Permeation protective agent as well as preparation method and application thereof

Abstract

The invention provides an osmotic protective agent with simple components. The osmotic protective agent is selected from a single osmotic protective agent consisting of a surfactant or a compound osmotic protective agent consisting of a surfactant and glycine betaine. The penetration protective agent provided by the invention can significantly improve the survival rate of bacteria or cells through a simple and short-time co-culture mode with the bacteria or cells. In addition, the raw materials of the permeation protective agent are mild, non-toxic, low in cost, simple and easy to obtain, the preparation method is simple, and the permeation protective agent has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an osmoprotectant, a preparation method thereof, and an application thereof. Background Art

[0002] Maintaining fluid balance is a necessary condition for organisms to ensure survival. Organisms all have a life system that can sense and respond to osmotic pressure caused by fluid imbalance at the cellular level. When an organism encounters drought, extreme cold, extreme heat, and high-salt environments, the water utilization of the organism's cells is restricted, and the osmotic pressure of the cell sap is imbalanced. This phenomenon is called osmotic stress. Severe osmotic stress will lead to the inhibition of the growth and development of organisms or even death. At the cellular level, osmotic stress will cause the genetic material of cells to be damaged, and cells will adhere and apoptose. Therefore, effective regulation of osmotic stress is crucial for improving the survival rate of organisms.

[0003] When facing osmotic stress, organisms mainly actively regulate through two means. The rapid response relies on activating ion channels to exchange ions, and the long-term regulation depends on the synthesis and accumulation of organic osmolytes. Depending on these two methods, organisms need to undergo a metabolic response of several hours to re-establish osmotic balance. Introducing osmotic regulators to help organisms quickly achieve osmotic balance is also an effective means, but related research is very scarce. There is an urgent need to develop simple, effective, and rapid cell osmotic stress regulators. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides an osmoprotectant, which is selected from Protectant A or Protectant B:

[0005] Protectant A: Composed of a surfactant (single-component osmoprotectant);

[0006] Protectant B: Composed of a surfactant and glycine betaine (betaine) (compound osmoprotectant).

[0007] According to an embodiment of the present invention, the osmoprotectant is applicable to various bacteria and cells; for example, Gram-negative bacteria, such as Escherichia coli.

[0008] According to an embodiment of the present invention, the bacteria are in a state of osmotic stress, such as hypoosmotic stress, hyperosmotic stress, etc. According to an embodiment of the present invention, the osmoprotectant is a hypoosmotic stress protectant or a hyperosmotic stress protectant.

[0009] According to an embodiment of the present invention, the surfactant is selected from non-ionic surfactants. According to an embodiment of the present invention, the non-ionic surfactant is selected from alkyl glycoside non-ionic surfactants and poloxamer non-ionic surfactants.

[0010] According to an embodiment of the present invention, the alkyl polyglycoside nonionic surfactant is selected from C8-20 alkyl polyglycoside nonionic surfactants, preferably C10-18 alkyl polyglycoside nonionic surfactants, such as C12, C14 or C16 alkyl polyglycoside nonionic surfactants.

[0011] According to an embodiment of the present invention, the alkyl polyglycoside nonionic surfactant is selected from alkyl maltoside nonionic surfactants and alkyl glucoside nonionic surfactants.

[0012] According to an embodiment of the present invention, the alkyl polyglycoside nonionic surfactant is selected from dodecyl glucoside and dodecyl maltoside.

[0013] According to an embodiment of the present invention, the molecular weight range of the poloxamer nonionic surfactant is 5000 Da to 15000 Da. According to an embodiment of the present invention, the poloxamer nonionic surfactant is selected from poloxamer F127, poloxamer P123, poloxamer P188, poloxamer P407, and poloxamer F68.

[0014] According to an embodiment of the present invention, the cryoprotectant optionally further includes a solvent; preferably, the solvent is water.

[0015] According to an embodiment of the present invention, in cryoprotectant A, the concentration (working concentration) of the surfactant is 0.1 μM to 100 μM, preferably 0.5 μM to 80 μM, more preferably 1.0 μM to 60 μM, such as 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 10.0 μM, 15.0 μM, 20.0 μM, 25.0 μM, 30.0 μM, 35.0 μM, 40.0 μM, 45.0 μM, 50.0 μM.

[0016] According to an embodiment of the present invention, in cryoprotectant B, the molar ratio of the surfactant to glycine betaine is 0.1-100:1000, preferably 0.5-80:1000, more preferably 1-60:1000, such as 1:1000, 3:1000, 5:1000, 10:1000, 15:1000, 20:1000, 25:1000, 30:1000, 35:1000, 40:1000, 45:1000, 50:1000, 55:1000, 60:1000.

[0017] According to an embodiment of the present invention, in protective agent B, the concentration (working concentration) of the surfactant is 0.1 μM to 100 μM, preferably 0.5 μM to 80 μM, more preferably 1.0 μM to 60 μM, such as 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 10.0 μM, 15.0 μM, 20.0 μM, 25.0 μM, 30.0 μM, 35.0 μM, 40.0 μM, 45.0 μM, 50.0 μM.

[0018] According to an embodiment of the present invention, in protective agent B, the concentration (working concentration) of glycine betaine is 0.01 mM to 20 mM, preferably 0.05 mM to 10 mM, more preferably 0.1 mM to 5 mM, such as 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 3.0 mM, 4.0 mM, 5.0 mM.

[0019] According to an embodiment of the present invention, the said osmoprotectant can significantly improve the survival rate of bacteria or cells.

[0020] According to an embodiment of the present invention, the hypotonic stress protectant is selected from any one of the following:

[0021] 1) Composed of 1.0 μM to 20 μM surfactant (such as 5 μM surfactant);

[0022] 2) Composed of 1.0 μM to 50 μM surfactant and 0.1 mM to 5 mM glycine betaine (such as 5 μM to 40 μM surfactant and 1 mM glycine betaine).

[0023] According to an embodiment of the present invention, the hypertonic stress protectant is selected from any one of the following:

[0024] 1) Composed of 1.0 μM to 20 μM surfactant (such as 5 μM surfactant);

[0025] 2) Composed of 1.0 μM to 20 μM surfactant and 0.1 mM to 5 mM glycine betaine (such as 5 μM surfactant and 1 mM glycine betaine).

[0026] The present invention also provides a preparation method of the above osmoprotectant, and the preparation method includes:

[0027] Prepare a surfactant solution, and the said osmoprotectant is obtained;

[0028] Or, prepare a surfactant solution and a glycine betaine solution respectively, and mix them to obtain the said osmoprotectant.

[0029] The present invention also provides a method for using the above-mentioned osmoprotectant, and the method comprises the following steps: adding the osmoprotectant into bacteria or cells under osmotic stress, and co-culturing them.

[0030] According to an embodiment of the present invention, the bacteria are selected from Gram-negative bacteria, such as Escherichia coli.

[0031] According to an embodiment of the present invention, the osmotic stress state is selected from a hypotonic stress state and a hypertonic stress state.

[0032] According to an embodiment of the present invention, the co-culturing time is 1 min to 60 min, preferably 5 min to 40 min, such as 10 min, 15 min, 20 min, 25 min, 30 min.

[0033] According to an embodiment of the present invention, the method can significantly improve the survival rate of bacteria or cells.

[0034] The present invention also provides an application of the above-mentioned osmoprotectant in treating bacteria or cells.

[0035] According to an embodiment of the present invention, the bacteria are selected from Gram-negative bacteria, such as Escherichia coli.

[0036] Beneficial effects

[0037] The present invention provides an osmoprotectant with simple components, and the osmoprotectant is selected from a single-component osmoprotectant composed of a surfactant or a compound osmoprotectant composed of a surfactant and glycine betaine.

[0038] The osmoprotectant provided by the present invention can significantly improve the survival rate of bacteria or cells by a simple and short-time co-culture method with bacteria or cells. Moreover, the raw materials of the osmoprotectant are mild, non-toxic, low-cost, easily available, the preparation method is simple, and it has good application prospects. Description of the drawings

[0039] Figure 1 It is a statistical result diagram of different states of Escherichia coli corresponding to Examples 1-6 and Control Examples 1-2 under hypotonic stress based on flow cytometry statistics.

[0040] Figure 2 It is a statistical result diagram of different states of Escherichia coli corresponding to Examples 7-8 and Control Examples 3-4 under hypertonic stress based on flow cytometry statistics. Detailed implementation manners

[0041] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for exemplarily illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0042] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods.

[0043] "mM" represents a concentration of mmol / L, and "μM" represents a concentration of μmol / L.

[0044] In the following examples and comparative examples, the statistical results of different states of Escherichia coli were obtained by the following method:

[0045] Use a commercially available SYTO-9 / PI bacterial viability and apoptosis fluorescence dye kit to label Escherichia coli under different treatment conditions. Among them, SYTO-9 can label viable bacteria, and PI can label dead and apoptotic bacteria. Use a flow cytometer to statistically analyze the fluorescence signals of the stained bacteria and obtain the proportion of bacteria in different states.

[0046] The method for treating Escherichia coli with hypotonic stress is as follows: Freshly cultured Escherichia coli is centrifuged to remove the culture medium and resuspended in pure water. This operation is repeated three times to ensure complete removal of nutrients and buffer salts. After completing the above operations, it is left for use. The method for treating Escherichia coli with hypertonic stress is as follows: Freshly cultured Escherichia coli is centrifuged to remove the culture medium and resuspended in a high-concentration glucose solution. This operation is repeated three times to ensure complete removal of nutrients and buffer salts. After completing the above operations, it is left for use.

[0047] Example 1

[0048] Prepare an aqueous solution of dodecyl glucoside at 20 μM and an aqueous solution of glycine betaine at 4 mM. Mix them evenly in equal volumes at room temperature to obtain osmoprotectant 1, and the molar ratio of the two is 5:1000. Escherichia coli under hypotonic stress is mixed and cultured with osmoprotectant 1 in equal volumes at room temperature for 15 min, where the concentration of glycine betaine is 1 mM and the concentration of dodecyl glucoside is 5 μM. Subsequently, the proportion of Escherichia coli in different states was obtained by the above-mentioned statistical method for different states of Escherichia coli. The results are as Figure 1 shown, and the proportion of viable Escherichia coli is 69.7%.

[0049] Example 2

[0050] Prepare an aqueous solution of poloxamer (F127) at 20 μM, and prepare an aqueous solution of glycine betaine at 4 mM. Mix them evenly in equal volumes at room temperature to obtain cryoprotectant 2, and the molar ratio of the two is 5:1000. Escherichia coli under hypoosmotic stress is mixed and cultured with cryoprotectant 2 in equal volumes at room temperature for 15 min, where the concentration of glycine betaine is 1 mM and the concentration of poloxamer (F127) is 5 μM. Subsequently, the proportion of different states of Escherichia coli is obtained by using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 1 shown, and the proportion of surviving Escherichia coli is 59.9%.

[0051] Example 3

[0052] Prepare an aqueous solution of dodecyl glucoside at 100 μM, and prepare an aqueous solution of glycine betaine at 4 mM. Mix them evenly in equal volumes at room temperature to obtain cryoprotectant 3, and the molar ratio of the two is 25:1000. Escherichia coli under hypoosmotic stress is mixed and cultured with cryoprotectant 3 in equal volumes at room temperature for 15 min, where the concentration of glycine betaine is 1 mM and the concentration of dodecyl glucoside is 25 μM. Subsequently, the proportion of different states of Escherichia coli is obtained by using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 1 shown, and the proportion of surviving Escherichia coli is 58.1%.

[0053] Example 4

[0054] Prepare an aqueous solution of poloxamer (F127) at 160 μM, and prepare an aqueous solution of glycine betaine at 4 mM. Mix them evenly in equal volumes at room temperature to obtain cryoprotectant 4, and the molar ratio of the two is 40:1000. Escherichia coli under hypoosmotic stress is mixed and cultured with cryoprotectant 4 in equal volumes at room temperature for 15 min, where the concentration of glycine betaine is 1 mM and the concentration of poloxamer (F127) is 40 μM. Subsequently, the proportion of different states of Escherichia coli is obtained by using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 1 shown, and the proportion of surviving Escherichia coli is 40.1%.

[0055] Example 5

[0056] Prepare an aqueous solution of dodecyl glucoside at 10 μM at room temperature to obtain cryoprotectant 5. Escherichia coli under hypoosmotic stress is mixed and cultured with cryoprotectant 5 in equal volumes at room temperature for 15 min, where the concentration of dodecyl glucoside is 5 μM. Subsequently, the proportion of different states of Escherichia coli is obtained by using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 1 shown, and the proportion of surviving Escherichia coli is 70.2%.

[0057] Example 6

[0058] Prepare an aqueous solution of poloxamer (F127) at 10 μM to obtain cryoprotectant 6 at room temperature. Escherichia coli under hypotonic stress is mixed with cryoprotectant Example 8 in equal volumes and cultured at room temperature for 15 min, where the working concentration of poloxamer (F127) is 5 μM. Subsequently, the proportions of different states of Escherichia coli are obtained using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 1 shown, and the proportion of surviving Escherichia coli is 40.4%.

[0059] Example 7

[0060] Prepare an aqueous solution of dodecyl glucoside at 20 μM and an aqueous solution of glycine betaine at 4 mM, and mix them evenly in equal volumes at room temperature to obtain cryoprotectant 1, with a molar ratio of 5:1000 between the two. Escherichia coli under hypertonic stress is mixed with cryoprotectant 1 in equal volumes and cultured at room temperature for 15 min, where the working concentration of glycine betaine is 1 mM and the working concentration of dodecyl glucoside is 5 μM. Subsequently, the proportions of different states of Escherichia coli are obtained using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 2 shown, and the proportion of surviving Escherichia coli is 35.2%.

[0061] Example 8

[0062] Prepare an aqueous solution of poloxamer (F127) at 20 μM and an aqueous solution of glycine betaine at 4 mM, and mix them evenly in equal volumes at room temperature to obtain cryoprotectant 2, with a molar ratio of 5:1000 between the two. Escherichia coli under hypertonic stress is mixed with cryoprotectant 2 in equal volumes and cultured at room temperature for 15 min, where the working concentration of glycine betaine is 1 mM and the working concentration of poloxamer (F127) is 5 μM. Subsequently, the proportions of different states of Escherichia coli are obtained using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 2 shown, and the proportion of surviving Escherichia coli is 22.3%.

[0063] Control Example 1

[0064] Escherichia coli under hypotonic stress is mixed with pure water in equal volumes and cultured at room temperature for 15 min, and then the proportions of different states of Escherichia coli are obtained using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 1 shown, and the proportion of surviving Escherichia coli is 29.2%.

[0065] Control Example 2

[0066] An Escherichia coli solution under hypotonic stress was mixed with 2 mM glycine betaine in equal volume and cultured at room temperature for 15 min, with the concentration of glycine betaine being 1 mM. Subsequently, the proportion of different states of Escherichia coli was obtained by using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 1 shown, and the proportion of surviving Escherichia coli is 47.5%.

[0067] Control Example 3

[0068] Escherichia coli under hypertonic stress was mixed with pure water in equal volume and cultured at room temperature for 15 min. Subsequently, the proportion of different states of Escherichia coli was obtained by using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 2 shown, and the proportion of surviving Escherichia coli is 7.0%.

[0069] Control Example 4

[0070] An Escherichia coli solution under hypertonic stress was mixed with 2 mM glycine betaine in equal volume and cultured at room temperature for 15 min, with the concentration of glycine betaine being 1 mM. Subsequently, the proportion of different states of Escherichia coli was obtained by using the above-mentioned statistical methods for different states of Escherichia coli. The results are as Figure 2 shown, and the proportion of surviving Escherichia coli is 11.7%.

[0071] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A penetration protectant, which is selected from protectant A or protectant B: Protectant A: Composed of surfactants; Protectant B: Composed of surfactants and glycine betaine.

2. The penetration protective agent according to claim 1, characterized in that, The penetration protectant is applicable to various bacteria and cells; Preferably, the bacteria are in a state of osmotic stress; Preferably, the penetration protectant is a hypotonic stress protectant or a hypertonic stress protectant.

3. The penetration protective agent according to claim 1 or 2, characterized in that, The surfactant is selected from non-ionic surfactants; Preferably, the non-ionic surfactant is selected from alkyl glycoside non-ionic surfactants and poloxamer non-ionic surfactants; Preferably, the alkyl glycoside non-ionic surfactant is selected from C8-20 alkyl glycoside non-ionic surfactants; Preferably, the alkyl glycoside non-ionic surfactant is selected from alkyl maltoside non-ionic surfactants and alkyl glucoside non-ionic surfactants; Preferably, the alkyl glycoside non-ionic surfactant is selected from dodecyl glucoside and dodecyl maltoside; Preferably, the molecular weight range of the poloxamer non-ionic surfactant is 5000Da - 15000Da; Preferably, the poloxamer non-ionic surfactant is selected from poloxamer F127, poloxamer P123, poloxamer P188, poloxamer P407, and poloxamer F68.

4. The penetration protective agent according to any one of claims 1 to 3, characterized in that, The penetration protectant optionally further includes a solvent; preferably, the solvent is water.

5. The penetration protective agent according to any one of claims 1-4, characterized in that, In protectant A, the concentration of the surfactant is 0.1 μM - 100 μM, preferably 0.5 μM - 80 μM.

6. The penetration protective agent according to any one of claims 1-5, characterized in that, In protectant B, the molar ratio of the surfactant to glycine betaine is 0.1 - 100:1000, preferably 0.5 - 80:1000; Preferably, in protectant B, the concentration of the surfactant is 0.1 μM - 100 μM, preferably 0.5 μM - 80 μM; Preferably, in protectant B, the concentration of glycine betaine is 0.01 mM - 20 mM, preferably 0.05 mM - 10 mM.

7. The penetration protective agent according to claim 2, characterized in that, The hypotonic stress protectant is selected from any one of the following: 1) Composed of 1.0 μM - 20 μM surfactant; 2) Composed of 1.0 μM - 50 μM surfactant and 0.1 mM - 5 mM glycine betaine; Preferably, the hypertonic stress protectant is selected from any one of the following: 1) Composed of 1.0 μM - 20 μM surfactant; 2) Composed of 1.0 μM - 20 μM surfactant and 0.1 mM - 5 mM glycine betaine.

8. The preparation method of the penetration protectant according to any one of claims 1 - 7, the preparation method comprising: Preparing a surfactant solution to obtain the penetration protectant; Or, separately preparing a surfactant solution and a glycine betaine solution, and mixing them to obtain the penetration protectant.

9. The usage method of the penetration protectant according to any one of claims 1 - 7, the usage method comprising the following steps: Adding the penetration protectant according to any one of claims 1 - 7 to bacteria or cells in a state of osmotic stress, and co-culturing; Preferably, the bacteria are selected from Gram-negative bacteria, such as Escherichia coli; Preferably, the osmotic stress state is selected from a hypoosmotic stress state and a hyperosmotic stress state; Preferably, the co-culture time is 1 min to 60 min, preferably 5 min to 40 min.

10. Use of the osmoprotectant according to any one of claims 1-7 in treating bacteria or cells.

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