A strain of Lactobacillus helveticus capable of improving memory and cognitive function and its application

Abstract

The invention discloses a strain of Lactobacillus helveticus with improved memory and cognitive function and its application, belonging to the field of dairy processing and fermentation technology. The invention screened a strain of Lactobacillus helveticus GDMCCNo.60811 from Physalis oleraceus in Dali, Yunnan Province. This strain is a food-grade safe strain, and can significantly improve memory and cognitive ability, significantly reduce the level of scopolamine in serum, from 27.0mg / L to 8.3mg / L, down-regulated by 3.3 times. In addition, the Lactobacillus helveticus GDMCCNo.60811 has good tolerance to the intestinal tract. Therefore, the Lactobacillus helveticus GDMCCNo.60811 can be used to prepare various foods, microbial preparations, and can also be used to prepare medicines for diseases such as depression, anxiety symptoms, PTSD symptoms and Alzheimer's disease related to choline metabolism.

Description

Technical Field

[0001] The invention relates to a strain of Lactobacillus helveticus capable of improving memory and cognitive functions and application thereof, belonging to the technical field of food processing and fermentation. Background Art

[0002] In today's society, young people have memory loss due to heavy study burdens and high pressure, adults have intermittent amnesia due to social pressure and fierce competition, and the elderly have fragmented amnesia due to aging or neurodegeneration. These memory declines or cognitive degeneration have become non-pathological diseases. If exogenous stimuli such as oxidative stress or inflammatory damage accumulate over time, aggravating memory damage, it may lead to lesions in the hippocampus of the brain and imbalance of the neurotransmitter system, and eventually cause memory-related diseases such as Alzheimer's disease, Parkinson's disease and vascular dementia. Therefore, it is of great significance to explore active substances that can prevent and improve memory decline and cognitive degeneration.

[0003] Lactobacillus helveticus is one of the important fermentation strains in the development of the dairy industry. Most of the research on Lactobacillus helveticus focuses on its fermentation characteristics, such as its effects on the proteolysis and flavor formation of fermented milk and cheese. As a commonly used food fermentation strain, it will be of great significance if Lactobacillus helveticus can be further explored in the regulation of host metabolism after ingestion. Summary of the invention

[0004] The inventors screened a strain of Lactobacillus helveticus from Physalis alkekengi in Dali, Yunnan. This strain can significantly improve memory and cognitive abilities, which provides theoretical guidance for the study of strain diversity and application of Lactobacillus helveticus.

[0005] The first object of the present invention is to provide a strain of Lactobacillus helveticus, which is screened from Physalis alkekengi collected in Dali, Yunnan, my country, and has been preserved in Guangdong Provincial Microbiological Culture Collection Center on October 16, 2019, with a preservation number of GDMCC No.60811, and a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] In one embodiment, the Lactobacillus helveticus GDMCC No.60811 strain has the following properties:

[0007] (1) Significantly improve memory and cognitive abilities;

[0008] (2) Significantly reduce serum scopolamine levels;

[0009] (3) It has good intestinal tolerance and in vivo proliferation ability.

[0010] The second object of the present invention is to provide a method for culturing the Lactobacillus helveticus GDMCC No.60811, wherein the Lactobacillus helveticus is inoculated into an MRS culture medium and cultured at 35-40°C.

[0011] The third object of the present invention is to provide a composition containing the Lactobacillus helveticus GDMCC No.60811.

[0012] In one embodiment, the composition contains one or more substances selected from the group consisting of a live strain of Lactobacillus helveticus GDMCC No. 60811, a dried strain of Lactobacillus helveticus GDMCC No. 60811, a metabolite of Lactobacillus helveticus GDMCC No. 60811, an inactivated Lactobacillus helveticus GDMCC No. 60811, a fermentation broth of Lactobacillus helveticus GDMCC No. 60811, a lysate of Lactobacillus helveticus GDMCC No. 60811, or an extract of Lactobacillus helveticus GDMCC No. 60811.

[0013] In one embodiment, the number of viable Lactobacillus helveticus in the composition is not less than 5×10 9 CFU / mL or 5×10 9 CFU / g.

[0014] A fourth object of the present invention is to provide a product containing the composition.

[0015] In one embodiment, the product includes but is not limited to a microbial agent, a food or a medicine.

[0016] In one embodiment, the product is used to prevent and / or improve cognitive impairment or improve memory and cognitive function.

[0017] In one embodiment, the medicine contains the Lactobacillus helveticus GDMCC No. 60811, the composition, a drug carrier and / or a pharmaceutical excipient.

[0018] In one embodiment, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0019] In one embodiment, the pharmaceutical excipient comprises an excipient and / or an additive.

[0020] In one embodiment, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0021] In one embodiment, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

[0022] In one embodiment, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0023] In one embodiment, the viable count of Lactobacillus helveticus GDMCC No.60811 in the product is not less than 5×10 9 CFU / mL or 5×10 9 CFU / g.

[0024] In one embodiment, the food is a health food; or the food is a dairy product, bean product or fruit and vegetable product produced using a starter containing the Lactobacillus helveticus GDMCC No.60811; or the food is a beverage or snack containing the Lactobacillus helveticus GDMCC No.60811.

[0025] In one embodiment, the fermented food is produced by fermentation using the Lactobacillus helveticus GDMCC No. 60811, including solid food, liquid food, and semi-solid food.

[0026] In one embodiment, the fermented food includes dairy products, bean products or fruit and vegetable products; the dairy products include milk, yogurt, sour cream or cheese; the bean products include soy milk and sour soy milk; the raw materials of the fruit and vegetable products include yellow peaches, hawthorns, cucumbers, carrots, beets, celery or cabbage products.

[0027] In one embodiment, the method for preparing the starter is to inoculate the above-mentioned Lactobacillus helveticus GDMCC No.60811 into the culture medium at an inoculation amount of 2-4% of the total mass of the culture medium, and culture it at 37°C for 18 hours to obtain a culture solution; centrifuge the culture solution to obtain bacterial cells; wash the bacterial cells with physiological saline for 3 times and then resuspend them with a freeze-drying protective agent to obtain a resuspension; freeze-dry the resuspension by a vacuum freezing method to obtain a starter.

[0028] The present invention also provides the use of the Lactobacillus helveticus GDMCC No. 60811 or the composition in preparing a product for reducing scopolamine.

[0029] Beneficial effects of the present invention:

[0030] 1. The Lactobacillus helveticus GDMCC No.60811 of the present invention can significantly improve memory and cognitive ability, and improve at least one of spatial memory ability, working memory ability, reference memory ability, and recognition ability;

[0031] 2. Lactobacillus helveticus GDMCC No.60811 can significantly reduce serum scopolamine levels from 27.0 mg / L to 8.3 mg / L, a 3.3-fold decrease, with good intestinal tolerance and in vivo proliferation ability;

[0032] 3. Lactobacillus helveticus GDMCC No.60811 is a safe strain that can be used in food. It is safe and healthy and can be used to prepare various foods, medicines, and microbial preparations.

[0033] Biomaterial Deposit

[0034] The Lactobacillus helveticus provided by the present invention is taxonomically named Lactobacillus helveticus, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 16, 2019, with a deposit number of GDMCC No: 60811, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a Gram-stained photo of Lactobacillus helveticus GDMCC No.60811 observed under a 1000x microscope;

[0036] FIG2 shows how Lactobacillus helveticus GDMCC No.60811 improves the spatial memory ability of mice;

[0037] Figure 3 Lactobacillus helveticus GDMCC No.60811 improves the working memory and reference memory abilities of mice;

[0038] Figure 4 The improvement of recognition ability of mice by Lactobacillus helveticus GDMCC No.60811;

[0039] Figure 5 The results show that Lactobacillus helveticus GDMCC No.60811 reduces serum scopolamine levels;

[0040] Figure 6 To evaluate the intestinal tolerance and in vivo proliferation of Lactobacillus helveticus GDMCC No.60811. DETAILED DESCRIPTION

[0041] definition

[0042] As used herein, the following terms have the following definitions.

[0043] As used herein, the term "strain" refers to a microorganism of a particular species having common characteristics. Unless otherwise indicated, the terms "strain" and "cell" are used interchangeably herein.

[0044] The term "plate" used in this article refers to plate culture medium, which is the most commonly used form of solid culture medium for obtaining pure culture of microorganisms. It is a solid surface of culture medium formed in a sterile culture dish after cooling and solidification, and is often referred to as a culture plate or plate.

[0045] The term "medium" as used herein refers to a culture medium comprising the chemical elements required for the growth of the microorganisms together with at least one carbon source and one nitrogen source.

[0046] (I) The culture medium involved in the following embodiments:

[0047] MRS liquid culture medium (1L): peptone 10.0g, beef extract 10.0g, yeast extract powder 5.0g, diamine hydrogen citrate 2.0g, glucose 20.0g, Tween-80 1.0mL, sodium acetate 2.0g, magnesium sulfate heptahydrate 0.5g, manganese sulfate monohydrate 0.25g, dipotassium hydrogen phosphate 2.0g. Add distilled water to completely dissolve until the pH is between 6.8 and 7.2. Sterilize at 115℃ for 20min.

[0048] MRS solid medium (1L): Prepare according to MRS liquid medium, then add 15g agar powder. Sterilize at 115℃ for 20min.

[0049] (II) Experimental animals involved in the following examples

[0050] Male ICR / 4 mice were purchased from Beijing Weitonglihua Company.

[0051] (III) Strains involved in the following examples

[0052] Control strain ATCC15009: a commercial fermentation culture purchased from the China Industrial Microbiological Culture Collection. It has been reported in the literature that ATCC15009 has a certain in vivo colonization ability and a certain in vivo regulatory effect. Therefore, in the study of host regulation of Lactobacillus helveticus, ATCC15009 was used as a control strain.

[0053] (IV) Experimental methods involved in the following examples

[0054] Serum metabolite detection method: Carefully draw 100 μL of serum into a 1.5 mL centrifuge tube; add 400 μL of methanol (pre-cooled at -20°C in advance) to precipitate protein, which will produce a large amount of white floccules; vortex for 30 seconds, place the sample in a -20°C refrigerator for 1 hour to increase the protein precipitation rate; centrifuge at 15000 rpm for 15 minutes at 4°C; take 400 μL of supernatant and evaporate to dryness. Add 100 μL of methanol: water (4:1, v / v) and vortex for 30 seconds to re-dissolve; centrifuge at 15000 rpm for 15 minutes at 4°C, transfer an appropriate volume of supernatant into a sample vial for testing; QC preparation: transfer an equal volume from the prepared sample to a new sample vial and mix evenly; Blank preparation: 1 mL of methanol: water (4:1, v / v) to the sample vial and mix evenly. Metabolome analysis was performed using an UltiMate 3000 HPLC system (Thermo Fisher Scientific, MA, USA) coupled to a high-resolution Q-Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA) in full scan mode. Chromatographic separation of metabolites was performed using a Waters Acquity UPLCHSS reverse phase T3 column (Waters, M, USA; 2.1×100 mm×1.8 μm) at 35°C.

[0055] The present invention is described in detail below in conjunction with the embodiments described in the accompanying drawings, wherein the same numerals in all the accompanying drawings represent the same features. Although specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0056] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the following description of suitable methods and material examples is illustrative only and does not limit the present invention in any way.

[0057] Second, "one embodiment" or "an embodiment" as referred to herein refers to a particular feature, structure, or characteristic that may be included in at least one implementation of the present invention.

[0058] Example 1: Lactobacillus helveticus GDMCC No.60811 strain isolation and identification method

[0059] (1) Prepare appropriate sample dilution gradient and culture

[0060] Take out the lactic acid Physalis from Dali, Yunnan, which is stored in a -80℃ refrigerator, and thaw it on ice. After shaking and mixing, take 0.5mL of the sample and add it to 4.5mL of sterile saline to complete a 10-fold dilution. After shaking and mixing, take out 0.5mL of the dilution and add it to 4.5mL of sterile saline to complete a second 10-fold dilution, and so on, until the dilution reaches 10. -6 , take 100 μL from each gradient dilution, spread it evenly on the MRS solid culture medium plate, invert it, culture it at 37℃ under anaerobic conditions for 48 hours, and observe it in time.

[0061] (2) Line separation and purification

[0062] After taking out the plates with colonies, select the gradient plates with obvious single colonies, pick colonies with different colony morphologies, and perform secondary streaking until all single colonies are purified.

[0063] (3) Gram staining and catalase test

[0064] Pick a single colony onto a glass slide, and examine under a microscope after smearing, drying, fixing, primary staining, washing, mordanting, washing, decolorization, restaining, washing, and drying. Record the Gram staining results (see Figure 1 ); and pick a single colony on a slide, add 3% hydrogen peroxide solution, observe whether bubbles are generated, and record the catalase contact results.

[0065] (4) Preservation of bacterial strains

[0066] After purification, a single colony of each strain was picked into 5 mL of liquid MRS medium, and cultured at 37°C under anaerobic conditions for 24 h. 1 mL of bacterial solution was transferred to a bacterial preservation tube, and centrifuged at 6000 rpm for 3 min. The supernatant was discarded, and 1 mL of 37% sterile glycerol solution was added, resuspended, and stored at -80°C.

[0067] (5) 16S rDNA sequence amplification

[0068] Pipette 1mL of bacterial solution and centrifuge at 6000rpm for 3min, discard the supernatant, wash twice with water, centrifuge and discard the supernatant to obtain bacterial sludge, which is used as a template for PCR amplification. The process is as follows:

[0069] 1) Amplification system 20 μL:

[0070] The amount of template was 1 μL, the amount of bidirectional primers was 1 μL each, the amount of Taq enzyme MasterMix was 10 μL, and the amount of ddH2O was 7 μL.

[0071] The primers used were 27F and 1492R.

[0072] 27F: AGA GTT TGA TCC TGG CCT CA (SEQ ID No. 1),

[0073] 1492R: GGT TAC CTT GTT ACG ACT T (SEQ ID No. 2).

[0074] 2) Amplification conditions:

[0075] Pre-denaturation: 95℃3min,

[0076] First step denaturation: 94℃1min,

[0077] Second step annealing: 60℃37s,

[0078] The third step extension: 72℃2min,

[0079] Number of cycles: 37 cycles,

[0080] Step 4: Final extension: 72℃5min,

[0081] Step 5: Maintain: 12℃ for 10min.

[0082] (6) Agarose gel electrophoresis

[0083] Weigh 80mL of agarose and add it to a conical flask, add 80mL of 1×TAE, heat intermittently in a microwave for 4 minutes until the liquid is clear and transparent, cool slightly, add 4μL of nucleic acid dye, shake well, and pour into a gel plate tank. After cooling for 40 minutes to solidify, place it in an electrophoresis tank, remove bubbles, add PCR amplification products in sequence, add 2μL of PCR amplification products to each well, run the gel at 120V for 15 minutes, take out after the end, place it in a gel electrophoresis imager to take a picture and save it, record the number of the successful PCR sample, and store the successful PCR product in a -20℃ refrigerator.

[0084] (7) Sequencing and identification

[0085]

[0086] A BLAST search was performed in the National Center for Biotechnology Information (NCBI) sequence database (http: / / www.ncbi.nlm.nih.gov / blast), and the strain information with the highest matching degree was selected for result recording. After analysis and identification, the strain provided by the present invention is Lactobacillus helveticus.

[0087] Example 2: Lactobacillus helveticus GDMCC No.60811 improves memory and cognitive ability in mice

[0088] (1) Animal experiment groups

[0089] Thirty-two 4-week-old male ICR mice were selected. After one week of adaptive feeding, they were randomly divided into a blank group, a model group, a Lactobacillus helveticus ATCC15009 intervention group, and a Lactobacillus helveticus GDMCC No.60811 intervention group according to body weight, with 8 mice in each group. Mice in all groups were free to eat and drink water. The blank group and the model group were gavaged with sterilized 0.9% saline, and the gavage volume was 0.2 mL; the number of viable bacteria in the Lactobacillus helveticus intervention group was 5×10 9 CFU / mL of Lactobacillus helveticus, with an intragastric volume of 0.2 mL.

[0090] (2) Inducing memory and cognitive impairment

[0091] On the 21st to 35th day of the experiment, 30 minutes after intragastric administration, the model group, Lactobacillus helveticus ATCC15009 intervention group and Lactobacillus helveticus GDMCC No.60811 intervention group were intraperitoneally injected with scopolamine to induce memory impairment in mice. The concentration of scopolamine was 0.125 mg / mL, and the injection volume was 8 mL / kg body weight according to the weight of the mice. The blank group was injected with 0.9% saline at a volume of 8 mL / kg body weight. The mice in each experimental group were intragastrically administered for 20 consecutive days.

[0092] (3) Behavioral evaluation

[0093] From 21 days on, behavioral evaluation of all mice was performed, including water maze test, novel object recognition test and Y maze test. All behavioral experiments started on time 30 minutes after intraperitoneal injection. Days 21 to 26 were the water maze training period, day 27 was the water maze test period, day 32 was the novel object recognition test, day 34 was the Y maze test, and normal gavage and intraperitoneal injection were performed during the interval.

[0094] (4) Behavioral experimental methods and results

[0095] Morris water maze is used to evaluate the spatial memory ability of mice. The experimental device is a pool with a diameter of 1.20m and a height of 0.6m. During the experiment, 0.40m deep water was injected into the pool, and the pool was divided into four quadrants. The experiment was divided into three stages, including platform visibility, positioning navigation, and spatial exploration. In the platform visibility stage, the platform position was adjusted to be 1cm above the water surface, and eye-catching signs were posted on the four quadrants of the pool wall for mice to refer to. In the platform visibility stage, the mouse was placed in the water facing the pool wall, and the time it took for the mouse to find the platform was recorded. The test time was 1min. The mouse was placed in the water from different quadrants and 4 trainings were completed. In the positioning navigation stage, the platform position was adjusted to be 1cm underwater, and the platform visibility stage training was repeated to guide the mice that did not find the platform to reach the platform and stay on it for 15s. The training was continued for 5 days. The 7th day of the experiment was the spatial exploration stage. The platform in the pool was removed, and the mouse was placed in the water facing the pool wall. The frequency of platform crossing and other indicators of the mouse were recorded. The test time was 1min, and the movement trajectory was tracked and recorded by the infrared camera above the pool.

[0096] As can be seen from Figure 2, the spatial memory ability of mice in the model group was significantly lower than that of the blank group, as shown by the scattered swimming trajectories during the test, no obvious stay in the platform quadrant, and low platform crossing frequency, which was only 2.00 times. Compared with the model group, the Lactobacillus helveticus GDMCC No. 60811 intervention group was able to significantly improve the spatial memory ability of mice, as shown by the concentrated swimming trajectories during the test, obvious stay in the platform quadrant, and extremely significant increase in crossing frequency, which reached 5.40 times, while the crossing frequency of mice in the control strain ATCC15009 intervention group was only 1.00 times. Therefore, Lactobacillus helveticus GDMCC No. 60811 can significantly improve the spatial memory ability of mice.

[0097] The Y-maze behavior test is used to evaluate working memory and reference memory. During the experiment, the mouse is put down from any arm, and consecutive entry into three different arms is defined as one alternation. The test time is 5 minutes, and the infrared camera above the device records the frequency of the mouse entering the three arms and other indicators.

[0098] Depend on Figure 3 It can be seen that compared with the model group mice, the Lactobacillus helveticus GDMCC No. 60811 intervention group can significantly increase the rotation frequency of mice in the Y maze, from 5.17 times in the model group to 14.00 times, and is significantly higher than the blank group (6.83) and the control strain ATCC15009 intervention group (8.63 times). Therefore, Lactobacillus helveticus GDMCC No. 60811 can significantly improve the working memory and reference memory ability of mice.

[0099] The novel object recognition behavior experiment is used to evaluate recognition ability, which is mainly divided into three stages, including the adaptation stage, the familiarization stage and the test stage. In the adaptation stage, the mice were placed in the experimental box to explore the environment freely for 6 minutes. After an interval of 24 hours, the familiarization stage began. Two identical building blocks were placed in the experimental box. The mice explored freely in the box for 6 minutes. After an interval of 24 hours, the test stage began. One of the building blocks in the experimental box was replaced with a cone-shaped building block. The infrared camera above the experimental box tracked and recorded the time the mice explored the new and old objects, the frequency of nose tip touches, the cumulative time and other parameters. The test time was 6 minutes.

[0100] Depend on Figure 4 It can be seen that there was no significant difference in the thermal imaging of the touch of two objects by mice in different groups during the familiarization stage, while in the test stage, the cumulative touch time of the model group to the new objects was significantly reduced compared with the blank group, indicating that scopolamine significantly reduced the recognition ability of mice; compared with the model group, the Lactobacillus helveticus GDMCC No. 60811 intervention group could significantly increase the cumulative touch time of mice to the new objects from 21.14 seconds in the model group to 36.44 seconds, which was significantly higher than the control strain ATCC15009 (27.04 seconds), so Lactobacillus helveticus GDMCC No. 60811 could significantly improve the recognition ability of mice.

[0101] Example 3: Lactobacillus helveticus GDMCC No.60811 reduces scopolamine levels in mouse serum

[0102] According to the animal experiment design of Example 2, on the 35th day, the mice that had completed the gavage were fasted for 12 hours, anesthetized, and the eyeballs were quickly removed to collect blood, and then the mice were killed by cervical dislocation. Blood samples were collected from the mice after the sacrifice, and the blood samples were left to stand at 4°C for 1 hour, centrifuged at 3000 rpm for 15 minutes, and the upper serum was carefully collected. The serum metabolites were detected, and the measured scopolamine content in the serum was as shown in Figure 5 Compared with the model group, Lactobacillus helveticus GDMCC No.60811 can significantly reduce the content of scopolamine in mouse serum, from 27.0 mg / L in the model group to 8.3 mg / L, a 3.3-fold decrease. At the same time, it is significantly greater than the 22.6 mg / L of the control strain ATCC15009. Therefore, Lactobacillus helveticus GDMCC No.60811 has the function of significantly reducing scopolamine in serum.

[0103] Example 4: Lactobacillus helveticus GDMCC Evaluation of intestinal tolerance and in vivo proliferation of No.60811

[0104] (1) Determination of tolerance to simulated intestinal fluid

[0105] To prepare simulated intestinal fluid, weigh a certain amount of trypsin (Sigma, p1500) and bile salt (oxoid, LP0055), dissolve them in 2 / 3 of the required volume of normal saline (0.5% w / v), adjust the pH to 8.0 with 0.1 mol / L NaOH, filter with a 0.22 μm sterile filter membrane and adjust the volume to the required volume to achieve final concentrations of 1 g / L and 0.3%, respectively. Prepare the simulated intestinal fluid and use it immediately.

[0106] Lactobacillus helveticus GDMCC No.60811 stored at -80°C was inoculated into MRS liquid medium and cultured at 37°C for 24 h. The culture was subcultured 2 to 3 times until the bacterial concentration reached 10 8 ~10 9 CFU / mL; take out 10μL of bacterial solution activated in MRS medium and add it to 0.99mL of simulated intestinal fluid, vortex mixer 3 times at medium speed. Incubate the bacterial solution added to simulated intestinal fluid at 37℃ for 0h, and perform live bacteria count experiment for 2h.

[0107] Depend on Figure 6 It can be seen that compared with the control strain, Lactobacillus helveticus GDMCC No. 60811 has a higher in vitro bile salt tolerance, and the survival rate reaches 92.22%, indicating that Lactobacillus helveticus GDMCC No. 60811 has good intestinal tolerance.

[0108] (2) Determination of intestinal proliferation ability of Lactobacillus helveticus

[0109] According to the animal experiment design of Example 2, feces were collected on days 0, 7, 14, and 35 to detect the amount of Lactobacillus helveticus in the feces. 3 CFU, 1×10 6 CFU, 1×10 9 CFU, 1×10 12 CFU bacteria, extract DNA, and use Nanodrop to measure the amount of nucleic acid under 260 / 280 conditions, and construct standard curves of single Lactobacillus helveticus nucleic acid amount and colony count. Weigh the freeze-dried feces sample, extract DNA from it, and use Nanodrop to measure the amount of nucleic acid under 260 / 280 conditions to calculate the total amount of nucleic acid per unit feces. In the clean data of metagenomic sequencing, use SOAPaligner software to screen reads from the target Lactobacillus helveticus genome, obtain the proportion of target strain gene fragments in the total sequencing reads, calculate the nucleic acid mass of the target strain per unit feces, and calculate the total number of colonies of the target strain per unit feces based on the standard curve of the target strain nucleic acid amount and colony count. Figure 6As shown in the figure, the proliferation ability of Lactobacillus helveticus in vivo increased significantly 7 days before gavage, and continued to increase from 7 to 14 days, and gradually stabilized after 14 days. On days 7, 14, and 35, the distribution content of Lactobacillus helveticus GDMCC No. 60811 was higher than that of the control strain ATCC15009. Therefore, Lactobacillus helveticus GDMCC No. 60811 has a good proliferation ability in the intestine.

[0110] Example 5: GDMCC containing Lactobacillus helveticus Preparation of microbial agent No.60811

[0111] Lactobacillus helveticus GDMCC No.60811 was streaked on MRS solid medium and cultured at 37°C for 48 hours to obtain a single colony; a single colony was picked and inoculated into MRS liquid medium and cultured at 37°C for 18 hours for activation. The seed solution was obtained by continuous activation at 37°C for two generations. The seed solution was inoculated into MRS medium at a volume ratio of 2% to 4%, and anaerobically cultured at 37°C for 15 to 20 hours to collect the bacterial solution. The bacterial solution was centrifuged, the supernatant was removed, and the precipitate was resuspended in physiological saline containing a freeze-dried protective agent to a bacterial concentration of 1×10 9 CFU / mL to obtain a liquid bacterial agent; the liquid bacterial agent is freeze-dried to obtain bacterial powder to obtain a solid bacterial agent.

[0112] 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 strain of Lactobacillus helveticus ( Lactobacillus helveticus ), characterized in that, The Lactobacillus helveticus was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 16, 2019, with the deposit number GDMCC No.60811.

2. A composition comprising the Lactobacillus helveticus according to claim 1.

3. The composition according to claim 2, characterized in that The composition contains live strains of Lactobacillus helveticus or dry strains of Lactobacillus helveticus.

4. The composition according to claim 2 or 3, characterized in that The number of viable Lactobacillus helveticus in the composition is not less than 5×10 9 CFU / mL or 5×10 9 CFU / g.

5. A product containing the composition according to any one of claims 2 to 4, characterized in that: The product is a medicine, which is used to prevent and / or improve cognitive impairment or improve memory and cognitive functions, including improving at least one of spatial memory ability, working memory ability, reference memory ability, and recognition ability.

6. The product according to claim 5, characterized in that The medicine contains the Lactobacillus helveticus described in claim 1, the composition described in claim 2 or 3, a drug carrier and / or a pharmaceutical excipient.

7. A product containing the composition according to any one of claims 2 to 4, characterized in that: The product is a microbial agent or food.

8. The product according to claim 7, characterized in that The food is a health product; or the food is a dairy product, a bean product or a fruit and vegetable product prepared using the Lactobacillus helveticus described in claim 1 or a starter containing the Lactobacillus helveticus described in claim 1; or the food is a beverage or a snack containing the Lactobacillus helveticus described in claim 1.

9. Use of the Lactobacillus helveticus according to claim 1, or the composition according to claim 2 or 3, in the preparation of a product for reducing scopolamine.

10. Use of the Lactobacillus helveticus according to claim 1 or the composition according to claim 2 or 3 in the preparation of a product for preventing and / or improving cognitive impairment or improving memory and cognitive function, characterized in that: The product is a medicine, which is used to improve at least one of the memory and cognitive abilities of spatial memory, working memory, reference memory, and recognition ability.

11. Use of the Lactobacillus helveticus according to claim 1 or the composition according to claim 2 or 3 in the preparation of microbial agents or foods.

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