Culture method of spore-producing functional bacteria

Abstract

The invention relates to the technical field of microorganisms, and provides a method for culturing spore-producing functional bacteria, which comprises the following steps: S1, activating a strain, and culturing the strain in a seed culture medium to obtain a seed solution; s2, inoculating the seed solution into a fermentation culture medium, and after fermentation culture, centrifugally collecting thalli to obtain the spore-producing functional bacteria, the strains comprise bacillus subtilis, trichoderma, streptomyces and rhodopseudomonas palustris, and the mass ratio of the bacillus subtilis to the trichoderma to the streptomyces to the rhodopseudomonas palustris is (4-6): 2: 1: 1. Through the technical scheme, the problems of low sporulation rate and low thallus biomass of sporulation functional bacteria in related technologies are solved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for culturing spore-producing functional bacteria. Background Art

[0002] With the growing demand for bio-clean energy and the high attention paid to sustainable development, the high-value utilization of biogas residues and liquids has become a key issue. However, the current large-scale production technology of microbial agents is not yet mature, and the product shelf life is short, which greatly limits its industrial production. In order to solve this problem, promote the development of bio-clean energy, increase farmers' benefits and achieve sustainable economic development, it is crucial to carry out research on the creation process of bioactive microbial agents for the high-value utilization of biogas residues and liquids. Therefore, the development of an efficient cultivation method for spore-producing functional bacteria has important practical significance and broad application prospects for improving the level of high-value utilization of biogas residues and liquids, promoting the development of bio-clean energy and sustainable agricultural development. Summary of the invention

[0003] The present invention provides a method for culturing spore-forming functional bacteria, which solves the problems of low spore production rate and low bacterial biomass of spore-forming functional bacteria in the related art.

[0004] The technical solution of the present invention is as follows: The present invention provides a method for culturing spore-forming functional bacteria, comprising the following steps: S1, after the bacteria are activated, they are cultured in a seed culture medium to obtain a seed solution; S2, inoculating the seed liquid into the fermentation medium, and collecting the bacterial cells by centrifugation after fermentation to obtain the spore-forming functional bacteria; The bacterial species include Bacillus subtilis, Trichoderma, Streptomyces, and Rhodopseudomonas palustris, and the mass ratio of the Bacillus subtilis, the Trichoderma, the Streptomyces, and the Rhodopseudomonas palustris is 4-6:2:1:1.

[0005] As a further technical solution, the seed culture medium includes the following components in weight percentage: yeast extract 0.5%~1.5%, peptone 0.1%~1%, (NH4)2SO4 0.1%~1%, NaCl 0.1%~1%, nutritional supplement 0.5%~2%, and the rest is purified water.

[0006] As a further technical solution, the seed culture medium includes the following components in weight percentage: 1% yeast extract, 0.5% peptone, 0.5% (NH4)2SO4, 0.5% NaCl, 1% nutritional supplement, and the rest is purified water.

[0007] As a further technical solution, the fermentation medium includes the following components in weight percentage: CMC-Na 0.5%~1.5%, yeast extract 0.5%~1.5%, peptone 0.5%~1.5%, NaCl 0.1%~1%, and the rest is purified water.

[0008] As a further technical solution, the fermentation medium includes the following components in weight percentage: CMC-Na 1%, yeast extract 1%, peptone 1%, NaCl 0.5%, and the rest is purified water.

[0009] As a further technical solution, the nutritional supplement includes microcrystalline cellulose, dextrin, ferrous sulfate, and amino acids, and the mass ratio of the microcrystalline cellulose, the dextrin, the ferrous sulfate, and the amino acids is 30:40:1:9~29.

[0010] In the present invention, the mass ratio of each nutritional supplement is reasonably adjusted to meet the needs of different strains, avoid the waste of nutrients, make the nutritional supplement most effectively utilized, and increase the spore production amount and bacterial biomass.

[0011] As a further technical solution, the amino acids include one or more of tryptophan, cysteine, and lysine.

[0012] As a further technical solution, the pH of the seed culture medium and the fermentation culture medium is 6.0, and they are sterilized at 121° C. for 15 minutes.

[0013] As a further technical solution, the fermentation temperature is 28-32° C., and the fermentation time is 5-7 days.

[0014] As a further technical solution, the stirring speed of the fermentation culture is 150~200r / min.

[0015] The working principle and beneficial effects of the present invention are: In the present invention, Bacillus subtilis, Trichoderma, Streptomyces and Rhodopseudomonas palustris are mixed and cultured to give play to the synergistic effect between different bacterial species, promote each other's growth through a symbiotic relationship, thereby increasing the biomass of the bacteria; and regulate the proportion of each bacterial species to ensure that each bacterial species has enough space and resources to produce spores in the mixed culture system, thereby improving the efficiency and quantity of spore production. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0017] In the following examples and comparative examples: Bacillus subtilis: content ≥ 100 billion (%), model 01-01, manufacturer Shandong Dehe Mingxing Biotechnology Co., Ltd. Trichoderma: Trichoderma harzianum, content ≥ 100 billion (%); model 01-03, manufacturer Shandong Dehe Mingxing Biotechnology Co., Ltd. Streptomyces: Streptomyces loucheri, content ≥ 100 (%); model 01-08, manufacturer Shandong Dehe Mingxing Biotechnology Co., Ltd.; Rhodopseudomonas palustris: limited content is 10 billion (%), the manufacturer is Shandong Xinzhuoyuan Biotechnology Co., Ltd.; The % in the culture medium composition refers to the mass percentage.

[0018] Example 1 S1. After activating Bacillus subtilis, Trichoderma, Streptomyces, and Rhodopseudomonas palustris, respectively, the cells of Bacillus subtilis, Trichoderma, Streptomyces, and Rhodopseudomonas palustris are taken from the activated culture medium, respectively, and added to a seed culture medium containing 0.5% yeast extract, 0.1% peptone, 0.1% (NH4)2SO4, 0.1%, NaCl, 0.5% nutritional supplement, and the rest purified water for cultivation to obtain Bacillus subtilis seed solution, Trichoderma seed solution, Streptomyces seed solution, and Rhodopseudomonas palustris seed solution, wherein the 0.5% nutritional supplement includes 0.195% microcrystalline cellulose, 0.26% dextrin, 0.0065% ferrous sulfate, and 0.0385% tryptophan; S2. Inoculate 12% of the seed liquid into a fermentation medium, the pH of the fermentation medium is 6.0, and sterilize at 121° C. for 15 min. The fermentation medium comprises the following components in weight percentage: 0.5% CMC-Na, 0.5% yeast extract, 0.5% peptone, 0.1% NaCl, and the remainder is purified water. Ferment at 28° C. for 7 days with a stirring speed of 150 r / min. After fermentation and cultivation, collect the bacteria by centrifugation to obtain spore-forming functional bacteria; wherein the 12% seed liquid comprises 4% Bacillus subtilis seed liquid, 4% Trichoderma seed liquid, 2% Streptomyces seed liquid, and 2% Rhodopseudomonas palustris seed liquid.

[0019] Example 2 S1. After activating Bacillus subtilis, Trichoderma, Streptomyces, and Rhodopseudomonas palustris, respectively, the cells of Bacillus subtilis, Trichoderma, Streptomyces, and Rhodopseudomonas palustris are taken from the activated culture medium, respectively, and added to a seed culture medium containing 1% yeast extract, 0.5% peptone, 0.5% (NH4)2SO4, 0.5% NaCl, 1% nutritional supplement, and the rest purified water for cultivation to obtain Bacillus subtilis seed solution, Trichoderma seed solution, Streptomyces seed solution, and Rhodopseudomonas palustris seed solution, wherein the 1% nutritional supplement includes 0.39% microcrystalline cellulose, 0.52% dextrin, 0.013% ferrous sulfate, and 0.077% cysteine; S2. Inoculate 12% of the seed liquid into a fermentation medium, the pH of the fermentation medium is 6.0, and sterilize at 121° C. for 15 min. The fermentation medium includes the following components in weight percentage: 1% CMC-Na, 1% yeast extract, 1% peptone, 0.5% NaCl, and the rest is purified water. Ferment at 30° C. for 6 days with a stirring speed of 175 r / min. After fermentation and cultivation, collect the bacteria by centrifugation to obtain spore-forming functional bacteria; wherein the 12% seed liquid includes 4% Bacillus subtilis seed liquid, 4% Trichoderma seed liquid, 2% Streptomyces seed liquid, and 2% Rhodopseudomonas palustris seed liquid.

[0020] Example 3 S1. After activating Bacillus subtilis, Trichoderma, Streptomyces, and Rhodopseudomonas palustris, respectively, the cells of Bacillus subtilis, Trichoderma, Streptomyces, and Rhodopseudomonas palustris are taken from the activated culture medium, respectively, and added to a seed culture medium containing 1.5% yeast extract, 1% peptone, 1% (NH4)2SO4, 1% NaCl, 2% nutritional supplement, and the rest purified water for cultivation to obtain Bacillus subtilis seed solution, Trichoderma seed solution, Streptomyces seed solution, and Rhodopseudomonas palustris seed solution, wherein the nutritional supplement 1% includes 0.78% microcrystalline cellulose, 1.04% dextrin, 0.026% ferrous sulfate, and 0.154% cysteine; S2. Inoculate 12% of the seed liquid into a fermentation medium, the pH of the fermentation medium is 6.0, and sterilize at 121° C. for 15 min. The fermentation medium includes the following components in weight percentage: 1.5% CMC-Na, 1.5% yeast extract, 1.5% peptone, 1% NaCl, and the rest is purified water. Ferment at 32° C. for 5 days with a stirring speed of 200 r / min. After fermentation and cultivation, collect the bacteria by centrifugation to obtain spore-forming functional bacteria; wherein the seed liquid 12% includes 4% Bacillus subtilis seed liquid, 4% Trichoderma seed liquid, 2% Streptomyces seed liquid, and 2% Rhodopseudomonas palustris seed liquid.

[0021] Example 4 Compared with Example 2, the difference of Example 4 is that the seed solution 12% in the fermentation medium includes 6% of Bacillus subtilis seed solution, 3% of Trichoderma seed solution, 1.5% of Streptomyces seed solution, and 1.5% of Rhodopseudomonas palustris seed solution.

[0022] Example 5 Compared with Example 2, the difference of Example 5 is that the seed liquid 12% in the fermentation medium includes 7.2% of Bacillus subtilis seed liquid, 2.4% of Trichoderma seed liquid, 1.2% of Streptomyces seed liquid, and 1.2% of Rhodopseudomonas palustris seed liquid.

[0023] Example 6 Compared with Example 2, the difference of Example 6 is that the seed liquid 12% in the fermentation medium includes 8% of Bacillus subtilis seed liquid, 2% of Trichoderma seed liquid, 1% of Streptomyces seed liquid, and 1% of Rhodopseudomonas palustris seed liquid.

[0024] Example 7 Compared with Example 5, the difference of Example 7 is that the nutritional supplement 1% includes 0.375% of microcrystalline cellulose, 0.5% of dextrin, 0.0125% of ferrous sulfate, and 0.1125% of cysteine.

[0025] Example 8 Compared with Example 5, the difference of Example 8 is that the nutritional supplement 1% includes 0.3% microcrystalline cellulose, 0.4% dextrin, 0.01% ferrous sulfate, and 0.29% cysteine.

[0026] Example 9 Compared with Example 5, the difference of Example 9 is that the nutritional supplement 1% includes 0.27% microcrystalline cellulose, 0.36% dextrin, 0.009% ferrous sulfate, and 0.361% cysteine.

[0027] Example 10 Compared with Example 9, Example 10 is different in that cysteine ​​is replaced by an equal amount of tryptophan.

[0028] Embodiment 11 Compared with Example 9, Example 11 is different in that cysteine ​​is replaced by an equal amount of lysine.

[0029] Comparative Example 1 Compared with Example 2, the difference in Comparative Example 1 is that 12% of the seed liquid in the fermentation medium is only Bacillus subtilis seed liquid.

[0030] Comparative Example 2 Compared with Example 2, the difference of Comparative Example 2 is that the seed liquid 12% in the fermentation medium includes 6% of Trichoderma seed liquid, 3% of Streptomyces seed liquid, and 3% of Rhodopseudomonas palustris seed liquid.

[0031] The microorganisms in the spore-forming functional bacteria fermentation medium prepared in Examples 1 to 11 and Comparative Examples 1 to 2 were tested according to the following method: 1. Spore count: Use the plate dilution coating method and observe the spore production rate of the test sample under an oil immersion microscope after malachite green staining.

[0032] 2. Bacterial biomass: The bacterial biomass of the test sample was obtained by plate dilution and spreading method and plate counting.

[0033] The test results are shown in the following table: Table 1 Performance test results of microorganisms in the spore-forming functional bacteria fermentation medium obtained in Examples 1 to 11 and Comparative Examples 1 to 2

[0034] Compared with Example 2, the seed liquid in the fermentation medium of Comparative Example 1 is only Bacillus subtilis seed liquid, and the seed liquid in the fermentation medium of Comparative Example 2 includes Trichoderma seed liquid, Streptomyces seed liquid, and Rhodopseudomonas palustris seed liquid. As a result, the spore production rate and bacterial biomass of Example 2 are greater than those of Comparative Example 1 and Comparative Example 2, indicating that when the fermentation medium contains Bacillus subtilis seed liquid, Trichoderma seed liquid, Streptomyces seed liquid, and Rhodopseudomonas palustris seed liquid at the same time, the microbial spore production rate and bacterial biomass in the spore-producing functional bacteria culture medium are more. Compared with Example 2, seed liquids of different mass ratios are added to the fermentation medium of Examples 4 to 6, and the spore production rate and bacterial biomass of Examples 4 and 5 are greater than those of Examples 2 and 6, indicating that when the mass ratio of Bacillus subtilis seed liquid, Trichoderma seed liquid, Streptomyces seed liquid, and Rhodopseudomonas palustris seed liquid in fermentation culture is 4 to 6:2:1:1, the microbial spore production rate and bacterial biomass in the spore-producing functional bacteria culture medium can be improved.

[0035] Compared with Example 5, Examples 7 to 9 added microcrystalline cellulose, dextrin, ferrous sulfate, and cysteine ​​in different mass ratios. As a result, the spore production rate and bacterial biomass of Examples 7 and 8 were greater than those of Examples 5 and 9, indicating that when the mass ratio of microcrystalline cellulose, dextrin, ferrous sulfate, and cysteine ​​was 30:40:1:9 to 29, the spore production rate and bacterial biomass of the microorganisms in the obtained spore-forming functional bacteria culture medium were higher.

[0036] Compared with Example 9, Examples 10 and 11 added different types of amino acids. As a result, the spore production rate and bacterial biomass of Example 9 were greater than those of Examples 10 and 11, indicating that when the amino acid added to the fermentation medium was cysteine, the spore production rate and bacterial biomass of the microorganisms in the obtained spore-forming functional bacteria culture medium were higher.

[0037] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for culturing spore-forming functional bacteria, characterized in that: The following steps are involved: S1, after the bacteria are activated, they are cultured in a seed culture medium to obtain a seed solution; S2, inoculating the seed liquid into the fermentation medium, and collecting the bacterial cells by centrifugation after fermentation to obtain the spore-forming functional bacteria; The bacterial species include Bacillus subtilis, Trichoderma, Streptomyces, and Rhodopseudomonas palustris, and the mass ratio of the Bacillus subtilis, the Trichoderma, the Streptomyces, and the Rhodopseudomonas palustris is 4-6:2:1:

1.

2. The method for culturing spore-forming functional bacteria according to claim 1, characterized in that: The seed culture medium comprises the following components in weight percentage: 0.5% to 1.5% yeast extract, 0.1% to 1% peptone, 0.1% to 1% (NH4)2SO4, 0.1% to 1% NaCl, 0.1% to 1% nutritional supplement, and the rest is purified water.

3. The method for culturing spore-forming functional bacteria according to claim 2, characterized in that: The seed culture medium comprises the following components in weight percentage: 1% yeast extract, 0.5% peptone, 0.5% (NH4)2SO4, 0.5% NaCl, 1% nutritional supplement, and the rest is purified water.

4. The method for culturing spore-forming functional bacteria according to claim 1, characterized in that: The fermentation medium comprises the following components in weight percentage: CMC-Na 0.5%-1.5%, yeast extract 0.5%-1.5%, peptone 0.5%-1.5%, NaCl 0.1%-1%, and the rest is purified water.

5. The method for culturing spore-forming functional bacteria according to claim 4, characterized in that: The fermentation medium comprises the following components in weight percentage: 1% CMC-Na, 1% yeast extract, 1% peptone, 0.5% NaCl, and the rest is purified water.

6. The method for culturing spore-forming functional bacteria according to claim 2, characterized in that: The nutritional supplement includes microcrystalline cellulose, dextrin, ferrous sulfate, and amino acids, and the mass ratio of the microcrystalline cellulose, the dextrin, the ferrous sulfate, and the amino acids is 30:40:1:9-29.

7. The method for culturing spore-forming functional bacteria according to claim 6, characterized in that: The amino acids include one or more of tryptophan, cysteine ​​and lysine.

8. The method for culturing spore-forming functional bacteria according to claim 1, characterized in that: The pH of the seed culture medium and the fermentation culture medium was 6.0, and they were sterilized at 121° C. for 15 min.

9. The method for culturing spore-forming functional bacteria according to claim 1, characterized in that: The fermentation temperature is 28-32° C., and the fermentation time is 5-7 days.

10. The method for culturing spore-forming functional bacteria according to claim 1, characterized in that: The stirring speed of the fermentation culture is 150-200 r / min.

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