CN117122619A - Use of Akkermansia muciniphila for improving cognitive function - Google Patents

Use of Akkermansia muciniphila for improving cognitive function Download PDF

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CN117122619A
CN117122619A CN202311047605.1A CN202311047605A CN117122619A CN 117122619 A CN117122619 A CN 117122619A CN 202311047605 A CN202311047605 A CN 202311047605A CN 117122619 A CN117122619 A CN 117122619A
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akkermansia muciniphila
mice
cfu
disease
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王炳华
喻扬
郁雪平
马新
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Thankcome Biotechnology Suzhou Co ltd
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Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of Akkermansia muciniphila in improving cognitive functions. The invention provides an application of Akkermansia muciniphila in improving cognitive function, wherein Akkermansia muciniphila is preserved in CGMCC with the preservation number of CGMCC No.20955. The Akkermansia muciniphila can obviously improve cognitive functions, increase the content of the anti-inflammatory factors IL-4 and IL-10, obviously lower the expression quantity of the inflammatory factors and the apoptosis factors, obviously up-regulate the expression quantity of Abeta degrading enzyme IDE, protect the normal form of colon and have no toxicity to liver.

Description

Use of Akkermansia muciniphila for improving cognitive function
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of Akkermansia muciniphila in improving cognitive functions.
Background
Neurodegenerative diseases are a group of diseases in which the motor or psychological functions of the central or peripheral nervous system are gradually impaired, and their pathological features are abnormal aggregation and deposition of proteins in the brain and spinal cord. Neurodegenerative diseases mainly include Parkinson's Disease (PD), alzheimer's Disease (AD), huntington's Disease (HD), amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS), frontotemporal dementia (Frontotemporal dementia, FTD), and the like. The histopathological features of these diseases are similar, with common cellular and molecular mechanisms. The neurodegenerative diseases known today are divided into two categories according to the condition of the patient at the time of onset: can impair the memory and cognitive ability of the patient, ultimately causing dementia in the patient; the second is to influence the patient's movement and balance.
Huntington's disease, the onset of which is generally the main cause of the middle-aged, can impair the memory and cognitive abilities of the patient, and can also affect the motor abilities of the patient. Mainly manifested as choreography and neurological dysfunction, patients usually die within 10 to 20 years after onset. Frontotemporal dementia, which generally occurs in the early senile stage, mainly involves degeneration and atrophy of frontal and temporal lobe areas of the brain of the patient, thereby impairing the linguistic and motor abilities of the patient. Dementia in patients can result three to four years after frontotemporal lobar degeneration has occurred. Amyotrophic lateral sclerosis is a neurodegenerative disease that is a major occurrence in adults, and a part of cases of this disease may be associated with genetic and familial genetic defects. ALS is mainly characterized by the death of motor neurons, and with the progression of the disease, symptoms such as respiratory failure, muscle atrophy, dysphagia, etc. appear. Patients will typically die from respiratory failure 3 to 5 years after onset.
Alzheimer's Disease (AD) is one of the most common types of senile dementia, and is clinically manifested by deterioration of cognitive function and memory capacity, accounting for 50% -75% of all dementias, and is also the most frequently occurring neurodegenerative disease (Thies, et al 2012Alzheimer's disease facts and figures. [ J ]. Alzheimer & Dementia the Journal of the Alzheimers Association,2012,8 (2): 131-168.). The brains of AD patients exhibit total brain atrophy, with dense Senile Plaques (SP) formed by the deposition of aggregates of beta amyloid polypeptide (Amyloid beta peptide, aβ) and a substantial loss of neurofibrillary tangles (Neurofibrillary tangles, NFT) and neurons caused by hyperphosphorylation of Tau protein (microcable-associated protein Tau) in the brains (jakobroetone, et al, alzheimer's disease: from pathology to therapeutic approaches. [ J ]. Angew Chem Int Ed Engl,2010,40 (25): 3030-3059.).
There are numerous theories about the pathogenesis of Alzheimer's disease, and the "amyloid cascade hypothesis" is now relatively well-established. The amyloid hypothesis was first proposed by Hardy et al in 1992 that one of the characteristic pathological changes in AD is the formation of senile plaques whose core component is beta-amyloid. Abnormal deposition of aβ with neurotoxicity in the brain can form senile plaques. Abnormal accumulation and aggregation of Abeta can initiate pathological cascades, induce Tau aberrant phosphorylation followed by formation of neurofibrillary tangles, induce neuroinflammation and oxidative stress in the brain, etc., followed by extensive neuronal loss (Hardy J A, et al, alzheimer 'S disease: the amyloid cascade hypothesis. [ J ]. Science,1992,256 (5054): 184-185.; hardy, et al, alzheimer' S disease: the amyloid cascade hypothesis: an update and reappraisal. [ J ]. Journal of Alzheimers Disease,2006,9 (S3): 151-153.).
The aging is a main risk factor for inducing the neurodegenerative diseases, and the neurodegenerative diseases seriously endanger the health of the old, so that the search for a safe and effective treatment method has important significance.
In recent years, with the intensive research of intestinal flora, the theory of microorganism-intestinal-brain axis is proposed, and intestinal microorganisms regulate gastrointestinal functions and simultaneously can influence central nervous system functions through a nerve-immune-endocrine system. The study found that intestinal microbiomes changed in various neurodegenerative diseases, indicating that the occurrence and development of neurodegenerative diseases had a significant effect on intestinal microbiomes (LOPEZ-thin C, et al, the hallmarks of aging. [ J ]. Cell,2013,153 (6): 1194.121). Thus, the use of microorganisms to improve and/or treat neurodegenerative diseases is a new option.
U.S. patent application US17780436 discloses a composition and method for treating or preventing progression of neurodegenerative disease, the composition comprising a bacterial composition having an effective amount of a live non-pathogenic microorganism, a live non-pathogenic bacterium, wherein at least one of the bacteria is a predatory bacterium, such as bdellovibrio bacteriovorus; the disclosed bacterial compositions may be used to treat or prevent progression of neurodegenerative diseases such as ALS, alzheimer's disease, huntington's disease, and parkinson's disease.
Chinese patent application 201880039628.9 discloses a composition comprising a bacterial strain of the genus megacoccus for use in the treatment or prevention of neurodegenerative disorders, for use in the treatment or prevention of early-onset neurodegenerative disorders, and for use in the prevention or delay of onset or progression of neurodegenerative disorders.
In the field of neurodegenerative diseases, in particular alzheimer's disease, there is still a need for more methods and medicaments for the treatment and/or prevention of neurodegenerative diseases with microorganisms.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an application of Akkermansia muciniphila PROBIO (hereinafter referred to as AKK PROBIO) in improving cognitive functions.
The inventor creatively separates a strain Akkermansia muciniphila from healthy human bodies, and the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.20955 and the preservation date of 10 months and 26 days in 2020, and performs morphological classification and biochemical genetic function on the strain to identify and characterize the strain, thereby exploring the application of the strain in different fields (other patent applications are filed on the same day).
The inventors surprisingly found that AKK proxio achieved unexpected positive effects in a number of fields of anti-tumor, cognitive, metabolic, inflammatory, osteoarticular, cardiovascular and the like. However, in view of the practice of the chinese patent law, the protection is now applied separately for each indication in which there is no uniqueness.
The application aims at protecting the application of AKK PROBIO in improving cognitive functions.
Based on the practice of Chinese patent law, the application is created for avoiding the same application submitted on the same day. The applicant only records the related experimental data such as the morphological classification identification and the biochemical genetic property characterization of the microorganism in the microorganism patent application filed on the same day, and the application does not record the related information of the microorganism or protect the microorganism. In order to facilitate the examination work of the present application and to quickly understand the previous work of the inventors, information about AKK profile will now be briefly disclosed in the summary of the present application.
1. Basic information of AKK PROBIO
The conclusive information in this section is for facilitating the examination of the present invention and is not intended to limit the scope of the invention. The specific experimental process and experimental results are recorded in the microbial patents submitted on the same day as the invention.
1. Akkermansia muciniphila CGMCC No.20955 obtained by screening and separation by the inventor;
2. akkermansia muciniphila CGMCC No. 20955;
3. akkermansia muciniphila CGMCC No.20955 16s rRNA;
4. akkermansia muciniphila CGMCC No.20955 physiological and biochemical properties:
(1) Hydrophobicity: rise with time, and reach more than 30% at 60 min;
(2) Self-aggregation: rise with time, and tend to be stable at 20h, and remain at about 52%;
(3) Gastrointestinal fluid tolerance:
the survival rate in gastric juice is in a decreasing trend along with the time extension, and the survival rate is about 85% at 240 min;
the survival rate in intestinal juice is in a decreasing trend as a whole, and the survival rate is more than 80% at 240 min;
(4) Biofilm formation ability: weakness;
(5) Acute toxicity experiment:
the result of a bacterial back mutation test shows that the strain has no mutagenicity;
the mice have no death phenomenon through acute gastric lavage of Akkermansia muciniphila CGMCC No.20955 with high, medium and low doses;
No significant difference was observed between the daily weight change of the mice in the test group and the control saline group;
during acute gastric lavage, there was no significant difference between daily changes in food intake of the mice tested and the mice in the control saline group;
during acute gastric lavage, there was no significant difference between daily blood glucose changes in the mice tested and the control saline group mice;
the blood indexes of the mice in the acute gastric lavage group and the control physiological saline group have no obvious difference;
in the liver, the triglyceride level in the liver of the tested mice is extremely lower than that of the mice in the control physiological saline group;
the triglyceride level in serum was not significantly different in the test mice from the control mice;
the cholesterol levels of the mice in the test group and the mice in the control group have no significant difference in serum and liver;
the bile acid levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;
the glucose levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;
the total protein levels of the mice in the tested group and the mice in the control group are not significantly different in serum and liver;
the glutamic pyruvic transaminase enzyme activities of the tested mice and the mice of the control group are not significantly different in serum and liver;
The glutamic-oxaloacetic transaminase enzyme activities of the mice in the tested group and the mice in the control group have no significant difference in serum and liver;
the creatinine content of the mice in the test group and the creatinine content of the mice in the control group are not significantly different in serum;
the urea nitrogen concentration of the mice in the test group and the mice in the control group are not significantly different in serum;
the weight of main organs such as heart, liver, spleen, kidney, thymus, brain, testis, lung, stomach, intestine and the like of the tested mice and the mice of the control group are basically not obviously different;
the liver and kidney of the mice in the tested group and the mice in the control group have no obvious pathological damage.
(6) Sub-chronic toxicity test:
mice were perfused with AKK PROBIO at high, medium, and low doses for 90 consecutive days without mortality;
no significant difference was observed between weekly weight changes in mice in the test group and the control saline group;
during the continuous gastric lavage period, no significant difference exists between the feed intake change of the tested mice and the mice in the control physiological saline group every week;
during continuous lavage, there was a significant difference between the blood glucose change of the weekly mid-dose lavage mice and the control saline group mice, and no significant difference between the other groups and the control group;
the blood indexes of the mice in the gastric lavage group and the control physiological saline group have no obvious difference;
The triglyceride levels of the mice in the test group and the mice in the control group have no significant difference in liver and serum;
the cholesterol levels of the mice in the test group and the mice in the control group have no significant difference in liver and serum;
the bile acid levels of the mice in the intragastric dosage and the mice in the control group have significant differences in the liver, while the other groups have no significant differences in serum and liver from the control group;
the glucose levels of the mice in the test group and the mice in the control group are not significantly different in liver and serum;
the total protein content of the mice in the tested group and the mice in the control group has no significant difference in liver and serum;
the glutamic pyruvic transaminase enzyme activities of the tested mice and the mice of the control group are not significantly different in serum and liver;
the glutamic-oxaloacetic transaminase enzyme activities of the mice in the tested group and the mice in the control group have no significant difference in serum and liver;
the creatinine content of the mice in the test group and the creatinine content of the mice in the control group are not significantly different in serum;
the urea nitrogen concentration of the mice in the test group and the mice in the control group are not significantly different in serum;
the weight of main organs such as heart, liver, spleen, kidney, thymus, brain, testis, lung, pancreas, stomach and intestine of the tested mice and the mice of the control group are basically not significantly different;
The liver and the kidney of the mice in the tested group and the mice in the control group have no obvious pathological damage;
the blood sugar regulating ability of the tested mice and the mice of the control group is not significantly different.
The technical object of the present invention is to provide a microorganism which can improve cognitive function.
(7) Drug sensitivity analysis:
the AKK PROBIO is sensitive to ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline, moxifloxacin and chloramphenicol.
Intestinal microbiota (glut microbiota), which is a microorganism designated to be planted in the digestive tract of the human body, is a large number and complex species, and is composed of bacteria, fungi, viruses and protozoa. Since bacterial studies are most widespread, the intestinal microbiota is also commonly known as intestinal microbiota (backsed F, et al host-bacterial mutualismin the human intestine [ J ]. Science,2005,307 (5717):1915-1920.). The intestinal flora not only assists the host in digesting food and providing nutritional ingredients, but also has a profound effect on the health of the host. Studies have shown that: the structure and composition of different intestinal microorganisms affect a variety of important physiological activities such as nutrient processing, energy balance, immune function, gastrointestinal development and the like of a host, and intestinal flora plays an important role in various aspects such as growth, development, metabolism and the like of a host (Wen L, et al, innate immunity and intestinal microbiota in the development of Type 1diabetes, J. Nature,2008,455 (7216):1109-1113.).
The aim of improving the health state of human body can be achieved by adjusting the structure of intestinal flora based on the important role of the intestinal flora in the regulation of host metabolism and low-grade inflammation, and the probiotics developed based on the intestinal home bacteria are expected to become a new mode for treating a plurality of diseases. Probiotics (Probiotics) refers to the general term for beneficial microorganisms that survive in the human intestinal tract after ingestion by a host in sufficient quantity and are capable of producing a definite health effect. The different species of probiotics exert their respective different functions, and the probiotics are broadly classified into conventional lactic acid probiotics, non-lactic acid probiotics and next generation probiotics, the conventional probiotics mainly play roles in prevention and health care, and the research direction of the next generation probiotics is focused on the treatment and cure of diseases (Botina SG, et al, classification of domestic probiotic cultures of Lactobacillus genus. [ J ]. Zh Mikrobiol Epidemiol Immunobiol,2010 (5): 3-7.). Wherein the next generation probiotics mainly comprise Acremonium, bacteroides fragilis, clostridium tenecum and the like. Akkermansia muciniphila is taken as a potential probiotic, is a main research object of next-generation probiotics, and has wide medical application prospect.
Akkermansia muciniphila is an elliptic, flagellum-free, spore-free gram-negative anaerobic bacterium with a single cell major axis of 0.6 μm-1.0 μm, which can grow alone or in pairs, and which can also grow in clusters in mucin-containing media. Colonies appear as white films, the colonies are connected by a filamentous structure, and the size of a single colony is about 0.7mm (Derrien M.et al. Akkermansia muciniciphilia gen. Nov., sp. Nov., a human intestinal mucin-degrading bacterium. [ J ]. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY,2004,54 (5): 1469-1476.). Akkermansia muciniphila is a typical intestinal anaerobic bacterium, the difficulty of separation and purification is great, and the successfully separated strain is very limited. Currently, there are only two species of Acremonium (A. Glycophla) separated from human faeces Akkermansia muciniphila and Acremonium (A. Glycophla) separated from net boa faeces (Ouwerkerk J P, et al Akkermandia glycophla sp. Nov., an anaerobic mucin-degrading bacterium isolated from reticulated python faeces [ J ]. International Journal of Systematic and Evolutionary Microbiology,2016,66 (11): 4614-4620). Akkermansia muciniphila is however a potential probiotic, and obtaining a pure culture thereof is essential for elucidating the function of the bacterium.
In order to achieve the technical purpose, the invention provides the following technical scheme:
in one aspect, the invention provides an application of Akkermansia muciniphila in improving cognitive function, wherein Akkermansia muciniphila is preserved in CGMCC with the preservation number of CGMCC No.20955.
In one embodiment, the Akkermansia muciniphila improves cognitive function by at least one of the following mechanisms:
(1) Promoting expression of insulin degrading enzyme IDE;
(2) Increasing the content of anti-inflammatory factors IL-4 and IL-10;
(3) Down-regulating the expression of pro-inflammatory factors IL-1 beta, IL-18, CCL2, NLRP3 and chemokine CXCL 1;
(4) Down-regulating the expression level of Abeta protein.
In yet another aspect, akkermansia muciniphila is used in the preparation of a product for the prevention, treatment and/or adjuvant treatment of neurodegenerative diseases, wherein Akkermansia muciniphila is preserved in CGMCC with the preservation number CGMCC No.20955.
In one embodiment, the product improves cognitive function by at least one of the following mechanisms:
1) Promoting expression of insulin degrading enzyme IDE;
2) Increasing the content of anti-inflammatory factors IL-4 and IL-10;
3) Down-regulating the expression of pro-inflammatory factors IL-1 beta, IL-18, CCL2, NLRP3 and chemokine CXCL 1;
4) Down-regulating the expression level of Abeta protein.
In one embodiment, the neurodegenerative disease comprises alzheimer's disease, parkinson's disease, huntington's disease, frontotemporal dementia, or amyotrophic lateral sclerosis.
In a preferred embodiment, the neurodegenerative disease may be Alzheimer's disease.
In one embodiment, the product comprises a viable or dead form of Akkermansia muciniphila, and isolates and/or secretions thereof.
In one embodiment, the viable form of Akkermansia muciniphila can be any biologically active Akkermansia muciniphila, such as: viable bacteria in fermentation broth, viable bacteria in solid culture medium, viable bacteria in fermentation broth precipitate, viable bacteria in fermentation broth supernatant, viable bacteria naturally existing in nature, viable bacteria existing in human or animal intestinal tract, etc.
In one embodiment, the dead form of Akkermansia muciniphila can be any Akkermansia muciniphila that is not biologically active, such as: dead bacteria in fermentation broth, dead bacteria in solid culture medium, dead bacteria in fermentation broth sediment, dead bacteria in fermentation broth supernatant, dead bacteria naturally existing in nature, dead bacteria existing in human or animal intestinal tracts, etc.
In one embodiment, the food, drug, pharmaceutical material or food material.
In one embodiment, the food product may comprise a health food or a dietary supplement, i.e. a health product.
In one embodiment, the food product may include a condiment, dairy product, beverage, biscuit, frozen drink, confectionery product, wine or food additive, and the like.
In a preferred embodiment, the dairy product comprises:
liquid milk, for example: pasteurized milk, reconstituted milk, sterilized milk, fermented milk, and the like;
milk powder, for example: whole milk powder, skim milk powder, partially skim milk powder, modified milk powder, whey powder, and the like;
other dairy products, for example: condensed milk, cream, anhydrous cream, cheese, processed cheese, specialty dairy products, and concentrated milk.
In a preferred embodiment, the beverage comprises:
fruit and vegetable juices and beverages thereof, for example: fruit juice, vegetable juice, fruit pulp, composite fruit and vegetable juice and concentrated fruit and vegetable juice;
protein beverages, for example: milk-containing beverages, vegetable protein beverages, and complex protein beverages;
solid beverages, for example: protein solid beverage, fruit and vegetable solid beverage, tea solid beverage, coffee solid beverage;
Other beverages, for example: coffee beverages, plant beverages, flavored beverages, sports beverages, nutrient beverages, energy beverages, electrolyte beverages, and beverage purees.
In a preferred embodiment, the biscuit comprises:
crisp biscuits, tough biscuits, fermented biscuits, compressed biscuits, cookies, sandwich (filled) biscuits, wafers, egg rolls, pancakes, decorative biscuits and bubble biscuits.
In a preferred embodiment, the frozen drink comprises:
ice cream, popsicle, slush, frozen stick, edible ice, and sweet ice.
In a preferred embodiment, the confectionery article comprises:
candy, for example: hard candy, toffee candy, sandwich candy, crisp candy, burnt candy (toffee candy), aerated candy, gel candy, gum base candy, pressed candy, fluid candy, film candy, fancy candy;
chocolate and its products;
cocoa butter substitute chocolate and its preparation;
jelly, for example: fruit juice type jelly, pulp type jelly, fruit type jelly, and milk-containing type jelly.
In yet another aspect, the present invention provides a probiotic formulation that may be used as a food product or as a health product.
In one embodiment, the probiotic formulation comprises an effective amount of a viable or dead form of Akkermansia muciniphila, and isolates and/or secretions thereof, said Akkermansia muciniphila having a preservation number of cgmccno.20955.
In one embodiment, the probiotic formulation further comprises at least one of a food acceptable adjuvant, a food acceptable compound, a food acceptable protein and/or polypeptide.
In a preferred embodiment, the food acceptable auxiliary material is selected from at least one of erythritol, D-mannitol, fumaric acid, glycerol, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L (+) -tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty glyceride, agar-agar, lactic acid, sodium lactate, sorbic acid and its potassium salts, sorbitol, acid red, calcium carbonate, sodium bicarbonate, betalain, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate, alpha-cyclodextrin, gamma-cyclodextrin and dextrin.
In a preferred embodiment, the food acceptable compound may be in the form of a compound of trace elements beneficial to the human body and/or derivatives thereof, such as ferric sulfate, succinic acid, zinc sulfate, zinc gluconate, and the like; compounds beneficial to the human body, such as beta-carotene, polyphenols antioxidant substances, dietary fibers, and the like.
In a preferred embodiment, the food acceptable proteins and/or polypeptides include soy protein isolate, meat protein, cereal protein, milk protein, plant polypeptides, animal polypeptides, and the like.
In one embodiment, the formulation of the probiotic formulation comprises a liquid formulation, a solid formulation, a semi-solid formulation, or a gaseous formulation.
In a preferred embodiment, the liquid formulation may be a liquid condiment, a liquid dairy product, a liquid beverage, or the like.
Wherein the liquid dairy product can be pasteurized milk, modified milk, sterilized milk, fermented milk, condensed milk, butter, dilute butter, anhydrous butter, special dairy product, concentrated milk, etc.;
the liquid beverage can be fruit juice, vegetable juice, fruit pulp, compound fruit and vegetable juice, concentrated fruit and vegetable juice, milk beverage, vegetable protein beverage, compound protein beverage, coffee beverage, vegetable beverage, flavored beverage, sport beverage, nutrient beverage, energy beverage, electrolyte beverage, etc.
In a preferred embodiment, the solid state formulation may be a solid condiment, a solid dairy product, a solid beverage, a biscuit, a confectionery product, a frozen drink, or the like.
Wherein the solid dairy product can be whole milk powder, skim milk powder, partially skim milk powder, modified milk powder, whey powder, cheese, processed cheese, etc.;
the solid beverage can be protein solid beverage, fruit and vegetable solid beverage, tea solid beverage, and coffee solid beverage;
in a preferred embodiment, the biscuits include crisp biscuits, tough biscuits, fermented biscuits, compressed biscuits, cookies, sandwich (filled) biscuits, wafers, wafer biscuits, egg rolls, pancakes, decorative biscuits and water-soaked biscuits.
In a preferred embodiment, the frozen drink comprises ice cream, slush, popsicle, food ice, sweet ice.
In one embodiment, the semi-solid formulation comprises a beverage syrup, ice cream, or the like.
In one embodiment, the probiotic formulation comprises at least about 10 per unit of said Akkermansia muciniphila 8 CFU/g or CFU/mL.
Where each unit may refer to a unit per tablet, dose, pill, drop, capsule, etc., it will be appreciated by those skilled in the art that the unit depends on the dosage form/carrier form of the formulation and does not constitute a limitation on the scope of the present invention.
In a preferred embodiment, the probiotic formulation comprises said Akkermansia muciniphila of about 10 per unit 8 -10 12 CFU/g or CFU/mL.
In a preferred embodiment, the probiotic formulation comprises said Akkermansia muciniphila of about 10 per unit 9 -10 12 CFU/g or CFU/mL.
In a preferred embodiment, the probiotic formulation comprises said Akkermansia muciniphila of about 10 per unit 9 -10 11 CFU/g or CFU/mL.
In a preferred embodiment, the probiotic formulation may comprise said Akkermansia muciniphila of about 10 per unit 9 -10 10 CFU/g or CFU/mL.
In a preferred embodiment, the probiotic formulation may comprise said Akkermansia muciniphila of about 1.0X10 per unit 9 、1.1×10 9 、1.2×10 9 、1.3×10 9 、1.4×10 9 、1.5×10 9 、1.6×10 9 、1.7×10 9 、1.8×10 9 、1.9×10 9 、2.0×10 9 、2.1×10 9 、2.2×10 9 、2.3×10 9 、2.4×10 9 、2.5×10 9 、2.6×10 9 、2.7×10 9 、2.8×10 9 、2.9×10 9 、3.0×10 9 、3.1×10 9 、3.2×10 9 、3.3×10 9 、3.4×10 9 、3.5×10 9 、3.6×10 9 、3.7×10 9 、3.8×10 9 、3.9×10 9 、4.0×10 9 、4.1×10 9 、4.2×10 9 、4.3×10 9 、4.4×10 9 、4.5×10 9 、4.6×10 9 、4.7×10 9 、4.8×10 9 、4.9×10 9 、5.0×10 9 、5.1×10 9 、5.2×10 9 、5.3×10 9 、5.4×10 9 、5.5×10 9 、5.6×10 9 、5.7×10 9 、5.8×10 9 、5.9×10 9 、6.0×10 9 、6.1×10 9 、6.2×10 9 、6.3×10 9 、6.4×10 9 、6.5×10 9 、6.6×10 9 、6.7×10 9 、6.8×10 9 、6.9×10 9 、7.0×10 9 、7.1×10 9 、7.2×10 9 、7.3×10 9 、7.4×10 9 、7.5×10 9 、7.6×10 9 、7.7×10 9 、7.8×10 9 、7.9×10 9 、8.0×10 9 、8.1×10 9 、8.2×10 9 、8.3×10 9 、8.4×10 9 、8.5×10 9 、8.6×10 9 、8.7×10 9 、8.8×10 9 、8.9×10 9 、9.0×10 9 、9.1×10 9 、9.2×10 9 、9.3×10 9 、9.4×10 9 、9.5×10 9 、9.6×10 9 、9.7×10 9 、9.8×10 9 、9.9×10 9 、1.0×10 10 CFU/g or CFU/mL.
In a further aspect, the present invention provides a medicament for the prophylaxis and/or treatment of neurodegenerative diseases, which comprises an effective amount of a viable or dead form of Akkermansia muciniphila, and an isolate and/or secretion thereof, said Akkermansia muciniphila having a deposit number of CGMCC No.20955.
In one embodiment, the neurodegenerative disease may be alzheimer's disease.
In one embodiment, the viable form of Akkermansia muciniphila can be any biologically active Akkermansia muciniphila, such as: viable bacteria in fermentation broth, viable bacteria in solid culture medium, viable bacteria in fermentation broth precipitate, viable bacteria in fermentation broth supernatant, viable bacteria naturally existing in nature, viable bacteria existing in human or animal intestinal tract, viable bacteria in lyophilized powder, etc.
In one embodiment, the dead form of Akkermansia muciniphila can be any Akkermansia muciniphila that is not biologically active, such as: dead bacteria in fermentation liquor, dead bacteria in solid culture medium, dead bacteria in fermentation liquor sediment, dead bacteria in fermentation liquor supernatant, dead bacteria naturally existing in nature, dead bacteria existing in human or animal intestinal tracts, dead bacteria in freeze-dried powder and the like.
In a preferred embodiment, the pharmaceutically acceptable auxiliary material is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsifying accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, and antioxidants.
In a preferred embodiment, the pharmaceutical dosage form is selected from at least one of powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, powder mists, aerosols, suppositories, drops and drop pills.
In one embodiment, each unit of medicament comprises at least about 10 of said Akkermansia muciniphila 8 CFU/g or CFU/mL.
Where each unit may refer to a unit per tablet, dose, pill, drop, capsule, etc., those skilled in the art will recognize that the unit depends on the dosage form/carrier form of the drug and does not limit the scope of the invention.
In a preferred embodiment, each unit of medicament may contain about 10 of said Akkermansia muciniphila 8 -10 12 CFU/g or CFU/mL.
In a preferred embodiment, each unit of medicament may contain about 10 of said Akkermansia muciniphila 9 -10 12 CFU/g or CFU/mL.
In a preferred embodiment, each unit of medicament may contain about 10 of said Akkermansia muciniphila 9 -10 11 CFU/g or CFU/mL.
In a preferred embodiment, each unit of medicament may contain about 10 of said Akkermansia muciniphila 9 -10 10 CFU/g or CFU/mL.
In a preferred embodiment, the probiotic formulation may comprise said Akkermansia muciniphila of about 1.0X10 per unit 9 、1.1×10 9 、1.2×10 9 、1.3×10 9 、1.4×10 9 、1.5×10 9 、1.6×10 9 、1.7×10 9 、1.8×10 9 、1.9×10 9 、2.0×10 9 、2.1×10 9 、2.2×10 9 、2.3×10 9 、2.4×10 9 、2.5×10 9 、2.6×10 9 、2.7×10 9 、2.8×10 9 、2.9×10 9 、3.0×10 9 、3.1×10 9 、3.2×10 9 、3.3×10 9 、3.4×10 9 、3.5×10 9 、3.6×10 9 、3.7×10 9 、3.8×10 9 、3.9×10 9 、4.0×10 9 、4.1×10 9 、4.2×10 9 、4.3×10 9 、4.4×10 9 、4.5×10 9 、4.6×10 9 、4.7×10 9 、4.8×10 9 、4.9×10 9 、5.0×10 9 、5.1×10 9 、5.2×10 9 、5.3×10 9 、5.4×10 9 、5.5×10 9 、5.6×10 9 、5.7×10 9 、5.8×10 9 、5.9×10 9 、6.0×10 9 、6.1×10 9 、6.2×10 9 、6.3×10 9 、6.4×10 9 、6.5×10 9 、6.6×10 9 、6.7×10 9 、6.8×10 9 、6.9×10 9 、7.0×10 9 、7.1×10 9 、7.2×10 9 、7.3×10 9 、7.4×10 9 、7.5×10 9 、7.6×10 9 、7.7×10 9 、7.8×10 9 、7.9×10 9 、8.0×10 9 、8.1×10 9 、8.2×10 9 、8.3×10 9 、8.4×10 9 、8.5×10 9 、8.6×10 9 、8.7×10 9 、8.8×10 9 、8.9×10 9 、9.0×10 9 、9.1×10 9 、9.2×10 9 、9.3×10 9 、9.4×10 9 、9.5×10 9 、9.6×10 9 、9.7×10 9 、9.8×10 9 、9.9×10 9 、1.0×10 10 CFU/g or CFU/mL.
In a further aspect, the present invention provides a method of preventing, treating and/or co-treating a subject suffering from a neurodegenerative disease, administering to said subject an effective amount of a viable or dead form of Akkermansia muciniphila, and isolates and/or secretions thereof, said probiotic preparation or said medicament;
Wherein the preservation number of the Akkermansia muciniphila is CGMCC No.20955.
In one embodiment, the neurodegenerative disease may be alzheimer's disease.
Compared with the prior art, the Akkermansia muciniphila provided by the invention has at least the following beneficial effects:
(1) The cognitive function is obviously improved, and in the water maze experiment, the latency period is shortened by at least 10s relative to a model group;
(2) Significantly elevated levels of anti-inflammatory factors IL-4, IL-10, down-regulated levels of pro-inflammatory factors CXCL1, CCL2 and Abeta, about 2.5-fold elevated IL-4, about 1.4-fold elevated IL-10, about 51% down-regulated levels of CXCL1, about 25% down-regulated levels of CCL2 and about 50% down-regulated Abeta relative to the model group;
(3) Significantly down-regulating the expression levels of pro-inflammatory factors (IL-1β, IL-18, NLRP 3) and apoptosis factor Caspase-1, with respect to the model group, IL-1β down-regulated by about 46%, IL-18 down-regulated by 44%, NLRP3 down-regulated by about 40%, caspase-1 down-regulated by about 47%; the expression quantity of Abeta degrading enzyme IDE is obviously up-regulated, and the expression quantity is increased by 2 times relative to a model group;
(4) Significantly down-regulating the expression level of aβ protein in hippocampal tissue;
(5) Can protect normal morphology of colon and has no toxicity to liver.
Drawings
Fig. 1 shows the latency statistics of the Morris water maze experiment.
FIG. 2 is a trace diagram of Morris water maze experiment. Wherein A is a track diagram of the WT group mice from being placed in a water tank until a platform is found, B is a track diagram of the APP group mice from being placed in the water tank until the platform is found, and C is a track diagram of the APP+AKK PROBIO group mice from being placed in the water tank until the platform is found.
FIG. 3 shows the results of measurement of the A.beta.protein content of mice in different treatment groups. Where letters a, b, c represent significant differences, the same lowercase letters indicate no significant differences between the corresponding two groups, and the different lowercase letters indicate significant differences between the corresponding two groups (p < 0.05).
FIG. 4 is a graph of Western Blot results of Abeta proteins from mice of different treatment groups.
Fig. 5 is a graph of liver sections of mice from different treatment groups. Wherein A is the liver slice of the WT group mice, B is the liver slice of the APP group mice, and C is the liver slice of the APP+AKK PROBIO group mice. Scale bar 100 μm.
Fig. 6 is a graph of colon sections of mice from different treatment groups. Wherein A is the colon slice of the WT group mice, B is the colon slice of the APP group mice, and C is the colon slice of the APP+AKK PROBIO group mice. The scale bar is 500 μm.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.
The term "cognitive function" as used herein refers to the ability to recognize, learn things, and specifically includes memory, computation, understanding thinking, judgment reasoning, language expression, executive capability, spatiotemporal orientation, and the like.
The terms "Akkermansia muciniphila", "AKK PROBIO" or "Akkermansia muciniphila" as used herein refer to Akkermansia muciniphila having the preservation number CGMCC No. 20955.
The term "neurodegenerative disease" as used herein is a type of disease in which neurons of the brain and spinal cord develop over time with progressive damage, characterized by massive loss of specific neurons, and is a type of complex disease that progresses and eventually becomes fatal.
The term "viable form of Akkermansia muciniphila" as used herein refers to Akkermansia muciniphila having at least one of culturability, transcriptability, metabolic activity, and cell membrane integrity. Wherein culturability refers to the ability of bacteria to form colonies on a medium; transcriptability refers to the ability of a cell of a bacterium to produce mRNA, and metabolic activity refers to the ability of the bacterium to exhibit life phenomena related to its metabolism, such as redox ability, respiration, ability to take up active enzymes and substrates, etc.; cell membrane integrity means that the cell membrane of the bacteria is not disrupted.
Otherwise, the death form of Akkermansia muciniphila is the "death form".
The term "isolate" as used herein refers to a microbial substance that a particular microbial individual has separated from a population or from a mixed population of microorganisms and is present in a medium. Wherein the medium comprises fermentation broth supernatant, fermentation broth sediment, bacterial suspension, freeze-dried powder and the like.
The term "secretion" as used herein refers to the products secreted by a particular microbial individual during growth or metabolism, including metabolites such as methanol, ethanol, propanol, isopropanol, n-butanol, succinic acid, tartaric acid, and the like; extracellular products such as proteins, fats, starches, celluloses, antibiotics, and the like.
The term "food product" as used herein refers to various finished and raw materials intended for human consumption or drinking as well as articles that are traditionally both food and traditional Chinese medicinal materials, but do not include articles intended for therapeutic purposes.
The terms "health product", "dietary supplement" or "health food" as used herein refer to a category of food that has the commonality of general foods and can regulate the function of the human body, suitable for consumption by a particular population, but not for the purpose of treating a disease.
The term "food material" as used herein refers to all materials used in food processing, including food ingredients, food additives.
The term "pharmaceutical raw material" or "pharmaceutical raw material" as used herein refers to a raw material drug containing a pharmaceutical active ingredient and biological and chemical substances as its source.
The term "probiotic preparation" as used herein refers to a preparation prepared according to the principle of microecology using normal beneficial microorganisms or promoting substances thereof beneficial to a host, which has the effects of maintaining or adjusting microecological balance, preventing and treating diseases and improving the health of the host.
The term "subject" as used herein refers to a subject, including a patient, healthy subject, who is engaged in a clinical trial and who is the recipient of a drug for the trial. Preferably, the subject is a patient.
The term "adjuvant therapy" as used herein refers to a therapy that is not directly used to prevent or combat a disease, and is used to reduce the side effects of the therapy, increase patient tolerance, and enhance the efficacy of the therapy. Products for adjuvant therapy generally refer to products that help promote primary drug absorption, prevent or reduce adverse reactions, and enhance body function.
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention. Such structures and techniques are also described in a number of publications.
EXAMPLE 1 cultivation of AKK PROBIO
Primary culture: AKK PROBIO frozen in 2mL glycerol pipe is taken, inoculated in an inoculum size of 10% into a culture medium containing 9mL brain heart infusion Broth (BHI), anaerobically cultured at 37 ℃ for 24-48h, and primary culture is carried out to obtain primary culture.
Secondary culture: the primary culture was inoculated into a flask containing 90mL at an inoculum size of 5%, and anaerobic cultured at 37℃for 24 hours to obtain a secondary culture.
Tertiary culture: the secondary culture was inoculated into a 300mL Erlenmeyer flask at an inoculum size of 5%, and anaerobically cultured at 37℃for 13 hours.
Alternatively, the tertiary culture is frozen at-80℃with 20% glycerol.
EXAMPLE 2 use of AKK PROBIO in improving cognitive function
The main experimental reagent is prepared:
preparing AKK PROBIO bacterial suspension: taking 5mL AKK PROBIO frozen stock solution, performing activation culture, centrifuging at 4000r/min for 10min, cleaning for 2 times, adding 5mL physiological saline after cleaning, and forming 1×10 concentration 10 CFU/mL of bacterial suspension.
The experiments of this example were performed in parallel with three measurements of tissue samples, respectively, and then the average value was calculated. The data were averaged and analyzed using SPSS software (SPSS v.25for Windows, IBM Software Group, chicago, IL, USA). Differences between the mean values of the individual groups were assessed by one-way analysis of variance using the Duncan multi-range test. Differences of p <0.05 are considered statistically significant.
2.1 animal Experimental design
In this example, 20 APP/PS1 double transgenic mice (MJ 0324-058 mice, MJ 2-058 mice) (Alzheimer's disease (AD) model) were randomly divided into 2 APP groups (model groups), APP+AKK PROBIO groups (AKK PROBIO was administered to AD model mice), 10 each; another 10 wild type C57 BL/6J mice with the same litter were used as blanks and used as WT groups. All mice were separately subjected to gavage and injection at the beginning of 12 months of age for 30 days, see in particular table 1. After the experiment is finished, the orbital veins of the mice are bled, the mice are killed by neck removal, partial brain, liver and colon tissues of the mice are respectively taken in 4% paraformaldehyde, and the rest tissues are placed in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for storage.
Table 1 lavage group and dose
Grouping Stomach lavage
WT Equivalent physiological saline for daily gastric lavage
APP Equivalent physiological saline for daily gastric lavage
APP+AKK PROBIO The daily gastric lavage concentration is 1×10 9 CFU/day/AKK PROBIO alone physiological saline
2.2 determination of AKK PROBIO in improving spatial memory
After the end of experimental dosing, a Morris water maze experiment was performed for a period of 4 d. The water maze consisted of a circular pool (120 cm diameter) and was equally divided into four quadrants. During the experiment, the mice are carried into a laboratory two hours in advance and are familiar with the environment in advance. Regulating water temperature to 21-22deg.C, and pouring milk into water tank to make it milky. Day 1-3 is training period, the mice facing the pool wall prompt direction are gently put into water from the first quadrant, the second quadrant, the third quadrant and the fourth quadrant respectively, swimming tracks are recorded for 1min, and the mice without finding the platform within the specified time need to be guided onto the platform. Each mouse was trained 1 time per quadrant per day for 3 days in succession. Day 4 is an experimental period, each mouse randomly selects one quadrant to be put into a pool, the time required for searching a platform within 60s, namely the escape latency period, is recorded, and the swimming track of each mouse is recorded by a video acquisition system; if the mice failed to find a plateau within 60s after water entry, escape latency was recorded as 60s.
The experimental results showed that the escape latency showed a decreasing trend after each group of mice had undergone a Morris water maze experiment for 4 days in succession (fig. 1). Compared with the last day test result of the APP group, the escape latency of the app+akk PROBIO group mice is shortened by about 22.5%, and the path to find the platform is also significantly shortened (fig. 1 and 2).
2.3 determination of the Effect of AKK PROBIO on the inflammation index
The experiments were performed by taking the hippocampal tissue of each group of mice in this example, homogenizing with PBS, and then sucking the supernatant. ELISA detection kit Specification detects the levels of IL-4 (interleukin-4, nanjing institute of bioengineering, cat# H005-1-2), IL-10 (interleukin-10, nanjing institute of bioengineering, cat# H009-1-2), CCL2 (chemokine ligand 2, nanjing institute of bioengineering, cat# H318-1) and CXCL1 (CXC chemokine ligand 1, nanjing institute of bioengineering, cat# H306-1) in hippocampal tissue.
Results As shown in Table 2, CXCL1 and CCL2 levels in the WT hippocampal tissue were the lowest in the four groups and IL-4 and IL-10 levels were the highest in the four groups. Whereas APP groups had IL-4 and IL-10 levels that were the lowest of the four groups and CXCL1 and CCL2 levels that were the highest of the four groups. CXCL1 and CCL2 levels in the APP+AKK PROBIO group tended to be downregulated compared to the APP group, and IL-4 and IL-10 levels tended to be increased compared to the APP group.
CCL-2 is involved in the formation of inflammatory responses, with which the latter migration is abnormally increased when microglia are treated, which microglia activate the factor, thereby mediating immune injury and the inflammatory response of neural tissue cells. CXCL1 belongs to the CXC chemokine family, is widely present in the nervous system, and is one of the important inflammatory mediators of nervous system lesions such as brain and spinal cord trauma, neurodegenerative diseases, and the like. The anti-inflammatory factor IL-4 has the main functions of promoting humoral immunity and inhibiting cellular immunity, thereby enhancing the killing effect of cells. The anti-inflammatory factor IL-10 has the main functions of inhibiting immune response, inhibiting inflammatory response and inhibiting apoptosis. Thus, the above results indicate that AKK PROBIO significantly upregulates the expression of the anti-inflammatory factors IL-4 and IL-10, significantly down regulates the levels of the pro-inflammatory factors CXCL1 and CCL2, and improves the inflammatory response of the nervous system by modulating the inflammatory factors.
Compared to the model group, the app+akk PROBIO group had an increased level of IL-4 by about 2.5 times, the IL-10 by about 1.4 times, the CXCL1 by about 51%, the CCL2 by about 25% and the aβ by about 50% in the hippocampus (fig. 3).
TABLE 2 measurement results of the inflammatory index of mice of this example
* Wherein the same lowercase letter indicates no significant difference between the corresponding two groups and the different lowercase letter indicates a significant difference (p < 0.05) between the corresponding two groups; wherein pg in pg/mL is the mass of IL-4, IL-10, CXCL1 or CCL2 and mL is the volume of serum; ng is the mass of Abeta in ng/mL and mL is the volume of serum.
2.4 determination of the Effect of AKK PROBIO on the expression of inflammatory factor-related Gene in hippocampal tissue
The hippocampal tissues of each group of mice in this example were taken and mRNA expression amounts of IL-1β, caspase-1, IL-18, IDE and NLRP3 in the hippocampal tissues of the mice were detected by qPCR. The hippocampal tissue of the mice was homogenized in a homogenization tube containing homogenized beads, and 1mL of TRIzol reagent was added thereto. 200 mu L of chloroform was added to the slurry, and the mixture was left to stand on ice for 15 minutes after being thoroughly mixed. Centrifuging to obtain supernatant, adding isopropanol, mixing, standing for 20min, centrifuging at 14000r/min at 4deg.C for 20min, collecting white precipitate at bottom of tube to obtain RNA, pouring supernatant, adding 75% ethanol, cleaning, and washing at 14000r at 4deg.CCentrifuging for 15min, collecting the liquid in the tube, placing the tube on an ultra-clean workbench for drying for 3min, and adding 20 mu L DEPC water to dissolve RNA to obtain RNA stock solution. The RNA is transcribed into cDNA using a transcription kit. To the octant was added 10. Mu. L SYBR Green PCR Master Mix, 4. Mu.L of sterile ultra pure water, 2. Mu.L of primer, and 2. Mu.L of cDNA in this order. The Real-time PCR instrument is pre-denatured at 95 ℃ for 10min, denatured at 95 ℃ for 15min, annealed at 60 ℃ for 30s, extended at 60 ℃ for 30s, operated for forty cycles, and finally GAPDH is taken as a housekeeping gene, and 2- ΔΔCt The expression level of mRNA of the target gene was calculated by the method. The primer sequences used are shown in Table 3. The test results are shown in Table 4.
TABLE 3 primers for detecting expression of inflammatory factor related genes
TABLE 4 determination of inflammatory factor related Gene expression
* Where the same lowercase letters indicate no significant differences between the corresponding two groups and the different lowercase letters indicate significant differences between the corresponding two groups (p < 0.05).
The results showed that the expression of inflammatory factors IL-1 beta, IL-18 and NLRP3, apoptosis factor Caspase-1 was significantly increased in the APP group, by about 3, 4, 6 and 4-fold, respectively, compared to the normal group. In addition, the level of the Abeta degrading enzyme IDE in the APP group is lowest, the Abeta degrading enzyme IDE is down-regulated by about 79 percent compared with the normal group, and the active expression of the IDE is obviously enhanced after AKK PROBIO is performed, and the active expression is increased by about 2 times compared with the model group (APP group). Whereas the APP+AKK PROBIO group significantly down-regulated IL-1β, IL-18, NLRP3 and Caspase-1 levels compared to the APP group, wherein IL-1β was down-regulated by about 46%, IL-18 was down-regulated by 44%, NLRP3 was down-regulated by about 40% and Caspase-1 was down-regulated by about 47%.
2.5 Effect of AKK PROBIO on hippocampal Abeta protein expression
The hippocampal tissue of each group of mice in this example was taken, homogenized in 1mL of RIPA (thermofimer, waltham, MA, USA) and 10. Mu.L of PMSF (thermofimer), and centrifuged at 12000 Xg for 5min at 4 ℃. Proteins were quantified using BCA protein assay kit (thermosusher). Protein samples were mixed with sample buffer (thermosusher) at 4:1 and heating at 95 ℃ for 5min, then spotting the samples into SDS-PAGE gel wells and running the gel at 100V. Bands on SDS-PAGE gels were transferred to PVFD membranes, the PVDF membranes were blocked with 5% skim milk for 1h, then incubated with primary antibody (thermosipher) overnight at 4℃after which the antibodies (thermosipher) were added and incubated for 1 h. Images were obtained at iBright (Thermofisher) after chemiluminescence using western ECL substrate (thermosusher) for subsequent analysis.
The results show that the expression of aβ protein in the app+akk PROBIO group is significantly down-regulated compared to the APP group but still higher than in the WT group (fig. 4).
Example 3 Effect of AKK PROBIO on mouse liver and colon tissue
The feeding, molding and grouping of mice of this example were the same as in example 2.
Livers of mice in each group were taken, and pathological sections of the livers were prepared and observed. The results showed that the hepatic lobular structures of each group of mice were ordered, with clear central veins and hepatic sinus (fig. 5). This indicates that the administration of AKK PROBIO is non-toxic to the liver of mice and has better safety.
Intestinal tissue pathology sections of mice were taken and observed. The results show that the intestinal tissue structure of the WT group mice is complete and the morphology is normal. The muscle layer cells of the intestinal tissue of the APP group mice are shrunken, the crypt cells are damaged, the mucous membrane tissue is shrunken, and the fold inflammation is enriched. Whereas the app+akk PROBIO group intestinal myocytes recovered, crypt and mucosal tissues tended to be normal (fig. 6). This suggests that app+akk PROBIO groups are less enriched in inflammation than APP groups.
Comparative example
In this comparative example, the common AKK bacteria (purchased from ATCC, CAT#: ATCC-BAA 835) were selected as a control group to compare the difference in spatial memory improvement with AKK PROBIO.
The experiments of this comparative example were performed three times separately for measurement of tissue samples, and then an average value was calculated. The data were averaged and analyzed using SPSS software (SPSS v.25for Windows, IBM Software Group, chicago, IL, USA). Differences between the mean values of the individual groups were assessed by one-way analysis of variance using the Duncan multi-range test. Differences of p <0.05 are considered statistically significant.
The mouse model was constructed as in example 2.
Experimental grouping: 30 12 month old APP/PS1 double transgenic mice were randomly divided into APP group (model group), APP+AKK PROBIO group (AKK PROBIO was administered to AD model mice), control group (ATCC-BAA 835 strain was administered to AD model mice), and 10 wild type C57 BL/6J mice were taken as blank. The WT group is filled with normal saline for daily stomach, the APP group is filled with normal saline for daily stomach, and the APP+AKK PROBIO group is filled with normal saline for daily stomach with concentration of 1×10 9 Physiological saline of CFU/day/AKK PROBIO solution, and the daily gastric lavage concentration of control group is 1×10 9 CFU/day/ATCC-BAA 835 solution in normal saline.
The water maze test conditions were the same as in example 2.
The results are shown in table 5, and the escape latency showed a decreasing trend after each group of mice underwent the Morris water maze test of 4 d in succession. The escape latency of the app+akk PROBIO group mice was reduced by about 24% compared to the last day test results of APP group. Whereas the escape latency of app+atcc-BAA835 group mice was only reduced by 10.3%. The AKK PROBIO bacteria provided by the invention has stronger capacity of improving spatial memory than common Akkermansia muciniphila.
TABLE 5 determination of escape latency for mice of each group (seconds, s)
* Where the same lowercase letters indicate no significant differences between the corresponding two groups and the different lowercase letters indicate significant differences between the corresponding two groups (p < 0.05).
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims (14)

  1. The application of 1.Akkermansia muciniphila in improving cognitive function is characterized in that Akkermansia muciniphila is preserved in CGMCC with the preservation number of CGMCC No.20955.
  2. The application of 2.Akkermansia muciniphila in preparing products for preventing, treating and/or assisting in treating neurodegenerative diseases is characterized in that Akkermansia muciniphila is preserved in CGMCC with the preservation number of CGMCC No.20955;
    preferably, the neurodegenerative disease comprises alzheimer's disease, parkinson's disease, huntington's disease, frontotemporal dementia and amyotrophic lateral sclerosis;
    more preferably, the neurodegenerative disease is alzheimer's disease.
  3. 3. Use according to claim 2, wherein the product comprises a viable or dead form of Akkermansia muciniphila, and isolates and/or secretions thereof;
    preferably, the product comprises food, pharmaceutical and food materials;
    More preferably, the food product comprises a health product, a condiment, a dairy product, a beverage, a biscuit, a frozen drink, a confectionery product, an alcohol and a food additive.
  4. 4. A probiotic preparation comprising an effective amount of a viable or dead form of Akkermansia muciniphila, and isolates and/or secretions thereof, said Akkermansia muciniphila having a preservation number CGMCC No.20955.
  5. 5. The probiotic formulation according to claim 4, further comprising at least one of a food acceptable adjuvant, a food acceptable compound, a food acceptable protein and/or polypeptide;
    preferably, the food acceptable auxiliary material is selected from at least one of erythritol, D-mannitol, fumaric acid, glycerol, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L (+) -tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty glyceride, agar-agar, lactic acid, sodium lactate, sorbic acid and potassium salts thereof, sorbitol, acid red, calcium carbonate, sodium bicarbonate, betalain, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate, alpha-cyclodextrin, gamma-cyclodextrin and dextrin;
    Preferably, the formulation of the probiotic preparation comprises a liquid, solid, semi-solid or gaseous preparation.
  6. 6. The probiotic preparation according to claim 4 or 5, characterized in that per unit of probiotic preparation at least comprises the Akkermansia muciniphila 10 8 CFU/g or CFU/mL.
  7. 7. The probiotic formulation of claim 6, wherein each unit of probiotic formulation comprises the Akkermansia muciniphila 10 8 -10 12 CFU/g or CFU/mL.
  8. 8. The probiotic formulation according to claim 7, characterized in that per unit of probiotic formulation comprises the Akkermansia muciniphila 10 9 -10 12 CFU/g or CFU/mL.
  9. 9. A medicament for the prevention and/or treatment of neurodegenerative diseases, which comprises an effective amount of a surviving or dead form of Akkermansia muciniphila, and isolates and/or secretions thereof, said Akkermansia muciniphila having a preservation number of CGMCC No.20955;
    preferably, the neurodegenerative disease is alzheimer's disease.
  10. 10. The medicament of claim 9, further comprising at least one of a pharmaceutically acceptable excipient, a pharmaceutically acceptable compound, a pharmaceutically acceptable protein, and/or polypeptide;
    Preferably, the pharmaceutically acceptable auxiliary material is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsification accelerators, buffers, absorbents, colorants, fragrances, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, and antioxidants;
    preferably, the dosage form of the medicament is at least one selected from powder, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, powder mists, aerosols, suppositories, drops and drop pills.
  11. 11. The medicament according to claim 9 or 10, characterized in that it comprises at least said Akkermansia muciniphila 10 per unit of medicament 8 CFU。
  12. 12. The medicament according to claim 11, wherein each unit of medicament comprises the Akkermansia muciniphila 10 8 -10 12 CFU/g or CFU/mL.
  13. 13. The medicament of claim 12, wherein each unit of medicament comprises the Akkermansia muciniphila 10 9 -10 12 CFU/g or CFU/mL.
  14. 14. A method of preventing and/or treating a subject suffering from a neurodegenerative disease, characterized in that an effective amount of a viable or dead form of Akkermansia muciniphila, and isolates and/or secretions thereof, a probiotic preparation according to any of claims 4-8 or a medicament according to any of claims 9-13, is administered to the subject;
    Wherein the preservation number of the Akkermansia muciniphila is CGMCC No.20955;
    preferably, the neurodegenerative disease is alzheimer's disease.
CN202311047605.1A 2023-08-18 2023-08-18 Use of Akkermansia muciniphila for improving cognitive function Pending CN117122619A (en)

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