Lactobacillus plantarum and application thereof in preparation of insomnia relieving product

Abstract

The invention belongs to the technical field of food microorganisms, and relates to lactobacillus plantarum and application thereof in preparation of products for relieving insomnia. The preservation number of the lactobacillus plantarum is GDMCC No: 65061. The lactobacillus plantarum can be applied to efficient fermentation of spina date seed juice, the content of GABA, polypeptide, total phenols and total flavonoids in the spina date seed juice is increased, and the content of spinosin, 6 ''-feruloyl spinosin, jujuboside A, jujuboside B and betulinic acid is increased; the reduction of whole brain coefficient and the increase of liver coefficient caused by long-term insomnia are relieved, the bad condition caused by long-term insomnia in a hippocampus DG region in the brain of the mouse is improved, the sleep time of the mouse is prolonged, and the insomnia symptom of the mouse is relieved; the content of inhibitory neurotransmitters 5-hydroxytryptamine, melatonin and gamma-aminobutyric acid in the hypothalamus of the mouse is increased, the content of excitatory neurotransmitter glutamic acid is reduced, and the reduction of organism immunity and oxidative stress of brain tissues caused by insomnia are relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of food microbiology, and particularly relates to a Lactobacillus plantarum and its application in the preparation of products for relieving insomnia. Background Art

[0002] Lactobacillus plantarum is a kind of lactic acid bacteria. Lactic acid bacteria are the most representative probiotics and are widely present in traditional fermented products. The organic acids and some other small-molecule chemical substances produced by them can play a role in inhibiting the growth of other bacteria. In plant-based fermented beverages, lactic acid bacteria fermentation can play a role in breaking the cell wall, thereby improving the efficiency of the dissolution of nutrients and beneficial substances in plant cells; the protease produced by lactic acid bacteria will decompose proteins to produce rich polypeptides, including some polypeptides with special physiological effects, and can also improve the utilization rate of proteins. Lactic acid bacteria are also physiological bacteria in the human intestine. It can adjust the complex flora in the human intestine and relieve intestinal diseases. Therefore, the development of functional products of lactic acid bacteria has gradually become a research hotspot.

[0003] γ-aminobutyric acid (GABA for short) is a non-protein natural amino acid widely present in vertebrates, plants and microorganisms. In the human body, GABA is an important inhibitory neurotransmitter in the central nervous system, has high physiological activity, participates in the regulation of various life activities, and has health-care effects such as lowering blood pressure, anti-arrhythmia, and anti-anxiety. As a bioactive substance, it has been widely used in various drugs and foods.

[0004] Semen Ziziphi Spinosae is the dried mature seed of the plant Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow of the family Rhamnaceae, and has various effects such as nourishing the liver, calming the mind, sedating, hypnotizing, arresting sweating, promoting the production of body fluids, lowering blood pressure, lowering blood lipids, and reducing fever. Semen Ziziphi Spinosae has a long history of medicinal use and belongs to the first batch of dual-purpose substances of medicine and food promulgated by the Ministry of Health, which is safe and effective for the human body. The chemical constituents of Semen Ziziphi Spinosae are very rich. So far, more than 130 bioactive substances have been identified, including saponins, flavonoids, alkaloids, fatty acids, polysaccharide compounds, etc. Among them, flavonoids are important active ingredients in Semen Ziziphi Spinosae, and their content can reach 1.59%. At present, more than 50 flavonoid compounds have been isolated from Semen Ziziphi Spinosae, mainly flavone-C-glycosides such as spinosin and its acylated derivatives, and flavone-O-glycosides such as rutin and hesperidin, all of which have the effects of enhancing memory, relieving depression and reducing anxiety, and also have certain help for improving human immunity and anti-aging.

[0005] Saponin components in wild jujube seeds not only have high contents but also diverse structures. They are the most complex and widely studied chemical components, with strong central inhibitory effects and are generally considered the main substances responsible for the sedative and hypnotic effects of wild jujube seeds. The main saponins in wild jujube seeds are dammarane-type triterpenoid saponins, which consist of aglycones and sugars, uronic acids or other organic acids. The sugar groups are usually linked to the C-3 of the aglycone. According to the differences in the aglycone structures generated after hydrolysis, jujuboside is divided into tetracyclic triterpenoids and pentacyclic triterpenoids. So far, researchers have isolated four triterpenoid saponin components from wild jujube seeds, including jujuboside A, jujuboside B, jujuboside G and jujuboside A1, and have also identified various pentacyclic triterpenoid saponins from wild jujube seeds, such as betulinic acid, oleanolic acid, pomolic acid, betulinic acid, lupeol, etc. The good physiological effects of wild jujube seeds make them have broad application prospects in the food and pharmaceutical fields, and their enhancement and transformation are the focus of modern food research. Summary of the Invention

[0006] The object of the present invention is to provide a Lactobacillus plantarum GD 06 strain, which can improve the GABA content, polypeptide content, and the contents of sedative and sleep-aiding active substances such as total phenols, total flavonoids and spinosin in wild jujube seeds through fermentation.

[0007] The second object of the present invention is to provide a product for relieving insomnia prepared by using the above-mentioned Lactobacillus plantarum.

[0008] In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

[0009] The present invention provides a Lactobacillus plantarum strain that produces γ-aminobutyric acid and is suitable for fermenting wild jujube seed juice. The strain is Lactobacillus plantarum GD 06, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on August 27, 2024, with the deposit number GDMCC No: 65061.

[0010] The present invention also provides the application of the Lactobacillus plantarum GD 06, the culture solution or bacterial suspension containing the Lactobacillus plantarum GD 06 in the preparation of a product for relieving insomnia. In a specific embodiment, the product is a food or a health product. More specifically, the functional product is fermented wild jujube seed juice.

[0011] The present invention further provides a method for fermenting wild jujube seed juice with the Lactobacillus plantarum GD 06, which includes the following steps:

[0012] (1) Strain domestication: Prepare a domestication medium, which includes wild jujube seed juice and MRS liquid medium; use the gradual domestication method to domesticate and culture Lactobacillus plantarum GD 06, gradually increasing the concentration of wild jujube seed juice in the domestication medium, that is, inoculate Lactobacillus plantarum GD 06 from the domestication medium with low-concentration wild jujube seed juice to the domestication medium with high-concentration wild jujube seed juice step by step, and obtain a strain with good fermentation ability in pure wild jujube seed juice;

[0013] (2) Fermentation of wild jujube seed juice: Inoculate the domesticated Lactobacillus plantarum GD 06 in (1) into wild jujube seed juice for fermentation; the inoculation amount of Lactobacillus plantarum GD 06 is 6.5 - 7.0 log CFU / mL

[0014] Furthermore, in step (1), the domestication temperature of the strain is 35 - 37 °C, and the domestication duration of each domestication medium is 20 - 24 h; in step (2), the fermentation temperature is 35 - 37 °C, and the fermentation duration is 24 - 48 h.

[0015] Furthermore, the preparation method of the wild jujube seed juice is as follows: Add distilled water to wild jujube seed powder, with a material-to-water ratio of 1:18. After ultrasonic treatment, add sucrose and ascorbic acid. By weight, the wild jujube seed powder:sucrose:ascorbic acid = 5:1:0.01.

[0016] Furthermore, the preparation method of the wild jujube seed powder is as follows: Crush wild jujube seeds with a crusher, sieve through a 60-mesh sieve, and then dry to obtain it.

[0017] The present invention also provides a fermented wild jujube seed juice, which is obtained by fermenting wild jujube seed juice with Lactobacillus plantarum GD 06.

[0018] Furthermore, the viable count of Lactobacillus plantarum GD 06 in the fermented wild jujube seed juice is 8.0 - 8.75 log CFU / mL.

[0019] The beneficial effects of the present invention include:

[0020] Lactobacillus plantarum GD 06 of the present invention has good tolerance to simulated gastric juice and simulated intestinal juice, and the GABA yield is significantly higher than that of the positive control strain Lactobacillus plantarum JYLP-326 (Lactobacillus plantarum JYLP-326 is a commercially applied strain with the functions of producing GABA and improving sleep). After the domestication experiment, the acid production ability and viable count of Lactobacillus plantarum GD 06 in pure wild jujube seed juice have been significantly improved compared with those before domestication.

[0021] The Lactobacillus plantarum GD 06 of the present invention can be applied to ferment wild jujube seed juice and increase the contents of polypeptides, total flavonoids, total phenols, spinosin, 6'''-feruloylspinosin, jujuboside A, jujuboside B and betulinic acid in the wild jujube seed juice. The Lactobacillus plantarum GD 06 of the present invention can significantly increase the content of active substances with the effect of improving insomnia in the wild jujube seed beverage. The fermented wild jujube seed juice can relieve the decrease of the whole brain coefficient and the increase of the liver coefficient caused by long-term insomnia, improve the reduction of cell number and the sparse arrangement in the DG area of the hippocampus in the mouse brain caused by long-term insomnia, prolong the sleeping time of the mouse and relieve the insomnia symptoms of the mouse; increase the contents of inhibitory neurotransmitters 5-hydroxytryptamine, melatonin and γ-aminobutyric acid in the hypothalamus of the mouse, reduce the content of excitatory neurotransmitter glutamate, and relieve the decrease of body immunity and oxidative stress of brain tissue caused by insomnia. Description of the Drawings

[0022] Figure 1 : Primary screening result diagram of γ-aminobutyric acid-producing strains; Lane 1 - GABA standard; Lane 8 - Strain G1.2; Lane 9 - Strain GD 06; Lane 20 - Strain M2.2.

[0023] Figure 2 : Re-screening result diagram of γ-aminobutyric acid-producing strains.

[0024] Figure 3 : A is the colony morphology diagram of Lactobacillus plantarum GD 06; B is the microscopic examination result diagram of Lactobacillus plantarum GD 06.

[0025] Figure 4 : A is the growth curve of Lactobacillus plantarum GD 06 in MRS medium; B is the change of pH of Lactobacillus plantarum GD 06 in MRS medium.

[0026] Figure 5 : Tolerance comparison of Lactobacillus plantarum GD 06 and Lactobacillus plantarum JYLP-326 in simulated gastric juice and simulated intestinal juice containing bile salts.

[0027] Figure 6 : A is the determination of pH difference during the domestication of Lactobacillus plantarum GD 06 in wild jujube seed juice; B is the determination of viable cell count during the domestication of Lactobacillus plantarum GD 06 in wild jujube seed juice.

[0028] Figure 7 : A is the determination of pH difference of Lactobacillus plantarum GD 06 in wild jujube seed juice before and after domestication; B is the determination of viable cell count of Lactobacillus plantarum GD 06 in wild jujube seed juice before and after domestication.

[0029] Figure 8: A shows the change in the viable cell count of Lactobacillus plantarum GD 06 after domestication in wild jujube seed juice; B shows the change in pH value of Lactobacillus plantarum GD 06 after domestication in wild jujube seed juice; C shows the change in titratable acidity of Lactobacillus plantarum GD 06 after domestication in wild jujube seed juice.

[0030] Figure 9 : Change in GABA content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice.

[0031] Figure 10 : Change in total sugar content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice.

[0032] Figure 11 : Change in total phenol content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice.

[0033] Figure 12 : Change in soluble protein content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice.

[0034] Figure 13 : Change in polypeptide content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice.

[0035] Figure 14 : Change in total flavonoid content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice.

[0036] Figure 15 : A shows the change in spinosin content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice; B shows the change in 6'''-feruloylspinosin content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice; C shows the change in jujuboside A content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice; D shows the change in jujuboside B content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice; E shows the change in betulinic acid content of Lactobacillus plantarum GD 06 after domestication during the fermentation of wild jujube seed juice.

[0037] Figure 16 : Brain HE staining section diagram.

[0038] Figure 17 : a Tail suspension test - first immobility time; b Tail suspension test - immobility duration.

[0039] Figure 18 : a Sleep cooperation test - sleep latency; b Sleep cooperation test - sleep duration.

[0040] Figure 19: a Serotonin content in the hypothalamus of mice; b Melatonin content in the hypothalamus of mice; c Dopamine content in the hypothalamus of mice.

[0041] Figure 20 : a Gamma-aminobutyric acid content in the hypothalamus of mice; b Glutamic acid content in the hypothalamus of mice; c Gamma-aminobutyric acid / glutamic acid ratio in the hypothalamus of mice.

[0042] Figure 21 : a IL-6 content in the whole brain of mice; b IL-1β content in the whole brain of mice; c TNF-α content in the whole brain of mice.

[0043] Figure 22 : a MDA content in the whole brain of mice; b SOD activity in the whole brain of mice; c GSH-Px activity in the whole brain of mice.

[0044] Preservation information:

[0045] Lactiplantibacillus plantarum GD 06:

[0046] Preservation time: August 27, 2024;

[0047] Name of the preservation unit: Guangdong Microbial Culture Collection Center;

[0048] Preservation number: GDMCC No: 65061;

[0049] Address of the preservation unit: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province;

[0050] Taxonomic name: Lactiplantibacillus plantarum GD 06. Detailed implementation manners

[0051] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0052] Example 1

[0053] I. Screening of lactic acid bacteria producing gamma-aminobutyric acid

[0054] 1. Primary screening

[0055] Twenty-two lactic acid bacteria samples were isolated from traditional fermented foods for screening. The lactic acid bacteria were activated for two generations and inoculated into MRS medium supplemented with 2% sodium glutamate at an inoculum size of 6.8 log CFU / mL, and cultured statically at 37 °C for 48 h and shaken (180 r / min) at 37 °C for 48 h, respectively. The fermentation broth was centrifuged (6000 r, 4 °C, 15 min), and the supernatant was taken for thin layer chromatography analysis.

[0056] The thin layer chromatography analysis was to detect GABA in the supernatant by thin layer chromatography (silagel 60F254 thin layer chromatography plate). The developing agent used was: n-butanol: glacial acetic acid: water (volume ratio) = 2:1:1, and 0.8% ninhydrin was added as the developer. The specific steps were as follows:

[0057] Take 1 μL of the fermentation supernatant and spot it at 1 cm from the bottom of the thin layer chromatography plate. Pour 0.7 cm of the developing agent into the chromatography tank, then place the thin layer chromatography plate into it, cover the lid, and wait until the developing agent has expanded to 1 cm from the upper edge of the thin layer chromatography plate. Take it out, dry it, and put it into an oven at 95 °C for color development for 5 min.

[0058] As Figure 1 shown, after color development, it was found that the fermentation broths of 3 strains of bacteria (lanes 8, 9, and 20) had darker colored spots at the same position as the GABA standard (lane 1) in terms of the retention factor (Rf). Therefore, these 3 strains of bacteria were preliminarily screened to have the ability to produce GABA.

[0059] 2. Re-screening

[0060] The content of GABA was determined by high performance liquid chromatography:

[0061] Mobile phase preparation: Mobile phase A: Weigh 8.205 g of sodium acetate and dissolve it in 900 mL of ultrapure water, add 0.5 mL of triethylamine, 0.7 mL of acetic acid, and 5.0 mL of acetonitrile, adjust the pH to 5.8, and make up the volume to 1000 mL.

[0062] Mobile phase B: Acetonitrile: water (60:40, volume ratio).

[0063] Chromatographic conditions: Chromatographic column Venysil ASB C18 (4.6 mm × 250 mm, 5 μm); detection wavelength 254 nm; injection volume 20 μL, column temperature 25 °C. Mobile phase A:B = 80:20, with linear isocratic elution at 0.6 mL / min.

[0064] Derivatization method: Take 100 μL of the standard solution or the supernatant of the sample fermentation broth and add it to a 1.5 mL conical centrifuge tube. Add 50 μL of acetonitrile - water - triethylamine - PITC (7:1:1:1, volume ratio). After mixing and standing at room temperature for 1 h, add 150 μL of mobile phase A - mobile phase B (80:20, volume ratio), shake on a vortex oscillator for 1 min, and then stand for 10 min. Aspirate the lower layer solution with a syringe, filter it through a 0.22 μm aqueous filter membrane and inject it into a liquid phase vial.

[0065] As Figure 2 shown, the GABA production of the strains cultured statically was significantly higher than that of the strains cultured on a shaker at 180 r / min. The strain numbered GD06 had the highest production, reaching 0.84 mg / mL.

[0066] II. Strain identification

[0067] 1. Colony characteristics

[0068] As Figure 3 shown in A, after culturing the strain GD06 on MRS agar medium for 48 h, the colony diameter of GD06 was between 0.3 - 1.0 mm. The colonies were round, with a raised middle, neat edges, smooth and moist, and milky white with neat edges.

[0069] 2. Morphology under the microscope

[0070] As Figure 3 shown in B, the strain GD06 was a straight bacillus with round ends, single, in pairs or in short chains, without flagella and not producing spores.

[0071] 3. 16S rDNA identification

[0072] The selected strain GD06 was identified by 16S rDNA gene sequencing. The target genomic DNA was extracted using a DNA extraction kit (Tiangen Biotech Co., Ltd.). PCR experiments for 16S rDNA were carried out using two universal primers, 27F and 1492R. The PCR amplification products were detected and photographed by agarose gel electrophoresis, and the amplified fragment length was about 1.5 kb. The PCR products were sent to BGI Tech Solutions Co., Ltd. in Shenzhen for sequencing and compared with the NCBI BLAST sequence database to identify the strain at the species level. The 16S rDNA sequence of strain GD06, after comparison, had a homology of more than 99% with Lactobacillus plantarum. The 16S rDNA sequence (5' - 3') of strain GD06 is as follows:

[0073] GGAGGTGGCGGCGTGCTATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTG

[0074] CATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCC

[0075] CAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGC

[0076] ATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTA

[0077] GCTAGATGGTGAGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTA

[0078] ATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGA

[0079] ATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTT

[0080] CGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTG

[0081] ACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG

[0082] GTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTC

[0083] TGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGT

[0084] GCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGGG

[0085] ACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATG

[0086] GGTAGGCGAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGGTGAATGCTAAG

[0087] GTGTTGGAGGGTTTCGCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGG

[0088] GGGAGTACGGCCCCAAGGCTAAACTCAGAGGATTTGACGAAGCCCGCACAAGCGGTGG

[0089] AGCGTGTGGGTTTAGTTCGAATCTACGCGAGGAGCCGTGACCAAGTCTGACGTGGGTAT

[0090] GCGAGATCTAAAAGTAACATCGCGTCGGTCACTCGATGCAGGTGGTTCATGGAGGTCGT

[0091] CGGCTCGGGACTGAAATGTGTGTGTAAGTGCCGGGGGCGAGCCCACCATAAGTATCAGT

[0092] TCAGGAATAGGTTGCCACTCGGGGAGGGAGCAGGTGAGAGGCGGGGGGCAATTTGTCA

[0093] CTTAGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGGGTTACAAACTCTCATG

[0094] GTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGC

[0095] GATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAAT

[0096] GGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGC

[0097] ACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCG

[0098] GTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGG

[0099] GTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATG

[0100] CACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTC

[0101] AAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGC

[0102] GGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGC

[0103] TTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGT

[0104] TTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGC

[0105] GTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATT

[0106] TCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGAT

[0107] GCACTTCTTCGGTTGAGCCGAAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTC

[0108] GCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGCTGCTGGCA

[0109] CGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCATACCTGACAGTACTCTCAGATATGT

[0110] TCTTCTTAACACAGAGTTTACGAGCGAAACCCTCTCACTCACGCGGCGTGCTCATCGACTTTCGTCCAATGTGAGATCCCTACTGCTGCCTCCCGTAGGAGTTTGGCCGTGTTCTCATC。

[0111] Combined with the sequence alignment results and physiological and biochemical results of strain GD 06, it was determined that the screened lactic acid bacterium GD 06 was Lactiplantibacillus plantarum.

[0112] III. Determination of probiotic characteristics

[0113] The positive control strain selected in this example was the commercial strain Lactiplantibacillus plantarum JYLP-326 that produces GABA and has the effect of relieving insomnia, which was isolated and purified from Jiajia High-Pressure Tablet Candy (Zhengzhou Linuo Pharmaceutical Co., Ltd., γ-aminobutyric acid compound probiotic tablet candy, production date April 9, 2024).

[0114] 1. Determination of acid production and growth curve of Lactiplantibacillus plantarum GD 06

[0115] Lactiplantibacillus plantarum GD 06 was activated and passaged, and inoculated into MRS liquid medium (pH = 6.2) at an inoculum size of 6.8 log CFU / mL, and cultured at 37 °C for 24 hours. The pH value and OD 600 value were measured every 2 h. As Figure 4 shown in A, Lactiplantibacillus plantarum GD 06 and Lactiplantibacillus plantarum JYLP-326 reached the stationary phase at 18 h, and the proliferation and growth tended to level off. As Figure 4 shown in B, the pH value of Lactiplantibacillus plantarum GD06 and Lactiplantibacillus plantarum JYLP-326 decreased rapidly within 12 h, and remained basically unchanged after 12 h. The growth and acid production ability of Lactiplantibacillus plantarum GD 06 were better than those of Lactiplantibacillus plantarum JYLP-326.

[0116] 2. Determination of the ability of Lactiplantibacillus plantarum GD 06 to tolerate simulated gastrointestinal fluids

[0117] Preparation of bacterial suspension: Lactobacillus plantarum GD 06 and Lactobacillus plantarum JYLP-326 were activated for two generations and then centrifuged at 4000 r / min and 4 °C for 10 min. The precipitate was taken, washed three times with 0.85% sterile saline by mass fraction, and then resuspended. The concentration of the bacterial suspension was uniformly adjusted to 1×10 8 CFU / mL, and the obtained bacterial suspension was that of Lactobacillus plantarum GD 06 and Lactobacillus plantarum JYLP-326.

[0118] Simulated gastric juice: The pH of 0.1 mol / L potassium phosphate buffer was adjusted to 2.5, sterilized, and then 10 g / L pepsin was added and dissolved thoroughly. It was preheated at 37 °C before use.

[0119] Simulated intestinal juice: The pH of 0.1 mol / L potassium phosphate buffer was adjusted to 8.0 and then sterilized. 10 g / L trypsin and 3 g / L porcine bile salt were added, and the mixture was dissolved thoroughly to obtain simulated intestinal juice. It was preheated at 37 °C before use.

[0120] 1 mL of the bacterial suspension of Lactobacillus plantarum GD 06 was respectively pipetted and inoculated into 9 mL of simulated gastric juice and simulated intestinal juice. After incubating at 37 °C for 3 h and 4 h respectively, Lactobacillus plantarum GD 06 was determined by the plate counting method. The tolerance rate was calculated according to formula Ⅰ:

[0121]

[0122] As Figure 5 shown, Lactobacillus plantarum GD 06 showed good tolerance in simulated gastric juice with a pH of 2.5 and simulated intestinal juice with a pH of 8.0 containing bile salts, and the tolerance rate in simulated gastric juice was significantly higher than that of the positive control Lactobacillus plantarum JYLP-326. Therefore, Lactobacillus plantarum GD 06 has good performance in tolerating the adverse gastrointestinal environment.

[0123] Example 2

[0124] Preparation method of wild jujube seed juice, including the following steps:

[0125] (1) Preparation of wild jujube seed powder: The wild jujube seeds were pulverized by a pulverizer for 25 s, passed through a 60-mesh sieve, dried overnight in an oven at 45 °C, and then placed in a drying tank for standby.

[0126] (2) Preparation of wild jujube seed juice: Weigh 10 g of the dried wild jujube seed powder, add 180 mL of distilled water, with a material-to-water ratio of 1:18, perform ultrasonic treatment for 40 min, add 2.0 g of sucrose and 0.02 g of ascorbic acid, and sterilize at 121 °C for 20 min for standby.

[0127] Example 3

[0128] Method for strain domestication, including the following steps:

[0129] Strain domestication: The strain was domestically cultured by the gradual domestication method. The concentration of wild jujube seed juice in the domestication medium was gradually increased, and the strain was inoculated step by step from the domestication medium with low-concentration wild jujube seed juice to the domestication medium with high-concentration wild jujube seed juice, so as to obtain a strain with good fermentation ability in pure wild jujube seed juice.

[0130] Process of strain domestication: Six groups of domestication media were prepared, numbered 1-6 in sequence. 0, 10, 20, 30, 40, and 50 mL of wild jujube seed juice and 50, 40, 30, 20, 10, and 0 mL of MRS liquid medium were added to the domestication media 1-6 respectively.

[0131] The initial pH values of the domestication media 1-6 before fermentation were measured and recorded respectively; 2 mL of the GD 06 bacterial suspension prepared in Example 1 was aspirated and added to the domestication medium 1. After static culture at 37 °C for 24 h, the pH value of the fermentation broth was measured, and 0.4 mL of the bacterial liquid was aspirated and gradient diluted with 4.6 mL of normal saline and then spread on the MRS solid medium. After culturing at 37 °C for 48 h, the viable bacteria count was calculated. At the same time, 2 mL of the bacterial suspension after fermentation of the domestication medium 1 was aspirated and added to the domestication medium 2, and domesticated and cultured at 37 °C for 24 h. The pH value and viable bacteria count after fermentation were measured, and the method was the same as the above operation. This operation was repeated and inoculated to the domestication medium 6 in turn to obtain a strain with good fermentation ability in pure wild jujube seed juice.

[0132] As Figure 6 shown in A, as the proportion of wild jujube seeds gradually increased during the domestication process, the pH difference showed a gradually increasing trend before the fifth generation and decreased when cultured in pure wild jujube seed juice of the sixth generation. The pH difference of Lactobacillus plantarum GD 06 was always higher than that of Lactobacillus plantarum JYLP-326 during the domestication process, indicating that the acid-producing ability of Lactobacillus plantarum GD 06 in wild jujube seed juice was higher than that of Lactobacillus plantarum JYLP-326.

[0133] As Figure 6 shown in B, as the proportion of wild jujube seed juice gradually increased during the domestication process, the viable bacteria count showed a slight decrease. The final viable bacteria counts of Lactobacillus plantarum GD 06 and Lactobacillus plantarum JYLP-326 in pure wild jujube seed juice reached 8.44 and 8.64 log CFU / mL respectively.

[0134] From Figure 7 A, it can be seen that after the domesticated Lactobacillus plantarum GD 06 and Lactobacillus plantarum JYLP-326 were inoculated into pure wild jujube seed juice, the pH differences increased by 0.33 and 0.34 respectively compared with the non-domesticated group.

[0135] As Figure 7As shown in Figure B, after domestication, the viable count of Lactobacillus plantarum GD 06 was significantly higher than that of the non-domesticated group after fermentation in wild jujube seed juice, indicating that after domestication, Lactobacillus plantarum GD 06 and Lactobacillus plantarum JYLP-326 were more adaptable to the pure wild jujube seed juice environment, with strong abilities in metabolism, acid production, and proliferation in wild jujube seed juice, and an improved fermentation ability for wild jujube seed juice.

[0136] Example 4

[0137] A method for preparing defatted freeze-dried powder of fermented wild jujube seed juice, comprising the following steps:

[0138] (1) Inoculate the domesticated strain in Example 3 into the prepared wild jujube seed juice at an inoculum size of 6.8 log CFU / mL, and perform static fermentation at 37°C. Place the fermented wild jujube seed juice at the fermentation end point (0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h) into an ultra-low temperature refrigerator at -80°C and freeze for 5 h.

[0139] (2) Freeze-dry the frozen fermented wild jujube seeds at -50°C for 48 h.

[0140] (3) Defat the freeze-dried powder of the fermented wild jujube seeds: Add the freeze-dried powder to a conical flask, add 5 times the volume of n-hexane, place it in a water bath shaker at 45°C, and defat at 140 r / min for 5 h. Then filter and collect the powder on the filter paper, and dry it in an oven at 45°C. The defatted freeze-dried powder of the fermented wild jujube seed juice is obtained.

[0141] The viable count, pH value, and acidity were measured during fermentation as follows:

[0142] The acidity calculation formula is shown in Equation II:

[0143]

[0144] X ——— The acidity of the sample (°T);

[0145] c ——— The concentration of the sodium hydroxide standard solution (mol / L);

[0146] V 1 —— The volume of sodium hydroxide consumed in the titration (mL);

[0147] V 0 —— The volume of sodium hydroxide consumed in the blank experiment (mL);

[0148] 9 —— The lactic acid conversion coefficient;

[0149] m —— The weight of the sample taken (g).

[0150] From Figure 8It can be seen that Lactobacillus plantarum GD 06 grows rapidly during the 0-14 h of fermenting wild jujube seed juice, and it is difficult to observe the lag phase, indicating that Lactobacillus plantarum GD 06 adapts to the environment of wild jujube seed juice after domestication and reaches the maximum viable count at 14 h. After 14 h, the viable count begins to gradually decrease.

[0151] As Figure 8 shown in B, the pH value of the fermentation broth changes rapidly within 0-14 h. After 14 h, with the increase in the number of bacteria, the pH value of the fermentation broth decreases rapidly. As the fermentation time extends, the accumulation of metabolites and the consumption of nutrients lead to a slowdown in the growth rate of the experimental strains and a slowdown in the decrease rate of the pH value.

[0152] As Figure 8 shown in C, the titratable acidity increases rapidly within 0-18 h. At this time, the metabolism of lactic acid bacteria is the most vigorous and the acid production rate is the fastest. Subsequently, within 18-48 h, the increase rate of the titratable acidity decreases. By measuring the viable count, pH value and titratable acidity in the fermentation broth, it shows that the domesticated Lactobacillus plantarum GD 06 has strong proliferation and acid production abilities in wild jujube seed juice and has fully adapted to grow and metabolize in pure wild jujube seed juice, and is suitable for fermenting wild jujube seed juice.

[0153] The contents of GABA, total sugar, total phenols, soluble protein, polypeptide, total flavonoids, total saponins, spinosin, 6'''-feruloylspinosin, jujuboside A, jujuboside B, and betulinic acid in the defatted freeze-dried powder of the above fermented wild jujube seed juice were measured, and the results are as Figures 9 - 15 shown. In the figure, the control group is the blank control of the defatted freeze-dried powder of pure wild jujube seed juice.

[0154] As Figure 9 shown, during the fermentation process, the GABA content shows a trend of first increasing and then decreasing, reaching the maximum value at 12 h and then gradually decreasing. The GABA content in the wild jujube seed juice fermented by Lactobacillus plantarum GD 06 is significantly higher than that of the positive control strain Lactobacillus plantarum JYLP-326. It shows that Lactobacillus plantarum GD 06 can significantly increase the GABA content in wild jujube seed juice through fermentation.

[0155] As Figure 10 shown, during the fermentation process, the total sugar content shows a rapid decreasing trend within 0-12 hours, and then the total sugar content changes slowly within 12-60 hours. There is no significant difference in the total sugar content between Lactobacillus plantarum GD 06 and Lactobacillus plantarum JYLP-326 during the fermentation process, indicating that Lactobacillus plantarum GD 06 utilizes the carbohydrate substances in wild jujube seed juice during the fermentation process.

[0156] As Figure 11As shown in the figure, the total phenolic content in the sour jujube kernel juice fermented by Lactobacillus plantarum GD 06 was significantly higher than that of Lactobacillus plantarum JYLP-326 and the blank control group from 24 to 60 hours, indicating that Lactobacillus plantarum GD 06 can increase the total phenolic content in sour jujube kernel juice and is suitable for fermenting sour jujube kernel juice.

[0157] As Figure 12 shown in the figure, the soluble protein content in the sour jujube kernel juice showed a sharp downward trend within 0-12 hours of fermentation and no significant change within 12-60 hours, indicating that the two strains of Lactobacillus plantarum produced protease during the fermentation of sour jujube kernels and enzymatically hydrolyzed the soluble protein in the sour jujube kernel juice.

[0158] As Figure 13 shown in the figure, the polypeptide content in the sour jujube kernel juice showed a trend of first increasing and then decreasing during the fermentation process. Combining Figure 12 it can be speculated that the soluble protein was decomposed into polypeptide substances with smaller molecular weights under the action of protease produced by the two strains of Lactobacillus plantarum, which is beneficial to improving the utilization rate of protein digestion and absorption in the human body.

[0159] As Figure 14 shown in the figure, the total flavonoid content showed a trend of first decreasing and then increasing during the fermentation process, which was Figure 12 similar to the trend. A short-term downward trend occurred in the early stage of fermentation, and then the total flavonoid content increased rapidly and basically remained unchanged after 36h. The sour jujube kernel juice fermented by Lactobacillus plantarum GD 06 reached the maximum value at 36 hours and was significantly higher than that of the Lactobacillus plantarum JYLP-326 fermentation group, increasing by 26.21% compared with the blank group. Flavonoid components are one of the main active components of sour jujube kernels. Flavonoid components play a major sedative and hypnotic role and have a wide range of pharmacological effects, indicating that the fermentation of Lactobacillus plantarum GD 06 has the potential to improve the sedative and sleep-promoting effect of sour jujube kernel juice.

[0160] As Figure 15 shown in A and B, the flavonoid sedative and sleep-promoting substances spinosin and 6'''-feruloylspinosin in sour jujube kernels showed a similar change trend to the total flavonoids during the fermentation process and both reached the highest value within 36 hours. The contents of spinosin and 6'''-feruloylspinosin in the fermentation group of Lactobacillus plantarum GD 06 were significantly higher than those of the Lactobacillus plantarum JYLP-326 group, increasing by 22.29% and 14.34% respectively compared with the blank group. Spinosin and 6'''-feruloylspinosin are flavonoid active substances with relatively high contents in sour jujube kernels and have been proven to have sedative and sleep-promoting effects, which can improve insomnia, indicating that the sour jujube kernel juice fermented by Lactobacillus plantarum GD 06 of the present invention can improve the sedative and sleep-promoting effect of sour jujube kernel juice.

[0161] As Figure 15As shown in Figures C and D, during the fermentation process, the contents of jujuboside A and jujuboside B both showed an increasing trend and then a decreasing trend after 36 h. The content in the Lactobacillus plantarum GD 06 fermentation group was significantly higher than that in the Lactobacillus plantarum JYLP-326 group. Jujuboside A and jujuboside B are the two saponin components with the highest contents in jujube seeds and have a wide range of pharmacological effects. For example, jujuboside A can significantly prolong sleep time and also has the effect of improving myocardial function. Jujuboside B has significant neuroprotective properties and has become a candidate component for preventing Alzheimer's disease. After the jujube seed juice is fermented by the Lactobacillus plantarum GD06 of the present invention, the contents of jujuboside A and jujuboside B are significantly increased.

[0162] As Figure 15 shown in Figure E, during the fermentation process, betulinic acid showed a trend of first increasing and then decreasing, and reached the maximum value at 36 hours. After fermenting for 36 hours, the content of betulinic acid in the Lactobacillus plantarum GD 06 group increased by 0.55 mg / mL, which was significantly higher than that in the control group of Lactobacillus plantarum JYLP-326. Betulinic acid has various functions, such as anti-tumor effect, anti-inflammatory effect, antibacterial effect, antiviral effect, anti-hyperlipidemia effect, anti-diabetic effect, antioxidant stress activity, etc. After the jujube seed juice is fermented by the Lactobacillus plantarum GD06 of the present invention, the content of betulinic acid is significantly increased.

[0163] Example 5 Verification Experiment on the Efficacy of Improving Insomnia

[0164] (1) Preparation of the medicament for establishing the insomnia animal model:

[0165] Preparation of the p-chlorophenylalanine (PCPA) suspension: PCPA was suspended in sterile physiological saline to prepare a 10 g / L suspension.

[0166] (2) Preparation of the gavage materials:

[0167] Jujube seed juice: The preparation method was as in Example 2.

[0168] Lactic acid bacteria-fermented jujube seed juice: The preparation method was as in Example 4, and the fermentation end point was selected at 36 h.

[0169] Bacterial suspension: Lactobacillus plantarum GD 06 was cultured in MRS liquid medium for 18 h, centrifuged at 6000 r for 10 min, washed three times with physiological saline, and resuspended to adjust the viable bacteria count to 2.5×10 8 CFU / mL for immediate use.

[0170] (3) Adaptive feeding of mice and model establishment

[0171] Eighty-four SPF female Kunming mice at four weeks of age were housed in an animal room at a temperature of (26±2)°C, a humidity of 50%-60%, a noise level below 60 decibels, and a lighting period of 12 hours (from 8:00 to 20:00). After 7 days of adaptation, they were randomly divided into two groups: a control group (12 mice) and a model group (72 mice). Mice in the model group were intraperitoneally injected with a PCPA suspension (6 mg PCPA / 20 g mouse) at 8:00 for three consecutive days. On the third day of modeling, they were randomly divided into six groups. According to the following grouping and doses, the mice were intervened by gavage.

[0172] Control group (NC): Purified water (0.8 mL / 30 g mouse),

[0173] Model group (MG): Purified water (0.8 mL / 30 g mouse),

[0174] Low-dose unfermented group (LDU): Unfermented wild jujube seed juice diluted twofold with purified water (0.8 mL / 30 g mouse),

[0175] Low-dose fermented group (LDF): Fermented wild jujube seed juice diluted twofold with purified water (0.8 mL / 30 g mouse),

[0176] High-dose unfermented group (HDU): Unfermented wild jujube seed juice (0.8 mL / 30 g mouse),

[0177] High-dose fermented group (HDF): Fermented wild jujube seed juice (0.8 mL / 30 g mouse),

[0178] Bacterial suspension group (LBS): Bacterial suspension (0.8 mL / 30 g mouse).

[0179] (4) Tail suspension test

[0180] The tail suspension test was conducted at 3 weeks of intervention. The mouse's tail (about 1 cm from the tip) was fixed to a hook with medical tape, allowing the mouse's head to hang naturally, about 30 cm from the ground. A camera device was directed at the mouse to record the mouse's state for 5 minutes. This experiment mainly evaluated the sedative state of the mouse by the continuous struggle of the mouse in the hanging state and the cumulative intermittent relative rest time. The time to reach the first stationary state and the duration of the relative rest period (specifically referring to the state where only small amplitude tremors or complete stillness of the limbs) were recorded.

[0181] (5) Evaluation of direct sleep effect

[0182] Thirty minutes after the last dose, the number of mice falling asleep was observed to evaluate the direct sleep effect of each group of drugs on the mice. The disappearance of the righting reflex for 60 s was used as the standard for entering the sleep state.

[0183] (6) Sleep synergy experiment

[0184] After the last administration, a chloral hydrate sleep cooperation experiment was conducted. Chloral hydrate (2.6%; 0.1 mL / 10 g) was injected intraperitoneally, and a video recording device was used to record. After the experiment, the sleep latency and sleep duration were recorded. The disappearance of the righting reflex (lateral position, dorsal position) for 60 s was defined as the onset of sleep.

[0185] (7) Anatomical sampling

[0186] At the end of the experiment, the mice were weighed and then sacrificed by cervical dislocation for dissection. The whole brain (excluding the olfactory bulb), liver, kidney, and spleen were taken. They were washed with pre-cooled normal saline, weighed, photographed, and the organ coefficient was calculated according to formula (Ⅲ). The whole brain and colon were fixed with 4% PFA fixative and photographed after HE staining.

[0187]

[0188] The hypothalamus was isolated from the whole brain and homogenized in an ice bath with pre-cooled normal saline at a ratio of 1:9. After centrifugation at 4000 r / min for 10 min, the supernatant was aliquoted and stored at -80 °C.

[0189] (8) Hypothalamic neurotransmitter detection

[0190] The glutamate, γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), melatonin (MT), and dopamine in the supernatant of the hypothalamic homogenate of mice were measured according to the instructions of the mouse glutamate (Glu) ELISA KIT, mouse γ-aminobutyric acid (GABA) ELISA KIT, mouse 5-hydroxytryptamine (5-HT) ELISA KIT, mouse melatonin (MT) ELISA KIT, and mouse dopamine (DA) ELISA KIT. The kits were purchased from Wuhan Sepei Biotechnology Co., Ltd. The interleukin 6 (IL-6), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), malondialdehyde (MDA), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-PX) in the supernatant of the whole brain homogenate of mice were measured according to the instructions of the mouse interleukin 6 (IL-6) ELISA KIT, mouse interleukin 1β (IL-1β) ELISA KIT, mouse tumor necrosis factor α (TNF-α) ELISA KIT, malondialdehyde (MDA) assay kit (TBA method), total superoxide dismutase (T-SOD) assay kit (WST-1 method), and glutathione peroxidase (GSH-PX) assay kit (colorimetric method). The kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0191] Table 1. Organ coefficient

[0192] Group Brain Liver NC <![CDATA[0.0136±0.0008 b > <![CDATA[0.0416±0.0033 b* > MG <![CDATA[0.0126±0.0012 c > <![CDATA[0.0452±0.0035 a* > LDU <![CDATA[0.0145±0.0014 ab > <![CDATA[0.0423±0.0028 ab* > LDF <![CDATA[0.0143±0.0012 ab > <![CDATA[0.0400±0.0044 b* > HDU <![CDATA[0.0148±0.0009 a > <![CDATA[0.0413±0.0033 b* > HDF <![CDATA[0.0145±0.0008 ab > <![CDATA[0.0396±0.0036 b* > LBS <![CDATA[0.0146±0.0010 ab > <![CDATA[0.0426±0.0037 ab* >

[0193] As can be seen from Table 1, compared with the control group (NC group), after PCPA modeling, the whole brain coefficient of mice in the MG group decreased significantly, and the liver coefficient increased significantly, indicating that insomnia caused damage to the brains and livers of mice. After intervention with wild jujube seed juice, fermented wild jujube seed juice, and Lactobacillus plantarum GD 06 bacterial suspension, the whole brain coefficients of mice in each group were improved. Among them, except for the high-dose unfermented (HDU) group, there were no significant differences between other intervention groups and the control group (NC), indicating that wild jujube seed juice, fermented wild jujube seed juice, and Lactobacillus plantarum GD 06 bacterial suspension all had the effect of alleviating the decrease in the whole brain coefficient caused by insomnia. After intervention, the liver coefficients of mice in the LDF group, HDU group, and HDF group decreased significantly and there were no significant differences from the control group, indicating that both wild jujube seed juice and fermented wild jujube seed juice could alleviate the increase in liver index caused by insomnia.

[0194] As Figure 16 shown, compared with the control group (NC group), after PCPA modeling, mice were in a state of sleep disorder for a long time, and the number of cells in the DG area of the hippocampus in the brain HE staining sections of mice became fewer and the arrangement became sparse (MG group). Compared with the MG group, among the 5 intervention groups, the number and arrangement of cells in the DG area of the hippocampus were improved to a certain extent. Among them, the number of cells in the hippocampus area of the HDF group was the largest and the arrangement was tight and orderly, being closest to the NC group. This shows that the wild jujube seed juice fermented by high-dose Lactobacillus plantarum GD 06 has the best effect on improving the damage of the DG area of the hippocampus caused by insomnia, being superior to unfermented wild jujube seed juice.

[0195] As Figure 17 , in the tail suspension test, compared with the control group (NC group), the PCPA-modeled mice showed a more restless state during the experiment, the time to first immobility was significantly prolonged, and the duration of immobility was significantly shortened (MG group). Compared with the MG group, the time to first immobility in the 5 intervention groups was significantly shortened, and the duration of immobility after administration was significantly increased, with the HDF group having the best effect. This shows that after fermentation by Lactobacillus plantarum GD 06, the sedative effect of wild jujube seed juice is significantly improved.

[0196] As shown in Table 2, 30 minutes after the last administration, the direct sleep rate of each group was zero, indicating that the samples in each group were safe and had no direct sleep effect.

[0197] Table 2. Direct sleep experiment

[0198] Group Total number Direct sleep number Direct sleep rate NC 12 0 0 MG 12 0 0 LDU 12 0 0 LDF 12 0 0 HDU 12 0 0 HDF 12 0 0 LBS 12 0 0

[0199] As Figure 18As shown, in the sleep extension experiment, compared with the control group (NC group), after PCPA modeling in mice, the sleep latency of the MG group was significantly prolonged, and the sleep time was significantly shortened. Compared with the MG group, only the HDF group and the LBS group significantly shortened the sleep latency; the HDU group and the HDF group significantly prolonged the sleep time, and the HDF group was significantly higher than the HDU group, indicating that the fermentation of Lactobacillus plantarum GD06 can improve the sleep-promoting effect of sour jujube juice.

[0200] As Figure 19 shown in a, compared with the control group (NC group), after PCPA modeling, the content of serotonin in the hypothalamus of mice in the MG group was significantly decreased. Serotonin is an inhibitory neurotransmitter that plays a role in promoting sleep and regulating the sleep cycle. A decrease in serotonin levels can lead to sleep disorders, which is also the most direct manifestation of successful modeling. Compared with the MG group, the content of serotonin in the 5 intervention groups was significantly increased. Among them, the LDF group was significantly higher than the LDU group, and the HDF group was significantly higher than the HDU group, indicating that the ability of fermented sour jujube juice to increase serotonin is better than that of unfermented sour jujube juice. In addition, there was no significant difference between the LBS group and the HDF group, indicating that Lactobacillus plantarum GD 06 itself also has a strong ability to increase serotonin.

[0201] As Figure 19 shown in b, compared with the control group (NC group), after PCPA modeling, the content of melatonin in the hypothalamus of mice in the MG group was significantly decreased. Melatonin is a hormone converted from serotonin, which has the functions of promoting sleep, regulating the biological clock, enhancing immune function, antioxidant and anti-aging, regulating hormone secretion, and maintaining the stability of the internal environment of the body. Long-term low levels of melatonin can cause health problems such as insomnia and reduced immunity. Compared with the MG group, the content of melatonin in the 5 intervention groups was significantly increased, and the HDF group had the best effect, which was better than unfermented sour jujube juice. It shows that the effect of fermented sour jujube juice by Lactobacillus plantarum GD 06 in increasing the content of melatonin in the hypothalamus has been improved.

[0202] As Figure 19 shown in c, compared with the control group (NC group), after PCPA modeling, there was no significant difference in the dopamine content in the hypothalamus of mice in the MG group compared with the NC group. Dopamine is an important neurotransmitter that can transmit the information of "happiness". When the dopamine level increases, the body can feel pleasure and relaxation. The dopamine content in the 5 intervention groups was significantly increased, and the HDF group and the LBS group had the best effect in increasing dopamine, indicating that the fermented sour jujube juice has the effect of increasing dopamine content, and Lactobacillus plantarum GD 06 itself also has a strong effect of increasing dopamine.

[0203] As Figure 20As shown, compared with the control group (NC group), after PCPA modeling, the level of gamma-aminobutyric acid (GABA) in the hypothalamus of MG group mice decreased ( Figure 20 a), the level of glutamate ( Figure 20 b) increased, and the GABA / glutamate balance was disrupted ( Figure 20 c), which may lead to decreased memory, anxiety, mood instability, aggressive behaviors such as self-harm, and damage to nerve cells, especially in brain regions such as the amygdala-hippocampus region. Compared with the MG group, the content and ratio of GABA and glutamate in the 5 intervention groups were significantly improved. Among them, the HDF group had the best effect and was closest to the NC group. This indicates that compared with unfermented wild jujube seed juice, the effect of wild jujube seed juice fermented by Lactobacillus plantarum GD 06 in regulating the GABA / glutamate balance in the hypothalamus was significantly improved.

[0204] As Figure 21 shown, compared with the control group (NC group), after PCPA modeling, the cytokines IL-6, IL-1β, and TNF-α in the whole brain of MG group mice were significantly decreased, and the cytokine levels were lower than the normal level, indicating that the decrease in hormone levels including melatonin caused by long-term insomnia inhibited the activity of immune cells, resulting in reduced immunity. In the 5 intervention groups, the levels of the 3 cytokines were improved, and the HDF group and the LBS group had the best improvement effects.

[0205] As Figure 22 shown, compared with the control group (NC group), after PCPA modeling, the MDA level in the whole brain of MG group mice was significantly increased, the SOD activity was significantly decreased, and the GSH-Px activity was significantly decreased. Long-term insomnia led to increased oxidative stress and weakened antioxidant capacity in the brain, and also indicated damage to brain cells. Compared with the MG group, the levels of MDA, SOD, and GSH-Px in the HDF group were significantly improved, better than unfermented wild jujube seed juice, and closest to the control group.

[0206] In summary, Lactobacillus plantarum GD 06 has a strong ability to produce GABA, and the nutritional properties of the wild jujube seed juice fermented by Lactobacillus plantarum GD 06 are significantly improved. The contents of γ-aminobutyric acid, polypeptide, total phenols, total flavonoids, spinosin, 6'''-feruloylspinosin, jujuboside A, B and betulinic acid are all significantly increased. In the verification experiment of improving insomnia efficacy, the wild jujube seed juice fermented by Lactobacillus plantarum GD 06 can alleviate the decrease of the whole brain coefficient and the increase of the liver coefficient caused by long-term insomnia, relieve the reduction of cell number and sparse arrangement in the DG area of the hippocampus in the mouse brain caused by long-term insomnia, prolong the sleeping time of the mouse, and improve the insomnia symptoms; increase the content of inhibitory neurotransmitters in the hypothalamus of the mouse, reduce the content of excitatory neurotransmitters, and relieve the decrease of body immunity and oxidative stress of brain tissue caused by insomnia. Therefore, the wild jujube seed juice fermented by Lactobacillus plantarum GD06 of the present invention has the efficacy of improving insomnia and can be developed into related functional foods.

[0207] All experiments of the present invention were repeated at least 3 times, and the data were expressed as mean ± standard deviation and were analyzed by SPSS25 software for comparison between groups, and one-way analysis of variance was used. Paired t-test was used for analysis within the group. Different letters represent significant differences between the two (p<0.05).

[0208] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Lactobacillus plantarum, characterized in that The plant lactobacillus was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 27, 2024, with the deposit number GDMCC No: 65061.

2. A culture solution or bacterial suspension containing the plant lactobacillus according to claim 1.

3. Use of the plant lactobacillus according to claim 1 or the culture solution or bacterial suspension according to claim 2 in preparing a product for alleviating insomnia.

4. The use according to claim 3, characterized in that: The product is food or health product.

5. The use according to claim 3, characterized in that: The product is fermented sour jujube kernel juice.

6. A method for fermenting wild jujube seed juice, characterized in that: The following steps are involved: (1) strain domestication: based on the gradual domestication method, the plant lactobacillus according to claim 1 is domesticated and cultured using a domestication medium, wherein the domestication medium comprises spiny jujube seed juice and MRS liquid medium; (2) Fermentation of sour jujube kernel juice: The domesticated Lactobacillus plantarum prepared in (1) is inoculated into sour jujube kernel juice for fermentation; the inoculation amount of the Lactobacillus plantarum is 6.5-7.0 log CFU / mL.

7. The method according to claim 6, characterized in that The gradual acclimation method in step (1) is: gradually increasing the concentration of the jujube seed juice in the acclimation medium.

8. The method according to claim 7, characterized in that The acclimation culture temperature in step (1) is 35-37°C, and the acclimation time of each acclimation culture medium is 20-24 h.

9. The method for preparing fermented spiny jujube seed juice according to claim 5, characterized in that: The fermentation temperature in step (2) is 35-37°C and the fermentation time is 24-48 hours.

10. A fermented spiny jujube seed juice, characterized in that: The fermented sour jujube seed juice is obtained by fermenting sour jujube seed juice with the plant lactobacillus according to claim 1, and the viable bacterial count of the plant lactobacillus in the fermented sour jujube seed juice is 8.0-8.75 log CFU / mL.

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