Application of bacillus amyloliquefaciens for resisting inflammation and inducing autophagy

Abstract

The invention discloses an application of bacillus amyloliquefaciens for resisting inflammation and inducing autophagy. The invention provides novel application of the bacillus amyloliquefaciens WF2020 strain in anti-inflammatory and autophagy induction, and research shows that the WF2020 strain has remarkable lipopolysaccharide degradation capacity, can reduce the level of inflammatory factors NO generated by lipopolysaccharide induction and prolong the service life of lipopolysaccharide induced caenorhabditis elegans; meanwhile, the anti-inflammatory action target spot of the WF2020 strain is obtained by activating a daf-2 gene mediated insulin signal channel IIS pathway and a sek-1 gene mediated p38MAPK signal channel, so that the WF2020 strain has a relatively good anti-inflammatory action effect. Besides, the WF2020 strain can increase LGG-1: GFP expression of autophagy research model animals and promote cell autophagy, and can be used for inducing autophagy, so that not only is new application of bacillus amyloliquefaciens expanded, but also more choices are provided for development of anti-inflammatory and autophagy promoting products.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the application of a strain of Bacillus amyloliquefaciens for anti-inflammation and inducing autophagy. Background Art

[0002] Lipopolysaccharide (LPS) is a component of the outer wall of the cell wall of Gram-negative bacteria. It is a substance composed of three parts: O-antigen, core polysaccharide and lipid A. When the bacteria die, lipopolysaccharide will fall off by dissolving and destroying the cells, and exert its toxicity by acting on animal cells, etc. When the dose is too high, it will cause inflammation of Gram-negative sepsis in humans or animals, and low doses of lipopolysaccharide will cause low-grade inflammation of metabolic disorders in humans or animals. With the research of microbial strains, the diversity of microorganisms provides rich resources for the research and development of new anti-inflammatory products. Microorganisms have high synthesis efficiency and selectivity, and can synthesize complex anti-inflammatory substances under relatively mild conditions, avoiding damage to anti-inflammatory substances under conditions such as high temperature and high pressure; compared with traditional chemical synthesis methods, microbial pharmaceuticals can use biological waste and renewable raw materials to reduce the impact on the environment. Existing research has used beneficial microbial strains to develop live biological drugs for the treatment of inflammatory bowel disease, diabetes and other diseases, but there is still a lack of microbial agents and products that can be used to treat inflammation in the body caused by LPS.

[0003] Most of the microbial strains that can degrade LPS reported so far can only be used to remove LPS in vitro, and their in vivo safety is poor. There are very few probiotics that can be used for in vivo anti-inflammatory. In addition, the strains that have the function of degrading LPS in existing studies are difficult to be industrially transformed and used for in vivo treatment. Existing research lacks microbial products that can be used to promote cell autophagy. Autophagy is a process of engulfing one's own cytoplasmic proteins or organelles and encapsulating them into vesicles, fusing with lysosomes to form autophagic lysosomes, and degrading the contents it encapsulates, thereby achieving the metabolic needs of the cell itself and the renewal of certain organelles. Promoting autophagy can reduce oxidative stress, improve metabolism, reduce inflammation, remove damaged cells, maintain cell homeostasis and stress resistance, and help resist infection and disease. By removing old and damaged cells and producing new and more powerful cells, it reduces inflammation and disease risks; autophagy also helps to promote the removal of waste in the body, reduce the accumulation of waste and toxins in the body, reduce damage to the body, and promote health and longevity. Currently available autophagy promoters such as hederin, rapamycin, spermidine, chlorogenic acid and other drugs can alleviate diseases by inducing autophagy, but there are few microbial agents and products that can promote cellular autophagy.

[0004] Bacillus amyloliquefaciens is a Gram-positive aerobic bacterium of the genus Bacillus. It can produce a series of metabolites that can inhibit fungi and bacteria during its growth and is widely present in nature. In recent years, many scholars have isolated and identified Bacillus amyloliquefaciens with antagonistic effects on a variety of pathogens from different materials. At the same time, the prior art also discloses Bacillus amyloliquefaciens that can be used for in vitro degradation of lipopolysaccharide LPS, but it is still unknown whether this strain can be used for in vivo inflammation treatment. At present, there is still a lack of Bacillus amyloliquefaciens that can be used for in vivo treatment and relieve inflammation induced by lipopolysaccharide LPS and can induce autophagy. In order to expand the microbial strain resource library with more functions, it is necessary to screen out more, more efficient and more functional Bacillus amyloliquefaciens. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of existing microbial strains that can be used for in vivo treatment and relief of lipopolysaccharide (LPS)-induced inflammation and induction of autophagy, and to provide an application of Bacillus amyloliquefaciens for anti-inflammatory and autophagy-inducing.

[0006] The purpose of the present invention is to provide a new application of Bacillus amyloliquefaciens WF2020 strain.

[0007] Another object of the present invention is to provide a method for degrading lipopolysaccharide for purposes other than disease diagnosis and treatment.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a new application of the Bacillus amyloliquefaciens WF2020 strain. The WF2020 strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 5, 2022, with a deposit number of GDMCC No: 62190. Studies have shown that the WF2020 strain has the ability to efficiently degrade lipopolysaccharides, and can completely remove 90EU of lipopolysaccharide LPS in 4 hours, and has a good in vitro effect of significantly reducing lipopolysaccharide LPS; at the same time, the experiment of lipopolysaccharide-induced mouse mononuclear macrophages RAW264.7 showed that the use of Bacillus amyloliquefaciens WF2020 cell-free supernatant can significantly reduce the inflammatory marker NO induced by LPS, and has an anti-inflammatory effect; and in the model animal nematode, the WF2020 strain also has a good anti-inflammatory effect, which can increase the survival rate of nematodes after LPS induction, and prolong the lifespan of nematodes after lipopolysaccharide LPS induction; further, the WF2020 strain was fed with daf-2 and sek-1 gene-deficient Caenorhabditis elegans, showing that the anti-inflammatory targets of the WF2020 strain are the IIS pathway of the insulin signaling pathway mediated by the daf-2 gene and the p38 MAPK signaling pathway mediated by the sek-1 gene. In addition, feeding the autophagy research model DA2123 Caenorhabditis elegans with the WF2020 strain can increase the expression of LGG-1::GFP, thereby inducing autophagy, promoting cellular autophagy, and being used to prepare autophagy promoters, thereby expanding the new applications of Bacillus amyloliquefaciens. It also provides more choices for microbial strains that can be used for in vivo treatment and relief of lipopolysaccharide (LPS)-induced inflammation and inducing autophagy.

[0010] Therefore, the present invention provides the use of Bacillus amyloliquefaciens WF2020 strain in the preparation of anti-inflammatory products.

[0011] Furthermore, the product can reduce the level of NO, a marker of inflammation.

[0012] Furthermore, the product can alleviate or treat inflammation induced by lipopolysaccharide.

[0013] The present invention provides the use of Bacillus amyloliquefaciens WF2020 strain in degrading lipopolysaccharide for the purpose of non-disease diagnosis and treatment.

[0014] The present invention provides application of Bacillus amyloliquefaciens WF2020 strain in preparing lipopolysaccharide degradation products.

[0015] The present invention provides use of the Bacillus amyloliquefaciens WF2020 strain in preparing a product that promotes autophagy.

[0016] The present invention also provides a method for degrading lipopolysaccharide for non-disease diagnosis and treatment purposes, wherein the sample is processed using the Bacillus amyloliquefaciens WF2020 strain and / or its bacterial solution.

[0017] Preferably, the product is food, medicine, feed or an additive thereof.

[0018] More preferably, the product is in the form of liquid, jelly, stick, powder, tablet, granule or capsule.

[0019] The present invention has the following beneficial effects:

[0020] The present invention provides a new application of Bacillus amyloliquefaciens WF2020 strain in anti-inflammatory and autophagy induction. Studies have shown that the WF2020 strain has a significant lipopolysaccharide degradation ability, can completely remove 90EU of lipopolysaccharide in 4h, and can reduce the inflammatory factor NO level produced by RAW264.7 induced by lipopolysaccharide and prolong the life span of Caenorhabditis elegans induced by lipopolysaccharide LPS; at the same time, since the WF2020 strain cannot prolong the life span of daf-2 and sek-1 gene-deficient Caenorhabditis elegans after lipopolysaccharide LPS induction, it is shown that the anti-inflammatory target of the WF2020 strain is the insulin signaling pathway IIS pathway mediated by the daf-2 gene and the p38 MAPK signaling pathway mediated by the sek-1 gene, so it has a good anti-inflammatory effect. In addition, the Bacillus amyloliquefaciens WF2020 strain can increase the expression of LGG-1::GFP in the autophagy research model DA2123 Caenorhabditis elegans, induce cellular autophagy, and can be used to prepare products that promote autophagy, expanding new applications of Bacillus amyloliquefaciens. It also provides more choices for microbial strains that can be used for in vivo treatment and relief of lipopolysaccharide (LPS)-induced inflammation and inducing autophagy. Bacillus amyloliquefaciens WF2020 can be used to prepare anti-inflammatory products and develop autophagy-inducing products. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the endotoxin standard curve of the automatic analyzer at 0h.

[0022] Figure 2 This is the 4h endotoxin standard curve of the automatic analyzer.

[0023] Figure 3 This is the test result of lipopolysaccharide (LPS) degradation (CONTROL in the figure is the sterile water group without inoculated bacteria, and WF2020 is the sterile water turbid liquid group containing only bacteria).

[0024] Figure 4 The effect of Bacillus amyloliquefaciens WF2020 on the inflammatory marker NO produced by mouse mononuclear macrophages RAW264.7 induced by lipopolysaccharide (LPS).

[0025] Figure 5The effect of Bacillus amyloliquefaciens WF2020 on the lifespan of wild-type Caenorhabditis elegans N2 induced by lipopolysaccharide (LPS).

[0026] Figure 6 The effect of Bacillus amyloliquefaciens WF2020 on the lifespan of daf-2 gene-deficient Caenorhabditis elegans CB1370 induced by lipopolysaccharide (LPS).

[0027] Figure 7 The effect of Bacillus amyloliquefaciens WF2020 on the lifespan of sek-1 gene-deficient Caenorhabditis elegans AU1 induced by lipopolysaccharide (LPS).

[0028] Figure 8 These are the fluorescence imaging results of LGG-1::GFP protein expression in DA2123 Caenorhabditis elegans (A: OP50, which is the Escherichia coli OP50 group; B: WF2020, which is the Bacillus amyloliquefaciens WF2020 group).

[0029] Fig. 9 The effect of Bacillus amyloliquefaciens WF2020 on the expression of LGG-1::GFP in DA2123 Caenorhabditis elegans. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] The research model mouse mononuclear macrophage RAW264.7 used in the present invention is a mouse macrophage cell line, which was originally isolated by the National Pox Institute (NCI) of the United States in the 1970s and was taken from tumor tissue induced by Abelson mouse leukemia virus. It has a round or spindle shape (the spindle shape belongs to the polarized state), belongs to the wall-attached cell, and will grow pseudopodia under certain conditions. Due to the characteristics of endocytosis and phagocytosis, it has become one of the most commonly used inflammatory cell models in the field of scientific research, and common applications include immune response research, inflammatory and infectious disease research, drug screening and toxicity evaluation, etc.

[0033] The research model animal Caenorhabditis elegans used in the present invention is a simple invertebrate model with the advantages of short life cycle, simple structure, strong reproductive ability, and many genes being highly conserved / homological with mammals (including humans) in evolution. The anti-inflammatory and autophagy mechanisms can be studied from the behavioral, molecular and genetic levels, and the anti-inflammatory and autophagy mechanisms have been confirmed in nematodes. Therefore, Caenorhabditis elegans is an important model organism for the study of inflammation and autophagy, and products that have anti-inflammatory and autophagy-inducing effects on nematodes can usually be used in other animals and are also believed to have the same effects on humans.

[0034] The reagents or formulations used in the following examples are:

[0035] NGM medium: 3g sodium chloride, 2.5g tryptone, 17g agar powder, 25mL potassium phosphate buffer (1mol / L), 1mL CaCl2 solution (1mol / L), 1mL MgSO4 solution (1mol / L), distilled water to 1000mL, autoclave at 121℃ for 20min. When the temperature drops to about 60℃, add 1mL cholesterol solution (5mg / mL, dissolved in 95% ethanol) under sterile conditions, shake well, and pour into a plate.

[0036] LB liquid medium: 5 g yeast powder, 10 g tryptone, 10 g NaCl, add distilled water to 1000 mL, and sterilize at 121°C for 20 min.

[0037] DMEM high-glucose medium, fetal bovine serum (FBS), and mouse mononuclear macrophage RAW264.7 were purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0038] Wild-type Caenorhabditis elegans N2, CB1370 (daf-2 gene defective), AU1 (sek-1 gene defective) and DA2123 (LGG-1::GFP), Escherichia coli OP50, etc. were purchased from the Caenorhabditis Genetics Center (CGC).

[0039] The Bacillus amyloliquefaciens WF2020 strain used in the present invention is the preliminary research result of the applicant's research team. The WF2020 strain has been proven to have good in vivo safety in preliminary studies, does not affect the reproductive ability of nematodes, and is safe and has no toxic side effects on nematodes; the Bacillus amyloliquefaciens WF2020 strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on January 5, 2022, with a deposit number of: GDMCC NO: 62190, classified and named Bacillus sp., deposit unit: Guangdong Provincial Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province, and recorded in the existing patent document: CN114644995A.

[0040] Example 1 Lipopolysaccharide degradation by WF2020 strain

[0041] 1. Lipopolysaccharide test method:

[0042] The dynamic colorimetric method using horseshoe crab reagent was obtained from Zhanjiang Andus Biological Co., Ltd.

[0043] 2. The degradation rate is defined as:

[0044] The percentage of the mass of the degraded part of the degradable component to the initial mass of the degradable component.

[0045] 3. Strain culture:

[0046] 100 μL of Bacillus amyloliquefaciens WF2020 frozen at -80°C was inoculated into 5 mL of LB liquid culture medium, and cultured at 37°C and 180 r / min with shaking, and used after activation for two generations.

[0047] 4. Sample processing:

[0048] Lipopolysaccharide comes from the working standard of endotoxin of Andus bacteria in Zhanjiang 90EU. 5mL of bacterial solution was taken from the cultured bacterial solution and placed in a pyrogen-free centrifuge tube. The centrifugation condition was 4000rmp for 10min. The supernatant was transferred to a pyrogen-free test tube. The bacterial sludge was resuspended 3 times with LPS-free sterile water to wash away the residual culture medium, and 5mL was supplemented with lipopolysaccharide-free sterile water. The following experimental groups were set up respectively:

[0049] The WF2020 group consisted of a sterile aqueous solution containing only WF2020 bacteria;

[0050] The CONTROL group consisted of sterile water without bacterial solution.

[0051] Add 1 mL of 90EU lipopolysaccharide to the WF2020 group and the CONTROL group, respectively, and react at 30-37°C for 0 h and 4 h, respectively; take the 0 h reaction solution and the 4 h reaction solution, dilute them 400 times, 800 times, and 1600 times, respectively, and then take 0.1 mL of the diluted solution and add it to the pyrogen removal microplate, add 3 wells for each concentration, and then add 0.1 mL of horseshoe crab reagent, shake at medium speed for 10 s to mix, and then put the microplate into the preheated endotoxin automatic analyzer ELx808 for detection, and establish a standard curve at the same time, using standard endotoxins at concentrations of 0.005EU / mL, 0.05EU / mL, 0.5EU / mL, and 5EU / mL. Each concentration of endotoxin solution has at least 3 parallel wells, and the negative control has 2 parallel wells. The test results are the average of the results of three parallel treatments. The standard curve is as follows: Figure 1 and Figure 2 As shown, it was used to calculate the endotoxin concentration.

[0052] 5. Test results:

[0053] Test results such as Figure 3 As shown, in the CONTROL group, after 90EU lipopolysaccharide was added at 0h, the total lipopolysaccharide content was determined to be 81.74EU, which was slightly decreased. The lipopolysaccharide content was determined to be 79.87EU after 4h (lipopolysaccharide was not removed); in the WF2020 group, after the bacterial solution was added to treat lipopolysaccharide at 0h, the lipopolysaccharide content rapidly decreased to 6.54EU. As the treatment time increased, the lipopolysaccharide was completely removed after 4h, indicating that Bacillus amyloliquefaciens WF2020 can completely degrade lipopolysaccharide.

[0054] Example 2 Anti-inflammatory effect of WF2020 strain

[0055] 1. NO test method:

[0056] Using a Griess kit (Biyuntian Biotech) and according to the method of the kit, 50 μL of cell culture supernatant was added to a 96-well plate, followed by the addition of Griess reagent I and Griess reagent II, and then the absorbance was measured at 540 nm using an ELISA reader.

[0057] 2. Strain culture and preparation of cell-free supernatant lyophilized powder:

[0058] Take 100 μL of Bacillus amyloliquefaciens WF2020 frozen at -80°C, inoculate it in 5 mL of LB liquid culture medium, shake and culture at 37°C and 180 r / min, and use it after activation for two generations. Cultivate Bacillus amyloliquefaciens WF202 in liquid culture medium to the logarithmic phase, centrifuge at 8000 r / min for 10 min at 4°C, remove the supernatant, and filter with a sterile 0.22 μm filter membrane to obtain a cell-free supernatant. Freeze the cell-free supernatant of Bacillus amyloliquefaciens WF2020 at -80°C for 24 hours, then freeze-dry the cell-free supernatant at -40°C to -30°C and 0.2 mbar for 24 hours, and store the freeze-dried powder at -20°C.

[0059] 3. Culture and passage of mouse mononuclear macrophage RAW264.7:

[0060] Mouse mononuclear macrophage RAW264.7 cells frozen at -80°C were inoculated into DMEM liquid high-glucose medium containing 10% fetal bovine serum FBS, cultured at 37°C, 95% air + 5% carbon dioxide, and used after 3 generations.

[0061] 4. Determination of NO produced by mouse mononuclear macrophage RAW264.7 induced by lipopolysaccharide (LPS):

[0062] The cells were plated at 50 × 10 4 Each well was inoculated with 2 mL of culture medium in a 6-well plate and cultured for 24 hours. After 24 hours of culture, different experimental groups were set up as follows: the CONTROL group was replaced with new DMEM medium, the LPS group was replaced with DMEM medium containing 1 μg / mL LPS, and the WF2020+LPS group was replaced with DMEM medium dissolved with WF2020 cell-free supernatant freeze-dried powder and 1 μg / mL LPS. After 24 hours of culture, the cell supernatant of each group was taken to test NO using the Griess reagent method.

[0063] 5. Test results:

[0064] The results are as follows Figure 4 As shown, the inflammatory marker NO produced by mouse monocyte macrophages RAW264.7 after lipopolysaccharide LPS induction was significantly increased; while the inflammatory marker NO produced by RAW264.7 was significantly reduced after treatment with the cell-free supernatant of Bacillus amyloliquefaciens WF2020. It was calculated that WF2020 significantly reduced the NO produced by RAW264.7 after lipopolysaccharide LPS induction by 56.8%, indicating that the WF2020 strain has a significant anti-inflammatory effect.

[0065] Example 3 Anti-inflammatory effect of WF2020 strain in vivo

[0066] 1. Activation of bacteria:

[0067] 100 μL of Bacillus amyloliquefaciens WF2020 frozen at -80°C was inoculated into 5 mL of LB liquid culture medium, and cultured at 37°C and 180 r / min with shaking, and used after activation for two generations.

[0068] 2. Cultivation and passaging of Caenorhabditis elegans:

[0069] Escherichia coli OP50 (OD 600 =0.5) as nematode food, then pick wild-type Caenorhabditis elegans N2 and place it on the surface of NGM culture medium, and place it in a 20°C biochemical incubator for culture. During the culture period, the nematodes need to be transferred to a fresh NGM plate coated with E. coli OP50 every day. If a single nematode is subcultured, the picking needle needs to be burned with an alcohol lamp and then cooled to pick a nematode to a new NGM culture medium; if a large number of nematodes are subcultured, the nematodes in the culture medium are washed with M9 buffer, and after standing and settling, the supernatant is removed and the worm body is transferred to a new NGM culture medium.

[0070] 3. Synchronization of Caenorhabditis elegans:

[0071] Collect the L4 stage of nematodes (day 3) into a centrifuge tube, add 1 mL of M9 buffer to wash away excess Escherichia coli OP50, use a centrifuge to centrifuge and wash 3 times to remove the supernatant, then add 1 mL of lysis buffer and shake thoroughly for 1 min. After removing the supernatant, centrifuge (3000 r / min, 1 min) and wash 3 times with M9 buffer to remove excess supernatant and retain the eggs. Transfer the eggs to NGM plates containing Bacillus amyloliquefaciens WF2020 or Escherichia coli OP50, incubate in a 20°C biochemical incubator, and synchronize L4 adults after 72 hours.

[0072] 4. Determination of lifespan of Caenorhabditis elegans:

[0073] Bacillus amyloliquefaciens WF2020 and Escherichia coli OP50 were cultured in liquid culture medium to the logarithmic phase, centrifuged at 8000r / min for 10min at 4℃, the supernatant was removed and the precipitate was washed 3-4 times with sterile water. After obtaining pure cells, sterile water was added and blown evenly to adjust the concentration to OD 600 =1.0, and then 100 μL of the bacterial solution was spread on NGM plates, and the NGM plates with only E. coli OP50 added were used as controls.

[0074] The L4 wild-type C. elegans N2 synchronized in the above steps was washed with sterile M9 buffer and transferred to a 1.5 mL centrifuge tube containing M9 buffer solution, washed 3 times with M9 buffer, added with 900 μL of liquid NGM without OP50, added with 100 μL of lipopolysaccharide (1000 μg / mL) (final concentration of 100 μg / mL), transferred to a 35 mm culture dish, and shaken at 20°C and 90 rpm for 24 hours. After 24 hours, the C. elegans was washed 3 times with M9 buffer and transferred to an NGM plate. The number of nematode deaths was recorded every 1 day until all nematodes died and a nematode lifespan curve was drawn.

[0075] 5. Test results:

[0076] The lifespan curve of nematodes after LPS treatment is as follows Figure 5 As shown, the average lifespan of nematodes fed with E. coli OP50 without lipopolysaccharide (M9) induction was 16 days, while the average lifespan of nematodes under lipopolysaccharide LPS (100 μg / mL) induction was 9 days, and it was calculated that the lifespan of nematodes induced by lipopolysaccharide (100 μg / mL) was significantly reduced (43.75%). Under LPS induction, the average lifespan of nematodes fed with Bacillus amyloliquefaciens WF2020 was extended by 8 days compared with nematodes fed with E. coli OP50, and by 1 day compared with the blank control group fed with E. coli OP50 without LPS induction, which shows that Bacillus amyloliquefaciens can restore the normal lifespan of nematodes under LPS induction without LPS induction, and the average lifespan can be extended by 1 day.

[0077] After feeding with Bacillus amyloliquefaciens WF2020, the survival rate of nematodes was significantly improved, and their lifespan was significantly prolonged compared with the control group using Escherichia coli OP50. It was calculated that Bacillus amyloliquefaciens WF2020 significantly increased the lifespan of nematodes induced by lipopolysaccharide (100 μg / mL) by 88.89%, which can effectively alleviate the effect of LPS on nematodes and improve the survival rate and lifespan of nematodes.

[0078] Example 4 Effects of WF2020 strain on lipopolysaccharide-induced nematodes with different gene defects

[0079] 1. Determination of lifespan of daf-2 and sek-1 gene-deficient Caenorhabditis elegans:

[0080] The same method as in Example 3 was used to obtain L4 stage daf-2 and sek-1 gene-deficient Caenorhabditis elegans fed with Bacillus amyloliquefaciens WF2020 or Escherichia coli OP50 (the daf-2 gene-deficient nematode used was CB1370 nematode, the daf-2 gene was located in the IIS pathway of the insulin signaling pathway, and the deletion of the daf-2 gene affected the IIS pathway of the insulin signaling pathway; the sek-1 gene-deficient nematode was AU1 nematode, the sek-1 gene was located in the p38 MAPK signaling pathway, and the deletion of sek-1 affected the p38 MAPK signaling pathway), the Caenorhabditis elegans was washed with sterile M9 buffer, and transferred to a 1.5 mL centrifuge tube containing M9 buffer solution, washed 3 times with M9 buffer, 900 μL of liquid NGM without OP50 was added, 100 μL of lipopolysaccharide (1000 μg / mL) was added (final concentration was 100 μg / mL), transferred to a 35 mm culture dish, and shaken at 20° C. and 90 rpm for 24 hours. After 24 hours, the C. elegans were washed three times with M9 buffer and transferred to NGM plates. The number of dead nematodes was recorded every 1 day until all the nematodes died and the nematode life span curve was drawn.

[0081] 2. Test results:

[0082] The results are as follows Figure 6 and Figure 7 As shown, it was observed whether the life span of different nematodes was different from that of N2 wild-type nematodes due to the loss of daf-2 gene or sek-1 gene. The average life span of daf-2 gene-deficient nematodes fed with Escherichia coli OP50 was 16 days under the induction of lipopolysaccharide LPS (100 μg / mL), and the average life span of daf-2 gene-deficient nematodes fed with Bacillus amyloliquefaciens WF2020 was 17 days under the induction of lipopolysaccharide LPS. The average life span of sek-1 gene-deficient nematodes fed with Escherichia coli OP50 was 15 days under the induction of lipopolysaccharide LPS (100 μg / mL), and the average life span of sek-1 gene-deficient nematodes fed with Bacillus amyloliquefaciens WF2020 was 16 days under the induction of lipopolysaccharide LPS. The results showed that Bacillus amyloliquefaciens WF2020 could not prolong the lifespan of daf-2 and sek-1 gene-deficient nematodes after lipopolysaccharide (LPS) induction. Compared with N2 wild-type nematodes, due to the absence of daf-2 gene or sek-1 gene, Bacillus amyloliquefaciens WF2020 could not prolong the lifespan of the gene-deficient nematodes under LPS induction, and the average lifespan differed by only 1 day. It was concluded that Bacillus amyloliquefaciens WF2020 alleviated and treated inflammation induced by lipopolysaccharide (LPS) by activating the IIS pathway of the insulin signaling pathway mediated by the daf-2 gene and the p38 MAPK signaling pathway mediated by the sek-1 gene.

[0083] Example 5 Autophagy test of Bacillus amyloliquefaciens WF2020 on Caenorhabditis elegans

[0084] 1. Autophagy fluorescence assay of Caenorhabditis elegans LGG-1::GFP:

[0085] The same method as in Example 3 was used to obtain L4 stage DA2123 Caenorhabditis elegans fed with Bacillus amyloliquefaciens WF2020 or Escherichia coli OP50 (the autophagy research model animal DA2123 nematode (LGG-1::GFP) was used, the autophagy process includes the induction of autophagosomes, the nucleation and extension of vesicles, and LGG-1 is a homolog of the microtubule-associated protein light chain 3 (LC3) that mediates the nucleation of autophagosome vesicles and nematodes, and is used to monitor autophagy. If autophagy is successfully induced, the nematode can show fluorescent spots under GFP fluorescence, which are protein aggregations of LGG-1), rinsed 3 times with sterile M9 buffer, the throat was blocked with 25 mM levamisole hydrochloride, and the green fluorescence (emission wavelength 529 nm) was observed by fluorescence microscopy, photographed and recorded, and the fluorescent photos were quantitatively calculated using Image J software.

[0086] 2. Test results:

[0087] The results are as follows Figure 8 As shown, the fluorescence imaging results showed that when Bacillus amyloliquefaciens WF2020 was used to feed DA2123 Caenorhabditis elegans, the fluorescent spots formed by LGG-1::GFP protein aggregation increased significantly ( Figure 8 AB), indicating that autophagy has occurred. The fluorescence photos were quantitatively calculated using Image J software, as shown in Fig. 9 As shown, Bacillus amyloliquefaciens WF2020 increased the number of fluorescent spots formed by LGG-1::GFP protein aggregation in DA2123 Caenorhabditis elegans by 44.30%. This indicates that Bacillus amyloliquefaciens WF2020 has the effect of promoting autophagy, and can be used to prepare products that promote or induce autophagy, which are used to reduce oxidative stress, improve metabolism, reduce inflammation, remove damaged cells, maintain cell homeostasis and resist stress, and help fight infection and disease. By removing old and damaged cells and producing new and more functional cells, inflammation and disease risks are reduced. Autophagy also helps promote the removal of waste in the body, reduce the accumulated waste and toxins in the body, reduce damage to the body, and promote health and longevity.

[0088] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of Bacillus amyloliquefaciens WF2020 strain in the preparation of anti-inflammatory products.

2. The application according to claim 1, characterized in that: The product can reduce the level of NO, a marker of inflammation.

3. The application according to claim 1, characterized in that: The product can alleviate or treat inflammation induced by lipopolysaccharide.

4. Application of Bacillus amyloliquefaciens WF2020 strain in the degradation of lipopolysaccharide for non-disease diagnosis and treatment purposes.

5. Application of Bacillus amyloliquefaciens WF2020 strain in the preparation of lipopolysaccharide degradation products.

6. Application of Bacillus amyloliquefaciens WF2020 strain in the preparation of products that promote autophagy.

7. A method for degrading lipopolysaccharide for non-disease diagnosis and treatment purposes, characterized in that: The samples were treated with Bacillus amyloliquefaciens WF2020 strain and / or its bacterial solution.

8. The use according to any one of claims 1 to 6 or the method according to claim 7, characterized in that: The WF2020 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 5, 2022, with the deposit number GDMCC No: 62190.

9. The use according to any one of claims 1 to 6, characterized in that: The product is food, medicine, feed or an additive thereof.

10. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the product is liquid, jelly, stick, powder, tablet, granule or capsule.

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