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Effect of Lactobacillus rhamnosus hsryfm 1301 on the Gut Microbiota and Lipid Metabolism in Rats Fed a High-Fat Diet
Effect of Lactobacillus rhamnosus hsryfm 1301 on the Gut Microbiota and Lipid Metabolism in Rats Fed a High-Fat Diet
Journal of Microbiology and Biotechnology. 2015. May, 25(5): 687-695
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : September 26, 2014
  • Accepted : November 22, 2014
  • Published : May 28, 2015
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About the Authors
Dawei Chen
Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, P.R. China
Zhenquan Yang
Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, P.R. China
Xia Chen
Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, P.R. China
Yujun Huang
Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, P.R. China
Boxing Yin
Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, P.R. China
Feixiang Guo
Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, P.R. China
Haiqing Zhao
Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, P.R. China
Jiadi Huang
Royal Dairy (Guangxi) Co., Ltd., Nanning, Guangxi 530007, P.R. China
Yun Wu
Royal Dairy (Guangxi) Co., Ltd., Nanning, Guangxi 530007, P.R. China
Ruixia Gu
Key Laboratory of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, P.R. China
Guruixia1963@163.com

Abstract
Accumulating evidence indicates that lactic acid bacteria could improve host physiology and lipid metabolism. To investigate the effect of the gut microbiota on host lipid metabolism, a hyperlipidemic rat model was established by feeding rats a high-fat diet for 28 days, and the gut microbiota of the rats was analyzed using real-time PCR before and after administration of Lactobacillus rhamnosus hsryfm 1301 and its fermented milk for 28 days. The findings showed that the Lactobacillus spp., Bifidobacterium spp., Bacteroides spp., and Enterococcus spp. content in the hyperlipidemic rats gut was increased significantly ( p < 0.05), while the Clostridium leptum and Enterobacter spp. content was decreased significantly after intervening with L. rhamnosus hrsyfm 1301 and its fermented milk for 28 days ( p < 0.05). Furthermore, the lipid levels of the serum and the liver were decreased significantly ( p < 0.05) and the fecal water content was increased significantly ( p < 0.05) in the hyperlipidemic rats after the intervention, and hepatocyte fatty degeneration of liver tissues was also prevented. A positive correlation was observed between the Clostridium leptum content and the level of serum cholesterol, triglycerides, low-density lipoprotein, and high-density lipoprotein, and a negative correlation was observed between the Enterobacter spp. content and the Lactobacillus spp. and Bifidobacterium spp. content in the hyperlipidemic rats gut. These results suggest that the gut microbiota and lipid metabolism of hyperlipidemic rats could be improved by supplementation with L. rhamnosus hsryfm 1301 and its fermented milk.
Keywords
Introduction
The human gut harbors a large number of complex microbial communities, and growing evidence [32 , 33] shows that the gut microbiota affects the physiological functions of the host, such as nutrient digestion and absorption, energy production, and lipid metabolism; moreover, the microbiome is rich in nutrient metabolism genes, which are involved in the metabolism of carbohydrates, amino acids, etc ., most of which humans do not have [31] .
Lactobacillus spp. and Bifidobacterium spp. are important probiotics in the human gut, and maintain the microbiota balance of the human intestine and enhance human immunity [34] . Studies [6 , 14] have also found that Bacteroides spp. and Clostridium leptum are the major bacteria found in the healthy human gut. Bacteroides spp. can absorb polysaccharides that humans cannot degrade, and can also regulate the generation of bacterial toxins and enhance the innate immune response in the host [7 , 37] . Clostridium leptum has the ability to degrade cellulose and produce butyrate and plays an important role in the energy metabolism and development of colonic epithelial cells, which contributes to the inhibition of colon cancer [9 , 30] . Because these microorganisms are anaerobic or facultatively anaerobic, it is difficult to study their changes in the gut by culturing methods; therefore, molecular techniques such as fluorescence quantitative Polymerase Chain Reaction and Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis technologies are increasingly being used in the study of these microbes [4] .
Diet is one of the most important factors that affects the composition and function of the gut microbiota [35] . High levels of serum lipid are closely associated with atherosclerosis, coronary heart disease, and other cardiovascular diseases [36] . Human and animal studies [1 , 10 , 15] show that Lactobacillus can regulate lipid metabolism by adjusting the intestinal microbiota. However, there are huge differences in growth conditions in vivo and in vitro , and scholars believe that the ideal probiotic must be from our own gut. Therefore, the strain having the hypolipidemic ability was screened from gut samples taken from individuals living in Bama longevity Village, Guangxi Province in China, and its effects on the gut microbiota and lipid metabolism were studied in order to explore the possible hypolipidemic mechanism of Lactobacillus in vivo .
Materials and Methods
- Bacteria and Growth
Lactobacillus rhamnosus hsryfm 1301 was screened from gut samples taken from individuals living in Bama longevity Village, Guangxi Province in China. The bacteria were cultured in MRS medium at 37℃ in an anaerobic jar (Ruskinn Technologies Ltd., South Wales, UK) for 24 h.
- Bacterial Collection and Fermentation of Milk
The L. rhamnosus hsryfm 1301 strain was collected by centrifugation of MRS medium at 4,000 × g for 10 min at 4℃ the precipitate was suspended in 10% (w/v) sterile skim milk to reach a viable count of 10 9 CFU/ml, and stored at 4℃. Ten-fold serial dilutions of the suspension were performed and plated on MRS agar (pH, 6.8 ± 0.2) in triplicate; the plates were cultured in an anaerobic jar (Ruskinn Technologies Ltd.) at 37℃ for 48 h, and then the colonies formed were counted.
L. rhamnosus hsryfm 1301 was inoculated in 10% heated skim milk with 3% (v/v) inoculum and fermented at 42℃ until the skim milk curdled; the fermentation was stopped when the viable count was 10 9 CFU/ml. The fermented milk was then stored at 4℃.
- Animal Experiments
Forty-two male Wistar rats (BetterBiotechnology Co., Ltd., Jiangsu, China), aged 5 weeks and weighing 125 ± 4.5 g, were individually housed in metal cages under controlled temperature (23 ± 1℃) and humidity (50 ± 5%) levels and were exposed to a 12 h light/12 h dark cycle. The care and use of rats in this study were approved by the Ethics Committee of Yang Zhou University and the protocol followed our institutional and national guidelines.
The rats were fed a normal diet, which included 20% (w/w) flour, 10% rice flour, 20% corn, 26% drum head, 20% bean, 2% fish powder, and 2% bone powder (XieTong, Organism Inc., Jiangsu, China) for 1 week. After this adaptation period, the rats were divided into four groups—control and model groups of 13 rats each, and two treatment groups (hsry1301 and hsry1301-f groups) of eight rats each; the initial average body weight of the rats in each group was similar. The control group was fed the normal diet; the model group was fed a high-fat diet; the hsry1301 group was fed a high-fat diet and L. rhamnosus hsryfm 1301 skim milk suspension; and the hsry1301-f group was fed a high-fat diet and L. rhamnosus hsryfm 1301 skim fermented milk. The high-fat diet included 10% (w/w) lard oil, 1% cholesterol, 0.2% sodium cholate, and 78.8% normal diet (XieTong Organism Inc.). Rats had free access to water and their diet. The rats in the hsry1301 and hsry1301-f groups were administered 2 ml (10 9 CFU/ml) daily of L. rhamnosus hsryfm 1301 skim milk suspension and its fermented milk for 28 days, respectively, after a hyperlipidemic rat model was established by feeding the high-fat diet for 28 days. The rats in the control and model groups received an equivalent volume of saline, and their weight was measured weekly.
- Calculation of Physiological Indexes
Measurement of visceral organ indexes. After euthanasia, the liver, kidney, cardiac, spleen, and fat tissue from the peritoneal tissue, testis, and kidney of the rats were removed and washed with physiological saline solution before blotting and weighing, and the visceral organ indexes were calculated using the following formula:
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Measurement of fecal water content. Fecal samples were collected, packed in airtight bags, and weighed on days 28 and 56 before administration of L. rhamnosus hsryfm 1301 and its fermented milk, and stored at -20℃ during the study. The fecal samples were freeze-dried (Labconco, Kansas City, Missouri, USA) until a constant weight was achieved within 24 h and then reweighed [17] . Fecal water content was calculated using the following formula:
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- Lipid Analysis
Measurement of serum lipids. On day 28, five rats each from the control and model groups were selected randomly, deprived of food for 12 h, and then were euthanized. Blood samples were collected from the celiac vein and transferred to non-heparinized vacuum collection tubes, and kept stationary at 0℃ for 30 min before centrifugation at 2,000 × g for 10 min at 4℃. The obtained serum was used to analyze the lipid levels.
The triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels of the serum were analyzed using commercial kits (Maker; Biotechnology Inc., Sichuan, China) and a chemical analyzer (model 7020; Hitachi, Tokyo, Japan). All the rats were weighed before they were euthanized, and their TC, TG, HDL-C, and LDL-C levels of the serum were measured as described above after administration of L. rhamnosus hsryfm 1301 and its fermented milk for 28 days.
Measurement of liver TC, TG, and fecal cholesterol, and bile acids content. After euthanasia, the middle lobes of the rat livers were removed and washed with physiological saline solution before blotting and weighing. The TC and TG content of the liver was detected after homogenization with Folch solution (chloroform:methanol, 2:1 (v/v)) [38] .
Fecal samples were collected on day 56 before oral administration; a 0.5 g freeze-dried (Labconco) fecal sample was extracted with Folch solution and anhydrous ethanol after homogenization, and fecal neutral and acidic sterols and bile acids were detected using the methods of Kim and Shin [20] and Kajiura et al . [18] .
- Electron Microscope Observation of Rat Liver Slices
The liver slices of rats were made as Meki et al . [25] and Ohtani et al . [28] described, and were observed under a CM100 Transmission Electron Microscope (TEM) (Philips, Amsterdam, Holland).
- Gut Microbiota Analysis
Fresh fecal samples were collected from each group on days 1, 28, and 56 before oral administration. DNA was extracted using the QIAamp DNA stool mini kit (Qiagen Inc., Hilden, Germany) according to the manufacturer’s instructions. The amount of DNA was determined using BioPhotometer plus (Eppendorf, Hamburg, Germany), and the integrity of the DNA was assessed using 1.0% agarose gel electrophoresis and ethidium bromide staining.
The primers used to detect Lactobacillus spp., Bifidobacterium spp., Bacteroides spp., Clostridium leptum , Enterococcus spp., and Enterobacter spp. were based on the 16S rRNA gene sequences [23 - 25] : Lac-F, 5’-GGAAACAGGTGCTAATACCG-3’; Lac-R, 5’-CACCGCTACACATGGAG-3’; Bif-F, 5’-CTCCTGGAAACGGG TGG-3’; Bif-R, 5’-GGTGTTCTTCGATATCTACA-3’; Bac-F, 5’-GAAGGTCCCCCACATTG-3’; Bac-R 5’-CAATCGGACTTCGTG-3’; Clept-F, 5’-GCACAAGCAGTGGAGT-3’; Clept-R, 5’-CTTCTTCCTCCGTTTTGTCAA-3’; Enco-F, 5’-CCCTTATTGTTAGTTGCCATCATT-3’; Enco-R, 5’-ACTCGTTGTACTTCCCATTGT-3’; Eco-F, 5’-CATTGACGTTACCCGCGAGAAGAAGC-3’; Eco-R, 5’-CTCTACGAGCTCAAGCTTGC-3’. Quantitative PCR analysis was performed with a StepOnePlus Real-time PCR system (Applied Biosystems, Foster City, CA, USA). Amplification reactions (20 µl) were performed with 10 µl of 2× iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), 0.5 µl of each of the specific primers at a concentration of 10 µM, and 60 ng of bacterial template DNA.
The quantitative PCR amplification reactions were carried out as follows: 95℃ for 3 min, followed by 40 cycles of 95℃ for 15 sec, 60℃ for 1 min, and 72℃ for 40 sec. Melting curves were constructed by heating the PCR mixtures from 55℃ to 90℃ (an increase of 1℃ per cycle of 10 sec). Plasmid DNAs containing the corresponding amplicon for each set of primers were extracted using the PureYield Plasmid Miniprep (Promega, WI, USA), and their concentrations were determined by Biophotometer plus (Eppendorf), and were then converted into 16S rRNA gene copy numbers. Quantitation of the unknown samples was performed using standard curves constructed from known concentrations of plasmid DNA containing the corresponding amplicon for each set of primers [12] . Reactions were carried out in triplicate along with a non-template control. Ct values were calculated under default settings for absolute quantification, using the software provided with the instrument.
- Statistical Analysis
The SPSS 19.0 software (IBM Corp, Armonk, NY, USA) was used to analyze the serum lipid data. Copy numbers of 16S rRNA genes of Lactobacillus spp., Bifidobacterium spp., Bacteroides spp., Clostridium leptum , Enterococcus spp., and Enterobacter spp. per gram of sample were transformed into logarithms, and were subjected to analyze its relationship to the serum lipid levels.
Results
- Effects ofL. rhamnosushsryfm 1301 and Its Fermented Milk on the Physiological Indexes of Rats
Weight gain in the hsry1301 and hsry1301-f groups was significantly lower than that in the model group ( p < 0.05; Table 1 ). The liver index of the model group was significantly higher than that of the control group ( p < 0.05; Table 1 ); conversely, the liver index of the hsry1301 and hsry1301-f groups was significantly lower than that of the model group after administration of L. rhamnosus hsryfm 1301 and its fermented milk for 28 days ( p < 0.05; Table 1 ). No significant difference in the kidney, cardiac, and spleen indexes was observed among the four groups ( p > 0.05; Table 1 ). The fat index of the model group was significantly higher than that of the other groups ( p < 0.05; Table 1 ), which could be one of the reasons for the increase in the weight of body and liver in the model group.
Visceral organ indexes and weight gain in the rats.
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Control group: normal diet; model group: high-fat diet; hsry1301 group: high-fat diet + L. rhamnosus hsryfm 1301; hsry1301-f: high-fat diet + L. rhamnosus hsryfm 1301 fermented milk. The data are shown as the mean ± standard deviation (n = 8); different superscript letters in the same row of each group indicate significant differences (p < 0.05).
In the control group, the structural integrity of the rat liver was normal and fat drops were not observed ( Fig. 1 A). In the model group, fat drops were observed and the mitochondria swelled and were arranged disorderly in the rat liver, which may suggest hepatocyte fatty degeneration ( Fig. 1 B). Histological changes in the liver of the hsry1301 group were similar to the control group ( Fig. 1 C), and the liver tissue showed normal structural integrity and no fatty degeneration. The number of mitochondria in the hsry1301-f group was lower than that in the control group, but mitochondrial swelling was not observed ( Fig. 1 D). These findings indicate that administration of L. rhamnosus hsryfm 1301 and its fermented milk can prevent hepatocyte fatty degeneration in the rat liver.
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TEM of rat liver subsequent to treatment with L. rhamnosus hsryfm 1301 and its fermented milk (original magnification, ×1,650). (A) Control group: normal diet; (B) model group: high-fat diet, showing fat drops (arrows) and the swelling and disordered arrangement of mitochondrial (M) in rat liver; (C) hsry1301 group: high-fat diet + L. rhamnosus hsryfm 1301; (D) hsry1301-f: high-fat diet + L. rhamnosus hsryfm 1301 fermented milk.
- Effects ofL. rhamnosushsryfm 1301 and Its Fermented Milk on the Lipid Levels in Rats
The serum TC, TG, and LDL-C level in the model group was significantly higher than that in the control group ( p < 0.05; Fig. 2 A), which indicated that the hyperlipidemic rat model was successfully established. The serum TC, TG, HDL-C, and LDL-C levels in the hsry1301 and hsry1301-f groups were significantly lower than that in the model group ( p < 0.05; Fig. 2 B), which suggested that the serum lipid levels in the hyperlipidemic rats were decreased significantly after administration of L. rhamnosus hsryfm 1301 and its fermented milk for 28 days ( p < 0.05). We also found that the TC and TG levels in the hyperlipidemic rats liver were decreased significantly by the administration ( p < 0.05; Fig. 2 C).
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Effects of L. rhamnosus hsryfm 1301 and its fermented milk on lipid levels in rats fed a high-fat diet. Control group: normal diet; model group: high-fat diet; hsry1301 group: high-fat diet + L. rhamnosus hsryfm 1301; hsry1301-f: high-fat diet + L. rhamnosus hsryfm 1301 fermented milk. The data are shown as the mean ± standard deviation (n = 8); different superscript letters for the same index indicate significant differences (p < 0.05).
- Effects ofL. rhamnosushsryfm 1301 and Its Fermented Milk on the Gut Microbiota in Rats
Significant differences in the content of Lactobacillus spp., Bifidobacterium spp , Bacteroides spp., Clostridium leptum , Enterococcus spp., and Enterobacter spp. were observed among the four groups on day 1, which was attributable to individual differences in the intestinal microbiota of the rats ( Fig. 3 ). During the study, the Lactobacillus spp., Bifidobacterium spp., Clostridium leptum , and Enterobacter spp. content in the rat gut was decreased slightly ( Figs. 3 A, 3 B, 3 D, and 3 F), while the Bacteroides spp. and Enterococcus spp. content increased significantly after the rats were fed a normal diet for 56 days ( p < 0. 05; Figs. 3 C and 3 E). The Clostridium leptum , Enterococcus spp., and Enterobacter spp. content was increased significantly ( p < 0.05; Fig. 3 D, 3 E, and 3 F), while the Lactobacillus spp., Bifidobacterium spp., and Bacteroides spp. content was decreased significantly after the rats was fed a high-fat diet for 28 and 56 days ( p < 0.05; Figs. 3 A, 3 B, and 3 C). After the hsry1301 and hsry1301-f groups were orally administered L. rhamnosus hsryfm 1301 and its fermented milk for 28 days, respectively, the Clostridium leptum and Enterobacter spp. content was reduced significantly ( p < 0.05; Figs. 3 D and 3 F), and the Lactobacillus spp., Bifidobacterium spp., Bacteroides spp., and Enterococcus spp. content was increased significantly ( p < 0. 05; Fig. 3 A, 3 B, 3 C, and 3 E). These results suggest that the gut microbiota in rats was effected by administration of 10 9 CFU/ml L. rhamnosus hsryfm 1301 for 28 days.
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Effects of L. rhamnosus hsryfm 1301 and its fermented milk on the gut microbiota in rats fed a high-fat diet. Control group: normal diet; model group: high-fat diet; hsry1301 group: high-fat diet + L. rhamnosus hsryfm 1301; hsry1301-f: high-fat diet + L. rhamnosus hsryfm 1301 fermented milk. The FQ in A, B, C, D, E, and F is expressed as copy number of 16S rRNA genes for Lactobacillus spp., Bifidobacterium spp., Bacteroides spp., Clostridium leptum, Enterococcus spp., and Enterobacter spp. per gram of fecal sample, respectively. The value of starting 16S rRNA gene copy numbers of plasmid DNA containing the corresponding amplicon of genus-specific primers of Lactobacillus spp., Bifidobacterium spp., Bacteroides spp., Clostridium leptum, Enterococcus spp., and Enterobacter spp. is 1. The data are shown as the mean ± standard deviation (n = 8); different superscript letters in each panel for the same day indicate significant differences (p < 0.05).
Great influences in the Bacteroides spp. and Enterococcus spp. were observed in the gut after rats were fed the highfat diet for 28 days ( p < 0.05; Figs. 3 C and 3 E), and the Bifidobacterium spp. was greatly influenced on day 56 ( p < 0.05; Fig. 3 B); great influences in the Lactobacillus spp. and Clostridium leptum were also observed in the rat gut after administration of L. rhamnosus hsryfm 1301 for 28 days ( p < 0.05; Figs. 3 A and 3 D); the Enterobacter spp. in the rat gut was greatly influenced after administration of L. rhamnosus hsryfm 1301 fermented milk for 28 days ( p < 0.05; Fig. 3 F).
- Effects ofL. rhamnosusHsryfm 1301 and Its Fermented Milk on the Water, Cholesterol, and Bile Acid Content of Rat Feces
No significant differences in the fecal water content were observed between the four groups on day 28 ( p > 0. 05; Fig. 4 A), but the fecal water content of the hsry1301 and hsry1301-f groups was significantly higher than that of the model group on day 56 ( p < 0.05; Fig. 4 A). The cholesterol and bile acid content in feces of the hsry1301 group and the cholesterol content in feces of the hsry1301-f group were significantly higher than those of the model group, respectively ( p < 0.05; Fig. 4 B). These results indicate that the decrease of lipid levels in the hyperlipidemic rats may be associated with an increase in the content of cholesterol and bile acids excreted.
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Water, cholesterol, and bile acid content of fecal matter. Control group: normal diet; model group: high-fat diet; hsry1301 group: high-fat diet + L. rhamnosus hsryfm 1301; hsry1301-f: high-fat diet + L. rhamnosus hsryfm 1301 fermented milk. The data are shown as the mean ± standard deviation (n = 8); different superscript letters in each panel for the same day indicate significant differences (p < 0.05).
Discussion
This study investigated the gut microbiota and lipid level changes in the hyperlipidemic rats by intervention with L. rhamnosus hsryfm 1301 and its fermented milk. With regard to the content of different bacterial species, the content of Lactobacillus spp., Bifidobacterium spp., Clostridium leptum , and Enterobacter spp. was observed to change slightly after the rats were fed a normal diet for 56 days ( Figs. 3 A, 3 B, 3 D, and 3 F), which may indicate that these bacteria are relatively stable at 5 weeks of age; however, the content of Bacteroides spp. and Enterococcus spp. increased significantly ( p < 0.05; Figs. 3 C and 3 E), which was probably promoted by the carbohydrates and polysaccharides present in the normal diet [3 , 22] .
Negative correlations were observed between the content of Enterobacter spp. and the content of Lactobacillus spp. and Bifidobacterium spp. ( Table 2 ), which indicate an inhibitory relationship between probiotics and harmful bacteria in the hyperlipidemic rats gut. The content of Enterobacter spp. was increased significantly after rats were fed the high-fat diet for 56 days ( p < 0.05; Fig. 3 F), which is consistent with Kim et al . [19] , and may indicate that the metabolites of high-fat diet in vivo could favor the Enterobacter spp. growth. The bile salts present in high-fat diets are known to have deleterious effects on the beneficial bacteria in the gut [5 , 19] , including bile stress, DNA damage, intracellular acidification, and oxidative and osmotic stresses; this may have led to the decrease in the content of Lactobacillus spp. and Bifidobacterium spp. in the high-fat diet-fed rats ( p < 0.05; Figs. 3 A and 3 B). However, the Enterobacter spp. content was reduced significantly ( p < 0 05; Fig. 3 F), while the content of Lactobacillus spp. and Bifidobacterium spp. was increased significantly in the hyperlipidemic rat gut by the intervention for 28 days ( p < 0.05; Fig. 3 A and 3 B), which may indicate that L. rhamnosus hsryfm 1301 and its fermented milk could remove the harmful metabolites of high-fat diets and promote Lactobacillus spp. and Bifidobacterium spp. growth in the rat gut. At the same time, the Enterobacter spp. are inhibited by Lactobacillus spp. and Bifidobacterium spp. that colonize gut epithelial cells, which compete with the harmful bacteria for the adsorption sites on epithelial cells and results in the better growth of other beneficial bacteria [11 , 16] .
Correlation between serum lipid levels and gut microbiota in rats.
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Pearson correlation analysis using the SPSS software was used to calculate Pearson’s correlation coefficient (r). *indicates a significance level of 0.05, **indicates a significance level of 0.01. Data for the serum lipid levels and gut microbiota on day 56 were used (n = 8).
Enterobacter spp. may be associated with the endotoxemia and inflammation associated with obesity [8] , which increased the lipid levels and weight of the model group ( Fig. 2 B; Table 1 ). The mucosa and immune system of the host could be improved by Lactobacillus spp., which enhance the function of the thymus so as to recover and balance the gut ecology [27] ; the emulsification efficiency of dietary lipids was decreased by the recovered gut microbiota that promoted deconjugation of bile acids in the gut [2] , and the serum lipid levels of the hsry 1301 and hsry1301-f groups were decreased significantly ( p < 0 05; Fig. 2 B). Moreover, Lactobacillus spp. and Bifidobacterium spp. play a key role in sugar and protein digestion for short-chain fatty acid synthesis in the intestine [29] , which inhibits the hepatic lipogenic enzyme activity [21] , and the rat lipid levels were also reduced significantly ( p < 0.05; Fig. 2 B).
The content of Clostridium leptum was observed to negatively correlate to the TC, TG, LDL-C, and HDL-C levels in the serum ( Table 2 ), which may suggest that Clostridium leptum could increase the serum lipid levels in hyperlipidemic rats. The calories present in food are absorbed by Clostridium leptum , which increases fat storage in the host body [32] ; thus, the increased Clostridium leptum content resulted in fat accumulation in hepatic tissue and induced an increase in the serum and liver lipid levels ( p < 0.05; Fig. 3 D, 2 B, and 2 C). However, the content of Clostridium leptum was decreased significantly in the hyperlipidemic rats gut by the intervention ( p < 0 05; Fig. 3 D), while the content of Bacteroides spp. increased significantly ( p < 0.05; Fig. 3 C), which could improve the efficiency of dietary utilization by absorbing and metabolizing certain polysaccharides and steroids [37] ; moreover, the expression of the microbiome in the rat gut is improved by Lactobacillus fermented milk, which participated in lipid and carbohydrate metabolism [26] , and resulted in the decrease of serum lipid levels of the hsry 1301 and hsry1301-f groups ( p < 0.05; Fig. 2 B).
Lipid levels in both the serum and the liver were reduced significantly by the intervention ( p < 0.05; Fig. 2 B and 2 C), which indicated that the serum TC and TG levels of the hsry1301 and hsry1301-f groups were actually reduced and not redistributed between the blood and liver. We found that the bile acid concentration in the feces of the hsry1301 group was significantly higher than that in the model group ( p < 0.05; Fig. 4 B), which may suggest that L. rhamnosus hsryfm 1301 reduced the cholesterol concentration by inducing an increase in the deconjugation and precipitation of bile acids in the intestinal lumen and consequently an increase in its fecal excretion. The fecal cholesterol concentration of the hsry1301 and hsry1301-f groups was significantly higher than that in the model group by the intervention ( p < 0.05; Fig. 4 B), which may be because cholesterol in vivo is probably assimilated by incorporation into L. rhamnosus hsryfm 1301 cellular membranes or cell walls and then excreted through the feces.
On day 56, the fecal water content in the hsry1301 and hsry1301-f groups was significantly higher than that in the model group ( p < 0.05; Fig. 4 A). As the fecal water content is an index of feces elimination, the results suggested that L. rhamnosus hsryfm 1301 and its fermented milk had laxative potential and may stimulate bowel movements, and may additionally reduce the time required for digestion of cholesterol in the gut. This may be another reason for the in vivo cholesterol-reducing ability of L. rhamnosus hsryfm 1301 and its fermented milk in vivo .
In conclusion, the content of Lactobacillus spp., Bifidobacterium spp., Bacteroides spp., Clostridium leptum , Enterococcus spp., and Enterobacter spp. in the hyperlipidemic rats gut was significantly altered after administration of L. rhamnosus hsryfm 1301 and its fermented milk. A positive correlation was observed between the content of Clostridium leptum and the serum levels of TC, TG, LDL-C, and HDL-C, and a negative correlation was observed between the content of Enterobacter spp. and the content of Lactobacillus spp. and Bifidobacterium spp. The lipid levels in the serum and the liver of hyperlipidemic rats were reduced significantly by supplementation with L. rhamnosus hsryfm 1301 and its fermented milk, and hepatocyte fatty degeneration in the rat liver could also be prevented.
Acknowledgements
This study was supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2013BAD18B12), the Key University Science Research Project of Jiangsu Province (12KJA550003), and National Natural Science Foundation of China (31201393).
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