Lactobacillus rhamnosus capable of enhancing memory ability, fermented food of lactobacillus rhamnosus and application of lactobacillus rhamnosus

Abstract

The invention discloses a lactobacillus rhamnosus HL8 capable of enhancing memory ability as well as a fermented food and application thereof, and belongs to the technical field of microorganisms. The lactobacillus rhamnosus HL8 can be used for preparing functional food, health care products and medicines for improving individual memory ability or improving memory impairment, the invention further provides fermented food, the fermented food is prepared by fermenting the lactobacillus rhamnosus, 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 discloses Lactobacillus rhamnosus HL8 for enhancing memory ability, fermented food thereof and application thereof, and belongs to the technical field of microorganisms. Background Art

[0002] In a rapidly changing modern society, having good learning and memory abilities can help us adapt to new environments and cope with new challenges. However, people of different age groups suffer from learning and memory disorders: teenagers may suffer from study pressure; adults may suffer from anxiety, depression or organic brain diseases; the elderly may suffer from aging of the memory-related hippocampus or central nervous system degenerative diseases such as Alzheimer's disease. Severe learning and memory disorders not only affect the individual's life and social adaptability, but also bring heavy pressure and economic burden to the family and society. However, the effects of current intelligence-enhancing products for improving the learning and memory abilities of infants, teenagers, adults and the elderly are very limited, especially there is no effective treatment for memory damage caused by neurodegenerative diseases such as dementia.

[0003] In recent years, it has been found that intestinal flora plays an important role in nutrient absorption, energy supply, metabolism, immunity and protection against pathogens. Its role not only affects the physiological function of the gastrointestinal tract, but also plays an indispensable role in the central nervous system through the "microorganism-gut-brain" axis. Its functional mechanism is that the various signals generated in the intestine regulate the functions of specific brain regions through corresponding signal molecules; with the deepening of the understanding of the signal transmission and mutual regulation between the intestine and the brain, the concept of the gut-brain axis has been proposed and widely recognized.

[0004] It can be seen that regulating intestinal flora through probiotics to improve brain function and cognitive ability has become a new possible method. Currently, the known functions of probiotics are mainly to regulate intestinal function, improve host nutrient metabolism, etc., and there are very few known probiotics that can improve 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 Lactobacillus rhamnosus HL8 for enhancing memory ability, its fermented food and its application, 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 there is an urgent need to screen out a probiotic that can effectively improve memory.

[0006] The first technical solution provided by the present invention is a rhamnosus Lactobacillus (Lacticaseibacillusrhamnosus) HL8, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 13, 2024, with a collection number of GDMCC.No: 65131, 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 Lactobacillus rhamnosus HL8 described in the first technical solution.

[0008] In one embodiment of the present invention, the concentration of Lactobacillus rhamnosus HL8 in the microbial preparation is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0009] Preferably, in the microbial preparation, the concentration of Lactobacillus rhamnosus HL8 is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0010] The third technical solution provided by the present invention is a starter culture, wherein the starter culture contains the Lactobacillus rhamnosus HL8 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 Lactobacillus rhamnosus HL8 added to the starter is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0012] Preferably, the amount of Lactobacillus rhamnosus HL8 added to the starter is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0013] The third technical solution provided by the present invention is a product, which is a product containing the rhamnosus Lactobacillus HL8 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 Lactobacillus rhamnosus HL8 described in the first technical solution or the microbial preparation described in the second technical solution, 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 Lactobacillus rhamnosus HL8.

[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 Lactobacillus rhamnosus HL8 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 Lactobacillus rhamnosus HL8 added 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 amount of Lactobacillus rhamnosus HL8 added 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 rhamnosus Lactobacillus HL8 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) Improve the individual's discrimination index and cognitive index;

[0027] (2) Enhance individual spatial memory ability;

[0028] (3) Enhance the individual's non-hippocampal dependent memory;

[0029] (4) changes in individual serum metabolites;

[0030] (5) Upregulate the expression of glutamate receptors in the individual brain and / or downregulate the expression of TLR4 receptors that promote neuronal apoptosis and neuroinflammation.

[0031] In one embodiment of the present invention, the product comprises a functional food, a medicine or a health product.

[0032] In one embodiment of the present invention, the medicine further contains a drug carrier and / or a pharmaceutical excipient.

[0033] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0034] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0035] In one embodiment of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0036] In one embodiment of the present invention, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

[0037] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0038] The sixth technical solution provided by the present invention is the use of the rhamnosus Lactobacillus HL8 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.

[0039] In one embodiment of the present invention, the regulating serum metabolites includes upregulating the content of dihydrojasmonate, undecylenic acid, LPS20:5 and the like.

[0040] In one embodiment of the present invention, the medicine further contains a drug carrier and / or a pharmaceutical excipient.

[0041] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0042] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0043] In one embodiment of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0044] In one embodiment of the present invention, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

[0045] In one embodiment of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0046] The seventh technical solution provided by the present invention is the use of the rhamnosus Lactobacillus HL8 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a regulator of glutamate receptors and / or immune receptor TLR4, wherein the regulator can upregulate the expression of glutamate receptors in the individual's brain and downregulate the expression of TLR4 receptors that promote neuronal apoptosis and neuroinflammation.

[0047] The technical effects of the present invention are as follows:

[0048] The Lactobacillus rhamnosus HL8 provided by the present invention has good intestinal colonization ability and can significantly improve the memory ability of individual mice:

[0049] Taking the Lactobacillus rhamnosus HL8 of the present invention can significantly improve the exploration desire of individual mice for new objects. The resolution index and cognitive index of mice intervened by Lactobacillus rhamnosus HL8 are 1.8 times and 1.3 times of the memory index of mice in the control group, respectively; taking the Lactobacillus rhamnosus HL8 of the present invention can significantly improve the spatial memory ability of individual mice. The exploration time of mice intervened by Lactobacillus rhamnosus HL8 in the novel arm is 2.2 times that of the control group; taking the Lactobacillus rhamnosus HL8 of the present invention can also significantly improve the non-hippocampus-dependent memory of individual mice. The conditioned fear stiffness ratio of mice intervened by Lactobacillus rhamnosus HL8 is 3.4 times that of the control group. At the same time, taking the Lactobacillus rhamnosus HL8 of the present invention can increase dihydrojasmonate (2.40 times), undecylenic acid (1.98 times), and LPS20:5 (1.71 times) in serum. These substances may affect the expression of neural receptors in the brain, promote neural signal transmission, and improve memory ability.

[0050] The Lactobacillus rhamnosus HL8 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.

[0051] Biomaterial Deposit

[0052] Lacticaseibacillus rhamnosus HL8, taxonomically named Lacticaseibacillus rhamnosus, was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 13, 2024, with the deposit number GDMCC.No:65131, and the deposit address is Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The figure shows the survival rate of Lactobacillus rhamnosus HL8 after being treated with gastrointestinal fluid, while the survival rate without being treated with gastrointestinal fluid was used as the control.

[0054] Figure 2 The content of intestinal bacteria in the mice in the Lactobacillus rhamnosus HL8 intervention group was detected by qPCR; *P<0.05.

[0055] Figure 3 Represents the discrimination index (DI) in the novel object recognition experiment; (A) Lactobacillus rhamnosus HL8 intervention group, (B) control strain L. gasseri intervention group, *P<0.05.

[0056] Figure 4 Represents the recognition index (RI); (A) Lactobacillus rhamnosus HL8 intervention group, (B) control strain L. gasseri intervention group, *P<0.05.

[0057] Figure 5 Represents the ability to explore the novel arm of the Y-maze; (A) Lactobacillus rhamnosus HL8 intervention group, (B) control strain L. gasseri intervention group; *P<0.05.

[0058] Figure 6 The conditioned fear freezing ratio is shown in (A) the Lactobacillus rhamnosus HL8 intervention group and (B) the control strain L. gasseri intervention group; *P<0.05.

[0059] Figure 7 It indicates that the Lactobacillus rhamnosus HL8 intervention group can significantly change serum metabolites.

[0060] Figure 8 Indicates the intervention group of Lactobacillus rhamnosus HL8 ( Figure 8 A) and control strain L. gasseri intervention group ( Figure 8 B) Overall distribution of differential metabolites. Compared with the control group, the serum methyl dihydrojasmonate in the Lactobacillus rhamnosus HL8 intervention group increased by 2.4 times ( Figure 8 C), the content of undecylenic acid increased by 1.98 times ( Figure 8 D), LPS20:5 increased by 1.71 times ( Figure 8 E), *P<0.05.

[0061] Fig. 9 Represents the effect of Lactobacillus rhamnosus HL8 on the expression of neural receptors in mouse brain, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0062] 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.

[0063] Test method:

[0064] 1. New object recognition experiment: The day before training, the tested animals were allowed to explore the test box for 10 min to adapt; during training, two identical objects were placed in the same test box, and the tested animals were allowed to explore the test box for 5 min; 24 h later, one of the objects was replaced with a new object, and then the tested animals were placed in the test box for 5 min of testing and video was recorded. The video was analyzed using VisuTrack software to examine whether the animals could recognize that one of the objects was a new object. The time the mice spent sniffing each object was recorded, and the discrimination index (DI) and recognition index (RI) were evaluated (DI = new object time - old object time / (new object time + old object time), RI = number of new object explorations / (number of new object explorations + number of old object explorations)).

[0065] 2. Y-maze test: It includes two tests, with an interval of 2 hours. The first test 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 test (recall phase) is conducted 2 hours later, in which all arms are opened and the animals are allowed to move freely in the three arms for 5 minutes. The movement of the mice is tracked using VisuTrack software, and the time and distance explored in each arm are recorded. The recorded parameters include the number of times the mice enter each arm, the time and distance explored in each arm.

[0066] 3. Conditioned fear experiment:

[0067] 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.

[0068] After 48 hours, the mice with conditioned fear were placed in the original box and no stimulation was given for 3 minutes.

[0069] 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, they were given sound stimulation of the same intensity for 3 minutes, and the sound-induced freezing behavior (cue freezing, which mainly reflects non-hippocampus-dependent memory) of the mice was recorded within 3 minutes.

[0070] 4. 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.

[0071] The raw materials used in the examples are:

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 4. C57BL / 6J male mice were obtained from Spefoc (Suzhou) Biotechnology Co., Ltd.

[0076] 5. Lactobacillus gasseri BN9 has been disclosed in patent CN118703356A.

[0077] Example 1 Strain Screening and Identification

[0078] (I) Isolation and screening of Lactobacillus rhamnosus HL8:

[0079] (1) Take an appropriate amount of fresh infant feces and dilute it in a gradient manner, then spread it on MRS solid culture medium and culture it at 37°C in an anaerobic environment for 72 hours;

[0080] (2) Observe and record the colony morphology, pick colonies and purify them by streaking;

[0081] (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.

[0082] (II) Molecular biological identification of Lactobacillus rhamnosus HL8:

[0083] (l) Extract single bacterial genomic DNA using EZNAStool DNA kit (Omega Bio-tek);

[0084] (2) Amplify 16S rDNA using universal primers for bacterial species identification;

[0085] (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;

[0086] (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 the strain was identified as Lactobacillus rhamnosus and named Lactobacillus rhamnosus HL8. The strain was stored at -80°C for future use.

[0087] Example 2 In vitro simulated digestion of Lactobacillus rhamnosus HL8

[0088] (I) Preparation of digestive juice

[0089] (1) In vitro simulated gastric fluid: Adjust the pH value of 0.1 mol / L potassium phosphate buffer to 1.5, add pepsin (10 g / L) in a clean bench after sterilization, mix to fully dissolve, and filter through a 0.22 μm filter to obtain simulated gastric fluid. Preheat to 37°C before use.

[0090] (2) In vitro simulated intestinal fluid: adjust the pH of potassium phosphate buffer to 6.8, sterilize, add trypsin (10 g / L) and porcine bile salt (3 g / L) in a clean bench, mix to fully dissolve, and filter through a 0.22 μm filter to obtain simulated intestinal fluid. Preheat to 37°C before use.

[0091] (II) Simulated digestion

[0092] (1) Take 1 mL of bacterial solution and add it to 20.0 mL of simulated gastric fluid. Simulate gastric digestion at 37°C under anaerobic conditions for 3 h. Determine the number of viable bacteria by plate count method.

[0093] (2) Take 1 mL of bacterial solution and add it to 80.0 mL of simulated intestinal fluid. Simulate intestinal digestion under anaerobic conditions at 37°C for 3.0 h, and determine the number of viable bacteria using the plate count method.

[0094] The results showed that the survival rate of Lactobacillus rhamnosus HL8 was still high after digestion with gastrointestinal fluid, and there was no significant difference compared with that without gastrointestinal fluid treatment. This indicates that Lactobacillus rhamnosus HL8 may have good intestinal colonization ability ( Figure 1 ).

[0095] Example 3 qPCR measurement of intestinal bacteria content

[0096] Preparation of PBS solution containing bacteria: Activated Lactobacillus rhamnosus HL8 was cultured anaerobically at 37°C for 48 hours in MRS liquid medium. Fresh bacterial culture solution in the stable phase was centrifuged, washed with PBS, and then the bacterial precipitate was dissolved in PBS solution to make the bacterial concentration reach OD 600 = 1.0.

[0097] This example 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 Lactobacillus rhamnosus HL8 intervention group and a control strain Lactobacillus gasseri BN9 (L.gasser)i intervention group. The experiment is divided into three stages: adaptation period (1 week), antibiotic sweep (2 weeks) and probiotic intervention (8 weeks). All groups of mice are fed a normal diet, and after 1 week of adaptation, they are subjected to a 2-week antibiotic sweep (mice are gavaged with 0.1mL / 10g animal body weight antibiotic solution, once in the morning and evening), and the intervention group mice are gavaged with 0.2mL of PBS solution containing Lactobacillus rhamnosus HL8 (OD600=1) or control strain L.gasseri (OD600=1) daily, and the control group mice are gavaged with the corresponding volume of sterile water. Behavioral testing began at week 12 after the probiotic intervention. 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 rhamnosus HL8 in the feces were detected by real-time fluorescence quantitative PCR.

[0098] DNA was extracted from mouse feces using the EZNAStool DNA kit (Omega Bio-tek), and the total bacterial count and the content of Lactobacillus rhamnosus HL8 in mouse feces and intestines were detected using TBGreen Premix Ex Taq II FAST qPCR (Takara) fluorescent dye. Bacterial universal primers and Lactobacillus rhamnosus HL8-specific amplification primers were purchased from Suzhou Jinweizhi Biotechnology Co., Ltd. The primer sequences used are shown in Table 1.

[0099] Table 1 Primer sequences for fluorescence quantitative PCR

[0100]

[0101] The results showed that after 2 weeks of antibiotic treatment (before HL8 intervention), there was no significant difference in the total bacterial count in the feces of mice in the control group and the Lactobacillus rhamnosus HL8 intervention group, both of which were 10 5 CFU / g feces; after 8 weeks of intervention with Lactobacillus rhamnosus HL8, the total bacterial count in the control group (without HL8 intervention) was approximately 10 7 CFU / g feces, the total bacterial count in the HL8 intervention group was approximately 10 9 CFU / g feces was significantly higher than that of the control group, indicating that Lactobacillus rhamnosus HL8 can promote the recovery of intestinal flora and may play an important role in maintaining the balance of intestinal flora ( Figure 2 A). In addition, a small amount of Lactobacillus rhamnosus HL8 was detected in the intestines of mice in the HL8 intervention group before intervention, with a bacterial count of about 10 2 CFU / g. After HL8 intervention, the amount of Lactobacillus rhamnosus HL8 in the intestine of the intervention group mice increased significantly to 10 5 CFU / g, ( Figure 2 B), indicating that Lactobacillus rhamnosus HL8 can colonize the mouse intestine.

[0102] Example 4 Effect of Lactobacillus rhamnosus HL8 on the memory ability of individual mice

[0103] The construction and grouping of the animal model refer to Example 3. In this example, novel object recognition, Y-maze, and conditioned fear experiments were used to evaluate the effect of Lactobacillus rhamnosus HL8 on the memory ability of mice, that is, behavioral tests were performed starting at week 12 after the probiotic intervention.

[0104] The results are as follows:

[0105] (1) The results of the novel object recognition experiment showed that Lactobacillus rhamnosus HL8 could significantly improve the discrimination index and cognitive index of mice ( Figure 3 A and Figure 4A), which were 1.8 and 1.3 times higher than the memory index of the control mice, indicating that they had a desire to explore new objects. Figure 3 B and Figure 4 B) The discrimination index of the intervention mice was significantly decreased compared with the cognitive index and the control group, that is, L. gasseri intervention could not improve the spatial memory ability of mice.

[0106] (2) Y-maze test: Figure 5 (A) shows that Lactobacillus rhamnosus HL8 can significantly increase the exploration time of mice in the novel arm, which is 2.2 times that of the control group. It can be seen that Lactobacillus rhamnosus HL8 can significantly enhance the spatial memory ability of mice. The control strain L. gasseri ( Figure 5 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.

[0107] (3) The results of the conditioned fear experiment showed that the proportion of conditioned fear freezing in the Lactobacillus rhamnosus intervention group was 3.4 times that of the control group ( Figure 6 A), it can be seen that Lactobacillus rhamnosus HL8 can significantly enhance the non-hippocampal dependent memory of mice. However, there was no significant difference in the proportion of conditioned fear freezing in mice treated with the control strain L. gasseri BN9 and the control group, that is, L. gasseri BN9 intervention could not improve the non-hippocampal dependent memory of mice ( Figure 6 B).

[0108] Example 5 Effect of Lactobacillus rhamnosus HL8 on Metabolites and Contents in Serum

[0109] The construction and grouping of the animal model refer to Example 3. In this example, after the behavioral experiment was completed, the mice were killed by cervical dislocation after anesthesia at week 14, and the eyeballs were removed to collect the blood of the mice for serum metabolite detection.

[0110] The results showed that feeding Lactobacillus rhamnosus HL8 could significantly change serum metabolites ( Figure 7 , the results of partial least squares discriminant analysis showed that the control group and the Lactobacillus rhamnosus HL8 intervention group could be distinguished). Figure 7 As shown in the figure, compared with the control group, there were 99 differential metabolites in the Lactobacillus rhamnosus HL8 intervention group, of which 56 metabolites were up-regulated and 43 metabolites were down-regulated ( Figure 8 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 8B). The results showed that the most differential metabolites in the serum of mice in the Lactobacillus rhamnosus HL8 intervention group were lipids and lipid molecules, accounting for 31.3%, followed by organic acids and their derivatives, accounting for 11%. Compared with the control group, the level of methyl dihydrojasmonate in the serum of mice in the Lactobacillus rhamnosus HL8 intervention group increased by 2.40 times ( Figure 8 C), the content of undecylenic acid increased by 1.98 times ( Figure 8 D), LPS20:5 increased by 1.71 times ( Figure 8 E).

[0111] Example 6 Effect of Lactobacillus rhamnosus HL8 on the expression of neural receptors in the brain

[0112] Animal grouping and intervention, refer to Example 3. After the behavioral experiment, the mice were killed by cervical dislocation after anesthesia at week 14. The brain tissues of the mice in the control group and the Lactobacillus rhamnosus HL8 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 GreenSupermix kit (Bio-Rad Laboratories) was used for real-time fluorescence quantitative PCR to detect the mRNA expression of neuroreceptors mGlu2 and TLR4 in brain tissue. The housekeeping gene β-Actin was used as an internal reference gene, and the primer sequences used are shown in Table 1.

[0113] Table 2 Fluorescence quantitative PCR primer sequences

[0114]

[0115] The results are as follows Fig. 9 As shown, compared with the control group mice, the mRNA expression of the brain neuroreceptors mGlu2 and TLR4 in the mice intervention group of Lactobacillus rhamnosus HL8 changed. The relative expression of mGlu2 mRNA in the brain of mice after Lactobacillus rhamnosus HL8 treatment increased by 1.3 times, and the relative expression of TLR4 receptor mRNA related to promoting neuronal apoptosis and neuroinflammation decreased by 0.5 times, indicating that Lactobacillus rhamnosus HL8 can affect signal transduction by affecting the expression of neuroreceptors, thereby affecting memory ability.

[0116] Example 7 Application of Lactobacillus rhamnosus HL8

[0117] Preparation of Lactobacillus rhamnosus HL8 bacterial agent:

[0118] The activated Lactobacillus rhamnosus HL8 was cultured anaerobically at 37°C in MRS liquid medium for 48 hours; 30 mL of Lactobacillus rhamnosus HL8 bacterial solution was placed in a centrifuge tube, centrifuged at 4°C and 4000 rpm for 10 minutes, the supernatant was discarded, the bacterial mud was collected, washed 2-3 times with strictly sterilized physiological saline, an appropriate amount of protective agent was 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 was immediately collected in a sterile packaging bag and sealed, and the Lactobacillus rhamnosus HL8 bacterial agent was obtained.

[0119] Fresh milk and white sugar were mixed and homogenized (10:1), sterilized at 140°C for 2 seconds, cooled to 35°C, and then inoculated with the Lactobacillus rhamnosus HL8 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).

[0120] The present invention can be used to produce other fermented foods by fermentation of Lactobacillus rhamnosus HL8, 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.

[0121] 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 Lactobacillus rhamnosus HL8, characterized in that It was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 13, 2024, with the deposit number GDMCC.No:65131.

2. A microbial preparation containing the Lactobacillus rhamnosus HL8 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the concentration of Lactobacillus rhamnosus HL8 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 Lactobacillus rhamnosus HL8 according to claim 1 or the microbial preparation according to claim 2 or 3.

5. A product, characterized in that: The product contains the Lactobacillus rhamnosus HL8 described in claim 1 or the microbial preparation described in claim 2 or 3, or the product is a product obtained by fermentation using the starter described in claim 4; optionally, the product is a feed additive, food, medicine or health product.

6. Use of the Lactobacillus rhamnosus HL8 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a product for improving individual memory ability or ameliorating memory impairment.

7. The use according to claim 6, characterized in that: The application includes at least one of the following functions: (1) Improve the individual's discrimination index and cognitive index; (2) Enhance individual spatial memory ability; (3) Enhance the individual's non-hippocampal dependent memory; (4) changes in individual serum metabolites; (5) Upregulate the expression of glutamate receptors in the individual brain and / or downregulate the expression of TLR4 receptors that promote neuronal apoptosis and neuroinflammation.

8. Use of the Lactobacillus rhamnosus HL8 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 methyl dihydrojasmonate, undecylenic acid and / or LPS20:

5.

10. Use of the Lactobacillus rhamnosus HL8 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a regulator of glutamate receptors and / or immune receptor TLR4, characterized in that: The regulator can upregulate the expression of glutamate receptors in the brain of an individual and downregulate the expression of TLR4 receptors that promote neuronal apoptosis and neuroinflammation.

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