Lactiplantibacillus plantarum JN-7, bacterial powder, preparation method, probiotic composition and use thereof

Abstract

Lactiplantibacillus plantarum JN-7, a bacterial powder, a preparation method, a probiotic composition and the use thereof. According to the present application, a new strain of Lactiplantibacillus plantarum named Lactiplantibacillus plantarum JN-7 with the deposit number of CGMCC No. 30605 is obtained by means of screening. By means of basic characteristic analysis (growth curve, acid resistance, bile salt resistance, and gastrointestinal fluid resistance), safety evaluation (antibiotic sensitivity, hemolytic capacity, toxigenic capacity, and cytotoxicity), adhesion evaluation, anti-inflammatory effect evaluation, production performance evaluation (fermentation yield and lyophilization yield), and stability tests (an accelerated storage stability test) of this strain, it is found that Lactiplantibacillus plantarum JN-7 is a safe strain with an excellent production performance, high stability, and an anti-inflammatory efficacy. The bacterial powder produced therefrom can remain stable and active for a long time.

Description

Lactiplantibacillus plantarum JN-7, bacterial powder, preparation method, probiotic composition and use thereof

[0001] This application claims priority to the Chinese patent application No. 202410775747.8, filed on June 14, 2024, and entitled “Lactiplantibacillus plantarum JN-7, bacterial powder, preparation method, probiotic composition and use thereof”, the content of which is to be understood as incorporated herein by reference. TECHNICAL FIELD

[0002] The present disclosure generally relates to the technical field of biological applications, and in particular to a Lactiplantibacillus plantarum JN-7, bacterial powder, preparation method, probiotic composition and use thereof. BACKGROUND

[0003] Probiotics are a class of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the microbial community in a certain part of the host. Probiotics can promote the absorption of nutrients and maintain intestinal health by regulating the immune function of the host's mucosa and systemic immune function or by regulating the balance of intestinal flora, thereby producing single microorganisms or mixed microorganisms with clear composition that are beneficial to health.

[0004] Lactiplantibacillus plantarum is a common probiotic that is widely used in fermented dairy products, meat products, plant products and baked foods. Its main role is to produce lactic acid, reduce pH, prevent the proliferation of spoilage bacteria, and improve product flavor and texture. In recent years, research has found that Lactiplantibacillus plantarum has good probiotic function, can inhibit gastrointestinal pathogenic bacteria including Helicobacter pylori, improve gastrointestinal function and metabolism, enhance intestinal barrier function, and reduce the risk of infection, etc.

[0005] The stability and activity of probiotics in food are closely related to the health benefits that probiotics can achieve, and there is an urgent need to develop probiotics with good stability and long-term preservation. SUMMARY

[0006] The present disclosure provides a Lactiplantibacillus plantarum JN-7, wherein the Lactiplantibacillus plantarum JN-7 is classified as Lactiplantibacillus plantarum, and the preservation number is CGMCC No. 30605.

[0007] In another aspect, the present disclosure also provides a bacterial powder prepared from the Lactiplantibacillus plantarum JN-7 described herein.

[0008] In another aspect, the present disclosure also provides a preparation method of the bacterial powder described herein, comprising the following steps:

[0009] 1) fermenting the Lactiplantibacillus plantarum JN-7 described herein;

[0010] 2) centrifuging the fermentation product to precipitate, freeze-drying to obtain the bacterial powder.

[0011] In another aspect, the present application also provides a probiotic composition comprising the Lactiplantibacillus plantarum JN-7 described herein or the bacterial powder described herein.

[0012] In another aspect, the present application also provides the use of the Lactiplantibacillus plantarum JN-7 described herein, the bacterial powder described herein or the probiotic composition described herein in the preparation of an anti-inflammatory drug.

[0013] In another aspect, the present application also provides the use of the Lactiplantibacillus plantarum JN-7 described herein, the bacterial powder described herein or the probiotic composition described herein in the preparation of a food or health product.

[0014] The number of viable cells surviving in the probiotic powder is 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, thereby helping to improve the quality of the probiotic powder and reduce the cost.

[0015] The present application provides a Lactiplantibacillus plantarum with good yield, stability, safety, acid, bile salt and gastrointestinal tolerance, and antibiotic sensitivity, which is named Lactiplantibacillus plantarum JN-7. It is preserved in the China General Microbiological Culture Collection Center, located at No. 3, Yuanmingyuan Road, Beijing, China, on May 13, 2024. The preservation number is CGMCC No. 30605.

[0016] Through basic property analysis (growth curve, acid tolerance, bile salt tolerance, gastrointestinal fluid tolerance) of the strain, 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 is found that the Lactiplantibacillus plantarum JN-7 is a safe strain with excellent production performance, high stability and anti-inflammatory efficacy. The bacterial powder produced therefrom can remain stable and active for a long time.

[0017] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the present application. Other advantages of the present application can be realized and obtained by means of the solutions described in the specification and the attached drawings. SUMMARY

[0018] The accompanying drawings are used to provide an understanding of the technical solutions of the present application, constitute a part of the specification, and are used together with the embodiments of the present application to explain the technical solutions of the present application, and do not constitute a limitation on the technical solutions of the present application.

[0019] Figure 1 is a circular map of the genome of Lactiplantibacillus plantarum JN-7 in the embodiments of the present application. The circular map shows seven kinds of information from outside to 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 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 fragment repeat sequence information on the genome (marked in orange).

[0020] Figure 2 is a 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.

[0021] Figure 3 is the hemolytic ability results of Lactiplantibacillus plantarum JN-7 in the embodiments of the present application.

[0022] Figure 4 shows the interaction of Lactiplantibacillus plantarum JN-7 with intestinal epithelial cells HT-29 in the embodiments of the present application. Wherein, a: Lactiplantibacillus plantarum JN-7 does not reduce the viability of HT-29 cells. Data represent the survival rate of cells after 18 hours of co-incubation with Lactiplantibacillus plantarum JN-7 and cells in pure medium (n=6). b: Adhesion ratio of Lactiplantobacillus rhamnosus LGG and Lactiplantibacillus plantarum JN-7 to HT-29 cells. Values represent the adhesion rate of LGG and Lactiplantibacillus plantarum JN-7 after 4 hours of incubation with HT-29. Data are expressed as Mean ± Sem, *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 5 shows the anti-inflammatory ability of Lactiplantibacillus plantarum JN-7 to regulate the production of inflammatory factors by LPS-stimulated THP-1 cells in the embodiments of the present application. Wherein, a: interleukin 6 (IL)-6 mRNA; b: IL-8 mRNA; c: IL-10 mRNA. Data are expressed as Mean ± Sem, *P<0.05, **P<0.01, ***P<0.001.

[0024] Figures 6A-6D are the viable cell counts and water activity of Lactiplantibacillus plantarum JN-7 powder (Figure 6A), Lactiplantobacillus rhamnosus LGG (Figure 6B) and other 15 strains of Lactiplantibacillus plantarum powder (Figures 6C-6D) during storage at 40°C and 75% humidity in the embodiments of the present application. Details

[0025] Unless otherwise indicated, the technical and scientific terms used herein have the same meaning as those which are commonly understood by one of ordinary skill in the art to which this application belongs. When a range, preferably a range, or a preferred upper value, and a preferred lower value are stated, it is intended to literally incorporate all ranges of values lying between the upper value and the lower value, and all individual values comprising the range, whether specifically stated or not. Unless otherwise specified, numerical ranges expressed in the format "from x to y" are understood to include x and y. When for a specific feature multiple preferred ranges are described in the format "from x, to y", it is understood that all ranges combining the different endpoints are also contemplated.

[0026] The terms "about" and "approximately" when used in connection with a numerical value, generally mean that the recited numerical value and all numerical values within experimental error (e.g., within 95% of the mean) or within ±10% of the stated numerical value, or within a broader range.

[0027] The expressions "comprising" or "comprise", and grammatical variants thereof, are used inclusively and not exclusivity, that is, "comprising A or B" mean A or B as well as A and B. The expressions "consisting of" and grammatical variants thereof, are used exclusivity, that is, "consisting of A" means A only and not A nor B nor the inclusion of any element not specified. The expression "consisting essentially of" and grammatical variants thereof, means the specified elements, plus an optional additional element or elements that do not materially affect the basic and novel characteristics of the claimed subject matter. It is understood that the expression "comprising" encompasses the expressions "consisting of" and "consisting essentially of".

[0028] The expressions "at least one" or "one or more" mean one, two, three, four, five, six, seven, eight, nine, or more.

[0029] Most of the researches on improving the stability of functional strains are achieved by the development of bacterial powder protectants. Due to the inherent stability of each strain, the protectants have limited effect on stabilizing the probiotic powder. Developing a strain with good stability for the complex of commercial probiotic powder is another way to solve the problem of low bacterial activity of probiotic products within the shelf life.

[0030] Therefore, the application provides a Lactiplantibacillus plantarum JN-7, wherein the Lactiplantibacillus plantarum JN-7 is named as Lactiplantibacillus plantarum, and the preservation number is CGMCC No.30605.

[0031] In another aspect, the application also provides a bacterial powder prepared from the Lactiplantibacillus plantarum JN-7 described herein.

[0032] In another aspect, the application also provides a preparation method of the bacterial powder described herein, comprising the following steps:

[0033] 1) fermenting the Lactiplantibacillus plantarum JN-7 described herein;

[0034] 2) centrifuging the fermentation product to precipitate, freeze-drying, to obtain the bacterial powder.

[0035] In some embodiments, the method of preparing the bacterial powder described herein comprises the following steps:

[0036] 1) fermenting the Lactiplantibacillus plantarum JN-7 described herein in a bioreactor, pH 4.5-5.3, temperature 35-40°C, agitator speed 160-200 rpm;

[0037] 2) centrifuging the fermentation product to precipitate, mixing the precipitate with a protective agent, freeze-drying in a vacuum freeze-dryer and pulverizing into powder, to obtain the bacterial powder.

[0038] In another aspect, the present application also provides a probiotic composition comprising the Lactiplantibacillus plantarum JN-7 described herein or the bacterial powder described herein.

[0039] In some embodiments, the probiotic composition further comprises one or more probiotics selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus, Ligilactobacillus, Latilactobacillus, Streptococcus, Lactococcus, Propionibacterium, Acidipropionibacterium, Weizmannia, Mammaliicoccus, Staphylococcus, Kluyveromyces, Leuconostoc, Pediococcus and Bacillus subtilis DE111.

[0040] In some embodiments, the probiotic composition further comprises one or more probiotic bacteria 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 kefiranofaciens subsp. kefiranofaciens, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactococcus lactis, Pediococcus acidilactici, Pediococcus pentosaceus, Streptococcus salivarius, and combinations thereof.Lactobacillus kefiranofaciens, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus sakei, Streptococcus salivarius subsp. thermophilus, Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. lactis biovar. The following bacteria are listed: *Lactococcus cremoris*, *Propionibacterium freudenreichii* subsp. *shermanii*, *Acidipropionibacterium acidipropionici*, *Leuconostoc mesenteroides* subsp. *mesenteroides*, *Pediococcus acidilactici*, *Pediococcus pentosaceus*, *Weizmannia coagulans*, *Mammaliicoccus vitulinus*, *Staphylococcus xylosus*, *Staphylococcus carnosus*, *Kluyveromyces marxianus*, and *Bacillus subtilis* DE111. .

[0041] In another aspect, the present application also provides use of the Lactobacillus plantarum JN-7 described herein, the bacterial powder described herein, or the probiotic composition described herein in the preparation of an anti-inflammatory medicament. In some embodiments, the medicament is administered orally.

[0042] In another aspect, the present application also provides use of the Lactobacillus plantarum JN-7 described herein, the bacterial powder described herein, or the probiotic composition described herein in the preparation of a food or a health product.

[0043] In some embodiments, the food or health product is a dairy product, a legume product, a meat product, a fruit / vegetable product, a beverage, or a snack. In some embodiments, the food or health product further comprises a food additive.

[0044] In some embodiments, the food is a health food; or the food comprises a dairy product, a legume product, a meat product, or a fruit / vegetable product; or the food is a beverage or a snack. In some embodiments, the food comprises the Lactobacillus plantarum JN-7 described herein and a food additive.

[0045] The present application describes multiple embodiments, but the description is exemplary rather than limiting, and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the embodiments described in the present application. Although many combinations of possible features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are possible. Unless specifically limited, any feature of any embodiment can be utilized with any other feature of any other embodiment, or can be replaced by any other feature.

[0046] 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 can also be combined with any conventional features to form unique inventive schemes defined by the claims. Any feature of any embodiment can also be combined with features from other inventive schemes to form another unique inventive scheme 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 appropriate combination. Accordingly, the embodiments are not to be restricted, except as by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0047] Furthermore, in describing representative embodiments, the specification can have presented the method and / or process as a particular sequence of steps. However, to the extent that the method or process depends on the performance of such steps, the method or process should not be limited to the

[0048] The experimental methods in the following examples, unless otherwise specified, were generally in accordance with the national standards. The experimental materials in the following examples, unless otherwise specified, were all commercially available raw materials. The equipment used in each step in the following examples was conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standard, the conventional condition, or according to the condition suggested by the manufacturer. Unless otherwise defined or explained, 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 equivalent to those described can be applied to the method of the present application.

[0049] Example 1. Obtaining and identification of Lactiplantibacillus plantarum JN-7

[0050] The Lactiplantibacillus plantarum JN-7 provided in the present application is isolated from self-pickled pickles in Chengdu, Sichuan.

[0051] 1. 16s sequencing of Lactiplantibacillus plantarum JN-7

[0052] The JN-7 bacterial genomic DNA was extracted using Takara Mini-BEST Bacterial Genomic DNA Extraction Kit version 3.0. The 16S rRNA gene was amplified by universal primers 27F: 5'- AGAGTTTTGATCCTGTCCAG-3' (SEQ ID NO: 1) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2). The PCR cycle was: initial activation at 94°C for 2 min; the cycle steps were: denaturation at 94°C for 30 s; annealing at 55°C for 1 min; extension at 72°C for 1 min; and finally at 72°C for 10 min. The PCR product was purified for first-generation sequencing, and the measured 16s rRNA sequence is shown as SEQ ID NO: 3.

[0053] After Blast comparison of Lactiplantibacillus plantarum on NCBI website, it was found that it had 99.93% similarity with the model strain Lactiplantibacillus plantarum SRCM100442. Combined with the physiological and biochemical indexes and molecular biology identification results of strain JN-7, and its morphological characteristics, we named it as Lactiplantibacillus plantarum JN-7, which was preserved in China General Microbiological Culture Collection Center on May 13, 2024, and its preservation number was CGMCC No. 30605.

[0054] 2. Whole genome sequencing of Lactiplantibacillus plantarum JN-7

[0055] To perform genome sequencing, Lactiplantibacillus plantarum JN-7 was cultured in MRS broth under anaerobic conditions at 37°C for 24 hours. Single colonies were cultured in MRS broth overnight. The broth was centrifuged at 8000 x g, 4°C, 5 min. The precipitate was sent to GENEWIZ sequencing company (China) for sequencing, assembly, annotation and bioinformatics analysis.

[0056] Whole genome sequencing was performed on the Illumina 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, using the PacBio Sequel System, the DNA fragments were sequenced to generate 20,000 reads, each with an average length of 10,000 bases. Express Template Preparation Kit 2.0 to build the library. Briefly, DNA sheared fragments were subjected to single-strand overhang removal, DNA damage repair, end repair, A-tailing, and barcoded overhang adapter ligation. The library was quantified using Qubit 3.0 fluorometer (Invitrogen, Carlsbad, CA) and checked for size using Agilent 2100 Bioanalyzer system. The subsequent steps were followed to prepare the SMRTbell library according to the manufacturer’s instructions. The library was sequenced using the PacBio Sequel platform. PacBio reads were assembled using Hifiasm / Canu. The genome was then re-corrected using previous Illumina data using Pilon software. Prodigal / Augustus gene finder software was used to find the coding genes. Transfer RNAs (tRNAs) were detected in the genome using the program tRNAscan-SE and default parameter settings. rRNAs were identified by using Barrnap. Other RNAs were identified by the rfam database. Coding genes were annotated by Diamond using the National Center for Biotechnology Information (NCBI) nr database. The genes were then annotated for their functions by the GO (Gene Ontology) database and pathways by the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. The proteins encoded by the genes were phylogenetically classified by the COG (Clusters of Orthologous Groups) database. Protein sequences with E<1e-5 were retrieved in the CAZy database, Swiss_Prot database, Pfam database, CARD database, VFDB database, or DFVF database using Diamond.

[0057] The genome sequence of P. lactis JN-7 was assembled and analyzed. The basic genomic information sequence is shown in Table 1. The genome length is 3255213 bp, and there are 3218 genes, 3090 protein-coding genes. The Circos (version 0.69) software was used to create a genome circle diagram showing the information of genes, ncRNAs, GC content, and repeat sequences, as shown in Figure 1.

[0058] Table 1 Basic genomic information of P. lactis JN-7

[0059] 3. Identification of P. lactis JN-7

[0060] The identification of Lactiplantibacillus plantarum JN-7 was performed by calculating the Average Nucleotide Identity (ANI) between the JN-7 genome sequence and the Lactiplantibacillus plantarum LMG 100442 standard strain using the online ANI calculator (http: / / enve-omics.ce.gatech.edu / ani / ). The ANI calculator uses the best hit between two genome datasets (one-way ANI) and the reciprocal best hit (two-way ANI) to estimate the average nucleotide identity. By comparing JN-7 and the Lactiplantibacillus plantarum reference strain, an ANI result of 99.21% was obtained, indicating that the JN-7 strain belongs to the Lactiplantibacillus plantarum species.

[0061] A database of 1215 (retrieved on 2024.2.20) Lactiplantibacillus plantarum genomes selected from NCBI was created using the Makeblastdb software. The criteria for Lactiplantibacillus plantarum selection was a complete genome or a genome draft already uploaded to NCBI. The assembled JN-7 genome was compared to the Lactiplantibacillus plantarum genome database by Blastn, generating the number of mismatches and the number of non-matching regions in the comparison entry. No reference was found that was completely identical to the assembly result or allowed up to 1000 mismatches, indicating that Lactiplantibacillus plantarum JN-7 is a strain never reported in NCBI.

[0062] Example 2. Characterization of Lactiplantibacillus plantarum JN-7

[0063] Characteristics of Lactiplantibacillus plantarum JN-7

[0064] Lactiplantibacillus plantarum JN-7 was subjected to fermentation and the following characteristics of the strain were determined

[0065] 1) Growth curve of Lactiplantibacillus plantarum JN-7

[0066] The growth curve of JN-7 was plotted using Growth profiler 960 (Enzyscreen B.V., Heemstede, Netherlands) at a temperature of 37°C. The procedure was as follows: JN-7 was inoculated in fresh MRS broth after 16 hours of MRS liquid culture and the final OD 600 was adjusted to 0.1. The adjusted OD bacterial solution was added to a sterile 96-well plate, ensuring that 250 μL was added per well, and a total of 11 groups of parallels (A1-A11) were performed. The culture was performed under aerobic conditions at 200 rpm and 37°C for 48 hours, and the OD value was collected every 30 minutes.

[0067] As shown in Figure 2, a typical quadratic curve was fitted by Growth Profiler 960. Lactobacillus plantarum JN-7 with initial OD of 0.1 reached exponential growth phase within 18 hours and entered stationary phase within the following 48 hours under the condition of 37°C, 200 rpm.

[0068] 2) Acid, bile salt and gastrointestinal tolerance

[0069] Fresh bacterial suspension cultured in MRS broth for 16 hours was centrifuged at 12000 rpm, 4°C for 2 minutes, and the precipitate was collected. The precipitate was washed twice with sterile PBS buffer, resuspended in sterile PBS (pH = 7) and adjusted to a bacterial concentration of 10 8 CFU / mL. The bacterial suspension was inoculated into MRS (N0), MRS (N1) with pH 3.0 or MRS (N1) containing 0.3% bile salt (w / v, Sigma, USA) at an addition amount of 5%. After 4 hours at 37°C, the viable cells of Lactobacillus plantarum JN-7 in different media were counted.

[0070] To detect the gastrointestinal tolerance of Lactobacillus plantarum JN-7, fresh single colony was inoculated into MRS broth and cultured at 37°C for 16 hours. 5 mL of bacterial suspension was centrifuged at 12000 rpm, 4°C for 2 minutes. The precipitate was washed with sterile PBS (pH = 7) and resuspended to a bacterial concentration of 10 7 CFU / mL. 100 μL of bacterial suspension was added to 900 μL of simulated gastric juice consisting 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 3 hours of incubation at 37°C, 250 μL of gastric juice mixture was added to 6 mL of simulated intestinal juice consisting of 45 mM NaCl, 1 g / L trypsin (Sigma, USA) and 3 g / L bile salt (Sigma, USA), and the pH was adjusted to 8.0 with NaOH, followed by 3 hours of incubation at 37°C.

[0071] The survival rate of Lactobacillus plantarum JN-7 under acid, bile salt and gastrointestinal treatment was calculated as follows:

[0072] Survival rate (%) = (N1 ÷ N0) x 100%

[0073] Wherein, N1 is the viable cell count after 6 hours of co-treatment with MRS (pH 3.0, 0.3% bile salt) or gastrointestinal juice; N0 is the viable cell count after 0 hours of treatment with MRS and gastrointestinal juice.

[0074] The results showed that the survival rate of L. plantarum JN-7 in MRS at pH 3.0 and 0.3% bile salt was 28.47% and 44.68%, respectively. The survival ability of JN-7 in gastrointestinal juice was evaluated in simulated gastric juice for 3 hours, and then in simulated intestinal juice for 3 hours. After 6 hours of treatment, the number of viable cells of JN-7 decreased to 12.32% of that before treatment.

[0075] 3) Sensitivity to antibiotics and MIC

[0076] Seven kinds of clinically commonly used antibiotics, gentamicin, ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin and clindamycin, were selected for antibiotic sensitivity analysis of the strain. Antibiotic stock solutions of 256 mg / L were prepared and diluted by two-fold. Fresh bacterial solution after 16 hours of culture was diluted to 0.0002 OD, and finally inoculated into the above different concentration antibiotic solutions at a ratio of 1:1. The results were observed after 24 hours of culture at 37°C to determine the minimum inhibitory concentration (MIC), which was compared and evaluated according to the regulations on bacterial resistance to antibiotics formulated by the European Food Safety Authority (EFSA).

[0077] The MIC of ampicillin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline and chloramphenicol for L. plantarum JN-7 is shown in Table 2. The results showed that the MIC values of all seven antibiotics were lower than the critical value of antibiotics specified in the EFSA guidelines, indicating that the strain was not resistant to antibiotics and had antibiotic safety.

[0078]

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

[0080] 4) Hemolytic activity

[0081] The absence of hemolytic activity and antibiotic resistance is considered a safety prerequisite for selecting probiotic strains (FAO / WHO, 2002).

[0082] After two generations of activation, L. plantarum JN-7 was streaked on Columbia blood agar medium (3400071, Haibo Co., China) containing 5% defibrinated sheep blood and incubated at 37°C for 48 hours. If a grass green hemolytic ring appeared, it was α-hemolytic; if a colorless transparent hemolytic ring appeared, it was β-hemolytic; if no hemolytic ring appeared, it was γ-hemolytic.

[0083] The results are shown in Figure 3. After incubation of L. plantarum JN-7 on blood agar, no hemolytic ring appeared around the colonies, indicating γ-hemolysis. This indicates that L. plantarum JN-7 has no hemolytic ability.

[0084] 5) Analysis of the production of D-lactic acid and L-lactic acid and the L-lactic acid / D-lactic acid ratio.

[0085] Fresh single colonies were picked and incubated in MRS broth at 37°C for 18 hours. D,L-lactic acid was analyzed in the culture supernatant using a commercially available D,L-lactic acid quantitative kit (Jingmei Co., China) according to the manufacturer's instructions via enzymatic methods.

[0086] After culturing the strain in MRS broth at 37°C for 18 hours, the D-lactic acid and L-lactic acid produced by *Lactobacillus plantarum* JN-7 were analyzed. JN-7 produced 1.2 nmol / L D-lactic acid and 4.28 nmol / L L-lactic acid during fermentation. The L-lactic acid / D-lactic acid ratio was 3.57 (see Table 3).

[0087] Table 3. D-lactic acid and L-lactic acid content and L-lactic acid / D-lactic acid ratio

[0088] 6) Detection of biogenic amines

[0089] The strain was cultured overnight in MRS liquid medium, then added 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 subcultured every 24 hours for a total of five times. The above liquid medium was then inoculated at a 2% inoculum into MRS medium containing 1% histamine, tyrosine, cadaverine, and putrescine, and cultured for 72 hours, with the color change of the medium observed. A purple color indicated a positive result, while a yellow color indicated a negative result. *Lactobacillus rhamnosus* LGG was used as a negative control, and *Escherichia coli* as a positive control.

[0090] The results showed that JN-7 turned yellow after three days of growth in four different biogenic amine detection media, indicating that the strain does not produce putrescine, cadaverine, histamine, or tyramine.

[0091] 7) API 50CH sugar fermentation experiment

[0092] The fresh bacterial culture, cultured for 16 hours, was centrifuged at 12,000 rpm for 2 minutes. The supernatant was discarded, and the precipitate was washed twice with sterile PBS, then resuspended in sterile PBS and the OD was adjusted to 0.3, resulting in bacterial culture solution a. 1 mL of bacterial culture solution a was added to 10 mL of 50 CHL medium (50410, API, France), resulting in bacterial culture solution b. 150 μL of bacterial culture solution b was added sequentially to the wells of a 50 CH test strip (50300, API, France), and the mixture was incubated at 37°C for 48 hours. The results were determined by color change; a yellow color compared to the control group indicated a positive result (purple for esculin positive), while no color change indicated a negative result.

[0093] The results show that B. plantarum JN-7 can metabolize D-ribose (5), D-galactose (10), D-glucose (11), D-fructose (12), D-mannitol (13), mannitol (18), sorbitol (19), methyl-a-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-melibiose (33), D-manninotriose (34), D-raffinose (35), D-gentiobiose (39), D-turanose (40), D-tagatose (42), and potassium gluconate (47), and ferment to produce acid.

[0094] Example 3. Cytotoxicity and adhesion of B. plantarum JN-7

[0095] Experimental methods:

[0096] Culture of HT-29 cells

[0097] HT-29 is a human intestinal epithelial cell line used for bacterial cytotoxicity and adhesion tests. HT-29 is cultured in RPMI 1640 medium containing 10% FBS and 1% P / S (penicillin / streptomycin). The cell density reaches about 90% after about 36-72 h of culture in a 37°C incubator with 5% CO2, and the cells are passaged and plated by trypsin digestion.

[0098] Cell viability detection

[0099] CCK-8 is a general method for detecting cell proliferation and toxicity. In this experiment, the CCK-8 kit (C0037, Biyun Tian) was used to detect the proliferation activity of HT-29 cells. The cells were seeded in a 96-well plate at an appropriate density, and 1 / 10 of the total volume of CCK-8 solution was added. The plate was incubated in the dark at 37°C for 2 hours to remove air bubbles, and the OD value at 450 nm was measured by a microplate reader.

[0100] Adhesion detection

[0101] HT-29 cells were seeded in a 24-well plate at a concentration of 5x10 5 cells / mL from a culture flask, and the medium was replaced with antibiotic-free medium for culture. After the cells were completely attached, the experiment was performed. Before adding bacteria, the cells in the well plate were washed twice with sterile PBS, and 500 μL of bacteria at a concentration of 10 8CFU / mL (V0). The 24-well plates were transferred to a 37 °C incubator with 5% CO2for 4 h to allow adhesion. The cells were washed 5 times with PBS solution per well to elute the non-adherent bacteria and metabolic secretions. 200 μL of 1% Triton X-100 was added to each well for digestion, and then the solution in each well was collected for gradient dilution and counting (V1). The adhesion rate (%) was calculated as follows:

[0102] Adhesion rate (%) = (V1 / V0) x 100%

[0103] To evaluate the potential cytotoxic effect of P. lactis JN-7 on HT-29 intestinal epithelial cells, a CCK-8 assay was performed after 18 h of co-culture with P. lactis JN-7 bacterial cells. The multiplicity of infection (MOI) of this assay was approximately 1 :200. As shown in Figure 4a, P. lactis JN-7 had no effect on the survival of intestinal epithelial cells at this MOI. The adhesion capacity of P. lactis JN-7 was compared with that of the commercial probiotic Lactobacillus rhamnosus (LGG) (Figure 4b). The results showed that LGG adhered twice as efficiently as JN-7.

[0104] Example 4. Anti-inflammatory effect of P. lactis JN-7

[0105] Experimental methods:

[0106] Culture and treatment of THP-1

[0107] THP-1 is a human monocytic leukemia cell that can be induced to differentiate into macrophages by phorbol ester (PMA) and is used to test the anti-inflammatory effect of bacteria. THP-1 is cultured in RPMI 1640 medium with 10% FBS and 1% P / S. The cells are cultured in a 37 °C incubator with 5% CO2, and when the cell density reaches 8-10 x 10 5 The cells are passaged when they reach 80-90% confluence, and the normal state of the cells is to be grown in suspension for semi-confluent medium passage.

[0108] THP-1 cells were seeded at 5 x 10 5 The cells were seeded in 6-well plates at 5 x 10

[0109] Real-time quantitative PCR (qRT-PCR)

[0110] Total mRNA was isolated from cells using Trizol reagent (R701-01-AA, Novogene) according to the manufacturer's instructions. Reverse transcription PCR (RT-PCR) was performed using HiScript II select qRT supermix (R222-01, Novogene) in a final volume of 20 μΐ containing 1 μg of RNA. qRT-PCR was performed using gene-specific primers and SYBR Green (Q712-02, Novogene) on a quantitative Real-time PCR (qRT-PCR) detection system. All primers were designed by ourselves and synthesized by Beijing GenScript Biotech Co., Ltd. (Beijing, China). The data were analyzed using the 2-ΔΔt method with β-actin as the reference gene.

[0111] The specific operation steps are as follows:

[0112] Extraction of RNA: (1) Collect cells in a 1.5 mL enzyme-free EP tube, add 200 μΐ of Trizol reagent, homogenize and fully lyse the cells. After incubation at room temperature for 5 min, centrifuge at 4°C, 12000 rpm for 5 min. (2) Transfer the supernatant to a new EP tube, add 4 / 5 volume of isopropanol, invert shake 10 times to mix well, and then stand at room temperature for 10 min. (3) Centrifuge at 4°C, 12000 rpm for 10 min, discard the supernatant, and a white feather-like precipitate appears, which is RNA. Wash twice with 800 μΐ of DEPC water containing 75% ethanol, discard the supernatant, and dry the precipitate. Dissolve the precipitate with 20 μΐ of DEPC water on ice to obtain RNA. (4) Measure the concentration of RNA with a NanoDrop microspectrophotometer.

[0113] RT-PCR synthesis of cDNA: Synthesize cDNA using Novogene's RT-PCR kit (HiScript II Q RT SuperMix for qPCR).

[0114] Table 4 Preparation of first-strand cDNA synthesis reaction solution

[0115] Gently mix and centrifuge at 500 rpm for 1 min to the bottom.

[0116] Table 5 First-strand cDNA synthesis reaction is performed under the following conditions

[0117] The obtained 20 μΐ of product can be immediately used for qRT-PCR reaction, or stored at -20°C (usable within half a year); long-term storage is recommended to be stored at -80°C after aliquoting and to avoid repeated freezing and thawing.

[0118] qRT-PCR:

[0119] Table 6 The 20 μL reaction system is as follows:

[0120] After mixing, centrifuge the liquid to the bottom, and amplify on a real-time fluorescence quantitative PCR instrument.

[0121] The reaction conditions are 94°C for 30 s; 94°C for 5 s; 60°C for 30 s, repeated for 40 cycles. The Bio-Rad CFX Manager Software automatically records amplification data and melting curves, and the amplification data is expressed as threshold cycle (CT value). The 2-ΔΔt method is used for data analysis. The primer sequences (SEQ ID NO: 4-11) are shown in the following table.

[0122] Table 7 Primer sequences

[0123] Anti-inflammatory effect of JN-7 on LPS-stimulated PMA-differentiated THP-1 cells

[0124] JN-7 down-regulates the expression of pro-inflammatory factor mRNA in LPS-stimulated cells (as shown in FIG. 5). JN-7 and LGG were co-cultured with macrophages differentiated from THP-1, and the anti-inflammatory effect of the strains was evaluated by detecting the expression levels of pro-inflammatory factors IL-6, IL-8 and anti-inflammatory cytokine IL-10 mRNA. Compared with the LPS stimulation control group, the expression of IL-6 and IL-8 mRNA in cells containing JN-7 and LGG after LPS stimulation was reduced, and the expression of IL-10 mRNA was increased. In addition, the down-regulation effect of LGG on LPS-stimulated IL-8 mRNA expression was better than that of JN-7. The ability of JN-7 to promote IL-10 release was higher than that of LGG, suggesting that JN-7 has an anti-inflammatory effect like LGG.

[0125] Example 5. Production and stability analysis of Bacillus velezensis JN-7 powder

[0126] 1) Fermentation and powder production of JN-7

[0127] Bacillus velezensis JN-7 was inoculated into MRS broth and cultured at 37°C for 18 h, activated twice and inoculated into a 5L bioreactor (New Brunswick Scientific, USA) containing 3L MRS medium. The culture was incubated at 37°C, 200 rpm and 0.5 vvm for 48 h. The bacteria were cultured in 5L bioreactor for 12 hours. After centrifugation, 41.5g of P. freudenreichii precipitate was collected. The precipitate was mixed with protective agents and the mixture was freeze-dried to produce 6.6g of bacteria powder, which had a viable cell count of 7.07x10

[0128] The bacteria were cultured in 5L bioreactor for 12 hours. After centrifugation, 41.5g of P. freudenreichii precipitate was collected. The precipitate was mixed with protective agents and the mixture was freeze-dried to produce 6.6g of bacteria powder, which had a viable cell count of 7.07x10 11 CFU / g.

[0129] 2) Evaluation of the stability of P. freudenreichii JN-7 bacteria powder

[0130] The packaged P. freudenreichii JN-7 bacteria powder was stored in an aluminum foil bag in a constant humidity chamber at 40°C and 75% humidity for accelerated testing, while commercially available Lactobacillus rhamnosus LGG bacteria powder and 15 strains of P. freudenreichii (bacteria powder production process same as JN-7) isolated from fermented vegetables were also stored under the same temperature and humidity for comparison. Within 90 days, samples of JN-7 bacteria powder, LGG bacteria powder, and 15 strains of P. freudenreichii bacteria powder were taken, and the number of lactic acid bacteria in the bacteria powder was detected according to the national standard GB 4789.35, and the water activity of the bacteria powder was measured.

[0131] The viable cell count and water activity of P. freudenreichii JN-7 (Figure 6A), Lactobacillus rhamnosus LGG (Figure 6B), and the other 15 strains of P. freudenreichii bacteria powder (Figure 6C) during storage are shown in Figures 6A-6C. As can be seen from the figures, the stability of JN-7 bacteria powder was significantly higher than that of the other 16 strains. The viable cell count of JN-7 decreased from 7.07x10 11 CFU / g to 1.73x10 11 CFU / g within 70 days, and the corresponding water activity increased from 0.024 to 0.053. The viable cell count of the control group LGG decreased from 3.53x10 11 CFU / g to 3.92x10 8 CFU / g within 70 days, and the corresponding water activity increased from 0.091 to 0.134. Through 70 days of testing, a linear fit was made for the reduction of viable bacteria of JN-7 (r 2 = 0.80). Through the fitting, it can be predicted that the time required for a 1-log reduction in viable bacteria under the conditions of the accelerated stability test is 136.99 days, which is 112.36 days longer than the 24.63 days required for the fitting prediction of LGG.

[0132] In general, the present application isolates and identifies a Lactobacillus plantarum JN-7 from a natural fermentation product. After whole genome sequencing and comparison with all Lactobacillus plantarum genomes available in NCBI, we found that the strain Lactobacillus plantarum JN-7 is a new strain. Through the basic characteristic analysis (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) of the strain, it is found that Lactobacillus 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 plant Lactiplantibacillus JN-7 is Lactiplantibacillus plantarum, and the preservation number is CGMCC No. 30605.

2. A bacterial powder prepared from the plant Lactiplantibacillus JN-7 of claim 1.

3. A method for preparing the bacterial powder of claim 2, comprising the following steps: 1) fermenting the plant Lactiplantibacillus JN-7; 2) centrifuging the fermentation product to precipitate, and freeze-drying to obtain the bacterial powder.

4. A probiotic composition comprising the plant Lactiplantibacillus JN-7 of claim 1 or the bacterial powder of claim 2.

5. The probiotic composition according to claim 4, wherein, The probiotic composition further comprises one or more probiotics selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus, Ligilactobacillus, Latilactobacillus, Streptococcus, Lactococcus, Propionibacterium, Acidipropionibacterium, Weizmannia, Mammaliicoccus, Staphylococcus, Kluyveromyces, Leuconostoc, Pediococcus, and Bacillus subtilis DE111.

6. Use of the plant Lactiplantibacillus JN-7 of claim 1, the bacterial powder of claim 2, or the probiotic composition of claim 4 or 5 in the preparation of an anti-inflammatory drug.

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

8. Use of the plant Lactiplantibacillus JN-7 of claim 1, the bacterial powder of claim 2, or the probiotic composition of claim 4 or 5 in the preparation of a food or health product.

9. Use according to claim 8, wherein, The food or health product is a dairy product, a bean product, a meat product, a fruit and vegetable product, a beverage, or a snack.

10. Use according to claim 8, wherein, The food or health product further comprises an edible excipient.

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