WO2020228144A1 - Application of breast milk-derived lactobacillus reuteri in lowering lipid and regulating lipid metabolism rhythm - Google Patents
Application of breast milk-derived lactobacillus reuteri in lowering lipid and regulating lipid metabolism rhythm Download PDFInfo
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- WO2020228144A1 WO2020228144A1 PCT/CN2019/098529 CN2019098529W WO2020228144A1 WO 2020228144 A1 WO2020228144 A1 WO 2020228144A1 CN 2019098529 W CN2019098529 W CN 2019098529W WO 2020228144 A1 WO2020228144 A1 WO 2020228144A1
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- lactobacillus reuteri
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Definitions
- the invention relates to an application of lactobacillus reuteri derived from breast milk for reducing lipid and regulating the rhythm of lipid metabolism, and belongs to the fields of microbial technology and food science.
- rhythmic bacteria mainly belong to the Clostridium, Lactobacillus and Bacteroides It accounts for about 60% of the total number of intestinal bacteria.
- rhythmic changes of bacteria in the intestines of humans and mammals will expose the intestinal epithelial cells to different numbers and types of bacteria at different time periods, and these rhythmic changes of bacteria will pass on the intestinal epithelial cells. Metabolites reach remote tissues such as the liver, thereby causing rhythmic changes in gene expression in remote tissues such as the liver, which in turn causes rhythmic changes in lipid metabolism in humans and mammals, that is, the rhythm of lipid metabolism.
- the rhythm of lipid metabolism is very important to the health of humans and mammals. Once the rhythm of lipid metabolism is disordered, it will affect the absorption and storage of fat in food by humans and mammals, leading to a large amount of liver fat synthesis in humans and mammals, causing human and Rhythm disorder of serum triglycerides in mammals. Serum triglycerides are mainly synthesized by the liver, adipose tissue and small intestine, and their rhythmic disturbances can accelerate the occurrence of atherosclerosis, fatty liver, cerebral vascular blockage and insulin resistance in humans and mammals.
- High-energy diets such as high-fat diets and high-sugar diets will cause disorders in the composition of the human intestinal flora and the customized mucus layer of the flora, thus causing The human body’s gene expression is disordered, which in turn makes the body’s lipid metabolism rhythm disorder, and ultimately increases the body’s probability of suffering from diseases such as atherosclerosis, fatty liver, cerebral blood vessel blockage, and insulin resistance.
- the present invention provides a Lactobacillus reuteri FN041, which was deposited in the Guangdongzhou Microbial Culture Collection on January 29, 2019 The center, the deposit number is GDMCC No. 60546, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.
- the Lactobacillus reuteri FN041 is obtained by first targeting the secretory immunoglobulin A (sIgA) in conjunction with the physiological characteristics of the symbiotic bacteria, and using the immunomagnetic bead method to obtain information from people in Lintan County, Gannan Vietnamese Autonomous Prefecture, Gansu province.
- the milk is enriched with breast milk IgA-binding bacteria, and then oriented and separated according to the resistance of Lactobacillus reuteri to vancomycin and the high temperature cultivable characteristics.
- the colony of Lactobacillus reuteri FN041 on the MRS agar medium is round, smooth and white, with a diameter of about 1 mm.
- the Lactobacillus reuteri FN041 has the following characteristics:
- the survival rate after staying in an environment with a pH of 3.5 for 2 hours is higher than 90%;
- the present invention also provides the application of the above-mentioned Lactobacillus reuteri FN041 in the preparation of a product for preventing and/or treating lipid metabolism rhythm disorders.
- the lipid metabolism rhythm refers to the rhythmic changes in human and mammal lipid metabolism caused by the rhythmic changes in human and mammalian distal tissue gene expression caused by the rhythmic changes in bacterial abundance in the intestines of humans and mammals;
- the bacterial abundance refers to the percentage of the number of a certain kind of bacteria in the intestines of humans and mammals to the total number of bacteria in the intestines of humans and mammals;
- the distal tissues include the liver; the genes expressed by the liver include the clock gene Clock , Bmal1 and Per2.
- the lipid metabolism rhythm disorder refers to the disorder of the rhythmic changes of human and mammal lipid metabolism caused by the disorder of the rhythmic changes of bacterial abundance in the intestines of humans and mammals.
- the number of viable bacteria of Lactobacillus reuteri FN041 is not less than 1 ⁇ 10 6 CFU/mL or 1 ⁇ 10 6 CFU/g.
- the product includes food, medicine or health care products.
- the dosage form of the medicine includes granules, capsules, tablets, pills or oral liquids.
- the medicine contains Lactobacillus reuteri FN041, a drug carrier and/or pharmaceutical excipients.
- the present invention also provides a product for preventing and/or treating lipid metabolism rhythm disorders, the product containing the above-mentioned Lactobacillus reuteri FN041.
- the number of viable bacteria of Lactobacillus reuteri FN041 is not less than 1 ⁇ 10 6 CFU/mL or 1 ⁇ 10 6 CFU/g.
- the product includes food, medicine or health care products.
- the dosage form of the medicine includes granules, capsules, tablets, pills or oral liquids.
- the medicine contains Lactobacillus reuteri FN041, a drug carrier and/or pharmaceutical excipients.
- the present invention also provides a cryopreservation agent for Lactobacillus reuteri (Lactobacillus reuteri) FN041, in which the number of viable bacteria of the above-mentioned Lactobacillus reuteri FN041 is not less than 1 ⁇ 10 10 CFU/mL.
- the preparation method of the cryopreservation agent is to first wash the above-mentioned Lactobacillus reuteri FN041 cells in the stable phase with a phosphate buffer with a pH of 7.0 to 7.4. ⁇ 2 times, and then add the washed Lactobacillus reuteri FN041 cells to the protective agent to obtain Lactobacillus reuteri FN041 cryopreservation agent; the protective agent contains 1g/L Cysteine hydrochloride and 200g/L glycerol.
- the present invention has screened out a Lactobacillus reuteri FN041.
- This Lactobacillus reuteri FN041 can prevent and/or treat lipid metabolism rhythm disorders, which is specifically embodied in:
- Gavage of Lactobacillus reuteri FN041 can make the body weight and weight growth rate of mice fed with high-fat diet compared with the high-fat feed of Lactobacillus reuteri FN041 without gavage Feeding mice decreased significantly. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly reduce the weight gain under the influence of high fat;
- Gavage of Lactobacillus reuteri (Lactobacillus reuteri) FN041 can make the peritesticular adipose tissue index of mice fed with high-fat diets compared with the high-fat diet of Lactobacillus reuteri FN041 without gavage. Feeding mice decreased significantly. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly reduce the abnormal increase in peritesticular adipose tissue index under the influence of high fat;
- Gavage of Lactobacillus reuteri (Lactobacillus reuteri) FN041 can make the liver fat infiltration and testicular fat cell area of mice fed high-fat diet higher than that of non-gavage Lactobacillus reuteri FN041 Fat diet fed mice significantly decreased. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly reduce liver fat infiltration and abnormal increase of testicular fat cell area under the influence of high fat;
- Gavage of Lactobacillus reuteri (Lactobacillus reuteri) FN041 can feed mice serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), The content of high-density lipoprotein cholesterol (HDL-C) was significantly lower than that of mice fed with high-fat diet of Lactobacillus reuteri FN041 without gavage.
- TG serum triglycerides
- TC total cholesterol
- LDL-C low-density lipoprotein cholesterol
- HDL-C high-density lipoprotein cholesterol
- Lactobacillus reuteri FN041 Treatment can significantly reduce the abnormal increase in serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) content in serum under the influence of high fat;
- TG serum triglyceride
- TC total cholesterol
- LDL-C low-density lipoprotein cholesterol
- HDL-C high-density lipoprotein cholesterol
- Gavage of Lactobacillus reuteri (Lactobacillus reuteri) FN041 can make the blood plasma FD4 content and serum endotoxin (LPS), endotoxin binding protein (LBP) and tumor necrosis factor in mice fed high-fat diet
- LPS serum endotoxin
- LBP endotoxin binding protein
- TNF- ⁇ tumor necrosis factor
- Lactobacillus reuteri FN041 can significantly improve The permeability of the murine intestinal epithelial barrier inhibits the abnormal increase in plasma FD4 content in the blood of mice caused by FD4 entering the blood caused by high-fat diet feeding, and Lactobacillus reuteri FN041 has played a role in protecting mice
- the role of the mucosal barrier inhibits the abnormal increase of endotoxin (LPS) and endotoxin binding protein (LBP) in the serum of mice caused by intestinal bacteria entering the blood caused by high-fat feed.
- Lactobacillus reuteri ( Lactobacillus reuteri) FN041 treatment can significantly inhibit the abnormal increase in serum tumor necrosis factor alpha (TNF- ⁇ ) under the influence of high fat;
- Secretory immunoglobulin A is an antibody molecule rich in human intestinal mucosal surface. It can combine with beneficial bacteria such as Lactobacillus that are symbiotic in the intestine to form a complex to promote beneficial bacteria to play a beneficial role in the human body. This beneficial effect is mainly reflected in:
- the combination of beneficial bacteria can promote its colonization in the mucous layer;
- the formation of complexes can promote the anchoring of beneficial bacteria on the top surface of intestinal epithelial cells, promote the phosphorylation of tight junction proteins of epithelial cells, maintain cell-cell interactions, thereby enhance the mucosal barrier, and induce the production of anti-inflammatory chemokines , To maintain a non-inflammatory environment of the mucosa;
- Lactobacilli can be recognized by dendrites or macrophages to regulate immune responses.
- These Lactobacillus-recognizing dendrites or macrophages mainly exist in the submucosal barrier, including the lamina intestinal and Peyer's collective lymph node (PP ), where the sIgA receptor on the surface of the intestinal lumen of PP can help transport sIgA-bound beneficial bacteria into PP.
- PP Peyer's collective lymph node
- beneficial bacteria that enter PP can interact with the dendritic cell subsets in PP to promote T cells to produce IL-10 and Anti-inflammatory cytokines such as TGF- ⁇ ;
- the beneficial bacteria can be shielded by the sIgA binding and package, preventing the bacterial surface antigen from inducing a strong inflammatory response.
- the Lactobacillus reuteri FN041 screened in the present invention can bind to secretory immunoglobulin A (sIgA). Therefore, the Lactobacillus reuteri FN041 of the present invention can be closer. Regulate the physiological activities of epithelial cells near the mucous layer, or release higher concentrations of metabolites locally in the mucus to regulate the metabolic rhythm.
- SIgA secretory immunoglobulin A
- the Lactobacillus reuteri FN041 screened in the present invention is derived from human milk. Therefore, the Lactobacillus casei CCFM1038 of the present invention does not cause any harm to the human body.
- the survival rate of Lactobacillus reuteri FN041 screened in the present invention after staying in an environment with a pH of 3.5 for 2 hours is higher than 90%, in a bile solution with a concentration of 3g/kg and 4g/kg, respectively
- the survival rate after staying for 4 hours is higher than 82% and 68% respectively, can withstand high temperature of 45°C, and has good physiological characteristics.
- Lactobacillus reuteri FN041, taxonomically named Lactobacillus reuteri has been deposited in the Guangdong Provincial Microbial Culture Collection on January 29, 2019, the deposit number is GDMCCNo.60546, and the deposit address is Guangzhou City 5th Floor, Building 59, Yard 100, Xianlie Middle Road.
- Figure 1 Flow chart of enrichment of IgA-binding bacteria in breast milk and selective isolation of Lactobacillus reuteri.
- MRS agar medium peptone 10g/L, yeast extract 5g/L, glucose 20g/L, anhydrous sodium acetate 2g/L, hydrogen citrate diamine 2g/L, K 2 HPO 4 ⁇ 3H 2 O 2.6g/ L, MgSO 4 ⁇ 7H 2 O 0.5g/L, MnSO 4 ⁇ 7H 2 O 0.25g/L, Tween-80 1g/L, agar 20g/L, distilled water 1000g/L.
- MRS liquid medium peptone 10g/L, yeast extract 5g/L, glucose 20g/L, anhydrous sodium acetate 2g/L, hydrogen citrate diamine 2g/L, K 2 HPO 4 ⁇ 3H 2 O 2.6g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, MnSO 4 ⁇ 7H 2 O 0.25g/L, Tween-80 1g/L, distilled water 1000g/L.
- Example 2 Collection of breast milk flora and analysis of Lactobacillus reuteri
- the solid matter obtained by centrifugation of breast milk is suspended in peptone buffer, bovine serum albumin is added to block non-specific binding (final concentration is 10%, 0.05 to 0.5%, respectively), and biotin-labeled rabbit anti-human IgA serum is added , Incubate for 15-30 minutes, add streptavidin-modified magnetic beads (additional amount is 0.1-1.0 mg/mL), adsorb bacteria with a magnet, wash twice with peptone buffer to obtain IgA-bound flora.
- the bacterial colony obtained in Example 3 was gradually diluted with PBS buffer solution (pH 6.8) under aseptic conditions, and 100 microliters of the appropriate dilution solution (containing about 100 bacteria per mL) was applied to it containing vancomycin (50 ⁇ g/mL) MRS agar medium plate, the plate is placed upside down into an anaerobic incubator, and cultured at 37°C for 36 ⁇ 72h, observe and record the colony morphology; pick different colonies on the MRS agar medium plate for streaking separation After culturing at 37°C for 48 hours, pick out single colonies of different morphologies on the MRS agar medium plate again for streaking, until a pure single colony with consistent morphology is obtained; pick the pure colonies on the MRS agar medium plate and inoculate it on Incubate in 5mL MRS liquid medium at 37°C for 18h; take 1mL of bacterial solution in a sterile centrifuge tube, centrifuge at 8000r/min for 3min,
- the isolated strains were subjected to PCR amplification of 16S rDNA, and the PCR products were sent to Huada Gene Sequencing Co., Ltd. for sequencing.
- the sequencing results were compared in ezbiocloud for nucleic acid sequence comparison.
- the nucleotide sequence of one strain was compared with The similarity of Lactobacillus reuteri JCM 1112 reached 99.72%, and this strain was determined to be Lactobacillus reuteri, named Lactobacillus reuteri FN041 (the 16S rDNA sequence of FN041 is as SEQ ID NO .1).
- Lactobacillus reuteri FN041 was placed in physiological saline with pH 3.5 for 2 hours, and it was found that the survival rate after 2 hours in an environment with pH 3.5 was higher than 90%.
- Lactobacillus reuteri FN041 was placed in a bile solution with a concentration of 3g/kg and 4g/kg for 4h, and it was found to stay in a bile solution with a concentration of 3g/kg and 4g/kg for 4h.
- the survival rates were higher than 82% and 68%.
- the Lactobacillus reuteri FN041 was inserted into the MRS liquid medium and cultured at 35, 40, 45, and 50°C for 36 to 72 hours, and then observed its growth curve. It was found that it could tolerate the high temperature of 45°C. It can grow well in the temperature range of 35 ⁇ 45°C.
- Example 5 Application of Lactobacillus reuteri FN041 in the prevention and/or treatment of metabolic rhythm disorders caused by high-energy diet
- mice Three-week-old healthy male C57BL/6J mice were randomly divided into cages and pre-raised for one week. The formal experiment began. The mice were randomly divided into normal diet groups (CON group, 20) and fed with low-fat diet (12% of energy was derived from Fat); high-fat diet group (HFD group, 80 animals), fed high-fat diet (45% of energy comes from fat); high-fat diet and normal diet formulas are shown in Table 1.
- the rearing temperature of the mice is 24 ⁇ 3°C, the humidity is 60 ⁇ 10%, the animal room is turned on for 12 hours a day, and 12 hours is dark; weekly weighing, recording the weight, food intake and water intake of the mice each week; The gastric experiment was started after 7 weeks of formal feeding.
- mice in the high-fat diet group were randomly divided into 2 groups (20 mice in each group), and phosphate buffered saline (PBS, pH7.3) (HFD group) and Lactobacillus reuteri FN041 bacterial suspension (HFD+R group); Among them, the concentration of Lactobacillus reuteri FN041 bacterial suspension is 8.0 ⁇ 10 8 CFU/mL, and the gastric volume is 200 ⁇ L/only, gavage time is 16:00 in the afternoon.
- PBS phosphate buffered saline
- HFD+R group Lactobacillus reuteri FN041 bacterial suspension
- the concentration of Lactobacillus reuteri FN041 bacterial suspension is 8.0 ⁇ 10 8 CFU/mL
- the gastric volume is 200 ⁇ L/only
- gavage time is 16:00 in the afternoon.
- mice On the last day of the experiment, each group of mice was divided into four batches (5 mice in each batch). Three batches of mice were sacrificed at 2:00, 8:00, and 20:00, and the other group of mice was irrigated at 10:00 Gastric fluorescein FITC-dextran (FD4), intragastric dose of 0.6mg/g body weight, 4h after intragastric administration (14:00), sacrificed, peripheral blood was collected, and plasma was detected with fluorescent microplate reader (excitation light 485nm, emission light 535nm) FD4; After all animals were sacrificed, the serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) content and endotoxin (LPS) were measured ), endotoxin binding protein (LBP) and serum tumor necrosis factor alpha (TNF- ⁇ ) content, and use Acrophase software to perform cosine fitting of blood lipid circadian rhythm to analyze blood
- TG serum triglyceride
- TC total cholesterol
- LDL-C low-density lipoprotein cholesterol
- HDL-C high-density lipoprotein cholesterol
- LPS endotoxin
- LBP endotoxin binding protein
- TNF- ⁇ serum tumor necrosis factor alpha
- the changes in blood lipid circadian rhythm are analyzed by the following methods:
- RNA of clock genes Clock, Bmal1 and Per2 in mouse liver measure its purity (OD260/280) and concentration with NanoDrop, adjust the RNA concentration to about 1000ng/ ⁇ L, that is, OD 260/280 is in the range of 1.8 ⁇ 2.0
- the reverse transcription process is divided into two steps. In the first step, the system is mixed at 85°C, 5min, and quickly cooled on an ice bath. Then, after the second step is added to the system, the cDNA is obtained in a water bath at 37°C for 1h, 95°C, 3min. ; Use reverse-transcribed cDNA as a template for fluorescent quantitative PCR to obtain RNA, and the gene primer sequence is:
- the nucleotide sequence is shown in SEQ ID NO. 2 F: AGCACACACACTTCCTCTCTGACAT;
- the nucleotide sequence is shown in SEQ ID NO.3: R: ATCAAGGGACTGAACACTCAAGACC;
- the primer of the liver's biological clock gene Bmal1 (brain and muscle ARNT-like-1) (NCBI Gene ID: 11865):
- the nucleotide sequence is F shown in SEQ ID NO.4: AGTCAGATTGAAAAGAGGCGTCG;
- the nucleotide sequence is R shown in SEQ ID NO. 5: AGAAATGTTGGCTTGTAGTTTGCTT;
- the nucleotide sequence is shown in SEQ ID NO. 6 F: TTCTCTGCTGTTCTTGTATCCTTTT;
- the nucleotide sequence is shown in SEQ ID NO.7 R: GCTTTCTGCTGGGAGCTAATG;
- the amplification conditions of fluorescence quantitative PCR are: 95°C, 5min; 95°C, 20s, 62°C, 30s, 72°C, 20s; 72°C, 2min; ⁇ -actin is used as the internal reference, and the data is determined by the 2- ⁇ Ct method
- the weight of mice in the HFD group increased significantly compared with mice in the CON group (P ⁇ 0.01), an increase of about 14%; the weight gain of mice in the HFD+R group was significantly lower than that of mice in the HFD group, and a decrease of approximately 7%; as shown in Figure 2B, the weight growth rate of the HFD group mice increased significantly compared with the CON group mice, an increase of about 56%; the weight growth rate of the HFD+R group mice was significantly lower than that of the HFD group mice (P ⁇ 0.05), a decrease of about 21%. It can be seen that Lactobacillus reuteri FN041 treatment can significantly reduce the weight gain under the influence of high fat.
- the peritesticular adipose tissue index of mice in the HFD group was significantly increased compared to that of the CON group (P ⁇ 0.01), with an increase of about 100%; the peritesticular adipose tissue index of the HFD+R group was smaller than that of the HFD group Mice decreased significantly by about 30%. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly reduce the abnormal increase in peritesticular adipose tissue index under the influence of high fat.
- the liver fat infiltration and testicular fat cell area of the HFD+R group mice were significantly lower than that of the HFD group mice (P ⁇ 0.05), with a decrease of about 10% and 28%, respectively. It can be seen that Lactobacillus reuteri (Lactobacillus reuteri) FN041 treatment can significantly reduce liver fat infiltration and abnormal increase in testicular fat cell area under the influence of high fat.
- the serum triglyceride content of mice in the HFD group was at 8:00 (P ⁇ 0.05), 14:00 (P ⁇ 0.01), and 20:00 (P ⁇ 0.05) compared with those in the CON group.
- the serum triglyceride content of HFD+R group mice was significantly lower than that of HFD group mice. It can be seen that Lactobacillus reuteri FN041 treatment can significantly reduce serum glycerol under the influence of high fat Triester (TG) content increased abnormally.
- TG high fat Triester
- the serum low-density lipoprotein cholesterol content of HFD group mice was significantly higher than that of CON group mice; the serum low-density lipoprotein cholesterol content of HFD+R group mice Compared with mice in the HFD group, there was a significant decrease, especially at 8:00 and 2:00 (P ⁇ 0.01). It can be seen that the treatment of Lactobacillus reuteri FN041 can significantly reduce the low density of serum under the influence of high fat. The content of lipoprotein cholesterol (LDL-C) is abnormally increased.
- the serum high-density lipoprotein cholesterol content of HFD group mice was significantly lower than that of CON group mice; the serum high-density lipoprotein cholesterol content of HFD+R group mice was significantly higher than that of HFD group mice, especially At 14:00 (P ⁇ 0.05), it can be seen that Lactobacillus reuteri FN041 treatment can significantly reduce the abnormal increase in serum high-density lipoprotein cholesterol (HDL-C) content under the influence of high fat.
- HDL-C serum high-density lipoprotein cholesterol
- the plasma FD4 content in the blood of the HFD group mice was significantly higher than that of the CON group mice; the plasma FD4 content in the blood of the HFD+R group mice was significantly lower than that of the HFD group mice, almost the same as the CON group mice Flat, it can be seen that Lactobacillus reuteri FN041 treatment can significantly improve the permeability of the mouse intestinal epithelial barrier, and inhibit the abnormal increase of plasma FD4 content in the blood of mice caused by high-fat diet feeding FD4 into the blood. .
- the serum tumor necrosis factor alpha (TNF- ⁇ ) content in the serum of the HFD group mice was significantly higher than that of the CON group mice; the serum tumor necrosis factor alpha (TNF- ⁇ ) in the serum of the HFD+R group mice The content of ⁇ ) was significantly lower than that of mice in the HFD group. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly inhibit the abnormal increase in serum tumor necrosis factor ⁇ (TNF- ⁇ ) content under the influence of high fat.
- CON control group
- HFD high-fat feed fed control group
- HFD+R high-fat feed fed group treated with Lactobacillus reuteri FN041.
- CON control group
- HFD high-fat feed fed control group
- HFD+R high-fat feed fed group treated with Lactobacillus reuteri FN041.
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Abstract
An application of breast milk-derived lactobacillus reuteri in lowering lipid and regulating lipid metabolism rhythm, relating to the fields of microbial technology and food science. The lactobacillus reuteri FN041 can prevent and/or treat lipid metabolism rhythm disorders and has the functions specifically embodied in: (1) significantly reducing weight gain under the influence of high lipid; (2) significantly reducing abnormal increase of peritesticular adipose tissue indexes under the influence of high lipid; (3) significantly reducing hepatic fatty infiltration and abnormal increase of testicular fat cell area under the influence of high lipid; (4) significantly reducing abnormal increase of the content of TG, TC, LDL-C, and HDL-C in serum under the influence of high lipid; (5) significantly reducing abnormal increase of plasma FD4 content in blood and of the content of LPS, LBP, and TNF-α in serum under the influence of high lipid; and (6) restoring the content of triglyceride in serum and the rhythm of the expressions of biological clock genes Clock, Bmal1, and Per2 in a liver under the influence of high lipid.
Description
本发明涉及一种母乳来源罗伊氏乳杆菌降脂及调节脂代谢节律的应用，属于微生物技术和食品科学领域。The invention relates to an application of lactobacillus reuteri derived from breast milk for reducing lipid and regulating the rhythm of lipid metabolism, and belongs to the fields of microbial technology and food science.
有研究表明，人和哺乳动物肠道中15％以上的细菌的丰度会在白天和黑夜呈周期性起伏的节律性变化，这些节律性变化的细菌主要属于梭菌目、乳杆菌目以及拟杆菌目，约占肠道细菌总数的60％。也有研究表明，人和哺乳动物肠道中细菌的节律性变化会使得肠道上皮细胞在不同时间段暴露于不同数量和不同种类的细菌下，这些节律性变化的细菌会通过肠道上皮细胞传递其代谢产物至肝脏等远端组织，从而使得肝脏等远端组织的基因表达产生节律性变化，进而使得人和哺乳动物的脂代谢发生节律性变化，即脂代谢节律。Studies have shown that the abundance of more than 15% of the bacteria in the intestines of humans and mammals will periodically fluctuate during the day and night. These rhythmic bacteria mainly belong to the Clostridium, Lactobacillus and Bacteroides It accounts for about 60% of the total number of intestinal bacteria. Studies have also shown that the rhythmic changes of bacteria in the intestines of humans and mammals will expose the intestinal epithelial cells to different numbers and types of bacteria at different time periods, and these rhythmic changes of bacteria will pass on the intestinal epithelial cells. Metabolites reach remote tissues such as the liver, thereby causing rhythmic changes in gene expression in remote tissues such as the liver, which in turn causes rhythmic changes in lipid metabolism in humans and mammals, that is, the rhythm of lipid metabolism.
脂代谢节律对人和哺乳动物的健康十分重要，脂代谢节律一但紊乱，就会影响人和哺乳动物对食物中脂肪的吸收和储存，导致人和哺乳动物肝脏脂肪的大量合成，引起人和哺乳动物体内血清甘油三酯等的节律性紊乱。血清甘油三酯主要由肝、脂肪组织及小肠合成，其节律性紊乱会加速人和哺乳动物动脉粥样硬化、脂肪肝、脑部血管堵塞和胰岛素抵抗的发生。The rhythm of lipid metabolism is very important to the health of humans and mammals. Once the rhythm of lipid metabolism is disordered, it will affect the absorption and storage of fat in food by humans and mammals, leading to a large amount of liver fat synthesis in humans and mammals, causing human and Rhythm disorder of serum triglycerides in mammals. Serum triglycerides are mainly synthesized by the liver, adipose tissue and small intestine, and their rhythmic disturbances can accelerate the occurrence of atherosclerosis, fatty liver, cerebral vascular blockage and insulin resistance in humans and mammals.
然而，当今社会，人群热衷于高脂饮食、高糖饮食等的高能饮食，高脂饮食、高糖饮食等的高能饮食会使得人体肠道菌群构成和菌群定制粘液层产生紊乱，从而使得人体的基因表达产生紊乱，进而使得人体的脂代谢节律产生紊乱，最终增加人体罹患动脉粥样硬化、脂肪肝、脑部血管堵塞和胰岛素抵抗等疾病的概率。However, in today’s society, people are keen on high-energy diets such as high-fat diets and high-sugar diets. High-energy diets such as high-fat diets and high-sugar diets will cause disorders in the composition of the human intestinal flora and the customized mucus layer of the flora, thus causing The human body’s gene expression is disordered, which in turn makes the body’s lipid metabolism rhythm disorder, and ultimately increases the body’s probability of suffering from diseases such as atherosclerosis, fatty liver, cerebral blood vessel blockage, and insulin resistance.
据预测，2020年全球将有2400万人死于动脉粥样硬化引起的心脑血管疾病，其中，35～45岁人群的死亡率明显增加，发病年龄提前了10～20年。也有数据表明，仅在中国，就有10％左右的人群饱受脂肪肝的困扰。It is predicted that in 2020, 24 million people will die from cardiovascular and cerebrovascular diseases caused by atherosclerosis in the world. Among them, the death rate of people aged 35 to 45 has increased significantly, and the age of onset is 10 to 20 years earlier. There are also data showing that in China alone, about 10% of the population suffers from fatty liver.
目前，人们常通过服用他汀类药物治疗血脂紊乱，但是，此方法具有一定的副作用，在服用剂量较高时有可能导致人体发生以转氨酶升高为特征的肝损害和以肌酸激酶升高为特征的横机纹溶解，因此，急需找到一种新的可治疗脂代谢节律紊乱的药物或方法以避免这些问题。At present, people often take statins to treat dyslipidemia. However, this method has certain side effects. At higher doses, it may lead to liver damage characterized by elevated transaminase and elevated creatine kinase. The characteristic flat knitting pattern dissolves, therefore, it is urgent to find a new medicine or method that can treat the disorder of lipid metabolism to avoid these problems.
发明内容Summary of the invention
为解决上述问题，本发明提供了一种罗伊氏乳杆菌(Lactobacillus reuteri)FN041，所述罗伊氏乳杆菌(Lactobacillus reuteri)FN041已于2019年1月29日保藏于广东省微生物菌种 保藏中心，保藏编号为GDMCC No.60546，保藏地址为广州市先烈中路100号大院59号楼5楼。In order to solve the above-mentioned problems, the present invention provides a Lactobacillus reuteri FN041, which was deposited in the Guangdong Province Microbial Culture Collection on January 29, 2019 The center, the deposit number is GDMCC No. 60546, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.
所述罗伊氏乳杆菌(Lactobacillus reuteri)FN041是通过先针对分泌型免疫球蛋白A(sIgA)结合共生菌的生理特征，利用免疫磁珠法从来源于甘肃省甘南藏族自治州临潭县的人乳中富集母乳IgA结合菌群，再根据罗伊氏乳杆菌对万古霉素的抗性和高温可培养特性定向分离得到的。The Lactobacillus reuteri FN041 is obtained by first targeting the secretory immunoglobulin A (sIgA) in conjunction with the physiological characteristics of the symbiotic bacteria, and using the immunomagnetic bead method to obtain information from people in Lintan County, Gannan Tibetan Autonomous Prefecture, Gansu Province. The milk is enriched with breast milk IgA-binding bacteria, and then oriented and separated according to the resistance of Lactobacillus reuteri to vancomycin and the high temperature cultivable characteristics.
所述罗伊氏乳杆菌(Lactobacillus reuteri)FN041在MRS琼脂培养基上的菌落为圆形、光滑、白色，直径约1mm。The colony of Lactobacillus reuteri FN041 on the MRS agar medium is round, smooth and white, with a diameter of about 1 mm.
所述罗伊氏乳杆菌(Lactobacillus reuteri)FN041具有以下特性：The Lactobacillus reuteri FN041 has the following characteristics:
(1)在pH为3.5的环境中停留2h后的存活率高于90％；(1) The survival rate after staying in an environment with a pH of 3.5 for 2 hours is higher than 90%;
(2)在浓度分别为3g/kg和4g/kg胆汁溶液中停留4h后的存活率分别高于82％和68％；(2) The survival rates after staying in bile solutions with concentrations of 3g/kg and 4g/kg for 4 hours are respectively higher than 82% and 68%;
(3)可耐受45℃高温。(3) Can withstand high temperature of 45℃.
本发明还提供了上述一种罗伊氏乳杆菌(Lactobacillus reuteri)FN041在制备预防和/或治疗脂代谢节律紊乱的产品中的应用。所述脂代谢节律是指由人和哺乳动物肠道中细菌丰度的节律性变化引起的人和哺乳动物远端组织基因表达的节律性变化所造成的人和哺乳动物脂代谢的节律性变化；所述细菌丰度是指人和哺乳动物肠道中某一种细菌的数量占人和哺乳动物肠道中细菌总数量的百分比；所述远端组织包含肝脏；所述肝脏表达的基因包含生物钟基因Clock、Bmal1和Per2。所述脂代谢节律紊乱是指由人和哺乳动物肠道中细菌丰度节律性变化的紊乱造成的人和哺乳动物脂代谢节律性变化的紊乱。The present invention also provides the application of the above-mentioned Lactobacillus reuteri FN041 in the preparation of a product for preventing and/or treating lipid metabolism rhythm disorders. The lipid metabolism rhythm refers to the rhythmic changes in human and mammal lipid metabolism caused by the rhythmic changes in human and mammalian distal tissue gene expression caused by the rhythmic changes in bacterial abundance in the intestines of humans and mammals; The bacterial abundance refers to the percentage of the number of a certain kind of bacteria in the intestines of humans and mammals to the total number of bacteria in the intestines of humans and mammals; the distal tissues include the liver; the genes expressed by the liver include the clock gene Clock , Bmal1 and Per2. The lipid metabolism rhythm disorder refers to the disorder of the rhythmic changes of human and mammal lipid metabolism caused by the disorder of the rhythmic changes of bacterial abundance in the intestines of humans and mammals.
本发明的一种实施方式中，所述产品中，罗伊氏乳杆菌(Lactobacillus reuteri)FN041的活菌数为不低于1×10
6CFU/mL或1×10
6CFU/g。
In one embodiment of the present invention, in the product, the number of viable bacteria of Lactobacillus reuteri FN041 is not less than 1×10 6 CFU/mL or 1×10 6 CFU/g.
本发明的一种实施方式中，所述产品包含食品、药品或保健品。In one embodiment of the present invention, the product includes food, medicine or health care products.
本发明的一种实施方式中，所述药品的剂型包含颗粒剂、胶囊剂、片剂、丸剂或口服液。In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills or oral liquids.
本发明的一种实施方式中，所述药品含有罗伊氏乳杆菌(Lactobacillus reuteri)FN041、药物载体和/或药用辅料。In one embodiment of the present invention, the medicine contains Lactobacillus reuteri FN041, a drug carrier and/or pharmaceutical excipients.
本发明还提供了一种用于预防和/或治疗脂代谢节律紊乱的产品，所述产品含有上述罗伊氏乳杆菌(Lactobacillus reuteri)FN041。The present invention also provides a product for preventing and/or treating lipid metabolism rhythm disorders, the product containing the above-mentioned Lactobacillus reuteri FN041.
本发明的一种实施方式中，所述产品中，罗伊氏乳杆菌(Lactobacillus reuteri)FN041的活菌数为不低于1×10
6CFU/mL或1×10
6CFU/g。
In one embodiment of the present invention, in the product, the number of viable bacteria of Lactobacillus reuteri FN041 is not less than 1×10 6 CFU/mL or 1×10 6 CFU/g.
本发明的一种实施方式中，所述产品包含食品、药品或保健品。In one embodiment of the present invention, the product includes food, medicine or health care products.
本发明的一种实施方式中，所述药品的剂型包含颗粒剂、胶囊剂、片剂、丸剂或口服液。In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills or oral liquids.
本发明的一种实施方式中，所述药品含有罗伊氏乳杆菌(Lactobacillus reuteri)FN041、药物载体和/或药用辅料。In one embodiment of the present invention, the medicine contains Lactobacillus reuteri FN041, a drug carrier and/or pharmaceutical excipients.
本发明还提供了一种罗伊氏乳杆菌(Lactobacillus reuteri)FN041冻存剂，所述冻存剂中，上述罗伊氏乳杆菌(Lactobacillus reuteri)FN041的活菌数为不低于1×10
10CFU/mL。
The present invention also provides a cryopreservation agent for Lactobacillus reuteri (Lactobacillus reuteri) FN041, in which the number of viable bacteria of the above-mentioned Lactobacillus reuteri FN041 is not less than 1×10 10 CFU/mL.
本发明的一种实施方式中，所述冻存剂的制备方法为先将处于稳定期的上述罗伊氏乳杆菌(Lactobacillus reuteri)FN041菌体用pH为7.0～7.4的磷酸盐缓冲液清洗1～2次，然后将清洗后的罗伊氏乳杆菌(Lactobacillus reuteri)FN041菌体加入保护剂中，得到罗伊氏乳杆菌(Lactobacillus reuteri)FN041冻存剂；所述保护剂含有1g/L的半胱氨酸盐酸盐以及200g/L的甘油。In one embodiment of the present invention, the preparation method of the cryopreservation agent is to first wash the above-mentioned Lactobacillus reuteri FN041 cells in the stable phase with a phosphate buffer with a pH of 7.0 to 7.4. ~ 2 times, and then add the washed Lactobacillus reuteri FN041 cells to the protective agent to obtain Lactobacillus reuteri FN041 cryopreservation agent; the protective agent contains 1g/L Cysteine hydrochloride and 200g/L glycerol.
[技术效果][Technical effect]
1、本发明筛选出了一种罗伊氏乳杆菌(Lactobacillus reuteri)FN041，此罗伊氏乳杆菌(Lactobacillus reuteri)FN041可预防和/或治疗脂代谢节律紊乱，具体体现在：1. The present invention has screened out a Lactobacillus reuteri FN041. This Lactobacillus reuteri FN041 can prevent and/or treat lipid metabolism rhythm disorders, which is specifically embodied in:
(1)灌胃此罗伊氏乳杆菌(Lactobacillus reuteri)FN041可使高脂饲料饲喂小鼠的体重以及体重增长率较未灌胃此罗伊氏乳杆菌(Lactobacillus reuteri)FN041的高脂饲料饲喂小鼠显著下降，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的体重增长；(1) Gavage of Lactobacillus reuteri FN041 can make the body weight and weight growth rate of mice fed with high-fat diet compared with the high-fat feed of Lactobacillus reuteri FN041 without gavage Feeding mice decreased significantly. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly reduce the weight gain under the influence of high fat;
(2)灌胃罗伊氏乳杆菌(Lactobacillus reuteri)FN041可使高脂饲料饲喂小鼠的睾丸周脂肪组织指数较未灌胃此罗伊氏乳杆菌(Lactobacillus reuteri)FN041的高脂饲料饲喂小鼠显著下降，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的睾丸周脂肪组织指数异常增加；(2) Gavage of Lactobacillus reuteri (Lactobacillus reuteri) FN041 can make the peritesticular adipose tissue index of mice fed with high-fat diets compared with the high-fat diet of Lactobacillus reuteri FN041 without gavage. Feeding mice decreased significantly. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly reduce the abnormal increase in peritesticular adipose tissue index under the influence of high fat;
(3)灌胃罗伊氏乳杆菌(Lactobacillus reuteri)FN041可使高脂饲料饲喂小鼠的肝脂肪浸润和睾丸脂肪细胞面积较未灌胃此罗伊氏乳杆菌(Lactobacillus reuteri)FN041的高脂饲料饲喂小鼠显著下降，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的肝脂肪浸润和睾丸脂肪细胞面积异常增加；(3) Gavage of Lactobacillus reuteri (Lactobacillus reuteri) FN041 can make the liver fat infiltration and testicular fat cell area of mice fed high-fat diet higher than that of non-gavage Lactobacillus reuteri FN041 Fat diet fed mice significantly decreased. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly reduce liver fat infiltration and abnormal increase of testicular fat cell area under the influence of high fat;
(4)灌胃罗伊氏乳杆菌(Lactobacillus reuteri)FN041可使高脂饲料饲喂小鼠血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)的含量较未灌胃此罗伊氏乳杆菌(Lactobacillus reuteri)FN041的高脂饲料饲喂小鼠显著降低，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C) 含量异常增加；(4) Gavage of Lactobacillus reuteri (Lactobacillus reuteri) FN041 can feed mice serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), The content of high-density lipoprotein cholesterol (HDL-C) was significantly lower than that of mice fed with high-fat diet of Lactobacillus reuteri FN041 without gavage. It can be seen that Lactobacillus reuteri FN041 Treatment can significantly reduce the abnormal increase in serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) content in serum under the influence of high fat;
(5)灌胃罗伊氏乳杆菌(Lactobacillus reuteri)FN041可使高脂饲料饲喂小鼠血液中血浆FD4的含量以及血清中内毒素(LPS)、内毒素结合蛋白(LBP)、肿瘤坏死因子α(TNF-α)的含量较未灌胃此罗伊氏乳杆菌(Lactobacillus reuteri)FN041的高脂饲料饲喂小鼠显著降低，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著改善小鼠肠上皮屏障通透性，抑制了高脂饲料饲喂引起的FD4进入血液导致的小鼠血液中血浆FD4含量异常增加，并且，罗伊氏乳杆菌(Lactobacillus reuteri)FN041发挥了保护小鼠粘膜屏障的作用，抑制了高脂饲料饲喂引起的肠道细菌进入血液导致的小鼠血清中内毒素(LPS)、内毒素结合蛋白(LBP)含量异常增加，另外，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著抑制高脂影响下的血清中肿瘤坏死因子α(TNF-α)含量异常增加；(5) Gavage of Lactobacillus reuteri (Lactobacillus reuteri) FN041 can make the blood plasma FD4 content and serum endotoxin (LPS), endotoxin binding protein (LBP) and tumor necrosis factor in mice fed high-fat diet The content of α (TNF-α) is significantly lower than that of mice fed with high-fat diet of Lactobacillus reuteri FN041 without gavage. It can be seen that the treatment of Lactobacillus reuteri FN041 can significantly improve The permeability of the murine intestinal epithelial barrier inhibits the abnormal increase in plasma FD4 content in the blood of mice caused by FD4 entering the blood caused by high-fat diet feeding, and Lactobacillus reuteri FN041 has played a role in protecting mice The role of the mucosal barrier inhibits the abnormal increase of endotoxin (LPS) and endotoxin binding protein (LBP) in the serum of mice caused by intestinal bacteria entering the blood caused by high-fat feed. In addition, Lactobacillus reuteri ( Lactobacillus reuteri) FN041 treatment can significantly inhibit the abnormal increase in serum tumor necrosis factor alpha (TNF-α) under the influence of high fat;
(6)小鼠血清中甘油三酯的含量以及小鼠肝脏的生物钟基因Clock、Bmal1和Per2的表达存在明显的节律性，但是，在高脂影响下，小鼠血清中甘油三酯的含量以及小鼠肝脏的生物钟基因Clock、Bmal1和Per2的表达会丧失节律性，而罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可恢复此节律性。(6) The triglyceride content in mouse serum and the expression of clock genes Clock, Bmal1 and Per2 in the liver of mice have obvious rhythmicity. However, under the influence of high fat, the content of triglyceride in mouse serum and The expression of clock genes Clock, Bmal1 and Per2 in the liver of mice loses rhythm, and treatment with Lactobacillus reuteri FN041 can restore this rhythm.
2、分泌型免疫球蛋白A(sIgA)是人肠粘膜表面含量丰富的抗体分子，其可以与肠道共生的如乳杆菌等的有益菌结合形成复合体促进有益菌在人体内发挥有益作用，此有益作用主要体现在：2. Secretory immunoglobulin A (sIgA) is an antibody molecule rich in human intestinal mucosal surface. It can combine with beneficial bacteria such as Lactobacillus that are symbiotic in the intestine to form a complex to promote beneficial bacteria to play a beneficial role in the human body. This beneficial effect is mainly reflected in:
第一，由于sIgA主要富集在肠道粘膜表面的粘液层，有益菌与之结合可促进其在粘液层定植；First, because sIgA is mainly concentrated in the mucous layer of the intestinal mucosa, the combination of beneficial bacteria can promote its colonization in the mucous layer;
第二，形成复合体可促进有益菌锚定于肠道上皮细胞顶端表面，促进上皮细胞紧密连接蛋白磷酸化，维持细胞与细胞的相互作用，从而增强粘膜屏障，并诱导抗炎趋化因子产生，保持粘膜的非炎环境；Second, the formation of complexes can promote the anchoring of beneficial bacteria on the top surface of intestinal epithelial cells, promote the phosphorylation of tight junction proteins of epithelial cells, maintain cell-cell interactions, thereby enhance the mucosal barrier, and induce the production of anti-inflammatory chemokines , To maintain a non-inflammatory environment of the mucosa;
第三，大多数乳杆菌可被树突或巨噬细胞识别而调节免疫反应，这些可识别乳杆菌的树突或巨噬细胞主要存在于粘膜屏障下层，包括固有层和派尔集合淋巴结(PP)，其中，PP的肠腔表面存在的sIgA受体可帮助转运sIgA结合的有益菌进入PP，这些进入PP的有益菌可和PP中的树突细胞亚群作用促进T细胞产生IL-10和TGF-β等抗炎细胞因子；Third, most Lactobacilli can be recognized by dendrites or macrophages to regulate immune responses. These Lactobacillus-recognizing dendrites or macrophages mainly exist in the submucosal barrier, including the lamina propria and Peyer's collective lymph node (PP ), where the sIgA receptor on the surface of the intestinal lumen of PP can help transport sIgA-bound beneficial bacteria into PP. These beneficial bacteria that enter PP can interact with the dendritic cell subsets in PP to promote T cells to produce IL-10 and Anti-inflammatory cytokines such as TGF-β;
第四，有益菌被sIgA结合包裹后可屏蔽细菌表面抗原，阻止其诱导过强炎性反应，Fourth, the beneficial bacteria can be shielded by the sIgA binding and package, preventing the bacterial surface antigen from inducing a strong inflammatory response.
而本发明筛选得到的罗伊氏乳杆菌(Lactobacillus reuteri)FN041可与分泌型免疫球蛋白A(sIgA)相结合，因此，本发明的罗伊氏乳杆菌(Lactobacillus reuteri)FN041可以更近的距离调节近粘液层上皮细胞的生理活动，或在粘液局部释放较高浓度代谢物以调节代谢节律。The Lactobacillus reuteri FN041 screened in the present invention can bind to secretory immunoglobulin A (sIgA). Therefore, the Lactobacillus reuteri FN041 of the present invention can be closer. Regulate the physiological activities of epithelial cells near the mucous layer, or release higher concentrations of metabolites locally in the mucus to regulate the metabolic rhythm.
3、本发明筛选得到的罗伊氏乳杆菌(Lactobacillus reuteri)FN041来源于人乳，因此，本发明的干酪乳杆菌(Lactobacillus casei)CCFM1038对人体不会产生任何伤害。3. The Lactobacillus reuteri FN041 screened in the present invention is derived from human milk. Therefore, the Lactobacillus casei CCFM1038 of the present invention does not cause any harm to the human body.
4、本发明筛选得到的罗伊氏乳杆菌(Lactobacillus reuteri)FN041在pH为3.5的环境中停留2h后的存活率高于90％，在浓度分别为3g/kg、和4g/kg胆汁溶液中停留4h后的存活率分别高于82％和68％，可耐受45℃高温，具有良好的生理特性。4. The survival rate of Lactobacillus reuteri FN041 screened in the present invention after staying in an environment with a pH of 3.5 for 2 hours is higher than 90%, in a bile solution with a concentration of 3g/kg and 4g/kg, respectively The survival rate after staying for 4 hours is higher than 82% and 68% respectively, can withstand high temperature of 45℃, and has good physiological characteristics.
生物材料保藏Biological material preservation
一株罗伊氏乳杆菌(Lactobacillus reuteri)FN041，分类学命名为Lactobacillus reuteri，已于2019年1月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCCNo.60546，保藏地址为广州市先烈中路100号大院59号楼5楼。A strain of Lactobacillus reuteri FN041, taxonomically named Lactobacillus reuteri, has been deposited in the Guangdong Provincial Microbial Culture Collection on January 29, 2019, the deposit number is GDMCCNo.60546, and the deposit address is Guangzhou City 5th Floor, Building 59, Yard 100, Xianlie Middle Road.
图1：富集母乳IgA结合菌群并选择性分离罗伊氏乳杆菌的流程图。Figure 1: Flow chart of enrichment of IgA-binding bacteria in breast milk and selective isolation of Lactobacillus reuteri.
图2：罗伊氏乳杆菌FN041对高脂饲料喂养小鼠增重和脂肪积累的影响；其中，A：体重变化，B：体增重率，C：器官指数，D：睾周脂肪和肝脏组织HE染色；数据为平均值±标准差(n＝5)；*p<0.05，**p<0.01，与CON对比；
#p<0.05，
##p<0.01，与HFD组比较；采用单因素方差分析进行比较，随后进行Tukey多重比较；CON：对照组，HFD：高脂饲料饲喂对照组，HFD+R：罗伊氏乳杆菌FN041处理的高脂饲料饲喂组。
Figure 2: The effect of Lactobacillus reuteri FN041 on the weight gain and fat accumulation of mice fed high-fat diet; A: body weight change, B: body weight gain rate, C: organ index, D: testicular fat and liver Tissue HE staining; data are mean±standard deviation (n=5); *p<0.05, **p<0.01, compared with CON; # p<0.05, ## p<0.01, compared with HFD group; using single Factor analysis of variance was used for comparison, followed by Tukey multiple comparison; CON: control group, HFD: high-fat feed fed control group, HFD+R: high-fat feed fed group treated with Lactobacillus reuteri FN041.
图3：罗伊氏乳杆菌FN041对高脂饲料喂养小鼠血脂节律性变化的影响；其中，数据为平均值±标准差(n＝5)；*p<0.05，**p<0.01，与CON对比；
##p<0.01，与HFD组比较；采用单因素方差分析进行比较，随后进行Tukey多重比较；CON，对照组；HFD，高脂饲料饲喂对照组；HFD+R，罗伊氏乳杆菌FN041处理的高脂饲料饲喂组；TG：血清中甘油三酯含量，TC：血清总胆固醇含量，LDL-C：血清低密度脂蛋白胆固醇含量，HDL-C：血清高密度脂蛋白胆固醇含量。
Figure 3: The effect of Lactobacillus reuteri FN041 on the rhythmic changes of blood lipids in mice fed high-fat diet; among them, the data is the mean ± standard deviation (n=5); *p<0.05, **p<0.01, and CON contrast; ## p<0.01, compared with HFD group; use one-way analysis of variance for comparison, followed by Tukey multiple comparison; CON, control group; HFD, high-fat diet fed control group; HFD+R, Roy's High-fat feed fed group treated with Lactobacillus FN041; TG: serum triglyceride content, TC: serum total cholesterol content, LDL-C: serum low-density lipoprotein cholesterol content, HDL-C: serum high-density lipoprotein cholesterol content.
图4：罗伊氏乳杆菌FN041对高脂饲料喂养小鼠粘膜屏障的影响；其中，数据为平均值±标准差(n＝5)；*p<0.05，**p<0.01，与CON对比；
#p<0.05，
##p<0.01，与HFD组比较；采用单因素方差分析进行比较，随后进行Tukey多重比较；CON：对照组，HFD：高脂饲料饲喂对照组，HFD+R：罗伊氏乳杆菌FN041处理的高脂饲料饲喂组。
Figure 4: The effect of Lactobacillus reuteri FN041 on the mucosal barrier of mice fed high-fat diet; among them, the data is the mean ± standard deviation (n=5); *p<0.05, **p<0.01, compared with CON ; # P<0.05, ## p<0.01, compared with the HFD group; use one-way analysis of variance for comparison, followed by Tukey multiple comparisons; CON: control group, HFD: high-fat feed fed control group, HFD+R: High-fat diet fed with Lactobacillus reuteri FN041.
下面结合具体实施例对本发明进行进一步的阐述。The present invention will be further described below in conjunction with specific embodiments.
下述实施例中涉及的培养基如下：The media involved in the following examples are as follows:
MRS琼脂培养基：蛋白胨10g/L、酵母提取物5g/L、葡萄糖20g/L、无水乙酸钠2g/L、 柠檬酸氢二胺2g/L、K
2HPO
4·3H
2O 2.6g/L、MgSO
4·7H
2O 0.5g/L、MnSO
4·7H
2O 0.25g/L、吐温-80 1g/L、琼脂20g/L、蒸馏水1000g/L。
MRS agar medium: peptone 10g/L, yeast extract 5g/L, glucose 20g/L, anhydrous sodium acetate 2g/L, hydrogen citrate diamine 2g/L, K 2 HPO 4 ·3H 2 O 2.6g/ L, MgSO 4 ·7H 2 O 0.5g/L, MnSO 4 ·7H 2 O 0.25g/L, Tween-80 1g/L, agar 20g/L, distilled water 1000g/L.
MRS液体培养基(g/L)：蛋白胨10g/L、酵母提取物5g/L、葡萄糖20g/L、无水乙酸钠2g/L、柠檬酸氢二胺2g/L、K
2HPO
4·3H
2O 2.6g/L、MgSO
4·7H
2O 0.5g/L、MnSO
4·7H
2O 0.25g/L、吐温-80 1g/L、蒸馏水1000g/L。
MRS liquid medium (g/L): peptone 10g/L, yeast extract 5g/L, glucose 20g/L, anhydrous sodium acetate 2g/L, hydrogen citrate diamine 2g/L, K 2 HPO 4 ·3H 2 O 2.6g/L, MgSO 4 ·7H 2 O 0.5g/L, MnSO 4 ·7H 2 O 0.25g/L, Tween-80 1g/L, distilled water 1000g/L.
实施例1：母乳样品采集Example 1: Collection of breast milk samples
具体步骤如下：Specific steps are as follows:
选择体检和体征调查证明健康的哺乳母亲，在收到样品采集知情同意书的情况下开始采集母乳；用吸奶器采集母乳前洗净双手，使用尺寸合适的吸乳护罩，将乳头放置于吸乳护罩中心部位，保持密闭性，选择适合压力(170～60mmHg)；母乳保存于存乳袋，在冰上于2h内运送到分析场所。Select breastfeeding mothers who are healthy by physical examination and physical sign survey, and start collecting breast milk after receiving the informed consent for sample collection; wash your hands before collecting breast milk with a breast pump, use a breast shield of appropriate size, and place the nipple on Keep the center of the breast shield airtight and select a suitable pressure (170～60mmHg); breast milk is stored in a milk storage bag and transported to the analysis site within 2 hours on ice.
实施例2：母乳菌群的收集和罗伊氏乳杆菌分析Example 2: Collection of breast milk flora and analysis of Lactobacillus reuteri
具体步骤如下：Specific steps are as follows:
将母乳于8000～12000rpm离心15min(4℃)，去除液体，收集离心管底部的固型物；取部分固型物提取DNA，用罗伊氏乳杆菌特异性PCR分析罗伊氏乳杆菌的存在情况。Centrifuge the breast milk at 8000～12000rpm for 15min (4℃), remove the liquid, collect the solids at the bottom of the centrifuge tube; take part of the solids to extract DNA, and analyze the existence of Lactobacillus reuteri by specific PCR of Lactobacillus reuteri Happening.
实施例3：IgA结合菌富集Example 3: Enrichment of IgA-binding bacteria
具体步骤如下：Specific steps are as follows:
如图1，将母乳离心获得的固形物悬浮于蛋白胨缓冲液，加入牛血清白蛋白封闭非特异性结合(终浓度分别为10％、0.05～0.5％)，加入生物素标记的兔抗人IgA血清，孵育15～30min，再加入链霉亲和素修饰磁珠(添加量为0.1-1.0mg/mL)，用磁铁吸附菌体，用蛋白胨缓冲液洗两次，即得IgA结合菌群。As shown in Figure 1, the solid matter obtained by centrifugation of breast milk is suspended in peptone buffer, bovine serum albumin is added to block non-specific binding (final concentration is 10%, 0.05 to 0.5%, respectively), and biotin-labeled rabbit anti-human IgA serum is added , Incubate for 15-30 minutes, add streptavidin-modified magnetic beads (additional amount is 0.1-1.0 mg/mL), adsorb bacteria with a magnet, wash twice with peptone buffer to obtain IgA-bound flora.
实施例4：罗伊氏乳杆菌的分离Example 4: Isolation of Lactobacillus reuteri
具体步骤如下：Specific steps are as follows:
1、筛选1. Screening
将实施例3获得的菌群在无菌条件下用PBS缓冲溶液(pH6.8)梯度稀释，吸取合适的稀释度溶液(每mL含有100个左右细菌)100微升涂布于含有万古霉素(50μg/mL)的MRS琼脂培养基平板，平板倒置放入厌氧培养箱，37℃培养36～72h，观察并记录菌落形态；挑取MRS琼脂培养基平板上不同形态的菌落进行划线分离，经37℃培养48h后，再次挑取MRS琼脂培养基平板上不同形态的单菌落进行划线分离，直至得到形态一致的纯的单菌落；挑取MRS琼脂培养基平板上的纯菌落接种于5mL MRS液体培养基中，37℃培养18h；取1mL 菌液于无菌离心管中，8000r/min离心3min后弃去上层培养基，菌泥重悬于30％甘油溶液中置于-80℃中保藏，得到菌株。The bacterial colony obtained in Example 3 was gradually diluted with PBS buffer solution (pH 6.8) under aseptic conditions, and 100 microliters of the appropriate dilution solution (containing about 100 bacteria per mL) was applied to it containing vancomycin (50μg/mL) MRS agar medium plate, the plate is placed upside down into an anaerobic incubator, and cultured at 37°C for 36～72h, observe and record the colony morphology; pick different colonies on the MRS agar medium plate for streaking separation After culturing at 37°C for 48 hours, pick out single colonies of different morphologies on the MRS agar medium plate again for streaking, until a pure single colony with consistent morphology is obtained; pick the pure colonies on the MRS agar medium plate and inoculate it on Incubate in 5mL MRS liquid medium at 37°C for 18h; take 1mL of bacterial solution in a sterile centrifuge tube, centrifuge at 8000r/min for 3min, discard the upper medium, and resuspend the bacterial paste in 30% glycerol solution and place at -80°C Save in the medium to obtain the strain.
2、鉴定2. Identification
对分离得到的菌株进行PCR扩增16S rDNA，PCR产物送至华大基因测序有限公司进行测序，将测序得到的结果在ezbiocloud中进行核酸序列比对，其中，有一株菌的核苷酸序列与罗伊氏乳杆菌(Lactobacillus reuteri)JCM 1112的相似性达到99.72％，判定此菌株为罗伊氏乳杆菌，命名为罗伊氏乳杆菌(Lactobacillus reuteri)FN041(FN041的16S rDNA序列如SEQ ID NO.1所示)。The isolated strains were subjected to PCR amplification of 16S rDNA, and the PCR products were sent to Huada Gene Sequencing Co., Ltd. for sequencing. The sequencing results were compared in ezbiocloud for nucleic acid sequence comparison. Among them, the nucleotide sequence of one strain was compared with The similarity of Lactobacillus reuteri JCM 1112 reached 99.72%, and this strain was determined to be Lactobacillus reuteri, named Lactobacillus reuteri FN041 (the 16S rDNA sequence of FN041 is as SEQ ID NO .1).
3、培养3. Training
将罗伊氏乳杆菌(Lactobacillus reuteri)FN041接入MRS琼脂培养基平板上37℃培养36～72h后，观察其菌落，发现其菌落为圆形、光滑、白色，直径约1mm。After inserting Lactobacillus reuteri FN041 into the MRS agar medium plate and culturing at 37°C for 36-72 hours, the colonies were observed and found to be round, smooth and white, with a diameter of about 1 mm.
将罗伊氏乳杆菌(Lactobacillus reuteri)FN041置于pH为3.5的生理盐水中停留2h，发现其在pH为3.5的环境中停留2h后的存活率高于90％。Lactobacillus reuteri FN041 was placed in physiological saline with pH 3.5 for 2 hours, and it was found that the survival rate after 2 hours in an environment with pH 3.5 was higher than 90%.
将罗伊氏乳杆菌(Lactobacillus reuteri)FN041分别置于浓度为3g/kg、和4g/kg胆汁溶液中停留4h，发现其在浓度分别为3g/kg、和4g/kg胆汁溶液中停留4h后的存活率分别高于82％和68％。Lactobacillus reuteri FN041 was placed in a bile solution with a concentration of 3g/kg and 4g/kg for 4h, and it was found to stay in a bile solution with a concentration of 3g/kg and 4g/kg for 4h. The survival rates were higher than 82% and 68%.
将罗伊氏乳杆菌(Lactobacillus reuteri)FN041接入MRS液体培养基中，分别于35、40、45、50℃培养36～72h后，观察其生长曲线，发现其可耐受45℃高温，于35～45℃的温度范围内均能良好生长。The Lactobacillus reuteri FN041 was inserted into the MRS liquid medium and cultured at 35, 40, 45, and 50°C for 36 to 72 hours, and then observed its growth curve. It was found that it could tolerate the high temperature of 45°C. It can grow well in the temperature range of 35～45℃.
实施例5：罗伊氏乳杆菌FN041在预防和/或治疗高能饮食引起的代谢节律紊乱方面的应用Example 5: Application of Lactobacillus reuteri FN041 in the prevention and/or treatment of metabolic rhythm disorders caused by high-energy diet
具体步骤如下：Specific steps are as follows:
1)动物实验分组1) Animal experiment group
将3周龄健康雄性C57BL/6J小鼠，随机分笼预饲养一周结束后开始正式实验，小鼠随机分成正常膳食组(CON组，20只)，饲喂低脂膳食(12％能量来源于脂肪)；高脂日粮组(HFD组，80只)，饲喂高脂日粮(45％能量来源于脂肪)；高脂日粮和正常膳食的配方见表1。Three-week-old healthy male C57BL/6J mice were randomly divided into cages and pre-raised for one week. The formal experiment began. The mice were randomly divided into normal diet groups (CON group, 20) and fed with low-fat diet (12% of energy was derived from Fat); high-fat diet group (HFD group, 80 animals), fed high-fat diet (45% of energy comes from fat); high-fat diet and normal diet formulas are shown in Table 1.
小鼠的饲养温度为24±3℃，湿度60±10％，动物房每日12h开灯光，12h黑暗；每周称重，记录小鼠每周的体重、采食量和摄水量；灌胃实验在正式饲养7周后开始，将高脂日粮组小鼠随机分2组(每组20只)，每日分别灌胃磷酸盐缓冲液(PBS，pH7.3)(HFD组)和罗伊氏乳杆菌(Lactobacillus reuteri)FN041菌悬液(HFD+R组)；其中，罗伊氏乳杆菌 (Lactobacillus reuteri)FN041菌悬液的浓度为8.0×10
8CFU/mL，灌胃体积为200μL/只，灌胃时间为下午16:00。
The rearing temperature of the mice is 24±3℃, the humidity is 60±10%, the animal room is turned on for 12 hours a day, and 12 hours is dark; weekly weighing, recording the weight, food intake and water intake of the mice each week; The gastric experiment was started after 7 weeks of formal feeding. The mice in the high-fat diet group were randomly divided into 2 groups (20 mice in each group), and phosphate buffered saline (PBS, pH7.3) (HFD group) and Lactobacillus reuteri FN041 bacterial suspension (HFD+R group); Among them, the concentration of Lactobacillus reuteri FN041 bacterial suspension is 8.0×10 8 CFU/mL, and the gastric volume is 200μL/only, gavage time is 16:00 in the afternoon.
表1 动物饲料配方Table 1 Animal feed formula
注：(a)混合矿物质配方(1kg)：FeSO
4·H
2O(256g)，GuSO
4·H
2O(27.9g)，MnSO
4·H
2O(243.8g)，ZnSO
4·H
2O(137g)，Na
2SeO
3(0.5g)，KI(0.7g)。
Note: (a) Mixed mineral formula (1kg): FeSO 4 ·H 2 O (256g), GuSO 4 ·H 2 O (27.9g), MnSO 4 ·H 2 O (243.8g), ZnSO 4 ·H 2 O (137g), Na 2 SeO 3 (0.5 g), KI (0.7 g).
实验最后一天，将每组小鼠分为四批(5只每批)，其中三批小鼠分别在2:00、8:00和20:00处死，另外一批小鼠在10:00灌胃荧光素FITC-dextran(FD4)，灌胃量为0.6mg/g体重，灌胃4h后(14:00)处死，取外周血，用fluorescent microplate reader(激发光485nm，发射光535nm)检测血浆FD4；所有动物处死后测定血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)的含量以及内毒素(LPS)、内毒素结合蛋白(LBP)和血清中肿瘤坏死因子α(TNF-α)的含量，并使用Acrophase软件进行血脂生理节律的余弦拟合以对血脂生理节律进行分析；On the last day of the experiment, each group of mice was divided into four batches (5 mice in each batch). Three batches of mice were sacrificed at 2:00, 8:00, and 20:00, and the other group of mice was irrigated at 10:00 Gastric fluorescein FITC-dextran (FD4), intragastric dose of 0.6mg/g body weight, 4h after intragastric administration (14:00), sacrificed, peripheral blood was collected, and plasma was detected with fluorescent microplate reader (excitation light 485nm, emission light 535nm) FD4; After all animals were sacrificed, the serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) content and endotoxin (LPS) were measured ), endotoxin binding protein (LBP) and serum tumor necrosis factor alpha (TNF-α) content, and use Acrophase software to perform cosine fitting of blood lipid circadian rhythm to analyze blood lipid circadian rhythm;
其中，血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)的含量通过购自南京建成的试剂盒进行检测；Among them, the serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) content was tested by a kit purchased from Nanjing ；
内毒素(LPS)、内毒素结合蛋白(LBP)、血清中肿瘤坏死因子α(TNF-α)的含量通过购自厦门慧嘉的酶联免疫试剂盒进行检测；The levels of endotoxin (LPS), endotoxin binding protein (LBP), and serum tumor necrosis factor alpha (TNF-α) were detected by an enzyme-linked immunoassay kit purchased from Xiamen Huijia;
血脂生理节律变化通过如下方法分析：The changes in blood lipid circadian rhythm are analyzed by the following methods:
采用Trizol法提取小鼠肝脏中生物钟基因Clock、Bmal1以及Per2的RNA，用NanoDrop测定其纯度(OD260/280)和浓度，调节RNA浓度至1000ng/μL左右，即OD
260/280在1.8～2.0范围内；反转录过程分两步，第一步体系混匀后85℃，5min，冰浴上迅速冷却，随后加入第 二步体系混匀后，37℃水浴1h，95℃，3min，获得cDNA；以反转录后的cDNA作为荧光定量PCR的模板，获得RNA，基因引物序列为：
Use Trizol method to extract RNA of clock genes Clock, Bmal1 and Per2 in mouse liver, measure its purity (OD260/280) and concentration with NanoDrop, adjust the RNA concentration to about 1000ng/μL, that is, OD 260/280 is in the range of 1.8～2.0 The reverse transcription process is divided into two steps. In the first step, the system is mixed at 85°C, 5min, and quickly cooled on an ice bath. Then, after the second step is added to the system, the cDNA is obtained in a water bath at 37°C for 1h, 95°C, 3min. ; Use reverse-transcribed cDNA as a template for fluorescent quantitative PCR to obtain RNA, and the gene primer sequence is:
肝脏的生物钟基因Clock(NCBIGeneID:12753)的引物：Primers of the liver's biological clock gene Clock (NCBIGeneID: 12753):
核苷酸序列如SEQ ID NO.2所示的F：AGCACACACACTTCCTCTCTGACAT；The nucleotide sequence is shown in SEQ ID NO. 2 F: AGCACACACACTTCCTCTCTGACAT;
核苷酸序列如SEQ ID NO.3所示的R：ATCAAGGGACTGAACACTCAAGACC；The nucleotide sequence is shown in SEQ ID NO.3: R: ATCAAGGGACTGAACACTCAAGACC;
肝脏的生物钟基因Bmal1(brain and muscle ARNT-like-1)(NCBI Gene ID:11865)的引物：The primer of the liver's biological clock gene Bmal1 (brain and muscle ARNT-like-1) (NCBI Gene ID: 11865):
核苷酸序列如SEQ ID NO.4所示的F：AGTCAGATTGAAAAGAGGCGTCG；The nucleotide sequence is F shown in SEQ ID NO.4: AGTCAGATTGAAAAGAGGCGTCG;
核苷酸序列如SEQ ID NO.5所示的R：AGAAATGTTGGCTTGTAGTTTGCTT；The nucleotide sequence is R shown in SEQ ID NO. 5: AGAAATGTTGGCTTGTAGTTTGCTT;
肝脏的生物钟基因Per2(Period2)(NCBI Gene ID:18627)的引物：Primers of the liver's biological clock gene Per2 (Period2) (NCBI Gene ID: 18627):
核苷酸序列如SEQ ID NO.6所示的F：TTCTCTGCTGTTCTTGTATCCTTTT；The nucleotide sequence is shown in SEQ ID NO. 6 F: TTCTCTGCTGTTCTTGTATCCTTTT;
核苷酸序列如SEQ ID NO.7所示的R：GCTTTCTGCTGGGAGCTAATG；The nucleotide sequence is shown in SEQ ID NO.7 R: GCTTTCTGCTGGGAGCTAATG;
荧光定量PCR的扩增条件为：95℃，5min；95℃，20s，62℃，30s，72℃，20s；72℃，2min；以β-actin作为内参，采用2
-△△Ct法对数据进行分析，数学模型为：Y＝M+Acos(ωt+θ)；其中，M是中值，A是节律振幅，ω是节律角频率，θ是峰值相位，通常情况下P<0.05则证明节律存在。
The amplification conditions of fluorescence quantitative PCR are: 95°C, 5min; 95°C, 20s, 62°C, 30s, 72°C, 20s; 72°C, 2min; β-actin is used as the internal reference, and the data is determined by the 2- △△Ct method For analysis, the mathematical model is: Y=M+Acos(ωt+θ); where M is the median, A is the rhythm amplitude, ω is the rhythm angular frequency, and θ is the peak phase. Normally, P<0.05 indicates the rhythm exist.
2)实验结果2) Experimental results
如图2A所示，HFD组小鼠的体重相对CON组小鼠显著增加(P<0.01)，涨幅约14％；HFD+R组小鼠的体重增长幅度相对HFD组小鼠显著下降，降幅约7％；如图2B所示，HFD组小鼠的体重增长率相对CON组小鼠显著增加，涨幅约56％；HFD+R组小鼠的体重增长率相对HFD组小鼠显著下降(P<0.05)，降幅约21％，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的体重增长。As shown in Figure 2A, the weight of mice in the HFD group increased significantly compared with mice in the CON group (P<0.01), an increase of about 14%; the weight gain of mice in the HFD+R group was significantly lower than that of mice in the HFD group, and a decrease of approximately 7%; as shown in Figure 2B, the weight growth rate of the HFD group mice increased significantly compared with the CON group mice, an increase of about 56%; the weight growth rate of the HFD+R group mice was significantly lower than that of the HFD group mice (P< 0.05), a decrease of about 21%. It can be seen that Lactobacillus reuteri FN041 treatment can significantly reduce the weight gain under the influence of high fat.
如图2C所示，HFD组小鼠的睾丸周脂肪组织指数相对CON组小鼠显著增加(P<0.01)，涨幅约100％；HFD+R组小鼠的睾丸周脂肪组织指数相对HFD组小鼠显著下降，降幅约30％，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的睾丸周脂肪组织指数异常增加。As shown in Figure 2C, the peritesticular adipose tissue index of mice in the HFD group was significantly increased compared to that of the CON group (P<0.01), with an increase of about 100%; the peritesticular adipose tissue index of the HFD+R group was smaller than that of the HFD group Mice decreased significantly by about 30%. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly reduce the abnormal increase in peritesticular adipose tissue index under the influence of high fat.
如图2D所示，HFD+R组小鼠的肝脂肪浸润和睾丸脂肪细胞面积相对HFD组小鼠显著下降(P<0.05)，降幅分别约10％和28％，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的肝脂肪浸润和睾丸脂肪细胞面积异常增加。As shown in Figure 2D, the liver fat infiltration and testicular fat cell area of the HFD+R group mice were significantly lower than that of the HFD group mice (P<0.05), with a decrease of about 10% and 28%, respectively. It can be seen that Lactobacillus reuteri (Lactobacillus reuteri) FN041 treatment can significantly reduce liver fat infiltration and abnormal increase in testicular fat cell area under the influence of high fat.
如图3A所示，HFD组小鼠血清中甘油三酯的含量相对CON组小鼠在8:00(P＜0.05)、 14:00(P＜0.01)、20:00时(P＜0.05)时明显升高；HFD+R组小鼠血清中甘油三酯的含量相对HFD组小鼠显著下降，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的血清中甘油三酯(TG)含量异常增加。As shown in Figure 3A, the serum triglyceride content of mice in the HFD group was at 8:00 (P＜0.05), 14:00 (P＜0.01), and 20:00 (P＜0.05) compared with those in the CON group. The serum triglyceride content of HFD+R group mice was significantly lower than that of HFD group mice. It can be seen that Lactobacillus reuteri FN041 treatment can significantly reduce serum glycerol under the influence of high fat Triester (TG) content increased abnormally.
如图3B所示，HFD组小鼠血清中总胆固醇的含量相对CON组小鼠在8:00和14:00时显著升高(P＜0.05)；HFD+R组小鼠血清中总胆固醇的含量相对CON组小鼠无明显升高，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的血清中总胆固醇(TC)含量异常增加。As shown in Figure 3B, the content of total cholesterol in serum of HFD group mice was significantly higher than that of CON group mice at 8:00 and 14:00 (P<0.05); the content of total cholesterol in serum of HFD+R group mice Compared with mice in the CON group, the content did not increase significantly. It can be seen that Lactobacillus reuteri FN041 treatment can significantly reduce the abnormal increase in serum total cholesterol (TC) content under the influence of high fat.
如图3C所示，除14:00外，HFD组小鼠血清中低密度脂蛋白胆固醇的含量相对CON组小鼠均显著升高；HFD+R组小鼠血清中低密度脂蛋白胆固醇的含量相对HFD组小鼠显著下降，尤其是在8:00和2:00时(P＜0.01)，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的血清中低密度脂蛋白胆固醇(LDL-C)含量异常增加。As shown in Figure 3C, except for 14:00, the serum low-density lipoprotein cholesterol content of HFD group mice was significantly higher than that of CON group mice; the serum low-density lipoprotein cholesterol content of HFD+R group mice Compared with mice in the HFD group, there was a significant decrease, especially at 8:00 and 2:00 (P<0.01). It can be seen that the treatment of Lactobacillus reuteri FN041 can significantly reduce the low density of serum under the influence of high fat. The content of lipoprotein cholesterol (LDL-C) is abnormally increased.
如图3D所示，HFD组小鼠血清中高密度脂蛋白胆固醇的含量相对CON组小鼠显著下降；HFD+R组小鼠血清中高密度脂蛋白胆固醇的含量相对HFD组小鼠显著升高，尤其是在14:00时(P＜0.05)，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著降低高脂影响下的血清中高密度脂蛋白胆固醇(HDL-C)含量异常增加。As shown in Figure 3D, the serum high-density lipoprotein cholesterol content of HFD group mice was significantly lower than that of CON group mice; the serum high-density lipoprotein cholesterol content of HFD+R group mice was significantly higher than that of HFD group mice, especially At 14:00 (P<0.05), it can be seen that Lactobacillus reuteri FN041 treatment can significantly reduce the abnormal increase in serum high-density lipoprotein cholesterol (HDL-C) content under the influence of high fat.
如图4A所示，HFD组小鼠血液中血浆FD4的含量相对CON组小鼠显著上升；HFD+R组小鼠血液中血浆FD4的含量相对HFD组小鼠显著下降，几乎与CON组小鼠持平，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著改善小鼠肠上皮屏障通透性，抑制了高脂饲料饲喂引起的FD4进入血液导致的小鼠血液中血浆FD4含量异常增加。As shown in Figure 4A, the plasma FD4 content in the blood of the HFD group mice was significantly higher than that of the CON group mice; the plasma FD4 content in the blood of the HFD+R group mice was significantly lower than that of the HFD group mice, almost the same as the CON group mice Flat, it can be seen that Lactobacillus reuteri FN041 treatment can significantly improve the permeability of the mouse intestinal epithelial barrier, and inhibit the abnormal increase of plasma FD4 content in the blood of mice caused by high-fat diet feeding FD4 into the blood. .
如图4B、4C所示，HFD组小鼠血清中内毒素(LPS)、内毒素结合蛋白(LBP)的含量相对CON组小鼠显著上升；HFD+R组小鼠血清中内毒素(LPS)、内毒素结合蛋白(LBP)含量相对HFD组小鼠显著下降，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041发挥了保护小鼠粘膜屏障的作用，抑制了高脂饲料饲喂引起的肠道细菌进入血液导致的小鼠血清中内毒素(LPS)、内毒素结合蛋白(LBP)含量异常增加。As shown in Figure 4B and 4C, the levels of endotoxin (LPS) and endotoxin binding protein (LBP) in the serum of HFD group mice were significantly higher than those of the CON group; the levels of endotoxin (LPS) in the serum of HFD+R group mice The content of endotoxin binding protein (LBP) was significantly lower than that of HFD group mice. It can be seen that Lactobacillus reuteri FN041 played a role in protecting the mucosal barrier of mice and inhibited the intestinal tract caused by high-fat feed. Bacteria entering the blood caused an abnormal increase in the levels of endotoxin (LPS) and endotoxin binding protein (LBP) in mouse serum.
如图4B、4C所示，HFD组小鼠血清中内毒素(LPS)、内毒素结合蛋白(LBP)的含量相对CON组小鼠显著上升；HFD+R组小鼠血清中内毒素(LPS)、内毒素结合蛋白(LBP)含量相对HFD组小鼠显著下降，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041发挥了保护小鼠粘膜屏障的作用，抑制了高脂饲料饲喂引起的肠道细菌进入血液导致的小鼠血清中内毒素(LPS)、内毒素结合蛋白(LBP)含量异常增加。As shown in Figure 4B and 4C, the levels of endotoxin (LPS) and endotoxin binding protein (LBP) in the serum of HFD group mice were significantly higher than those of the CON group; the levels of endotoxin (LPS) in the serum of HFD+R group mice The content of endotoxin binding protein (LBP) was significantly lower than that of HFD group mice. It can be seen that Lactobacillus reuteri FN041 played a role in protecting the mucosal barrier of mice and inhibited the intestinal tract caused by high-fat feed. Bacteria entering the blood caused an abnormal increase in the levels of endotoxin (LPS) and endotoxin binding protein (LBP) in mouse serum.
如图4D所示，HFD组小鼠血清中血清中肿瘤坏死因子α(TNF-α)的含量相对CON组 小鼠显著上升；HFD+R组小鼠血清中血清中肿瘤坏死因子α(TNF-α)含量相对HFD组小鼠显著下降，可见，罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可显著抑制高脂影响下的血清中肿瘤坏死因子α(TNF-α)含量异常增加。As shown in Figure 4D, the serum tumor necrosis factor alpha (TNF-α) content in the serum of the HFD group mice was significantly higher than that of the CON group mice; the serum tumor necrosis factor alpha (TNF-α) in the serum of the HFD+R group mice The content of α) was significantly lower than that of mice in the HFD group. It can be seen that treatment with Lactobacillus reuteri FN041 can significantly inhibit the abnormal increase in serum tumor necrosis factor α (TNF-α) content under the influence of high fat.
由表2-3可知，小鼠血清中甘油三酯的含量以及小鼠肝脏的生物钟基因Clock、Bmal1和Per2的表达存在明显的节律性，但是，在高脂影响下，小鼠血清中甘油三酯的含量以及小鼠肝脏的生物钟基因Clock、Bmal1和Per2的表达会丧失节律性，而罗伊氏乳杆菌(Lactobacillus reuteri)FN041处理可恢复此节律性。It can be seen from Table 2-3 that the triglyceride content in mouse serum and the expression of clock genes Clock, Bmal1 and Per2 in mouse liver have obvious rhythm. However, under the influence of high fat, triglyceride in mouse serum The content of esters and the expression of clock genes Clock, Bmal1 and Per2 in the liver of mice will lose rhythm, and treatment with Lactobacillus reuteri FN041 can restore this rhythm.
表2 罗伊氏乳杆菌FN041对高脂饲料喂养小鼠血脂昼夜节律变化的影响Table 2 The effect of Lactobacillus reuteri FN041 on the circadian rhythm of blood lipids in mice fed with high-fat diet
注：CON：对照组，HFD：高脂饲料饲喂对照组，HFD+R：罗伊氏乳杆菌FN041处理的高脂饲料饲喂组。Note: CON: control group, HFD: high-fat feed fed control group, HFD+R: high-fat feed fed group treated with Lactobacillus reuteri FN041.
表3 罗伊氏乳杆菌FN041对高脂日粮小鼠肝脏生物钟基因昼夜节律表达的影响Table 3 The effect of Lactobacillus reuteri FN041 on the circadian rhythm expression of liver biological clock genes in high-fat diet mice
| To | 拟合优度goodness of fit | 振幅(mmol/L)Amplitude (mmol/L) | 峰值相位(hr)Peak phase (hr) | 相变(hr)Phase change (hr) |
| ClockClock | To | To | To | To |
| CONCON | 0.0430.043 | 0.5590.559 | 99 | -- |
| HFDHFD | 0.0880.088 | 0.1550.155 | -- | -- |
| HFD+RHFD+R | 0.0420.042 | 0.2800.280 | 33 | -6-6 |
| Bmal1Bmal1 | To | To | To | To |
| CONCON | 0.0490.049 | 0.5690.569 | 99 | -- |
| HFDHFD | 0.0590.059 | 0.2900.290 | -- | -- |
| HFD+RHFD+R | 0.0460.046 | 0.3030.303 | 99 | 00 |
| Per2Per2 | To | To | To | To |
| CONCON | 0.0400.040 | 4.9154.915 | 21twenty one | -- |
| HFDHFD | 0.0760.076 | 3.8383.838 | -- | -- |
| HFD+RHFD+R | 0.0080.008 | 5.2155.215 | 21twenty one | 00 |
注：CON：对照组，HFD：高脂饲料饲喂对照组，HFD+R：罗伊氏乳杆菌FN041处理的高脂饲料饲喂组。Note: CON: control group, HFD: high-fat feed fed control group, HFD+R: high-fat feed fed group treated with Lactobacillus reuteri FN041.
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed as above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. The protection scope of the present invention should be defined by the claims.
Claims (19)
- 一种罗伊氏乳杆菌(Lactobacillus reuteri)，其特征在于，所述罗伊氏乳杆菌(Lactobacillus reuteri)已于2019年1月29日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60546，保藏地址为广州市先烈中路100号大院59号楼5楼。A Lactobacillus reuteri, characterized in that the Lactobacillus reuteri has been deposited in the Guangdong Provincial Microbial Culture Collection on January 29, 2019, and the deposit number is GDMCC No .60546, the preservation address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.
- 如权利要求1所述的一种罗伊氏乳杆菌(Lactobacillus reuteri)在制备预防和/或治疗脂代谢节律紊乱中的应用。The use of a Lactobacillus reuteri according to claim 1 in the preparation of prevention and/or treatment of lipid metabolism rhythm disorders.
- 如权利要求1所述的一种罗伊氏乳杆菌(Lactobacillus reuteri)在制备预防和/或治疗脂代谢节律紊乱的产品中的应用。The use of a Lactobacillus reuteri according to claim 1 in the preparation of a product for preventing and/or treating a lipid metabolism rhythm disorder.
- 如权利要求3所述的一种罗伊氏乳杆菌(Lactobacillus reuteri)在制备预防和/或治疗脂代谢节律紊乱的产品中的应用，其特征在于，所述产品中，罗伊氏乳杆菌(Lactobacillus reuteri)的活菌数为不低于1×10 6CFU/mL或1×10 6CFU/g。 The use of a Lactobacillus reuteri in the preparation of a product for preventing and/or treating a lipid metabolism rhythm disorder according to claim 3, characterized in that, in the product, Lactobacillus reuteri ( Lactobacillus reuteri) the number of viable bacteria is not less than 1×10 6 CFU/mL or 1×10 6 CFU/g.
- 如权利要求3或4所述的一种罗伊氏乳杆菌(Lactobacillus reuteri)在制备预防和/或治疗脂代谢节律紊乱的产品中的应用，其特征在于，所述产品包含食品、药品或保健品。The use of a Lactobacillus reuteri according to claim 3 or 4 in the preparation of a product for preventing and/or treating lipid metabolism rhythm disorders, characterized in that the product comprises food, medicine or health care Product.
- 如权利要求5所述的一种罗伊氏乳杆菌(Lactobacillus reuteri)在制备预防和/或治疗脂代谢节律紊乱的产品中的应用，其特征在于，所述药品的剂型包含颗粒剂、胶囊剂、片剂、丸剂或口服液。The use of a Lactobacillus reuteri in the preparation of a product for preventing and/or treating a lipid metabolism rhythm disorder according to claim 5, wherein the dosage form of the drug comprises granules and capsules , Tablets, pills or oral liquid.
- 如权利要求5所述的一种罗伊氏乳杆菌(Lactobacillus reuteri)在制备预防和/或治疗脂代谢节律紊乱的产品中的应用，其特征在于，所述药品含有罗伊氏乳杆菌(Lactobacillus reuteri)、药物载体和/或药用辅料。The use of a Lactobacillus reuteri in the preparation of a product for preventing and/or treating a lipid metabolism rhythm disorder according to claim 5, characterized in that the medicine contains Lactobacillus reuteri reuteri), drug carriers and/or pharmaceutical excipients.
- 一种用于预防和/或治疗脂代谢节律紊乱的产品，其特征在于，所述产品含有如权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)。A product for preventing and/or treating lipid metabolism rhythm disorders, characterized in that the product contains the Lactobacillus reuteri as claimed in claim 1.
- 如权利要求8所述的一种用于预防和/或治疗脂代谢节律紊乱的产品，其特征在于，所述产品中，罗伊氏乳杆菌(Lactobacillus reuteri)的活菌数为不低于1×10 6CFU/mL或1×10 6CFU/g。 A product for preventing and/or treating lipid metabolism rhythm disorders according to claim 8, wherein the number of viable bacteria of Lactobacillus reuteri is not less than 1 in the product. ×10 6 CFU/mL or 1×10 6 CFU/g.
- 如权利要求8或9所述的一种用于预防和/或治疗脂代谢节律紊乱的产品，其特征在于，所述产品包含食品、药品或保健品。A product for preventing and/or treating lipid metabolism rhythm disorders according to claim 8 or 9, characterized in that the product comprises food, medicine or health care products.
- 如权利要求10所述的一种用于预防和/或治疗脂代谢节律紊乱的产品，其特征在于，所述药品的剂型包含颗粒剂、胶囊剂、片剂、丸剂或口服液。The product for preventing and/or treating a lipid metabolism rhythm disorder according to claim 10, wherein the dosage form of the medicine comprises granules, capsules, tablets, pills or oral liquids.
- 如权利要求10所述的一种用于预防和/或治疗脂代谢节律紊乱的产品，其特征在于，所述药品含有罗伊氏乳杆菌(Lactobacillus reuteri)、药物载体和/或药用辅料。The product for preventing and/or treating lipid metabolism rhythm disorders according to claim 10, wherein the medicine contains Lactobacillus reuteri, a drug carrier and/or pharmaceutical excipients.
- 一种罗伊氏乳杆菌(Lactobacillus reuteri)冻存剂，其特征在于，所述冻存剂中，权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)的活菌数为不低于1×10 10CFU/mL。 A cryopreservation agent for Lactobacillus reuteri, wherein the number of viable bacteria of Lactobacillus reuteri according to claim 1 is not less than 1. ×10 10 CFU/mL.
- 如权利要求13所述的一种罗伊氏乳杆菌(Lactobacillus reuteri)冻存剂，其特征在于，所述冻存剂的制备方法为先将处于稳定期的权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)菌体用pH为7.0～7.4的磷酸盐缓冲液清洗1～2次，然后将清洗后的罗伊氏乳杆菌(Lactobacillus reuteri)菌体加入保护剂中，得到罗伊氏乳杆菌(Lactobacillus reuteri)冻存剂；所述保护剂含有1g/L的半胱氨酸盐酸盐以及200g/L的甘油。The cryopreservation agent of Lactobacillus reuteri according to claim 13, wherein the preparation method of the cryopreservation agent is to first combine the Reuteri according to claim 1 in the stable phase. Lactobacillus reuteri cells are washed 1 to 2 times with a phosphate buffer with a pH of 7.0 to 7.4, and then the washed Lactobacillus reuteri cells are added to the protective agent to obtain Reuteri Lactobacillus reuteri cryopreservation agent; the protective agent contains 1 g/L cysteine hydrochloride and 200 g/L glycerin.
- 一种制备预防和/或治疗脂代谢节律紊乱的产品的方法，其特征在于，所述方法为使用权利要求1所述的罗伊氏乳杆菌(Lactobacillus reuteri)。A method for preparing a product for preventing and/or treating a lipid metabolism rhythm disorder, wherein the method is to use the Lactobacillus reuteri according to claim 1.
- 如权利要求15所述的一种制备预防和/或治疗脂代谢节律紊乱的产品的方法，其特征在于，所述产品中，罗伊氏乳杆菌(Lactobacillus reuteri)的活菌数为不低于1×10 6CFU/mL或1×10 6CFU/g。 A method for preparing a product for preventing and/or treating a lipid metabolism rhythm disorder according to claim 15, wherein the number of viable bacteria of Lactobacillus reuteri in the product is not less than 1×10 6 CFU/mL or 1×10 6 CFU/g.
- 如权利要求15或16所述的一种制备预防和/或治疗脂代谢节律紊乱的产品的方法，其特征在于，所述产品包含食品、药品或保健品。The method for preparing a product for preventing and/or treating a lipid metabolism rhythm disorder according to claim 15 or 16, characterized in that the product comprises food, medicine or health care product.
- 如权利要求17所述的一种制备预防和/或治疗脂代谢节律紊乱的产品的方法，其特征在于，所述药品的剂型包含颗粒剂、胶囊剂、片剂、丸剂或口服液。The method for preparing a product for preventing and/or treating a lipid metabolism rhythm disorder according to claim 17, wherein the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.
- 如权利要求17所述的一种制备预防和/或治疗脂代谢节律紊乱的产品的方法，其特征在于，所述药品含有罗伊氏乳杆菌(Lactobacillus reuteri)、药物载体和/或药用辅料。The method for preparing a product for preventing and/or treating a lipid metabolism rhythm disorder according to claim 17, wherein the drug contains Lactobacillus reuteri, a drug carrier and/or pharmaceutical excipients .
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| CN111000246A (en) * | 2019-12-27 | 2020-04-14 | 汤臣倍健股份有限公司 | Probiotic dietary fiber composition for assisting in reducing triglyceride, application thereof and health-care product |
| CN111265553B (en) * | 2020-01-23 | 2021-06-22 | 湖南菲勒生物技术有限公司 | Application of lactobacillus reuteri from breast milk to adjustment of maternal and infant immune functions |
| CN113430153B (en) * | 2021-08-10 | 2022-04-01 | 浙江大学 | Lactobacillus reuteri ZJuuds 09 for reducing blood pressure and application thereof |
| CN114107088B (en) * | 2021-10-20 | 2023-07-21 | 山西大学 | Lactobacillus reuteri LRSY523 and application thereof |
| CN113841737A (en) * | 2021-10-22 | 2021-12-28 | 青岛大学 | Application of lactobacillus reuteri strain derived from breast milk to prevention and treatment of atopic dermatitis |
| CN114344344B (en) * | 2022-01-05 | 2023-06-13 | 东北农业大学 | Application of lactobacillus reuteri in relieving obesity function of high-fat diet-induced mice and compound containing lactobacillus reuteri |
| CN114990004B (en) * | 2022-04-19 | 2023-09-15 | 尚品健康科技(青岛)有限公司 | Secretory immunoglobulin A-packaged lactobacillus reuteri and application thereof in preventing and treating gestational diabetes |
| CN115478029B (en) * | 2022-09-22 | 2023-09-29 | 中国农业科学院北京畜牧兽医研究所 | Lactobacillus reuteri LRB5, microbial inoculum and application |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2662710A1 (en) * | 2006-09-07 | 2008-03-13 | Mcgill University | Oral polymeric membrane feruloyl esterase producing bacteria formulation |
| WO2009112455A1 (en) * | 2008-03-10 | 2009-09-17 | Nestec S.A. | Medium chain dicarboxylic acids, their derivates and metabolic disorders |
| WO2011060488A1 (en) * | 2009-11-18 | 2011-05-26 | Murray Goulburn Co-Operative Co. Limited | Recombinant microorganisms |
| CN106399154A (en) * | 2016-08-29 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Lactobacillus probiotics CGMCC NO. 12422 and application of lactobacillus probiotics in preparing lipid-lowering drug |
| CN106434427A (en) * | 2016-08-29 | 2017-02-22 | 中国疾病预防控制中心传染病预防控制所 | Lactic acid bacillus probiotic CGMCC NO.12421 and applicaiton to preparing lipid-lowering drugs |
| CN107523526A (en) * | 2017-10-17 | 2017-12-29 | 无限极（中国）有限公司 | A kind of lactobacillus reuteri and application thereof |
| CN108210525A (en) * | 2018-02-08 | 2018-06-29 | 中国疾病预防控制中心传染病预防控制所 | Applications of the Bacillus acidi lactici probiotics CGMCC NO.12422 in slimming medicine is prepared |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6942857B2 (en) * | 2002-08-09 | 2005-09-13 | Bioneer Corporation | Microorganisms for preventing and/or treating obesity or diabetes mellitus |
| CN104771416B (en) * | 2014-01-10 | 2020-11-10 | 景岳生物科技股份有限公司 | Lactobacillus reuteri GMNL-263 composition for controlling body weight and application thereof |
| CN104887716A (en) * | 2014-03-07 | 2015-09-09 | 景岳生物科技股份有限公司 | Lactobacillus reuteri GMNL-89 composition for treating type II diabetes mellitus and use thereof |
| CN105853467B (en) * | 2015-01-20 | 2019-07-26 | 景岳生物科技股份有限公司 | Lactobacillus reuteri GMNL-263 is used to prepare the purposes of blood-fat reducing composition |
| KR102125548B1 (en) * | 2015-02-10 | 2020-06-24 | 주식회사 지니스 | Microorganism having Anti-Obesity Ability and Pharmaceutical Composition Containing the same |
-
2019
- 2019-05-10 CN CN201910389419.3A patent/CN110205261B/en active Active
- 2019-07-31 WO PCT/CN2019/098529 patent/WO2020228144A1/en active Application Filing
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2662710A1 (en) * | 2006-09-07 | 2008-03-13 | Mcgill University | Oral polymeric membrane feruloyl esterase producing bacteria formulation |
| WO2009112455A1 (en) * | 2008-03-10 | 2009-09-17 | Nestec S.A. | Medium chain dicarboxylic acids, their derivates and metabolic disorders |
| WO2011060488A1 (en) * | 2009-11-18 | 2011-05-26 | Murray Goulburn Co-Operative Co. Limited | Recombinant microorganisms |
| CN106399154A (en) * | 2016-08-29 | 2017-02-15 | 中国疾病预防控制中心传染病预防控制所 | Lactobacillus probiotics CGMCC NO. 12422 and application of lactobacillus probiotics in preparing lipid-lowering drug |
| CN106434427A (en) * | 2016-08-29 | 2017-02-22 | 中国疾病预防控制中心传染病预防控制所 | Lactic acid bacillus probiotic CGMCC NO.12421 and applicaiton to preparing lipid-lowering drugs |
| CN107523526A (en) * | 2017-10-17 | 2017-12-29 | 无限极（中国）有限公司 | A kind of lactobacillus reuteri and application thereof |
| CN108210525A (en) * | 2018-02-08 | 2018-06-29 | 中国疾病预防控制中心传染病预防控制所 | Applications of the Bacillus acidi lactici probiotics CGMCC NO.12422 in slimming medicine is prepared |
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