Fermented lactobacillus mucus D22 capable of enhancing memory ability as well as fermented food and application thereof

Abstract

The invention discloses fermented lactobacillus mucilaginosus D22 capable of enhancing memory ability, fermented food thereof and application of the fermented food, and belongs to the technical field of microorganisms. The fermented lactobacillus mucus D22 can be used for preparing functional food, health care products and medicines for improving individual memory ability and improving memory impairment, the invention further provides fermented food, the fermented food is prepared by fermenting the fermented lactobacillus mucus D22, and the fermented food comprises solid food, liquid food and semi-solid food. And the method has a very wide application prospect.

Description

Technical Field

[0001] The invention relates to fermented mucus lactobacillus D22 for enhancing memory ability, fermented food thereof and application thereof, and belongs to the technical field of microorganisms. Background Art

[0002] Learning and memory disorders are a degenerative disease of the central nervous system. Many factors such as lack of sleep, stress, aging and disease can cause learning and memory disorders. In severe cases, it will not only affect the individual's life and social adaptability, but also bring heavy pressure and economic burden to the family and society. Therefore, the development of functional products to improve individual memory has a very broad market prospect. However, there are currently few types of functional products for improving the learning and memory abilities of infants, teenagers, adults and the elderly, the safety is unreliable, and the effect is very limited.

[0003] The gut-brain axis is a two-way information exchange system between the intestinal flora and the brain, through which the intestinal flora can affect the host's brain function and behavior. Supplementation with probiotics helps restore the intestinal flora, change microbial metabolites, reduce inflammation, and maintain the integrity of the intestinal barrier. The gut-brain axis is interconnected through multiple pathways such as the immune system, tryptophan metabolism, vagus nerve, and enteric nervous system, involving microbial metabolites such as short-chain fatty acids, branched-chain amino acids, and peptidoglycan. Many studies have shown that intestinal flora disorders may lead to cognitive impairment, accompanied by damage to hippocampal function. It can be seen that regulating the intestinal flora through probiotics to improve brain function and cognitive ability is a new possible method.

[0004] Currently, the known functions of probiotics are mainly to regulate intestinal function and improve host nutrient metabolism, while there are very few known probiotics that can improve cognitive impairment. Taking specific probiotics to improve cognitive function can help further reveal the complex relationship between intestinal microorganisms and the brain, and can provide new therapeutic targets for the prevention and treatment of cognitive impairment. Therefore, screening probiotics that improve individual memory ability and developing functional products that assist in improving individual memory have extremely broad application prospects. Summary of the invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides fermented mucus Lactobacillus D22 for enhancing memory ability, fermented foods thereof and applications thereof, aiming to solve the technical problem that the function of currently known probiotics is mainly to regulate intestinal function, and the types of probiotics that can improve individual memory ability are very few, and it is urgent to screen out a probiotic that can effectively improve individual memory.

[0006] The first technical solution provided by the present invention is a fermented mucus lactobacillus (Limosilactobacillusfermentum) D22, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 28, 2024, with a collection number of GDMCC.No: 65071, and the collection address is Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The second technical solution provided by the present invention is a microbial preparation containing the fermented mucus Lactobacillus D22 described in the first technical solution.

[0008] In one embodiment of the present invention, in the microbial preparation, the concentration of the fermented mucus Lactobacillus D22 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0009] Preferably, in the microbial preparation, the concentration of the fermented mucus Lactobacillus D22 is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0010] The present invention also provides a third technical solution, which is a starter culture, wherein the starter culture contains the fermented mucus lactobacillus D22 described in the first technical solution or the microbial preparation described in the second technical solution.

[0011] In one embodiment of the present invention, the amount of the fermented Lactobacillus mucilaginosus D22 added to the starter is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0012] Preferably, the amount of the fermented Lactobacillus mucilaginosus D22 added in the fermentation agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0013] The fourth technical solution provided by the present invention is a product, which is a product containing the fermented mucus Lactobacillus D22 described in the first technical solution or the microbial preparation described in the second technical solution, or the product is a product obtained by fermentation using the starter described in the third technical solution.

[0014] In one embodiment of the present invention, the product is a feed additive, food, medicine or health product.

[0015] In one embodiment of the present invention, the food is a fermented food containing the fermented Lactobacillus mucilaginosus D22 or the microbial preparation, or the food is a fermented food obtained by fermentation using the starter described in the third technical solution.

[0016] In one embodiment of the present invention, the fermented food includes dairy products, bean products, fruit and vegetable products or other fermented foods containing fermented Lactobacillus mucilaginosus D22.

[0017] In one embodiment of the present invention, the dairy product comprises milk, sour cream, cheese.

[0018] In one embodiment of the present invention, the fruit and vegetable products include products of one or more of cucumber, carrot, beet, celery, cabbage and other edible fruits and vegetables.

[0019] In one embodiment of the present invention, the fermented food further contains additives, and the additives are selected from one or a combination of two or more of spices, fruit and vegetable juices, scented tea juices, colorants, acidity regulators, preservatives, antioxidants, thickeners, and sweeteners.

[0020] In one embodiment of the present invention, the processed form of the fermented food includes solid food, liquid food or semi-solid food.

[0021] In one embodiment of the present invention, the feed additive contains the fermented mucus Lactobacillus D22 described in the first technical solution or the microbial preparation described in the second technical solution.

[0022] In one embodiment of the present invention, the amount of the fermented mucus Lactobacillus D22 in the feed additive is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0023] Preferably, in the feed additive, the added amount of the fermented mucus Lactobacillus D22 is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0024] The fifth technical solution provided by the present invention is the use of the fermented mucus Lactobacillus D22 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a product that improves individual memory ability or improves memory impairment.

[0025] In one embodiment of the present invention, the application includes at least one of the following functions:

[0026] (1) Enhance individual spatial memory ability;

[0027] (2) Enhance the individual's non-hippocampal dependent memory;

[0028] (3) changing the individual's serum metabolites and increasing the content of glycerophospholipids in the individual's serum metabolites;

[0029] (4) Upregulate the expression of glutamate receptors and / or dopamine DRD1 receptors in the individual's brain.

[0030] In one embodiment of the present invention, the product comprises a functional food, a medicine or a health product.

[0031] In one embodiment of the present invention, the medicine further contains a drug carrier and / or a pharmaceutical excipient.

[0032] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0033] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0034] In one embodiment of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0035] In one embodiment of the present invention, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

[0036] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0037] The sixth technical solution provided by the present invention is the use of the fermented mucus Lactobacillus D22 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of medicines for regulating serum metabolites.

[0038] In one embodiment of the present invention, the regulating serum metabolites includes upregulating the content of glycerophospholipids such as LPA 22:6 and LPS20:4.

[0039] In one embodiment of the present invention, the medicine further contains a drug carrier and / or a pharmaceutical excipient.

[0040] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0041] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0042] In one embodiment of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0043] In one embodiment of the present invention, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

[0044] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0045] The seventh technical solution provided by the present invention is the use of the fermented mucus Lactobacillus D22 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a promoter for glutamate receptors and / or dopamine DRD1 receptors.

[0046] The technical effects of the present invention are as follows:

[0047] The fermented mucus Lactobacillus D22 provided by the present invention has good intestinal colonization ability and can significantly improve the memory ability of individual mice:

[0048] In the animal experiment of mice, taking the fermented mucus lactobacillus D22 of the present invention can significantly improve the spatial memory ability of individual mice, and the exploration time of mice in the novel arm intervened by fermented mucus lactobacillus D22 is 2.4 times that of the control group; taking the fermented mucus lactobacillus D22 of the present invention can also significantly improve the non-hippocampus-dependent memory of individual mice, and the proportion of conditioned fear rigidity of mice in the fermented mucus lactobacillus D22 intervention group is 2.6 times that of the control group. At the same time, taking the fermented mucus lactobacillus D22 of the present invention can increase the content of memory-benefitting substances such as glycerophospholipids LPA22:6 (1.51 times) and LPS20:4 (2.1 times) in the blood, affect the expression of neural receptors in the brain, and promote neural signal transmission.

[0049] The fermented mucus lactobacillus D22 of the present invention can be used to prepare functional foods, health products and medicines that can enhance and improve memory ability or improve memory damage, and has very broad application prospects.

[0050] Biomaterial Deposit

[0051] A fermentative mucus lactobacillus (Limosilactobacillus fermentum) D22, taxonomically named Limosilactobacillus fermentum, was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 28, 2024, with a deposit number of GDMCC No: 65071. The deposit address is Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The figure is a comparison chart of the survival rate after gastrointestinal fluid treatment, with the survival rate without gastrointestinal fluid treatment as the control.

[0053] Figure 2 qPCR was used to detect the content of intestinal bacteria in mice; *P<0.05.

[0054] Figure 3 The exploration time of the novel arm of the individual in the Y-maze test; (A) Lactobacillus fermentans D22 intervention group; (B) L. gasseri intervention group; *P<0.05.

[0055] Figure 4 The proportion of conditioned fear freezing in individuals in the conditioned fear experiment; (A) Lactobacillus mucilaginosus D22 intervention group; (B) L. gasseri intervention group; *P<0.05.

[0056] Figure 5 To investigate the effect of Lactobacillus mucilaginosus D22 intervention on serum metabolites in mice.

[0057] Figure 6 The Lactobacillus mucilaginosus D22 intervention group ( Figure 6 A) and control strain L. gasseri intervention group ( Figure 6 B) Overall distribution of differential metabolites. The serum LPA22:6 in the mice intervention group of fermented Lactobacillus mucilaginosus D22 increased by 1.51 times ( Figure 6 C) LPS20:4 increased by 2.14 times ( Figure 6 D), *P<0.05.

[0058] Figure 7 This is a graph showing the effect of fermented Lactobacillus mucilaginosus D22 on the expression of neuroreceptors in the mouse brain, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0059] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0060] Test method:

[0061] 1. Y-maze test: It includes two experiments, 2 hours apart. The first experiment is the acquisition phase, in which one arm is closed and the animals are allowed to freely explore the other two arms for 5 minutes. The second experiment (recall phase) is conducted 2 hours later, in which all arms are opened and the animals are allowed to move freely in three arms for 5 minutes. The movement of mice is tracked using VisuTrack software, and the parameters recorded include the number of times they enter each arm, the time and distance explored in each arm.

[0062] 2. Conditioned fear experiment:

[0063] Establishment of conditioned fear: On the first day of the experiment, mice were placed in an experimental box (with a copper bar fence at the bottom of the box, which could be electrified). After acclimation for 3 minutes, they were given a single-frequency sound stimulus (3.0KHZ, 65db, 28s), and then an inescapable foot shock (0.7mA, 2s) was given at the same time. The sound and the shock ended at the same time. After the experiment, the mice were allowed to stay in the box for another 2 minutes and then returned to the cage. After each experiment, the bottom of the box was wiped with 75% alcohol.

[0064] After 48 hours, the mice with conditioned fear were placed in the original box and no stimulation was given for 3 minutes.

[0065] After 2 hours, the environment in the box was changed (a white board was placed at the bottom of the box, and the color of the box wall was changed), and the mice were put in again. After adaptation, the same intensity of sound stimulation was given for 3 minutes, and the sound-induced freezing behavior of the mice was recorded within 3 minutes. Freezing behavior was defined as no other movement behavior except breathing. The percentage of freezing time induced by environment and sound was recorded.

[0066] 3. After the collected blood was cooled and allowed to stand for 2 hours, it was centrifuged at 3000rpm for 10 minutes to collect serum and placed in an EP tube. An 80% methanol aqueous solution containing 0.1% formic acid was added. After vortexing, it was placed in an ice bath for 5 minutes, centrifuged at 15000g and 4°C for 10 minutes, and a certain amount of supernatant was taken and diluted with mass spectrometry grade water to a methanol content of 53%. It was placed in a centrifuge tube and centrifuged at 15000g and 4°C for 10 minutes, the supernatant was collected, and the sample was injected into LC-MS for analysis. Fold Change and VIP were used to screen differential metabolites, and the thresholds were set as VIP>1.0, FC>1.2 or FC<0.833 and P-value<0.05.

[0067] The raw materials used in the embodiment:

[0068] 1. MRS solid medium: tryptone 10.00 g / L, yeast extract 5.00 g / L, beef extract 10.00 g / L, glucose 20.00 g / L, ammonium citrate 2.00 g / L, anhydrous sodium acetate 5.00 g / L, magnesium sulfate 0.10 g / L, manganese sulfate 0.05 g / L, potassium hydrogen phosphate 2.00 g / L, Tween 80 1.00 mL / L, agar powder 15.00 g / L, the pH of the medium was adjusted to 6.5. All reagents used were analytically pure reagents purchased from Sinopharm Chemical Reagent Co., Ltd.

[0069] 2. MRS liquid culture medium: tryptone 10.00 g / L, yeast extract 5.00 g / L, beef extract 10.00 g / L, glucose 20.00 g / L, triammonium citrate 2.00 g / L, anhydrous sodium acetate 5.00 g / L, magnesium sulfate 0.10 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2.00 g / L, Tween 80 1.00 mL / L, the pH of the culture medium was adjusted to 6.5. All reagents used were analytically pure reagents purchased from Sinopharm Chemical Reagent Co., Ltd.

[0070] 3. Antibiotic solution: 108.0 mg bacitracin, 108.0 mg neomycin, 43.2 mg penicillin, 21.6 mg meropenem, and 6.48 mg vancomycin dissolved in 4.5 mL distilled water.

[0071] 4. C57BL / 6J male mice were obtained from Spefoc (Suzhou) Biotechnology Co., Ltd.

[0072] 5. Lactobacillus gasseri BN9 has been disclosed in patent CN118703356A.

[0073] Example 1 Strain Screening and Identification

[0074] (I) Isolation and screening of fermentative mucus lactobacillus:

[0075] (1) Fresh camel milk was diluted in gradients and applied to MRS solid culture medium, and then cultured at 37°C in an anaerobic environment for 72 hours;

[0076] (2) Observe and record the colony morphology, pick colonies and purify them by streaking;

[0077] (3) Pick a single colony and culture it in MRS liquid medium at 37°C for 48 h before collecting the bacteria for strain identification.

[0078] (II) Molecular biological identification of fermentative Lactobacillus mucilaginosus:

[0079] (1) Single bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology);

[0080] (2) Amplify 16S rDNA using universal primers for bacterial species identification;

[0081] (3) Prepare 1% agarose gel, mix the PCR product with loading buffer and load it, run at 150 V and 100 mA for 20 min, observe and cut the gel;

[0082] (4) The obtained PCR product was sent to a professional sequencing company for sequencing, and the obtained sequencing results were searched and compared for similarity in the GeneBank database using BLAST, and identified as Limosilactobacillusfermentum;

[0083] (5) Whole genome sequencing: The whole genome extracted in (1) was sent to a professional sequencing company, and the whole genome of the bacteria was sequenced using a second-generation sequencer. The obtained sequence results were searched and compared for similarity in the GeneBank database using BLAST. The sequencing results identified the strain as Lactobacillus fermentans and named it Lactobacillus fermentans D22. The strain was stored at -80°C for future use.

[0084] Example 2: In vitro simulated digestion of fermented Lactobacillus mucilaginosus D22

[0085] (I) Preparation of digestive juice

[0086] (1) In vitro simulated gastric fluid: Adjust the pH value of 0.1 mol / L potassium phosphate buffer to 1.5, sterilize, add pepsin (10 g / L), mix to fully dissolve, and obtain simulated gastric fluid. Preheat to 37°C before use.

[0087] (2) In vitro simulated intestinal fluid: adjust the pH of potassium phosphate buffer to 6.8 and sterilize, add trypsin (10 g / L) and porcine bile salt (3 g / L), mix and fully dissolve to obtain simulated intestinal fluid. Preheat to 37°C before use.

[0088] (II) Simulated digestion

[0089] (1) Take 1 mL of bacterial solution and add it to 20.0 mL of simulated gastric fluid. Simulate gastric digestion under anaerobic conditions at 37°C and 90 rpm for 3 h. Determine the number of viable bacteria by plate count method.

[0090] (2) Take 1 mL of bacterial solution and add it to 80.0 mL of simulated intestinal fluid. Incubate the mixture at 37°C and 120 rpm for 3 h to simulate intestinal digestion. Determine the number of viable bacteria using the plate count method.

[0091] The results showed that the survival rate of fermented Lactobacillus mucilaginosus D22 was still high after digestion with gastrointestinal fluid, and there was no significant difference compared with that without gastrointestinal fluid treatment. This indicates that fermented Lactobacillus mucilaginosus D22 may have good intestinal colonization ability ( Figure 1 ).

[0092] Example 3 qPCR measurement of intestinal bacteria content

[0093] Preparation of PBS solution containing bacteria: Activated fermented Lactobacillus mucilaginosus D22 was cultured anaerobically at 37°C for 48 hours in MRS liquid medium. Fresh stable bacterial culture solution was centrifuged, washed twice with PBS, and then the bacterial precipitate was dissolved in PBS solution to make the bacterial concentration reach OD600=1.0.

[0094] This embodiment uses 30 C57BL / 6J male mice of 6 weeks old and weighing 20±1g, and the mice are randomly divided into three groups (n=10), including a control group, a fermented mucus lactobacillus D22 intervention group and a control strain gasseri lactobacillus BN9 (L.gasseri) intervention group. The experiment is divided into three stages: adaptation period (1 week), antibiotic sweep (2 weeks) and probiotic intervention (8 weeks). Each group of mice is fed a normal diet, and after 1 week of adaptation, a 2-week antibiotic sweep (0.1mL / 10g animal body weight antibiotic solution is administered to mice, once in the morning and evening), and the mice in the intervention group are gavaged daily with 0.2mL of PBS solution containing fermented mucus lactobacillus D22 (OD600=1) or control strain L.gasseri (OD600=1), and the mice in the control group are gavaged with the corresponding volume of sterile water. After the probiotic intervention is completed, behavioral tests are performed starting from the 12th week. The feces of mice were collected before and after the behavioral test (week 11 and week 13), and the total bacterial count and the abundance of Lactobacillus mucosa fermentative D22 in the feces were detected by real-time fluorescence quantitative PCR.

[0095] DNA was extracted from mouse feces using the EZNAStool DNA kit (Omega Bio-tek), and the total bacterial count and the content of fermentative Lactobacillus mucosa D22 in mouse feces and intestines were detected using TBGreen Premix Ex Taq II FAST qPCR (Takara) fluorescent dye. Bacterial universal primers and fermentative Lactobacillus mucosa-specific amplification primers were purchased from Suzhou Jinweizhi Biotechnology Co., Ltd. The primer sequences used are shown in Table 1.

[0096] Table 1 Primer sequences for fluorescence quantitative PCR

[0097]

[0098] The results showed that after 2 weeks of antibiotic treatment (before D22 intervention), the total bacterial count in the feces of mice in the control group and the fermented mucus lactobacillus D22 intervention group was approximately 10 5 CFU / g feces, there was no significant difference between the two groups; after 8 weeks of intervention with fermented mucus lactobacillus D22, the total bacterial count in the control group (without D22 intervention) was about 10 7 CFU / g feces, the total bacterial count in the D22 intervention group was approximately 10 9CFU / g feces, which was significantly higher than that of the control group, indicating that fermented Lactobacillus mucilaginosus D22 is helpful for the repair of intestinal flora ( Figure 2 A). In addition, before D22 intervention, the amount of Lactobacillus mucilaginosus D22 fermentation in the intestines of mice in the D22 intervention group was about 10 2 CFU / g. After D22 intervention, the amount of fermented Lactobacillus mucilaginosus D22 in the intestine of the intervention group mice increased significantly to 10 5 CFU / g, ( Figure 2 B), indicating that Lactobacillus muciflorus D22 can colonize the mouse intestine.

[0099] Example 4 Effect of fermented Lactobacillus mucilaginosus D22 on the memory ability of individual mice

[0100] The construction and grouping of the animal model refer to Example 3. In this example, the Y-maze and conditioned fear test were used to evaluate the effect of fermented Lactobacillus mucilaginosus D22 on the memory ability of mice. That is, behavioral tests were performed starting from the 12th week after the probiotic intervention.

[0101] The results are as follows:

[0102] (1) The results of the Y-maze experiment showed that fermented Lactobacillus mucilaginosus D22 could significantly increase the exploration time of mice in the novel arm ( Figure 3 A), which is 2.4 times that of the control group. It can be seen that fermented mucus lactobacillus D22 intervention can significantly enhance the spatial memory ability of mice. The control strain L. gasseri ( Figure 3 B) There was no significant difference in the exploration time of the intervened mice in the novel arm compared with the control group, which means that L. gasseri intervention could not improve the spatial memory ability of mice.

[0103] (2) The results of the conditioned fear experiment showed that the proportion of conditioned fear freezing in the mice in the fermented Lactobacillus mucilaginosus D22 intervention group was 2.6 times that of the control group ( Figure 4 A), it can be seen that the intervention of fermented mucus lactobacillus D22 can significantly enhance the non-hippocampal dependent memory of mice. However, there is no significant difference between the proportion of conditioned fear freezing in mice treated with the control strain L. gasseri and the control group, that is, L. gasseri intervention cannot improve the non-hippocampal dependent memory of mice ( Figure 4 B).

[0104] After the behavioral experiment, the mice were killed by cervical dislocation after anesthesia at week 13, and their eyeballs were removed to collect blood for serum metabolite detection.

[0105] The results showed that the fermented Lactobacillus mucilaginosus D22 intervention group ( Figure 5 ) could significantly change serum metabolites, and the results of partial least squares discriminant analysis showed that the two groups could be distinguished. Figure 4As shown in the figure, compared with the control group, there were 52 differential metabolites in the fermented Lactobacillus mucilaginosus D22 intervention group, of which 23 metabolites were up-regulated, mainly lipids and lipid molecules, and 29 metabolites were down-regulated, mainly organic acids and their derivatives ( Figure 6 A). There were 73 differential metabolites in the control strain L. gasseri intervention group, of which 53 metabolites were up-regulated and 20 metabolites were down-regulated ( Figure 6 B). The results showed that the most differential metabolites in the serum of mice in the fermented Lactobacillus mucilaginosus D22 intervention group were lipids and lipid molecules, accounting for 19.2%, followed by organic acids and their derivatives, accounting for 13.5%. Compared with the control group, fermented Lactobacillus mucilaginosus D22 could significantly increase the content of glycerophospholipids in lipids, for example, LPA22:6 increased by 1.51 times ( Figure 6 C) LPS20:4 increased by 2.14 times ( Figure 6 D).

[0106] Example 5 Effect of fermented Lactobacillus mucilaginosus D22 on neuroreceptor expression in the brain

[0107] Animal grouping and intervention, refer to Example 3. After the behavioral experiment, the mice were killed by cervical dislocation after anesthesia at week 13. The brain tissues of the mice in the control group and the fermented Lactobacillus mucosa D22 intervention group were dissected, and the total RNA of the brain tissues was extracted using the SV Total RNA Isolation System Kit (Promega). The iScript TM cDNA was reverse transcribed using Advanced cDNA Synthesis Kit (Bio-Rad Laboratories) and then expressed using iTaq TM Universal Real-time fluorescence quantitative PCR was performed using the Green Supermix kit (Bio-Rad Laboratories) to detect the mRNA expression of the neuroreceptors mGlu2 and Drd1 in brain tissue. The housekeeping gene β-Actin was used as an internal reference gene, and the primer sequences used are shown in Table 2.

[0108] Table 2 Fluorescence quantitative PCR primer sequences

[0109]

[0110]

[0111] The results are as follows Figure 7As shown, compared with the control group mice, the expression of glutamate receptors in the brain of mice intervened by fermented Lactobacillus mucus D22 increased by 1.3 times, and the expression of dopamine DRD1 receptors increased by 1.8 times, indicating that fermented Lactobacillus mucus D22 can affect signal transmission by affecting the expression of neural receptors, thereby affecting memory ability.

[0112] Example 6 Application of fermented Lactobacillus mucilaginosus D22

[0113] Preparation of fermented Lactobacillus mucilaginosus D22 bacterial agent:

[0114] The activated fermented mucus lactobacillus D22 bacteria are cultured anaerobically at 37°C in an MRS liquid medium for 48 hours; 30 mL of fermented mucus lactobacillus D22 bacterial solution is placed in a centrifuge tube, centrifuged at 4°C and 4000 rpm for 10 minutes, the supernatant is discarded, the precipitate is collected, washed 2-3 times with strictly sterilized physiological saline, an appropriate amount of protective agent is added and mixed, and freeze-dried after precooling at -80°C for 4 hours, under the conditions of: cold trap temperature -70°C, drying time 24 hours, vacuum degree 1 Pa. After freeze drying, the bacterial powder is immediately collected in a sterile packaging bag and sealed, to obtain a fermented mucus lactobacillus D22 bacterial agent.

[0115] Preparation of fermented milk with Lactobacillus mucilaginosus D22:

[0116] Fresh milk and white sugar were mixed and homogenized (10:1), sterilized at 140°C for 2 seconds, cooled to 35°C, and then added with the fermented Lactobacillus mucilaginosus D22 bacterial agent starter prepared by the present invention, and fermented at 35°C for 4 hours, and then transferred to a 4°C refrigerator for 12 hours for post-ripening to obtain the final fermentation product (bacteria concentration 10 8 CFU / mL or more).

[0117] The present invention can be used to ferment and produce other fermented foods using fermented Lactobacillus mucilaginosus D22, and the fermented foods include solid foods, liquid foods and semi-solid foods. The fermented foods include dairy products, bean products and fruit and vegetable products, the dairy products include milk, sour cream and cheese; the fruit and vegetable products include cucumber, carrot, beet, celery and cabbage products.

[0118] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A fermented mucus lactobacillus (Limosilactobacillus fermentum) D22, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 28, 2024, with the deposit number GDMCC No: 65071.

2. A microbial preparation containing the fermented mucus Lactobacillus D22 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the concentration of the fermented mucus lactobacillus D22 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A leavening agent, characterized in that The starter contains the fermented Lactobacillus mucilaginosus D22 according to claim 1 or the microbial preparation according to claim 2 or 3.

5. A product, characterized in that: The product is a product containing the fermented mucus Lactobacillus D22 according to claim 1 or the microbial preparation according to claim 2 or 3, or the product is a product obtained by fermentation using the starter according to claim 4; optionally, the product is a feed additive, food, medicine or health product.

6. Use of the fermented mucus Lactobacillus D22 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a product that improves individual memory ability or improves memory impairment.

7. The use according to claim 6, characterized in that: The application includes at least one of the following functions: (1) Enhance individual spatial memory ability; (2) Enhance the individual's non-hippocampal dependent memory; (3) changing the individual's serum metabolites and increasing the content of glycerophospholipids in the individual's serum metabolites; (4) Upregulate the expression of glutamate receptors and / or dopamine DRD1 receptors in the individual brain.

8. Use of the fermented Lactobacillus mucilaginosus D22 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a medicine for regulating serum metabolites.

9. The use according to claim 8, characterized in that: Modulating serum metabolites includes upregulating the levels of LPA 22:6 and / or LPS 20:4 glycerophospholipids.

10. Use of the fermented mucus lactobacillus D22 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a promoter for glutamate receptors and / or dopamine DRD1 receptors.

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