CN110923180A

CN110923180A - Bacillus megaterium liquid high-density fermentation medium and supplementary culture method thereof

Info

Publication number: CN110923180A
Application number: CN202010028822.6A
Authority: CN
Inventors: 李昆仑; 李宝君; 赵晨; 赵林; 岳秋林; 苏乐; 孙欣
Original assignee: Shandong Zhuoran Biotechnology Co Ltd; Hang Chen Bio Tech Ltd Jinan
Current assignee: Shandong Zhuoran Biotechnology Co Ltd; Hang Chen Bio Tech Ltd Jinan
Priority date: 2020-01-11
Filing date: 2020-01-11
Publication date: 2020-03-27

Abstract

A bacillus megaterium and a fermentation technology for performing liquid high-density fermentation by using the bacillus megaterium, and greatly improving the fermentation level by supplementing bean pulp and corn starch hydrolysate. The high-density fermentation liquor with high spore rate is obtained through solid flat plate expanding culture, shake flask seed liquor preparation, fermentation tank liquid high-density fermentation and feeding processes. The method has the advantages of low cost, simple process and simple and convenient operation, and the obtained bacterial spores have high application value in the agricultural field.

Description

Bacillus megaterium liquid high-density fermentation medium and supplementary culture method thereof

Technical Field

The invention relates to bacillus and a method for performing high-density liquid fermentation by using the same, in particular to bacillus megatherium and a method for performing high-density liquid fermentation and supplementary fermentation by using the same.

Background

Bacillus is a ubiquitous dominant microbial population in soil microbiology. The strain has high stress resistance and antibacterial effect, and can be used for biological control of various soil-borne diseases. The bacillus successfully colonizes to the rhizosphere, the body surface or the body of the plant, competes with pathogenic bacteria for nutrient substances in the environment, and secretes antibacterial substances so as to inhibit the growth of the pathogenic bacteria, and the metabolite of the bacillus can also induce a plant defense system to jointly resist the invasion of pathogenic fungi, thereby achieving the biological control effect of plant diseases.

The bacillus megaterium is an aerobic, spore-producing and rod-shaped gram-positive bacterium, is nontoxic and harmless to people and livestock, and has 36 times of radiation resistance of spores of E.coli. The bacillus megaterium has good effect of degrading organic phosphorus in soil and is a common strain for producing biological organic fertilizer, so that the bacillus megaterium can be used for producing phosphorus bacterial fertilizer in agriculture. In recent years, researches and reports that bacillus megaterium is a biocontrol strain with a wider antibacterial spectrum and has prevention and treatment effects on pathogenic fungi and bacteria of different plants. As a functional bacterium, the compound can improve the breeding environment and reduce the occurrence of livestock and poultry diseases in livestock breeding. Meanwhile, the strain is also a common strain for preparing the water body treating agent.

In conclusion, the bacillus megaterium has good effects of phosphate solubilizing, field fertilizing, biocontrol and bacteriostasis, and can be used as a fertilizer additive to improve the biocontrol capability of crops and agricultural chemicals, reduce the use amount of chemical fertilizers and pesticides and reduce the damage of the agricultural chemicals to the ecological environment.

Disclosure of Invention

The invention aims to provide a fermentation method for carrying out high-density liquid fermentation of bacillus megaterium with high efficiency and low cost. The method is simple to operate, low in cost and high in feasibility.

The technical scheme of the invention is as follows:

the liquid fermentation stage is divided into two stages, wherein the first stage is a thallus natural growth stage, and the second stage is a material supplementing stage.

The natural growth stage of the thalli, the culture temperature is 37 ℃, and the rotating speed is 200 rpm. The composition of the used liquid culture medium is that the unit is g/L, glucose is 2-6, soybean meal is 40-80, corn starch is 25-30, corn steep liquor is 4-8, sodium chloride is 2-6, dipotassium phosphate is 2-6, manganese sulfate is 0.25, ferric trichloride is 0.01 and defoaming agent is 1, and the pH value is 7.5; and (3) a material supplementing stage, namely preparation of a material supplementing liquid: dissolving 30-70% of the material by 2-10 times of water, treating with high temperature amylase or high temperature saccharifying enzyme at 90-100 deg.C for 0.5-2 hr, and treating with alkaline protease at 30-60 deg.C for 0.5-2 hr. And after the enzymolysis is finished, centrifuging the enzymolysis liquid, taking the supernatant as a feed supplement liquid, and sterilizing for later use.

During the fermentation tank culture process, the pH value is firstly reduced and then increased. In the feeding stage, the OD value of fermentation liquor in the logarithmic growth phase is rapidly increased, the pH value is increased back to about 7 and is taken as a starting point, and the feeding time is preferably controlled to be 6-8 hours.

After the completion of the feeding, the culture is continued for about 12 hours according to the original parameters until the OD value is not increased any more and the spore rate is more than or equal to 90 percent, and the fermentation is finished.

The invention has the following advantages:

the invention provides a method for fermenting high-density liquid of bacillus megaterium, which has the advantages of simple operation, low cost and high operability and can meet the relevant requirements of industrial mass production.

The invention provides a method for stimulating growth and reproduction of bacillus megaterium thallus by enzymatic hydrolysate feeding operation in the middle and later stages of bacillus megaterium liquid fermentation, so as to realize rapid increase of the bacterial load.

The invention has the advantages of short fermentation period of about 24-36 hours, high fermentation speed, saving a large amount of manpower and material resources, low requirement on operation level, large bacillus megaterium quantity, good spore formation and industrial development value.

Detailed Description

The invention is described by way of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.

The numerical unitless of the formulations in the examples generally represents weight volume percent, in g/L.

Example 1:

inoculating bacillus megaterium preserved on a slant into a 1L triangular flask filled with a liquid seed culture medium, wherein the liquid loading capacity is 20%, and the bacillus megaterium is cultured at the constant temperature of 37 ℃ for 12 hours at the rotating speed of 200 rpm; the composition of the liquid culture medium is calculated by weight volume, the unit is g/L, wherein glucose is 6, soybean meal powder is 40, corn starch is 25, manganese sulfate is 0.25, and the pH value is 7.5; inoculating the seed liquid into a 50L seed tank after sterilization (sterilization conditions are 121 ℃ and 30 minutes), fermenting the liquid seeds, wherein the inoculation amount is 5 percent, the liquid loading amount is 60 percent, culturing at the constant temperature of 37 ℃ for 8-12 hours, rotating speed is 300rpm, the ventilation ratio is initially 0.5vvm, subsequently gradually increasing to 1.0vvm, and the pH is controlled to be more than 6.0; the fermentation medium comprises glucose 6, soybean meal powder 40, corn starch 25, manganese sulfate 0.25 and a defoaming agent 1 in unit of g/L in terms of weight volume, and the pH value is 7.5;

culturing the seed tank fermentation liquor for 8-12 hours, transferring the seed liquor into a sterilized 500L fermentation tank (under the sterilization condition of 121 ℃ and 30 minutes) when the OD value is more than 30, performing liquid fermentation, wherein the inoculum size is 10 percent, the liquid loading amount is 70 percent, culturing the seed liquor at the constant temperature of 37 ℃ for 24-36 hours, the rotating speed is 300rpm at most, the ventilation ratio is gradually increased from the initial 0.5vvm to 1.6vvm, and the pH value is controlled to be more than 6.0; the fermentation medium comprises glucose 2, soybean meal powder 40, corn starch 25, corn steep liquor 4, sodium chloride 2, dipotassium hydrogen phosphate 4, manganese sulfate 0.25, ferric trichloride 0.01 and a defoaming agent 1 in a unit of g/L in terms of weight volume, and the pH value is 7.5;

preparation of a feed liquid: dissolving 50% of the material by 9 times of water, treating with high temperature amylase or high temperature saccharifying enzyme at 90 deg.C for 1 hr, and treating with alkaline protease at 50 deg.C for 1 hr. And after the enzymolysis is finished, centrifuging the enzymolysis liquid, taking the supernatant as a feed supplement liquid, and sterilizing for later use.

The fermentation time is 6 hours, the pH value is raised back to 6.7, and the feeding is started to stimulate the growth of thalli and sporulation. After the completion of the feeding, the culture is continued for 10 hours according to the original parameters, the pH is controlled to be above 6.0, the culture is carried out until the OD value is not increased any more, the spore rate is more than or equal to 90%, and the fermentation is stopped.

The fermentation unit of the thallus is 1.12 multiplied by 10 by the national standard counting method¹⁰cfu/ml。

Example 2

In the same manner as in example 1, the corn starch was changed to potato starch of the same mass concentration, and the glucose was changed to sucrose of the same mass concentration during the feeding stage.

After the fermentation is finished, the analysis result shows that the fermentation unit in the fermentation liquor is 1.01 multiplied by 10¹⁰cfu/ml。

Example 3

The corn starch in the feeding phase was changed to the same quality of glucose as in example 1.

After the fermentation is finished, the analysis result shows that the fermentation unit in the fermentation liquor is 6.6 multiplied by 10⁹cfu/ml, sporulation rate < 80%.

Example 4

In the same way as in example 1, the amount of corn starch and soybean meal used in the feeding stage was changed to 30% of the amount of the base material.

After the fermentation is finished, the analysis result shows that the fermentation unit in the fermentation liquor is 8.7 multiplied by 10⁹cfu /ml。

Example 5

In the same way as in example 1, the starting point of the feeding phase was chosen such that the amount of corn starch and soybean meal was changed to 70% of the amount of the bottom material when the pH of the fermentation broth had risen to 7.5.

After the fermentation is finished, the analysis result shows that the fermentation unit in the fermentation liquor is 5.3 multiplied by 10⁹cfu/ml, sporulation rate < 50%.

Claims

1. A high-density fermentation culture medium of a bacillus megaterium liquid and a supplementary culture method thereof are characterized in that: the liquid fermentation is divided into two stages, wherein the first stage is a high-density viable bacteria natural growth stage, and the second stage is a stage for increasing the fermentation density by supplementing soybean meal and corn starch hydrolysate.

2. The process of claim 1, wherein the fermentation medium has a composition (in g/L) of: 2-4 parts of glucose, 40-60 parts of soybean meal, 20-30 parts of corn starch, 4-8 parts of corn steep liquor, 2-3 parts of sodium chloride, 4-8 parts of dipotassium phosphate, 0.25 part of manganese sulfate, 0.01 part of ferric trichloride and 1 part of defoaming agent;

in the feeding stage, as described in patent claim 1, in the logarithmic growth phase, the OD value of viable bacteria is rapidly increased, and the initial point is that the pH value is increased to about 7;

in the feeding stage, as described in patent claim 1, the material enzymolysis supernatant with 30-70% of the amount of the bottom material is fed in batches and is completed within 6-8 hours, so that the growth of the thallus is accelerated; after the completion of the feeding, the culture is continued according to the original parameters until the spore rate is more than or equal to 90 percent, and the fermentation is finished.

3. The logarithmic phase feed process of claim 3, characterized by: the raw material is one or a mixture of soybean meal, corn starch and potato starch;

the logarithmic phase feed process of claim 3, characterized by: the hydrolase is high-temperature amylase, high-temperature saccharifying enzyme, alkaline protease and the like.

4. The process of claim 5, wherein the enzymatic hydrolysis is carried out by dissolving the material in 5 to 10 volumes of water, treating with high temperature amylase or glucoamylase at 90 ℃ to 100 ℃ for 0.5 to 2 hours, and then treating with alkaline protease at 40 ℃ to 60 ℃ for 0.5 to 2 hours; and after the enzymolysis is finished, centrifuging the enzymolysis liquid, taking the supernatant as a feed supplement liquid for sterilization and standby.

5. As claimed in claim 3, DO is maintained at a level of 20% or more after mid-late stage of fermentation sporulation.

6. As stated in patent claim 3, the pH of the fermentation process should not be lower than 6.0.