A lactobacillus reuteri zklr0413 and a zizyphus jujuba fermenting liquid for improving sleep

Abstract

This invention relates to the field of sleep improvement products, specifically to a *Lactobacillus reuteri* ZKLr0413 jujube seed fermentation broth and *Lactobacillus reuteri* ZKLr0413 itself, which improves sleep. The *Lactobacillus reuteri* ZKLr0413 jujube seed fermentation broth for improving sleep is composed of *Lactobacillus reuteri* (… Lactobacillus reuteri The fermentation broth of this invention was prepared by fermenting jujube seed extract with Lactobacillus reuteri ZKLr0413. Lactobacillus reuteri ZKLr0413 was deposited on May 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30546, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The jujube seed extract was obtained by soaking and decocting jujube seed powder. Compared with mixed-culture fermentation or mixed fermentation of multiple medicinal and edible materials, the fermentation broth of this invention has a simple composition and significant advantages in safety and process stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sleep improvement products, and in particular to a sleep-improving Lactobacillus reuteri ZKLr0413 Zizyphus jujuba Mill. seed fermenting liquid and Lactobacillus reuteri ZKLr0413. BACKGROUND

[0002] Zizyphus jujuba Mill. seed is the dried seed of Ziziphus jujuba Mill. of the Rhamnaceae family, with a sweet taste and a neutral nature, and has the effects of calming the heart and soothing the nerves, nourishing the liver, and stopping sweating and generating fluid. It was first recorded in Shennong Bencao Jing. In the Bencao Gangmu written by Li Shizhen in the Ming Dynasty, it is mentioned that Zizyphus jujuba Mill. can be used to treat insomnia caused by gallbladder deficiency, and can also be used to treat symptoms of thirst and sweating, and is a Foot Lesser Yang and Earth Yin medicine. Modern research shows that Zizyphus jujuba Mill. has the effects of improving sleep disorders, antioxidant, anti-depression, anti-anxiety, and enhancing learning and memory. The flavonoids and saponins contained in Zizyphus jujuba Mill. are effective components for improving sleep. Therefore, how to improve the bioavailability of these effective components, promote drug absorption, and increase the content of active ingredients is a problem that needs to be solved in the process of Zizyphus jujuba Mill. resource development and utilization.

[0003] Microbial fermentation of Zizyphus jujuba Mill. can improve the above-mentioned problems existing in the utilization of Zizyphus jujuba Mill. to some extent. For example, CN116725179 A discloses a sleep-aiding and spirit-soothing herbal enzyme, which uses Zizyphus jujuba Mill. and ingredients such as mulberry, lily, and black date as raw materials, pretreats the raw material components by steaming, obtains a raw material liquid, and then inoculates a specific proportion of mixed strains of Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus into the raw material liquid for fermentation, and obtains the herbal enzyme by filtration and centrifugation.

[0004] CN115990212 A discloses a composition made by fermenting Zizyphus jujuba Mill. ultra-fine powder with a mixed bacterial liquid of Lactobacillus plantarum and Lactobacillus casei, which has the function of regulating GABA-A receptors.

[0005] CN120000741 A discloses a probiotic and traditional Chinese medicine composition that can be used for treating insomnia, which uses up to 18 kinds of probiotics, and the required number of live bacteria for fermentation is as high as 200 billion CFU / g or more. The traditional Chinese medicine composition includes Zizyphus jujuba Mill., lily, Poria cocos, lotus seed, and mulberry.

[0006] It can be seen that the current means to enhance the sleep-aiding effect of Chinese milk vetch mainly focuses on mixed microbial fermentation or the addition of Chinese medicinal materials with synergistic effects. However, these strategies have obvious limitations in practical application. For the mixed microbial fermentation strategy, the competition among multiple strains easily leads to fluctuations in fermentation efficiency. The preference or competition for nutrients among different strains may result in insufficient conversion of flavonoids and saponins in Chinese milk vetch. Bacteriocins secreted by different strains or acidic substances produced by metabolism may also inhibit the growth of each other, causing an imbalance in the proportion of strains and significant fluctuations in product quality between batches. For the strategy of adding Chinese medicinal materials with synergistic effects, although the effects can be theoretically superimposed, the physicochemical properties of each component often differ. If a pretreatment scheme is designed for each material separately, the production cost will be significantly increased. If uniform process conditions are used, the components of some materials may be destroyed. In addition, the mixing of multiple components easily produces unknown compounds or precipitates, and even introduces complex risks of pesticide residues and heavy metal pollution, increasing the difficulty of component analysis and quality control and making it difficult to trace and control safety hazards.

[0007] Therefore, the development of a preparation of Chinese milk vetch fermented by a single strain can avoid metabolic conflicts between strains and simplify the production process, which is conducive to ensuring product safety and process stability. SUMMARY

[0008] In view of the technical problems of mixed microbial fermentation or the addition of Chinese medicinal materials with synergistic effects, such as competition between strains, low conversion efficiency and easy destruction of components, difficulty in uniform process conditions, high cost, and complex components difficult to control, the present application provides a sleep-improving Lactobacillus reuteri ZKLr0413 Chinese milk vetch fermentation broth and Lactobacillus reuteri ZKLr0413. The Lactobacillus reuteri ZKLr0413 can be used alone for fermentation of Chinese milk vetch extract to efficiently release sleep-aiding active substances such as flavonoids and saponins in Chinese milk vetch. Compared with mixed microbial fermentation or fermentation with multiple food and drug homologous materials, the components of the fermentation broth of the present application are simpler, and the safety and process stability advantages are significant.

[0009] The technical scheme of the present application is as follows:

[0010] In a first aspect, a sleep-improving Lactobacillus reuteri ZKLr0413 Chinese milk vetch fermentation broth is prepared by fermenting Chinese milk vetch extract with Lactobacillus reuteri (Lactobacillus reuteri) ZKLr0413; Lactobacillus reuteri

[0011] In a first aspect, a sleep-improving Lactobacillus reuteri ZKLr0413 Chinese milk vetch fermentation broth is prepared by fermenting Chinese milk vetch extract with Lactobacillus reuteri (Lactobacillus reuteri) ZKLr0413; Lactobacillus reuteri

[0012] ​​The jujube seed extract is obtained by soaking and decocting jujube seed powder.

[0013] Further, the preparation method of the jujube seed extract is as follows:

[0014] The jujube seed powder is put into a decoction bag and then soaked in water to obtain jujube seed soaking liquid; the jujube seed soaking liquid is decocted and then filtered to separate the liquid and residue, and the liquid is concentrated by evaporation to obtain jujube seed extract with a crude drug content of 1.0 g / mL.

[0015] Further, the particle size of the jujube seed powder is 70-300 mesh.

[0016] Further, the Lactobacillus reuteri ZKLr0413 is inoculated into the jujube seed extract at a ratio of 1×10 10 CFU / L of the jujube seed extract, and then cultured at 37℃ under constant temperature and oxygen, to obtain Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid after the culture is completed.

[0017] Further, before the Lactobacillus reuteri ZKLr0413 is inoculated, the jujube seed extract is first sterilized.

[0018] Further, the freeze-dried powder of the Lactobacillus reuteri ZKLr0413 is added into the jujube seed extract for fermentation, and the viable bacterial count of the freeze-dried powder of the Lactobacillus reuteri ZKLr0413 is 1×10 11 CFU / g.

[0019] Further, after the fermentation is completed, the viable bacterial count of the Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid is 1.58×10 9 CFU / mL.

[0020] Further, the total flavonoid content of the Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid is 82.39±7.42 mg / mL.

[0021] Further, the total saponin content of the Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid is 73.57±4.23 mg / mL.

[0022] In a second aspect, the present application also provides a Lactobacillus reuteri ZKLr0413, which has been preserved in the China General Microbiological Culture Collection Center on May 13, 2024, with a preservation number of CGMCC No.30546 and a classification name of Lactobacillus reuteri. Lactobacillus reuteri .

[0023] The present application has the following beneficial effects:

[0024] The Lactobacillus reuteri ZKLr0413 used in the application has excellent fermentation effect on the jujube kernel extract, can tolerate the low pH environment caused by jujube kernel fermentation, and keep the viable count in the fermentation liquor at a high level; meanwhile, the Lactobacillus reuteri ZKLr0413 can efficiently promote the dissolution of sleep-aiding active substances such as flavonoids and saponins in the jujube kernel extract, and the dissolution effect is better than that of common probiotic single or mixed fermentation, thereby ensuring the fermentation efficiency and improving the product safety and process stability.

[0025] The experimental results show that the jujube kernel fermentation liquor fermented by the Lactobacillus reuteri ZKLr0413 significantly improves the sleep effect, can significantly improve the sleep rate, shorten the sleep latency, prolong the total sleep time, and stimulate the brain to release 5-hydroxytryptamine and gamma-aminobutyric acid. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows, and obviously, other drawings can also be obtained by those skilled in the art without creative labor on the premise of the drawings.

[0027] Figure 1 It is a colony morphology photo of the isolated strain in Example 1.

[0028] Figure 2 It is a gram staining picture of the isolated strain in Example 1. DETAILED DESCRIPTION

[0029] In order to enable the personnel in the technical field to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described as follows, and obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the protection scope of the present application.

[0030] The MRS solid medium and the MRS liquid medium used in the following embodiments are purchased from Beijing Aoboxing Biotechnology Co., Ltd.; the MRS-CaCO3 solid medium is prepared by adding 1.5% of CaCO3 to the MRS solid medium and sterilizing.

[0031] The DNA extraction kit used in the following embodiments is purchased from Beijing Tiangeng Biochemical Technology Co., Ltd.

[0032] Example 1: Isolation and identification of Lactobacillus reuteri ZKLr0413

[0033] 1. Sample source: collected from Songbai Town of Shennongjia Forest Region in Hubei Province in October 2013.

[0034] 2. Strain isolation: the collected sample was diluted with physiological saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 times, respectively, 100 μL of the diluent was taken and uniformly coated on MRS-CaCO3 solid culture medium, and cultured at 37°C for 24-48 h under anaerobic inversion. The growth of the colonies was observed. After the colonies were formed, single colonies with calcium rings were picked and three-zone streaked three times. The streaking was repeated multiple times until the colonies were purified. The index of colony purification was that the colony morphology was single, and the gram staining morphology was single. The purified strain was inoculated into MRS liquid culture medium for culture. The culture liquid of the strain and 50% sterile glycerol were mixed at a volume ratio of 1:1, and stored in a-80°C refrigerator.

[0035] 3. Strain identification:

[0036] (1) Colony morphology identification

[0037] ① The isolated strain was anaerobically cultured on MRS solid culture medium for 48 h, and the colony morphology and color were observed and recorded. The colony morphology is shown in Figure 1 . The colony is round, smooth, convex, with neat edges, opaque, milky white, and occasionally light yellow.

[0038] ② The colonies cultured on MRS solid culture medium were picked and smeared, fixed, crystal violet primary stained, mordant stained, decolorized, washed with water, safranine re-stained, and dried in turn. Then the dried colonies were examined under an oil lens with a magnification of 1000 times. From the examination results Figure 2 , it can be seen that the colonies are stained blue-purple and short rod-shaped.

[0039] (2) 16S rDNA sequence determination

[0040] The culture liquid of the isolated strain was centrifuged at 5000 r / min for 10 min. After centrifugation, the supernatant was discarded, and the pure culture bacteria were collected. The genomic DNA of the strain was extracted according to the instructions of the DNA extraction kit.

[0041] According to the conserved region of the 16S rDNA sequence of Lactobacillus, the universal primer 27F / 1492R was used for PCR amplification. The PCR amplification conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s; annealing at 55°C for 35 s; extension at 72°C for 90 s; 32 cycles, and finally extension at 72°C for 5 min.

[0042] The PCR products were identified by 1.0% agarose gel electrophoresis, and the amplified products were sequenced. The sequencing work was completed by Beijing Genesee Biotechnology Co., Ltd. The 16S rDNA sequence (SEQ ID NO. 1) of the strain is as follows:

[0043]

[0044] The sequencing results were analyzed by BLAST comparison analysis through the NCBI database, and a phylogenetic tree was constructed to determine that the strain was Lactobacillus reuteri. Lactobacillus reuteri The strain was named Lactobacillus reuteri ZKLr0413 and was preserved, and the preservation information is as follows:

[0045] Preservation time: May 13, 2024;

[0046] Preservation unit: China General Microbiological Culture Collection Center;

[0047] Preservation address: No. 3, Beichen West Road, Chaoyang District, Beijing;

[0048] Preservation number: CGMCC No. 30546;

[0049] Classification name: Lactobacillus reuteri Lactobacillus reuteri .

[0050] Example 2: Experiment of Lactobacillus reuteri ZKLr0413 on gastrointestinal environment tolerance

[0051] 1. Tolerance of Lactobacillus reuteri ZKLr0413 to artificial gastric juice

[0052] According to the preparation method recorded in Chinese Pharmacopoeia, take dilute hydrochloric acid 16.4 mL, add water 800 mL and pepsin 10 g, stir evenly, add water to 1000 mL, filter sterilization and reserve. Adjust the pH of artificial gastric juice to 2.5 and 3.0 using dilute hydrochloric acid.

[0053] Activate the preserved Lactobacillus reuteri ZKLr0413 twice, centrifuge (10000xg, 5 min, 4℃) 10 mL of Lactobacillus reuteri ZKLr0413 bacterial liquid to obtain bacterial slurry, wash twice with PBS buffer (pH=7.2), resuspend in 10 mL of PBS buffer to obtain bacterial suspension; take 1 mL of bacterial suspension and add to 9 mL of pH=2.5 and pH=3.0 artificial gastric juice, respectively, and incubate at 37℃, sample at 0 h and 3 h to determine the viable cell count, and calculate the survival rate according to the following formula:

[0054]

[0055] In the formula, N0 is the viable cell count at 0 h of artificial gastric juice treatment, and N1 represents the viable cell count after 3 h of artificial gastric juice treatment.

[0056] The final calculation results are shown in Table 1. It can be seen that Lactobacillus reuteri ZKLr0413 has good gastric acid resistance. The survival rate can reach more than 80% after being treated in artificial gastric juice with pH = 2.5 for 3 h, and the survival rate can reach more than 90% after being treated in artificial gastric juice with pH = 3.0 for 3 h.

[0057] Table 1 Survival of Lactobacillus reuteri ZKLr0413 in artificial gastric juice

[0058]

[0059] 2. Tolerance of Lactobacillus reuteri ZKLr0413 to artificial intestinal juice

[0060] According to the preparation method recorded in Chinese Pharmacopoeia, 6.8 g of potassium dihydrogen phosphate was added to 500 mL of water, and the pH value was adjusted to 6.8 with 0.4% sodium hydroxide solution; 10 g of pancreatin was dissolved in an appropriate amount of water, and the two liquids were mixed and then diluted to 1000 mL with water, and filtered to sterilize for use.

[0061] The stored Lactobacillus reuteri ZKLr0413 was activated twice, 10 mL of Lactobacillus reuteri ZKLr0413 bacterial solution was centrifuged (10000xg, 5 min, 4°C) to obtain bacterial slurry, which was washed twice with PBS buffer (pH = 7.2) and resuspended in 10 mL of PBS buffer to obtain bacterial suspension; 1 mL of bacterial suspension was added to 9 mL of artificial intestinal juice and cultured at 37°C, and the viable cell count was determined at 0 h and 3 h, respectively, and the survival rate was calculated according to the following formula:

[0062]

[0063] In the formula, C0 is the viable cell count at 0 h of artificial intestinal juice treatment, and C1 represents the viable cell count after 3 h of artificial intestinal juice treatment.

[0064] The final calculation results are shown in Table 2. It can be seen that the survival rate of Lactobacillus reuteri ZKLr0413 in artificial intestinal juice for 3 h can reach more than 90%, and it has good intestinal juice resistance.

[0065] Table 2 Survival of Lactobacillus reuteri ZKLr0413 in artificial intestinal juice

[0066]

[0067] In summary, Lactobacillus reuteri ZKLr0413 has good tolerance to the gastrointestinal environment, which creates conditions for preparing sleep-improving products from Zizyphus jujuba Mill. extract fermented by Lactobacillus reuteri ZKLr0413.

[0068] Example 3 Preparation of Zizyphus jujuba Mill. fermentation broth of Lactobacillus reuteri ZKLr0413

[0069] The preparation method of the Lactobacillus reuteri ZKLr0413 fermented liquid of Zizyphus jujuba Mill. includes two processes, first preparing Zizyphus jujuba Mill. extract liquid, and then fermenting the Zizyphus jujuba Mill. extract liquid using Lactobacillus reuteri ZKLr0413. The specific steps are as follows:

[0070] Step S1, preparing Zizyphus jujuba Mill. extract liquid:

[0071] Step S11, using a traditional Chinese medicine crusher to crush Zizyphus jujuba Mill. to prepare Zizyphus jujuba Mill. powder with a particle size of 70-300 mesh;

[0072] Step S12, putting 1 part by weight of Zizyphus jujuba Mill. powder into a decoction bag, and then soaking it in 8-10 parts by weight of water for 0.5-1 h to obtain Zizyphus jujuba Mill. soaking liquid;

[0073] Step S13, first boiling the Zizyphus jujuba Mill. soaking liquid with a large fire, and then continuing to decoct for 1-2 h, and then filtering and separating the liquid and residue;

[0074] Step S14, concentrating the liquid by evaporation to obtain Zizyphus jujuba Mill. extract liquid with a crude drug content of 1.0 g / mL;

[0075] Step S15, sterilizing the Zizyphus jujuba Mill. extract liquid to obtain sterilized Zizyphus jujuba Mill. extract liquid.

[0076] Step S2, fermenting the Zizyphus jujuba Mill. extract liquid using Lactobacillus reuteri ZKLr0413:

[0077] Step S21, freeze-drying Lactobacillus reuteri ZKLr0413 bacterial liquid to prepare Lactobacillus reuteri ZKLr0413 freeze-dried powder;

[0078] Step S22, inoculating 1×10 10 CFU / L of sterilized Zizyphus jujuba Mill. extract liquid into the sterilized Zizyphus jujuba Mill. extract liquid of step S15, shaking well, and then incubating at 37℃ under constant temperature and aerobic conditions, and after the incubation is completed, obtaining Lactobacillus reuteri ZKLr0413 Zizyphus jujuba Mill. fermented liquid.

[0079] In this embodiment, Lactobacillus reuteri ZKLr0413 Zizyphus jujuba Mill. fermented liquid one and Lactobacillus reuteri ZKLr0413 Zizyphus jujuba Mill. fermented liquid two were prepared by adjusting the process parameters.

[0080] Lactobacillus reuteri ZKLr0413 Zizyphus jujuba Mill. fermented liquid one was obtained under the following process parameters:

[0081] The jujube seed powder with a particle size of 70 meshes was put into a decoction bag and then was soaked in 8 times of water for 1 h to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled with a large fire, and then was slowly boiled with a small fire for 2 h; during the slow boiling process, the jujube seed soaking liquid was kept slightly boiling; then, the liquid and the residue were separated by filtration; the liquid was concentrated by boiling; a jujube seed extract liquid with a crude drug content of 1.0 g / mL was obtained; the jujube seed extract liquid was sterilized at 121 ℃ for 15 min to obtain a sterilized jujube seed extract liquid;

[0082] The Lactobacillus reuteri ZKLr0413 bacterial liquid was freeze-dried at -50 ℃ for 40 h to obtain a freeze-dried powder of Lactobacillus reuteri ZKLr0413 with a viable bacterial count of 1×10 11 CFU / g; the freeze-dried powder of Lactobacillus reuteri ZKLr0413 was inoculated into the sterilized jujube seed extract liquid at an inoculation amount of 0.1 g / L, and then was uniformly shaken and cultured at 37 ℃ for 24 h; after the culture, a Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid one was obtained, which was referred to as fermentation liquid 1.

[0083] The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid two was obtained under the following process parameters:

[0084] The jujube seed powder with a particle size of 300 meshes was put into a decoction bag and then was soaked in 10 times of water for 0.5 h to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled with a large fire, and then was slowly boiled with a small fire for 1 h; during the slow boiling process, the jujube seed soaking liquid was kept slightly boiling; then, the liquid and the residue were separated by filtration; the liquid was concentrated by boiling; a jujube seed extract liquid with a crude drug content of 1.0 g / mL was obtained; the jujube seed extract liquid was sterilized at 121 ℃ for 15 min to obtain a sterilized jujube seed extract liquid;

[0085] The Lactobacillus reuteri ZKLr0413 bacterial liquid was freeze-dried at -50 ℃ for 40 h to obtain a freeze-dried powder of Lactobacillus reuteri ZKLr0413 with a viable bacterial count of 1×10 11 CFU / g; the freeze-dried powder of Lactobacillus reuteri ZKLr0413 was inoculated into the sterilized jujube seed extract liquid at an inoculation amount of 0.1 g / L, and then was uniformly shaken and cultured at 37 ℃ for 24 h; after the culture, a Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid two was obtained.

[0086] Comparative Example 1: Fermentation of jujube seed extract liquid using a single strain or mixed strains of probiotics

[0087] 1. Preparation of Lactobacillus plantarum RZKLp100 jujube seed fermentation liquid

[0088] Lactobacillus plantarum RZKLp100 was used instead of Lactobacillus reuteri ZKLr0413 to ferment the acid jujube extract. Lactobacillus plantarum RZKLp100 is a probiotic bacteria collected and preserved by the applicant and has been disclosed in Chinese invention patent CN 118374549 B. Previous studies have found that Lactobacillus plantarum RZKLp100 has good effects on fermenting licorice.

[0089] The specific steps are as follows:

[0090] The acid jujube powder with a particle size of 70 mesh was placed in a decoction bag and then soaked in 8 times the mass of water for 1 h to obtain an acid jujube soaking solution. The acid jujube soaking solution was boiled with a large fire, and then slowly simmered with a small fire for 2 h. During the slow simmering process, the acid jujube soaking solution was kept slightly boiling. Then, the liquid and residue were separated by filtration. The liquid was concentrated by evaporation to obtain an acid jujube extract with a crude drug content of 1.0 g / mL. The acid jujube extract was sterilized at 121℃ for 15 min to obtain a sterilized acid jujube extract.

[0091] The Lactobacillus plantarum RZKLp100 bacterial solution was freeze-dried at -50℃ for 40 h to obtain Lactobacillus plantarum RZKLp100 freeze-dried powder with a viable count of 1×10 11 CFU / g. The Lactobacillus plantarum RZKLp100 freeze-dried powder was inoculated into the sterilized acid jujube extract at an inoculation amount of 0.1 g / L, and then shaken uniformly. The mixture was incubated at 37℃ for 24 h under constant temperature and aerobic conditions. After incubation, a Lactobacillus plantarum RZKLp100 acid jujube fermentation solution, referred to as fermentation solution 2, was obtained.

[0092] 2. Preparation of Lactobacillus acidophilus RZKLa0701 acid jujube fermentation solution

[0093] Lactobacillus acidophilus RZKLa0701 was used instead of Lactobacillus reuteri ZKLr0413 to ferment the acid jujube extract. Lactobacillus acidophilus RZKLa0701 is a probiotic bacteria collected and preserved by the applicant and has been disclosed in Chinese invention patent CN 117305188 B.

[0094] The specific steps are as follows:

[0095] The acid jujube powder with a particle size of 70 mesh was placed in a decoction bag and then soaked in 8 times the mass of water for 1 h to obtain an acid jujube soaking solution. The acid jujube soaking solution was boiled with a large fire, and then slowly simmered with a small fire for 2 h. During the slow simmering process, the acid jujube soaking solution was kept slightly boiling. Then, the liquid and residue were separated by filtration. The liquid was concentrated by evaporation to obtain an acid jujube extract with a crude drug content of 1.0 g / mL. The acid jujube extract was sterilized at 121℃ for 15 min to obtain a sterilized acid jujube extract.

[0096] The Lactobacillus acidophilus RZKLa0701 bacterial solution was freeze-dried at -50℃ for 40 h to obtain Lactobacillus acidophilus RZKLa0701 freeze-dried powder with a viable count of 1×1011 Lactobacillus acidophilus RZKLa0701 freeze-dried powder with a CFU of 1 x 10

[0097] 3. Preparation of Lactobacillus casei ZKLc1221 Zizyphus jujuba Mill. fermentation broth

[0098] Lactobacillus casei ZKLc1221 was used to replace Lactobacillus reuteri ZKLr0413 to ferment Zizyphus jujuba Mill. extract. Lactobacillus casei ZKLc1221 is a probiotic bacteria collected and preserved by the applicant and has been disclosed in Chinese Invention Patent CN 119614462 B.

[0099] The specific steps are as follows:

[0100] Zizyphus jujuba Mill. powder with a particle size of 70 mesh was placed in a decoction bag and soaked in 8 times the mass of water for 1 h to obtain Zizyphus jujuba Mill. soaking liquid. The Zizyphus jujuba Mill. soaking liquid was boiled with a large fire, and then slowly boiled with a small fire for 2 h. During the slow boiling process with a small fire, the Zizyphus jujuba Mill. soaking liquid was kept slightly boiling, and then the liquid and residue were separated by filtration. The liquid was concentrated by evaporation to obtain Zizyphus jujuba Mill. extract with a crude drug content of 1.0 g / mL. The Zizyphus jujuba Mill. extract was sterilized at 121℃ for 15 min to obtain sterilized Zizyphus jujuba Mill. extract.

[0101] Lactobacillus casei ZKLc1221 bacterial liquid was freeze-dried at -50℃ for 40 h to obtain Lactobacillus casei ZKLc1221 freeze-dried powder with a viable count of 1 x 10 11 Lactobacillus casei ZKLc1221 freeze-dried powder with a CFU of 1 x 10

[0102] 4. Preparation of Lactobacillus plantarum RZKLp100 / Lactobacillus casei ZKLc1221 Zizyphus jujuba Mill. fermentation broth

[0103] According to the prior art, the mixed bacterial liquid of Lactobacillus plantarum and Lactobacillus casei has the effect of improving the dissolution rate of saponins and flavonoids active ingredients in Zizyphus jujuba Mill. Therefore, the mixed bacterial liquid of Lactobacillus plantarum RZKLp100 and Lactobacillus casei ZKLc1221 was used to replace Lactobacillus reuteri ZKLr0413 to ferment Zizyphus jujuba Mill. extract.

[0104] The specific steps are as follows:

[0105] The jujube seed powder with a particle size of 70 meshes was put into a decoction bag, and then was put into 8 times of water by mass to soak for 1 h to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled with a large fire, and then was slowly boiled with a small fire for 2 h; the jujube seed soaking liquid was kept slightly boiling during the slow boiling process with a small fire, and then was filtered to separate a liquid and a residue; the liquid was concentrated by evaporation to obtain a jujube seed extract liquid with a crude drug content of 1.0 g / mL, and then was sterilized at 121 ℃ for 15 min to obtain a sterilized jujube seed extract liquid;

[0106] The Lactobacillus plantarum RZKLp100 bacterial liquid was freeze-dried at -50 ℃ for 40 h to prepare a freeze-dried powder of Lactobacillus plantarum RZKLp100 with a viable bacterial count of 1×10 11 CFU / g; the Lactobacillus casei ZKLc1221 bacterial liquid was freeze-dried at -50 ℃ for 40 h to prepare a freeze-dried powder of Lactobacillus casei ZKLc1221 with a viable bacterial count of 1×10 11 CFU / g; the freeze-dried powder of Lactobacillus plantarum RZKLp100 was inoculated into the sterilized jujube seed extract liquid at an inoculation amount of 0.067 g / L, the freeze-dried powder of Lactobacillus casei ZKLc1221 was inoculated into the sterilized jujube seed extract liquid at an inoculation amount of 0.033 g / L, and then was shaken and uniformly mixed, and then was incubated at 37 ℃ for 24 h under aerobic conditions, to obtain a Lactobacillus plantarum RZKLp100 / Lactobacillus casei ZKLc1221 jujube seed fermentation liquid, which is referred to as fermentation liquid 5.

[0107] Example 4: Test on components of fermentation liquid

[0108] 1. Test on pH value and viable bacterial count of fermentation liquid

[0109] The pH value and viable bacterial count of the fermentation liquid 1-fermentation liquid 5 prepared in the above example 3 and comparative example 1 were determined, and the results are shown in Table 3. It can be seen that the viable bacterial count of the fermentation liquid 1 is higher than that of the fermentation liquid 2-fermentation liquid 5, which indicates that the Lactobacillus reuteri ZKLr0413 provided in the application has a better fermentation effect on jujube seed.

[0110] Table 3: pH value and viable bacterial count of fermentation liquid 1-fermentation liquid 5

[0111]

[0112] 2. Test on total flavonoid content and total saponin content of fermentation liquid

[0113] (1) Test on total flavonoid content

[0114] ① Reagents:

[0115] Petroleum ether (boiling range 60-90℃, analytical pure), ethanol (analytical pure), methanol (analytical pure) were purchased from National Pharmaceutical Group; Sodium nitrite (analytical pure), aluminum nitrate (analytical pure), sodium hydroxide (analytical pure) were purchased from Chengdu Kelong Chemical Reagent Factory; Spinonin (analytical pure) was purchased from Shanghai Yuan Ye Biological Co., Ltd.

[0116] 2. Preparation of solution:

[0117] Accurately take 70 mL of anhydrous ethanol into a 100 mL volumetric flask, add distilled water to constant volume, mix well, and get 70% ethanol.

[0118] Accurately take 5.0 g of sodium nitrite into a 100 mL volumetric flask, add water to constant volume, mix well, and get 5% sodium nitrite solution.

[0119] Accurately take 10.0 g of aluminum nitrate into a 100 mL volumetric flask, add water to constant volume, mix well, and get 10% aluminum nitrate solution.

[0120] Accurately take 4.0 g of sodium hydroxide into a 100 mL volumetric flask, add water to constant volume, mix well, and get 4% sodium hydroxide solution.

[0121] Accurately take 12.00 mg of spinonin, place it in a 10 mL volumetric flask, add methanol to constant volume, mix well, and get a concentration of 1.20 mg / mL of reference solution.

[0122] Accurately take 10 mL of fermentation broth 1-fermentation broth 5, respectively, into a Soxhlet extractor, add an appropriate amount of petroleum ether, heat reflux for 6 h, after reflux, discard the petroleum ether liquid, and fully evaporate the solvent in the residue. Transfer the residue to a round-bottom flask, add 60 mL of 70% ethanol, and extract at 100℃ water bath for 3 times, with extraction times of 1 h, 1 h, and 40 min, respectively. After extraction, combine the extract, and concentrate the extract at 60℃ using a rotary evaporator. Finally, transfer the concentrated liquid to a 10 mL volumetric flask, add methanol to constant volume, and get test solution 1-5 for subsequent determination of total flavonoid content.

[0123] Accurately take 10 mL of sterilized Chinese date seed extract solution (preparation method see Comparative Example 1), place it in a Soxhlet extractor, add an appropriate amount of petroleum ether, heat and reflux for 6 h. After the reflux is completed, discard the petroleum ether liquid, and fully evaporate the solvent in the residue. Transfer the residue to a round-bottom flask, add 60 mL of 70% ethanol, and extract under the condition of a 100°C water bath for 3 times, with extraction times of 1 h, 1 h, and 40 min respectively. After the extraction is completed, combine the extract solutions and concentrate the extract solutions at 60°C using a rotary evaporator. Finally, transfer the concentrated solution to a 10 mL volumetric flask, add methanol to the calibration mark, and obtain a test solution 6 for subsequent determination of total flavonoid content.

[0124] ③Preparation of standard curve:

[0125] Accurately pipette different volumes (0.50, 0.75, 1.00, 1.25, 1.5 mL) of the control solution into 10 mL volumetric flasks, respectively, and add 3 mL of methanol to each. Then add 0.50 mL of 5% sodium nitrite solution, shake well, and let stand for 6 min. Add 0.50 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Next, add 4 mL of 4% sodium hydroxide solution, and finally dilute to the calibration mark with distilled water. Shake well and let stand for 15 min.

[0126] Using distilled water as a blank control, measure the absorbance at a wavelength of 396 nm on a UV-6100 ultraviolet spectrophotometer. Plot the standard curve with the content of the control (mg) as the abscissa and the absorbance as the ordinate. The regression equation of the standard curve is Y = 0.0435X - 0.0018, with a correlation coefficient R 2 = 0.9999, indicating a good linear relationship within the concentration range of 0.600-1.800 mg.

[0127] ④Determination of total flavonoid content:

[0128] Accurately pipette 1.0 mL of test solution 1-6 into 10 mL volumetric flasks, and follow the same solution treatment steps as in the standard curve preparation process, i.e. add 3 mL of methanol, 0.5 mL of 5% sodium nitrite solution, shake well and let stand for 6 min, add 0.5 mL of 10% aluminum nitrate solution, shake well and let stand for 6 min, add 4 mL of 4% sodium hydroxide solution, and finally dilute to the calibration mark with distilled water. Shake well and let stand for 15 min.

[0129] Again, using distilled water as a blank control, measure the absorbance at a wavelength of 396 nm on a UV-6100 ultraviolet spectrophotometer. Finally, according to the standard curve plotted earlier, calculate the total flavonoid content (mg / mL).

[0130] ⑤Test results:

[0131] The total flavone contents of the fermentation liquor 1-5 and the sterilized Chinese date kernel extract are shown in Table 4. It can be seen that the total flavone content of the fermentation liquor 1 is increased by 91.78% compared with the sterilized Chinese date kernel extract, and is also significantly higher than the total flavone contents of the fermentation liquor 2-5, indicating that the fermentation ability of the Lactobacillus reuteri ZKLr0413 provided in the application to the Chinese date kernel extract is better than that of other common lactobacillus single bacteria or mixed fermentation effect.

[0132] Table 4 Total flavone contents of the fermentation liquor 1-5 and the sterilized Chinese date kernel extract

[0133]

[0134] Note: Different letters in the table represent significant differences, P <0.05.

[0135] (2) Total saponin content test

[0136] ① Reagent:

[0137] Glacial acetic acid (analytical pure), perchloric acid (analytical pure) were purchased from China National Pharmaceutical Group; vanillin (analytical pure), Chinese date kernel saponin A standard (analytical standard, item number: J114068-20 mg) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0138] ② Solution preparation:

[0139] Accurately take 70 mL of anhydrous ethanol in a 100 mL volumetric flask, add distilled water to constant volume, mix well, and obtain 70% ethanol.

[0140] Accurately take 5.0 g of vanillin in a 100 mL volumetric flask, add glacial acetic acid to constant volume, mix well, and obtain 5% vanillin glacial acetic acid solution.

[0141] Accurately take 2.50 mg of Chinese date kernel saponin A standard, place it in a 10 mL volumetric flask, add methanol to constant volume, mix well, and obtain a control solution with a concentration of 0.25 mg / mL.

[0142] Accurately take 10 mL of fermentation broth 1-fermentation broth 5, respectively, in a Soxhlet extractor, add an appropriate amount of petroleum ether, heated to reflux for 6 h, after reflux, discard the petroleum ether liquid, the solvent in the drug residue is fully dry. The drug residue was transferred to a round-bottom flask, 60 mL of 70% ethanol was added, and 3 times of extraction was carried out under the condition of 100℃ water bath, the extraction time was 1 h, 1 h, 40 min respectively. After extraction, the extract was concentrated at 60℃ by rotary evaporator. The concentrated liquid was placed in a 50℃ air drying oven and dried into powder. Finally, the powder was transferred to a 5 mL volumetric flask, and methanol was added to the mark to obtain the test solution 1-5 for the determination of total saponin content.

[0143] Accurately take 10 mL of sterilized Zizyphus jujuba extract (preparation method see comparative example 1), place it in a Soxhlet extractor, add an appropriate amount of petroleum ether, heat to reflux for 6 h, after reflux, discard the petroleum ether liquid, the solvent in the drug residue is fully dry. The drug residue was transferred to a round-bottom flask, 60 mL of 70% ethanol was added, and 3 times of extraction was carried out under the condition of 100℃ water bath, the extraction time was 1 h, 1 h, 40 min respectively. After extraction, the extract was concentrated at 60℃ by rotary evaporator. The concentrated liquid was placed in a 50℃ air drying oven and dried into powder. Finally, the powder was transferred to a 5 mL volumetric flask, and methanol was added to the mark to obtain the test solution 6 for the determination of total saponin content.

[0144] ③Standard curve drawing:

[0145] Accurately take different volumes (0.10, 0.15, 0.20, 0.30, 0.40, 0.50 mL) of the control solution, respectively, in a test container, dry the solvent, and add 0.20 mL of 5% vanillin glacial acetic acid solution; then add 0.80 mL of perchloric acid, seal and heat in a 70℃ water bath for 20 min, take out immediately and cool in an ice water bath for 5 min, add 5 mL of glacial acetic acid and shake well.

[0146] The absorbance was measured at 546 nm wavelength on a UV-6100 type ultraviolet spectrophotometer with methanol as a blank control. The content (mg) of the control was taken as the abscissa and the absorbance as the ordinate to draw the standard curve. The regression equation of the standard curve was Y=3.8406X+0.0101, the correlation coefficient R 2 =0.9999, indicating that the linear relationship was good within the concentration range of 0.025-0.125 mg.

[0147] ④Total saponin content determination:

[0148] Precise 0.10 mL of the test solution 1-6 was taken and placed in the test container, and the subsequent operation steps were the same as the processing steps of the solution in the standard curve preparation process, that is, 0.20 mL of 5% vanillin glacial acetic acid solution was added; then 0.80 mL of perchloric acid was added, sealed and heated at 70℃ in a water bath for 20 min, immediately cooled in an ice water bath for 5 min, and 5 mL of glacial acetic acid was added and shaken well.

[0149] Similarly, methanol was used as a blank control, and the absorbance was measured at 546 nm wavelength on a UV-6100 type ultraviolet spectrophotometer. Finally, according to the standard curve drawn before, the total saponin content (mg / mL) was calculated.

[0150] ⑤Test results:

[0151] The total saponin contents in the fermentation broth 1-5 and the sterilized Chinese date kernel extract are shown in Table 5. It can be seen that the total saponin content of the fermentation broth 1 is increased by 89.66% compared with the sterilized Chinese date kernel extract without fermentation, and is also significantly higher than the total saponin contents of the fermentation broth 2-5, indicating that the fermentation ability of the Lactobacillus reuteri ZKLr0413 provided by the present application on the Chinese date kernel extract is better than that of other common lactobacillus single bacteria or mixed fermentation effect.

[0152] Table 5 Total saponin contents of fermentation broth 1-5 and sterilized Chinese date kernel extract

[0153]

[0154] Note: Different letters in the table represent significant differences, P <0.05.

[0155] Example 5 Sleep improvement effect of fermentation broth on insomnia rats

[0156] 1. Reagents:

[0157] p-chlorophenylalanine (PCPA) was purchased from Sigma Company of the United States; sodium pentobarbital was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; and diazepam was purchased from Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.

[0158] 2. Modeling:

[0159] 90 Wistar rats of 8-9 weeks old and weighing 180-220 g were purchased. The rats were adaptively fed for 7 days, and the rats were free to eat feed and water, and the litter was kept dry; after 7 days, the body weight was weighed, and the rats were randomly divided into a blank group (10 rats) and a modeling group (80 rats), and the modeling group was intraperitoneally injected with PCPA for 2 days, and PCPA was prepared into a suspension with physiological saline before use according to the dosage of 400 mg / kg of the body weight of the rats, and the blank group was intraperitoneally injected with the same volume of physiological saline for 2 days.

[0160] The phenomenon of circadian rhythm loss was observed in the modeling group rats, which showed continuous activity during the day and night. Through the pentobarbital sodium reversal experiment, it was found that there was a statistical significance in sleep latency and sleep time between the blank group and the modeling group, which indicated the success of modeling.

[0161] 3. Experimental grouping and administration:

[0162] Blank group: 0.4 mL of normal saline was administered to each rat per day for 14 consecutive days.

[0163] The 80 modeling successful rats were randomly divided into a model group, a fermentation broth 1 group, a fermentation broth 2 group, a fermentation broth 3 group, a fermentation broth 4 group, a fermentation broth 5 group, an extract 1 group, and a diazepam positive control group.

[0164] Model group: 0.4 mL of normal saline was administered to each rat per day for 14 consecutive days.

[0165] Fermentation broth 1 group: 0.4 mL of the fermentation broth 1 prepared in Example 3 was administered to each rat per day for 14 consecutive days.

[0166] Fermentation broth 2 group: 0.4 mL of the fermentation broth 2 prepared in Comparative Example 1 was administered to each rat per day for 14 consecutive days.

[0167] Fermentation broth 3 group: 0.4 mL of the fermentation broth 3 prepared in Comparative Example 1 was administered to each rat per day for 14 consecutive days.

[0168] Fermentation broth 4 group: 0.4 mL of the fermentation broth 4 prepared in Comparative Example 1 was administered to each rat per day for 14 consecutive days.

[0169] Fermentation broth 5 group: 0.4 mL of the fermentation broth 5 prepared in Comparative Example 1 was administered to each rat per day for 14 consecutive days.

[0170] Extract group: 0.4 mL of sterilized Semen Ziziphi Spinosae extract (prepared by the same method as in Comparative Example 1) was administered to each rat per day for 14 consecutive days.

[0171] Positive control group: 0.4 mL of a diazepam normal saline solution with a concentration of 1 mg / mL was administered to each rat per day for 14 consecutive days.

[0172] 4. Experimental methods and results

[0173] After the last administration of 30 min, each group of rats was intraperitoneally injected with 25.00 mg of pentobarbital sodium / kg of body weight in a quiet environment at 24~25℃.

[0174] The number of rats falling asleep within 30 min (the number of rats whose righting reflex disappeared for more than 1 min) was recorded, the sleep conditions of the rats in each group were counted, and the sleep rate was calculated. The results are shown in Table 6. It can be seen that, compared with the model group, the sleep rate of the rats in the fermentation liquor 1 group was significantly increased, close to that of the blank group, and higher than that of the fermentation liquor 2 group, the fermentation liquor 3 group, the fermentation liquor 4 group, the fermentation liquor 5 group and the extract group. This shows that the ZKLr0413 Zizyphus jujuba Mill. fermentation liquor provided by the application has the strongest ability to improve the sleep rate of insomnia rats.

[0175] Table 6: Sleep rate statistics of rats in each group

[0176]

[0177] The time from pentobarbital sodium injection to disappearance of righting reflex and the time from disappearance of righting reflex to recovery were recorded to observe the effect of the test substance on pentobarbital sodium sleep latency and sleep time. The results are shown in Table 7. It can be seen that, compared with the model group, the sleep latency of the rats in the fermentation liquor 1 group was significantly reduced, close to that of the blank group, and significantly lower than that of the fermentation liquor 2 group, the fermentation liquor 3 group, the fermentation liquor 4 group, the fermentation liquor 5 group and the extract group. Compared with the model group, the sleep time of the rats in the fermentation liquor 1 group was significantly increased, close to that of the blank group, and significantly higher than that of the fermentation liquor 2 group, the fermentation liquor 3 group, the fermentation liquor 4 group, the fermentation liquor 5 group and the extract group. This shows that the ZKLr0413 Zizyphus jujuba Mill. fermentation liquor provided by the application has a significant sleep-inducing effect.

[0178] Table 7: Sleep latency and sleep time statistics of rats in each group

[0179]

[0180] Note: Different letters in the table represent significant differences, P <0.05.

[0181] After the rats were sacrificed, the whole brain was removed by craniotomy in an ice bath environment, and the brain was placed on an ice flat dish for separation. The brain was rinsed with ice 0.9% sodium chloride solution to remove blood, and then wiped dry with filter paper. After weighing the brain, 9 times the amount of ice 0.9% sodium chloride solution was added, and homogenization was performed at 4℃. The homogenized slurry was transferred to a 2 mL centrifuge tube, centrifuged at 3000 r / min for 15 min at 4℃, and the supernatant was taken. According to the related operation steps of the kit (purchased from Wuhan Pure Biotechnology Co., Ltd.), the contents of 5-hydroxytryptamine and gamma-aminobutyric acid in the supernatant were determined.

[0182] The results are shown in Table 8 below. It can be seen that, compared with the model group, the content of 5-hydroxytryptamine and gamma-aminobutyric acid in the brain of the rats in the fermentation liquor 1 group was significantly increased, close to that of the blank group, and significantly higher than that of the fermentation liquor 2 group, the fermentation liquor 3 group, the fermentation liquor 4 group, the fermentation liquor 5 group and the extraction liquor group. This shows that the Lactobacillus reuteri ZKLr0413 Zizyphus jujuba fermentation liquor provided by the application can significantly promote the release of 5-hydroxytryptamine and gamma-aminobutyric acid in the brain of the insomnia rats.

[0183] Table 8: Content of 5-hydroxytryptamine and gamma-aminobutyric acid in the brain of rats in each group

[0184]

[0185] Note: Different letters in the table represent significant differences, P <0.05.

[0186] In summary, compared with other single-strain fermentation or mixed fermentation processes of probiotics, the Lactobacillus reuteri ZKLr0413 single-strain fermentation of Zizyphus jujuba extract liquid used in the application has significant advantages in multiple indicators: the number of viable bacteria, the content of total flavonoids and total saponins in the Lactobacillus reuteri ZKLr0413 Zizyphus jujuba fermentation liquor are higher, and these components have a positive effect on sleep regulation. The rat experiment further verifies the sleep improvement effect of the Lactobacillus reuteri ZKLr0413 Zizyphus jujuba fermentation liquor: compared with the model group, the sleep rate of the rats administered with the Lactobacillus reuteri ZKLr0413 Zizyphus jujuba fermentation liquor is significantly improved, higher than that of the rats administered with fermentation liquor 2-fermentation liquor 5 and sterilized Zizyphus jujuba extract liquid, and closer to that of the normal rats. This series of results show that the Lactobacillus reuteri ZKLr0413 provided by the application has good adaptability to Zizyphus jujuba extract liquid, can ferment Zizyphus jujuba extract liquid in a targeted manner, fully stimulate the active ingredients of Zizyphus jujuba, and thus effectively improve the sleep quality, providing a new effective solution for insomnia-related problems.

[0187] Although the application has been described in detail with reference to the preferred embodiments, the application is not limited to this. Those skilled in the art can make various equivalent modifications or replacements to the embodiments of the application without departing from the spirit and essence of the application, and these modifications or replacements should be within the scope of the application. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the application, which should be covered by the protection scope of the application.

Claims

1. A *Lactobacillus reuteri* ZKLr0413 jujube seed fermentation broth for improving sleep, characterized in that, The lyophilized powder of Lactobacillus reuteri ZKLr0413 was prepared at a ratio of 1×10⁻⁶. 10 The ratio of CFU / L to jujube seed extract, the viable count of lyophilized Lactobacillus reuteri ZKLr0413 inoculated into jujube seed extract, was 1×10⁻⁶. 11 CFU / g, and then cultured at 37℃ under constant temperature and aerobic conditions. After the culture was completed, the fermentation broth of Lactobacillus reuteri ZKLr0413 jujube seed was obtained. After fermentation, the viable count of Lactobacillus reuteri ZKLr0413 in the jujube seed fermentation broth was 1.58 × 10⁻⁶. 9 The total flavonoid content of the fermented broth of *Lactobacillus reuteri* ZKLr0413 in jujube seed was 82.39±7.42 mg / mL, and the total saponin content of the fermented broth of *Lactobacillus reuteri* ZKLr0413 in jujube seed was 73.57±4.23 mg / mL. Lactobacillus reuteri ZKLr0413 was deposited on May 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30546, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Lactobacillus reuteri. Lactobacillus reuteri ; The jujube seed extract is obtained by soaking and decocting jujube seed powder.

2. The *Lactobacillus reuteri* ZKLr0413 jujube seed fermentation broth as described in claim 1, characterized in that, The preparation method of jujube seed extract is as follows: The jujube seed powder was placed in a decoction bag and soaked in water to obtain a jujube seed soaking solution. The jujube seed soaking solution was then decocted, and the liquid and residue were separated by filtration. The liquid was then concentrated by steaming to obtain a jujube seed extract with a crude drug content of 1.0 g / mL.

3. The *Lactobacillus reuteri* ZKLr0413 jujube seed fermentation broth as described in claim 1 or 2, characterized in that, The particle size of jujube seed powder is 70-300 mesh.

4. The *Lactobacillus reuteri* ZKLr0413 jujube seed fermentation broth as described in claim 1 or 2, characterized in that, Before inoculating with Lactobacillus reuteri ZKLr0413, the jujube seed extract was first sterilized.

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