Phytobacterium plantarum B534 and application thereof in preparation of functional product helpful for improving sleep and regulating emotion

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a plant lactobacillus B534 strain and application thereof in preparation of functional products beneficial to sleep improvement and emotion regulation. The lactobacillus plantarum B534 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the lactobacillus plantarum B534 is CGMCC No.33031. The lactobacillus plantarum B534 is named as lactobacillus plantarum B534. The plant lactobacillus B534 provided by the invention is a natural strain and has food safety; the lactobacillus plantarum B534 has relatively strong acetylcholin esterase inhibition activity, and also has relatively strong capability of synthesizing and secreting GABA (gamma-aminobutyric acid), 3-HB (3-hydroxybutyric acid) and 5-HT (5-hydroxytryptamine); meanwhile, the plant lactobacillus B534 has relatively strong acid resistance and cholate resistance and also has relatively strong tolerance to simulated gastrointestinal fluid, so that the plant lactobacillus B534 has relatively good intestinal colonization ability and has important application value in the fields of products beneficial to sleep improvement, emotion regulation products and the like.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a strain of Lactobacillus plantarum B534 and its application in preparing functional products that help improve sleep and regulate mood. Background Art

[0002] Depression, also known as depressive disorder, is a mental disorder with a high incidence, high clinical cure rate, but low treatment acceptance rate and high recurrence rate. Its main clinical manifestations are persistent low mood, mental depression and fatigue, and may also cause sleep and mental movement disorders, guilt, low self-esteem, and have a great impact on the patient's daily life and social interaction.

[0003] For individuals, facing depression, one of the most disabling mental disorders, the pathogenesis of depression remains unclear due to its complexity and heterogeneity determined by genetic and environmental factors. At present, the theories about depression mainly focus on the monoamine neurotransmitter depletion hypothesis, the neuroplasticity hypothesis, and the hypothalamus-pituitary-adrenal axis hypothesis. However, in recent years, with the in-depth study of intestinal flora, it has been found that the influence of intestinal flora on people is multifaceted, including the gut-liver axis, the gut-skin axis, the gut-kidney axis, etc. Among them, the gut-brain axis has also been widely reported. At present, a large number of studies believe that the gut flora directly or indirectly affects depression through itself and its metabolites. For example, the analysis of the gut flora of patients with depression found that several strains of Bacteroidetes were unique to patients with depression, and the appearance of these strains significantly increased people's susceptibility to depression.

[0004] The study also found that metabolites derived from the intestinal flora of patients with depression play an important role in their pathogenesis. Among them, short-chain fatty acids (SCFAs: acetate, butyrate and propionate) are depleted in patients with depression. Conventional drug treatment is to improve intestinal permeability and hypothalamic-pituitary-adrenal axis responsiveness associated with depression by allowing patients to ingest SCFAs, especially the intake of butyrate and neurotransmitters (such as 5-hydroxytryptamine and γ-aminobutyric acid) with antidepressant effects.

[0005] Gamma-aminobutyric acid (GABA) is an essential neurotransmitter in the brain and is very important for regulating the enteric nervous system and gut-brain dual communication. Bui Duc Tri et al. prepared poly (GABA) in the form of nanoparticles and proved that poly (GABA) is a promising candidate for the treatment of depression through behavioral tests and stress biomarkers. Christopher Ritter's research is more convincing, by measuring the GABA concentration in the prefrontal cortex of healthy people, people who have suffered from depression and people who are currently suffering from depression. The results show that compared with the control group, the concentration of GABA in the prefrontal cortex of people who have suffered from depression and are currently suffering from depression is relatively low.

[0006] Serotonin (5-HT), which also has a great impact on depression, has also been extensively studied. M. Uzbekov's study used drug intervention on 22 patients with depression, in which the drug ingredient was the 5-HT-norepinephrine inhibitor - milnacipran. The results showed that the drug has the potential to become an antidepressant. Due to the effectiveness of symptomatic treatment and easy preparation, the current drug ingredients for depression mainly include monoamine oxidase inhibitors, tricyclic antidepressants, selective 5-HT re-extraction inhibitors (SSRI), 5-HT and norepinephrine re-extraction inhibitors (SNRI), etc. However, most antidepressants have obvious side effects. For example, monoamine oxidase inhibitors have been discontinued due to their large side effects, while SSRI and SNRI have side effects such as gastrointestinal symptoms, agitation symptoms, and sexual dysfunction.

[0007] 3-Hydroxybutyric acid (3-HB) is an inhibitory neurotransmitter in the central nervous system like GABA. It can inhibit excessive excitement of neurons. Therefore, it has an auxiliary sedative effect and can also relieve anxiety, tension and other emotions.

[0008] Patients with depression may eventually suffer from cognitive impairment due to difficulty in interacting with others. Acetylcholine is an important neurotransmitter for cognitive function in the human body, so it is necessary to measure the inhibitory activity of probiotics on its decomposing enzyme, AchE.

[0009] Therefore, most researchers are currently shifting their treatment of depression patients from direct oral medication to the screening of edible strains that produce high 5-HT. By consuming strains that produce high 5-HT, the side effects of the drugs are reduced, and at the same time, the production of 5-HT can be fed back through the gut-brain axis. At present, there are relatively few technical solutions for screening edible strains that produce high 5-HT in my country. Even if edible strains that produce 5-HT are screened out, their 5-HT production level is still at a low level. For example In the Chinese invention patent application with publication number CN116004455A, a fermented lactobacillus strain A21196 with the function of secreting 5-hydroxytryptamine is disclosed, wherein the 5-HT output of fermented lactobacillus A21196 is 3.84 ug / mL. In the Chinese invention patent application with publication number CN118792218A, a Pediococcus pentosaceus strain A20001 with anti-inflammatory and 5-HT secretion functions and its derivative products and applications are disclosed, wherein the output of 5-HT of Pediococcus pentosaceus is 2.848 ug / mL.

[0010] In summary, there are relatively few publicly available technical solutions for screening high-yield 5-HT edible strains, especially for natural and safe strains that have a strong inhibitory effect on acetylcholinesterase, a strong ability to synthesize and secrete GABA, 3-HB and 5-HT, and good tolerance to gastric juice and intestinal juice. There is no relevant research and reports.

[0011] Therefore, how to develop a natural and safe bacterial strain that has a strong inhibitory effect on acetylcholinesterase, has a strong ability to synthesize and secrete GABA, 3-HB and 5-HT, and has good tolerance to gastric juice and intestinal juice is exactly the problem that this field is committed to solving. Summary of the invention

[0012] In order to address the deficiencies of the prior art mentioned in the above background technology, the present application provides a Lactobacillus plantarum B534, which is a natural strain. While having significant inhibitory activity on acetylcholinesterase, it can synthesize and secrete GABA, 3-HB and 5-HT, and is resistant to gastric acid and bile salts, has good intestinal colonization ability, and has important application value in the fields of products that help improve sleep, mood regulation products, etc.

[0013] The present application provides a strain of Lactobacillus plantarum B534, whose Latin name is: Lactiplantibacillus plantarum , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: December 11, 2024, deposit number: CGMCC No.33031.

[0014] The present application also provides the use of the Lactobacillus plantarum B534 as described above in the preparation of a product for inhibiting acetylcholinesterase activity.

[0015] The present application also provides a method for preparing the bacterial suspension of Lactobacillus plantarum B534 as described above: inoculating the Lactobacillus plantarum B534 into an MRS liquid culture medium, culturing at (30-40)°C for 24-36 hours, separating the culture medium into solid and liquid, and resuspending the solid to obtain a bacterial suspension having acetylcholinesterase inhibitory activity.

[0016] The present application also provides the use of the Lactobacillus plantarum B534 as described above in preparing a fermentation product with a high level of γ-aminobutyric acid and / or a high level of 3-hydroxybutyric acid and / or a high level of 5-hydroxytryptamine.

[0017] The present application also provides a method for preparing the fermentation broth of Lactobacillus plantarum B534 as described above: inoculating Lactobacillus plantarum B534 into an MRS liquid culture medium, culturing at (30-40)°C for 24-36 hours, taking the culture solution for solid-liquid separation, collecting the liquid, and obtaining a fermentation broth; wherein the fermentation broth is a fermentation broth containing γ-aminobutyric acid, 3-hydroxybutyric acid, and 5-hydroxytryptamine.

[0018] The present application also provides a Lactobacillus plantarum B534 agent, the components of which include the Lactobacillus plantarum B534 as described in claim 1.

[0019] In some embodiments, the components of the bacterial agent include fermentation broth of Lactobacillus plantarum B534 and / or bacterial powder of Lactobacillus plantarum B534.

[0020] The present application also provides the use of the Lactobacillus plantarum B534 as described above in the preparation of a functional product that helps to improve sleep and / or regulate mood.

[0021] The present application also provides the use of the above-mentioned Lactobacillus plantarum B534 in the preparation of functional products, wherein the functional products are functional foods or health products, such as solid beverage products, fermented beverage products, etc.

[0022] In some embodiments, the functional food includes one of a functional food that helps improve sleep, a functional food that regulates mood, an acid-resistant and bile-resistant food, and a functional food that inhibits the activity of acetylcholinesterase.

[0023] In some embodiments, the Lactobacillus plantarum B534 agent includes a fermentation broth of Lactobacillus plantarum B534 and / or a bacterial powder of Lactobacillus plantarum B534. In some embodiments, the fermentation broth of Lactobacillus plantarum B534 includes one or more combinations of fermentation supernatant, cell-free extract, and bacterial cell metabolites.

[0024] Based on the above characteristics, this application has the following beneficial effects: The Lactobacillus plantarum B534 provided in the present application is a natural strain with food safety; it not only has significant inhibitory activity on acetylcholinesterase, but also has a strong ability to synthesize and secrete GABA, 3-HB and 5-HT, and is resistant to gastric acid and bile salts, and has good intestinal colonization ability, and has important application value in the fields of products that help improve sleep, mood regulation products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0026] Figure 1 This is a colony morphology diagram of Lactobacillus plantarum of Example 1 of the present application; Figure 2 This is a comparison chart of the enzymatic rate of acetylcholinesterase of the control group and the B534 strain and the acetylcholinesterase inhibitory activity data of the B534 strain in Example 1 of the present application; Figure 3 This is a staining display of Lactobacillus plantarum of Example 1 of the present application; Figure 4 The standard curve of GABA and the concentration data of GABA in B534 fermentation broth; Figure 5 It is the standard curve data diagram of 3-HB; Figure 6 This is a data graph of the survival rate of B534 in the simulated gastrointestinal fluid experiment; Figure 7 This is a physical picture of the inhibition zone of different antibiotics against B534 strain in the drug sensitivity test; Figure 8 This is a data chart of the behavioral experimental results of B534 in improving zebrafish sleep; Fig. 9 This is a data chart showing the molecular biology experimental results of B534 in improving zebrafish sleep. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0028] The present application also provides the following operation examples and embodiments: The present application provides a strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum )B534, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.33031.

[0029] Source: Lactobacillus plantarum B534 was isolated and screened from the intestines of infants and young children breastfed by a healthy pregnant woman in Xiamen, Fujian Province.

[0030] Colony morphology: The colony morphology of Lactobacillus plantarum B534 is as follows Figure 1 As shown, it is round, milky white, with a smooth and raised surface, neat edges and is opaque.

[0031] The specific process of isolating and screening Lactobacillus plantarum B534 from the intestinal tract of infants and young children breastfed by a healthy pregnant woman in Xiamen, Fujian Province is as follows: 5 g of intestinal samples from infants breastfed by a healthy pregnant woman in Xiamen, Fujian Province were taken by the spread plate method and placed in a sterile homogenizing bag. The bag was marked and 45 mL of 0.85% saline was added and then patted to mix thoroughly.

[0032] Then, 100 μL of the sample was aspirated for 10-fold serial dilutions. 3 times, 10 4 times, 10 5 100 μL of the sample was spread on a plate containing 2.5% CaCO 3 The strain was placed on an MRS plate and inverted at 37°C for 48 hours. The colonies with good growth and large calcium dissolution zone were selected and repeatedly separated and purified by plate streaking separation method until a single colony was obtained. The isolated strain was named B534 and stored in a bacteria bank at -80°C glycerol.

[0033] Example 1: Screening of strains (i.e. Lactobacillus plantarum B534) that efficiently inhibit acetylcholinesterase 1. Determination of acetylcholinesterase inhibitory activity Lactic acid bacteria frozen in glycerol tubes from more than 60 lactic acid bacteria strains were activated for 2 generations from the bacteria library and inoculated into MRS liquid culture medium at an inoculum size of 3% (v / v). The culture was cultured at 37°C for 24 h, and 1 mL of the fermentation broth was centrifuged at 8000 r / min and 4°C for 10 min. The bacterial sludge precipitate was retained, and 1 mL of 0.85% saline was added to the bacterial sludge precipitate to resuspend it to obtain a bacterial sludge resuspension. Add 25 μL of bacterial sludge resuspension of different strains, 25 μL of 8 U / mL acetylcholinesterase, 50 μL of 5 mmol / L 5,5-dithiobis(2-nitrobenzoic acid), and 100 μL of 10 mmol / L PBS buffer (pH 7.4) to the 96-well plate, and react at 37°C for 5 min. After the reaction is completed, add 50 μL of 20 mmol / L iodinated thioacetylcholine. Immediately place it in the microplate reader and measure the absorbance at 412 nm at 0 min and 15 min. At the same time, set up a control group (PBS instead of sample), a control blank group (PBS instead of acetylcholinesterase in the sample), and a sample blank group (PBS instead of acetylcholinesterase in the sample).

[0034] Enzyme reaction rate v (OD / min) = (A 15 -A 0 ) / 15; (1) Acetylcholinesterase inhibition rate (%) = (1-v / v 0 )*100%; (2) Where: A 15 OD after 15 min incubation 412 Absorbance, A 0 The OD at 0 min is 412 absorbance, v0 is the enzymatic reaction rate of the control group, and v0 is the enzymatic reaction rate of the control group.

[0035] The data obtained from the above experiments were processed by formulas (1) and (2) to obtain the enzyme inhibition rate of each strain. That is, among the more than 60 lactic acid bacteria in the company's bacterial library, the lactic acid bacteria with the highest acetylcholinesterase inhibition rate was the lactic acid bacteria numbered B534 extracted, separated and cultured from the intestinal samples of infants and young children fed with breast milk from healthy pregnant women. The lactic acid bacteria were cultured in MRS liquid medium for the second generation to obtain Lactobacillus plantarum B534. Among them, it was tested that in the above fermentation process with an inoculation amount of 3%, a fermentation temperature of 37°C, and a fermentation time of 24h, the inhibition rate of Lactobacillus plantarum B534 on acetylcholinesterase reached 46.78±8.49%. The specific data are as follows Figure 2 As shown, Figure 2 “Con” is the abbreviation of “control”, which stands for control group.

[0036] 2. Identification of Example 2 strain B534 (1) Colony morphology identification Take a tube of glycerol bacteria numbered B534 from the bacteria library, inoculate it into MRS liquid culture medium at an inoculum size of 3% (v / v) for activation, culture it in a constant temperature incubator at 37°C for 24 h, then streak it on an MRS solid culture medium plate, and culture it upside down in a constant temperature incubator at 37°C for 24 h to obtain a single colony.

[0037] The colony morphology of Lactobacillus plantarum B534 is as follows Figure 1 As shown, it is round, milky white, with a smooth and raised surface, neat edges and is opaque.

[0038] (2) Physiological and biochemical experiments The strain B534 isolated and purified by the plate streak method was selected for Gram staining of single colonies with good growth, and its physiological and biochemical indices were measured. The experimental results were compared with the "Berger's Manual of Systematic Bacteriology, Eighth Edition" for preliminary identification of the strain, specifically: After staining, Lactobacillus plantarum Figure 3 As shown, the experiment showed that the strain B534 was purple in Gram stain, positive, and its cell shape was rod-shaped.

[0039] (3) 16S rRNA identification 1) Extract the strain gene DNA. The specific process is to extract the strain DNA according to the instructions of the bacterial genomic DNA extraction kit of Tiangen Biochemical Technology Co., Ltd.

[0040] 2) PCR amplification of 16S rRNA gene using gene DNA as template.

[0041] The universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') were used as amplification primers.

[0042] The PCR reaction system was: DNA 1 μL, 27F 1 μL, 1492R 1 μL, Premix Ex Taq 10 μL, ddH2O 7 μL.

[0043] PCR reaction conditions: pre-denaturation at 95°C for 8 min; denaturation at 95°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 1 min30 s, 30 cycles; final extension at 72°C for 5 min.

[0044] Then, the PCR amplification products were subjected to DNA sequencing (the sequencing company was Sangon Biotechnology Co., Ltd.).

[0045] The sequencing results were searched for similar sequences in the NCBI database using Blast software, and the strain B534 sequence was compared with the 16S rRNA gene sequence of related species obtained from the gene bank. The gene sequence is as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1).

[0046] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0047] The results of 16S rRNA gene sequencing are as follows:

[0048] In summary, based on the comprehensive analysis of the colony morphology, cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence and other data of the strain B534, with reference to the Bergey's Manual of Systematic Bacteriology, 8th Edition, the strain B534 was identified as Lactobacillus plantarum ( Lactiplantibacillus plantarum ).

[0049] Example 2 Determination of GABA secretion ability of strain B534 GABA, as an important inhibitory neurotransmitter of the central nervous system, has physiological effects such as improving sleep quality and lowering blood pressure. Therefore, the ability of strains to improve sleep and regulate emotions was evaluated based on their GABA synthesis and secretion capabilities.

[0050] The B534 strain was cultured to the second generation by colorimetry. The culture process was as follows: plant lactobacillus B534 was inoculated into MRS liquid medium, cultured at 37°C for 24h, the culture solution was separated into solid and liquid, and the liquid was collected to obtain the fermentation broth. 1 mL of the fermentation broth was centrifuged at 8000 r / min and 4°C for 10 min, and the fermentation broth was retained. 0.3 mL of the fermentation broth was added to the test tube, followed by 0.2 mL of 0.2 mol / L borate buffer (pH 10.0), 0.1 mL of 6% (v / v%) phenol solution, and 0.8 mL of 5% NaClO solution, and mixed evenly. Placed in a boiling water bath for 10 min, placed in an ice bath for 5 min after the boiling water bath, and added 2 mL of 60% ethanol when the solution turned blue-green. After mixing, the absorbance at 645 nm was measured using an enzyme marker.

[0051] The same steps as above were used to complete the determination of the GABA standard curve. The concentration range of the standard solution was 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL and 1.0 mg / mL. The obtained standard curve was as follows: Figure 4 Shown in the middle left picture.

[0052] Substituting the absorbance data at 645 nm measured by the above experiment into the standard curve formula, the GABA concentration of the fermentation broth of strain B534 was 0.1021±0.0129 mg / mL. Figure 4 As shown in the middle right picture, Figure 4 In the middle right figure, the experimental group numbered "MRS" is a blank control group using MRS culture medium instead of fermentation medium.

[0053] Example 3 Determination of the ability of strain B534 to secrete 3-HB 3-HB, like GABA, is an inhibitory neurotransmitter in the central nervous system. It can inhibit excessive excitement of neurons and thus has an auxiliary sedative effect. It can also relieve anxiety, tension and other emotions.

[0054] In this example, the ability of B534 strain to secrete 3-HB was determined using a 3-HB content detection kit from Beijing Solebow Technology Co., Ltd. According to the instructions, 8 mg / mL of sodium 3-hydroxybutyrate standard solution was diluted with distilled water to 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, 0.015625 mg / mL, and 0.0078125 mg / mL as the concentrations of the standard curve, and the standard curve was measured as follows: Figure 5 As shown, the 3-HB content of the B534 strain fermentation broth was then determined according to the instructions.

[0055] Substituting the data of the samples measured in the above experiment into the standard curve formula, the 3-HB concentration of the fermentation broth of strain No. B534 was obtained to be 2.5075±0.4548 μg / mL.

[0056] Example 4 Determination of the ability of strain B534 to secrete 5-HT 5-HT, also known as serotonin, is widely present in mammalian tissues, especially in the cerebral cortex and nerve processes. 5-HT is both an inhibitory neurotransmitter and an excitatory neurotransmitter, and is an active ingredient in many antidepressant drugs.

[0057] In this example, the ability of the B534 strain to secrete 5-HT was determined by a rapid fluorescence assay. The B534 strain was cultured to the second generation, 1 mL of the fermentation broth was centrifuged at 8000 r / min and 4° C. for 10 min, and the fermentation broth was retained.

[0058] Add 4.5 mL of acidic n-butanol to 0.5 mL of the strain fermentation broth, vortex on a vortexer for 5 min, centrifuge the mixed solution at 3000 r / min for 10 min, take 3 mL of the supernatant, add 3 mL of n-heptane and 1 mL of 0.1 M hydrochloric acid, vortex and extract for 5 min to extract the interfering fluorescent substances into the n-heptane liquid phase (about 0.2 mL of n-heptane is dissolved in the aqueous phase here), then centrifuge at 3000 r / min for 5 min. At this time, the lower liquid phase contains 5-HT. MRS liquid culture medium was used instead of fermentation broth as a control.

[0059] According to different groups, reagents were added in sequence according to Table 1. After mixing, the mixture was placed in a water bath at 100°C for 10 min. After the water bath was over, ice water was used for cooling. Within 90 min, the fluorescence intensity was measured at an excitation wavelength (Ex) of 350 nm and an emission wavelength (Em) of 477 nm.

[0060] Table 15-HT assay sample addition order

[0061] Among them, T, B 1 , S, B 2 They are represented as sample group, measurement blank group, standard sample group and standard blank group respectively.

[0062] The calculation formula for the concentration of 5-HT in a specific sample is shown in the following formula (3): ; (3) In the formula, the F value is expressed as the fluorescence intensity at the emission wavelength (Em) of 477 nm, where F(T), F(B1), F(S), and F(B2) represent the fluorescence intensities at 477 nm of the sample group, the measurement blank group, the standard sample group, and the standard blank group, respectively.

[0063] Substituting the data obtained by the microplate reader into the formula, the 5-HT concentration in the B534 fermentation broth was calculated to be 5.2063±0.4394 μg / mL.

[0064] Example 5 In vitro digestion resistance test of strain B534 in simulated gastric and intestinal fluids Food eaten by the human body needs to be decomposed by gastric juice and digested by intestinal juice. Studies have shown that the pH value of gastric juice in normal humans is generally between 0.9 and 1.5, and the mass concentration of bile salts in intestinal juice is generally between 0.3 and 3 g / L. Simulated artificial digestive juice was prepared to fully simulate the digestive environment of human gastric juice and intestines to determine the tolerance of strain B534.

[0065] (1) Preparation of simulated artificial gastric juice: Pepsin (p7000) was prepared into a pepsin solution with a concentration of 2 g / L using sterilized 0.85% saline, and the pH value was adjusted to 3 using 4 mol / L hydrochloric acid solution. The solution was then filtered through a 0.22 μm microporous filter membrane for sterilization and set aside.

[0066] (2) Preparation of simulated artificial intestinal fluid: Trypsin (USP (P7545)) was prepared into a 2 g / L solution with sterilized 0.85% saline, and ox bile salt was added to make the concentration 0.2%. The pH value was adjusted to 8.0 with 1 mol / L sodium hydroxide, and then filtered through a 0.22 μm microporous filter membrane for sterilization before use.

[0067] (3) Artificial gastric juice tolerance test: The B534 bacterial suspension (i.e., the fermentation liquid of Example 2) after being cultured at 37°C for 24 hours was centrifuged and resuspended with physiological saline. 1 mL of the B534 resuspended liquid was added to 9 mL of the prepared artificial gastric juice. After being fully mixed, 1 mL of the mixed liquid was diluted tenfold to a certain gradient. After being shaken and mixed, 100 μL was taken and the number of viable bacteria was measured by the pouring plate method. At the same time, the simulated artificial gastric juice was incubated at 37°C for 2 hours. Then 1 mL of the mixed liquid was diluted tenfold to a certain gradient. After being shaken evenly, 100 μL was taken and the number of viable bacteria was measured by the pouring plate method. The survival rate was calculated as follows: Strain survival rate (%) = b / a × 100%; (6) Where: a is the number of viable bacteria at 0 h after adding 1 mL of bacterial suspension to 9 mL of artificial gastric fluid, unit: CFU / mL; b is the number of viable bacteria after incubation in artificial gastric fluid for 2 h, CFU / mL.

[0068] like Figure 6 As shown in the figure, the experimental results show that the growth of strain B534 in the simulated artificial gastric juice is relatively good, with a survival rate of 97.58%. This shows that after strain B534 enters the gastric juice environment, the pH in the gastric juice will cause a small number of bacteria to die, while a considerable number of bacteria survive, indicating that strain B534 has a strong tolerance to both acid and pepsin and can survive well in the simulated gastric juice environment.

[0069] (4) Artificial intestinal fluid tolerance test: After incubating in artificial gastric fluid for 2 h, 1 mL of the B534 bacterial solution was evenly shaken and added to 9 mL of the prepared artificial intestinal fluid. After fully mixing, the mixture was incubated at 37°C for 1 h. Then 1 mL of the mixed solution was diluted tenfold to a certain gradient. After evenly shaking, 100 μL was taken and the number of viable bacteria was measured by the pour plate method. The survival rate was calculated using the following formula: Strain survival rate (%) = c / b × 100%; (7) Where: b is the number of viable bacteria after 2 h of incubation in artificial gastric juice, CFU / mL; c is the number of viable bacteria after 1 h of incubation in artificial intestinal juice, CFU / mL.

[0070] like Figure 6 As shown in the experimental results, the growth of strain B534 in the simulated artificial intestinal fluid was good, with a survival rate of 99.86%. This shows that after strain B534 entered the intestinal fluid environment, the high concentration of bile salts in the intestinal fluid caused a very small number of bacteria to die, while the majority of the remaining bacteria survived. The specific data are as follows Figure 6 shown.

[0071] Example 6 Determination of drug sensitivity of strain B534 In order to prove that the screened B534 strain does not have strong drug resistance, Example 8 determined its drug sensitivity, and selected 6 antibiotics including tetracycline, azithromycin, amoxicillin, erythromycin, chloramphenicol and ampicillin for the experiment.

[0072] After strain B534 was activated for two generations with an inoculum of 3% (v / v), it was inoculated into MRS liquid culture medium and cultured in a 37°C constant temperature incubator for 24 h. The bacterial suspension cultured for 24 h was diluted tenfold with physiological saline, and 100 μL was drawn from the diluted bacterial suspension and injected onto the MRS solid agar plate. The bacterial suspension was evenly spread with a coating stick and placed in front of an alcohol lamp to dry. When there was no obvious liquid on the surface of the plate, the drug-sensitive paper (tetracycline, azithromycin, amoxicillin, erythromycin, chloramphenicol and ampicillin, etc.) was clamped and placed flat on the MRS agar plate, and then the plate was inverted and cultured in a 37°C constant temperature incubator for 24 h. After the culture was completed, the size of the inhibition zone was observed.

[0073] Since the inhibition zone is less than 10 mm, it is insensitive. Figure 7 As shown, it can be seen that strain B534 is sensitive to six antibiotics including tetracycline, azithromycin, amoxicillin, erythromycin, chloramphenicol and ampicillin, proving that it does not have drug resistance.

[0074] Example 7 Evaluation of the efficacy of Lactobacillus plantarum B534 in improving sleep Based on similarities in genetics, pharmacology, and neuroanatomy, the ontogeny of zebrafish and human sleep cycles is very similar.

[0075] In this example, the insomnia model of zebrafish was induced by using PTZ (Pentetrazol, a tetrazole derivative, an inhibitor of γ-aminobutyric acid and its receptor complex), an inhibitor of GABA, and melatonin was used as the positive control group and B534 was used as the experimental group to conduct an experiment on the efficacy of improving sleep. First, the maximum experimental concentration of B534 was determined. The maximum experimental concentration of Lactobacillus plantarum B534 on zebrafish was 1000ug / mL through the MTC (maximum detection concentration) experiment of B534 on zebrafish. 8 CFU / mL, the oral concentration of B534 in subsequent behavioral experiments was set at 10 8 CFU / mL. There are 3 groups in the behavioral experiment. One group is the probiotic group (i.e. the experimental group, i.e. the sample is B534 bacterial solution), and the experimental concentration of probiotics is 10 8 CFU / mL, and a normal control group, a model control group, and a probiotic group were set up. The probiotic treatment process was gavage into the zebrafish. After the probiotic treatment, the zebrafish were placed under a behavior analyzer to detect the amount and duration of awakening activity. The experimental results are as follows Figure 8 shown.

[0076] The results showed that Lactobacillus plantarum B534 has the effect of improving sleep, specifically by reducing the amount of wakefulness activity and the total wakefulness time.

[0077] Example 8 Effect of Lactobacillus plantarum B534 on the relative expression of GABAA receptor gene and melatonin receptor gene Both GABA and melatonin have the functions of improving sleep and regulating mood, among which melatonin's sleep-inducing effect is most widely known.

[0078] Therefore, this embodiment uses the QPCR method to perform genetic testing on zebrafish treated with Lactobacillus plantarum B534 strain, and GABA and melatonin receptors are key to their function in the human body, so by testing the relevant receptor genes gabra1 and mtnr1aa The relative expression of B534 was used to evaluate the sleep-inducing ability of B534. After the MTC experiment of B534 on zebrafish was obtained through behavioral experiments, QPCR was performed with melatonin as the positive control group and B534 as the experimental group.

[0079] QPCR method: quantitative PCR (real-time polymerase chain reaction, Real-time Polymerase Chain Reaction, referred to as Real-time PCR, real-time PCR), also known as quantitative real-time polymerase chain reaction (Quantitative real time polymerase chain reaction, referred to as Q-PCR / qPCR / rt-qPCR, quantitative real-time PCR, real-time quantitative PCR) There are 3 groups in the QPCT experiment. One probiotic group (i.e., the experimental group, i.e., the sample is B534 bacterial solution), the experimental concentration of the probiotic group is 10 8 CFU / mL, and set up normal control group, model control group and probiotics group.

[0080] After the probiotic treatment, QPCR experiment was performed, and the experimental results were as follows Fig. 9 The results showed that Lactobacillus plantarum B534 has the effect of improving sleep, which is manifested by increasing gabra1 and mtnr1aa Relative gene expression.

[0081] Example 9 Preparation of Lactobacillus plantarum B534 bacterial agent After the strain B534 was activated for two generations with an inoculum of 3% (v / v), it was inoculated into MRS liquid culture medium, cultured in a constant temperature incubator at 37°C for 24 h, centrifuged at 8000 r / min for 5 min at 4°C, and the supernatant was removed to retain the bacterial mud. The bacterial mud was mixed with a freeze-drying protective agent in a mass ratio of 1:(1-2), and then embedded to obtain embedded bacterial mud, which was stored at -80°C overnight. The pre-frozen embedded bacterial mud was freeze-dried in a vacuum freeze dryer for 48 h to obtain a freeze-dried bacterial powder of Lactobacillus plantarum B534 with a viable count of 200 billion CFU / g.

[0082] The freeze-dried protective agent was prepared as follows: 8 g skim milk powder, 2 g sucrose, 100 g distilled water was added, stirred to dissolve, sterilized at 115 °C for 15 min, and used after cooling.

[0083] It should be noted that the freeze-drying protective agent can adopt other protective agent compositions or formulas, including but not limited to the above-mentioned embodiment schemes.

[0084] Example 10 Application of Lactobacillus plantarum B534 in solid beverages By mass percentage, 2% of the Lactobacillus plantarum B534 powder obtained in Example 9, 30% of alkalized cocoa powder, 8% of sorbitol, 29% of maltodextrin, 1% of xanthan gum, and 30% of oligofructose were mixed (the percentage is the mass ratio) to obtain a probiotic solid beverage with the effects of improving sleep, resisting depression, and regulating mood.

[0085] It should be noted that this embodiment is only an example of the application of Lactobacillus plantarum B534 in a probiotic solid beverage. Lactobacillus plantarum B534 can also be applied to probiotic solid beverages of other formulations, including but not limited to the probiotic solid beverage shown in the above embodiment 10.

[0086] In summary: The plant lactobacillus B534 provided in the present application is a natural strain with food safety; it not only has significant inhibitory activity on acetylcholinesterase, but also has a strong ability to synthesize and secrete GABA, 3-HB and 5-HT, and is resistant to gastric acid and bile salts, and has good prebiotic properties, so it has important application value in the field of food or health products that help improve sleep, anti-depression and mood regulation. For example: using plant lactobacillus B534 as a raw material component of a microbial agent to prepare a microbial agent with the above functions; plant lactobacillus B534 is fermented to obtain a fermented product with the above functions.

[0087] In summary, the Lactobacillus plantarum B534 provided in this application has the following beneficial effects: The Lactobacillus plantarum B534 provided in this application is isolated and screened from the intestinal samples of infants and young children breastfed by a healthy pregnant woman in Xiamen, Fujian Province. It is a natural strain and has food safety. Under the conditions of 3% inoculation, second-generation liquid activation culture, fermentation temperature of 37℃, and fermentation time of 24 h, the inhibitory activity of the bacterial suspension on acetylcholinesterase was measured to be as high as 46.78±8.49%, and the concentrations of GABA, 3-HB and 5-HT in the fermentation broth were 0.1021±0.0129 mg / mL, 2.5075±0.4548 μg / mL and 5.2063±0.4394 μg / mL, respectively. It has strong acetylcholinesterase inhibitory activity and high levels of γ-aminobutyric acid, 3-hydroxybutyric acid and 5-hydroxytryptamine in the fermentation broth. Moreover, B534 has an ideal survival rate in simulated gastrointestinal fluid and can be used to produce products that help improve sleep, anti-depression and mood regulation, and has certain commercial value.

[0088] It should be noted that: MRS medium is a commonly used microbial culture medium used to isolate and culture lactic acid bacteria and other acidic strains, also known as lactic acid bacteria medium.

[0089] The culture medium involved in the examples of the present application is specifically: MRS liquid medium: 20.0 g glucose, 10.0 g tryptone, 10.0 g beef extract, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g potassium dihydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate monohydrate, 1 L deionized water, pH 6.5 (add 1.5% agar powder to make MRS solid medium); Unless otherwise specified, the experimental operation methods involved in the embodiments of the present application are conventional experimental operation methods in the art, and the reagents or instruments involved can be purchased from regular channels.

[0090] In addition, plant lactobacillus B534 has a strong ability to synthesize and secrete GABA, 3-HB and 5-HT. Based on the correlation between its above-mentioned ability and characteristics and the directional effects such as improving sleep, anti-depression, and mood regulation, the plant lactobacillus B534 of the present application can be used as a raw material component of probiotic products, fermented products, and functional foods with directional effects such as improving sleep, anti-depression, and mood regulation. Therefore, if the acetylcholinesterase inhibitory activity and the high-level ability to synthesize and secrete GABA, 3-HB and 5-HT are also associated with other directional effects, it can also be applied to the preparation of other directional products, including but not limited to helping to improve sleep, anti-depression, The effects of mood regulation. The application of plant lactobacillus B534 of the present application, specifically acts on the activity of inhibiting acetylcholinesterase and maintaining a high level of various excitatory neurotransmitters, has general applicability for people such as anxiety, depression and insomnia.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) B534, characterized in that, Its deposit number is CGMCC No.33031.

2. Application of Lactobacillus plantarum B534 in the preparation of products for inhibiting acetylcholinesterase activity, characterized in that: The Lactobacillus plantarum B534 is the Lactobacillus plantarum B534 as claimed in claim 1.

3. A method for preparing a bacterial suspension of Lactobacillus plantarum B534 according to claim 1, characterized in that: The plant lactobacillus B534 is inoculated into an MRS liquid culture medium, cultured at (30-40)°C for 24-36 hours, the culture medium is separated into solid and liquid, and the solid is resuspended to obtain a bacterial suspension having acetylcholinesterase inhibitory activity.

4. Use of Lactobacillus plantarum B534 in the preparation of a fermented product having a high level of γ-aminobutyric acid and / or a high level of 3-hydroxybutyric acid and / or a high level of 5-hydroxytryptamine, characterized in that: The Lactobacillus plantarum B534 is the Lactobacillus plantarum B534 as claimed in claim 1.

5. A method for preparing a fermentation liquid of Lactobacillus plantarum B534 as claimed in claim 1, characterized in that: Inoculate Lactobacillus plantarum B534 into MRS liquid culture medium, culture at (30-40)°C for 24-36 hours, separate the culture medium into solid and liquid, collect the liquid, and obtain the fermentation liquid; Wherein, the fermentation broth is a fermentation broth containing γ-aminobutyric acid, 3-hydroxybutyric acid and 5-hydroxytryptamine.

6. Lactobacillus plantarum B534 bacterial agent, characterized in that: Its components include the Lactobacillus plantarum B534 as claimed in claim 1.

7. The bacterial agent according to claim 6, characterized in that: The components thereof include fermentation liquid of Lactobacillus plantarum B534 and / or bacterial powder of Lactobacillus plantarum B534.

8. Use of Lactobacillus plantarum B534 in the preparation of a functional product that helps improve sleep and / or regulate mood, characterized in that: The Lactobacillus plantarum B534 is the Lactobacillus plantarum B534 as claimed in claim 1.

9. Application of Lactobacillus plantarum B534 in the preparation of functional products, characterized in that: The functional product is a functional food or a health product; The Lactobacillus plantarum B534 is the Lactobacillus plantarum B534 as claimed in claim 1.

10. The use according to claim 9, characterized in that: The functional food includes one of a functional food that helps improve sleep, a functional food that regulates mood, an acid-resistant and bile-resistant food, and a functional food that inhibits the activity of acetylcholinesterase.

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