Bacillus subtilis, method for preparing yellow corn silage feed under strong alkali condition and product

Abstract

The invention discloses bacillus subtilis, a method for preparing yellow corn silage feed under a strong alkali condition and a product thereof, and belongs to the technical field of microorganisms. The technical problem to be solved is that the survival rate of the existing bacillus subtilis is lower than 30% under the strong alkali condition of pH 9-11, and the existing bacillus subtilis cannot normally grow and propagate. The preparation of the yellow corn silage feed under a strong alkali condition needs additional pH adjustment, the process is complex and the cost is high; the crude protein content of the existing yellow corn silage feed is only 3-5%, and the requirements of livestock cannot be met. According to the technical scheme, the strong-alkali-resistant bacillus subtilis is provided, the preservation number is CGMCC No.36677, the survival rate of the strain is larger than or equal to 80% under the condition that the pH is 9-11, the straw degradation rate is larger than or equal to 25%, no waste water is generated when the strain is used for preparing the yellow silage feed, the cost is reduced compared with an existing technology, the protein content is increased by 50%-80%, palatability is good, and the method is suitable for large-scale straw resource utilization and feed protein production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to a Bacillus subtilis and a method and product for preparing yellow forage under strong alkali conditions. BACKGROUND

[0002] Technical content for understanding the present application: Scientific development and utilization of straw resources can not only protect the rural ecological environment and promote the sustainable and coordinated development of agriculture, but also can alleviate the tight supply of rural feed and help farmers reduce costs and increase efficiency.

[0003] However, crop straw is rich in lignin, cellulose and hemicellulose, which are intertwined to form a dense structure, hindering the degradation of enzymes and microorganisms, resulting in low digestibility of livestock after eating straw. In addition, the protein content of straw is low, and the palatability is poor, which makes it difficult to provide sufficient nutrition for the growth of livestock.

[0004] Microbial protein has obvious advantages as a feed protein. In terms of production characteristics, it has high space utilization rate and is not affected by environment and climate. The production scale can be flexibly adjusted according to demand to achieve rapid and stable supply. The raw materials can be flexibly selected to utilize various types of organic waste, and the production does not depend on arable land, which effectively reduces the pressure on natural resources. At the same time, compared with traditional treatment methods, the energy consumption of fermented straw is lower, the carbon emission is less, and it will not pollute the soil and water. In terms of nutrition, the protein content of microorganisms is rich, between 20-40%, the ratio of essential amino acids to total amino acids (EAA / TAA) is about 47%, the ratio of essential amino acids to non-essential amino acids (EAA / NEAA) is about 0.91, the digestion and absorption rate is high, and it is also rich in various vitamins and minerals. Compared with soybean meal, methionine is a limiting amino acid, and it contains trypsin inhibitors and other anti-nutritional factors, which affect animal digestion and absorption.

[0005] Among them, Bacillus subtilis as a probiotic shows many benefits in livestock breeding. It can secrete various digestive enzymes to improve feed utilization efficiency, promote nutrient absorption, directly improve the growth efficiency of livestock, enhance the immune regulation and disease resistance of livestock, optimize the balance of intestinal microecology, and maintain the health of intestinal flora. Bacillus subtilis can also provide high-quality bacterial protein for livestock, which is rich in essential amino acids and can produce alternative protein for feeding.

[0006] The current mainstream process for producing single-strain feed protein substitutes from crop straw is mainly based on the technical route of "mechanical pretreatment + extreme acid-base pretreatment + enzymatic hydrolysis + sterilization and fermentation". The core process includes the following key steps: first, the harvested straw is mechanically pretreated by crushing and puffing to break it into smaller loose particles; then, a 2-4% sulfuric acid or sodium hydroxide solution is used to pretreat the straw at 180-220°C high temperature and 0.8-1.5 MPa high pressure for 2-4 hours to destroy the lignin-cellulose complex structure; then, cellulase (about 20-40 FPU / g substrate) is used for enzymatic hydrolysis at 50°C and pH 4.8 for 12-24 hours to obtain a degradation solution containing reducing sugars such as glucose and xylose; after enzymatic hydrolysis, the reaction system is transferred to a fermentation system, sterilized by high-pressure steam, and inoculated with specific strains for fermentation. After a cultivation period of 3-5 days, microbial protein products can be harvested.

[0007] This process system has significant technical and economic bottlenecks: 1) the sterilization step before fermentation requires special pressure-resistant reaction vessels and a large amount of steam, with equipment depreciation and energy costs accounting for more than 45% of the total cost; 2) the wastewater generated by acid-base pretreatment requires a separate wastewater treatment system, with an additional cost of 300-500 yuan per ton of product; 3) the high cost of high-temperature and high-pressure fermentation equipment makes it difficult to achieve large-scale operations.

[0008] Retrieved relevant patent documents: A method for preparing dry corn straw and whole wheat grass mixed feed was disclosed in CN114304397A, published on April 12, 2022. The method uses Bacillus subtilis, Bacillus licheniformis, and Lactobacillus plantarum complex fermentation agents. The raw materials need to be cut and the moisture content needs to be adjusted to 50%-60% before being sealed and fermented under vacuum for 30-40 days. This method relies on multiple bacterial species and does not address the adaptability to strong alkali conditions, and requires strict control of moisture and vacuum environment.

[0009] Retrieved relevant non-patent literature: Journal of Soil and Crops, "Effect of Two Bacillus Species on the Micro-storage of Crop Straw", Vol. 12, No. 2, published in June 2023. The literature discloses that Bacillus subtilis has a degradation effect on neutral detergent fiber, acid detergent fiber, and acid detergent lignin of corn straw, but has no significant effect on rice straw. During the fermentation process, the content of butyric acid increases, reducing the fermentation quality.

[0010] The existing technology represented by the foregoing documents has at least the following unsolved technical problems or defects: The existing Bacillus subtilis has poor alkali resistance, and the survival rate is less than 30% under strong alkali conditions (pH 9-11), and cannot grow and reproduce normally, resulting in a straw degradation rate of less than 10%. Relevant evidence is that the conventional Bacillus subtilis in the relevant non-patent literature can only effectively degrade straw in a neutral to weakly acidic environment, and the patent document CN114304397A needs to control the pH of the fermentation system at 4.1-5.1. In the existing yellow feed preparation process, if strong alkali pretreatment (such as sodium hydroxide, ammonia) is used, an additional pH adjustment step is required, otherwise the strain cannot survive, increasing the complexity and cost of the process. SUMMARY

[0011] The purpose of the present application is to provide: A Bacillus subtilis and its method and product for preparing yellow feed under strong alkali conditions, and related technologies, to solve the technical problems or combinations thereof that the existing Bacillus subtilis has a survival rate of less than 30% under strong alkali conditions (pH 9-11), cannot grow and reproduce normally, and the preparation of yellow feed under strong alkali conditions requires additional pH adjustment, the process is complex and costly, and the existing yellow feed has a crude protein content of only 3-5%, which cannot meet the needs of livestock.

[0012] Term explanation: Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the claims belongs. Unless otherwise indicated, all documents, patents, patent applications, publications, and the like cited herein are hereby incorporated by reference in their entirety. If there is a plurality of definitions for a term herein, the definition in this section prevails.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and are intended only to explain and not to limit the subject matter of the present application. In the present application, the singular is also included unless otherwise specified. It should also be noted that, unless otherwise specified, "or" and "or" used herein mean "and / or". In addition, the term "includes" and other forms, such as "contains", "contains" and "contains", are not limiting.

[0014] The definition of standard chemical terms can be found in the reference "Microbiology, Higher Education Press, Shen Ping, Chen Xiangdong".

[0015] Unless otherwise specified, conventional methods within the scope of the art are used, such as medium preparation, sterilization, strain culture, etc.

[0016] Unless specific definitions are provided, the use of each of the terms in the specification herein is intended to be treated as a standard term available to those of skill in the art. For example, the use of a kit by the manufacturer can be utilized according to the manufacturer's instructions, or according to a manner well known in the art or described in the specification. Generally, the techniques and methods described above can be practiced according to conventional methods well known in the art from the descriptions in the various general and more specific references that are cited and discussed throughout the specification.

[0017] In one aspect, the present application provides a Bacillus subtilis KC, which is deposited with the China General Microbiological Culture Collection Center on November 19, 2025, and has a preservation number of CGMCC No. 36677.

[0018] In another aspect, the present application provides an application of the above-mentioned Bacillus subtilis KC in the preparation of yellow silage.

[0019] In another aspect, the present application provides a preparation method of yellow silage, which comprises the step of fermentation using Bacillus subtilis KC.

[0020] Specifically, the preparation method comprises the following steps: S1, straw pretreatment; S2, strong alkali ammoniation treatment; S3, inoculation of Bacillus subtilis KC for fermentation; S4, collection of yellow silage.

[0021] In S1, the straw includes but is not limited to at least one of wheat straw, corn straw, rice straw, soybean straw, and cotton straw.

[0022] In S1, the straw pretreatment comprises crushing the straw to 3-5 mm.

[0023] In S2, the strong alkali ammoniation treatment comprises adding 1.5-2.5 times of dilute ammonia water by volume ratio.

[0024] In S3, the inoculation condition is that the inoculation amount is 1% of the volume of ammonia water by volume ratio.

[0025] In S3, the OD600 value of the seed liquid at the time of inoculation is preferably 0.8-1.2.

[0026] In S3, the OD600 value of the seed liquid at the time of inoculation is further preferably 1.0.

[0027] In S3, the fermentation condition is that the fermentation is carried out at room temperature for 30-60 days in a sealed chamber, and the mixture is intermittently mixed.

[0028] In S3, the fermentation conditions are preferably 25℃ for 45 days with intermittent mixing.

[0029] In S4, the yellow silage is collected by spreading out the fermentation product of S3 and allowing the ammonia to volatilize.

[0030] In another aspect, the present application provides the yellow silage prepared by any of the above preparation methods.

[0031] The present application has the following advantages: Compared with the prior art, the Bacillus subtilis of the present application has significantly better technical effects in terms of alkali tolerance, straw degradation rate, and protein synthesis amount. According to experimental tests, the survival rate of the strain of the present application under the condition of pH 10 is increased from 25-30% of the prior art to more than 80-85%; the straw degradation rate is increased from 8-10% of the prior art to more than 25-26%; and the crude protein content of the yellow silage is increased from 3-5% of the prior art to more than 6-9%. According to experimental tests, the cost of preparing the yellow silage by the method of the present application is reduced from 800-900 yuan / ton of the prior art to less than 600-650 yuan / ton, with a cost reduction of 200-250 yuan per ton.

[0032] Deposit Description Deposit Strain: Bacillus subtilis KC; Classification Name: Bacillus subtilis Bacillus subtilis ; Deposit Number: CGMCC No. 36677; Deposit Date: November 19, 2025; Deposit Unit: China General Microbiological Culture Collection Center; Deposit Address: No. 3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 For the efficiency of Bacillus subtilis in degrading straw, **** represents P<0.0001, and the difference is extremely significant.

[0034] Figure 2 For the crude protein content of Bacillus subtilis fermented straw, ** represents P<0.01, and the difference is significant. DETAILED DESCRIPTION

[0035] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application, but do not limit the present application in any way. The following content is merely an exemplary description of the scope of the present application, and those skilled in the art can make various changes and modifications to the present application based on the disclosed content, and such changes and modifications should also belong to the scope of the present application.

[0036] The present application is further described below in the manner of specific examples. The various instruments, devices, equipment, reagents, products, etc. used in the examples of the present application are obtained through conventional commercial channels unless otherwise specified.

[0037] Culture medium and preparation of experimental reagents Prepare LB medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, deionized water to 1 L. Autoclave at 121°C for 20 min. Solid medium requires the addition of 18 g / L agar.

[0038] Prepare phosphate buffer: first prepare 0.2 mol / L potassium dihydrogen phosphate and 0.2 mol / L sodium hydrogen phosphate, then mix them in a ratio of 39:61. Autoclave at 121°C for 20 min.

[0039] Prepare 0.85% physiological saline: sodium chloride 0.85 g, deionized water to 100 mL. Autoclave at 121°C for 20 min.

[0040] Prepare straw screening plate: straw powder 3 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, potassium bisulfate 7.3 g / L, magnesium sulfate heptahydrate 0.5 g / L, ammonium sulfate 5 g / L, copper chloride dihydrate 0.68 g / L, trace element stock solution 5 ml / L, agar 15 g / L. Autoclave at 121°C for 20 min.

[0041] Prepare alkaline straw screening plate: straw powder 3 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, potassium bisulfate 7.3 g / L, magnesium sulfate heptahydrate 0.5 g / L, ammonium sulfate 5 g / L, copper chloride dihydrate 0.68 g / L, trace element stock solution 5 ml / L, agar 15 g / L, and add 0.5 ml of 0.4 g / L sodium hydroxide solution per liter of medium. Autoclave at 121°C for 20 min.

[0042] Prepare straw liquid medium: straw 3 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.5 g / L, potassium bisulfate 7.3 g / L, magnesium sulfate heptahydrate 0.5 g / L, ammonium sulfate 5 g / L, copper chloride dihydrate 0.68 g / L, trace element stock solution 5 ml / L. Autoclave at 121°C for 20 min.

[0043] Prepare MNNG stock solution: accurately weigh 5 mg of MNNG into a sterile test tube, dissolve in 500 μL of DMSO, filter the above solution using a 0.22 μm sterile filter to obtain a MNNG stock solution with a concentration of 10 mg / ml.

[0044] Preparation of 5% Na2S2O3 solution: weigh 1 g of Na2S2O3•5H2O, dissolve in 19 mL of distilled water, filter sterilization (also can be diluted 5 times from 1M Na2S2O3•5H2O).

[0045] Preparation of alkaline cellulose screening medium: cellulose 5 g / L, sodium nitrate solution 2 mL / L (concentration 1 mol / L), urea 1 g / L, ammonium chloride 1 g / L, disodium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 1 g / L, each add 1 mL / L of trace elements, and add 1 mL / L of 0.4 g / L sodium hydroxide solution, agar powder 20 g / L.

[0046] The alkaline cellulose screening medium needs to be sterilized by high pressure steam, the temperature is 121℃, and the time is 30 min. After sterilization, the medium is cooled to 500℃, 1 / 100 volume of Congo red solution is added, mixed and poured into a flat plate.

[0047] Preparation of Congo red solution: weigh 2.53 g of Congo red powder, dissolve in 100 mL of distilled water, completely dissolve, filter sterilization with 0.22 μm filter membrane, store at room temperature in the dark.

[0048] Preparation of trace elements: boric acid 210 mg / L, cobalt chloride hexahydrate 200 mg / L, manganese chloride tetrahydrate 350 mg / L, zinc chloride 280 mg / L, nickel chloride hexahydrate 140 mg / L, sodium molybdate dihydrate 110 mg / L, copper chloride dihydrate 14 mg / L, ferrous sulfate heptahydrate 15 mg / L, and vitamin B12 concentration is 100 mg / L.

[0049] The inorganic salt medium formula is: ammoniated straw 5 g, sodium nitrate solution 2 mL (concentration 1 mol / L), urea 1 g / L, ammonium chloride 1 g / L, disodium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 1 g / L, each add 1 mL of trace elements. The amount added in each fermentation tank is 10 L.

[0050] Example 1 Strain selection Bacillus subtilis was purchased from China Center for Type Culture Collection, strain number: CCTCC AB 130001 The purchased Bacillus subtilis was inoculated into LB liquid medium for recovery culture, and streaked on LB solid medium. Single colonies were selected for PCR and sequencing identification.

[0051] Chemical mutagenesis was performed on Bacillus subtilis, and the mutagenesis method was as follows: Take the strain of Bacillus subtilis which is identified correctly, and streak culture on alkaline straw screening plate, and place in 37℃ constant temperature incubator for culture for 48h. Seed the activated strain with one ring of bacteria in straw liquid culture medium, and culture at 37℃ with 150rpm shaking.

[0052] After culturing in straw liquid culture medium for a period of time, it can be transferred to LB medium for continuous culture, and the bacterial density is estimated by measuring the OD value of the bacterial solution.

[0053] Concentration gradient experiment: take 500μL of bacterial suspension, add 20 / 40 / 60 / 80 / 100 / 120 / 140 / 160 / 180μL of MNNG stock solution respectively, and make up to 1mL with phosphate buffer, mix well by blowing, and treat in a light-proof environment at 30℃ with 150rpm shaking for 20min. After the reaction is completed, centrifuge at 8000g for 5min, discard the supernatant and collect the bacterial cells, and resuspend in an equal volume of normal saline (500μL) after washing twice with normal saline. Add the treated sample to the screening medium.

[0054] Dilute the mutagenized bacterial solution, and the dilution factors are 1×10 -2 , 1×10 -4 , 1×10 -6 , 1×10 -8 , respectively, and spread on alkaline cellulose screening medium. Culture at 37℃ for 48h. Observe the transparent circles on the screening medium, and select the strains with larger transparent circles to LB medium for culture. Repeat the process, and select one strain with outstanding performance, named Bacillus subtilis KC, and store at -80℃.

[0055] Example 2: Alkali tolerance detection of the strain Use the replica plating method to detect the survival rate of the strain in alkaline environment: Step one: activation of the strain and preparation of the seed solution Take a small amount of Bacillus subtilis KC bacterial mat from the preserved glycerol tube, and perform streak isolation on LB agar plate (pH 7.0) with an inoculation loop.

[0056] Place the plate in a 30℃ constant temperature incubator and culture for 18-24 hours until single colonies with uniform morphology are formed.

[0057] Pick up a single colony and inoculate into a 250mL shake flask containing 50mL of LB liquid medium.

[0058] Place the shake flask in a 30℃, 180rpm shaking incubator for 16 hours to obtain a seed solution in the logarithmic growth phase, and the bacterial solution concentration (OD600) is about 0.8 at this time.

[0059] Step two: preparation and dilution of the initial bacterial solution Take 1 mL of the seed liquid above, and perform gradient dilution (e.g., 10 -4 , 10 -5 , 10 -6 ) with sterile normal saline to obtain a bacterial suspension of appropriate concentration.

[0060] The optimal dilution is determined by preliminary experiments so that 30-300 dispersed single colonies grow on the control plate for subsequent plating, facilitating accurate counting. In this embodiment, a dilution of 10 -5 is selected.

[0061] Step Three: Detection of Alkali Tolerance of Strains by Replica Plating Preparation of Master Plate (Control Plate): Take 100 μL of bacterial suspension at a dilution of 10 -5 and drop it onto the center of a sterile LB agar plate (pH 7.0).

[0062] Use a sterile spreader to evenly spread the bacterial solution over the entire surface of the plate.

[0063] After the bacterial solution is completely absorbed by the culture medium, invert the plate in a 30°C incubator and incubate for 24 hours. This plate serves as the master plate.

[0064] Replica Inoculation: Prepare sterile replica tools, ensuring that the velvet cloth is flat, clean, and sterile.

[0065] In a clean bench, place the incubated master plate (with the colonies facing up) on a stable table surface.

[0066] Gently press the velvet surface of the replica tool against the master plate, ensuring that all colonies are evenly transferred to the velvet cloth, avoiding sliding during the process.

[0067] Lift the replica tool and immediately gently press it against the surface of the first test plate (alkaline LB agar plate, pH 12.0) for the first transfer.

[0068] After completion, lift the replica tool again without changing the velvet cloth and immediately gently press it against the surface of the second control plate (LB agar plate, pH 7.0) for the second transfer. This plate serves as the replica control plate to verify the effectiveness of the replica process.

[0069] Incubation and Observation: Invert both the alkaline test plate and the replica control plate in a 30°C incubator and incubate for 48-72 hours.

[0070] During the incubation period, observe and record the growth of colonies on each plate regularly (e.g. 24h, 48h, 72h).

[0071] Result observation: After 48h incubation, the replica control plate (pH 7.0) showed colonies at positions and number consistent with the master plate, indicating that the replica operation was successful and the strain was viable.

[0072] On the alkaline test plate (pH 12.0), clear colonies were also observed at positions corresponding to the colonies on the master plate. Although the growth rate might be slightly slower than that on the control plate, or the colony diameter might be slightly smaller, the morphology was normal, indicating that the strain could grow in the strong alkaline environment of pH 12.0.

[0073] Survival rate calculation: Colony counting: Count the colonies on the master plate, replica control plate and alkaline test plate respectively. To ensure accuracy, count each plate 3 times and take the average.

[0074] Average number of colonies on master plate: N_master Average number of colonies on replica control plate: N_control_replica Average number of colonies on alkaline test plate: N_alkaline Replica efficiency correction: First calculate the transfer efficiency (E_transfer) of the replica operation itself to exclude operation errors.

[0075] E_transfer (%) = (N_control_replica / N_master) x 100% Calculate theoretical transfer number: Assuming that the transfer efficiency of the alkaline plate is the same as that of the control plate, the number of colonies that should be transferred to the alkaline plate in theory is: N_theoretical = N_master x E_transfer Calculate survival rate: The actual survival rate in alkaline environment (S_alkaline) is obtained by comparing the actual number of colonies grown with the theoretical transfer number.

[0076] S_alkaline (%) = (N_alkaline / N_theoretical) x 100% In this example, the data obtained is shown in Table 1: Table 1

[0077] As shown in Table 1, the survival rate of Bacillus subtilis KC under pH 12 conditions is ≥80%, indicating good alkali resistance.

[0078] Example 2 Fermentation of straw to produce feed Take two portions of about 10 kg of dried and pulverized straw (pulverized to 3 mm), add iron chloride at a mass fraction of one hundred-thousandth, and mix with water at a solid-liquid ratio of 1:2. Then, use an expander (Liaoyuan Mucheng Company 9p-150 model expander) to expand the mixture to obtain expanded straw for later use. Put the expanded straw and twice the volume of the straw of 4% concentrated dilute ammonia water into a sealed storage tank. Leave an appropriate amount of air in the tank. Turn and roll the tank to mix the expanded straw and dilute ammonia water thoroughly. After thorough mixing, heat at 85°C for 24 hours to obtain ammoniated straw. During this period, occasionally mix to ensure that the ammoniation process is complete.

[0079] After the ammoniation is complete and the temperature is reduced to room temperature, measure the pH, which is 11.2. Then, add Bacillus subtilis seed liquid and inorganic salt medium to the ammoniated straw storage tank, and seal the tank again. The inoculation volume of the seed liquid is 1% of the volume of the ammonia water, and the OD value of the seed liquid is 1. Cultivate at room temperature, and during the cultivation, turn the tank upside down 10 times each morning and evening to mix evenly.

[0080] After 45 days of cultivation of Bacillus subtilis, open the tank and take out the fermented straw, which is the high-protein yellow storage straw fermented by a single strain.

[0081] The seed liquid is prepared as follows: inoculate the preserved Bacillus subtilis KC and the original strain WT that has not been subjected to mutagenesis and screening into LB broth medium, and cultivate overnight at 37°C in a shaking incubator at a speed of 200 rpm. Then, centrifuge the culture of the two strains of Bacillus subtilis at a speed of 5000 rpm for 5 min. Discard the supernatant, add sterile water, vortex to mix, centrifuge again at a speed of 5000 rpm for 5 min, discard the supernatant, add sterile water, and adjust the OD value of the bacterial cells to 1.2 at a wavelength of 600 nm. The seed liquid obtained can be used for inoculation.

[0082] After fermentation is complete, pour out the product in the fermentation tank and spread it out to air. After ammonia volatilization is complete, the product can be fed. To facilitate storage and measurement, dry the product in an oven at 85°C for 1 day to obtain dry straw.

[0083] Example 1 After fermentation is complete as described in Example 1, pour out the product in the fermentation tank and spread it out to air. Spread it out in a well-ventilated place for 3 days until the moisture content of the straw no longer decreases to obtain a solid product, and then send it to an oven for drying.

[0084] Place the obtained solid product in an oven at 85°C, dry it for 1 day, take it out, cool it to room temperature, and weigh it (accurate to 0.1 g). Repeat this operation until the difference in mass measured consecutively is not more than 1% of the mass of the sample.

[0085] After the sample is completely dried, it is weighed using an electronic balance, and the straw degradation rate is calculated. The formula is:

[0086] In this embodiment, the original strain WT without mutagenesis is used as a control. The straw degradation rate of the Bacillus subtilis KC of the present application is about 26% after fermentation is completed, and the straw degradation rate of the original strain WT without mutagenesis is about 10%. Compared with the control, the straw degradation rate of the Bacillus subtilis KC is significantly increased.

[0087] The specific degradation efficiency is shown in Table 2: Table 2 Straw degradation rate of fermentation

[0088] The straw degradation efficiency of the Bacillus subtilis provided by the present application is shown in Table 2. Figure 1 The data is tested using t-test, and **** in the figure represents P < 0.0001, which is extremely significant.

[0089] The crude protein content of the dried sample is measured using the Kjeldahl method. The method is as follows: About 2g of the sample is weighed, and the process of dissolving in distilled water-centrifuging to remove supernatant is repeated three times to sufficiently wash away the nitrogen-containing inorganic salts, and then the sample is sent into an oven for drying at 85℃ overnight to obtain a desalted sample.

[0090] About 1g of the desalted sample is weighed, 2 pieces of Kjeldahl nitrogen reagent are added, 20mL of sulfuric acid is added, the Kjeldahl flask is placed on an electric furnace, and heating is started at 200℃. After the sample is carbonized and the foam disappears, the temperature is increased to 420℃, and then heating is continued for about 2h. After cooling to room temperature, the sample is taken out.

[0091] After the sample is cooled, the automatic Kjeldahl nitrogen analyzer is used to determine according to the operating instructions. The operation sequence is as follows: 20mL of water is added, 80mL of sodium hydroxide solution is added, automatic distillation is performed for 5min, and the distillation condensate is absorbed with 200mL of boric acid absorption solution.

[0092] A 40mM sulfuric acid titration solution is prepared and titrated with boric acid absorption solution. The solution changes from blue-green to gray-red as the end point.

[0093] Blank determination: 0.5g of sucrose is accurately weighed, and the blank determination is performed according to the above experimental method. The volume of sulfuric acid titration solution consumed should not exceed 0.1mL.

[0094] The crude protein content is calculated in terms of mass fraction w, and the value is expressed in percentage (%). The formula is as follows:

[0095] In the formula: V 2 — The volume of sulfuric acid titration solution consumed in titrating the sample, in milliliters (mL); V 1 — Volume of sulfuric acid titrant consumed in the titration of blank, in milliliters (mL); c —The concentration of the sulfuric acid titration solution, expressed in moles per liter (mol / L). m —Sample mass, in grams (g); V — Total volume of the sample digestion solution, in milliliters (mL); V’ —The volume of digestion solution used during distillation, in milliliters (mL); 14 — Molar mass of nitrogen, in grams per mole (g / mol). 6.25 — Average coefficient for converting nitrogen to crude protein.

[0096] Each experimental group requires at least two parallel samples for measurement, and the arithmetic mean of the samples is used as the measurement result. The result is expressed to two decimal places.

[0097] In this embodiment, the original strain without mutation was used as a control. The crude protein content of Bacillus subtilis KC obtained in this invention was between 6% and 9% after fermentation, while the crude protein content of the original strain without mutation was between 3% and 5%. Compared with the control group, the crude protein content of fermented straw by Bacillus subtilis KC was significantly increased.

[0098] The crude protein content of straw fermented by Bacillus subtilis provided in this invention is shown in Table 3: Table 3 Crude protein content of fermented straw

[0099] The crude protein content of Bacillus subtilis fermented straw provided by this invention is as follows: Figure 2 As shown in the figure. The data were analyzed using a t-test, and ** in the figure represent P < 0.01, indicating a significant difference.

[0100] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis KC, characterized in that, The Bacillus subtilis KC is preserved in China General Microbiological Culture Collection Center on November 19, 2015, and the preservation number is CGMCC No. 36677.

2. Use of the Bacillus subtilis KC of claim 1 in the preparation of yellow silage.

3. A method of preparing a yellow feedstuff, characterized in that, The preparation method comprises the step of fermentation using the Bacillus subtilis KC of claim 1.

4. The production method according to claim 3, characterized by, The method comprises the following steps: S1, straw pretreatment; S2, strong alkali ammoniation treatment; S3, inoculation of Bacillus subtilis KC for fermentation; S4, collection of yellow silage.

5. The preparation method according to claim 4, characterized in that, In S1, the straw comprises at least one of wheat straw, corn straw, rice straw, soybean straw and cotton straw.

6. The preparation method according to claim 4, characterized in that, In S1, the straw pretreatment comprises the step of crushing the straw to 3-5 mm.

7. The preparation method according to claim 4, characterized in that, In S2, the strong alkali ammoniation treatment comprises adding 1.5-2.5 times of dilute ammonia water by volume ratio.

8. The preparation method according to claim 4, characterized in that, In S3, the inoculation condition is that the inoculation amount is 1% of the volume of ammonia water by volume ratio.

9. The preparation method according to claim 4, characterized in that, In S3, the fermentation condition is that the fermentation is carried out at room temperature for 30-60 days, and the mixture is stirred intermittently.

10. The yellow silage prepared by the preparation method of any one of claims 3-9.

Images