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WO2020228144A1 - Application de lactobacillus reuteri dérivé du lait maternel dans la réduction des lipides et la régulation du rythme du métabolisme lipidique - Google Patents

Application de lactobacillus reuteri dérivé du lait maternel dans la réduction des lipides et la régulation du rythme du métabolisme lipidique Download PDF

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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
product
lipid metabolism
preventing
treating
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孙进
李双祁
齐策
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Fine Guangzhou Biotechnology Co Ltd
Fine Hunan Biotechnology Co Ltd
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Fine Guangzhou Biotechnology Co Ltd
Fine Hunan Biotechnology Co Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • 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

Application de Lactobacillus reuteri dérivé du lait maternel dans la réduction des lipides et la régulation du rythme du métabolisme lipidique. Le lactobacillus reuteri FN041 peut prévenir et/ou traiter des troubles du rythme du métabolisme lipidique et a les fonctions spécifiquement mises en œuvre dans: (1) la réduction significative du gain de poids sous l'influence d'un taux élevé de lipides; (2) la réduction significative de l'augmentation anormale des indices de tissu adipeux péritesticulaire sous l'influence d'un taux élevé de lipides; (3) la réduction significative de l'infiltration graisseuse hépatique et d'une augmentation anormale de la zone des cellules adipeuses testiculaires sous l'influence d'un taux élevé de lipides; (4) la réduction significative de l'augmentation anormale de la teneur en TG, TC, LDL-C et HDL-C dans le sérum sous l'influence d'un taux élevé de lipides; (5) la réduction significative d'une augmentation anormale de la teneur en FD4 du plasma dans le sang et de la teneur en LPS, LBP, et TNF-alpha dans le sérum sous l'influence d'un taux élevé de lipides; et (6) la restauration de la teneur en triglycéride dans le sérum et le rythme des expressions des gènes d'horloge biologique Clock, Bmal1 et Per2 dans un foie sous l'influence d'un taux élevé de lipides.
PCT/CN2019/098529 2019-05-10 2019-07-31 Application de lactobacillus reuteri dérivé du lait maternel dans la réduction des lipides et la régulation du rythme du métabolisme lipidique Ceased WO2020228144A1 (fr)

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