CN117264829A - Lactobacillus plantarum for preventing and treating hypercholesterolemia, fermented product and application thereof - Google Patents

Lactobacillus plantarum for preventing and treating hypercholesterolemia, fermented product and application thereof Download PDF

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CN117264829A
CN117264829A CN202311255496.2A CN202311255496A CN117264829A CN 117264829 A CN117264829 A CN 117264829A CN 202311255496 A CN202311255496 A CN 202311255496A CN 117264829 A CN117264829 A CN 117264829A
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lactobacillus plantarum
hypercholesterolemia
fermented product
strain
weight
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蒲小平
邓燕
谢建将
景晓青
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SICHUAN GAOFUJI BIOLOGICAL TECHNOLOGY CO LTD
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SICHUAN GAOFUJI BIOLOGICAL TECHNOLOGY CO LTD
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    • 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
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/38—Other non-alcoholic beverages
    • A23L2/382—Other non-alcoholic beverages fermented
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • 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
    • 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
    • C12R2001/25—Lactobacillus plantarum
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A lactobacillus plantarum for preventing and treating hypercholesterolemia, a fermented product and application thereof are provided, wherein the lactobacillus plantarum is lactobacillus plantarum 543 with a preservation number of CCTCC NO: M2021353, and a 16S rRNA gene sequence is shown as SEQ ID NO: 1. The invention also discloses the application of the lactobacillus plantarum 543 and/or the fermented product containing the lactobacillus plantarum 543 in preparing medicaments for reducing weight, reducing blood sugar, preventing and treating hypercholesterolemia and/or resisting inflammation. The lactobacillus plantarum 543 has high bioactivity and good acid production property, can resist artificial gastric acid and bile salt, can efficiently reduce cholesterol level in vivo and in vitro, can obviously reduce insulin resistance index, total cholesterol, total triglyceride and low-density lipoprotein cholesterol of a high-fat diet mouse, can improve the high-density lipoprotein cholesterol level, promotes synthesis of tryptophan metabolites, and has wide application prospects in aspects of weight reduction, blood glucose reduction, anti-inflammatory and hypercholesterolemia management.

Description

Lactobacillus plantarum for preventing and treating hypercholesterolemia, fermented product and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum for preventing and treating hypercholesterolemia and application thereof in preparing medicaments for reducing weight, reducing blood glucose, preventing and treating hypercholesterolemia and/or resisting inflammation.
Background
The blood lipid metabolism disorder is closely related to the occurrence of atherosclerosis-related cardiovascular diseases, and is mainly manifested by hypercholesterolemia, hypertriglyceridemia, high density lipoprotein reduction or low density lipoprotein cholesterol elevation. The control of serum cholesterol levels is effective in preventing the occurrence of cardiovascular and cerebrovascular diseases. At present, the occurrence rate of hypercholesterolemia and high-low density lipoprotein hyperlipidemia of people over 35 years old in China is 7.5% and 6% respectively. Meanwhile, along with social and economic development, life style and diet structure of people are changed, and obesity becomes global epidemic. The publication of month 8 in 2023, journal Diabetes Obesity and Metabolism, entitled "prevalence of obesity in China" and related complications: cross-section real world study of 1580 ten thousand adults reports that according to BMI (body Mass index) classification standards of overweight and obesity in China, the overweight population accounts for 34.8 percent, the obese population accounts for 14.1 percent and the most common obesity-related complications in the whole population account for 24.9 percent, so that the obesity and the control situation of hypercholesterolemia in China are still very severe.
Currently, the weight loss methods include intermittent fasting, exercise, weight loss surgery and drug treatment, and many people cannot persist for a long time in the fasting and exercise methods, and the weight loss drugs have great side effects, such as semeglutide drugs including nausea, vomiting, diarrhea, constipation and abdominal pain, and may also induce serious side effects such as pancreatitis, kidney injury and diabetic retinopathy; the medicine for treating hypercholesterolemia mainly comprises statin medicines, can effectively reduce cholesterol content and reduce occurrence risk of cerebrovascular diseases and coronary heart diseases, but common side effects of the statin medicines comprise gastrointestinal reaction, liver function damage and rhabdomyolysis, so that the medicine for treating hypercholesterolemia has small adverse reaction, and can regulate weight and prevent and treat hypercholesterolemia, and is very necessary.
In recent years, more and more researches show that obesity and hypercholesteremia are closely related to intestinal microorganisms, and intestinal flora changes are often accompanied by inflammatory immune response, metabolic changes and the like, and intestinal flora may participate in the occurrence and development of obesity and hypercholesteremia through the changes. However, the prior published literature and techniques mainly intervene on high fat diet induced obesity for probiotics alone or prebiotics alone, as disclosed in CN113005060B bifidobacterium adolescentis CCFM1173, which strain by increasing leptin levels in serum; lowering the expression level of lipid synthesis genes in the liver; the expression of thermogenic genes and genes related to lipid decomposition in brown fat is obviously improved, non-flutter thermogenesis in brown fat is activated, and lipid consumption is increased; reduce the weight of visceral white fat and spleen to achieve the aim of weight reduction. CN 112662791B discloses enterococcus faecium 132 and lactobacillus paracasei 201, both strains can reduce cholesterol concentration in vitro cholesterol liquid medium, and have good blood lipid reducing effect in animal body.
Specific tryptophan metabolites can enter intestinal tracts and systemic circulation and activate an Aromatic Hydrocarbon Receptor (AHR) signal channel, and at present, no report on the weight reduction and prevention and treatment of hypercholesterolemia of strains and metaplasias of high-yield tryptophan metabolites is seen, so that new strains and compositions thereof for targeting weight reduction and cholesterol regulation are developed, and the novel strains and compositions are used for preparing safe and efficient microecological preparations, functional foods or medicines for relieving and treating obesity and cholesterol, and have important significance and market value.
Disclosure of Invention
The invention aims to solve the technical problems that: overcomes the defects of the prior art, provides a plant source and a lactobacillus plantarum 543 which can be separated, purified and cultivated, further provides foods/medicines rich in lactobacillus plantarum 543 strain and fermented products thereof, and exploits new applications of lactobacillus plantarum 543 and the fermented products containing the same in weight reduction, prevention and treatment of hypercholesterolemia and the like.
One of the technical schemes adopted for solving the technical problems is as follows:
a Lactobacillus plantarum for preventing and treating hypercholesterolemia is named Lactobacillus plantarum (Lactobacillus plantarum) 543, and is preserved in China center for type culture collection (CCTCC NO: M2021353) of China 4-month 12-year 2021.
Biological preservation description: lactobacillus plantarum (Lactobacillus plantarum) 543, deposited at chinese collection of typical cultures, deposit address: eight-path 299 of Wuchang district in Wuhan, hubei province, the preservation organization is abbreviated as: cctccc, date of preservation: 2021, 4, 12 (2021, 4, 12 received registration book, 4, 19 detected as viable and preserved), biological preservation number cctccc NO: m2021353, strain number: lactobacillus plantarum 543.
Wherein the lactobacillus plantarum 543 is isolated from a plant source, in particular to lactobacillus plantarum 543 which is obtained by screening and separating from allium chinense of Sichuan Chengdu farmer.
The biological properties of the lactobacillus plantarum 543 of the present invention are as follows:
1) Morphological features: the colony on the MRS agar culture medium is round, medium in size, milk white, convex upwards, tidy in edge, moist and easy to pick; positive after gram staining.
2) Biological identification: the 16S rRNA gene sequence of the lactobacillus plantarum 543 is shown as SEQ ID NO. 1, the 16S rRNA sequence of the lactobacillus plantarum 543 is subjected to NCBI BLAST comparison, the similarity with lactobacillus plantarum (Lactobacillus plantarum) in Genebank is more than 99%, and based on the 16S rRNA gene sequence comparison result, a Neighbor-Joining phylogenetic tree constructed by taking Lactobacillus floricola Ryu-2 (AB 523780.1) as an outer branch is identified as the lactobacillus plantarum (Lactobacillus plantarum).
The tolerance test of the lactobacillus plantarum 543 to gastric acid and bile salt shows that the survival rate of the lactobacillus plantarum 543 reaches 65.32% when the gastric juice is pH 2.5; when the concentration of bile salt reaches 3.0g/L, the survival rate of the strain reaches 97.43 percent. Lactobacillus plantarum 543 has very good gastric acid and bile salt tolerance, and is suitable for oral administration.
The other technical scheme adopted by the invention for solving the technical problems is as follows:
a fermented product containing Lactobacillus plantarum for hypercholesterolemia is prepared by using Lactobacillus plantarum 543 and/or fermentation.
The fermented product is one or more of the live strain of Lactobacillus plantarum 543, inactivated strain of Lactobacillus plantarum 543, strain metabolite or Lactobacillus plantarum 543 metazoan.
The invention provides a post-biotics of lactobacillus plantarum 543, which refers to lactobacillus thallus and metabolic components after processing/inactivating, concentrating and drying a lactobacillus plantarum 543 fermentation liquid, wherein the lactobacillus thallus and metabolic components are generally called as the lactobacillus thallus components and metabolic products.
Use of lactobacillus plantarum in the preparation of fermented food.
The fermented food is any one of ferment, pickle, solid beverage, pill, tablet or microcapsule crystal ball.
The application of lactobacillus plantarum and/or lactobacillus plantarum-containing fermented products in preparing medicaments for reducing weight, reducing blood sugar, preventing and treating hypercholesterolemia and/or resisting inflammation.
The medicament also contains a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carriers include, but are not limited to: one or more of a filler, binder, wetting agent, disintegrant, or lubricant.
The filler is one or more of fucoidin, trehalose, lactose, chitosan, starch or dextrin; the adhesive is one or more of liquid glucose, starch paste or syrup; the wetting agent is one or more of glycerol or ethanol; the disintegrating agent is one or more of crospovidone, sodium carboxymethyl starch or sodium cross-linked carboxymethyl starch; the lubricant is one or more of silicon dioxide magnesium stearate or sodium stearate fumarate.
The lactobacillus plantarum 543 of the invention can efficiently produce indole-3-formaldehyde, indole-3-acetic acid and indole-3-lactic acid by degrading cholesterol levels in intestinal tracts, and tryptophan metabolites produced by the lactobacillus plantarum 543 enter the intestinal tracts and the systemic circulation and activate an Aromatic Hydrocarbon Receptor (AHR) signal pathway to reduce insulin resistance, total cholesterol, total triglyceride, low density lipoprotein cholesterol and intestinal permeability, increase high density lipoprotein cholesterol levels and reduce body inflammation.
Based on the above-described characteristics of the lactobacillus plantarum 543 of the present invention, one of applications of the lactobacillus plantarum of the present invention and its fermented product is:
the application of lactobacillus plantarum 543 in preparing medicaments for reducing weight, reducing blood sugar, preventing and treating hypercholesterolemia and/or resisting inflammation; or the fermented product containing lactobacillus plantarum 543 is applied to medicaments for reducing weight, reducing blood sugar, preventing and treating hypercholesterolemia and/or resisting inflammation.
The medicine comprises lactobacillus plantarum 543 or a fermented product containing lactobacillus plantarum 543 and a pharmaceutically acceptable carrier.
In one embodiment, the pharmaceutically acceptable carrier includes, but is not limited to: one or more of a filler, binder, wetting agent, disintegrant, or lubricant.
Preferably, the medicament is an oral medicament.
In one exemplary embodiment, lactobacillus plantarum 543 is used in an amount of 1X 10 8 ～5×10 10 CFU/day, lactobacillus plantarum 543 eradicationThe usage amount of the live strain is 1-8 multiplied by 10 10 The amount of the metabolite of the lactobacillus plantarum 543 is 100-250 mg/day, and the amount of the metazoan of the lactobacillus plantarum 543 is 50-120 mg/day.
Or, the lactobacillus plantarum and/or the lactobacillus plantarum-containing fermented product are applied to preparing weight-reducing and blood glucose-reducing functional foods required for non-disease treatment. The functional food is health product, dietary supplement, solid beverage or microbial preparation.
The lactobacillus plantarum 543 of the invention can ferment and produce acid by using substrates such as L-arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, saligenin, cellobiose, maltose, lactose, sucrose, trehalose, melezitose, trehaltose, D-melezitose and the like, and has wide carbon source utilization capacity; the method has the advantages of strong acid production capacity, rapid acid production in the initial stage of fermentation, maximum acid production, and stable acid production along with the increase of fermentation time. Based on the above characteristics, the lactobacillus plantarum and the further application of the metazoan thereof are provided:
Lactobacillus plantarum 543 and application of a fermented product thereof as a starter in preparation of fermented food.
The lactobacillus plantarum 543 is used in an amount of 1×10 7 ～1×10 9 CFU/mL, the dosage of the post-metazoan of the lactobacillus plantarum 543 is 1-30 mg/kg.
The lactobacillus plantarum has the beneficial effects that:
the Lactobacillus plantarum 543 has high bioactivity, wide carbon source utilization capacity, good acid production property, and tolerance to gastric acid and bile salts.
The lactobacillus plantarum 543 has higher activity of bile salt hydrolase, can efficiently reduce cholesterol level in vivo and in vitro, can ensure that the high-fat and cholesterol-containing feed is fed to obesity and mice accompanied with hypercholesterolemia, obviously reduces insulin resistance index, total cholesterol, total triglyceride and low-density lipoprotein cholesterol, and simultaneously improves the high-density lipoprotein cholesterol level; lactobacillus plantarum 543 and fermented products (such as metazoan) thereof can improve the content of indole derivatives (such as indole-3-formaldehyde, indole-3-acetic acid and indole-3-lactic acid) in tryptophan metabolites in human bodies, and the tryptophan metabolites enter the intestinal tract and the systemic circulation and activate an Aromatic Hydrocarbon Receptor (AHR) signal path to reduce weight and regulate high cholesterol, so that the lactobacillus plantarum has wide application prospect in the aspects of weight reduction, blood glucose reduction, anti-inflammatory and hypercholesterolemia management.
Lactobacillus plantarum 543 and its fermented product (such as metaplasia) can reduce intestinal permeability caused by high-fat diet, remarkably reduce serum inflammatory factor TNF-alpha and IL-1β levels, and has health effects of regulating immunity and inhibiting inflammatory reaction, thereby relieving intestinal barrier leakage of mice caused by high-fat diet.
The lactobacillus plantarum 543 has good safety, is widely applied, and can be applied to preparation of medicaments for reducing weight, reducing blood glucose, preventing and treating hypercholesterolemia and/or resisting inflammation or used as a starter for preparing fermented foods and health-care foods.
Drawings
FIG. 1 is a colony chart of Lactobacillus plantarum 543 in MRS agar medium;
FIG. 2 is a diagram showing the morphology of a colony of Lactobacillus plantarum 543 under a microscope (1000X) after culture in MRS agar medium;
FIG. 3 is a phylogenetic tree of Lactobacillus plantarum 543;
FIG. 4 is a graph showing the growth and acid production profile of Lactobacillus plantarum 543;
FIG. 5 is a graph showing the activity of bile salt hydrolase in Lactobacillus plantarum 543;
FIG. 6 is a graph showing the change in body weight of Lactobacillus plantarum 543 and its metazoan, tryptophan metabolites, in groups of mice with a high fat diet;
FIG. 7 is a graph showing the analysis of the increase in body weight of Lactobacillus plantarum 543 and its metazoan, tryptophan metabolites, in groups of high fat diet mice;
FIG. 8 is a graph showing statistical analysis of insulin resistance index of each group of Lactobacillus plantarum 543 and its metants, tryptophan metabolites, and high fat diet mice;
FIG. 9 is a graph showing comparison of changes in serum TC and TG levels in various groups of Lactobacillus plantarum 543 and its metazoan, tryptophan metabolites, and mice with a high fat diet;
FIG. 10 is a graph showing analysis of changes in serum LDL-C and HDL-C levels of Lactobacillus plantarum 543 and its metazoans, tryptophan metabolites, in groups of mice that interfere with high fat diet;
FIG. 11 is a graph showing intestinal tract permeability of Lactobacillus plantarum 543 and its metazoan, tryptophan metabolites, in groups of mice with high fat diet;
FIG. 12 is a graph showing serum inflammatory factor levels of Lactobacillus plantarum 543 and its metazoan, tryptophan metabolites, in groups of mice with high fat diet;
FIG. 13 is a graph showing the comparison of the concentration of tryptophan metabolite in Lactobacillus plantarum 543 and its metants, tryptophan metabolite intervention high fat diet mice.
The significance between groups in fig. 6-13 is indicated as: HFD group compared with CON group, # represents p <0.05, # represents p <0.01, 543 group, POS group, TYM group compared with HFD group, # represents p <0.05, # represents p <0.01.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The formula of the culture medium in each embodiment of the invention comprises the following components:
MRS medium (lactobacillus plantarum 543): 10.0g/L peptone, 5.0g/L beef powder, 4.0g/L yeast powder, 20.0g/L glucose, 1.0g/L Tween 80, K 2 HPO 4 ·7H 2 O2.0 g/L, anhydrous sodium acetate 5g/L, diammine citrate 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O0.038 g/L, (15 g/L of agar powder is added as a solid culture medium).
tryptophan+MRS medium (Lactobacillus plantarum 543) (abbreviated as TRP+MRS): 10.0g/L peptone, 5.0g/L beef powder, 4.0g/L yeast powder, 20.0g/L glucose, 1.0g/L Tween 80, K 2 HPO 4 ·7H 2 O2.0 g/L, anhydrous sodium acetate 5g/L, diammine citrate 2.0g/L, mgSO 4 ·7H 2 O 0.2g/L，MnSO 4 ·H 2 O0.038 g/L and tryptophan 0.5-2g/L (15 g/L of agar powder is added as solid culture medium).
Example 1
Lactobacillus plantarum 543 (Lactobacillus plantarum 543) of the invention, accession number: cctccc No. M2021353.
1) Isolation and screening of Lactobacillus plantarum 543
Microorganism screening samples are collected from Sichuan Chengdu allium chinense, 1g of the collected samples are weighed after being sheared, the samples are put into 9mL of sterile physiological saline, and after being fully and evenly mixed by shaking, the samples are diluted by 10 times of gradient and coated in MRS solid culture medium, and are cultured for 48 hours at 37 ℃. Visually observing, picking single colonies with different forms and sizes in the culture medium, and repeatedly streaking, purifying and culturing; then the lactobacillus is primarily determined by microscopic observation and a calcium dissolving method, and the purified strain is stored in a refrigerator at the temperature of minus 80 ℃ for standby.
a) Morphological observation
The purified Lactobacillus plantarum543 was streaked on MRS agar medium, and after culturing for 48 hours at 37℃in an inverted manner, the colony morphology of the strain was observed, and the results are shown in FIG. 1: the strain grows well on an MRS agar culture medium, and the bacterial colony is round, medium in size, milky white, convex upwards, tidy in edge, moist and easy to pick; positive after gram staining; the shape under the microscope is rod-shaped (as shown in fig. 2).
b) Molecular biology identification of strains
The purified strain is sent to China center for 16S rRNA identification, NCBI BLAST alignment is carried out on the measured 16S rRNA sequence, the similarity with lactobacillus plantarum (Lactobacillus plantarum) in Genebank is greater than 99%, and the strain can be primarily identified as lactobacillus plantarum (Lactobacillus plantarum). The 16S rRNA identification sequence of the strain is shown as SEQ ID NO. 1, and a Neighbor-training phylogenetic tree (figure 3) constructed by taking Lactobacillus floricola Ryu-2 (AB 523780.1) as an outer branch based on the 16S rRNA gene sequence comparison result is named as lactobacillus plantarum543 (Lactobacillus plantarum 543).
2) Lactobacillus plantarum543 growth capacity and performance measurement
(1) Lactobacillus plantarum543 growth and acid production capacity determination
Lactobacillus plantarum 543 strainInoculating into MRS liquid culture medium according to 5% inoculum size, activating at 37deg.C for 24 hr, and activating twice. Inoculating the activated 543 fungus solution into MRS liquid culture medium according to 5% of inoculation amount, mixing uniformly, and sub-packaging into sterile test tubes (18 mm×180mm test tubes) according to 8 ml/branch; placing the packaged lactobacillus plantarum 543 bacterial liquid in a constant temperature incubator at 37 ℃ for static culture; at regular intervals, taking 3 test tubes to measure 543 bacterial liquid absorbance OD 600 And total acid, calculating an average value; absorbance value OD with time on the abscissa 600 And acid production is taken as an ordinate, and a growth and acid production curve is drawn.
The lactobacillus plantarum 543 strain growth curve is shown in figure 4, and the strain grows rapidly and has large bacterial body quantity, and OD at 8h can be seen from figure 4 600 Up to 5.42, the OD is up to 12.955 when the acid production reaches 1.23g/100g and 20h, and the acid production reaches 1.82g/100g, which indicates that the lactobacillus plantarum 543 can grow rapidly and produce acid rapidly and is stable in the later period.
(2) Substrate utilization ability
The ability of Lactobacillus plantarum 543 to utilize carbon sources of glycerol, erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, beta-methyl-D-xyloside, galactose, glucose, fructose, mannose, sorbose, rhamnose, dulcitol, sorbitol, alpha-methyl-D-mannoside, alpha-methyl-D-glucoside, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salix, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, trehalulose, D-lyxose, D-tagatose, D-rock, L-rock, D-arabitol, L-arabitol, glucose salts was examined, the lactic acid bacteria to be tested were inoculated in a fresh slant culture medium by puncturing or after the addition of a fresh medium to a mixed culture medium of which a 10% of a 10% strain of E.1C was placed in an agar medium was subjected to a medium to a dilution of a temperature of about 37% of the medium after the medium was subjected to a dilution of the medium, and the strain was subjected to a culture medium was subjected to a culture for a uniform culture for a temperature of about 37% of a culture medium to be subjected to a culture of a strain of fluid to a strain of fluid, and a strain of the strain was subjected to a strain of strain was subjected to a culture to a test.
The experimental result shows that lactobacillus plantarum 543 strain can utilize a plurality of carbon source substrates of L-arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, saligenin, cellobiose, maltose, lactose, sucrose, trehalose, melezitose, trepanose and D-melezitose for growth and fermentation to produce acid.
(3) Measurement of in vitro cholesterol degradation capability of Lactobacillus plantarum 543
Preparing MRS culture solution (MRS-CHOL culture medium) containing cholesterol and MRS culture solution without cholesterol.
MRS-CHOL medium: consists of MRS culture solution, sodium thioglycolate, bile salt and cholesterol, wherein the concentration of the sodium thioglycolate in the MRS-CHOL culture solution is 2g/L, the concentration of the bile salt in the MRSO-CHOL culture solution is 0.2 weight part, and the concentration of the cholesterol is 150 mug/mL.
Lactobacillus plantarum 543 strain is inoculated into MRS liquid culture medium according to an inoculum size of 5 percent, and is subjected to activating culture for 24 hours at 37 ℃ and is continuously activated twice. Inoculating the activated bacterial liquid into MRS-CHOL culture solution (experimental group) and MRS culture solution without cholesterol (control group) respectively according to 5% inoculum size, culturing at 37 ℃ for 32h at constant temperature, measuring cholesterol content in supernatant (culture solution centrifuging at 5000r/min, 4 ℃ for 10 min) with o-phthalaldehyde color developing agent every 8h, and calculating cholesterol degradation capacity,
Wherein, in the formula: m is m 1 Cholesterol mass in supernatant per μg of fermentation for 0 hours. m is m 2 The supernatant was fermented for cholesterol mass/. Mu.g at various time points.
The test results are shown in Table 1, and the Lactobacillus plantarum 543 strain 8h has 37.7% of cholesterol degradation capacity, and the 24h degradation capacity is 67.52%, and later tends to be stable with time, which indicates that the Lactobacillus plantarum 543 has good in vitro cholesterol degradation capacity.
TABLE 1 Lactobacillus plantarum 543 cholesterol degrading ability
Cultivation time (h) 0 8 16 24 32
Cholesterol content (μg/mL) 150 93.45 50.13 48.72 48.15
Cholesterol removal rate (%) / 37.7 25.53 67.52 67.9
(4) Lactobacillus plantarum 543 bile salt hydrolase activity determination
Bacterial strain bile hydrolase (BSH) activity assay. Preparing a bile salt hydrolase screening culture medium: adding ox gall salt (final mass concentration of 2 g/L), sodium thioglycolate (final mass concentration of 2 g/L) and CaCl into MRS plate 2 (final mass concentration was 0.37 g/L). Screening sterilized BSHThe medium was poured into sterile plates and after the medium had solidified, a sterile filter paper sheet was placed evenly over the medium. And (3) absorbing 10 mu L of activated strain bacterial liquid to be detected on a filter paper sheet with the diameter of about 3-4 mm, culturing for 72h under anaerobic condition at 37 ℃ after the bacterial liquid is no longer flowing, observing whether white sediment is generated around the cultured filter paper sheet, measuring a white sediment ring by using a vernier caliper, and verifying that the strain contains BSH if the sediment ring appears.
BSH hydrolyzes bile salts in bound form into bile salts in unbound form, which have low solubility in water and are capable of reacting with Ca in the medium under acidic conditions 2+ And combine to form a precipitate. Therefore, if Lactobacillus plantarum 543 has BSH activity, a precipitation circle can be observed in the BSH-screening medium. From the results shown in FIG. 5, it was found that white precipitates were produced around Lactobacillus plantarum 543, the diameter was 9.+ -.1 mm, and apparent granular precipitates were seen around the colonies, which, in combination with the known reports, revealed that Lactobacillus plantarum 543 had very high bile salt hydrolase BSH activity.
(5) Lactobacillus plantarum 543 test for gastric acid tolerance
The tolerance of Lactobacillus plantarum 543 of the present invention to gastric acid was examined by simulating gastric acid conditions. Lactobacillus plantarum 543 strain is inoculated into MRS liquid culture medium according to an inoculum size of 5 percent, and is subjected to activating culture for 24 hours at 37 ℃ and is continuously activated twice. Inoculating the activated 543 fungus liquid into MRS liquid culture medium according to 5% of inoculation amount, and standing and culturing for 15h at 37 ℃ in a constant temperature incubator. Centrifuging the cultured 543 fungus solution at 5000rpm for 10min to collect thalli, and shaking the thalli uniformly with sterile physiological saline; adding the bacterial liquid with uniform vibration into sterile physiological saline with pH value of 2.5, 3.0, 3.5 and 4.0 according to the addition amount of 10%, and incubating for 2 hours in a constant temperature incubator at 37 ℃ by taking the sterile physiological saline with pH value of 6.0 as a control. Taking out the incubated bacterial liquid, immediately diluting the bacterial liquid by 10 times, adding sterilized normal saline, beating and uniformly mixing, detecting the number of lactobacillus, counting the detected viable lactobacillus, and calculating the survival rate, wherein the survival rate of the strain is=test group/control group multiplied by 100 percent.
The test results show that the survival rates of the strains at pH values of 2.5, 3.0, 3.5 and 4.0 are 65.32 percent, 89.90 percent, and 94.35 and 99.13 percent respectively have high survival rates; the survival rate at pH 3.0 still remained 89.90%. The pH of gastric juice is generally about 3.5 after eating, indicating that Lactobacillus plantarum 543 can tolerate gastric acid after eating.
(6) Lactobacillus plantarum 543 test for bile salt tolerance
Lactobacillus plantarum 543 was inoculated into MRS liquid medium at an inoculum size of 5%, and subjected to activation culture at 37℃for 24 hours, followed by two consecutive activations. Inoculating the activated 543 fungus solution into MRS liquid culture medium according to an inoculum size of 5%, and standing and culturing for 15h at 37 ℃ in a constant temperature incubator. And (3) centrifuging the cultured bacterial liquid at 5000rpm for 10min to collect bacterial bodies, and vibrating the bacterial bodies uniformly with sterile physiological saline.
Adding the bacterial liquid with uniform vibration into MRS culture medium with bile salt concentration of 1.0g/L, 2.0g/L, 3.0g/L and 0.0g/L (initial bacterial liquid) according to the addition amount of 10%, and taking the bile salt concentration of 0.0g/L as a control group. Then incubated in a constant temperature incubator at 37℃for 3h. Taking out the incubated bacterial liquid, immediately diluting according to 10 times, adding sterile physiological saline, beating and uniformly mixing, and detecting the number of lactic acid bacteria; counting the detected live lactobacillus and calculating the survival rate, wherein the calculation formula is as follows:
Strain survival (%) = test/control x 100%.
The 543 strain bile salt tolerance data indicated that: the survival rates of the strains were 100% and 99.57% when the bile salt concentrations were 1.0g/L and 2.0g/L, respectively, but the survival rates of the strains still reached 97.43% when the bile salt concentrations reached 3.0 g/L. Because lactobacillus plantarum is separated from Sichuan pickle, the lactobacillus plantarum has better bile salt and salt tolerance, and the concentration of the bile salt in the intestinal tract is between 0.3 and 3.0g/L, which shows that the 543 strain has very excellent bile salt tolerance.
(7) Determination of high-yield tryptophan metabolite of lactobacillus plantarum 543
Activating lactobacillus plantarum 543 in an MRS culture medium twice, inoculating the activated lactobacillus plantarum 543 and bacterial solutions of other strains into the MRS culture medium according to an inoculation amount of 5%, uniformly mixing, and sub-packaging into sterile test tubes (18 mm multiplied by 180 mm) according to 8 ml/branch; will be divided intoPlacing the packed candidate strain bacterial liquid in a constant temperature incubator at 37 ℃ for static culture HPLC (high performance liquid chromatography) to measure tryptophan and metabolites thereof at different times, wherein the conversion rate of tryptophan metabolites is calculated as follows: wherein S is 0 Represents the initial tryptophan content, S 1 Represents tryptophan content at the end, where T 1 Represents the total tryptophan metabolite content, T 0 Representing the total tryptophan metabolite content initially.
TABLE 2 Tryptophan metabolite detection results a
Annotation: a. tryptophan and metabolites were quantified in mg/L.
b. Represents the sum of the 3-IAId, IAA and ILA contents.
Lactobacillus plantarum 543 gives table 2 the tryptophan metabolite yields at different times, wherein the tryptophan concentration tended to decrease with increasing fermentation time, indole-3-carbaldehyde (3-IAId), indole-3-acetic acid (IAA), indole-3-lactic acid (ILA) increased continuously with increasing fermentation time, the highest levels at 20h were 0.92mg/L indole-3-carbaldehyde, 6.85mg/L indole-3-acetic acid, 16.43mg/L indole-3-lactic acid, respectively, the tryptophan metabolite sum was 24.2mg/L, tryptophan metabolite conversion was 5.07%.
In summary, lactobacillus plantarum 543 is capable of producing indole-3-carbaldehyde (3-IAId), indole-3-acetic acid (IAA), and indole-3-lactic acid (ILA) at high levels.
Example 2
A fermented product containing lactobacillus plantarum is a lactobacillus plantarum 543 metagen in the embodiment 1, and the preparation method of the lactobacillus plantarum 543 metagen comprises the following steps: fermenting 543 Lactobacillus plantarum in MRS liquid culture medium (37deg.C, 25 h) to obtain fermentation broth, concentrating, spray drying to obtain solid powder containing thallus and metabolite, The Lactobacillus plantarum 543 metazoan contains 10 Lactobacillus plantarum 543 biomass 5.0 x 10/g, free amino acid > 1mg/g, tryptophan metabolite sum (3-IAId, IAA，ILA)＞100mg/kg。
based on the fact that the lactobacillus plantarum 543 can degrade cholesterol in vitro, has good tolerance to gastric acid and bile salts, and can ferment high-yield tryptophan metabolite, namely indole derivatives, the applicant further develops animal experiments that the live lactobacillus plantarum 543 powder described in the embodiment 1 and the post-metazoan lactobacillus plantarum 543 described in the embodiment 2 are used for preventing and treating obesity and hypercholesterolemia of high-fat diet mice, and is convenient for expanding new application directions of fermented products containing the lactobacillus plantarum 543.
3) Lactobacillus plantarum 543 and fermented product thereof for preventing obesity and hypercholesterolemia of high-fat diet mice
Sample to be tested: lactobacillus plantarum 543 live bacteria powder produced by Sichuan Gao Fu, biological technology Co., ltd., batch number 20220226, and live bacteria number 5×10 10 CFU/g (described in example 1), lactobacillus plantarum 543 metazoan (described in example 2), lot 20220305, 10 the number of the bacterial cells is 5.0 x 10/g，Tryptophan metabolite (3-IAId, IAA, ILA) > 100mg/kg(described in example 2).
Feeding conditions and feed: the temperature is 23+/-2 ℃, the relative humidity is 50-55%, the 12h of day and night circulation is controlled in a pathogen-free room, and 8:00 points are controlled time for free feeding and drinking;
Test grouping: male SPF class C57BL/6 mice, 8 weeks old, were randomly divided into 5 groups of 6 mice each, and 3 mice each were housed in the same cage. The grouping situation is as follows:
CON group: feeding with common maintenance feed, drinking water, and filling with maltodextrin with equal amount for 12 weeks;
HDF group: high Fat feed feeding (Fat 60%, cholesterol 1%), daily gastric lavage of equivalent maltodextrin, for 12 weeks;
lactobacillus plantarum 543 live bacteria group (hereinafter referred to as 543 group): high Fat feed (Fat 60%, cholesterol 1%), 1×10 viable count of Lactobacillus plantarum 543 per day 9 CFU/g, continuous feeding for 12 weeks;
lactobacillus plantarum 543 metagenome (hereinafter: POS group): feeding with high Fat feed (Fat 60%, cholesterol 1%), and daily feeding with Lactobacillus plantarum 543 metazoan, wherein the number of Lactobacillus plantarum 543 cells is 5×10 9 CFU/g, continuous feeding for 12 weeks;
tryptophan metabolite group (hereinafter referred to as TYM group): high Fat feed (Fat 60%, cholesterol 1%) was fed for 12 weeks at 100mg/kg day of daily gastric lavage tryptophan metabolite (3-IAId: IAA: ila=1:7:14).
The results of the test for controlling obesity and hypercholesterolemia of the high fat diet mice by lactobacillus plantarum 543 of this example are expressed as mean ± standard error; inter-group significance is indicated in the drawings: HFD group is compared with CON group, p <0.05, p <0.01, 543 group, POS group, TYM group is compared with HFD group, #p <0.05, #p <0.01.
1) Effect of Lactobacillus plantarum 543 on weight of high fat diet mice
The experimental mice were weighed and recorded weekly, and the body weights of the mice of each group at the end of the experiment are shown in fig. 6. The body weight of the HFD group mice is obviously increased, and the modeling is successful; after the intervention of group 543, POS and TYM, the weight of the high-fat diet mice is obviously reduced at the end of the experiment; the weight gain of mice in group 543 and POS was significantly reduced compared to the model group (p < 0.01), respectively, and the TYM was reduced compared to the model group (p < 0.05), indicating that: lactobacillus plantarum 543 and metaplasia and tryptophan metabolite thereof can obviously inhibit weight increase caused by high-fat diet of mice; secondly, the minimum increase in body weight of group 543 and POS was lower than that of TYM group (FIG. 7), indicating that Lactobacillus plantarum 543 and its metants had a stronger inhibitory effect on body weight gain by high-fat diet of mice than tryptophan metabolite, which was associated with inhibitory effect on weight gain by high-fat diet by Lactobacillus plantarum 543 cells (live strain or dead strain).
2) Effect of Lactobacillus plantarum 543 on insulin resistance index in high fat diet mice
The feed was cut off 24 hours before blood glucose testing and the intervention of probiotics or metazoans or tryptophan metabolites was stopped, water was supplied only, then blood was taken from the tail vein, and blood glucose was tested using a glucometer. The blood was placed in a sterile desiccation tube, left standing at room temperature for half an hour, centrifuged at 4000rpm at room temperature for 10 minutes, and the upper yellowish liquid was collected to obtain serum, and the insulin resistance index was calculated by formula 1).
Insulin resistance index = fasting serum insulin (mU/L) times fasting blood glucose (mmol/L)/22.5 formula 1
The serum insulin resistance index of mice in each experimental group is shown in fig. 8, and after the intervention treatment of lactobacillus plantarum 543 live strain, the metabolites thereof and tryptophan metabolites, the insulin resistance index of the high-fat diet mice 543 group and the POS group is extremely obviously reduced (p < 0.01), the insulin resistance index of the mice after the intervention of the TYM group is obviously reduced, and p <0.05, which indicates that the serum insulin resistance index of the high-fat diet mice is more favorable to be reduced compared with the conventional tryptophan metabolites.
3) Effects of Lactobacillus plantarum 543 on total cholesterol, triglycerides, high density lipoprotein cholesterol, and Low density lipoprotein cholesterol in high fat diet mice
The test of Total Cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and the like in mouse serum using the kit (nanjing build) showed a trend of decreasing TC in group 543, POS, and TYM, compared to HFD group, in which POS group decreased extremely (p < 0.01), and both of 543 and TYM decreased significantly (p < 0.05), as shown in fig. 9: the lactobacillus plantarum 543 bacterial body and tryptophan metabolite in the lactobacillus plantarum 543 metagenome play a role in mutual synergy on the reduction of the total cholesterol of the mice; the significantly reduced TG in group 543, POS and TYM compared to HFD groups, wherein the significantly reduced TG in group 543 and POS (p < 0.01), significantly reduced TG in group TYM (p < 0.05), and the lowest TG concentration in group 543, indicated that the viable lactobacillus plantarum 543 has the strongest effect on the degradation of Triglyceride (TG), and that the dead lactobacillus plantarum 543 in the post-natant of lactobacillus plantarum 543 and tryptophan metabolites produced by the same have a synergistic effect on the degradation of Triglyceride (TG).
The results of HDL-C and LDL-C measurements are shown in FIG. 10, in which LDL-C was decreased in the group 543, POS and TYM, respectively, in comparison with the HFD group, and in which the decrease in the POS group was extremely remarkable (p < 0.01), and the decrease in the group 543 and TYM was remarkable (p < 0.05), indicating that: the Lactobacillus plantarum 543 cell and tryptophan metabolite in the Lactobacillus plantarum 543 metatuple have synergistic effect on the reduction of mouse low density lipoprotein cholesterol (LDL-C); the high density lipoprotein cholesterol (HDL-C) of group 543, POS group and TYM group was significantly increased compared with HFD group, wherein the increase of group 543 was extremely significant (p < 0.01), the increase of POS group and TYM group was significant (p < 0.05), and the HDL-C concentration of group 543 was highest, near the level of normal group, indicating that the formation of high density lipoprotein cholesterol (HDL-C) was most promoted by live Lactobacillus plantarum 543, and the formation of high density lipoprotein cholesterol (HDL-C) was synergistically promoted by dead bacteria of Lactobacillus plantarum 543 and tryptophan metabolite produced by them in the metazoan of Lactobacillus plantarum 543.
4) Lactobacillus plantarum 543 alleviates high fat diet induced leakage of the intestinal barrier in mice
In vivo evaluation of intestinal barrier permeability levels were determined using the oral fluorescein FITC-dextran. Blood was collected from the experiment: after FITC is orally taken for 4 hours, the mice are anesthetized and fixed, eyeballs are rapidly extracted by forceps, blood flows into a sterile EP tube from the eyesocket, the mixture is kept still for more than half an hour at 37 ℃, a centrifuge is opened, the mixture is centrifuged for 10 minutes at 4000rpm at room temperature, and a yellowish liquid of the supernatant is collected to obtain serum; detecting intestinal permeability at excitation wavelength of 493nm by using a multifunctional enzyme-labeled instrument, and detecting serum fluorescence intensity at absorption wavelength of 518 nm; the levels of inflammatory factors TNF-alpha and IL-1 beta in the serum of each group of mice were detected using ELISA kit, and the indole-3-carbaldehyde, indole-3-acetic acid, indole-3-lactic acid content in the serum was detected using HPLC.
Referring to fig. 11 to 13, the model high fat diet mice of HFD group had significantly increased intestinal permeability compared to the control group, significantly increased levels of expression of inflammatory factors tnfα and IL-1β in serum, decreased levels of indole-3-carbaldehyde, indole-3-acetic acid, indole-3-lactic acid, indicating that the intestinal barrier was disrupted and blood circulation had inflammation occurred, and decreased tryptophan metabolites negatively associated with obesity and hypercholesterolemia. Lactobacillus plantarum 543 and metazoan tryptophan metabolites dry and then the intestinal permeability of high fat diet mice is significantly reduced, wherein POS group reduction is extremely significant (p < 0.01), and 543 group and POS group reduction is significant (p < 0.05), indicating: the lactobacillus plantarum 543 in the metazoan of the lactobacillus plantarum 543 and indole derivatives in the metabolites have synergistic effect on improving the intestinal permeability of mice; the levels of inflammatory factors TNF-alpha and IL-1β in group 543 were extremely reduced (p < 0.01), while the levels of inflammatory factors TNF-alpha and IL-1β in TYM group were only significantly reduced (p < 0.05), the levels of inflammatory factors TNF-alpha in POS group were extremely reduced (p < 0.01), and IL-1β levels were only significantly reduced (p < 0.05); description: the Lactobacillus plantarum 543 live strain has the strongest promotion effect on the reduction of the levels of inflammatory factors TNF-alpha and IL-1 beta in serum, and the weakest promotion effect on tryptophan metabolites; tryptophan metabolite results in serum indicate that: compared with HFD group, the indole-3-formaldehyde and indole-3-acetic acid in group 543 and TYM group are obviously increased (p < 0.05), the POS group is extremely obviously increased (p < 0.01), and compared with relative lactobacillus plantarum 543, the tryptophan metabolite contained in the metazoan of lactobacillus plantarum 543 is rich in indole-3-formaldehyde and indole-3-acetic acid, and relative TYM group, the indole-3-formaldehyde and indole-3-acetic acid are extremely obviously increased in the lactobacillus plantarum 543 thallus in the metazoan of lactobacillus plantarum 543; the indole-3-lactic acid in the POS group and TYM group is obviously increased (p is less than 0.05), and the indole-3-lactic acid in the 543 group is obviously increased (p is less than 0.01), which proves that the lactobacillus plantarum 543 live bacteria powder is more beneficial to the generation of indole-3-lactic acid in the high-fat diet mice.
In summary, compared with the CON group and the HFD group, the 543 group, the POS group and the TYM group can significantly inhibit the weight gain of the high-fat diet mice, and simultaneously reduce the insulin resistance index, the total cholesterol, the total triglyceride and the low-density lipoprotein cholesterol level of the mice and improve the high-density lipoprotein cholesterol level; meanwhile, the intestinal permeability is obviously reduced, the expression level of inflammatory factors TNFa and IL-1 beta in serum of mice is obviously reduced, the content of tryptophan metabolites, namely indole-3-formaldehyde, indole-3-acetic acid and indole-3-lactic acid is obviously increased, lactobacillus plantarum 543 and metaplasia are used for efficiently generating indole-3-formaldehyde, indole-3-acetic acid and indole-3-lactic acid, the tryptophan metabolites enter the intestinal canal and the systemic circulation and activate an Aromatic Hydrocarbon Receptor (AHR) signal path, the insulin resistance, the total cholesterol, the total triglyceride, the low-density lipoprotein cholesterol and the intestinal permeability are reduced, the high-density lipoprotein cholesterol level is increased, the body inflammation is reduced, the theoretical basis is provided for the intervention and the treatment of metabolic diseases such as hypercholesterolemia, and the like, and the novel application direction is provided for lactobacillus plantarum 543 and fermentation products thereof, such as the application in the preparation of medicaments for reducing weight, reducing sugar, preventing and treating hypercholesterolemia and/or anti-inflammatory diseases.
Example 3
Use of a live strain of lactobacillus plantarum 543 for the manufacture of a medicament for weight loss, blood glucose reduction, prevention and treatment of hypercholesterolemia and/or anti-inflammatory, said live strain of lactobacillus plantarum 543 being as described in example 1.
Example 4
A lactobacillus plantarum-containing fermented product of the present embodiment includes a lactobacillus plantarum 543 live strain and a lactobacillus plantarum 543 metazoan, wherein the lactobacillus plantarum 543 live strain is as described in example 1; lactobacillus plantarum 543 metazoan is described in example 2; the weight ratio of live strain of Lactobacillus plantarum 543 to inactivated strain of Lactobacillus plantarum 543 was 2:1.
As is clear from 2) — (3), (7) in example 1 and experiment 3) in example 2, the live strain of Lactobacillus plantarum 543 and the metazoan of Lactobacillus plantarum 543 can both reduce cholesterol in vivo and in vitro, and are rich in tryptophan metabolites, so the composition of this example can be used for preventing/treating hypercholesterolemia with good safety.
The application of the lactobacillus plantarum-containing fermented product in preparing medicaments for preventing/treating hypercholesterolemia is that the lactobacillus plantarum 543 in the composition is 2.0 x 10 when the lactobacillus plantarum-containing fermented product is used 10 CFU/day, lactobacillus plantarum 543 metazoan amount 5.0 x 10 10 Personal/day.
Example 5
The lactobacillus plantarum-containing fermented product of this example includes a live lactobacillus plantarum 543 strain and an inactivated lactobacillus plantarum 543 strainWherein the live strain of Lactobacillus plantarum 543 has a live strain content of 5.0X10 as described in example 1 10 CFU/g (cell number 5.0X10) 10 CFU/g, lot number: 20230522 A) is provided; the preparation method of the inactivated strain of lactobacillus plantarum 543 comprises the following steps: fermenting lactobacillus plantarum 543 to obtain fermentation broth, centrifuging the fermentation broth to obtain precipitate, re-suspending, cell lysing, inactivating at high temperature (121deg.C, 30 min), centrifuging, concentrating, and spray drying to obtain the final product (cell number 3.5X10) 10 Number of lots/g: 20230524 A) is provided; preferably, the live strain 543 and the inactivated strain 543 of lactobacillus plantarum have a content ratio of 2, based on the number of cells contained therein: 1.
the application of the composition in the embodiment in preparing the medicine for reducing weight and preventing and treating hypercholesterolemia is that the dosage of the lactobacillus plantarum 543 is 5.0 x 10 10 CFU/day, inactivated Lactobacillus plantarum 543 2.5×10 10 Personal/day.
Example 6
The lactobacillus plantarum-containing fermented product of the embodiment comprises a lactobacillus plantarum 543 live strain, a lactobacillus plantarum 543 inactivated strain and a lactobacillus plantarum 543 metaplasia with a mass ratio of 2:1:1.
The lactobacillus plantarum-containing fermented product of the embodiment has the effects of reducing cholesterol and diminishing inflammation, and is applied to the preparation of medicines for preventing and treating hypercholesterolemia and resisting inflammation, and when the lactobacillus plantarum 543 live strain is used, the dosage is 2 multiplied by 10 10 CFU/day, inactivated strain of Lactobacillus plantarum 543 1×10 10 The amount of metagen is 50mg/day.
Example 7
A fermented product containing Lactobacillus plantarum 543 is prepared by fermenting Lactobacillus plantarum 543 of example 1 on MRS culture medium for 30 hr to obtain fermentation broth, centrifuging the fermentation broth, and lyophilizing the obtained precipitate to obtain lyophilized powder (batch number: 20230612) containing Lactobacillus plantarum 543 live strain, wherein the lyophilized powder contains Lactobacillus plantarum 543 live strain 1.0×10 11 CFU/g。
According to the animal experiments, the Lactobacillus plantarum 543 live strain in the composition has strong effect of reducing inflammatory factors in bloodAction of sub level, application of Lactobacillus plantarum 543 fermentation product of this embodiment in preparing anti-inflammatory drug, wherein the usage amount of Lactobacillus plantarum 543 live strain is 1.0X10 10 CFU/day。
Example 8
A fermented product containing Lactobacillus plantarum 543 is a fermentation broth obtained by fermenting Lactobacillus plantarum 543 of example 1 on MRS medium for 20 hr, wherein the thallus concentration of Lactobacillus plantarum 543 is 1×10 or more 8 CFU/mL。
According to example 1, the live strain of Lactobacillus plantarum 543 in the composition has a strong acid production ability, and the fermented product containing Lactobacillus plantarum 543 of this example is used as a starter in preparing fermented food and health food.
The fermented food is pickle, and the application method of the composition as a starter in preparing pickle is as follows:
cleaning fresh vegetables, adding into 4-5 times of drinking water, adding edible glucose 1% of total volume and edible sodium chloride 0.4-0.6% of total volume, and inoculating Lactobacillus plantarum 543 prepared in this example to reach concentration of 10 8 Fermenting at room temperature for 5-15 hr to obtain fermented sauerkraut containing Lactobacillus plantarum 543 and Lactobacillus plantarum 543 strain metabolite. The fermented pickle has crisp and unique flavor, contains lactobacillus plantarum 543 thalli and metabolites, and has good safety and probiotic functions.
Example 9
The fermented product containing Lactobacillus plantarum 543 of this example comprises Lactobacillus plantarum 543 powder (2.0X10 10 CFU/g) 10 parts, lactobacillus plantarum 543 inactivated bacteria powder (3.0X10) 10 21 parts by weight, lactobacillus plantarum 543 metazoan (cell number 4.0X10) 10 2 parts per gram) and auxiliary materials, wherein the auxiliary materials comprise 1 part of magnesium stearate, 23 parts of lactose, 4 parts of fucoidin, 14 parts of resistant starch, 5 parts of microcrystalline cellulose, 10 parts of maltodextrin, 8 parts of glucose, 1 part of vitamin C and 1 part of folic acid.
Firstly, 23 parts by weight of lactose, 4 parts by weight of fucoidin, 14 parts by weight of resistant starch, 5 parts by weight of microcrystalline cellulose, 10 parts by weight of maltodextrin, 8 parts by weight of glucose, 1 part by weight of vitamin C and 1 part by weight of folic acid are weighed and uniformly mixed, a 20-mesh screen with the concentration of 30% alcohol wet method is adopted to granulate into wet granules, the wet granules are dried at 55 ℃ for 3.5 hours, the 20-mesh screen is adopted to granulate, and then lactobacillus plantarum 543 bacterial powder (2.0 multiplied by 10) is added 10 CFU/g) 10 parts, lactobacillus plantarum 543 inactivated bacteria powder (3.0X10) 10 21 parts by weight, lactobacillus plantarum 543 metazoan (cell number 4.0X10) 10 2 parts per gram) and 1 part of magnesium stearate, and tabletting by a rotary tablet press after being uniformly mixed to obtain the tablet with the lactobacillus plantarum 543 dietary supplement.
The fermented product containing lactobacillus plantarum 543 of the embodiment is used for preparing weight-reducing and blood glucose-reducing dietary supplements (tablets), improves human metabolism, and plays a role in preventing and treating human metabolic diseases.
Example 10
A fermented product containing Lactobacillus plantarum 543 of this example comprises, by weight, lactobacillus plantarum 543 (3.0X10 10 CFU/g) 20 parts by weight, maltodextrin 20 parts by weight, sorbitol 11 parts by weight, galacto-oligosaccharide 13 parts by weight, corn peptide 10 parts by weight, anserine 1 part by weight, soybean peptide 5 parts by weight, xylo-oligosaccharide 4 parts by weight, broccoli seed aqueous extract 4 parts by weight, selenium-rich yeast 3 parts by weight, sucralose 2 parts by weight, malic acid 2 parts by weight, glutathione 2 parts by weight, vitamin E1 part by weight, vitamin C1 part by weight, folic acid 1 part by weight.
The raw materials of the composition are firstly sieved by a 40-mesh screen, then uniformly mixed according to a proportion, and packaged by a screw back-packaging machine to prepare 2 g/bag solid beverage with weight and sugar reduction.
Example 11
A metabolite of lactobacillus plantarum 543, and a preparation method thereof: the fermentation broth obtained by fermenting Lactobacillus plantarum 543 (37 ℃ C., 30 h) in MRS medium as described in example 1 is adopted, supernatant obtained by centrifugation is subjected to high-temperature inactivation (121 ℃ C., 30 min), concentration and spray drying to obtain (free amino acid > 1.2mg/g, tryptophan metabolite sum (3-IAId, IAA, ILA) > 200mg/kg, batch number: 20230615).
The application of the metabolite of the lactobacillus plantarum 543 in preparing the medicine for preventing and treating the hypercholesteremia is that the metabolite dosage of the lactobacillus plantarum 543 is 150mg/day.
Example 12
The use of Lactobacillus plantarum 543 metagen in preparing medicine for reducing weight, reducing blood sugar and resisting inflammation is that the dosage of Lactobacillus plantarum 543 metagen is 100mg/day.
Wherein Lactobacillus plantarum 543 metazoan was obtained by the preparation method as described in example 2.

Claims (10)

1. The lactobacillus plantarum for preventing and treating the hypercholesterolemia is characterized in that the lactobacillus plantarum is named as lactobacillus plantarum (Lactobacillus plantarum) 543 and is preserved in the China center for type culture collection of Wuhan in China, wherein the preservation number is CCTCC NO: M2021353, and the month 12 of 2021.
2. The lactobacillus plantarum for preventing and treating hypercholesterolemia according to claim 1, wherein the 16S rRNA gene sequence of the lactobacillus plantarum 543 is shown in SEQ ID No. 1.
3. A fermented product containing lactobacillus plantarum, characterized in that the fermented product is produced by using lactobacillus plantarum 543 of claim 1 and/or fermentation.
4. A lactobacillus plantarum containing fermented product according to claim 3 comprising one or more of a live strain of lactobacillus plantarum 543, an inactivated strain of lactobacillus plantarum 543, a strain metabolite or a post-natal of lactobacillus plantarum 543.
5. Use of lactobacillus plantarum for the prevention and treatment of hypercholesterolemia according to claim 1 or 2 in the preparation of fermented foods.
6. Use of lactobacillus plantarum for the control of hypercholesterolemia according to claim 1 or 2 and/or lactobacillus plantarum containing fermented product according to claim 3 or 4 for the preparation of a medicament for weight reduction, blood glucose reduction, control of hypercholesterolemia and/or anti-inflammatory.
7. The use according to claim 6, wherein the lactobacillus plantarum meets the requirements of the use by at least one of the following routes:
1) Decreasing insulin resistance index and weight gain rate;
2) Lowering total cholesterol, total triglycerides and low density lipoprotein cholesterol levels, and increasing high density lipoprotein cholesterol levels;
3) Reducing the level of serum inflammatory factor TNF-alpha and IL-1 beta;
4) High-yielding tryptophan metabolites include, but are not limited to, indole-3-carbaldehyde, indole-3-acetic acid, indole-3-lactic acid.
8. The use according to claim 6, wherein the medicament comprises lactobacillus plantarum 543 or a fermented product containing lactobacillus plantarum 543 and a pharmaceutically acceptable carrier.
9. The use according to claim 6, wherein the medicament is an oral medicament.
10. Use of lactobacillus plantarum for the control of hypercholesterolemia according to claim 1 or 2 and/or lactobacillus plantarum containing fermented product according to claim 3 or 4 for the preparation of weight-and sugar-reducing functional food products required for non-disease treatment.
CN202311255496.2A 2023-09-27 2023-09-27 Lactobacillus plantarum for preventing and treating hypercholesterolemia, fermented product and application thereof Pending CN117264829A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117547029A (en) * 2024-01-11 2024-02-13 东北农业大学 Lactobacillus plantarum J26 metazoan freeze-dried powder with weight-losing and lipid-lowering effects, and preparation method and application thereof
CN117645965A (en) * 2024-01-29 2024-03-05 潍坊康地恩生物科技有限公司 Lactobacillus casei rhamnosus with intestinal indole degrading and chronic renal failure relieving functions

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117547029A (en) * 2024-01-11 2024-02-13 东北农业大学 Lactobacillus plantarum J26 metazoan freeze-dried powder with weight-losing and lipid-lowering effects, and preparation method and application thereof
CN117547029B (en) * 2024-01-11 2024-04-05 东北农业大学 Lactobacillus plantarum J26 metazoan freeze-dried powder with weight-losing and lipid-lowering effects, and preparation method and application thereof
CN117645965A (en) * 2024-01-29 2024-03-05 潍坊康地恩生物科技有限公司 Lactobacillus casei rhamnosus with intestinal indole degrading and chronic renal failure relieving functions

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