CN118581008A - A strain of Bifidobacterium animalis subspecies lactis MY9 and its application in the preparation of laxative and intestinal protective food and medicine - Google Patents

Abstract

The present invention belongs to the technical field of probiotics and their application, and specifically relates to a strain of animal Bifidobacterium lactis subspecies MY9 and its application in the preparation of laxative and intestinal protection food and medicine. The present invention separates and purifies a strain of animal Bifidobacterium lactis subspecies (Bifidobacterium animalis subsp.lactis) MY9 strain from the feces of a healthy adult in Guangdong Province, China. The strain not only has superior protease activity and cellulase activity, but also can inhibit acetylcholinesterase activity, and has the functions of promoting digestion and absorption of protein food, improving protein allergy; promoting intestinal absorption, peristalsis and laxative; promoting the absorption of cellulose, protecting the intestine and improving constipation. It can be seen that the animal Bifidobacterium lactis subspecies MY9 strain newly isolated by the present invention has multiple probiotic effects, can be used in the fields of laxative and intestinal protection, and has important application value and economic value.

Description

A strain of animal Bifidobacterium lactis subspecies MY9 and its application in the preparation of laxative and intestinal protective food and medicine

Technical Field

The invention belongs to the technical field of probiotics and their applications, and specifically relates to a strain of animal Bifidobacterium lactis subspecies MY9 and its application in the preparation of laxative and intestinal protecting food and medicine.

Background Art

Bifidobacterium animalis subsp. lactis is a Gram-positive bacillus belonging to the genus Bifidobacterium. Its colonies are smooth, convex, with complete edges, milky white, shiny and soft. Bifidobacterium animalis subsp. lactis does not form spores, is non-motile, and is an anaerobic organism. Its optimum growth temperature is 3-41°C; its optimum fermentation temperature is 35-40°C; its minimum growth temperature is 25-28°C; and its maximum growth temperature is 43-45°C. Bifidobacterium animalis subsp. lactis is mainly distributed in the human intestines, and is also present in large quantities in the digestive tracts of some warm-blooded animals (such as pigs, dogs, mice and bees), indicating that Bifidobacterium animalis subsp. lactis is a normal strain present in the human body. In addition, Bifidobacterium animalis subsp. lactis has good acid and bile resistance, can survive in the stomach and intestines of humans and animals, and is widely present in the intestines of humans and animals.

Animal Bifidobacterium lactis subspecies is one of the strains that can be used for production and processing, and is widely present in the intestinal tract of humans and animals and is discharged with feces. As a probiotic in the intestinal flora, it is also an edible lactic acid bacteria. Animal Bifidobacterium lactis subspecies can also help intestinal peristalsis, and can also enhance the digestive ability of the human body and improve immunity. Therefore, animal Bifidobacterium lactis subspecies has become a hot topic of research in recent years as a probiotic bifidobacterium with great potential. Thereby animal Bifidobacterium lactis subspecies is widely used in the production of probiotic preparations for humans and animals as a probiotic, and is constantly being used to make probiotic preparations suitable for humans and animals.

Studies have shown that different strains of Bifidobacterium animalis subsp. lactis have different probiotic functions: (1) Bifidobacterium animalis subsp. lactis BB-12, isolated from fermented dairy products collected by Chr. Hansen, has been used in infant formula, complementary foods and fermented dairy products around the world. BB-12 exhibits high gastric acid and bile tolerance and has adhesion in the intestines. Therefore, it has potential application value in maintaining intestinal microecological balance, improving intestinal dysfunction, and alleviating symptoms and signs of allergic diseases (Jungersen M, Wind A, Johansen E, et al. The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis Microorganisms.2014Mar 28;2(2):92-110.doi:10.3390/microorganisms2020092.PMID:27682233; PMCID:PMC5029483.). (2) Bifidobacterium animalis subsp. lactis strain V9 has good tolerance to gastrointestinal digestive juices and antagonistic effects on intestinal pathogens. It has probiotic functions such as significantly improving the recovery rate of diarrhea and regulating immunity (Sun Z, Chen X, Wang J, et al. Complete genome sequence of probiotic Bifidobacterium animalis subsp. lactis strain V9. J Bacteriol. 2010 Aug; 192(15): 4080-1. doi: 10.1128/JB.00369-10. Epub 2010 May 28. PMID: 20511504; PMCID: PMC2916379.). (3) Bifidobacterium animalis subsp. lactis XLTG11 has the effect of preventing and treating colitis (Wang N, Wang S, Xu B, et al. Alleviation Effects of Bifidobacterium animalis subsp. lactis XLTG11 on Dextran Sulfate Sodium-Induced Colitis in Mice. Microorganisms. 2021 Oct 3; 9(10): 2093. doi: 10.3390/microorganisms9102093. PMID: 34683415; PMCID: PMC8539219.). (4) As people's living standards continue to improve, the nutrition they consume is becoming more abundant, which leads to an increase in serum cholesterol in the body, thereby causing a series of diseases such as hypertension, coronary heart disease, and arteriosclerosis. Animal Bifidobacterium lactis subsp. HCS04-002 has good resistance to adverse environments and can withstand the effects of artificial gastric juice and artificial intestinal juice, and can enter the intestine with a higher survival rate to effectively play a probiotic role. It not only has a high lactase activity, which can alleviate lactose intolerance, but also has triglyceride degradation function, and has certain application prospects in the preparation of lipid-lowering products (Yu Ping, Zhao Di, Zhang Chunyu, et al. Research on the probiotic properties of animal Bifidobacterium lactis subsp. HCS04-002 [J]. China Brewing, 2021, 40(05): 86-90.).

Since animal Bifidobacterium lactis subspecies has diverse sources, it has genetic diversity and functional diversity. However, there are still few studies on the isolation and identification, probiotic properties and metabolic mechanisms of animal Bifidobacterium lactis subspecies, which to a certain extent affects the development and utilization of animal Bifidobacterium lactis subspecies. Therefore, it is necessary to explore more new strains based on the different sources of animal Bifidobacterium lactis subspecies, and then determine their efficacy based on the function of the strain or the probiotic metabolites, clarify their application prospects, and enable them to better play a probiotic role. In summary, the research and application of probiotic animal Bifidobacterium lactis subspecies has broad development space.

Summary of the invention

In order to overcome the shortcomings of the above-mentioned prior art, the present invention isolates and purifies a Bifidobacterium animalis subsp. lactis MY9 strain from the feces of a healthy adult in Guangdong Province, China. The strain not only has excellent protease activity and cellulase activity, but also can inhibit acetylcholinesterase activity, and has important potential application value in the fields of laxative and intestinal protection.

In order to achieve the above object, the technical solution adopted by the present invention is:

The first aspect of the present invention provides a Bifidobacterium animalissubsp.lactis MY9 strain, which was deposited in the China Center for Type Culture Collection on October 25, 2023, with the accession number: CCTCC NO:M 20232016; the 16SrDNA sequence of the Bifidobacterium animalissubsp.lactis MY9 strain is shown in SEQ ID No: 1.

The second aspect of the present invention provides use of the Bifidobacterium animalis subsp. lactis MY9 strain described in the first aspect in producing protease.

Research has found that the probiotic Bifidobacterium animalis subsp. lactis MY9 strain can produce protease, suggesting that Bifidobacterium animalis subsp. lactis MY9 strain is expected to be used to produce protease, and through this property of producing protease, it can be used to promote the human body's digestion and absorption of protein in food, anti-allergy, and help animals digest and absorb nutrients.

The third aspect of the present invention provides use of the Bifidobacterium animalis subsp. lactis MY9 strain described in the first aspect in producing cellulase.

Research has found that the probiotic Bifidobacterium animalis subspecies MY9 strain can produce cellulase, suggesting that Bifidobacterium animalis subspecies MY9 strain can be used to produce cellulase, and through this characteristic of producing cellulase, it can be used to promote the human body's digestion and absorption of protein in food, improve the absorption of small peptides and amino acids, and anti-allergy.

The fourth aspect of the present invention provides use of the Bifidobacterium animalis subsp. lactis MY9 strain described in the first aspect in the preparation of an acetylcholinesterase inhibitor.

Studies have shown that the probiotic Bifidobacterium animalis subspecies MY9 can effectively inhibit the activity of acetylcholinesterase, suggesting that Bifidobacterium animalis subspecies MY9 can be used to enhance cognitive ability and memory, dilate blood vessels, and excite skeletal and smooth muscles by inhibiting the activity of acetylcholinesterase.

The fifth aspect of the present invention provides a method for preparing protease, specifically: inoculating the Bifidobacterium animalis subsp. lactis MY9 strain described in the first aspect into MRS liquid culture medium, collecting the bacterial suspension after culturing to a stable period, and finally separating and purifying the protease in the bacterial suspension.

Preferably, the culture conditions are: 37°C constant temperature anaerobic.

Preferably, the Bifidobacterium animalis subsp. lactis MY9 strain is first cultured in MRS liquid culture medium to a stable phase, and then expanded into a new MRS liquid culture medium at a dilution multiple of 1:20-100.

The sixth aspect of the present invention provides an acetylcholinesterase inhibitor, wherein the inhibitor uses the Bifidobacterium animalis subsp. lactis MY9 strain described in the first aspect as a main active ingredient.

Preferably, in the field of medical applications, the inhibitor further includes a pharmaceutically acceptable excipient.

More preferably, the excipients include carriers and excipients. The excipients refer to diluents, adhesives, lubricants, disintegrants, solubilizers, stabilizers, etc. that can be used in the pharmaceutical field, as well as some pharmaceutical matrices. The carrier is a functional pharmaceutical excipient available in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

Preferably, in the field of medical applications, the dosage form of the inhibitor includes but is not limited to tablets, granules, capsules, pellets, sustained-release agents, oral liquid preparations, and injections. The above dosage forms refer to dosage forms commonly used in clinical practice. The pharmaceutical preparation can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). If some drugs are unstable under gastric conditions, they can be prepared into enteric-coated tablets.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention separates and purifies a strain of Bifidobacterium animalis subsp. lactis MY9 from the feces of a healthy adult in Guangdong Province, China. The strain has multiple prebiotic effects, including superior protease activity and cellulase activity, and can inhibit acetylcholinesterase activity. Therefore, the Bifidobacterium animalis subsp. lactis MY9 strain has multiple functions such as promoting digestion and absorption of protein food, improving protein allergy; promoting intestinal absorption, peristalsis and laxative; promoting absorption of cellulose, protecting the intestine and improving constipation. It can be seen that the newly isolated Bifidobacterium animalis subsp. lactis MY9 strain of the present invention has multiple prebiotic effects and can be used in the fields of laxative and intestinal protection. For example, it can be made into a laxative and intestinal protection medicine, which has important application value and economic value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a phylogenetic tree of Bifidobacterium animalis subsp. lactis MY9 strain (all strains are derived from the Genome database of NCBI);

Figure 2 is a degradation experiment of milk plate by Bifidobacterium lactis subspecies MY9 strain (a is a blank control group; b is an experimental group);

Figure 3 shows that the fermentation liquid of Bifidobacterium animalis subsp. lactis MY9 can secrete substances that significantly inhibit the activity of acetylcholinesterase;

FIG. 4 shows the degradation experiment of Bifidobacterium animalis subsp. lactis MY9 strain on cellulose plate (left side, blank control group; right side, experimental group).

DETAILED DESCRIPTION

The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

The experimental materials involved in the following examples are as follows:

(1) Bifidobacterium animalis subsp. lactis MY9 strain was isolated from the feces of a healthy adult in Guangdong, China by the Runze Laboratory of the Intestinal Microbiology Research Group and stored in glycerol tubes at -80°C. Generally, colonies can be obtained by inoculating it on the surface of an MRS solid culture medium plate and inverting it in a 37°C constant temperature anaerobic incubator for 24 hours, or by shaking it in an MRS liquid culture medium in a 37°C constant temperature anaerobic incubator for 24-48 hours to obtain a fermentation liquid.

(2) Kit: Acetylcholinesterase inhibitor screening kit (abnova, Cat: KA6219).

(3) MRS plate: beef extract 10 g, peptone 10 g, yeast extract 5 g, triammonium citrate 2 g, sodium acetate 5 g, glucose 20 g, dipotassium phosphate 2 g, Tween 80 1 mL, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, agar 15 g, ddH ₂ O to 1 L, adjust pH to 6.2-6.6, autoclave at 121°C for 20 min, and prepare MRS plate.

(4) MRS liquid medium: beef extract 10 g, peptone 10 g, yeast extract 5 g, triammonium citrate 2 g, sodium acetate 5 g, glucose 20 g, dipotassium phosphate 2 g, Tween 80 1 mL, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, make up to 1 L with ddH ₂ O, adjust the pH to 6.2-6.6, and sterilize at 121°C for 20 min to prepare MRS liquid medium.

(5) MP plate: 10 g skim milk powder, 1 g sodium chloride, 10 g beef extract, 10 g peptone, 5 g yeast extract, 20 g glucose, 2 g triammonium citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.5 mL Tween 80, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 15 g agar, make up to 1 L with ddH ₂ O, adjust pH to 6.2-6.6, sterilize by high pressure at 121°C for 20 min, and prepare MP plate.

(6) CMC plate: 10 g sodium carboxymethyl cellulose, 1.5 g ammonium sulfate, 0.3 g manganese sulfate, 0.2 g calcium chloride, 5 g sodium chloride, 0.3 g urea, 10 g beef extract, 10 g peptone, 5 g yeast extract, 20 g glucose, 2 g triammonium citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.5 mL Tween 80, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 15 g agar, make up to 1 L with _ddH2O , adjust pH to 6.2-6.6, sterilize by high pressure at 121°C for 20 min, and prepare CMC plate.

Example 1 Isolation and Identification of Bifidobacterium animalis subsp. lactis MY9 Strain

Bifidobacterium animalis subsp. lactis MY9 strain was isolated from the feces of a healthy adult (BMI=21.1) in Guangdong Province, China, as follows:

The fecal sample was repeatedly washed 3 times with sterile water, placed in a mortar, 500uL sterile water was added for every 100mg of feces, the homogenate was thoroughly ground, an appropriate amount of grinding liquid was taken with a pipette, and it was spread on an MRS plate and cultured at room temperature for 3 days. Then the colonies to be streaked and purified in the separation experiment plate were numbered with a marker pen, and the strain number was marked on the plate accordingly. After marking, the colonies were picked and inoculated on the MRS plate, and the strains were purified by the plate streaking method. If this method cannot separate the strains, it is necessary to pick the colonies from the enrichment plate, dilute them with the MRS liquid medium gradient, and spread them on the culture medium. Finally, referring to the Bergey Bacterial Identification Manual (Eighth Edition) and the Fungal Classification and Identification Manual, the strains belonging to bacteria were first distinguished. A purified strain was initially isolated, the strain number was MY9, and after 48 hours of culture, the colonies of the strain were observed to be small, smooth, convex, with complete edges and milky white.

Next, the isolated MY9 strain was molecularly identified by 16S rDNA universal primers (27F: AGAGTTTGATCCTGGCTCAG, 1492R: TACGGCTACCTTGTTACGACTT), and then Beijing Biomaike Biotechnology Co., Ltd. performed whole genome sequencing on the isolated MY9 strain. The obtained sequence 16S rDNA sequence (SEQID No: 1) was subjected to BLAST comparison in the Genome database of NCBI. The results showed that the homology between the MY9 strain and the known 16S rDNA sequence of Bifidobacterium animalis subsp. lactis was >99%, and evolutionary analysis was performed with homologous strains (Figure 1), confirming that MY9 is a different strain of the same species of Bifidobacterium animalis subsp. lactis.

Finally, the strain MY9 was deposited, and the deposit information is as follows: deposit time: October 25, 2023; name of the depository: China Center for Type Culture Collection (CCTCC); deposit number: CCTCC NO:M 20232016; address of the depository: Wuhan University, Wuhan, China; classification name: Bifidobacterium animalis subsp.lactis.

Bifidobacterium animalis subsp. lactis is a probiotic strain with a wide range of probiotic effects, but strains from different sources have different effects, indicating that the new Bifidobacterium animalis subsp. lactis MY9 isolated from human feces in the present invention can be used as a probiotic and may have new effects and functions.

Bifidobacterium animalis subsp.Lactis MY9 16S rDNA sequence (1397bp, SEQ ID No: 1):

GCAAGTCGAACGGGATCCCTGGCAGCTTGCTGTCGGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCAACCTGCCCTGTGCACCGGAATAGCTCCTGGAAACGGGTGGTAATACCGGATGCTCCGCTCCATCGCATGGTGGGGTGGGAAATGCTTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGTTGGCGGGGTGATGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGGTG ACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGT GCGGGATGGAGGCCTTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGTTTCGGCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGG TAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAG CGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTGCCGGATCGCCGTGGAGACACGGT TTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAA CCCTCGCCGCATGTTGCCAGCGGGTGATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGCGGTGCGACACGGTGACGTGGGGCGGATCGCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACG CCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGTAGCACCCGAAGCCGGTGGCCCGACCCTTGTGGGG.

Example 2 Function and application of Bifidobacterium animalis subsp. lactis MY9 strain

(1) Animal Bifidobacterium lactis subspecies MY9 strain can produce protease

The ability of animal Bifidobacterium lactis subspecies MY9 to secrete proteases to hydrolyze proteins was identified and measured using skim milk plate medium (MP plate) according to the agar well diffusion test. During the test, 3uL of animal Bifidobacterium lactis subspecies MY9 bacterial solution with a concentration of 10Abs was added to the MP plate of the experimental group, and 3uL of blank MRS medium was added to the control group, and then inverted cultured in a 37°C constant temperature anaerobic incubator for 3 days. The results showed that compared with the control with blank culture medium added, strain MY9 can significantly degrade protein and form an obvious degradation circle (Figure 2). Instructions Animal Bifidobacterium lactis subspecies MY9 strain can produce proteases that degrade milk protein.

Animal Bifidobacterium lactis subsp. MY9 strain can produce protease, which can be used as a probiotic strain to promote the digestion and absorption of protein in food by the human body and improve the absorption of small peptides and amino acids. At the same time, it can also be used for anti-allergy (improving food allergies caused by protein indigestion or non-absorption). In addition, it can also be used for the extraction of protease and applied to the production of protease in the food industry or the washing industry; it can also be used in microbial feed to help animals digest and absorb nutrients and improve the utilization rate of feed.

(2) The fermentation liquid of Bifidobacterium animalis subspecies MY9 can effectively inhibit the activity of acetylcholinesterase

Animal Bifidobacterium lactis subspecies MY9 cultured to the stable phase in MRS liquid medium was expanded to a new MRS liquid medium at a dilution multiple of 1:30, and the bacterial suspension was collected when the stable phase was reached for 24 hours. The fermentation supernatant was collected after centrifugation at 10,000×g and 4°C for 10 minutes, and then the fermentation supernatant was determined by the acetylcholinesterase inhibitor screening kit (KA6219) to determine the inhibitory ability of the fermentation supernatant on acetylcholinesterase (AchE) activity. The results showed that compared with the blank medium MRS without inhibition, the fermentation supernatant of strain MY9 significantly inhibited the activity of acetylcholinesterase, and the inhibition rate was about 13.74%, indicating that the fermentation broth of animal Bifidobacterium lactis subspecies MY9 can effectively inhibit the activity of acetylcholinesterase (Figure 3).

Acetylcholinesterase inhibitors reversibly inhibit acetylcholinesterase, allowing choline to accumulate in synapses under the action of acetylcholine released from nerve fiber endings, stimulating choline receptors, prolonging and increasing the effect of acetylcholine. It is reported that acetylcholinesterase inhibitors can enhance cognitive ability and memory (improve Alzheimer's disease, improve cognitive impairment), dilate blood vessels (improve tachycardia), and stimulate skeletal and smooth muscles (improve constipation, postoperative abdominal distension, and postoperative urinary retention).

Therefore, the probiotic animal Bifidobacterium lactis subsp. MY9 strain has the effects of inhibiting acetylcholinesterase activity and stimulating skeletal muscle and intestinal smooth muscle, which makes it a potential probiotic for promoting gastrointestinal absorption and motility.

(3) Bifidobacterium animalis subsp. lactis MY9 strain can produce cellulase

The ability of animal Bifidobacterium lactis subsp. MY9 to degrade cellulose was identified and determined using cellulose plate medium (CMC plate) according to the agar well diffusion method. During the experiment, 3uL of animal Bifidobacterium lactis subsp. MY9 bacterial solution with a concentration of 10Abs was added to the CMC plate of the experimental group, and 3uL of blank MRS medium was added to the control group. Congo red staining was performed after inverted culture in a 37°C constant temperature anaerobic incubator for 3 days. The results showed that compared with the control with blank culture medium added, strain MY9 could significantly degrade cellulose and form an obvious degradation circle (Figure 4). This shows that animal Bifidobacterium lactis subsp. MY9 strain can produce cellulase.

Since the animal Bifidobacterium lactis MY9 strain can produce cellulase, the probiotic animal Bifidobacterium lactis MY9 strain can play the following multiple roles through the effect of producing cellulase: (1) It is used to promote the digestion and absorption of dietary ingredients; (2) It improves constipation. Probiotics break down cellulose in the intestine to produce a certain amount of water, which has the effect of softening stool; (3) Lowering cholesterol is the most important effect of cellulose. After being absorbed by the human body, soluble fiber can inhibit the body's absorption of cholesterol, and can combine with cholesterol in the intestine to accelerate the metabolism of cholesterol in the body, preventing the body from inducing hyperlipidemia due to high cholesterol, and can reduce the incidence of arteriosclerosis and coronary heart disease; (4) Cellulose, a non-sweet "sugar", can slow down the blood's absorption of glucose, balance blood sugar concentration, and promote the sensitivity of muscle and fat cells to insulin, thereby preventing and assisting in the treatment of diabetes.

In addition, the animal Bifidobacterium lactis subsp. MY9 strain can also be used for the fermentation extraction of cellulase; for degrading the cell walls of pathogenic fungi and controlling diseases; for the decomposition of cellulose in compost; and for the preparation of livestock and poultry feed, such as feed for monogastric animals such as pigs and chickens, to overcome their inability to utilize cellulose.

In summary, the newly isolated Bifidobacterium animalis subsp. lactis MY9 of the present invention has multiple probiotic effects: (1) has excellent protease activity; (2) can inhibit acetylcholinesterase activity; (3) has excellent cellulase activity. It can be seen that the newly isolated Bifidobacterium animalis subsp. lactis MY9 strain of the present invention has important application value and economic value in the fields of laxative and intestinal protection.

The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims (8)

1. Use of a strain of bifidobacterium animalis subsp.lactis (Bifidobacterium animalis subsp. Lactis) MY9 in the production of a protease, wherein said strain of bifidobacterium animalis subsp.my 9 was deposited in the chinese collection of typical cultures at 10 months and 25 days 2023 under the accession number: CCTCC NO: M20232016; the 16S rDNA sequence of the animal bifidobacterium lactis MY9 strain is shown as SEQ ID No: 1.

2. Use of a strain of bifidobacterium animalis subsp.lactis (Bifidobacterium animalis subsp. Lactis) MY9 in cellulase production, wherein said strain of bifidobacterium animalis MY9 was deposited in the chinese collection of typical cultures at 10 and 25 of 2023 under the accession number: CCTCC NO: M20232016; the 16S rDNA sequence of the animal bifidobacterium lactis MY9 strain is shown as SEQ ID No: 1.

3. Use of a strain of bifidobacterium animalis subsp.lactis (Bifidobacterium animalis subsp. Lactis) MY9 for the preparation of an acetylcholinesterase inhibitor, wherein said strain of bifidobacterium animalis subsp.my 9 was deposited in the chinese collection of typical cultures at 10 months and 25 days 2023 under the accession number: CCTCC NO: M20232016; the 16S rDNA sequence of the animal bifidobacterium lactis MY9 strain is shown as SEQ ID No: 1.

4. A method for preparing protease is characterized in that an animal bifidobacterium subsp (Bifidobacterium animalis subsp.lacti) MY9 strain is inoculated in an MRS liquid culture medium, a bacterial suspension is collected after the culture is carried out until the stationary phase, and finally the protease in the bacterial suspension is separated and purified to obtain the protease; the animal bifidobacterium lactis MY9 strain is preserved in China center for type culture Collection (China, with the preservation number: CCTCC NO: M20232016; the 16S rDNA sequence of the animal bifidobacterium lactis MY9 strain is shown as SEQ ID No: 1.

5. The method for producing a protease according to claim 4, wherein the conditions for the cultivation are: anaerobic at a constant temperature of 37 ℃.

6. A method for preparing protease according to claim 4, wherein the strain MY9 of Bifidobacterium animalis subsp.lactis (Bifidobacterium animalis subsp. Lactis) is cultured in MRS liquid medium to a stationary phase, and then expanded in new MRS liquid medium at a dilution of 1:20-100.

7. An acetylcholinesterase inhibitor, characterized in that the inhibitor comprises a strain of bifidobacterium animalis subsp.lactis (Bifidobacterium animalis subsp.lactis) MY9 as a main active ingredient; the animal bifidobacterium lactis MY9 strain is preserved in China center for type culture Collection (China, with the preservation number: CCTCC NO: M20232016; the 16S rDNA sequence of the animal bifidobacterium lactis MY9 strain is shown as SEQ ID No: 1.

8. The acetylcholinesterase inhibitor according to claim 7, wherein in the field of medical use, the inhibitor further comprises a pharmaceutically acceptable excipient.