A vacuum freeze-drying protectant for Bacillus velezensis and its application

Abstract

The present invention relates to a microbial vacuum freeze-drying protective agent, specifically to a vacuum freeze-drying protective agent for Bacillus velezensis, its preparation method and its application. The present invention discloses a vacuum freeze-drying protective agent for Bacillus velezensis. The vacuum freeze-drying protective agent contains the following components by mass: 100×10 3 -200×10 3 parts of skim milk powder, 260×10 3 -370×10 3 parts of mannitol, 50×10 3 -90×10 3 parts of sodium glutamate, 1-3 parts of PQQ and 120×10 3 -230×10 3 parts of tryptone. Under the protection of the vacuum freeze-drying protective agent, the survival rate of microorganisms after vacuum freeze-drying can be significantly improved, providing a basis for the wide application of functional microorganisms.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial vacuum freeze-drying protectants, and particularly relates to a vacuum freeze-drying protectant for Bacillus velezensis, a preparation method thereof, and an application thereof. Background Art

[0002] Vacuum freeze-drying technology is a new drying technology that combines vacuum and vacuum freezing. In freeze-drying technology, a water-containing material is vacuum-frozen into a solid, and under low-temperature and low-pressure conditions, the sublimation property of water is utilized to dehydrate the material at low temperature to achieve the purpose of drying.

[0003] Bacillus velezensis belongs to a new species of the genus Bacillus, and is a Gram-positive aerobic bacterium. The cells are rod-shaped, with a size of 0.5×(1.5 - 3.5) μm, and form endospores. It is widely distributed in natural water bodies, soil, air, plant roots, plant surfaces, and animal intestines. In recent years, there have been more and more reports on Bacillus velezensis at home and abroad. The research mainly focuses on promoting the growth of animals and plants, antagonizing pathogenic bacteria, inducing systemic resistance, identifying antibacterial substances and their gene clusters, and the mechanism of antagonistic action. This bacterium plays an important role in biological control, drug research and development, food fermentation, and industrial applications.

[0004] Chinese invention "A Bacillus velezensis and Its Application in Controlling Apple Diseases" (CN109880764A) discloses using glycerol, skim milk, vegetable oil, sodium alginate, or chitosan as a protectant for Bacillus velezensis to obtain a paste-like microbial inoculant; Chinese invention patent "Bacillus velezensis K01 and Its Application" (CN112746043A) uses a protectant containing dextrin, soluble starch, and diatomaceous earth for protection in microbial centrifugal spray drying; in Chinese invention patent "A Bacterial Agent Containing Bacillus velezensis SUNO-18S-36 and Its Application" (CN113322206A), a UV protectant containing one or more of flavonoids, lecithin, fluorescent brighteners, robinin, and hemp seed oil is used to protect Bacillus velezensis from UV damage.

[0005] The reasons affecting the survival rate of microorganisms after vacuum freeze-drying are mainly in two aspects. On the one hand, it is the microorganisms themselves, and on the other hand, it is the environmental conditions for vacuum-freezing, drying, and storing microorganisms. The relatively low survival rate of Bacillus velezensis after vacuum freeze-drying has limited, to a certain extent, applications such as biological control, drug research and development, food fermentation, and industrial applications. The addition of a protectant can largely make up for this disadvantage. Summary of the Invention

[0006] To solve the technical problem that the survival rate of microorganisms, especially Bacillus velezensis and its compound with other functional microorganisms, is low after vacuum freeze-drying, the present invention discloses the following technical solutions:

[0007] A vacuum freeze-drying protectant for Bacillus velezensis, the microbial vacuum freeze-drying protectant comprising skim milk powder, mannitol and sodium glutamate.

[0008] Preferably, the vacuum freeze-drying protectant for Bacillus velezensis further comprises PQQ.

[0009] Preferably, the vacuum freeze-drying protectant for Bacillus velezensis further comprises tryptone.

[0010] Preferably, the vacuum freeze-drying protectant for Bacillus velezensis comprises the following components by mass: 100×10 3 -200×10 3 parts of skim milk powder, 260×10 3 -370×10 3 parts of mannitol, 50×10 3 -90×10 3 parts of sodium glutamate, 1-3 parts of PQQ and 120×10 3 -230×10 3 parts of tryptone.

[0011] A method for preparing a vacuum freeze-drying protectant for Bacillus velezensis, dissolving the components of the vacuum freeze-drying protectant for Bacillus velezensis in distilled water, sterilizing and cooling to obtain the vacuum freeze-drying protectant for Bacillus velezensis.

[0012] Preferably, the mass fraction of the vacuum freeze-drying protectant for Bacillus velezensis is 8%.

[0013] An application of a vacuum freeze-drying protectant for Bacillus velezensis in microbial vacuum freeze-drying.

[0014] Preferably, the microorganism is Bacillus velezensis.

[0015] Preferably, the Bacillus velezensis is Bacillus velezensis XY40-1, deposited at the China Center for Type Culture Collection on March 29, 2022, with the deposit number CCTCC NO: M 2022342 and the deposit address: Wuhan University, Wuhan, China.

[0016] Advantages of the present invention:

[0017] The present invention discloses a vacuum freeze-drying protectant for Bacillus velezensis, wherein the vacuum freeze-drying protectant comprises the following components by mass: 100×10 3 -200×10 3 parts of skim milk powder, 260×10 3 -370×10 3 parts of mannitol, 50×10 3 -90×10 3 parts of sodium glutamate, 1-3 parts of PQQ, and 120×10 3 -230×10 3 parts of tryptone. Mixing the microorganism with the vacuum freeze-drying protectant can significantly improve the survival rate of the microorganism after vacuum freeze-drying, providing a basis for the wide application of functional microorganisms. Detailed implementation manners

[0018] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0019] The Bacillus velezensis XY40-1 involved in the present invention has a nucleotide sequence of 16S rRNA as shown in SEQ ID NO:1. It was isolated from the pepper leaves of the Xiangyan pepper base in Changsha City, Hunan Province. The Bacillus velezensis XY40-1 was deposited at the China Center for Type Culture Collection on March 29, 2022, with the deposit number CCTCC NO:M 2022342 and the deposit address: Wuhan University, China.

[0020] Fermentation medium or liquid medium (g / L): 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 15 g / L of agar powder. Dissolve in water and finally make up to 1000 mL, pH 7.0±0.2, autoclave (121°C, 30 min).

[0021] Example 1 Preparation of protectants with different components

[0022] Mix the protectants with different components according to the formula shown in Table 1.

[0023] Table 1 Combinations of protectants with different formulas

[0024]

[0025]

[0026] Example 2 Preparation of Microbial Lyophilized Preparation

[0027] (1) Seed liquid preparation. The activated strain was inoculated into 100 mL of liquid medium (inoculation amount 1%), and cultured in a tabletop constant temperature oscillator at 26 °C and 220 r / min for 48 h to obtain the seed liquid. Then the seed liquid was inoculated into 100 mL of liquid fermentation medium (inoculation amount 1%), and cultured under the same conditions for 96 h to obtain the bacterial liquid.

[0028] (2) Preparation of bacterial suspension. Take 1000 mL of the bacterial liquid in a centrifuge tube, centrifuge at 5000 r / min for 8 min at 4 °C, discard the supernatant, add physiological saline to supplement to the volume before centrifugation, repeat 3 times to obtain a clear supernatant, and finally suspend the bacterial sludge in physiological saline, and mix well by vortex oscillation.

[0029] (3) Preparation of cryoprotectant. The cryoprotectants in group numbers 1-11 in Table 1 were dissolved in distilled water according to the formula respectively to prepare a cryoprotectant solution with a concentration of 8% (mass fraction). After sterilization, it was mixed with the bacterial suspension.

[0030] (4) Vacuum freeze-drying. Add the previously prepared cryoprotectant to the bacterial suspension at a volume ratio of 1:1, remove the centrifuge tube cap, replace it with a sealing film, and pre-freeze at -80 °C for 4 h. After taking out the pre-frozen sample, quickly put it into the cold trap, and freeze-dry the sample with a vacuum freeze-dryer. After it completely becomes powdery, perform vacuum packaging and store it in a dry place for standby.

[0031] Example 2 Determination of the Survival Rate of Microorganisms after Vacuum Freeze-Drying

[0032] Dissolve the bacterial powder prepared in Example 1 in the liquid medium, count the bacteria after streak culture, and calculate the total viable bacteria number in the bacterial powder. Among them, the survival rate (%) = total viable bacteria number after vacuum freeze-drying / total viable bacteria number before vacuum freeze-drying × 100%. The results are shown in Table 2:

[0033] Table 2 Survival Rates of Bacillus velezensis under Different Cryoprotectant Treatments

[0034]

[0035] From the data of group numbers 1 - 4 in Table 2, it can be seen that on the basis of the basic formula (skim milk powder + mannitol + sodium glutamate), the combination of the present invention with PQQ can significantly improve the survival rate of Bacillus velezensis after vacuum freeze - drying compared with not using a vacuum freeze - drying protectant (group 12). The survival rate of group number 5 (using PQQ as a protectant) is 67.8%, the survival rate of group number 11 (using skim milk powder, mannitol, sodium glutamate and tryptone as protectants) is 72.0%, and the survival rate of group number 2 (using PQQ, skim milk powder, mannitol, sodium glutamate and tryptone as protectants) is 83.5%, indicating that the compounding of PQQ, skim milk powder, mannitol, sodium glutamate and tryptone has a synergistic effect.

[0036] Through a large number of experiments, the inventor of the present invention has explored a protectant that can significantly improve the protection of Bacillus velezensis after vacuum freeze - drying, providing a solid foundation for the application of Bacillus velezensis in biological control, drug research and development, food fermentation and industrial applications, etc.

Claims

1. Application of a vacuum freeze-drying protectant for Bacillus velezensis in vacuum freeze-drying Bacillus velezensis, characterized in that, the vacuum freeze-drying protectant for Bacillus velezensis comprises the following components by mass parts: 100×10³ - 200×10³ parts of skim milk powder, 260×10³ - 370×10³ parts of mannitol, 50×10³ - 90×10³ parts of sodium glutamate, 1 - 3 parts of PQQ, and 120×10³ - 230×10³ parts of tryptone; the preparation method of the vacuum freeze-drying protectant for Bacillus velezensis is: dissolving the components of the vacuum freeze-drying protectant for Bacillus velezensis in distilled water, sterilizing and cooling to obtain the vacuum freeze-drying protectant for Bacillus velezensis.

2. The application according to claim 1, characterized in that, the vacuum freeze-drying protectant for Bacillus velezensis is formulated into a protectant solution with a mass fraction of 8% for use.

3. The application according to claim 1, characterized in that, The Bacillus velezensis is Bacillus velezensis ( Bacillus velezensis ) XY40-1, and the preservation number is CCTCC NO: M 2022342.

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