CN115386516A - Method for producing microbial agent by using sugar-making waste honey - Google Patents
Method for producing microbial agent by using sugar-making waste honey Download PDFInfo
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- CN115386516A CN115386516A CN202210965145.XA CN202210965145A CN115386516A CN 115386516 A CN115386516 A CN 115386516A CN 202210965145 A CN202210965145 A CN 202210965145A CN 115386516 A CN115386516 A CN 115386516A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/07—Bacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/11—Bacillus megaterium
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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Abstract
The invention relates to the technical field of recycling of sugar making waste honey, in particular to a method for producing a microbial agent by using sugar making waste honey, which comprises the following steps: s1, strain activation: selecting Paenibacillus mucilaginosus, bacillus laterosporus, bacillus megaterium, bacillus mucilaginosus and Saccharomyces cerevisiae to be respectively activated; s2, amplifying the strains activated by the S1; s3, mixing the strains subjected to amplification in the S2 in proportion to prepare an initial mixed microbial inoculum; s4, preparing a culture medium by using the sugar-making waste honey; and S5, putting the initial mixed microbial inoculum in the S3 into a culture medium of the S4 for fermentation to obtain a microbial inoculum. The invention can prepare the microbial agent which is beneficial to nitrogen fixation, purification and absorption of heavy metals in soil and improvement of soil texture, and improves the quality of crops.
Description
Technical Field
The invention relates to the technical field of recycling of sugar refining waste honey, in particular to a method for producing a microbial agent by using sugar refining waste honey.
Background
The sugar-making waste honey contains a large amount of various sugars and proteins, is a cheap and excellent raw material in the fermentation brewing industry, such as various minerals, vitamins and amines, and is an ideal compound raw material for the fermentation of microorganisms. At present, main products obtained by fermenting waste molasses at home and abroad comprise: alcohol, citric acid and monosodium glutamate are produced in bulk. In addition, molasses can be used for producing useful products such as yeast, glycerol lactic acid, acetone, butanol, fumaric acid, butanediol, butyric acid and lysine by fermentation.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for producing a microbial agent by utilizing sugar-making waste honey, which can be used for preparing the microbial agent which is beneficial to nitrogen fixation, purification and absorption of heavy metals in soil, improvement of soil texture and improvement of crop quality.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for producing microbial inoculum by using sugar refining waste honey comprises the following steps:
s1, strain activation: selecting Paenibacillus mucilaginosus, bacillus laterosporus, bacillus megaterium, bacillus mucilaginosus and Saccharomyces cerevisiae to be respectively activated;
s2, amplifying the activated strain of the S1;
s3, mixing the strains subjected to amplification in the S2 in proportion to prepare an initial mixed microbial inoculum;
s4, preparing a culture medium by using the sugar-making waste honey;
and S5, putting the initial mixed microbial inoculum in the S3 into a culture medium of the S4 for fermentation to obtain a microbial inoculum.
Preferably, the mixing ratio of each strain in step S2 is as follows: 25 to 35 percent of paenibacillus mucilaginosus, 27 to 32 percent of bacillus laterosporus, 15 to 25 percent of bacillus megaterium, 8 to 14 percent of bacillus mucilaginosus and 6 to 12 percent of saccharomyces cerevisiae.
Preferably, the culture medium in step S4 is composed of the following raw materials in parts by weight: 80 to 120 parts of dry cow dung, 30 to 40 parts of coal cinder, 40 to 60 parts of crushed straw, 1 to 3 parts of urea, 2 to 4 parts of gypsum, 1 to 2 parts of sugar waste honey and a proper amount of water.
Preferably, the fermentation time of the strain in the culture medium in step S5 is 7 to 8 days.
Preferably, at least two amplification media are used for amplification culture in step S2.
The invention has the beneficial effects that:
the invention can prepare the microbial agent which is beneficial to nitrogen fixation, purification and absorption of heavy metals in soil and improvement of soil texture, and improves the quality of crops.
Detailed Description
The present invention will be further described with reference to the following examples for facilitating understanding of those skilled in the art, and the description of the embodiments is not intended to limit the present invention.
Example 1
And taking out the paenibacillus jelly, the bacillus laterosporus, the bacillus megaterium, the bacillus mucilaginosus and the saccharomyces cerevisiae which are preserved in the glycerol freezing tube from the refrigerator, melting in an ultra-clean workbench sterilized by ultraviolet, and aseptically sucking 1ml of the bacillus mucilaginosus and inoculating the bacillus mucilaginosus into 10ml of the sterilized liquid seed activation culture medium by a liquid transfer gun.
The culture medium for activating the paenibacillus mucilaginosus comprises, by mass, 0.9% of starch, 0.4% of sugar-making waste honey, 0.5% of dipotassium hydrogen phosphate, 0.5% of magnesium sulfate, 0.2% of bean cake powder, 0.1% of zinc sulfate and a proper amount of water, the pH value of the culture medium is adjusted to 7.0, and the culture medium is sterilized at 115 ℃ for 20 minutes and then is used for activating the paenibacillus mucilaginosus.
The culture medium for activating the bacillus laterosporus is a nutrient agar culture medium, the pH value of the culture medium is adjusted to 7.0, and the culture medium is sterilized for 20 minutes at the temperature of 115 ℃ and then is used for activating the bacillus laterosporus.
The culture medium for activating the bacillus megaterium is LB slant culture medium, and the LB slant culture medium is sterilized for 15 minutes at 120 ℃ and then activated by the bacillus megaterium.
The components of the culture medium for activating the saccharomyces cerevisiae comprise 1.0 percent of yeast extract, 2.0 percent of peptone and 2 percent of sucrose in percentage by mass, and the culture medium is sterilized at the temperature of 115 ℃ for 20 minutes and then is used for activating the saccharomyces cerevisiae.
Example 2
Inoculating Paenibacillus mucilaginosus into a liquid seed activation culture medium, and then activating for 12 hours at the temperature of 37 ℃; inoculating Bacillus pumilus to liquid seed activating culture medium, and activating at 37 deg.c for 12 hr; after bacillus megaterium is inoculated into a liquid seed activation culture medium, activating for 12 hours at the temperature of 32 ℃; after the saccharomyces cerevisiae is inoculated into the liquid seed activation culture medium, the activation is carried out for 24 hours at the temperature of 30 ℃.
1mL of each activated strain was aspirated under aseptic conditions, and inoculated into 50mL of amplification medium for primary culture. Culturing Paenibacillus mucilaginosus and Bacillus pumilus for about 12 hours at 37 ℃ and 50 rpm; culturing the bacillus megaterium at 35 ℃ and 200rpm for about 12 hours; saccharomyces cerevisiae was cultured at 30 ℃ and 50rpm for 18 hours. Wherein the amplification medium for the first-stage culture of the paenibacillus jelly and the bacillus pumilus lateral spore comprises the following components: 1 percent of sugar-making waste honey, 0.5 percent of yeast extract and 1 percent of sodium chloride, and the PH value is adjusted to 7.0; the amplification medium for the primary culture of the saccharomyces cerevisiae comprises the following components: 2 percent of sugar-making waste honey and 1 percent of yeast extract. The amplification medium was sterilized at 115 ℃ for 20 minutes and then used for the first-stage culture.
Inoculating the seed solution obtained by the primary culture into a fresh amplification culture medium by the inoculation amount of 2% for secondary culture, and culturing the paenibacillus mucilaginosus and the bacillus pumilus for about 10 hours at the conditions of 37 ℃ and 200 rpm; culturing the bacillus megaterium at 30 ℃ and 100rpm for about 10 hours; saccharomyces cerevisiae was cultured at 200rpm at 30 ℃ for 15 hours. Wherein the amplification medium for the secondary culture of the paenibacillus jelly and the bacillus pumilus laterosporus comprises the following components: 1 percent of sugar-making waste honey, 0.5 percent of yeast extract and 1 percent of sodium chloride, and the PH value is adjusted to 7.0; the amplification medium for the secondary culture of the bacillus megaterium comprises MRS; the amplification medium for the secondary culture of the saccharomyces cerevisiae comprises the following components: 2 percent of sugar-making waste honey and 1 percent of yeast extract. The amplification medium was sterilized at 115 ℃ for 20 minutes and then used for secondary culture.
And mixing the secondary seed liquid obtained by secondary culture according to 30% of paenibacillus jelly, 30% of bacillus laterosporus, 20% of bacillus megaterium, 10% of bacillus mucilaginosus and 10% of saccharomyces cerevisiae to prepare the initial microorganism mixed microbial inoculum.
Example 3
Inoculating the initial microbial mixed microbial inoculum into a molasses-containing inorganic salt culture medium, wherein the molasses-containing inorganic salt culture medium is prepared from the following raw materials in parts by weight: 100 parts of dried cow dung, 35 parts of coal cinder, 50 parts of crushed straw, 2 parts of urea, 3 parts of gypsum, 1.5 parts of sugar-making waste honey and a proper amount of water. Then the microbial inoculum of the invention is obtained after micro-aerobic fermentation for 7 days.
Tests show that the viable bacteria concentration of the microbial agent is more than 109CFU/mL, the microbial agent can be stably stored for 12 months at room temperature, and the microbial agent can be mixed according to the proportion, can exert the condition with the optimal effect, and can adapt to the restoration treatment of various phosphorus-containing soils. The optimal application conditions are as follows: the temperature is 20-40 ℃; the pH value is 6.5-8.5.
The microbial agents prepared by the method are respectively tested, microbial agent fertilizers with different concentrations are prepared according to the actual conditions of heavy metals such as phosphorus and the like in soil, and pre-buried treatment is carried out on the soil. Test results show that the microbial agent has the functions of dissolving phosphorus and potassium; has the function of activating the secondary elements of silicon, calcium and magnesium in the soil; has the effect of improving the supply of iron, manganese, copper, zinc, molybdenum and boron; the fertilizer efficiency is improved or prolonged, and the fertilizer consumption is reduced; can also improve the stress resistance of crops and prevent or reduce diseases.
All the technical features in the embodiment can be modified according to actual needs.
The above embodiments are preferred implementations of the present invention, and the present invention can be implemented in other ways without departing from the spirit of the present invention.
Claims (5)
1. A method for producing microbial inoculum by utilizing sugar refining waste honey is characterized by comprising the following steps: the method comprises the following steps:
s1, strain activation: selecting Paenibacillus mucilaginosus, bacillus laterosporus, bacillus megaterium, bacillus mucilaginosus and Saccharomyces cerevisiae to be respectively activated;
s2, amplifying the activated strain of the S1;
s3, mixing the strains subjected to amplification in the S2 in proportion to prepare an initial mixed microbial inoculum;
s4, preparing a culture medium by using the sugar-making waste honey;
and S5, putting the initial mixed microbial inoculum in the S3 into a culture medium of the S4 for fermentation to obtain a microbial inoculum.
2. The method for producing microbial agents by using molasses as claimed in claim 1, wherein: the mixing ratio of each strain in the step S2 is as follows: 25 to 35 percent of paenibacillus jelly, 27 to 32 percent of bacillus pumilus, 15 to 25 percent of bacillus megaterium, 8 to 14 percent of bacillus mucilaginosus and 6 to 12 percent of saccharomyces cerevisiae.
3. The method for producing microbial agents by using molasses as claimed in claim 1, wherein: the culture medium in the step S4 comprises the following raw materials in parts by weight: 80-120 parts of dry cow dung, 30-40 parts of coal cinder, 40-60 parts of crushed straw, 1-3 parts of urea, 2-4 parts of gypsum, 1-2 parts of sugar-making waste honey and a proper amount of water.
4. The method for producing microbial agents by using molasses as claimed in claim 1, wherein: in the step S5, the fermentation time of the strain in the culture medium is 7-8 days.
5. The method for producing microbial agents by using molasses as claimed in claim 1, wherein: in step S2, at least two amplification culture media are adopted for amplification culture.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210965145.XA CN115386516A (en) | 2022-08-12 | 2022-08-12 | Method for producing microbial agent by using sugar-making waste honey |
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| CN202210965145.XA CN115386516A (en) | 2022-08-12 | 2022-08-12 | Method for producing microbial agent by using sugar-making waste honey |
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