CN114317358A - Preservative for actinomycete strains, application and preservation method - Google Patents
Preservative for actinomycete strains, application and preservation method Download PDFInfo
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- CN114317358A CN114317358A CN202111663275.XA CN202111663275A CN114317358A CN 114317358 A CN114317358 A CN 114317358A CN 202111663275 A CN202111663275 A CN 202111663275A CN 114317358 A CN114317358 A CN 114317358A
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- 239000003755 preservative agent Substances 0.000 title claims abstract description 81
- 230000002335 preservative effect Effects 0.000 title claims abstract description 79
- 241001446247 uncultured actinomycete Species 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 60
- 238000004321 preservation Methods 0.000 title claims abstract description 37
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 107
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 69
- 241000186361 Actinobacteria <class> Species 0.000 claims abstract description 42
- 239000000843 powder Substances 0.000 claims abstract description 31
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Abstract
The invention discloses a preservative for actinomycetes strains, application and a preservation method, relates to the field of microbial preservation, and solves the problems of preservation of actinomycetes by a glycerol cold storage method, a liquid paraffin sealing method and a freeze drying method in the prior art. The preservative for the actinomycete strains comprises the following components in concentration: 10 to 30 percent of dimethyl sulfoxide, 20 to 60 percent of glycerol and 10 to 30 percent of skimmed milk powder, and the preservative is a liquid composite preservative prepared from the dimethyl sulfoxide, the glycerol and the skimmed milk powder. The preservative for the actinomycete strains comprises dimethyl sulfoxide, and can improve the permeability of cell membranes, thereby reducing the damage of the actinomycete strains at low temperature or in the drying process; the glycerol can reduce the activity of free water and the like in thallus cells, thereby achieving the purpose of inhibiting cell metabolism; the bacterial strain also comprises skimmed milk powder which can form a protein film outside the bacterial strain so as to protect cells and improve the heat resistance and the storage stability of the bacterial strain.
Description
Technical Field
The invention relates to the technical field of microorganism preservation, in particular to a preservative for actinomycetes strains, application of the preservative and a preservation method of the actinomycetes strains.
Background
Biological prevention and control is used as a prevention and control measure capable of promoting agricultural health sustainable development, and the application range of the biological prevention and control measure is wider and wider. The biological prevention and control of various soil-borne diseases refers to the prevention and control of outbreak of soil-borne diseases by inhibiting or eliminating plant pathogenic bacteria in a mode of inducing the systemic resistance operation of plants to pathogenic bacteria by using antagonistic substances secreted by functional microorganisms. Therefore, the actinomycetes are separated from the soil sample collected in the field for multiple times, and the actinomycetes are obtained through strain purification and separation, so that the actinomycetes strain with the bactericidal effect is screened out. Through an antagonistic test, the actinomycete strain has certain potential value in preventing and controlling agricultural diseases by direct utilization. The stability research of the actinomycete strain and the fermentation liquor thereof shows that the culture characteristics of the actinomycete strain are stable in the continuous passage process, the preservation process under different temperature conditions or the culture process by different culture media, and the actinomycete strain can be put into actual production and field application.
In the laboratory research process, the well-screened and identified strains are often used, so that the strains are imperative to be preserved. At present, methods for preserving actinomycetes in a laboratory mainly comprise: glycerin refrigeration method, liquid paraffin sealing method, and freeze drying method. The glycerol refrigeration method is very simple and easy to store strains, but in the storage process, the storage life is indefinite, the storage life cannot be accurately determined, and the method is not suitable for actinomycetes needing long-term storage. The liquid paraffin sealing method is required to be vertically placed during preservation, occupied space is large, carrying is inconvenient, on the other hand, after bacteria liquid is taken from the liquid paraffin and activated, the inoculating loop is burnt on an alcohol burner, the bacteria liquid is easy to splash together with residual liquid paraffin to form aerosol, and certain risk is brought to biosafety protection. The freeze-drying method has long storage life, but has complex operation, needs equipment such as a freeze dryer and the like, and has long preparation process and preparation time.
The preservative is added in the strain preservation process, so that the physical environment and the chemical environment of the microorganism during drying can be changed, the damage to cells in the drying or rehydration process is reduced or eliminated, the original various physiological and biochemical characteristics and biological activity of the strain are kept as far as possible, and the purpose of protecting the strain is further achieved. In addition, the same preservative has different protective effects on bacterial cells under moist heat conditions depending on the amount of the preservative added. Therefore, for the long-term preservation of actinomycetes, a preservative and a preservation method which are simple, fast and excellent in effect are also required to be explored.
Disclosure of Invention
The invention aims to provide a preservative for actinomycetes, an application and a preservation method, and solves the technical problems that the preservation period cannot be accurately determined when the actinomycetes are preserved by adopting a glycerol refrigeration method, the space occupied by the actinomycetes preserved by adopting a liquid paraffin sealing method is large and has certain risk, and the actinomycetes preserved by a freeze-drying method is complex to operate and needs long time in the prior art. The various technical effects that can be produced by the preferred technical solution of the present invention are described in detail below.
In order to achieve the purpose, the invention provides the following technical scheme:
the preservative for the actinomycete strains comprises the following components in concentration: the preservative comprises 10-30% of dimethyl sulfoxide, 20-60% of glycerol and 10-30% of skimmed milk powder, and the preservative is a liquid composite preservative prepared from the dimethyl sulfoxide, the glycerol and the skimmed milk powder.
According to a preferred embodiment, said preservative for actinomycete species comprises the following components in the following concentrations: dimethyl sulfoxide with the concentration of 20%, glycerol with the concentration of 40% and skimmed milk powder with the concentration of 10%.
According to a preferred embodiment, the weight ratio of dimethyl sulfoxide, glycerol and skimmed milk powder is 1: 1.
According to a preferred embodiment, the dimethyl sulfoxide, the glycerol and the skimmed milk powder are respectively sterilized and then compounded according to the weight ratio of 1: 1.
The preservative of any technical scheme of the invention is applied to culture and preservation of actinomycetes.
The method is used for preserving the actinomycete strains, and the method is used for preserving the actinomycete strains by using the preservative of any technical scheme in the invention.
According to a preferred embodiment, the strain preservation solution is prepared by mixing the preservative with actinomycete strains, and the strain preservation solution is packaged in a strain tube and stored at normal temperature.
According to a preferred embodiment, the preparation of the seed stock comprises the following steps: and mixing the preservative and the actinomycete strain suspension according to the weight ratio of 1: 1 to prepare a preservation solution.
According to a preferred embodiment, the preparation of the seed stock further comprises the steps of: taking sand as a carrier, uniformly mixing the sand and the preservation solution according to the weight ratio of 5: 1, and drying the preservation solution and sand after fully dispersing.
According to a preferred embodiment, the preservation solution and the sand are dried at a temperature of 45 ℃ for 2 hours.
The preservative for the actinomycete strain, the application and the preservation method provided by the invention at least have the following beneficial technical effects:
the preservative for the actinomycete strains comprises dimethyl sulfoxide, and the dimethyl sulfoxide is used as a component of the preservative and can improve the permeability of cell membranes due to small relative molecular weight and strong permeability of the dimethyl sulfoxide, so that the damage of the actinomycete strains at low temperature or in a drying process is reduced; the preservative for the actinomycete strains also comprises glycerol, wherein the glycerol can be diffused into the thallus cells, and the aim of inhibiting cell metabolism can be fulfilled by reducing the activity of free water and the like in the thallus cells; the preservative for the actinomycete strains also comprises the skim milk powder, and the skim milk powder can form a protein film outside the strains so as to protect the cells and improve the heat resistance and the storage stability of the strains. The preservative for the actinomycetes strains is prepared from 10-30% of dimethyl sulfoxide, 20-60% of glycerol and 10-30% of skimmed milk powder, and has a good protection effect on the actinomycetes.
The preservative of any technical scheme of the invention is applied to the culture of the actinomycetes, so that the cultured actinomycetes has better tolerance no matter in the freezing or drying process, and the yield of the actinomycetes can be improved. The preservative of any technical scheme of the invention is applied to the preservation of actinomycetes, can ensure the activity of strains, does not influence the biochemical characteristics of microorganisms, does not need freeze-drying, can be preserved at normal temperature, and is very convenient to use and operate.
The preservation method for the actinomycete strains, disclosed by the invention, has the advantages that the preservative is uniformly mixed with the actinomycete strains, so that the protection effect on the actinomycete strains is good; on the other hand, the method for preserving the actinomycete strains has the preservation mode that the actinomycete strains are uniformly mixed with sandy soil, can comprehensively inhibit the growth and the propagation of the actinomycete under the conditions of dryness, oxygen deficiency and no nutrient substances, and achieves the purpose of prolonging the preservation period under the normal temperature condition at one time; therefore, the method for preserving the actinomycete strain can ensure the activity of the strain, does not influence the biochemical characteristics of microorganisms, does not need freeze-drying, can be preserved at normal temperature, is very convenient to use and operate, can stably survive for more than five years at normal temperature, and is convenient for long-term preservation of the actinomycete strain.
The invention relates to a storage agent and a storage method for actinomycetes, which solve the technical problems that the storage period cannot be accurately determined when the actinomycetes are stored by adopting a glycerol refrigeration method, the space occupied by the actinomycetes stored by adopting a liquid paraffin sealing method is large, certain risks are caused, and the actinomycetes stored by a freeze-drying method are complex to operate and long in required time in the prior art.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the effect of storing actinomycetes with the preservatives of different test numbers in example 1;
FIG. 2 is a comparative graph showing the effect of storing actinomycetes by the storage method of the different treatment method in example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The preservative for the actinomycete strains comprises the following components in concentration: dimethyl sulfoxide with the concentration of 10-30%, glycerin with the concentration of 20-60% and skimmed milk powder with the concentration of 10-30%, and the preservative is a liquid composite preservative prepared from the dimethyl sulfoxide, the glycerin and the skimmed milk powder. Preferably, the preservative for actinomycete species comprises the following components in concentrations: dimethyl sulfoxide with the concentration of 20%, glycerol with the concentration of 40% and skimmed milk powder with the concentration of 10%.
According to a preferred embodiment, the weight ratio of dimethyl sulfoxide, glycerol and skimmed milk powder is 1: 1. Preferably, the dimethyl sulfoxide, the glycerol and the skimmed milk powder are respectively sterilized and then compounded according to the weight ratio of 1: 1. Specifically, prepared dimethyl sulfoxide, glycerol and skimmed milk powder are respectively sterilized, then uniformly mixed according to the weight ratio of 1: 1, and then placed into a refrigerator at 4 ℃ for refrigeration for later use. According to the preferred technical scheme, the components are respectively sterilized and then compounded, so that the three components are mutually soluble, no precipitate is generated, and the preparation of the preservative is facilitated.
The preservative for the actinomycete strains comprises dimethyl sulfoxide, and the dimethyl sulfoxide is used as a component of the preservative and can improve the permeability of cell membranes due to small relative molecular weight and strong permeability of the dimethyl sulfoxide, so that the damage of the actinomycete strains at low temperature or in a drying process is reduced; the preservative for the actinomycete strains also comprises glycerol, wherein the glycerol can be diffused into the thallus cells, and the aim of inhibiting cell metabolism can be fulfilled by reducing the activity of free water and the like in the thallus cells; the preservative for the actinomycete strains also comprises the skim milk powder, and the skim milk powder can form a protein film outside the strains so as to protect the cells and improve the heat resistance and the storage stability of the strains. The preservative for the actinomycetes strains is prepared from 10-30% of dimethyl sulfoxide, 20-60% of glycerol and 10-30% of skimmed milk powder, and has a good protection effect on the actinomycetes.
The preservative of any technical scheme of the invention can be used for culturing actinomycetes. The preservative of any technical scheme of the invention is applied to the culture of the actinomycetes, so that the cultured actinomycetes has better tolerance no matter in the freezing or drying process, and the yield of the actinomycetes can be improved.
The preservative of any technical scheme of the invention can be used for storing actinomycetes. The preservative of any technical scheme of the invention is applied to the preservation of actinomycetes, can ensure the activity of strains, does not influence the biochemical characteristics of microorganisms, does not need freeze-drying, can be preserved at normal temperature, and is very convenient to use and operate.
The preservation method for the actinomycete strains is used for preserving the actinomycete strains by using the preservative of any one technical scheme in the invention. Preferably, the strain preservation solution is prepared by mixing the preservative and actinomycete strains, and the strain preservation solution is packaged in a strain tube and is stored at normal temperature.
According to a preferred embodiment, the preparation of the seed stock comprises the following steps: mixing the preservative and the actinomycete strain suspension according to the weight ratio of 1: 1 to prepare a preservation solution; taking sand as a carrier, uniformly mixing the sand and the preservation solution according to the weight ratio of 5: 1, fully dispersing the preservation solution and sand, and drying at 45 ℃ for 2 hours. Preferably, the sandy soil is 60-mesh sandy soil which is sterilized at least three times.
The preservation method for the actinomycete strains, disclosed by the invention, has the advantages that the preservative is uniformly mixed with the actinomycete strains, so that the protection effect on the actinomycete strains is good; on the other hand, the method for preserving the actinomycete strains has the preservation mode that the actinomycete strains are uniformly mixed with sandy soil, can comprehensively inhibit the growth and the propagation of the actinomycete under the conditions of dryness, oxygen deficiency and no nutrient substances, and achieves the purpose of prolonging the preservation period under the normal temperature condition at one time; therefore, the method for preserving the actinomycete strain can ensure the activity of the strain, does not influence the biochemical characteristics of microorganisms, does not need freeze-drying, can be preserved at normal temperature, is very convenient to use and operate, can stably survive for more than five years at normal temperature, and is convenient for long-term preservation of the actinomycete strain.
Compared with the traditional sand-soil tube preservation method, the preservation method for the actinomycete strain has at least the following advantages: mixing the preservative with actinomycete strains to prepare a strain preservation solution, wherein the heat resistance of actinomycete strains can be improved by the preservation solution in the drying process, and meanwhile, the cells of the strains can be protected from being damaged, and the activity and the basic characteristics of the strains are kept unchanged; on the other hand, the preservation solution is dried at the temperature of 45 ℃, so that the water content in the sand can be reduced, the air is isolated, the contamination of bacteria is prevented, and the preservation time of the actinomycetes is prolonged to 5 years.
The preservative for actinomycete strains, the use thereof and the preservation method of the present invention will be specifically described below with reference to inventions 1 to 3.
Example 1
This example illustrates the concentrations of the components of the preservative for actinomycete species of the present invention.
This example explores the concentrations of the various components of the preservative using the following steps:
the method comprises the following steps: operating in a laboratory clean bench, inoculating actinomycetes to be stored on a solid LB culture medium, culturing for 48h in an incubator at 25 ℃, washing with 2mL sterile deionized water after actinomycetes sporophyte grows out to obtain actinomycetes suspension with the concentration of 200 multiplied by 108cfu/mL。
Step two: and (4) preparing a preservative. The design and result analysis of an orthogonal test table are carried out by using a sps statistical analysis software, the concentration of the dimethyl sulfoxide is selected from three points of 10%, 20% and 30%, the concentration of the glycerol is selected from three points of 20%, 40% and 60%, the concentration of the skim milk powder is selected from three points of 10%, 20% and 30%, and the orthogonal table is shown in table 1.
Step three: sterilizing the components of different test number pairs in the orthogonal table respectively, and compounding according to the weight ratio of 1: 1 to prepare the preservative.
Step four: and uniformly mixing the bacterial suspension and a preservative according to the weight ratio of 1: 1 to form a strain preservation solution.
Step five: after 1 month, the bacterial load of 9 test numbers was measured, and the results are shown in FIG. 1.
TABLE 1 orthogonal table of different concentrations of components in preservative
And (3) test analysis: during the preparation and sterilization test of the preservative, the mutual solubility of glycerol, dimethyl sulfoxide and skimmed milk powder when different concentrations are prepared is found; but has obvious chemical reaction in the sterilization process, so glycerin, dimethyl sulfoxide and skimmed milk powder are prepared in proportion after being sterilized respectively, and the specific experimental operation process is shown in example 2. As is clear from Table 1 and FIG. 1, the concentration of the strain of test No. 8 was the highest in the orthogonal test, indicating that the strain preservation effect and the strain survival rate were higher with this preservative.
Therefore, the concentration of dimethyl sulfoxide in the preservative is preferably 20%, the concentration of glycerol is preferably 40%, and the concentration of skimmed milk powder is preferably 10%.
Example 2
This example illustrates the timing of sterilization of the components of the preservative for actinomycete species of the present invention.
The example explores the timing of sterilization of the preservative components using the following steps:
the method comprises the following steps: the components in the preservative are prepared respectively.
Preparation of 10%, 20%, 30% dimethyl sulfoxide concentration: 10mL, 20mL and 30mL of Dimethyl sulfoxide (Dimethyl sulfoxide, the content of C2H6OS is not less than 99.5 percent, analytically pure) are taken by a measuring cylinder and placed in a 100mL volumetric flask, deionized water is added, the volume is fixed to 100mL, and the mixture is fully and uniformly shaken.
Configuration with glycerol concentrations of 20%, 40%, 60%: 20mL, 40mL and 60mL of Glycerol (Glycerol, the content of C3H8O3 is not less than 99.0 percent, analytical purity) are taken by a measuring cylinder and placed in a 100mL volumetric flask, deionized water is added, the volume is fixed to 100mL, and the mixture is fully shaken up.
The preparation of the skim milk powder with the concentration of 10%, 20% and 30%: 10g, 20g and 30g of skimmed milk powder are taken by weighing paper and placed in a 250mL beaker, 100g of deionized water is added, and the mixture is stirred and shaken uniformly.
Step two: and performing sterilization comparison tests before and after compounding of the prepared components. Specifically, after sterilizing the components respectively, mixing the components uniformly according to the ratio of 1: 1; and uniformly mixing according to the ratio of 1: 1, and then sterilizing.
Step three: the bacterial suspension is respectively and uniformly mixed with the preservatives in the two treatment modes according to the ratio of 1: 1 to form the strain preservation solution.
And (3) test analysis: the two treatment modes of sterilizing after compounding the components and compounding after sterilizing are obviously different. Specifically, after the components are compounded and sterilized, the phenomena of flocculent precipitate and agglomeration appear, and the three components generate chemical reaction in the sterilization process, so that the three components are insoluble and cannot be used as a preservative of the actinomycetes; the components are respectively sterilized and then compounded, and the three components are mutually soluble and have no precipitate. Therefore, the components in the preservative should be compounded after being sterilized respectively.
Example 3
This example describes the method for preserving actinomycete species according to the present invention.
The following steps are adopted in this embodiment to explain the preservation method:
the method comprises the following steps: 100g of sandy soil is taken to pass through a 60-mesh sieve, 85g of sandy soil under the sieve is taken to be put into a 250mL conical flask, the conical flask is repeatedly sterilized in a sterilization pot at 121 ℃ for three times, and 4mL of sterilized sandy soil is respectively taken from a super clean bench and is subpackaged into 10mL centrifuge tubes for standby.
Step two: the sterile sandy soil and the strain preservation solution are uniformly mixed according to the weight ratio of 5: 1, so that the strain preservation solution and the sandy soil are fully dispersed.
Step three: performing a storage comparison test, respectively placing in a drying oven at 45 deg.C for 2h, and storing at normal temperature; and directly storing at room temperature without drying.
Step four: the strain activity and the strain amount were measured at 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months of storage, respectively, and the results are shown in FIG. 2.
And (3) test analysis: as can be seen from FIG. 2, the strain preservation solution and sand were not dried, and the activity of the strain was significantly reduced after 3 months of preservation at room temperature. The strain preservation solution and the sandy soil are dried for 2 hours at the temperature of 45 ℃, the strain is preserved at normal temperature, and the concentration of the actinomycetes tends to be stable, which indicates that the strain preservation solution and the sandy soil are dried, so that the strain activity is higher and the preservation effect is good.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. A preservative for actinomycete strains, which is characterized by comprising the following components in concentration: the preservative comprises 10-30% of dimethyl sulfoxide, 20-60% of glycerol and 10-30% of skimmed milk powder, and the preservative is a liquid composite preservative prepared from the dimethyl sulfoxide, the glycerol and the skimmed milk powder.
2. A preservative for actinomycete species as claimed in claim 1, which includes the following components in the concentrations: dimethyl sulfoxide with the concentration of 20%, glycerol with the concentration of 40% and skimmed milk powder with the concentration of 10%.
3. A preservative for actinomycete species according to claim 1 or claim 2, wherein the weight ratio of dimethyl sulfoxide, glycerol and skim milk powder is 1: 1.
4. The preservative for actinomycete strains according to claim 3, wherein the dimethyl sulfoxide, the glycerol and the skim milk powder are respectively sterilized and then compounded in a weight ratio of 1: 1.
5. Use of the preservative of any one of claims 1 to 4 for the culture and preservation of actinomycetes.
6. A method for preserving an actinomycete species, wherein the method comprises preserving the actinomycete species with the preservative of any one of claims 1 to 4.
7. The method for preserving actinomycete species according to claim 6, wherein the preservative is mixed with actinomycete species to prepare a species preservation solution, and the species preservation solution is packaged in a seed tube and stored at room temperature.
8. The method for preserving actinomycete species according to claim 7, wherein the step of preparing the species preservative solution includes the steps of: and mixing the preservative and the actinomycete strain suspension according to the weight ratio of 1: 1 to prepare a preservation solution.
9. The method for preserving actinomycete species according to claim 8, wherein the step of preparing the species preservative solution further comprises the steps of: taking sand as a carrier, uniformly mixing the sand and the preservation solution according to the weight ratio of 5: 1, and drying the preservation solution and sand after fully dispersing.
10. The preservation method for actinomycete species according to claim 9, wherein the preservation solution and sand are dried at a temperature of 45 ℃ for 2 hours.
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2021
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