CN115927062A - Bacillus licheniformis, screening method, product and preparation method - Google Patents

Abstract

The invention discloses bacillus licheniformis, a screening method, a product and a preparation method, wherein the bacillus licheniformis is bacillus licheniformis (SLY 1812), which has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC25162. The fermentation product of the bacillus licheniformis provided by the embodiment of the invention has extremely high neutral and alkaline protease activities, and can efficiently catalyze protein hydrolysis within the pH range of 6.0-11.0. The strain is adopted to obtain a fermented feed raw material which has high neutral and alkaline protease activity and is rich in nutritional ingredients such as protein, small peptide, dietary fiber and the like through solid state fermentation.

Description

Bacillus licheniformis, screening method, product and preparation method

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus licheniformis, a screening method, a product and a preparation method.

Background

Proteases are a generic term for a class of enzymes that hydrolyze peptide chains of proteins. It refers to a hydrolase that can degrade proteins into small peptides and amino acids, accounting for over 60% of the entire industrial enzyme market. The protease can be divided into acid protease, neutral protease and alkaline protease according to its optimum reaction pH, and its optimum pH is pH2.0-5.0, pH6.0-8.0, and pH9.0-11.0 respectively.

Researches show that under the background of low-protein daily ration, the addition of the protease in the feed can improve the apparent digestibility of nutrient substances and reduce the nitrogen content in excrement, thereby effectively reducing the feed cost and lightening the pollution of the breeding industry to the environment. The alkaline protease has the characteristics of low substrate specificity requirement, high hydrolysis degree and low cost; neutral protease has the advantages of good flavor of hydrolysate, small bitter taste and the like, a plurality of proteases are separated and purified from a plurality of microbial metabolites, but the microorganisms of high-activity neutral protease and alkaline protease applied to feed production still account for a few. In addition, the protease additive commonly used in the feed at present only has single acidic, neutral or alkaline protease activity, and does not contain nutritional ingredients such as protein, dietary fiber and the like required by animals.

Disclosure of Invention

The invention aims to provide bacillus licheniformis, a screening method, a product and a preparation method, which are used for solving the problems that the existing protease additive only has single protease activity and does not contain nutritional ingredients such as protein, dietary fiber and the like required by animals.

In a first aspect, an embodiment of the present invention provides a Bacillus licheniformis, where the Bacillus licheniformis is Bacillus licheniformis (SLY 1812), which has been deposited in the common microorganism center of the china committee for culture collection management of microorganisms, and the preservation number is CGMCC No:25162.

in a second aspect, the invention discloses a method for screening bacillus licheniformis, which comprises the following steps:

separating aerobic bacterial strains from the excrement of the laying hens;

transferring the separated aerobic bacterial strain to an LB culture medium for activation, inoculating the aerobic bacterial strain to a casein agar culture medium by a point inoculation method, culturing for 24 hours at 37 ℃, and selecting a bacterial strain with a transparent ring size reaching a preset range;

culturing the selected strain on solid culture medium at 37 deg.C for 40 hr, drying the culture in drying oven at 40 deg.C until the water content is 6-8%, measuring the activities of neutral protease and alkaline protease of the fermented product, and selecting Bacillus licheniformis (Bacillus licheniformis) SLY1812 with highest neutral protease activity.

According to one embodiment of the invention, the matrix of the solid medium contains 85% wheat bran and 15% rice hull, and the water content is 45%.

In a third aspect, the invention discloses a preparation of Bacillus licheniformis, which comprises the Bacillus licheniformis (SLY 1812) described in the first aspect.

In a fourth aspect, the present invention discloses a method for preparing a Bacillus licheniformis product, wherein the Bacillus licheniformis (Bacillus licheniformis) SLY1812 of claim 1, the method comprises: performing slant culture on the Bacillus licheniformis (SLY 1812), wherein the components of the culture medium comprise 4-5g/L of yeast powder, 8-10g/L of sodium chloride, 8-10g/L of peptone, 1.5-2g/L of glucose, 15-20g/L of agar, and the pH value of the culture medium is 6.8-7.2, and the sterilization mode is sterilization at 121 ℃ for 30min; the culture condition is that the inoculated seeds are cultured for 20 to 30 hours at the constant temperature of 37 ℃; and performing liquid strain fermentation culture on the Bacillus licheniformis (SLY 1812) after the slant culture until the spore rate of the Bacillus licheniformis (SLY) is detected to be more than 90%, and finishing the culture to obtain a liquid product of the Bacillus licheniformis.

According to an embodiment of the fourth aspect of the invention, the liquid seed culture fermentation comprises:

fermenting and culturing a liquid primary strain, wherein the component content of a culture medium in the fermenting and culturing of the liquid primary strain is 4-5g/L of yeast powder, 8-10g/L of sodium chloride, 8-10g/L of peptone, 1.5-2g/L of glucose, the balance of water, the total amount of 1L, the pH value of the culture medium is 6.8-7.2, and the sterilization mode is that the sterilization is carried out for 30min at 121 ℃;

the culture condition is that at 37 deg.C, a 1L volume triangular flask is adopted, each flask is filled with 200-250mL liquid culture medium, strains are inoculated from a solid inclined test tube, the rotation speed of a shaker is 200-250rpm, and the culture is carried out for 22-28 hours.

According to an embodiment of the fourth aspect of the present invention, the liquid seed culture further comprises:

liquid secondary strain fermentation culture, wherein the component content of a culture medium in the liquid secondary strain fermentation culture is 60-75g/L of soybean meal, 50-60g/L of corn starch, 4-6g/L of glucose, 5-8g/L of corn steep liquor, 2-4g/L of light calcium carbonate, 0.7-1.2g/L of potassium carbonate, 1.5-2.5g/L of calcium chloride, 2-3g/L of dipotassium phosphate, 2-4 ten thousand U/L of neutral protease, 1000-2000U/L of medium temperature amylase and pH6.8-7.2; sterilizing at 121-123 deg.C for 20-30min under 0.12MPa; the culture temperature is 37-38 ℃.

According to one embodiment of the fourth aspect of the present invention, the process for preparing the medium in the fermentation culture of the liquid secondary strain comprises: putting the soybean meal powder, the corn starch, the glucose and the corn steep liquor in the components of the culture medium into an aerobic fermentation tank for constant volume, and heating to 45-50 ℃; adding neutral protease dissolved in advance, and stirring for 1.5h; heating to 65-68 deg.C, adding pre-dissolved medium temperature amylase after temperature is stable, and continuing enzymolysis for 1 hr; and after the enzymolysis is finished, adding the light calcium carbonate, the potassium carbonate, the calcium chloride and the potassium dihydrogen phosphate, and heating and sterilizing at 121-123 ℃ for 20-30min.

According to an embodiment of the fourth aspect of the invention, the method further comprises: performing solid fermentation on the strain subjected to fermentation culture of the liquid strain, wherein the solid fermentation culture medium comprises the following components in percentage by solid: 60-80% of wheat bran, 0-15% of soybean meal, 10-20% of rice hull and 45-55% of initial moisture content of matrix.

According to an embodiment of the fourth aspect of the present invention, the culture conditions for the solid state fermentation are: sterilizing the culture medium at 121 deg.C for 20-30min, cooling to 60-70 deg.C, inoculating liquid strain at a ratio of 4-8%, mixing, maintaining the temperature of material at 35-42 deg.C, aerobic fermenting for 30-40 hr until the microscopic spore rate of strain reaches above 90%, and drying at 70 deg.C to obtain solid product of Bacillus licheniformis.

The embodiment of the invention has at least the following beneficial effects:

according to the bacillus licheniformis, the screening method, the product and the preparation method provided by the embodiment of the invention, 1 bacillus licheniformis strain capable of generating high neutral protease and alkaline protease activities is screened by the method, the fermentation product of the bacillus licheniformis strain has extremely high neutral and alkaline protease activities, and the bacillus licheniformis strain can efficiently catalyze protein hydrolysis within the pH range of 6.0-11.0. The solid state fermentation is carried out by adopting the strain and taking wheat bran, bean pulp and rice hull as main matrixes, so that a fermented feed raw material which has high neutral and alkaline protease activity and is rich in nutritional ingredients such as protein, small peptide and dietary fiber can be obtained.

Drawings

FIG. 1 is a flat plate diagram of the morphological characteristics of the strain SLY1812 of the example of the present invention;

FIG. 2 is a morphological view of the strain SLY1812 under a microscope;

FIG. 3 is a structural diagram of a phylogenetic tree of the strain SLY1812 according to the example of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

First, bacillus licheniformis in accordance with an embodiment of the present invention will be described.

The Bacillus licheniformis of the embodiment of the invention is Bacillus licheniformis (SLY 1812), which has been preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation addresses are as follows: the preservation date of No. 3 Xilu Beijing Xiyan No. 1 Beijing, chaoyang district is: the result is survival at 23/6 2022, and the preservation number is CGMCC25162.

The method for screening Bacillus licheniformis SLY1812 according to the embodiment of the present invention will be described in detail with reference to the accompanying drawings.

Aerobic bacterial strains are separated from the excrement of the free-range laying hens, the separated bacterial colonies are transferred to an LB culture medium for activation, then the bacterial colonies are inoculated to a casein agar culture medium by a point inoculation method, the size of a transparent ring is observed after the bacterial colonies are cultured for 24 hours at 37 ℃, and the bacterial strains capable of generating a large transparent ring are selected.

Respectively placing the selected strains in a solid matrix containing 85% of wheat bran, 15% of rice hull solid matrix and 45% of water content, culturing for 40 hours at a constant temperature of 37 ℃ according to the charging amount of 50g/1000mL triangular flask, drying the culture at a constant temperature in a constant temperature air drying oven at 40 ℃ until the water content is 6-8%, determining the activities of neutral protease and alkaline protease of the fermentation product according to a method for determining neutral protease of a protease preparation in GB/T23527-2009, and finally screening to obtain the strain SLY1812 with the highest neutral protease activity.

The morphological characteristics of strain SLY1812 are shown in FIG. 1 as a flat-sheet: an off-white nearly circular colony is formed on an LB solid culture medium, is semitransparent, has a wet and smooth surface and is jagged at the edge. The strain SLY1812 shown in FIG. 2 was observed in the form of a fine rod under a microscope, which was 0.6-0.7X 1.8-3.2. Mu.m. The developmental trees may be referred to the structural diagram of the phylogenetic tree of strain SLY1812 shown in FIG. 3.

Identification of the strains

After observing the strain morphology, physiological and biochemical tests are carried out according to Bojie's Manual of identification of bacteria and general identification method of bacteria, and then the three strains are all thermophilic bacillus. The molecular biological identification is carried out, and the 16S rDNA is amplified by adopting an upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and a downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction conditions were: 5min at 95 ℃; 1min at 94 ℃, 1min at 55 ℃, 90s at 72 ℃ and 30 cycles; 10min at 72 ℃. The PCR product was detected by electrophoresis on 1% agarose gel and purified by PCR product purification kit, stored at-20 ℃ for further use, and the amplified product was detected by electrophoresis on 1% agarose gel 100V for 40min. Inputting the sequencing result into NCBI for BLAST comparison, constructing a phylogenetic tree of the target bacteria by using a Neighbor-Joining method, and identifying the SLY1812 as a Bacillus licheniformis (Bacillus licheniformis) SLY1812 according to the comparison result.

Physiological and biochemical characteristics of strain SLY 1812: gram reaction positive, 7% NaCl growth positive, phenylalanine deamination negative, citrate utilization weak positive, propionate utilization negative, casein hydrolysis positive, starch hydrolysis positive, D-glucose acid production positive, L-arabinose acid production positive, D-mannitol acid production positive, D-xylose acid production positive, glucose gas production negative, fermentation xylose positive, fermentation mannitol positive, V-P reaction positive, nitrate reduction positive, gelatin hydrolysis positive.

The 16S rDNA sequence of the strain SLY1812 of Bacillus licheniformis (Bacillus licheniformis) is shown in the attached table.

The fermentation product of the Bacillus licheniformis (Bacillus licheniformis) SLY1812 provided by the embodiment of the invention has extremely high neutral and alkaline protease activities, and can efficiently catalyze protein hydrolysis within the pH range of 6.0-11.0. The solid state fermentation is carried out by adopting the strain and taking wheat bran, bean pulp and rice hull as main matrixes, so that a fermented feed raw material which has high neutral and alkaline protease activity and is rich in nutritional ingredients such as protein, small peptide and dietary fiber can be obtained.

The invention also discloses a preparation method of the product of Bacillus licheniformis (SLY 1812), which comprises the following steps:

step 01, performing slant culture on the Bacillus licheniformis (SLY 1812), wherein the component content of a culture medium is 4-5g/L of yeast powder, 8-10g/L of sodium chloride, 8-10g/L of peptone, 1.5-2g/L of glucose, 15-20g/L of agar, pH6.8-7.2 of the culture medium, and the sterilization mode is sterilization at 121 ℃ for 30min; the culture condition is that the inoculated seeds are cultured for 20 to 30 hours at the constant temperature of 37 ℃;

and step 02, performing liquid strain fermentation culture on the Bacillus licheniformis (SLY 1812) after slant culture until the spore rate of the Bacillus licheniformis (SLY) is detected to be more than 90%, and finishing the culture to obtain the Bacillus licheniformis product.

Wherein, the liquid strain fermentation culture process may comprise a primary liquid strain fermentation culture process. For example, the culture can be finished by one-time liquid primary strain fermentation culture, or by liquid primary strain fermentation culture and liquid secondary strain fermentation culture, or by liquid primary strain fermentation culture, liquid secondary strain fermentation culture, liquid tertiary strain fermentation culture and liquid quaternary strain fermentation culture until the spore rate of the thalli is detected to be more than 90% by a microscope.

In one embodiment, the process of obtaining the product through two-sided liquid strain fermentation culture is described as an example,

1. preparing a strain slant and a plate culture medium: the formula of the culture medium is as follows: 4-5g/L of yeast powder, 8-10g/L of sodium chloride, 8-10g/L of peptone, 1.5-2g/L of glucose, 15-20g/L of agar, pH6.8-7.2, and sterilizing for 30min at 121 ℃.

2. Strain slant culture conditions: after inoculation, the culture is carried out for 20 to 30 hours at the constant temperature of 37 ℃.

3. The liquid primary strain fermentation medium is prepared from the following raw materials (component content): 4-5g/L of yeast powder, 8-10g/L of sodium chloride, 8-10g/L of peptone and 1.5-2g/L of glucose, wherein the water is added to the volume of 1L, the pH value is 6.8-7.2, and the sterilization condition is 121 ℃ for 30min.

4. The liquid first-level strain culture conditions are as follows: under the condition of 37 ℃, adopting a triangular flask with the volume of 1L, filling 200-250mL of liquid culture medium into each flask, inoculating strains from a solid inclined plane test tube, culturing for 22-28 hours at the rotating speed of a shaking table of 200-250rpm, and finishing when the spore rate of the strains reaches more than 90% through microscope detection;

5. the liquid secondary strain fermentation medium is prepared from the following raw materials (component content): 60-75g/L of soybean meal, 50-60g/L of corn starch, 4-6g/L of glucose, 5-8g/L of corn steep liquor, 2-4g/L of light calcium carbonate, 0.7-1.2g/L of potassium carbonate, 1.5-2.5g/L of calcium chloride, 2-3g/L of dipotassium hydrogen phosphate, 2-4 ten thousand U/L of neutral protease, 1000-2000U/L of medium-temperature amylase and 6.8-7.2 of pH; sterilizing at 121-123 deg.C under 0.12MPa for 20-30min.

6. The liquid secondary strain culture conditions are as follows:

a culture container: 100L/1000L/10000L liquid stirring type aerobic fermentation tank

Pretreatment of enzymolysis: (1) After the culture medium (soybean meal powder, corn starch, glucose and corn steep liquor) is put into the culture medium, the volume is determined, and the temperature is raised to 45-50 ℃; then adding neutral protease dissolved in advance, and stirring for 1.5h;

(2) After the first step of enzymolysis is finished, the temperature is increased to 65-68 ℃, after the temperature is stable, the medium temperature amylase dissolved in advance is added, and the enzymolysis is continued for 1 hour;

(3) After the enzymolysis is finished; adding inorganic salt (light calcium carbonate, potassium carbonate, calcium chloride, potassium dihydrogen phosphate), heating and sterilizing;

(4) After sterilization, cooling to the set culture temperature (37-38 ℃), adding ammonia water to adjust the pH to 7.0, and waiting for seeding;

the culture temperature is as follows: 37-38 ℃, ventilation: liquid: air ratio of 1: (0.6-1): 1.2, stirring speed is 180-240r/min;

inoculation: a 100L fermentation tank is inoculated with the first-level liquid strain according to the inoculation amount of 2 percent, and a 1000L/10000L fermentation tank is inoculated with the 100L/1000L fermentation tank bacterial liquid according to the inoculation amount of 10 percent;

pH: within the first 8 hours after inoculation, when the pH value of the culture medium is lower than 6.8, the pH value is adjusted back to 7.0 by using concentrated ammonia water;

and (3) finishing the culture: stopping fermentation when spore formation rate reaches 90-95%, heating to 65 deg.C, and maintaining for 20-30min to obtain liquid product.

In other embodiments of the present invention, the method may further comprise performing scale-up culture, i.e. culture by solid state fermentation, on the strain obtained through the liquid strain fermentation culture process. Wherein, the solid state fermentation medium is prepared from the following raw materials: 60-80% of wheat bran (solid content), 0-15% of soybean meal (solid content), 10-20% of rice hull (solid content), and 45-55% of initial water content of matrix.

The solid state fermentation culture process conditions are as follows: sterilizing at 121 deg.C for 20-30min, cooling to 60-70 deg.C, inoculating liquid strain at a ratio of 4-8%, mixing, maintaining the temperature of material at 35-42 deg.C, ventilating, aerobic fermenting for 30-40 hr, terminating fermentation when the microscopic spore rate of thallus in the material reaches above 90%, and drying at low temperature below 70 deg.C.

The method has simple preparation process, and can obtain large amount of Bacillus licheniformis (SLY 1812) with high purity.

The invention also discloses a feed, which comprises Bacillus licheniformis (SLY 1812).

According to the feed provided by the embodiment of the invention, the Bacillus licheniformis (Bacillus licheniformis) SLY1812 is added, compared with the common industrial enzyme combined into the feed, the common industrial enzyme enables the cost of neutral protease and alkaline protease of the feed to be 8000-10000 yuan/ton when the activities of the neutral protease and the alkaline protease respectively reach 12000U/g and 24000U/g, and the cost of a fermentation product produced by aerobic solid fermentation of the strain is only 3000-4000 yuan/ton under the condition of the same enzyme activity, and the fermentation product additionally contains 15-18% of crude protein, 2-4% of small peptide and other nutritional ingredients. The application of the fermentation product (Bacillus licheniformis SLY 1812) of the invention can greatly reduce the feed cost and obviously improve the feed nutrition quality.

The process of scaling up the culture of Bacillus licheniformis SLY1812 to produce products in large quantities is described below with reference to specific examples.

Example one

1. Preparing a strain slant and a plate culture medium: the formula of the culture medium is as follows: 5g/L of yeast powder, 10g/L of sodium chloride, 10g/L of peptone, 2g/L of glucose, 16g/L of agar, pH7.0, and sterilizing at 121 ℃ for 30min.

Wherein, the culture conditions of the strain slant are as follows: after inoculation, the cells were incubated at 37 ℃ for 22 hours.

2. The liquid primary strain fermentation medium is prepared from the following raw materials (component content): 5g/L of yeast powder, 10g/L of sodium chloride, 10g/L of peptone and 2g/L of glucose, wherein the volume of water is up to 1L, the pH value is 6.8-7.2, and the sterilization condition is 121 ℃ for 30min.

Wherein, the liquid first-level strain culture conditions are as follows: inoculating strains from a solid inclined test tube into a triangular flask with the volume of 1L at the temperature of 37 ℃ by using 250mL of liquid culture medium in each flask, culturing for 22 hours at the rotating speed of 220rpm of a shaking table, and finishing the culture when the spore rate of the strains is detected to be more than 90% by a microscope.

3. The liquid secondary strain fermentation medium is prepared from the following raw materials (component content): 60g/L of soybean meal, 60g/L of corn starch, 5g/L of glucose, 5g/L of corn steep liquor, 3g/L of light calcium carbonate, 1g/L of potassium carbonate, 2g/L of calcium chloride, 2g/L of dipotassium phosphate, 3 ten thousand U/L of neutral protease, 32000U/L of medium-temperature amylase and pH7.0; the sterilization condition is 0.12MPa, and the sterilization is carried out for 30min at 121 ℃.

Wherein, the secondary strain culture conditions are as follows:

a culture container: 100L liquid stirring type aerobic fermentation tank

Pretreatment in enzymolysis: (1) After the culture medium (soybean meal powder, corn starch, glucose and corn steep liquor) is put into the container, the volume is fixed, and the temperature is raised to 45 ℃; then adding neutral protease dissolved in advance, and stirring for 1.5h;

(2) After the first step of enzymolysis is finished, the temperature is raised to 65 ℃, after the temperature is stable, medium temperature amylase dissolved in advance is added, and enzymolysis is continued for 1 hour;

(3) After the enzymolysis is finished; adding inorganic salt (light calcium carbonate, potassium carbonate, calcium chloride, potassium dihydrogen phosphate), heating and sterilizing;

(4) Cooling to 36 deg.C after sterilization, and adjusting pH to 7.0 with ammonia water;

wherein, the culture temperature is: 37-38 ℃, ventilation: liquid: air ratio of 1: (0.6-1): 1.2, stirring at a rotating speed of 200r/min;

inoculation: inoculating the first-stage liquid strain according to the inoculation amount of 2%.

pH: within the first 8 hours after inoculation, when the pH value of the culture medium is lower than 6.8, the pH value is adjusted back to 7.0 by using concentrated ammonia water;

and (3) finishing the culture: after culturing for 14 hours, microscopic observation shows that the spore rate of the thalli reaches 90%, the fermentation is stopped, the temperature is raised to 65 ℃, and the temperature is kept for 30min.

4. Liquid three-stage strain culture:

inoculation and culture: inoculating the second-level strain into 1000L fermentation tank sterilized culture medium with 10% inoculation amount, wherein the culture medium components and culture conditions are the same as those of the second-level strain.

And (3) finishing the culture: after culturing for 12 hours, microscopic observation shows that the spore rate of the thalli reaches 92%, the fermentation is stopped, the temperature is raised to 65 ℃, and the temperature is kept for 30min.

5. Liquid-state four-stage strain culture:

inoculation and culture: the third-level strain is inoculated into 10000L fermentation tank sterilization culture medium according to the inoculation amount of 10%, and the components and the culture conditions of the culture medium are the same as those of the second-level strain and the third-level strain.

And (3) finishing the culture: after culturing for 12 hours, microscopic observation shows that the spore rate of the thalli reaches 90%, the fermentation is stopped, the temperature is raised to 65 ℃, and the temperature is kept for 30min.

6. Solid-state culture:

the solid fermentation medium is prepared from the following raw materials: 80% of wheat bran (solid content), 10% of soybean meal (solid content), 10% of rice hull (solid content), and 52% of initial moisture content of the matrix.

The solid state fermentation culture process conditions are as follows: sterilizing at 121 ℃ for 30min, cooling the material to 70 ℃, inoculating four-stage strains according to the proportion of 8%, uniformly mixing, transferring into a disc starter propagation machine with the diameter of 16 m for culture, supporting the bottom of the material to be a porous stainless steel plate, wherein the thickness of a material layer is 37cm, the single batch feeding amount is 12 tons (dry weight), ventilating and cooling to 35 ℃ when the temperature of the material rises to more than 42 ℃, culturing for 34 hours, and detecting related indexes after the microscopic spore rate of thalli in the material reaches more than 90 percent, ending fermentation and naturally ventilating and drying.

Index measurement: 7.9 percent of water, 13960U/g of neutral protease activity, 24885U/g of alkaline protease activity, 17.2 percent of crude protein and 3.4 percent of small peptide.

Example two:

1. preparing a strain slant and a plate culture medium: the formula of the culture medium is as follows: 4g/L of yeast powder, 8g/L of sodium chloride, 8g/L of peptone, 1.5g/L of glucose, 20g/L of agar and pH7.0, and sterilizing for 30min at 121 ℃.

Wherein, the culture conditions of the strain slant are as follows: after inoculation, the cells were incubated at 37 ℃ for 30 hours.

2. The liquid first-level strain fermentation medium is prepared from the following raw materials (component content): 4g/L of yeast powder, 8g/L of sodium chloride, 8g/L of peptone and 1.5g/L of glucose, wherein the volume of water is up to 1L, the pH value is 6.8, and the sterilization condition is that sterilization is carried out for 30min at 121 ℃.

Wherein, the liquid first-level strain culture conditions are as follows: inoculating strains from a solid inclined test tube in a triangular flask with the volume of 1L and liquid culture medium of 200mL per bottle at the temperature of 37 ℃, culturing for 28 hours at the rotating speed of 250rpm of a shaking table, and finishing the culture when the spore rate of the strains is detected to be more than 90% by a microscope.

3. The liquid secondary strain fermentation medium is prepared from the following raw materials (component content): 75g/L of soybean meal, 50g/L of corn starch, 6g/L of glucose, 8g/L of corn steep liquor, 4g/L of light calcium carbonate, 1.2g/L of potassium carbonate, 2.5g/L of calcium chloride, 3g/L of dipotassium hydrogen phosphate, 3 ten thousand U/L of neutral protease, 32000U/L of medium-temperature amylase and pH7.0; the sterilization condition is 0.12MPa, and the sterilization is carried out for 30min at 121 ℃.

Wherein, the secondary strain culture conditions are as follows:

a culture container: 100L liquid stirring type aerobic fermentation tank

Pretreatment of enzymolysis: (1) After the culture medium (soybean meal powder, corn starch, glucose and corn steep liquor) is put into the container, the volume is fixed, and the temperature is raised to 50 ℃; then adding neutral protease dissolved in advance, and stirring for 1.5h;

(2) After the first step of enzymolysis is finished, the temperature is increased to 68 ℃, after the temperature is stable, medium temperature amylase dissolved in advance is added, and enzymolysis is continued for 1 hour;

(3) After the enzymolysis is finished, adding inorganic salt (light calcium carbonate, potassium carbonate, calcium chloride and potassium dihydrogen phosphate), heating and sterilizing;

(4) Cooling to 38 ℃ after sterilization, and adding ammonia water to adjust the pH value to 7.0;

wherein, the culture temperature is: 37-38 ℃, ventilation: liquid: air ratio of 1: (0.6-1): 1.2, stirring speed is 200r/min;

inoculation: inoculating the first-stage liquid strain according to the inoculation amount of 2%.

pH: within the first 8 hours after inoculation, when the pH value of the culture medium is lower than 6.8, the pH value is adjusted back to 7.0 by using concentrated ammonia water;

and (3) finishing the culture: after culturing for 14 hours, microscopic observation shows that the spore rate of the thalli reaches 90%, the fermentation is stopped, the temperature is raised to 65 ℃, and the temperature is kept for 30min.

4. Liquid three-stage strain culture:

inoculation and culture: inoculating the second-level strain into 1000L fermentation tank sterilized culture medium with 10% inoculation amount, wherein the culture medium components and culture conditions are the same as those of the second-level strain.

And (3) finishing the culture: after culturing for 12 hours, microscopic observation shows that the spore rate of the thalli reaches 92%, the fermentation is stopped, the temperature is raised to 65 ℃, and the temperature is kept for 30min.

5. Liquid-state four-stage strain culture:

inoculation and culture: inoculating the third-level strain into 10000L fermentation tank sterilization culture medium according to the inoculation amount of 10%, wherein the components and the culture conditions of the culture medium are the same as those of the second-level strain and the third-level strain.

And (3) finishing the culture: after culturing for 12 hours, microscopic observation shows that the spore rate of the thalli reaches 90%, the fermentation is stopped, the temperature is raised to 65 ℃, and the temperature is kept for 30min.

6. Solid strain culture:

solid-state fermentation raw materials: 85% of wheat bran (solid content), 15% of rice hull (solid content), and 50% of initial water content of the matrix.

The solid state fermentation culture process conditions are as follows: sterilizing at 121 ℃ for 30min, cooling the material to 70 ℃, inoculating a fourth-class strain according to the proportion of 8%, uniformly mixing, transferring into a 16-meter-diameter disc koji making machine for culturing, supporting the bottom of the material to be a porous stainless steel plate, wherein the thickness of a material layer is 40cm, the single-batch feeding amount is 11.5 tons (dry weight), ventilating and cooling to 35 ℃ when the temperature of the material rises to more than 42 ℃, culturing for 30 hours, and detecting related indexes after the microscopic spore rate of thalli in the material reaches more than 90%, ending fermentation and naturally ventilating and drying.

Index measurement: 7.3% of water, 15423U/g of neutral protease activity, 25920U/g of alkaline protease activity, 15.6% of crude protein and 2.9% of small peptide.

Example three:

(1) The culture process for preparing the strain slant and the plate culture medium and the liquid first-level strain is the same as that of the first embodiment.

(2) Solid state fermentation culture

Solid fermentation raw material: 85% of wheat bran (solid content), 5% of soybean meal (solid content), 10% of rice hull (solid content), and 47% of initial moisture content of the matrix.

The solid state fermentation culture process conditions are as follows: the mixed and unsterilized materials are loaded into a full-automatic solid-state koji machine in layers, the thickness of each layer of the materials is 1.5cm, the bottom of the materials is supported by a metal gauze, the single-batch fermentation feeding amount is 110kg (dry weight), the materials are sterilized for 30min at 121 ℃, cooled to 70 ℃, first-class strains are inoculated in a spraying mode according to the proportion of 10 percent, when the temperature of the materials is raised to over 38 ℃, the temperature is reduced to 36 ℃ by ventilation, the culture time is 36 hours, the microscopic spore rate of the strains in the materials reaches over 95 percent, the fermentation is finished, and relevant indexes are detected after natural ventilation drying.

Index measurement: 8.2% of water, 16023U/g of neutral protease activity, 27550U/g of alkaline protease activity, 16.1% of crude protein and 3.1% of small peptide.

While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. The Bacillus licheniformis is Bacillus licheniformis (SLY 1812), which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC25162.

2. A method for screening Bacillus licheniformis, which is characterized by comprising the following steps:

separating aerobic bacterial strains from the excrement of the laying hens;

transferring the separated aerobic bacterial strain to an LB culture medium for activation, inoculating the aerobic bacterial strain to a casein agar culture medium by a point inoculation method, culturing for 24 hours at 37 ℃, and selecting a bacterial strain with a transparent ring size reaching a preset range;

culturing the selected strain on solid culture medium at 37 deg.C for 40 hr, drying the culture in drying oven at 40 deg.C until the water content is 6-8%, measuring the activities of neutral protease and alkaline protease of the fermented product, and selecting Bacillus licheniformis (Bacillus licheniformis) SLY1812 with highest neutral protease activity.

3. The method for screening bacillus licheniformis according to claim 2, characterized in that the solid medium comprises 85% wheat bran and 15% rice hull in the matrix and has a water content of 45%.

4. A Bacillus licheniformis preparation comprising the Bacillus licheniformis (SLY 1812) according to claim 1.

5. A method for preparing a Bacillus licheniformis product, wherein the Bacillus licheniformis is the Bacillus licheniformis (SLY 1812) of claim 1, the method comprising:

performing slant culture on the Bacillus licheniformis (SLY 1812), wherein the components of the culture medium comprise 4-5g/L of yeast powder, 8-10g/L of sodium chloride, 8-10g/L of peptone, 1.5-2g/L of glucose, 15-20g/L of agar, and the pH value of the culture medium is 6.8-7.2, and the sterilization mode is sterilization at 121 ℃ for 30min; the culture condition is that the inoculated seeds are cultured for 20 to 30 hours at the constant temperature of 37 ℃;

and performing liquid strain fermentation culture on the Bacillus licheniformis (SLY 1812) after the slant culture until the spore rate of the Bacillus licheniformis (SLY) is detected to be more than 90%, and finishing the culture to obtain the Bacillus licheniformis product.

6. The method of claim 5, wherein the liquid fermentation culture of the bacterial species comprises:

fermenting and culturing a liquid primary strain, wherein the component content of a culture medium in the fermenting and culturing of the liquid primary strain is 4-5g/L of yeast powder, 8-10g/L of sodium chloride, 8-10g/L of peptone, 1.5-2g/L of glucose, the balance of water, the total amount of 1L, the pH value of the culture medium is 6.8-7.2, and the sterilization mode is that the sterilization is carried out for 30min at 121 ℃;

the culture condition is that at 37 deg.C, a 1L volume triangular flask is adopted, each flask is filled with 200-250mL liquid culture medium, strains are inoculated from a solid inclined test tube, the rotation speed of a shaker is 200-250rpm, and the culture is carried out for 22-28 hours.

7. The method of claim 6, wherein the liquid fermentation culture further comprises:

liquid secondary strain fermentation culture, wherein the component content of a culture medium in the liquid secondary strain fermentation culture is 60-75g/L of soybean meal, 50-60g/L of corn starch, 4-6g/L of glucose, 5-8g/L of corn steep liquor, 2-4g/L of light calcium carbonate, 0.7-1.2g/L of potassium carbonate, 1.5-2.5g/L of calcium chloride, 2-3g/L of dipotassium phosphate, 2-4 ten thousand U/L of neutral protease, 1000-2000U/L of medium temperature amylase and pH6.8-7.2; sterilizing at 121-123 deg.C for 20-30min under 0.12MPa; the culture temperature is 37-38 ℃.

8. The method of claim 7, wherein the step of preparing the culture medium for the fermentation culture of the liquid secondary strain comprises:

putting the soybean meal powder, the corn starch, the glucose and the corn steep liquor in the components of the culture medium into an aerobic fermentation tank for constant volume, and heating to 45-50 ℃; adding neutral protease dissolved in advance, and stirring for 1.5h;

heating to 65-68 deg.C, adding pre-dissolved medium temperature amylase after temperature is stable, and continuing enzymolysis for 1h;

and after the enzymolysis is finished, adding the light calcium carbonate, the potassium carbonate, the calcium chloride and the potassium dihydrogen phosphate, and heating and sterilizing at 121-123 ℃ for 20-30min.

9. A process for the preparation of a Bacillus licheniformis product according to any of the claims 5-8 further comprising: performing solid fermentation on the strain subjected to fermentation culture of the liquid strain, wherein the solid fermentation culture medium comprises the following components in percentage by solid: 60-80% of wheat bran, 0-15% of soybean meal, 10-20% of rice hull and 45-55% of initial moisture content of matrix.

10. The method for preparing a bacillus licheniformis product according to claim 9, characterized in that the solid state fermentation is carried out under the following conditions: sterilizing the culture medium at 121 deg.C for 20-30min, cooling to 60-70 deg.C, inoculating liquid strain at a ratio of 4-8%, mixing, maintaining the temperature of material at 35-42 deg.C, aerobic fermenting for 30-40 hr until the microscopic spore rate of strain reaches above 90%, and drying at 70 deg.C to obtain solid product of Bacillus licheniformis.

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