Method for reducing loss rate of viable count in spore liquid agricultural microbial agent

Abstract

The invention provides a method for reducing the loss rate of viable count in a spore liquid agricultural microbial agent, and belongs to the technical field of microbial preparations. The method comprises the following steps: after bacillus grows to a stable phase, raising the temperature of a bacillus culture medium to 41-46 DEG C, continuously culturing for 8-14 hours, and packaging as a liquid agricultural microbial agent. According to the method, firstly, bacillus is cultured to the later stage of a stable phase, nutrient substances in a culture medium are consumed massively at the moment, then bacillus strain sporation is accelerated in a physical heating mode, after the temperature is reduced, the nutrient substances in the culture medium cannot support the bacillus to be converted into an active bacterial cell state from a dormant spore state, and the bacillus strain sporation is accelerated. Therefore, the loss rate of the viable count of the packaged liquid agricultural microbial agent in the storage period can be effectively reduced, and the loss rate of the viable count is only 8.0-17.91% after the liquid agricultural microbial agent is stored for one month at room temperature.

Description

Technical Field

[0001] The invention relates to the technical field of microbial preparations, and in particular to a method for reducing the loss rate of live bacteria count in spore-based liquid agricultural microbial inoculants. Background Art

[0002] Microbial inoculants in agriculture (GB 20287-2006 Agricultural microbial inoculants product standard) refers to live bacterial preparations made by industrially producing and multiplying target microorganisms (effective bacteria). It has the functions of directly or indirectly improving soil, restoring soil fertility, maintaining the balance of rhizosphere microbial flora, and degrading toxic and harmful substances. It is applied to agricultural production, and through the life activities of the microorganisms contained therein, it increases the supply of plant nutrients or promotes plant growth, improves the quality of agricultural products and the agricultural ecological environment.

[0003] There are three types of agricultural microbial agents: liquid, powder and granule. One of the product indicators is the effective viable count. Liquid agricultural microbial agents have a high water content, which leads to a short microbial decay cycle and a high viable count loss rate. Therefore, it is of great significance to provide a method for reducing the viable count loss rate in liquid agricultural microbial agents.

[0004] Spores, also known as endospores, are dormant bacterial bodies. Spores have extremely low water content and strong resistance to stress. They can withstand high temperatures, ultraviolet rays, ionizing radiation, and a variety of chemical substances. When spore-forming bacteria lack nutrients or are in an environment that is not suitable for growth, they can form round or oval dormant spore bodies in the bacteria, and when conditions are suitable, they will become a bacterium (nutrient cell) again.

[0005] At present, most of the existing technologies add chemical additives that inhibit the growth of Bacillus cells, such as potassium sorbate, to liquid agricultural microbial agents, so that the cells are in a dormant state and a large number of cell deaths are reduced during storage. However, chemical additives have certain damage to the activity and probiotic effects of Bacillus, affecting the application effect of liquid agricultural microbial agents. Therefore, it is necessary to provide a simple, feasible and safe method for reducing the loss rate of live bacteria in spore-based liquid agricultural microbial agents. Summary of the invention

[0006] The object of the present invention is to provide a simple, feasible and safe method for reducing the loss rate of viable bacteria in a spore-based liquid agricultural microbial inoculant.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for reducing the loss rate of live bacteria count in a spore-based liquid agricultural microbial agent. After the bacillus grows to a stable period, the temperature of the bacillus culture medium is increased to 41-46°C, and the culture is continued for 8-14 hours, and the liquid agricultural microbial agent is packaged.

[0009] Preferably, the Bacillus includes any one or more of Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis and Bacillus thuringiensis.

[0010] Preferably, the stable period is the late stable period.

[0011] Preferably, the temperature of the Bacillus culture medium is raised to 42-45° C., and the culture is continued for 10-12 hours.

[0012] Preferably, the Bacillus culture medium contains 8-12 g / L peptone, 2-4 g / L beef extract powder and 3-7 g / L sodium chloride.

[0013] Preferably, the spore rate of the liquid agricultural microbial agent during packaging is greater than 90%, and the number of viable bacteria is 1×10 9 CFU / mL~5×10 9 CFU / mL.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides a method for reducing the loss rate of live bacteria count in a spore-based liquid agricultural microbial inoculant. When the bacillus grows to the late stable period and has not reached the decay period, the sporeization of the bacillus strain is accelerated by physical heating to make the spore rate reach more than 90%. At this time, the nutrients in the culture medium are insufficient. When the temperature is lowered, the culture medium cannot provide sufficient nutritional conditions for the bacillus, and the bacillus cannot re-form active bacteria (nutrient cells). The loss rate of live bacteria count of the packaged liquid agricultural microbial inoculant during the storage period can be effectively reduced, thereby achieving the purpose of increasing the shelf life of the liquid agricultural microbial inoculant product. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 The figure shows the change of spore rate when the Bacillus in Example 5 was cultured in medium at 35°C and 45°C. DETAILED DESCRIPTION

[0018] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0019] Example 1

[0020] A method for reducing the loss rate of viable bacteria in a spore-based liquid agricultural microbial inoculant, comprising the following steps:

[0021] (1) Dissolve 10 g of peptone, 3 g of beef extract powder, and 5 g of sodium chloride in 1 L of water to obtain a Bacillus culture medium, which is sterilized at 121°C for 30 min and set aside.

[0022] (2) Inoculate Bacillus licheniformis into a stirred fermenter containing the culture medium prepared in step (1), and culture at 37° C. and 150 rpm.

[0023] (3) After the Bacillus licheniformis grows to the late stable phase, the temperature of the culture medium is raised to 45°C and the culture is continued for 12 hours. The liquid agricultural microbial agent is packaged. At this time, the spore rate in the liquid agricultural microbial agent is 97% and the number of viable bacteria is 3.35×10 9 CFU / mL.

[0024] After being stored at room temperature for one month, the number of live bacteria in the liquid agricultural microbial agent was counted and the loss rate of live bacteria was calculated. After being stored at room temperature for one month, the number of live bacteria in the liquid agricultural microbial agent was 2.75×10 9 CFU / mL, and the viable bacteria loss rate was 17.91%.

[0025] Example 2

[0026] A method for reducing the loss rate of viable bacteria in a spore-based liquid agricultural microbial inoculant, comprising the following steps:

[0027] (1) Dissolve 10 g of peptone, 3 g of beef extract powder, and 5 g of sodium chloride in 1 L of water to obtain a Bacillus culture medium, which is sterilized at 121°C for 30 min and set aside.

[0028] (2) Inoculate Bacillus subtilis into a stirred fermenter containing the culture medium prepared in step (1), and culture at 37° C. and 150 rpm.

[0029] (3) After the Bacillus subtilis grows to the late stable phase, the temperature of the culture medium is raised to 45°C and the culture is continued for 12 hours. The liquid agricultural microbial agent is packaged. At this time, the spore rate in the liquid agricultural microbial agent is 98% and the number of viable bacteria is 3.2×10 9 CFU / mL.

[0030] After being stored at room temperature for one month, the number of live bacteria in the liquid agricultural microbial agent was counted and the loss rate of live bacteria was calculated. After being stored at room temperature for one month, the number of live bacteria in the liquid agricultural microbial agent was 2.944×10 9 CFU / mL, and the loss rate of viable bacteria was 8.0%.

[0031] Example 3

[0032] A method for reducing the loss rate of viable bacteria in a spore-based liquid agricultural microbial inoculant, comprising the following steps:

[0033] (1) Dissolve 10 g of peptone, 3 g of beef extract powder, and 5 g of sodium chloride in 1 L of water to obtain a Bacillus culture medium, which is sterilized at 121°C for 30 min and set aside.

[0034] (2) Inoculate Bacillus subtilis into a stirred fermenter containing the culture medium prepared in step (1), and culture at 37° C. and 150 rpm.

[0035] (3) After the Bacillus subtilis grows to the late stable phase, the temperature of the culture medium is raised to 41°C and the culture is continued for 10 hours. The liquid agricultural microbial agent is packaged. At this time, the spore rate in the liquid agricultural microbial agent is 95% and the number of viable bacteria is 3.2×10 9 CFU / mL.

[0036] After being stored at room temperature for one month, the number of live bacteria in the liquid agricultural microbial agent was counted and the loss rate of live bacteria was calculated. After being stored at room temperature for one month, the number of live bacteria in the liquid agricultural microbial agent was 2.89×10 9 CFU / mL, and the viable bacteria loss rate was 9.69%.

[0037] Example 4

[0038] A method for reducing the loss rate of viable bacteria in a spore-based liquid agricultural microbial inoculant, comprising the following steps:

[0039] (1) Dissolve 10 g of peptone, 3 g of beef extract powder, and 5 g of sodium chloride in 1 L of water to obtain a Bacillus culture medium, which is sterilized at 121°C for 30 min and set aside.

[0040] (2) Inoculate Bacillus subtilis into a stirred fermenter containing the culture medium prepared in step (1), and culture at 37° C. and 150 rpm.

[0041] (3) After the Bacillus subtilis grows to the late stable phase, the temperature of the culture medium is raised to 45°C and the culture is continued for 8 hours. The liquid agricultural microbial agent is packaged. At this time, the spore rate in the liquid agricultural microbial agent is 93% and the number of viable bacteria is 3.2×10 9 CFU / mL.

[0042] After being stored at room temperature for one month, the number of live bacteria in the liquid agricultural microbial agent was counted and the loss rate of live bacteria was calculated. After being stored at room temperature for one month, the number of live bacteria in the liquid agricultural microbial agent was 2.81×10 9 CFU / mL, and the viable bacteria loss rate was 12.19%.

[0043] Example 5

[0044] The inventors also studied the effect of culture solution temperature on the formation of Bacillus spores, and the specific method is as follows:

[0045] (1) Dissolve 10 g of peptone, 3 g of beef extract powder, and 5 g of sodium chloride in 1 L of water to obtain a Bacillus culture medium, which is sterilized at 121°C for 30 min and set aside.

[0046] (2) Inoculate Bacillus subtilis into a stirred fermenter containing the culture medium prepared in step (1), and culture at 37° C. and 150 rpm.

[0047] (3) After Bacillus subtilis grew to the late stable phase, the temperature of the culture medium was raised to 45°C in the treatment group and adjusted to 35°C in the control group. The culture was continued and the spore rate of Bacillus subtilis in the culture medium was counted at 3, 6 and 12 h of culture.

[0048] The results are as follows Figure 1 As shown. Figure 1 It can be seen that when the temperature of the culture medium is increased to 45°C, the spore rate of Bacillus subtilis in the culture medium increases significantly, indicating that increasing the temperature of the culture medium to 45°C is conducive to the spore formation of Bacillus subtilis.

[0049] It can be seen from the above embodiments that the present invention first cultured the Bacillus to the late stable period, at which time the nutrients in the culture medium were consumed in large quantities, and then accelerated the spore formation of the Bacillus strain by physical heating. When the temperature was lowered, the nutrients in the culture medium could not support the transformation of the Bacillus from the dormant spore state to the active bacterial cell state, thereby effectively reducing the loss rate of the number of live bacteria in the packaged liquid agricultural microbial inoculant during the storage period. After being stored at room temperature for one month, the loss rate of the number of live bacteria was only 8.0-17.91%.

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for reducing the loss rate of viable bacteria in a spore-based liquid agricultural microbial inoculant, characterized in that: After the Bacillus grows to a stable period, the temperature of the Bacillus culture medium is raised to 41-46° C., and the culture is continued for 8-14 hours before being packaged as a liquid agricultural microbial agent.

2. The method according to claim 1, characterized in that The Bacillus includes any one or more of Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis and Bacillus thuringiensis.

3. The method according to claim 1, characterized in that The stable period is the late stable period.

4. The method according to claim 1, characterized in that Increase the temperature of the Bacillus culture medium to 42-45°C and continue culturing for 10-12 hours.

5. The method according to claim 4, characterized in that The bacillus culture medium contains 8-12 g / L of peptone, 2-4 g / L of beef extract powder and 3-7 g / L of sodium chloride.

6. The method according to any one of claims 1 to 5, characterized in that: The spore rate of the liquid agricultural microbial agent during packaging is greater than 90%, and the number of live bacteria is 1×10 9 CFU / mL~5×10 9 CFU / mL.

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