Lactobacillus reuteri ZKLr0413 spina date seed fermentation liquor for improving sleep and lactobacillus reuteri ZKLr0413

Abstract

The invention relates to the technical field of products with a sleep improving function, in particular to lactobacillus reuteri ZKLr0413 semen ziziphi spinosae fermentation liquor for improving sleep and lactobacillus reuteri ZKLr0413. Lactobacillus reuteri ZKLr0413 spina date seed fermentation liquor capable of improving sleep is prepared by fermenting a spina date seed extracting solution through lactobacillus reuteri ZKLr0413, the lactobacillus reuteri ZKLr0413 is preserved in the China General Microbiological Culture Collection Center on May 13, 2024, the preservation number is CGMCC No.30546, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the semen ziziphi spinosae extracting solution is obtained by soaking and decocting semen ziziphi spinosae powder. Compared with mixed bacteria fermentation or mixed fermentation of multiple medicinal and edible materials, the fermentation liquor is simple in component, and the advantages of safety and process stability are remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of products with the function of improving sleep, and in particular to a Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid and Lactobacillus reuteri ZKLr0413 for improving sleep. Background Art

[0002] Ziziphus jujuba seeds are the dried seeds of the plant Ziziphus jujuba, a member of the Rhamnaceae family. They are sweet and neutral in nature, and have been shown to calm the mind, nourish the liver, and promote sweating and fluid production. They were first mentioned in the Shennong's Classic of Materia Medica. In the Ming Dynasty's Compendium of Materia Medica, Li Shizhen stated that jujube seeds, when cooked, "treat insomnia due to gallbladder deficiency, thirst, and spontaneous sweating; and when used raw, treat gallbladder heat and promote sleep. Both are remedies for the Jueyin and Shaoyang meridians of the foot." Modern research indicates that jujube seeds have benefits such as improving sleep disorders, providing antioxidant benefits, antidepressant properties, anxieties, and learning and memory enhancement. The flavonoids and saponins they contain are active ingredients in improving sleep. Therefore, improving the bioavailability of these active ingredients, promoting drug absorption, and increasing their content are pressing challenges in the development and utilization of jujube seed resources.

[0003] Microbial fermentation of Chinese jujube seeds can, to a certain extent, alleviate the aforementioned issues with their utilization. For example, CN116725179 A discloses a herbal enzyme for sleep and tranquility. The enzyme uses Chinese jujube seeds in combination with mulberries, lilies, and black dates as raw materials. These ingredients are pretreated by steaming to produce a liquid stock. A mixed strain of Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus in a specific ratio is then introduced into the liquid stock for fermentation. The enzyme is then filtered and centrifuged to produce the herbal enzyme.

[0004] CN 115990212 A discloses a composition prepared by fermenting ultrafine powder of Chinese date seeds with a mixed bacterial liquid of Lactobacillus plantarum and Lactobacillus casei. The composition has the function of regulating GABA-A receptors.

[0005] CN 120000741 A discloses a probiotic and traditional Chinese medicine composition that can be used to treat insomnia. The probiotics used are as many as 18 types, and the number of viable bacteria required for fermentation is as high as 200 billion CFU / g. The traditional Chinese medicine composition includes Chinese jujube seeds, lily, poria, lotus seeds and mulberries.

[0006] It can be seen that the current means of enhancing the sleep-inducing effect of Chinese jujube seeds mainly focus on microbial mixed fermentation or increasing the synergistic efficacy of traditional Chinese medicines. However, these strategies have exposed obvious limitations in practical applications. For the microbial mixed fermentation strategy, competition among multiple bacterial species can easily lead to fluctuations in fermentation efficiency. The differences in preferences or competition among different bacterial species for nutrient substrates may result in the inability to fully convert the effective ingredients of flavonoids and saponins in Chinese jujube seeds. The bacteriocins secreted by different bacterial species or the acidic substances produced by metabolism will inhibit each other's growth, causing an imbalance in the proportion of bacterial populations and resulting in significant quality fluctuations between product batches. For the strategy of increasing the synergistic efficacy of traditional Chinese medicines, although the efficacy can be superimposed in theory, the physical and chemical properties of each component are often different. If a pretreatment scheme is designed for each material separately, the production cost will be greatly increased; if the process conditions are unified, the components of some materials will be destroyed. In addition, the mixing of multiple components is prone to produce unknown complexes or precipitation, and even introduces complex risks of pesticide residues and heavy metal pollution, which increases the difficulty of component analysis and quality control, making it difficult to trace and control safety hazards.

[0007] Based on this, the development of preparations using fermented jujube seeds with a single strain of bacteria can not only avoid metabolic conflicts between strains, but also simplify the production process, which is conducive to ensuring product safety and process stability. Summary of the Invention

[0008] To address technical issues such as interspecies competition, low and easily destroyed component conversion efficiency, difficulty standardizing process conditions, high costs, and complex and difficult-to-control ingredients that often arise from mixed microbial fermentation or the use of traditional Chinese medicines to enhance synergistic efficacy, the present invention provides a sleep-enhancing fermentation broth of Chinese jujube seeds using Lactobacillus reuteri ZKLr0413 and Lactobacillus reuteri ZKLr0413. Lactobacillus reuteri ZKLr0413 can be used alone in the fermentation of Chinese jujube seed extracts to efficiently release the sleep-promoting flavonoids and saponins found in the seeds. Compared to mixed fermentation or mixed fermentation of multiple medicinal and edible materials, the fermentation broth of the present invention has simpler ingredients and offers significant advantages in safety and process stability.

[0009] The technical solutions of the present invention are as follows: In the first aspect, a sleep-improving Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid is prepared from Lactobacillus reuteri ( Lactobacillus reuteri ) ZKLr0413 fermented jujube seed extract; Among them, Lactobacillus reuteri ZKLr0413 has been deposited in the General Microbiology Center of China Culture Collection Administration on May 13, 2024, with the deposit number CGMCC No.30546, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as Lactobacillus reuteri Lactobacillus reuteri ; The Chinese jujube seed extract is obtained by soaking and decocting Chinese jujube seed powder.

[0010] Furthermore, the preparation method of the Chinese date seed extract is as follows: The Chinese jujube seed powder is put into a decoction bag and then soaked in water to obtain a Chinese jujube seed soaking liquid; the Chinese jujube seed soaking liquid is decocted, and then the liquid and the residue are separated by filtration, and the liquid is steamed and concentrated to obtain a Chinese jujube seed extract with a crude drug content of 1.0 g / mL.

[0011] Furthermore, the particle size of the Suanzaorensis seed powder is 70-300 meshes.

[0012] Furthermore, Lactobacillus reuteri ZKLr0413 was added at a concentration of 1×10 10 The ratio of CFU / L of jujube seed extract was inoculated into the jujube seed extract, and then cultured aerobically at a constant temperature of 37°C. After the culture was completed, the jujube seed fermentation liquid of Lactobacillus reuteri ZKLr0413 was obtained.

[0013] Furthermore, before inoculating Lactobacillus reuteri ZKLr0413, the jujube seed extract was first sterilized.

[0014] Furthermore, the freeze-dried powder of Lactobacillus reuteri ZKLr0413 was added to the jujube seed extract for fermentation. The viable cell count of the freeze-dried powder of Lactobacillus reuteri ZKLr0413 was 1×10 11 CFU / g.

[0015] Furthermore, after the fermentation was completed, the viable bacterial count of the fermentation liquid of Lactobacillus reuteri ZKLr0413 was 1.58×10 9 CFU / mL.

[0016] Furthermore, the total flavonoids content of the fermentation liquid of jujube seeds of Lactobacillus reuteri ZKLr0413 was 82.39±7.42 mg / mL.

[0017] Furthermore, the total saponin content of the fermentation liquid of jujube seeds of Lactobacillus reuteri ZKLr0413 was 73.57±4.23 mg / mL.

[0018] In a second aspect, the present invention also provides a strain of Lactobacillus reuteri ZKLr0413, which has been deposited in the General Microbiology Center of the China Culture Collection Administration Committee on May 13, 2024, with a deposit number of CGMCC No. 30546 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as Lactobacillus reuteri. Lactobacillus reuteri .

[0019] The beneficial effects of the present invention are: The Lactobacillus reuteri ZKLr0413 used in the present invention has an excellent fermentation effect on the Chinese jujube seed extract, can withstand the low pH environment caused by the fermentation of the Chinese jujube seed, and maintains the number of live bacteria in the fermentation liquid at a high level; at the same time, the Lactobacillus reuteri ZKLr0413 can effectively promote the dissolution of sleep-inducing active substances such as flavonoids and saponins in the Chinese jujube seed extract, and its dissolution effect is better than the dissolution effect of single-bacteria fermentation or mixed fermentation of commonly used probiotics. While ensuring the fermentation efficiency, the product safety and process stability are improved.

[0020] The experimental results show that the fermented jujube seed liquid fermented by Lactobacillus reuteri ZKLr0413 can significantly improve sleep, significantly increase sleep rate, shorten sleep latency, prolong total sleep time, and stimulate the brain to release serotonin and γ-aminobutyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0022] Figure 1 This is a photo of the colony morphology of the isolated strain in Example 1.

[0023] Figure 2 This is a Gram staining picture of the isolated strain in Example 1. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0025] The MRS solid culture medium and MRS liquid culture medium used in the following examples were purchased from Beijing Aoboxing Biotechnology Co., Ltd.; MRS-CaCO3 solid culture medium was prepared by adding 1.5% CaCO3 to MRS solid culture medium and then sterilizing it.

[0026] The DNA extraction kit used in the following examples was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0027] Example 1 Isolation and identification of Lactobacillus reuteri ZKLr0413 1. Sample source: Collected from Songbai Town, Shennongjia Forest District, Hubei Province in October 2013.

[0028] 2. Strain isolation: dilute the collected samples with physiological saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Draw 100 µL of each dilution evenly onto MRS-CaCO₃ solid medium. Incubate inverted at 37°C anaerobically for 24–48 hours and observe colony growth. Once colonies have formed, select a single colony with a calcium ring and perform three streaking operations. Repeat this streaking operation multiple times until the colonies are purified. Purification is determined by uniform colony morphology and Gram staining. Inoculate the purified strain into MRS liquid medium. Mix the culture medium with 50% sterile glycerol in a 1:1 volume ratio and store at -80°C.

[0029] 3. Strain Identification: (1) Colony morphology identification ① The isolated strains were cultured anaerobically on MRS solid medium for 48 h, and the colony morphology and color were observed and recorded. Figure 1 As shown, the colonies are round, smooth, convex, with neat edges, opaque, milky white, and occasionally light yellow.

[0030] ②Pick the colonies cultured on MRS solid medium and carry out smearing, fixation, crystal violet primary staining, mordanting, decolorization, water washing, safranin counterstaining and drying in sequence. Then examine the dried colonies under oil microscope at a magnification of 1000 times. Figure 2 ) It can be seen that the colonies are stained blue-purple and are short rod-shaped.

[0031] (2) 16S rDNA sequence determination The culture medium of the isolated strain was centrifuged at 5000 r / min for 10 min. The supernatant was discarded after centrifugation, and the pure cultured bacteria to be tested were collected. The genomic DNA of the strain was extracted using a DNA extraction kit according to the instructions.

[0032] Based on the conserved region of the Lactobacillus 16S rDNA sequence, bacterial universal primers 27F / 1492R were 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; after 32 cycles, the final extension was at 72°C for 5 min.

[0033] The PCR products were identified by 1.0% agarose gel electrophoresis and then sequenced. The sequencing work was completed by Beijing Qingke Biotechnology Co., Ltd. The 16S rDNA sequence of the strain (SEQ ID NO. 1) is as follows:

[0034] The sequencing results were compared with the NCBI database by BLAST analysis, and a phylogenetic tree was constructed to determine that the strain was Lactobacillus reuteri ( Lactobacillus reuteri The strain was named Lactobacillus reuteri ZKLr0413 and deposited. The deposit information is as follows: Deposit date: May 13, 2024; Depository: General Microbiology Center, China Culture Collection Administration; Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No.30546; Classification name: Lactobacillus reuteri Lactobacillus reuteri .

[0035] Example 2 Experiment on the tolerance of Lactobacillus reuteri ZKLr0413 to the gastrointestinal environment 1. Tolerance of Lactobacillus reuteri ZKLr0413 to artificial gastric fluid According to the Chinese Pharmacopoeia, prepare 16.4 mL of dilute hydrochloric acid, add 800 mL of water, and 10 g of pepsin. Stir thoroughly, then dilute to 1000 mL with water. Filter and sterilize the solution until ready for use. Use dilute hydrochloric acid to adjust the pH of artificial gastric fluid to 2.5 and 3.0.

[0036] The preserved Lactobacillus reuteri ZKLr0413 was activated twice. 10 mL of the Lactobacillus reuteri ZKLr0413 bacterial suspension was centrifuged (10,000 × g, 5 min, 4°C) to obtain bacterial slurry. The slurry was washed twice with PBS buffer (pH = 7.2) and resuspended in 10 mL of PBS buffer to obtain a bacterial suspension. 1 mL of the bacterial suspension was added to 9 mL of artificial gastric juice at pH = 2.5 and pH = 3.0, respectively. The suspensions were cultured at 37°C. Samples were taken at 0 h and 3 h to determine the number of viable cells. The survival rate was calculated according to the following formula:

[0037] Where N0 is the number of viable bacteria at 0 h of artificial gastric juice treatment, and N1 is the number of viable bacteria after 3 h of artificial gastric juice treatment.

[0038] The final calculation results are shown in Table 1. It can be seen that Lactobacillus reuteri ZKLr0413 has good gastric acid resistance. After being treated in artificial gastric juice with a pH of 2.5 for 3 hours, the survival rate can reach over 80%; after being treated in artificial gastric juice with a pH of 3.0 for 3 hours, the survival rate can reach over 90%.

[0039] Table 1 Survival of Lactobacillus reuteri ZKLr0413 in artificial gastric fluid

[0040] 2. Tolerance of Lactobacillus reuteri ZKLr0413 to artificial intestinal fluid According to the preparation method recorded in the Chinese Pharmacopoeia, add 6.8 g of potassium dihydrogen phosphate to 500 mL of water and adjust the pH to 6.8 with 0.4% sodium hydroxide solution. Dissolve 10 g of pancreatic enzyme in an appropriate amount of water. Mix the two solutions and add water to 1000 mL. Filter and sterilize the solution for later use.

[0041] The preserved Lactobacillus reuteri ZKLr0413 was activated twice. 10 mL of the Lactobacillus reuteri ZKLr0413 bacterial suspension was centrifuged (10,000 × g, 5 min, 4°C) to obtain bacterial slurry. The slurry was washed twice with PBS buffer (pH = 7.2) and resuspended in 10 mL of PBS buffer to obtain a bacterial suspension. 1 mL of the bacterial suspension was added to 9 mL of artificial intestinal fluid and incubated at 37°C. Samples were taken at 0 h and 3 h to determine the number of viable bacteria. The survival rate was calculated according to the following formula:

[0042] Where C0 is the number of viable bacteria at 0 h of artificial intestinal fluid treatment, and C1 is the number of viable bacteria after 3 h of artificial intestinal fluid treatment.

[0043] The final calculation results are shown in Table 2. It can be seen that the survival rate of Lactobacillus reuteri ZKLr0413 after being treated in artificial intestinal fluid for 3 h can reach more than 90%, and it has good resistance to intestinal fluid.

[0044] Table 2 Survival of Lactobacillus reuteri ZKLr0413 in artificial intestinal fluid

[0045] In summary, Lactobacillus reuteri ZKLr0413 has good tolerance to the gastrointestinal environment, which creates conditions for its fermentation of jujube seed extract to prepare sleep-improving products.

[0046] Example 3 Preparation of fermentation broth of Lactobacillus reuteri ZKLr0413 from Chinese date seeds The method for preparing the fermentation liquid of Chinese jujube seeds with Lactobacillus reuteri ZKLr0413 includes two steps: first, preparing the Chinese jujube seed extract, and then fermenting the Chinese jujube seed extract with Lactobacillus reuteri ZKLr0413. The specific steps are as follows: Step S1, preparing a jujube seed extract: Step S11, using a traditional Chinese medicine grinder to grind the Chinese jujube seeds to prepare a Chinese jujube seed powder with a particle size of 70-300 mesh; Step S12, after adding 1 part by weight of the Chinese jujube seed powder into a decoction bag, soaking it in 8-10 parts by weight of water for 0.5-1 hour to obtain a Chinese jujube seed soaking liquid; Step S13: first boil the sour jujube kernel soaking liquid over high heat, then continue to boil for 1-2 hours, and then filter to separate the liquid and the residue; Step S14, steaming and concentrating the feed liquid to obtain a jujube seed extract with a crude drug content of 1.0 g / mL; Step S15: sterilize the Chinese jujube seed extract to obtain a sterilized Chinese jujube seed extract.

[0047] Step S2, fermenting the jujube seed extract with Lactobacillus reuteri ZKLr0413: Step S21, freeze-drying the Lactobacillus reuteri ZKLr0413 bacterial liquid to obtain Lactobacillus reuteri ZKLr0413 freeze-dried powder; Step S22: freeze-dried powder of Lactobacillus reuteri ZKLr0413 was added at a concentration of 1×10 10 The inoculum amount of CFU / L sterilized jujube seed extract is inoculated into the sterilized jujube seed extract in step S15, shaken and cultured aerobically at a constant temperature of 37°C. After the culture is completed, the jujube seed fermentation liquid of Lactobacillus reuteri ZKLr0413 is obtained.

[0048] In this example, by adjusting the process parameters, Lactobacillus reuteri ZKLr0413 Suanzaorensis seed fermentation broth 1 and Lactobacillus reuteri ZKLr0413 Suanzaorensis seed fermentation broth 2 were prepared respectively.

[0049] Wherein, the fermentation liquid of Lactobacillus reuteri ZKLr0413 sour jujube kernel is obtained under the following process parameters: The 70-mesh jujube seed powder was placed in a decoction bag and then soaked in 8 times the weight of water for 1 hour to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled over high heat, and then slowly decocted over low heat for 2 hours, while keeping the jujube seed soaking liquid slightly boiling during the slow decocting process; the liquid and the residue were separated by filtration, and the liquid was steamed and concentrated to obtain a jujube seed extract with a crude drug content of 1.0 g / mL, and the extract was sterilized at 121°C for 15 minutes to obtain a sterilized jujube seed extract; The Lactobacillus reuteri ZKLr0413 bacterial solution was freeze-dried at -50 °C for 40 h to obtain a viable cell count of 1 × 10 11 CFU / g of Lactobacillus reuteri ZKLr0413 lyophilized powder, the Lactobacillus reuteri ZKLr0413 lyophilized powder was inoculated into the sterilized jujube seed extract at an inoculum size of 0.1 g / L, and the mixture was shaken and aerobically cultured at 37°C for 24 h. After the culture was completed, Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid 1 was obtained, which was referred to as fermentation liquid 1.

[0050] The second fermentation liquid of Lactobacillus reuteri ZKLr0413 was obtained under the following process parameters: The jujube seed powder with a particle size of 300 mesh was placed in a decoction bag and then soaked in 10 times the weight of water for 0.5 h to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled over high heat, and then slowly decocted over low heat for 1 h, while keeping the jujube seed soaking liquid slightly boiling during the decocting process; the liquid and the residue were separated by filtration, and the liquid was steamed and concentrated to obtain a jujube seed extract with a crude drug content of 1.0 g / mL, and the extract was sterilized at 121°C for 15 min to obtain a sterilized jujube seed extract; The Lactobacillus reuteri ZKLr0413 bacterial solution was freeze-dried at -50 °C for 40 h to obtain a viable cell count of 1 × 10 11 CFU / g of Lactobacillus reuteri ZKLr0413 lyophilized powder, the Lactobacillus reuteri ZKLr0413 lyophilized powder was inoculated into the sterilized jujube seed extract at an inoculum size of 0.1 g / L, and the mixture was shaken and aerobically cultured at 37°C for 24 h. After the culture was completed, the Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid II was obtained.

[0051] Comparative Example 1: Using probiotics to ferment the extract of jujube seeds 1. Preparation of fermentation broth of jujube seeds with Lactobacillus plantarum RZKLp100 Lactobacillus plantarum RZKLp100 was used instead of Lactobacillus reuteri ZKLr0413 to ferment the jujube seed extract. Lactobacillus plantarum RZKLp100 is a probiotic 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 a good effect on fermenting licorice.

[0052] The specific steps are as follows: The 70-mesh jujube seed powder was placed in a decoction bag and then soaked in 8 times the weight of water for 1 hour to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled over high heat, and then slowly decocted over low heat for 2 hours, while keeping the jujube seed soaking liquid slightly boiling during the slow decocting process; the liquid and the residue were separated by filtration, and the liquid was steamed and concentrated to obtain a jujube seed extract with a crude drug content of 1.0 g / mL, and the extract was sterilized at 121°C for 15 minutes to obtain a sterilized jujube seed extract; The Lactobacillus plantarum RZKLp100 bacterial solution was freeze-dried at -50 °C for 40 h to obtain a viable cell count of 1 × 10 11CFU / g of Lactobacillus plantarum RZKLp100 freeze-dried powder, the Lactobacillus plantarum RZKLp100 freeze-dried powder was inoculated into the sterilized jujube seed extract at an inoculum size of 0.1 g / L, shaken and cultured aerobically at 37°C for 24 h. After the culture was completed, the jujube seed fermentation liquid of Lactobacillus plantarum RZKLp100 was obtained, which was referred to as fermentation liquid 2.

[0053] 2. Preparation of fermentation broth of jujube seeds with Lactobacillus acidophilus RZKLa0701 Lactobacillus acidophilus RZKLa0701 is used instead of Lactobacillus reuteri ZKLr0413 to ferment the jujube seed extract. Lactobacillus acidophilus RZKLa0701 is a probiotic collected and preserved by the applicant and has been disclosed in Chinese invention patent CN 117305188 B.

[0054] The specific steps are as follows: The 70-mesh jujube seed powder was placed in a decoction bag and then soaked in 8 times the weight of water for 1 hour to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled over high heat, and then slowly decocted over low heat for 2 hours, while keeping the jujube seed soaking liquid slightly boiling during the slow decocting process; the liquid and the residue were separated by filtration, and the liquid was steamed and concentrated to obtain a jujube seed extract with a crude drug content of 1.0 g / mL, and the extract was sterilized at 121°C for 15 minutes to obtain a sterilized jujube seed extract; The Lactobacillus acidophilus RZKLa0701 bacterial solution was freeze-dried at -50 °C for 40 h to obtain a viable cell count of 1 × 10 11 CFU / g of Lactobacillus acidophilus RZKLa0701 freeze-dried powder, the Lactobacillus acidophilus RZKLa0701 freeze-dried powder was inoculated into the sterilized jujube seed extract at an inoculum size of 0.1 g / L, shaken and cultured aerobically at 37°C for 24 h. After the culture was completed, the Lactobacillus acidophilus RZKLa0701 jujube seed fermentation liquid was obtained, referred to as fermentation liquid 3.

[0055] 3. Preparation of fermentation broth of Lactobacillus casei ZKLc1221 and jujube seeds Lactobacillus casei ZKLc1221 was used instead of Lactobacillus reuteri ZKLr0413 to ferment the jujube seed extract. Lactobacillus casei ZKLc1221 is a probiotic collected and preserved by the applicant and has been disclosed in Chinese invention patent CN 119614462 B.

[0056] The specific steps are as follows: The 70-mesh jujube seed powder was placed in a decoction bag and then soaked in 8 times the weight of water for 1 hour to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled over high heat, and then slowly decocted over low heat for 2 hours, while keeping the jujube seed soaking liquid slightly boiling during the slow decocting process; the liquid and the residue were separated by filtration, and the liquid was steamed and concentrated to obtain a jujube seed extract with a crude drug content of 1.0 g / mL, and the extract was sterilized at 121°C for 15 minutes to obtain a sterilized jujube seed extract; The Lactobacillus casei ZKLc1221 bacterial solution was freeze-dried at -50 °C for 40 h to obtain a viable cell count of 1 × 10 11 CFU / g of Lactobacillus casei ZKLc1221 freeze-dried powder, the Lactobacillus casei ZKLc1221 freeze-dried powder was inoculated into the sterilized jujube seed extract at an inoculum size of 0.1 g / L, shaken and cultured aerobically at 37°C for 24 h. After the culture was completed, the Lactobacillus casei ZKLc1221 jujube seed fermentation liquid was obtained, referred to as fermentation liquid 4.

[0057] 4. Preparation of fermentation broth from jujube seeds with Lactobacillus plantarum RZKLp100 / Lactobacillus casei ZKLc1221 According to prior art records, a mixed bacterial liquid of Lactobacillus plantarum and Lactobacillus casei has the effect of increasing the dissolution rate of saponins and flavonoids in jujube kernel. Therefore, a mixed bacterial liquid of Lactobacillus plantarum RZKLp100 and Lactobacillus casei ZKLc1221 is used instead of Lactobacillus reuteri ZKLr0413 to ferment the jujube kernel extract.

[0058] The specific steps are as follows: The 70-mesh jujube seed powder was placed in a decoction bag and then soaked in 8 times the weight of water for 1 hour to obtain a jujube seed soaking liquid; the jujube seed soaking liquid was boiled over high heat, and then slowly decocted over low heat for 2 hours, while keeping the jujube seed soaking liquid slightly boiling during the slow decocting process; the liquid and the residue were separated by filtration, and the liquid was steamed and concentrated to obtain a jujube seed extract with a crude drug content of 1.0 g / mL, and the extract was sterilized at 121°C for 15 minutes to obtain a sterilized jujube seed extract; The Lactobacillus plantarum RZKLp100 bacterial solution was freeze-dried at -50 °C for 40 h to obtain a viable cell count of 1 × 10 11 CFU / g of Lactobacillus plantarum RZKLp100 freeze-dried powder; Lactobacillus casei ZKLc1221 bacterial solution was freeze-dried at -50℃ for 40 h to obtain a viable cell count of 1×10 11CFU / g of Lactobacillus casei ZKLc1221 freeze-dried powder; Lactobacillus plantarum RZKLp100 freeze-dried powder was inoculated into sterilized jujube seed extract at an inoculum size of 0.067 g / L, and Lactobacillus casei ZKLc1221 freeze-dried powder was inoculated into sterilized jujube seed extract at an inoculum size of 0.033 g / L. The mixture was shaken and aerobically cultured at 37°C for 24 h. After the culture was completed, Lactobacillus plantarum RZKLp100 / Lactobacillus casei ZKLc1221 jujube seed fermentation liquid was obtained, referred to as fermentation liquid 5.

[0059] Example 4 Fermentation Broth Component Test 1. pH value and viable cell count test of fermentation broth The pH values ​​and viable bacterial counts of fermentation broths 1 to 5 prepared in Example 3 and Comparative Example 1 were measured, and the results are shown in Table 3. It can be seen that the viable bacterial count in fermentation broth 1 was higher than that in fermentation broths 2 to 5, indicating that the Lactobacillus reuteri ZKLr0413 provided by the present invention has a good fermentation effect on jujube seeds.

[0060] Table 3 pH value and viable bacterial count of fermentation broth 1 to fermentation broth 5

[0061] 2. Test of total flavonoids and total saponins in fermentation broth (1) Total flavonoid content test ① Reagents: Petroleum ether (boiling range 60–90°C, analytical grade), ethanol (analytical grade), and methanol (analytical grade) were purchased from Sinopharm Group; sodium nitrite (analytical grade), aluminum nitrate (analytical grade), and sodium hydroxide (analytical grade) were purchased from Chengdu Kelon Chemical Reagent Factory; and spinosin (analytical grade) was purchased from Shanghai Yuanye Biological Co., Ltd.

[0062] ②Solution preparation: Accurately measure 70 mL of anhydrous ethanol into a 100 mL volumetric flask, add distilled water to the mark, and mix to obtain 70% ethanol.

[0063] Accurately weigh 5.0 g of sodium nitrite into a 100 mL volumetric flask, add water to the mark, and mix to obtain a 5% sodium nitrite solution.

[0064] Accurately weigh 10.0 g of aluminum nitrate into a 100 mL volumetric flask, add water to the mark, and mix to obtain a 10% aluminum nitrate solution.

[0065] Accurately weigh 4.0 g of sodium hydroxide into a 100 mL volumetric flask, add water to the mark, and mix to obtain a 4% sodium hydroxide solution.

[0066] Accurately weigh 12.00 mg of spinosum and place it in a 10 mL volumetric flask. Add methanol to the mark and mix to obtain a reference solution with a concentration of 1.20 mg / mL.

[0067] Accurately weigh 10 mL of fermentation broth 1-5 and place them in a Soxhlet extractor. Add an appropriate amount of petroleum ether and heat under reflux for 6 hours. After reflux, discard the petroleum ether and thoroughly evaporate the solvent from the medicinal residue. Transfer the medicinal residue to a round-bottom flask and add 60 mL of 70% ethanol. Extract in a 100°C water bath three times for 1 hour, 1 hour, and 40 minutes, respectively. After extraction, combine the extracts and concentrate them using a rotary evaporator at 60°C. Finally, transfer the concentrate to a 10 mL volumetric flask and dilute to the mark with methanol to obtain test solutions 1-5 for subsequent determination of total flavonoid content.

[0068] Accurately weigh 10 mL of sterilized Ziziphus jujuba seed extract (preparation method, see Comparative Example 1) and place it in a Soxhlet extractor. Add an appropriate amount of petroleum ether and heat under reflux for 6 hours. After reflux, discard the petroleum ether and thoroughly evaporate the solvent from the medicinal residue. Transfer the medicinal residue to a round-bottom flask and add 60 mL of 70% ethanol. Extract three times in a 100°C water bath for 1 hour, 1 hour, and 40 minutes, respectively. After extraction, combine the extracts and concentrate them using a rotary evaporator at 60°C. Finally, transfer the concentrate to a 10 mL volumetric flask and dilute to the mark with methanol to obtain test solution 6, which will be used for subsequent determination of total flavonoid content.

[0069] ③ Drawing of standard curve: Accurately pipette different volumes (0.50, 0.75, 1.00, 1.25, 1.5 mL) of reference solution into 10 mL volumetric flasks, add 3 mL of methanol to each flask, then add 0.50 mL of 5% sodium nitrite solution, shake well and let it stand for 6 min; then add 0.50 mL of 10% aluminum nitrate solution, shake well and let it stand for 6 min; then add 4 mL of 4% sodium hydroxide solution, and finally dilute to the mark with distilled water, shake well and let it stand for 15 min.

[0070] Distilled water was used as a blank control and the absorbance was measured at a wavelength of 396 nm using a UV-6100 ultraviolet spectrophotometer. A standard curve was drawn with the content (mg) of the reference substance as the horizontal axis and the absorbance as the vertical axis. The regression equation of the standard curve was calculated to be Y=0.0435X-0.0018, and the correlation coefficient R 2 =0.9999, indicating that the linear relationship is good in the concentration range of 0.600-1.800 mg.

[0071] ④Determination of total flavonoids content: Accurately measure 1.0 mL of test solution 1-6 and place them in 10 mL volumetric flasks respectively. The subsequent operation steps are exactly the same as the solution treatment steps in the preparation of the standard curve, that is, add 3 mL of methanol to each solution, add 0.5 mL of 5% sodium nitrite solution, shake well and let it stand for 6 min, add 0.5 mL of 10% aluminum nitrate solution, shake well and let it stand for 6 min, add 4 mL of 4% sodium hydroxide test solution, and then add distilled water to the scale, shake well and let it stand for 15 min.

[0072] Similarly, using distilled water as a blank control, the absorbance was measured at 396 nm on a UV-6100 spectrophotometer. Finally, the total flavonoid content (mg / mL) was calculated based on the previously drawn standard curve.

[0073] ⑤Test results: The total flavonoid contents in fermentation broths 1 to 5 and the sterilized Chinese jujube seed extract are shown in Table 4 below. As can be seen, the total flavonoid content in fermentation broth 1 was increased by 91.78% compared to the unfermented sterilized Chinese jujube seed extract, and was significantly higher than the total flavonoid content in fermentation broths 2 to 5, indicating that the fermentation ability of Lactobacillus reuteri ZKLr0413 provided by the present invention on Chinese jujube seed extract is superior to that of other common lactobacilli either alone or in mixed fermentations.

[0074] Table 4 Total flavonoid content of fermentation broth 1 to 5 and sterilized jujube seed extract

[0075] Note: Different letters in the table represent significant differences. P <0.05.

[0076] (2) Total saponin content test ① Reagents: Glacial acetic acid (analytical grade) and perchloric acid (analytical grade) were purchased from Sinopharm Group; vanillin (analytical grade) and jujuboside A standard (analytical standard, product number: J114068-20 mg) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0077] ②Solution preparation: Accurately measure 70 mL of anhydrous ethanol into a 100 mL volumetric flask, add distilled water to the mark, and mix to obtain 70% ethanol.

[0078] Accurately weigh 5.0 g of vanillin into a 100 mL volumetric flask, add glacial acetic acid to the mark, and mix well to obtain a 5% vanillin glacial acetic acid solution.

[0079] Accurately weigh 2.50 mg of jujuboside A standard, place it in a 10 mL volumetric flask, add methanol to the mark, and mix to obtain a reference solution with a concentration of 0.25 mg / mL.

[0080] Accurately weigh 10 mL of fermentation broth 1-5 and place them in a Soxhlet extractor. Add an appropriate amount of petroleum ether and heat under reflux for 6 hours. After reflux, discard the petroleum ether and thoroughly evaporate the solvent from the medicinal residue. Transfer the medicinal residue to a round-bottom flask and add 60 mL of 70% ethanol. Extract in a 100°C water bath three times for 1 hour, 1 hour, and 40 minutes, respectively. After extraction, combine the extracts and concentrate them using a rotary evaporator at 60°C. Dry the concentrate in a 50°C forced air drying oven to a powder. Finally, transfer the powder to a 5 mL volumetric flask and dilute to the mark with methanol to obtain test solutions 1-5 for subsequent determination of total saponin content.

[0081] Accurately weigh 10 mL of sterilized Ziziphus jujuba seed extract (preparation method, see Comparative Example 1) and place it in a Soxhlet extractor. Add an appropriate amount of petroleum ether and heat under reflux for 6 hours. After reflux, discard the petroleum ether and thoroughly evaporate the solvent from the medicinal residue. Transfer the medicinal residue to a round-bottom flask and add 60 mL of 70% ethanol. Extract three times in a 100°C water bath for 1 hour, 1 hour, and 40 minutes, respectively. After extraction, combine the extracts and concentrate them using a rotary evaporator at 60°C. Dry the concentrate in a 50°C forced air drying oven to a powder. Finally, transfer the powder to a 5 mL volumetric flask and dilute to the mark with methanol to obtain test solution 6, which will be used for subsequent determination of total saponin content.

[0082] ③ Drawing of standard curve: Accurately pipette different volumes (0.10, 0.15, 0.20, 0.30, 0.40, and 0.50 mL) of reference solution into test containers. Evaporate the solvent and add 0.20 mL of 5% vanillin glacial acetic acid solution to each container. Then add 0.80 mL of perchloric acid. Seal the container and heat in a 70°C water bath for 20 min. Remove the container and immediately cool in an ice-water bath for 5 min. Add 5 mL of glacial acetic acid and shake well.

[0083] Methanol was used as a blank control and the absorbance was measured at a wavelength of 546 nm using a UV-6100 spectrophotometer. A standard curve was drawn with the content (mg) of the reference substance as the horizontal axis and the absorbance as the vertical axis. The regression equation of the standard curve was calculated to be Y=3.8406X+0.0101, and the correlation coefficient R 2 =0.9999, indicating that the linear relationship is good in the concentration range of 0.025-0.125 mg.

[0084] ④Determination of total saponin content: Accurately measure 0.10 mL of test solution 1-6 and place them in the test container respectively. The subsequent operation steps are exactly the same as the solution processing steps in the preparation of the standard curve, that is, add 0.20 mL of 5% vanillin glacial acetic acid solution to each; then add 0.80 mL of perchloric acid, seal and heat at a constant temperature of 70℃ water bath for 20 min, take out and immediately cool in ice water bath for 5 min, add 5 mL of glacial acetic acid, and shake well.

[0085] Methanol was also used as a blank control, and the absorbance was measured at 546 nm on a UV-6100 spectrophotometer. Finally, the total saponin content (mg / mL) was calculated based on the previously drawn standard curve.

[0086] ⑤Test results: The total saponin contents in fermentation broths 1 to 5 and the sterilized Chinese jujube seed extract are shown in Table 5 below. As can be seen, the total saponin content in fermentation broth 1 was increased by 89.66% compared to the unfermented sterilized Chinese jujube seed extract, and was significantly higher than the total saponin content in fermentation broths 2 to 5, indicating that the fermentation ability of Lactobacillus reuteri ZKLr0413 provided by the present invention on Chinese jujube seed extract is superior to the fermentation effects of other common lactobacilli alone or in mixed cultures.

[0087] Table 5 Total saponin content of fermentation broth 1 to fermentation broth 5 and sterilized jujube seed extract

[0088] Note: Different letters in the table represent significant differences. P <0.05.

[0089] Example 5 Effect of Fermented Broth on Improving Sleep in Insomnia Rats 1. Reagents: Parachlorophenylalanine (PCPA) was purchased from Sigma, USA; sodium pentobarbital was purchased from Sinopharm Chemical Reagent Co., Ltd.; and diazepam was purchased from Shenyang First Pharmaceutical Co., Ltd., Northeast Pharmaceutical Group.

[0090] 2. Modeling: Ninety male Wistar rats, 8-9 weeks old and weighing 180-220 g, were purchased. The rats were adaptively fed for 7 days with free access to feed and water, and the bedding was kept dry. After 7 days, the rats were weighed and randomly divided into a blank group (10 rats) and a modeling group (80 rats). The modeling group received an intraperitoneal injection of PCPA for 2 consecutive days. Immediately prior to administration, PCPA was prepared as a suspension in normal saline at a dose of 400 mg / kg rat body weight. The blank group received an intraperitoneal injection of an equal volume of normal saline for 2 consecutive days.

[0091] A circadian rhythm deficit was observed in the modeling rats, with continuous activity during both daytime and nighttime. A sodium pentobarbital righting test revealed statistically significant differences in sleep latency and sleep duration between the blank and modeling groups, indicating successful modeling.

[0092] 3. Experimental Grouping and Dosing: Blank group: Each rat was gavaged with 0.4 mL of normal saline every day for 14 consecutive days.

[0093] The 80 rats with successful modeling were randomly divided into model group, fermentation liquid group 1, fermentation liquid group 2, fermentation liquid group 3, fermentation liquid group 4, fermentation liquid group 5, extract liquid group 1, and diazepam positive control group.

[0094] Model group: Each rat was gavaged with 0.4 mL of normal saline every day for 14 consecutive days.

[0095] Fermentation broth group 1: Each rat was orally administered 0.4 mL of the fermentation broth 1 prepared in Example 3 every day for 14 consecutive days.

[0096] Fermentation broth group 2: Each rat was orally administered 0.4 mL of fermentation broth 2 prepared in Comparative Example 1 every day for 14 consecutive days.

[0097] Fermentation broth group 3: Each rat was orally administered 0.4 mL of fermentation broth 3 prepared in Comparative Example 1 every day for 14 consecutive days.

[0098] Fermentation broth group 4: Each rat was orally administered 0.4 mL of fermentation broth 4 prepared in Comparative Example 1 every day for 14 consecutive days.

[0099] Fermentation broth group 5: Each rat was orally administered 0.4 mL of fermentation broth 5 prepared in Comparative Example 1 every day for 14 consecutive days.

[0100] Extract group: Each rat was gavaged with 0.4 mL of sterilized jujube seed extract (prepared by the same method as in Comparative Example 1) every day for 14 consecutive days.

[0101] Positive control group: Each rat was gavaged with 0.4 mL of 1 mg / mL diazepam saline solution every day for 14 consecutive days.

[0102] 4. Experimental Methods and Results Thirty minutes after the last oral gavage, rats in each group were intraperitoneally injected with 25.00 mg sodium pentobarbital / kg body weight in a quiet environment at 24-25°C.

[0103] The number of rats that fell asleep within 30 minutes (those with loss of righting reflex for at least 1 minute) was recorded, and the sleep status of each group was statistically analyzed to calculate the sleep onset rate. The results are shown in Table 6 below. As can be seen, compared with the model group, the sleep onset rate of rats in fermentation broth group 1 was significantly increased, approaching that of the blank group. Furthermore, the sleep onset rate was higher than that in fermentation broth groups 2, 3, 4, 5, and the extract group. This indicates that the Lactobacillus reuteri ZKLr0413 jujube seed fermentation broth provided by the present invention is the most effective in improving the sleep onset rate in rats with insomnia.

[0104] Table 6 Statistics of sleeping rate of rats in each group

[0105] The time from the injection of sodium pentobarbital to the disappearance of the righting reflex and the time from the disappearance of the righting reflex to recovery were recorded to observe the influence of the test substance on the sleep latency of sodium pentobarbital and sleep time. The results are shown in Table 7 below. As can be seen, compared with the model group, the sleep latency of fermented liquid group 1 rats significantly decreased, approached the blank group, and was significantly lower than fermented liquid group 2, fermented liquid group 3, fermented liquid group 4, fermented liquid group 5 and extract group. Compared with the model group, the sleep time of fermented liquid group 1 rats significantly increased, approached the blank group, and was significantly higher than fermented liquid group 2, fermented liquid group 3, fermented liquid group 4, fermented liquid group 5 and extract group. This shows that Lactobacillus reuteri ZKLr0413 Semen Ziziphi Spinosae fermented liquid provided by the present invention has a significant sleep-inducing effect.

[0106] Table 7 Statistics of sleep latency and sleep time of rats in each group

[0107] Note: Different letters in the table represent significant differences. P <0.05.

[0108] After the rats were sacrificed, the craniotomy was immediately performed in an ice bath and the whole brain was removed. The brain was placed on an ice plate for dissection, rinsed with ice-cold 0.9% sodium chloride solution to remove blood, and then dried with filter paper. After weighing the brain, 9 times the volume of ice-cold 0.9% sodium chloride solution was added and homogenized at 4°C. The homogenized slurry was transferred to a 2 mL centrifuge tube and centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected. The supernatant was assayed for 5-hydroxytryptamine and γ-aminobutyric acid levels according to the instructions of the kit (purchased from Wuhan Pure Biotechnology Co., Ltd.).

[0109] The results are shown in Table 8 below. As can be seen, compared with the model group, the levels of 5-HT and γ-aminobutyric acid in the brains of rats in fermentation broth group 1 were significantly elevated, approaching those in the blank group, and significantly higher than those in fermentation broth groups 2, 3, 4, 5, and the extract group. This demonstrates that the Lactobacillus reuteri ZKLr0413 jujube seed fermentation broth provided by the present invention can significantly promote the release of 5-HT and γ-aminobutyric acid in the brains of rats with insomnia.

[0110] Table 8 Statistics of 5-HT and γ-aminobutyric acid levels in the brains of rats in each group

[0111] Note: Different letters in the table represent significant differences. P <0.05.

[0112] In summary, compared with other probiotic single-strain or mixed fermentation processes, the present invention's fermentation of Chinese jujube kernel extract using Lactobacillus reuteri ZKLr0413 demonstrates significant advantages across multiple metrics: The Lactobacillus reuteri ZKLr0413 Chinese jujube kernel fermentation broth contains higher levels of viable bacteria, total flavonoids, and total saponins, all of which have a positive effect on sleep regulation. Experiments with rats further validated the sleep-improving efficacy of the present invention's Lactobacillus reuteri ZKLr0413 Chinese jujube kernel fermentation broth: Compared with the model group, rats gavaged with Lactobacillus reuteri ZKLr0413 Chinese jujube kernel fermentation broth showed a significantly improved sleep onset rate, exceeding that of rats gavaged with fermentation broths 2-5 and sterilized Chinese jujube kernel extract, and closer to that of normal rats. This series of results shows that the Lactobacillus reuteri ZKLr0413 provided by the present invention has good compatibility with the Chinese jujube seed extract, can specifically ferment the Chinese jujube seed extract, and fully stimulate the active ingredients of the Chinese jujube seed, thereby effectively improving sleep quality and providing a new and effective solution to insomnia-related problems.

[0113] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be readily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A Lactobacillus reuteri ZKLr0413 fermented jujube seed broth for improving sleep, characterized in that: Lactobacillus reuteri ( Lactobacillus reuteri ) ZKLr0413 fermented jujube seed extract; Among them, Lactobacillus reuteri ZKLr0413 has been deposited in the General Microbiology Center of China Culture Collection Administration on May 13, 2024, with the deposit number CGMCC No.30546, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as Lactobacillus reuteri Lactobacillus reuteri ; The Chinese jujube seed extract is obtained by soaking and decocting Chinese jujube seed powder.

2. The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid according to claim 1, wherein The preparation method of the Chinese date seed extract is as follows: The Chinese jujube seed powder is put into a decoction bag and then soaked in water to obtain a Chinese jujube seed soaking liquid; the Chinese jujube seed soaking liquid is decocted, and then the liquid and the residue are separated by filtration, and the liquid is steamed and concentrated to obtain a Chinese jujube seed extract with a crude drug content of 1.0 g / mL.

3. The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid according to claim 1 or 2, wherein The particle size of the sour jujube seed powder is 70-300 meshes.

4. The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid according to claim 1 or 2, wherein Lactobacillus reuteri ZKLr0413 was added at a rate of 1×10 10 The ratio of CFU / L of jujube seed extract was inoculated into the jujube seed extract, and then cultured aerobically at a constant temperature of 37°C. After the culture was completed, the jujube seed fermentation liquid of Lactobacillus reuteri ZKLr0413 was obtained.

5. The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid according to claim 4, characterized in that Before inoculating Lactobacillus reuteri ZKLr0413, the jujube seed extract was first sterilized.

6. The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid according to claim 4, wherein The freeze-dried powder of Lactobacillus reuteri ZKLr0413 was added to the extract of Ziziphus jujuba seeds for fermentation. The viable cell count of the freeze-dried powder of Lactobacillus reuteri ZKLr0413 was 1×10 11 CFU / g.

7. The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid according to claim 1, wherein After the fermentation, the number of viable bacteria of Lactobacillus reuteri ZKLr0413 in the fermentation liquid of jujube seeds was 1.58×10 9 CFU / mL.

8. The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid according to claim 1, wherein The total flavonoids content of the fermentation broth of jujube seeds produced by Lactobacillus reuteri ZKLr0413 was 82.39±7.42 mg / mL.

9. The Lactobacillus reuteri ZKLr0413 jujube seed fermentation liquid according to claim 1, wherein The total saponin content of the fermentation broth of jujube seeds grown with Lactobacillus reuteri ZKLr0413 was 73.57±4.23 mg / mL.

10. A Lactobacillus reuteri strain ZKLr0413, characterized in that: Lactobacillus reuteri ZKLr0413 was deposited in the General Microbiology Center of China Culture Collection Administration on May 13, 2024, with the deposit number CGMCC No. 30546. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the classification name is Lactobacillus reuteri Lactobacillus reuteri .

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