Protective agent for prolonging survival time of rhizobium and application thereof

Abstract

The invention relates to the technical field of microbial fermentation, and particularly discloses a protective agent for prolonging the survival time of rhizobium and application of the protective agent. The protective agent comprises the following components: 0.2 to 0.5 g / L of polyvinylpyrrolidone; 0.5 to 1 g / L of sorbitol; 0.1 mL / L to 0.5 mL / L of dimethyl sulfoxide; 0.2 to 0.5 g / L of nano carbon powder; 1-2 g / L of sodium dodecyl sarcosinate; 18 to 22 g / L of guanidine isothiocyanate; vancomycin is 0.1 to 0.5 mg / L; and 2-5g / L of sodium alginate. According to the application, the protective agent is added in the fermentation stage of the rhizobium, so that the preservation time of the rhizobium liquid can be prolonged under the condition of ensuring that the number of the rhizobium in the rhizobium liquid is relatively high.

Description

Technical Field

[0001] The present application relates to the technical field of microbial fermentation, and more specifically, to a protective agent for prolonging the survival period of rhizobia and an application thereof. Background Art

[0002] Since rhizobia are parasitic microorganisms, their ability to survive independently is low and they can only survive for a long time at low temperatures, but not at room temperature. Therefore, the storage methods of rhizobia products are mainly low-temperature storage, freeze-drying, or storage after adsorption with solid adsorbents.

[0003] At present, most rhizobium products exist in solid form, and there are fewer rhizobium products in the form of bacterial liquid. Usually, the shelf life of rhizobium products is only about 1 month, and the number of rhizobia preserved in rhizobium products is only 200-500 million / ml.

[0004] Therefore, it is necessary to find a new method to improve rhizobium products in order to increase the shelf life of rhizobium products and the number of rhizobia preserved in the products. Summary of the invention

[0005] The present application provides a protective agent for prolonging the survival period of rhizobia and its application. The present application adds a protective agent during the rhizobium fermentation stage, thereby prolonging the storage time of the rhizobium solution while ensuring that the number of rhizobia in the rhizobium solution is high.

[0006] The present application uses the rhizobium with the deposit number CGMCC No. 29321 as an example for testing. Adding the protective agent provided by the present application during the rhizobium fermentation stage can ensure that the rhizobium liquid can maintain an effective live bacterial count of more than 7 billion / ml after being stored at room temperature for 3 months.

[0007] In a first aspect, the present application provides a protective agent for prolonging the survival period of rhizobia, using the following technical solution:

[0008] A protective agent for prolonging the survival period of rhizobia, the protective agent comprising the following components: 0.2-0.5 g / L of polyvinyl pyrrolidone; 0.5-1 g / L of sorbitol; 0.1-0.5 mL / L of dimethyl sulfoxide; 0.2-0.5 g / L of nano carbon powder; 1-2 g / L of sodium lauryl sarcosinate; 18-22 g / L of guanidine isothiocyanate; 0.1-0.5 mg / L of vancomycin; and 2-5 g / L of sodium alginate.

[0009] Optionally, the concentration of vancomycin is 0.2-0.4 mg / L.

[0010] Optionally, the concentration of sodium alginate is 3-4 g / L.

[0011] Optionally, the polyvinyl pyrrolidone is K60.

[0012] Optionally, the protective agent is added to the culture medium of the rhizobium, and the culture medium includes the following components: 16-18 g / L glucose; 3-5 g / L yeast; 0.2-0.4 g / L calcium chloride; 0.3-0.5 g / L magnesium sulfate heptahydrate; 0.5-1 g / L potassium dihydrogen phosphate; 0.5-1 g / L dipotassium hydrogen phosphate; 0.1-0.2 g / L sodium chloride; 5-7 ml / L Rh trace element solution; 0.0008-0.001 g / L ferrous sulfate; 0.5-1 g / L dithiothreitol; and 5-10% bovine serum.

[0013] Optionally, any one or more of the guanidine thiocyanate and the vancomycin are added after fermentation.

[0014] Optionally, the rhizobium (Bradyrhizobium elkanii) is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.29321.

[0015] Optionally, the Rh trace element solution includes boron at a concentration of 0.005 g / L and molybdenum at a concentration of 0.005 g / L.

[0016] Optionally, the guanidine isothiocyanate is sterilized by treating it with 0.1% diethyl pyrocarbonate and then boiling it to remove the diethyl pyrocarbonate.

[0017] In a second aspect, the present application provides a use of the above-mentioned protective agent in the preparation of a rhizobium liquid product for increasing the number of preserved rhizobia.

[0018] In summary, this application has the following beneficial effects:

[0019] 1. This application uses rhizobium with a deposit number of CGMCC No. 29321 as an example for testing. Adding the protective agent provided by this application during the rhizobium fermentation stage can extend the storage time of the rhizobium solution while ensuring that the number of rhizobia in the rhizobium solution is high.

[0020] 2. In the related art, rhizobia are mostly produced in the form of freeze-dried powder or other solid forms. The present application provides a rhizobium product in the form of bacterial liquid, and the rhizobium liquid product has a high bacterial count and a stable shelf life. It can be stored at room temperature for 6 months, and the bacterial count can still reach more than 7 billion / ml. DETAILED DESCRIPTION

[0021] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0022] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0023] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0024] The present application provides a protective agent for prolonging the survival period of rhizobia, which comprises the following components: 0.2-0.5 g / L of polyvinyl pyrrolidone; 0.5-1 g / L of sorbitol; 0.1-0.5 mL / L of dimethyl sulfoxide; 0.2-0.5 g / L of nano carbon powder; 1-2 g / L of sodium lauryl sarcosinate; 18-22 g / L of guanidine isothiocyanate; 0.1-0.5 mg / L of vancomycin; and 2-5 g / L of sodium alginate.

[0025] The protective agent provided by the present application is added to a culture medium of rhizobia, which includes the following components: 16-18 g / L of glucose; 3-5 g / L of yeast; 0.2-0.4 g / L of calcium chloride; 0.3-0.5 g / L of magnesium sulfate heptahydrate; 0.5-1 g / L of potassium dihydrogen phosphate; 0.5-1 g / L of dipotassium hydrogen phosphate; 0.1-0.2 g / L of sodium chloride; 5-7 ml / L of Rh trace element solution; 0.0008-0.001 g / L of ferrous sulfate; 0.5-1 g / L of dithiothreitol; and 5-10% of bovine serum.

[0026] Furthermore, any one or more of the guanidine thiocyanate and the vancomycin are added after fermentation.

[0027] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be interpreted as limiting the present application.

[0028] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0029] The bovine serum used was produced by VivaCell, model number C04002-500.

[0030] The present application is further described in detail below in conjunction with the embodiments and test results.

[0031] Preparation Example

[0032] Preparation Example 1-34

[0033] Preparation Examples 1-34 respectively provide a protective agent for prolonging the survival period of rhizobia. The specific formula of the protective agent is shown in Table 1.

[0034] The differences between the above protective agents are as follows:

[0035] (1) The difference between Preparation Examples 1-4 is whether sodium lauryl sarcosinate and / or guanidine isothiocyanate are added. The details are as follows:

[0036] In Preparation Example 1, sodium lauryl sarcosinate and guanidine isothiocyanate were not added.

[0037] In Preparation Example 2, only sodium lauryl sarcosine was added.

[0038] In Preparation Example 3, only guanidine isothiocyanate was added.

[0039] Preparation Example 4: Sodium lauryl sarcosinate and guanidine isothiocyanate were added.

[0040] (2) The difference between Preparation Examples 3 and 5-15 is whether erythromycin, vancomycin, sodium alginate and / or polyethylene glycol are added. The details are as follows:

[0041] The difference between Preparation Example 5 and Preparation Example 4 is that erythromycin is added.

[0042] The difference between Preparation Example 6 and Preparation Example 4 is that vancomycin is added.

[0043] The difference between Preparation Example 7 and Preparation Example 4 is that sodium alginate is added.

[0044] The difference between Preparation Example 8 and Preparation Example 4 is that polyethylene glycol is added.

[0045] The difference between Preparation Example 9 and Preparation Example 4 is that erythromycin and sodium alginate are added.

[0046] The difference between Preparation Example 10 and Preparation Example 4 is that erythromycin and polyethylene glycol are added.

[0047] The difference between Preparation Example 11 and Preparation Example 4 is that vancomycin and sodium alginate are added.

[0048] The difference between Preparation Example 12 and Preparation Example 4 is that vancomycin and polyethylene glycol are added.

[0049] The difference between Preparation Example 13 and Preparation Example 4 is that vancomycin, sodium alginate and polyethylene glycol are added.

[0050] The difference between Preparation Example 14 and Preparation Example 4 is that erythromycin, sodium alginate and polyethylene glycol are added. The difference between Preparation Example 15 and Preparation Example 11 is that sodium lauryl sarcosinate and guanidine isothiocyanate (3) are not added. The difference between Preparation Examples 11, 16-19 is the amount of vancomycin added.

[0051] (4) The difference between Preparation Examples 11 and 16-19 is the amount of sodium alginate added.

[0052] (5) The difference between Preparation Examples 21 and 24-28 is the addition of nano-carbon powder. The details are as follows:

[0053] The difference between Preparation Example 24 and Preparation Example 21 is that no nano-carbon powder is added.

[0054] The difference between Preparation Examples 25-27 and Preparation Example 21 is the amount of nano-carbon powder added.

[0055] The difference between Preparation Example 28 and Preparation Example 21 is that activated carbon is used instead of nano carbon powder.

[0056] (6) The difference between Preparation Examples 25 and 29-31 lies in the type of polyvinyl pyrrolidone.

[0057] (7) The difference between Preparation Examples 25 and 32-34 is the addition method of guanidine thiocyanate and / or vancomycin. The difference between Preparation Example 32 and Preparation Example 25 is that guanidine thiocyanate and vancomycin are added after the fermentation is completed. The difference between Preparation Example 33 and Preparation Example 25 is that guanidine thiocyanate is added after the fermentation is completed.

[0058] The difference between Preparation Example 34 and Preparation Example 25 is that vancomycin is added after the fermentation is completed.

[0059] Table 1 Components of protective agents in Preparation Examples 1-34

[0060]

[0061]

[0062]

[0063] Example

[0064] Examples 1-34

[0065] Examples 1-34 respectively provide a method for preparing a Bradyrhizobium sojae liquid, wherein the deposit number of Bradyrhizobium sojae is CGMCC No.29321.

[0066] The above preparation method specifically comprises the following steps:

[0067] (1) Prepare the fermentation medium according to the following formula, sterilize at high temperature, and set aside.

[0068] The fermentation medium formula is as follows: glucose 16-18g / L; yeast 3-5g / L; calcium chloride 0.2-0.4g / L; magnesium sulfate heptahydrate 0.3-0.5g / L; potassium dihydrogen phosphate 0.5-1g / L; dipotassium hydrogen phosphate 0.5-1g / L; sodium chloride 0.1-0.2g / L; Rh trace element solution (consisting of boron and molybdenum) 5-7ml / L; ferrous sulfate 0.0008-0.001g / L; dithiothreitol 0.5-1g / L; bovine serum 5-10%; natural pH.

[0069] After adding the protective agents provided in the preparation examples respectively, high temperature sterilization is carried out.

[0070] Among them, since guanidine isothiocyanate is very easy to denature in an environment of 121°C and cannot be directly sterilized by high-temperature steam, it needs to be treated with 0.1% diethyl pyrocarbonate for sterilization, and then boiled to remove the diethyl pyrocarbonate. After sterilization in this way, it is added to the sterilized culture medium.

[0071] The addition "after fermentation" in Table 1 refers to the addition of the sterilized substance after the fermentation is completed, and there is no need to add the substance before fermentation.

[0072] (2) Seed liquid culture: Select soybean bradyrhizobium and inoculate it into YMA liquid culture medium (formula: mannitol 10 g / L, yeast 1 g / L, dipotassium hydrogen phosphate 0.25 g / L, potassium dihydrogen phosphate 0.25 g / L, magnesium sulfate 0.2 g / L, sodium chloride 0.1 g / L; pH 6.8-7.2), place it in a shaking incubator at 200 r / min and 28°C and culture for 7 days to obtain seed liquid.

[0073] (3) Inoculation: Use a pipette to take 3 mL of the diluted seed solution and add it to the above fermentation medium, and shake it thoroughly to prepare an initial fermentation medium with an inoculation volume of 3%.

[0074] (4) Culture of bacterial strains: The initial fermentation medium was immediately placed in a shaker at 200 rpm and 28°C for 5 days.

[0075] Comparative Example

[0076] The comparative example provides a method for preparing a soybean bradyrhizobium bacterial liquid. The difference between this comparative example and Example 1 is that no protective agent is added to the fermentation medium. The rest is consistent with Example 1.

[0077] Test 1

[0078] The rhizobium bacterial liquid products prepared in the above examples and comparative examples were stored at room temperature (25° C.), and the number of rhizobia in the rhizobium bacterial liquid products was detected by a spectrophotometer at different storage times. The detection results are shown in Table 2.

[0079] Table 2 Test results

[0080]

[0081]

[0082] As shown in Table 2, Examples 11, 13, 17, 20-22, 25, 31-34 still have an effective live bacterial count higher than 2 billion / ml when stored for 6 months, among which Example 32 has the highest bacterial count, and Example 11 has a slightly lower bacterial count than Example 32. However, due to the presence of substances added after fermentation in Example 32, the difficulty of operation is increased while also increasing the probability of contamination of the rhizobium bacterial solution, which is more difficult in actual production applications. Therefore, all substances can be added in the early stage of fermentation, and there is no need for excessive subsequent operations. In comparison, there are fewer operations in Example 11, and no substances need to be added after the end, which is relatively safer and has a higher bacterial count. The final formula is determined to be Example 11.

[0083] Test 2

[0084] The protective agent and the method for preparing rhizobium solution of the present application were used to culture rhizobia known in the related art, and the number of rhizobia at different storage times was detected. The detection results are shown in Table 3.

[0085] Table 3 Detection results of different types of rhizobia

[0086]

[0087]

[0088] Under the same formula and protective agent, other types of rhizobia were used for preservation, and another rhizobium strain could also achieve an effective viable count of 2 billion / ml for 6 months, indicating that this formula has a certain degree of general applicability, not only for AMSD1.

[0089] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A protective agent for prolonging the survival period of rhizobia, characterized in that: The protective agent comprises the following components: polyvinyl pyrrolidone 0.2-0.5 g / L; sorbitol 0.5-1 g / L; dimethyl sulfoxide 0.1-0.5 mL / L; nano carbon powder 0.2-0.5 g / L; sodium lauryl sarcosinate 1-2 g / L; guanidine isothiocyanate 18-22 g / L; vancomycin 0.1-0.5 mg / L; and sodium alginate 2-5 g / L.

2. The protective agent for prolonging the survival period of rhizobia according to claim 1, characterized in that: The concentration of vancomycin is 0.2-0.4 mg / L.

3. The protective agent for prolonging the survival period of rhizobia according to claim 1, characterized in that: The concentration of the sodium alginate is 3-4 g / L.

4. The protective agent for prolonging the survival period of rhizobia according to claim 1, characterized in that: The concentration of the nano carbon powder is 0.2-0.4 g / L.

5. The protective agent for prolonging the survival period of rhizobia according to claim 1, characterized in that: The polyvinyl pyrrolidone is K60.

6. The protective agent for prolonging the survival period of rhizobia according to claim 1, characterized in that The protective agent is added to the culture medium of the rhizobium, and the culture medium includes the following components: 16-18 g / L of glucose; 3-5 g / L of yeast; 0.2-0.4 g / L of calcium chloride; 0.3-0.5 g / L of magnesium sulfate heptahydrate; 0.5-1 g / L of potassium dihydrogen phosphate; 0.5-1 g / L of dipotassium hydrogen phosphate; 0.1-0.2 g / L of sodium chloride; 5-7 ml / L of Rh trace element solution; 0.0008-0.001 g / L of ferrous sulfate; 0.5-1 g / L of dithiothreitol; and 5-10% of bovine serum.

7. The protective agent for prolonging the survival period of rhizobia according to claim 6, characterized in that: Any one or more of the guanidine thiocyanate and the vancomycin are added after fermentation.

8. The protective agent for prolonging the survival period of rhizobia according to claim 6, characterized in that The rhizobium ( Bradyrhizobium elkanii ) is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No.29321.

9. The culture medium according to claim 6, characterized in that The Rh trace element solution includes boron with a concentration of 0.005 g / L and molybdenum with a concentration of 0.005 g / L.

10. Use of the protective agent according to any one of claims 1 to 9 for increasing the number of preserved Rhizobium bacteria in the preparation of a liquid Rhizobium product.

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