Fermented milk whey having memory and cognition improving function and use thereof

Abstract

Fermented milk whey having a memory and cognition improving function and a use thereof. The fermented milk whey is obtained by using Lactobacillus helveticus CCFM1263 as a fermentation starter for the fermentation of skimmed milk and performing operations such as precipitation, centrifugation, and membrane filtration to remove proteins. The Lactobacillus helveticus CCFM1263 fermented milk whey can alleviate memory and cognitive impairments and effectively relieve damage to hippocampal neurons.

Description

Fermented milk whey with improved memory and cognitive functions and application thereof Technical Field

[0001] The invention relates to fermented milk whey with the function of improving memory and cognition and application thereof, belonging to the technical field of dairy product processing and fermentation. Background Art

[0002] As living conditions improve, consumer demand for food is constantly evolving, with nutrition and health being a key focus for many. Fermented milk, made from cow's milk fermented with lactic acid bacteria, is an effective way to supplement nutrients like protein and minerals. However, the current homogeneity of commercially available fermented milk products is a serious issue, hindering the development of dairy companies. The rise of functional dairy products has changed this dynamic. Because they align with consumers' pursuit of a healthier lifestyle, they have garnered significant attention and become a hotbed of competition among dairy companies.

[0003] "Cognition" refers to all the brain's activities and processes for acquiring, storing, retrieving, and processing information, including memory, learning, comprehension, judgment, and execution. Cognitive impairment refers to impairment in one or more of these cognitive activities, manifesting as difficulty remembering, learning new things, concentrating, or making daily decisions. Cognitive impairment ranges from mild to severe. With mild impairment, people may gradually begin to notice changes in cognitive function but remain able to carry out daily activities. With severe impairment, people lose the ability to understand the meaning or significance of things, leading to the inability to function normally. Statistics from Jia et al. (2020) indicate that in China, as of 2020, the prevalence of cognitive impairment among people aged 60 and over was approximately 15.5%, of which approximately 6.0% had dementia. The World Alzheimer's Report 2016 further indicates that in 2015, there were approximately 46.8 million people with dementia worldwide, and this number is projected to increase to 131 million by 2050. As the aging population intensifies, cognitive impairment has become a global public health issue. Furthermore, with increasing stress from daily life and studies, coupled with poor sleep quality, more and more young people are experiencing memory loss. Therefore, improving memory and cognitive function is attracting increasing attention from researchers.

[0004] Fermented milk whey is a by-product produced during the preparation of fermented milk. It contains a variety of nutrients, including protein, amino acids, vitamins, minerals, etc. The main treatment methods are to use it directly as animal feed or directly treat it as wastewater, which leads to additional environmental pollution and costs, and also constitutes a loss of resources. In order to effectively utilize fermented milk whey, it is necessary to develop a functional fermented milk whey product. However, the differences in fermentation strains will affect the functional components and functional properties of fermented milk whey, such as bioactive peptides. In the prior art, no Lactobacillus helveticus that can ferment and produce fermented milk whey with improved memory and cognitive functions has been found. Therefore, it is of great significance to develop Lactobacillus helveticus that can produce functional fermented milk whey, and to develop more fermented milk whey products with unique characteristics and functional properties to meet the needs of different consumers.

[0005] Summary of the Invention

[0006] The present invention provides a strain of Lactobacillus helveticus (Lactobacillus helveticus) CCFM1263, which was screened from Qula collected in Xining, Qinghai, my country. It was deposited with the Guangdong Provincial Microbial Culture Collection on May 17, 2022, with a deposit number of GDMCC No. 62417. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The present invention also provides a microbial preparation.

[0008] In one embodiment, the microbial preparation contains Lactobacillus helveticus CCFM1263, or a fermentation broth containing Lactobacillus helveticus CCFM1263, or a lysate containing Lactobacillus helveticus CCFM1263, or a lysate containing Lactobacillus helveticus CCFM1263, or an extract containing Lactobacillus helveticus CCFM1263.

[0009] In one embodiment, the microbial preparation contains Lactobacillus helveticus with a cell count of ≥1×10 3 CFU / ml or ≥1×10 3 CFU / g.

[0010] The present invention also provides a fermented milk whey with the function of improving memory and cognitive function. The fermented milk whey is produced by fermenting the Lactobacillus helveticus CCFM1263 or the microbial preparation.

[0011] The present invention also provides the use of Lactobacillus helveticus CCFM1263 or the fermented milk whey of the microbial preparation in the preparation of a health product for assisting in improving memory.

[0012] The present invention also provides the use of Lactobacillus helveticus CCFM1263 or the fermented milk whey of the microbial preparation in the preparation of medicines for improving memory and cognition.

[0013] In one embodiment, the drug further comprises a drug carrier or a pharmaceutical excipient.

[0014] In one embodiment, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0015] In one embodiment, the pharmaceutical excipient comprises an excipient and / or an additive.

[0016] In one embodiment, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0017] The present invention also provides the use of Lactobacillus helveticus CCFM1263 or the fermented milk whey of the microbial preparation in the preparation of food for improving memory and cognition.

[0018] In one embodiment, the food comprises dairy products, soy products or fruit and vegetable products.

[0019] In one embodiment, the improvement of memory and cognition includes but is not limited to the following aspects:

[0020] (1) Significantly shorten the first latency of the water maze spatial exploration period;

[0021] (2) Significantly improve the ability to recognize new objects;

[0022] (3) Significantly improve the spontaneous alternation rate in the Y-maze;

[0023] (4) Significant improvement in anxiety-like behaviors;

[0024] (5) Effectively alleviate hippocampal neuron damage.

[0025] The present invention also provides a method for preparing the fermented milk whey, which comprises using Lactobacillus helveticus CCFM1263 (original number DQHXN-Q32M42) or the microbial preparation as a starter, fermenting skim milk at 30-42° C. for 12-72 hours, and then removing protein through operations such as precipitation, centrifugation, and membrane filtration to obtain the fermented milk whey.

[0026] In one embodiment, the skim milk is prepared by dissolving 5-20% (w / w) skim milk powder in water.

[0027] In one embodiment, the method is to culture the purified Lactobacillus helveticus CCFM1263 in MRS medium and skim milk system adaptively until the bacterial concentration reaches 10 8 ~10 9cfu / mL.

[0028] In one embodiment, the method comprises: 8 The concentration of CFU / mL was inoculated into skim milk for fermentation, and the inoculation amount was 2% to 4% (v / v).

[0029] The present invention also provides use of the Lactobacillus helveticus, or the microbial preparation, or the fermented milk whey in preparing a product for alleviating hippocampal tissue damage. Beneficial effects:

[0030] 1. The Lactobacillus helveticus CCFM1263 used in the present invention is a food-safe and healthy strain that can be used to prepare various foods, medicines, and microbial preparations.

[0031] 2. The Lactobacillus helveticus CCFM1263 fermented milk whey of the present invention can significantly enhance the inhibitory activity of acetylcholinesterase. Compared with ATCC15009 fermented milk whey, the inhibitory activity of acetylcholinesterase is enhanced by 456%; compared with GDMCC No.60811 fermented milk whey, the inhibitory activity of acetylcholinesterase is enhanced by 77%; and compared with cerebrolysin, the inhibitory activity of acetylcholinesterase is enhanced by 32%.

[0032] 3. The lactobacillus helveticus CCFM1263 fermented milk whey of the present invention can significantly enhance the antioxidant capacity. Compared with the lactobacillus helveticus fermented milk whey of GDMCC No. 60811, the DPPH and ABTS free radical scavenging rates are enhanced by 21% and 17%, respectively.

[0033] 4. The lactobacillus helveticus CCFM1263 fermented milk whey of the present invention can significantly improve the memory and cognitive impairment of mice induced by scopolamine. The CCFM1263 fermented milk whey can significantly shorten the time to reach the platform for the first time in the water maze spatial exploration by 178%, and significantly increase the spontaneous alternation rate in the Y maze test by 15%.

[0034] 5. The lactobacillus helveticus CCFM1263 fermented milk whey of the present invention can effectively alleviate the damage of hippocampal tissue in mice.

[0035] Biomaterial Deposit

[0036] The Lactobacillus helveticus CCFM1263 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, with a deposit number of GDMCC No: 62417, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1: Colony morphology of Lactobacillus helveticus CCFM1263;

[0038] Figure 2: Evaluation of the acetylcholinesterase inhibitory and antioxidant capacity of Lactobacillus helveticus fermented milk whey. Data are presented as mean ± SD, n = 3. Different letters (a, b, c, d) above the bars indicate statistically significant differences (p < 0.05).

[0039] Figure 3: Representative trajectories during the spatial exploration phase of the water maze experiment for mice in different treatment groups;

[0040] Figure 4: The first latency of the spatial exploration phase of the water maze experiment in mice of different treatment groups;

[0041] Figure 5: Representative trajectories of the novel object recognition experiment in mice of different treatment groups (the concentric circles on the left represent the new objects, and the concentric circles on the right represent the old objects)

[0042] Figure 6: Spontaneous alternation rate in the Y-maze of mice in different treatment groups;

[0043] Figure 7: Hippocampal tissue morphology of mice in different treatment groups (Nissl staining);

[0044] Among them, * indicates comparison with the model group, *: p < 0.05, **: p < 0.01, ***: p < 0.001. DETAILED DESCRIPTION

[0045] Technical terms:

[0046] The "inoculation amount" in the present invention refers to the ratio of the volume of the seed solution added to the volume of the culture solution after inoculation.

[0047] The term "improvement" in the present invention refers to reversing, alleviating, minimizing, inhibiting, stopping and / or stopping one or more clinical symptoms of functional impairment after functional impairment.

[0048] In the present invention, "memory and cognitive disorders" refer to abnormalities in the brain's high-level intelligent processing processes related to learning, memory, and thinking and judgment, such as forgetting, memory loss, and memory errors; the memory loss refers to a general decline in recognition, retention, recognition, and recall.

[0049] In the present invention, "spatial exploration ability" refers to the ability to explore the shape and / or position of an object. The spatial exploration ability includes observing, thinking, imagining, recognizing and / or exploring the shape and / or position of an object.

[0050] The reagents used to prepare MRS liquid / solid culture medium in the following examples were purchased from Sinopharm Chemical Reagent Co., Ltd.; the skim milk powder involved in the following examples was purchased from Bright Dairy Co., Ltd.; the SPF-grade 6-week-old male ICR mice involved in the following examples were purchased from Weitonglihua Experimental Animal Co., Ltd.

[0051] The culture medium involved in the following examples is as follows:

[0052] MRS solid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, agar 17 g / L.

[0053] MRS liquid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L.

[0054] Example 1: Isolation and Identification Method of Lactobacillus helveticus CCFM1263 Strain

[0055] 1. Prepare appropriate sample dilution gradient and incubate

[0056] Take out Qula from Xining, Qinghai, which is stored in a -80℃ refrigerator and slowly thaw it on ice. After shaking and mixing, take 0.5mL of 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 dilution from the dilution and add it to 4.5mL of sterile saline to complete a second 10-fold dilution. Repeat this process until the fifth 10-fold dilution is completed (diluted to 10). -5 ), 100 μL was taken from each gradient dilution, evenly spread on MRS solid culture medium plates, and inverted to culture at 37°C for 48 h.

[0057] 2. Line separation and purification

[0058] Remove the plate with colonies, select the gradient plate with obvious single colonies, pick colonies with different colony morphologies, and perform secondary streaking until all single colonies are purified.

[0059] 3. Strain preservation

[0060] After purification, a single colony of each strain was picked into 5 mL of MRS liquid medium and cultured at 37°C for 24 h. After mixing, 1 mL of the 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 30% (v / v) sterile glycerol solution was added, resuspended, and stored at -80°C.

[0061] 4. 16S rDNA sequence amplification

[0062] After mixing, aspirate 1 mL of the bacterial suspension and centrifuge at 6000 rpm for 3 minutes. The supernatant was decanted, washed twice with sterile water, and centrifuged to discard the supernatant. This bacterial slurry was used as a template for PCR amplification. The amplification system consisted of 20 μL of the template, 1 μL of each bidirectional primer, 0.5 μL of each primer, 10 μL of Taq MasterMix, and 8 μL of ddH2O. Primers used were 27F: AGAGTT TGATCC TGG CCT CA (SEQ ID No. 1) and 1492R: GGT TAC CTT GTT ACG ACT T (SEQ ID No. 2). Amplification conditions were as follows: initial denaturation at 94°C for 10 minutes; denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds; extension at 72°C for 2 minutes; 30 cycles; a final extension at 72°C for 10 minutes; and a hold at 12°C for 10 minutes.

[0063] 5. Sequencing and identification

[0064] The PCR sample was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing, and the amplified nucleotide sequence sequencing results of the strain number DQHXN-Q32M42 ​​were obtained. The sequencing results were compared with the National Center for Biotechnology Information (NCBI) sequence database (http: / / www.ncbi.nlm.nih.gov / blast) by BLAST, and it was determined that the strain provided by the present invention was Lactobacillus helveticus.

[0065] Example 2: Preparation method of Lactobacillus helveticus CCFM1263 fermented milk whey

[0066] The Lactobacillus helveticus CCFM1263 obtained in Example 1 was inoculated into MRS liquid culture medium, cultured at 37°C for 18-24 hours, and subcultured 2-3 times until the bacterial concentration reached 10 8 ~10 9 cfu / mL. Take out the bacterial suspension activated in MRS, centrifuge at 6000×g for 5 min, resuspend to the same volume with 11% (w / w) skim milk, inoculate into 11% (w / w) skim milk at a 2% (v / v) inoculum, subculture 2 to 3 times until the bacterial concentration reaches 10 8 ~109 cfu / mL. Wash with Tris acid to remove protein and adjust the viable count to 10 8 CFU / mL~10 9 CFU / mL, inoculated at an inoculum size of 2% to 4% (v / v) into 11% (w / w) skim milk, and fermented at 30-42°C for 12-72 hours to obtain Lactobacillus helveticus CCFM1263 fermented milk. 1 mol / L sodium hydroxide solution was added to the Lactobacillus helveticus CCFM1263 fermented milk to adjust the sample pH to 4.5-4.7, followed by pasteurization in an autoclave. The sterilized Lactobacillus helveticus CCFM1263 fermented milk was centrifuged at 10,000 × g at 4°C for 10 minutes, and the supernatant was collected and filtered through a 0.45 μm organic filter membrane to obtain the whey of the Lactobacillus helveticus CCFM1263 fermented milk.

[0067] Example 3 Acetylcholinesterase Inhibitory and Antioxidant Capacity of Whey Fermented Milk by Lactobacillus helveticus CCFM1263

[0068] 1. Inhibition of acetylcholinesterase

[0069] 30 μL of iodothioacetylcholine, 125 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 40 μL of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and 50 μL of sample or cerebrolysin were added to a 96-well plate, mixed, and incubated at 37°C for 15 minutes. The samples in each group were: cerebrolysin (10 mg / mL cerebrolysin), raw milk (11% (w / w) skim milk), ATCC15009 fermented milk whey (ATCC15009 fermented milk whey), CCFM1263 fermented milk whey (CCFM1263 fermented milk whey), and GDMCC No. 60811 fermented milk whey (GDMCC No. 60811 fermented milk whey). Next, 30 μL of acetylcholinesterase (AChE) (0.05 U / mL) was added to activate the reaction. After 15 minutes, the absorbance of each reaction well at a wavelength of 412 nm was measured using a microplate reader. The AChE inhibitory activity of the sample was calculated as follows: AChE inhibitory activity (%) = (1-(A sample - A sample blank) / (A control - A control blank)) × 100. The sample blank group consisted of a control group using HEPES instead of AChE, a control group using HEPES instead of AChE and the sample, and a control blank group using HEPES instead of the sample. The AChE inhibitory activity of each group was measured. The preparation methods of ATCC 15009 and GDMCC No. 60811 fermented milk whey were similar to those in Example 2.

[0070] As shown in Figure 2A, the CCFM1263 fermented milk whey group showed significantly stronger AChE inhibitory activity than the GDMCC No. 60811 fermented milk whey group and the cerebrolysin group, while the ATCC15009 fermented milk whey group showed the weakest. The CCFM1263 fermented milk whey group showed a 77% increase over the GDMCC No. 60811 fermented milk whey group, a 456% increase over the ATCC15009 fermented milk whey group, and a 32% increase over the cerebrolysin group.

[0071] 2. Antioxidant capacity determination

[0072] Accurately weigh 3.9432 mg of DPPH and dissolve it in 10 mL of anhydrous ethanol to prepare a 1 mM DPPH working solution. Add 100 μL of sample and 100 μL of DPPH working solution to a 96-well plate and incubate in the dark at room temperature. After 30 minutes, measure the absorbance of each well at 510 nm using a microplate reader. Accurately weigh 39.5 mg of ABTS and dissolve it in 10 mL of water to prepare a 7.2 mM ABTS solution. Also, weigh 6.8 mg of potassium persulfate and dissolve it in 10 mL of water to prepare a 2.5 mM potassium persulfate solution. Incubate the resulting ABTS and potassium persulfate solutions in the dark for 16 hours to obtain the ABTS working solution, which is ready for use. Before use, dilute the ABTS working solution to an absorbance of 0.8 at 734 nm. Add 100 μL of sample and 100 μL of ABTS working solution to a 96-well plate and incubate in the dark at room temperature. After 15 minutes, the absorbance of each reaction well was measured at 734 nm using a microplate reader. The DPPH and ABTS free radical scavenging rates of the glutathione group (1 mg / mL), the raw milk group (11% (w / w) skim milk), the ATCC15009 fermented milk whey group, the CCFM1263 fermented milk whey group, and the GDMCC No. 60811 fermented milk whey group were measured.

[0073] As shown in Figures 2B and C, the CCFM1263 fermented milk whey group had significantly higher DPPH and ABTS free radical scavenging rates than the GDMCC No. 60811 fermented milk whey group, with the ATCC15009 fermented milk whey group showing the weakest results. The CCFM1263 fermented milk whey group had 21% and 17% higher DPPH and ABTS free radical scavenging rates, respectively, compared to the GDMCC No. 60811 fermented milk whey group.

[0074] Example 4: Effect of Lactobacillus helveticus CCFM1263 fermented milk whey on the behavior of mice with cognitive impairment induced by scopolamine

[0075] 1. Animal Experimental Protocol

[0076] Thirty-two SPF-grade 6-week-old male ICR mice were housed in a barrier with equal day and night intervals and constant temperature and humidity. The experiment was divided into an adaptation period, a Lactobacillus helveticus fermented milk whey intervention period, and a parallel period of Lactobacillus helveticus fermented milk whey intervention and scopolamine modeling. After one week of adaptive feeding, the Lactobacillus helveticus fermented milk whey intervention period began. All mice were randomly divided into four groups with 8 mice in each group: a blank group, a model group, an ATCC15009 fermented milk whey group, a GDMCC No.60811 fermented milk whey group, and a CCFM1263 fermented milk whey group. They were gavaged daily according to Table 1. Among them, ATCC15009 is the model strain of Lactobacillus helveticus. On the 22nd day of gavage, the Lactobacillus helveticus fermented milk whey intervention and scopolamine modeling parallel period began. A 7-day water maze test was first conducted. After a 5-day rest, a 2-day novel object recognition test was conducted. At this time, gavage was continued daily. Thirty minutes after gavage, intraperitoneal injection was performed to establish the model according to Table 1. If behavioral experiments were required on the same day, they were initiated 30 minutes after intraperitoneal injection. Specific experimental animal groupings and treatments are shown in Table 1.

[0077] Table 1 Animal experimental groups and treatment methods

[0078] Note: The 1 mg in the modeling dose of 1 mg / kg·BW refers to the scopolamine content, not the scopolamine solution content.

[0079] 2. Behavioral evaluation indicators and results

[0080] The Morris water maze is used to test the spatial exploration and learning and memory abilities of experimental mice. Specifically, it tests the stable spatial position cognition that animals have developed through repeated training. Because the animals' entry into the water changes, they cannot rely on their own position and instead rely on spatial information (external cues) to locate the hidden platform. This is a form of reference cognition that uses objects as a reference, forming a form of spatial reference memory. The experimental apparatus consists of a 120 cm diameter, 60 cm high pool and a 12 cm diameter, height-adjustable, removable platform. During the experiment, the pool was filled with 30 cm of water and stirred with black ink until it turned a deep black. The amount of ink added was determined by the platform becoming invisible underwater. The water temperature was maintained at approximately 21°C. Four cardboard pieces of different colors and shapes were placed equidistantly around the pool as markers. The experiment consisted of three phases: a platform visibility period (1 day), a navigation period (days 2–6), and a spatial exploration period (day 7). The test duration for all three phases was set to 60 seconds. Cameras recorded the mice's swimming trajectories for subsequent digital analysis. During the localization cruise phase, if the mice failed to find the platform within 60 seconds, they were guided to the platform and remained there for at least 15 seconds. During the spatial exploration phase, the mice were allowed to explore freely for 60 seconds.

[0081] As shown in the composite heatmaps in Figure 3, the blank group was able to remember the platform's location relatively accurately, circling near it. The model group, however, exhibited memory discrepancies or failed to remember the platform's location, rarely remaining near it and remaining largely within its path. The control group (ATCC15009 fermented milk whey group) performed similarly to the model group, significantly deviating from the platform. The CCFM1263 fermented milk whey and GDMCC No. 60811 fermented milk whey groups showed similar results to the blank group, circling within a small area of ​​the platform's attachment area. Furthermore, representative trajectory plots indicate that the blank group adopted a linear exploration strategy, while the model group's wandering paths were irregular, characterized by marginal and random movements. Compared to the model group, the ATCC15009 fermented milk whey group showed some improvement in their exploration strategy, adopting a directional approach. The CCFM1263 fermented milk whey group performed better than the GDMCC No. 60811 fermented milk whey group, approaching the blank group's, adopting a linear approach.

[0082] The results in Figure 4 also demonstrate that scopolamine-induced modeling causes memory and cognitive impairment in mice, as evidenced by a significant increase in the time it took the model group mice to reach the platform (i.e., first latency) during the water maze exploration phase compared to the blank control group. Compared to the model group, GDMCC No. 60811 fermented milk whey and CCFM1263 fermented milk whey significantly shortened the first latency during the water maze exploration phase in mice by 160% and 178%, respectively, and were significantly lower than those in the ATCC15009 fermented milk whey group.

[0083] The novel object recognition test assesses learning and memory abilities based on an animal's natural tendency to explore novel objects. The experiment lasted two days and consisted of an acclimation period, a familiarization period, and a test period. The first day was the acclimation period, during which mice freely explored a 40 cm × 40 cm × 30 cm black open-field box for 6 minutes. The next morning, the familiarization period began. Two identical hexagonal wooden blocks were placed in the black open-field box, each 10 cm from the wall. The mice freely explored the box for 6 minutes. After the mice returned to their original cages and rested for 6 hours, the test period began. One of the hexagonal blocks was replaced with a cone-shaped block, and the mice were again allowed to freely explore. An infrared camera recorded the mice's exploration trajectory over the 6-minute period. After each experiment, the Y-maze was wiped clean with 75% alcohol to eliminate odors.

[0084] As shown in Figure 5, during the novel object test phase, compared with the blank group, the model group mice were more likely to walk close to the wall, exhibiting higher anxiety levels and a less pronounced tendency to explore the novel object. The ATCC15009 fermented milk whey group exhibited similar performance to the model group; however, the GDMCC No. 60811 fermented milk whey and CCFM1263 fermented milk whey groups showed significantly reduced anxiety levels, with clear signs of exploration in the central area. They also tended to explore the left novel object area more than the right old object area, and their overall performance was similar to that of the model group.

[0085] Spontaneous alternation is a measure of spatial working memory, driven by rodents' curiosity about unknown areas. It can be assessed by allowing mice to explore all three arms of a Y-maze. Mice with intact working memory remember which arms they have previously visited and thus alternate between the three arms. The results in Figure 6 show that compared to the model group, only the CCFM1263 fermented milk whey group significantly increased spontaneous alternation rate in the Y-maze test by 15%, suggesting that CCFM1263 fermented milk whey can significantly improve short-term memory.

[0086] In summary, the behavioral experimental results (Morris water maze spatial exploration period, novel object recognition test period and Y maze spontaneous alternation period) showed that CCFM1263 fermented milk whey can effectively improve scopolamine-induced memory and cognitive impairment.

[0087] Example 5: Effect of Lactobacillus helveticus CCFM1263 fermented milk whey on the hippocampal tissue morphology of mice with scopolamine-induced cognitive impairment

[0088] Following the animal experiment design described in Example 4, mice were sacrificed the day after the concurrent Lactobacillus helveticus fermented milk whey intervention and scopolamine modeling. Mice were anesthetized, their eyes rapidly enucleated for blood collection, and then sacrificed by cervical dislocation. Following sacrifice, the mice were dissected, and their brains were removed and fixed in 4% paraformaldehyde. After 48 hours of fixation, the brains were paraffin-embedded and sectioned, and hippocampal tissue morphology was observed using Nissl staining.

[0089] Figure 7 shows Nissl staining results in the blank group, showing uniform Nissl bodies in the CA1 and DG regions of the hippocampus, with regular cell shapes. In the model group, however, intact, surviving neurons in the CA1 and DG regions of the hippocampus were low in density, scattered, hyperchromatic, and abnormally morphologically characterized by pyknosis and diamond-shaped and triangular nuclei. In the CCFM1263 fermented milk whey group, neurons showed regular morphology, a clear nuclear-cytoplasmic boundary, and significantly less hyperchromasia, demonstrating effective mitigation of this injury. While the damage in the ATCC15009 fermented milk whey group was slightly less severe than in the model group, it remained severe. These results suggest that CCFM1263 fermented milk whey can alleviate scopolamine-induced hippocampal neuronal damage in mice, thereby improving cognitive impairment.

[0090] Although the present invention has been disclosed above in terms of preferred embodiments, 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 CCFM1263, characterized in that: The Lactobacillus helveticus CCFM1263 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 17, 2022, with the collection number GDMCCNo: 62417.

2. A microbial preparation, characterized in that: The microbial preparation contains the Lactobacillus helveticus CCFM1263 described in claim 1, or a fermentation broth containing Lactobacillus helveticus CCFM1263, or a freeze-dried powder containing Lactobacillus helveticus CCFM1263, or an inactivated bacterial cell of Lactobacillus helveticus CCFM1263, or a lysate of Lactobacillus helveticus CCFM1263, or an extract of Lactobacillus helveticus CCFM1263.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation containing Lactobacillus helveticus CCFM1263, or containing the freeze-dried powder of Lactobacillus helveticus CCFM1263, or containing the inactivated bacteria of Lactobacillus helveticus CCFM1263, the number of bacteria is ≥ 1×10 3 CFU / ml or ≥1×10 3 CFU / g.

4. A fermented milk whey for improving memory and cognitive function, characterized in that: The fermented milk whey is obtained by fermenting the Lactobacillus helveticus CCFM1263 described in claim 1 or the microbial preparation described in claim 2 or 3.

5. Use of the Lactobacillus helveticus according to claim 1, or the microbial preparation according to claim 2 or 3, or the fermented milk whey according to claim 4 in the preparation of a health product for assisting in improving memory.

6. Use of the Lactobacillus helveticus according to claim 1, or the microbial preparation according to claim 2 or 3, or the fermented milk whey according to claim 4 in the preparation of medicines for improving memory and cognition.

7. The use according to claim 6, characterized in that: The medicine further comprises a drug carrier or a pharmaceutical excipient.

8. The use according to claim 7, characterized in that: The drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

9. The use according to claim 8, characterized in that: The pharmaceutical excipients include excipients and / or additives.

10. The use according to claim 9, characterized in that: The dosage form of the medicine is powder, granule, capsule, tablet, pill or oral solution.

11. Use of the Lactobacillus helveticus according to claim 1, or the microbial preparation according to claim 2 or 3, or the fermented milk whey according to claim 4 in the preparation of foods for improving memory and cognition.

12. The use according to claim 11, characterized in that: The food includes dairy products, bean products or fruit and vegetable products.

13. The method for preparing fermented milk whey according to claim 4, characterized in that: The skim milk is fermented at 30-42° C. for 12-72 hours using the Lactobacillus helveticus CCFM1263 described in claim 1 or the microbial preparation described in claim 2 or 3 as a starter, and the protein is removed.

14. The method according to claim 13, characterized in that The inoculation amount of the fermentation agent is 2% to 4% by volume, and the concentration of Lactobacillus helveticus CCFM1263 in the fermentation agent is (1 to 5)×10 8 cfu / mL.

15. Use of the Lactobacillus helveticus CCFM1263 according to claim 1, or the microbial preparation according to claim 2 or 3, or the fermented milk whey according to claim 4 in preparing a product for alleviating hippocampal tissue damage.

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