A *Pediococcus pentosaceus* strain SUN02 with acetylcholinesterase inhibitory activity and its application

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Pediococcus pentosaceus* SUN02 with acetylcholinesterase inhibitory activity and its applications. This *Pediococcus pentosaceus* SUN02 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 29705. The *Pediococcus pentosaceus* SUN02 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 pentosaceus* SUN02, 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 pentosaceus SUN02 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 an enzymatic product of its precursor protein, β-amyloid precursor protein (APP). Secreted by cells, it accumulates in the cytosol and exhibits strong neurotoxicity, being a major 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] None of the publicly disclosed *Pediococcus pentosaceus* strains possess acetylcholinesterase inhibitory activity. Therefore, those skilled in the art are dedicated to developing probiotic *Pediococcus pentosaceus* strains with more sources and functions, such as probiotics possessing acetylcholinesterase inhibitory activity, γ-aminobutyric acid (GABA) production, and antioxidant functions. This would broaden the application areas of probiotic *Pediococcus pentosaceus* strains, particularly showing greater potential 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 pentosaceus SUN02.

[0009] This invention provides a strain of Pediococcus pentosaceus SUN02 ( Pediococcus pentosaceus It 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. 29705.

[0010] The Pediococcus pentosaceus SUN02 strain was isolated from Antarctic krill hydrolysate. 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 pentosaceus* SUN02 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 pentosaceus* SUN02 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 Pediococcus pentosacchari SUN02 bacterial powder, the components of which include the freeze-dried Pediococcus pentosacchari SUN02 bacterial powder described above.

[0013] In some embodiments, the viable count of *Pediococcus pentosaceus* SUN02 in the freeze-dried bacterial powder is (1–5) × 10⁻⁶. 11CFU / g.

[0014] The present invention also provides the use of Pediococcus pentosaceus SUN02 as described above in the preparation of acetylcholinesterase inhibitors.

[0015] The present invention also provides the use of Pediococcus pentosacchari SUN02 as described above in the production of γ-aminobutyric acid.

[0016] This invention also provides the application of *Pediococcus pentosaceus* SUN02, as described above, in the preparation of fermented plant extracts. The application of *Pediococcus pentosaceus* SUN02 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 Pediococcus pentosacchari SUN02 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 Pentosacchariformis SUN02 provided by the present invention has the following beneficial effects:

[0023] The *Pediococcus pentosaceus* SUN02 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 pentosaceus* SUN02 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, enhance learning and memory abilities, regulate mood, and improve sleep.

[0024] In summary, the *Pediococcus pentosaceus* SUN02 provided by this invention exhibits stronger acetylcholinesterase inhibition rates in both its bacterial suspension and inactivated 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 pentosacchari SUN02;

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

[0028] Figure 3 Graph showing the DPPH free radical scavenging activity of Pediococcus pentosaceus SUN02;

[0029] Figure 4 The results of ABTS free radical scavenging activity of Pediococcus pentosaceus SUN02 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 Pentosaccharidococcus ( Pediococcus pentosaceus SUN02:

[0033] Pediococcus pentosaceus ( Pediococcus pentosaceusSUN02 is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 29705;

[0034] Source: The Pediococcus pentosaccharis SUN02 strain was isolated from Antarctic krill hydrolysate. 16S rDNA sequencing of this strain was performed, and Blast sequence alignment showed that it was highly homologous to Pediococcus pentosaccharis. Therefore, it was named Pediococcus pentosaccharis SUN02.

[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 pentosaceus SUN02 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 inoculate with twice-activated Pediococcus pentosacchari SUN02 at an inoculation rate of 3% (v / v, unit ml / ml, i.e., 300ul of Pediococcus pentosacchari SUN02 bacterial solution per 10ml of sterilized plant extracts). 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 Pediococcus pentosaceus SUN02 seed culture;

[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 Pediococcus pentosaceus SUN02 freeze-dried 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 pentosaceus SUN02

[0050] 1. Separation and screening:

[0051] Aseptic sampling was performed on the hydrolysate of Antarctic krill using the plate coating method. 5g of the hydrolysate sample was placed in a sterile homogenizing bag, and 45mL of 0.85% physiological saline was added and 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 -4 100 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 pentosaccharis SUN02 and stored in a bacterial bank at -80°C with glycerol.

[0053] The colony morphology of the isolated and purified Pediococcus pentosaceus SUN02 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 pentosaccharis SUN02 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] ① Extraction of SUN02 bacterial genomic DNA: Bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.;

[0062] ②PCR 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 10XEasyTag@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] ③ 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 SUN02 was obtained as follows, and the strain was identified as Pediococcus pentosaceus SUN02.

[0068] 16S rDNA sequence:

[0069] AGGCAGTGGGGGCTGCTATACATGCAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCCGCCTGGGGAGTACGACCGCCAGGTTGAAACTCAAAGATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTTGATTTAATCCAAGC。

[0070] Blast alignment analysis of gene sequences was performed in the NCBI database, combined with colony morphology observation, Gram staining and catalase test, and homology analysis in the 16S rDNA phylogenetic tree (e.g., Figure 1 As shown), SUN02 was identified as Pediococcus pentosaceus (as shown). Pediococcus pentosaceus ) bacterial strains.

[0071] Example 2: Preparation of freeze-dried Pediococcus pentosaceus SUN02 bacterial powder

[0072] The activated Pediococcus pentosaceus SUN02 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, and incubated at 37°C for 24 h. After centrifugation (4°C, 8000 r / min, 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 pentosaceus SUN02 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 be other 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 pentosaceus SUN02

[0079] 1. Acid resistance

[0080] The Pediococcus pentosaceus SUN02 lyophilized bacterial powder obtained in Example 2 was inoculated at a rate of 1% (w / v, g / ml) into MRS liquid medium with pH 6.0, pH 4.0 and pH 2.0, respectively. After incubation at 37°C for 3 h, viable bacteria were counted using the plate count method. The results are shown in Table 1.

[0081] Table 1. Study on acid resistance of Pediococcus pentosacchari SUN02

[0082]

[0083] It can be seen that under pH 2.0 conditions, Pediococcus pentosaceus SUN02 still has a sufficient number of viable bacteria that can pass through the gastric environment after 3 hours, indicating 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 Pediococcus pentosaceus SUN02 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 pentosacchari SUN02

[0088]

[0089] It can be seen that at a bile salt concentration of 0.3%, Pediococcus pentosaceus SUN02 still retains a sufficient number of viable bacteria after 3 hours, indicating 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 Pediococcus pentosaceus SUN02 lyophilized bacterial powder obtained in Example 2 was inoculated into MRS liquid medium and artificial gastric fluid at an inoculation rate of 1% (w / v, g / ml) and cultured at 37°C for 3 h. Then, the fermentation broth in artificial gastric fluid was inoculated into artificial intestinal fluid at an inoculation rate of 10% (v / v, ml / ml) and cultured at 37°C for 3 h. Viable bacteria were counted using the plate count method. The results are shown in Table 3.

[0094] Table 3. Tolerance of Pediococcus pentosacchari SUN02 in simulated gastric and intestinal fluids.

[0095]

[0096] It can be seen that after passing through artificial gastric fluid and then artificial intestinal fluid, Pediococcus pentosaceus SUN02 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 pentosacetylcholinesterase SUN02

[0098] 1. Preparation of Pediococcus pentosaceus SUN02 bacterial suspension and inactivated bacterial cell samples

[0099] Take the glycerol cryopreservation tubes of Pediococcus pentosaceus SUN02 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 phosphate-buffered saline (PBS, 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 (using PBS instead of samples), blank control groups (using PBS instead of samples and acetylcholinesterase), and sample blank groups (using PBS instead of acetylcholinesterase in the sample groups) were set up.

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

[0105]

[0106]

[0107] Among them, in the enzyme-catalyzed reaction rate v In the calculation, △A 15 The absorbance value refers to the difference between the absorbance value of the control group or sample group and the corresponding absorbance value of the blank group at 15 min, while ΔA0 refers to the difference between the absorbance value of the control group or sample group and the corresponding absorbance value of the blank group at 0 min. In the calculation of the inhibition rate, v i The enzyme reaction rate of the sample group. v 0 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 pentosaceus SUN02 inhibited acetylcholinesterase by 29.41% and 18.56%, respectively.

[0110] 10 9 10 inactivated bacteria / mL, 10 8 The inhibition rates of inactivated Pediococcus pentosaceus SUN02 cells / mL against acetylcholinesterase were 25.43% and 18.14%, respectively.

[0111] Therefore, both the bacterial suspension and inactivated cells of *Pediococcus pentosaceus* SUN02 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 pentosaceus* SUN02 can be used in the preparation of acetylcholinesterase inhibitors, and they have the advantages of safety and fewer side effects. The bacterial suspension and inactivated cells of *Pediococcus pentosaceus* SUN02 can be used in the preparation of acetylcholinesterase inhibitors and functional products with corresponding functions.

[0112] Example 5: Study on the production of γ-aminobutyric acid by Pediococcus pentosaceus SUN02

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

[0114] Plotting the γ-aminobutyric acid (GABA) standard curve: Take 1 mL of γ-aminobutyric acid standard solutions 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 NaCO3 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.

[0115] Determination of γ-aminobutyric acid (GABA) content in the sample: Similar to the fermentation broth of *Pediococcus pentosaccharis* SUN02 activated to the second generation in Example 4, *Pediococcus pentosaccharis* SUN02 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 of the standard curve.

[0116] The test results showed that the γ-aminobutyric acid (GABA) content in the fermentation supernatant of Pediococcus pentosaceus SUN02 was 0.30 mg / mL, indicating that Pediococcus pentosaceus SUN02 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.

[0117] Example 6: Determination of antioxidant activity of Pediococcus pentosaceus SUN02

[0118] 1. Determination of the DPPH free radical scavenging ability of Pediococcus pentosaceus SUN02

[0119] Following the method in Example 4, Pediococcus pentosaceus SUN02 was activated to the second generation to obtain the strain's fermentation broth.

[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 phosphate-buffered saline (PBS, 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 then 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 bacterial 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 pentosaceus* SUN02 bacterial suspension, inactivated cells, and fermentation supernatant were 30.43%, 24.50%, and 103.71%, respectively. Even at the lowest concentration, *Pediococcus pentosaceus* SUN02 bacterial suspension, inactivated cells, and fermentation supernatant also exhibited some scavenging ability. This indicates that *Pediococcus pentosaceus* SUN02 possesses a strong DPPH free radical scavenging ability.

[0128] 2. Determination of the ABTS free radical scavenging ability of Pediococcus pentosaceus SUN02

[0129] Following the method in the DPPH free radical scavenging experiment, bacterial suspensions, inactivated cells, and fermentation supernatants of Pediococcus pentosaceus SUN02 at different concentrations were prepared.

[0130] 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.

[0131] 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:

[0132] ABTS free radical scavenging rate = [1-(A 样 -A 空 / A 对 )]×100%.

[0133] 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.

[0134] 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.

[0135] The measurement results are as follows Figure 4 As shown:

[0136] At the highest concentration, the scavenging rates of ABTS free radicals by *Pediococcus pentosaceus* SUN02 bacterial suspension, inactivated cells, and fermentation supernatant were 89.40%, 82.39%, and 97.01%, respectively. Even at the lowest concentration, *Pediococcus pentosaceus* SUN02 bacterial suspension, inactivated cells, and fermentation supernatant also exhibited some scavenging ability. This indicates that *Pediococcus pentosaceus* SUN02 possesses a strong ABTS free radical scavenging ability.

[0137] Example 7 Application of Pediococcus pentosaceus SUN02 in the preparation of fermented plant extracts

[0138] 1. Preparation of plant extract from *Pediococcus pentosaceus* SUN02 fermentation:

[0139] Weigh 25 g of wolfberries, add distilled water, heat, bring to a boil, then maintain a gentle boil for 1 hour. Filter through a 300-mesh filter cloth, collect the filtrate, add distilled water to a final volume of 200 g, and cool to room temperature to obtain the wolfberry extract. Prepare jujube seed and licorice extracts using the same method.

[0140] 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, and inoculate with twice-activated Pediococcus pentosacchariformis SUN02 bacterial suspension at an inoculation rate of 3% (v / v, ml / ml). Incubate at 37℃ for 36 h to obtain fermented plant extracts.

[0141] 2. Determination of the inhibition rate of acetylcholinesterase:

[0142] 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.

[0143] The test results show that:

[0144] The supernatants of unfermented wolfberry, jujube seed, and licorice extracts showed inhibition rates of 34.13%, 14.29%, and 19.05% against acetylcholinesterase, respectively.

[0145] The supernatants of *Pediococcus pentosaceus* SUN02 fermented extracts of wolfberry, jujube seed, and licorice showed inhibition rates of 65.08%, 67.46%, and 27.78% against acetylcholinesterase, respectively. This indicates that fermentation with *Pediococcus pentosaceus* SUN02 significantly enhances the inhibition rate of acetylcholinesterase in these extracts. *Pediococcus pentosaceus* SUN02 was then applied to the fermentation of extracts of wolfberry, jujube seed, and licorice.

[0146] Example 8: Application of Pediococcus pentosaceus SUN02 in probiotic solid beverages

[0147] A probiotic solid beverage is obtained by mixing 2% of the freeze-dried Pediococcus pentosaceus SUN02 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 pentosaccharis SUN02 in probiotic solid beverages. Pediococcus pentosaccharis SUN02 can also be applied to probiotic solid beverages with other formulations, including but not limited to the probiotic solid beverage shown in Example 8 above.

[0149] Example 9: Application of Pediococcus pentosaceus SUN02 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 Pediococcus pentosaceus SUN02 freeze-dried bacterial 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 pentosacchari SUN02 in fermented beverages. Pediococcus pentosacchari SUN02 can also be applied to fermented beverages in 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 *Pediococcus pentosaccharide* SUN02 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 juice and artificial intestinal juice, and can smoothly reach the human intestine;

[0155] 2. It has a high inhibition rate against acetylcholinesterase, 10 9 CFU / mL, 10 8 The CFU / mL suspension of *Pediococcus pentosaceus* SUN02 showed inhibition rates of 29.41% and 18.56% against acetylcholinesterase, respectively. 9 10 inactivated bacteria / mL, 10 8 The inhibition rates of inactivated Pediococcus pentosaceus SUN02 cells / mL against acetylcholinesterase were 25.43% and 18.14%, respectively.

[0156] 3. Pediococcus pentosaceus SUN02 has the ability to produce γ-aminobutyric acid (GABA), and the GABA content in the fermentation supernatant of this strain is 0.30 mg / mL;

[0157] 4. The bacterial suspension, inactivated cells, and fermentation supernatant of Pediococcus pentosaceus SUN02 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 pentosaceus SUN02 showed significantly enhanced inhibitory activity against acetylcholinesterase. These plant extracts include, but are not limited to, extracts from wolfberry, jujube seed, and licorice.

[0159] In summary, compared with the prior art, the Pediococcus pentosaceus SUN02 provided by the present invention has the following beneficial effects:

[0160] The Pediococcus pentosacchari strain SUN02 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 pentosaceus SUN02 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 therein. Such 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 pentosaceus ( Pediococcus pentosaceus SUN02, characterized in that: Its accession number is CGMCC No. 29705.

2. A freeze-dried Pediococcus pentosaceus SUN02 bacterial powder, characterized in that: Its components include Pediococcus pentosaceus SUN02 as described in claim 1.

3. The application of Pediococcus pentosaceus SUN02 in the inhibition of acetylcholinesterase for non-disease diagnostic or therapeutic purposes, characterized by: The Pediococcus pentosaccharis SUN02 is the Pediococcus pentosaccharis SUN02 as described in claim 1.

4. The application of Pediococcus pentosaceus SUN02 in the production of γ-aminobutyric acid, characterized in that: The Pediococcus pentosaccharis SUN02 is the Pediococcus pentosaccharis SUN02 as described in claim 1.

5. The application of Pediococcus pentosaceus SUN02 in the preparation of fermented plant extracts, characterized by: The Pediococcus pentosaccharis SUN02 is the Pediococcus pentosaccharis SUN02 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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