CN110923179A

CN110923179A - Bacillus subtilis liquid high-density fermentation medium and culture method thereof

Info

Publication number: CN110923179A
Application number: CN202010028820.7A
Authority: CN
Inventors: 李昆仑; 李宝君; 赵晨; 赵林; 岳秋林; 苏乐; 孙欣
Original assignee: Shandong Chen Zhang Biotechnology Co Ltd; Hang Chen Bio Tech Ltd Jinan
Current assignee: Shandong Chen Zhang 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 subtilis and a fermentation technology for performing liquid high-density fermentation by using the bacillus subtilis. The high-density fermentation liquor with high spore rate is obtained through solid flat plate expanding culture, shake flask seed liquor preparation and fermentation tank liquid high-density fermentation. 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 subtilis liquid high-density fermentation medium and 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 subtilis and a method for performing high-density liquid 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 subtilis is an aerobic, spore-producing and rod-shaped gram-positive bacterium, is nontoxic and harmless to people and livestock, can produce various antibiotics and biological enzymes, and has broad-spectrum antibacterial activity and extremely strong stress resistance. In literature reports, a plurality of bacillus subtilis biocontrol strains with excellent properties are researched for preventing and treating diseases in greenhouses or fields, and have good preventing and treating effects on diseases of crops such as cucumbers, hot peppers, rice, wheat and the like.

The bacillus subtilis not only has wider application in the fermentation of biological fertilizers, but also has wider application in sewage treatment and the manufacture of feeds or fermentation beds, and is a multifunctional microorganism.

1. Can be mixed with various strains, and has important function in agricultural production;

2. municipal and industrial sewage treatment, industrial circulating water treatment, treatment of septic tanks, septic tanks and the like, treatment of livestock breeding animal waste and odor, manure treatment systems, treatment of garbage, manure pits, septic tanks and the like;

3. livestock raising, poultry, special animals and pets breeding, and aquaculture;

4. helping digestion and digestive tract probiotics.

In conclusion, the bacillus subtilis has good biocontrol and bacteriostasis effects, 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 subtilis 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:

preparing a seed solution: inoculating the bacillus subtilis preserved on the inclined plane into a conical flask filled with a liquid seed culture medium, wherein the liquid filling amount is 20%, and the bacillus subtilis is cultured at the constant temperature of 37 ℃ for 8-16 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 6-10, soybean meal is 40-80, corn starch is 25-50, manganese sulfate is 0.25-0.5, and the pH value is 7.0-8.0;

and (3) fermentation in a tank: culturing the seed solution for 8-16 hours, transferring the seed solution into a sterilized fermentation tank (the seed tank can be transferred according to the requirement and then transferred into a large fermentation tank) when the OD value is more than 30 (the sterilization condition is 121 ℃, and is 30 minutes), performing liquid fermentation, wherein the inoculation amount is 10%, the liquid loading amount is 70%, culturing the seed solution at the constant temperature of 37 ℃ for 24-36 hours at the maximum rotation speed of 300rpm, the ventilation ratio is gradually increased from the initial 0.5vvm to 1.5vvm, and the pH value is controlled to be more than 6.0; the fermentation medium comprises 2-6 parts of glucose, 20-40 parts of soybean meal, 25-50 parts of corn starch, 4-8 parts of corn steep liquor, 2-6 parts of sodium chloride, 2-6 parts of dipotassium hydrogen phosphate, 0.25-0.5 part of manganese sulfate and 1 part of defoaming agent in unit g/L (weight volume), and the pH value is 7.5;

in the process of fermentation tank culture, the pH is firstly reduced and then raised, the pH is controlled to be 6.0-7.5 within 12 hours, the pH is naturally raised after 12 hours, 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 invention has the following advantages:

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

2) The final fermentation level of the fermentation technology provided by the invention is more than 250 hundred million/ml, and belongs to the domestic leading level.

3) 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 subtilis bacterial quantity, good spore formation and industrial development value.

Detailed Description

The invention is described below by means 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 below generally represents weight volume percent, in g/L.

Example 1

Inoculating bacillus subtilis 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 subtilis 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 10 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 20, corn starch 25, manganese sulfate 0.25 and a defoaming agent 1 in unit g/L in terms of weight volume, and the pH value is 7.5;

culturing the seed tank fermentation liquor for 10 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 at the constant temperature of 37 ℃ for 24 hours, the rotating speed is 300rpm at most, the ventilation ratio is gradually increased from 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 20, corn starch 25, corn steep liquor 4, sodium chloride 2, dipotassium phosphate 2, manganese sulfate 0.25 and a defoaming agent 1 in a unit of g/L in terms of weight volume, and the pH value is 7.5;

the fermentation unit of the thallus is 2.62 multiplied by 10 by the national standard counting method¹⁰cfu/ml。

Example 2

In the same way as in example 1, the corn starch was changed to potato starch of the same quality.

After the fermentation is finished, the analysis result shows that the fermentation unit in the fermentation liquor is 2.78 multiplied by 10¹⁰cfu/ml。

Example 3

In the same manner as in example 1, the soybean meal was changed to the same quality.

After the fermentation is finished, the analysis result shows that the fermentation unit in the fermentation liquor is 2.28 multiplied by 10¹⁰cfu /ml。

Example 4

In the same manner as in example 1, all of the corn starch was replaced with glucose of equal mass.

After the fermentation is finished, the analysis result shows that the fermentation unit in the fermentation liquor is 8.7 multiplied by 10⁹cfu/ml, sporulation rate < 50%.

Claims

1. A bacillus subtilis liquid high-density fermentation culture medium and a culture method thereof are characterized in that a seed culture medium consists of the following components (unit g/L): 2-8 parts of glucose, 40-60 parts of soybean meal, 20-30 parts of corn starch, 0.2-0.5 part of manganese sulfate and 1 part of defoaming agent, wherein the pH value after the sterilization is 7.0-8.0.

2. A bacillus subtilis liquid high-density fermentation medium and a culture method thereof are characterized in that the fermentation medium consists of the following components (unit g/L): 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, 2-8 parts of dipotassium phosphate, 0.2-0.5 part of manganese sulfate and 1 part of defoaming agent, wherein the pH value after the sterilization is 7.0-8.0.

3. As described in claim 2, DO levels should be maintained at least 20% after the emergence of spores in the middle and late stages of fermentation.

4. As described in claim 2, the pH during the fermentation culture is not lower than 6.0.