A solid fermentation medium for Bacillus licheniformis

Abstract

The invention relates to the technical field of bacillus licheniformis solid fermentation culture medium, in particular to a bacillus licheniformis solid fermentation culture medium, which is composed of the following components: 10-30 weight parts of composite active enzyme, 120-140 weight parts of potato peel paste, 230-240 weight parts of sheep manure, 15-25 weight parts of yeast powder, 1-3 weight parts of potassium dihydrogen phosphate, 4-6 weight parts of magnesium sulfate, 0.1-0.3 weight parts of manganese sulfate, 40-50 weight parts of calcium carbonate, 8-12 weight parts of sodium chloride and 25-35 weight parts of beef extract, wherein the composite active enzyme is prepared by mixing alpha-amylase and maltase at a mass ratio of 1:0.5-2 and then being embedded and modified by sodium alginate, and the composite active enzyme is used to interact with the potato peel paste to prepare a bacillus licheniformis solid fermentation culture medium, which can provide a more suitable growth environment and nutrients required for growth for microorganisms, and effectively improve the number of live bacteria of the bacillus licheniformis.

Description

Technical Field

[0001] The invention relates to the technical field of Bacillus licheniformis solid fermentation culture medium, in particular to a Bacillus licheniformis solid fermentation culture medium. Background Art

[0002] Bacillus licheniformis, belonging to the genus Bacillus, is a Gram-positive bacterium that is widely distributed in nature. It is characterized by the ability to form endospores with strong stress resistance, which enables it to survive in extreme environments, such as high temperature, dryness, radiation and the presence of chemicals. It can still remain active. Bacillus licheniformis has multiple uses due to its unique biological characteristics. It can adjust the imbalance of intestinal flora, promote the proliferation of beneficial bacteria, inhibit the growth of pathogens, and help treat digestive diseases such as acute and chronic enteritis and diarrhea. Adding Bacillus licheniformis to aquaculture and feed can promote digestion, enhance immunity, and improve the growth performance and disease resistance of farmed objects. In addition, it has a strong inhibitory effect on a variety of plant pathogens, such as Fusarium, Sclerotinia, Rice blast pathogen and Rice sheath blight pathogen. It has strong antibacterial activity and shows good biocontrol potential.

[0003] At present, the main ingredients of the fermentation medium formula for producing Bacillus licheniformis are corn starch, peptone, beef powder or beef extract and some other trace elements. The ingredients of the fermentation medium have a great influence on the bacterial content of the microbial fermentation liquid. However, the existing fermentation medium for Bacillus licheniformis has the problem of low number of viable bacteria. In view of this, we propose a solid fermentation medium for Bacillus licheniformis. Summary of the invention

[0004] The object of the present invention is to provide a solid fermentation medium for Bacillus licheniformis to solve the problems raised in the above background technology.

[0005] To achieve the above purpose, the present invention provides a Bacillus licheniformis solid fermentation medium, which is composed of the following components: 10-30 weight parts of composite active enzyme, 120-140 weight parts of potato peel paste, 230-240 weight parts of sheep manure, 15-25 weight parts of yeast powder, 1-3 weight parts of potassium dihydrogen phosphate, 4-6 weight parts of magnesium sulfate, 0.1-0.3 weight parts of manganese sulfate, 40-50 weight parts of calcium carbonate, 8-12 weight parts of sodium chloride and 25-35 weight parts of beef extract, wherein the composite active enzyme is prepared by mixing alpha-amylase and maltase in a mass ratio of 1:0.5-2 and then embedding and modifying them with sodium alginate.

[0006] Preferably, the preparation method of the composite active enzyme is as follows:

[0007] The α-amylase and maltase are mixed and prepared with physiological saline to form a mixed solution with a mass concentration of 30-60 mg / L, and the solution is placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 minutes to prepare a sodium alginate solution with a mass ratio of 0.5-1%, and the solution is stirred in a water bath to dissolve. After the sodium alginate solution is cooled to room temperature, the solution is mixed with the enzyme mixed solution to obtain a sodium alginate composite solution, calcium chloride powder is weighed to prepare a calcium chloride solution, and the sodium alginate composite solution is added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads. Finally, the gel beads are washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzymes.

[0008] α-Amylase is an endo-enzyme that can randomly hydrolyze α-1,4-glycosidic bonds in starch and glycogen, but does not decompose α-1,6-glycosidic bonds, which means that it can cut starch macromolecules into smaller fragments, such as maltose, maltotriose and other oligosaccharides and glucose; maltase, also known as α-glucosidase, is an exo-enzyme that specifically hydrolyzes α-1,4-glycosidic bonds, but unlike α-amylase, it can act on smaller sugar chains such as maltose and dextrin to decompose them into glucose. Therefore, maltase is usually used after the initial hydrolysis of starch to further convert the larger oligosaccharides produced by α-amylase into glucose; since α-amylase and maltase are complementary in function, after mixing the two, sodium alginate is used as a carrier and the embedding technology is used to combine the two with each other, which can not only maintain the hydrolysis effect of the enzyme and promote enzyme activity, but also improve the reusability and stability of the enzyme, providing microorganisms with a stable nutrient glucose, while small molecule glucose is also more conducive to the absorption of microbial nutrients.

[0009] Sodium alginate is a viscous polymer compound. Unlike starch and cellulose, it has carboxyl groups and is a high-polysaccharide uronic acid formed by the aldehyde group of β-D-mannuronic acid with a glycosidic bond. Sodium alginate is highly hydrophilic and can be dissolved in both cold and warm water to form a very viscous solution. After adding α-amylase and maltase, it can form a uniform solution. At the same time, since the sodium alginate molecular chain contains a large number of hydroxyl and carboxyl groups, when the sodium alginate mixed solution is dripped into the calcium oxide solution, the calcium oxide acts as a cross-linking agent. The alginate anions electrolyzed by the sodium alginate solution and the divalent calcium ions generated by the calcium oxide solution are attracted to each other by electrostatic action, thereby agglomerating and precipitating to form a porous calcium alginate polymer. Since the higher the concentration of sodium alginate, the smaller the pore size of the gel formed, it is necessary to select a suitable concentration of sodium alginate.

[0010] Preferably, the α-amylase and maltase are mixed in a mass ratio of 1:0.5-2.

[0011] Preferably, the water bath stirring dissolution temperature is 60-80°C.

[0012] Preferably, the sodium alginate composite solution is obtained by mixing a sodium alginate solution and an enzyme mixed solution in a mass ratio of 90:5-10.

[0013] Preferably, the mass concentration of the calcium chloride solution is 1.5-4.5 mg / L.

[0014] Preferably, the curing time is 1-3 hours and the curing temperature is 25°C-30°C.

[0015] In another aspect, the present invention provides a Bacillus licheniformis fermentation method, using any one of the Bacillus licheniformis solid fermentation medium described above, comprising the following steps:

[0016] S1.1. Prepare 10-30 parts by weight of composite active enzyme, 120-140 parts by weight of potato peel paste, 230-240 parts by weight of sheep manure, 15-25 parts by weight of yeast powder, 1-3 parts by weight of potassium dihydrogen phosphate, 4-6 parts by weight of magnesium sulfate, 0.1-0.3 parts by weight of manganese sulfate, 40-50 parts by weight of calcium carbonate, 8-12 parts by weight of sodium chloride and 25-35 parts by weight of beef extract.

[0017] S1.2. Use a disinfected and sterilized flask, add the above raw materials in sequence, shake for 10-15 minutes, adjust the moisture and pH value of the raw materials, filter the mixed solution in the flask with filter paper to obtain a culture medium, sterilize at 120°C for 1 hour, and after the culture medium is cooled to room temperature, inoculate Bacillus licheniformis, place the inoculated culture medium in a 30°C constant temperature incubator for static fermentation, tap the flask for ventilation every 1 hour, and collect the fermentation product after the fermentation is completed.

[0018] Preferably, the potato peel paste is obtained by crushing potato peels, mixing with water, heating at 65-67° C. for gelatinization, and finally drying the moisture.

[0019] Due to its longer molecular chain structure and larger molecular weight, the decomposition process of intact starch molecules requires more enzymes and time to gradually decompose it into monosaccharides. Since potato peels contain starch, the dextrin produced after starch gelatinization is easier to be decomposed by microorganisms than starch. This is because dextrin is composed of shorter glucose molecular chains and is a product of partial hydrolysis of starch. Its molecular weight is smaller than starch and its structure is simpler. α-amylase and maltase can attack dextrin molecules more efficiently and quickly, breaking them down into smaller molecules, which is beneficial to the direct absorption and utilization of microorganisms and more conducive to the growth of microorganisms. Therefore, the product of potato peel gelatinization is selected as the carbon source.

[0020] Preferably, the water content of the culture medium is 55%.

[0021] Since the moisture content will directly affect the yield of the final product, it is crucial to choose an appropriate moisture content. This is because if the moisture content is too high, it will not be conducive to ventilation and cooling. After sterilization, the culture medium will easily clump and the ventilation will be poor, which is not conducive to enzyme production. If the moisture content is too low, the porosity will increase, and the water loss caused by ventilation and heat dissipation will affect the growth of microorganisms and the yield of the final product.

[0022] Preferably, the initial pH value is adjusted to 6-7 by a sodium hydroxide solution with a molar concentration of 10 mol / L.

[0023] The initial pH value is an important part of optimizing fermentation conditions. An appropriate pH value can significantly increase fermentation yield. A high or low pH value will lead to changes in cell membrane permeability, which will affect the absorption and utilization of nutrients in the environment by Bacillus licheniformis. Therefore, a pH value of 6-7 is selected as the optimal initial pH value.

[0024] Preferably, the inoculation amount of the Bacillus licheniformis culture medium is 7-9%.

[0025] Since too high or too low inoculation size is not conducive to the growth of microorganisms, too large an inoculation size will bring in waste from the previous culture solution, and too small an inoculation size will slow down the growth of bacteria, so choosing the appropriate inoculation size is also an important part of optimizing fermentation conditions.

[0026] Preferably, the culture time of the Bacillus licheniformis culture medium is 120-130 hours.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] In the Bacillus licheniformis solid fermentation medium and the culture method thereof, sodium alginate is used as a carrier, and an embedding technology is adopted to combine alpha-amylase and maltase with each other to form a porous composite active enzyme, which can not only maintain the hydrolysis effect of the enzyme and promote the enzyme activity, but also improve the reusability and stability of the enzyme. The Bacillus licheniformis solid fermentation medium prepared by using potato peel paste as a carbon source to interact with the composite active enzyme can provide a more suitable growth environment and nutrients required for growth for microorganisms, effectively increase the number of live bacteria of the Bacillus licheniformis, enable it to be better applied to production practice, and improve production efficiency. DETAILED DESCRIPTION

[0029] 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 making creative work are within the scope of protection of the present invention.

[0030] The invention discloses a Bacillus licheniformis solid fermentation medium, which is composed of the following components: 10-30 weight parts of composite active enzyme, 120-140 weight parts of potato peel paste, 230-240 weight parts of sheep manure, 15-25 weight parts of yeast powder, 1-3 weight parts of potassium dihydrogen phosphate, 4-6 weight parts of magnesium sulfate, 0.1-0.3 weight parts of manganese sulfate, 40-50 weight parts of calcium carbonate, 8-12 weight parts of sodium chloride and 25-35 weight parts of beef extract, wherein the composite active enzyme is prepared by mixing alpha-amylase and maltase in a mass ratio of 1:0.5-2 and then embedding and modifying the mixture with sodium alginate.

[0031] Example 1: A solid fermentation medium for Bacillus licheniformis:

[0032] Preparation of composite active enzyme: α-amylase and maltase were mixed in a mass ratio of 1:1 and then mixed with physiological saline to form a mixed solution with a mass concentration of 45 mg / L, placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 min, a sodium alginate solution with a mass ratio of 0.5% was prepared, and dissolved by stirring in a water bath at a temperature of 80°C. After the sodium alginate solution was cooled to room temperature, the sodium alginate solution and the enzyme mixed solution were mixed in a mass ratio of 90:7 to obtain a sodium alginate composite solution, calcium chloride powder was weighed to prepare a calcium chloride solution with a mass concentration of 3 mg / L, and the sodium alginate composite solution was added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads, the solidification time was 1 h, and the solidification temperature was 30°C. Finally, the gel beads were washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzyme;

[0033] A Bacillus licheniformis fermentation method is as follows:

[0034] S1.1, prepare 10 parts by weight of composite active enzyme, 130 parts by weight of potato peel paste, 235 parts by weight of sheep manure, 20 parts by weight of yeast powder, 2 parts by weight of potassium dihydrogen phosphate, 5 parts by weight of magnesium sulfate, 0.2 parts by weight of manganese sulfate, 45 parts by weight of calcium carbonate, 10 parts by weight of sodium chloride and 30 parts by weight of beef extract;

[0035] S1.2. Use a disinfected and sterilized flask to add the above raw materials in sequence. After oscillating for 15 minutes, adjust the moisture content of the raw materials to 55%. Adjust the pH to 6 with a sodium hydroxide solution with a molar concentration of 10 mol / L. Filter the mixed solution in the flask with filter paper to obtain a culture medium. Sterilize it at 120°C for 1 hour. After the culture medium is cooled to room temperature, inoculate Bacillus licheniformis with an inoculation amount of 8%. Place the inoculated culture medium in a constant temperature incubator at 30°C for static fermentation for 120 hours. Tap the flask for ventilation every 1 hour. Collect the fermentation product after the fermentation is completed.

[0036] Example 2: A solid fermentation medium for Bacillus licheniformis:

[0037] Preparation of composite active enzyme: α-amylase and maltase were mixed in a mass ratio of 1:1 and then prepared with physiological saline to form a mixed solution with a mass concentration of 45 mg / L, placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 min, and a sodium alginate solution with a mass ratio of 0.5% was prepared. The solution was dissolved by stirring in a water bath at a temperature of 80°C. After the sodium alginate solution was cooled to room temperature, the sodium alginate solution and the enzyme mixed solution were mixed in a mass ratio of 90:7 to obtain a sodium alginate composite solution. Calcium chloride powder was weighed to prepare a calcium chloride solution with a mass concentration of 3 mg / L. The sodium alginate composite solution was added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads. The solidification time was 1 h and the solidification temperature was 30°C. Finally, the gel beads were washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzyme.

[0038] A Bacillus licheniformis fermentation method is as follows:

[0039] S1.1, prepare 20 parts by weight of composite active enzyme, 130 parts by weight of potato peel paste, 235 parts by weight of sheep manure, 20 parts by weight of yeast powder, 2 parts by weight of potassium dihydrogen phosphate, 5 parts by weight of magnesium sulfate, 0.2 parts by weight of manganese sulfate, 45 parts by weight of calcium carbonate, 10 parts by weight of sodium chloride and 30 parts by weight of beef extract;

[0040] S1.2. Use a disinfected and sterilized flask to add the above raw materials in sequence. After oscillating for 15 minutes, adjust the moisture content of the raw materials to 55%. Adjust the pH to 6 with a sodium hydroxide solution with a molar concentration of 10 mol / L. Filter the mixed solution in the flask with filter paper to obtain a culture medium. Sterilize it at 120°C for 1 hour. After the culture medium is cooled to room temperature, inoculate Bacillus licheniformis with an inoculation amount of 8%. Place the inoculated culture medium in a constant temperature incubator at 30°C for static fermentation for 120 hours. Tap the flask for ventilation every 1 hour. Collect the fermentation product after the fermentation is completed.

[0041] Example 3: A solid fermentation medium for Bacillus licheniformis:

[0042] Preparation of composite active enzyme: α-amylase and maltase were mixed in a mass ratio of 1:1 and then prepared with physiological saline to form a mixed solution with a mass concentration of 45 mg / L, placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 min, and a sodium alginate solution with a mass ratio of 0.5% was prepared. The solution was dissolved by stirring in a water bath at a temperature of 80°C. After the sodium alginate solution was cooled to room temperature, the sodium alginate solution and the enzyme mixed solution were mixed in a mass ratio of 90:7 to obtain a sodium alginate composite solution. Calcium chloride powder was weighed to prepare a calcium chloride solution with a mass concentration of 3 mg / L. The sodium alginate composite solution was added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads. The solidification time was 1 h and the solidification temperature was 30°C. Finally, the gel beads were washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzyme.

[0043] A Bacillus licheniformis fermentation method is as follows:

[0044] S1.1, prepare 30 parts by weight of composite active enzyme, 130 parts by weight of potato peel paste, 235 parts by weight of sheep manure, 20 parts by weight of yeast powder, 2 parts by weight of potassium dihydrogen phosphate, 5 parts by weight of magnesium sulfate, 0.2 parts by weight of manganese sulfate, 45 parts by weight of calcium carbonate, 10 parts by weight of sodium chloride and 30 parts by weight of beef extract;

[0045] S1.2. Use a disinfected and sterilized flask to add the above raw materials in sequence. After oscillating for 15 minutes, adjust the moisture content of the raw materials to 55%. Adjust the pH to 6 with a sodium hydroxide solution with a molar concentration of 10 mol / L. Filter the mixed solution in the flask with filter paper to obtain a culture medium. Sterilize it at 120°C for 1 hour. After the culture medium is cooled to room temperature, inoculate Bacillus licheniformis with an inoculation amount of 8%. Place the inoculated culture medium in a constant temperature incubator at 30°C for static fermentation for 120 hours. Tap the flask for ventilation every 1 hour. Collect the fermentation product after the fermentation is completed.

[0046] Example 4: A solid fermentation medium for Bacillus licheniformis:

[0047] α-amylase and maltase were mixed in a mass ratio of 1:1 and then prepared with physiological saline to form a mixed solution with a mass concentration of 45 mg / L. The mixture was placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 min to prepare a sodium alginate solution with a mass ratio of 0.5%. The mixture was stirred and dissolved in a water bath at a temperature of 80°C. After the sodium alginate solution was cooled to room temperature, the sodium alginate solution and the enzyme mixed solution were mixed in a mass ratio of 90:7 to obtain a sodium alginate composite solution. Calcium chloride powder was weighed to prepare a calcium chloride solution with a mass concentration of 3 mg / L. The sodium alginate composite solution was added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads. The solidification time was 1 h and the solidification temperature was 30°C. Finally, the gel beads were washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzymes.

[0048] A Bacillus licheniformis fermentation method is as follows:

[0049] S1.1, prepare 10 parts by weight of composite active enzyme, 130 parts by weight of potato peel paste, 235 parts by weight of sheep manure, 20 parts by weight of yeast powder, 2 parts by weight of potassium dihydrogen phosphate, 5 parts by weight of magnesium sulfate, 0.2 parts by weight of manganese sulfate, 45 parts by weight of calcium carbonate, 10 parts by weight of sodium chloride and 30 parts by weight of beef extract;

[0050] S1.2. Use a disinfected and sterilized flask to add the above raw materials in sequence. After oscillating for 15 minutes, adjust the moisture content of the raw materials to 55%. Adjust the pH to 6.5 with a sodium hydroxide solution with a molar concentration of 10 mol / L. Filter the mixed solution in the flask with filter paper to obtain a culture medium. Sterilize it at 120°C for 1 hour. After the culture medium is cooled to room temperature, inoculate Bacillus licheniformis with an inoculation amount of 8%. Place the inoculated culture medium in a 30°C constant temperature incubator for static fermentation for 120 hours. Tap the flask for ventilation every 1 hour. Collect the fermentation product after the fermentation is completed.

[0051] Example 5: A solid fermentation medium for Bacillus licheniformis:

[0052] α-amylase and maltase were mixed in a mass ratio of 1:1 and then prepared with physiological saline to form a mixed solution with a mass concentration of 45 mg / L. The mixture was placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 min to prepare a sodium alginate solution with a mass ratio of 0.5%. The mixture was stirred and dissolved in a water bath at a temperature of 80°C. After the sodium alginate solution was cooled to room temperature, the sodium alginate solution and the enzyme mixed solution were mixed in a mass ratio of 90:7 to obtain a sodium alginate composite solution. Calcium chloride powder was weighed to prepare a calcium chloride solution with a mass concentration of 3 mg / L. The sodium alginate composite solution was added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads. The solidification time was 1 h and the solidification temperature was 30°C. Finally, the gel beads were washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzymes.

[0053] A Bacillus licheniformis fermentation method is as follows:

[0054] S1.1, prepare 10 parts by weight of composite active enzyme, 130 parts by weight of potato peel paste, 235 parts by weight of sheep manure, 20 parts by weight of yeast powder, 2 parts by weight of potassium dihydrogen phosphate, 5 parts by weight of magnesium sulfate, 0.2 parts by weight of manganese sulfate, 45 parts by weight of calcium carbonate, 10 parts by weight of sodium chloride and 30 parts by weight of beef extract;

[0055] S1.2. Use a disinfected flask and add the above raw materials in sequence. After oscillating for 15 minutes, adjust the moisture content of the raw materials to 55%. Adjust the pH to 7 with a sodium hydroxide solution with a molar concentration of 10 mol / L. Filter the mixed solution in the flask with filter paper to obtain a culture medium. Sterilize it at 120°C for 1 hour. After the culture medium is cooled to room temperature, inoculate Bacillus licheniformis with an inoculation amount of 8%. Place the inoculated culture medium in a 30°C constant temperature incubator and ferment it for 120 hours. Tap the flask for ventilation every 1 hour. Collect the fermentation product after the fermentation is completed.

[0056] Example 6: A solid fermentation medium for Bacillus licheniformis:

[0057] Preparation of composite active enzyme: α-amylase and maltase were mixed in a mass ratio of 1:1 and then prepared with physiological saline to form a mixed solution with a mass concentration of 45 mg / L, placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 min, and a sodium alginate solution with a mass ratio of 0.5% was prepared. The solution was dissolved by stirring in a water bath at a temperature of 80°C. After the sodium alginate solution was cooled to room temperature, the sodium alginate solution and the enzyme mixed solution were mixed in a mass ratio of 90:7 to obtain a sodium alginate composite solution. Calcium chloride powder was weighed to prepare a calcium chloride solution with a mass concentration of 3 mg / L. The sodium alginate composite solution was added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads. The solidification time was 1 h and the solidification temperature was 30°C. Finally, the gel beads were washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzyme.

[0058] A Bacillus licheniformis fermentation method is as follows:

[0059] S1.1, prepare 20 parts by weight of composite active enzyme, 120 parts by weight of potato peel paste, 230 parts by weight of sheep manure, 15 parts by weight of yeast powder, 1 part by weight of potassium dihydrogen phosphate, 4 parts by weight of magnesium sulfate, 0.1 parts by weight of manganese sulfate, 40 parts by weight of calcium carbonate, 8 parts by weight of sodium chloride and 25 parts by weight of beef extract;

[0060] S1.2. Use a disinfected and sterilized flask to add the above raw materials in sequence. After oscillating for 15 minutes, adjust the moisture content of the raw materials to 55%. Adjust the pH to 6 with a sodium hydroxide solution with a molar concentration of 10 mol / L. Filter the mixed solution in the flask with filter paper to obtain a culture medium. Sterilize it at 120°C for 1 hour. After the culture medium is cooled to room temperature, inoculate Bacillus licheniformis with an inoculation amount of 8%. Place the inoculated culture medium in a constant temperature incubator at 30°C for static fermentation for 120 hours. Tap the flask for ventilation every 1 hour. Collect the fermentation product after the fermentation is completed.

[0061] Example 7: A solid fermentation medium for Bacillus licheniformis:

[0062] Preparation of composite active enzyme: α-amylase and maltase were mixed in a mass ratio of 1:1.5, and then mixed with physiological saline to form a mixed solution with a mass concentration of 50 mg / L, and placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 min to prepare a sodium alginate solution with a mass ratio of 0.5%, and dissolved by stirring in a water bath at a temperature of 60°C. After the sodium alginate solution was cooled to room temperature, the sodium alginate solution and the enzyme mixed solution were mixed in a mass ratio of 90:8 to obtain a sodium alginate composite solution. Calcium chloride powder was weighed to prepare a calcium chloride solution with a mass concentration of 4.0 mg / L, and the sodium alginate composite solution was added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads. The solidification time was 2 h and the solidification temperature was 25°C. Finally, the gel beads were washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzyme.

[0063] A Bacillus licheniformis fermentation method is as follows:

[0064] S1.1, prepare 20 parts by weight of composite active enzyme, 140 parts by weight of potato peel paste, 240 parts by weight of sheep manure, 25 parts by weight of yeast powder, 3 parts by weight of potassium dihydrogen phosphate, 6 parts by weight of magnesium sulfate, 0.3 parts by weight of manganese sulfate, 50 parts by weight of calcium carbonate, 12 parts by weight of sodium chloride and 35 parts by weight of beef extract;

[0065] S1.2. Use a disinfected and sterilized flask to add the above raw materials in sequence. After oscillating for 10 minutes, adjust the moisture content of the raw materials to 55%. Adjust the pH to 6 with a sodium hydroxide solution with a molar concentration of 10 mol / L. Filter the mixed solution in the flask with filter paper to obtain a culture medium. Sterilize it at 120°C for 1 hour. After the culture medium is cooled to room temperature, inoculate Bacillus licheniformis with an inoculation amount of 7%. Place the inoculated culture medium in a 30°C constant temperature incubator and let it ferment for 130 hours. Tap the flask for ventilation every 1 hour. Collect the fermentation product after the fermentation is completed.

[0066] Comparative Example 1: The method of Example 2 was adopted, but no composite active enzyme was added to the solid fermentation medium of Bacillus licheniformis.

[0067] The present invention adopts a Bacillus licheniformis solid fermentation medium prepared by using a composite active enzyme, wherein the performance index inspection items and inspection standards of the Bacillus licheniformis solid fermentation medium are as follows:

[0068] The viable count of Bacillus licheniformis refers to the number of live Bacillus licheniformis cells that can grow and form colonies in a certain amount of sample. This indicator is one of the important parameters for measuring product efficacy, environmental pollution level or microbial activity in research. The test is carried out using the spread plate method. First, the sample is appropriately diluted, and nutrient agar is used as the culture medium. The diluted sample is taken up in a certain dilution degree using a sterile pipette and evenly spread on the surface of the sterilized and cooled nutrient agar plate. The spread plate is placed at 37°C and cultured for 18-24 hours. The number of colonies formed is counted to estimate the number of viable bacteria. Finally, the number of viable bacteria per milliliter or per gram of sample is calculated using a formula based on the dilution degree and the number of spread plates.

[0069] According to the above standards, a solid fermentation medium of Bacillus licheniformis prepared in the above Examples 1-7 and Comparative Example 1 was tested, and the obtained data are shown in Table 1:

[0070] Table 1 Performance data of Examples 1-7 and Comparative Example 1

[0071] Example Viable bacteria count (CFU / mL) Example 1 <![CDATA[8.16×10 10 ]]> Example 2 <![CDATA[8.54×10 10 ]]> Example 3 <![CDATA[8.02×10 10 ]]> Example 4 <![CDATA[8.11×10 10 ]]> Example 5 <![CDATA[8.08×10 10 ]]> Example 6 <![CDATA[8.42×10 10 ]]> Example 7 <![CDATA[8.36×10 10 ]]> Comparative Example 1 <![CDATA[2.53×10 10 ]]>

[0072] The above data fully show that compared with Comparative Example 1, Examples 1-7 can fully show the effect of the composite active enzyme on increasing the number of viable bacteria in a solid fermentation medium of Bacillus licheniformis.

[0073] Since the present invention uses a composite active enzyme to prepare a Bacillus licheniformis solid fermentation medium, the decomposition rate of the carbon source is increased through the interaction between the composite active enzyme and the carbon source, the growth of microorganisms is promoted, and the number of viable microorganisms is effectively increased, as shown below:

[0074] It can be seen from Examples 1-3 that:

[0075] With the continuous increase of the content of composite active enzyme, the number of live bacteria in a solid fermentation medium of Bacillus licheniformis first increased and then decreased. This is because α-amylase can hydrolyze starch, and maltase can further convert the larger oligosaccharides produced by α-amylase into glucose. When sodium alginate is used to combine the two, the composite enzyme gel ball formed can maintain the hydrolysis effect of the two enzymes, while improving the reusability and stability of the enzyme. Therefore, potato peel paste is used as a carbon source, and the enzyme of the composite enzyme gel ball is hydrolyzed, which promotes the decomposition of the carbon source and decomposes it into smaller molecules, providing stable nutrient glucose for microorganisms, promoting the direct absorption and utilization of microorganisms, and is beneficial to The growth of microorganisms increases the number of live bacteria of Bacillus licheniformis. However, when the content of complex active enzymes is too high, the carbon source will be over-decomposed too quickly and over-decomposed, resulting in excessive consumption of the carbon source, causing an abnormal increase in the sugar content in the culture medium. On the one hand, it will change the conditions of the culture medium, affect the growth rate, metabolic pathways and synthesis of the final products of the microorganisms, and even cause the growth of the microorganisms to be hindered due to nutrient imbalance. On the other hand, it will increase the osmotic pressure of the culture medium, produce osmotic stress on the microbial cells, and damage the cell membrane. At the same time, because the enzymatic reaction process can only produce protons or hydroxide ions, the pH value of the culture medium will change, affecting the growth environment of the microorganisms, and thus the number of live bacteria of the microorganisms will decrease.

[0076] It can be seen from Examples 1, 4 and 5 that:

[0077] As the pH value increases, the number of live microorganisms decreases slightly. This is because different microorganisms have different pH preferences. The activity of their enzymes is highest within a specific range. When the pH value deviates from the most suitable pH value for microbial growth, the activity of related enzymes will decrease, affecting the microorganism's utilization of nutrients and the synthesis of metabolites, thereby inhibiting the growth and reproduction of microorganisms and causing a decrease in the number of microorganisms. At the same time, changes in pH will affect the charge state of the microbial cell membrane and change its permeability, which not only affects the absorption of nutrients, but may also interfere with the excretion of metabolites, thereby affecting the balance of material exchange inside and outside the cell and having a negative impact on the survival and proliferation of microorganisms. Therefore, pH is an important part of optimizing fermentation conditions. An appropriate pH value can increase the number of live microorganisms.

[0078] It can be seen from Examples 2, 6 and 7 that:

[0079] With the change of the content of potato peel paste, sheep manure, yeast powder, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, calcium carbonate sodium chloride and beef extract in the fermentation medium, the number of live bacteria in a solid fermentation medium of Bacillus licheniformis has no obvious change. This is because Bacillus licheniformis is a bacterium with strong environmental adaptability, and these components only need to provide sufficient nutrients as a whole, and have little effect on Bacillus licheniformis, so the number of live bacteria has no obvious change. According to the above test experiments, a solid fermentation medium of Bacillus licheniformis prepared according to Example 2 has the best performance, so Example 2 is taken as the best example;

[0080] By comparing Example 2 with Comparative Example 1, it can be seen that:

[0081] In Comparative Example 1, no composite active enzyme was added to the solid fermentation medium for Bacillus licheniformis, and the number of live bacteria decreased significantly. This is because without the composite active enzyme to further accelerate the decomposition of the carbon source, the production rate of small molecules slowed down, and the nutrients that the microorganisms can directly absorb decreased, so the growth rate slowed down, and the number of live bacteria of Bacillus licheniformis tested in the same period of time was also greatly reduced.

[0082] In summary, a solid fermentation medium for Bacillus licheniformis is prepared through the interaction of composite active enzymes and carbon sources, which can promote the decomposition of carbon sources into smaller molecules, provide stable nutrients such as glucose for microorganisms, and promote direct absorption and utilization by microorganisms. At the same time, the activity of enzymes in microorganisms is highest within a specific pH range, which can promote the utilization of nutrients and the synthesis of metabolites by microorganisms, provide microorganisms with more suitable nutrients and growth environment for growth, and effectively increase the number of viable bacteria of Bacillus licheniformis.

[0083] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only preferred examples of the present invention, and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A Bacillus licheniformis solid fermentation medium, characterized in that: The invention is composed of the following components: 10-30 parts by weight of composite active enzyme, 120-140 parts by weight of potato peel paste, 230-240 parts by weight of sheep manure, 15-25 parts by weight of yeast powder, 1-3 parts by weight of potassium dihydrogen phosphate, 4-6 parts by weight of magnesium sulfate, 0.1-0.3 parts by weight of manganese sulfate, 40-50 parts by weight of calcium carbonate, 8-12 parts by weight of sodium chloride and 25-35 parts by weight of beef extract; The composite active enzyme is prepared by mixing α-amylase and maltase in a mass ratio of 1:0.5-2 and then modified by sodium alginate encapsulation.

2. The Bacillus licheniformis solid fermentation medium according to claim 1, characterized in that: The preparation method of the composite active enzyme is as follows: The α-amylase and maltase are mixed and prepared with physiological saline to form a mixed solution with a mass concentration of 30-60 mg / L, and the solution is placed in a constant temperature shaker at 30°C and 120 rpm / min for 10 minutes to prepare a sodium alginate solution with a mass ratio of 0.5-1%, and the solution is stirred in a water bath to dissolve. After the sodium alginate solution is cooled to room temperature, the solution is mixed with the enzyme mixed solution to obtain a sodium alginate composite solution, calcium chloride powder is weighed to prepare a calcium chloride solution, and the sodium alginate composite solution is added dropwise to the calcium chloride solution to solidify the sodium alginate to form gel beads. Finally, the gel beads are washed with clean water and dried to obtain composite enzyme gel beads, i.e., composite active enzymes.

3. The Bacillus licheniformis solid fermentation medium according to claim 2, characterized in that: The water bath stirring dissolving temperature is 60-80°C.

4. The Bacillus licheniformis solid fermentation medium according to claim 2, characterized in that: The sodium alginate composite solution is obtained by mixing a sodium alginate solution and an enzyme mixed solution in a mass ratio of 90:5-10.

5. The Bacillus licheniformis solid fermentation medium according to claim 2, characterized in that: The mass concentration of the calcium chloride solution is 1.5-4.5 mg / L.

6. The Bacillus licheniformis solid fermentation medium according to claim 2, characterized in that: The curing time is 1-3 hours, and the curing temperature is 25°C-30°C.

7. A Bacillus licheniformis fermentation method, using the Bacillus licheniformis solid fermentation medium according to any one of claims 1 to 6, characterized in that: The steps include: S1.1, prepare 10-30 parts by weight of composite active enzyme, 120-140 parts by weight of potato peel paste, 230-240 parts by weight of sheep manure, 15-25 parts by weight of yeast powder, 1-3 parts by weight of potassium dihydrogen phosphate, 4-6 parts by weight of magnesium sulfate, 0.1-0.3 parts by weight of manganese sulfate, 40-50 parts by weight of calcium carbonate, 8-12 parts by weight of sodium chloride and 25-35 parts by weight of beef extract; S1.

2. Use a disinfected and sterilized flask, add the above raw materials in sequence, shake for 10-15 minutes, adjust the moisture and pH value of the raw materials, filter the mixed solution in the flask with filter paper to obtain a culture medium, sterilize at 120°C for 1 hour, and after the culture medium is cooled to room temperature, inoculate Bacillus licheniformis, place the inoculated culture medium in a 30°C constant temperature incubator for static fermentation, tap the flask for ventilation every 1 hour, and collect the fermentation product after the fermentation is completed.

8. A Bacillus licheniformis fermentation method according to claim 7, characterized in that: The potato peel paste is obtained by crushing potato peels, adding water to mix, heating at 65-67° C. for gelatinization, and finally drying the moisture.

9. A Bacillus licheniformis fermentation method according to claim 8, characterized in that: The water content of the culture medium is 55%; the initial pH value is adjusted to 6-7 by a sodium hydroxide solution with a molar concentration of 10 mol / L; the inoculation amount of the Bacillus licheniformis culture medium is 7-9%; and the culture time of the Bacillus licheniformis culture medium is 120-130 hours.