CN114404459B - Application of lactobacillus reuteri CCFM1135 in reducing plasma trimethylamine oxide - Google Patents
Application of lactobacillus reuteri CCFM1135 in reducing plasma trimethylamine oxide Download PDFInfo
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- 229940001882 lactobacillus reuteri Drugs 0.000 title claims abstract description 59
- 241000186604 Lactobacillus reuteri Species 0.000 title claims abstract description 58
- UYPYRKYUKCHHIB-UHFFFAOYSA-N trimethylamine N-oxide Chemical compound C[N+](C)(C)[O-] UYPYRKYUKCHHIB-UHFFFAOYSA-N 0.000 title claims abstract description 28
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- 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
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A23V2400/00—Lactic or propionic acid bacteria
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- A23V2400/173—Reuteri
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Abstract
The invention discloses an application of lactobacillus reuteri CCFM1135 in reducing plasma trimethylamine oxide, and belongs to the technical field of microorganisms. The lactobacillus reuteri CCFM1135 is capable of reducing plasma TMAO levels; can improve the structure of intestinal flora, recover intestinal flora disorder caused by high choline, improve the abundance of beneficial bacteria Roseburia, reduce the abundance of harmful bacteria Ruminococcaceae UCG-009, and reduce the risks of intestinal stress syndrome, obesity, allergy, neurological diseases, type II diabetes, nonalcoholic fatty liver, hyperlipidemia and hypertension. Therefore, the lactobacillus reuteri CCFM1135 has wide application value in preparing products for preventing and/or treating cardiovascular diseases and improving intestinal flora.
Description
Technical Field
The invention relates to an application of lactobacillus reuteri CCFM1135 in reducing plasma trimethylamine oxide, and belongs to the technical field of microorganisms.
Background
Cardiovascular disease is the leading cause of morbidity and mortality worldwide, atherosclerosis (AS) being the pathological basis of cardiovascular disease. Atherosclerosis is a chronic inflammatory disease with pathological features including monocyte and macrophage aggregation, smooth muscle cell proliferation and migration, fibroblast proliferation, cholesterol crystallization and free cholesterol and connective tissue deposition at the lesion. Currently, the accepted initiating factor for AS is arterial wall endothelial injury and lipid deposition; risk factors are hypertension, elevated blood lipid, inflammation, oxidized high choline, obesity, smoking, etc.
The intestinal canal of human body contains more than 1000A total of about 10 for a plurality of microorganisms 14 ～10 15 The mass of the powder can reach 1-1.5 kg. The total number of these intestinal microorganism-encoding genes exceeds 330 ten thousand, about 100 times the total number of human self-encoding genes, so that the intestinal microorganism is regarded as the second genome of the human body. The intestinal microbial genome, together with the human genome, affects a variety of important physiological functions such as food digestion and metabolism, immune response and inflammation, neural activity, etc. of the host through interaction with environmental factors. The interaction between the intestinal flora and its metabolites and the host is essential for maintaining the health of the host. Disorders of intestinal microecology are associated with a number of diseases including diabetes, obesity, inflammatory bowel disease, neurodegenerative diseases and tumors, and the like.
Studies have shown that intestinal microorganisms affect atherosclerosis mainly through bacterial infection, regulation of cholesterol and lipid metabolism, food and microbial metabolites. TMAO produced via the dietary-intestinal microbial-liver-trimethylamine oxide (TMAO) pathway may promote the development of cardiovascular disease. The microbial-dependent Trimethylamine (TMA)/TMAO pathway has been shown to be associated with the pathogenesis of cardiovascular disease and is an important diagnostic and therapeutic target for cardiovascular disease. Intervention in the metabolism of the intestinal flora, or possibly one of the methods for preventing and treating cardiovascular diseases.
Probiotics have been widely accepted by consumers as dietary supplements. The supplementing probiotics can directly inject a large amount of beneficial bacteria into human intestinal tracts, so as to help improve the metabolic function of the bacteria. Numerous scientific studies and clinical experiments have demonstrated that probiotics have a significant improving effect on constipation, enteritis, lactose intolerance, anti-infection, inflammation, allergy, and glycolipid metabolic disorders.
Disclosure of Invention
A first object of the present invention is to provide the use of lactobacillus reuteri (Lactobacillus reuteri) CCFM1135 for the preparation of a product for the prevention and/or treatment of cardiovascular diseases and for improving intestinal flora.
In one embodiment, the Lactobacillus reuteri (Lactobacillus reuteri) CCFM1135 has been deposited at the Guangdong province microorganism strain collection at 24/7/2020 with accession number GDMCC No.61102 at the Guangdong province microbiological institute of building 5, hirschq 100, guangzhou City
In one embodiment, the amount of lactobacillus reuteri CCFM1135 in the product is not less than 1 x 10 6 CFU/mL or ≡1X10. 6 CFU/g。
In one embodiment, the product has at least one of the following effects:
(1) Lowering plasma TMAO levels in high choline diet mammals;
(2) Increasing the abundance of the high choline diet mammal Roseburia.
(3) Decreasing the abundance of high choline dietary mammals Ruminococcaceae UCG-009.
In one embodiment, the mammal includes, but is not limited to, a human.
In one embodiment, the product comprises a food or pharmaceutical product.
In one embodiment, the pharmaceutical product contains lactobacillus reuteri CCFM1135 and/or a pharmaceutically acceptable carrier.
In one embodiment, the carrier comprises microcapsules, microspheres, nanoparticles, and/or liposomes.
In one embodiment, the pharmaceutical product is in the form of powder, granule, capsule, tablet, pill or oral liquid.
In one embodiment, the food product comprises a dairy product, a soy product, a fruit and vegetable product.
In one embodiment, the dairy product comprises milk, sour cream, cheese.
In one embodiment, the fruit and vegetable product comprises one or more of cucumber, carrot, beet, celery, cabbage and other edible fruits and vegetables.
In one embodiment, the food further comprises an additive selected from one or a combination of two or more of spices, fruit and vegetable juices, flower juices, colorants, acidity regulators, preservatives, antioxidants, thickeners, sweeteners.
In one embodiment, the processed form of the food comprises a solid food, a liquid food, a semi-solid food.
The beneficial effects are that:
1. in the high choline model apoE-/-mouse experiment, the plasma TMAH level can be obviously reduced by taking the lactobacillus reuteri CCFM1135, and the plasma TMAH level of the mice in the lactobacillus reuteri CCFM1135 group is reduced by 29.65 percent compared with that of the mice in the model group.
2. In an apoE-/-mouse experiment of a high choline model, the abundance of beneficial bacteria Roseburia is improved by taking lactobacillus reuteri CCFM1135, the relative abundance of beneficial bacteria Roseburia in the mice of the lactobacillus reuteri CCFM1135 group is improved by 8.43 times compared with that of the beneficial bacteria Roseburia in the model group, the abundance of beneficial bacteria Roseburia is improved, and the risks of irritable bowel syndrome, obesity, allergy, neurological diseases and type II diabetes are further reduced.
3. In an apoE-/-mouse experiment of a high choline model, the administration of lactobacillus reuteri CCFM1135 reduces the abundance of harmful bacteria Ruminococcaceae UCG-009, and compared with the mice of the model group, the relative abundance of the harmful bacteria Ruminococcaceae UCG-009 of the lactobacillus reuteri CCFM1135 is reduced by 71.33%, the abundance of the harmful bacteria Ruminococcaceae UCG-009 is reduced, so that the risks of obesity, non-alcoholic fatty liver, hyperlipidemia and hypertension are further reduced.
4. The lactobacillus reuteri CCFM1135 provided by the invention not only has the function of regulating intestinal flora, but also can effectively reduce the blood plasma TMAO level, expands the application range of lactobacillus reuteri as probiotics, and has very important significance for deep excavation of the functions of probiotics and development of probiotics with higher health care value.
Biological preservation material
Lactobacillus reuteri (Lactobacillus reuteri) CCFM1135, classified as Lactobacillus reuteri, was deposited at the Guangdong province microorganism strain collection on 7 months 24 in 2020, at the accession number GDMCC No.61102 from the Guangdong province microorganism institute, building 5, hirschq 100, guangzhou City.
Drawings
FIG. 1 shows colony morphology of Lactobacillus reuteri CCFM 1135.
FIG. 2 is the effect of Lactobacillus reuteri CCFM1135 on plasma TMAO in choline-fed apoE-/-mice; wherein P <0.01 and P <0.0001.
FIG. 3 is the effect of Lactobacillus reuteri CCFM1135 on cecum Roseburia of choline fed apoE-/-mice; wherein P <0.05.
FIG. 4 is a graph showing the effect of Lactobacillus reuteri CCFM1135 on cecum Ruminococcaceae UCG-009 of choline fed apoE-/-mice; wherein P <0.05.
FIG. 5 is the effect of different Lactobacillus reuteri on plasma TMAO in choline-fed apoE-/-mice; wherein P <0.01 and P <0.0001.
Detailed Description
Example 1: influence of Lactobacillus reuteri CCFM1135 on plasma TMAO
The colony morphology of lactobacillus reuteri CCFM1135 is shown in figure 1.
Lactobacillus reuteri CCFM1135 and Lactobacillus reuteri FHNXY12L1 (reported in Ni Caixin. Influence of Lactobacillus on hyperuricemia and route of action were explored [ D ]]Culturing in MRS culture medium at 37deg.C, collecting thallus, suspending in physiological saline to give a concentration of 1×10 9 CFU/mL of bacterial suspension.
The lactobacillus reuteri CCFM1135 obviously reduces the level of plasma TMAO, and compared with apoE-/-mice in a model group, the apoE-/-mice in the gastric lavage lactobacillus reuteri CCFM1135 bacterial liquid reduce the plasma TMAO by 29.65%;
healthy female apolipoprotein E knockout mice (apoE-/-mice) weighing 18-20g were taken in 24, acclimatized for 1 week, randomly divided into 4 groups of 6 apoE-/-mice each: control, model (Choline), lactobacillus reuteri CCFM1135 (CCFM 1135), lactobacillus reuteri FHNXY12L1 Control (FHNXY 12L 1). The grouping and treatment methods of the experimental animals are shown in table 1:
table 1 experimental animal groups
| Group of | Number/group of only | Duration of treatment | Feed stuff | Treatment method |
| Control | 6 | 2-24 weeks | Common feed | 0.2mL physiological saline is infused into stomach every day |
| Choline | 6 | 2-24 weeks | Choline feed | 0.2mL physiological saline is infused into stomach every day |
| CCFM1135 | 6 | 2-24 weeks | Choline feed | Gastric lavage 0.2mL of lactobacillus reuteri CCFM1135 bacterial suspension per day |
| FHNXY12L1 | 6 | 2-24 weeks | Choline feed | Gastric lavage per day 0.2mL lactobacillus reuteri FHNXY12L1 bacterial suspension |
Week 2-24: the control group apoE-/-mice were fed normal diet and the remaining three groups apoE-/-mice were fed Choline diet. Common feeds (LAD 3001M, choline content 0.1%) and choline feeds (LAD 3001M, choline content 1.0%) were purchased from south-through terlafei feed technologies.
Four groups of apoE-/-mice were fasted and kept water for 4h before the end of the trial, and blood was drawn through the periorbital capillaries. Blood samples were centrifuged at 4000 Xg at 4℃for 15min, the supernatant was frozen in a-80℃refrigerator, 20. Mu.L of plasma samples were taken and 80. Mu.L (V: V, 1:4) acetonitrile was added to precipitate proteins in the plasma samples, while deuterated trimethylamine oxide (d 9-TMAO) was added to the plasma samples at a final concentration of 2.0. Mu.M as an internal standard. Mixing, standing at-80deg.C for 2 hr, sucking supernatant into sample bottle at 4deg.C for 12000 Xg 15min, storing in-80deg.C refrigerator, and measuring plasma TMAO level by HPLC-MS/MS.
Plasma TMAO experimental results As shown in FIG. 2, the plasma TMAO of the model group apoE-/-mice is significantly higher than that of the control group, the level of plasma TMAO is significantly reduced by the Lactobacillus reuteri CCFM1135 compared with the model group apoE-/-mice, and the plasma TMAO is reduced by 29.65% by the apoE-/-mice of the Lactobacillus reuteri CCFM1135 compared with the model group apoE-/-mice. Lactobacillus reuteri FHNXY12L1 had no significant effect on TMAO levels in plasma.
Example 2: effect of Lactobacillus reuteri CCFM1135 on mouse intestinal Roseburia
Grouping, modeling and treatment of apoE-/-mice were as in example 2.
At the end of the test, four groups of apoE-/-mice are fasted and forbidden for 12 hours, after the anesthesia of 10% chloral hydrate solution is injected into the abdominal cavity, cecum is taken, cecum DNA is extracted according to a fecal DNA kit method, and a second-generation sequencer is used for carrying out 16S rDNA flora analysis on the V3-V4 region of the cecum.
The experimental results are shown in FIG. 3. The relative abundance of the cecum Roseburia of the model group apoE-/-mice is obviously lower than that of the control group, compared with the model group apoE-/-mice, the relative abundance of the cecum Roseburia of the lavage lactobacillus reuteri CCFM1135 is obviously improved, compared with the model group apoE-/-mice, the relative abundance of the cecum Roseburia of the lavage lactobacillus reuteri CCFM1135 is improved by 8.43 times, and the risks of irritable bowel syndrome, obesity, allergy, neurological diseases and type II diabetes can be reduced.
Example 3: effect of Lactobacillus reuteri CCFM1135 on the mouse intestinal tract Ruminococcaceae UCG-009 genus
Grouping, modeling and treatment of apoE-/-mice were as in example 2.
At the end of the test, four groups of apoE-/-mice are fasted and forbidden for 12 hours, after the anesthesia of 10% chloral hydrate solution is injected into the abdominal cavity, cecum is taken, cecum DNA is extracted according to a fecal DNA kit method, and a second-generation sequencer is used for carrying out 16S rDNA flora analysis on the V3-V4 region of the cecum.
The experimental results are shown in FIG. 4. The relative abundance of the cecum Ruminococcaceae UCG-009 of the model group apoE-/-mice is significantly higher than that of the control group, the relative abundance of the cecum Ruminococcaceae UCG-009 of the lactobacillus reuteri CCFM1135 is significantly reduced compared with the model group apoE-/-mice, the relative abundance of the cecum Ruminococcaceae UCG-009 of the lactobacillus reuteri CCFM1135 is reduced by 71.33% compared with the model group apoE-/-mice, and the risks of obesity, non-alcoholic fatty liver, hyperlipidemia and hypertension can be reduced.
Example 4: comparison of the effect of different Lactobacillus reuteri on apoE-/-mouse plasma TMAO
The apoE-/-mice were subjected to the same molding and treatment as in example 1, and 16 different strains of Lactobacillus reuteri were used to perfuse the apoE-/-mice with the respective strains, and plasma TMAO was detected, as shown in FIG. 5, only CCFM1135 had a significant reduction in the apoE-/-mice plasma TMAO, a reduction of 29.65%, and none of the remaining Lactobacillus reuteri had a significant reduction in the apoE-/-mice plasma TMAO.
Example 5: fermented food prepared from Lactobacillus reuteri CCFM1135
Cleaning fresh fructus Mali Pumilae, squeezing, performing high-temperature instant sterilization, and sterilizing at 140deg.CAfter 2 seconds of high-temperature sterilization, the temperature is immediately reduced to 37 ℃, and then the lactobacillus reuteri CCFM1135 microbial inoculum starter prepared by the invention is inoculated to ensure that the concentration reaches 10 percent 8 And (3) refrigerating and preserving the mixture at the temperature of 4 ℃ above CFU/mL, so as to obtain the fruit and vegetable beverage containing the lactobacillus reuteri CCFM1135 viable bacteria.
The lactobacillus reuteri CCFM1135 can be used for producing other fermented foods by fermentation, wherein the fermented foods comprise solid foods, liquid foods and semi-solid foods. The fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
The fermented food prepared by the invention is fed to a high choline model mouse, and can also reduce the level of plasma TMAO; can improve the structure of intestinal flora, recover intestinal flora disorder caused by high choline, improve Pielou uniformity index, observed species richness index and shannon diversity index, improve the abundance of beneficial bacteria Roseburia, reduce the abundance of harmful bacteria Ruminococcaceae UCG-009, and reduce the risks of intestinal stress syndrome, obesity, allergy, neurological diseases, type II diabetes, nonalcoholic fatty liver, hyperlipidemia and hypertension.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. Lactobacillus reuteri @Lactobacillus reuteri) Use of CCFM1135 in the manufacture of a medicament for the prevention and/or treatment of cardiovascular disease; the lactobacillus reuteri CCFM1135 is preserved in the microorganism strain preservation center of Guangdong province at 7 months and 24 days in 2020, and the preservation address is 59 th floor 5 building Guangdong province microorganism research institute of Mitsui No. 100 in Guangzhou city, and the preservation number is GDMCC No.61102.
2. The use according to claim 1, wherein the amount of lactobacillus reuteri CCFM1135 in the medicament is not less than 1 x 10 6 CFU/mL or ≡1X10. 6 CFU/g。
3. The use according to claim 1, wherein the pharmaceutical product has at least one of the following actions:
(1) Lowering plasma TMAO levels in high choline diet mammals;
(2) Improving high choline diet mammalRoseburiaAbundance of genus;
(3) Reducing high choline diet in mammalsRuminococcaceaeAbundance of UCG-009.
4. The use according to claim 1, wherein the medicament comprises lactobacillus reuteri CCFM1135 and/or a pharmaceutically acceptable carrier.
5. The use according to claim 4, wherein the carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
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