Composition containing plant lactobacillus and application thereof

Abstract

The invention discloses a composition containing three plant lactobacillus plantarum strains and application of the composition, the composition comprises the plant lactobacillus plantarum AUSA002, the plant lactobacillus plantarum AUSA004 and the plant lactobacillus plantarum FL001, and the preservation numbers of the plant lactobacillus plantarum AUSA002, the plant lactobacillus plantarum AUSA004 and the plant lactobacillus plantarum FL001 in the Guangdong Microbial Culture Collection Center (CDMCC) are GDMCC.No64417, GDMCC.No64418 and GDMCC.No64420 respectively. The composition provided by the invention has the effects of synergistically improving learning memory and cognitive impairment and preventing senile dementia, and also has the effect of improving gastrointestinal functions.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition containing Lactiplantibacillus plantarum and its use, belonging to the field of microbial technology. BACKGROUND

[0002] In the current field of medicine and biotechnology, probiotic formulations are an important research direction, and their development aims to enhance health and treat diseases by modulating the host's microbiota. However, this field faces many challenges, including how to accurately position and regulate the balance of the microecosystem, and how to balance safety, stability, and efficacy.

[0003] Probiotics can treat and prevent a series of digestive system diseases by maintaining and restoring the balance of the intestinal microecosystem, but there are still many problems to be overcome in practical application, such as strain selectivity and individual response differences.

[0004] In recent years, the research of probiotics has also expanded to the improvement of neurodegenerative diseases, especially in the prevention and intervention of Alzheimer's disease. Early studies have shown that regulating the intestinal microecosystem may affect brain health and cognitive function through the "gut-brain axis". However, these studies are still limited to basic scientific research and small-scale clinical trials, and their effects have not been fully recognized. The drugs approved for Alzheimer's disease (AD) mainly have some improvement effect on mild to moderate AD, and there is a lot of controversy due to unclear action targets or too many side effects. In addition, the development of drugs for AD is very expensive, and the benefits are very small. The causes of AD are multiple, and the disease has a long incubation period (10 years or even more than 30 years), so the intervention strategy for AD should be shifted from drug treatment after the onset to prevention in the pre-AD stage, i.e., the preclinical symptom period and the mild cognitive impairment period. For the early prevention of AD, the existing intervention methods including drugs obviously have certain limitations, at least ignoring the role of nutrient imbalance and intestinal flora imbalance on dementia.

[0005] Lactiplantibacillus plantarum is a gram-positive, short rod-shaped facultative anaerobic cell, originally named Lactobacillus plantarum, and now named Lactiplantibacillus plantarum. Lactiplantibacillus plantarum is applied in various functional foods and medical products. This strain has attracted attention due to its potential benefits in regulating intestinal microecology, activating immune response, etc. Recent research progress has also found that Lactiplantibacillus plantarum may help inhibit pathogenic bacteria and improve the host's nutrient absorption and metabolic health. SUMMARY

[0006] The technical solution of the present application is:

[0007] A composition comprising:

[0008] (1) Lactiplantibacillus plantarum AUSA002, deposited in the Guangdong Microbial Digital Culture Collection Center on March 15, 2024, with the deposit number GDMCC No: 64417, and the classification name Lactiplantibacillus plantarum. The deposit address is: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, China, 5th Floor, Building, Experimental Building;

[0009] (2) Lactiplantibacillus plantarum AUSA004, deposited in the Guangdong Microbial Digital Culture Collection Center on March 15, 2024, with the deposit number GDMCC No: 64418, and the classification name Lactiplantibacillus plantarum. The deposit address is: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, China, 5th Floor, Building, Experimental Building;

[0010] (3) Lactiplantibacillus plantarum FL001, deposited in the Guangdong Microbial Digital Culture Collection Center on March 15, 2024, with the deposit number GDMCC No: 64420, and the classification name Lactiplantibacillus plantarum. The deposit address is: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, China, 5th Floor, Building, Experimental Building.

[0011] In the application, the composition can comprise a protective agent, and the protective agent composition comprises 10-15% skimmed milk powder, 3-10% sucrose, 1-5% lactose, 1-5% trehalose, and 3-10% fructooligosaccharide. As a preferred, the protective agent composition comprises 12% skimmed milk powder, 5% sucrose, 3% lactose, 3% trehalose, and 5% fructooligosaccharide.

[0012] In the application, the composition can further comprise a prebiotic and an auxiliary material. The prebiotic is selected from one or more of inulin, malt dextrin, isomaltooligosaccharide, polydextrose, lactitol, fructooligosaccharide, isomaltitol, xylooligosaccharide, stachyose, and galactooligosaccharide.

[0013] In the composition of the application, the mass ratio of Lactiplantibacillus plantarum AUSA002, Lactiplantibacillus plantarum AUSA004, and Lactiplantibacillus plantarum FL001 is 1-3:1-3:1-3, and preferably the mass ratio is 1:1:1.

[0014] In the application, the composition is prepared into tablets, capsules, powders, oil drops, liquids, pills, or granules.

[0015] In the composition described in the present application, the total number of viable bacteria of Lactobacillus plantarum AUSA002, Lactobacillus plantarum AUSA004 and Lactobacillus plantarum FL001 is 1x10 6 CFU / g ~ 1.5x10 11 CFU / g; preferably, the total number of viable bacteria of Lactobacillus plantarum AUSA002, Lactobacillus plantarum AUSA004 and Lactobacillus plantarum FL001 is 1x10 9 CFU / g ~

[0016] 1x10 10 CFU / g.

[0017] Use of the composition described in the present application for preparing a product for improving learning and memory and cognitive impairment.

[0018] Use of the composition described in the present application for preparing a product for preventing senile dementia.

[0019] Use of the composition described in the present application for preparing a product for preventing gastrointestinal diseases, including regulating intestinal flora balance, relieving diarrhea, improving constipation.

[0020] The preparation method of the composition described in the present application comprises the following steps:

[0021] 1) Lactobacillus plantarum AUSA002, Lactobacillus plantarum AUSA004 and Lactobacillus plantarum FL001 are respectively subjected to primary seed fermentation to obtain primary seed culture solution;

[0022] 2) The primary seed culture solution of Lactobacillus plantarum AUSA002, Lactobacillus plantarum AUSA004 and Lactobacillus plantarum FL001 is inoculated into a secondary fermentation tank for fermentation culture to obtain secondary fermentation culture solution; or the secondary fermentation culture solution is further inoculated into a tertiary fermentation tank for fermentation culture to obtain tertiary fermentation culture solution;

[0023] 3) The secondary fermentation culture solution or the tertiary fermentation culture solution is centrifuged to obtain Lactobacillus active bacteria slurry; the active bacteria slurry is mixed with a protective agent at a weight ratio of 1:2, and freeze-dried to obtain the powdery composition of Lactobacillus.

[0024] The composition containing Lactobacillus plantarum AUSA002 isolated from Tibetan kefir and Lactobacillus plantarum AUSA004 and Lactobacillus plantarum FL001 isolated from breast milk described in the present application has the advantages of strong colonization ability in human body, not easy to be eliminated, and the ability to improve learning and memory and cognitive impairment, prevent senile dementia, improve gastrointestinal function, good safety, high patient compliance and other significant advantages. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The colony morphology chart of Lactobacillus plantarum AUSA002 provided by the present application on MRS plate.

[0026] Figure 2 The colony morphology chart of Lactobacillus plantarum AUSA004 provided by the present application on MRS plate.

[0027] Figure 3 The colony morphology chart of Lactobacillus plantarum FL001 provided by the present application on MRS plate.

[0028] Figure 4 The gram staining chart of Lactobacillus plantarum AUSA002 provided by the present application.

[0029] Figure 5 The gram staining chart of Lactobacillus plantarum AUSA004 provided by the present application.

[0030] Figure 6 The gram staining chart of Lactobacillus plantarum FL001 provided by the present application. DETAILED DESCRIPTION

[0031] The present application is further described below in conjunction with specific embodiments, but is not limited to the present application, and any equivalent replacement in the art according to the content of the present application belongs to the protection scope of the present application.

[0032] Example 1: Isolation and identification of Lactobacillus plantarum in the composition provided by the present application

[0033] 1. The isolation step of Lactobacillus plantarum in the composition provided by the present application is:

[0034] (1) Sample collection: milk from a lactating woman in Shenzhen area with good physical condition, and Tibetan kefir mushroom from Tibet Lingzhi area.

[0035] (2) Isolation and purification of strains: 1 g of collected breast milk or Tibetan kefir mushroom sample was added to 9 mL of sterile normal saline, mixed thoroughly, 1 mL of the suspension was added to 9 mL of sterile normal saline, and a series of ten-fold dilutions were made, 0.1 mL of each of 10 -5 , 10 -6 and 10 -7 mL of bacterial solution was inoculated on MRS plate, three repeats were made for each dilution, and 37℃ anaerobic culture was carried out for 48h.

[0036] (3) Strain morphology observation: After Gram staining, the morphology of the screened strains was observed under a microscope. The pure culture of Gram-positive bacteria was determined as a suspected lactic acid bacteria, mixed with 50% glycerol at a ratio of 2:1, and stored at -80°C for future use.

[0037] (4) Strain screening:

[0038] 1) The above isolated strains were screened in a folic acid-free medium (FACM). 1 g of each Tibetan kefir mushroom and breast milk sample was homogenized in 9 mL of sterile normal saline (0.9% w / v NaCl). The homogenate was serially diluted and diluted to 10 -5 -10 -6 , inoculated in duplicate with 2% (v / v) sterile FACM, and incubated at 30°C under aerobic conditions for 48 h.

[0039] 2) The visible growth of the bacterial solution was diluted and plated (10 -5 and 10 -6 ) on sterile FACM plates and incubated at 30°C for 72 hours. Two plates were prepared for each dilution. A blank plate was left as a control to check for contamination.

[0040] 3) Single colonies with different morphologies were picked and streaked on fresh FACM medium 1-2 times. After morphological examination (cell and colony morphology and Gram staining), all strains were stored in FACM at -80°C in glycerol (80%) until the strain was identified.

[0041] 2. Strain identification

[0042] The above screened strain isolated from Tibetan kefir mushroom, numbered AUSA002, two strains isolated from breast milk, numbered AUSA004 and FL001, were extracted using a bacterial genomic DNA extraction kit. The 16S rDNA was amplified by PCR technology and sequenced for analysis to identify the species of the strain.

[0043] (1) The 16S rDNA gene sequence of the strain isolated from Tibetan kefir mushroom, numbered AUSA002, is as follows:

[0044]

[0045] (2) The 16S rDNA gene sequence of the strain isolated from breast milk, numbered AUSA004, is as follows:

[0046]

[0047] (3) The 16S rDNA gene sequence of strain FL001 isolated from breast milk is as follows:

[0048]

[0049] The obtained sequence results were searched and similarity aligned in GENBANK using BLAST, and the sequencing results of the strain numbered AUSA002 were identified as Lactiplantibacillus plantarum strain Heal19; the sequencing results of the strain numbered AUSA004 were identified as Lactiplantibacillus plantarum strain cqf-43; and the sequencing results of the strain numbered FL001 were identified as Lactobacillus plantarum strain SLDL-130. The three strains of Lactiplantibacillus plantarum were mixed with 50% glycerol at a ratio of 2:1, and stored at -80°C for standby use.

[0050] Whole genome sequence analysis:

[0051] The bacterial genomic DNA extraction kit was used to extract strain DNA for whole genome sequencing analysis to further identify the genomic characteristics of the strain. The whole genome sequence of the Lactiplantibacillus plantarum AUSA002 has been uploaded to the National Center for Biotechnology Information (NCBI) with Accession No: CP160858; and the whole genome sequence of the Lactiplantibacillus plantarum AUSA004 has been uploaded to the National Center for Biotechnology Information (NCBI) with Accession No: CP160637.

[0052] Example 2: Preparation method of the composition powder fungicide of the present application

[0053] (1) The Lactiplantibacillus plantarum AUSA002, AUSA004 and FL001 strains were respectively streaked on MRS slant for 20-24h, and single colonies were picked up with a inoculating loop in a sterile operation table and inoculated in 5mL of MRS liquid medium, which was shaken and cultured for 20-24h, then inoculated in 80mL of MRS broth at an inoculation amount of 1%, and cultured at a temperature of 35-40, ℃ and a rotation speed of 120-220r / m for 10-30h to prepare 240mL of primary seed culture. The culture conditions were preferably 37, ℃, 50r / m, and cultured for 14h.

[0054] (2) The 240mL of primary seed culture was inoculated in a 10L fermenter with a liquid volume of 8L, and cultured at a temperature of 37, ℃ and a stirring speed of 50r / m for 10-30h to obtain a fermentation culture, and the effective viable bacterial count of Lactiplantibacillus plantarum in the fermentation broth reached 5.2×10 9CFU / mL. The medium components in the 10 L fermenter were as follows: peptone 15 g / L, beef powder 7.5 g / L, yeast powder 6.0 g / L, glucose 30 g / L, Tween 1 ml / L, potassium phosphate dibasic 1.4 g / L, sodium acetate 3 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, and the balance was water, pH 6.6 ± 0.2.

[0055] (3) The fermentation broth obtained in step (2) was centrifuged by a 4500 r / m centrifuge for 8-15 min to obtain active bacterial slurry of the P. lumipes. The active bacterial slurry was mixed with a protective agent (the protective agent was prepared by mixing 12 g of skim milk powder, 5 g of sucrose, 3 g of lactose, 3 g of trehalose, and 5 g of fructooligosaccharide in 100 ml of water) at a weight ratio of 1:2, uniformly stirred in a blender, adjusted to a humidity of 30-45%, freeze-dried to a water content of ≤6%, and sieved through a 20-mesh sieve to obtain powdered bacterial agents of P. lumipes AUSA002, AUSA004, and FL001, respectively. The viable bacterial counts of the three powdered bacterial agents were all 4.0 x 10 11 CFU / g.

[0056] Examples 3-8: Preparation method of the powder of the composition of the present application

[0057] Formulation: 1000 units of the preparation

[0058]

[0059] (1) The powdered bacterial agents of P. lumipes AUSA002, P. lumipes AUSA004, and P. lumipes FL001 prepared in Example 2 were taken in the amounts shown in the table above, (2) fructooligosaccharide, maltodextrin, and isomaltooligosaccharide were dried to a water activity of ≤0.2, (3) the P. lumipes powder, fructooligosaccharide, maltodextrin, and isomaltooligosaccharide were uniformly mixed, and then divided into composite film bags according to a certain filling amount to obtain the powder product.

[0060] Examples 9-14: Preparation method of the tablet of the composition of the present application

[0061] Formulation: 1000 units of the preparation

[0062] Component Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 AUSA002 2.50g 8.33g 10.00g 25.00g 20.00g 75.00g AUSA004 2.50g 8.33g 30.00g 25.00g 40.00g 25.00g FL001 7.50g 8.33g 10.00g 25.00g 40.00g 25.00g Fructooligosaccharides 50g 50g 50g 50g 50g 50g Isomalt 1572.5g 1560.0g 1535.0g 1510.0g 1485.0g 1460.0g Isomaltulose 250g 250g 250g 250g 250g 250g Microcrystalline cellulose 100g 100g 100g 100g 100g 100g Flavour 5g 5g 5g 5g 5g 5g Magnesium stearate 10g 10g 10g 10g 10g 10g

[0063] (1) according to the above table, take the plantarum AUSA002, plantarum AUSA004, plantarum FL001 powder prepared in example 2, (2) fructooligosaccharide, microcrystalline cellulose, isomalt, dry to water activity ≤0.2, (3) the plantarum powder, fructooligosaccharide, microcrystalline cellulose, isomalt, isomalt, essence, magnesium stearate are mixed uniformly, according to a certain piece weight, double layer tablet or single layer tablet is pressed by tablet press, tablet product is obtained.

[0064] Example 15-20: preparation method of capsule of the composition of the application

[0065] Formulation: 1000 preparation units

[0066] Component Example 15 Example 16 Example 17 Example 18 Example 19 Example 20 AUSA002 7.50g 8.33g 10.00g 25.00g 25.00g 50.00g AUSA004 2.50g 8.33g 30.00g 25.00g 50.00g 50.00g FL001 2.50g 8.33g 10.00g 25.00g 50.00g 150.00g Fructooligosaccharides 25g 25g 25g 25g 25g 25g Maltodextrin 857.5g 845.0g 820.0g 795.0g 745.0g 620.0g Isomaltulose 100g 100g 100g 100g 100g 100g Magnesium stearate 5g 5g 5g 5g 5g 5g

[0067] (1) according to the above table, take the plantarum AUSA002, plantarum AUSA004, plantarum FL001 powder prepared in example 2, (2) fructooligosaccharide, malt dextrin, isomalt dry to water activity ≤0.2, (3) the plantarum powder, fructooligosaccharide, malt dextrin, isomalt, magnesium stearate are mixed uniformly, according to a certain amount, capsule filling machine is made into capsule, capsule product is obtained.

[0068] Example 21: the influence of the composition provided by the application on the gastrointestinal function of rats

[0069] SPF level Wistar rats, half male and half female, 7-8 weeks old, are raised in the environment of room temperature 18-28, relative humidity 40%-70%, and free water. According to the following table, the corresponding plantarum powder fermented in the above preparation examples is given, and the viable cell count reaches 4.0×10 11 CFU / g. Dilute with normal saline to the corresponding viable cell content of each group in the following table. AUSA002+AUSA004+FL001 high dose+prebiotic group is given prebiotic solution (prebiotic solution is prepared according to the mixture containing 0.5 mg of fructooligosaccharide, 15.6 mg of isomalt, 2.5 mg of isomalt per milliliter). The volume of gavage is calculated according to 10 ml / kg, and gavage is performed once a day. The model group and the control group are given the same volume of normal saline.

[0070] The dose design of each group refers to the equivalent dose conversion method in “pharmacological experimental method” compiled by Xu Shuyun, and the intake of 1-10 g of probiotic preparation per day in human body is converted to the corresponding dose of rats according to the body surface area.

[0071] After intragastric administration for one week, the rats except the control group were given senna leaf decoction on the 8th day. The diarrhea of rats was counted from 3h after intragastric administration, and the diarrhea rate and loose stool rate were calculated. Diarrhea rate (%) = 100 x number of animals with diarrhea / total number of animals in the group. The loose stool rate was the ratio of the number of loose stools to the total number of stools per rat.

[0072] The interaction between each component in the composition was calculated by Kim Jong-Geun Q value method:

[0073] To prove the scientificity of the probiotic combination provided by the present application, it is illustrated that the three components of the strain composition are reasonably matched, and can exert a synergistic effect by mutual combination, rather than a simple pharmacological effect superposition. Kim Jong-Geun Q value method, also known as probability addition method, is used to calculate the pharmacological effect of three drugs used in combination and the pharmacological effect of three drugs used alone according to the following calculation formula: Q = E A+B+C / (E A +E B +E C -E A *E B -E A *E C -E B *E C -E A *E B *E C ), wherein the numerator represents the "measured combined effect", and the denominator represents the "expected combined effect", (in order to meet the analysis of the pharmacological effect relationship of the components and the composition, the pharmacological effects of the components and the composition are converted into effects that can directly reflect the strength of the pharmacological effects, and the calculation formula is: E i = 1-P i / P 模型组 , P i is the pharmacological index of each component, and P 模型组 is the pharmacological index of the model group), and Q is the ratio of the two: Q is less than 0.85, indicating that the two drugs are in antagonistic action; less than 1.15 and greater than 0.85, indicating that they are in additive action; and greater than 1.15, indicating that they are in synergistic action.

[0074] The results were statistically processed by GraphPad Prism 5 statistical analysis software; the data results were represented by mean ± standard deviation, t test was performed between groups, P<0.05 was statistically significant, and P>0.05 was not statistically significant.

[0075] Table 1: Diarrhea of rats in each group n = 9-10

[0076]

[0077]

[0078] Note: compared with the control group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0079] Except for the control group, the rats in each group orally administered with senna leaf decoction all had diarrhea, the diarrhea rate of the model group was 100%, and the loose stool rate was 87%, indicating that the modeling of the diarrhea rats was successful. Compared with the model group, the diarrhea rate and loose stool rate of the rats in each single component administration group decreased, but only in the high-dose groups of the three single components were there significant differences (P<0.05). In the AUSA002+AUSA004+FL001 low-dose group and

[0080] the AUSA002+AUSA004+FL001 high-dose group, compared with the model group, the diarrhea rate and loose stool rate were significantly reduced; compared with the same dose of single strain, the AUSA002+AUSA004+FL001 low-dose group and the AUSA002+AUSA004+FL001 high-dose group also had the effect of further reducing the diarrhea rate and loose stool rate, and had a synergistic effect (Q≥1.15) in reducing the average loose stool rate of rats. On the basis of

[0081] the AUSA002+AUSA004+FL001 low-dose group and the high-dose group, increasing the simultaneous intragastric administration of prebiotics had the effect of further improving the rat diarrhea function.

[0082] Example 22: Effect of the composition provided by the present application on constipated rats

[0083] SPF Wistar rats, male, weighing 210-260 g, were raised in an environment with room temperature of 18-28 ℃ and relative humidity of 40%-70%, and were allowed to eat and drink freely.

[0084] Ten rats were selected as the control group and were fed with ordinary feed. The remaining rats were fed with low-fiber feed (41.5% corn starch, 24.5% milk casein protein, 10% sucrose, 10% dextrin, 7% minerals, 6% corn oil, and 1% folic acid-free vitamins), and were fed for 4 weeks. During the feeding period, the rats were divided into groups according to the table below and were given the corresponding plant lactobacillus plantarum powder prepared by fermentation in the preparation examples described above, and the viable bacterial count reached 4.0×10 11 CFU / g. The physiological saline was diluted to the corresponding viable bacterial content of each group in the table below.

[0085] The AUSA002+AUSA004+FL001 high-dose + prebiotic group was simultaneously administered a prebiotic solution via gavage (prebiotic solution prepared as a mixture containing 0.5 mg of fructooligosaccharide, 15.6 mg of isomaltitol, and 2.5 mg of isomaltose per milliliter), with a gavage volume calculated at 10 ml / kg body weight. The model group and control group received an equal volume of physiological saline. Gavage was performed once daily for 4 weeks.

[0086] The dosage design for each group was based on the equivalent dose coefficient conversion method in "Pharmacological Experimental Methodology" edited by Xu Shuyun. The dosage was converted between species according to the daily intake of 1-10g of probiotic preparations in humans and the corresponding dosage for rats based on body surface area.

[0087] After gavage, all rats were placed in separate metabolic cages, with no food or water, and their feces were collected within 12 hours. The fecal water content was then determined according to the Chinese Pharmacopoeia method.

[0088] Table 2: Number of fecal particles and fecal water content of rats in each group ( n = 9 to 10)

[0089]

[0090]

[0091] Note: Compared with the control group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0092] Compared with the control group, the number of fecal particles and the water content of rats in the model group were significantly reduced, indicating that the rat constipation model was successfully established. Compared with the model group, when using a single strain of *Lactobacillus plantarum* AUSA002, AUSA004, or FL001, regardless of the low-dose group or the high-dose group, the number of fecal particles and the water content of rats increased to some extent, but only in the three single high-dose groups were there significant differences (P<0.05). In the combination of the three strains of *Lactobacillus plantarum*, regardless of the low-dose group...

[0093] 1×10 7 CFU / mL + 1×10 7 CFU / mL + 1×10 7 CFU / mL or high-dose group 2×10 8 CFU / mL + 2 × 10 8 CFU / mL + 6 × 10 8CFU / mL, more fecal particles and higher fecal water content than the same dose of single strain, and the combination of three strains has a synergistic effect (Q≥1.15). On the basis of the low-dose group and the high-dose group of AUSA002+AUSA004+FL001, increasing the prebiotics has the effect of further improving the function of relieving constipation in rats.

[0094] Example 23: Effect of the composition of the present application on the behavior of scopolamine dementia mice

[0095] In order to verify the efficacy of the plant lactobacillus composition provided by the present application in preventing and improving the symptoms of Alzheimer's disease, a Morris water maze test was designed to detect the learning and memory function of scopolamine dementia mice through behavior.

[0096] Experimental animals and grouping: SPF Kunming (KM) mice, half male and half female, weighing 20±2g, were raised in an environment with room temperature of 18-28℃ and relative humidity of 40%-70%, and were allowed to eat and drink freely. The mice were adaptively fed with ordinary feed for 1 week, and then randomly divided into the following groups, 10 mice in each group. The mice in each group were given the corresponding plant lactobacillus powder prepared by fermentation in the above preparation examples, with a viable bacterial count of 4.0×10 11 CFU / g. The physiological saline was diluted to the corresponding viable bacterial content of each group in the table, and the mice in the AUSA002+AUSA004+FL001 high-dose+prebiotic group were also given prebiotic solution (the prebiotic solution was prepared by mixing 0.25mg of fructooligosaccharide, 6.2mg of maltodextrin and 1mg of isomaltooligosaccharide per milliliter) by gavage at the same time. The gavage volume was calculated according to 10ml / kg, and the mice were given the drug once a day. The modeling was performed after 7 days of continuous administration.

[0097] The dose design of each group was based on the equivalent dose conversion method in Xu Shuyun, ed. Pharmacological Experimental Methods, and the daily intake of 1-10g of probiotic preparation in humans was converted to the corresponding dose in rats according to the body surface area.

[0098] Preparation of scopolamine dementia mouse model: During the experiment, the mice in the control group were injected with scopolamine 5mg / kg once a day, and the mice in the control group were injected with the same amount of normal saline. The mice in each group were continuously injected for 7 days, and the administration of the mice in each group was unchanged during the whole experiment.

[0099] After modeling, the mice were continuously given the corresponding test substances according to the table. After 4 weeks of continuous administration, the Morris water maze behavior test was performed, and the method was as follows:

[0100] ① Place navigation experiment: used to detect the spatial learning ability of mice, the experiment lasted for 6 days, each quadrant was tested once a day in the afternoon, each test lasted for 60 s, and the time used to find the platform in 60 s (escape latency) was recorded. If the mouse did not find the platform within 60 s, the experimenter pulled it to the platform with his hand and stayed for 10 s, and the escape latency was recorded as 60 s, and the interval between two experiments was 20 s. The latency of the mouse was recorded. ② Spatial exploration experiment: used to detect the spatial memory ability of mice. On the 7th day of the above experiment, the platform was removed, the mouse faced the pool wall from the opposite quadrant of the platform quadrant, and the mouse searched for the platform in memory without the platform, and the time spent in the target quadrant and the number of times crossing the original platform in 60 s were recorded.

[0101] Table 3: Average escape latency (s) of each group of mice in the water maze orientation navigation experiment n = 9-10

[0102]

[0103]

[0104] Note: compared with the control group, # P < 0.05, ## P < 0.01; compared with the model group, * P < 0.05, ** P < 0.01.

[0105] As shown in Table 3, in the place navigation experiment, with the training, from the 2nd to the 6th day, the latency of the mice in the model group was significantly longer than that in the control group, indicating that the learning ability of the mice was significantly reduced, and there was a significant statistical difference (P < 0.01). Compared with the model group, each group taking single-strain plant lactobacillus had a certain degree of reduction in the average escape latency of mice from the 2nd day to the 6th day of the experiment, but neither the low-dose group nor the high-dose group had a significant difference compared with the model group.

[0106] Compared with the model group, from the 2nd day, the average escape latency of the AUSA002+AUSA004+FL001 low-dose group and the AUSA002+AUSA004+FL001 high-dose group was significantly decreased (P < 0.05, P < 0.01), and decreased day by day as the experiment progressed, and the difference was the largest on the 6th day of the experiment (P < 0.01).

[0107] Compared with the same amount of single Lactobacillus plantarum, the combination of the three Lactobacillus plantarum provided by the present application has a synergistic effect of reducing the average escape latency of the model (Q≥1.15) in both the low-dose group and the high-dose group, proving that the composition provided by the present application can synergistically improve the learning and memory ability, and is suitable for preventing early symptoms of senile dementia. On the basis of the low-dose group and the high-dose group of AUSA002+AUSA004+FL001, increasing the prebiotics can further reduce the average escape latency of the mice, thereby further improving the learning and memory function of the mice.

[0108] Table 4: Water maze space exploration experiment of mice in each group n = 9-10

[0109]

[0110]

[0111] Note: Compared with the control group, # P < 0.05, ## P < 0.01; compared with the model group, * P < 0.05, ** P < 0.01.

[0112] As shown in the experimental results in Table 4, in the space exploration experiment, the average number of times of crossing the platform and the target quadrant residence time of the mice in the model group were significantly lower than those in the control group (P < 0.01). Compared with the model group, the average number of times of crossing the platform and the target quadrant residence time of the mice in each group using single strain increased, but only in the case of using a higher dose was there a significant difference (P < 0.05).

[0113] Compared with the same amount of single Lactobacillus plantarum, the combination of the three Lactobacillus plantarum provided by the present application has a synergistic effect of reducing the average escape latency of the model (Q≥1.15) in both the low-dose group (3×10 7 CFU / mL+1×10 7 CFU / mL+1×10 7 CFU / mL) and the high-dose group (1×10 8 CFU / mL+1×10 8 CFU / mL+1×10 7CFU / mL of the composition synergistically increased the average number of platform crossings and prolonged the average escape latency (Q≥1.15), demonstrating that the composition provided by this invention can synergistically improve learning and memory abilities and spatial cognitive abilities, and has a significant effect in preventing Alzheimer's disease. Adding prebiotics to the low-dose and high-dose AUSA002+AUSA004+FL001 groups further increased the average number of platform crossings and prolonged the average escape latency in mice, indicating that adding prebiotics to AUSA002+AUSA004+FL001 further improves the learning and memory abilities and spatial exploration functions of mice.

Claims

1. A composition, characterized in that, contain: (1) Lactobacillus plantarum AUSA002, deposited at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No: 64417; (2) Lactobacillus plantarum AUSA004, deposited at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No: 64418; (3) Lactobacillus plantarum FL001, deposited at Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 64420.

2. The composition according to claim 1, characterized in that, The composition comprises a protective agent, the protective agent comprising: 10-15% skim milk powder, 3-10% sucrose, 1-5% lactose, 1-5% trehalose, and 3-10% fructooligosaccharides; preferably, the protective agent comprises: 12% skim milk powder, 5% sucrose, 3% lactose, 3% trehalose, and 5% fructooligosaccharides.

3. The composition according to claim 1, characterized in that, The composition comprises a prebiotic and excipients, wherein the prebiotic is selected from one or more of inulin, maltodextrin, isomaltooligosaccharide, polydextrose, lactitol, fructooligosaccharide, isomaltitol, xylooligosaccharide, stachyose, galactooligosaccharide, etc.

4. The composition according to claim 1, characterized in that, The mass ratio of *Lactobacillus plantarum* AUSA002, *Lactobacillus plantarum* AUSA004, and *Lactobacillus plantarum* FL001 is 1–3:1–3:1–3, preferably 1:1:

1.

5. The composition according to claim 1, characterized in that, The composition is prepared into tablets, capsules, powders, oil drops, oral liquids, pills, and granules.

6. The composition according to any one of claims 1 to 5, characterized in that, The total viable count of *Lactobacillus plantarum* AUSA002, *Lactobacillus plantarum* AUSA004, and *Lactobacillus plantarum* FL001, based on the total weight of the composition, is 1 × 10⁻⁶. 6 CFU / g ~ 1.5 × 10 11 CFU / g; preferably, the total viable count of *Lactobacillus plantarum* AUSA002, *Lactobacillus plantarum* AUSA004, and *Lactobacillus plantarum* FL001 is 1×10⁻⁶. 9 CFU / g ~ 1×10 10 CFU / g.

7. Use of the composition according to any one of claims 1 to 6 in preparing a product for improving learning, memory, and cognitive impairment.

8. Use of the composition according to any one of claims 1 to 6 in preparing a product for the prevention of Alzheimer's disease.

9. A product for the prevention of gastrointestinal diseases prepared from the composition according to any one of claims 1 to 6. The intended uses include regulating the balance of intestinal flora, relieving diarrhea, and improving constipation.

10. A method for preparing the composition according to claim 1, comprising the following steps: 1) The *Lactobacillus plantarum* AUSA002, *Lactobacillus plantarum* AUSA004, and *Lactobacillus plantarum* FL001 were subjected to primary seed fermentation to obtain primary seed culture medium. 2) Inoculate the primary seed culture of *Lactobacillus plantarum* AUSA002, *Lactobacillus plantarum* AUSA004, and *Lactobacillus plantarum* FL001 into a secondary fermenter and ferment to obtain a secondary fermentation culture; or further inoculate the secondary fermentation culture into a tertiary fermenter and ferment to obtain a tertiary fermentation culture. 3) Centrifuge the secondary or tertiary fermentation culture to obtain active bacterial sludge of *Lactobacillus plantarum*; mix the active bacterial sludge with a protectant at a weight ratio of 1:2, freeze-dry, and obtain a powdered composition of *Lactobacillus plantarum*.

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