A strain of Micrococcus sucrose MEP0410 and its application in Dendrobium officinale seedling cultivation

Abstract

The present invention discloses a strain of Microcera sucrose MEP0410 and its application in Dendrobium officinale seedling cultivation. The Microcera sucrose MEP0410 is classified and named Microcera sucrose ( Kosakonia sugar ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on March 18, 2024, and its deposit number is CGMCC NO.30045. The sucrose microorganism MEP0410 of the present invention has potential growth-promoting abilities such as nitrogen fixation, siderophore production, cellulase production, and ACC deaminase activity production, and can significantly increase the content of secondary metabolites such as polysaccharides, flavonoids, and alkaloids of Dendrobium officinale; it can be used for artificial cultivation of Dendrobium officinale and improve the quality of Dendrobium officinale seedlings, which helps to solve the current problem of shortage of Dendrobium officinale resources and inability to guarantee quality in the market, promotes the industrialization development of Dendrobium officinale, and has important application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Micrococcus saccharosus MEP0410 and application of the strain in Dendrobium officinale seedling cultivation. Background Art

[0002] Dendrobium candidum Dendrobium officinale Kimura et Migo, also known as black knot grass, is a perennial herbaceous plant of the genus Dendrobium in the Orchidaceae family. It grows on cliff rocks or trunks in dense forests at an altitude of 800-1600 meters. Dendrobium officinale is a traditional precious Chinese medicinal material. It is known as the "life-saving fairy grass" among the people and enjoys the reputation of being the first of the "Nine Fairy Grasses in China". It is mainly distributed in Yunnan, Guangxi, Fujian, Guizhou, Zhejiang and other places in my country. As a plant with both medicinal and edible properties, Dendrobium officinale is often used as a dry product (such as decoction pieces, maple buckets, etc.). Its fresh stems can be eaten directly, or in the form of soup, juice, and wine. Due to its extremely high medicinal and economic value, the phenomenon of over-exploitation of Dendrobium officinale is extremely serious. In addition, its low reproduction rate and slow growth under natural conditions have caused the wild resources of Dendrobium officinale to be on the verge of exhaustion. Artificial cultivation is an effective way to meet the huge market demand, but in order to pursue high yield and shorten the growth cycle, there is currently a phenomenon of abusing fertilizers and chemical agents in artificial cultivation of Dendrobium officinale, which leads to a shortened growth period and poor quality of artificially cultivated Dendrobium officinale. Therefore, finding an effective and green way to improve the quality of artificially cultivated Dendrobium officinale is of great significance to ensure the quality of Dendrobium officinale and the safety of its use.

[0003] Plant endophytes refer to a type of microorganisms that live in healthy plant tissues or organs at a certain stage or throughout their life cycle and do not cause obvious disease symptoms to the host plant. They mainly include endophytic bacteria, endophytic actinomycetes, and endophytic fungi. As a very important type of microbial resource, they play an important role in promoting plant growth, accumulating secondary metabolites, and improving plant stress resistance. In recent years, exploring the interaction between endophytes and medicinal plants and using endophytes to improve the quality and yield of medicinal plants has become a research hotspot. For example, Schmidt et al. isolated Miscanthus × giganteus The endophytes of orchids were detected and their plant growth-promoting properties in the host were tested. It was found that all endophytes produced plant hormones, and some bacteria also produced iron carriers, dissolved phosphorus and showed ACC deaminase activity in vitro, which promoted the growth of host plants. Song et al. isolated an endophytic bacterium LB 5-3 from ginseng roots, which can improve the accumulation of ginsenosides in adventitious roots. There are many important endophytes living inside orchids that affect their growth and development. If the quality of artificially cultivated Dendrobium candidum can be improved by applying functional endophytes with quality-improving effects, this will have broad application prospects in ensuring the effectiveness and safety of its medicinal use. Summary of the invention

[0004] In view of the technical difficulties such as the abuse of chemical fertilizers in the current artificial cultivation of Dendrobium officinale, which leads to poor quality of Dendrobium officinale and the inability to ensure its medicinal quality, as well as environmental pollution, the present invention provides an endophyte MEP0410 of Dendrobium officinale and its application in promoting the growth of Dendrobium officinale, especially in promoting the accumulation of secondary metabolites of Dendrobium officinale.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A strain of Microcystis sucrose Kosakonia sacchari ) MEP0410, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO. 30045, the deposit date is March 18, 2024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The strain MEP0410 of the present invention is obtained by separation and purification of endophytic bacteria from healthy wild Dendrobium officinale collected from Jinxiu County, Laibin City, Guangxi Zhuang Autonomous Region, and is identified by morphology and molecular identification as Osteoporosis sucroseus ( Kosakonia sacchari ). The specific separation and purification method is as follows:

[0008] (1) Take the roots, stems and leaves of wild Dendrobium officinale plants and rinse them under running water for 30 minutes until the surface is clean. Then use absorbent paper to absorb the moisture on the surface of the tissue. The cleaned samples were surface disinfected in an ultra-clean workbench: first soaked in 75% ethanol for 1 minute, then rinsed with sterile water twice, then soaked in 5% sodium hypochlorite for 10 minutes, and rinsed with sterile water 4 times. The surface disinfected samples (roots, stems, leaves) were placed in a mortar, 9 mL of sterile water was added, and the grinding liquid was diluted with sterile water in a gradient manner (10 -1 , 10 -2 , 10 -3 ), then grind the stock solution and dilution (10 -1 , 10 -2 , 10 -3 ) were spread on ISP2 solid medium and cultured at 28℃ for 3-5 days until single colonies grew.

[0009] (2) Pick a single colony and streak it onto the same ISP2 plate for purification, then culture it at 28°C for 3-5 days;

[0010] (3) Repeat step (2) until a pure culture is obtained.

[0011] The application of the above-mentioned Osteoporosis sucrose MEP0410 in promoting the growth of Dendrobium officinale.

[0012] Furthermore, the Oscillopora sucrose MEP0410 promotes the accumulation of secondary metabolites of Dendrobium officinale. Preferably, the secondary metabolites include polysaccharides, flavonoids, and alkaloids.

[0013] The application of the above-mentioned Osteoporosis sucrose MEP0410 in the preparation of products promoting the growth of Dendrobium officinale.

[0014] The bacterial agent prepared by the above-mentioned Osteoporosis sucrose MEP0410.

[0015] Furthermore, the bacterial agent is prepared by the following method: the activated bacterial liquid of the above-mentioned Osteoporosis sucrose MEP0410 is transferred to ISP2 liquid culture medium, cultured on a shaker at 28°C and 200 rpm for 4 days, centrifuged to collect the bacteria, washed with sterile water and then centrifuged to collect the bacteria, and resuspended the bacteria with sterile water to obtain the bacterial liquid, which is the bacterial agent.

[0016] In one embodiment of the present invention, the activated bacterial solution is obtained by inoculating the strain of Osteoporosis sucrose MEP0410 into ISP2 liquid culture medium and culturing it at 28° C. and 200 rpm for 4 days for activation.

[0017] The application of the above-mentioned bacterial agent in cultivating high-quality seedlings of Dendrobium officinale.

[0018] A method for cultivating high-quality Dendrobium officinale seedlings comprises soaking the roots of the Dendrobium officinale tissue culture seedlings with the above-mentioned bacterial agent, and then transferring the Dendrobium officinale tissue culture seedlings into MS basal culture medium for cultivation.

[0019] The present invention provides a strain of Microsaccharomyces sucrose isolated from the root of Dendrobium officinale ( Kosakonia sacchari ) MEP0410, which has potential growth-promoting abilities such as nitrogen fixation, siderophore production, and ACC deaminase activity, and can effectively promote the accumulation of secondary metabolites of Dendrobium officinale. The present invention can provide excellent bacterial strain resources for the development of bacterial fertilizers to improve the quality of artificially cultivated Dendrobium officinale, and is of great significance for promoting the rapid development of Dendrobium officinale industrialization.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] (1) The isolated and obtained Osmunda sucrose bacteria ( Kosakonia sacchari ) MEP0410 has the growth-promoting potential of nitrogen fixation, siderophore production, and ACC deaminase activity, and can effectively increase the content of secondary metabolites such as polysaccharides, flavonoids, and alkaloids in Dendrobium officinale. This strain can be developed as a microbial agent for the cultivation of high-quality Dendrobium officinale seedlings and for improving the quality of artificially cultivated Dendrobium officinale, producing green, ecological, and high-quality Dendrobium officinale medicinal materials, which can ensure the effectiveness and safety of its medicinal materials, and at the same time can reduce environmental pollution caused by the abuse of fertilizers.

[0022] (2) The sucrose bacteria disclosed in the present invention ( Kosakonia sacchari ) MEP0410 has simple culture conditions, is easy to preserve, is genetically stable, and is easy to industrialize, and has broad development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The morphology of strain MEP0410 on ISP2 medium, where A is the front side of the plate and B is the back side of the plate.

[0024] Figure 2 The physiological and biochemical characteristics of strain MEP0410. Among them: A is the result of the glucose fermentation experiment of strain MEP0410; B is the result of the xylose fermentation experiment of strain MEP0410; C is the result of the gelatin biochemical experiment of strain MEP0410; D is the result of the urease experiment of strain MEP0410; E is the result of the VP reaction of strain MEP0410.

[0025] Figure 3 The results of the pro-liferative activity identification of strain MEP0410 are shown in Figure 1. A is the nitrogen fixation activity of strain MEP0410; B is the siderophore production activity of strain MEP0410; C is the cellulase production activity of strain MEP0410; D is the protease production activity of strain MEP0410; and E is the ACC deaminase production activity of strain MEP0410.

[0026] Figure 4 This is the result of the effect of strain MEP0410 on the polysaccharide content of Dendrobium officinale.

[0027] Figure 5 This is the result of the effect of strain MEP0410 on the flavonoids content in Dendrobium officinale.

[0028] Figure 6 This is the result of the effect of strain MEP0410 on the alkaloid content of Dendrobium officinale. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] The culture medium used in the following examples includes:

[0033] ISP2 liquid culture medium: malt extract powder 10 g; yeast extract powder 4 g; glucose 4 g; distilled water 1000 mL, pH 7.0-7.2.

[0034] ISP2 solid culture medium: malt extract powder 10 g; yeast extract powder 4 g; glucose 4 g; agar 20 g; distilled water 1000 mL, pH 7.0-7.2. Example 1

[0035] Isolation, purification and identification of strain MEP0410

[0036] 1. Isolation and purification of strains

[0037] (1) The strain was isolated from healthy wild Dendrobium officinale collected from Jinxiu County, Laibin City, Guangxi Zhuang Autonomous Region. An appropriate amount of roots, stems, and leaves of wild Dendrobium officinale were washed under running water for 30 min until the surface was clean, and then the surface moisture of the tissue was absorbed with absorbent paper.

[0038] (2) The cleaned samples were surface disinfected in an ultra-clean workbench: first soaked in 75% ethanol for 1 min, washed twice with sterile water, and then soaked in 5% sodium hypochlorite for 10 min. After washing four times with sterile water, 80 μL of the sterile water used for the last wash of each tissue was aspirated and spread on ISP2 solid culture medium, and then placed in an inverted culture in a 28°C incubator for 7 days to verify whether the surface disinfection is reliable.

[0039] (3) Isolation of endophytes: Place the surface-sterilized samples (roots, stems, and leaves) in a mortar, add 9 mL of sterile water, and grind evenly. Then, dilute the grinding solution with sterile water in a gradient manner (10 -1 , 10 -2 , 10 -3 ), then grind the stock solution and dilution (10 -1 , 10 -2 , 10 -3 ) were spread on ISP2 solid medium and cultured at 28℃ for 3-5 d.

[0040] (4) Purification and preservation of endophytic bacteria: Select colonies of different colors and morphologies and perform repeated purification on new plates by streaking multiple times until no foreign bacteria can be observed with the naked eye and single colonies of the same morphology appear. After the purified single colonies are expanded and cultured in ISP2 liquid medium, the bacterial solution and sterile 60% glycerol are added to the seed preservation tube in a ratio of 1:1 and placed in a -80℃ refrigerator for long-term storage.

[0041] 2. Identification of strains

[0042] The colony, morphological, physiological and biochemical characteristics of strain MEP0410 are as follows:

[0043] (1) Morphological characteristics of strains

[0044] like Figure 1 As shown, the purified strain MEP0410 grew well on ISP2 solid medium. The colonies were round, white, moist, with irregular edges. The front and back of the colonies were different colors, white on the front and light yellow on the back.

[0045] (2) Physiological and biochemical characteristics of strains

[0046] like Figure 2 As shown, strain MEP0410 can utilize glucose, hydrolyze xylose and gelatin, and its urease reaction and VP reaction are negative.

[0047] (3) 16S rRNA gene sequence analysis of strain MEP0410

[0048] The strain DNA was prepared into the corresponding reaction system using the high-purity heat-resistant DNA polymerase from Novozymes Biotech for PCR amplification (amplification according to the conventional PCR method), and the PCR product was sent to General Biotech (Anhui) Co., Ltd. for sequencing. The 16SrRNA gene sequencing result is shown in SEQ ID NO.1. The sequencing results were compared with the source sequence of NCBI's Blast detection system and statistically summarized. The comparison results showed that strain MEP0410 and Kosakonia sacchari The similarity was 99.86%. The strain MEP0410 was identified as Microsaccharomyces sucroseus ( Kosakonia sacchari ).

[0049] 3. Identification of growth-promoting potential

[0050] (1) Nitrogen fixation activity

[0051] After activating the strain, streak the strain in a Z shape on the Ashby nitrogen-free medium and culture it upside down in a constant temperature incubator at 28°C for 7 days. If it can grow, perform continuous subculture. If it can continue to grow on the nitrogen-free medium after three continuous subcultures, it is preliminarily considered that the strain has the potential to fix nitrogen.

[0052] (2) Cellulase activity

[0053] After activating the strain, the strain to be tested was spotted on the cellulase activity screening medium and inverted in a constant temperature incubator at 28°C for 7 days; an appropriate amount of 0.5% Congo red solution was added to the plate for staining, and the plate was allowed to stand for 10 min before being poured out; an appropriate amount of 1 mol / L NaCl solution was added to the plate for decolorization, and the plate was left to stand and then poured out; it was observed whether a transparent circle was produced. If so, it was preliminarily considered that the strain was able to produce cellulase. The ratio of the transparent circle diameter (D) to the strain diameter (d) reflected the activity. The larger the ratio, the higher the activity, and vice versa.

[0054] (3) Protease activity

[0055] After activating the strain, the strain to be tested was spotted on the protease activity screening medium and inverted in a constant temperature incubator at 28°C for 5 days to observe whether a transparent circle was produced. If so, it was preliminarily considered that the strain was able to produce protease. The ratio of the transparent circle diameter (D) to the strain diameter (d) reflected the strength of the activity. The larger the ratio, the higher the activity, and vice versa.

[0056] (4) Producing ACC deaminase activity

[0057] After activating the strain, the strain to be tested was streaked in a Z shape on the ADF medium and inverted in a constant temperature incubator at 28°C for 7 days. If it can grow, it was serially subcultured. If it can continue to grow on the ADF medium after three consecutive subcultures, it was preliminarily considered that the strain had the potential to produce ACC deaminase.

[0058] (5) Siderophore production activity

[0059] After activating the strain, the strain to be tested was spotted on the CAS detection medium and incubated upside down in a constant temperature incubator at 28°C for 7 days. It was observed whether a transparent circle was formed around the spotted strain on the CAS detection plate. If so, it was preliminarily considered that the strain was able to produce iron carriers. The ratio of the diameter of the transparent circle (D) to the diameter of the strain (d) reflected the strength of the activity. The larger the ratio, the higher the activity, and vice versa.

[0060] like Figure 3 As shown, strain MEP0410 has nitrogen fixation, siderophore production, cellulase production, ACC deaminase production, and protease production activities. Example 2

[0061] Effects of strain MEP0410 on the polysaccharide, flavonoid and alkaloid contents of Dendrobium officinale

[0062] The strain used in this example was from the Micrococcus saccharosacchari (MEP0410) obtained by previous separation, purification and identification in Example 1; the sterile tissue culture seedlings of Dendrobium officinale were obtained from the Jiangsu Provincial Key Laboratory of Medicinal Plant Biotechnology of Jiangsu Normal University.

[0063] (1) Treatment of Dendrobium officinale tissue culture seedlings with bacterial strains

[0064] In the clean bench, take the strong seedlings of Dendrobium officinale in the seedling stage with consistent growth and transfer them to MS basic medium (MS powder 4.74 g; sucrose 30 g; agar 7.5 g; distilled water 1000 mL), 8 plants per bottle, and place them in a tissue culture room with a temperature of (25±2)℃, a light intensity of 2000 Lx, and a daily light of 12 h for 2 weeks to adapt to the MS basic medium. The roots of the adapted tissue culture seedlings were immersed in MEP0410 bacterial solution (the specific preparation method of MEP0410 bacterial solution is: strain MEP0410 was inoculated into fresh ISP2 liquid medium, cultured at 28℃, 200 rpm for 4 days, and the strain was activated; then the activated bacterial solution was transferred to ISP2 liquid medium at a ratio of 1%, and cultured at 28℃, 200 rpm for 4 days, centrifuged to collect the bacteria, washed 3 times with sterile water, centrifuged again to collect the bacteria, and resuspended the bacteria with sterile water, and the bacterial solution concentration was adjusted to OD 600 =0.5) for 20 min, and the blank control group (CK) was treated with sterile water. Then, 8 strains per bottle were transferred to new MS basal medium for culture. Samples were taken from both the treatment group and the control group after they were transferred to the new MS basal medium for culture for 15 d, 30 d, and 45 d, thus obtaining 6 groups of materials.

[0065] (2) Determination of polysaccharide content by phenol-sulfuric acid method

[0066] Drawing of standard curve: prepare 0.9 mg / mL solution of glucose reference substance dried to constant weight, and then dilute 10 times to obtain 90 μg / mL solution. Accurately add 0.2, 0.4, 0.6, 0.8, and 1 mL of glucose reference substance solution to 10 mL test tubes, respectively. Make up to 1 mL with deionized water, add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid, shake well, and place in a 90 ℃ water bath for 20 min. Take out and cool in an ice bath for 5 min. Measure the absorbance at 488 nm, use 1 mL of water as blank according to the same color development operation, and use the calculated polysaccharide concentration as the horizontal axis. The absorbance value (OD 488 ) as the vertical axis to draw a standard curve.

[0067] Polysaccharide extraction: The seedlings of Dendrobium officinale in each group were taken out, dried and ground, 0.1 g was weighed into a 25 mL test tube, 10 mL of distilled water was added, and ultrasonic treatment was performed for 30 min. Then, the mixture was placed in a constant temperature water bath and boiled in boiling water for 3 h. After cooling, the supernatant was centrifuged and poured into a 10 mL centrifuge tube and made up to volume with distilled water. Subsequently, 2 mL was placed in a 50 mL volumetric flask and made up to volume with deionized water.

[0068] Determination of the polysaccharide content of Dendrobium candidum: Take 1 mL of the above extract in a clean test tube, then add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid, shake well, place in a 90℃ water bath for 20 min, take out and place in an ice bath for 5 min, then use an ultraviolet spectrophotometer to measure the absorbance at 488nm, and use 1 mL of water as a blank in the same color development operation. Then calculate the results.

[0069] The polysaccharide detection values ​​of Dendrobium officinale co-cultured with strain MEP0410 for different time periods were brought into the drawn glucose standard curve to obtain the polysaccharide content of each treatment group. The results are as follows: Figure 4 As shown in the data, the polysaccharide content in the treatment group was significantly higher than that in the CK group. At 30 days of treatment, the polysaccharide content was most significantly higher than that in the CK group, reaching 152.98 mg / g, indicating that strain MEP0410 can effectively increase the polysaccharide content in Dendrobium officinale.

[0070] (3) Determination of flavonoid content

[0071] Preparation of standard curve: accurately weigh 10 mg of rutin standard dried to constant weight, add a small amount of 95% ethanol, shake well, fully dissolve under ultrasound assistance, and then dilute to 50 ml with 60% ethanol. Add 0, 1, 2, 3, 4, and 5 mL of the solution to 6 25 mL volumetric flasks respectively; add 1 mL of NaNO 2 The solution was mixed and placed at room temperature for 6 min, and 1 mL of Al(NO 3 ) 3 solution, mix well and let stand at room temperature for 6 min; finally, add 10.00 mL of 4% NaOH solution, mix well and let stand at room temperature for 15 min, and make up to volume with 60% ethanol solution; detect the absorbance of the solution at 510 nm and draw a standard curve.

[0072] Determination of flavonoids content in Dendrobium officinale: weigh 0.20 g of dried powdered Dendrobium officinale sample, add 10 mL of 60% ethanol, extract in a 60°C water bath for 3 h, cool and filter, take the filtrate and make up to 20 mL with 60% ethanol for testing, then substitute into the standard curve to calculate the total flavonoids content in Dendrobium officinale.

[0073] The flavonoids detection values ​​of Dendrobium officinale co-cultured with strain MEP0410 for different time periods were brought into the drawn rutin standard curve to obtain the flavonoids content of each treatment group. The results are as follows: Figure 5 As shown in the results, the flavonoid content in the treatment group was significantly higher than that in the CK group at different time periods, and the flavonoid content changed most significantly compared with the CK group at 45 days of treatment, indicating that strain MEP0410 can effectively promote the accumulation of flavonoids in Dendrobium officinale.

[0074] (3) Determination of alkaloid content

[0075] Sample processing: accurately weigh 0.1 g of dried and ground Dendrobium officinale into a round-bottom flask, add 10 mL of 0.05% formic acid-methanol solution and weigh it, heat and reflux for 3 h and weigh it, make up the volatilized weight with 0.05% formic acid-methanol solution, shake and filter, accurately measure 2 mL of the filtrate, place it in a 5 mL volumetric flask, and then send it to Nanjing Jiangbei New Area Biomedicine Public Service Platform Co., Ltd. for testing.

[0076] The samples were tested by high performance liquid chromatography to determine the alkaloid content of each group of Dendrobium officinale samples at different time periods. Figure 6 As shown in the data, after the tissue culture seedlings of Dendrobium officinale were treated with the bacterial solution of strain MEP0410, the content of alkaloids was significantly increased compared with that of the CK group, and the increase was most significant at 30 days and 45 days compared with the CK group, indicating that strain MEP0410 can effectively promote the accumulation of alkaloids in Dendrobium officinale.

Claims

1. A strain of Microcystis sucrose ( Kosakonia sacchari ) MEP0410, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC NO. 30045, the deposit date is March 18, 2024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Osteoporosis sucrose MEP0410 described in claim 1 in promoting the growth of Dendrobium officinale.

3. The use according to claim 2, characterized in that: The Oscillopora sucrose MEP0410 promotes the accumulation of secondary metabolites of Dendrobium officinale, and the secondary metabolites include polysaccharides, flavonoids and alkaloids.

4. Use of the Osteoporosis sucrose MEP0410 described in claim 1 in the preparation of a product for promoting the growth of Dendrobium officinale.

5. The bacterial agent prepared by the Osteoporosis sucrose MEP0410 according to claim 1.

6. The bacterial agent according to claim 5, characterized in that The preparation method is as follows: the sucrose microorganism MEP0410 described in claim 1 is activated and inoculated into ISP2 liquid culture medium, cultured on a shaking table at 28°C and 200 rpm for 4 days, the bacteria are collected by centrifugation, washed with sterile water and then collected by centrifugation, and the bacteria are resuspended with sterile water to obtain the bacterial liquid, which is the bacterial agent.

7. The bacterial agent according to claim 6, characterized in that: The activation is performed by inoculating the strain of Osteoporosis sucrose MEP0410 described in claim 1 into ISP2 liquid culture medium, and culturing for 4 days at 28° C. and 200 rpm in a shaking incubator for activation.

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