Lactobacillus helveticus YM1922 for promoting brain development, improving concentration and nourishing nerves as well as metabolite, complex flora, metabolite and application of lactobacillus helveticus YM1922

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus helveticus YM1922 for promoting brain development, improving concentration and nourishing nerves as well as a metabolite, a complex flora, a metabolite and application. The lactobacillus helveticus YM1922 is preserved in the China General Microbiological Culture Collection Center on September 10, 2024, and the preservation number of the lactobacillus helveticus YM1922 is CGMCC No. 31928. Fermentation metabolites of the strain can promote secretion of brain-derived neurotrophic factors, and thalli can inhibit production of intestinal tract D-gluconic acid in intestinal tracts. Therefore, the strain and the metabolite thereof have application potentials of promoting brain development, improving cognitive level, improving concentration and nourishing nerves, and can be prepared into products for playing corresponding roles.

Description

Technical Field

[0001] The present invention belongs to the field of microbiological technology, and particularly relates to a Lactobacillus helveticus YM1922 strain that promotes brain development, improves concentration, nourishes nerves, as well as its metabolites, complex flora, postbiotics and applications. Background Art

[0002] In recent years, with the in-depth development of the brain-gut axis theory, research on the influence of gut microbiota on central nervous system function through metabolic products, immune regulation, and neural signal transmission has attracted much attention. As the core component of gut microecological regulation, the functions of probiotics have gradually expanded from the traditional field of gut health to the field of neuroscience. For example, Lactobacillus helveticus ( Lactobacillus helveticus ), due to its unique proteolytic ability and metabolite diversity, shows potential in regulating immunity, inhibiting pathogenic bacteria, and producing bioactive peptides. However, strains with targeted nerve functions are scarce. Although some studies have reported the potential effects of probiotics on nerve functions, most strains have single functions and lack targeted research on improving concentration and promoting nerve development. For example, although some Lactobacillus helveticus strains can improve stress-induced depression, they do not show regulatory effects on neurotrophic factors (such as BDNF) in healthy brain cells; although the exopolysaccharides (EPS) of some Lactobacillus helveticus strains can promote the proliferation of probiotics and generate short-chain fatty acids (SCFAs), they do not show direct effects on neurons. Summary of the Invention

[0003] To address the above technical problems, the present invention provides a Lactobacillus helveticus YM1922 strain that promotes brain development, improves concentration, nourishes nerves, as well as its metabolites, complex flora, postbiotics and applications. The fermentation metabolites of the Lactobacillus helveticus YM1922 strain can promote the secretion of brain-derived neurotrophic factor (BDNF), and the bacterial cells can inhibit the production of intestinal D-gluconic acid in the intestine. Therefore, it has the application potential of promoting brain development, improving cognitive level, enhancing concentration, and nourishing nerves.

[0004] To achieve the above invention objective, the present invention adopts the following technical solutions: In the first aspect of the present invention, a Lactobacillus helveticus YM1922 strain is provided, and its classification name is Lactobacillus helveticus ( Lactobacillus helveticus ), which was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 10, 2024, with the deposit number CGMCC No. 31928; the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The Lactobacillus helveticus strain YM1922 provided by the present invention was isolated from the traditional dairy product "nai gedda", and its fermentation metabolites can promote the secretion of BDNF, and the bacteria can inhibit the production of intestinal D-gluconic acid in the intestine.

[0006] BDNF is a key neurotrophic protein widely distributed in the central nervous system (especially in regions such as the hippocampus and prefrontal cortex). Its core functions include: promoting neuron survival and differentiation, enhancing synaptic plasticity, and regulating the neurotransmitter system. For healthy adults, BDNF levels are closely related to cognitive function. Promoting its secretion helps improve memory, attention, and learning ability, and delays cognitive decline. For adults suffering from neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), an increase in BDNF may help slow down the progression of the disease and protect neurons from damage. For patients with brain injuries (such as stroke and traumatic brain injury), an increase in BDNF helps promote nerve repair and functional recovery. For children and infants in the critical developmental window period, BDNF can shape an efficient neural network by regulating synaptic density and efficiency, and promote the cognitive development of children and infants, as well as improve their learning ability, memory, decision-making ability, and attention concentration, by enhancing synaptic connections between neurons in the prefrontal cortex (PFC).

[0007] Reactive oxygen species in breast milk are crucial for the formation of the neonatal intestinal flora. A decrease in hydrogen peroxide levels disrupts the intestinal flora and leads to the production of abnormal metabolite D-gluconic acid, which inhibits the formation of hippocampal myelin in infancy and may cause abnormal behaviors in adulthood. In addition, D-gluconic acid may also change the acid-base balance in the intestine, interfere with the normal function of the intestinal mucosal barrier, and then trigger intestinal inflammation or even a systemic inflammatory response. Moreover, the intestinal flora dysbiosis and inflammatory response caused by D-gluconic acid will also affect the brain development of infants and young children through the gut-brain axis, resulting in impaired cognitive function.

[0008] The Lactobacillus helveticus strain YM1922 provided by the present invention can achieve the effects of promoting brain development, improving cognitive level, enhancing concentration, and nourishing nerves by promoting BDNF and inhibiting the production of intestinal D-gluconic acid.

[0009] The second aspect of the present invention provides the use of the metabolites of the above-mentioned Lactobacillus helveticus strain YM1922 in the preparation of a product for promoting the secretion of brain-derived neurotrophic factor.

[0010] Preferably, the product is a product for improving cognition, memory, decision-making ability, and / or attention concentration.

[0011] Preferably, the product is a product for healthy people.

[0012] The third aspect of the present invention provides the use of the above-mentioned Lactobacillus helveticus YM1922 strain in the preparation of a product for inhibiting the production of D-gluconic acid in the intestine.

[0013] Preferably, the product is a product for promoting hippocampal myelination in infancy.

[0014] Preferably, the product is a product for improving cognition.

[0015] Preferably, the product is a product for healthy people.

[0016] The fourth aspect of the present invention provides the metabolite of the above-mentioned Lactobacillus helveticus YM1922 strain.

[0017] Preferably, the preparation method of the metabolite is: inoculating the Lactobacillus helveticus YM1922 bacterial liquid into MRS liquid medium, activating it continuously for 3 generations, centrifuging, and taking the supernatant to obtain the metabolite.

[0018] The fifth aspect of the present invention provides a complex flora containing the above-mentioned Lactobacillus helveticus YM1922 strain.

[0019] The sixth aspect of the present invention provides a postbiotic prepared from the above-mentioned Lactobacillus helveticus YM1922 strain. According to the well-known concept of "postbiotic" in the art, the postbiotic provided by the present invention refers to the metabolites and bacterial cells of the Lactobacillus helveticus YM1922 strain after processing, including inactivated bacterial cells, bacterial cell fragments, cell lysates, and metabolites secreted by the strain in the cell-free supernatant.

[0020] The beneficial effects of the present invention are as follows: The metabolite of the Lactobacillus helveticus YM1922 strain provided by the present invention can promote the secretion of BDNF, which has the effects of improving memory, attention and learning ability, and delaying cognitive decline in healthy adults. For adults suffering from neurodegenerative diseases, it helps to slow down the progression of the disease. For patients with brain injuries, it helps to promote nerve repair and functional recovery. For children and infants, it can promote their cognitive development, improve their learning ability, memory, decision-making ability and attention concentration. The Lactobacillus helveticus YM1922 strain also has a significant effect of inhibiting the production of intestinal D-gluconic acid in the intestine, thus helping to maintain the balance of the intestinal flora and the intestinal mucosal barrier, and having an indirect preventive effect on cognitive function impairment caused by intestinal flora imbalance and inflammatory response. More importantly, for the growth stage in infancy, the inhibition of intestinal D-gluconic acid is also helpful for the formation of hippocampal myelin, thus avoiding abnormal behaviors in adulthood caused by delayed hippocampal myelination. Therefore, the strain and its metabolite have the application potential of promoting brain development, improving cognitive level, enhancing concentration and nourishing nerves, and can be made into products for exerting corresponding effects. Description of the Drawings

[0021] Figure 1 It is the cell morphology of Lactobacillus helveticus YM1922 in Example 1; Figure 2 It is the colony morphology of Lactobacillus helveticus YM1922 in Example 1. Detailed implementation manners

[0022] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0023] The solutions of the present invention will be described below through specific embodiments.

[0024] The components and preparation methods of the culture media used in the following examples are as follows: MRS liquid culture medium: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate (MgSO4·7H2O) 0.1 g / L, manganese sulfate (MnSO4·4H2O) 0.05 g / L, dipotassium hydrogen phosphate 2.0 g / L, Tween-80 1.0 g / L, pH 6.5, and the solvent is water; DMEM / F12 Medium: L-alanine 4.45 mg / L, L-arginine dihydrochloride 31.29 mg / L, L-aspartic acid 6.65 mg / L, L-asparagine monohydrate 7.5 mg / L, L-cysteine hydrochloride monohydrate 17.56 mg / L, glycine 18.75 mg / L, L-glutamic acid 7.35 mg / L, L-glutamine 365 mg / L, L-histidine hydrochloride monohydrate 31.48 mg / L, L-hydroxyproline 54.47 mg / L, L-leucine 59.05 mg / L, L-lysine hydrochloride 91.25 mg / L, L-methionine 17.24 mg / L, L-phenylalanine 35.48 mg / L, L-proline 17.25 mg / L, L-serine 26.25 mg / L, L-threonine 53.45 mg / L, L-tryptophan 9.02 mg / L, L-tyrosine disodium salt dihydrate 55.79 mg / L, L-valine 52.85 mg / L, biotin 0.0035 mg / L, choline chloride 8.98 mg / L, D-calcium pantothenate 2.24 mg / L, folic acid 2.24 mg / L, nicotinamide 2.65 mg / L, pyridoxine hydrochloride 2.02 mg / L, riboflavin 0.219 mg / L, thiamine hydrochloride 2.17 mg / L, anhydrous CaCl2 116.6 mg / L, CuSO4·5H2O 0.0013 mg / L, Fe(NO3)3·9H2O 0.05 mg / L, FeSO4·7H2O 0.417 mg / L, anhydrous MgCl2 28.64 mg / L, anhydrous MgSO4 48.84 mg / L, KCl 311.8 mg / L, NaHCO3 1200 mg / L, NaCl 6995.5 mg / L, anhydrous NaH2PO4 54.3 mg / L, ZnSO4·7H2O 0.432 mg / L, D-glucose 3151 mg / L, sodium pyruvate 0.5 mM, HEPES buffer 15 mM, phenol red indicator 8.1 mg / L.

[0025] Colonic Model Medium (CMGM): Starch 5.0 g / L; Pectin 2.0 g / L; Guar gum 1.0 g / L; Mucin (porcine stomach type III) 4.0 g / L; Xylan 2.0 g / L; Arabinogalactan 2.0 g / L; Inulin 1.0 g / L; Casein 3.0 g / L; Peptone water 5.0 g / L; Tryptone 5.0 g / L; Bile salts 0.4 g / L; Yeast extract 4.5 g / L; Ferrous sulfate (FeSO4·7H2O) 0.005 g / L; Sodium chloride 4.5 g / L; Potassium chloride 4.5 g / L; Potassium dihydrogen phosphate (KH2PO4) 0.5 g / L; Magnesium sulfate (MgSO4·7H2O) 1.25 g / L; Calcium chloride (CaCl2·6H2O) 0.15 g / L; Sodium bicarbonate (NaHCO3) 1.5 g / L; Cysteine 0.8 g / L; Heme 0.05 g / L; Tween 80, 1.0 g / L.

[0026] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents, etc. used in the following examples can all be obtained through commercial channels.

[0027] Example 1 The embodiment of the present invention provides Lactobacillus helveticus strain YM1922, its isolation process and identification results.

[0028] 1. Isolation and identification of Lactobacillus helveticus strain YM1922 1.1 Strain isolation Take 1 g of traditional dairy product koumiss produced in Taxkorgan Tajik Autonomous County (altitude 4000 m, latitude 35°37′ - 38°40′, longitude 71°20′ - 77°01′), add it to 10 mL of physiological saline, and sequentially dilute it 6 times with a 10-fold gradient. Take 1 mL of each of the obtained different gradient dilutions and spread them on MRS plates, and incubate them anaerobically and inverted at 37°C for 48 h. Pick different morphological single colonies and streak culture them three times repeatedly until purified single colonies are obtained. Pick the purified single colonies into MRS liquid medium, and after culturing for 24 h, mix the culture solution with 30% glycerol and store it at -80°C.

[0029] 1.2 Colony morphology observation Cell morphology: Rod-shaped, without flagella, without spores (as Figure 1 shown); Colony morphology: Round, milky white, with neat edges (as Figure 2 shown); Gram staining: Positive.

[0030] 1.3 Strain 16S rDNA identification After extracting the genomic DNA of the strain using the Ezup column bacterial genomic DNA extraction kit, PCR amplification was performed with 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) as primers. After purifying the amplification product, 16S rDNA sequencing was carried out.

[0031] The 16S rDNA sequence is (as shown in SEQ ID No.1):

[0032] By performing BLAST sequence alignment in the NCBI database, this strain was identified as Lactobacillus helveticus ( Lactobacillus helveticus ).

[0033] This strain was named Lactobacillus helveticus YM1922 and was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101), with the deposit number CGMCC No. 31928.

[0034] Example 2 This example provides a fermentation broth of Lactobacillus helveticus YM1922 strain, and its preparation method is as follows: Inoculate the Lactobacillus helveticus YM1922 bacterial solution into MRS liquid medium, activate it continuously for 3 generations, centrifuge at 12000 rpm for 3 min, and take the supernatant to obtain the fermentation broth of Lactobacillus helveticus YM1922 strain.

[0035] Example 3 This example provides a YM1922 probiotic powder, and its preparation method is as follows: Add Lactobacillus helveticus YM1922 into the medium for fermentation culture at an inoculation amount of 3%, and the culture conditions are: 37 °C, 12 hours; after fermentation is completed, centrifuge at 6000 r / min for 30 min to obtain bacterial sludge, mix the bacterial sludge evenly with the freeze-drying protectant and then freeze-dry, and then pulverize to obtain YM1922 probiotic powder (the viable count is 1.0×10 10 CFU / g).

[0036] Preparation method of the medium: Mix 40 g of glucose, 4 g of soy peptone, 3 g of yeast extract powder, 2 g of diammonium citrate, 6 g of anhydrous sodium acetate, 3 g of dipotassium hydrogen phosphate, 1 g of isomaltooligosaccharide, 3 g of starch and other food-grade materials, add 1000 g of pure water and mix until there are no visible particles to the naked eye. After melting the materials, adjust the pH to 5.0 - 6.8, and sterilize at 117.5 - 118.5 °C for 20 min to obtain the medium.

[0037] Preparation method of the freeze-drying protectant: Mix 100 g of starch, 20 g of glucose, 60 g of maltodextrin, 30 g of sucrose, 10 g of lactose, 50 g of skim milk powder and other food raw materials evenly, add 1000 g of pure water and dissolve until there are no visible particles to the naked eye. After melting the materials, adjust the pH to 5.0 - 6.8, sterilize at 117.5 - 118.5 °C for 20 min, and after sterilization, adjust the pH to 6.0 - 7.3 to obtain the freeze-drying protectant.

[0038] Example 4 This example identified the ability of Lactobacillus helveticus YM1922 to promote the secretion of neurotrophic factor (BDNF).

[0039] 1. Culture method and treatment of human neuroblastoma SH-SY5Y cells Discard the old medium of commercially available SH-SY5Y cells, add 1 - 2 ml of 0.25% trypsin to the culture flask, place it in a 37°C incubator for digestion for 2 - 3 minutes, then observe the cell digestion under a microscope. After most cells become round and detached, quickly take it back to the operating table, gently tap the culture flask, and then add 3 - 6 mL of special medium for SH-SY5Y cells to terminate digestion. Gently pipette the cells, and after complete detachment, aspirate them into a centrifuge tube and centrifuge (1000 rpm, 5 min). Discard the supernatant, and resuspend with 1 - 3 mL of medium. Transfer the resuspended cell suspension to two new T25 culture flasks, and supplement with fresh complete medium to 8 - 10 mL per flask.

[0040] 2. Treatment of the culture supernatant of Lactobacillus helveticus YM1922 Take the bacterial solution of Lactobacillus helveticus YM1922 (1.0×10 9 CFU / mL) and inoculate it into 10 mL of MRS liquid medium at an inoculation amount of 3%. Incubate statically at 37°C for 24 h, activate for 3 consecutive generations, centrifuge at 12000 rpm for 3 min, take the supernatant, and filter it with a 0.22 μm filter membrane to obtain the sterile filtrate of YM1922 for standby.

[0041] 3. Differentiation and treatment of SH-SY5Y cells SH-SY5Y cells are cultured in DMEM / F12 medium containing 10% v / v fetal bovine serum, 1×10 5 U / L penicillin and 100 mg / L streptomycin at 37°C and 5% CO2. Inoculate the cells into a 6-well plate or 24-well plate coated with polylysine solution. After 24 h, change to DMEM / F12 medium containing 0.5% B27, 1×10 5 U / L penicillin, 100 mg / L streptomycin, and 10 μmol / L all-trans retinoic acid to induce differentiation, and change half of the medium every other day. After 7 days of cell differentiation culture, divide the cells into three groups (control group, YM1922 group, MRS group), and add 1% v / v of PBS, the sterile filtrate of YM1922, and MRS liquid medium respectively. Continue to culture for 24 h and then conduct detection.

[0042] 4. Detection of BDNF expression level After collecting the cells, extract the total RNA by the Trizol method. After reverse transcription into cDNA, detect the content of BDNF mRNA by real-time quantitative PCR.

[0043] BDNF primer sequences, F: 5’-AGCTGAGCGTGTGTGACAGTATTAG-3’, R: 5’-TCTCGCTCCTGGAAGATGGTGATG-3’.

[0044] Using GAPDH as an internal reference, the 2 -∆∆Ct method was used for relative quantitative analysis of each component of the target gene, and the results are shown in Table 1.

[0045] Table 1 Relative expression levels of BDNF mRNA

[0046] Note: a indicates comparison with the control group P <0.01, b indicates comparison between the YM1922 group and the MRS group P <0.05.

[0047] RT-PCR results showed that the expression level of BDNF mRNA in SH-SY5Y cells differentiated after stimulation with YM1922 metabolites increased significantly.

[0048] Example 5 This example identified the ability of Lactobacillus helveticus YM1922 to inhibit the production of intestinal D-gluconic acid.

[0049] 1. Cultivation of the intestinal bacterial population in an in vitro fermentation system and detection of D-gluconic acid content Using a three-stage continuous culture intestinal model system, the human colon three-stage continuous culture model consists of three fermenters, simulating the proximal (V1, 280 mL), transverse (V2, 300 mL), and distal colon (V3, 320 mL). The three series-connected fermenters are maintained at 37 °C, and the pH values are maintained at 5.5 (V1), 6.2 (V2), and 6.8 (V3), and anaerobic conditions are introduced by continuous N2 introduction. V1 is connected to the intestinal model medium (CMGM medium) supplemented with D-gluconic acid (100 mg / L) by a peristaltic pump. Fresh infant fecal samples are collected and stored in an anaerobic cabinet (10% H2, 10% CO2, 80% N2), and a fecal dilution is prepared in anaerobic PBS (0.1 mol / L PBS, pH 7.4) at a solid-liquid ratio of 1:5 (w / w) within 15 minutes. Each stage of the colon model is inoculated with 10 mL of fecal slurry. According to the average retention time of healthy individuals, the total system throughput time is set to 48 h. After inoculation, the colon model runs as a batch culture for 24 h to stabilize the bacterial population before the start of medium infusion. After 24 h, the system runs 8 full-volume turnovers to achieve a steady state. The blank control group is not intervened, and in the experimental group (YM1922 group), the YM1922 probiotic powder prepared in Example 3 is added to V1 at 1% (1 g / 100 mL) every day, and then eight volume turnovers are carried out to reach steady state 3, and then sampling is carried out continuously for three days, and the D-gluconic acid content in the system after inoculation is detected using an improved cupric ion colorimetric method. The results are shown in Table 2. The intervention with viable YM1922 bacteria can significantly reduce the production of D-gluconic acid in the intestine.

[0050] Table 2 D-gluconic acid content in the system after inoculation

[0051] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Lactobacillus helveticus strain YM1922, characterized in that, Its classification name is Lactobacillus helveticus ( Lactobacillus helveticus ), which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 10, 2024, with the deposit number of CGMCC No. 31928; the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the metabolite of Lactobacillus helveticus strain YM1922 as claimed in claim 1 in the preparation of a product for promoting the secretion of brain-derived neurotrophic factor.

3. The application according to claim 2, wherein The product is a product for improving cognition, enhancing memory, decision-making ability and / or attention concentration; and / or The product is a product for healthy people.

4. Use of the Lactobacillus helveticus strain YM1922 as claimed in claim 1 in the preparation of a product for inhibiting the production of D-gluconic acid in the intestine.

5. The application according to claim 4, characterized in that, The product is a product for promoting hippocampal myelination in infancy; and / or The product is a product for improving cognition; and / or The product is a product for healthy people.

6. The metabolite of the Lactobacillus helveticus strain YM1922 as claimed in claim 1.

7. The metabolite according to claim 6, wherein The preparation method of the metabolite is: inoculating the Lactobacillus helveticus YM1922 bacterial liquid into MRS liquid medium, activating continuously for 3 generations, centrifuging, and taking the supernatant to obtain the metabolite.

8. A composite microbial community, characterized in that, Containing the Lactobacillus helveticus strain YM1922 as claimed in claim 1.

9. A postbiotic, characterized in that, Prepared from the Lactobacillus helveticus strain YM1922 as claimed in claim 1.

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