CN110295113B - Marine protease bacillus stability protective agent - Google Patents
Marine protease bacillus stability protective agent Download PDFInfo
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- CN110295113B CN110295113B CN201910694814.2A CN201910694814A CN110295113B CN 110295113 B CN110295113 B CN 110295113B CN 201910694814 A CN201910694814 A CN 201910694814A CN 110295113 B CN110295113 B CN 110295113B
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Abstract
The invention relates to a bacillus marinus stability protective agent, belonging to the technical field of microorganisms. The protective agent comprises Tris-HCl buffer solution, glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate, wherein the Tris-HCl buffer solution, the glycerol, the mannitol, the EDTA, the potassium chloride, the magnesium sulfate, the disodium hydrogen phosphate and the 2-ethyl diphenyl antimony hexanoate are prepared into a liquid protective agent according to a certain proportion. The stability protective agent for the marine protease bacillus overcomes the problems of denaturation and low survival rate of marine protease bacillus strains under the condition of low-temperature refrigeration, and can stabilize the survival rate of the strains to be more than 90%.
Description
Technical Field
The invention relates to the field of microbial preservation, in particular to a protective agent for the stability of bacillus marinus.
Background
With the rapid development of large-scale and intensive aquaculture industry, diseases of aquaculture frequently occur. In recent years, beneficial microorganisms, especially enzyme-producing probiotics in the ocean, are used in breeding production to improve the breeding ecological environment, improve the immunity of breeding animals, and inhibit pathogenic microorganisms, so as to reduce the occurrence of diseases. Wherein, bacillus derived from marine protease is developed into various using modes due to the special property of protease, can be directly added into aquaculture water or added into feed for use, can act on marine aquaculture animals through bait organisms, and can be used through injection or dipping bath.
Since the protease-producing bacillus is derived from the sea, the growth and survival conditions of the protease-producing bacillus are similar to the marine ecological environment, but the strains cultured under the environmental conditions simulated in a laboratory usually have the problems of low survival rate, reduced activity and the like during cold storage. Therefore, the search for a protective agent capable of maintaining the stability of marine protein-producing bacillus is a subject of constant research by related researchers.
Disclosure of Invention
The invention aims to provide a bacillus marinus stability protective agent, which is used for enhancing the activity of bacteria and improving the survival rate of the bacteria.
In order to achieve the technical purpose, the invention adopts the technical scheme that: a bacillus stability protective agent for marine protease is characterized in that: the protective agent is prepared by adding glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate into Tris-HCl buffer solution.
Further, the Tris-HCl buffer refers to 10 mM Tris-HCl buffer, and the pH value of the buffer is 7.5.
Further, the concentration of the glycerol is 25-35% (v/v), the concentration of the mannitol is 5.5-7.5% (w/v), the concentration of the EDTA is 1.5-3.5% (w/v), the concentration of the potassium chloride is 0.5-0.7% (w/v), the concentration of the magnesium sulfate is 0.35-0.55% (w/v), the concentration of the disodium hydrogen phosphate is 0.25-0.45% (w/v), the concentration of the antimony diphenyl2-ethylhexanoate is 0.03-0.08% (v/v), and the concentrations of the above components are calculated by taking the volume of the protective agent as a reference.
Further, the concentration of glycerol is 32% (v/v), the concentration of mannitol is 5.5% (w/v), the concentration of EDTA is 1.5% (w/v), the concentration of potassium chloride is 0.55% (w/v), the concentration of magnesium sulfate is 0.35% (w/v), the concentration of disodium hydrogen phosphate is 0.32% (w/v), and the concentration of antimony diphenyl2-ethylhexanoate is 0.05% (v/v).
Further, the preparation method of the stabilizer comprises the following steps: glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate are proportionally added into a solution of 10 mM Tris-HCl buffer solution (pH 7.5), stirred uniformly at normal temperature to completely dissolve solute, and after standing for 10 min, the volume is determined by using a solution of 10 mM Tris-HCl buffer solution (pH 7.5).
The invention discloses a bacillus marinus stability protective agent for protease, which is characterized in that glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and diphenyl antimony 2-ethylhexanoate are added into a Tris-HCl buffer solution as a base solution. Wherein, the Tris-HCl buffer solution is used for providing a stable liquid environment, so that the pH value of the solution is kept stable, and the pH value is 7.6 close to the growth pH environment of the marine protease bacillus in seawater; the glycerol is used for freely diffusing into the cells, reducing the water activity in the cells and inhibiting the biochemical reaction of the strains; mannitol is used for balancing osmotic pressure inside and outside the bacterial strain cells; EDTA as chelating antiseptic for inhibiting the growth of mixed bacteria; potassium chloride, magnesium sulfate and disodium hydrogen phosphate can provide inorganic salt ions required by the survival of the bacillus marinus in the marine environment; the antimony diphenyl 2-ethylhexanoate can effectively inhibit strain degeneration and is an important biological stabilizer. The activity and the survival rate of the protease bacillus can be greatly improved by applying the protective agent.
Detailed Description
The present invention provides a bacillus marinus stability protective agent which is further illustrated by the following specific examples.
Example 1
A bacillus marinus stability protective agent for marine protease is prepared by adding glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate into a solution of 10 mM Tris-HCl buffer solution (pH 7.5) in proportion, stirring uniformly at normal temperature to completely dissolve solute, standing for 10 min, and diluting with a solution of 10 mM Tris-HCl buffer solution (pH 7.5) to constant volume to obtain the final protective agent with the concentration of each component: 32% glycerol (v/v), 5.50% mannitol (w/v), 1.50% EDTA (w/v), 0.55% potassium chloride (w/v), 0.35% magnesium sulfate (w/v), 0.32% disodium hydrogen phosphate (w/v), 0.05% diphenylantimony 2-ethylhexanoate (v/v). Where mM units mean millimoles per liter.
Taking live bacillus solution of marine protease-producing bacillus with live bacteria content of 50 hundred million cfu/mL, adding 5% of stability protective agent into the solution, preserving the solution at 4 ℃ for 30 days, taking out the solution every 5 days, coating the solution on a 2216E flat plate, placing the flat plate in a constant-temperature incubator at 30 ℃ for 24 hours, counting the live bacteria, and counting the survival rate as shown in the table.
Example 2
A bacillus marinus stability protective agent for marine protease is prepared by adding glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate into a solution of 10 mM Tris-HCl buffer solution (pH 7.5) in proportion, stirring uniformly at normal temperature to completely dissolve solute, standing for 10 min, and diluting with a solution of 10 mM Tris-HCl buffer solution (pH 7.5) to constant volume to obtain the final protective agent with the concentration of each component: 35% glycerol (v/v), 6.2% mannitol (w/v), 3.20% EDTA (w/v), 0.50% potassium chloride (w/v), 0.47% magnesium sulfate (w/v), 0.30% disodium hydrogen phosphate (w/v), 0.06% diphenylantimony 2-ethylhexanoate (v/v), and made up in 10 mM Tris-HCl buffer (pH 7.5). Where mM units mean millimoles per liter.
Taking live bacillus solution of marine protease-producing bacillus with live bacteria content of 50 hundred million cfu/mL, adding 5% of stability protective agent into the solution, preserving the solution at 4 ℃ for 30 days, taking out the solution every 5 days, coating the solution on a 2216E flat plate, placing the flat plate in a constant-temperature incubator at 30 ℃ for 24 hours, counting the live bacteria, and counting the survival rate as shown in the table.
Example 3
A bacillus marinus stability protective agent for marine protease is prepared by adding glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate into a solution of 10 mM Tris-HCl buffer solution (pH 7.5) in proportion, stirring uniformly at normal temperature to completely dissolve solute, standing for 10 min, and diluting with a solution of 10 mM Tris-HCl buffer solution (pH 7.5) to constant volume to obtain the final protective agent with the concentration of each component: 27% glycerol (v/v), 5.8% mannitol (w/v), 1.8% EDTA (w/v), 0.60% potassium chloride (w/v), 0.55% magnesium sulfate (w/v), 0.45% disodium hydrogen phosphate (w/v), 0.07% diphenylantimony 2-ethylhexanoate (v/v), and made up in 10 mM Tris-HCl buffer (pH 7.5). Where mM units mean millimoles per liter.
Taking live bacillus solution of marine protease-producing bacillus with live bacteria content of 50 hundred million cfu/mL, adding 5% of stability protective agent into the solution, preserving the solution at 4 ℃ for 30 days, taking out the solution every 5 days, coating the solution on a 2216E flat plate, placing the flat plate in a constant-temperature incubator at 30 ℃ for 24 hours, counting the live bacteria, and counting the survival rate as shown in the table.
Example 4
A bacillus marinus stability protective agent for marine protease is prepared by adding glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate into a solution of 10 mM Tris-HCl buffer solution (pH 7.5) in proportion, stirring uniformly at normal temperature to completely dissolve solute, standing for 10 min, and diluting with a solution of 10 mM Tris-HCl buffer solution (pH 7.5) to constant volume to obtain the final protective agent with the concentration of each component: 30% glycerol (v/v), 6.5% mannitol (w/v), 3.4% EDTA (w/v), 0.47% potassium chloride (w/v), 0.45% magnesium sulfate (w/v), 0.35% disodium hydrogen phosphate (w/v), 0.08% diphenylantimony 2-ethylhexanoate (v/v), and made up in 10 mM Tris-HCl buffer (pH 7.5). Where mM units mean millimoles per liter.
Taking live bacillus solution of marine protease-producing bacillus with live bacteria content of 50 hundred million cfu/mL, adding 5% of stability protective agent into the solution, preserving the solution at 4 ℃ for 30 days, taking out the solution every 5 days, coating the solution on a 2216E flat plate, placing the flat plate in a constant-temperature incubator at 30 ℃ for 24 hours, counting the live bacteria, and counting the survival rate as shown in the table.
Example 5
A bacillus marinus stability protective agent for marine protease is prepared by adding glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate into a solution of 10 mM Tris-HCl buffer solution (pH 7.5) in proportion, stirring uniformly at normal temperature to completely dissolve solute, standing for 10 min, and diluting with a solution of 10 mM Tris-HCl buffer solution (pH 7.5) to constant volume to obtain the final protective agent with the concentration of each component: 25% glycerol (v/v), 5.8% mannitol (w/v), 3.5% EDTA (w/v), 0.70% potassium chloride (w/v), 0.50% magnesium sulfate (w/v), 0.35% disodium hydrogen phosphate (w/v), 0.04% diphenylantimony 2-ethylhexanoate (v/v), and made up in 10 mM Tris-HCl buffer (pH 7.5). Where mM units mean millimoles per liter.
Taking live bacillus solution of marine protease-producing bacillus with live bacteria content of 50 hundred million cfu/mL, adding 5% of stability protective agent into the solution, preserving the solution at 4 ℃ for 30 days, taking out the solution every 5 days, coating the solution on a 2216E flat plate, placing the flat plate in a constant-temperature incubator at 30 ℃ for 24 hours, counting the live bacteria, and counting the survival rate as shown in the table.
The inventor finds that the stability protective agent for the bacillus marinus can keep the survival rate of the bacillus marinus to be more than 90% by comparing the analysis results of the above examples, wherein the effect of example 1 is the best and reaches 94.16%.
Claims (2)
1. A bacillus stability protective agent for marine protease is characterized in that: the protective agent is prepared by adding glycerol, mannitol, EDTA, potassium chloride, magnesium sulfate, disodium hydrogen phosphate and 2-ethyl diphenyl antimony hexanoate into a Tris-HCl buffer solution;
the Tris-HCl buffer solution is 10 mM Tris-HCl buffer solution, and the pH value of the buffer solution is 7.5;
the concentration of the glycerol is 25-35% v/v, the concentration of the mannitol is 5.5-7.5% w/v, the concentration of the EDTA is 1.5-3.5% w/v, the concentration of the potassium chloride is 0.5-0.7% w/v, the concentration of the magnesium sulfate is 0.35-0.55% w/v, the concentration of the disodium hydrogen phosphate is 0.25-0.45% w/v, the concentration of the antimony diphenyl 2-ethylhexanoate is 0.03-0.08% v/v, and the concentration of the components is calculated by taking the volume of the protective agent as a reference.
2. The bacillus marinus stability protective agent according to claim 1, which is characterized in that: the concentration of glycerol is 32% v/v, the concentration of mannitol is 5.5% w/v, the concentration of EDTA is 1.5% w/v, the concentration of potassium chloride is 0.55% w/v, the concentration of magnesium sulfate is 0.35% w/v, the concentration of disodium hydrogen phosphate is 0.32% w/v, and the concentration of antimony diphenyl 2-ethylhexanoate is 0.05% v/v.
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| CN201910694814.2A CN110295113B (en) | 2019-07-30 | 2019-07-30 | Marine protease bacillus stability protective agent |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA870647A (en) * | 1971-05-11 | R. Leebrick John | Diphenylantimony 2-ethylhexanoate and preparation | |
| WO2000007606A2 (en) * | 1998-08-07 | 2000-02-17 | Ganeden Biotech, Inc. | Methods for increasing the solubility of nutritional materials using probiotic lactic acid-producing bacteria |
| JP2003320000A (en) * | 2002-05-02 | 2003-11-11 | Fuji Silysia Chemical Ltd | Sterilizing method and sterilizing apparatus |
| WO2004063036A1 (en) * | 2003-01-09 | 2004-07-29 | Baxter Healthcare S.A. | Safety containers for biologically active substances and method for producing said container |
| CN101511950A (en) * | 2006-08-29 | 2009-08-19 | 3M创新有限公司 | Resin systems including reactive surface-modified nanoparticles |
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- 2019-07-30 CN CN201910694814.2A patent/CN110295113B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA870647A (en) * | 1971-05-11 | R. Leebrick John | Diphenylantimony 2-ethylhexanoate and preparation | |
| WO2000007606A2 (en) * | 1998-08-07 | 2000-02-17 | Ganeden Biotech, Inc. | Methods for increasing the solubility of nutritional materials using probiotic lactic acid-producing bacteria |
| JP2003320000A (en) * | 2002-05-02 | 2003-11-11 | Fuji Silysia Chemical Ltd | Sterilizing method and sterilizing apparatus |
| WO2004063036A1 (en) * | 2003-01-09 | 2004-07-29 | Baxter Healthcare S.A. | Safety containers for biologically active substances and method for producing said container |
| CN101511950A (en) * | 2006-08-29 | 2009-08-19 | 3M创新有限公司 | Resin systems including reactive surface-modified nanoparticles |
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