WO2020006663A1 - Nouveau lactobacillus paracasei gks6 pour atténuer le syndrome métabolique, milieu de culture associé, procédé de culture, utilisation, composition pharmaceutique et composition comestible - Google Patents

Nouveau lactobacillus paracasei gks6 pour atténuer le syndrome métabolique, milieu de culture associé, procédé de culture, utilisation, composition pharmaceutique et composition comestible Download PDF

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WO2020006663A1
WO2020006663A1 PCT/CN2018/094085 CN2018094085W WO2020006663A1 WO 2020006663 A1 WO2020006663 A1 WO 2020006663A1 CN 2018094085 W CN2018094085 W CN 2018094085W WO 2020006663 A1 WO2020006663 A1 WO 2020006663A1
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gks6
lactobacillus paracasei
strain
paracasei gks6
fat
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Chinese (zh)
Inventor
陈劲初
陈炎炼
许胜杰
林珊
林诗伟
李丽雅
吴思颖
陈彦博
王启宪
侯毓欣
石仰慈
林静雯
陈雅君
江佳琳
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Grape King Bio Ltd
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Priority to CN201880002040.6A priority patent/CN111164201B/zh
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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; 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

Definitions

  • the invention relates to a lactic acid bacterium, in particular to a novel Lactobacillus paracasei GKS6 for improving metabolic syndrome, a culture medium, a culture method, a use thereof, a pharmaceutical composition and an edible composition.
  • the World Health Organization has listed obesity as the most important public health and preventive medicine topic in the 21st century.
  • the WHO defines obesity as a body mass index (BMI, weight / height 2 (kg / m 2 )) greater than 30 as obese, and between 25 and 29.9 as overweight.
  • the cause of obesity is mainly that the long-term calorie intake is greater than the calorie consumption, which causes fat to accumulate in the body, and thus forms obesity.
  • the formation of obesity is divided into primary and spontaneous, in which primary obesity is caused by environmental factors such as diet and exercise; spontaneous obesity is caused by genetic or disease.
  • Obesity as defined by cell biology refers to the increase in the number and size of preadipocytes that differentiate into adipocytes in adipose tissue.
  • Adipose tissue can secrete a variety of substances, including hormones, growth factors, enzymes, cytokines, complements, and matrix proteins. Its main functions are to regulate metabolism, reproduction, immunity, blood pressure, and angiogenesis. Therefore, obesity can induce a variety of diseases throughout the body.
  • a high oil and fat diet will cause obesity, which in turn will cause liver cells to accumulate excess fat particles.
  • the fat accumulates more than 5% of the weight of the liver, it is called fatty liver, which causes symptoms such as hepatomegaly.
  • causes of hepatomegaly include viral hepatitis, cirrhosis, toxicity, and drug-induced hepatitis, stasis, and fatty liver, tumors, or leukemia caused by metabolic abnormalities.
  • ALT alanine aminotransferase
  • GPT pyruvate transaminase
  • AST aspartate aminotransferase
  • GOT glutamate oxaacetate transaminase
  • liver index examples include viral-induced hepatitis, drug-induced hepatitis, explosive hepatitis, liver suppuration, liver cancer, decreased blood pressure, acute myocardial infarction, hyperthyroidism, and fatty liver caused by obesity.
  • Fat in blood is mainly divided into two categories, namely cholesterol and triglycerides, and it is further subdivided into chylomicrons (CM), very low density lipoprotein (VLDL), and low density lipoprotein ( LDL) and high-density lipoprotein (HDL). If there is too little good or bad cholesterol in the blood, it is called hyperlipidemia.
  • Primary hyperlipidemia is a family inheritance, and secondary hyperlipidemia is caused by endocrine diseases (such as diabetes, hypothyroidism, or liver and kidney disease), lifestyle habits, and drugs. Hyperlipidemia accumulates cholesterol in the blood vessels and forms atherosclerotic arteriosclerosis, resulting in poor blood flow, which can cause symptoms such as myocardial infarction, stroke, and peripheral arterial blockage.
  • Ketone bodies include acetone, acetoacetic acid, and ⁇ -hydroxybutyric acid.
  • acetone bodies include acetone, acetoacetic acid, and ⁇ -hydroxybutyric acid.
  • pathological conditions such as diabetes
  • fat metabolism increases, and a large amount of fatty acids are absorbed and oxidized by liver cells.
  • gluconeogenesis in the body will also be activated , which in turn generates ketone bodies.
  • uric acid contained in food produces uric acid after ingestion and decomposition. Uric acid is excreted through the kidneys or intestines. When too much uric acid is produced in the body or renal dysfunction occurs, uric acid cannot be smoothly excreted from the body, and the uric acid content in the blood is too high, which is called hyperuricemia. Furthermore, the accumulation of uric acid in the blood in the joints or tissues will cause swelling and deformation, that is, gout. Genetic, improper hyperpurine diet, drugs, alcohol, etc. may lead to primary hyperuricemia, and ketone bodies produced by lipolysis will inhibit uric acid excretion. Therefore, obese people may have symptoms of excessive uric acid, which may lead to Related diseases.
  • the present invention screens and isolates a novel Lactobacillus paracasei GKS6 from the feces of healthy female infants, the GKS6 strain and its powder, pharmaceutical composition, and edible composition.
  • the form is resistant to acids and bile salts, which can effectively reduce body weight, reduce organ (heart, liver, kidney) hypertrophy, eliminate body fat, lower blood fat, reduce liver enlargement, lower liver function index (ALT and / or AST), Reducing the content of visceral adipose tissue and / or subcutaneous adipose tissue, reducing the content of liver lipids (triglycerides and / or cholesterol), can be used to prevent and / or treat diseases associated with metabolic syndrome, reduce obesity caused by high-fat diets, increase Exhaustion of body fat reduces obesity caused by high-fat diets.
  • the present invention discloses a novel Lactobacillus paracasei GKS6, which was deposited at the General Microbiological Center (CGMCC) of the China Microbial Species Collection and Management Committee on August 25, 2017, and the deposit number is CGMCC No. 14566, and in 2017 The survival test was passed on August 26,
  • the GKS6 strain includes a whole genome having a nucleotide sequence of SEQ ID NO: 3, a pheS gene having a nucleotide sequence of SEQ ID ID NO: 1 and a recN having a nucleotide sequence of SEQ ID NO: 2
  • the gene and the nucleotide sequence are SEQ ID NO: 4 plastids.
  • the GKS6 strain has polysaccharides composed of glucose and lactose in a ratio of about 3: 1.
  • the GKS6 strain can tolerate gastric acidity as low as pH 2.0, and can tolerate at least 0.3% of bile salts in the digestive tract.
  • the present invention also discloses an edible composition
  • an edible composition comprising the aforementioned novel GKS6 strain.
  • the GKS6 strain is presented as a bacterial powder.
  • the edible composition also includes a protective agent including, but not limited to, trehalose, milk powder, polydextrin, monosodium glutamate, pyrophosphate, vitamins, spermine, and combinations thereof.
  • trehalose trehalose
  • milk powder polydextrin
  • monosodium glutamate pyrophosphate
  • vitamins, spermine and combinations thereof.
  • the aforementioned ingredients and ratios can be modified and adjusted by those with ordinary knowledge in the technical field according to their familiar skills.
  • the invention also discloses a culture medium of a novel GKS6 strain.
  • the culture medium includes: a carbon source composed of glucose and lactose at a ratio of 3: 1, and a nitrogen source containing yeast extract.
  • the nitrogen source further includes beef extract.
  • the invention also discloses a method for cultivating a novel GKS6 strain, comprising: aseptically inoculating the GKS6 strain into a culture medium and culturing the culture medium at 37 ° C, wherein the culture medium includes a carbon source and a carbon source, and the carbon source is composed of glucose and lactose as 3; : 1 ratio, and the nitrogen source contains yeast extract.
  • the invention also discloses the use of a novel GKS6 strain to prepare a medicinal composition for treating or preventing obesity caused by metabolic syndrome.
  • the GKS strain can promote fat excretion in the body, effectively reduce obesity caused by high-fat foods, and further develop it into a medical composition or an edible composition for treating or preventing obesity.
  • FIG. 1 shows the evolutionary tree diagram of the pheS gene of Lactobacillus paracasei GKS6 and other lactic acid bacteria strains of the present invention.
  • FIG. 2 is a schematic diagram showing the evolutionary tree of the recN gene of Lactobacillus paracasei GKS6 and other lactic acid bacteria strains according to the present invention.
  • FIG. 3 shows the acid resistance of Lactobacillus paracasei GKS6 according to the present invention and other commercially available Lactobacillus paracasei.
  • FIG. 4 shows the bile-salt-resistant ability of Lactobacillus paracasei GKS6 according to the present invention and other commercially available Lactobacillus paracasei.
  • FIG. 5 shows a schematic diagram of the initial weight, final weight, and weight change of rats in the control group (ND), the high-fat diet group (HFD), and the high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • Figure 6 shows schematic diagrams of heart, liver, spleen, lung, and kidney weight of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6). .
  • Figure 7 shows the different parts of the rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6) (perirenal, epiphyseal, mesenteric, Extra-peritoneal and groin) Adipose tissue weight versus fat weight of rats.
  • Figure 8 shows different types of adipose tissue (total fat, visceral adipose tissue) of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6). And subcutaneous adipose tissue) relative to the body weight of the rat.
  • Figure 9 shows total liver lipids, triglycerides, and cholesterol of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6). Content diagram.
  • FIG. 10 is a schematic diagram showing the contents of serum AST and ALT in rats of the control group (ND), the high-fat diet group (HFD), and the high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • Figure 11 shows the serum triglyceride, cholesterol, and high-density lipids of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • ND control group
  • HFD high-fat diet group
  • HFD + GKS6 high-fat diet group
  • HFD + GKS6 Lactobacillus paracasei GKS6 group
  • FIG. 12 is a schematic diagram showing the contents of serum ketone bodies of rats in the control group (ND), the high-fat diet group (HFD), and the high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • FIG. 13 shows a schematic diagram of serum uric acid content of rats in the control group (ND), the high-fat diet group (HFD), and the high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • FIG. 14 shows a schematic diagram of lipid contents in dry feces of rats in the control group (ND), the high-fat diet group (HFD), and the high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • FIG. 15 is a schematic diagram showing changes in blood glucose levels of an oral glucose tolerance test in C57BL / 6JNarl mice after tube feeding for 14 days.
  • FIG. 16 shows the change in the area under the 120-minute blood glucose curve (AUC 120min ) of the oral glucose tolerance test in C57BL / 6JNarl mice after tube feeding for 14 days.
  • FIG. 17 is a graph showing changes in the blood glucose rise value of the oral glucose tolerance test in C57BL / 6JNarl mice after tube feeding for 14 days.
  • FIG. 18 shows the change in area under the 120-minute blood glucose rise curve (iAUC 120 min ) of the oral glucose tolerance test in C57BL / 6JNarl mice after tube feeding for 14 days.
  • GKS6 novel Lactobacillus paracasei GKS6
  • GKS6 novel Lactobacillus paracasei GKS6
  • Maternal age is less than 35 years old, this is the most suitable age to nurse a child.
  • the mother is not treated with medication due to illness during pregnancy, and the mother has good immunity and health.
  • the mother has a good physique without allergic symptoms.
  • Maternal check-ups are normal, no fertility medications are used, and the fetus is healthy and healthy during pregnancy.
  • the newborns were selected within one week of birth to facilitate the screening of the original strains that grew in the intestine.
  • the novel Lactobacillus paracasei GKS6 of the present invention was screened from about 300 strains of lactic acid bacteria by using an acid resistance test and an interferon- ⁇ (IFN- ⁇ ) inducing ability of a peripheral blood mononuclear cell (PBMC) screening platform as a selection index. It has the best high acid resistance of 90% and the ability to induce IFN- ⁇ (91pg / mL).
  • IFN- ⁇ interferon- ⁇
  • the IFN- ⁇ induction ability is 36.5 times better than the known Lactobacillus rhamnosus rhamnosus GG (referred to as LGG bacteria).
  • the induction capacity of pg / mL IFN- ⁇ was about 2.5 times higher.
  • GKS6 is isolated and screened from the feces of a newborn baby girl. It can grow well in MRS (De Man, Rogosa and Sharpe) selective (agar) medium at 37 ° C. The GKS6 strain was streaked in MRS medium with aseptic technique and activated, and then a single GKS6 colony was inoculated into MRS medium with aseptic technique for expansion, and fresh GKS6 cells were collected every other day. Genomic DNA (gDNA) of the strain was extracted by hexadecyl trimethyl-ammonium bromide (CTAB) method, and gDNA quality was confirmed by 0.6% agar colloid electrophoresis.
  • CAB hexadecyl trimethyl-ammonium bromide
  • the whole-genome analysis of the GKS6 strain was performed using the third-generation sequencing technology platform PacBio RSII (Pacific Biosciences of California, Inc., CA, USA), where the entire genome length of the GKS6 strain was 3,076,381 bp (SEQ ID ID: NO: 3), and contains a 81,241 bp plastid (SEQ ID NO: 4).
  • the coding of the GKS6 whole genome sequence was sent to the whole genome database for comparison, and the pheS gene sequence (SEQ ID NO: 1) and recN gene sequence (SEQ ID NO: 2) of the GKS6 strain were obtained. Then the pheS gene sequence was sent to the National Center for Biotechnology Information (NCBI) database for comparison.
  • NCBI National Center for Biotechnology Information
  • the GKS6 strain was randomly selected and purchased from 5 strains of Lactobacillus casei BCRC 16094, BCRC 17005, BCRC 17475, BCRC 17482 and BCRC 80881 for comparison.
  • the gDNA of the GKS6 strain and 5 other commercially available Lactobacillus paracasei strains were extracted, and the primer pair pheS-F (5'-TTAACCCTCCTGGCTGAATTG-3 ', SEQ ID NO: 5) and pheS-R (5' -ATGGATCTTCAAACCAARC TTGA-3 ', SEQ ID NO: 6) and recN-F (5'-TTAACTCATG CGTCCATGTT T-3', SEQ ID NO: 7) and recN-R (5'-ATGTTACAAG AGGTTAGCGAT TCATG-3 ', SEQ ID NO: 8), polymerase chain reaction was performed at 94 ° C for 3 minutes, then at 94 ° C for 30 seconds, 52 ° C for 30 seconds, 72 ° C for 1 minute and 30 seconds (35 cycles), and 72 ° C for 5 minutes. The products of the reaction were sequenced into the pheS and recN gene sequences of the GKS6 strain and these 5 commercially available Lac
  • Lactobacillus casei 12A (Lactobacillus casei 12A) (gene bank number: CP006690) and the entire genome of L. paracasei subsp. JCM 8130 (gene bank number) were downloaded from the NCBI database. : AP012541), Lactobacillus paracasei KL1 (L.paracasei KL1) whole genome (GenBank No .: CP013921), Lactobacillus paracasei ATCC 334 (L.paracasei ATCC 334) Whole Genome (Genbank No .: CP000423), and casein milk Sequence analysis of the pheS and recN genes in the entire genome (Genbank No. AP012544) of L. casei ATCC 393 Neighbor-Joining mode of software "Bootstrap Test of Phylogeny" draws evolution tree.
  • FIG. 1 and FIG. 2 are schematic diagrams of pheS genes and pheS genes of Lactobacillus paracasei GKS6 and other lactic acid bacteria strains of the present invention, respectively.
  • the GKS6 strain is independent of other lactic acid bacteria strains, showing that the GKS6 strain is a novel Lactobacillus paracasei strain.
  • the Lactobacillus paracasei GKS6 of the present invention was deposited on August 25, 2017 at the General Microbiology Center of the China Microbial Strain Collection Management Committee (CGMCC, No. 3, Beichen West Road, Chaoyang District, Beijing), and its deposit The serial number is CGMCC No. 14566, and it passed the survival test on August 26, 2017.
  • the novel GKS6 strain of the present invention is a probiotic strain with special mucopolysaccharides with high activity performance.
  • the polysaccharides in the GKS6 strain were composed of glucose and galactose in a ratio of about 3: 1. Therefore, in the fermentation culture of the GKS6 strain, a fermentation test was performed with 3% glucose and 1% lactose. In addition, the combination of 4% glucose and 4% lactose and the addition ratio of glucose and lactose (3: 1) were tested and compared. There was no significant difference. Therefore, glucose and lactose were used as the carbon source for fermentation at a ratio of 3: 1.
  • the nitrogen source for cultivating the novel GKS6 strain of the present invention is selected from the group consisting of non-modified yeast extract, soybean isolated hydrolyzed protein, beef extract, or a combination thereof. After orthogonal screening and cross-screening comparison test, the optimal conditions are that the ratio of yeast extract and beef extract is 1%: 1%, and the number of GKS6 bacteria can reach 4.3 ⁇ 10 9 cfu / mL. When using 2% yeast extract as a nitrogen source, the number of GKS6 bacteria can reach 3.7 ⁇ 10 9 cfu / mL; while when soybean protein is used to isolate and hydrolyze protein as a nitrogen source, the number of GKS6 bacteria is only 6.4 ⁇ 10 8 cfu / mL. Therefore, soybean proteins are not suitable for the cultivation of GKS6 strains. When considering that the GKS6 strain product must meet the requirements of vegans, the yeast extract is used as a nitrogen source for high-density fermentation of the GKS6 strain.
  • the GKS6 strain was aseptically inoculated into a medium containing a complex carbohydrate carbon source (3% glucose and 1% lactose) and 2% yeast extract and subjected to high-density fermentation at 37 ° C, the viable number of GKS6 reached 1.3 ⁇ 10 10 cfu / mL, achieving a 400% improvement. Therefore, in the subsequent experiments of the present invention, unless otherwise specified, the novel GKS6 strain was cultured under this culture condition.
  • a complex carbohydrate carbon source 3% glucose and 1% lactose
  • yeast extract 2% yeast extract
  • a protective agent may be added during the freeze-drying process of the GKS strain, which includes but is not limited to trehalose, milk powder, polydextrin, monosodium glutamate, pyrophosphate, vitamins, arginine, and combinations thereof. Therefore, the GKS6 strain can be made into an edible composition in the form of bacterial powder.
  • the GKS6 strain can be combined with a pharmaceutically acceptable carrier or excipient to prepare a pharmaceutical composition.
  • the pharmaceutical composition in an effective number of GKS6 strains is preferably suitable for oral administration to a desired individual for the treatment, prevention or improvement of its metabolic syndrome.
  • Example 3 acid resistance test of lactic acid bacteria
  • the novel GKS6 strain of the present invention is more acid-resistant than the commercially available Lactobacillus paracasei BCRC 16094, 17005, 17475, 17482, and 80881.
  • the pH of the MRS broth was adjusted to pH 3.2, 2.4, or 2.0 with hydrochloric acid after the strain was activated, and the number of bacteria was counted after 3 hours of incubation at 37 ° C.
  • FIG. 3 is a schematic diagram showing the acid resistance of Lactobacillus paracasei GKS6 and other commercially available Lactobacillus paracasei in the present invention.
  • the GKS6 strain and the BCRC 16094, 17005, 17475, 17482, and 80881 strains can grow to about 10 10 under the original pH of the MRS medium (about pH 6.5). After adjusting the pH value of the MRS broth to pH 2.4 and 2.0, it was found that the unit count of BCRC 16094, 17005, 17475, 17482, and 80881 were significantly lower than the unit count of GKS6 strain (P ⁇ 0.05). Therefore, the GKS6 strain of the present invention has better acid resistance than other known Lactobacillus paracasei, and better ability to pass gastric acid.
  • Example 4 Bile salt tolerance test of lactic acid bacteria
  • the novel GKS6 strain of the present invention is compared with the commercially available Lactobacillus paracasei BCRC 16094, 17005, 17475, 17482, and 80881 against bile salt tolerance.
  • the strain was activated, the strain was inoculated into MRS culture solution containing 0.3% bile salt at 37 ° C, and the number of bacteria was counted after soaking for 0.5 hour.
  • FIG. 4 shows the bile-salt-resistant ability of Lactobacillus paracasei GKS6 according to the present invention and other commercially available Lactobacillus paracasei.
  • the unit number of GKS6 strain and BCRC 16094, 17005, 17475, 17482, and 80881 are close to 9 ⁇ 10 9.
  • the BCRC16094, BCRC 17005, BCRC 17475, BCRC 17482, and BCRC 80881 were significantly lower than the GKS6 bacteria (P ⁇ 0.05). It can be seen that the GKS6 strain is more resistant to bile salts than other Lactobacillus paracasei. The ability of the bile salts of the digestive tract is better.
  • the experimental animals of this example are 18 male Wistar rats 6 weeks old and weighing 201-225 grams.
  • the rats were kept in stainless steel squirrel cages, while the temperature of the animal room was controlled at 22 ⁇ 2 °C, the humidity was controlled at 60-80%, and the light and darkness were each 12 hours (07: 00-19: 00 is the light period; 19: 00-07 : 00 is the dark period).
  • rats were free to take in feed and distilled water.
  • the culture solution in the culture medium was centrifuged to obtain GKS6 cells, and the GKS6 cells were freeze-dried to form GKS6 bacteria powder.
  • protective agents including but not limited to trehalose, milk powder, polydextrin, monosodium glutamate, pyrophosphate, vitamins, arginine, and combinations thereof
  • GKS6 strain used in subsequent experiments was GKS6 bacterial powder.
  • Wistar rats were fed with normal adult rat feed and drinking distilled water. After adapting to the environment for 1 week, they were randomly divided into three groups. The test samples were fed orally and grouped as follows:
  • the feed formula of the high-fat diet group was based on AIN93G, supplemented by adjusting the fat ratio.
  • the high-fat diet group contained 68% feed solids, 7% soybean oil, and 25% lard.
  • 102.8 mg / kg rat / day GKS6 strain was orally fed for 6 weeks.
  • the initial weight of the rats was recorded, and then every 2 days, the weight was precisely measured and the changes were observed.
  • the test sample was given for 6 weeks. Rat feces were collected 3 days before sacrifice, and fasted 12 hours before the end of the test. The rats were sacrificed with carbon dioxide, and the final body weight and weight change of the rats were recorded. In addition, blood was collected from the veins of rats for subsequent serum biochemical analysis.
  • organ tissues herein, liver, spleen, lungs and kidneys
  • adipose tissues peripheral adipose tissue issues
  • epididymal adipose tissue issues mesenteric adipose tissue issues
  • Extraperitoneal adipose tissue and inguinal adipose tissue was washed and wiped with normal saline, and the weight of each organ and adipose tissue was accurately weighed and recorded.
  • the total weight of various adipose tissues was total fat Weight, the total fat weight divided by the final weight is the body fat percentage.
  • the weighed organs and adipose tissue are covered with aluminum foil paper, frozen with liquid nitrogen, and stored at -80 ° C for experimental analysis.
  • FIG. 5 shows the initial weight, final weight, and weight of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • Change amount diagram The amount of weight change is calculated by the following formula (1):
  • Amount of weight change (g) final weight (g)-initial weight (g). Equation (1)
  • FIG. 6 shows the heart, liver, spleen, and lungs of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • ND control group
  • HFD high-fat diet group
  • HFD + GKS6 high-fat diet group
  • FIG. 6 shows the weight of the spleen and lungs of the rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • the weight of the spleen and lungs of the rats did not change significantly between the groups; the weight of the heart, liver, and kidneys in the high-fat diet group was higher than that in the normal diet group, while the heart, liver, and Kidney weight decreased significantly compared with the high-fat diet group (p> 0.05). Therefore, taking the novel GKS6 strain of the present invention can effectively reduce the weight of the heart,
  • FIG. 7 shows the control group (ND), the high-fat diet group (HFD), and the high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6) to different parts (perirenal, ⁇ , mesenteric, extraperitoneal, and groin) Adipose tissue weight relative to the fat mass of the rat.
  • adipose tissue is divided into visceral fat and subcutaneous fat, of which peri-renal fat, para-fat fat, and mesenteric fat are visceral fat, groin fat and extraperitoneal fat are subcutaneous fat, and the total weight of various adipose tissue is the total Fat mass.
  • the amount of visceral fat and subcutaneous fat is calculated by formula (2) and formula (3) respectively, and the total fat amount is calculated by formula (4).
  • Visceral fat mass (mg / g rat) [peri-renal fat mass (mg) + para-fat fat mass (mg) + mesenteric fat mass (mg)] ⁇ final weight (g): (2)
  • Subcutaneous fat mass (mg / g rat) [inguinal fat (mg) + extraperitoneal fat mass (mg)] ⁇ final body weight (g)
  • Total fat mass (mg / g rat) [peri-renal fat mass (mg) + para-fat fat mass (mg) + mesenteric fat mass (mg) + inguinal fat (mg) + extraperitoneal fat mass (mg)] ⁇ Final body weight (g): (4)
  • FIG. 8 shows different types of adipose tissue (total lipids) of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • ND control group
  • HFD high-fat diet group
  • HFD + GKS6 high-fat diet group
  • Visceral adipose tissue and subcutaneous adipose tissue relative to the body weight of a rat.
  • the weight of visceral adipose tissue in rats induced by a high-fat diet was significantly higher than that of the normal diet group (p ⁇ 0.05); the group with a high-fat diet combined with GKS6 compared with the group with a high-fat diet alone.
  • the weights of visceral fat and subcutaneous adipose tissue were significantly lower than those in the high-fat diet group (p ⁇ 0.05); indicating that the novel GKS6 strain of the present invention can effectively reduce fat accumulation caused by a high-fat diet, regardless of visceral fat or subcutaneous fat. .
  • chloroform: methanol 2: 1 (v / v)
  • chloroform: methanol 2: 1 (v / v)
  • FIG. 9 shows total liver lipids, triacids of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • ND control group
  • HFD high-fat diet group
  • HFD + GKS6 Lactobacillus paracasei GKS6 group
  • FIG. 10 shows the contents of serum AST and ALT in rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • the serum ALT and AST of rats in the high-fat diet group were significantly higher than those in the normal diet group (p ⁇ 0.05), indicating a higher degree of liver inflammation; however, rats induced by the high-fat diet combined with GKS6 Compared with the high-fat diet group, the serum ALT and AST concentrations can be significantly reduced (p ⁇ 0.05), indicating that taking the novel GKS6 strain of the present invention can effectively reduce the serum ALT and AST concentrations to improve liver function.
  • the high-fat diet-induced rats combined with GKS6 significantly increased the high-density lipoprotein cholesterol content, indicating that taking the novel GKS6 strain of the present invention can effectively increase the concentration of high-density lipoprotein cholesterol in serum .
  • This test uses commercially available analysis kits (DiaSys, Diagonostic Systems, GmbH, Holzheim, Germany) to measure uric acid in rat serum.
  • the experimental results are shown in Figure 13.
  • the results showed that the serum uric acid content of the rats in the high-fat diet group was significantly higher than that in the normal diet group; the group with high-fat diet combined with GKS6 significantly reduced the serum uric acid concentration compared with the high-fat diet group (p ⁇ 0.05)
  • the level to the normal diet group indicates that the administration of the novel GKS6 strain of the present invention can effectively reduce the uric acid concentration in the serum.
  • FIG. 14 shows the lipid content in dry feces of rats in the control group (ND), high-fat diet group (HFD), and high-fat diet group given Lactobacillus paracasei GKS6 group (HFD + GKS6).
  • ND control group
  • HFD high-fat diet group
  • HFD + GKS6 high-fat diet group given Lactobacillus paracasei GKS6 group
  • the purpose of this example is to test the ability of the GKS6 strain to improve glucose sensitivity.
  • the experimental animals were 20 male C57BL / 6JNarl mice. After a one-week observation period, mice were randomized into groups of 5 animals per cage.
  • the litter for breeding mice was sterilized wood dust litter (ASPEN shavings, Northeastern Products Corp., Warrensburg, NY 12885, U.S.A.), which was changed twice a week.
  • the mice were reared according to the general experimental animal breeding management method.
  • the temperature of the animal room was controlled at 23 ⁇ 2 ° C, the relative humidity was controlled at 50 ⁇ 10%, and the light and darkness were each 12 hours. During the experiment, the mice had free access to feed and distilled water.
  • the GKS6 strain of the experimental group was fed with a dose of 500 mg / kg of mouse weight, and the feeding volume was 0.1 mL / 10 g body weight / day for 14 days.
  • the control group was given normal feed and distilled water, but not GKS6 strain.
  • oral glucose tolerance test oral glucose test, OGTT
  • mice were fasted for 16 hours, blood samples were collected from the tail end, and fasting was measured by a Glucosure II blood glucose meter (ApexBio Inc., Taiwan) with a blood glucose test strip (glucose oxidase method) (0 minutes) )Blood sugar level.
  • Glucose solution (2g / kg mouse weight) was administered to the tube, and blood glucose was measured at 30, 60 and 120 minutes.
  • mice were sacrificed after OGTT, and liver, kidney and spleen were removed and weighed.
  • the 30-minute blood glucose value of the GKS6 experimental group was significantly lower than that of the control group (Table 4, Figure 15).
  • the area under curve (AUC 120min ) of the GKS6 experimental group at 120 minutes was also slightly lower than that of the control group (p ⁇ 0.05) (Table 4, Figure 16).
  • the 30-minute blood glucose rise value (i30 minutes) and the area under the 120-minute blood glucose rise curve ( iAUC 120mn ) in the GKS6 experimental group were significantly lower than those in the control group (Table 5, Figure 17, Figure 18), and the GKS6 experimental group had 120
  • the minute blood glucose rise value (i120 minutes) was also significantly lower than that of the control group (Table 5, Fig. 17).
  • the GKS6 strain has the potential to improve glucose sensitivity at a dose of 500 mg / kg mouse weight of the GKS6 strain.
  • a strain of Lactobacillus paracasei GKS6 was isolated from the feces of healthy baby girls.
  • the strain GKS6 has acid and bile salt resistance and can reach the intestine through the digestive tract after consumption, thereby assisting the digestion and decomposition of food. , To increase the amount of lipid excretion, and effectively reduce body fat and blood lipids.
  • the above also illustrates the relationship between the intestinal bacterial phase and digestion and absorption, and not all probiotics can successfully reach the intestine after taking it. It must be resistant to gastric acid, bile salts, and have excellent intestinal adsorption capacity. Can reach the intestine alive, otherwise it will become dead bacteria after entering the intestine, then the original advertised effect cannot be fully exerted.
  • the present invention is an incompetent innovation, which is of great industrial value.
  • the present invention can be modified in any way by those skilled in the art without departing from the scope of protection as claimed in the appended claims.

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Abstract

L'invention concerne un nouveau Lactobacillus paracasei GKS6 pour atténuer le syndrome métabolique, ayant un numéro d'accès CGMCC No. 14566. L'invention concerne également un milieu de culture d'une souche GKS6, un procédé de culture, une utilisation, une composition pharmaceutique et une composition comestible. Une souche GKS6 est résistante aux acides et résistante aux sels biliaires, peut réduire efficacement le poids, réduire l'hypertrophie d'organe, diminuer le lipides dans le foie, diminuer les lipides dans le sang, réduire l'hypertrophie du foie, réduire l'indice de fonction hépatique, diminuer la teneur en tissu adipeux viscéral et/ou en tissu adipeux sous-cutané, et éliminer la graisse corporelle, et peut être utilisée pour prévenir et/ou traiter des maladies liées au syndrome métabolique, réduire l'obésité provoquée par un régime riche en graisses, et augmenter l'évacuation des graisses in vivo pour réduire l'obésité provoquée par un régime riche en graisses.
PCT/CN2018/094085 2018-07-02 2018-07-02 Nouveau lactobacillus paracasei gks6 pour atténuer le syndrome métabolique, milieu de culture associé, procédé de culture, utilisation, composition pharmaceutique et composition comestible Ceased WO2020006663A1 (fr)

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