3 group was lower than that in MC_LRS_2 group, mainly including the reduction of HDCA, MDCA and LCA. The excretion of HDCA, MDCA and LCA in feces of hyperlipidemic rats was reduced by intragastric administration of LRS. As a new type of dietary fiber, LRS could promote the direct excretion of primary bile acids, and reduce the conversion of primary bile acids to secondary bile acids in hyperlipidemic rats [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effect of LRS on fecal primary bile acids profiles\u003c/h2\u003e \u003cp\u003eFurther, the influence of LRS on the primary bile acids profiles of normal rats and hyperlipidemic rats were explored, and the bile acids changes at detection points 2 and 3 were discussed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, it is the difference of rat feces primary bile acids at detection point 3 and point 2. The difference above the X-axis indicates the increase of bile acids content; otherwise, it indicates the decrease of bile acids content. In normal rats, compared with NC_2 group, the contents of α-MCA, β-MCA, CA and CDCA in feces of NC_3 group were decreased, while the contents of TCA and T-β-MCA were increased, with significant changes in α-MCA and β-MCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (a)). Compared with NC_LRS_2 group, the contents of α-MCA, β-MCA, CA and CDCA in feces of rats in NC_LRS_3 group were decreased, while the contents of TCA and T-β-MCA were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (b)). The change trend of fecal primary bile acids profiles in NC_LRS group was the same as that in NC group. However, the amplitude of change was different, mainly reflected in α-MCA, CA and CDCA, suggesting that the excretion of fecal CA and CDCA could be promoted by gavage of LRS in normal rats. Previous study had also shown that the bile acid profile was closely related to dietary fiber in the diet [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn hyperlipidemic rats, compared with MC_2 group, the contents of TCA, β-MCA, T-β-MCA and CA in feces of rats in MC_3 group were increased, while the content of CDCA was decreased, with great changes in CDCA and CA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (c)). The contents of α-MCA, β-MCA and CA in feces of rats in MC_LRS_3 group were increased, while the content of CDCA was decreased compared with those in MC_LRS_2 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (d)). The variation trend or amplitude of fecal primary bile acids profiles of rats in MC_LRS group were different from those in MC group, mainly manifested in α-MCA, β-MCA, T-β-MCA, CA and CDCA. These results indicated that the intragastric administration of LRS could promote the excretion of free primary bile acids (α-MCA, β-MCA, CA, and CDCA) and reduce the excretion of combined primary bile acids (TCA, T-β-MCA) in hyperlipidemic rats. Promoting the excretion of free primary bile acids may be one of the effective ways for LRS to reduce cholesterol in hyperlipidemic rats [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, LRS could promote the excretion of free primary bile acids (CA and CDCA) in rats, which may be because resistant starch had certain adsorption effect on bile acids and could accelerate their excretion in the body [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effect of LRS on fecal secondary bile acids profiles\u003c/h2\u003e \u003cp\u003eIn order to explore the effect of LRS on the secondary bile acids profiles of normal rats and hyperlipidemic rats, the top 10 secondary bile acids in two kinds of rat feces were analyzed according to their contents. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, it is the difference of secondary bile acids in rat feces at detection point 3 and point 2. The difference above the X-axis indicates the increase of bile acids content; otherwise, it indicates the decrease of bile acids content. In normal rats, compared with NC_2 group, the contents of DCA, HDCA, ω-MCA, MDCA, LCA and isoLCA in feces of NC_3 group were decreased, while the content of Dehydro-LCA was increased, and ω-MCA and LCA were significantly changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (a)). Although the variation trends of DCA, HDCA, ω-MCA, MDCA and LCA in feces of rats in NC_LRS group were the same as those in NC group, the variation amplitude of secondary bile acids profiles was different. They were mainly reflected in DCA, HDCA, ω-MCA, LCA and isoLCA. Meanwhile, the trend of Dehydro-LCA in feces of rats in NC_LRS group was different from that in NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (b)). The results showed that the excretion of DCA, HDCA, LCA and isoLCA could be promoted and the contents of ω-MCA and Dehydro-LCA could be reduced in normal rats by gavage of LRS. Secondary bile acids are closely related to gut microbiota, and previous study had shown that a diet rich in resistant starch resulted in altered microbiota-dependent secondary bile acids pool size and composition [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn hyperlipidemic rats, compared with MC_2 group, the contents of DCA, HDCA, ω-MCA, MDCA, HCA and 12-keto-CDCA in feces of MC_3 group were increased, while the contents of UDCA, LCA, UCA and 7-keto-DCA were decreased, among which HDCA and ω-MCA had great changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (c)). Compared with MC_LRS_2 group, the contents of HDCA, ω-MCA, MDCA, LCA and HCA were decreased, while the contents of UDCA, UCA and 7-keto-DCA were increased in feces of rats in MC_LRS_3 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (d)). There were differences in the change trend of secondary bile acids profiles between MC_LRS group and MC group, mainly UDCA, HDCA, ω-MCA, MDCA, LCA, HCA, UCA and 7-keto-DCA. Intragastric administration of LRS in hyperlipidemic rats could promote the excretion of UDCA, UCA and 7-keto-DCA, and reduce the contents of HDCA, ω-MCA, MDCA, LCA and HCA in feces. The effects of LRS on secondary bile acids profiles in feces of normal rats and hyperlipidemic rats were different, which might be because resistant starch had different regulation effects on the bacterial community of the two kinds of rats, and different bacterial community structure would affect the type and content of secondary bile acids [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Correlation analysis between fecal flora and bile acids\u003c/h2\u003e \u003cp\u003eAccording to the composition of fecal flora and bile acids profiles, LRS had different effects on fecal flora and bile acids profiles of normal rats and hyperlipidemic rats. Bile acids are closely related to intestinal flora, so pearson correlation coefficient was further used to analyze the correlation among fecal flora, primary bile acids and secondary bile acids of rats. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, it is the correlation heat map between the top 15 bacteria genera with the relative abundance of normal rat fecal flora and the primary and secondary bile acids, among which the secondary bile acids are the top 10 according to the content in normal rat feces. In normal rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (a)), free primary bile acids were mainly positively correlated with \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eTuricibacter\u003c/em\u003e, and negatively correlated with \u003cem\u003enorank_f__norank_o__Clostridia_UCG-014\u003c/em\u003e. Among them, β-MCA had significant positive correlation with \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eTuricibacter\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), α-MCA and CDCA were positively correlated with \u003cem\u003eBifidobacterium\u003c/em\u003e (0.001\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while β-MCA and α-MCA were negatively correlated with \u003cem\u003enorank_f__norank_o__Clostridia_UCG-014\u003c/em\u003e (0.001\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Further, as can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (b), HDCA was significantly positively correlated with \u003cem\u003enorank_f__norank_o__Clostridia_UCG-014\u003c/em\u003e (0.001\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Meanwhile HDCA was positively correlated with \u003cem\u003eRuminococcus\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and had a significant negative correlation with \u003cem\u003eBifidobacterium\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results indicated that the relative abundance of \u003cem\u003enorank_f__norank_o__Clostridia_UCG-014\u003c/em\u003e in normal rat feces was related to the type and content of bile acids [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. After the intervention of LRS, the relative abundance of \u003cem\u003enorank_f__norank_o__Clostridia_UCG-014\u003c/em\u003e in normal rats could be adjusted. The regulation of intestinal flora by resistant starch had been previously reported [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs can be seen form Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e (a), free primary bile acids (CDCA and CA) were significantly correlated with fecal flora in hyperlipidemic rats. Among them, CDCA was positively correlated with \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e and \u003cem\u003eStaphylococcus\u003c/em\u003e. CA was positively correlated with \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eAllobaculum\u003c/em\u003e, while had a significant negative correlated with \u003cem\u003eBifidobacterium\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, α-MCA was positively correlated with \u003cem\u003eRuminococcus\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and was significantly negative correlated with \u003cem\u003ePrevotella\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, in hyperlipidemic rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e (b)), UDCA were significantly positive correlated with \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eTuricibacter\u003c/em\u003e and \u003cem\u003eStaphylococcus\u003c/em\u003e (0.001\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and were positively correlated with \u003cem\u003eRoseburia\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was a significant negative correlation between UCA and \u003cem\u003ePrevotella\u003c/em\u003e, and a significant positive correlation between UCA and \u003cem\u003eRomboutsia\u003c/em\u003e (0.001\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the feeding process, \u003cem\u003eRomboutsia\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e in feces of hyperlipidemic rats were closely related to bile acids. This result was consistent with previous studies that \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e are bile acids metabolism related bacteria [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Correlation analysis of blood lipid indicators, fecal flora and bile acids\u003c/h2\u003e \u003cp\u003eFurther, correlation analysis was carried out on the blood lipid indicators, fecal flora and bile acids of rats, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. The blood lipid indicators (TCHO, TG, LDL-C, HDL-C) were obtained by measuring the abdominal arterial blood of rats in each group after the end of the experimental period. The data can be viewed from previous study, which showed that oral administration of LRS in hyperlipidemic rats could ameliorate their blood lipid levels [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. TCHO, LDL-C were negatively correlated with CA, CDCA, UDCA and UCA, and TCHO was positively correlated with \u003cem\u003ePrevotella\u003c/em\u003e. At the same time, TG was negatively correlated with CA, CDCA, 7-keto-DCA and UCA, while HDL-C was positively correlated with α-MCA. There was a certain correlation between fecal bile acids and bacteria. For example, CDCA was negatively correlated with \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, UDCA was negatively correlated with \u003cem\u003eBifidobacterium\u003c/em\u003e, and 7-keto-DCA was positively correlated with \u003cem\u003eRuminococcus\u003c/em\u003e. One of the effective methods for LRS to ameliorated blood lipids in hyperlipidemic rats may be to regulate the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e, and promote the direct excretion of primary bile acids (CA, CDCA, α-MCA) and secondary bile acids such as UDCA and UCA, and then accelerate the transformation of liver cholesterol into primary bile acids. Reducing blood lipids by accelerating the production and expulsion of bile acids from the liver had been previously reported [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, 16S rRNA technology and bile acids targeted metabolomics technology were used to determine the changes of fecal flora and bile acids profiles in normal rats and hyperlipidemic rats at different periods, aiming to reveal the hypolipidemic effect of LRS. Our findings suggested that LRS regulated the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eunclassified_f__Lachnospiraceae\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e in hyperlipidemic rats, and promoted the direct excretion of primary bile acids (CA, CDCA, α-MCA) and secondary bile acids such as UDCA and UCA. Regulating the gut microbiota and accelerating the transformation of liver cholesterol into primary bile acids for excretion from the body was one of the effective means for LRS to reduce blood lipid levels in hyperlipidemic rats. The present study provides a theoretical basis for the hypolipidemic mechanism of LRS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACA, apocholic acid; Allo-CA, allocholic acid； \u0026alpha;-MCA, alpha-muricholic acid; \u0026beta;-MCA, beta-muricholic acid; CA, cholic acid; CDCA, chenodeoxycholic acid; CDCA-3\u0026beta;-D-G, chenodeoxycholic acid-3\u0026beta;-D-glucuronide; CDCA-24-Acyl-\u0026beta;-D-G, chenodeoxycholic acid 24-acyl-\u0026beta;-D-glucuronide; DCA, deoxycholic acid; Dehydro-LCA, dehydrolithocholic acid; Dehydro-CA, dehydrocholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GDCA, glycodeoxycholic acid; GHCA, glycohyocholic acid; GLCA, glycolithocholic acid; GUDCA, glycoursodeoxycholic acid; HCA, hyocholic acid; HDCA, hyodeoxycholic acid; HDL-C, high density lipoprotein cholesterol; isoDCA, isodeoxycholic acid; isoLCA, isolithocholic acid; LCA, lithocholic acid; LCA-3-S, lithocholic acid-3-sulfate; LDL-C, low density lipoprotein cholesterol; LRS, lotus seed resistant starch; MDCA, murideoxycholic acid; \u0026omega;-MCA, omega-murichoclic acid; T-\u0026alpha;-MCA, tauro-\u0026alpha;-muricholic acid; T-\u0026beta;-MCA, tauro-\u0026beta;-muricholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TCHO, total cholesterol; TDCA, taurodeoxycholate acid; TG, triglycerides; THCA, taurohyocholic acid; THDCA, taurohyodeoxycholic acid; TLCA, taurolithocholic acid; TUDCA, tauroursodeoxycholic acid; UCA, ursocholic acid; UDCA, ursodeoxycholic acid; 3-Dehydro-CA, 3-dehydrocholic acid; 3\u0026beta;-CA, 3\u0026beta;-cholic acid; 3\u0026beta;-UDCA, 3\u0026beta;-ursodeoxycholic acid; 7-keto-DCA, 7-ketodeoxycholic acid; 7-keto-LCA, 7-ketolithocholic acid; 7,12-Diketo-LCA, 7,12-diketolithocholic acid; 12-keto-CDCA, 12-ketochenodeoxycholicacid; 12-keto-LCA, 12-ketolithocholic acid; 23-nor-CA, 23-norcholic acid; 23-nor-DCA, 23-nordeoxycholic acid.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuzhen Lei:\u003c/strong\u003e Conceptualization, formal analysis, Writing - Original Draft. \u003cstrong\u003eYijun Jiang\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Software, Methodology.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eXiaoliang Cai\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eInvestigation, Validation.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eZhixiong Lin\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Formal analysis. \u003cstrong\u003eYi Zhang:\u0026nbsp;\u003c/strong\u003eProject administration, supervision.\u003cstrong\u003e\u0026nbsp;Hongliang Zeng:\u003c/strong\u003e Conceptualization, Writing - Review \u0026amp; Editing, Supervision, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthic\u003c/strong\u003e\u003cstrong\u003eal\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies were performed in compliance with the \u003cem\u003eGuidelines for the Care and Use of Laboratory Animals\u003c/em\u003e (NIH Publication 85-23, 1996) published by the U.S. National Institutes of Health. Meanwhile all animal experimental procedures were approved by the Animal Ethics Committee of Fujian Academy of Chinese Medicine (Approval number: FJATCM-IAEC2021002).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant number: 31972076), the Program for Leading Talent in Fujian Provincial University (grant number: 660160190).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang Y, Ding W X, Li T (2018) Cholesterol and bile acid-mediated regulation of autophagy in fatty liver diseases and atherosclerosis. 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Basis Dis. 1868:166320. https://doi.org/10.1016/j.bbadis.2021.166320.\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":"Lotus seed resistant starch, Fecal flora, Bile acids, Hypolipidemic effect, Correlation network","lastPublishedDoi":"10.21203/rs.3.rs-4210834/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4210834/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOur research group had shown that lotus seed resistant starch (LRS) had hypolipidemic effect, but its mechanism is still being studied. Bile acids are important metabolic pathway of cholesterol, accelerating the conversion of cholesterol into bile acids and excreting them in the fecal may be one of the effective ways to reduce cholesterol levels in the body. This study aimed to reveal the lipid-lowering effect of LRS from the perspectives of fecal microbiota and bile acids. Herein, a rat model of hyperlipidemia was established and intervened with LRS. Fecal samples from different periods were collected to study the changes in microbiota and bile acids, and the correlation network diagram was established to reveal the lipid-lowering mechanism of LRS. The results showed that LRS inhibited the growth of \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eAllobaculum\u003c/em\u003e in hyperlipidemic rats. Meanwhile LRS promoted the excretion of cholic acid (CA), chenodeoxycholic acid (CDCA), alpha-muricholic acid (α-MCA), ursodeoxycholic acid (UDCA), ursocholic acid (UCA), 7-ketodeoxycholic acid (7-keto-DCA) in hyperlipidemic rats. Furthermore, total cholesterol (TCHO), low-density lipoprotein cholesterol (LDL-C) were negatively correlated with CA, CDCA, UDCA and UCA, and TCHO was positively correlated with \u003cem\u003ePrevotella\u003c/em\u003e. Triglycerides (TG) was negatively correlated with CA, CDCA, 7-keto-DCA and UCA, while high-density lipoprotein cholesterol (HDL-C) was positively correlated with α-MCA. Regulating the gut microbiota such as \u003cem\u003ePrevotella\u003c/em\u003e and accelerating the transformation of liver cholesterol into primary bile acids (CA, CDCA) for excretion from the body was one of the effective means for LRS to ameliorate blood lipid levels in hyperlipidemic rats.\u003c/p\u003e","manuscriptTitle":"The dynamic revolution of intestinal flora and bile acids profiles revealed the hypolipidemic effect of lotus seed resistant starch","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 09:17:42","doi":"10.21203/rs.3.rs-4210834/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":"b5649ceb-825d-44bb-b200-23ab814422bf","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-11T18:30:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-10 09:17:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4210834","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4210834","identity":"rs-4210834","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}