CN118048265A

CN118048265A - Lactobacillus rhamnosus DACN1224 and application thereof in preventing/improving Alzheimer's disease

Info

Publication number: CN118048265A
Application number: CN202410239840.7A
Authority: CN
Inventors: 索化夷
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2024-03-04
Filing date: 2024-03-04
Publication date: 2024-05-17

Abstract

The invention discloses a lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224 and a microbial inoculum thereof, wherein the DACN1224 is preserved in China center for type culture collection, and the preservation number is CCTCCNO: m20232453. Also disclosed is the use of DACN1224 or a microbial inoculum thereof in the manufacture of a product for the prevention and/or amelioration of alzheimer's disease or related neurodegenerative diseases. The DACN1124 can be used for preventing and/or treating Alzheimer's disease and related neurodegenerative diseases, can be used as functional probiotics to be applied to foods, food additives, health products or medicines, and has good application prospect.

Description

Lactobacillus rhamnosus DACN1224 and application thereof in preventing/improving Alzheimer's disease

Technical Field

The invention relates to the technical field of microorganisms, in particular to a lactobacillus rhamnosus DACN1224 and application thereof in preventing/improving Alzheimer's disease.

Background

Alzheimer's Disease (AD) is the most common neurodegenerative disease and is the main cause of senile dementia, accounting for about 60% -70% of cases of dementia, and is becoming a global threat to human health. It is counted that about 5000 tens of thousands of people worldwide have dementia, and this figure is expected to double every 20 years. AD is a destructive and ubiquitous multi-causative degenerative disease of the central nervous system that results in loss of synaptic connections between brain cells and neuronal cells, and thus in a decrease in progressive cognitive ability. The main symptoms of AD patients include: language disorder, reduced understanding ability, self life failure, reduced exercise ability, memory deficit or loss, and the like, and is accompanied by changes in emotion such as anxiety, depression, and the like. The physiopathology of Alzheimer's disease suggests: alzheimer's disease is associated with the manifestations of multiple aspects of Abeta deposition, tau protein hyperphosphorylation, neuroinflammation, synaptic dysfunction, and dysfunction of the intestinal-brain axis.

The drugs currently applied to the treatment of Alzheimer's disease mainly comprise two main categories: 1) Cholinesterase inhibitors (AChEI): it is based on classical cholinergic hypothesis, considering the key role of acetylcholine (ACh) in human brain cognitive function, on the other hand, decreased function of muscarinic M1 receptor subtype and nicotinic receptor in brain also leads to impaired cholinergic neurotransmission in AD due to aβ deposition, and thus AChEI relieves AD by inhibiting acetylcholine decomposition, current AChEI drugs are donepezil hydrochloride, galantamine, rivastigmine, etc.; 2) N-methyl-D-aspartate (NMDA) receptor antagonists: such a drug is mainly memantine, a non-competitive NMDA receptor antagonist, approved for the treatment of mild to severe AD. It can reduce excitotoxicity and neurodegeneration caused by excessive glutamatergic neurotransmission. It can also reduce Tau hyperphosphorylation and prevent aβ -induced neuronal toxicity. However, these drugs are symptomatic therapeutic drugs at present, drugs affecting disease processes are still in the preliminary research stage, and most of these drugs have side effects. Therefore, it is particularly important to explore an intervention method capable of delaying the disease process in the early stage of the onset of Alzheimer's disease, and the method has a wide market potential. The "gut-brain axis" is a new concept proposed in recent years, and research finds that the brain and the gut communicate bidirectionally through a neural pathway, a hormonal pathway and an immunological pathway, while gut microorganisms play a key role in the process of communicating between the gut and the brain.

Lactic acid bacteria are common "mental probiotics" which are mainly derived from natural organisms such as fermented foods, healthy human bodies and the like. At present, a plurality of preclinical and clinical researches show that probiotics can play a role in relieving Alzheimer's disease through mechanisms such as regulating intestinal microorganism composition, protecting intestinal barrier, regulating organism immunity and the like. The lactobacillus casei LTL1361 can increase intestinal villus length, reduce inflammatory infiltration, up regulate mRNA expression of tight junction proteins Claudin-1 and zonula occludens-1 (ZO-1) in small intestine and colon, further exert the protection effect on brain and intestinal tract, alleviate age-related cognitive decline in AD, and have a certain degree of alleviation on AD. Akkermansiamuciniphila can reduce hippocampal microglial proliferation and pro-inflammatory cytokine expression, restore neuronal development and synaptic plasticity and thereby improve AD progression and its associated cognitive decline. However, the currently known "mental probiotics" species useful for controlling neurodegenerative diseases are relatively poor, and due to the complexity of the intestinal microbiota and its difficulty of study, there are few clinical studies currently based on modulating the intestinal microbiota to alleviate neurodegenerative diseases, the mechanism by which they act is not yet defined. Therefore, the method screens out the probiotics which can relieve the Alzheimer's disease and deeply digs the action mechanism of the probiotics, is very important to develop the probiotics products with higher health care value, has very broad prospect and is worthy of intensive research.

Disclosure of Invention

The invention aims at solving the problems and provides a lactobacillus rhamnosus DACN1224 and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

The first aspect of the invention provides a strain of Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus)

DACN1224, deposited at the chinese collection of typical cultures with the accession number CCTCCNO:

M20232453。

In a second aspect, the invention provides a microbial agent, the active ingredient of which comprises one or more of the above-mentioned lactobacillus rhamnosus DACN1224, lactobacillus rhamnosus DACN1224 inactivated bacteria, metabolites of lactobacillus rhamnosus DACN1224, and lactobacillus rhamnosus DACN1224 lyophilized strains.

The microbial agent, lactobacillus rhamnosus DACN1224, is used alone or in combination with other strains.

The third aspect of the invention provides a preparation method of the microbial inoculum, which comprises the following steps: inoculating the lactobacillus rhamnosus DACN1224 into a culture medium for culture, and preferably culturing in the MRS culture medium at 30-38 ℃ for 18-24h.

In a fourth aspect, the invention provides the use of lactobacillus rhamnosus DACN1224 as described above or of a microbial agent as described above for the manufacture of a product for the prevention and/or amelioration of alzheimer's disease or related neurodegenerative diseases including dementia, cognitive disorders, memory disorders.

Said application, said product having at least one of the following functions:

(1) Preventing or ameliorating Alzheimer's disease, preventing or ameliorating cognitive and behavioral dysfunction in a subject;

(2) Preventing or improving Alzheimer's disease, preventing or relieving colon and Hippocampus injury of the subject, preventing or relieving intestinal inflammation and protecting intestinal tract;

(3) Preventing or improving Alzheimer's disease, and reducing intestinal permeability;

(4) Preventing or improving Alzheimer's disease, preventing or improving systemic inflammation in a subject;

(5) Preventing or improving Alzheimer's disease, protecting neurons in brain of a subject, and preventing or improving neuroinflammatory injury of the subject.

The product is a medicine, a functional food, a health-care product, a food additive, a health-care product additive or a feed.

The pharmaceutical product also includes a pharmaceutically acceptable carrier.

The dosage forms of the medicine comprise powder, granules, capsules, tablets, pills or solutions.

In the application technical scheme, according to the raw material source of the food, the food comprises dairy products, bean products, fruit and vegetable products and fermented food; the food comprises solid food, liquid food and semi-solid food according to the state of the food.

The invention screens lactobacillus with excellent antioxidant capacity through in vitro test combined with in vivo animal test, and the test result of alleviating mouse Alzheimer's disease shows that: lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224 has excellent antioxidant capacity; in an Alzheimer's disease mouse model test, the cognitive and behavioral dysfunction of the Alzheimer's disease mouse can be obviously relieved by taking DACN1224, and evaluation indexes are open field experiments, new object recognition experiments and nesting experiments; DACN1224 administered significantly reduced colon and hippocampal injury in mice with alzheimer's disease, assessed as colon and hippocampal H & E staining section analysis, reduced inflammation and protected intestinal tract; the administration of DACN1224 can obviously reduce the permeability of intestinal tracts, and the evaluation indexes are the expression of colon zonulin ZO-1, occludin, claudin-1 and Claudin-5; DACN1224 can obviously improve the occurrence of the systemic inflammation of mice with Alzheimer's disease, and the evaluation indexes are the gene expression of colon Interleukin (IL) -6, interleukin (IL) -beta, tumor Necrosis Factor (TNF) -alpha and IL-10, and the level of serum IL-6, IL-beta, TNF-alpha and IL-10, and the gene expression of hippocampal IL-6, IL-beta, TNF-alpha and IL-10; DACN1224 was administered to protect neurons in the brain of mice with alzheimer's disease, and was evaluated for gene expression of hippocampal BDNF, PSD 95. The DACN1124 can be used for preventing and/or treating Alzheimer's disease and related neurodegenerative diseases, can be used as functional probiotics to be applied to foods, food additives, health products or medicines, and has good application prospect.

Drawings

FIG. 1 shows colony morphology (A) of isolated strains and gram staining results (B).

FIG. 2 shows the API 50CH reaction results of Lactobacillus rhamnosus DACN 1224.

FIG. 3 shows the results of the measurement of the ability of lactic acid bacteria to produce gamma-aminobutyric acid.

FIG. 4 is a schematic representation of the behavioral changes of DACN1224 in mice with Alzheimer's disease 8 weeks after intervention, (A) open field experiments; (B) new object recognition experiments; and (C) nesting experiments.

Fig. 5 shows colon H & E staining results 8 weeks after DACN1224 intervention in alzheimer's disease mice.

FIG. 6 shows the results of H & E staining of hippocampus 8 weeks after DACN1224 intervention in Alzheimer's disease mice.

FIG. 7 shows the expression of the colonic zonulin ZO-1, occludin, claudin-1, claudin-5 after 8 weeks of DACN1224 intervention in Alzheimer's disease mice.

FIG. 8 shows gene expression of serum albumin (IL) -6, interleukin (IL) -beta, tumor Necrosis Factor (TNF) -alpha and IL-10 after DACN1224 intervention in Alzheimer's disease mice for 8 weeks.

FIG. 9 shows levels of colon Interleukin (IL) -6, interleukin (IL) -beta, tumor Necrosis Factor (TNF) -alpha and IL-10.

FIG. 10 shows gene expression of hippocampal Interleukin (IL) -6, interleukin (IL) -beta, tumor Necrosis Factor (TNF) -alpha and IL-10.

FIG. 11 shows gene expression of BDNF and PSD95 in hippocampus.

In FIGS. 4-11, con- -control, mod- -model, DACN1224- -intervention, don- -donepezil hydrochloride intervention.

Detailed Description

The invention is further illustrated, but is not limited, by the following examples.

The experimental methods in the following examples are conventional methods unless otherwise specified; unless otherwise indicated, all biological and chemical reagents used are conventional in the art and are commercially available.

Example 1 in vitro antioxidant Activity Screen of lactic acid bacteria DACN1224

1 Experimental materials

The lactobacillus rhamnosus DACN1224 strain source: screening traditional fermented foods such as pickle and yogurt from the south of the west (Yun Gui Chongqing, etc.) and residents in Qinghai-Tibet plateau, screening DACN1224, and storing in Soxhlet flos Magnoliae laboratory strain library of food science college of the university of West.

The remaining 25 strains of lactobacillus in table 2 are all lactobacillus deposited by the national food science institute of soxhlet laboratory screening of the university of southwest: lactobacillus plantarum (Lactobacillusplantarum), lactobacillus mucilaginosus (Lactobacillusfermentans), lactobacillus rhamnosus (Lacticaseibacillus rhamnosus), lactobacillus paracasei (Lactobacillusparacasei), lactobacillus casei (Lactobacillus casei), lactobacillus delbrueckii (Lactobacillus bulgaricus), lactobacillus salivarius (Lactobacillussalivarius) and lactobacillus curvatus (Lactobacillus curvatus).

The DACN1224 strain deposit information is as follows:

The strain DACN1224 is preserved in China center for type culture collection (CCTCC for short) for 12 months in 2023, and the preservation date is in the eight-path 299-No. Wuhan university school in Wuhan district, wuhan, hubei province: 2023, 12, 04 th month, deposit number: cctccc NO: m20232453; the classification is named: lactobacillus rhamnosus DACN1224 (Lacticaseibacillus rhamnosusDACN 1224).

2 Experimental methods

2.1 Morphological identification of lactic acid bacteria

Lactic acid bacteria were stained according to the instructions of the gram staining kit, and then the morphology was observed under a 100-fold microscope with a binocular microscope.

2.2PCR amplification of 16S rDNA sequences

The bacterial genome DNA extraction kit is adopted to extract the DNA of the strain. PCR amplification was performed using a 25. Mu.L reaction system, and after the completion of the reaction, detection was performed by agarose gel electrophoresis. The qualified samples were sequenced by the large gene technologies, inc., and the sequencing results were analyzed for homology by BLAST program in NCBI.

2.3 Biochemical identification of lactic acid bacteria API 50CH kit

The isolated strain is cultured in MRS liquid culture medium at 37 ℃ for 18 hours, and the thalli are collected by centrifugation under the conditions of 3000r/min and 15min, washed by sterile normal saline and resuspended into bacterial suspension. Reference Mei Liai (france) API 50CH kit instructions.

2.4 Preparation of lactic acid bacteria DACN1224

The strain DACN1224 stored in the glycerol tube was taken out from the refrigerator at-80℃and inoculated into 10mL MRS broth liquid tube at an inoculum size of 2%, cultured at 37℃for 24 hours at 100r/min, and activated for two consecutive generations. 5mL of strain culture solution is sucked, and centrifugation is carried out for 10min under 6000r/min to collect thalli, the collected thalli is washed three times by 1X PBS, then the thalli is resuspended in PBS, and the concentration of the thalli is regulated to 1.0X10 ⁹ CFU/mL by PBS for standby.

2.5 Detection of the resistance of lactic acid bacteria to bile salts of Artificial gastric juice

Detection of the tolerance of lactic acid bacteria to artificial gastric juice: firstly, preparing artificial gastric juice, namely, 0.2 percent of NaCl, 0.35 percent of pepsin (1:10000), and adjusting the pH value to 3.0 by using 1M HCl, and filtering the gastric juice by using a filter head in a sterile operation table for later use. Taking 5mL of activated strain culture solution, pouring the strain culture solution into a sterilized 10mL centrifuge tube in a sterile ultra-clean operation table, centrifuging at 4 ℃ and 4000rpm for 10min to collect thalli, adding 5mL of sterilized normal saline (0.9%) and uniformly mixing to prepare bacterial suspension, taking 1mL of bacterial suspension, mixing with 9mL of artificial gastric juice with pH of 3.0, shaking uniformly, placing in a constant temperature oscillator for culture (37 ℃ C., 100 r/min), sampling (10 mu L) at 0h and 3h respectively, taking four gradients after dilution, and pouring with MRS agar culture medium for culture at 37 ℃ for 48h. Finally, the viable count is measured by a plate counting method, and the survival rate (%) is calculated. The calculation formula is as follows:

survival rate (%) =number of viable bacteria for 3 h/number of viable bacteria for 0h×100%

The bacterial strain with the artificial gastric juice survival rate result of more than 60 percent is subjected to the next bile salt tolerance test.

Bile salt tolerance test: 0.3% bile salt and 0.2% sodium thioglycolate are added into MRS liquid culture medium to prepare 0.3% bile salt culture medium (MRS-THIO culture medium). 5mL of the activated strain is inoculated into MRS-THIO culture medium containing 0.0% (namely blank) and 0.3% respectively according to the inoculum size (v/v) of 2%. After culturing for 24h at 37 ℃ and 100r/min in a constant temperature shaker, 200uL of blank culture medium (unvaccinated 0.0% and 0.3% MRS-THIO culture medium) is taken as a control, OD ₆₀₀ values of the culture mediums with different concentrations are measured in an ELISA plate, and the tolerance of the strain to bile salts is calculated.

The calculation formula is as follows:

0.3% bile salt tolerance= (0.3% inoculation OD value-0.3% blank OD value)/(0.0% inoculation OD value-0.0% blank OD value) ×100%

Strains with an artificial gastric juice tolerance of > 60% and bile salt tolerance of > 10% were used in the subsequent experiments.

2.6DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging Capacity detection

DPPH free radical scavenging experiments are commonly used for evaluating the antioxidant capacity, and DPPH is dissolved in 95% ethanol to prepare a solution with the concentration of 0.1mmol/L for later use. Mixing 500 mu L of a sample to be detected with 500 mu LDPPH (1:1, v/v) solution, shaking and uniformly mixing for 1min, standing at room temperature in a dark place for 30min, centrifuging at 4 ℃ and 8000r/min for 3min to obtain a supernatant, and measuring absorbance at the wavelength of 517 nm. Each sample to be tested was treated in 3 replicates with 0.5mg/mL Vc solution as positive control. And the DPPH clearance was calculated by the following formula. The calculation formula is as follows:

R(％)＝[1-(Ai-Aj)/Ac)]×100％ (1)

In the formula (1): ai-sample to be measured+DPPH solution absorbance at 517 nm;

Aj-absorbance of the sample to be measured +95% ethanol solution at 517 nm;

Ac-DPPH solution+PBS absorbance at 517 nm.

2.7 Detection of ABTS (2, 2-Di-aza-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) free radical scavenger capability

ABTS free radical scavenging experiments, which are one of the commonly used methods for evaluating antioxidant capacity, are increasingly applied to the identification and evaluation of antioxidant capacity of lactic acid bacteria, and refer to Li et al for [Characterization,antioxidant and immunomodulatory activities ofpolysaccharides fromprunella vulgaris linn;2015; journal: int J Biol Macromol ] and slightly adjusted. The ABTS working solution was prepared as it is and overnight to prepare the radical cation, 2.45mM potassium persulfate was homogeneously mixed with 7mM ABTS (1:1 v/v) and stabilized against light at room temperature for 24 hours. In the experiment, the ABTS working solution is diluted by distilled water, so that the absorbance of the solution at the position of 734nm reaches 0.7+/-0.005, and the ABTS working solution can be obtained. Then, 50 mu L of bacteria liquid to be detected and 100 mu L of ABTS reaction liquid are evenly mixed, incubated for 10min at room temperature, the absorbance of the reactant is measured at the wavelength of 734nm after incubation is finished, and the calculation is carried out according to the following formula, and each sample to be detected is subjected to three parallel treatments. The calculation formula is as follows:

R(％)＝[Ab-(As-Ac)]/Ab×100％ (2)

In the formula (2): ab-distilled water+ABTS solution absorbance at 734 nm;

the absorbance of the As-sample to be detected+ABTS solution at 734 nm;

ac-sample to be tested+PBS absorbance at 734 nm.

2.8 Thin layer chromatography for detecting the ability of the Strain to produce gamma-aminobutyric acid

The strain was inoculated in an inoculum size of 3% in MRS medium containing 1% L-glutamic acid and cultured at 37℃for 48 hours. 1mL of the fermentation broth was centrifuged (16000 g, 15 min), and the supernatant was collected. And taking a proper amount of fermentation supernatant by using a capillary tube, and spotting the fermentation supernatant on a G-type silica gel thin layer plate. Preparing developing agent with n-butanol, glacial acetic acid and water=4:1:3, adding 0.4% ninhydrin, sealing, developing, and developing at 90deg.C for 15min. Meanwhile, L-glutamic acid liquid culture medium, 1g/L gamma-aminobutyric acid standard solution and 1g/L L-glutamic acid standard solution are used as control.

2.9 Data and statistical analysis

All experimental data were plotted using the Duncan method (P < 0.05) using the one-wayANOVA method in SPSS26.0 software, using Graphpad8.0. The results of the experiments are expressed as mean ± standard deviation, three replicates per experiment.

3 Results and analysis

3.1 Colony morphology and cell morphology of Strain

The morphology of the strain was observed using plate streaking, gram staining and scanning electron microscopy. Strains are single colonies with single forms on MRS plates, and most of the colonies are round in shape, regular in edge, white and moist and smooth in surface. After gram staining, the strain is observed by a microscope to be blue-violet under the microscope, short rods are connected together and have uniform morphology, and the strain is judged to be pure gram-positive bacteria. Among them, the colony morphology and gram staining results of DACN1224 are shown in FIG. 1.3.2 PCR amplification results of Strain 16SrDNA sequence

PCR amplification and detection of PCR amplification products by 1.2% agarose gel electrophoresis, after qualified sequencing of the amplification products, inputting the sequencing results into NCBI database, and carrying out homology comparison by Blast, wherein the comparison result shows that the sequence (SEQ ID NO. 1) of the 16S rDNAPCR amplification product of the strain DACN1224 is 100% of that of the Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus, accession number: NZ_CP 040780) in the Gene Bank database, and has the highest homology in the Gene Bank.

The sequence of the 16S rDNAPCR amplification product of DACN1224 (SEQ ID NO. 1) is as follows ：CCATTTGGTCACTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACACAGTTACTCTGCCGACCATTCTTCTCCAACACAGAGTTTTACGACCCGAAAGCCTTCTCACTCACGCGCGTGCTCATCAGACTTGCGTCCATGTGAGATCCCTACTGCTGCCTCCCGTAGAGTTTGGGCCGGTGTTCCTCAGTCCCAATGTGGCCGAATCAAC.

3.3 Results of identification of Biochemical Properties of Strain

The API 50CH is mainly based on the fermentation test of the strain on carbohydrate as a main basis for judging the level phenotype of lactobacillus species. There are 49 different carbohydrates in the API 50CH kit, and the strain is identified by its use of the compounds in 49. FIG. 2 and Table 1 show the results of fermentation tests of strain DACN1224 on 49 carbohydrates. The results show that strain DACN1224 is able to utilize 28 of these 49 carbohydrates, identified by API detection, strain DACN1224 is lactobacillus rhamnosus (Lactobacillus rhamnosus) with an ID value of 99.90% and a T value of 0.96, meeting the identification requirements (ID value of 99,0% and T value of 0.5 or more), and thus strain DACN1224 is identified as lactobacillus rhamnosus (Lactobacillus rhamnosus).

TABLE 1 results of fermentation test of DACN1224 on 49 carbohydrates

3.4 Artificial gastric juice bile salt tolerance of lactic acid bacteria

Gastric juice is considered one of the primary physiological challenges faced by probiotic strains due to the low pH and antibacterial effects of pepsin, while survival of probiotics in the small intestine is considered another challenge due to the presence of bile salts and pancreatin in the small intestine. Therefore, the tolerance of the strain to gastric bile salts can reflect whether the strain can survive passing through the upper gastrointestinal tract after entering the host body, and then reaches the intestinal tract to exert beneficial effects. All strains used in the study are well-tolerated strains which have been subjected to gastric acid bile salt screening and self screening in the laboratory, wherein the strains with a survival rate of more than 60% and a bile salt growth rate of more than 10% at ph=3.0 are target strains. Table 2 shows the test results of the test for the tolerance of the strain in the present test by screening for bile salts of artificial gastric juice: 60 strains are screened, wherein 23 strains with better gastric acid bile salt tolerance are selected, and the strains comprise 7 strains of lactobacillus plantarum, 8 strains of lactobacillus fermentum, 1 strain of lactobacillus rhamnosus, 3 strains of lactobacillus paracasei, 1 strain of lactobacillus casei, 1 strain of lactobacillus delbrueckii, 1 strain of lactobacillus salivarius and 1 strain of lactobacillus curvatus. The 23 strains meet the screening conditions that the survival rate of the strains reaches more than 60 percent and the growth rate of 0.3 percent bile salt reaches more than 10 percent when the pH=3.0 gastric acid, and the 23 strains are primarily judged to have the capacity of resisting gastric bile salt and reach the intestinal tract after survival of the upper gastrointestinal tract, so that the probiotic effect is further exerted.

Table 2 artificial gastric juice bile salt tolerance of strains

3.5 In vitro antioxidant Activity of lactic acid bacteria

Oxidative stress is widely considered to be closely related to the onset of Alzheimer's disease, and attempts have been made to evaluate the antioxidant ability of lactic acid bacteria in vitro by searching for strains having an antioxidant effect, i.e., an antioxidant effect, which alleviate oxidative stress, while DPPH and ABTS free radical scavenging ability are key indicators. The strain with strong resistance activity of the artificial gastric juice bile salt screened in the earlier stage is evaluated for the oxidation resistance through DPPH and ABTS clearance experiments. The radical scavenging rate varies greatly between different strains, which also reflects the strength of their antioxidant capacity. As shown in Table 3, the comprehensive evaluation of DPPH and ABTS clearance shows that lactobacillus plantarum 2018SWU.B05.08004, lactobacillus mucilaginosus 2017SWU.B05.13002, lactobacillus rhamnosus DACN1224, lactobacillus paracasei 2018SWU.B05.03001 and lactobacillus salivarius 2021SWU.B05.31022 have stronger antioxidant capacity (DPPH clearance (%): 30.32+/-1.02, 47.58 +/-2.50, 64.86 +/-3.83, 37.96 +/-1.10, 41.71 +/-1.17 and ABTS clearance (%) 20.00+/-0.42, 19.73+/-2.24, 51.46 +/-1.91, 35.06 +/-2.38 and 42.38 +/-2.26), so that animal pre-experiments are carried out on the strains, and the improvement effect on cognitive performance disorder of the strains on mice with the Alzheimer disease model is analyzed. Animal pre-experiment results show that the lactobacillus rhamnosus DACN1224 has a positive effect on improving cognitive behavioral dysfunction of mice with Alzheimer's disease, and has more obvious effect compared with other strains.

TABLE 3 results of DPPH and ABTS radical scavenging Rate for strains

3.6 Ability of lactic acid bacteria to produce gamma-aminobutyric acid

Gamma-aminobutyric acid is an inhibitory neurotransmitter that catalyzes the synthesis of glutamate by glutamate decarboxylase and is commonly distributed in animals and plants. The research shows that the gamma-aminobutyric acid has the functions of reducing neurogenic property and preventing nerve cell overheating, and also has the physiological effects of preventing arteriosclerosis, regulating arrhythmia, reducing blood fat, enhancing liver function, improving sleep and the like. Lactic acid bacteria are normal flora in the intestinal tract of animals, are safe and beneficial to human bodies, and are considered as the first choice for synthesizing gamma-aminobutyric acid in the food and pharmaceutical industries. Therefore, the capacity of measuring the lactobacillus strain to produce the gamma-aminobutyric acid is considered, and a foundation is provided for subsequent researches. As shown in figure 3, the qualitative detection of the thin-layer chromatography shows that 7 strains have better gamma-aminobutyric acid production capacity and ：2021SWU.B05.06001、2028SWU.B05.03001、2018SWU.B05.08004、2021SWU.B05.31022、2017SWU.B05.13002、2020SWU.B05.16002、DACN1224. thin-layer chromatography results show that the relative content is mainly shown, and the results show that the strains have stronger gamma-aminobutyric acid production capacity compared with other strains.

The lactobacillus rhamnosus DACN1224 strain has excellent antioxidation and gamma-aminobutyric acid production capacity, which are helpful for stabilizing the body health, and the gamma-aminobutyric acid is reported to have sleep improving effect, so that the lactobacillus rhamnosus DACN1224 strain has a certain potential in the preparation of sleep-aiding or sleep-improving products.

EXAMPLE 2 animal experiment 1 of Lactobacillus rhamnosus DACN1224 establishment of mouse model for Alzheimer's disease and administration method

Male C57BL/6J mice of 10 weeks of age were taken and the experimental mice were acclimatized for one week before the initiation of the formal experiment. After the adaptive feeding is completed, all mice are weighed and subjected to a formal experiment after being randomly grouped according to the average body weight. Mice were randomly assigned to the normal group (Con), model group (Mod), lacticaseibacillus rhamnosus DACN group 1224 (DACN 1224), donepezil hydrochloride (Donepezil Hydrochloride) group (Don), 8 animals each. The method of Song et al is referred to and slightly improved for modeling of the Alzheimer's disease model mice, and the results of preliminary experiments are combined to determine that three groups except for Con groups are used for constructing an AD mouse model by injecting D-gal (120 mg.kg-1.d ^-1) into the abdominal cavity of the mice, and simultaneously filling stomach AlCl ₃(20 mg·kg-1·d^-1, and the modeling period is 8 weeks, so as to ensure successful modeling of the Alzheimer's disease, and the Con groups of mice are injected with physiological saline and the stomach double distilled water into the abdominal cavity according to the same volume. After the initiation of the formal experiments, the modeling and administration of mice were performed simultaneously, the corresponding doses of the drugs were administered by intragastric administration in each group, and the equal volumes of physiological saline were administered by Con and Mod groups, and the mice were free to drink water during the experiments. The animals were grouped and the mode of administration is shown in Table 4.

TABLE 4 grouping and administration of animals

The preparation method of DACN1224 bacterial suspension comprises the following steps: taking a glycerin tube preserved at the temperature of minus 80 ℃, inoculating a strain DACN1224 into an MRS liquid culture medium with an inoculum size of 2% (v/v) for culture at the temperature of 37 ℃ for 24 hours, collecting thalli by centrifugation for 10min at 8000r/min after two generations of activation, precipitating the thalli 3 times by using physiological saline, and regulating the concentration of the strain by using the physiological saline to ensure that the final gastric lavage live bacteria concentration is 1 multiplied by 10 ⁹ cfu/mL and the gastric lavage volume is 0.1mL/10 g/min.

D-galactose: weighing 2.4g D-galactose in a volumetric flask, fixing the volume to 100mL with normal saline, keeping the final concentration of the solution at 24 mg/mL, filtering with a 0.22um sterile filter membrane, packaging, and storing in a refrigerator at-20deg.C for use.

AlCl ₃ solution: weighing 0.4g AlCl ₃ in a volumetric flask, metering the volume to 100mL with double distilled water, and filtering and packaging with a 0.22um sterile filter membrane, and storing in a refrigerator at-20deg.C for use.

All mice were subjected to behavioural tests, during which modeling and dosing were performed normally, starting at week 9, and after the behavioural tests were completed, the mice were sacrificed and related tissues and serum were collected.

2 Animal experiment method

2.1 Mouse behavioural test

(1) Open field experiment (Open FIELD TEST, OFT)

The open field test is a popular task for evaluating the autonomous behavior of experimental animals in unfamiliar environments, and can be used for evaluating anxiety-like behavior of experimental mice, curiosity and spontaneous exploration behavior of the outside world. The open field experimental device consists of four pure white square box bodies (40 cm multiplied by 40 cm) and a digital acquisition system. A camera is arranged right above the device to record the movement track and movement condition of the mice in the whole course, and soft light is kept in the whole course of the experiment so as to reduce the stimulation to the experimental mice. After the experiment is started, each mouse is placed at the center of the bottom to freely move, and is tracked and recorded by professional equipment, each mouse is recorded for 5-6min, and meanwhile, the experimenter should be kept quiet in the whole course and keep a proper distance from an open field activity box. After each mouse is tested, the test device is cleaned by 75% alcohol, so that the influence of odor and excrement on the next mouse is avoided, meanwhile, the defecation amount of the mouse can be recorded, and the anxiety degree of the tested mouse is reflected to a certain extent. After the experiment is finished, the motion trail of the mice is captured by Noldus Etho Vision XT 8.5.5 software, and the total motion distance, the center region activity distance and the standing times of the mice are analyzed.

(2) New object identification experiment (Novel object recognition NOR)

Experimental mice are more prone to old objects than new objects, which have been studied by behavioral pharmacologists and neurosciences for learning and memory, while new object recognition experiments are mainly aimed at examining the learning and memory abilities of mice and the exploratory ability for novelty. The test was carried out in a sound-proof chamber, at a temperature of 22-25 ℃, and consisted of three stages: habituation, training and testing phases. The experimental device consists of four pure white square boxes (40 cm multiplied by 40 cm), wherein the first stage is habituation, the mice are placed into a test box from the middle position towards the wall to be freely explored for 5min, and then are placed back into a mouse cage; after 24 hours, entering a training stage, placing two objects with identical materials, shapes and colors in the middle of two ends of a test box, wherein the objects are at the same distance from the boundary of the test box, and at the same time, the positions placed by the mice are at the same distance, then placing the mice facing the wall into the test box, and freely exploring the same objects for 5 minutes, wherein the whole experiment process is tracked and recorded by professional equipment; and after the training is finished for 24 hours, starting a testing stage, replacing one object in the testing box with a new object with different materials, shapes and colors, putting the mouse into the testing box facing the wall, freely exploring for 5 minutes, and recording the times of exploring the new object and the old object by the mouse respectively. After each mouse completes the experiment, 75% alcohol is needed to clean the experimental device, so that the influence of odor and excrement on the next mouse is avoided. The new object identification index (Novel object exploration index, RI%) is calculated as follows: RI (%) = (number of times new object is explored/total number of times new and old object is explored) ×100%

(3) Nesting experiment (Nestingbehavioral tests)

Nesting is an instinctive social behavior of rodents that reflects to some extent the cognitive ability of mice, and is also demonstrated to be sensitive to brain injury, genetic mutation, and brain injury or aging can cause damage to nesting ability. This is a simple and easy to accomplish test. The experimental mice were transferred to separate cages with corncob bedding placed thereon at least one hour in advance before the dark phase began. A nest compressed cotton block (3.0 g) was placed in each cage. The next morning, the remaining nests were scored according to the scoring system described previously and using a well-defined 5-point bird nest rating scale (1: no obvious contact or >90% intact for the nest, 2: 50-90% intact for the nest, 3: 50-90% crushed but no identifiable nest location, 4: 90% crushed for the nest, the nest lying flat in a 1/4 cage, 5: a (near) perfect nest with a higher nest wall height than the body height of the mouse, about 50% of the perimeter of the nest. Unwarped nest fragments were weighed. Unwarped fragments were defined as greater than about 0.1 gram.

2.2 Tissue H & E section preparation

Taking the brain after the mice are sacrificed, and fixing the whole half brain of the mouse brain; the colon part is fixed by taking about 1cm in length, the tissues are washed clean by normal saline, the water is sucked by filter paper, and the tissues are completely immersed into 4% paraformaldehyde for fixation. And (5) preparing paraffin sections through gradient ethanol dehydration, paraffin dipping and paraffin embedding. After embedding, sequentially performing operations of xylene dewaxing, gradient-decreasing alcohol hydration, hematoxylin and eosin staining, gradient-increasing alcohol dehydration, xylene transparency and neutral resin sealing, preparing a sea horse tissue slice, performing HE staining, observing pathological changes of sea horse tissue and colon tissue under a fluorescence normal microscope, and photographing and recording.

2.3 Biochemical index determination

And taking a proper amount of mouse serum, and detecting the IL-6, IL-beta, TNF-alpha and IL-10 levels in the serum according to the instruction of the kit.

2.4 QRT-PCR detection of expression of related genes in tissues

Total RNA was extracted from hippocampus and colon using Trizol Reagent. RNA purity was determined by absorbance ratio (260/280 nm), with absorbance ratio of 1.8-2.0 for all samples. After diluting the total RNA to a proper concentration, a high-capacity cDNA reverse transcription kit is used for generating cDNA, and the cDNA is stored at-80 ℃ for standby. qPCR was performed using SYBR GREEN MASTER Mix and detected on a fluorescent real-time quantitative PCR detection system, PCR reaction procedure: firstly, pre-denaturing for 1min at 95 ℃, and then, at 95 ℃ for 20s;50 ℃ for 20s; cycling was performed 40 times at 72℃for 30 s. The CT values of the respective reactions were provided, and the change in transcription level of the internal reference target gene was normalized, as calculated by the following formula, target gene relative mRNA level (control fold) =2 ^-ΔΔCT, and β -actin as the internal reference gene. The primer sequences are shown in Table 5.

TABLE 5 primer sequences

2.5 Data statistics and analysis

3 Results and analysis

3.1 Modification of the behavior of Lactobacillus rhamnosus in mice with Alzheimer's disease

The open field experiment can reflect the autonomous activity ability of the experimental mice, the exploration ability of the new environment and the anxiety emotion. Fig. 4A shows representative motion trajectories for each group of mice. As shown in FIG. 4B, D-galactose was combined with AlCl compared to Con group ₃

Induced Mod mice showed a significant decrease in erection in open field, while DACN 1224-supplemented mice showed an increase in erection, and Mod mice showed a significant decrease in total locomotion distance (p < 0.05) compared to Con mice, while DACN1224 mice showed a significant increase in total locomotion distance (p < 0.05) in open field. It was observed that Mod group defecation was higher than Con group mice, and that the amount of defecation in the new environment was proportional to their anxiety, and that DACN1224 was supplemented to reduce the amount of defecation in the mice. Furthermore, both the time and distance that Mod mice stay in the central region were significantly shortened (p < 0.05) compared to Con groups, however the intervention of DACN1224 torqued this trend. The results indicate that DACN1224 may have positive effects of alleviating anxiety and enhancing voluntary locomotion ability, and can restore to some extent the curiosity and exploratory ability of mice to the outside world.

To investigate the effect of DACN1224 on D-galactose in combination with AlCl ₃ -induced cognitive decline in mice, we performed a new object recognition experiment. Figure 4A shows representative trajectories of activity and exploration for each group of mice. As shown in fig. 4B, the cognitive ability and exploratory ability of the Mod group mice were reduced compared to the Con group, demonstrating that the Mod group mice were more exposed to old objects than new objects, the new object recognition index was significantly reduced (p < 0.05) which suggests that the Mod group mice were cognitively impaired, while the new object recognition index was significantly increased (p < 0.05) and was close to the Con group (p > 0.05) in addition to DACN 1224. The result shows that the DACN1224 can relieve the condition of the cognitive impairment of the mice induced by the D-galactose combined with AlCl ₃ to a certain extent, and has positive protective effect on the cognitive ability.

Nesting behavior is an instinctive social behavior of mice that is regulated by multiple brain regions and neurotransmitter activities. Aging and injury of the brain can reduce nesting ability, so that the nesting ability reflects brain health and cognitive ability of experimental mice to a certain extent. Fig. 4C is a representative nesting situation for each group of mice in the nesting experiment. The nesting performance test shows that compared with Con group, the nesting ability of the D-galactose combined with AlCl ₃ induced Mod group mice is impaired, the nesting score is obviously lower than that of Con group (p < 0.05), and the weight of the undivided nesting fragments is obviously higher than that of Con group (p < 0.05). While DACN 1224-supplemented mice had higher nesting ability than Mod group mice, with no significant differences in nesting score and unworn chip weight (p > 0.05) from the normal group. These results indicate that DACN1224 supplementation is effective in alleviating the cognitive deficit caused by D-galactose in combination with AlCl ₃.

3.2 Results of staining the colon and hippocampus H & E of Alzheimer's disease mice with Lactobacillus rhamnosus

The effect of lactobacillus DACN1224 on the colon pathological tissue structure of the experimental mice was analyzed by H & E staining, and the results are shown in fig. 5. H & E results show that the colon tissue structure of Con group is complete, no sign of inflammatory infiltration is basically seen, the colon structure of Mod group mice is severely damaged, the crypt structure is abnormal or disappears, inflammatory cell infiltration is obvious, and colonic mucosa is edematous. From previous studies, we can speculate that disruption of colonic integrity, which may also lead to disruption of the colonic barrier and further induce release of inflammatory factors within the colon, disrupting the state of body balance. The colon tissue of the mice taking the lactobacillus DACN1224 is slightly infiltrated by inflammation, the phenomenon of mucosa edema is obviously reduced, and the colon tissue morphology of the mice with the AD model is restored to a certain extent. Red arrows indicate inflammatory infiltration of the colon of the mice and blue arrows indicate colonic mucosal edema of the mice.

The main distribution areas of neurons in the hippocampus of mice are the dentate (Dentate Gyrus, DG), the CA1 and CA3 regions, so we mainly observe the changes of neurons in these three brain regions. The H & E staining results are shown in FIG. 6, the neurons in each region of the hippocampus of Con mice are in good morphology, are orderly arranged, the number of the neurons is not reduced, the cell structures in each brain region are clear, and no obvious degeneration is seen. Compared with Con group, D-galactose and AlCl ₃ combined Mod group mice have abnormal tissue structure of Hippocampus, and neuronal cell shrinkage and alkalophilic deep staining of a large number of neuronal cells appear in DG region of Hippocampus, as shown by red arrow in figure; partial neuronal degeneration is seen in the CA1 region, with cell shrinkage and deep staining, as indicated by the blue arrow in the figure for the CA1 region; whereas the arrangement of neurons in the CA3 region of the hippocampus is loosely disturbed, the number is reduced, partial visual degeneration occurs, the cell body is wrinkled, and the shape is irregular, and the CA3 region is shown by yellow arrows in the figure. In contrast to the Mod group, DACN 1224-supplemented mice were prevented to some extent from the above-described histopathological lesions, with small numbers of neuronal degeneration seen in the DG region of brain tissue in DACN1224 group mice, and the CA1 and CA3 regions exhibited a normal structure approaching that of the Con group.

3.3 Effect of Lactobacillus rhamnosus on improving intestinal permeability of Alzheimer's disease mice

The intestinal barrier, which is composed of a tight junction of transmembrane protein (Claudin, occludin) and perimembranous protein (ZO), is the first line of defense against the entry of intestinal harmful substances into the body. Impaired intestinal barriers increase neurotoxicity and penetration of pro-inflammatory microbial metabolites (such as lipopolysaccharides), while tight junction proteins are important structures to maintain the mechanical barrier and permeability of the intestinal mucosal epithelium. Therefore, the expression of the colon zonulin ZO-1, occludin, claudin-1 and Claudin-5 by the real-time fluorescence quantitative analysis technique is shown in the figure 7, and the experimental result shows that the administration of DACN1224 can reverse the reduction of the expression of the ZO-1, occludin, claudin-1 and Claudin-5 genes in colon tissues caused by Alzheimer's disease. Wherein DACN1224 has an improved effect on the expression of colon claudin over donepezil hydrochloride.

3.4 Improving Effect of Lactobacillus rhamnosus on systemic inflammation in Alzheimer's disease mice

One of the key factors in alzheimer's disease is oxidative stress, which causes inflammation in our body and damages the link between neurons, resulting in cognitive dysfunction, further inducing alzheimer's disease. Inflammatory cytokines in the blood can be transferred to various parts of the body through the blood circulation, and exert a strong inflammatory effect on the whole body including the brain. The experimental results are shown in fig. 8, 9 and 10, and the experimental results show that the administration of DACN1224 can reverse the decrease of the expression level and the content of the IL-10 gene in colon, hippocampal tissues and blood caused by the alzheimer disease and relieve the increase of the expression level and the content of the IL-6, IL-1 beta and TNF-alpha gene caused by the alzheimer disease.

3.5 Protection of brain neurons in Alzheimer's disease mice by Lactobacillus rhamnosus DACN1224

BDNF and PSD95 are important for protecting neurons, and the content of BDNF and PSD95 in the brain is closely related to Alzheimer's disease. The experimental results are shown in fig. 11, and the experimental results show that the administration of DACN1224 can restore BDNF and PSD95 levels in the hippocampus of mice with alzheimer's disease, which is close to the normal group.

In conclusion, the results show that the lactobacillus rhamnosus DACN1224 has excellent antioxidant capacity, can relieve the cognitive behavioral dysfunction induced by the combination of D-galactose and AlCl ₃, can improve the intestinal permeability and systemic inflammation of mice with Alzheimer's disease, and can improve the neurotrophic factors in the brain of the brain, so the lactobacillus rhamnosus DACN1224 has a certain effect of relieving the Alzheimer's disease.

Application example 1 preparation of lactic acid bacteria Using Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224

The method comprises the following steps:

S1, storing original strain of lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224 in 30 wt% glycerol suspension at-75deg.C or freeze-drying powder at 4deg.C.

S2, preparing the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224 working starter according to the invention by adopting any one of two methods:

The first method comprises inoculating Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224 original strain into 12% (weight) skimmed milk, sterilizing at 110deg.C for 10min, culturing at 37deg.C for 14-16 hr to curd, and continuously culturing and activating for two generations to obtain mother starter; inoculating the mother starter in sterilized milk at 3-5% (by volume), culturing for 14-16 hr to obtain curd, wherein viable bacteria in the curd is about 10 ⁹ cfu/mL to obtain the working starter, which can be directly added into food or used together with symbiotic commercial starter for fermented milk preparation such as Lactobacillus bulgaricus and Streptococcus thermophilus to prepare fermented milk.

The second method is that 9/10 pages of the original strain of the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224 is inoculated in MRS liquid culture medium, cultured for 12-16 hours at 37 ℃ for activation, then activated for two generations, the activated culture is inoculated in the MRS culture medium according to 2-4% (volume), cultured for 16-18 hours, centrifuged for 15 minutes at 4000r/min at 4 ℃, the supernatant is removed, cell sediment is obtained, and a certain amount of aseptic skim milk is used for preparing the sediment into suspension to obtain the working starter for standby.

S3, sterilizing raw milk at 95 ℃ for 20min or at 140 ℃ for 2S, cooling to 4 ℃, adding the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224 working starter to enable the concentration to reach more than 10 ⁶ cfu/ml, and refrigerating and preserving at 4 ℃ to obtain the lactobacillus milk beverage containing lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224 viable bacteria.

In the present invention, the MRS liquid medium is well known to those skilled in the art, and is a medium for lactobacillus culture sold by Soy Corp.

The heat sterilization is performed, for example, by using a TW10D1000 tubular sterilizer sold by Shanghai Wo Di Automation Equipment Co., ltd.

The high-temperature sterilization is performed by using a YC-104 plate type ultrahigh-temperature sterilization machine sold by Beijing Yongjia mechanical equipment Co., ltd.

Application example 2 preparation of milk powder Using Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224

The lactobacillus milk beverage is prepared according to the preparation method of the lactobacillus milk beverage in application example 1, except that raw milk is sterilized by heating at 95 ℃ for 20min or by high temperature sterilization at 140 ℃ for 2s, then cooled to 37 ℃, inoculated with the lactobacillus pentosus working starter in application example 1 by 4% of the inoculation amount of the volume of the raw milk, and fermented for 16h at 37 ℃ to obtain lactobacillus pentosus fermented milk; then adding lactobacillus pentosus fermented milk into the sterilized raw material milk according to the ratio of 1:3 (V: V), homogenizing, concentrating in vacuum, and spray drying to obtain lactobacillus pentosus-containing milk powder.

The homogenization is performed, for example, using a GJB500-40 mini-homogenizer sold by Changzhou homogenizer Limited. The concentration is performed, for example, using a vacuum concentration pot sold by Shanghai Wei light industry machinery Co., ltd. The spray drying is carried out, for example, using an experimental spray dryer sold by Shanghai Wo Di technologies, inc.

Application example 3 preparation of Capsule preparation Using Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224

Sterilizing raw milk at 140 ℃ for 2s, cooling to 37 ℃, inoculating lactobacillus pentosus working starter in application example 1 with a bacterial inoculation amount of 4% of the volume of the raw milk, and fermenting at 37 ℃ for 16h to obtain lactobacillus rhamnosus fermented milk. Adding lactobacillus rhamnosus fermented milk into sterilized raw milk at a ratio of 1:3 (V: V) for homogenizing, vacuum concentrating, spray drying to obtain milk powder, and encapsulating to obtain capsule product.

Application example 4 preparation of fermented milk Using Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224

The preparation method of the lactobacillus milk beverage in application example 1 is carried out by heating and sterilizing raw milk at 95deg.C for 20min or high-temperature sterilizing at 140deg.C for 2s, cooling to 37deg.C, adding into lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) working starter according to 3-5% (volume), adding into 3-5% (volume) symbiotic fermented milk commodity starter, mixing, fermenting at 37deg.C until titrating acidity to 0.6-0.7% of lactic acid, cooling to 4deg.C, and refrigerating.

The commercial starter is preferably lactobacillus bulgaricus or streptococcus thermophilus.

In the sense of the present invention, the raw milk is one or more raw milk selected from skim milk, fresh milk and reconstituted milk.

The above description is merely illustrative of the embodiments of the present invention, but the present invention is not limited thereto, and any changes or modifications easily made by those skilled in the art within the scope of the present invention are intended to be included in the claims of the present invention.

Claims

1. Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) DACN1224, which is preserved in the China center for type culture collection with a preservation number of cctcno: m20232453.

2. A microbial agent comprising as an active ingredient one or more combinations of the lactobacillus rhamnosus DACN1224, lactobacillus rhamnosus DACN1224 inactivated bacteria, metabolites of lactobacillus rhamnosus DACN1224, and lactobacillus rhamnosus DACN1224 lyophilized strains of claim 1.

3. The microbial agent of claim 2, wherein: lactobacillus rhamnosus DACN1224 is used alone or in combination with other strains.

4. A method for preparing the microbial inoculum of claim 2 or 3, which is characterized in that: the lactobacillus rhamnosus DACN1224 of claim 1 is inoculated in a culture medium and cultured, preferably in an MRS medium at 30-38 ℃ for 18-24h.

5. Use of lactobacillus rhamnosus DACN1224 according to claim 1 or the microbial agent according to claim 2 or 3 for the manufacture of a product for the prevention and/or amelioration of alzheimer's disease or related neurodegenerative diseases including dementia, cognitive disorders, memory disorders.

6. The use according to claim 5, characterized in that: the product has at least one of the following functions:

7. The use according to claim 5, characterized in that: the product is a medicine, a functional food, a health-care product, a food additive, a health-care product additive or a feed.

8. The use according to claim 7, characterized in that: the pharmaceutical product also includes a pharmaceutically acceptable carrier.

9. The use according to claim 7, characterized in that: the dosage forms of the medicine comprise powder, granules, capsules, tablets, pills or solutions.

10. The use according to claim 7, characterized in that: according to the raw material source of the food, the food comprises dairy products, bean products, fruit and vegetable products and fermented food; the food comprises solid food, liquid food and semi-solid food according to the state of the food.