Small molecule peptide for culture medium for virus-free seedlings and preparation method of small molecule peptide

Abstract

The invention relates to a small molecule peptide for a culture medium for virus-free seedlings and a preparation method of the small molecule peptide, and belongs to the technical field of small molecule peptides. The small molecule peptide provided by the invention is obtained by fermenting bacillus amyloliquefaciens and paecilomyces hepialid. The invention also provides a preparation method of the small molecule peptide, and the preparation method comprises the steps of microbial fermentation, separation, enzymolysis and purification to finally obtain the small molecule peptide. The small molecule peptide provided by the invention can be used for preparing a culture medium for virus-free seedlings, and when the culture medium prepared by utilizing the small molecule peptide is used for culturing the virus-free seedlings, the browning rate of the virus-free seedlings can be effectively reduced, and the occurrence degree of vitrified seedlings can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of small molecule peptides, and relates to a small molecule peptide for a culture medium for virus-free seedlings and a preparation method thereof. Background Art

[0002] In agricultural production, after several generations of cultivation, the quality of some crops generally shows varying degrees of degradation, which is particularly prominent in crops such as potatoes, vegetables, fruit trees, strawberries, and flowers. According to long-term research by scientists, the main reason for the decline in crop quality is the infection of plant viruses. To obtain high yields, virus-free seedlings, that is, detoxified seedlings, need to be cultivated in production. Virus-free seedlings refer to the plant virus detoxification technology, which is to use methods such as high-temperature treatment and shoot tip tissue culture to remove the viruses infected by plants, and cultivate virus-free plants under ultra-clean and aseptic conditions, and carry out vegetative propagation to rapidly breed and produce virus-free seedlings and seed potatoes for application in field production.

[0003] Browning is one of the three major adverse problems in plant tissue culture, and its occurrence mechanism and influencing conditions are complex. It is the most common but the most difficult to solve. It mainly occurs during the primary culture period of explants, and often occurs in other periods, easily leading to poor growth or even death of itself. A number of studies have shown that browning can cause changes in the morphological structure of plant tissue cells, such as chlorosis and discoloration, increased starch grains, thickened cell walls, increased fibrous structure, chaotic arrangement of callus cells, increased cell membrane permeability, and blockage of leaf vascular bundles, seriously affecting tissue culture.

[0004] Vitrification refers to a semi-transparent phenomenon similar to waterlogging that occurs during the growth of plant seedlings, which is different from the "vitrification" in encapsulation-vitrification cryopreservation and vitrification cryo-detoxification. Vitrification is a pathological phenomenon of plant physiological disorder. After vitrification occurs during tissue culture, seedlings will show symptoms such as poor growth, low differentiation ability, and difficulty in survival.

[0005] Research has shown that in addition to common plant hormones such as auxin, signal small peptides also play an important role in short-distance signal communication between plant cells. As a new type of plant hormone, signal small peptides participate in multiple processes of plant growth and development. Existing research has shown that active peptides such as alanine, histidine, and carnosine can promote plant growth and development, inhibit membrane lipid peroxidation, effectively scavenge free radicals in cells, and increase plant regeneration ability.

[0006] At present, there is no relevant application of small molecule peptides in the cultivation of virus-free seedlings. Summary of the Invention

[0007] The main object of the present invention is to provide a small molecule peptide and a preparation method thereof. The small molecule peptide provided by the present invention can be used for tissue culture of virus-free seedlings, and can significantly reduce the browning and vitrification phenomena of virus-free seedlings.

[0008] The present invention adopts the following technical solutions to achieve the above object:

[0009] A small molecule peptide for the culture medium of detoxified seedlings, wherein the small molecule peptide is obtained by fermenting Bacillus amyloliquefaciens and Paecilomyces hepiali.

[0010] The present invention provides a preparation method of the above small molecule peptide, comprising the following steps:

[0011] Step 1: Inoculate the Bacillus amyloliquefaciens seed liquid and Paecilomyces hepiali seed liquid into a fermentation medium prepared from glucose, corn steep liquor, calcium chloride, potassium dihydrogen phosphate, pentosan, and water, and culture at 30 °C and 150 r / min for 8 - 10 h to obtain a primary fermentation liquid;

[0012] Step 2: Add turkesterone, corn silk extract, magnesium sulfate heptahydrate, and zinc chloride to the primary fermentation liquid, dissolve and mix well, then raise the temperature to 35 °C and continue to culture for 20 - 24 h to obtain a final fermentation liquid. Centrifuge the fermentation liquid and take the supernatant to obtain a crude peptide extract;

[0013] Step 3: Add shikimic acid to the obtained crude peptide extract, mix well and let stand for 1.5 - 2 h, then add anhydrous ethanol to adjust the alcohol content to 70 - 75%, let stand at 4 - 5 °C for 2 - 3 h, centrifuge, and take the precipitate;

[0014] Step 4: After redissolving the obtained precipitate in water, add subtilisin, streptomyces protease, and nattokinase, enzymatically hydrolyze at 40 °C for 2 - 3 h, inactivate the enzyme, and concentrate to obtain an enzymatic hydrolysate;

[0015] Step 5: Separate the enzymatic hydrolysate by a gel column, elute with a methanol - water mixed solution as an eluent, detect the obtained eluate, intercept the eluate containing small molecule peptides with a molecular weight of 500 - 800 Da, combine the eluates, remove methanol, concentrate, and dry to obtain the small molecule peptide.

[0016] Further, in Step 1, the inoculation amount of the Bacillus amyloliquefaciens seed liquid is 5 - 8%, and the inoculation amount of the Paecilomyces hepiali seed liquid is 10 - 12%.

[0017] Further, the preparation method of the fermentation medium in Step 1 is: weigh 10 - 12 parts by weight of glucose, 13 - 15 parts by weight of corn steep liquor, 1 - 1.2 parts by weight of calcium chloride, 0.5 - 0.6 parts by weight of potassium dihydrogen phosphate, 3 - 5 parts by weight of pentosan, add 1000 parts by weight of water, dissolve and mix well, adjust the pH to 6.5, and sterilize at high temperature to obtain the fermentation medium.

[0018] Further, the addition amounts of turkesterone, corn silk extract, magnesium sulfate heptahydrate, and zinc chloride in step 2 are 3 - 3.5 g / L of turkesterone, 5 - 8 g / L of corn silk extract, 0.2 - 0.3 g / L of magnesium sulfate heptahydrate, and 0.6 - 0.65 g / L of zinc chloride, respectively.

[0019] Further, in step 3, the addition amount of shikimic acid is 1 - 1.5 g / L.

[0020] Further, in step 4, the addition amount of subtilisin is 0.2 - 0.3% of the precipitate mass, the addition amount of pronase is 0.05 - 0.08% of the precipitate mass, and the addition amount of nattokinase is 0.1 - 0.15% of the precipitate mass.

[0021] Further, in step 5, the volume ratio of methanol to water in the methanol - water mixed solution is (0.3 - 0.5):1.

[0022] The present invention provides that the preparation method of the corn silk extract in step 2 is as follows:

[0023] Step A: Crush the corn silk, soak it with petroleum ether for 2.5 - 3 h, filter to remove the petroleum ether, add an ethanol solution with a mass fraction of 60 - 65%, carry out reflux extraction, filter, and reserve the extract.

[0024] Step B: Extract the extract with ethyl acetate, take the upper layer solution, concentrate it, carry out silica gel column chromatography separation, elute with a chloroform - methanol solvent system, combine the eluates, and remove the organic solvents to obtain the corn silk extract.

[0025] Further, in step B, the volume ratio of chloroform to methanol is 100:(3 - 5).

[0026] The present invention provides the specific use of the obtained small molecule peptide, that is, the small molecule peptide can be used to prepare a culture medium for detoxified seedlings.

[0027] The present invention has the following beneficial effects:

[0028] 1. The present invention provides a small molecule peptide obtained by fermenting Bacillus amyloliquefaciens and Paecilomyces hepiali, followed by separation, enzymatic hydrolysis, and purification. This small molecule peptide can be used to prepare a culture medium for tissue culture of detoxified seedlings. When using the culture medium prepared with this small molecule peptide for tissue culture, it can effectively avoid the occurrence of browning and vitrification phenomena in tissue culture, improve the quality of tissue culture, and reduce costs.

[0029] 2. In the preparation process of the small molecule peptides of the present invention, fermentation strains were first screened. During the research process, it was found that the peptides obtained by co-fermentation of Bacillus amyloliquefaciens and Paecilomyces hepiali had the effect of preventing browning and vitrification of tissue culture seedlings after separation, enzymatic hydrolysis, and purification; during the fermentation process, a medium containing pentosan was selected as the basic fermentation medium. After culturing for a period of time, tukestosterone and corn silk extract were added to induce peptide production; for the obtained peptide-containing fermentation broth, ethanol was used to precipitate proteins. However, in this process, ethanol would react with the chemical bonds in the peptides, change the conformation of the peptides, thus destroying the structure of the peptides and causing the peptides to lose certain activity. Therefore, in this process, shikimic acid was added to stabilize and protect the peptides and prevent them from inactivating; after enzymatic hydrolysis, the peptides were separated and purified by the action of a gel column molecular sieve, and small molecule peptides with a molecular weight of 500 - 800 Da were intercepted. After experimental verification, the obtained small molecule peptides could effectively improve the browning and vitrification degrees of tissue culture seedlings. Detailed implementation mode

[0030] The following further illustrates the present invention with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art fall within the scope protected by the claims of this application.

[0031] Example 1

[0032] Preparation of corn silk extract:

[0033] Step A: Take dry corn silk, crush it, and pass it through a 100-mesh sieve; add petroleum ether according to a solid-liquid ratio of 1:10 (kg / L), soak for 3 h, filter, and naturally volatilize to remove the residual petroleum ether; then add an ethanol solution with a mass fraction of 65% according to a solid-liquid ratio of 1:10 (kg / L), reflux and extract 2 times, 1 h each time, filter, combine, and concentrate to obtain an extract for standby.

[0034] Step B: Add the extract to an equal volume of ethyl acetate and extract 3 times. Take the upper extraction solution, combine, concentrate, load it onto a silica gel column for separation, elute with a chloroform-methanol (100:3) solvent system, combine the eluates, remove the organic solvents under reduced pressure, concentrate, and dry to obtain the corn silk extract.

[0035] Preparation of small molecule peptides:

[0036] Step 1: After activating Bacillus amyloliquefaciens with the preservation number CGMCC1.1414 and Paecilomyces hepiali with the preservation number CGMCC3.14870 purchased from the China General Microbiological Culture Collection Center according to the instructions respectively, inoculate them into the seed medium for subculture, and then dilute and prepare them into an effective viable count of 3.0×109 Bacillus amyloliquefaciens seed solution with a cfu / mL of 1.0×10 9 Paecilomyces hepiali seed solution with a cfu / mL of 1.0×10. Prepare the fermentation medium according to the following method: Weigh 10 g of glucose, 15 g of corn steep liquor, 1 g of calcium chloride, 0.6 g of potassium dihydrogen phosphate, and 3 g of pentosan, add 1000 g of water, dissolve and mix well, adjust the pH to 6.5, and sterilize at high temperature to obtain the fermentation medium. Inoculate the prepared Bacillus amyloliquefaciens seed solution and Paecilomyces hepiali seed solution into the fermentation medium at inoculation amounts of 8% and 10% respectively, and culture at 30 °C and 150 r / min for 10 h to obtain the primary fermentation broth;

[0037] Step 2, add 3.5 g / L of turkesterone, 5 g / L of corn silk extract, 0.2 g / L of magnesium sulfate heptahydrate, and 0.65 g / L of zinc chloride to the primary fermentation broth. After dissolving and mixing well, raise the temperature to 35 °C and continue to culture at 150 r / min for 24 h to obtain the final fermentation broth. Centrifuge the fermentation broth and take the supernatant to obtain the crude peptide extract;

[0038] Step 3, add 1 g / L of shikimic acid to the obtained crude peptide extract, mix well and let stand for 2 h, then add anhydrous ethanol to adjust the alcohol content to 75%, let stand at 5 °C for 2 h, centrifuge, and take the precipitate;

[0039] Step 4, after redissolving 100 g of the obtained precipitate in water, add 0.2 g of subtilisin with an enzyme activity of 150000 U / g, 0.08 g of pronase with an enzyme activity of 7000 U / g, and 0.1 g of nattokinase with an enzyme activity of 20000 U / g, and enzymatically hydrolyze at 40 °C for 3 h, inactivate the enzyme, and concentrate to obtain the enzymatic hydrolysate;

[0040] Step 5, load the enzymatic hydrolysate onto a Sephadex G-25 gel column for separation, use a methanol-water mixed solution with a volume ratio of 0.5:1 as the eluent for elution, detect the obtained eluate, and according to the detection results, intercept the eluate of small molecular peptides with a molecular weight cut-off of 500 - 800 Da, combine the eluates, remove methanol, concentrate, and dry to obtain the small molecular peptides.

[0041] Example 2

[0042] Preparation of corn silk extract:

[0043] Step A, take dry corn silk, crush it, and pass through a 100-mesh sieve; add petroleum ether according to a solid-liquid ratio of 1:10 (kg / L), soak for 2.5 h, filter, and naturally volatilize to remove the residual petroleum ether; then add an ethanol solution with a mass fraction of 60% according to a solid-liquid ratio of 1:10 (kg / L), reflux and extract 2 times, 1 h each time, filter, combine and concentrate to obtain the extract for standby;

[0044] Step B: Add the extract to an equal volume of ethyl acetate and extract three times. Take the upper extraction solution, combine and concentrate it, load it onto a silica gel column for separation, elute it with a chloroform-methanol (100:5) solvent system, combine the eluates, remove the organic solvents under reduced pressure, concentrate and dry to obtain the corn silk extract.

[0045] Preparation of small molecule peptides:

[0046] Step 1: After activating Bacillus amyloliquefaciens with the preservation number CGMCC 1.1414 and Paecilomyces hepiali with the preservation number CGMCC 3.14870 purchased from the China General Microbiological Culture Collection Center according to the instructions respectively, inoculate them into the seed medium for scale-up culture, and then dilute and prepare a Bacillus amyloliquefaciens seed solution with an effective viable count of 3.0×10 9 cfu / mL and a Paecilomyces hepiali seed solution with an effective viable count of 1.0×10 9 cfu / mL. Prepare the fermentation medium according to the following method: Weigh 12 g of glucose, 13 g of corn steep liquor, 1.2 g of calcium chloride, 0.5 g of potassium dihydrogen phosphate, and 5 g of pentosan, add 1000 g of water, dissolve and mix evenly, adjust the pH to 6.5, and sterilize at high temperature to obtain the fermentation medium. Inoculate the prepared Bacillus amyloliquefaciens seed solution and Paecilomyces hepiali seed solution into the fermentation medium at inoculation amounts of 5% and 12% respectively, and culture at 30 °C and 150 r / min for 8 h to obtain the primary fermentation broth;

[0047] Step 2: Add 3 g / L of turkesterone, 8 g / L of corn silk extract, 0.3 g / L of magnesium sulfate heptahydrate, and 0.6 g / L of zinc chloride to the primary fermentation broth. After dissolving and mixing evenly, raise the temperature to 35 °C and continue to culture at 150 r / min for 20 h to obtain the final fermentation broth. Centrifuge the fermentation broth and take the supernatant to obtain the crude peptide extract;

[0048] Step 3: Add 1.5 g / L of shikimic acid to the obtained crude peptide extract, mix evenly and let it stand for 1.5 h, then add absolute ethanol to adjust the alcohol content to 70%, let it stand at 4 °C for 3 h, and centrifuge to take the precipitate;

[0049] Step 4: After redissolving 100 g of the obtained precipitate in water, add 0.3 g of subtilisin with an enzyme activity of 150000 U / g, 0.05 g of pronase with an enzyme activity of 7000 U / g, and 0.15 g of nattokinase with an enzyme activity of 20000 U / g, and enzymatically hydrolyze at 40 °C for 2 h, inactivate the enzyme, and concentrate to obtain the enzymatic hydrolysate;

[0050] Step 5: Load the enzymolysis solution onto a Sephadex G-25 gel column for separation, elute with a methanol-water mixed solution with a volume ratio of 0.3:1 as the eluent, detect the obtained eluate, according to the detection results, intercept the eluate of small molecule peptides with a molecular weight cut-off of 500 - 800 Da, combine the eluates, remove methanol, concentrate, and dry to obtain the small molecule peptides.

[0051] Example 3

[0052] Preparation of corn silk extract:

[0053] Step A: Take dry corn silk, crush it, and pass through a 100-mesh sieve; add petroleum ether according to a solid-liquid ratio of 1:10 (kg / L), soak for 3 h, after filtration, naturally volatilize to remove the residual petroleum ether; then add an ethanol solution with a mass fraction of 65% according to a solid-liquid ratio of 1:10 (kg / L), reflux and extract 2 times, each time for 1 h, filter, combine and concentrate to obtain the extract for standby;

[0054] Step B: Add the extract to an equal volume of ethyl acetate and extract 3 times, take the upper layer of the extraction solution, combine and concentrate, load onto a silica gel column for separation, elute with a chloroform-methanol (100:4) solvent system, combine the eluates, remove the organic solvents under reduced pressure, concentrate, and dry to obtain the corn silk extract.

[0055] Preparation of small molecule peptides:

[0056] Step 1: After activating Bacillus amyloliquefaciens with the preservation number of CGMCC 1.1414 and Paecilomyces hepiali with the preservation number of CGMCC 3.14870 purchased from the China General Microbiological Culture Collection Center according to the instructions respectively, inoculate them into the seed medium for subculture respectively, and then dilute and prepare a Bacillus amyloliquefaciens seed solution with an effective viable count of 3.0×10 9 cfu / mL and a Paecilomyces hepiali seed solution with an effective viable count of 1.0×10 9 cfu / mL. Prepare the fermentation medium according to the following method: Weigh 11 g of glucose, 14 g of corn steep liquor, 1.1 g of calcium chloride, 0.6 g of potassium dihydrogen phosphate, 4 g of pentosan, add 1000 g of water, dissolve and mix evenly, adjust the pH to 6.5, and sterilize at high temperature to obtain the fermentation medium. Inoculate the prepared Bacillus amyloliquefaciens seed solution and Paecilomyces hepiali seed solution into the fermentation medium according to the inoculation amounts of 6% and 11% respectively, and culture at 30 °C and 150 r / min for 9 h to obtain the primary fermentation broth;

[0057] Step 2: Add 3.3 g / L of turkesterone, 6 g / L of corn silk extract, 0.25 g / L of magnesium sulfate heptahydrate, and 0.6 g / L of zinc chloride to the primary fermentation broth. After dissolving and mixing evenly, heat up to 35°C and continue culturing at 150 r / min for 24 h to obtain the final fermentation broth. Centrifuge the fermentation broth and take the supernatant to obtain the crude peptide extract.

[0058] Step 3: Add 1.3 g / L of shikimic acid to the obtained crude peptide extract. After mixing evenly, let it stand for 2 h, then add absolute ethanol to adjust the alcohol content to 70%. Let it stand at 4°C for 3 h, centrifuge, and take the precipitate.

[0059] Step 4: After redissolving 100 g of the obtained precipitate in water, add 0.25 g of subtilisin with an enzyme activity of 150000 U / g, 0.06 g of pronase with an enzyme activity of 7000 U / g, and 0.12 g of nattokinase with an enzyme activity of 20000 U / g. Carry out enzymatic hydrolysis at 40°C for 2 h, inactivate the enzyme, and concentrate to obtain the enzymatic hydrolysate.

[0060] Step 5: Load the enzymatic hydrolysate onto a Sephadex G-25 gel column for separation, use a methanol-water mixed solution with a volume ratio of 0.4:1 as the eluent for elution, detect the obtained eluate. According to the detection results, intercept the eluate with small molecular peptides having a molecular weight cut-off of 500 - 800 Da, combine the eluates, remove methanol, concentrate, and dry to obtain the small molecular peptides.

[0061] Comparative Example 1

[0062] Step 1: After activating Bacillus amyloliquefaciens with the preservation number CGMCC 1.1414 and Paecilomyces hepiali with the preservation number CGMCC 3.14870 purchased from the China General Microbiological Culture Collection Center according to the instructions respectively, inoculate them into the seed medium for subculture, and then dilute and prepare a Bacillus amyloliquefaciens seed solution with an effective viable count of 3.0×10 9 cfu / mL and a Paecilomyces hepiali seed solution with an effective viable count of 1.0×10 9 cfu / mL. Prepare the fermentation medium according to the following method: Weigh 11 g of glucose, 14 g of corn steep liquor, 1.1 g of calcium chloride, 0.6 g of potassium dihydrogen phosphate, 4 g of pentosan, add 1000 g of water, dissolve and mix evenly, adjust the pH to 6.5, and sterilize at high temperature to obtain the fermentation medium. Inoculate the prepared Bacillus amyloliquefaciens seed solution and Paecilomyces hepiali seed solution into the fermentation medium at inoculation amounts of 6% and 11% respectively, and culture at 30°C and 150 r / min for 24 h to obtain the fermentation broth; centrifuge the fermentation broth and take the supernatant to obtain the crude peptide extract.

[0063] Step 2: Add 0.25 g / L of subtilisin with an enzyme activity of 150,000 U / g, 0.06 g / L of pronase with an enzyme activity of 7,000 U / g, and 0.12 g / L of nattokinase with an enzyme activity of 20,000 U / g to the obtained crude peptide extract, enzymatically hydrolyze at 40 °C for 2 h, inactivate the enzyme, concentrate, obtain the enzymatic hydrolysate, and dry it to obtain small molecule peptides.

[0064] Comparative Example 2

[0065] Prepare corn silk extract:

[0066] Take dry corn silk, crush it, and pass it through a 100-mesh sieve; add an ethanol solution with a mass fraction of 65% according to a solid-liquid ratio of 1:10 (kg / L), reflux and extract twice, 1 h each time, filter, combine and concentrate, and dry to obtain corn silk extract.

[0067] Prepare small molecule peptides:

[0068] Step 1: After activating Bacillus amyloliquefaciens with the preservation number of CGMCC 1.1414 and Paecilomyces hepiali with the preservation number of CGMCC 3.14870 purchased from the China General Microbiological Culture Collection Center according to the instructions respectively, inoculate them into the seed medium for subculture respectively, and then dilute and prepare a Bacillus amyloliquefaciens seed solution with an effective viable count of 3.0×10 9 cfu / mL and a Paecilomyces hepiali seed solution with an effective viable count of 1.0×10 9 cfu / mL. Prepare the fermentation medium according to the following method: Weigh 11 g of glucose, 14 g of corn steep liquor, 1.1 g of calcium chloride, and 0.6 g of potassium dihydrogen phosphate, add 1000 g of water, dissolve and mix evenly, adjust the pH to 6.5, and sterilize at high temperature to obtain the fermentation medium. Inoculate the prepared Bacillus amyloliquefaciens seed solution and Paecilomyces hepiali seed solution into the fermentation medium according to the inoculation amounts of 6% and 11% respectively, and culture at 30 °C and 150 r / min for 9 h to obtain the primary fermentation broth;

[0069] Step 2: Add 6 g / L of corn silk extract, 0.25 g / L of magnesium sulfate heptahydrate, and 0.6 g / L of zinc chloride to the primary fermentation broth, dissolve and mix evenly, then raise the temperature to 35 °C, and continue to culture at 150 r / min for 24 h to obtain the final fermentation broth. Centrifuge the fermentation broth and take the supernatant to obtain the crude peptide extract;

[0070] Step 3: Add absolute ethanol to the obtained crude peptide extract to adjust the alcohol content to 70%, let it stand at 4 °C for 3 h, centrifuge, and take the precipitate;

[0071] Step 4: After redissolving 100 g of the obtained precipitate in water, add 0.25 g of subtilisin with an enzyme activity of 150,000 U / g and 0.06 g of pronase with an enzyme activity of 7,000 U / g, and enzymatically hydrolyze at 40 °C for 2 h. Inactivate the enzyme, concentrate, and obtain an enzymatic hydrolysate;

[0072] Step 5: Load the enzymatic hydrolysate onto a Sephadex G-25 gel column for separation, elute with a methanol-water mixed solution with a volume ratio of 0.4:1 as the eluent, detect the obtained eluate, and according to the detection results, intercept the eluate of small peptides with a molecular weight cut-off of 500-800 Da. Combine the eluates, remove methanol, concentrate, and dry to obtain small peptides.

[0073] Comparative Example 3

[0074] Preparation of corn silk extract:

[0075] Step A: Take dry corn silk, crush it, and pass through a 100-mesh sieve; add petroleum ether according to a solid-liquid ratio of 1:10 (kg / L), soak for 3 h, filter, and naturally volatilize to remove the residual petroleum ether; then add an ethanol solution with a mass fraction of 65% according to a solid-liquid ratio of 1:10 (kg / L), reflux and extract twice, 1 h each time, filter, combine and concentrate to obtain an extract for standby;

[0076] Step B: Add the extract to an equal volume of ethyl acetate and extract 3 times. Take the upper extraction solution, combine and concentrate, load it onto a silica gel column for separation, elute with a chloroform-methanol (100:4) solvent system, combine the eluates, remove the organic solvent under reduced pressure, concentrate, and dry to obtain the corn silk extract.

[0077] Preparation of small peptides:

[0078] Step 1: After activating Bacillus amyloliquefaciens with a preservation number of CGMCC 1.1414 and Paecilomyces hepiali with a preservation number of CGMCC 3.14870 purchased from the China General Microbiological Culture Collection Center according to the instructions, inoculate them into a seed medium for scale-up culture respectively, and then dilute and prepare a Bacillus amyloliquefaciens seed solution with an effective viable count of 3.0×10 9 cfu / mL and a Paecilomyces hepiali seed solution with an effective viable count of 1.0×10 9 cfu / mL. Prepare the fermentation medium according to the following method: Weigh 11 g of glucose, 14 g of corn steep liquor, 1.1 g of calcium chloride, 0.6 g of potassium dihydrogen phosphate, and 4 g of pentosan, add 1000 g of water, dissolve and mix evenly, adjust the pH to 6.5, and sterilize at high temperature to obtain the fermentation medium. Inoculate the prepared Bacillus amyloliquefaciens seed solution and Paecilomyces hepiali seed solution into the fermentation medium at inoculation amounts of 6% and 11% respectively, and culture at 30 °C and 150 r / min for 9 h to obtain a primary fermentation broth;

[0079] Step 2: Add 3.3 g / L of turkesterone, 6 g / L of corn silk extract, 0.25 g / L of magnesium sulfate heptahydrate, and 0.6 g / L of zinc chloride to the primary fermentation broth. After dissolving and mixing evenly, heat up to 35°C and continue culturing at 150 r / min for 24 h to obtain the final fermentation broth. Centrifuge the fermentation broth and take the supernatant to obtain the crude peptide extract.

[0080] Step 3: Add absolute ethanol to the obtained crude peptide extract to adjust the ethanol content to 70%. Let it stand at 4°C for 3 h, then centrifuge and take the precipitate.

[0081] Step 4: After redissolving 100 g of the obtained precipitate in water, add 0.25 g of subtilisin with an enzyme activity of 150000 U / g, 0.06 g of pronase with an enzyme activity of 7000 U / g, and 0.12 g of nattokinase with an enzyme activity of 20000 U / g. Enzymatically hydrolyze at 40°C for 2 h, inactivate the enzyme, and concentrate to obtain the enzymatic hydrolysate.

[0082] Step 5: Load the enzymatic hydrolysate onto a Sephadex G-25 gel column for separation, and use a methanol-water mixed solution with a volume ratio of 0.4:1 as the eluent for elution. Detect the obtained eluate. According to the detection results, intercept the eluate of small peptides with a molecular weight cut-off below 500 Da and a molecular weight in the range of 800 - 1000 Da. Combine the eluates, remove methanol, concentrate, and dry to obtain the small peptides.

[0083] Performance Test

[0084] Test Experiment 1: Preventive Effect of the Small Peptides of the Present Invention on Browning of Strawberry Virus-Free Seedlings

[0085] 1. Materials and Methods

[0086] This experiment was carried out in a tissue culture room. The selected strawberry variety was "Miaoxiang".

[0087] This experiment was carried out in the following manner with reference to the prior art:

[0088] Select healthy strawberry stolons without pests and diseases, take the apical buds 4 - 5 cm long, rinse them thoroughly, disinfect them, wash them with sterile water, cut them into stem tips of 1 - 2 mm under a microscope, inoculate them onto the culture medium, and culture them at 25°C with a light intensity of 2000 lx, a light time of 12 h / d, and a dark time of 12 h / d. Inoculate 2 stem tips into each culture medium, and inoculate 15 bottles of each culture medium.

[0089] Culture Medium Preparation:

[0090] Blank Group 1: MS medium (added with 30 g / L of sucrose + 6 g / L of agar + 0.2 mg / L of naphthaleneacetic acid);

[0091] Control Group 1: Blank group + VC (50 mg / L);

[0092] Test Group 1-1: Blank group + small molecule peptide of Example 1 (30 mg / L)

[0093] Test Group 1-2: Blank group + small molecule peptide of Example 2 (30 mg / L)

[0094] Test Group 1-3: Blank group + small molecule peptide of Example 3 (30 mg / L)

[0095] Test Group 1-4: Blank group + small molecule peptide of Comparative Example 1 (30 mg / L)

[0096] Test Group 1-5: Blank group + small molecule peptide of Comparative Example 2 (30 mg / L)

[0097] Test Group 1-6: Blank group + small molecule peptide of Comparative Example 3 (30 mg / L)

[0098] Adjust the pH value of the above culture medium to 6.0 and use it after high-temperature sterilization.

[0099] After culturing for 20 d, count the germination rate, browning rate, and contamination rate of strawberry shoot tips.

[0100]

[0101] 2. Results and Analysis

[0102] As can be seen from the results in Table 1 below, the browning degrees of strawberry shoot tips treated differently are different. Among them, the strawberry shoot tips in the blank group are severely browned, and the overall browning rate is above 25%; in the control group and each test group, the browning of strawberry shoot tips is alleviated, but due to different treatment methods, the improvement degrees of strawberry browning are different. Using the culture media prepared with the small molecule peptides of Examples 1-3 of the present invention can significantly avoid the browning of strawberry shoot tips; there are obvious differences in the preparation methods of the small molecule peptides in Comparative Examples 1-3 and the present invention. In particular, the small molecule peptides intercepted in Comparative Example 3 are completely different from those of the present invention, and when used for the tissue culture of strawberry shoot tips, the improvement degree of strawberry shoot tip browning is small.

[0103] Table 1 Comparison of browning degrees in strawberry shoot tip culture

[0104] Group Germination rate (%) Contamination rate (%) Browning rate (%) Blank group 70.00 20 26.67 Control group 96.67 0 6.67 Test group 1-1 93.33 0 3.33 Test group 1-2 90.00 0 3.33 Test group 1-3 96.67 3.33 0.00 Test group 1-4 83.33 10.00 13.33 Test group 1-5 86.67 6.67 10.00 Test group 1-6 80.00 13.33 20.00

[0105] Test Experiment 2: Preventive effect of small molecule peptides on vitrification of virus-free seedlings

[0106] 1. Materials and Methods

[0107] Take the tender stems of ice plants and the stolons of "Hongyan" strawberries that are robust and free from diseases and pests as the initial materials. After rinsing them thoroughly and disinfecting, wash them with sterile water and set aside. Conduct the following experimental operations with reference to the existing technology.

[0108] 1.1 Cut the processed ice plant stem segments into stem segments about 1 cm long (with 1 axillary bud), inoculate them into the following media, inoculate 2 plants per bottle, and inoculate 20 bottles for each medium. Cultivate them under the conditions of 25 °C, light intensity of 2000 lx, and 12 h / d of light. After 15 days, observe the survival rate, contamination rate, and incidence of vitrified seedlings of the ice plants.

[0109] Medium preparation:

[0110] Blank group 2: MS medium (added with 30 g / L of sucrose + 6 g / L of agar + 5 g / L of sodium chloride);

[0111] Test group 2-1: Blank group + small molecule peptide of Example 1 (30 mg / L)

[0112] Test group 2-2: Blank group + small molecule peptide of Example 2 (30 mg / L)

[0113] Test group 2-3: Blank group + small molecule peptide of Example 3 (30 mg / L)

[0114] Test group 2-4: Blank group + small molecule peptide of Comparative Example 1 (30 mg / L)

[0115] Test group 2-5: Blank group + small molecule peptide of Comparative Example 2 (30 mg / L)

[0116] Test group 2-6: Blank group + small molecule peptide of Comparative Example 3 (30 mg / L)

[0117] Adjust the pH value of the above media to 5.8 and use them after autoclaving.

[0118]

[0119]

[0120] 1.2 Cut the strawberry stolons into shoot tips of 1-2 mm under a microscope and inoculate them into the primary medium (formula: MS medium + 0.5 mg / L of 6-BA + 6 g / L of agar + 30 g / L of sucrose, pH 5.8) for cultivation. After 30 days of primary cultivation, select the primary seedlings with basically the same growth vigor and inoculate them into the following media for subculture. Inoculate 4 plants per bottle and inoculate 10 bottles for each medium. Cultivate them under the conditions of 25 °C, light intensity of 2000 lx, and 12 h / d of light for 30 days, and count the vitrification rate of the strawberry tissue culture seedlings in different media.

[0121] Blank group 3: MS medium (supplemented with 30 g / L sucrose + 6 g / L agar + 0.2 mg / L IBA);

[0122] Control group 3: Blank group + VC (50 mg / L);

[0123] Test group 3-1: Blank group + small molecule peptide of Example 1 (30 mg / L)

[0124] Test group 3-2: Blank group + small molecule peptide of Example 2 (30 mg / L)

[0125] Test group 3-3: Blank group + small molecule peptide of Example 3 (30 mg / L)

[0126] Test group 3-4: Blank group + small molecule peptide of Comparative Example 1 (30 mg / L)

[0127] Test group 3-5: Blank group + small molecule peptide of Comparative Example 2 (30 mg / L)

[0128] Test group 3-6: Blank group + small molecule peptide of Comparative Example 3 (30 mg / L)

[0129] Adjust the pH value of the above medium to 6.0 and use it after autoclaving.

[0130]

[0131] 1.3 Randomly divide the vitrified strawberry seedlings that appear in the ordinary culture into 7 groups and inoculate them into the following media, 1 plant per bottle and 10 bottles for each medium. Cultivate for 30 d under the conditions of 25 °C, light intensity of 2000 lx, and 12 h / d of light, and count the reversal rate of the vitrified strawberry seedlings in different media.

[0132] Blank group 4: MS medium (supplemented with 30 g / L sucrose + 6 g / L agar + 0.2 mg / L IBA);

[0133] Test group 4-1: Blank group + small molecule peptide of Example 1 (50 mg / L)

[0134] Test group 4-2: Blank group + small molecule peptide of Example 2 (50 mg / L)

[0135] Test group 4-3: Blank group + small molecule peptide of Example 3 (50 mg / L)

[0136] Test group 4-4: Blank group + small molecule peptide of Comparative Example 1 (50 mg / L)

[0137] Test group 4-5: Blank group + small molecule peptide of Comparative Example 2 (50 mg / L)

[0138] Experimental groups 4 - 6: Blank group + small molecule peptide in Comparative Example 3 (50 mg / L)

[0139]

[0140] Inoculate the above rejuvenated strawberry seedlings into the culture medium respectively (formula: MS medium + 6 g / L agar + 30 g / L sucrose, pH 5.8), and culture them for 30 days under the conditions of 25°C, light intensity of 2000 lx, and 12 h / d of light, and count the proliferation rate of the rejuvenated strawberry seedlings in each group.

[0141]

[0142] 2. Results and analysis

[0143] As can be seen from the results in Table 2 and Table 3 below, the small molecule peptide of the present invention has an obvious improvement effect on the vitrification phenomenon in the tissue culture of ice plants and strawberries. When using the small molecule peptides obtained in Examples 1 - 3 of the present invention to prepare the culture medium for tissue culture, the vitrification rate of ice plants is below 5%, and the vitrification rate of strawberries is below 7.5%, with a relatively low vitrification rate; when using the culture medium prepared with the small molecule peptides obtained in Comparative Examples 1 - 3 for tissue culture, the vitrification rates of ice plants and strawberries are relatively high, both above 10%.

[0144] Table 2 Comparison of vitrification degrees of ice plants

[0145] Group Germination rate (%) Vitrification rate (%) Blank group 77.5 22.5 Control group 97.5 7.5 Test group 2-1 95.0 5 Test group 2-2 97.5 2.5 Test group 2-3 92.5 2.5 Test group 2-4 87.5 12.5 Test group 2-5 82.5 10 Test group 2-6 80.0 15

[0146] Table 3 Comparison of vitrification degrees of strawberry shoot tips

[0147]

[0148]

[0149] As can be seen from the results in Table 4 below, when using the culture medium prepared with the small molecule peptides of Examples 1 - 3 of the present invention for tissue culture, it can effectively reverse the vitrified state of strawberry seedlings, and the reversal rate is above 80%. When the rejuvenated strawberry seedlings are subjected to proliferation culture, their proliferation rate is relatively high, and the effect is significantly better than that of the small molecule peptides obtained in Comparative Examples 1 - 3.

[0150] Table 4 Comparison of reversal degrees of vitrified strawberry seedlings

[0151] Group Reversal rate (%) Proliferation rate (%) Blank group 10 3 Test group 4-1 80 3.63 Test group 4-2 80 3.88 Test group 4-3 90 2.89 Test group 4-4 40 2.75 Test group 4-5 50 3.40 Test group 4-6 30 2.67

Claims

1. A small molecule peptide for the culture medium of virus-free seedlings, characterized in that, The small molecule peptide is obtained by fermenting Bacillus amyloliquefaciens and Paecilomyces hepiali 2. A method for preparing the small molecule peptide according to claim 1, characterized in that, It includes the following steps: Step 1, inoculate the Bacillus amyloliquefaciens seed liquid and Paecilomyces hepiali seed liquid into a fermentation medium prepared from glucose, corn steep liquor, calcium chloride, potassium dihydrogen phosphate, pentosan, and water, and culture at 30°C and 150 r / min for 8 - 10 h to obtain a primary fermentation broth; Step 2, add turkesterone, corn silk extract, magnesium sulfate heptahydrate, and zinc chloride to the primary fermentation broth, dissolve and mix evenly, then raise the temperature to 35°C and continue to culture for 20 - 24 h to obtain a final fermentation broth. Centrifuge the fermentation broth and take the supernatant to obtain a crude peptide extract; Step 3, add shikimic acid to the obtained crude peptide extract, mix evenly and let stand for 1.5 - 2 h, then add absolute ethanol to adjust the alcohol content to 70 - 75%, and let stand at 4 - 5°C for 2 - 3 h, centrifuge and take the precipitate; Step 4, after redissolving the obtained precipitate in water, add subtilisin, streptomyces protease, and nattokinase, and enzymatically hydrolyze at 40°C for 2 - 3 h, inactivate the enzyme, concentrate to obtain an enzymatic hydrolysate; Step 5, separate the enzymatic hydrolysate using a gel column, elute with a methanol - water mixed solution as the eluent, detect the obtained eluate, intercept the eluate containing small molecule peptides with a molecular weight of 500 - 800 Da, combine the eluates, remove methanol, concentrate, and dry to obtain the small molecule peptide.

3. The preparation method according to claim 2, characterized in that, In step 1, the inoculation amount of the Bacillus amyloliquefaciens seed liquid is 5 - 8% and the inoculation amount of the Paecilomyces hepiali seed liquid is 10 - 12%.

4. The preparation method according to claim 2, characterized in that, The preparation method of the fermentation medium in step 1 is: weigh 10 - 12 parts by weight of glucose, 13 - 15 parts by weight of corn steep liquor, 1 - 1.2 parts by weight of calcium chloride, 0.5 - 0.6 parts by weight of potassium dihydrogen phosphate, 3 - 5 parts by weight of pentosan, add 1000 parts by weight of water, dissolve and mix evenly, adjust the pH to 6.5, and sterilize at high temperature to obtain the fermentation medium.

5. The preparation method according to claim 2, characterized in that, In step 2, the addition amounts of turkesterone, corn silk extract, magnesium sulfate heptahydrate, and zinc chloride are 3 - 3.5 g / L of turkesterone, 5 - 8 g / L of corn silk extract, 0.2 - 0.3 g / L of magnesium sulfate heptahydrate, and 0.6 - 0.65 g / L of zinc chloride respectively.

6. The preparation method according to claim 2, wherein, In step 3, the addition amount of shikimic acid is 1 - 1.5 g / L.

7. The preparation method according to claim 2, characterized in that, In step 4, the addition amount of subtilisin is 0.2 - 0.3% of the precipitate mass, the addition amount of streptomyces protease is 0.05 - 0.08% of the precipitate mass, and the addition amount of nattokinase is 0.1 - 0.15% of the precipitate mass.

8. The preparation method according to claim 2, characterized in that, In step 5, the volume ratio of methanol to water in the methanol - water mixed solution is (0.3 - 0.5):

1.

9. The preparation method according to claim 2, wherein, The preparation method of the corn silk extract in step 2 is: Step A, crush the corn silk and soak it in petroleum ether for 2.5 - 3 h, filter to remove the petroleum ether, add an ethanol solution with a mass fraction of 60 - 65%, reflux and extract, filter, and reserve the extract; Step B: The extract is extracted with ethyl acetate. The upper layer solution is taken, concentrated, separated by silica gel column chromatography, eluted with a chloroform-methanol solvent system, the eluates are combined, and the organic solvent is removed to obtain the corn stigma extract.

10. The preparation method according to claim 9, wherein In the step B, the volume ratio of chloroform to methanol is 100:(3 - 5).

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