Phytobacterium plantarum JN-7, bacterium powder, preparation method, probiotic composition and application thereof

Abstract

The invention relates to a plant lactobacillus JN-7, bacterial powder, a preparation method, a probiotic composition and application thereof. According to the application, a new plant lactobacillus strain is obtained through screening, the new plant lactobacillus strain is named as plant lactobacillus JN-7, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.30605. Basic characteristic analysis (growth curve, acid resistance, cholate resistance and gastrointestinal fluid resistance), safety evaluation (antibiotic sensitivity, hemolysis capability, toxin production capability and cytotoxicity), adhesiveness evaluation, anti-inflammatory action evaluation, production performance evaluation (fermentation yield and freeze-drying yield) and stability test (accelerated storage stability test) of the strain are carried out; it is found that the plant lactobacillus JN-7 is a safe strain and has excellent production performance, high stability and an anti-inflammatory effect. The bacterial powder produced by the bacterial strain can keep stable and active for a long time.

Description

Technical Field

[0001] This document generally relates to the field of biological application technologies, and particularly to a Lactiplantibacillus plantarum JN-7, a bacterial powder, a preparation method, a probiotic composition and its uses. Background Art

[0002] Probiotics are a class of beneficial active microorganisms that colonize the human body and change the composition of the flora in a certain part of the host. By regulating the mucosal and systemic immune functions of the host or by regulating the balance of the intestinal flora, they play a role in promoting nutrient absorption and maintaining intestinal health, thereby producing a single microorganism or a well-defined mixed microorganism that is beneficial to health.

[0003] Lactiplantibacillus plantarum is a common type of probiotic, which is widely used in fermented dairy products, meat products, plant products and baked foods. Its main function is to produce lactic acid, lower the pH, prevent the reproduction of spoilage bacteria, improve the flavor and texture of products, etc. In recent years, studies have found that Lactiplantibacillus plantarum has good probiotic functions, such as inhibiting gastrointestinal pathogenic bacteria including Helicobacter pylori, improving gastrointestinal function and metabolism, enhancing intestinal barrier function, and reducing the risk of infection.

[0004] The stability and viability of probiotics in food are closely related to the health effects that probiotics can achieve. There is an urgent need to develop probiotics with good stability and long-term preservation. Summary of the Invention

[0005] Based on the above background, the present application provides a Lactiplantibacillus plantarum JN-7, wherein the classification and naming of the Lactiplantibacillus plantarum JN-7 is Lactiplantibacillus plantarum, and the preservation number is CGMCC No. 30605.

[0006] On the other hand, the present application also provides a bacterial powder prepared from the Lactiplantibacillus plantarum JN-7 described herein.

[0007] On the other hand, the present application also provides a preparation method of the bacterial powder described herein, including the following steps:

[0008] 1) Ferment the Lactiplantibacillus plantarum JN-7 described herein;

[0009] 2) Centrifuge and precipitate the fermentation product, and freeze-dry it to obtain the product.

[0010] On the other hand, the present application also provides a probiotic composition containing the Lactiplantibacillus plantarum JN-7 described herein or the bacterial powder described herein.

[0011] On the other hand, the present application also provides the use of Lactiplantibacillus plantarum JN-7 described herein, the bacterial powder described herein, or the probiotic composition described herein in the preparation of anti-inflammatory drugs.

[0012] On the other hand, the present application also provides the use of Lactiplantibacillus plantarum JN-7 described herein, the bacterial powder described herein, or the probiotic composition described herein in the preparation of foods or health products.

[0013] The viable cells surviving in the probiotic powder are closely related to the quality and production cost of the probiotic product. Strains with good stability can increase the number of viable cells in the probiotic powder, thus contributing to improving the quality of the probiotic powder and reducing the cost.

[0014] The present application provides a strain of Lactiplantibacillus plantarum that is good in terms of yield, stability, safety, acid, bile salt and gastrointestinal tolerance, and antibiotic sensitivity, and it is named Lactiplantibacillus plantarum JN-7. It was deposited in: China General Microbiological Culture Collection Center, Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date was May 13, 2024. The deposit number is CGMCC No. 30605.

[0015] Through the analysis of the basic characteristics of this strain (growth curve, acid tolerance, bile salt tolerance, gastrointestinal fluid tolerance), safety evaluation (antibiotic sensitivity, hemolytic ability, toxin production ability, cytotoxicity), adhesion evaluation, anti-inflammatory effect evaluation, production performance evaluation (fermentation yield and freeze-drying yield), and stability test (accelerated storage stability test), it was found that Lactiplantibacillus plantarum JN-7 is a safe strain with excellent production performance, high stability and anti-inflammatory efficacy. The bacterial powder produced therefrom can maintain stability and activity for a long time.

[0016] Other features and advantages of the present application will be described in the subsequent specification, and, in part, will be obvious from the specification, or will be understood by implementing the present application. Other advantages of the present application can be realized and obtained through the solutions described in the specification and the drawings. Description of the Drawings

[0017] The drawings are used to provide an understanding of the technical solutions 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 solutions of the present application and do not constitute a limitation to the technical solutions of the present application.

[0018] Figure 1This is the circular genome map of Lactiplantibacillus plantarum JN-7 in the embodiments of the present application. The circular map shows seven types of information from the outside to the inside: the first circle is the genomic position information, the second circle is the GC content information, the third circle is the coding genes on the positive strand (marked in red), the fourth circle is the coding genes on the negative strand (marked in green), the fifth circle is the ncRNAs 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 fragment repeat sequence information on the genome (marked in orange).

[0019] Figure 2 This is the growth curve of Lactiplantibacillus plantarum JN-7 starting from OD 0.1 at 37 °C and 200 rpm in the embodiments of the present application.

[0020] Figure 3 This is the hemolytic ability result of Lactiplantibacillus plantarum JN-7 in the embodiments of the present application.

[0021] Figure 4 This shows the interaction between Lactiplantibacillus plantarum JN-7 and intestinal epithelial cells HT-29 in the embodiments of the present application. Among them, (a) Lactiplantibacillus plantarum JN-7 does not reduce the viability of HT-29 cells. The data represent the survival rate of cells after co-incubation with Lactiplantibacillus plantarum JN-7 for 18 hours compared with those in the pure culture medium group (n = 6). (b) The adhesion ratio of Lactobacillus rhamnosus LGG and Lactiplantibacillus plantarum JN-7 to HT-29 cells. The values represent the adhesion rates of LGG and Lactiplantibacillus plantarum JN-7 after incubation with HT-29 for 4 hours. The data are expressed as Mean±Sem, *P<0.05, **P<0.01, ***P<0.001.

[0022] Figure 5 This shows the anti-inflammatory ability of Lactiplantibacillus plantarum JN-7 in regulating the production of inflammatory factors in LPS-stimulated THP-1 cells in the embodiments of the present application. Among them, (a) interleukin 6 (IL)-6 mRNA; (b) IL-8 mRNA; (c) IL-10 mRNA. The data are expressed as Mean±Sem, *P<0.05, **P<0.01, ***P<0.001.

[0023] Figures 6A - 6D This is the bacterial powder of Lactiplantibacillus plantarum JN-7 in the embodiments of the present application ( Figure 6A ), Lactobacillus rhamnosus LGG ( Figure 6B ), and the bacterial powders of 15 other strains of Lactiplantibacillus plantarum ( Figures 6C - 6D ) during storage at 40 °C and 75% humidity in terms of the number of viable cells and water activity. Detailed implementation manners

[0024] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. When a quantity, 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 as specifically disclosing any range obtained by combining any pair of upper range limits or preferred numerical values with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise specified, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0025] The terms “about” or “approximately” when used in conjunction with a numerical variable generally refer to the value of that variable and all values of that variable within experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or within a wider range.

[0026] The expression “comprising” or similar expressions synonymous therewith, such as “including,” “containing,” and “having,” are open-ended and do not exclude additional unrecited elements, steps, or components. The expression “consisting of” excludes any unstated element, step, or component. The expression “consisting essentially of” means that the scope is limited to the specified elements, steps, or components, plus optionally existing elements, steps, or components that do not substantially affect the basic and novel features of the claimed subject matter. It should be understood that the expression “comprising” encompasses the expressions “consisting essentially of” and “consisting of.”

[0027] The expression “at least one” or “one or more” means 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0028] Most of the research on improving the stability of functional strains is achieved through the development of bacterial powder protectants. Due to the inherent stability of each strain, the role of protectants in stabilizing probiotic powders is limited. Developing strains with good stability for use in commercial probiotic powder complexes is another way to solve the problem of low bacterial activity in probiotic products during the shelf life.

[0029] Based on this, the present application provides a Lactiplantibacillus plantarum JN-7, wherein the taxonomic name of Lactiplantibacillus plantarum JN-7 is Lactiplantibacillus plantarum, and the deposit number is CGMCC No. 30605.

[0030] In another aspect, the present application also provides a bacterial powder prepared from Lactiplantibacillus plantarum JN-7 described herein.

[0031] In another aspect, the present application also provides a method for preparing the bacterial powder described herein, comprising the following steps:

[0032] 1) Ferment Lactiplantibacillus plantarum JN-7 described herein;

[0033] 2) Centrifuge and precipitate the fermentation product, and then freeze-dry it to obtain the product.

[0034] In some embodiments, the method for preparing the bacterial powder described herein includes the following steps:

[0035] 1) Ferment and culture Lactiplantibacillus plantarum JN-7 described herein in a bioreactor at a pH of 4.5 - 5.3, a temperature of 35 - 40 °C, and a stirrer speed of 160 - 200 rpm;

[0036] 2) Centrifuge and precipitate the fermentation product, mix the precipitate with a cryoprotectant, and then freeze-dry and pulverize it into a powder in a vacuum freeze dryer to obtain the product.

[0037] On the other hand, the present application also provides a probiotic composition comprising Lactiplantibacillus plantarum JN-7 described herein or the bacterial powder described herein.

[0038] In some embodiments, the probiotic composition further comprises one or more probiotics selected from the following: Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus mucosae, Lactiplantibacillus, Lactobacillus collinoides, Lactobacillus paraplantarum, Streptococcus, Lactococcus, Propionibacterium, Propionibacterium acidipropionici, Weissella, Aerococcus, Staphylococcus, Kluyveromyces, Leuconostoc, Pediococcus, and Bacillus subtilis DE111.

[0039] In some embodiments, the probiotic composition further comprises one or more probiotics selected from the following: 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 kefiranofaciens subsp. kefiranofaciens, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus mucosae fermentum, Lactobacillus mucosae reuteri, Lactiplantibacillus plantarum, Lactobacillus salivarius subsp. salicinius, Lactobacillus paraplantarum curvatus, Lactobacillus paraplantarum sakei, Streptococcus salivarius subsp. thermophilus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis (diacetyl type), Lactococcus cremoris, Propionibacterium freudenreichii subsp. shermanii, Propionibacterium acidipropionici, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella confusa, Aerococcus viridans, Staphylococcus xylosus, Staphylococcus carnosus, Kluyveromyces marxianus, and Bacillus subtilis DE111.

[0040] On the other hand, the present application also provides the use of Lactiplantibacillus plantarum JN-7 described herein, the bacterial powder described herein, or the probiotic composition described herein in the preparation of anti-inflammatory drugs. In some embodiments, the drug is administered orally.

[0041] On the other hand, the present application also provides the use of Lactiplantibacillus plantarum JN-7 described herein, the bacterial powder described herein, or the probiotic composition described herein in the preparation of foods or health products.

[0042] 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 food excipients.

[0043] In some embodiments, the food is a health food; or the food includes a dairy product, a soy product, a meat product, or a fruit and vegetable product; or the food is a beverage or a snack. In some embodiments, the food comprises Lactiplantibacillus plantarum JN-7 described herein and food excipients.

[0044] The present application describes a number of embodiments, but the description is exemplary rather than restrictive, and it will be apparent to those of ordinary skill in the art that there may be more embodiments and implementation schemes within the scope of the embodiments described in the present application. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically restricted, any feature of any embodiment can be combined with any other feature in any other embodiment, or can replace any other feature in any other embodiment.

[0045] The present application includes and contemplates combinations with features known to those of ordinary skill in the art. The embodiments and features already disclosed in the present application can also be combined with any conventional features to form a unique inventive solution defined by the claims. Any feature of any embodiment can also be combined with features from other inventive solutions to form another unique inventive solution defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in the present application can be implemented alone or in any suitable combination. Therefore, the embodiments are not limited except as defined by the appended claims and their equivalents. In addition, various modifications and changes can be made within the scope of the appended claims.

[0046] 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 depend on the specific order of the steps described herein, the method or process should not be limited to the specific order of steps described. As will be understood by those of ordinary skill in the art, other step sequences are possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation on the claims. In addition, the claims directed to the method and / or process should not be limited to performing their steps in the order written, as those skilled in the art can readily understand that these orders can vary and still remain within the spirit and scope of the embodiments of the present application.

[0047] For the experimental methods without specific conditions noted in the following examples, they are generally determined according to national standards. The experimental materials without sources noted in the following examples are all commercially available raw materials. The equipment used in each step of the following examples is all conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer. Unless otherwise defined or stated, 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 methods and materials similar or equivalent to the described content can be applied to the methods of this application.

[0048] Example 1 Obtaining and Identification of Lactiplantibacillus plantarum JN-7

[0049] The Lactiplantibacillus plantarum JN-7 provided in this application was isolated from self-made pickles in Chengdu, Sichuan.

[0050] 1. 16S Sequencing of Lactiplantibacillus plantarum JN-7

[0051] The genomic DNA of JN-7 bacteria was extracted using the Takara Mini-BEST Bacterial Genomic DNA Extraction Kit version 3.0. The 16S rRNA gene was amplified by the universal primers 27F: 5'-AGAGTTTTGATCCTGTCCAG-3’ (SEQ ID NO:1) and 1492R: 5'-GGTTACCTTGTTACGACTT-3’ (SEQ ID NO:2). PCR cycle: initial activation at 94°C for 2 min; denaturation step cycling at 94°C for 30 s; annealing at 55°C for 1 min; extension at 72°C for 1 min; and finally cycling 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.

[0052] After comparing with Lactiplantibacillus plantarum through the Blast on the NCBI website, it was found that its similarity with the type strain Lactiplantibacillus plantarum SRCM100442 reached 99.93%. Combining the relevant physiological and biochemical indexes and molecular biology identification results of strain JN-7, and considering its morphological characteristics, we named it Lactiplantibacillus plantarum JN-7, which was deposited in the China General Microbiological Culture Collection Center on May 13, 2024, with the deposit number of CGMCC No. 30605.

[0053] 2. Whole-genome sequencing of Lactiplantibacillus plantarum JN-7

[0054] For genome sequencing, Lactiplantibacillus plantarum JN-7 was cultured in MRS broth under anaerobic conditions at 37 °C for 24 hours. Single colonies were cultured overnight in MRS broth. The broth was centrifuged at 8000×g, 4 °C for 5 min. The pellet was sent to GENEWIZ Sequencing Company (China) for sequencing, assembly, annotation and bioinformatics analysis.

[0055] The whole-genome sequencing was carried out 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. Then The Express Template Preparation Kit 2.0 constructs a library. Briefly, the DNA sheared fragments are subjected to single-stranded overhang removal, DNA damage repair, end repair, A-tailing, and ligation of barcoded overhang adapters. The library is quantified using a Qubit 3.0 fluorometer (Invitrogen, Carlsbad, California), and the size of the library is examined using an Agilent 2100 Bioanalyzer system. Follow-up steps are carried out according to the manufacturer's instructions to prepare the SMRTbell library. The library is sequenced using the PacBio Sequel platform. PacBio reads are assembled using Hifiasm / Canu. Then the Pilon software is used to re-correct the genome using the previous Illumina data. The Prodigal / Augustus gene-finding software has been used to find the coding genes. Transfer RNA (tRNA) is detected in the genome using the program tRNAscan-SE with default parameter settings. rRNA is identified using Barrnap. Other RNAs are identified through the rfam database. Coding genes are annotated using Diamond with the National Center for Biotechnology Information (NCBI) nr database. Then the functions of the genes are annotated through the GO (Gene Ontology) database, and the pathways are annotated using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. Proteins encoded by genes are phylogenetically classified using the COG (Clusters of Orthologous Groups) database. Protein sequences with E < 1e-5 are retrieved using Diamond in the CAZy database, Swiss_Prot database, Pfam database, CARD database, VFDB database, or DFVF database.

[0056] The genomic sequence of Lactiplantibacillus plantarum JN-7 was assembled and analyzed. The basic genomic information sequences are shown in Table 1. The genome length is 3,255,213 bp, with a total of 3,218 genes and 3,090 protein-coding genes. The Circos (version 0.69) software was used to create a genomic circular map showing gene, ncRNA, GC content, and repeat sequence information, as shown in Figure 1 .

[0057] Table 1 Basic genomic information of Lactiplantibacillus plantarum JN-7

[0058]

[0059]

[0060] 3. Identification of Lactiplantibacillus plantarum JN-7

[0061] The identification of Lactiplantibacillus plantarum JN-7 was carried out by calculating the Average Nucleotide Identity (ANI) between the JN-7 genomic sequence and the standard strain of Lactiplantibacillus plantarum SRCM100442 using an online ANI calculator ( http: / / enve- omics.ce.gatech.edu / ani / ). The ANI calculator uses the best hit (one-way ANI) and the mutual best hit (two-way ANI) between two genomic datasets to estimate the average nucleotide identity. By comparing JN-7 with the reference strain of Lactiplantibacillus plantarum, an ANI result of 99.21% was obtained, indicating that the JN-7 strain belongs to the species Lactiplantibacillus plantarum.

[0062] A genomic database of 1,215 Lactiplantibacillus plantarum strains (retrieved on February 20, 2024) selected from NCBI was established using the Makeblastdb software. The criteria for screening Lactiplantibacillus plantarum were complete genomes or genome drafts that had been uploaded to NCBI. The assembled JN-7 genome was compared with the Lactiplantibacillus plantarum genomic database by Blastn to generate the mismatched regions and the number of mismatched regions in the comparison entries. No reference that was completely identical or tolerated at most 1,000 mismatches compared to the assembly results was found, indicating that Lactiplantibacillus plantarum JN-7 is a strain that has never been reported in NCBI.

[0063] Example 2. Characterization of Lactiplantibacillus plantarum JN-7

[0064] Characteristics of Lactiplantibacillus plantarum JN-7

[0065] Lactiplantibacillus plantarum JN-7 was fermentatively cultured, and the following characteristics of the strain were determined

[0066] 1) Growth curve of Lactiplantibacillus plantarum JN-7

[0067] The growth curve of JN-7 was plotted at 37 °C using a Growth profiler 960 (Enzyscreen B.V., Heemstede, Netherlands). The specific operation was as follows: After culturing JN-7 in MRS liquid for 16 hours, it was inoculated into fresh MRS broth and the final OD 600 was adjusted to 0.1. The bacterial solution with adjusted OD was added to a sterile 96-well plate, ensuring that the addition amount per well was 250 μL, and a total of 11 groups of parallels (A1 - A11) were carried out. Under aerobic conditions, it was cultured at 200 rpm and 37 °C for 48 hours, and the OD value was collected every 30 minutes.

[0068] As Figure 2As shown, a typical quadratic curve was fitted by Growth Profiler 960. Lactobacillus plantarum JN-7 with an initial OD of 0.1 reached the exponential growth phase within 18 hours and was in the stationary phase within the next 48 hours when cultured at 37 °C and 200 rpm.

[0069] 2) Acid, bile salt and gastrointestinal tolerance

[0070] The fresh bacterial solution cultured in MRS broth for 16 h was centrifuged at 12,000 rpm and 4 °C for 2 minutes, and the precipitate was retained. The precipitate was washed twice with sterile PBS buffer and resuspended in sterile PBS (pH = 7), and the cell concentration was adjusted to 10 8 CFU / mL. This bacterial solution was inoculated into MRS (N0), MRS with pH 3.0 (N1) or MRS liquid medium containing 0.3% bile salt (w / v, Sigma, USA) (N1) at an addition amount of 5%. After being placed at 37 °C for 4 h, the viable cells of Lactobacillus plantarum JN-7 in different media were counted.

[0071] To detect the gastrointestinal tolerance of Lactobacillus plantarum JN-7, a fresh single colony was picked and inoculated into MRS broth and cultured at 37 °C for 16 h. 5 mL of the bacterial solution was centrifuged at 12,000 rpm and 4 °C for 2 minutes. The precipitate was washed with sterile PBS (pH = 7) and resuspended to a cell concentration of 10 7 CFU / mL. 100 μL of the bacterial suspension was added to 900 μL of simulated gastric juice, which was composed of 125 mM NaCl, 7 mM KCl, 45 mM NaHCO3 and 3 g / L pepsin (Sigma, USA), and the pH was adjusted to 3.0 with HCl. After incubation at 37 °C for 3 h, 250 μL of the gastric juice mixture was added to 6 mL of simulated intestinal juice, which was composed of 45 mM NaCl, 1 g / L trypsin (Sigma, USA) and 3 g / L bile salt (Sigma, USA), and the pH value was adjusted to 8.0 with NaOH, and then incubated at 37 °C for 3 h.

[0072] The survival rate of Lactobacillus plantarum JN-7 under acid, bile salt and gastrointestinal treatments was calculated according to the following formula:

[0073] Survival rate (%) = (N1 ÷ N0) × 100%

[0074] where N1 is the viable cell count after 6 h of co-treatment with MRS (pH 3.0, 0.3% bile salt) or gastrointestinal fluid; N0 is the viable cell count after 0 h of treatment with MRS and gastrointestinal fluid.

[0075] The results showed that the survival rates of Lactiplantibacillus plantarum JN-7 in MRS at pH 3.0 and MRS with 0.3% bile salts were 28.47% and 44.68%, respectively. The survival ability of JN-7 in gastrointestinal fluids was evaluated in simulated gastric fluid for 3 hours and then in simulated intestinal fluid for 3 hours. After 6 hours of treatment, the number of viable cells of JN-7 decreased to 12.32% of that before treatment.

[0076] 3) Sensitivity to antibiotics and MIC

[0077] Seven commonly used clinical antibiotics, gentamicin, ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin, and clindamycin, were used to analyze the antibiotic sensitivity of the strain. Antibiotic stock solutions of 256 mg / L were prepared and serially diluted two-fold. The OD of the fresh bacterial solution after 16 h of culture was diluted to 0.0002, and finally inoculated into the above different concentrations of antibiotic solutions at an inoculation amount of 1:1. After culturing at 37 °C for 24 h, the results were observed to determine the minimum inhibitory concentration (MIC), and the comparison and evaluation were carried out according to the regulations on bacterial antibiotic resistance formulated by the European Food Safety Authority (EFSA).

[0078] The MICs of ampicillin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, and chloramphenicol against Lactiplantibacillus plantarum JN-7 are shown in Table 2. The results showed that the MIC values of all 7 antibiotics were lower than the antibiotic critical values specified in the EFSA guidelines, indicating that the strain is not resistant to antibiotics and has antibiotic safety.

[0079] Table 2 MIC (mg / L) of different antibiotics against Lactiplantibacillus plantarum JN-7

[0080]

[0081] 4) Hemolytic activity

[0082] The absence of hemolytic activity and antibiotic resistance are considered safe prerequisites for the selection of probiotic strains (FAO / WHO, 2002).

[0083] Lactiplantibacillus plantarum JN-7 after two generations of activation was streaked on Columbia blood agar medium containing 5% defibrinated sheep blood (3400071, Haibo Co., China) and cultured at 37 °C for 48 hours. If a greenish hemolytic zone appears, it is α-hemolysis; if a colorless and transparent hemolytic zone appears, it is β-hemolysis; if there is no hemolytic zone, it is γ-hemolysis.

[0084] The results are as Figure 3 shown. After culturing Lactiplantibacillus plantarum JN-7 on blood agar, no hemolytic zone appeared around the colonies, which was γ-hemolysis. This indicates that Lactiplantibacillus plantarum JN-7 has no hemolytic ability.

[0085] 5) Production of D-lactic acid, L-lactic acid and analysis of the L-lactic acid / D-lactic acid ratio

[0086] Pick fresh single colonies and culture them in MRS broth at 37 °C for 18 hours. Use a commercially available D,L-lactic acid quantification kit (Jingmei Co., China) to analyze the D,L-lactic acid in the culture supernatant by an enzymatic method according to the manufacturer's protocol.

[0087] After culturing the strain in MRS broth at 37 °C for 18 hours, the D-lactic acid and L-lactic acid produced by Lactiplantibacillus plantarum JN-7 were analyzed. During the fermentation of JN-7, 1.2 nmol / L D-lactic acid and 4.28 nmol / L L-lactic acid were produced. The L-lactic acid / D-lactic acid ratio was 3.57, as shown in Table 3.

[0088] Table 3 Contents of D-lactic acid, L-lactic acid and L-lactic acid / D-lactic acid ratio

[0089]

[0090] 6) Biogenic amine detection

[0091] Culture the strain overnight in MRS liquid medium, and add it to MRS liquid medium containing 0.1 g / L histidine, tyrosine, ornithine, lysine and 0.05 g / L pyridoxal-5-phosphate to a final concentration of OD 0.01 and passage it, once every 24 h for a total of five times. The above liquid medium was inoculated into MRS medium containing 1% histamine, tyrosine, cadaverine and putrescine at an inoculation amount of 2% and cultured for 72 h, and the color change of the medium was observed. If the medium color turns purple, it is positive; if the medium turns yellow, it is negative. Lactobacillus rhamnosus LGG is used as a negative control, and Escherichia coli is used as a positive control.

[0092] The results showed that JN-7 was yellow after growing in the four biogenic amine detection media for three days, indicating that the strain does not produce putrescine, cadaverine, histamine and tyramine.

[0093] 7) API 50CH sugar fermentation experiment

[0094] Centrifuge the freshly cultured bacterial solution for 2 min at 12,000 rpm after 16 h of cultivation. Discard the supernatant, wash the precipitate twice with sterile PBS, and then resuspend it in sterile PBS. Adjust the OD to 0.3 to obtain bacterial solution a. Take 1 mL of bacterial solution a and add it to 10 mL of 50CHL medium (50410, API, France) to obtain bacterial solution b. Add 150 μL of bacterial solution b to the test holes of a 50CH strip (50300, API, France) and incubate at 37 °C for 48 h. Determine the results by color change. Compared with the control group, yellow color indicates a positive result (where esculin positive is purple), and no change is recorded as a negative result.

[0095] The results showed that Lactiplantibacillus plantarum JN-7 could ferment D-ribose (5), D-galactose (10), D-glucose (11), D-fructose (12), D-mannitol (13), mannitol (18), sorbitol (19), methyl-α-D-mannopyranoside (20), N-acetylglucosamine (22), amygdalin (23), arbutin (24), esculin (25), salicin (26), D-cellobiose (27), D-maltose (28), D-lactose (29), D-melibiose (30), D-sucrose (31), D-trehalose (32), D-melezitose (34), D-raffinose (35), D-gentiobiose (39), D-talose (40), D-tagatose (42), and potassium gluconate (47) to produce acid.

[0096] Example 3. Cytotoxicity and adhesiveness of Lactiplantibacillus plantarum JN-7

[0097] Experimental method:

[0098] Cultivation of HT-29 cells

[0099] HT-29 is a human intestinal epithelial cell line used for testing the cytotoxicity and adhesiveness of bacteria. HT-29 is cultured in RPMI 1640 medium containing 10% FBS and 1% P / S (penicillin / streptomycin). Incubate in an incubator with 5% CO2 at 37 °C for about 36 h - 72 h until the cell density reaches about 90%. Digest the cells with trypsin and passage them for seeding.

[0100] Cell viability detection

[0101] CCK-8 is a general method for detecting cell proliferation and toxicity. In this experiment, a CCK-8 kit (C0037, Beyotime) was used to detect the proliferation activity of HT-29 cells. Seed the cells at an appropriate density into a 96-well plate, add 1 / 10 of the total volume of CCK-8 solution, incubate in the dark, incubate at 37 °C for 2 h, remove the bubbles, and measure the OD value at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0102] Adhesion detection

[0103] Inoculate HT-29 cells from the culture flask into a 24-well plate at a concentration of 5×10 5 cells / mL, change to a medium without antibiotics for culture, and the experiment can be carried out after the cells are completely adherent. Before adding bacteria, wash the cells in the well plate twice with sterile PBS, and then add 500 μL of bacteria with a concentration of 10 8 CFU / mL (V0) to each well. Transfer the 24-well plate to a 5% CO2 incubator at 37 °C for 4 h to allow adhesion. Wash the cells in each well 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, and then collect the solution in each well for serial dilution and counting (V1). The calculation method of the adhesion rate (%) is as follows:

[0104] Adhesion rate (%) = (V1 / V2) × 100%

[0105] To evaluate the potential cytotoxic effect of Lactiplantibacillus plantarum JN-7 on HT-29 intestinal epithelial cells, a CCK-8 assay was performed after co-culturing with Lactiplantibacillus plantarum JN-7 bacterial cells for 18 h. The multiplicity of infection (MOI) of this test was approximately 1:200. As shown in a of Figure 4 , Lactiplantibacillus plantarum JN-7 had no effect on the survival of intestinal epithelial cells at this MOI. The adhesion ability of Lactiplantibacillus plantarum JN-7 was compared with that of the commercial probiotic Lactobacillus rhamnosus (LGG) ( Figure 4 b). The results showed that the efficiency of LGG adhering to cells was twice that of JN-7.

[0106] Example 4. Anti-inflammatory effect of Lactiplantibacillus plantarum JN-7

[0107] Experimental method:

[0108] Culture and treatment of THP-1

[0109] THP-1 is a human monocytic leukemia cell line that can be induced to differentiate into macrophages by phorbol 12-myristate 13-acetate (PMA) and is used to detect the anti-inflammatory effect of bacteria. THP-1 is cultured in RPMI 1640 medium containing 10% FBS and 1% P / S. It is cultured in an incubator with 5% CO2 at 37 °C. When the cell density reaches 8-10×10 5 cells / mL, subculture is carried out. The normal state of the cells is suspension growth, and semi-medium subculture can be carried out.

[0110] Inoculate THP-1 cells at 5×10 5Cells were seeded at a density of

[0111] Quantitative real-time PCR (qRT-PCR)

[0112] Total mRNA was isolated from cells using Trizol reagent (R701-01-AA, Novoprotein) according to the manufacturer's instructions. Reverse transcription (RT-PCR) was performed using HiScript II Select qRT SuperMix (R222-01, Novoprotein) in a final volume of 20 μL containing 1 μg of RNA. qRT-PCR was carried out on a quantitative real-time PCR (qRT-PCR) detection system using gene-specific primers and SYBR Green (Q712-02, Novoprotein). All primers were designed by ourselves and synthesized by Genewiz Biotechnology Co., Ltd. (Beijing, China). β-actin was used as a reference gene, and data analysis was performed using the 2-ΔΔt method.

[0113] The specific operation steps are as follows:

[0114] RNA extraction: (1) Cells were collected in a 1.5 mL enzyme-free EP tube, and 200 μL of Trizol reagent was added to lyse the cells thoroughly by homogenization. After incubation at room temperature for 5 min, the mixture was centrifuged at 12,000 rpm for 5 min at 4°C. (2) The supernatant was transferred to a new EP tube, and 4 / 5 volume of isopropanol was added. The mixture was shaken up and down 10 times to mix well and then left to stand at room temperature for 10 min. (3) The mixture was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was discarded. A white feathery precipitate was the RNA, which was washed twice with 800 μL of DEPC water containing 75% ethanol. The supernatant was discarded, and the precipitate was air-dried. The precipitate was dissolved in 20 μL of DEPC water on ice to obtain RNA. (4) The concentration of RNA was measured using a NanoDrop micro-spectrophotometer.

[0115] Synthesis of cDNA by RT-PCR: cDNA was synthesized using the RT-PCR kit from Novoprotein (HiScript II Q RT SuperMix for qPCR).

[0116] Table 4 Preparation of the first-strand cDNA synthesis reaction mixture

[0117]

[0118] Mix gently and centrifuge at 500 rpm for 1 min to the bottom.

[0119] Table 5 The first-strand cDNA synthesis reaction was carried out under the following conditions

[0120]

[0121] The obtained 20 μL product can be immediately used for qRT-PCR reaction, or stored at -20 °C (usable within half a year); for long-term storage, it is recommended to aliquot and store at -80 °C and avoid repeated freezing and thawing.

[0122] qRT-PCR:

[0123] Table 6 The total 20 μL reaction system is as follows:

[0124]

[0125] Flick to mix well and centrifuge the liquid to the bottom, and amplify on a real-time fluorescence quantitative PCR instrument.

[0126] The reaction conditions were 94 °C for 30 s; 94 °C for 5 s; 60 °C for 30 s, repeated for 40 cycles. Bio-Rad CFXManager Software automatically recorded the amplification data and melting curve, etc. The amplification data was represented by threshold cycle (CT value), and the 2-ΔΔt method was used for data analysis. The primer sequences (SEQ ID NO: 4-11) are shown in the following table.

[0127] Table 7 Primer sequences

[0128]

[0129]

[0130] Anti-inflammatory effect of JN-7 on LPS-stimulated PMA-differentiated THP-1 cells

[0131] JN-7 downregulates the expression of pro-inflammatory cytokine mRNA in LPS-stimulated cells (such as Figure 5 ). JN-7 and LGG were co-cultured with macrophages differentiated from THP-1 respectively, and the anti-inflammatory effects of the strains were evaluated by detecting the expression levels of pro-inflammatory cytokines IL-6, IL-8 and anti-inflammatory cytokine IL-10 mRNA. Compared with the LPS-stimulated control group, the expression of IL-6 and IL-8 mRNA in cells containing JN-7 and LGG decreased after LPS stimulation, while the expression of IL-10 mRNA increased. In addition, LGG had a better downregulating effect on the expression of LPS-stimulated IL-8 mRNA than JN-7. The ability of JN-7 to promote IL-10 release was higher than that of LGG, suggesting that JN-7 has the same anti-inflammatory effect as LGG.

[0132] Example 5. Production and Stability Analysis of Lactiplantibacillus plantarum JN-7 Bacterial Powder

[0133] 1) Fermentation of JN-7 and Production of Bacterial Powder

[0134] Inoculate Lactiplantibacillus plantarum JN-7 into MRS broth and culture it at 37 °C for 18 h. Activate it twice and culture it in a 5 L bioreactor ( 320, eppendof) for 12 hours. After the fermentation is completed, collect the bacterial cells at 4 °C, 10000 rpm for 10 min. The obtained bacterial cells are mixed with a cryoprotectant (1 - 10 g / L polysaccharide, 20 - 50 g / L disaccharide, 1 - 20 g / L vitamin C or its salts, and 1 - 10 g / L peptone), and pre-frozen at -80 °C for 24 hours. The bacterial powder is freeze-dried by a vacuum freeze dryer (PO14416, Telstar LyoQuest-55plus), pulverized into powder, and then vacuum-packed in an aluminum foil bag.

[0135] After centrifugation after 12 hours of fermentation in a 5 L bioreactor, a total of 41.5 g of precipitate, Lactiplantibacillus plantarum, was collected. The precipitate was mixed with the cryoprotectant, and the mixture was freeze-dried to produce 6.6 g of bacterial powder with a viable cell count of 7.07×10 11 CFU / g.

[0136] 2) Evaluation of the Stability of Lactiplantibacillus plantarum JN-7 Bacterial Powder

[0137] The packaged Lactiplantibacillus plantarum JN-7 bacterial powder was placed in an aluminum foil bag and stored in a constant humidity incubator at 40 °C and 75% humidity for an accelerated test. At the same time, a commercially available Lactobacillus rhamnosus GG bacterial powder and 15 strains of Lactiplantibacillus plantarum isolated from fermented vegetables (the production process of the bacterial powder is the same as that of JN-7) were stored under the same temperature and humidity as controls. Samples of JN-7 bacterial powder, LGG bacterial powder, and 15 strains of Lactiplantibacillus plantarum bacterial powder were taken within 90 days, and the number of lactic acid bacteria in the bacterial powder was detected according to the national standard GB 4789.35, and the water activity of the bacterial powder was measured.

[0138] During storage, Lactiplantibacillus plantarum JN-7 ( Figure 6A ), Lactobacillus rhamnosus GG ( Figure 6B ), and 15 other strains of Lactiplantibacillus plantarum bacterial powder ( Figure 6C ), the viable cell counts and water activities are shown in Figures 6A - 6C . It can be seen from the figure that the stability of JN-7 bacterial powder is significantly higher than that of the other 16 kinds of bacterial powders. The viable cells of JN-7 decreased from 7.07×10 11 to 1.73×10 11 CFU / g within 70 days, and the corresponding water activity increased from 0.024 to 0.053. The viable cells of the control group LGG decreased from 3.53×1011 Reduced to 3.92×10 8 CFU / g, and the corresponding water activity increased from 0.091 to 0.134. Through 70 days of testing, a linear fit (r 2 = 0.80) was performed on the reduction of viable JN-7 bacteria. Through fitting, it can be predicted that under the conditions of accelerated stability test, the time required for the viable bacteria count to decrease by 1 log is 136.99 days, which is 112.36 days longer than the 24.63 days required for the predicted LGG by fitting.

[0139] Generally speaking, a Lactiplantibacillus plantarum JN-7 was isolated and identified from natural fermentation products in this application. After whole-genome sequencing and comparison with all Lactiplantibacillus plantarum genomes retrievable from NCBI, we found that the strain Lactiplantibacillus plantarum JN-7 is a new strain. Through the analysis of the basic characteristics of this strain (growth curve, acid resistance, bile salt resistance, gastrointestinal fluid resistance), safety evaluation (antibiotic sensitivity, hemolytic ability, toxin production ability, cytotoxicity), adhesion evaluation, anti-inflammatory effect evaluation, production performance evaluation (fermentation yield and freeze-drying yield), and stability test (accelerated storage stability test), it was found that Lactiplantibacillus plantarum JN-7 is a safe strain with excellent production performance, high stability, and anti-inflammatory efficacy.

Claims

1. A Lactiplantibacillus plantarum JN-7, wherein, The taxonomic name of the Lactiplantibacillus plantarum JN-7 is Lactiplantibacillus plantarum, and its deposit number is CGMCC No. 30605.

2. The bacterial powder prepared from the Lactiplantibacillus plantarum JN-7 described in claim 1.

3. The preparation method of the bacterial powder described in claim 2, comprising the following steps: 1) Fermenting the Lactiplantibacillus plantarum JN-7; 2) Centrifuging and precipitating the fermentation product, and freeze-drying to obtain the product.

4. A probiotic composition comprising the Lactiplantibacillus plantarum JN-7 described in claim 1 or the bacterial powder described in claim 2.

5. The probiotic composition according to claim 4, wherein, The probiotic composition further comprises one or more probiotics selected from the following: Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus mucosae, Lactiplantibacillus, Lactobacillus collinoides, Lactobacillus paraplantarum, Streptococcus, Lactococcus, Propionibacterium, Propionibacterium, Weissella, Aerococcus, Staphylococcus, Kluyveromyces, Leuconostoc, Pediococcus, and Bacillus subtilis DE111.

6. The use of the Lactiplantibacillus plantarum JN-7 described in claim 1, the bacterial powder described in claim 2, or the probiotic composition described in claim 4 or 5 in the preparation of anti-inflammatory drugs.

7. The use according to claim 6, wherein The drug is administered orally.

8. The use of the Lactiplantibacillus plantarum JN-7 described in claim 1, the bacterial powder described in claim 2, or the probiotic composition described in claim 4 or 5 in the preparation of foods or health products.

9. The use according to claim 8, wherein, The food or health product is dairy products, soy products, meat products, fruit and vegetable products, beverages or snacks.

10. The use according to claim 8, wherein The food or health product further comprises food additives.

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