A strain of *Pediococcus lactis* SUN01 with acetylcholinesterase inhibitory activity and its application.

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Pediococcus acidilactici* SUN01 with acetylcholinesterase inhibitory activity and its applications. This *Pediococcus acidilactici* SUN01 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29704. The *Pediococcus acidilactici* SUN01 provided by this invention exhibits strong acid and bile salt resistance, good tolerance in simulated gastric and intestinal fluids, and can successfully reach the human intestine. *Pediococcus acidilactici* SUN01, in its bacterial suspension and inactivated cells, possesses strong acetylcholinesterase inhibitory activity, produces γ-aminobutyric acid (GABA), and has antioxidant and other probiotic functions. Its application in probiotic products may help improve Alzheimer's disease, enhance learning and memory abilities, regulate mood, and improve sleep.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Pediococcus lactis SUN01 with acetylcholinesterase inhibitory activity and its applications. Background Technology

[0002] Alzheimer's disease (AD) is characterized by the extensive deposition of senile plaques, the formation of neurofibrillary tangles, and the degeneration and loss of neurons. Its main clinical manifestations include learning and memory impairment, cognitive dysfunction, and language impairment. Alzheimer's disease is currently the most widespread and prevalent neurodegenerative disease in the world.

[0003] The current pathogenesis mechanisms of Alzheimer's disease (AD) include: the β-amyloid (Aβ) hypothesis, the cholinergic injury hypothesis, the oxidative stress hypothesis, the tau protein hypothesis, and the apoptosis hypothesis. Among these, Aβ is the precursor protein of β-amyloid (Aβ). amyloid beta-protein precursor, APP The enzymatic hydrolysis products of α, secreted by cells, have strong neurotoxic effects after precipitating and accumulating in the cell matrix. They are the main cause of neuronal degeneration and death around senile plaques in the brains of AD patients.

[0004] The cholinergic system in the brain is closely related to human learning and memory. Defects in cholinergic transmission have potential impacts on various aspects of cognition and behavior, including information processing in the cortex and hippocampus. Acetylcholine (Ach) is an important neurotransmitter in the cholinergic system, and acetylcholinesterase (AchE) hydrolyzes acetylcholine into choline and acetic acid. Imbalances and degeneration of cholinergic neurons can lead to the deposition of Aβ, thereby exacerbating cognitive impairment in Alzheimer's disease (AD). Furthermore, central cholinergic degeneration can induce abnormal phosphorylation of Tau protein, neurological inflammation, apoptosis, and various pathological phenomena such as imbalances in neurotransmitter and neurohormonal regulation. Therefore, excessively high AchE activity promotes the development and progression of AD. Simultaneously, excessive reactive oxygen species produced by neuronal oxidative stress can cause irreversible neuronal damage; reducing AchE activity and alleviating oxidative stress are beneficial for improving AD.

[0005] The most prominent clinical manifestation of Alzheimer's disease (AD) is cognitive impairment. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system, which participates in various physiological activities in the human body and has the effects of promoting sleep, enhancing memory, and relieving anxiety. Disorders of the GABA system may be one of the causes of cognitive impairment.

[0006] Currently, the main acetylcholinesterase inhibitors used in the clinical treatment of Alzheimer's disease (AD) are donepezil hydrochloride, rivastin, tacrine, and galantamine. These drugs have been successfully used clinically to treat cognitive impairment, but they have significant side effects in AD patients, such as hypertension, nausea, diarrhea, and vomiting. In contrast, probiotics have relatively fewer toxic side effects and are considered safer. Therefore, developing probiotics that can improve AD function has significant application value in the medical and other fields.

[0007] Currently, there are relatively few probiotic strains with acetylcholinesterase inhibitory activity, such as *Lactobacillus plantarum* and *Lactobacillus paracasei*. *Pediococcus lactis* is safe and practical, with diverse sources, and has been widely used in the probiotic field. The diversity of *Pediococcus lactis* sources results in genetic and functional diversity. Current research has developed *Pediococcus lactis* strains capable of producing γ-aminobutyric acid (GABA), but none of these strains have disclosed acetylcholinesterase inhibitory activity. Therefore, those skilled in the art are dedicated to developing probiotics containing *Pediococcus lactis* from more sources and with more functions, such as developing probiotic *Pediococcus lactis* strains that combine acetylcholinesterase inhibitory activity, GABA production, and antioxidant functions. This would broaden the application areas of probiotic *Pediococcus lactis*, especially showing greater promise in improving Alzheimer's disease (AD). Summary of the Invention

[0008] To address the shortcomings of the prior art mentioned in the background section, the present invention provides a strain of Pediococcus lactis SUN01.

[0009] This invention provides a strain of Pediococcus lactis ( Pediococcus acidilactici SUN01 was deposited on January 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29704.

[0010] The *Pediococcus lactis* SUN01 strain was isolated from pickled eggplant in Yimen County, Yunnan Province. This strain has good tolerance to gastrointestinal fluids, strong acetylcholinesterase inhibitory activity, and probiotic functions such as γ-aminobutyric acid production and antioxidant activity. It can help improve Alzheimer's disease, improve people's learning and memory abilities, regulate mood, and improve sleep.

[0011] In summary, the *Pediococcus lactis* SUN01 provided by this invention has strong acid and bile salt resistance, exhibits good tolerance in artificial gastric and intestinal fluids, and can successfully reach the human intestine. The bacterial suspension and inactivated cells of *Pediococcus lactis* SUN01 all possess strong acetylcholinesterase inhibitory activity, γ-aminobutyric acid production, and antioxidant and other probiotic functions. Applying it to probiotic products can help improve Alzheimer's disease, regulate mood, and improve sleep.

[0012] The present invention also provides a freeze-dried Peptococcus lactis SUN01 bacterial powder, the components of which include Peptococcus lactis SUN01 as described above.

[0013] In some embodiments, the viable count of *Pediococcus lactis* SUN01 in the freeze-dried bacterial powder is (1–5) × 10⁻⁶. 11 CFU / g.

[0014] The present invention also provides the use of the above-described Pediococcus lactis SUN01 in the preparation of acetylcholinesterase inhibitors.

[0015] The present invention also provides the use of the above-described Pediococcus lactis SUN01 in the production of γ-aminobutyric acid.

[0016] This invention also provides the application of *Pediococcus lactis* SUN01, as described above, in the preparation of fermented plant extracts. The application of *Pediococcus lactis* SUN01 in the preparation of fermented plant extracts can significantly improve the inhibitory activity of the plant extracts against acetylcholinesterase. The fermented plant extracts include, but are not limited to, fermented wolfberry extract, fermented jujube seed extract, and fermented licorice extract.

[0017] The present invention also provides the application of Pietrococcus lactis SUN01 as described above in the preparation of functional products.

[0018] In some embodiments, the functional product includes at least one of the following functions:

[0019] (1) It has acetylcholinesterase inhibitory activity;

[0020] (2) Produces γ-aminobutyric acid;

[0021] (3) Antioxidant.

[0022] Based on the above, the *Pediococcus lactis* SUN01 provided by the present invention has the following beneficial effects:

[0023] The *Pediococcus lactis* SUN01 provided by this invention has strong acid and bile salt resistance, good tolerance in artificial gastric and intestinal fluids, and can successfully reach the human intestine. *Pediococcus lactis* SUN01 bacterial suspensions and inactivated cells all have strong acetylcholinesterase inhibitory activity, γ-aminobutyric acid production, antioxidant and other probiotic functions. Its application in probiotic products can help improve Alzheimer's disease, improve learning and memory abilities, regulate mood, and improve sleep.

[0024] In summary, the *Pediococcus lactis* SUN01 provided by this invention exhibits stronger acetylcholinesterase inhibition rates in both its bacterial suspension and inactivated bacterial cells, providing a theoretical basis for inactivated probiotic products to improve Alzheimer's disease. Furthermore, its fermented plant extracts can significantly enhance the acetylcholinesterase inhibition rate, offering a new approach to improving Alzheimer's disease using traditional fermented medicinal and edible plant extracts. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Unless otherwise specified, the positional relationships shown in the drawings in the following description are based on the direction in which the components are drawn in the figure.

[0026] Figure 1 Phylogenetic tree diagram of Pediococcus lactis SUN01;

[0027] Figure 2 Graph of γ-aminobutyric acid standard curve data;

[0028] Figure 3 Graph showing the DPPH free radical scavenging activity of Pediococcus lactis SUN01;

[0029] Figure 4 The results of ABTS free radical scavenging activity of Pediococcus lactis SUN01 are shown in the figure. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. The technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] In the description of this invention, it should be noted that all terms used in this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and should not be construed as limiting the invention; it should be further understood that the terms used in this invention should be understood to have the same meaning as those in the context of this specification and in the relevant field, and should not be understood in an idealized or overly formal sense, except as expressly defined in this invention.

[0032] This invention provides a type of Pietrococcus lactis ( Pediococcus acidilactici SUN01:

[0033] Pediococcus acidilactici ( Pediococcus acidilactici SUN01 is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 29704;

[0034] Source: The *Pediococcus lactis* SUN01 strain was isolated from pickled eggplant in Yimen County, Yunnan Province. 16S rDNA sequencing of this strain was performed, and Blast sequence alignment showed that it was highly homologous to *Pediococcus lactis*. Therefore, it was named *Pediococcus lactis* SUN01.

[0035] Colony morphology: In MRS solid medium, colonies are milky white, round, smooth, and have neat edges.

[0036] Physiological characteristics of the strain: It has strong acid and bile salt resistance, good tolerance in artificial gastric and intestinal fluids, and can successfully reach the human intestine.

[0037] Probiotic functions of the strain: Pediococcus lactis SUN01 has strong acetylcholinesterase inhibitory activity, and this strain also has probiotic functions such as producing γ-aminobutyric acid and anti-oxidation, which can help improve Alzheimer's disease, improve people's learning and memory abilities, regulate mood, and improve sleep.

[0038] This invention also provides an operational example of a method for preparing fermented plant extracts:

[0039] Weigh 25g of wolfberries, add distilled water, heat, bring to a boil, then maintain a gentle boil and continue boiling for 1 hour. Filter through a 300-mesh filter cloth, collect the filtrate, add distilled water to make up to 200g, and cool to room temperature to obtain wolfberry extract. Similarly, prepare jujube seed extract and licorice extract using the same method.

[0040] Add 2% (w / v, unit g / ml, i.e., 2g glucose per 100ml of extract) of glucose to the plant extracts (goji berry, jujube seed, and licorice extracts) as described above, dispense into containers, sterilize at 115℃ for 15min, cool to room temperature, and then inoculate the sterilized plant extracts with a 3% (v / v, unit ml / ml, i.e., 300ul of Pediococcus lactis SUN01 bacterial solution per 10ml of sterilized plant extracts) bacterial solution of secondary activated Pediococcus lactis SUN01 bacterial solution. Incubate at (30-40)℃ for 24-36h to obtain fermented plant extracts.

[0041] This invention also provides a method for preparing freeze-dried bacterial powder, comprising the following preparation steps:

[0042] 1) Preparation of seed culture of Pediococcus lactis SUN01;

[0043] 2) Seed culture expansion;

[0044] 3) Seed culture fermentation to obtain fermentation broth;

[0045] 4) Centrifuge the fermentation broth to obtain bacterial sludge;

[0046] 5) After mixing the bacterial sludge with the freeze-drying protectant, emulsify and embed it to obtain an emulsion;

[0047] 6) The emulsion is freeze-dried and pulverized to obtain freeze-dried Peptococcus lactis SUN01 bacterial powder.

[0048] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1: Isolation and identification of Pediococcus lactis SUN01

[0050] 1. Separation and screening:

[0051] Aseptic sampling was performed on pickled eggplants from Yimen County, Yunnan Province. Using the plate coating method, 5g of pickled eggplant sample was placed in a sterile homogenizing bag, 45mL of 0.85% physiological saline was added, and the mixture was homogenized to obtain the sample.

[0052] Perform a 10-fold serial dilution of 100 μL of sample, taking 10 μL of each diluted sample. -2 , 10 -3 , 10 -4100 μL of the sample was spread onto an MRS solid plate containing 2.5% CaCO3 and incubated upside down at 37°C for 24 h. Colonies with good growth and large calcium dissolution zones were picked and repeatedly isolated and purified by streak plating until single colonies were obtained. The isolated strain was named Pediococcus lactis SUN01 and stored in a bacterial bank at -80°C with glycerol.

[0053] The colony morphology of the isolated and purified Pediococcus lactis SUN01 was as follows: in MRS solid medium, the colonies were milky white, round, smooth, and had neat edges.

[0054] The formula for MRS liquid medium is as follows: 10.0g beef extract, 20.0g glucose, 10.0g tryptone, 5.0g yeast extract, 1.0g Tween 80, 2.0g dipotassium hydrogen phosphate, 2.0g ammonium citrate, 5.0g anhydrous sodium acetate, 0.58g magnesium sulfate, 0.28g manganese sulfate monohydrate, 1.0L deionized water, pH 6.5 (add 1.5% agar to make MRS solid medium).

[0055] 2. Strain identification:

[0056] 2.1 Morphological observation: The colony morphology of the isolated and purified Pediococcus lactis SUN01 was as follows: In MRS solid medium, the colonies were milky white, round, smooth, and with neat edges.

[0057] 2.2 Molecular biological identification:

[0058] The screened and purified strains were subjected to Gram staining and catalase tests, and their physiological and biochemical indicators were measured. The test results were compared with those in Bergey's Manual of Systematic Bacteriology, 8th Edition, for preliminary identification of the bacterial species.

[0059] The test showed that the selected strain stained purple with Lansell staining, indicating a positive result. It was spherical in shape, catalase-negative, and did not form spores.

[0060] DNA was extracted from the bacterial strain according to the instructions of the bacterial DNA extraction kit, and PCR amplification was performed. The amplification products were sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The extraction and amplification process was as follows:

[0061] Extraction of SUN01 bacterial genomic DNA: Genomic DNA was extracted using a bacterial genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0062] PCR amplification of the 16S rDNA sequence:

[0063] The 16S rDNA gene sequence was amplified using primers 27F (5'-AGAGTT TGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0064] PCR reaction system: 1 μL 27F (10 μM), 1 μL 1492R (10 μM), 5 μL 10X EasyTag@R Buffer (2.5 mM), 4 μL dNTPs (2.5 mM), 1 μL DNA template, 0.3 μL EasyTag@R DNA Polymerase (5 U / L), 37.7 μL ddH2O;

[0065] PCR amplification program: 94℃ for 5 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, 72℃ for 10 min, repeat steps 2 to 4 32 times, store at 4℃.

[0066] PCR product detection and sequencing analysis:

[0067] 50 μL of the PCR product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of SUN01 was obtained as follows, and the strain was identified as Pediococcus lactis SUN01.

[0068] 16S rDNA sequence:

[0069] .

[0070] Blast alignment analysis of 16S rDNA sequences was performed in the NCBI database, combined with colony morphology observation, Gram staining, catalase test, and other methods. Figure 1 Homology analysis in the 16S rDNA phylogenetic tree shown identified SUN01 as Pediococcus lactis ( Pediococcus acidilactici ) bacterial strains.

[0071] Example 2: Preparation of freeze-dried Powder of Pediococcus lactis SUN01

[0072] The activated *Pediococcus lactis* SUN01 bacterial suspension was inoculated at a rate of 3% (v / v, ml / ml) into a culture medium that had been sterilized at 115°C for 15 min and then cooled. The medium was then incubated at 37°C for 24 h. After centrifugation (4°C, 8000 rpm, 5 min), the supernatant was discarded to obtain bacterial sludge.

[0073] After mixing the bacterial sludge and freeze-drying protectant at a mass ratio of 1:(0.5~2.5), emulsification and encapsulation were carried out to obtain an emulsion, which was then pre-frozen at -40℃ for 12h.

[0074] The pre-frozen emulsion was freeze-dried in a vacuum freeze dryer for 36 hours. This yielded freeze-dried Pediococcus lactis SUN01 bacterial powder with a viable count of 250 billion CFU / g, i.e., freeze-dried bacterial powder.

[0075] The preparation of the freeze-drying protectant is as follows: Take 10g of skim milk powder and 2g of trehalose, add 88g of distilled water, stir to dissolve, sterilize at 115℃ for 15min, and cool for later use.

[0076] The culture medium consists of: 20g glucose, 15g bovine bone peptone, 5g yeast extract, 5g beef extract, 2.0g dipotassium hydrogen phosphate, 2.0g ammonium citrate, 5g anhydrous sodium acetate, 0.58g magnesium sulfate, 0.28g manganese sulfate, 1g Tween 80, 1.0L deionized water, and pH 6.8.

[0077] It should be noted that the freeze-drying protectant can also use other existing protectant components or formulations, including but not limited to the above-described embodiments.

[0078] Example 3: Study on the acid and bile salt resistance characteristics of Pediococcus lactis SUN01

[0079] 1. Acid resistance

[0080] The freeze-dried Pyrrosia lingua SUN01 bacterial powder obtained in Example 2 was inoculated into MRS liquid medium with pH 6.0, pH 4.0 and pH 2.0 at an inoculation amount of 1% (w / v, g / ml). After incubation at 37°C for 3 h, viable bacteria were counted by plate count method. The results are shown in Table 1.

[0081] Table 1. Study on acid resistance of Pietrococcus lactis SUN01

[0082]

[0083] It can be seen that under pH 2.0 conditions, Pseudococcus lactis SUN01 still has a sufficient number of live bacteria that can pass through the gastric environment after 3 hours, indicating that it has good acid resistance.

[0084] The formulation of the MRS liquid culture medium is the same as that in Example 1.

[0085] 2. Bile salt tolerance

[0086] The freeze-dried Pyrrosia lingua SUN01 lyophilized bacterial powder obtained in Example 2 was inoculated at a rate of 1% (w / v, g / ml) into MRS liquid medium with bile salt concentrations of 0% (w / v, g / ml) and 0.3% (w / v, g / ml), respectively. After incubation at 37°C for 3 hours, viable bacteria were counted using the plate count method. The results are shown in Table 2.

[0087] Table 2. Study on bile salt tolerance of Pediococcus lactis SUN01

[0088]

[0089] It can be seen that at a bile salt concentration of 0.3%, Pseudococcus lactis SUN01 still retains a sufficient number of viable bacteria after 3 hours, indicating that it has good bile salt tolerance.

[0090] The formulation of the MRS liquid culture medium is the same as that in Example 1.

[0091] 3. Tolerance in artificial gastric and intestinal fluids

[0092] A certain amount of pepsin was dissolved in a PBS buffer solution at pH 3.0, and its final concentration was adjusted to 3.0 g / L to obtain simulated artificial gastric fluid. A certain amount of trypsin was dissolved in a PBS buffer solution at pH 8.0, and its final concentration was adjusted to 1.0 g / L to obtain simulated artificial intestinal fluid. Both the simulated gastric and artificial intestinal fluids were filtered through a 0.22 μm filter membrane under sterile conditions. The PBS buffer solution is also known as phosphate buffer saline.

[0093] The freeze-dried Pyrococcus lactis SUN01 obtained in Example 2 was inoculated at 1% (w / v, g / ml) in MRS liquid medium and artificial gastric fluid and cultured at 37°C for 3 h. Then, the fermentation broth in artificial gastric fluid was inoculated into artificial intestinal fluid at 10% (v / v, ml / ml) and cultured at 37°C for 3 h. Viable bacteria were counted by plate count method. The results are shown in Table 3.

[0094] Table 3. Tolerance of Pediococcus lactis SUN01 in simulated gastric and intestinal fluids.

[0095]

[0096] It can be seen that after passing through artificial gastric fluid and then artificial intestinal fluid, Pyocortisone lactis SUN01 has good tolerance and can successfully reach the intestine to colonize and exert its effects.

[0097] Example 4: Study on the inhibitory activity of Pediococcus lactis SUN01 against acetylcholinesterase

[0098] 1. Preparation of Pediococcus lactis SUN01 bacterial suspension and inactivated bacterial cell samples

[0099] Take the glycerol cryopreservation tubes of *Pediococcus lactis* SUN01 from the -80℃ freezer, and after thawing, inoculate them into MRS liquid medium at an inoculation rate of 3% (v / v, ml / ml). Incubate at 37℃ for 24 h to obtain the first-generation seed culture. Then, inoculate the first-generation seed culture into MRS liquid medium at an inoculation rate of 3% (v / v, ml / ml) and incubate at 37℃ for 18 h to obtain the fermentation broth activated to the second generation.

[0100] Preparation of bacterial suspensions at different concentrations: The fermentation broth was centrifuged (8000 r / min, 5 min, 4℃), the supernatant was discarded, the bacterial sludge was collected, washed twice with physiological saline, and resuspended in PBS phosphate buffer (10 mmol / L, pH 7.4) to obtain 10 9 A bacterial suspension of CFU / mL, then 10 9 The bacterial suspension of CFU / mL was diluted 10-fold to obtain 10 8 A bacterial suspension of CFU / mL, the above bacterial suspension is the live bacterial sample.

[0101] Preparation of inactivated bacterial cells at different concentrations: The above 10 9 The bacterial suspension of CFU / mL was sterilized at 115°C for 15 min and cooled to room temperature to obtain 10 9 A sample of inactivated bacterial cells per mL was prepared and then diluted 10-fold to obtain 10... 8 One inactivated bacterial cell / mL of inactivated bacterial sample.

[0102] 2. The inhibitory activity of acetylcholinesterase (AChE) was determined using a modified Ellman method:

[0103] In 96-well plates, 25 μL of samples (inactivated bacterial cells or bacterial suspensions) of different concentrations, 25 μL of acetylcholinesterase (8 U / mL), 50 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (5 mmol / L), and 100 μL of PBS buffer (10 mmol / L, pH 7.4) were added sequentially. The plates were incubated at 37°C for 5 min, followed by the addition of 50 μL of thioacetylcholine iodide (20 mmol / L). The plates were immediately placed in a microplate reader, and the incubation temperature was set to 37°C. Kinetic analysis was performed, and the absorbance at 412 nm was measured at 0 min and 15 min. Simultaneously, control groups (with PBS replacing the samples), blank control groups (with PBS replacing the samples and acetylcholinesterase), and sample blank groups (with PBS replacing the acetylcholinesterase in the sample groups) were set up.

[0104] Calculate the enzyme reaction rate and inhibition rate using the following formulas:

[0105]

[0106]

[0107] In the calculation of the enzyme-catalyzed reaction rate v, ΔA 15 This refers to the absorbance value of the control group or sample group at 15 min minus the absorbance value of the corresponding blank group. △A0 refers to the absorbance value of the control group or sample group at 0 min minus the absorbance value of the corresponding blank group. In the calculation of the inhibition rate, v i v0 refers to the enzyme reaction rate of the sample group, while v0 refers to the enzyme reaction rate of the control group.

[0108] 3. The measurement results show:

[0109] 10 9 CFU / mL, 10 8 The CFU / mL suspension of *Pediococcus lactis* SUN01 showed inhibition rates of 30.41% and 21.13% against acetylcholinesterase, respectively.

[0110] 10 9 10 inactivated bacteria / mL, 10 8 The inhibition rates of inactivated *Pediococcus lactis* SUN01 samples with 1 inactivated bacterial cell / mL against acetylcholinesterase were 22.78% and 18.81%, respectively.

[0111] Therefore, both the bacterial suspension and inactivated cells of *Pediococcus lactis* SUN01 exhibit strong inhibitory activity against acetylcholinesterase, thereby reducing acetylcholine consumption and contributing to the improvement of Alzheimer's disease (AD). The bacterial suspension and inactivated cells of *Pediococcus lactis* SUN01 can be used in the preparation of acetylcholinesterase inhibitors, and they have the advantages of safety and fewer side effects. In conclusion, the bacterial suspension and inactivated cells of *Pediococcus lactis* SUN01 can be used in the preparation of acetylcholinesterase inhibitors and corresponding functional products.

[0112] Example 5: Study on the production of γ-aminobutyric acid by Pediococcus lactis SUN01

[0113] 1. The content of γ-aminobutyric acid was determined by colorimetric method.

[0114] Plotting the standard curve for γ-aminobutyric acid:

[0115] Take 1 mL of γ-aminobutyric acid (GABA) standard solutions at concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, add 0.1 mL of 1 mol / L Na₂CO₃ solution, 0.5 mL of 0.2 mol / L borate buffer (pH=10.0), and 1 mL of 6% phenol solution. Mix well, and within 5 min at room temperature, add 1.0 mL of 5.2% NaClO solution, mix well, and let stand for 6 min, then immediately incubate on ice for 20 min. After a blue-green color appears, add 2 mL of 60% ethanol solution, mix well, and incubate on ice for 30 min. Use a blank reagent as a reference and measure the OD value at λ=640 nm. The results are as follows: Figure 2 The standard curve for γ-aminobutyric acid shown is Y = 0.3799X + 0.0182, R0 2 =0.9907.

[0116] Determination of γ-aminobutyric acid (GABA) content in the sample: Similar to the fermentation broth of *Pediococcus lactis* SUN01 activated to the second generation in Example 4, *Pediococcus lactis* SUN01 was activated to the second generation. The fermentation broth was centrifuged (8000 r / min, 5 min, 4℃), and the supernatant was collected to determine the GABA content. The determination method was the same as that used for the standard curve.

[0117] The test results showed that the γ-aminobutyric acid (GABA) content in the fermentation supernatant of *Pediococcus lactis* SUN01 was 0.38 mg / mL, indicating that *Pediococcus lactis* SUN01 has the ability to produce GABA. The GABA produced by this strain helps improve learning and memory abilities, as well as regulate mood and improve sleep.

[0118] Example 6: Determination of antioxidant activity of Pediococcus lactis SUN01

[0119] The ability of *Pediococcus lactis* SUN01 to scavenge DPPH free radicals was determined according to the method in Example 4. *Pediococcus lactis* SUN01 was activated to the second generation to obtain the fermentation broth of the strain.

[0120] Preparation of bacterial suspensions at different concentrations: The fermentation broth was centrifuged (8000 r / min, 5 min, 4℃), the supernatant was discarded, the bacterial sludge was collected, washed twice with physiological saline, and resuspended in PBS phosphate buffer (10 mmol / L, pH 7.4) to obtain 10 9 A bacterial suspension of CFU / mL, then 10 9 The bacterial suspension of CFU / mL was serially diluted 10-fold to obtain 10 8 CFU / mL, 10 7 CFU / mL bacterial suspension.

[0121] Preparation of inactivated bacterial cells at different concentrations: The above 10 9 The bacterial suspension of CFU / mL was sterilized at 115°C for 15 min and cooled to room temperature to obtain 10 9 A sample of inactivated bacterial cells per mL was prepared and then serially diluted 10-fold to obtain 10... 8 10 inactivated bacteria / mL, 10 7 One inactivated bacterial cell / mL of inactivated bacterial sample.

[0122] Preparation of fermentation supernatants of different concentrations: The fermentation broth was serially diluted 10-fold to obtain 10... 8 10 7 Fermentation broth of CFU / mL, 10 9 CFU / mL, 10 8 CFU / mL, 10 7 Centrifuge the fermentation broth at CFU / mL (8000 r / min, 5 min, 4℃) and collect the supernatant.

[0123] The DPPH free radical scavenging rate was determined by measuring the DPPH free radical scavenging capacity of bacterial suspensions, inactivated cells, and fermentation supernatants at different concentrations.

[0124] Take 100 μL of the sample to be tested and 100 μL of DPPH ethanol solution (make up to 100 mL with 0.0078 g of DPPH using anhydrous ethanol, where the DPPH concentration is 0.2 mmol / L), mix well, and react in the dark for 40 min to obtain the sample group; simultaneously set up a control group (using anhydrous ethanol instead of the sample solution) and a blank group (using anhydrous ethanol instead of the DPPH solution), and measure the absorbance at a wavelength of 517 nm. The calculation formula is:

[0125] DPPH free radical scavenging rate = [1-(A 样 -A空 / A 对 )]×100%.

[0126] Among them, A 样 A represents the absorbance at 517 nm measured for the sample group. 空 A represents the absorbance at 517 nm measured in the blank group. 对 The absorbance value at a wavelength of 517 nm was measured for the control group.

[0127] The measurement results are as follows Figure 3 As shown, at the highest concentration, the DPPH free radical scavenging rates of *Pediococcus lactis* SUN01 bacterial suspension, inactivated cells, and fermentation supernatant were 34.06%, 26.69%, and 109.29%, respectively. Even at the lowest concentration, *Pediococcus lactis* SUN01 bacterial suspension, inactivated cells, and fermentation supernatant also exhibited some scavenging ability. This indicates that *Pediococcus lactis* SUN01 possesses a strong DPPH free radical scavenging ability.

[0128] The ability of *Pediococcus lactis* SUN01 to scavenge ABTS free radicals was determined by preparing bacterial suspensions, inactivated cells, and fermentation supernatants of *Pediococcus lactis* SUN01 at different concentrations according to the method in the DPPH free radical scavenging experiment.

[0129] The ABTS free radical scavenging rate was determined by measuring the ABTS free radical scavenging capacity of bacterial suspensions, inactivated bacterial cells, and fermentation supernatants at different concentrations.

[0130] Take 100 μL of the sample to be tested and 100 μL of ABTS working solution, mix well, and react in the dark for 10 min. Simultaneously set up a control group (using distilled water instead of the sample solution) and a blank group (using distilled water instead of ABTS working solution), and measure the absorbance at a wavelength of 734 nm. The calculation formula is:

[0131] ABTS free radical scavenging rate = [1-(A 样 -A 空 / A 对 )]×100%.

[0132] Among them, A 样 A represents the absorbance value measured at 734 nm for the sample group. 空 A represents the absorbance at 734 nm measured in the blank group. 对 The absorbance value at a wavelength of 734 nm is the control group.

[0133] The preparation process of the ABTS working solution is as follows: Dissolve 0.0066 g of potassium persulfate in 5 mL of PBS buffer solution (0.2 mol / L pH=7.4), and dissolve 0.0192 g of ABTS in 5 mL of PBS buffer solution (0.2 mol / L pH=7.4). Mix these two solutions at a volume ratio of 1:1, incubate thoroughly, and store at 4°C in the dark overnight (12-16 h) to obtain the ABTS stock solution. Dilute the ABTS stock solution with PBS buffer solution (0.2 mol / L pH=7.4), in the dark, and after reacting for 10 min, measure the absorbance at a wavelength of 734 nm. Select the OD value... 734 nm The concentration of ABTS working solution is 0.7 ± 0.002.

[0134] The measurement results are as follows Figure 4 As shown:

[0135] At the highest concentration, the scavenging rates of ABTS free radicals by *Pediococcus lactis* SUN01 bacterial suspension, inactivated cells, and fermentation supernatant were 81.19%, 73.73%, and 101.49%, respectively. Even at the lowest concentration, *Pediococcus lactis* SUN01 bacterial suspension, inactivated cells, and fermentation supernatant also exhibited some scavenging ability. This indicates that *Pediococcus lactis* SUN01 possesses a strong ABTS free radical scavenging ability.

[0136] Example 7 Application of *Pediococcus lactis* SUN01 in the preparation of fermented plant extracts

[0137] 1. Preparation of fermented plant extract from *Pediococcus lactis* SUN01:

[0138] Weigh 25g of goji berries, add distilled water, heat, bring to a boil, then maintain a gentle boil and continue cooking for 1 hour. Filter through a 300-mesh filter cloth, collect the filtrate, add distilled water to a final volume of 200g, and cool to room temperature to obtain the goji berry extract. system Prepare jujube seed and licorice extract.

[0139] Add 2% (w / v, g / ml) glucose to the plant extracts (goji berry, jujube seed, and licorice extracts) as described above, dispense into containers, sterilize at 115℃ for 15 min, cool to room temperature, inoculate with twice-activated Pediococcus lactis SUN01 bacterial suspension at an inoculation rate of 3% (v / v, ml / ml), and culture at 37℃ for 36 h to obtain fermented plant extracts.

[0140] 2. Determination of the inhibition rate of acetylcholinesterase:

[0141] The fermented plant extract and the unfermented plant extract were centrifuged (8000 r / min, 5 min, 4 °C), and the supernatant was collected. The inhibitory activity of the supernatant sample against acetylcholinesterase (AChE) was determined according to the method in Example 4.

[0142] The test results show that:

[0143] The supernatants of unfermented wolfberry, jujube seed, and licorice extracts showed inhibition rates of 34.13%, 14.29%, and 19.05% against acetylcholinesterase, respectively.

[0144] The supernatants of fermented wolfberry, jujube seed, and licorice extracts by *Pediococcus lactis* SUN01 showed inhibition rates of 42.06%, 46.03%, and 49.21% on acetylcholinesterase, respectively.

[0145] This indicates that fermentation of extracts from wolfberry, jujube seed, and licorice with *Pediococcus lactis* SUN01 significantly increases their inhibition rate against acetylcholinesterase. *Pediococcus lactis* SUN01 was applied to the fermentation of extracts from wolfberry, jujube seed, and licorice.

[0146] Example 8: Application of Pediococcus lactis SUN01 in probiotic solid beverages

[0147] A probiotic solid beverage is obtained by mixing 2% of the freeze-dried Pyrococcus lactis SUN01 bacterial powder obtained in Example 2, 8% of sorbitol, 30% of skim milk powder, 23% of maltodextrin, 15% of fructooligosaccharides, 15% of fermented blueberry powder, and 7% of strawberry powder (these percentages are by mass).

[0148] It should be noted that this embodiment is only an example of the application of Pediococcus lactis SUN01 in probiotic solid beverages. Pediococcus lactis SUN01 can also be applied to other formulations of probiotic solid beverages, including but not limited to the probiotic solid beverage shown in Example 8 above.

[0149] Example 9: Application of Pediococcus lactis SUN01 in fermented beverages

[0150] Step 1: Weigh 1g of sesame seeds, 2g of yam, 0.8g of almonds, and 2g of peanuts. Add 94.2g of water, bring to a boil, and maintain a gentle boil for 1 hour. Filter through a 300-mesh screen. Sterilize the filtrate at 115℃ for 15 minutes. After cooling, add 0.001g of the freeze-dried *Pediococcus lactis* SUN01 culture powder from Example 2 and ferment for 24 hours.

[0151] Step 2: Take 71.899% of the fermentation liquid from Step 1 by mass percentage, add 3% soybean peptide, 10% albumin peptide, 15% fructooligosaccharide, 0.1% vitamin C, and 0.001% vitamin D to obtain a fermented plant beverage.

[0152] It should be noted that this embodiment is only an example of the application of Pediococcus lactis SUN01 in fermented beverages. Pediococcus lactis SUN01 can also be applied to fermented beverages with other formulations, including but not limited to the fermented beverage shown in Example 9 above.

[0153] Based on the results of the above embodiments, the SUN01 lactic acid cocci provided by the present invention has the following properties and effects:

[0154] 1. It has strong resistance to acid and bile salts, and is well tolerated in artificial gastric and intestinal fluids, allowing it to reach the human intestine smoothly;

[0155] 2. It has a high inhibition rate against acetylcholinesterase, 10 9 CFU / mL, 10 8 The CFU / mL suspension of *Pediococcus lactis* SUN01 showed inhibition rates of 30.41% and 21.13% against acetylcholinesterase, respectively; 10 9 10 inactivated bacteria / mL, 10 8 The inhibition rates of inactivated Pediococcus lactis SUN01 cells / mL against acetylcholinesterase were 22.78% and 18.81%, respectively.

[0156] 3. Lactococcus lactis SUN01 has the ability to produce γ-aminobutyric acid (GABA), and the GABA content in the fermentation supernatant of this strain is 0.38 mg / mL;

[0157] 4. The bacterial suspension, inactivated cells, and fermentation supernatant of *Pediococcus lactis* SUN01 have strong scavenging ability against DPPH and ABTS free radicals and have good antioxidant activity.

[0158] 5. Compared with unfermented plant extracts, plant extracts fermented with Pediococcus lactis SUN01 showed significantly increased inhibitory activity against acetylcholinesterase; among which, plant extracts included, but were not limited to, extracts of wolfberry, jujube seed, and licorice.

[0159] In summary, compared with the prior art, the *Pediococcus lactis* SUN01 provided by this invention has the following beneficial effects:

[0160] The SUN01 strain of *Pediococcus lactis* provided by this invention has strong acid and bile salt resistance, good tolerance in artificial gastric and intestinal fluids, and can successfully reach the human intestine.

[0161] Pediococcus lactis SUN01 possesses strong acetylcholinesterase inhibitory activity, γ-aminobutyric acid (GABA) production, and antioxidant properties, thus helping to improve Alzheimer's disease, enhance learning and memory abilities, regulate mood, and improve sleep. It can be applied to functional products containing the following effects:

[0162] (1) It has acetylcholinesterase inhibitory activity;

[0163] (2) Produces γ-aminobutyric acid;

[0164] (3) Antioxidant.

[0165] In this application, functional products with the effects described above (1)-(3) are described, including but not limited to those with manifest effects such as improving Alzheimer's disease, improving people's learning and memory abilities, regulating emotions, improving sleep, and anti-oxidation. These manifest effects are based on the correlation between the effects and efficacy of the above (1)-(3). For example, excessive AChE activity can promote the occurrence and development of Alzheimer's disease (AD), and having acetylcholinesterase inhibitory activity is beneficial to improving Alzheimer's disease. γ-aminobutyric acid is the main inhibitory neurotransmitter of the central nervous system, participates in various physiological activities of the human body, and has the effects of promoting sleep, enhancing memory, and relieving anxiety. Therefore, based on the correlation between the effects of the above (1)-(3) and other related manifest effects, the functional products can also play other manifest effects of inhibiting the occurrence and development of diseases and benefiting human health, including but not limited to those with manifest effects such as improving Alzheimer's disease, improving people's learning and memory abilities, regulating emotions, improving sleep, and anti-oxidation.

[0166] It should be noted that:

[0167] The relevant existing technical means or terms involved in this application are as follows:

[0168] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through a analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. "ODx" is the optical density value measured when the wavelength is set to x nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures, usually used to indicate bacterial cell density. The method for measuring "OD" values ​​is existing technology, and its principles and methods will not be elaborated here.

[0169] DPPH is a general abbreviation for 1,1-diphenyl-2-trinitrophenylhydrazine.

[0170] ABTS is a general abbreviation for 2,2-adiazon-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Pediococcus lactis ( Pediococcus acidilactici SUN01, characterized in that: Its accession number is CGMCC No. 29704.

2. A freeze-dried Powder of Pediococcus lactis SUN01, characterized in that: Its components include Pediococcus lactis SUN01 as described in claim 1.

3. The application of *Pediococcus lactis* SUN01 in inhibiting acetylcholinesterase for non-disease diagnostic or therapeutic purposes, characterized by: The *Pediococcus lactis* SUN01 used is the *Pediococcus lactis* SUN01 as described in claim 1.

4. The application of *Pediococcus lactis* SUN01 in the production of γ-aminobutyric acid, characterized by: The *Pediococcus lactis* SUN01 used is the *Pediococcus lactis* SUN01 as described in claim 1.

5. The application of *Pediococcus lactis* SUN01 in the preparation of fermented plant extracts, characterized by: The Pediococcus lactis SUN01 is the Pediococcus lactis SUN01 as described in claim 1; The fermented plant extract includes fermented wolfberry extract, fermented jujube seed extract, or fermented licorice extract.

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