CN115624572A - Bifidobacterium breve CCFM683 capable of intervening in metabolic syndrome and application thereof - Google Patents

Bifidobacterium breve CCFM683 capable of intervening in metabolic syndrome and application thereof Download PDF

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CN115624572A
CN115624572A CN202211328357.3A CN202211328357A CN115624572A CN 115624572 A CN115624572 A CN 115624572A CN 202211328357 A CN202211328357 A CN 202211328357A CN 115624572 A CN115624572 A CN 115624572A
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bifidobacterium breve
metabolic syndrome
ccfm683
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杨波
陈卫
郑富莉
陈洋
陈海琴
刘小鸣
赵建新
张灏
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Jiangnan University
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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
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    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • 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

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Abstract

The invention discloses bifidobacterium breve CCFM683 capable of intervening in metabolic syndrome and application thereof, belonging to the technical field of microorganisms. The bifidobacterium breve CCFM683 screened by the invention can obviously reduce the fasting blood glucose of a mouse with metabolic syndrome; significantly reducing the insulin resistance index of a mouse with metabolic syndrome; significantly reducing the daily dietary intake of metabolic syndrome mice; significantly reducing the white fat index and increasing the brown fat index of a mouse with metabolic syndrome; can regulate serum low density lipoprotein cholesterol level, and reduce serum low density lipoprotein cholesterol level to normal level. The bifidobacterium breve CCFM683 can be used for preparing medicines for relieving metabolic syndrome, non-alcoholic fatty liver, diabetes and other diseases, and has very wide application prospect.

Description

Bifidobacterium breve CCFM683 capable of intervening in metabolic syndrome and application thereof
Technical Field
The invention relates to bifidobacterium breve CCFM683 capable of intervening in metabolic syndrome and application thereof, belonging to the technical field of microorganisms.
Background
The Metabolic Syndrome (MS) is a group of clinical syndromes that the manifestations of central obesity, hyperglycemia (diabetes or impaired glucose regulation), dyslipidemia (including high TG and/or low HDL-C blood disease) and hypertension exist simultaneously, multiple risk factors based on pathological changes of glucose metabolism and lipid metabolism are aggregated, and various cardiovascular and cerebrovascular diseases such as type II diabetes and atherosclerosis are promoted to develop. In recent years, with the continuous development of the economic and social level of China, the living standard of the public is continuously improved, the specific gravity of carbohydrates and grease in the dietary structure is increased day by day, and the people have a habit of sedentary sitting due to the limitation of various working conditions. The external environmental factors are combined with the genetic factors of the external environmental factors to greatly improve the prevalence rate of the metabolic syndrome of the population in China. It has been shown that about 7700 million patients with metabolic syndrome exist in the country, i.e. 1 in 8 adults have metabolic syndrome. The prevalence rate of metabolic syndrome of old people over 60 years old in China is 25%, and the prevalence rate gradually increases with age. Therefore, it is necessary to find a suitable means for relieving metabolic syndrome.
The treatment measures adopted aiming at the metabolic syndrome at present mainly comprise strengthening exercise and improving diet and combining drug therapy, and the aims are all various risk factors for controlling and improving the metabolic syndrome, and mainly comprise weight-reducing drugs such as orlistat and lorcaserin; insulin resistance reducing agents such as metformin, thiazolidinediones; fibrates that lower blood lipids such as fenofibrate; statins that lower cholesterol such as simvastatin; angiotensin converting enzyme inhibitor for lowering blood pressure such as captopril. These drugs have good efficacy against the single risk factors of metabolic syndrome, but long-term administration of these drugs causes side effects on gastrointestinal and hepatic renal functions.
A great deal of research reports that the intestinal flora plays an extremely important role in the physiological metabolism of human bodies, and the structural disorder of the intestinal flora is related to various diseases, including gastrointestinal diseases (colitis, constipation and the like) and metabolic diseases (obesity, hyperlipidemia, diabetes and the like). The occurrence of metabolic syndrome is closely related to the imbalance of intestinal flora, and common intestinal flora regulating preparations comprise probiotics, prebiotics and the like. The probiotics is edible microorganisms beneficial to human health, and has the potential functions of relieving blood sugar and dyslipidemia and regulating intestinal flora structure.
Disclosure of Invention
The invention provides application of Bifidobacterium breve (Bifidobacterium breve) CCFM683 in preparing medicaments for preventing and/or relieving metabolic syndrome; the Bifidobacterium breve (Bifidobacterium breve) CCFM683 is preserved in the China general microbiological culture collection center on 12 days 04 of 2015, the preservation number is CGMCC No.11828, and is disclosed in a patent application document with the publication number of CN 106038611A.
In one embodiment, the prevention and/or alleviation of metabolic syndrome is specifically embodied in:
(1) Significantly reducing fasting blood glucose in a mammal with metabolic syndrome;
(2) Significantly reducing the insulin resistance index in a mammal with metabolic syndrome;
(3) Significantly reducing the daily dietary intake of a mammal with metabolic syndrome;
(4) Significantly reduces the white fat index and increases the brown fat index of a mammal with metabolic syndrome;
(5) Regulating low density lipoprotein cholesterol content in blood serum of mammal with metabolic syndrome to reduce it to normal level.
In one embodiment, the symptoms exhibited by metabolic syndrome include hyperglycemia, hyperlipidemia, and/or hepatic oxidative stress.
In one embodiment, the content of Bifidobacterium breve CCFM683 in the medicament is more than or equal to 1 x 10 8 CFU/g or 1X 10 8 CFU/mL。
In one embodiment, the medicament further comprises a pharmaceutically acceptable excipient.
In one embodiment, the pharmaceutically acceptable excipients include one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents.
In one embodiment, the dosage form of the medicament is granules, capsules, tablets, pills or oral liquid.
In one embodiment, the medicament is a microbial preparation comprising wet cells or lyophilized cells of said bifidobacterium breve CCFM 683.
In one embodiment, the microbial preparation has a viable count of Bifidobacterium breve CCFM683 of 1 × 10 or more 6 CFU/g。
In one embodiment, the method of preparing the microbial preparation comprises:
(1) Culturing said bifidobacterium breve CCFM683 in a suitable culture medium;
(2) Collecting said Bifidobacterium breve CCFM683 cells, and mixing said Bifidobacterium breve CCFM683 cells with a protectant.
In one embodiment, the preparation method specifically comprises the following steps: inoculating Bifidobacterium breve CCFM683 into mMRS culture medium, culturing at 37 deg.C under anaerobic condition for 18-20 hr, collecting thallus, and resuspending thallus cells with protective agent to make the concentration of thallus reach 1 × 10 10 CFU/mL, culturing the suspension at 37 deg.C under anaerobic condition for 50-70min, and drying.
In one embodiment, the mrs medium contains per liter: 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 80 mL of tween and 0.5g of cysteine.
In one embodiment, the protective agent comprises one or more of skimmed milk powder, maltodextrin, trehalose.
In one embodiment, the protectant comprises: 100g/L-150g/L skimmed milk powder, 100g/L-150g/L maltodextrin and 140g/L-160g/L trehalose.
In one embodiment, the collected bacterial cells are washed 2-4 times with a phosphate buffer having a pH of 6.8-7.2.
In one embodiment, the drying may be performed by any one of a number of methods including, but not limited to, vacuum freeze drying.
In one embodiment, the vacuum freeze-drying is performed after pre-freezing at-15 to-20 ℃ for 8 to 14 hours.
The invention also provides application of the Bifidobacterium breve (Bifidobacterium breve) CCFM683 in preparing health care products for maintaining healthy blood fat level and/or healthy blood sugar level.
In one embodiment, the nutraceutical includes, but is not limited to, probiotic powder, probiotic fermented juice, and the like.
The invention also provides application of the bifidobacterium breve CCFM683 in preparing a medicament for preventing and/or relieving abnormal rise of blood sugar and/or abnormal rise of serum low-density cholesterol caused by high-fat diet.
Has the advantages that: the invention provides a new application of bifidobacterium breve CCFM683 in improving metabolic syndrome, and specifically, the improving metabolic syndrome comprises the following steps: reducing fasting blood glucose levels in individuals with metabolic syndrome; reducing the insulin resistance index in individuals with metabolic syndrome; reducing the daily dietary intake of a metabolic syndrome mouse; reducing white fat and increasing brown fat in individuals with metabolic syndrome; reducing serum low density lipoprotein cholesterol in individual with metabolic syndrome. The bifidobacterium breve CCFM683 can be used for preparing health care products or medicines for relieving metabolic syndrome and has very wide application prospect.
Drawings
FIG. 1 shows the effect of Bifidobacterium breve CCFM683 on fasting plasma glucose in mice with metabolic syndrome;
FIG. 2 shows the effect of Bifidobacterium breve CCFM683 on the insulin resistance index in mice with metabolic syndrome;
FIG. 3 shows the effect of Bifidobacterium breve CCFM683 on daily dietary intake in mice with metabolic syndrome;
FIG. 4 shows the effect of Bifidobacterium breve CCFM683 on the white and brown fat indices of mice with metabolic syndrome;
FIG. 5 shows the effect of Bifidobacterium breve CCFM683 on serum low density lipoprotein cholesterol (LDL-C) in mice with metabolic syndrome;
(Note: p <0.05, p <0.01, p <0.001, p < 0.0001).
Detailed Description
The invention will be better understood from the following examples.
In the present invention, "%" or percentages used to indicate concentrations or ratios are weight percentages unless otherwise specified.
Example 1: bifidobacterium breve CCFM683 regulating effect on fasting blood glucose of mice with metabolic syndrome
24 healthy male C57BL/6J mice weighing 20-21g were acclimated for 1 week and randomized into 3 groups: blank control group (NC), high fat model control group (HFD), bifidobacterium breve CCFM683 priming group (CCFM 683), with 8 mice per group. Culturing Bifidobacterium breve CCFM683, mixing bacterial sludge with protectant (100 g/L-150g/L skimmed milk powder, 100g/L-150g/L maltodextrin, 140g/L-160g/L trehalose) according to 1:2 (w/v, 1g bacterial sludge: 2mL protectant solution), pre-freezing at-15-20 deg.C for 8-14h, vacuum freeze-drying to obtain freeze-dried bacterial powder, suspending the freeze-dried bacterial powder in physiological saline to make the concentration of Lactobacillus reuteri in physiological saline suspension be 1 × 10 10 CFU/mL, used for gavage of mice, and the grouping and treatment method of experimental animals are shown in Table 1.
TABLE 1 groups of experimental animals
Group of Number/group only Often times of treatment Feed stuff Treatment method (intragastric administration every day)
NC 8 For 12 weeks Control feed 0.2mL of physiological saline + freeze-drying protective agent
HFD
8 For 12 weeks High-fat feed 0.2mL of physiological saline + freeze-drying protective agent
CCFM683
8 For 12 weeks High-fat feed 0.2mL of physiological saline suspension containing CCFM683
The body weight of the mice was monitored periodically during the experiment. At the end of the experiment, mice were fasted for 12h without water deprivation and fasting blood glucose was measured. After anesthesia by injecting 10% chloral hydrate solution into abdominal cavity, blood is collected from heart, and then killed by cervical dislocation. Centrifuging the blood sample at 4 deg.C for 15min at 3000 Xg, collecting supernatant, and freezing at-80 deg.C for measuring related serum index. Collecting part of liver, rapidly placing in pre-cooled normal saline for rinsing and removing blood, placing in paraformaldehyde for fixation, freezing the rest part of liver in liquid nitrogen at a medium speed, transferring to-80 ℃ for freezing storage, and subsequently preparing into liver homogenate for measuring related indexes, wherein the specific preparation method comprises the following steps: weighing a certain amount of liver tissue, and carrying out the following steps: adding physiological saline at a ratio of 9, grinding tissue, centrifuging at 3000r for 10min, and freezing the supernatant at-80 deg.C.
The results are shown in FIG. 1. After the model group mice are modeled, the fasting blood glucose reaches 6.32mmol/L. After the bifidobacterium breve CCFM683 is perfused, the fasting blood sugar of the mice is reduced to 5.60mmol/L which is close to 4.81mmol/L of the blank group, and the bifidobacterium breve CCFM683 has positive regulation effect on the blood sugar of the mice with the metabolic syndrome.
Example 2: bifidobacterium breve CCFM683 lowering insulin resistance index
Grouping, modeling and treatment methods of C57BL/6J mice were the same as in example 1, and insulin resistance index was measured (method is referred to Food funct.2021,12 (9): 3919-3930.). The results are shown in fig. 2, the insulin resistance index of the mice in the model group reaches 2.35, and after the administration of the bifidobacterium breve CCFM683, the insulin resistance index of the mice is reduced by 20 percent and reaches 1.88, which is close to 1.58 of the blank group, and the result indicates that the bifidobacterium breve CCFM683 has the function of regulating and controlling insulin and further has the function of improving blood sugar.
Example 3: bifidobacterium breve CCFM683 for reducing daily dietary intake of mice with metabolic syndrome
Grouping, modeling and treatment methods of C57BL/6J mice are the same as example 1. The dietary intake was calculated by weighing. The results are shown in FIG. 3. The daily dietary intake of the model group mice was significantly increased to 12.73 kcal/day/each, while the perigastric bifidobacterium breve CCFM683 significantly decreased the daily dietary intake of the mice, resulting in a dietary intake of 10.64 kcal/day/each, approaching 9.67 kcal/day/each of the blank group. The bifidobacterium breve CCFM683 is suggested to have a certain regulation effect on the food consumption of the mice with high fat diet.
Example 4: bifidobacterium breve CCFM683 decreases white fat index and increases brown fat index
The grouping, modeling and treatment methods of the C57BL/6J mice are the same as example 1. Dissecting, collecting epididymal fat and brown fat, weighing, and measuring white fat index and brown fat index. The results are shown in FIG. 4. The white fat index of the mice in the model group is obviously increased, the white fat index (white fat/body weight) of the mice is obviously reduced by the bifidobacterium breve through gastric lavage CCFM683, the white fat index (white fat/body weight) is reduced by 32.3 percent relative to the model group, the brown fat index (brown fat/body weight) is increased and is 1.35 times of that of the model group, and the white fat/brown fat value of the mice through gastric lavage is obviously reduced by 44.1 percent relative to the model group.
Example 5: bifidobacterium breve CCFM683 for reducing serum low density lipoprotein cholesterol (LDL-C) level of mice with metabolic syndrome
Grouping, modeling and treatment methods of C57BL/6J mice are the same as example 1, and at the end of the test, the mice are fasted and are not forbidden to be watered for 12 hours, and after 10% chloral hydrate solution is injected into the abdominal cavity for anesthesia, blood is collected from the heart. The blood sample was centrifuged at 3000 Xg at 4 ℃ for 15min, the supernatant was collected, and the content of low-density lipoprotein cholesterol (LDL-C) in the blood was measured by the detection method of the kit.
The results are shown in FIG. 5. The content of serum low-density lipoprotein cholesterol (LDL-C) of mice in a high-fat diet group is increased to 1.07mmol/L, and the content of serum low-density lipoprotein cholesterol can be reduced to 0.69mmol/L by virtue of the bifidobacterium breve CCFM683, which indicates that the bifidobacterium breve CCFM683 has a positive effect on blood lipid metabolism.
Example 6: preparation of microbial preparation containing Bifidobacterium breve CCFM683
mMRS culture medium: 10g of tryptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate monohydrate, 80 mL of Tween, 0.5g of cysteine, and sterilizing at the temperature of 119-123 ℃ for 15-25min, wherein the volume of water is up to 1000mL, the pH value is adjusted to 6.5, and the sterilization is carried out for 15-25min.
A protective agent: 100g/L-150g/L skimmed milk powder, 100g/L-150g/L maltodextrin, 140g/L-160g/L trehalose.
Inoculating Bifidobacterium breve CCFM683 into mMRS culture medium, culturing at 37 deg.C under anaerobic condition for 18-20 hr, collecting thallus, resuspending thallus cells with protective agent to make the concentration reach 10 10 CFU/mL, then culturing the suspension at 37 deg.C under anaerobic condition for 50-70min, and drying.
Optionally, the drying is vacuum freeze drying after pre-freezing for 8-14h at-15 to-20 ℃.
Example 7: capsule product prepared from bifidobacterium breve
The bifidobacterium breve CCFM683 is cultured on mMRS culture medium for 24 hours, centrifuged for 20 minutes at 4 ℃ and 4000r/min, washed twice by PBS, added with 4 percent of skimmed milk powder and 6 percent of lactose which are mixed according to the weight of the powder containing the bifidobacterium breve CCFM683 for 10 minutes, added with sterile 2 percent of calcium chloride and 3 percent of sodium alginate, stirred for 10 minutes at 150r/min, then statically solidified for 30 minutes, finally cleaned and filtered, and the obtained filtrate is frozen and dried for 20 hours to obtain powder containing the bifidobacterium breve CCFM683, and the powder is filled into commercial medicinal microcapsules to obtain the capsule product.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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 (10)

1. Application of Bifidobacterium breve (Bifidobacterium breve) CCFM683 in preparing medicines for preventing and/or relieving metabolic syndrome is characterized in that the preservation number of the Bifidobacterium breve (Bifidobacterium breve) CCFM683 is CGMCC No.11828.
2. Use according to claim 1, characterized by comprising at least one of the following functions:
(1) Lowering fasting blood glucose in a mammal with metabolic syndrome;
(2) Reducing the insulin resistance index in a mammal with metabolic syndrome;
(3) Reducing the daily dietary intake of a mammal with metabolic syndrome;
(4) Reducing the white fat index and increasing the brown fat index in a mammal with metabolic syndrome;
(5) Reducing the serum low density lipoprotein cholesterol level in mammals with metabolic syndrome.
3. The use according to claim 1 or 2, wherein the Bifidobacterium breve CCFM683 is present in the medicament in an amount of ≥ 1 x 10 8 CFU/g or 1X 10 8 CFU/mL。
4. The use according to any one of claims 1 to 3, wherein the medicament is a microbial preparation comprising wet or lyophilized cells of said Bifidobacterium breve CCFM 683.
5. The use according to claim 4, wherein the microbial preparation is prepared as follows:
(1) Culturing said bifidobacterium breve CCFM683 in a suitable culture medium;
(2) Collecting said Bifidobacterium breve CCFM683 cells, and mixing said Bifidobacterium breve CCFM683 cells with a protectant.
6. The use according to claim 5, wherein the protective agent comprises one or more of skimmed milk powder, maltodextrin, trehalose.
7. The use according to any one of claims 1 to 3, wherein the medicament further comprises a pharmaceutically acceptable excipient.
8. The composition of claim 7, wherein the pharmaceutically acceptable excipients comprise one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents.
9. The medicament according to any one of claims 1 to 7, wherein the dosage form of the medicament is granules, capsules, tablets, pills or oral liquid.
10. Application of Bifidobacterium breve (CCFM 683) in preparing health product for maintaining healthy level of blood lipid and/or blood glucose is provided.
CN202211328357.3A 2022-10-26 2022-10-26 Bifidobacterium breve CCFM683 capable of intervening in metabolic syndrome and application thereof Pending CN115624572A (en)

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