CN120330077A

CN120330077A - Lactobacillus plantarum JN-7, bacterial powder, preparation method, probiotic composition and use thereof

Info

Publication number: CN120330077A
Application number: CN202410775747.8A
Authority: CN
Inventors: 徐长云; 闫紫晴
Original assignee: Aidimeng Shanghai Management Co ltd
Current assignee: Aidimeng Shanghai Management Co ltd
Priority date: 2024-06-14
Filing date: 2024-06-14
Publication date: 2025-07-18
Also published as: WO2025256673A3; WO2025256673A2; AU2025230764A1

Abstract

Lactobacillus plantarum JN-7, bacterial powder, preparation method, probiotic composition and application thereof. The application obtains a new lactobacillus plantarum strain named as lactobacillus plantarum JN-7 through screening, and the preservation number is CGMCC No.30605. The lactobacillus plantarum JN-7 is found to be a safe strain and has excellent production performance, high stability and anti-inflammatory efficacy through 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 freeze-drying yield) and stability test (accelerated storage stability test) of the strain. The bacterial powder produced by the method can keep stability and activity for a long time.

Description

Lactobacillus plantarum JN-7, bacterial powder, preparation method, probiotic composition and application thereof

Technical Field

The present disclosure relates generally to the field of biological applications, and more particularly to lactobacillus plantarum JN-7, bacterial powder, a preparation method, a probiotic composition and uses thereof.

Background

Probiotics are a class of active microorganisms beneficial to a host by colonizing the human body and altering the flora composition of a part of the host. By regulating the immune function of host mucous membrane and system or regulating the balance of flora in intestinal tract, the effect of promoting nutrient absorption and maintaining intestinal health is achieved, so that single microorganism or mixed microorganism with definite composition beneficial to health is produced.

Lactobacillus plantarum (Lactiplantibacillus plantarum) is a common type of probiotic bacteria that is widely used in fermented dairy products, meat products, plant products and bakery products. The main functions of the method are that lactic acid can be produced, the pH is reduced, putrefying bacteria are prevented from propagating, and the flavor and the texture of the product are improved. In recent years, the research shows that the lactobacillus plantarum has good probiotic function, can inhibit gastrointestinal tract pathogenic bacteria including helicobacter pylori and the like, improve gastrointestinal tract function and metabolism, strengthen intestinal tract barrier function, reduce infection risk and the like.

The stability and activity of probiotics in food are closely related to the health care effect achieved by probiotics, and development of probiotics with good stability and long-term storage is needed.

Disclosure of Invention

Based on the background, the application provides the lactobacillus plantarum JN-7, wherein the lactobacillus plantarum JN-7 is classified and named as lactobacillus plantarum (Lactiplantibacillus plantarum), and the preservation number is CGMCC No.30605.

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

In another aspect, the application also provides a method of preparing the bacterial powder described herein, comprising the steps of:

1) Fermenting lactobacillus plantarum JN-7 described herein;

2) And centrifuging, precipitating, and freeze-drying the fermentation product to obtain the final product.

In another aspect, the application also provides a probiotic composition comprising lactobacillus plantarum JN-7 as described herein or the bacterial powder as described herein.

In another aspect, the application also provides the use of lactobacillus plantarum JN-7 as described herein, the bacterial powder as described herein or the probiotic composition as described herein for the preparation of an anti-inflammatory drug.

In another aspect, the application also provides the use of lactobacillus plantarum JN-7 as described herein, the bacterial powder as described herein or the probiotic composition as described herein for the preparation of a food or health care product.

Viable cells in the probiotic powder are closely related to the quality and production cost of the probiotic product. The strain with good stability can improve the number of living cells in the probiotic powder, thereby being beneficial to improving the quality of the probiotic powder and reducing the cost.

The application provides lactobacillus plantarum which is good in yield, stability, safety, acid, bile salt and gastrointestinal tract tolerance and antibiotic sensitivity, and the lactobacillus plantarum is named as lactobacillus plantarum JN-7. The strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) with the address of North Star Xilu, no. 1, 3 of the district of Korean Yang in Beijing, china national academy of sciences, and the preservation date is 2024, 05 and 13. The preservation number is CGMCC No.30605.

The lactobacillus plantarum JN-7 is found to be a safe strain and has excellent production performance, high stability and anti-inflammatory efficacy through 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 freeze-drying yield) and stability test (accelerated storage stability test) of the strain. The bacterial powder produced by the method can keep stability and activity for a long time.

Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application. Other advantages of the application may be realized and attained by the structure particularly pointed out in the written description and drawings.

Drawings

The accompanying drawings are included to provide an understanding of the principles of the application, and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain, without limitation, the principles of the application.

FIG. 1 is a circular diagram of the genome of Lactobacillus plantarum JN-7 in an embodiment of the application. Seven pieces of information are shown from outside to inside, the first circle is genomic position information, the second circle is GC content information, the third circle is the coding gene on the positive strand (marked red), the fourth circle is the coding gene on the negative strand (marked green), the fifth circle is the ncRNA on the positive strand (marked blue), the sixth circle is the ncRNA information on the negative strand (marked purple), and the seventh circle is long-segment repeat information on the genome (marked orange).

FIG. 2 shows the growth curve of Lactobacillus plantarum JN-7 starting from OD 0.1 at 37℃and 200rpm in the examples according to the application.

FIG. 3 shows the results of the hemolytic ability of Lactobacillus plantarum JN-7 in the examples of the present application.

FIG. 4 shows the interaction of Lactobacillus plantarum JN-7 with intestinal epithelial cells HT-29 in an embodiment of the application. Wherein (a) Lactobacillus plantarum JN-7 does not decrease HT-29 cell viability. The data represent the viability of cells after 18 hours of co-incubation with lactobacillus plantarum JN-7 with cells from the pure medium group (n=6). (b) Adhesion ratio of lactobacillus rhamnosus LGG and lactobacillus plantarum JN-7 to HT-29 cells. The values represent the adhesion rates of LGG and Lactobacillus plantarum JN-7 after 4 hours incubation with HT-29. Data are expressed in mean±sem P <0.05, P <0.01, P <0.001.

FIG. 5 shows the anti-inflammatory ability of Lactobacillus plantarum JN-7 to modulate LPS-stimulated THP-1 cell production of inflammatory factors in an embodiment of the application. Wherein, (a) interleukin 6 (IL) -6mRNA, (b) IL-8mRNA, and (c) IL-10mRNA. Data are expressed in mean±sem P <0.05, P <0.01, P <0.001.

FIGS. 6A-6D show the number of viable cells and water activity during storage at 40℃and 75% humidity of Lactobacillus plantarum JN-7 powder (FIG. 6A), lactobacillus rhamnosus LGG (FIG. 6B) and other 15 Lactobacillus plantarum powder (FIG. 6C-6D) according to the examples of the present application.

Detailed Description

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or an upper preferable range value, and a lower preferable range value, this is to be understood as equivalent to any range specifically disclosed by combining any pair of upper range values or preferred range values with any lower range value or preferred range value, regardless of whether the range is specifically disclosed. Unless otherwise indicated, the numerical ranges set forth herein are intended to include the endpoints of the ranges and all integers and fractions (fractions) within the range.

The terms "about", "about" when used in conjunction with a numerical variable generally refer to the value of the variable and all values of the variable being within experimental error (e.g., within a confidence interval of 95% for the average) or within + -10% of the specified value, or more broadly.

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

The expression "at least one" or "one (or more)" means 1, 2, 3,4, 5, 6, 7, 8, 9 (or more(s).

The research to improve the stability of functional strains has been mostly carried out by the development of bacterial powder protectants. Due to the inherent stability of each strain, the protective agent has limited effect in stabilizing the probiotic powder. The development of a stable strain for use in commercial probiotic powder complexes is another approach to solving the problem of low bacterial activity of probiotic products over their shelf life.

Based on the above, the application provides the lactobacillus plantarum JN-7, wherein the lactobacillus plantarum JN-7 is classified and named as lactobacillus plantarum (Lactiplantibacillus plantarum) and the preservation number is CGMCC No.30605.

1) Fermenting lactobacillus plantarum JN-7 described herein;

In some embodiments, the methods of preparing the bacterial powders described herein comprise the steps of:

1) Fermenting and culturing lactobacillus plantarum JN-7 described herein in a bioreactor, wherein the pH is 4.5-5.3, the temperature is 35-40 ℃, and the stirrer speed is 160-200rpm;

2) Centrifuging and precipitating the fermentation product, mixing the precipitate with a protective agent, freeze-drying in a vacuum freeze-dryer, and pulverizing into powder.

In some embodiments, the probiotic composition further comprises one or more probiotics selected from the group consisting of bifidobacterium, lactobacillus mucilaginosus, lactobacillus plantarum, lactobacillus co-mingii, lactobacillus cloacae, streptococcus, lactococcus, propionibacterium, wegenemania, zoococcus, staphylococcus, kluyveromyces, leuconostoc, pediococcus and bacillus subtilis DE111.

In some embodiments, the probiotic composition further comprises one or more probiotics selected from the group consisting of: bifidobacterium adolescentis, bifidobacterium animalis subspecies animalis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium longum subspecies longum, bifidobacterium longum subspecies infantis, lactobacillus acidophilus, lactobacillus crispatus, lactobacillus delbrueckii subspecies bulgaricus, lactobacillus delbrueckii subspecies lactis, lactobacillus grignard, lactobacillus helveticus, lactobacillus johnsonii, lactobacillus equi-like subspecies, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus fermentum, lactobacillus reuteri lactobacillus plantarum, lactobacillus salivarius, lactobacillus curvatus, lactobacillus cloacae, streptococcus salivarius thermophilus, lactobacillus lactis, lactococcus lactis (diacetyl), lactococcus cremoris, propionibacteria, leuconostoc mesenteroides, pediococcus acidilactici, pediococcus pentosaceus, wei Zhman's, zoococcus caldarius, staphylococcus xylosus, staphylococcus botulinum, kluyveromyces marxianus and bacillus subtilis DE111.

In another aspect, the application also provides the use of lactobacillus plantarum JN-7 as described herein, the bacterial powder as described herein or the probiotic composition as described herein for the preparation of an anti-inflammatory drug. In some embodiments, the medicament is administered orally.

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

In some embodiments, the food product is a health food product, or the food product comprises a dairy product, a soy product, a meat product, or a fruit and vegetable product, or the food product is a beverage or snack. In some embodiments, the food product comprises lactobacillus plantarum JN-7 described herein and an edible adjuvant.

The present application has been described in terms of several embodiments, but the description is illustrative and not restrictive, 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 described embodiments. 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 possible. Any feature of any embodiment may be used in combination with, or in place of, any other feature of any other embodiment, unless expressly limited otherwise.

The present application includes and contemplates combinations with features known to those of ordinary skill in the art. The disclosed embodiments and features of the application may also be combined with any conventional features to form a unique inventive arrangement as defined in the claims. Any feature of any embodiment may also be combined with features from other inventive arrangements to form another unique inventive arrangement as defined in the claims. It is therefore to be understood that any of the features shown and/or discussed in the present application may be implemented alone or in any suitable combination. Accordingly, the embodiments are not to be restricted except in light of the attached claims and their equivalents. Further, various modifications and changes may be made within the scope of the appended claims.

Furthermore, in describing representative embodiments, the specification may have presented the method and/or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. Other sequences of steps are possible as will be appreciated by those of ordinary skill in the art. Accordingly, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. Furthermore, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the embodiments of the present application.

The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. The experimental materials not shown in the examples below are all commercially available. The equipment used in each step in the following examples is conventional equipment. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer. Unless defined or otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described may be used in the methods of the present application.

Example 1 acquisition and identification of Lactobacillus plantarum JN-7

The lactobacillus plantarum JN-7 provided by the application is separated from pickled pickle of Sichuan Cheng-du family.

1. 16S sequencing of Lactobacillus plantarum JN-7

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 the universal primers 27F:5'-AGAGTTTTGATCCTGTCCAG-3' (SEQ ID NO: 1) and 142R:5 '-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2). The PCR cycle was 94℃for 2min of initial activation, the denaturation step was cycled at 94℃for 30s, 55℃for 1min of annealing, 72℃for 1min of extension, and finally at 72℃for 10 min. The PCR product is purified for first generation sequencing, and the sequence of the obtained 16s rRNA is shown as SEQ ID NO. 3.

After comparison with the NCBI website Blast, the lactobacillus plantarum has the similarity of 99.93 percent with the model strain Lactiplantibacillus plantarum SRCM100442, is named Lactiplantibacillus plantarum JN-7 by combining the physiological and biochemical indexes related to the strain JN-7 and the molecular biological identification results and combining the morphological characteristics, and is preserved in the China general microbiological culture Collection center (CGMCC) No.30605 at the 5-13-2024 month.

2. Whole genome sequencing of Lactobacillus plantarum JN-7

For genome sequencing, lactobacillus plantarum JN-7 hours was cultured using MRS broth under anaerobic conditions at 37 ℃. The single colonies were cultured overnight in MRS broth. The broth was centrifuged at 8000 Xg at 4℃for 5 min. The pellet was sent to GENEWIZ sequencing company (china) for sequencing, assembly, annotation and bioinformatics analysis.

Whole genome sequencing was performed on the illumine PE150 platform and PacBio sequence system. For the PacBio sequencing library, 5-10. Mu.g of genomic DNA was cut into 10-15Kb fragments using a g-TUBE device. And then useExpress Template Preparation Kit 2.0.0 library construction. Briefly, the DNA fragments were ligated via single-stranded overhang removal, DNA damage repair, end repair, a tailing, bar-coded overhang ends. Libraries were quantified using a Qubit 3.0 fluorometer (Invitrogen, carlsbad, california) and checked for library size using an Agilent2100 bioanalyzer system. Subsequent steps are performed to prepare SMRTbell libraries according to manufacturer's instructions. The library was sequenced using the PacBio sequence platform. PacBIO reads were assembled using Hifiasm/Canu. The genome was then recalibrated using the previous Illumina data using pilot software. Prodigal/Augustus gene search software has been used to search for coding genes. Transferred RNAs (trnas) were detected in the genome using program tRNAscan-SE and default parameter settings. rRNA was identified by using Barrnap. Other RNAs were identified by rfam database. The coding genes were annotated by Diamond with the National Center for Biotechnology Information (NCBI) nr database. The function of the gene was then annotated by GO (Gene Ontology) database and the pathway was annotated using KEGG (Kyoto Encyclopedia of Genes and Genomes) database. The proteins encoded by the genes were phylogenetically classified by COG (Clusters of Orthologous Groups) database. Protein sequences of E <1E-5 were retrieved using Diamond in CAZy database, swiss_prot database, pfam database, CARD database, VFDB database or DFVF database.

The genome sequence of Lactobacillus plantarum JN-7 was assembled and analyzed. The basic genomic information sequences are shown in table 1. The length of genome is 3255213bp, and the genome is total of 3218 genes and 3090 protein coding genes. Genome circles showing the information of gene, ncRNA, GC content and repetitive sequence were created using Circos (version 0.69) software, see fig. 1.

TABLE 1 basic genomic information of Lactobacillus plantarum JN-7

3. Identification of Lactobacillus plantarum JN-7

The identification of Lactobacillus plantarum JN-7 was performed by calculating the average nucleotide identity (Average Nucleotide Identity, ANI) between the JN-7 genomic sequence and the standard strain of Lactobacillus plantarum Lactiplantibacillus plantarum SRCM100442 using an on-line ANI calculator (http:// enve-omics. Ce. Gatech. Edu/ANI /). The ANI calculator uses the best hits between the two genome datasets (one-way ANI) and the mutual best hits (two-way ANI) to estimate average nucleotide identity. By comparing JN-7 with the Lactobacillus plantarum reference strain, 99.21% ANI results were obtained, indicating that the JN-7 strain belongs to the Lactobacillus plantarum species.

1215 Lactobacillus plantarum genome databases selected from NCBI (retrieved at 2024.2.20) were established using Makeblastdb software. The criteria for lactobacillus plantarum screening are complete genome or genome sketches that have been uploaded to NCBI. The assembled JN-7 genome was compared to the lactobacillus plantarum genome database by Blastn to generate the unmatched regions and the number of unmatched regions in the comparison entry. No reference was queried that was completely identical or allowed up to 1000 mismatches compared to the assembly results, indicating that lactobacillus plantarum JN-7 was a strain that was never reported in NCBI.

EXAMPLE 2 characterization of Lactobacillus plantarum JN-7

Characteristics of Lactobacillus plantarum JN-7

Lactobacillus plantarum JN-7 was fermented and the following characteristics of the strain were determined

1) Lactobacillus plantarum JN-7 growth curve

The growth curve for JN-7 was plotted using Growth profiler 960 (Enzyscreen b.v., HEEMSTEDE, netherlands) at a temperature of 37 ℃. The procedure was as follows, JN-7MRS broth cultured for 16 hours and inoculated into fresh MRS broth and the final OD ₆₀₀ was adjusted to 0.1. The OD-adjusted bacterial solutions were added to sterile 96-well plates to ensure an addition of 250. Mu.L per well, and 11 groups of replicates (A1-A11) were performed. OD was collected every 30 minutes by incubation at 200rpm at 37 ℃ for 48 hours under aerobic conditions.

A typical quadratic curve was fitted through a Growth Profiler 960, as shown in FIG. 2. Lactobacillus plantarum JN-7, with an initial OD of 0.1, reached the exponential growth phase within 18 hours and was in stationary phase within the next 48 hours, when cultured at 37℃and 200 rpm.

2) Acid, bile salts and gastrointestinal tract tolerance

Fresh bacterial liquid cultured in MRS broth for 16h was centrifuged at 12000rpm at 4℃for 2 minutes, and a precipitate was left. The pellet was washed twice with sterile PBS buffer, resuspended in sterile PBS (ph=7) and the cell concentration adjusted to 10 ⁸ CFU/mL. The bacterial solution was inoculated into MRS (N0), MRS (N1) having pH3.0 or MRS liquid medium (N1) containing 0.3% bile salts (w/v, sigma, USA) at an addition amount of 5%. After 4h at 37 ℃, viable cells of lactobacillus plantarum JN-7 in different media were counted.

To examine gastrointestinal tolerance of lactobacillus plantarum JN-7, fresh single colonies were picked up and inoculated into MRS broth and cultured at 37 ℃ for 16h. 5mL of the bacterial liquid was centrifuged at 12000rpm and 4℃for 2 minutes. The pellet was washed with sterile PBS (ph=7) and resuspended to a cell concentration of 10 ⁷ CFU/mL. mu.L of the bacterial suspension was added to 900. Mu.L of simulated gastric fluid consisting of 125mM NaCl, 7mM KCl, 45mM NaHCO ₃ and 3g/L pepsin (Sigma, USA) and the pH was adjusted to 3.0 with HCl. After 3 hours incubation at 37 ℃, 250 μl of gastric juice mixture was added to 6mL of simulated intestinal fluid consisting of 45mM NaCl, 1g/L trypsin (Sigma, usa) and 3g/L bile salts (Sigma, usa), pH adjusted to 8.0 with NaOH, then incubated for 3 hours at 37 ℃.

The survival rate of lactobacillus plantarum JN-7 under acid, bile salt and gastrointestinal treatments was calculated as follows:

survival (%) = (n1++n0) ×100%

Wherein N1 is MRS (pH 3.0, 0.3% bile salt), or viable cell count after gastrointestinal fluid has been treated for 6 hours, and N0 is viable cell count after MRS and gastrointestinal fluid have been treated for 0 hour.

The results showed that the survival rate of Lactobacillus plantarum JN-7 in MRS at pH3.0 and 0.3% bile salts was 28.47% and 44.68%, respectively. The viability of JN-7 in gastrointestinal fluids was assessed in simulated gastric fluid for 3 hours and then in simulated intestinal fluid for 3 hours. After 6h of treatment, the number of living cells of JN-7 was reduced to 12.32% before treatment.

3) Sensitivity to antibiotics and MIC

7 Clinical antibiotics of gentamicin, ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin and clindamycin are selected for carrying out antibiotic sensitivity analysis on the strain, 256mg/L of antibiotic mother liquor is respectively prepared and diluted twice, fresh bacterial liquid OD after 16h of culture is diluted to 0.0002, and finally, the bacterial liquid OD is inoculated into the antibiotic solutions with different concentrations according to the inoculum size of 1:1. After 24h incubation at 37 ℃, the Minimum Inhibitory Concentration (MIC) was determined and compared to the standard for antibiotic resistance of bacteria established by the European Food Safety Agency (EFSA).

The MICs of ampicillin, gentamicin, kanamycin, erythromycin, clindamycin, tetracycline and chloramphenicol for Lactobacillus plantarum JN-7 are shown in Table 2. The results show that the MIC values of all 7 antibiotics are lower than the critical value of the antibiotics specified by the EFSA guidelines, which indicates that the strain is intolerant to antibiotics and has antibiotic safety.

TABLE 2 MIC (mg/L) of different antibiotics for Lactobacillus plantarum JN-7

4) Hemolytic activity

The absence of hemolytic activity and antibiotic resistance is considered to be a safety prerequisite for the selection of probiotic strains (FAO/WHO, 2002).

The activated two-generation Lactobacillus plantarum JN-7 is streaked on Columbia blood agar medium (3400071, haibo Co., china) containing 5% defibrinated sheep blood, and cultured at 37 ℃ for 48 hours, wherein the alpha-hemolysis is obtained if a grass green hemolysis ring appears, the beta-hemolysis is obtained if a colorless transparent hemolysis ring appears, and the gamma-hemolysis is obtained if no hemolysis ring appears.

As shown in FIG. 3, after Lactobacillus plantarum JN-7 was cultured on blood agar, no hemolytic ring appeared around the colony, and the colony was gamma-hemolytic. Thus, it was demonstrated that Lactobacillus plantarum JN-7 had no hemolytic ability.

5) Production of D-lactic acid, L-lactic acid and analysis of L-lactic acid/D-lactic acid ratio

Fresh single colonies were picked and cultured in MRS broth at 37℃for 18 hours. Culture supernatants were subjected to D, L-lactic acid analysis by enzymatic methods using a D, L-lactic acid commercial quantification kit (Jingmei Co., china) according to the manufacturer's protocol.

After culturing the strain in MRS broth at 37℃for 18 hours, D-lactic acid and L-lactic acid produced by Lactobacillus plantarum JN-7 were analyzed. 1.2nmol/L D-lactic acid and 4.28nmol/L L-lactic acid were produced during the JN-7 fermentation. The L-lactic acid/D-lactic acid ratio was 3.57, as shown in Table 3.

TABLE 3D-lactic acid, L-lactic acid content and L-lactic acid/D-lactic acid ratio

6) Biogenic amine detection

The strain was cultured overnight in MRS liquid medium, added to MRS liquid medium containing 0.1g/L of histidine, tyrosine, ornithine, lysine and 0.05g/L of pyridoxal-5-phosphate, respectively, to a final concentration of OD 0.01 and passaged, once every 24 hours, five times in total. The above liquid medium was inoculated in an MRS medium containing 1% of histamine, tyrosine, cadaverine and putrescine in an inoculum size of 2% respectively for culturing for 72 hours and the color change of the medium was observed. The color of the medium was positive if it turned purple, and the color of the medium was negative if it turned yellow. Lactobacillus rhamnosus LGG was used as a negative control and escherichia coli was used as a positive control.

The results showed that JN-7 was yellow after three days of growth in the four biogenic amine detection media, indicating that the strain did not produce putrescine, cadaverine, histamine, and tyramine.

7) API 50CH sugar fermentation experiments

The fresh bacterial liquid cultured for 16 hours is centrifuged for 2min at 12000 rpm. The supernatant was discarded, and the pellet was washed twice with sterile PBS, resuspended in sterile PBS, and OD was adjusted to 0.3 to give broth a. 1mL of bacterial liquid a to 10mL of 50CHL medium (50410, API, france) was taken as bacterial liquid b. 150. Mu.L of bacterial liquid b was added to 50CH test strip (50300, API, france) and incubated at 37℃for 48h. The results were judged by color change, and yellow color represents positive (wherein esculin positive was purple) compared with the control group, and no change was recorded as negative.

The results show that lactobacillus plantarum JN-7 is capable of metabolizing 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-Toulon sugar (40), D-tagatose (42) and potassium gluconate (47) to produce acid by fermentation.

EXAMPLE 3 cytotoxicity and adhesion of Lactobacillus plantarum JN-7

The experimental method comprises the following steps:

culture of HT-29 cells

HT-29 is a human intestinal epithelial cell line cell used to conduct cytotoxicity and adhesion tests of bacteria. HT-29 was cultured with RPMI 1640 medium containing 10% FBS and 1% P/S (penicillin/streptomycin). Culturing in incubator containing 5% CO ₂ and 37 deg.C for 36-72 hr until cell density reaches 90%, and digesting the passaging plate with pancreatin.

Cell activity assay

CCK-8 is a general method for detecting cell proliferation and toxicity. The assay used for the detection of HT-29 cell proliferation activity uses the CCK-8 kit (C0037, biyun). Cell counts were seeded at the appropriate density in 96-well plates, added with 1/10 of the total volume of CCK-8 solution, incubated in the dark, incubated at 37 ℃ for 2 hours, air bubbles removed, and the OD at 450nm was measured by an microplate reader.

Adhesion test

HT-29 cells were seeded from flasks at a concentration of 5X 10 ⁵ cells/mL into 24-well plates and replaced with antibiotic-free medium for culture, and experiments were performed after cell attachment was complete. Prior to addition of bacteria, cells in the well plate were washed twice with sterile PBS and 500. Mu.L of bacteria at a concentration of 10 ⁸CFU/mL(V₀) were added to each well. The 24-well plate was transferred to a 37℃5% CO ₂ incubator and incubated for 4 hours to allow adhesion. The PBS solution was washed 5 times per well of cells to elute bacteria and metabolic secretions that were not adhered. mu.L of 1% Triton X-100 was added to each well for digestion, and the solution was collected for gradient dilution and counted (V ₁) per well. The adhesion (%) was calculated as follows:

adhesion (%) = (V ₁/V₂) ×100%

To evaluate the potential cytotoxic effect of lactobacillus plantarum JN-7 on HT-29 intestinal epithelial cells, CCK-8 assays were performed after 18 hours of co-culture with lactobacillus plantarum JN-7 bacterial cells. The fold infection (MOI) for this test was about 1:200. As shown in a of fig. 4, lactobacillus plantarum JN-7 had no effect on the survival of intestinal epithelial cells at this MOI. The adhesion capacity of lactobacillus plantarum JN-7 was compared to that of the commercial probiotic lactobacillus rhamnosus (LGG) (b in fig. 4). The results showed that LGG adherent cells were twice as efficient as JN-7.

EXAMPLE 4 anti-inflammatory action of Lactobacillus plantarum JN-7

The experimental method comprises the following steps:

Cultivation and treatment of THP-1

THP-1 is a human mononuclear leukemia cell that can differentiate into macrophages under the induction of phorbol ester (PMA) for detection of bacterial anti-inflammatory effects. THP-1 was cultured in RPMI 1640 medium containing 10% FBS and 1% P/S. The cells are cultured in an incubator containing 5% CO ₂ and at 37 ℃ and passaged when the cell density reaches 8-10 multiplied by 10 ⁵ cells/mL, and the cells can be passaged by half culture medium when the cells are grown in a normal state in suspension.

THP-1 cells were seeded at 5X 10 ⁵ cells/mL in 6-well plates and cultured overnight in a CO ₂ incubator at 37 ℃. And inducing differentiated cells by PMA with the concentration of 100ng/mL, wherein the cell wall is shaped like a fusiform or a polygon after 48 hours, and differentiating into macrophages.

Real-time fluorescence quantitative PCR (qRT-PCR)

Total mRNA was isolated from cells using Trizol reagent (R701-01-AA, norflud) according to the manufacturer's instructions. Reverse transcription (Reverse transcription PCR, RT-PCR) was performed using HISCRIPT II SELECT QRT supermix (R222-01, norflu) in a final volume of 20. Mu.L containing 1. Mu.g RNA. qRT-PCR was performed on a real-time fluorescent quantitative PCR (quantitative Real-timePCR, qRT-PCR) detection system using gene-specific primers and SYBR Green (Q712-02, norpran). All primers were designed by us themselves and synthesized at Jin Weizhi biotechnology limited (beijing, china). And (3) taking beta-actin as a reference gene, and adopting a 2-delta t method to perform data analysis.

The specific operation steps are as follows:

RNA extraction (1) cells were collected in 1.5mL enzyme-free EP tube, 200. Mu.L Trizol reagent was added, and the homogenate was used to lyse the cells. After 5min incubation at room temperature, centrifugation was performed at 12000rpm for 5min at 4 ℃. (2) Transfer the supernatant to a fresh EP tube, add 4/5 volume of isopropanol, shake 10 times upside down to mix well and then stand at room temperature for 10min. (3) Centrifuging at 12000rpm at 4deg.C for 10min, removing supernatant, collecting white feather-like precipitate to obtain RNA, washing with 800 μl DEPC water containing 75% ethanol twice, removing supernatant, volatilizing precipitate, and dissolving precipitate with 20 μl DEPC water on ice to obtain RNA. (4) measuring the concentration of RNA with a Nanodrop micro-spectrophotometer.

CDNA was synthesized by RT-PCR using a kit for RT-PCR of Reprandial (HISCRIPT IIQ RT SuperMix for qPCR).

TABLE 4 preparation of first strand cDNA Synthesis reaction solution

After gentle mixing at 500rpm, centrifuge for 1min to bottom.

TABLE 5 first strand cDNA synthesis reactions were performed under the following conditions

The 20. Mu.L product obtained can be used for qRT-PCR reaction immediately or stored at-20deg.C (available within half a year), and can be stored at-80deg.C after long-term storage and packaging.

qRT-PCR:

Table 6A total of 20. Mu.L of reaction system was as follows:

after being mixed evenly, the liquid is centrifuged to the bottom, and amplified on a real-time fluorescent quantitative PCR instrument.

The reaction conditions were 94℃for 30s, 94℃for 5s, 60℃for 30s, and 40 cycles were repeated. Bio-Rad CFX Manager Software automatically records amplification data expressed as threshold cycle (CT value) and dissolution curve, and uses 2-delta t method to analyze the data. The primer sequences (SEQ ID NOS: 4-11) are shown in the following table.

TABLE 7 primer sequences

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

JN-7 down-regulates LPS-stimulated expression of cellular pro-inflammatory factor mRNA (see fig. 5). JN-7 and LGG were co-cultured with macrophages formed by THP-1 differentiation, respectively, and the anti-inflammatory effect of the strain was evaluated by detecting the expression levels of proinflammatory factors IL-6, IL-8 and anti-inflammatory cytokines IL-10 mRNA. IL-6 and IL-8mRNA expression was decreased and IL-10mRNA expression was increased in cells containing JN-7 and LGG after LPS stimulation compared to LPS stimulated control. In addition, LGG has better effect of down-regulating IL-8mRNA expression stimulated by LPS than JN-7.JN-7 has a higher ability to promote IL-10 release than LGG, suggesting that JN-7 has an anti-inflammatory effect as LGG.

EXAMPLE 5 Lactobacillus plantarum JN-7 powder production and stability analysis

1) JN-7 fermentation and fungus powder production

Inoculating Lactobacillus plantarum JN-7 into MRS broth, culturing at 37deg.C for 18 hr, activating twice, and treating in 5L bioreactor320, Eppendorf) for 12 hours. After the fermentation was completed, the cells were collected at 4℃and 10000rpm for 10 min. The obtained thallus is mixed with protective agent (1-10 g/L polysaccharide, 20-50g/L disaccharide, 1-20g/L vitamin C or its salt substance, and 1-10g/L peptone), and pre-frozen at-80deg.C for 24 hr. Freeze-drying the bacterial powder by a vacuum freeze dryer (PO 14416, telstar LyoQuest-55 plus) and pulverizing into powder, and vacuum packaging in an aluminum foil bag.

After fermentation in a 5L bioreactor for 12 hours, a total of 41.5g of precipitate were collected after centrifugation. The pellet was mixed with a protectant and the mixture was freeze-dried to yield 6.6g of a bacterial powder having a viable cell count of 7.07 x 10 ¹¹ CFU/g.

2) Evaluation of stability of Lactobacillus plantarum JN-7 powder

Packaging lactobacillus plantarum JN-7 bacteria powder into an aluminum foil bag, storing the lactobacillus plantarum JN-7 bacteria powder in a constant humidity box with the temperature of 40 ℃ and the humidity of 75% for accelerated test, simultaneously using commercially available lactobacillus rhamnosus LGG bacteria powder and 15 lactobacillus plantarum (the bacteria powder production process is the same as that of JN-7) which are also separated from fermented vegetables for comparison under the same temperature and humidity, sampling the JN-7 bacteria powder, the LGG bacteria powder and 15 lactobacillus plantarum bacteria powder within 90 days, detecting the quantity of lactobacillus in the bacteria powder according to national standard GB 4789.35, and measuring the water activity of the bacteria powder.

Viable cell count and water activity of Lactobacillus plantarum JN-7 (FIG. 6A), lactobacillus rhamnosus LGG (FIG. 6B) and other 15 strains of Lactobacillus plantarum powder (FIG. 6C) during storage are shown in FIGS. 6A-6C. From the graph, the stability of the JN-7 bacterial powder is obviously higher than that of the other 16 bacterial powders. The JN-7 70 days old living cells decreased from 7.07×10 ¹¹ to 1.73×10 ¹¹ CFU/g, with a corresponding increase in water activity from 0.024 to 0.053. The control LGG reduced viable cells from 3.53X10 ¹¹ to 3.92X10 ⁸ CFU/g within 70 days, with a corresponding increase in water activity from 0.091 to 0.134. The reduction of JN-7 viable bacteria was linear fit (r ² =0.80) by a 70 day test. It can be predicted by fitting that under accelerated stability test conditions, the time required for 1 log reduction of viable bacteria count is 136.99 days longer than 24.63 days required for fitting predictive LGG, 112.36 days longer.

In general, the present application isolated and identified a Lactobacillus plantarum JN-7 from a natural fermentation product. After whole genome sequencing and comparison with all the Lactobacillus plantarum genomes retrievable by NCBI, we found that the strain Lactobacillus plantarum JN-7 was a new strain. The lactobacillus plantarum JN-7 is found to be a safe strain and has excellent production performance, high stability and anti-inflammatory efficacy through 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 freeze-drying yield) and stability test (accelerated storage stability test) of the strain.

Claims

1. The lactobacillus plantarum JN-7 is classified and named as lactobacillus plantarum (Lactiplantibacillus plantarum), and the preservation number is CGMCC No.30605.

2. A bacterial powder prepared from lactobacillus plantarum JN-7 of claim 1.

3. The method for preparing the bacterial powder of claim 2, comprising the following steps:

1) Fermenting the lactobacillus plantarum JN-7;

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

5. The probiotic composition of claim 4, wherein the probiotic composition further comprises one or more probiotics selected from the group consisting of: bifidobacterium, lactobacillus mucilaginosus, lactobacillus and lactobacillus in combination with Lactobacillus, streptococcus, lactococcus, lactobacillus, streptococcus, lactobacillus, streptococcus, in combination with Lactobacillus, lactobacillus guangdum, lactobacillus Streptococcus, lactococcus.

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

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

8. Use of lactobacillus plantarum JN-7 of claim 1, the bacterial powder of claim 2 or the probiotic composition of claim 4 or 5 for the preparation of a food or health care product.

9. The 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. The use according to claim 8, wherein the food or nutraceutical further comprises edible adjuvants.