Lactobacillus plantarum C067 capable of enhancing memory ability as well as fermented food and application of lactobacillus plantarum C067

Abstract

The invention discloses lactobacillus plantarum C067 capable of improving memory ability, fermented food thereof and application of the lactobacillus plantarum C067. The lactobacillus plantarum C067 can significantly improve the fear memory and spatial memory ability of individuals, increase the content of antioxidant substances in blood, reduce the level of inflammatory factors, influence the nerve receptor expression of the brain, promote neural signal transmission, regulate and control intestinal flora, increase the abundance of beneficial bacteria, and increase the acetic acid and butyric acid content of intestinal tracts; the fermented food also has the effect of remarkably improving the individual memory ability. The lactobacillus plantarum C067 disclosed by the invention can be used for preparing functional foods, health-care products and medicines for enhancing and improving the memory ability or improving memory impairment, and has a very wide application prospect.

Description

Technical Field

[0001] The invention relates to plant lactobacillus C067 for enhancing memory ability, fermented food thereof and application thereof, and belongs to the technical field of microorganisms. Background Art

[0002] Learning and memory are the basis of human cognition and are also necessary functions for individuals to adapt to the social environment and survive better. Cognitive function is crucial to an individual's daily life. It not only includes the ability to remember, learn, think and solve problems, but also affects emotions and behaviors. Studies have shown that cognitive function is closely related to overall health. Good cognitive function can improve the quality of life, delay the aging process, and reduce the risk of neurodegenerative diseases. Therefore, exploring methods to enhance and protect cognitive function is of great scientific and clinical significance.

[0003] In recent years, a large number of literatures have pointed out that the gut microbiome affects cognitive function through the gut-brain axis. The brain-gut axis is a complex two-way communication network that connects the gastrointestinal tract and the central nervous system through endocrine, immune and neural pathways. Experimental animals raised in a sterile environment and antibiotic-induced gut microbial imbalance models have demonstrated the causal relationship between disruption of the gut microbial community and memory impairment. Studies have shown that intestinal microorganisms may change the cognitive function of the brain by affecting intestinal permeability, the body's inflammation level, synaptic plasticity, and changing neurotransmitter signaling, affecting neuroreceptors and related metabolites.

[0004] It can be seen that regulating intestinal flora through probiotics to improve brain function and cognitive ability has become a new possible method. At present, the known functions of probiotics are mainly to regulate intestinal function, improve host nutritional metabolism, etc. There are very few reported probiotics that can improve cognitive ability, and most of them focus on disease states of cognitive decline, lacking strains that can improve the cognitive ability of healthy individuals. Strains with potential brain function regulation, such as Bifidobacterium, have high requirements for culture conditions and require a strict anaerobic environment, which also limits their application. In addition, the function of probiotics is highly strain-specific. What kind of probiotics can improve the cognitive ability of healthy individuals, and how they improve the memory and cognitive ability of healthy individuals are unknown. Therefore, screening probiotics that improve individual memory ability and have good environmental tolerance, and developing functional products that assist in improving individual memory have extremely broad application prospects. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides Lactobacillus plantarum C067 that can rapidly proliferate in an aerobic culture environment and has the ability to enhance memory, its fermented food and its application, with the aim of solving the technical problems that there are very few types of probiotics known to improve cognitive impairment, which probiotics can improve the cognitive ability of healthy individuals, and how to improve the memory and cognitive ability of healthy individuals are all unknown.

[0006] The first technical solution provided by the present invention is a plant lactobacillus (Lactiplantibacillus plantarum) C067, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 15, 2024, with a collection number of GDMCC No: 65484, 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 plantarum C067 described in the first technical solution.

[0008] In certain embodiments, in the microbial preparation, the concentration of Lactobacillus plantarum C067 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 plantarum C067 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 fermentation agent, wherein the fermentation agent contains the plant lactobacillus C067 described in the first technical solution or the microbial preparation described in the second technical solution.

[0011] In some embodiments, the amount of Lactobacillus plantarum C067 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 plantarum C067 added to 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 Lactobacillus plantarum C067 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 certain embodiments, the product is a feed additive, a food, a medicine or a health product.

[0015] In certain embodiments, the food is a fermented food containing Lactobacillus plantarum C067 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 certain embodiments, the fermented food includes dairy products, soy products, fruit and vegetable products, or other fermented foods containing Lactobacillus plantarum C067.

[0017] In certain embodiments, the dairy product comprises milk, sour cream, cheese.

[0018] In certain embodiments, the fruit and vegetable preparation includes one or more preparations of cucumber, carrot, beet, celery, cabbage and other edible fruits and vegetables.

[0019] In some embodiments, 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 certain embodiments, the processed form of the fermented food includes solid food, liquid food or semi-solid food.

[0021] In certain embodiments, the feed additive contains Lactobacillus plantarum C067 described in the first technical solution or the microbial preparation described in the second technical solution.

[0022] In some embodiments, the amount of Lactobacillus plantarum C067 added to 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 plantarum C067 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 Lactobacillus plantarum C067 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 some embodiments, the application includes at least one of the following effects:

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

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

[0028] (3) Increase the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) in the individual's serum;

[0029] (4) upregulating the expression of brain-derived nutritional factor, glutamate receptors and / or neuropeptide Y receptors in the hippocampus of the individual brain;

[0030] (5) Regulate individual brain immunity and reduce the content of inflammatory factors in individual hippocampal tissue;

[0031] (6) Regulate individual intestinal flora and intestinal metabolites.

[0032] In certain embodiments, the product comprises a functional food, a medicine or a health product.

[0033] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0034] In certain embodiments, the drug carrier comprises a microcapsule, a microsphere, a nanoparticle and / or a liposome.

[0035] In certain embodiments, the pharmaceutical excipient comprises an excipient and / or an additive.

[0036] In certain embodiments, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0037] In certain embodiments, the additional agent comprises a solubilizer, a co-solvent, a co-solvent and / or a preservative.

[0038] In certain embodiments, the drug is in the form of powder, granules, capsules, tablets, pills or oral liquid.

[0039] The sixth technical solution provided by the present invention is the use of Lactobacillus plantarum C067 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a medicine for regulating individual brain immunity.

[0040] In certain embodiments, regulating brain immunity in an individual comprises reducing the levels of TNF-α and IL-1β in hippocampal tissue of the individual.

[0041] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0042] In certain embodiments, the drug carrier comprises a microcapsule, a microsphere, a nanoparticle and / or a liposome.

[0043] In certain embodiments, the pharmaceutical excipient comprises an excipient and / or an additive.

[0044] In certain embodiments, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0045] In certain embodiments, the additional agent comprises a solubilizer, a co-solvent, a co-solvent and / or a preservative.

[0046] In certain embodiments, the drug is in the form of powder, granules, capsules, tablets, pills or oral liquid.

[0047] The seventh technical solution provided by the present invention is the use of Lactobacillus plantarum C067 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of a promoter of hippocampal brain-derived nutritional factor, glutamate receptor and / or neuropeptide Y receptor.

[0048] The technical effects of the present invention are as follows:

[0049] In the animal experiment on mice, taking the plant lactobacillus C067 of the present invention can significantly improve the fear memory and spatial memory ability of an individual, increase the content of antioxidant substances in the blood and reduce the level of inflammatory factors, affect the expression of neural receptors in the brain, promote neural signal transmission, regulate the intestinal flora to increase the abundance of beneficial bacteria, and increase the content of intestinal acetic acid and butyric acid; the fermented food also has the effect of significantly improving the memory ability of an individual.

[0050] The Lactobacillus plantarum C067 of the present invention can be used to prepare functional foods, health products and medicines that can enhance or improve memory ability or improve memory damage, and has very broad application prospects.

[0051] Biomaterial Deposit

[0052] A strain of Lactobacillus plantarum (Lactiplantibacillus plantarum) C067, taxonomically named Lactiplantibacillus plantarum, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 15, 2024, with the collection number GDMCCNO:65484, and the collection 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 effect of Lactobacillus plantarum C067 on the memory ability of mice. (A) The freezing time of mice in the conditioned fear experiment; (B) The average distance to the platform in the water maze experiment; (B) The number of times the platform was crossed in the water maze experiment; *P<0.05, **P<0.01.

[0054] Figure 2 The effect of Lactobacillus plantarum C067 on the redox level of mice. (A) Serum reduced glutathione (GSH) content; (B) Serum superoxide dismutase (SOD) content; *P<0.05, **P<0.01

[0055] Figure 3 The effect of Lactobacillus plantarum C067 on the levels of inflammatory factors in mouse serum. (A) TNA-α content in serum; (B) IL-1β content in serum; *P<0.05, **P<0.01

[0056] Figure 4 The effect of Lactobacillus plantarum C067 on the expression of neuroreceptors in the mouse brain. (A) Relative expression of brain-derived nutritional factor (BDNF) mRNA; (B) Relative expression of neuropeptide Y receptor 5 (NPY5R) mRNA; (C) Relative expression of metabotropic glutamate receptor 1 (mGluR1) mRNA; (C) Relative expression of metabotropic glutamate receptor 1 (mGluR3) mRNA; *P<0.05, **P<0.01.

[0057] Figure 5 The effect of Lactobacillus plantarum C067 on the intestinal flora of mice. (A) Relative abundance of the community (phylum level); (B) Relative abundance of the community (genus level); (C) β-diversity analysis of intestinal flora;

[0058] Figure 6 Effects of Lactobacillus plantarum C067 on intestinal metabolites in mice. (A) Acetic acid concentration; (B) Propionic acid concentration; (C) Butyric acid concentration; *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] The following examples relate to the test method:

[0061] 1. Morris water maze test

[0062] The experiment lasted for 6 days, with 4 training sessions every day at a fixed time period. On the first day, the mice were trained to stand on the platform. From the second to the fifth day, the platform was placed in the first quadrant, and the mice were placed into the pool facing the pool wall from any of the four starting points on the pool wall. The free video recording system recorded the time and swimming path of the mice to find the platform. The mice were placed into the water from four different starting points (different quadrants) for 4 training sessions. If the mice could not find the platform within 60 seconds, they were guided to the platform and rested on the platform for 10 seconds. The next training session was conducted after an interval of 1 minute. On the sixth day, the platform was removed, and the mice were placed into the water at the same entry point in a specific quadrant. The swimming path of the mice within 60 seconds was recorded, as well as the number of times the mice crossed the target quadrant platform, the swimming speed, etc.

[0063] 2. Conditioned fear experiment:

[0064] 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 powered). After acclimation for 2 minutes, they were given a single-frequency sound stimulus (3.0KHZ, 65db, 30s), and then an inescapable foot shock (0.4mA, 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 breeding cage. After each experiment, the bottom of the box was wiped with 75% alcohol. After 24 hours, the mice that had established conditioned fear were placed in the original box without any stimulation, and the environment-induced rigidity behavior was recorded within 3 minutes. 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 placed in it again. After acclimation, the same intensity of sound stimulation was given for 3 minutes, and the sound-induced rigidity behavior of the mice was recorded within 3 minutes. Rigidity behavior is defined as no other movement behavior except breathing.

[0065] 3. Analysis of mouse intestinal flora

[0066] Fresh feces of mice were used to extract total DNA from fecal samples using the Stool DNA Kit (OMEGA). The mouse fecal genome was used as a template and the V3-V4 region fragment of 16S rDNA was amplified using the upstream primer 338F and the downstream primer 806R. Three replicates were performed for each sample. The PCR products of the same sample were mixed and detected by 2% agarose gel electrophoresis. The PCR products were excised and recovered using the AxyPrep DNA gel recovery kit (AXYGEN) and PCR products were analyzed using QuantiFluor TM-ST blue fluorescence quantitative system (Promega) was used for quantitative detection. According to the fluorescence quantitative results and the sequencing requirements of each sample, each sample was mixed in the corresponding proportion. Then, the sequence was spliced ​​according to the overlapping relationship on the Illumina Miseq sequencer, and the sequence quality was controlled and filtered. The effective data of all samples were subjected to operational taxonomic unit (OTU) cluster analysis and species annotation with 97% consistency. Based on the results of OTU cluster analysis, various diversity index analyses can be performed on OTU, as well as the sequencing depth detection; based on taxonomic information, statistical analysis of community structure can be performed at various classification levels.

[0067] 4. Analysis of short-chain fatty acids in mouse feces

[0068] Accurately weigh 0.1g of mouse feces, add 1.5mL of deionized water, shake and mix, centrifuge at 4℃, 10,000r / min for 10min, and take the supernatant and filter it with a 0.45mm filter membrane. Then take 0.9mL of the sample solution and transfer it to a centrifuge tube containing 0.1mL of 50% sulfuric acid and 2mL of internal standard working solution (2-ethyl-butyric acid concentration is 2mmol / L). After mixing for 5 minutes, centrifuge at 10,000r / min for 10min and keep it at -20℃ for 20min, and take the supernatant for short-chain fatty acid determination. The short-chain fatty acids present in the supernatant are analyzed by gas chromatography system. Determination conditions: detector, hydrogen flame detector (FID); temperature program, 120℃ for 5min, increase to 250℃ at 15℃ / min, and keep it for 1min; FID temperature 255℃; carrier gas is N2; flow rate 1.5mL / min; sample injection volume 2μL; split ratio 10:1. Quantification was accomplished by measuring the peak areas of acetate, propionate, butyrate and valerate relative to 2-ethyl-butyrate and was expressed in mg / g feces.

[0069] The culture medium involved in the following examples is as follows:

[0070] MRS solid 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, agar powder 15.00 g / L, the pH of the culture medium was adjusted to 6.5.

[0071] 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.

[0072] The following examples relate to animals:

[0073] 1. C57BL / 6J male mice were obtained from Spefoc (Suzhou) Biotechnology Co., Ltd.

[0074] Example 1 Strain screening and identification:

[0075] (I) Isolation and screening of Lactobacillus plantarum from fresh camel milk:

[0076] (1) Fresh camel milk samples were selected and graded diluted with sterilized PBS buffer and then applied to a 1% CaCO 3 The cells were cultured on MRS solid medium in a 37°C incubator for 48 h.

[0077] (2) Pick a single white or milky white colony with a distinct transparent zone for Gram staining and microscopic examination, select a rod-shaped Gram-positive bacterial strain, and pick a colony for streaking purification;

[0078] (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.

[0079] (II) Molecular biological identification of lactobacilli:

[0080] (1) Extract genomic DNA using a bacterial genome extraction kit;

[0081] (2) PCR amplification of 16S rDNA using universal primers for bacterial species identification;

[0082] (3) 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 Lactobacillus plantarum in combination with its morphological characteristics;

[0083] (4) 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 plantarum and named it Lactobacillus plantarum C067. The strain was stored at -80°C for future use.

[0084] Example 2 Effect of Lactobacillus plantarum C067 on the memory ability of mice

[0085] 40 6-week-old male C57BL / 6J mice were randomly divided into four groups after one week of adaptation: normal control group, Lactobacillus plantarum C067 intervention group, antibiotic sweep control group, and Lactobacillus plantarum C067 intervention group after sweep, with 10 mice in each group. The specific animal groups are as follows:

[0086] Group 1 (control group): free diet and water, oral administration of control solvent;

[0087] Group 2 (Lactobacillus plantarum C067 intervention group): free diet and water, oral administration of Lactobacillus plantarum C067;

[0088] Group 3 (antibiotic control group): After 2 weeks of antibiotic treatment, the mice were allowed to eat and drink freely and were gavaged with a control solvent;

[0089] Group 4 (antibiotics + Lactobacillus plantarum C067 intervention group): after 2 weeks of antibiotic treatment, Lactobacillus plantarum C067 was gavaged;

[0090] Antibiotic treatment: The mice in each group were fed a normal diet. The mice in the antibiotic treatment group (groups 3 and 4) were gavaged with 0.1 mL / 10 g animal body weight 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), and the mice in the other groups were gavaged with the corresponding volume of solvent twice a day (once in the morning and once in the evening) for 2 weeks.

[0091] Lactic acid bacteria intervention: Activated Lactobacillus plantarum C067 was cultured in MRS liquid medium at 37°C for 48 hours. The fresh bacterial culture was centrifuged to collect the cells, the supernatant was discarded and the cells were resuspended in PBS to a bacterial concentration of 10 9 CFU / mL. Gavage once a day in the morning for 4 weeks, with a gavage volume of 0.1 mL / 10 g animal body weight.

[0092] After the lactic acid bacteria intervention, the mice were subjected to behavioral tests, including Morris water maze and conditioned fear test. The specific results are as follows:

[0093] (1) The results of the conditioned fear experiment are as follows Figure 1As shown, compared with the control group, feeding Lactobacillus plantarum C067 can significantly increase the fear memory of mice, and the freezing time increased from 41.42 seconds to 58.49 seconds, and the treatment group was 1.41 times that of the control group. The freezing time of mice in the antibiotic sweep group decreased, and feeding Lactobacillus plantarum C067 increased it by 1.39 times, from 26.45 seconds to 36.64 seconds. The results show that Lactobacillus plantarum C067 can significantly improve the fear memory ability of normal animals, and at the same time improve the memory of mice with antibiotic-induced flora disorders.

[0094] (2) The results of the Morris water maze test are as follows Figure 1 As shown, the average distance to the platform of the normal group and the antibiotic sweep group after intervention with Lactobacillus plantarum C067 was 35.97 cm and 37.71 cm, respectively, which was 14% and 12% less than that of the control group, respectively. The average number of times of crossing the platform was 6.40 times and 5.33 times, respectively, which was 1.42 times and 1.57 times of that of the control group, respectively. The results show that Lactobacillus plantarum C067 can significantly improve the spatial memory ability of animals.

[0095] Example 3: Effect of Lactobacillus plantarum C067 on the redox balance of mice

[0096] The construction and grouping of the animal model were as follows: refer to Example 2. The mice in each group were euthanized and the mouse serum was collected. The levels of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and nitric oxide (NO) in the serum were detected using a kit. The results showed that ( Figure 2 ), feeding Lactobacillus plantarum C067 can significantly change the levels of SOD and GSH in the serum of mice. After intervention with Lactobacillus plantarum C067, the SOD levels in the serum of the normal group and the antibiotic sweep group were 162.20U / mL and 161.81U / mL, respectively, which were 1.31 times and 1.26 times higher than those of the control group. The GSH levels were 24.96μmol / L and 17.95μmol / L, which were 1.46 times and 2.07 times higher than those of the control group, respectively. This shows that Lactobacillus plantarum C067 can promote the production of antioxidants in animals, regulate the body's redox balance, and thus enhance cognitive function.

[0097] Example 4: Effects of Lactobacillus plantarum C067 on the expression of neural receptors in the brain

[0098] The construction and grouping of the animal model were as follows: Referring to Example 2, the brain tissue of each group of mice was dissected, and the hippocampal tissue was isolated on ice. The total RNA of the tissue was extracted using the RNAiso Plus kit (Takara), and the PrimeScript TM cDNA was reverse transcribed using RTMaster Mix (Takara) and iTaqTM Universal Green Supermix kit (Bio-Rad Laboratories) was used for fluorescence quantitative PCR to detect the neuroreceptors in the posterior hippocampus. The housekeeping gene GAPDH was used as the internal reference gene, and the primer sequences used are shown in Table 1.

[0099] Table 1 Primer sequences for fluorescence quantitative PCR

[0100]

[0101] The results are as follows Figure 3 As shown in the results, Lactobacillus plantarum C067 intervention changed the expression of brain-derived nutritional factor (BDNF), neuropeptide Y receptor (NPY5R), and metabotropic glutamate receptors (mGluR1, mGluR3) in the mouse hippocampus. The expression of NPY5R and mGluR3 in the normal group of mice increased by 1.67 and 1.56 times, respectively, while the expression of BDNF, mGluR1, and mGluR3 in the antibiotic treatment group increased by 2.06 times, 1.46 times, and 1.99 times, respectively, indicating that Lactobacillus plantarum C067 affects signal transduction by affecting the expression of neural receptors, thereby affecting memory ability.

[0102] Example 5: Immunomodulatory effect of Lactobacillus plantarum C067 on mouse brain

[0103] The construction and grouping of the animal model were as follows: with reference to Example 2, the mice were euthanized, the mouse brain tissue was taken, and the hippocampal tissue was separated on ice, sterile PBS buffer was added, and the tissue homogenate was prepared with a tissue crusher, and the contents of IL-1β, IL-6, IL-10 and TNF-α were detected with an ELISA kit. The results showed that ( Figure 4 ), Lactobacillus plantarum C067 can significantly reduce the TNF-α and IL-1β content of mouse hippocampal tissue, the TNF-α of mice in the normal group and the antibiotic group was reduced to 494.89ng / ug and 317.84ng / ug, respectively, which was reduced by 2.75 times and 3.1 times, respectively, and the IL-1β of the antibiotic group was reduced from 11.43ng / ug to 2.36ng / ug, which was reduced by 4.84 times. It can be seen that Lactobacillus plantarum C067 can reduce the content of inflammatory factors in the mouse brain and has a significant anti-inflammatory effect.

[0104] Example 6: Effects of Lactobacillus plantarum C067 on intestinal flora and metabolites in mice

[0105] The construction and grouping of the animal model were carried out with reference to Example 2. Fresh feces of mice were taken to analyze the intestinal flora and metabolites of mice:

[0106] (1) The results of mouse intestinal flora analysis showed that ( Figure 5), principal coordinates analysis (PCoA) was used to evaluate the beta diversity of the microbiota. The results showed that Lactobacillus plantarum C067 intervention could change the structure of the intestinal flora and normalize the intestinal flora structure of antibiotic-treated mice. At the phylum level, feeding Lactobacillus plantarum C067 increased the Verrucomicrobia phylum in the healthy group and the antibiotic-treated group; the Actinobacteria phylum in the antibiotic-treated mice increased, and Lactobacillus plantarum C067 intervention normalized it, restoring the intestinal flora structure to normal. At the genus level, Lactobacillus plantarum C067 intervention increased the abundance of Akkermansia genus. Akkermansia muciniphila and other bacteria in the Akkermansia genus have the effects of strengthening the intestinal barrier, improving the metabolic health of the host body, regulating the immune system and anti-inflammatory, which also promotes the beneficial effects of Lactobacillus plantarum C067, thereby enhancing the host's cognitive ability.

[0107] (2) The results of short-chain fatty acid analysis in mouse feces showed that ( Figure 6 ), antibiotic treatment reduced the content of short-chain fatty acids in mouse feces, and Lactobacillus plantarum C067 intervention increased the acetic acid content in the healthy group and the antibiotic group to 14.23mg / g and 8.86mg / g, respectively, which increased by 1.37 times and 1.35 times compared with the control group, and the butyric acid content in the feces of mice in the antibiotic group increased from 0.522mg / g to 0.87mg / g, which increased by 1.67 times compared with the control group. The results further proved that Lactobacillus plantarum C067 can also regulate intestinal acetic acid and butyric acid content, regulate intestinal health, enhance the immune system, reduce inflammation, inhibit the growth of harmful bacteria, and promote the growth of beneficial bacteria, thereby maintaining the balance of intestinal flora.

[0108] Example 7: Application of Lactobacillus plantarum C067

[0109] 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 plant lactobacillus C067 bacterial agent starter prepared by the present invention. The mixture was sealed 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).

[0110] The present invention can be used to produce other fermented foods by fermentation using Lactobacillus plantarum C067, 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.

[0111] 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 plantarum C067, characterized in that It was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 15, 2024, with the deposit number GDMCC No: 65484.

2. A microbial preparation containing Lactobacillus plantarum C067 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the concentration of Lactobacillus plantarum C0672 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 plantarum C067 according to claim 1 or the microbial preparation according to claim 2 or 3.

5. A product, comprising the Lactobacillus plantarum C067 of claim 1 or the microbial preparation of claim 2 or 3, or the product obtained by fermentation using the starter of claim 4; optionally, the product is a feed additive, food, medicine or health product.

6. Use of the Lactobacillus plantarum C067 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) Enhance individual fear memory ability; (2) Enhance individual spatial memory ability; (3) Increase the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) in the individual's serum; (4) upregulating the expression of brain-derived nutritional factor, glutamate receptors and / or neuropeptide Y receptors in the hippocampus of the individual brain; (5) Regulate individual brain immunity and reduce the content of inflammatory factors in individual hippocampal tissue; (6) Regulate individual intestinal flora and intestinal metabolites.

8. Use of the Lactobacillus plantarum C067 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a medicine for regulating individual brain immunity.

9. The use according to claim 8, characterized in that: The regulating the individual's brain immunity includes reducing the content of TNF-α and IL-1β in the individual's hippocampal tissue.

10. Use of the Lactobacillus plantarum C067 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a promoter for hippocampal brain-derived nutritional factor, glutamate receptor and / or neuropeptide Y receptor.

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