A preparation method of microbial exopolysaccharide and its application in tissue culture medium

Abstract

The present invention relates to a method for preparing microbial exopolysaccharides and their use in tissue culture medium, belonging to the technical field of microbial polysaccharide preparation. The method for preparing microbial exopolysaccharides utilizes batch fermentation of different microorganisms, adds a beet extract and a supplement to the culture medium to promote microbial exopolysaccharide production, and further purifies, enzymatically hydrolyzes, and separates the exopolysaccharides. The resulting target microbial exopolysaccharides have a good effect on promoting the development and rooting of tissue culture seedlings and can be used to prepare tissue culture medium.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial polysaccharide preparation, and relates to a method for preparing microbial extracellular polysaccharide and application of the same in a culture medium for tissue culture. Background Art

[0002] Polysaccharides are widely present in nature, serving not only as an important form of energy storage but also as a key component of the cytoskeleton, possessing diverse functional properties. In addition to plants and algae, microorganisms (such as molds, yeasts, and bacteria) can also synthesize polysaccharides. These polysaccharides are characterized by strong environmental adaptability, low cost, and high biological activity, offering broader application prospects than plant-derived polysaccharides.

[0003] Microbial polysaccharides exist primarily in the form of exopolysaccharides, intracellular polysaccharides, and cell wall polysaccharides. Microbial exopolysaccharides offer significant advantages in safety, environmental protection, health, and cost. Their excellent physical and chemical properties and numerous potential functional properties offer broad development prospects in a wide range of fields.

[0004] Literature reports that adding Lactobacillus plantarum exopolysaccharides to schizonepeta seedlings under salt stress can promote seed germination and seedling growth. It also improves the seedlings' salt tolerance through osmotic regulation and activation of antioxidant enzyme defense systems, alleviating the effects of salt stress on the plants. Other literature reports indicate that PS04 exopolysaccharides can enhance the cold tolerance of tomato seedlings, increasing chlorophyll, SOD, CAT, and POD enzyme activity, and soluble protein content, while reducing malondialdehyde content. This has the effect of enhancing the antioxidant capacity of tomato seedlings, thereby improving their cold tolerance.

[0005] However, there is no related application of microbial exopolysaccharides in tissue culture in the prior art. Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for preparing microbial exopolysaccharide. The obtained microbial exopolysaccharide can be used to prepare tissue culture medium and has a good effect of promoting the development and rooting of tissue culture seedlings.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned purpose:

[0008] A method for preparing microbial exopolysaccharide mainly comprises the following steps:

[0009] Step A: inoculating Lactobacillus rhamnosus seed liquid into a fermentation medium containing soy peptone, glucose, sodium nitrate, calcium chloride, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate, and beet extract, and culturing at 35° C. for 72 hours to obtain an initial fermentation liquid; adding catechin, soy flour, mannose, and ferrous sulfate to the initial fermentation liquid, adjusting the pH to 6.0-6.2, inoculating Aspergillus polymorpha seed liquid, and culturing at 30° C. for 96 hours to obtain a final fermentation liquid;

[0010] Step B, filtering the final fermentation broth to obtain a filtrate; adding a citric acid solution with a mass fraction of 25-30% to the filtrate, adjusting the pH value of the supernatant to 4.0, standing for 10 hours, and centrifuging to obtain a supernatant;

[0011] Step C, adding anhydrous ethanol to the supernatant to adjust the alcohol content of the supernatant to 50% and 75% in sequence, standing for 8 hours after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0012] Step D, dissolving the precipitate in water, adding β-glucanase and xylanase, performing enzymolysis at 45-50°C for 1.5-2h, inactivating the enzyme, and drying to obtain the primary product;

[0013] In step E, the primary product is redissolved in water, loaded onto a Sephadex G-25 dextran gel column, and eluted using a methanol-water mixed solution as an eluent. Detection is performed during the elution process, and based on the detection results, a polysaccharide eluate with a molecular weight of 0.5 to 10 kDa is retained, methanol is removed, and the eluate is concentrated and dried to obtain the target microbial exopolysaccharide.

[0014] Furthermore, in step A, the inoculation amount of the Lactobacillus rhamnosus seed liquid is 5%.

[0015] Furthermore, in step A, the effective viable count of Lactobacillus rhamnosus seed solution is 5.0×10 9 CFU / mL.

[0016] Furthermore, the preparation method of the fermentation medium in step A is as follows: weigh the raw materials according to the ratio of 15 g / L soy peptone, 10 g / L glucose, 5 g / L sodium nitrate, 0.03 g / L calcium chloride, 1.5 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, and 3-5 g / L beet extract, add distilled water, adjust the pH value to 6.2-6.5, and sterilize at high temperature to obtain the fermentation medium.

[0017] Furthermore, in step A, the amounts of catechin, soybean powder, mannose, and ferrous sulfate added are 0.1-0.15 g / L of catechin, 5-8 g / L of soybean powder, 5-8 g / L of mannose, and 0.3-0.4 g / L of ferrous sulfate, respectively.

[0018] Furthermore, the inoculation amount of the Aspergillus polymorpha seed liquid in step A is 10%.

[0019] Furthermore, the amount of spores in the Aspergillus polymorpha seed solution in step A is 3.0×10 10 pieces / mL.

[0020] Furthermore, in step D, the amount of β-glucanase added is 1% to 1.5% of the mass of the precipitate, and the amount of xylanase added is 0.3% to 0.35% of the mass of the precipitate.

[0021] Furthermore, the enzyme activity of the β-glucanase is 500,000 U / g, and the enzyme activity of the xylanase is 100,000 U / g.

[0022] Furthermore, the volume ratio of methanol to water in the methanol-water mixed solution in step E is (0.8-1):10.

[0023] Furthermore, the preparation method of the beet extract comprises the following steps:

[0024] Step 1: Grind beetroot with water to prepare a homogenate, add cellulase, mix well, and then stand for 50-60 minutes. Then, reflux extract at 50-60°C for 2-3 hours, filter, and obtain an extract for later use;

[0025] Step 2, adding citric acid solution to the extract to adjust the pH value of the extract to 4.0, letting it stand for 10 hours, centrifuging, and obtaining supernatant I for later use;

[0026] Step 3, adding anhydrous ethanol to the supernatant I to adjust the alcohol content of the supernatant I to 75%, letting it stand for 10 hours, and centrifuging to obtain supernatant II for later use;

[0027] Step 4: After removing ethanol from the supernatant II, the supernatant II is concentrated and dried to obtain the beet extract.

[0028] Furthermore, in step 1 of the above-mentioned method for preparing the beet extract, the amount of cellulase added is 0.1-0.15% of the mass of the beet root.

[0029] Furthermore, in step 1 of the above-mentioned method for preparing beet extract, the enzymatic activity of cellulase is 200,000 U / g.

[0030] Furthermore, in step 2 of the above-mentioned method for preparing beet extract, the mass fraction of the citric acid solution is 25-30%.

[0031] The present invention provides a specific use of the microbial exopolysaccharide obtained by the above preparation method, namely, use of the microbial exopolysaccharide in preparing a culture medium for tissue culture.

[0032] Furthermore, the above-mentioned tissue culture medium includes but is not limited to primary culture medium, secondary culture medium or induction culture medium, proliferation culture medium, rooting culture medium, etc.

[0033] The present invention has the following beneficial effects:

[0034] 1. The microbial exopolysaccharide provided in the present invention has a good promoting effect on the growth and development of tissue culture seedlings in plant tissue culture. When used in the primary culture medium, it can significantly promote the germination and differentiation of tissues such as the stem tip and improve the survival rate of the stem tip; when used in the secondary culture medium, it can promote the division and proliferation of explant cells and increase the proliferation rate; it can also promote the rooting of plant adventitious buds and promote the growth and development of tissue culture seedlings.

[0035] 2. In the process of using microbial extracellular polysaccharides for tissue culture, it was found that not all polysaccharides have a promoting effect on plant tissue culture. Therefore, the inventors optimized the preparation method of microbial extracellular polysaccharides. In terms of fermentation strains, Lactobacillus rhamnosus and Aspergillus polymorpha were screened as the optimal fermentation strains. In the fermentation process, different strains were inoculated and fermented in batches. The two microorganisms were used for joint fermentation to produce various types of microbial extracellular polysaccharides. In the process of screening the fermentation medium, beet extract was added during the first fermentation process to promote Lactobacillus rhamnosus to produce extracellular polysaccharides. polysaccharides; in order to avoid the loss of polysaccharides caused by using the produced extracellular polysaccharides as a carbon source for the growth of the bacteria in the later stage, the present invention adopts a feeding method to add carbon sources and other nutrients, and at the same time inoculates Aspergillus polypolysaccharides, uses catechin as an inducer to further promote Aspergillus polypolysaccharides to produce extracellular polysaccharides, and in order to avoid the loss of polysaccharides, acid precipitation is adopted to remove components such as proteins; then, different types of polysaccharides are precipitated by adjusting different alcohol contents, and after enzymatic hydrolysis, the target polysaccharides are obtained by separation through gel column chromatography. Experimental verification shows that the target polysaccharides obtained by the present invention have good effects in plant tissue culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Effects of different polysaccharides on strawberry stem tip germination in experiment 1, where A is the S1-1 group, B is the S1-5 group, and C is the blank group;

[0037] Figure 2 : The effects of different polysaccharides on strawberry proliferation in Experiment 2, where A is the S1-1 group, B is the S1-5 group, and C is the blank group. DETAILED DESCRIPTION

[0038] The present invention is further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications to the various equivalent forms of the present invention by those skilled in the art all fall within the scope protected by the claims of this application. The contents disclosed and protected in the present invention are preliminary research results, the purpose of which is to obtain microbial extracellular polysaccharides that have a promoting effect on plant tissue culture. The specific components, structure and specific mechanism of action of the target microbial extracellular polysaccharides will be further studied in the later stage. The Lactobacillus rhamnosus and Aspergillus polymorpha seed solutions used in the present invention were obtained by laboratory expansion culture, and the strains were commercial strains purchased from the China General Microbial Culture Collection Center; the cellulase and other hydrolases used are existing commercially available enzyme products.

[0039] Example 1

[0040] Prepare beet extract:

[0041] Step 1: 10 kg of beetroot was added with water and ground into a homogenate, 15 g of cellulase (enzyme activity of 200,000 U / g) was added, the mixture was allowed to stand for 50 minutes, and then refluxed at 60° C. for 2 hours, filtered, and the extract was prepared for later use;

[0042] Step 2, adding 30% by mass of citric acid solution to the extract, adjusting the pH value of the extract to 4.0, standing for 10 hours, centrifuging, and obtaining supernatant I for later use;

[0043] Step 3, adding anhydrous ethanol to the supernatant I to adjust the alcohol content of the extract to 75%, letting it stand for 10 hours, and centrifuging to obtain supernatant II for later use;

[0044] Step 4: After removing ethanol from the supernatant II, concentrating and drying, the beet extract is obtained.

[0045] Preparation of microbial exopolysaccharides:

[0046] Step A: The purchased Lactobacillus rhamnosus strain numbered CGMCC1.549 was activated and expanded according to the instructions, and then diluted to make an effective viable cell count of 5.0×10 9 CFU / mL of Lactobacillus rhamnosus seed liquid; the purchased strain numbered CGMCC3.11449 was activated and expanded according to the instructions, and then diluted to make a spore volume of 3.0×10 10 / mL of Aspergillus polymorpha seed liquid; weigh the raw materials according to the ratio of 15g / L soy peptone, 10g / L glucose, 5g / L sodium nitrate, 0.03g / L calcium chloride, 1.5g / L dipotassium hydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 0.05g / L manganese sulfate monohydrate, and 3-5g / L beet extract, add distilled water, adjust the pH to 6.5, and sterilize at high temperature to obtain a fermentation medium; inoculate the prepared Lactobacillus rhamnosus seed liquid into the prepared fermentation medium at a rate of 5% and culture at 35°C for 72h to obtain an initial fermentation liquid; add catechin 0.15g / L, 5g / L soy flour, 8g / L mannose, and 0.3g / L ferrous sulfate to the initial fermentation liquid, adjust the pH to 6.2, inoculate the Aspergillus polymorpha seed liquid at a rate of 10%, and culture at 30°C for 96h to obtain a final fermentation liquid;

[0047] Step B, filtering the final fermentation broth to obtain a filtrate; adding a 30% by mass fraction citric acid solution to the filtrate, adjusting the pH value of the supernatant to 4.0, standing for 10 hours, and centrifuging to obtain a supernatant;

[0048] Step C, adding anhydrous ethanol to the supernatant to adjust the alcohol content of the supernatant to 50% and 75% in sequence, standing for 8 hours after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0049] Step D, after dissolving the precipitate in water, adding 1.5% of the precipitate mass of β-glucanase (enzyme activity of 500,000 U / g) and 0.3% of the precipitate mass of xylanase (enzyme activity of 100,000 U / g), enzymolysis at 50° C. for 1.5 hours, inactivating the enzyme, and drying to obtain the initial product;

[0050] In step E, the primary product is redissolved in water, loaded onto a Sephadex G-25 dextran gel column, and eluted with a mixed solution of methanol and water (1:10, v / v) as an eluent. Detection is performed during the elution process. Based on the detection results, a polysaccharide eluate with a molecular weight of 0.5 to 10 kDa is retained, methanol is removed, and the eluate is concentrated and dried to obtain the target microbial exopolysaccharide.

[0051] Example 2

[0052] Prepare beet extract:

[0053] Step 1: 10 kg of beetroot was added with water and ground into a homogenate, 10 g of cellulase (enzyme activity of 200,000 U / g) was added, the mixture was allowed to stand for 60 minutes, and then refluxed at 50° C. for 3 hours, filtered, and the extract was prepared for later use;

[0054] Step 2, adding 25% by mass of citric acid solution to the extract, adjusting the pH value of the extract to 4.0, letting it stand for 10 hours, centrifuging, and obtaining supernatant I for later use;

[0055] Step 3, adding anhydrous ethanol to the supernatant I to adjust the alcohol content of the extract to 75%, letting it stand for 10 hours, and centrifuging to obtain supernatant II for later use;

[0056] Step 4: After removing ethanol from the supernatant II, concentrating and drying, the beet extract is obtained.

[0057] Preparation of microbial exopolysaccharides:

[0058] Step A: The purchased Lactobacillus rhamnosus strain numbered CGMCC1.549 was activated and expanded according to the instructions, and then diluted to make an effective viable cell count of 5.0×10 9 CFU / mL of Lactobacillus rhamnosus seed liquid; the purchased strain numbered CGMCC3.11449 was activated and expanded according to the instructions, and then diluted to make a spore volume of 3.0×10 10 / mL of Aspergillus polymorpha seed liquid; weigh the raw materials according to the ratio of 15g / L soy peptone, 10g / L glucose, 5g / L sodium nitrate, 0.03g / L calcium chloride, 1.5g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 0.05g / L manganese sulfate monohydrate, and 3-5g / L beet extract, add distilled water, adjust the pH to 6.2, and sterilize at high temperature to obtain a fermentation medium; inoculate the prepared Lactobacillus rhamnosus seed liquid into the prepared fermentation medium at a rate of 5% inoculum, and culture at 35°C for 72h to obtain an initial fermentation liquid; add catechin 0.1g / L, soy flour 8g / L, mannose 5g / L, and ferrous sulfate 0.4g / L to the initial fermentation liquid, adjust the pH to 6.0, inoculate the Aspergillus polymorpha seed liquid at a rate of 10%, and culture at 30°C for 96h to obtain a final fermentation liquid;

[0059] Step B, filtering the final fermentation broth to obtain a filtrate; adding a 25% by mass citric acid solution to the filtrate, adjusting the pH value of the supernatant to 4.0, standing for 10 hours, and centrifuging to obtain a supernatant;

[0060] Step C, adding anhydrous ethanol to the supernatant to adjust the alcohol content of the supernatant to 50% and 75% in sequence, standing for 8 hours after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0061] Step D, after dissolving the precipitate in water, adding 1% of the precipitate mass of β-glucanase (enzyme activity of 500,000 U / g) and 0.35% of the precipitate mass of xylanase (enzyme activity of 100,000 U / g), enzymolysis at 45° C. for 2 h, inactivating the enzyme, and drying to obtain the initial product;

[0062] In step E, the primary product is redissolved in water, loaded onto a Sephadex G-25 dextran gel column, and eluted with a mixed solution of methanol and water (0.8:10, v / v) as an eluent. Detection is performed during the elution process. Based on the detection results, a polysaccharide eluate with a molecular weight of 0.5 to 10 kDa is retained, methanol is removed, and the eluate is concentrated and dried to obtain the target microbial exopolysaccharide.

[0063] Example 3

[0064] Prepare beet extract:

[0065] Step 1: 10 kg of beetroot was ground into a homogenate with water, 13 g of cellulase (enzyme activity of 200,000 U / g) was added, the mixture was allowed to stand for 60 minutes, and then refluxed at 60° C. for 2.5 hours. The extract was filtered to obtain a standby solution;

[0066] Step 2, adding 30% by mass of citric acid solution to the extract, adjusting the pH value of the extract to 4.0, standing for 10 hours, centrifuging, and obtaining supernatant I for later use;

[0067] Step 3, adding anhydrous ethanol to the supernatant I to adjust the alcohol content of the extract to 75%, letting it stand for 10 hours, and centrifuging to obtain supernatant II for later use;

[0068] Step 4: After removing ethanol from the supernatant II, concentrating and drying, the beet extract is obtained.

[0069] Preparation of microbial exopolysaccharides:

[0070] Step A: The purchased Lactobacillus rhamnosus strain numbered CGMCC1.549 was activated and expanded according to the instructions, and then diluted to make an effective viable cell count of 5.0×10 9 CFU / mL of Lactobacillus rhamnosus seed liquid; the purchased strain numbered CGMCC3.11449 was activated and expanded according to the instructions, and then diluted to make a spore volume of 3.0×10 10 / mL of Aspergillus polymorpha seed liquid; weigh the raw materials according to the ratio of 15g / L soy peptone, 10g / L glucose, 5g / L sodium nitrate, 0.03g / L calcium chloride, 1.5g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 0.05g / L manganese sulfate monohydrate, and 3-5g / L beet extract, add distilled water, adjust the pH to 6.2, and sterilize at high temperature to obtain a fermentation medium; inoculate the prepared Lactobacillus rhamnosus seed liquid into the prepared fermentation medium at a rate of 5%, and culture at 35°C for 72h to obtain an initial fermentation liquid; add catechin 0.12g / L, soy flour 6g / L, mannose 7g / L, and ferrous sulfate 0.3g / L to the initial fermentation liquid, adjust the pH to 6.0, inoculate the Aspergillus polymorpha seed liquid at a rate of 10%, and culture at 30°C for 96h to obtain a final fermentation liquid;

[0071] Step B, filtering the final fermentation broth to obtain a filtrate; adding a 30% by mass fraction citric acid solution to the filtrate, adjusting the pH value of the supernatant to 4.0, standing for 10 hours, and centrifuging to obtain a supernatant;

[0072] Step C, adding anhydrous ethanol to the supernatant to adjust the alcohol content of the supernatant to 50% and 75% in sequence, standing for 8 hours after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0073] Step D, after dissolving the precipitate in water, adding 1.3% of the precipitate mass of β-glucanase (enzyme activity of 500,000 U / g) and 0.32% of the precipitate mass of xylanase (enzyme activity of 100,000 U / g), enzymolysis at 50° C. for 1.5 h, inactivating the enzyme, and drying to obtain the initial product;

[0074] In step E, the primary product is redissolved in water, loaded onto a Sephadex G-25 dextran gel column, and eluted with a mixed solution of methanol and water (0.9:10, v / v) as an eluent. Detection is performed during the elution process. Based on the test results, a polysaccharide eluate with a molecular weight of 0.5 to 10 kDa is retained, methanol is removed, and the eluate is concentrated and dried to obtain the target microbial exopolysaccharide.

[0075] Comparative Example 1

[0076] The purchased Lactobacillus rhamnosus strain numbered CGMCC1.549 was activated and expanded according to the instructions, and then diluted to make an effective viable count of 5.0×10 9CFU / mL of rhamnosus Lactobacillus seed liquid; weigh the raw materials according to the ratio of 15g / L soy peptone, 10g / L glucose, 5g / L sodium nitrate, 0.03g / L calcium chloride, 1.5g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 0.05g / L manganese sulfate monohydrate, and 3-5g / L beet extract, add distilled water, adjust the pH value to 6.2, and sterilize at high temperature to obtain a fermentation medium; inoculate the prepared rhamnosus Lactobacillus seed liquid into the prepared fermentation medium at a rate of 5% and culture at 35°C for 72h to obtain an initial fermentation liquid; add catechin 0.12g / L, 6g / L soy flour, 7g / L mannose to the initial fermentation liquid. g / L, 0.3 g / L ferrous sulfate, adjust the pH to 6.2, and culture at 35°C for 96 hours to obtain the final fermentation broth; centrifuge the fermentation broth, take the supernatant and concentrate it to obtain a concentrate; add 5 times its volume of anhydrous ethanol to the concentrate, mix well, let it stand overnight, collect the precipitate by centrifugation, air-dry at room temperature and dissolve it with distilled water, add 3 times the volume of Sevag reagent to remove protein, until no protein is produced in the middle layer, add an equal volume of anhydrous ethanol to the filtrate, mix well and let it stand, collect the precipitate by centrifugation, wash the precipitate with acetone, ether and anhydrous ethanol in sequence, and finally dissolve the precipitate in distilled water and dialyze it with a dialysis bag for 48 hours. The obtained polysaccharide solution is concentrated and dried to obtain microbial extracellular polysaccharides.

[0077] Comparative Example 2

[0078] Step A: The purchased Lactobacillus rhamnosus strain numbered CGMCC1.549 was activated and expanded according to the instructions, and then diluted to make an effective viable cell count of 5.0×10 9 CFU / mL of Lactobacillus rhamnosus seed liquid; the purchased strain numbered CGMCC3.11449 was activated and expanded according to the instructions, and then diluted to make spores with a volume of 3.0×10 10 / mL of Aspergillus polymorpha seed liquid; weigh the raw materials according to the ratio of 15g / L soy peptone, 10g / L glucose, 5g / L sodium nitrate, 0.03g / L calcium chloride, 1.5g / L potassium hydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, and 0.05g / L manganese sulfate monohydrate, add distilled water, adjust the pH value to 6.2, and sterilize at high temperature to obtain a fermentation medium; inoculate the prepared Lactobacillus rhamnosus seed liquid into the prepared fermentation medium at a rate of 5% inoculation, and culture at 35°C for 72h to obtain an initial fermentation liquid; add 6g / L soy flour, 7g / L mannose, and 0.3g / L ferrous sulfate to the initial fermentation liquid, adjust the pH to 6.0, inoculate the Aspergillus polymorpha seed liquid at a rate of 10%, and culture at 30°C for 96h to obtain a final fermentation liquid;

[0079] Step B, filtering the final fermentation broth to obtain a filtrate; adding a 30% by mass fraction citric acid solution to the filtrate, adjusting the pH value of the supernatant to 4.0, standing for 10 hours, and centrifuging to obtain a supernatant;

[0080] Step C, concentrating the supernatant, adding 5 times the volume of anhydrous ethanol, mixing, letting it stand overnight, and centrifuging to obtain a precipitate for later use;

[0081] Step D, dissolving the precipitate in water, adding 2% of the precipitate mass of β-glucanase (enzyme activity of 500,000 U / g), enzymolysis at 50° C. for 1.5 h, inactivating the enzyme, and drying to obtain the initial product;

[0082] In step E, the primary product is redissolved in water, loaded onto a Sephadex G-25 dextran gel column, and eluted with a mixed solution of methanol and water (0.9:10, v / v) as an eluent. Detection is performed during the elution process. Based on the detection results, a polysaccharide eluate with a molecular weight of 0.5 to 10 kDa is retained, methanol is removed, and the product is concentrated and dried to obtain microbial extracellular polysaccharides.

[0083] Comparative Example 3

[0084] Prepare beet extract:

[0085] Step 1: 10 kg of beetroot was ground into a homogenate with water, 13 g of cellulase (enzyme activity of 200,000 U / g) was added, the mixture was allowed to stand for 60 minutes, and then refluxed at 60° C. for 2.5 hours. The extract was filtered to obtain a standby solution;

[0086] Step 2, adding 30% by mass of citric acid solution to the extract, adjusting the pH value of the extract to 4.0, standing for 10 hours, centrifuging, and obtaining supernatant I for later use;

[0087] Step 3, adding anhydrous ethanol to the supernatant I to adjust the alcohol content of the extract to 75%, letting it stand for 10 hours, and centrifuging to obtain supernatant II for later use;

[0088] Step 4: After removing ethanol from the supernatant II, concentrating and drying, the beet extract is obtained.

[0089] Preparation of microbial exopolysaccharides:

[0090] Step A: The purchased Lactobacillus rhamnosus strain numbered CGMCC1.549 was activated and expanded according to the instructions, and then diluted to make an effective viable cell count of 5.0×10 9 CFU / mL of Lactobacillus rhamnosus seed liquid; the purchased strain numbered CGMCC3.11449 was activated and expanded according to the instructions, and then diluted to make a spore volume of 3.0×10 10 / mL of Aspergillus polymorpha seed liquid; weigh the raw materials according to the ratio of 15g / L soy peptone, 10g / L glucose, 5g / L sodium nitrate, 0.03g / L calcium chloride, 1.5g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 0.05g / L manganese sulfate monohydrate, and 3-5g / L beet extract, add distilled water, adjust the pH to 6.2, and sterilize at high temperature to obtain a fermentation medium; inoculate the prepared Lactobacillus rhamnosus seed liquid into the prepared fermentation medium at a rate of 5%, and culture at 35°C for 72h to obtain an initial fermentation liquid; add catechin 0.12g / L, soy flour 6g / L, mannose 7g / L, and ferrous sulfate 0.3g / L to the initial fermentation liquid, adjust the pH to 6.0, inoculate the Aspergillus polymorpha seed liquid at a rate of 10%, and culture at 30°C for 96h to obtain a final fermentation liquid;

[0091] Step B, filtering the final fermentation broth to obtain a filtrate; adding a 30% by mass fraction citric acid solution to the filtrate, adjusting the pH value of the supernatant to 4.0, standing for 10 hours, and centrifuging to obtain a supernatant;

[0092] Step C, adding anhydrous ethanol to the supernatant to adjust the alcohol content of the supernatant to 50% and 75% in sequence, standing for 8 hours after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0093] Step D, after dissolving the precipitate in water, adding 1.3% of the precipitate mass of β-glucanase (enzyme activity of 500,000 U / g) and 0.32% of the precipitate mass of xylanase (enzyme activity of 100,000 U / g), enzymolysis at 50° C. for 1.5 h, inactivating the enzyme, and drying to obtain the initial product;

[0094] In step E, the primary product is redissolved in water, loaded onto a Sephadex G-25 dextran gel column, and eluted with a mixed solution of methanol and water (0.9:10, v / v) as an eluent. Detection is performed during the elution process. Based on the test results, the polysaccharide eluate with a molecular weight of less than 0.5 KDa and a molecular weight of greater than 10 KDa is retained, methanol is removed, and the product is concentrated and dried to obtain microbial extracellular polysaccharides.

[0095] Performance Testing

[0096] Experiment 1: Effects of adding microbial exopolysaccharides on strawberry stem tip germination

[0097] 1. Materials and Methods

[0098] This experiment was conducted in a tissue culture laboratory, and the strawberry variety was "Hongyan".

[0099] Test Method: Rinse freshly harvested Red Strawberry runners approximately 5 cm long with running water, disinfect with 75% alcohol, rinse with sterile water, disinfect with 4% sodium hypochlorite, rinse with sterile water, and blot dry with absorbent paper. Remove the stem tip, cut into pieces 0.3-0.5 mm in size, and inoculate them in the following culture media for shoot tip germination induction. Five stem tips were inoculated per bottle, and 10 bottles were inoculated per medium. Culture conditions were: room temperature approximately 25°C, light intensity 1500 Lux, 12 hours of light per day, and 12 hours of darkness per day. Survival rate and growth were measured 14 days after inoculation.

[0100]

[0101] Blank group: MS medium (added with 30 g / L sucrose and 7 g / L agar, pH 5.8);

[0102] Control group: blank group medium + 0.3 mg / L indoleacetic acid;

[0103] Group S1-1: blank group medium + 100 mg / kg polysaccharide of Example 1;

[0104] Group S1-2: blank group medium + 100 mg / kg polysaccharide of Example 2;

[0105] Group S1-3: blank group medium + 100 mg / kg polysaccharide of Example 3;

[0106] Group S1-4: blank group medium + 100 mg / kg polysaccharide of Comparative Example 1;

[0107] Group S1-5: blank group medium + 100 mg / kg polysaccharide from Comparative Example 2;

[0108] Group S1-6: blank group culture medium + 100 mg / kg polysaccharide of Comparative Example 3.

[0109] 2. Results and Analysis

[0110] As can be seen from the results in Table 1 below, strawberry tissue culture using the microbial exopolysaccharides obtained in Examples 1 to 3 of the present invention resulted in a high shoot tip survival rate of over 90%, and good growth of the shoot tips. Groups S1 to 3 had effects equivalent to those of plant hormones. Strawberry tissue culture using the microbial exopolysaccharides obtained in Comparative Examples 1 to 3 had a low survival rate, and the growth of the cut shoot tips was average. Groups S1 to 6 had a strawberry tissue culture effect equivalent to that of the blank group.

[0111] Table 1 Effects of different polysaccharides on the growth of strawberry stem tips

[0112] Group Number of inoculations (strains) Survival rate (%) Growth performance Blank group 50 66 Slow growth, few young leaves, and yellowish buds control group 50 96 Good growth, young leaves are more stretched, buds are green S1-1 group 50 92 Good growth, young leaves are more stretched, buds are green S1-2 group 50 90 Good growth, young leaves stretching, green buds Group S1-3 50 94 Good growth, young leaves are more stretched, buds are green Group S1-4 50 76 Growth is average, with young leaves stretching and buds yellow-green Group S1-5 50 80 Average growth, few young leaves, green buds S1-6 groups 50 70 Slow growth, few young leaves, yellow-green buds

[0113] Experiment 2: Effects of adding microbial exopolysaccharides on strawberry proliferation

[0114] 1. Materials and Methods

[0115] This experiment was conducted in a tissue culture laboratory, and the strawberry variety was "Hongyan".

[0116] Experimental method: Take about 5 cm of freshly picked red strawberry runners, rinse them with running water, disinfect them with 75% alcohol, rinse them with sterile water, disinfect them with 4% sodium hypochlorite, rinse them with sterile water, and dry them with absorbent paper; peel off the stem tip, cut the stem tip with a size of 0.3 to 0.5 mm, and inoculate it into MS culture medium (add 30 g / L sucrose and 7 g / L agar, pH 5.8) for primary culture. The culture conditions are: room temperature of about 25°C, light intensity of 1500 Lux, light time of 12 h / d, and dark time of 12 h / d.

[0117] Inoculate well-growing, essentially identical test tube seedlings into the following culture medium for shoot proliferation and differentiation. Inoculate three plants per bottle, using 10 bottles of each medium. Culture conditions: room temperature approximately 25°C, light intensity 1500 Lux, 12 hours of light per day, 12 hours of darkness per day. Analyze shoot proliferation coefficients and growth 14 days after inoculation.

[0118]

[0119] Blank group: MS medium (added with 30 g / L sucrose and 7 g / L agar, pH 5.8);

[0120] Control group: blank culture medium + 0.3 mg / L 6-benzyladenine;

[0121] Group S2-1: blank group medium + 150 mg / kg polysaccharide of Example 1;

[0122] Group S2-2: blank group medium + 150 mg / kg polysaccharide of Example 2;

[0123] Group S2-3: blank group medium + 150 mg / kg polysaccharide of Example 3;

[0124] Group S2-4: blank group culture medium + 150 mg / kg polysaccharide of Comparative Example 1;

[0125] Group S2-5: blank group culture medium + 150 mg / kg polysaccharide from Comparative Example 2;

[0126] Group S2-6: blank group culture medium + 150 mg / kg polysaccharide from Comparative Example 3.

[0127] 2. Results and Analysis

[0128] As can be seen from the results in Table 2 below, when the microbial extracellular polysaccharides obtained in Examples 1 to 3 of the present invention were used to prepare the subculture medium for proliferation culture, the proliferation multiples of the strawberry tissue culture seedlings were significantly increased, the effect was equivalent to that of plant hormones, and the proliferation and growth were good, with strong and numerous buds; when the microbial extracellular polysaccharides obtained in Comparative Examples 1 to 3 were used for subculture, the effect was not obvious, among which although the number of proliferated seedlings in Group S2-4 was large, the bud growth was average; the proliferation of strawberries in Group S2-6 was equivalent to that of the blank group.

[0129] Table 2 Effects of different polysaccharides on strawberry proliferation

[0130] Group Number of inoculations (strains) Number of proliferated seedlings proliferation multiples Growth performance Blank group 30 41 1.37 Slow growth, yellowish color, weak, few buds control group 30 95 3.17 Fast growing, tender green, strong, with many buds S2-1 Group 30 91 3.03 Fast growing, tender green, strong, with many buds S2-2 group 30 89 2.97 Fast growing, tender green, strong, with many buds S2-3 group 30 93 3.10 Fast growing, tender green, strong, with many buds S2-4 Group 30 62 2.07 Growth is average, color is green, small, and buds are average S2-5 Group 30 57 1.90 Growth is average, color is yellow-green, strong, and the buds are average S2-6 Group 30 45 1.50 Slow growth, yellow-green color, small, with few buds

[0131] Experiment 3: Effects of Adding Microbial Exopolysaccharides on Rooting of Adventitious Buds of Tissue-cultured Strawberry

[0132] 1. Materials and Methods

[0133] The strawberry adventitious buds required for this experiment were obtained from the tissue culture laboratory, and the strawberry variety was "Miaoxiang".

[0134] Experimental method: Strawberry adventitious buds of basically the same growth potential were inoculated into the following different rooting media, with 4 plants inoculated into each bottle and 10 bottles inoculated into each medium. The inoculated adventitious buds were placed in a sterile culture room for cultivation and observation. The culture conditions were: the sterile culture room temperature was around 20°C during the day and not lower than 15°C at night, the relative air humidity was 65-75%, the light intensity was 2000 Lux, the light period was 12 hours / day, and the dark period was 12 hours / day.

[0135] Blank group: MS medium (added with 30 g / L sucrose and 7 g / L agar);

[0136] Control group: blank group medium + 0.5 mg / L indolebutyric acid;

[0137] Group S3-1: blank group medium + 200 mg / kg polysaccharide of Example 1;

[0138] Group S3-2: blank group medium + 200 mg / kg polysaccharide from Example 2;

[0139] Group S3-3: blank group medium + 200 mg / kg polysaccharide of Example 3;

[0140] Group S3-4: blank group medium + 200 mg / kg polysaccharide of Comparative Example 1;

[0141] S3-5 group: blank group medium + 200 mg / kg polysaccharide of comparative example 2;

[0142] Group S3-6: blank group culture medium + 200 mg / kg polysaccharide of Comparative Example 3.

[0143] The rooting and growth status were counted on the 30th day after inoculation, specifically:

[0144]

[0145] SPSS22.0 data statistical software was used to process the above data.

[0146] 2. Results and Analysis

[0147] As can be seen from the results in Table 1, the microbial exopolysaccharides prepared in Examples 1 to 3 of the present invention have a significant promoting effect on the rooting of strawberry adventitious buds, and the number of roots, average number of roots, and average root length are significantly increased; the microbial exopolysaccharides obtained in Comparative Examples 1 to 3 have no obvious effect on the rooting of strawberry tissue culture seedlings.

[0148] Table 3 Effects of different polysaccharides on strawberry rooting

[0149]

[0150]

[0151] As can be seen from the above experimental results, the microbial exopolysaccharide prepared in the present invention has a good promoting effect on plant tissue culture, can promote shoot apex germination and proliferation, and promote adventitious bud rooting. It can be used in all stages of primary culture, subculture, and rooting culture, and its use effect is comparable to that of existing disclosed plant hormones. The preparation methods of microbial exopolysaccharides in Comparative Examples 1-3 are significantly different from those in the present invention. The polysaccharides obtained therefrom are different from those in the present invention and have poor effects in plant tissue culture. In particular, the microbial exopolysaccharide obtained in Comparative Example 3 has essentially no effect on plant tissue culture.

Claims

1. A method for preparing microbial exopolysaccharide, characterized in that: The preparation method comprises the following steps: Step A: inoculating Lactobacillus rhamnosus seed liquid into a fermentation medium containing soy peptone, glucose, sodium nitrate, calcium chloride, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate, and beet extract, and culturing at 35° C. for 72 hours to obtain an initial fermentation liquid; adding catechin, soy flour, mannose, and ferrous sulfate to the initial fermentation liquid, adjusting the pH to 6.0-6.2, inoculating Aspergillus polymorpha seed liquid, and culturing at 30° C. for 96 hours to obtain a final fermentation liquid; Step B, filtering the final fermentation broth to obtain a filtrate; adding a citric acid solution with a mass fraction of 25-30% to the filtrate, adjusting the pH value of the supernatant to 4.0, standing for 10 hours, and centrifuging to obtain a supernatant; Step C, adding anhydrous ethanol to the supernatant to adjust the alcohol content of the supernatant to 50% and 75% in sequence, standing for 8 hours after each adjustment, centrifuging, and obtaining a precipitate for later use; Step D, dissolving the precipitate in water, adding β-glucanase and xylanase, performing enzymolysis at 45-50°C for 1.5-2h, inactivating the enzyme, and drying to obtain the primary product; In step E, the primary product is redissolved in water, loaded onto a Sephadex G-25 dextran gel column, and eluted using a methanol-water mixed solution as an eluent. Detection is performed during the elution process, and based on the detection results, a polysaccharide eluate with a molecular weight of 0.5 to 10 kDa is retained, methanol is removed, and the eluate is concentrated and dried to obtain the target microbial exopolysaccharide.

2. The preparation method according to claim 1, characterized in that The inoculation amount of the Lactobacillus rhamnosus seed solution in step A is 5%; the effective viable bacteria count in the Lactobacillus rhamnosus seed solution is 5.0×10 9 CFU / mL.

3. The preparation method according to claim 1, characterized in that The fermentation medium in step A is prepared by weighing the raw materials according to the ratio of 15 g / L soy peptone, 10 g / L glucose, 5 g / L sodium nitrate, 0.03 g / L calcium chloride, 1.5 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, and 3-5 g / L beet extract, adding distilled water, adjusting the pH to 6.2-6.5, and sterilizing at high temperature to obtain the fermentation medium.

4. The preparation method according to claim 1, characterized in that In the step A, the amounts of catechin, soybean powder, mannose, and ferrous sulfate added are 0.1-0.15 g / L of catechin, 5-8 g / L of soybean powder, 5-8 g / L of mannose, and 0.3-0.4 g / L of ferrous sulfate, respectively.

5. The preparation method according to claim 1, characterized in that The inoculation amount of the Aspergillus polymorpha seed solution in step A is 10%; the spore amount in the Aspergillus polymorpha seed solution is 3.0×10 10 pieces / mL.

6. The preparation method according to claim 1, characterized in that In the step D, the amount of β-glucanase added is 1% to 1.5% of the mass of the precipitate, and the amount of xylanase added is 0.3% to 0.35% of the mass of the precipitate; the enzyme activity of the β-glucanase is 500,000 U / g, and the enzyme activity of the xylanase is 100,000 U / g.

7. The preparation method according to claim 1, characterized in that The volume ratio of methanol to water in the methanol-water mixed solution in step E is (0.8-1):

10.

8. The preparation method according to claim 1, characterized in that The preparation method of the beet extract comprises the following steps: Step 1: Grind beetroot with water to prepare a homogenate, add cellulase, mix well, and then stand for 50-60 minutes. Then, reflux extract at 50-60°C for 2-3 hours, filter, and obtain an extract for later use; Step 2, adding 25-30% by mass of citric acid solution to the extract, adjusting the pH value of the extract to 4.0, letting it stand for 10 hours, centrifuging, and obtaining supernatant I for later use; Step 3, adding anhydrous ethanol to the supernatant I to adjust the alcohol content of the supernatant I to 75%, letting it stand for 10 hours, and centrifuging to obtain supernatant II for later use; Step 4: After removing ethanol from the supernatant II, concentrating and drying, the beet extract is obtained.

9. The preparation method according to claim 8, characterized in that In step 1 of the beet extract preparation method, the amount of cellulase added is 0.1-0.15% of the mass of the beet root; and the enzymatic activity of the cellulase is 200,000 U / g.

10. Use of the microbial exopolysaccharide prepared by the preparation method according to any one of claims 1 to 9 in preparing a culture medium for tissue culture.

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