CN105255785A - Fermentation method of bacillus megatherium with high rate of sporation - Google Patents

Abstract

The invention relates to a method for fermenting Bacillus megaterium with a high spore rate, comprising the following steps: (1) inoculating the Bacillus megaterium on a solid medium after being activated, and cultivating it statically to obtain secondary activated Bacillus megaterium Bacillus; (2) inoculate the Bacillus megaterium after the secondary activation in the seed medium, and ferment and cultivate it to obtain the seed bacterial liquid; (3) inoculate the seed bacterial liquid in the fermentation medium, ferment and cultivate, and add feed liquid, and continue to ferment to obtain Bacillus megaterium fermentation liquid; the present invention increases the number of live Bacillus megaterium bacteria by adding a spore-forming accelerator of Bacillus megaterium during the fermentation process through the selection of culture conditions and medium components. and spore rate, the number of viable bacteria in the prepared fermentation broth can reach more than 8.0×10 ⁹ CFU/ml, and the spore rate is more than 95% when observed by oil microscope after staining with crystal violet, which can significantly improve the number of spores and product quality of the product.

Description

A kind of fermentation method of Bacillus megaterium with high spore rate

technical field

The invention relates to a method for fermenting Bacillus megaterium with a high spore rate, and belongs to the technical field of microbial fermentation engineering.

Background technique

Bacillus megaterium (Bacillus megaterium) is a Gram-positive bacterium, a plant root growth-promoting bacterium, and a common bacterial species in microbial fertilizers. Bacillus megaterium can be used to produce phosphorus-dissolving, potassium-dissolving, and nitrogen-fixing fertilizers. It has the effect of degrading phosphorus and potassium in the soil that cannot be used by plants. Applying it to tobacco leaves has a unique effect on improving the fermentation and aroma of tobacco leaves. It is not only a common strain for the production of microbial fertilizers, but also a common strain for the production of water treatment agents. Bacillus megaterium has many benefits to agricultural production, so it has broad application prospects in agriculture. Bacillus is mainly a special dormant body produced by bacteria in the genus Bacillus and Clostridium. It has strong stress resistance and strong tolerance to various adverse conditions. Due to the extremely strong stress resistance of the spores, the bacillus has a good application prospect, especially in the use of live bacterial preparations, which shows strong vitality, and is an ideal form of existence for the preparation of microbial bacterial preparations.

Bacillus megaterium also has the characteristic of producing spores, but compared with microbial strains that are easy to form spores such as Bacillus amyloliquefaciens and Bacillus subtilis, there are still low viable bacteria, low sporulation rate, long fermentation cycle, and production costs. advanced questions. Moreover, the premature or late generation of spores will seriously affect the production of some target products and the acquisition of high-concentration bacteria.

Contents of the invention

The present invention aims at the deficiencies of the Bacillus megaterium fermentation technology commonly used in the existing microbial fertilizers, resulting in the problem that the spore yield is not high, and provides a Bacillus megaterium fermentation method with a high spore rate to improve the spore content of the product, product quality and use. Effect.

Technical scheme of the present invention is as follows:

A fermentation method of Bacillus megaterium with high spore rate, comprising the following steps:

(1) After activating Bacillus megaterium, inoculate it on a solid medium, and culture it statically at 34-37°C for 2-3 days to obtain Bacillus megaterium after secondary activation;

(2) Inoculate the Bacillus megaterium after the secondary activation obtained in step (1) in the seed culture medium, at a temperature of 34 to 37° C., and a dissolved oxygen value of 20 to 70 mg/L, and ferment and cultivate it for 14 to 20 hours to prepare get the seed bacterium liquid;

Described seed culture medium is made up of following components by mass percentage:

Sucrose 1.40-1.80%, soybean meal 1.30-1.90%, calcium carbonate 0.02-0.05%, sodium chloride 0.30-0.60%, dipotassium hydrogen phosphate 0.40-0.80%, potassium dihydrogen phosphate 0.20-0.40%, yeast extract 0.02-0.06 %, magnesium sulfate 0.02-0.05%, manganese sulfate 0.01-0.03%, bran 0.30-0.80%, foam enemy 0.01-0.02%, the balance is water, pH 6.5-7.5;

(3) Inoculate the seed bacterial liquid prepared in step (2) in the fermentation medium, at a temperature of 34-37° C., a dissolved oxygen value of 20-70 mg/L, and ferment and cultivate for 18-22 hours, according to the volume percentage of the fermentation liquid 20% to 25% of the nutrient solution was added, and the fermentation was continued for 24 to 32 hours to obtain a Bacillus megaterium fermentation liquid;

The fermentation medium is composed of the following components according to mass percentage: 1.60-2.00% bran, 1.50-2.00% soybean meal, 0.05-0.1% calcium carbonate, 0.30-0.60% sodium chloride, 0.40-0.80 dipotassium hydrogen phosphate %, potassium dihydrogen phosphate 0.20-0.40%, magnesium sulfate 0.02-0.05%, manganese sulfate 0.01-0.03%, starch 0.50-1.00%, foam enemy 0.01-0.02%, the balance is water, pH 6.5-7.5;

Described feed liquid, component is as follows

Calcium carbonate 2.0-5.0g/L, wheat flour 30-80g/L, the balance is water.

Preferably according to the present invention, in the step (1), the activation condition is: activation culture at 34-37° C. for 2-3 days.

Preferably according to the present invention, in said step (1), the activated activation medium has the following components:

Beef extract 0.20-0.50%, sodium chloride 0.30-0.80%, peptone 0.80-1.20%, agar 1.80-2.00%, the balance is water, pH7.0-7.5;

Preferably according to the present invention, in said step (1), the solid medium is composed of the following components by mass percentage:

Beef extract 0.20-0.50%, sodium chloride 0.30-0.80%, peptone 0.80-1.20%, agar 1.80-2.00%, the balance is water, pH7.0-7.5;

Preferably according to the present invention, said bacillus megaterium (Bacillus megaterium) is selected from:

China General Microorganism Culture Collection Management Center, culture preservation number CGMCC1.223; China Agricultural Microbiology Culture Collection Management Center, culture preservation number: ACCC02991; China Agricultural Microbiology Culture Collection Management Center, culture preservation number: ACCC03031; China Agricultural Microorganism Culture Collection Management Center, strain preservation number: ACCC03044; one of China Agricultural Microbiology Culture Collection Management Center, strain preservation number: ACCC03116.

Those skilled in the art can predict that because Bacillus megaterium has common biological characteristics, the same technical effect can also be achieved after being replaced by other Bacillus megaterium.

A spore-promoting agent for Bacillus megaterium, the components are as follows, all in parts by weight:

2-5 parts of calcium carbonate, 30-80 parts of wheat flour.

Preferably according to the present invention, the described spore formation accelerator has the following components, all in parts by weight:

3-4 parts of calcium carbonate, 40-70 parts of wheat flour.

The sterilization conditions of the culture medium and feed solution described in this application can all adopt the conventional sterilization conditions in the field: 121°C, 0.11Mpa sterilization for 20-30min.

Beneficial effect

In the present invention, through the selection of culture conditions and medium components, and at the same time adding a spore-forming accelerator of Bacillus megaterium during the fermentation process, the number of viable bacteria and the rate of spores of Bacillus megaterium are increased, and the number of viable bacteria in the prepared fermentation liquid is It can reach more than 8.0×10 ⁹ CFU/ml, and the spore rate is more than 95% when observed by oil microscope after staining with crystal violet, which can significantly improve the number of spores and product quality of the product.

detailed description

The technical scheme of the present invention will be further described below in combination with implementation, but the scope of protection of the present invention is not limited thereto.

Source of biological material:

The Bacillus megaterium described in Example 1 comes from the China Agricultural Microorganism Culture Collection and Management Center, and the culture preservation number is: ACCC02991.

The Bacillus megaterium described in Example 2 comes from the China Agricultural Microorganism Culture Collection and Management Center, and the culture preservation number is ACCC03031.

The Bacillus megaterium described in Example 3 comes from the China Agricultural Microorganism Culture Collection and Management Center, and the culture preservation number is ACCC03044.

The Bacillus megaterium described in Example 4 comes from the China Agricultural Microorganism Culture Collection and Management Center, and the culture preservation number is ACCC03116.

The Bacillus megaterium described in Example 5 is sourced from China General Microorganism Culture Collection and Management Center, and the culture preservation number is: CGMCC1.223.

Example 1

A fermentation method of Bacillus megaterium with high spore rate, comprising the following steps:

(1) inoculate the slant of Bacillus megaterium stored at -4°C on the slant medium by streaking, and activate and culture at 37°C for 2 days to obtain activated cells;

The activation medium is composed of the following components according to mass percentage: beef extract 0.30%, sodium chloride 0.50%, peptone 0.90%, agar 2.00%, the balance is water, pH 7.2;

Then the activated bacterium was washed with sterile physiological saline and inoculated on the solid medium in the eggplant-shaped culture bottle, and cultured at 37°C for 3 days to obtain the secondary activated Bacillus megaterium;

The solid medium is composed of the following components according to mass percentage: beef extract 0.30%, sodium chloride 0.50%, peptone 0.90%, agar 2.00%, the balance is water, pH7.2;

(2) Inoculate the Bacillus megaterium after the secondary activation obtained in step (1) in the seed culture medium, at a temperature of 37° C., a dissolved oxygen value of 20 to 70 mg/L, and an aeration ratio: the initial aeration ratio is 1: 0.5. When the dissolved oxygen value is lower than 20mg/L, increase the ventilation ratio to 1:1. When the dissolved oxygen value rises above 70mg/L, reduce the ventilation ratio. Stirring speed: start at 150rpm, adjust according to the dissolved oxygen value (the dissolved oxygen value increases, and the stirring speed is reduced; the dissolved oxygen value decreases, and the stirring speed is increased) the maximum stirring is no more than 200rpm, and the fermentation is carried out for 16 hours to obtain the seed bacterial liquid;

Described seed culture medium is made up of following components by mass percentage:

Sucrose 1.50%, soybean meal 1.40%, calcium carbonate 0.03%, sodium chloride 0.50%, dipotassium hydrogen phosphate 0.40%, potassium dihydrogen phosphate 0.20%, yeast extract 0.03%, magnesium sulfate 0.02%, manganese sulfate 0.02%, bran 0.30%, bubble enemy 0.01%, the balance is water, pH6.5～7.5;

(3) Inoculate the seed bacterial liquid prepared in step (2) in the culture medium, the inoculum volume ratio is 5%, at a temperature of 37°C, the dissolved oxygen value is 20-70mg/L, ferment and cultivate for 20 hours, press the fermentation broth 25% of the volume percentage is added with feeding liquid, and the fermentation is continued for 30 hours to obtain a Bacillus megaterium fermentation liquid;

The fermentation medium is composed of the following components according to mass percentage: 2.00% bran, 2.00% soybean meal, 0.1% calcium carbonate, 0.30% sodium chloride, 0.40% dipotassium hydrogen phosphate, 0.20% potassium dihydrogen phosphate, sulfuric acid Magnesium 0.02%, manganese sulfate 0.02%, starch 1.00%, foam enemy 0.01%, the balance is water, pH7.0;

The feeding liquid has the following components:

Calcium carbonate 5.0g/L, wheat flour 60g/L, the balance is water.

After the fermentation was finished, the fermentation culture liquid was collected to obtain a liquid product of Bacillus megaterium with a high spore rate. The number of live bacteria was 8.5×10 ⁹ CFU/ml detected by serial dilution and smearing plate method, and the spore rate was 95% under oil microscope observation after crystal violet staining.

Example 2

A fermentation method of Bacillus megaterium with high spore rate, comprising the following steps:

(1) inoculate the slant of Bacillus megaterium stored at -4°C on the slant medium by streaking, and activate and culture at 36°C for 2 days to obtain activated cells;

The activation medium is composed of the following components according to mass percentage: beef extract 0.20%, sodium chloride 0.30%, peptone 1.20%, agar 1.80%, the balance is water, pH 7.0;

Then the activated thallus was washed with sterile physiological saline and then inoculated on the solid medium in the eggplant-shaped culture bottle, and cultured at 36°C for 3 days to obtain the secondary activated Bacillus megaterium;

The solid medium is composed of the following components according to mass percentage: beef extract 0.50%, sodium chloride 0.30%, peptone 0.80%, agar 1.90%, the balance is water, pH 7.5;

(2) Inoculate the Bacillus megaterium after the secondary activation obtained in step (1) in the seed culture medium, at a temperature of 36° C., a dissolved oxygen value of 20 to 70 mg/L, and an aeration ratio: the initial aeration ratio is 1: 0.5. When the dissolved oxygen value is lower than 20mg/L, increase the ventilation ratio to 1:1. When the dissolved oxygen value rises above 70mg/L, reduce the ventilation ratio. Stirring speed: start at 150rpm, adjust according to the dissolved oxygen value (the dissolved oxygen value increases, and the stirring speed is reduced; the dissolved oxygen value decreases, and the stirring speed is increased) the maximum stirring is no more than 200rpm, and the fermentation is carried out for 16 hours to obtain the seed bacterial liquid;

Described seed culture medium is made up of following components by mass percentage:

Sucrose 1.80%, soybean meal 1.50%, calcium carbonate 0.04%, sodium chloride 0.30%, dipotassium hydrogen phosphate 0.60%, potassium dihydrogen phosphate 0.30%, yeast extract 0.03%, magnesium sulfate 0.02%, manganese sulfate 0.02%, bran 0.40%, bubble enemy 0.01%, the balance is water, pH6.8;

(3) Inoculate the seed bacterium solution prepared in step (2) in the culture medium, the inoculum volume ratio is 5%, at a temperature of 36°C, the dissolved oxygen value is 20-70 mg/L, and the fermentation is carried out for 18 hours, and the fermentation liquid is pressed. 25% of the volume percentage is added with feed liquid, and the fermentation is continued for 25 hours to obtain a Bacillus megaterium fermentation liquid;

The fermentation medium is composed of the following components by mass percentage: 1.40% bran, 1.80% soybean meal, 0.08% calcium carbonate, 0.30% sodium chloride, 0.40% dipotassium hydrogen phosphate, 0.20% potassium dihydrogen phosphate, sulfuric acid Magnesium 0.02%, manganese sulfate 0.02%, starch 1.00%, foam enemy 0.01%, the balance is water, pH7.0;

The feeding liquid has the following components:

Calcium carbonate 3.0g/L, wheat flour 40g/L, the balance is water.

After the fermentation was finished, the fermentation culture liquid was collected to obtain a liquid product of Bacillus megaterium with a high spore rate. The number of live bacteria was 8.6×10 ⁹ CFU/ml detected by the serial dilution method, and the spore rate was 96% when observed with an oil microscope after staining with crystal violet.

Example 3

A fermentation method of Bacillus megaterium with high spore rate, comprising the following steps:

(1) inoculate the slant of Bacillus megaterium stored at -4°C on the slant medium by streaking, and activate and culture at 35°C for 3 days to obtain activated cells;

The activation medium is composed of the following components according to mass percentage: beef extract 0.40%, sodium chloride 0.50%, peptone 1.00%, agar 1.80%, the balance is water, pH 7.0;

Then the activated bacteria were washed with sterile physiological saline and then inoculated on the solid medium in the eggplant-shaped culture bottle, and cultured at 35°C for 2 days to obtain the secondary activated Bacillus megaterium;

The solid medium is composed of the following components according to mass percentage: beef extract 0.40%, sodium chloride 0.50%, peptone 1.00%, agar 1.80%, the balance is water, pH 7.0;

(2) Inoculate the Bacillus megaterium after the secondary activation obtained in step (1) in the seed culture medium, at a temperature of 35° C., a dissolved oxygen value of 20 to 70 mg/L, and an aeration ratio: the initial aeration ratio is 1: 0.5. When the dissolved oxygen value is lower than 20mg/L, increase the ventilation ratio to 1:1. When the dissolved oxygen value rises above 70mg/L, reduce the ventilation ratio. Stirring speed: start at 150rpm, adjust according to the dissolved oxygen value (the dissolved oxygen value increases, and the stirring speed is reduced; the dissolved oxygen value decreases, and the stirring speed is increased) the maximum stirring is no more than 200rpm, and the fermentation is carried out for 18 hours to obtain the seed bacterial liquid;

Described seed culture medium is made up of following components by mass percentage:

Sucrose 1.60%, soybean meal 1.50%, calcium carbonate 0.02%, sodium chloride 0.50%, dipotassium hydrogen phosphate 0.40%, potassium dihydrogen phosphate 0.20%, yeast extract 0.04%, magnesium sulfate 0.02%, manganese sulfate 0.02%, bran 0.40%, bubble enemy 0.01%, the balance is water, pH7.5;

(3) Inoculate the seed bacterial liquid prepared in step (2) in the culture medium, the volume ratio of the inoculum is 5%, at a temperature of 35°C, the dissolved oxygen value is 20-70 mg/L, and the fermentation is carried out for 19 hours, and the fermentation liquid is pressed. 23% of the volume percentage is added with feed liquid, and the fermentation is continued until 28 hours to obtain a Bacillus megaterium fermentation liquid;

The fermentation medium is composed of the following components according to mass percentage: 1.80% of bran, 2.00% of soybean meal, 0.10% of calcium carbonate, 0.30% of sodium chloride, 0.60% of dipotassium hydrogen phosphate, 0.30% of potassium dihydrogen phosphate, sulfuric acid Magnesium 0.02%, manganese sulfate 0.02%, starch 0.80%, foam enemy 0.01%, the balance is water, pH7.0;

The feeding liquid has the following components:

Calcium carbonate 4.0g/L, wheat flour 50g/L, the balance is water.

After the fermentation was completed, the fermentation culture liquid was collected to obtain a liquid product of Bacillus megaterium with a high spore rate. The number of live bacteria was 8.0×10 ⁹ CFU/ml detected by serial dilution method, and the spore rate was 95% when observed with an oil microscope after staining with crystal violet.

Example 4

A fermentation method of Bacillus megaterium with high spore rate, comprising the following steps:

(1) inoculate the slant of Bacillus megaterium stored at -4°C on the slant medium by streaking, and activate and culture at 35°C for 2 days to obtain activated cells;

The activation medium is composed of the following components according to mass percentage: beef extract 0.20%, sodium chloride 0.40%, peptone 1.00%, agar 1.80%, the balance is water, pH7.2;

Then the activated bacteria were washed with sterile physiological saline and then inoculated on the solid medium in the eggplant-shaped culture bottle, and cultured at 35°C for 2 days to obtain the secondary activated Bacillus megaterium;

The solid medium is composed of the following components according to mass percentage: beef extract 0.20%, sodium chloride 0.40%, peptone 1.00%, agar 1.80%, the balance is water, pH7.2;

(2) Inoculate the Bacillus megaterium after the secondary activation obtained in step (1) in the seed culture medium, at a temperature of 35° C., a dissolved oxygen value of 20 to 70 mg/L, and an aeration ratio: the initial aeration ratio is 1: 0.5. When the dissolved oxygen value is lower than 20mg/L, increase the ventilation ratio to 1:1. When the dissolved oxygen value rises above 70mg/L, reduce the ventilation ratio. Stirring speed: start at 150rpm, adjust according to the dissolved oxygen value (the dissolved oxygen value increases, and the stirring speed is reduced; the dissolved oxygen value decreases, and the stirring speed is increased) the maximum stirring is no more than 200rpm, and the fermentation is carried out for 18 hours to obtain the seed bacterial liquid;

Described seed culture medium is made up of following components by mass percentage:

Sucrose 1.60%, soybean meal 1.30%, calcium carbonate 0.04%, sodium chloride 0.40%, dipotassium hydrogen phosphate 0.80%, potassium dihydrogen phosphate 0.40%, yeast extract 0.03%, magnesium sulfate 0.02%, manganese sulfate 0.02%, bran 0.30%, bubble enemy 0.01%, the balance is water, pH7.2;

(3) Inoculate the seed bacteria solution prepared in step (2) in the culture medium, the inoculum volume ratio is 5%, at a temperature of 35°C, the dissolved oxygen value is 20-70mg/L, ferment and cultivate for 20 hours, press the fermentation broth 20% of the volume percentage is added with feeding liquid, and the fermentation is continued for 30 hours to obtain a Bacillus megaterium fermentation liquid;

The fermentation medium is composed of the following components according to mass percentage: 1.90% bran, 1.60% soybean meal, 0.1% calcium carbonate, 0.30% sodium chloride, 0.50% dipotassium hydrogen phosphate, 0.25% potassium dihydrogen phosphate, sulfuric acid Magnesium 0.02%, manganese sulfate 0.02%, starch 1.00%, foam enemy 0.01%, the balance is water, pH7.0;

The feeding liquid has the following components:

Calcium carbonate 5.0g/L, wheat flour 70g/L, the balance is water.

After the fermentation was finished, the fermented liquid was collected to obtain a liquid product of Bacillus megaterium with a high spore rate. The number of live bacteria was 8.2×10 ⁹ CFU/ml detected by serial dilution method, and the spore rate was 98% when observed with an oil microscope after staining with crystal violet.

Example 5

A fermentation method of Bacillus megaterium with high spore rate, comprising the following steps:

(1) Inoculate the slant of Bacillus megaterium stored at -4°C on the slant medium by streaking, and activate and culture at 37°C for 3 days to obtain activated cells;

The activation medium is composed of the following components according to mass percentage: beef extract 0.25%, sodium chloride 0.50%, peptone 1.00%, agar 1.80%, the balance is water, pH 7.2;

Then the activated bacterium was washed with sterile physiological saline and inoculated on the solid medium in the eggplant-shaped culture bottle, and cultured at 37°C for 3 days to obtain the secondary activated Bacillus megaterium;

The solid medium is composed of the following components according to mass percentage: beef extract 0.20%, sodium chloride 0.40%, peptone 1.00%, agar 1.80%, the balance is water, pH7.2;

(2) Inoculate the Bacillus megaterium after the secondary activation obtained in step (1) in the seed culture medium, at a temperature of 37° C., a dissolved oxygen value of 20 to 70 mg/L, and an aeration ratio: the initial aeration ratio is 1: 0.5. When the dissolved oxygen value is lower than 20mg/L, increase the ventilation ratio to 1:1. When the dissolved oxygen value rises above 70mg/L, reduce the ventilation ratio. Stirring speed: start at 150rpm, adjust according to the dissolved oxygen value (the dissolved oxygen value increases, and the stirring speed is reduced; the dissolved oxygen value decreases, and the stirring speed is increased) the maximum stirring is no more than 200rpm, and the fermentation is carried out for 16 hours to obtain the seed bacterial liquid;

Described seed culture medium is made up of following components by mass percentage:

Sucrose 1.80%, soybean meal 1.90%, calcium carbonate 0.04%, sodium chloride 0.30%, dipotassium hydrogen phosphate 0.80%, potassium dihydrogen phosphate 0.40%, yeast extract 0.05%, magnesium sulfate 0.02%, manganese sulfate 0.02%, bran 0.30%, bubble enemy 0.01%, the balance is water, pH7.2;

(3) Inoculate the seed bacterium solution prepared in step (2) in the culture medium, the inoculum volume ratio is 5%, at a temperature of 37°C, the dissolved oxygen value is 20-70mg/L, and the fermentation is carried out for 22 hours. 23% of the volume percentage is added with feeding liquid, and the fermentation is continued until 32 hours to obtain a Bacillus megaterium fermentation liquid;

The fermentation medium is composed of the following components according to mass percentage: 2.00% of bran, 2.00% of soybean meal, 0.06% of calcium carbonate, 0.30% of sodium chloride, 0.60% of dipotassium hydrogen phosphate, 0.3% of potassium dihydrogen phosphate, sulfuric acid Magnesium 0.02%, manganese sulfate 0.02%, starch 0.80%, foam enemy 0.01%, the balance is water, pH7.0;

The feeding liquid has the following components:

Calcium carbonate 5.0g/L, wheat flour 60g/L, the balance is water.

After the fermentation was finished, the fermented liquid was collected to obtain a liquid product of Bacillus megaterium with a high spore rate. The number of live bacteria was 8.8×10 ⁹ CFU/ml detected by serial dilution method, and the spore rate was 98% when observed with an oil microscope after staining with crystal violet.

Comparative example 1

As described in Example 1, the difference is that no feed solution was added in step (3).

After the fermentation, the fermented liquid was collected to obtain a liquid product of Bacillus megaterium with a high spore rate. The number of live bacteria was 6.9×10 ⁹ CFU/ml detected by serial dilution method, and the spore rate was 85% when observed with an oil microscope after staining with crystal violet.

Comparative example 2

As described in Example 1, the difference is that the optimized medium described in Wang Jiwen’s paper “Optimization of Bacillus megaterium C2 spore-producing culture conditions” published in the 2014 China Agricultural Science Bulletin: bran 1.5%, soybean meal 1.5% %, sodium chloride 0.5%, calcium carbonate 0.017%, manganese sulfate 0.017%, magnesium sulfate 0.017%.

The above-mentioned optimized culture medium was used as the fermentation medium instead of the fermentation medium in Example 1. After the fermentation was completed, the fermentation liquid was collected, and the number of viable bacteria was detected by serial dilution method to be 5.2×10 ⁹ CFU/ml, and observed with an oil microscope after staining with crystal violet , The spore rate is 90%.

Result analysis

From the results of the above examples and comparative examples, it can be seen that the present invention can stimulate the formation of spores through the optimization of medium components and the addition of feed components, and can make the number of viable bacteria reach more than 8.0×10 ⁹ CFU/ml, The yield of spores exceeds 95%, which can significantly improve the number of spores and the quality of products.

Claims (7)

1. a fermentation method of the bacillus megaterium of high spore rate, is characterized in that, comprises the following steps: (1) Inoculate the Bacillus megaterium on a solid medium after activation, and culture it statically at 34-37°C for 2-3 days to obtain the second-activated Bacillus megaterium; (2) Inoculate the Bacillus megaterium after the secondary activation obtained in step (1) in the seed culture medium, at a temperature of 34 to 37° C., and a dissolved oxygen value of 20 to 70 mg/L, and ferment and cultivate it for 14 to 20 hours to prepare get the seed bacterium liquid; Described seed culture medium is made up of following components by mass percentage: Sucrose 1.40-1.80%, soybean meal 1.30-1.90%, calcium carbonate 0.02-0.05%, sodium chloride 0.30-0.60%, dipotassium hydrogen phosphate 0.40-0.80%, potassium dihydrogen phosphate 0.20-0.40%, yeast extract 0.02-0.06 %, magnesium sulfate 0.02-0.05%, manganese sulfate 0.01-0.03%, bran 0.30-0.80%, foam enemy 0.01-0.02%, the balance is water, pH 6.5-7.5; (3) Inoculate the seed bacterial liquid prepared in step (2) in the fermentation medium, at a temperature of 34-37° C., a dissolved oxygen value of 20-70 mg/L, and ferment and cultivate for 18-22 hours, according to the volume percentage of the fermentation liquid 20% to 25% of the nutrient solution was added, and the fermentation was continued for 24 to 32 hours to obtain a Bacillus megaterium fermentation liquid; The fermentation medium is composed of the following components according to mass percentage: 1.60-2.00% bran, 1.50-2.00% soybean meal, 0.05-0.1% calcium carbonate, 0.30-0.60% sodium chloride, 0.40-0.80 dipotassium hydrogen phosphate %, potassium dihydrogen phosphate 0.20-0.40%, magnesium sulfate 0.02-0.05%, manganese sulfate 0.01-0.03%, starch 0.50-1.00%, foam enemy 0.01-0.02%, the balance is water, pH 6.5-7.5; The feeding liquid has the following components: Calcium carbonate 2.0-5.0g/L, wheat flour 30-80g/L, the balance is water. 2. The fermentation method according to claim 1, characterized in that, in the step (1), the activation condition is: activation culture at 34-37° C. for 2-3 days. 3. fermentation method as claimed in claim 1, is characterized in that, in described step (1), the activation culture medium of activation, component is as follows: Beef extract 0.20-0.50%, sodium chloride 0.30-0.80%, peptone 0.80-1.20%, agar 1.80-2.00%, the balance is water, pH 7.0-7.5. 4. fermentation method as claimed in claim 1, is characterized in that, in described step (1), solid culture medium is made up of following components by mass percentage: Beef extract 0.20-0.50%, sodium chloride 0.30-0.80%, peptone 0.80-1.20%, agar 1.80-2.00%, the balance is water, pH 7.0-7.5. 5. fermentation method as claimed in claim 1, is characterized in that, described bacillus megaterium (Bacillus megaterium) is selected from: China General Microorganism Culture Collection Management Center, culture preservation number CGMCC1.223; China Agricultural Microbiology Culture Collection Management Center, culture preservation number: ACCC02991; China Agricultural Microbiology Culture Collection Management Center, culture preservation number: ACCC03031; China Agricultural Microorganism Culture Collection Management Center, strain preservation number: ACCC03044; one of China Agricultural Microbiology Culture Collection Management Center, strain preservation number: ACCC03116. 6. A spore-forming accelerator of Bacillus megaterium, characterized in that, the components are as follows, all in parts by weight: 2-5 parts of calcium carbonate, 30-80 parts of wheat flour. 7. The spore-forming accelerator as claimed in claim 6, wherein the components are as follows, all in parts by weight: 3-4 parts of calcium carbonate, 40-70 parts of wheat flour.