A microbial vacuum freeze-drying protective agent and its preparation method and application

Abstract

The present invention relates to the technical field of microbial vacuum freeze drying protective agents, and in particular to a colorless bacillus protective agent, a preparation method and an application thereof. The present invention discloses a microbial vacuum freeze drying protective agent, comprising the following components in parts by mass: 5×10 3 ‑9×10 3 Sucrose, 12×10 3 ‑21×10 3 Dextrin, 4×10 3 ‑8×10 3 The vacuum freeze drying protective agent of the technical solution of the present invention can significantly improve the survival rate of Achromobacter, and the survival rate of Achromobacter after vacuum freeze drying can reach up to 89.4%, providing a basis for the wide application of Achromobacter.

Description

Technical Field

[0001] The invention relates to the technical field of microbial vacuum freeze-drying protective agents, and in particular to a colorless bacillus protective agent, a preparation method and an application thereof. Background Art

[0002] The vacuum freeze-drying technology was first invented by the British Wollaston in 1813. In 1909, Shsckell used this method to freeze-dry antitoxins, bacteria, rabies virus and other biological products, and achieved good results. During World War II, the huge demand for blood products greatly stimulated the development of vacuum freeze-drying technology, and since then, the technology has entered the stage of industrial application. Since then, the rapid development of refrigeration and vacuum equipment has provided strong material conditions for the rapid development of vacuum freeze-drying technology. The factors that affect the effect of vacuum freeze-drying of microorganisms are as follows: (1) the type and quality of bacteria; (2) protective agent; (3) drying speed; (4) the influence of air; (5) the influence of temperature; (6) the influence of water content.

[0003] Due to the influence of various factors in nature, the effect of a single bacterial agent is usually not very stable in a complex natural environment. Compound biocontrol agents can effectively make up for the shortcomings of a single strain and improve their efficacy.

[0004] The Chinese invention patent "A composite denitrification and denitrification agent, its preparation method and application" (CN107488621A) discloses a protective agent for a composite denitrification and denitrification agent (containing Achromobacter): β-cyclodextrin, maltodextrin and carboxymethyl cellulose. The Chinese invention patent "Compound microbial agent for chemical wastewater treatment and its screening and preparation method" (CN109402016A) discloses a strain protective agent for Achromobacter denitrificans: Tween-20, xanthan gum and glycerol. The Chinese invention patent "A special fertilizer for selenium-rich vegetables and its preparation method" (CN113372147A) discloses a vacuum freeze-dried protective agent for Achromobacter denitrificans: the protective agent includes the following components: 8-12 mg / L skim milk, 5-8 mg / L fructose and 0.05-0.1 mg / L poly-γ-glutamic acid. The Chinese invention patent "A highly efficient formaldehyde degrading bacterial agent, its preparation and application" (CN113773995A) discloses a protective agent for achromobacterium that degrades formaldehyde: 35% yeast powder; 35% fish protein; 30% trace elements.

[0005] Existing results show that after Achromobacter is mixed with a protective agent, the survival rate of the bacteria is not high after vacuum freeze-drying. Summary of the invention

[0006] In order to solve the technical problem in the prior art that the survival rate and activity of Achromobacter are low after vacuum freeze drying, the present invention provides the following technical solution:

[0007] A microbial vacuum freeze-drying protective agent, comprising PQQ.

[0008] Preferably, the microorganism vacuum freeze-drying protective agent further comprises sucrose, dextrin and sodium glutamate.

[0009] Preferably, the microbial vacuum freeze-drying protective agent comprises the following components by mass: 52×10 3 -93×10 3 Sucrose, 121×10 3 -215×10 3 Dextrin, 45×10 3 -85×10 3 parts of monosodium glutamate and 2-6 parts of PQQ.

[0010] Preferably, the method comprises: dissolving the components of the microbial vacuum freeze-drying protective agent in distilled water, sterilizing and cooling to obtain the vacuum freeze-drying protective agent.

[0011] Preferably, the mass fraction of the microorganism vacuum freeze-drying protective agent is 8%.

[0012] The invention discloses an application of the microorganism vacuum freeze drying protective agent in microorganism vacuum freeze drying.

[0013] Preferably, the microorganism is at least one of Achromobacter, Enterobacter and Pseudomonas.

[0014] Preferably, the ratio of the number of live bacteria of Achromobacter, Enterobacter and Pseudomonas in the microorganisms is 4:4:2.

[0015] Preferably, the colorless bacterium is Achromobacter insuavis SL8, deposited in the China Center for Type Culture Collection on March 18, 2022, with a deposit number of CCTCC NO: M 2022286; the Enterobacter is Enterobacter cancerogenus SL12, with a deposit number of CCTCC NO: M2022288, deposited in the China Center for Type Culture Collection (CCTCC) on March 18, 2022, and the deposit address is: Wuhan, China. Wuhan. Wuhan University; the Pseudomonas is Pseudomonas sp. NTO4, with a deposit number of CCTCC NO: M 20221479, a deposit date of September 23, 2022, and a deposit address of: Wuhan, China. Wuhan. Wuhan University.

[0016] Beneficial effects of the present invention:

[0017] The present invention obtains a vacuum freeze-drying protective agent capable of significantly improving the survival rate of microorganisms by compounding PQQ with sucrose, dextrin and sodium glutamate. By adopting the technical scheme of the present invention, the survival rate of the microorganisms after vacuum freeze-drying can reach up to 89.4%, thus saving costs and processing functional microorganisms into bacterial powder through freeze-drying. DETAILED DESCRIPTION

[0018] In order to make the purpose, 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 combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] The present invention relates to Achromobacter insuavis SL8, whose 16SrRNA nucleotide sequence is shown in SEQ ID NO: 1, and Enterobacter cancerogenus SL12, whose 16SrRNA nucleotide sequence is shown in SEQ ID NO: 2. The preservation number of Enterobacter cancerogenus SL12 is CCTCC NO: M2022288; the preservation number of Achromobacter insuavis SL8 is CCTCC NO: M 2022286. The above two strains of microorganisms were deposited in the China Center for Type Culture Collection (CCTCC) on March 18, 2022, and the preservation address is: Wuhan University, Wuhan, China.

[0020] The deposit number of Pseudomonas sp. NTO4 is CCTCC NO:M20221479, the nucleotide sequence of its 16S rRNA is shown in SEQ ID NO:3, the deposit date is September 23, 2022, and the deposit address is: Wuhan University, Wuhan, China.

[0021] Liquid fermentation medium or liquid culture medium (g / L): 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder, dissolve in water, and finally make up to 1000 mL, pH 7.0 ± 0.2, and sterilize under high pressure (121°C, 30 min).

[0022] Example 1 Configuration of protective agents of different components

[0023] The protective agents of different components were mixed according to the formula shown in Table 1.

[0024] Table 1 Combinations of protective agents with different formulations

[0025] Group No. Protective agent formula 1 10 parts sucrose + 26 parts dextrin + 5 parts sodium glutamate 2 <![CDATA[52×10 3 Sucrose, 121×10 3 Dextrin, 45×10 3 1 part sodium glutamate and 2 parts PQQ]]> 3 <![CDATA[93×10 3 Sucrose, 215×10 3 dextrin, 85×10 3 1 part sodium glutamate and 6 parts PQQ]]> 4 <![CDATA[60×10 3 Sucrose, 150×10 3 Dextrin, 60×10 3 1 part sodium glutamate and 4 parts PQQ]]> 5 PQQ (500nmol / L) 6 Maltodextrin 7 Tween-20 8 12mg / L skim milk, 8mg / L fructose and 0.1mg / L poly-gamma-glutamic acid 9 2 parts sucrose + 3 parts dextrin + 3 parts sodium glutamate 10 Yeast powder 35%; fish protein 35%; trace elements 30% 11 -(Distilled water control) 12 52 parts sucrose, 121 parts dextrin, 45 parts sodium glutamate

[0026] Example 2 Preparation of Freeze-dried Microbial Preparations

[0027] (1) Preparation of seed solution. The activated strain (Achromobacter SL8 and its combination with other microorganisms) was inoculated into 100 mL of liquid culture medium (inoculation amount 1%), and cultured in a desktop constant temperature oscillator at 26°C and 220 r / min for 48 hours to obtain seed solution. The seed solution was then inoculated into 100 mL of liquid fermentation medium (inoculation amount 1%), and cultured under the same conditions for 96 hours to obtain bacterial solution.

[0028] (2) Prepare bacterial suspension. Take 1000 mL of bacterial suspension in a centrifuge tube and centrifuge at 5000 r / min for 8 min at 4°C. Discard the supernatant and add physiological saline to the volume before centrifugation. Repeat this process three times to obtain a clear supernatant. Finally, suspend the bacterial sludge in physiological saline and mix it thoroughly by vortexing.

[0029] (3) Preparation of protective agent. The protective agents 1-10 and 12 in Table 1 were dissolved in distilled water according to the formula to prepare a protective agent solution with a concentration of 8% (mass fraction), and after sterilization, mixed with the bacterial suspension (the mixing of the protective agent and the bacterial suspension was repeated 3 times).

[0030] (4) Vacuum freeze drying. Add the protective agent prepared in advance 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 it at -80℃ for 4 hours. After taking out the pre-frozen sample, quickly put it into a freezing trap and freeze-dry it with a vacuum freeze dryer. After it is completely powdered, vacuum pack it and store it in a dry place for later use.

[0031] Example 2 Determination of the survival rate of microorganisms after vacuum freeze drying

[0032] The bacterial powder prepared in Example 1 was dissolved in LB liquid culture medium, and the bacteria were counted after streaking culture, and the total number of viable bacteria in the bacterial powder was calculated. Among them, the survival rate (%) = the total number of viable bacteria after vacuum freeze drying / the total number of viable bacteria before vacuum freeze drying × 100%. The results are shown in Table 2:

[0033] Table 2 Survival rate of microorganisms under different protective agents

[0034]

[0035] Note: In Table 2, the microorganisms in group numbers 1, 5-11 are Achromobacter; group numbers 2 and 12 are mixtures of Achromobacter, Enterobacter and Pseudomonas (4:4:2); group number 3 is a mixture of Achromobacter and Pseudomonas (4:2); group number 4 is a mixture of Achromobacter and Enterobacter (1:1).

[0036] From the data of group 5 in Table 2, it can be seen that the survival rate of microorganisms in the vacuum freeze-drying protective agent containing PQQ reached 65.3%; the survival rate of microorganisms in the vacuum freeze-drying protective agent containing sucrose, dextrin and sodium glutamate reached 80.3%; the data of group numbers 2-4 show that compounding PQQ with sucrose, dextrin and sodium glutamate can significantly increase the loss rate of microorganisms in the freeze-drying process and improve the crude survival rate.

[0037] The data from groups 6-10 and 12 showed that the use of maltodextrin, Tween-20, skim milk, skim milk, fructose, poly-γ-glutamic acid, yeast powder, fish protein, and 30% trace elements, compared with not using a vacuum freeze-drying protective agent (group 11), can also improve the survival rate of Colorless Bacillus after vacuum freeze-drying to a certain extent, but compared with the data from groups 1-5, the effect of improving the survival rate after vacuum freeze-drying is not good.

[0038] The inventors have obtained a protective agent through a large number of experiments, which can significantly improve the survival rate of Achromobacter and its compound bacteria after vacuum freeze drying, and provide a basis for the wide application of Achromobacter and its compound bacteria.

Claims

1. An application of a microbial vacuum freeze drying protective agent in microbial vacuum freeze drying, characterized in that: The microbial vacuum freeze drying protective agent comprises the following components by mass: 52×10 3 -93×10 3 Sucrose, 121×10 3 -215×10 3 Dextrin, 45×10 3 -85×10 3 1 part sodium glutamate and 2-6 parts PQQ; The preparation method of the microorganism vacuum freeze-drying protective agent is as follows: dissolving the components of the microorganism vacuum freeze-drying protective agent in distilled water, sterilizing and cooling to obtain the microorganism vacuum freeze-drying protective agent; The microorganism is at least one of Achromobacter, Enterobacter and Pseudomonas.

2. The use according to claim 1, characterized in that: The microorganism vacuum freeze-drying protective agent is configured to be used as a protective agent solution with a mass fraction of 8%.

3. The use according to claim 1, characterized in that: The ratio of the number of live bacteria of Achromobacter, Enterobacter and Pseudomonas in the microorganisms is 4:4:

2.

4. The use according to claim 1, characterized in that: The achromobacter is achromobacter ( Achromobacter insuavis )SL8, the deposit number is CCTCC NO:M 2022286; the Enterobacter is Enterobacter ( Enterobacter cancerogenus )SL12, deposit number: CCTCC NO:M 2022288; the Pseudomonas is Pseudomonas ( Pseudomonas sp. )NTO4, the deposit number is CCTCC NO:M 20221479.

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