Lactobacillus plantarum JN9, preparation, probiotic composition and use thereof

Abstract

A plant lactobacillus JN9, a preparation, a probiotic composition and its use. The present application obtains a new plant lactobacillus JN9 through screening, and the deposit number is: CCTCC NO: M 20241647. Through the basic characteristic analysis (growth curve, acid resistance, bile salt resistance, gastrointestinal fluid resistance), safety evaluation (antibiotic sensitivity, hemolytic ability, toxin production ability, cytotoxicity, ability to produce bioamines), adhesion evaluation, GABA production evaluation, alcohol tolerance and acetaldehyde dehydrogenase production ability evaluation of the strain, it is found that it is a safe strain with excellent GABA production performance, high alcohol tolerance and high acetaldehyde dehydrogenase enzyme activity. It can be used to improve mood such as relieving stress, anti-depression and improving the body's sleep quality, lowering blood pressure, anti-diabetes, anti-cancer, anti-oxidation, anti-inflammatory, anti-microbial, anti-allergic, and enhancing alcohol tolerance during drinking and relieving discomfort after drinking.

Description

Technical Field

[0001] The present invention generally relates to the field of biological application technology, and in particular to a Lactobacillus plantarum JN9, a preparation, a probiotic composition and uses thereof. Background Art

[0002] γ-Aminobutyric Acid (GABA) is a non-protein natural amino acid that is widely found in animals, plants and microorganisms. GABA is an important inhibitory neurotransmitter in the central nervous system that can regulate diseases related to depression and other mental health problems. Studies have shown that taking a certain amount of GABA can improve sleep, lower blood pressure, treat epilepsy and regulate emotions. Animal and clinical population trials have also confirmed that GABA has the effect of improving depressive-like behavior in mice and relieving depression in people. With the deepening of research, GABA has developed into a new functional factor and is widely used in industries such as medicine, food health care and agriculture.

[0003] GABA can be prepared by chemical synthesis or biosynthesis. The biosynthetic method of GABA may be more promising than the chemical synthesis method due to its simple reaction steps, high catalytic efficiency, mild reaction conditions, and good environmental compatibility.

[0004] Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the host's flora in a certain part. Probiotics promote nutrient absorption and maintain intestinal health by regulating the host's mucosal and systemic immune functions or by regulating the balance of intestinal flora, thereby producing single microorganisms or mixed microorganisms with clear composition that are beneficial to health. Finding a probiotic strain that is highly safe and can produce GABA is of great significance for improving mood, fighting depression and improving sleep quality. Summary of the invention

[0005] Based on the above background, the present application provides a Lactobacillus plantarum JN9, wherein the classification name of Lactobacillus plantarum JN9 is Lactiplantibacillus plantarum (Lactiplantibacillus plantarum), and the preservation number is CCTCC NO: M20241647.

[0006] In another aspect, the present application also provides a preparation prepared by the Lactobacillus plantarum JN9 described herein.

[0007] In another aspect, the present application also provides a probiotic composition comprising the Lactobacillus plantarum JN9 described herein or the preparation described herein.

[0008] In another aspect, the present application also provides use of Lactobacillus plantarum JN9 described herein, the preparation described herein, or the probiotic composition described herein in the preparation of a medicament for reducing stress, improving mood, resisting depression, improving sleep quality and / or lowering blood pressure.

[0009] In another aspect, the present application also provides use of the Lactobacillus plantarum JN9 described herein, the preparation described herein, or the probiotic composition described herein in the preparation of a medicament for protecting the liver and / or preventing and treating alcohol hangover.

[0010] In another aspect, the present application also provides use of the Lactobacillus plantarum JN9 described herein, the preparation described herein, or the probiotic composition described herein in the preparation of food or health products.

[0011] In another aspect, the present application also provides use of the Lactobacillus plantarum JN9 described herein in producing GABA.

[0012] The present application provides a strain of Lactobacillus plantarum that can produce GABA, tolerate alcohol, and has good safety, acid, bile salt and gastrointestinal tolerance, and antibiotic sensitivity, and is named Lactobacillus plantarum JN9. It is deposited in: China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit date is July 22, 2024. The deposit number is CCTCC NO: M 20241647.

[0013] Through the analysis of the basic characteristics of the strain (growth curve, acid resistance, bile salt resistance, gastrointestinal fluid resistance), safety evaluation (antibiotic sensitivity, hemolytic ability, toxin production ability, cytotoxicity, ability to produce bioamines), adhesion evaluation, GABA production evaluation, alcohol tolerance and acetaldehyde dehydrogenase production ability evaluation, it was found that plant lactobacillus JN9 is a safe strain with excellent GABA production performance, high alcohol tolerance and high acetaldehyde dehydrogenase activity. Based on the efficacy of the produced GABA, strain JN9 can be used to improve mood such as reducing stress, anti-depression and improving the body's sleep quality, lowering blood pressure, anti-diabetes, anti-cancer, anti-oxidation, anti-inflammatory, anti-microbial, and anti-allergic products; based on the strong tolerance of JN9 strain to alcohol and high acetaldehyde dehydrogenase activity, it can be used in products that enhance alcohol tolerance during drinking and relieve discomfort after drinking.

[0014] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0016] Figure 1 It is a circular diagram of the genome of Lactobacillus plantarum JN9 in the embodiment of the present application. The circular diagram shows seven kinds of information from the outside to the inside: the first circle is the genome position information, the second circle is the GC content information, the third circle is the coding gene on the positive strand (marked in red), the fourth circle is the coding gene on the negative strand (marked in green), the fifth circle is the ncRNA on the positive strand (marked in blue), the sixth circle is the ncRNA information on the negative strand (marked in purple), and the seventh circle is the long segment repeat sequence information on the genome (marked in orange).

[0017] Figure 2 The colony morphology and Gram staining results of Lactobacillus plantarum JN9 in the examples of the present application are shown in Figure 1. (a) is the colony morphology of Lactobacillus plantarum JN9, and (b) is the bacterial morphology of Lactobacillus plantarum JN9.

[0018] Figure 3 This is the API 50CH sugar fermentation test result of Lactobacillus plantarum JN9 in the examples of this application.

[0019] Figure 4 This is the growth curve of Lactobacillus plantarum JN9 in the example of the present application within 48 hours.

[0020] Figure 5 The tolerance evaluation of Lactobacillus plantarum JN9 in the examples of this application is shown in Figure 1. (a) is the survival rate of Lactobacillus plantarum JN9 after 3 hours in MRS with pH = 3.0 or containing 0.3% bile salt; (b) is the survival rate of Lactobacillus plantarum JN9 in a sterile electrolyte solution containing 100 mg / L lysozyme for 30 minutes and 1 hour.

[0021] Figure 6 This is a graph showing the hemolysis results of Lactobacillus plantarum JN9 in the examples of the present application.

[0022] Figure 7 The figure is a result of the cytotoxicity of Lactobacillus plantarum JN9 on intestinal epithelial cells HT-29 in the examples of the present application. The data are represented by Mean±Sem, n=6.

[0023] Figure 8 This is a graph showing the cell adhesion results of Lactobacillus plantarum JN9 in the examples of the present application.

[0024] Fig. 9The results of the GABA production capacity of Lactobacillus plantarum JN9 in the examples of the present application are shown in FIG. 1 , wherein (a) is a PCR amplification diagram of gadB; and (b) is a mass spectrometry diagram of a GABA standard sample and a fermentation broth of Lactobacillus plantarum JN9.

[0025] Fig.10 The present invention is the detection of the acetaldehyde dehydrogenase gene (aldH) and the alcohol dehydrogenase gene (adh) of Lactobacillus plantarum JN9 in the examples of the present application, wherein (a) is the adh gene amplification map, and (b) is the aldh gene amplification map. DETAILED DESCRIPTION

[0026] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the application belongs. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood to be equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with lower range limits or preferred numerical values, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0027] The terms "about" and "approximately" when used with a numerical variable generally refer to the value of that variable and all values ​​of that variable are within experimental error (e.g., within a 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0028] The expression "comprising" or its synonymous similar expressions "including", "containing" and "having" etc. are open-ended and do not exclude additional unrecited elements, steps or ingredients. The expression "consisting of excludes any element, step or ingredient not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or ingredients, plus the optional elements, steps or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0029] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0030] Most of the probiotic products on the market currently focus on gastrointestinal regulation, women's physiological health, pet health, etc., and there are no hot-selling probiotic products or leading brands that focus on emotional health, anxiety and depression relief, etc. This application develops strains with antidepressant effects for people with emotional problems such as insomnia, anxiety, depression, etc., and screens out probiotics that can convert monosodium glutamate, one of the commonly used condiments in people's daily diet, into GABA, which provides the possibility for discovering antidepressant probiotics that improve sleep, and supplements the combination of antidepressant probiotic products. In addition, plant lactobacillus JN9, in addition to having a higher GABA conversion ability, also has a strong tolerance to alcohol and high acetaldehyde dehydrogenase enzyme activity, which makes the strain not only have application potential in the anti-anxiety market for improving sleep, but also in the alcohol tolerance and relief of discomfort after drinking market.

[0031] The present application provides a Lactobacillus plantarum JN9, wherein the classification name of Lactobacillus plantarum JN9 is Lactiplantibacillus plantarum, and the deposit number is CCTCC NO: M 20241647. The strain is isolated from fermented food for the first time, and the whole genome sequencing shows that it is a Lactobacillus plantarum strain that has not been reported in NCBI.

[0032] In another aspect, the present application also provides a preparation prepared by the Lactobacillus plantarum JN9 described herein.

[0033] In some embodiments, the formulation is selected from one or more of the following:

[0034] 1) a bacterial agent of Lactobacillus plantarum JN9; 2) a bacterial suspension of Lactobacillus plantarum JN9; 3) a postbiotic of Lactobacillus plantarum JN9; 4) a metabolite of Lactobacillus plantarum JN9; and 5) a fermentation extract of Lactobacillus plantarum JN9.

[0035] In some embodiments, the Lactobacillus plantarum JN9 described herein is fermented and then prepared into the various formulations described herein. The preparation methods of the various formulations described herein are known to those skilled in the art.

[0036] In another aspect, the present application also provides a probiotic composition comprising the Lactobacillus plantarum JN9 described herein or the preparation described herein.

[0037] In some embodiments, the probiotic composition further comprises one or more probiotics selected from the group consisting of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus mucinus, Lactobacillus plantarum, Lactobacillus unisorbitalis, Lactobacillus, Streptococcus, Lactococcus, Propionibacterium, Propionibacterium, Weizmannella, Zoococcus, Staphylococcus, Kluyveromyces, Leuconostoc, Pediococcus, and Bacillus subtilis.

[0038] In some embodiments, the probiotic composition further comprises one or more probiotics selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus kumiss subsp. kumiss, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus. Bacteria, Lactobacillus fermentative, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Streptococcus salivarius thermophilus subsp., Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis (diacetyl type), Lactococcus cremoris, Propionibacterium freudenreichii subsp. shermanii, Propionibacterium acidigenicum, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weizmannia coagulans, Zoococcus xylosus, Staphylococcus carnosus, Kluyveromyces marxianus and Bacillus subtilis DE111.

[0039] On the other hand, the present application also provides use of Lactobacillus plantarum JN9 described herein, the preparation described herein, or the probiotic composition described herein in the preparation of a medicament for reducing stress, improving mood, resisting depression, improving sleep quality and / or lowering blood pressure.

[0040] In another aspect, the present application also provides use of the Lactobacillus plantarum JN9 described herein, the preparation described herein, or the probiotic composition described herein in the preparation of a medicament for protecting the liver and / or preventing and treating alcohol hangover.

[0041] In some embodiments, the drug is administered orally.

[0042] In another aspect, the present application also provides use of the Lactobacillus plantarum JN9 described herein, the preparation described herein, or the probiotic composition described herein in the preparation of food or health products.

[0043] In some embodiments, the food or health product is a food or health product for reducing stress, improving mood, fighting depression, improving sleep quality, lowering blood pressure and / or improving alcohol tolerance.

[0044] In some embodiments, the food or health product is a dairy product, a soy product, a meat product, a fruit and vegetable product, a beverage or a snack. In some embodiments, the food or health product further comprises an edible auxiliary material.

[0045] In some embodiments, the food is a health food; or the food includes dairy products, soy products, meat products or fruit and vegetable products; or the food is a beverage or a snack. In some embodiments, the food comprises Lactobacillus plantarum JN9 described herein and an edible supplement.

[0046] In another aspect, the present application also provides use of the Lactobacillus plantarum JN9 described herein in producing GABA.

[0047] The present application describes a plurality of embodiments, but the description is exemplary rather than restrictive, and it is apparent to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application. Although many possible feature combinations are shown in the drawings and discussed in the specific embodiments, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

[0048] The present application includes and contemplates combinations of features known to those of ordinary skill in the art. The embodiments and features disclosed in the present application may also be combined with any conventional features to form a unique invention scheme defined by the claims. Any feature of any embodiment may also be combined with features from other invention schemes to form another unique invention scheme defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in the present application may be implemented individually or in any appropriate combination. Therefore, except for the limitations made according to the attached claims and their equivalents, the embodiments are not subject to other restrictions. In addition, various modifications and changes may be made within the scope of protection of the attached claims.

[0049] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps described herein, the method or process should not be limited to the steps of the specific order described. As will be understood by those of ordinary skill in the art, other sequences of steps are also possible. Therefore, the specific sequence of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for the method and / or process should not be limited to the steps of performing them in the order written, and those skilled in the art can easily understand that these sequences can be changed and still remain within the spirit and scope of the embodiments of the present application.

[0050] The experimental methods in the following examples without specifying specific conditions are usually measured according to national standards. The experimental materials in the following examples without specifying the source are all commercially available raw materials. The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise defined or specified, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of this application.

[0051] Example 1 Acquisition and Identification of Lactobacillus plantarum Lactobacillus plantarum JN9

[0052] The Lactobacillus plantarum JN9 provided in the present application was isolated from a portion of kimchi in Sichuan.

[0053] 1. 16s rRNA Sequencing of Lactobacillus plantarum JN9

[0054] After culturing Lactobacillus plantarum JN9 at 37°C for 16 h, fresh cells were collected and DNA was extracted using a DNA extraction kit (9763, Takara, Japan). The genomic DNA was amplified by 16s rRNA PCR, purified, and sequenced. The 16s rRNA amplification primers were universal primers 27F: 5'-AGAGTTTTGATCCTGTCCAG-3' (SEQ ID NO: 1) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2). The PCR reaction conditions were denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 1 min, extension at 72°C for 1 min, 30 cycles in total, and extension at 72°C for 10 min. The PCR product was purified for first-generation sequencing, and the measured 16s rRNA sequence is shown in SEQ ID NO: 3.

[0055] After 16s rRNA sequencing of Lactobacillus plantarum JN9, it was found that its similarity with the standard strain of Lactobacillus plantarum SRCM100442 was 99.86%. Based on the physiological and biochemical indicators, morphological characteristics and molecular biological identification results of the strain Lactobacillus plantarum JN9, we named it Lactiplantibacillus plantarum JN9 (Lactiplantibacillus plantarum JN9), and it was deposited in the China Center for Type Culture Collection on July 22, 2024, and its preservation number is CCTCC NO: M20241647.

[0056] 2. Whole genome sequencing of Lactobacillus plantarum JN9

[0057] For genome sequencing, Lactobacillus plantarum JN9 was cultured in MRS broth at 37°C under anaerobic conditions for 24 h. Single colonies were cultured overnight in MRS broth. The broth was centrifuged at 8000×g, 4°C, 5 min. The precipitate was sent to GENEWIZ Sequencing (China) for sequencing, assembly, annotation, and bioinformatics analysis.

[0058] Whole genome sequencing was performed on the illumine PE150 platform and the PacBio Sequel system. For the PacBio sequencing library, 5-10 μg of genomic DNA was sheared into 10-15 Kb fragments using a g-TUBE device. Express Template Preparation Kit 2.0 was used to construct the library. In brief, the DNA sheared fragments were subjected to single-stranded overhang removal, DNA damage repair, end repair, A-tailing, and barcoded overhang adapter ligation. The library was quantified using a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, California), and the size of the library was checked using an Agilent 2100 bioanalyzer system. The manufacturer's instructions were followed for subsequent steps to prepare the SMRTbell library. The library was sequenced using the PacBio Sequel platform. PacBio reads were assembled using Hifiasm / Canu. The genome was then recalibrated using previous Illumina data using Pilon software. Prodigal / Augustus gene search software has been used to find coding genes. Transfer RNA (tRNA) was detected in the genome using the program tRNAscan-SE and default parameter settings. rRNA was identified using Barrnap. Other RNAs were identified using the rfam database. Coding genes were annotated using the National Center for Biotechnology Information (NCBI) nr database using Diamond. The functions of the genes were then annotated using the GO (Gene Ontology) database, and the pathways were annotated using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. The proteins encoded by the genes were phylogenetically classified using the COG (Clusters of Orthologous Groups) database. Diamond was used to search for protein sequences with E < 1e-5 in the CAZy database, Swiss_Prot database, Pfam database, CARD database, VFDB database, or DFVF database.

[0059] The genome sequence of Lactobacillus plantarum JN9 was assembled and analyzed. The basic genome information sequence is shown in Table 1. The genome length is 3187478 bp, with a total of 3151 genes and 3029 protein-coding genes. The Circos (version 0.69) software was used to create a genome circular map showing gene, ncRNA, GC content and repeat sequence information, see Figure 1 .

[0060] Table 1 Basic genome information of Lactobacillus plantarum JN9

[0061] feature value % of total Size (bp) 3187478 100 G+C content (bp) 1425066 44.71 Coding region (bp) 2687457 84.31 Total genes 3151 100 RNA genes 122 3.87 Protein coding genes 3029 96.13 Enzyme protein coding gene 1032 32.75 Genes assigned to COGs 2318 73.56 COG Cluster 1247 53.8 Genes containing signal peptides 163 5.17 Genes containing transmembrane helices 831 26.37

[0062] 3. Identification of Lactobacillus plantarum JN9

[0063] The average nucleotide identity (ANI) was estimated by calculating the best hit (one-way ANI) and mutual best hit (two-way ANI) between the Lactobacillus plantarum JN9 genome sequence and the Lactobacillus plantarum standard strain GCA_009913655.1 (ASM991365v1) using an online ANI calculator (http: / / enve-omics.ce.gatech.edu / ani / ) to determine the genus and species of Lactobacillus plantarum JN9.

[0064] A total of 1215 Lactobacillus plantarum genome sequences were obtained by querying the complete genome or draft genome of Lactobacillus plantarum in NCBI (retrieved on February 20, 2024). The Makeblastdb software was used to establish a database of the 1215 Lactobacillus plantarum genomes and the complete genome of Lactobacillus plantarum JN9 was compared with the database using Blastn. The mismatched regions in the comparison entries and the number of mismatched regions were generated according to 1000 mismatches.

[0065] The ANI value of the Lactobacillus plantarum JN9 genome sequence and the Lactobacillus plantarum reference strain GCA_009913655.1 obtained by the online ANI calculator was 99.10%, indicating that Lactobacillus plantarum JN9 is Lactobacillus plantarum. At the same time, the Lactobacillus plantarum JN9 genome was compared with 1215 strains of Lactobacillus plantarum genomes by Blastn, and no reference that was completely consistent with the assembly result or allowed up to 1000 mismatches was found, indicating that Lactobacillus plantarum JN9 is a strain that has never been reported in NCBI.

[0066] Example 2. Characterization of Lactobacillus plantarum JN9

[0067] The plant lactobacillus JN9 was fermented and the following characteristics of the strain were determined

[0068] 1) Morphological observation and sugar fermentation test of Lactobacillus plantarum JN9

[0069] The colony morphology of Lactobacillus plantarum JN9 was observed, and a single colony was picked for Gram staining and examined under a 100x microscope.

[0070] The sugar fermentation test of Lactobacillus plantarum JN9 was carried out by API 50CH (50300, API, France). Specific operation: After culturing Lactobacillus plantarum JN9 at 37°C for 16h, fresh cells were collected, and the cells were washed twice with sterile PBS and resuspended in sterile PBS, and the OD was adjusted to 0.3. Take 1mL of the resuspended bacterial solution and add it to 10mL of 50CHL culture medium. After mixing, take 150μL and add it to the test wells of 50CH test strips, and place it at 37°C for 48h. The results are determined by color changes. Compared with the control group, the color is yellow for positive (among which the positive color of esculin is black and red), and no change is recorded as negative.

[0071] The results showed that the colonies of Lactobacillus plantarum JN9 grown on MRS plates were milky white, round, with neat edges, smooth and moist surfaces, and a colony diameter of about 1-2 mm ( Figure 2 a in the figure). After selecting a single colony for Gram staining and observing it under an optical microscope, it was found to be a Gram-positive bacterium with a rod shape ( Figure 2 b) in the above.

[0072] The results of carbohydrate utilization by Lactobacillus plantarum JN9 are as follows Figure 3 As shown: Lactobacillus plantarum JN9 can metabolize D-ribose (5), D-galactose (10), D-glucose (11), D-fructose (12), D-mannitol (13), mannitol (18), methyl-α-D-pyranoside (20), methyl-α-D-pyranoside (21), N-acetylglucosamine (22), amygdalin (23), arbutin (24), esculin (25), salicin (26), D-cellobiose (27), D-maltose (28), D-lactose (29), D-melbiose (30), D-sucrose (31), D-trehalose (32), D-melezitose (34), D-gentiobiose (39), D-toulose (40) and potassium gluconate (47) to produce acid by fermentation.

[0073] 2) Growth curve determination of Lactobacillus plantarum JN9

[0074] Growth profiler 960 (Enzyscreen BV, Heemstede, Netherlands) was used to draw the growth curve of Lactobacillus plantarum JN9. The specific operation is as follows: Lactobacillus plantarum JN9 was cultured in MRS for 16 hours and then inoculated into fresh MRS broth, and the final OD600 was adjusted to 0.1. The bacterial solution with adjusted OD was added to a sterile 96-well plate, ensuring that the amount added to each well was 250 μL, and a total of 11 parallel groups (A1-B9) were performed. Under aerobic conditions, the culture was carried out at 200 rpm and 37 ° C for 48 hours, and the OD value was collected every 30 minutes. At the same time, MRS liquid culture medium was used as a blank control group.

[0075] Lactobacillus plantarum JN9 with an initial OD of 0.1 was cultured in MRS liquid medium at 37°C and 200 rpm, reaching an exponential growth phase within 16 hours and being in a stable phase within the next 48 hours. Figure 4 This is the growth curve of Lactobacillus plantarum JN9 fitted by Growth Profiler960.

[0076] 3) Tolerance of Lactobacillus plantarum JN9

[0077] Acid and bile resistance

[0078] Plant Lactobacillus plantarum JN9 was cultured in MRS broth at 37°C for 16 h, and the fresh bacterial solution was centrifuged at 4°C and 12,000 rpm for 2 minutes to collect the cells. The cells were washed twice with sterile PBS buffer, resuspended in PBS (pH = 7) and the cell concentration was adjusted to 10 8 CFU / mL. The resuspended bacterial solution was inoculated into MRS broth with pH=3.0 or containing 0.3% bile salt (w / v, Sigma, USA) at a 5% addition rate and cultured in a 37°C incubator for 3 h. The number of viable Lactobacillus plantarum JN9 in different culture media was recorded before culture (N0) and after culture for 3 h (N1).

[0079] Lysozyme resistance

[0080] Lactobacillus plantarum JN9 was cultured in MRS broth at 37°C for 16 h, and the fresh bacterial solution was centrifuged at 4°C and 12,000 rpm for 2 min to collect the cells. The cells were washed twice with sterile PBS buffer and resuspended in 2 mL of Ringer's solution (8.5 g / L NaCl, 0.4 g / L KCl, 0.34 g / L hydrated CaCl 2 ) to 10 8 -10 9 CFU / mL. The bacterial suspension was inoculated in a sterile electrolyte solution (SES) (0.22 g / L CaCl 2, 6.2g / L NaCl, 2.2g / L KCl, 1.2g / L NaHCO 3 ), lysozyme (Sigma, USA) was added to a final concentration of 100 mg / L and incubated at 37°C for 30 minutes or 1 hour. The bacterial suspension without lysozyme added to SES was used as a negative control. The number of viable cells of Lactobacillus plantarum JN9 before (N0) and after (N1) incubation was recorded.

[0081] Resistance to gastrointestinal fluid

[0082] To test the gastrointestinal tolerance of Lactobacillus plantarum JN9, fresh single colonies were picked and cultured in MRS broth at 37°C for 16 h. 5 mL of bacterial solution was centrifuged at 12000 rpm and 4°C for 2 minutes. The cells were washed with sterile PBS (pH = 7) and resuspended to adjust the cell concentration to 10 7 CFU / mL. Take 100 μL of bacterial suspension and add 900 μL of simulated gastric fluid (125 mM NaCl, 7 mM KCl, 45 mM NaHCO 3 and 3g / L pepsin, pH adjusted to 3.0 with HCl) and incubated at 37°C. After 3 hours, 250 μL of the gastric juice mixture was added to 6 mL of simulated intestinal fluid (45 mM NaCl, 1 g / L trypsin, 3 g / L bile salts, pH adjusted to 8.0 with NaOH), and incubated at 37°C for another 3 hours. The number of viable cells before incubation with gastric and intestinal fluids was recorded as N0, and the number of viable cells after 6 hours of incubation multiplied by the dilution factor of 25 was recorded as N1.

[0083] The survival rate of Lactobacillus plantarum JN9 under acid, bile salt, lysozyme and gastrointestinal tract treatment was calculated as follows:

[0084] Survival rate (%) = (N1 ÷ N0) × 100%

[0085] Among them, N1 is the viable cell count after 3 hours in MRS (pH 3.0 or 0.3% bile salt), 30 minutes or 1 hour in SES (100 mg / L lysozyme), or 6 hours in gastrointestinal fluid; N0 is the viable cell count of Lactobacillus plantarum JN9 treated for 0 hours under different conditions.

[0086] Tolerance assessment of Lactobacillus plantarum JN9

[0087] The survival rates of Lactobacillus plantarum JN9 in MRS with pH 3.0 and MRS containing 0.3% bile salt were 47.87% and 75.11%, respectively. Figure 5 a). After incubation in sterile electrolyte solution containing 100 mg / L lysozyme for 30 min and 1 h, the survival rates were 87.18% and 73.42%, respectively ( Figure 5b) After 6 hours of digestion in simulated gastric and intestinal fluids, the number of viable cells of Lactobacillus plantarum JN9 was reduced to 12.50% of that before treatment.

[0088] 4) Safety of Lactobacillus plantarum JN9

[0089] Susceptibility and MIC to antibiotics

[0090] Seven commonly used clinical antibiotics, including gentamicin, ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin and clindamycin, were selected to analyze the antibiotic sensitivity of the strain. The mother solutions containing 256 mg / L of different antibiotics were prepared and diluted twice in a gradient and added to a 96-well plate, 100 μL per well. The OD of the fresh bacterial solution after 16 hours of culture was diluted to 0.0002, and 100 μL / well was added to the antibiotic well plate, and cultured at 37 ° C for 24 hours. The minimum inhibitory concentration (MIC) of plant lactobacillus JN9 for each antibiotic was determined and compared with the critical values ​​of microbial antibiotics recommended by the European Food Safety Authority (EFSA) for feed additives or production.

[0091] The MICs of ampicillin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline and chloramphenicol against Lactobacillus plantarum JN9 are shown in Table 2. The results showed that the MIC values ​​of the seven antibiotics tested were all lower than the critical values ​​of antibiotics specified in the EFSA guidelines, indicating that the strain was sensitive to these seven antibiotics and had microbial antibiotic safety.

[0092] Table 2 MIC (mg / L) of different antibiotics against Lactobacillus plantarum JN9

[0093]

[0094] Hemolytic activity

[0095] The absence of hemolytic activity and antibiotic resistance are considered safety prerequisites for the selection of probiotic strains (FAO / WHO, 2002).

[0096] The activated Lactobacillus plantarum JN9 was streaked onto Columbia blood agar medium (3400071, Haibo, China) containing 5% defibrinated sheep blood and cultured at 37°C for 48 hours. If a grass-green hemolytic ring appeared, it was α-hemolysis; if a colorless and transparent hemolytic ring appeared, it was β-hemolysis; if there was no hemolytic ring, it was γ-hemolysis. At the same time, Lactobacillus rhamnosus LGG was used as a negative control.

[0097] The results are as follows Figure 6 As shown, after Lactobacillus plantarum JN9 was cultured on blood agar, no hemolytic ring appeared around the colonies, indicating γ-hemolysis. This shows that Lactobacillus plantarum JN9 has no hemolytic ability.

[0098] Determination of D-lactic acid and L-lactic acid production by Lactobacillus plantarum JN9

[0099] Fresh single colonies were picked and cultured in MRS broth at 37° C. for 16 hours. The culture supernatant was analyzed for D / L-lactic acid by enzymatic method using a commercial D / L-lactic acid quantitative kit (Jingmei Co., China) according to the product instructions.

[0100] The results showed that after Lactobacillus plantarum JN9 was cultured in MRS broth at 37°C for 16 hours, the concentrations of D-lactic acid and L-lactic acid in the supernatant were 2.28 nmol / L and 8.99 nmol / L, respectively, and the ratio of L-lactic acid to D-lactic acid was 3.94.

[0101] Bioamine production capacity test

[0102] After activation, plant lactobacillus JN9 was cultured overnight in MRS liquid medium, and added to MRS liquid medium containing 0.1g / L histidine, tyrosine, ornithine, lysine and 0.05g / L pyridoxal-5-phosphate to a final concentration of OD 0.01 and passaged, and passaged once every 24h according to 2% inoculum, for a total of five times. The above-mentioned liquid medium was inoculated in a medium containing 0.1% histidine, tyrosine, ornithine and lysine according to 2% inoculum and cultured for 72h, and the color change of the medium was observed. If the color of the medium becomes purple, it is positive, and if the medium becomes yellow, it is negative. Rhamnosus lactobacillus LGG was used as a negative control, and Escherichia coli was used as a positive control.

[0103] The results showed that Lactobacillus plantarum JN9 turned yellow after three days of growth in four biogenic amine detection culture media, indicating that the strain did not produce putrescine, cadaverine, histamine and tyramine.

[0104] Cytotoxicity

[0105] CCK-8 assay is a universal method for detecting cell proliferation and toxicity. HT-29 cells are human intestinal epithelial cell lines cultured in RPMI 1640 medium containing 10% FBS and 1% P / S (penicillin / streptomycin). Its cell proliferation activity was detected by the Bio-Time CCK-8 kit. HT-29 cells were cultured in 5% CO 2 , cultured in a 37°C incubator for about 36h-72h, the cell density reached about 90%, digested with trypsin and subcultured into 96-well plates at an appropriate density. After the cells adhered to the wall, 100 μL of 10 8CFU / mL of Lactobacillus plantarum JN9 was co-cultured with cells for 18 hours. The cell culture medium was washed with sterile PBS and 1 / 10 of the total volume of CCK-8 solution was added, incubated in the dark at 37°C for 2 hours, bubbles were removed, and the absorbance at 450nm was measured with an enzyme marker.

[0106] To evaluate the potential cytotoxic effects of Lactobacillus plantarum JN9 on HT-29 intestinal epithelial cells, CCK-8 assay was performed after 18 h of bacterial-cell co-culture. The multiplicity of infection (MOI) of this assay was approximately 1:200. Figure 7 As shown, at this MOI, the effect of Lactobacillus plantarum JN9 on the activity of cells was not significantly different from that of the blank control group (p>0.05), indicating that Lactobacillus plantarum JN9 had no negative impact on the survival of HT-29 intestinal epithelial cells.

[0107] 5) Intestinal adhesion of Lactobacillus plantarum JN9

[0108] HT-29 cells were cultured at 5 × 10 5 Inoculate the cells from the culture flask into a 24-well plate at a concentration of 10 cells / mL and replace with a medium without antibiotics. After the cells have fully attached to the wall, the experiment can be performed. Before adding bacteria, wash the cells in the plate twice with sterile PBS and add 500 μL of 10 8 The 24-well plate was transferred to a 37°C, 5% CO 2 Culture in an incubator for 4 hours to allow adhesion. Wash each well of cells 5 times with PBS solution to elute non-adherent bacteria and metabolic secretions. Add 200 μL of 1% Triton X-100 to each well for digestion, then collect the solution in each well for gradient dilution and count (V1). The adhesion rate (%) is calculated as follows:

[0109] Adhesion rate (%) = (V1 / V0) × 100%

[0110] To evaluate the cell adhesion of Lactobacillus plantarum JN9 to HT-29 intestinal epithelial cells, adhesion assay was performed after 4 hours of co-culture of bacterial cells. The infection multiplicity (MOI) of this test was about 1:200. The results showed that the proportion of Lactobacillus plantarum JN9 that could adhere to HT-29 cells after 4 hours of co-culture was 15.98%, and its adhesion efficiency was about 1.5 times that of LGG (10.50%). Figure 8 ).

[0111] Example 3. Fermentation of Lactobacillus plantarum JN9 and production of bacterial powder

[0112] After activation, Lactobacillus plantarum JN9 was inoculated into MRS broth and cultured at 37°C for 18 h. After two transfers, it was transferred into the bioreactor ( 320, ependof). The total amount of fermentation liquid is 6L. After fermentation for 12h, collect the bacteria at 4℃, 10000rpm, and 10min. The obtained bacteria are mixed with the protective agent (1-10g / L polysaccharide, 20-50g / L disaccharide, 1-20g / L vitamin C or its salts, and 1-10g / L peptone) at a dry weight of 1:1, and pre-frozen at -80℃ for 24 hours. The bacterial powder is freeze-dried and crushed into powder by a vacuum freeze dryer (PO14416, Telstar LyoQuest-55plus), and then vacuum-packed in aluminum foil bags.

[0113] The number of viable cells of Lactobacillus plantarum JN9 in the bioreactor was 3.23×10 9 -5.20×10 9 CFU / mL. 6L of bacterial solution was centrifuged at 10000rpm and 4℃ for 10min to collect 120-127g of bacteria. The bacteria were mixed with the protective agent and freeze-dried to finally obtain 50-60g of bacterial powder, with a viable cell count of 2.81×10 11 -3.53×10 11 CFU / g.

[0114] Example 4. GABA production capacity of Lactobacillus plantarum JN9

[0115] Detection of gadB gene in Lactobacillus plantarum JN9

[0116] After culturing Lactobacillus plantarum JN9 at 37°C for 16 h, fresh cells were collected and DNA was extracted according to the instructions of the DNA extraction kit (9763, Takara, Japan). The amplification primers for glutamate decarboxylase B gene (gadB gene) are shown in Table 3. PCR reaction system (25 μL): 2×Premix Taq TM (R004Q, Takara) 12.5μL, upstream and downstream primers (10μmol / L) 1μL each, template (10ng) 1μL, nuclease-free water to 25μL. PCR reaction conditions are 94℃ denaturation for 5min, 94℃ denaturation for 30s, 52℃ annealing for 30s, 72℃ extension for 1.5min, 30 cycles in total, 72℃ extension for 7min. PCR products are detected by gel electrophoresis to form and size bands: 1μL of the above PCR product is taken, 5μL DNA Loading buffer is added, the sample is added to the spotting well after mixing, 100V, 40min electrophoresis, and the bands are observed by gel imaging after completion.

[0117] Table 3 Primer sequences (SEQ ID NO: 4-9)

[0118]

[0119]

[0120] Determination of GABA production capacity of Lactobacillus plantarum JN9

[0121] After activation, plant lactobacillus JN9 (experimental group) and the commercial control bacteria plant lactobacillus SG5 (GDMCC 60020) with the ability to produce GABA were inoculated with single colonies in MRS liquid supplemented with 20g / L sodium L-glutamate at 37°C for 48h. The fresh bacterial liquid was centrifuged at 4°C and 12000rpm for 1 minute, 200μL of the supernatant was taken and placed in a centrifuge tube, 400μL of 4.2% sodium bicarbonate solution and 200uL of 1% 2,4-dinitrofluorobenzene solution were added and mixed, and placed in a 60°C water bath for 1h. After cooling, 9.2mL of 0.136% potassium dihydrogen phosphate solution was added, shaken evenly, filtered with a 0.22μm filter membrane and LC-MS determination was performed. Liquid phase conditions: C18 column, column temperature 35°C±5°C, gradient elution, mobile phase A was 0.41% anhydrous sodium acetate solution, and mobile phase C was acetonitrile. Flow rate: 0.8 mL / min. Detection wavelength: 360 nm, injection volume: 10 μL. Mass spectrometry conditions: ESI positive ion mode, mass spectrometry scanning mass range: 20-2000 m / z. ESI source conditions: capillary voltage: 3.5 kV, desolvation temperature: 400 °C, cone voltage: 30 V, desolvation gas flow rate: 700 L / h, cone gas flow rate: 50 L / h, collision energy: 6 / 20 V.

[0122] The results showed that gene amplification showed that the genome of Lactobacillus plantarum JN9 contained the gadB gene ( Fig. 9 The components in the fermentation broth of Lactobacillus plantarum JN9 were analyzed by LC-MS to confirm that GABA ( Fig. 9 b), and the output of GABA will increase as the fermentation time increases. After 24h fermentation, the GABA output of plant lactobacillus JN9 was 10.53±1.70g / L, and the GABA output of commercially available control group plant lactobacillus SG5 was 0.42±0.08g / L; after 48h fermentation, the GABA output of plant lactobacillus JN9 was increased to 13.54±0.80g / L, and the GABA output of commercially available control group plant lactobacillus SG5 was 0.56±0.03g / L.

[0123] It can be seen that the genome of Lactobacillus plantarum JN9 contains the gadB gene, has a high GABA production capacity, and can be used for the production of GABA. And because it has good safety and alcohol resistance, it can be used to prepare drugs for improving sleep and anti-depression, and can also be used to prepare a series of functional (anti-depression, improving sleep, protecting the liver, enhancing immunity and / or preventing alcohol hangover) health products or foods.

[0124] Example 5. Alcohol tolerance of Lactobacillus plantarum JN9

[0125] Aldehyde dehydrogenase gene (aldH) and alcohol dehydrogenase gene (adh) detection

[0126] After culturing Lactobacillus plantarum JN9 at 37°C for 16 h, fresh cells were collected and DNA was extracted according to the instructions of the DNA extraction kit (9763, Takara, Japan). The amplification primers of aldH and adh genes are shown in Table 3. PCR reaction system (25 μL): 2×Premix Taq TM (R004Q, Takara) 12.5μL, upstream and downstream primers (10μmol / L) 1μL each, template (10ng) 1μL, nuclease-free water to 25μL. PCR reaction conditions are 98℃ denaturation for 4min, 98℃ denaturation for 10s, 56℃ annealing for 15s, 72℃ extension for 30s, 32 cycles in total, 72℃ extension for 7min. PCR product is detected by electrophoresis band formation and size: 1μL of the above PCR product is taken, 5μL DNA Loading buffer is added, the sample is added to the spotting well after mixing, 100V, 40min electrophoresis, and the band is observed by gel imaging after completion.

[0127] Evaluation of tolerance of Lactobacillus plantarum JN9 to different concentrations of alcohol

[0128] After activation, Lactobacillus plantarum JN9 was inoculated into MRS broth and cultured at 37°C for 16 h, and the bacterial solution concentration was adjusted to 10 8 CFU / mL, the bacterial suspension was added to MRS liquid medium containing anhydrous ethanol at a ratio of 1:1000 to a final ethanol concentration of 3%, 7%, 12% and 15% and cultured at 37°C for 24 h. The growth of the strain was detected by the viable bacteria counting method.

[0129] Detection of acetaldehyde dehydrogenase activity of Lactobacillus plantarum JN9

[0130] Lactobacillus plantarum JN9 (experimental group), Lactobacillus rhamnosus LGG (control 1) and commercially available Lactobacillus plantarum YLA1 (CCTCC NO: M2020289, control 2) with acetaldehyde dehydrogenase activity were activated and inoculated into MRS broth, and the cells were collected by centrifugation after static culture at 37°C for 16h, washed twice with sterile PBS buffer and resuspended, and the OD was adjusted to 1. Ice bath ultrasonic crushing (power 300w, ultrasonic 5s, interval 7s, total time 15min) was used to obtain the sample to be tested, and PBS was used as a blank control. According to Table 4, the enzyme activity reaction systems of Lactobacillus plantarum JN9, LGG, YLA1 and blank samples were respectively configured and incubated at 37°C for 30min. Record the absorbance values ​​A1 and A2 at 340nm before and after the sample incubation, and calculate ΔA assay tube = A2 assay tube - A1 assay tube, ΔA blank tube = A2 blank tube - A1 blank tube, ΔA = ΔA assay tube - ΔA blank tube. Enzyme activity definition: One unit of enzyme activity is defined as the amount of NADH generated per milliliter of sample per minute.

[0131] ALDH enzyme activity (U / mL) = ΔA ÷ (ε × d) × 10 6 ×V total ÷ V sample ÷ T

[0132] ε: NADH molar extinction coefficient, 6.22×103 L / mol / cm; d: 96-well plate light path, 0.6 cm; V total: total volume of the reaction system; V sample: sample volume in the reaction system; T: reaction time, 30 min; 10 6 : Unit conversion factor, 1mol = 10 6 μmol

[0133] Table 4 Enzyme activity assay reaction system

[0134]

[0135]

[0136] By amplifying the adh and aldh genes of Lactobacillus plantarum JN9, the electrophoresis results of the PCR amplification products are as follows Fig.10As shown, both gene bands are single, and the sizes are about 500bp and 1400bp respectively, which are consistent with the expected size. It shows that aldh and adh are contained in the genome of plant lactobacillus JN9. Through the alcohol tolerance experiment, it is concluded that plant lactobacillus JN9 has good growth ability in MRS medium containing 3%, 7% and 12% ethanol, and can tolerate MRS medium containing 15% ethanol. After 16h incubation, the viable bacteria detection found that the survival rate of the bacteria was 16.67%. Through the acetaldehyde dehydrogenase activity assay, it is known that the acetaldehyde dehydrogenase activity of plant lactobacillus JN9 strain is 5.29 (μmol / mL), the commercially available control group plant lactobacillus YLA (control 2) contains adh and aldh genes and its acetaldehyde dehydrogenase activity is 3.22 (μmol / mL), and rhamnosus lactobacillus LGG (control 1) does not contain aldh gene and acetaldehyde dehydrogenase activity is not detected.

[0137] It can be seen that the genome of Lactobacillus plantarum JN9 strain contains acetaldehyde dehydrogenase gene (aldH) and alcohol dehydrogenase gene (adh), the strain has higher acetaldehyde dehydrogenase activity, has stronger alcohol tolerance, and can be used to prepare drugs for protecting the liver and / or preventing and treating alcohol hangover.

Claims

1. A Lactobacillus plantarum JN9, wherein: The classification of the plant lactobacillus JN9 is named plant lactobacillus ( Lactiplantibacillus plantarum ), the deposit number is CCTCC NO: M 20241647.

2. The preparation prepared by the plant lactobacillus JN9 according to claim 1, wherein The preparation is selected from one or two of the following: 1) an inoculum of Lactobacillus plantarum JN9; and 2) Bacterial suspension of Lactobacillus plantarum JN9.

3. A probiotic composition comprising the Lactobacillus plantarum JN9 according to claim 1 or the preparation according to claim 2.

4. The probiotic composition according to claim 3, wherein The probiotic composition further comprises one or more probiotics selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis subspecies animalis, Bifidobacterium animalis subspecies lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii subspecies bulgaricus, Lactobacillus delbrueckii subspecies lactis, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus kumiss subspecies kumiss, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and fermented Lactobacillus mucilaginosus, Lactobacillus mucilaginosus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Streptococcus salivarius thermophilus subsp., Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis (diacetyl type), Lactococcus cremoris, Propionibacterium freudenreichii subsp. shermanii, Propionibacterium acidigenicum, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weizmannia coagulans, Zoococcus calvae, Staphylococcus xylosus, Staphylococcus carnosus, Kluyveromyces marxianus and Bacillus subtilis DE111.

5. Use of the plant lactobacillus JN9 according to claim 1 in producing GABA.

6. Use of the Lactobacillus plantarum JN9 according to claim 1 in the preparation of a medicament for preventing and treating alcohol hangover.

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