Lactobacillus plantarum BJ-M8 and application thereof

Abstract

The invention relates to the field of microbial fermentation, and provides lactobacillus plantarum BJ-M8 and application thereof. The lactobacillus plantarum BJ-M8 is obtained through screening, and compared with other strains, the lactobacillus plantarum BJ-M8 has good growth capacity and acid production performance and is suitable for solid-state and liquid-state fermentation. The lactobacillus plantarum BJ-M8 has high yield of glucoside hydrolase, glycosyl transferase, carbohydrase and polysaccharide lyase, the dandelion is fermented, the alkaloid, phenolic acid and flavonoid composition of the dandelion can be effectively improved, and the content of target products of naringenin, hesperetin, apigenin, luteolin, citrus aurantium, kaempferol and quercetin is increased. Meanwhile, the broiler chicken is taken as a test animal, and the fermented dandelion can improve the average daily feed intake and average daily gain of the LPS challenge broiler chicken and reduce the feed-weight ratio. The fermented dandelion can be used as a feed additive, and has great significance in livestock and poultry production and application.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation, in particular to a strain of Lactobacillus plantarum BJ-M8 and application thereof. Background Art

[0002] my country has a wide variety of Chinese herbal medicine resources, and there are nearly 12,000 medicinal plant resources that can be used as the basis for the research and development of feed additives. So far, the "Catalogue of Feed Raw Materials" has included 117 natural plants with medicinal and edible properties as feed raw materials, and studies have shown that some Chinese herbal medicines have antibacterial and immunostimulatory activities, and have the effect of improving animal meat quality and reducing animal stress. The antibiotic substitutes we choose must not only improve feed utilization and breeding efficiency, but also meet health, safety and environmental protection requirements. These Chinese herbal medicines with medicinal and edible properties have received widespread attention as the only growth-promoting additives allowed to be added in livestock and poultry production, and have become one of the effective strategies to replace antibiotics in breeding production.

[0003] Dandelion, commonly known as Dandelion, also known as Huahualang, Huanghua Sanqi, Baidiaoding, etc., belongs to the genus Taraxacum of the Asteraceae family and is a perennial herb. Dandelion is widely distributed in Northeast my country, North China, Northwest China, Central China, and Southwest China, and its resources are abundant, cheap and easy to obtain. It has shown that it can improve the production performance of livestock and poultry production and has good development and application prospects. However, the dense structure of the dandelion plant cell wall hinders the release of its active substances. At the same time, the effective ingredients of dandelion itself are complex and low in content, which greatly weakens its role.

[0004] Although the addition of carbohydrate enzymes can promote the degradation of plant leaf cell walls, it is too expensive to be used on a large scale. Lactobacillus plantarum is one of the edible microorganisms widely used in fermentation, and can achieve high-value conversion of substrates through its rich enzyme activity. During the fermentation process, Lactobacillus plantarum can not only produce health-promoting compounds, but also promote the conversion of substrates, improving their nutrition and value. Fermentation of dandelions with Lactobacillus plantarum can increase the content of active substances, produce new active ingredients, and reduce the composition of toxic and harmful substances. In addition, the mechanism of biotransformation of dandelions by Lactobacillus plantarum has not yet been clarified, and further research is needed. Summary of the invention

[0005] The invention aims to provide a strain of Lactobacillus plantarum BJ-M8 and application thereof.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a strain of Lactobacillus plantarum BJ-M8 isolated and purified from sauerkraut, which is classified and named Lactobacillus plantarum, this strain has now been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a postal code of 100101. The deposit number is CGMCC No. 31307, and the deposit date is July 15, 2024.

[0007] In a second aspect, the present invention provides a bacterial agent containing the Lactobacillus plantarum.

[0008] In a third aspect, the present invention provides any one of the following applications of the Lactobacillus plantarum: 1) Fermentation to produce acid; 2) Used for preparing an antioxidant preparation; 3) Used for preparing a feed additive; 4) Used for plant fermentation; 5) Used for food fermentation.

[0009] In a fourth aspect, the present invention provides a dandelion fermentation process, using the Lactobacillus plantarum or a mixed bacterium containing the Lactobacillus plantarum for dandelion fermentation.

[0010] Furthermore, the optimal fermentation process includes: crushing dry dandelions, mixing them with water at a material-liquid ratio of 1 g:(1 - 2) mL as the fermentation substrate, inoculating the Lactobacillus plantarum bacterial solution into the fermentation substrate at a volume ratio of 4 - 6% (preferably 5%) for sealed fermentation; wherein, the content of Lactobacillus plantarum in the bacterial solution is 1×10 8 -10 10 CFU / mL.

[0011] Preferably, the fermentation conditions are: fermenting at 34 - 38 °C for 16 - 20 d.

[0012] In a fifth aspect, the present invention provides a dandelion fermentation product prepared according to the above process.

[0013] In a sixth aspect, the present invention provides the application of the dandelion fermentation product in preparing a feed additive.

[0014] In a seventh aspect, the present invention provides the application of the dandelion fermentation product in livestock and poultry breeding.

[0015] In a specific embodiment of the present invention, the dandelion fermentation product can be used as a feed additive to feed broilers.

[0016] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: The present invention screened and obtained a strain of Lactobacillus plantarum ( Lactobacillus plantarum) BJ-M8 has good growth ability and acid production performance and is suitable for solid-state fermentation. Lactobacillus plantarum BJ-M8 can effectively improve the composition of alkaloids, phenolic acids and flavonoids in dandelion through fermentation. The glycoside hydrolases, glycosyltransferases, carbohydrate enzymes and polysaccharide lyases produced by Lactobacillus plantarum promote the degradation of the cell wall of plant leaves and catalyze the hydrolysis of glycosidic bonds, thereby promoting the conversion of macromolecular glycosides in dandelion into small molecular aglycones and increasing the contents of target products such as naringenin, hesperetin, apigenin, luteolin, auraptene, kaempferol and quercetin. At the same time, fermented dandelion can increase the average daily feed intake and average daily weight gain of broilers challenged with LPS and reduce the feed-to-weight ratio. Fermented dandelion can be used as a feed additive to feed broilers, which has great significance in the application of poultry production. Description of the Drawings

[0017] Figure 1 This is the morphological colony of Lactobacillus plantarum BJ-M8 in the preferred embodiment of the present invention.

[0018] Figure 2 This is the genomic circular map of Lactobacillus plantarum BJ-M8 in the preferred embodiment of the present invention.

[0019] Figure 3 This is the potential carbohydrate-active enzyme genes of Lactobacillus plantarum BJ-M8 in the preferred embodiment of the present invention.

[0020] Figure 4 This is the 24-hour growth curve of Lactobacillus plantarum BJ-M8 in the preferred embodiment of the present invention.

[0021] Figure 5 This is the 24-hour acidity change of Lactobacillus plantarum BJ-M8 in the preferred embodiment of the present invention.

[0022] Figure 6 This is the gastric acid tolerance of Lactobacillus plantarum BJ-M8 in the preferred embodiment of the present invention.

[0023] Figure 7 This is the bile salt tolerance of Lactobacillus plantarum BJ-M8 in the preferred embodiment of the present invention.

[0024] Figure 8 This is the antioxidant capacity of Lactobacillus plantarum BJ-M8 in the preferred embodiment of the present invention.

[0025] Figure 9 This is the process flow chart of fermented dandelion in the preferred embodiment of the present invention.

[0026] Figure 10 This is the total ion chromatogram of QC samples in the preferred embodiment of the present invention. A is the negative ion mode and B is the positive ion mode.

[0027] Figure 11A and Figure 11BThis is a diagram of the categories and compositions of dandelion and fermented dandelion metabolites in a preferred embodiment of the present invention.

[0028] Figure 12 This is a clustering heat map of differential metabolites in a preferred embodiment of the present invention.

[0029] Figure 13A and Figure 13B This is a KEGG enrichment diagram of the metabolic pathways of differential metabolites in a preferred embodiment of the present invention.

[0030] Figure 14 This is a core metabolic pathway diagram of flavonoid biosynthesis in a preferred embodiment of the present invention. Detailed implementation manners

[0031] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0032] The dandelion used in the following examples was purchased from Changda Chinese Herbal Medicine Pieces Co., Ltd. (Baoding City, Hebei Province), pulverized and sieved through a 40-mesh sieve for standby.

[0033] Lactobacillus plantarum (BJ-M8) was isolated and identified by the Feed Resources and Biological Conversion Team Laboratory of the Feed Research Institute, Chinese Academy of Agricultural Sciences. It has excellent acid-producing performance. Lactobacillus plantarum BJ-M8 was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on July 15, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31307.

[0034] The MRS liquid medium was purchased from Shanghai Solarbio Science & Technology Co., Ltd.

[0035] The experimental data was statistically analyzed using SPSS 26.0 (IBM) software. The one-way analysis of variance (ANOVA) program was used for statistical analysis. The data was expressed as the mean and standard error of the mean (SEM). P<0.05 indicates significant difference. GraphPad Prism was used to make and edit column charts and line charts.

[0036] Example 1 Discovery, isolation and purification of target strains The bacteria-screening sample uses naturally fermented and safely edible Northeast sauerkraut as the raw material. Under aseptic conditions, weigh 5 g of the sample and place it in a triangular flask containing 45 mL of normal saline and glass beads. Shake at 180 rpm for about 20 min to fully disperse the microorganisms in the sample and prepare a microbial suspension. Then, perform gradient dilution of the suspension in a sterile operating table. Take 100 μL and spread it evenly on the MRS agar medium plate. Place it in a biochemical incubator at 37 °C and incubate it upside down for 48 h. Pick the most vigorously growing single colony and continue to purify it three generations on the MRS broth medium and MRS agar medium. Finally, number the isolated single colonies and store them frozen in the MRS broth medium containing 50% glycerol (cryoprotectant) at -80 °C for later use.

[0037] Figure 1 It is the morphological colony of Lactobacillus plantarum BJ-M8.

[0038] Example 2 Identification of Lactobacillus plantarum BJ-M8 16S rRNA gene sequencing and phylogenetic analysis were used to determine the molecular characteristics of strain BJ-M8. The strain was entrusted to a limited company for 16S rRNA gene sequencing, using universal bacterial primers (27F, 1492R), 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-CTACGGCTACCTTGTTACGA-3′. The obtained gene sequence (SEQ ID NO:1) was compared and analyzed with the data in the NCBI 16S database using the Blast program of the National Center for Biotechnology Information (NCBI). Strain BJ-M8 was identified as Lactobacillus plantarum ( Lactobacillus plantarum )

[0039] The strain BJ-M8 was entrusted to Beijing Biomarker Technologies Co., Ltd. for whole-genome sequencing. Figure 2 It is the genome circular map of Lactobacillus plantarum BJ-M8. Carbohydrate-active enzymes (CAZymes) are mainly responsible for the cleavage, modification, or formation of glycosidic bonds. The strain was entrusted to Beijing Biomarker Co., Ltd. for whole-genome sequencing, and it was found that the genome of Lactobacillus plantarum BJ-M8 contains 128 potential CAZy genes such as Figure 3Shown are 8 genes related to auxiliary activity (AA), which function in redox reactions synergistic with CAZy; 19 genes encoding carbohydrate esterase (CE), which function in the hydrolysis of carbohydrate esters; 15 genes of non-catalytic active carbohydrate-binding module (CBM), which function in carbohydrate adhesion and also belong to glycoside hydrolases; 49 genes encoding glycoside hydrolase (GH), which function in the hydrolysis or rearrangement of glycosidic bonds; 36 genes encoding glycosyl transferase (GT), which function in the formation of glycosidic bonds; and 1 gene of polysaccharide lyase (PL), which function in the degradation of polysaccharides.

[0040] Glycoside hydrolase (GH) has the potential to hydrolyze complex carbohydrates and is considered a key enzyme responsible for carbohydrate metabolism. The most abundant CAZy classification predicted in the genome of Lactobacillus plantarum BJ-M8 is GH, which contains 49 genes belonging to 18 different families, including GH1, GH2, GH4, GH113, GH20, GH25, GH31, GH32, GH36, GH38, GH42, GH65, GH73, GH78, GH92, GH109, and GH125. Nine genes belonging to the GH1 family, one gene belonging to the GH2 family, two genes belonging to the GH36 family, and two genes belonging to the GH78 family in the genome of Lactobacillus plantarum BJ-M8 can be used to hydrolyze the carbohydrates in dandelion cell walls; eight genes belonging to the GH13 family and two genes belonging to the GH4 family can be used to degrade starch and glycogen; four genes belonging to the GH25 family and three genes belonging to the GH73 family can be used to hydrolyze peptidoglycan; and one gene belonging to the GH32 family can be used to hydrolyze sucrose and fructan. As shown in Tables 1 and 2, Lactobacillus plantarum BJ-M8 has genes that can produce glycoside hydrolase, glycosyl transferase, carbohydrate enzymes, and polysaccharide lyase, which can promote the degradation of plant leaf cell walls and catalyze the hydrolysis of glycosidic bonds, and thus be applied to the fermentation of Chinese herbal medicines.

[0041] Table 1 Analysis of Carbohydrate-Active Enzymes in Lactobacillus plantarum BJ-M8

[0042] Table 2 Analysis of Carbohydrate-Active Enzymes in Lactobacillus plantarum BJ-M8

[0043] Identification of Physicochemical Properties of Lactobacillus plantarum BJ-M8 in Example 3 1. Growth Curve and Acidity Change Inoculate BJ-M8 into MRS broth medium at an inoculation amount of 1%, and incubate it at a constant temperature of 37 °C for 24 h. Take the bacterial liquid every 2 h, and measure the absorbance value at OD 600 nm and pH value. Use the OD 600 value as the ordinate and time as the abscissa to plot the 24-h growth curve of the strain; use the pH value as the ordinate and time as the abscissa to plot the 24-h acidity change curve of the strain. Each sampling is measured in parallel three times. The results are as shown in Figure 4 and Figure 5 . Lactobacillus plantarum BJ-M8 reaches the plateau after 12 h; the initial pH of Lactobacillus plantarum BJ-M8 is 6.13, the pH drops below 4 after the 14th hour, and the pH is stably maintained at about 3.85 after 18 h, that is, Lactobacillus plantarum BJ-M8 has a strong acid-producing ability.

[0044] 2. Gastric Acid Tolerance Inoculate the liquid culture that has been activated for 2 generations into a 10 mL test tube of MRS broth medium at an inoculation amount of 1%, and incubate it statically at 37 °C for 24 h for standby. Based on sterile normal saline, adjust its pH to 2.5 with hydrochloric acid with a volume fraction of 37% and mix it well with the standby fermentation broth of the strain to be tested (v / v, 10%) as the test group. Take samples after incubating and culturing at 37 °C in the dark for 1 h, 2 h, 3 h, and 4 h respectively, spread them on plates, and measure the viable count; use sterile normal saline to replace the hydrochloric acid treatment solution with pH 2.5 as the control group. Each test is set with three replicates. The tolerance of lactic acid bacteria strains to gastric acid (hydrochloric acid) is characterized by the survival rate. The survival rate calculation formula: Lactic acid bacteria survival rate (%) = (viable count in the test group medium / viable count in the control group medium) × 100. The results are as shown in Figure 6 . The survival rate at 1 h of gastric acid is 54.41%, the survival rate at 2 h is 30.88%, the survival rate at 3 h is 18.38%, and the survival rate at 4 h is 11.03%, that is, Lactobacillus plantarum BJ-M8 has the ability to tolerate gastric acid.

[0045] 3. Bile Salt Tolerance The activated liquid culture was inoculated into 10 mL of MRS broth medium at an inoculation amount of 1%, and statically cultured at 37 °C for 24 h for standby. After preparing a 0.3% sodium taurocholate solution based on sterile normal saline and fully mixing it with the standby fermentation broth of the strain to be tested (v / v, 10%), it was used as the experimental group. Samples were taken after incubation at 37 °C in the dark for 1 h, 2 h, 3 h, and 4 h respectively, plated, and the viable bacteria count was measured; sterile normal saline was used to replace the 0.3% sodium taurocholate solution as the control group. Each experiment was set with three replicates. The tolerance of lactic acid bacteria strains to bile salts was characterized by the survival rate, and the survival rate calculation formula was: lactic acid bacteria survival rate (%) = (number of viable bacteria in the experimental group medium / number of viable bacteria in the control group medium) × 100. The results are as Figure 7 shown. The survival rate of Lactobacillus plantarum in the sodium taurocholate solution was 63.28% at 1 h, 28.13% at 2 h, 7.81% at 3 h, and 0 at 4 h, that is, Lactobacillus plantarum BJ-M8 has a certain bile salt tolerance ability.

[0046] 4. Evaluation of antioxidant capacity Using the T-AOC assay kit and SOD assay kit from Nanjing Jiancheng, the total antioxidant capacity and SOD enzyme activity of the fermentation supernatant of the selected strains cultured for 24 h were evaluated. The test operations and calculation formulas were carried out according to the kit instructions, and each strain sample was measured three times repeatedly. The results are as Figure 8 shown. The SOD enzyme activity of Lactobacillus plantarum BJ-M8 was 99 U / mL; the T-AOC (total antioxidant capacity) of Lactobacillus plantarum BJ-M8 was 2.5 mmol / mL; that is, Lactobacillus plantarum BJ-M8 has relatively strong antioxidant capacity.

[0047] Example 4 Fermentation of dandelion by Lactobacillus plantarum BJ-M8 The frozen Lactobacillus plantarum stored at -20 °C was quickly thawed, inoculated into autoclaved MRS liquid medium respectively, and statically cultured at 37 °C for 24 h to observe the activation of the strains. Then, they were inoculated into MRS liquid medium at an inoculation amount of 5% respectively, and statically cultured at 37 °C for 24 h. After microscopic examination and counting, according to the pre-experiment results, the inoculation bacteria at 1×10 9 CFU / mL was used as the optimal inoculation amount, and its content was adjusted as the solid fermentation strain for standby.

[0048] The dry dandelion was crushed and passed through a 40-mesh sieve, divided into 12 groups, with 3 replicates in each group. Each replicate was sealed and fermented in a 35 mm × 45 mm plastic film fermentation bag for 30 d. As Figure 9As shown in the figure, Group A is the group with a dandelion material-to-water ratio of 1:1 (g:mL). 5% (volume percentage) of Lactobacillus plantarum bacterial liquid was inoculated into the fermentation substrate and cultured at 30 °C; Group B is the group with a dandelion material-to-water ratio of 1:1 (g:mL). 5% of Lactobacillus plantarum bacterial liquid was inoculated into the fermentation substrate and cultured at 37 °C; Group C is the group with a dandelion material-to-water ratio of 1:2 (g:mL). 5% of Lactobacillus plantarum bacterial liquid was inoculated into the fermentation substrate and cultured at 30 °C; Group D is the group with a dandelion material-to-water ratio of 1:2 (g:mL). 5% of Lactobacillus plantarum bacterial liquid was inoculated into the fermentation substrate and cultured at 37 °C; Group E is the group with a dandelion material-to-water ratio of 1:1 (g:mL). 0.2% (weight percentage) of cellulase (2.0×10 5 U / g, the CAS number of cellulase is 9012-54-8, purchased from Shandong Longkete Enzyme Preparation Co., Ltd.) was added to the fermentation substrate and cultured at 30 °C; Group F is the group with a dandelion material-to-water ratio of 1:1 (g:mL). 0.2% of cellulase was added to the fermentation substrate and cultured at 37 °C; Group G is the group with a dandelion material-to-water ratio of 1:2 (g:mL). 0.2% of cellulase was added to the fermentation substrate and cultured at 30 °C; Group H is the group with a dandelion material-to-water ratio of 1:2 (g:mL). 0.2% of cellulase was added to the fermentation substrate and cultured at 37 °C; Group I is the group with a dandelion material-to-water ratio of 1:1 (g:mL). 2.5% of Lactobacillus plantarum bacterial liquid and 0.1% of cellulase were added to the fermentation substrate and cultured at 30 °C; Group J is the group with a dandelion material-to-water ratio of 1:1 (g:mL). 2.5% of Lactobacillus plantarum bacterial liquid and 0.1% of cellulase were added to the fermentation substrate and cultured at 37 °C; Group K is the group with a dandelion material-to-water ratio of 1:2 (g:mL). 2.5% of Lactobacillus plantarum bacterial liquid and 0.1% of cellulase were added to the fermentation substrate and cultured at 30 °C; Group L is the group with a dandelion material-to-water ratio of 1:2 (g:mL). 2.5% of Lactobacillus plantarum bacterial liquid and 0.1% of cellulase were added to the fermentation substrate and cultured at 37 °C.

[0049] Example 5 Changes in Fermented Dandelion Flavonoid Content and pH 1. pH Measurement Samples were taken from multiple points in 4 test groups on days 0, 2, 4, 6, 8, 10, 12, 16, and 20 respectively. A total of 1 g was taken from 5 points, namely the upper, middle, lower, left, and right points in each group, and 10 mL of deionized water was added. After vortex mixing for 30 min, the pH was measured using a pH meter.

[0050] 2. Flavonoid Content Measurement (1) Prepare the extraction solution: 60% ethanol; (2) Add 10 mg of rutin to 1 mL of the standard dilution solution to prepare a 10 mg / mL standard solution; (3) Preheat the microplate reader for more than 30 min and adjust the wavelength to 470 nm; (4) Dilute the 10 mg / mL rutin standard solution with the standard dilution solution in a 1.5 mL centrifuge tube to 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, 0.039 mg / mL; (5) Dry the dandelion at 37 °C to constant weight, crush it, pass through a 40-mesh sieve, weigh about 0.1 g, add 1 mL of the extraction solution, and perform extraction by ultrasonic extraction method. The ultrasonic power is 300 W, the temperature is 60 °C, and the extraction time is 30 min. Centrifuge at 12000 rpm and 25 °C for 10 min, take the supernatant, and make up the volume to 1 mL with 60% ethanol of the extraction solution; (6) Add them to a 1.5 mL centrifuge tube in turn according to the flavonoid reagent instruction manual, vortex and mix well, place in a 37 °C water bath for an accurate reaction of 45 min, then centrifuge at 10000 g at room temperature for 10 min, take 200 μL of the supernatant and put it into a 96-well plate, and use a microplate reader to measure OD 470 .

[0051] The results are as Figure 9 shown. The total flavonoid content in group A is 17.87 mg / g, and the contents of kaempferol and auraptene are increased by 9.76 times; the total flavonoid content in group B is 20.45 mg / g, and the contents of kaempferol and auraptene are increased by 13.05 times; the total flavonoid content in group C is 16.49 mg / g, and the contents of kaempferol and auraptene are increased by 10.11 times; the total flavonoid content in group D is 19.13 mg / g, and the contents of kaempferol and auraptene are increased by 12.13 times; the total flavonoid content in group E is 14.65 mg / g, and the contents of kaempferol and auraptene are increased by 1.58 times; the total flavonoid content in group F is 15.14 mg / g, and the contents of kaempferol and auraptene are increased by 1.02 times; the total flavonoid content in group G is 15.23 mg / g, and the contents of kaempferol and auraptene are increased by 1.79 times; the total flavonoid content in group H is 15.62 mg / g, and the contents of kaempferol and auraptene are increased by 1.34 times; the total flavonoid content in group I is 17.38 mg / g, and the contents of kaempferol and auraptene are increased by 6.75 times; the total flavonoid content in group J is 18.71 mg / g, and the contents of kaempferol and auraptene are increased by 7.97 times; the total flavonoid content in group K is 16.56 mg / g, and the contents of kaempferol and auraptene are increased by 5.59 times; the total flavonoid content in group L is 16.77 mg / g, and the contents of kaempferol and auraptene are increased by 6.41 times.

[0052] Example 6 Targeted metabolomics of dandelion and fermented dandelion Take the sample and place it in a freeze dryer for vacuum freeze-drying for 63 h. After grinding it into a powder, weigh 50 mg of the sample powder, add 1200 μL of an internal standard extraction solution of 70% methanol-water precooled to -20 °C, vortex it 6 times for 30 seconds continuously, centrifuge (rotation speed 12000 rpm, 3 minutes), then aspirate the supernatant, filter it through a 0.22 μm microporous filter membrane, and store it in a sample injection vial for UPLC-MS / MS analysis.

[0053] The UPLC liquid phase uses an AgilentSB-C18 1.8 µm, 2.1 mm×100 mm chromatographic column; the mobile phase is that phase A is ultrapure water (added with 0.1% formic acid), and phase B is acetonitrile (added with 0.1% formic acid); the elution gradient is that the proportion of phase B is 5% at 0.00 min, linearly increases to 95% within 9.00 min, and is maintained at 95% for 1 min, from 10.00 - 11.10 min, the proportion of phase B drops to 5%, and is balanced at 5% until 14 min; the flow rate is 0.35 mL / min; the column temperature is 40 °C; the injection volume is 2 μL.

[0054] For MS / MS mass spectrometry, the electrospray ionization (ESI) source temperature is 500 °C; the ion spray voltage (IS) is 5500 V (positive ion mode) / -4500 V (negative ion mode); the source gas I (GSI), gas II (GSII) and curtain gas (CUR) are set to 50, 60 and 25 psi respectively, and the collision-induced ionization parameters are set to high. The QQQ scan uses the MRM mode, and the collision gas (nitrogen) is set to medium. Through further optimization of the declustering potential (DP) and collision energy (CE), the DP and CE of each MRM ion pair are completed. According to the metabolites eluted in each period, a specific set of MRM ion pairs is monitored in each period.

[0055] The results are shown in Table 3, Figure 10 、 Figure 11A and Figure 11BAs shown in the figure, in the identification and analysis of the secondary metabolites of dandelion, by performing total ion current map superposition analysis on QC samples, it was found that in both positive and negative ion modes, the total ion current curves of metabolite detection highly overlapped, and the retention times and peak intensities were consistent, indicating that the established analytical method has signal stability, data repeatability, and instrument stability. A total of 1,522 metabolites were identified in dandelion and fermented dandelion, with 300 up-regulated and 537 down-regulated. Among the metabolites, there were 394 flavonoid compounds, accounting for 25.89%; 258 phenolic acid compounds, accounting for 16.95%; 290 alkaloid compounds, accounting for 19.05%; 246 terpene compounds, accounting for 16.16%; 118 lignin and coumarin compounds, accounting for 7.75%; 30 quinone compounds, accounting for 1.97%; 13 tannin compounds, accounting for 0.85%; and 173 other compounds such as lactones, accounting for 11.37%.

[0056] Table 3 Some significantly changed metabolites in fermented dandelion

[0057] At the same time, in Figure 12 the clustering heat map of differential metabolites and target metabolites, it can be found that Lactobacillus plantarum fermentation of dandelion can effectively improve the composition of alkaloids, phenolic acids, and flavonoids in dandelion. At the same time, it was found that Lactobacillus plantarum can promote the conversion of macromolecular glycosides into small molecular aglycones, which may be due to the glycoside hydrolases, glycosyltransferases, carbohydrases, and polysaccharide lyases produced by Lactobacillus plantarum promoting the degradation of the plant leaf cell wall and catalyzing the hydrolysis of glycosidic bonds, thereby promoting the conversion of glycosides into aglycones.

[0058] According to Figure 13A and Figure 13B the KEGG enrichment map of differential metabolite metabolic pathways and Figure 14 the core metabolic pathway map analysis of flavonoid biosynthesis, most compounds were enriched in the synthesis of secondary metabolites and the phenylpropanoid biosynthesis pathway, among which the biosynthesis of flavonoids and flavonols and the biosynthesis of flavonoids were more obvious. Finally, the biosynthesis of flavonoids was determined to be the most dynamic metabolic pathway during the fermentation process. Naringenin can be converted into genistein through isoflavone biosynthesis. Naringenin is also oxidized to auraptene, which is further oxidized to taxifolin, and then oxidized and dehydroxylated to generate kaempferol and quercetin. Finally, quercetin reacts with glucose to form rutin. Similarly, naringenin can be oxidized to apigenin and eriodictyol, then oxidized to luteolin and chrysoeriol, and chrysoeriol is dehydrogenated to generate genistein, and luteolin reacts with glucose to form luteolin glycoside.

[0059] Naringin (downregulated by 0.088 times after fermentation) is hydrolyzed by the glycosidase of Lactobacillus plantarum B8-M8 to produce rhamnose and naringenin (upregulated by 1.711 times after fermentation). Naringenin can increase the permeability of bacteria by regulating gene expression and inhibiting the action of enzymes, reduce virulence and motility, thereby achieving the effect of anti-pathogenic bacteria; it can also regulate signal pathways such as TLR2 / NF-κB, Nrf2 / HO-1, and PI3K / AKT, initiate intracellular lysosomes and autophagy, and inhibit the polarization of T cells and macrophages to reduce the body's inflammatory response; it can also play a strong antioxidant role by directly scavenging free radicals, regulating the antioxidant enzyme system, and participating in the redox system. Quercetin (upregulated by 3.494 times after fermentation) produced by the hydrolysis of rutin (downregulated by 0.015 times after fermentation) is a natural antioxidant that scavenges free radicals in the body, stimulates the immune system through different signal pathways, regulates enzyme activity, changes the mitotic cycle, and changes gene expression, thus having a wide range of biological activities, such as reducing blood pressure, reducing capillary fragility, lowering blood lipids, dilating the coronary artery, and increasing coronary blood flow; it also has antioxidant, anti-diabetic, anti-inflammatory, antiviral, antibacterial, and anti-cancer effects. Hesperetin (upregulated by 20.927 times after fermentation) is produced by the hydrolysis of hesperidin (downregulated by 0.374 times after fermentation). Apigenin (upregulated by 2.364 times after fermentation) is produced by the hydrolysis of apiin (downregulated by 0.136 times after fermentation). Luteolin (upregulated by 1.498 times after fermentation) is produced by the decomposition of rutinose. Kaempferol (upregulated by 5.881 times after fermentation) and aurapten (upregulated by 7.169 times after fermentation) are produced by the hydrolysis of kaempferitrin (downregulated by 0.051 times after fermentation) by glycosidase. These glycosides are hydrolyzed into aglycones in the active glycoside ligand form, which can enhance the body's antioxidant capacity by directly scavenging free radicals and activating the Keap1-Nrf2-ARE signal pathway; they can also play an anti-inflammatory role by inhibiting inflammatory signal pathways such as Txnip / NLRP3, MAPK, and NF-κB.

[0060] In summary, during the fermentation process, the content of key precursor substances for the synthesis of flavonoid compounds such as naringenin, hesperetin, apigenin, luteolin, aurapten, kaempferol, and quercetin concerned in the present invention increased significantly, while the content of luteolin and rutin decreased significantly. These reflect the significant upregulation of flavonoid aglycones in fermented dandelion, effectively improving the antioxidant and anti-inflammatory activities of dandelion. Moreover, flavonoid aglycones are more easily absorbed by the gastrointestinal tract and have high bioavailability, which also indicates that Lactobacillus plantarum greatly improves the antioxidant and anti-inflammatory effects of dandelion.

[0061] Example 7 Animal Experiments on the Application of Dandelion and Fermented Dandelion Take 30 grams of fermented dandelion, add 300 mL of pure water respectively, soak in cold water, boil over high heat, and decoct over low heat for 30 minutes for the first decoction. Filter the medicinal liquid to obtain the medicinal stock solution containing active ingredients. The experiment was conducted at the Nankou Experimental Base of the Feed Research Institute, Chinese Academy of Agricultural Sciences.

[0062] For the broiler oxidative damage model, 40 one-day-old Arbor Acres broilers with similar health and body weight (0.047 kg) were selected. Using a single-factor randomized experimental design, they were divided into 4 groups, with 10 replicates in each group and 1 broiler in each replicate. Group A was the negative control group, Group B was the positive control group (LPS challenge), Group C was the dandelion experimental group (LPS + dandelion extract), and Group D was the fermented dandelion group (LPS + fermented dandelion extract). Among them, in Groups B, C, and D, LPS was intraperitoneally injected at a dose of 0.5 mg / kg body weight on days 19, 20, and 21 to establish an oxidative stress model; Group A was injected with medical saline. In Groups C and D, the extract was continuously administered by gavage 3 days before the challenge. On days 1, 19, and 21 of the experiment, the body weight was measured in replicates, the feed intake of the broilers from 1 to 21 days was recorded, and the number of dead and culled broilers was recorded. The corn-soybean meal-based antibiotic-free basal diet was formulated with reference to the Chinese Broiler Nutritional Requirements (NY / T 33 - 2004) and the American NRC (1994) broiler nutritional requirement standards. The composition and nutritional levels of the basal diet formula are shown in Table 4.

[0063] As shown in Table 5, after intraperitoneal injection of LPS, the average daily feed intake and average daily weight gain of AA broilers decreased, while after administering dandelion and fermented dandelion oral liquids by gavage, the average daily feed intake and average daily weight gain of the broilers increased.

[0064] Table 4 Composition and nutritional levels of the basal diet (air-dried basis) 1 The premix provided per kilogram of diet: VA, 9,000 IU; VD3, 4,000 IU; VE, 26 IU; VK3, 4 mg; VB1, 2 mg; VB2, 8.8 mg; VB6, 9.8 mg; VB12, 0.03 mg; biotin, 0.18 mg; folic acid, 1.2 mg; D-calcium pantothenate, 20 mg; nicotinic acid, 40 mg; Cu, 12.5 mg; Fe, 105 mg; Mn, 120 mg; Zn, 100 mg; I, 0.70 mg; Se, 0.30 mg, choline, 1,000 mg / kg.

[0065] 2 The nutritional levels of the diet are calculated values.

[0066] Table 5 Effects of dandelion and fermented dandelion on growth performance of LPS-challenged broilers

[0067] Although the present invention has been described in detail with general descriptions and specific embodiments above, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the present invention claimed.

Claims

1. Lactobacillus plantarum ( Lactobacillus plantarum )BJ-M8, the deposit number is CGMCC No. 31307.

2. A bacterial agent containing the plant lactobacillus according to claim 1.

3. Any of the following applications of the plant lactobacillus according to claim 1: 1) Fermentation to produce acid; 2) Used for preparing antioxidant preparations; 3) Used for preparing feed additives; 4) Used in plant fermentation; 5) Used for food fermentation.

4. Dandelion fermentation process, characterized in that: Dandelion fermentation is carried out using the plant lactobacillus or a mixed bacteria containing the plant lactobacillus according to claim 1.

5. The process according to claim 4, characterized in that The optimal fermentation process includes: crushing dry dandelion, mixing dandelion and water at a ratio of 1g:(1-2)mL as a fermentation substrate, inoculating Lactobacillus plantarum culture liquid at a volume ratio of 4-6% into the fermentation substrate for sealed fermentation; wherein the content of Lactobacillus plantarum in the culture liquid is 1×10 8 -10 10 CFU / mL.

6. The process according to claim 5, characterized in that Fermentation conditions: 34-38℃ for 16-20 days.

7. A dandelion fermented product prepared according to the process described in any one of claims 4 to 6.

8. Use of the dandelion fermented product according to claim 7 in the preparation of feed additives.

9. Use of the dandelion fermented product according to claim 7 in livestock and poultry breeding.

10. The use according to claim 9, characterized in that: The dandelion fermented product is used as a feed additive to feed broiler chickens.

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