Preparation and Application of a Bacillus subtilis Strain for Improving Utilizable Protein in Distillers Dried Grains with Solubles and Its Compound Bacterial Agent

Abstract

The present invention belongs to the technical field of microbial engineering, and particularly relates to a Bacillus subtilis for enhancing the available protein in alcoholic corn distillers grains and the preparation and application of its composite bacterium agent. The present invention provides a Bacillus subtilis with a preservation number of CGMCC No. 32291. The Bacillus subtilis of the present invention can secrete protease and cellulase, degrade macromolecular proteins in alcoholic corn distillers grains into small molecular weight proteins during fermentation, and reduce the content of prolamin. The present invention also provides a composite bacterium agent, which includes Lactobacillus plantarum and the Bacillus subtilis described in the above technical solution. The composite bacterium agent of the present invention can increase the viable bacteria count and the content of corresponding beneficial metabolites in fermented alcoholic corn distillers grains, enrich the nutrition of alcoholic corn distillers grains, and improve the utilization rate of alcoholic corn distillers grains.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial engineering, and specifically relates to a Bacillus subtilis for improving the available protein in alcoholic corn distillers grains and the preparation and application of its compound bacterium agent. Background Art

[0002] Alcoholic corn distillers grains are the corn distillers grains used for alcohol production and are derived from the process of corn fermentation to produce alcohol. During the process of corn fermentation to produce alcohol, a large amount of alcoholic corn distillers grains are generated, causing environmental pollution and waste of resources. In order to solve the problems such as the shortage of protein feed resources and the low utilization rate of corn, it is very necessary to improve the nutritional value of alcoholic corn distillers grains.

[0003] At present, solid-state fermentation technology is the key technology for improving the feeding value of alcoholic corn distillers grains. Solid-state fermentation can improve its nutritional value by increasing the digestibility, amino acid composition and flavor of protein feed, and at the same time improve the antioxidant capacity and immunity of the body to a certain extent, which is beneficial to the growth of animals.

[0004] Alcoholic corn distillers grains are extremely abundant in yield. In addition to containing a large amount of protein nutrients, they also contain rich carotenoids and are often used as animal feed. However, the protein in alcoholic corn distillers grains is mainly prolamin. Animals have a low absorption and utilization rate of most prolamins, and the taste of prolamin is poor, reducing the feeding value of alcoholic corn distillers grains and resulting in waste of resources. At the same time, after hydrolysis of alcoholic corn distillers grains, essential amino acids for the body are lacking, restricting its application in animal feed and still requiring further development.

[0005] In view of the environmental pollution caused by the corn processing by-product alcoholic corn distillers grains and the insufficient utilization of corn processing by-product resources, etc., how to reduce the content of prolamin in alcoholic corn distillers grains and improve the utilization rate of alcoholic corn distillers grains is a technical problem that needs to be solved urgently. Summary of the Invention

[0006] The purpose of the present invention is to provide a Bacillus subtilis for improving the available protein in alcoholic corn distillers grains and the preparation and application of its compound bacterium agent. The Bacillus subtilis provided by the present invention can reduce the content of prolamin in alcoholic corn distillers grains and improve the utilization rate of alcoholic corn distillers grains.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention provides a strain of Bacillus subtilis ( Bacillus subtilis ), and the preservation number is CGMCC No. 32291.

[0009] The present invention provides a compound bacterium agent, which includes Lactobacillus plantarum and Bacillus subtilis described in the above technical solution.

[0010] Preferably, the application form of the Bacillus subtilis includes a fermentation broth, and the viable count of the fermentation broth is ≥10 9 CFU / mL; the application form of the Lactobacillus plantarum includes a fermentation broth, and the viable count of the fermentation broth is ≥10 9 CFU / mL.

[0011] The present invention provides the application of the compound bacterium agent described in the above technical solution in fermenting alcoholic corn distillers grains and / or reducing the content of prolamin in alcoholic corn distillers grains.

[0012] The present invention provides a preparation method for fermenting alcoholic corn distillers grains, which includes: inoculating Bacillus subtilis in the compound bacterium agent described in the above technical solution into the raw material of alcoholic corn distillers grains for solid-state aerobic fermentation culture, and then inoculating Lactobacillus plantarum in the compound bacterium agent described in the above technical solution for solid-state anaerobic fermentation culture.

[0013] Preferably, the temperature of the solid-state aerobic fermentation culture is 28-35°C, and the time is 3-5 days; the temperature of the solid-state anaerobic fermentation culture is 30-40°C, and the time is 3-5 days.

[0014] Preferably, the initial water content of the raw material of alcoholic corn distillers grains is 60wt%-100wt%.

[0015] Preferably, the inoculation amount of the Bacillus subtilis is 4wt%-10wt% of the raw material of alcoholic corn distillers grains; the inoculation amount of the Lactobacillus plantarum is 4wt%-10wt% of the raw material of alcoholic corn distillers grains.

[0016] The present invention provides the application of the fermented alcoholic corn distillers grains obtained by the preparation method described in the above technical solution in any one or more of the following 1)-4),

[0017] 1) Livestock and poultry breeding;

[0018] 2) Improving the growth performance of livestock and poultry;

[0019] 3) Preparing livestock and poultry animal feed.

[0020] The present invention provides a feed additive, which includes the fermented alcoholic corn distillers grains obtained by the preparation method described in the above technical solution.

[0021] Advantages of the present invention: The present invention provides a strain of Bacillus subtilis with a preservation number of CGMCC No. 32291. The Bacillus subtilis of the present invention can secrete protease and cellulase, and can fully degrade macromolecular proteins in alcoholic corn distillers grains into small molecular weight proteins during the fermentation process, reducing the content of prolamin.

[0022] The present invention also provides a compound bacterium agent, which includes Lactobacillus plantarum and the Bacillus subtilis described in the above technical solution. Lactobacillus plantarum of the present invention can produce a large amount of acid by fermenting alcoholic corn distillers grains as a substrate, providing an acidic environment for fermentation, regulating the normal flora of the animal gastrointestinal tract, promoting animal growth, and maintaining microecological balance. The compound bacterium agent of the present invention can increase the viable bacteria count and the content of corresponding beneficial metabolites in fermented alcoholic corn distillers grains, enrich the nutrition of alcoholic corn distillers grains, and improve the utilization rate of alcoholic corn distillers grains. The results of the examples show that: using the compound bacterium agent to prepare fermented alcoholic corn distillers grains, and using the obtained fermented alcoholic corn distillers grains as a feed additive to feed chicks and weaned piglets can improve the growth performance of chicks and weaned piglets. Description of the drawings

[0023] Figure 1 It is a cell morphology diagram of Bacillus subtilis under an oil immersion microscope;

[0024] Figure 2 It is a phylogenetic tree of Bacillus subtilis, Figure 2 in which CGMCC No. 32291 represents the Bacillus subtilis of the present invention ( Bacillus subtilis );

[0025] Figure 3 It is the change in the prolamin content in alcoholic corn distillers grains before and after fermentation;

[0026] Figure 4 It is the change in the molecular weight of prolamin in alcoholic corn distillers grains before and after fermentation;

[0027] Figure 5 It is the change in the protein in alcoholic corn distillers grains before and after fermentation, Figure 5 The left side is the prolamin content of alcoholic corn distillers grains before fermentation, and the right side is the prolamin content of alcoholic corn distillers grains after fermentation;

[0028] Figure 6 It is the change in the structure of alcoholic corn distillers grains before and after fermentation, Figure 6 in which the red line represents the alcoholic corn distillers grains before fermentation, and the blue line is the alcoholic corn distillers grains after fermentation.

[0029] Figure 7 It is the change in the activities of acid protease and cellulase before and after fermentation.

[0030] Biological Deposit Description

[0031] Bacillus subtilis ( Bacillus subtilis ), classified and named as Bacillus subtilis ( Bacillus subtilis ), was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC) on October 21, 2024, with the deposit number CGMCC No. 32291. The address of the deposit unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation Modes

[0032] The present invention provides a strain of Bacillus subtilis ( Bacillus subtilis ), with the deposit number CGMCC No. 32291.

[0033] The Bacillus subtilis of the present invention was isolated from dry straw in Gongzhuling City, Changchun City, Jilin Province. The colonies of this strain are milky white, with irregular edges, convex and wrinkled. After Gram staining, the morphological characteristics of the cells were observed under an oil immersion lens (100×) of an optical microscope, indicating that the strain is a Gram-positive bacterium, with rod-shaped cells, blunt ends, and ellipsoidal central spores. Using the 16S rDNA of the Bacillus subtilis for Blast alignment, a phylogenetic tree as shown in Figure 2 was constructed according to the highest similarity sequence provided by GenBank, and it was found that the strain is Bacillus subtilis ( Bacillus subtilis ).

[0034] The Bacillus subtilis of the present invention has the functions of secreting protease and cellulase, and through mouse pathogenicity and toxicity tests, it is proved that the strain has no toxic and side effects and can be used as a feed additive for livestock and poultry breeding, improving growth performance, enhancing the intestinal barrier function of livestock and poultry, effectively improving the quality of livestock and poultry products, and having broad market application prospects.

[0035] The present invention provides a compound bacterium agent, which includes Lactobacillus plantarum and the Bacillus subtilis described in the above technical solution. The Lactobacillus plantarum and Bacillus subtilis of the present invention are preferably applied separately. When applying, the Bacillus subtilis is added first for aerobic fermentation, and then the Lactobacillus plantarum is added for anaerobic fermentation, which can reduce the content of zein in fermented alcoholic corn distillers grains.

[0036] As an alternative implementation mode, the application form of the Bacillus subtilis of the present invention includes a fermentation broth, and the viable bacteria count of the fermentation broth is ≥10 9 CFU / mL, more preferably 10 9 CFU / mL. The preparation method of the fermentation broth of the present invention includes: inoculating the seed liquid of the Bacillus subtilis into a fermentation medium for fermentation culture to obtain the fermentation broth.

[0037] As an alternative embodiment, the method for preparing the seed liquid of Bacillus subtilis of the present invention includes: inoculating the Bacillus subtilis in a seed culture medium for seed culture to obtain a Bacillus subtilis seed liquid. The present invention has no special limitation on the inoculation method, and a conventional method can be used. The present invention has no strict requirement for the inoculation amount of the seed culture. In a specific embodiment of the present invention, picking a loop of bacterial cells with an inoculation loop and putting it into the seed culture medium for the conventional operation of seed culture is sufficient. As an alternative embodiment, the temperature of the seed culture in the present invention is 30-37°C, or can also be 35-36°C; in a specific embodiment of the present invention, the temperature of the seed culture is 30, 32, 34, 35, 36 or 37°C. The time of the seed culture in the present invention is 20-30 h, or can also be 22-26 h; the rotation speed of the seed culture is 160-240 rpm, further can be 180-220 rpm, and more preferably 190 rpm. The present invention inoculating the Bacillus subtilis into the seed culture medium can achieve the activation of Bacillus subtilis. As an alternative embodiment, the inoculation amount of the Bacillus subtilis seed liquid in the present invention is 1%-3% of the volume of the culture medium. In a specific embodiment of the present invention, the inoculation amount can be 1%, 2% or 3% of the volume of the culture medium. The temperature of the fermentation culture in the present invention is 30-37°C, or can also be 35-36°C; in a specific embodiment of the present invention, the temperature of the fermentation culture is 30, 31, 32, 33, 34, 35, 36 or 37°C. The time of the fermentation culture in the present invention is 20-30 h, or can also be 22-26 h, and more preferably 24 h. The rotation speed of the fermentation culture in the present invention is 160-240 rpm, further can be 180-220 rpm, and more preferably 190 rpm. The seed culture medium and the fermentation culture medium of the present invention preferably include LB medium. The composition of the LB medium of the present invention preferably includes: peptone 1 wt%, yeast powder 0.3 wt%, glucose 0.3 wt%, sodium chloride 0.5 wt% and the balance of water. The LB medium of the present invention is preferably autoclaved at 121°C for 20 min before use.

[0038] As an alternative embodiment, the application form of the Lactobacillus plantarum of the present invention includes a fermentation broth, and the viable count of the fermentation broth is ≥10 9 CFU / mL, and more preferably 10 9 CFU / mL. The method for preparing the fermentation broth of the present invention includes: inoculating the seed liquid of the Lactobacillus plantarum in a fermentation culture medium for fermentation culture to obtain a fermentation broth.

[0039] As an alternative embodiment, the method for preparing the seed liquid of Lactobacillus plantarum of the present invention includes: inoculating the Lactobacillus plantarum into a seed culture medium for seed culture to obtain a Lactobacillus plantarum seed liquid. The present invention has no special limitation on the inoculation method, and a conventional method can be used. The present invention has no strict requirement for the inoculation amount of the seed culture, and a conventional method can be used. In a specific embodiment of the present invention, it is only necessary to pick a loop of bacterial cells with an inoculation loop and put them into the seed culture medium for conventional seed culture operations. As an alternative embodiment, the temperature of the seed culture in the present invention is 30-35 °C, or can also be 31-33 °C; in a specific embodiment of the present invention, the temperature of the seed culture is 30, 31, 32, 33, 34 or 35 °C. The time of the seed culture in the present invention is 40-50 h, or can also be 42-49 h, more preferably 48 h; the rotation speed of the seed culture is 160-240 rpm, further can be 180-220 rpm, more preferably 190 rpm. The present invention can activate Lactobacillus plantarum by inoculating the Lactobacillus plantarum into the seed culture medium.

[0040] As an alternative embodiment, the inoculation amount of the Lactobacillus plantarum seed liquid of the present invention inoculated into the fermentation culture medium is 1%-3% of the volume of the culture medium. In a specific embodiment of the present invention, the inoculation amount can be 1%, 2% or 3% of the volume of the culture medium. The temperature of the fermentation culture in the present invention is 30-35 °C, or can also be 32-33 °C; in a specific embodiment of the present invention, the temperature of the fermentation culture is 30, 31, 32, 33, 34 or 35 °C. The time of the fermentation culture in the present invention is 42-50 h, or can also be 44-49 h, more preferably 48 h; the rotation speed of the fermentation culture is 160-240 rpm, or can also be 180-220 rpm, more preferably 190 rpm.

[0041] The seed culture medium and fermentation culture medium of the present invention preferably include MRS culture medium. The composition of the MRS culture medium of the present invention preferably includes 2.5 wt% of casein peptone, 2.5 wt% of beef extract, 1.25 wt% of yeast powder, 1.25 wt% of glucose, 1.25 wt% of sodium acetate, 1 wt% of disodium citrate, 0.5 wt% of Tween 80, 1 wt% of K2HPO4, 0.5 wt% of MgSO4, 0.2 wt% of MnSO4·H2O and the balance of water. The MRS culture medium of the present invention is preferably autoclaved at 121 °C for 20 min before use. The present invention has no special limitation on the source of the Lactobacillus plantarum, and conventional strains can be used. In the specific embodiment of the present invention, the Lactobacillus plantarum numbered CICC 21790 is used for effect verification. The Lactobacillus plantarum of the present invention is a kind of lactic acid bacterium, which can produce a large amount of acid, promote animal growth, regulate the normal flora of the gastrointestinal tract and maintain the microecological balance. During the fermentation process, the Lactobacillus plantarum helps to reduce the antibacterial acidity of the fermentation and inhibit the proliferation of certain pathogenic microorganisms.

[0042] The present invention provides the application of the compound bacterium agent described in the above technical solution in fermenting alcoholic corn distillers grains and / or reducing the content of zein in alcoholic corn distillers grains.

[0043] The compound bacterium agent of the present invention can improve the quality of alcoholic corn distillers grains through fermentation, and use the fermented alcoholic corn distillers grains as a protein source in feed to improve the growth performance of livestock and poultry, thereby improving the aquaculture productivity level.

[0044] The present invention provides a preparation method for fermenting alcoholic corn distillers grains, including: inoculating Bacillus subtilis in the compound bacterium agent described in the above technical solution into the raw material of alcoholic corn distillers grains for solid-state aerobic fermentation culture, and then inoculating Lactobacillus plantarum for solid-state anaerobic fermentation culture. The Lactobacillus plantarum is an anaerobic bacterium and reproduces better in an anaerobic environment.

[0045] As an alternative embodiment, before inoculation, the present invention first mixes the raw material of alcoholic corn distillers grains with water to obtain a mixture; the initial water content of the mixture is 60 wt% - 100 wt%, more preferably 90 wt%. The function of adding water is to promote the growth and reproduction of Bacillus subtilis and Lactobacillus plantarum.

[0046] As an alternative embodiment, the Bacillus subtilis of the present invention is first prepared into a fermentation broth and then inoculated into the raw material of alcoholic corn distillers grains for solid-state aerobic fermentation culture. The preparation method of the fermentation broth of the present invention has been described above and will not be elaborated here. As an alternative embodiment, the inoculation amount of the Bacillus subtilis fermentation broth of the present invention is 4wt% - 10wt% of the initial mixture, more preferably 6wt%. The temperature of the solid-state aerobic fermentation culture of the present invention is 28 - 37°C, more preferably 37°C; the time is 3 - 5 days, or can also be 3 - 4 days, more preferably 3 days.

[0047] As an alternative embodiment, after the solid-state aerobic fermentation culture is completed, Lactobacillus plantarum is inoculated into the product obtained from the solid-state aerobic fermentation culture of the present invention for solid-state anaerobic fermentation culture. The Bacillus subtilis of the present invention is an aerobic bacterium and does not reproduce during the solid-state anaerobic fermentation process.

[0048] As an alternative embodiment, the Lactobacillus plantarum is first prepared into a fermentation broth and then inoculated into the raw material of alcoholic corn distillers grains for solid-state anaerobic fermentation culture. The preparation method of the fermentation broth of the present invention has been described above and will not be elaborated here. As an alternative embodiment, the inoculation amount of the Lactobacillus plantarum fermentation broth of the present invention is 4wt% - 10wt% of the initial mixture, more preferably 6wt%.

[0049] As an alternative embodiment, the temperature of the solid-state anaerobic fermentation culture of the present invention is 30 - 35°C, more preferably 30°C; the time is 3 - 5 days, or can also be 3 - 4 days.

[0050] After the solid-state anaerobic fermentation culture is completed, the present invention obtains a two-stage solid-state fermented product. As an alternative embodiment, the present invention performs drying treatment and pulverization treatment on the two-stage solid-state fermented product to obtain fermented alcoholic corn distillers grains. As an alternative embodiment, the temperature of the drying treatment of the present invention is 40 - 70°C, or can also be 50 - 60°C, more preferably 55°C. The drying treatment of the present invention only needs to reduce the water content of the solid-state fermented product to 15wt% - 18wt%. The present invention has no strict requirements on the drying treatment method, and conventional methods can be used. As an alternative embodiment, the present invention pulverizes the dried product, passes it through a 60-mesh sieve, and obtains the material under the sieve. The material under the sieve is the finished product of fermented alcoholic corn distillers grains. The pulverization of the present invention is preferably carried out using a pulverizer.

[0051] The present invention separately conducts seed culture and fermentation culture on Bacillus subtilis and Lactobacillus plantarum, and performs two-stage solid-state fermentation on the obtained Bacillus subtilis fermentation broth and Lactobacillus plantarum fermentation broth, creating a process for producing fermented distillers' grains with alcohol for feed by two-stage solid-state fermentation, which can increase the number of viable fermenting bacteria and the yield of corresponding beneficial metabolites in the fermented distillers' grains with alcohol product. When performing two-stage solid-state fermentation, the complex bacteria in the present invention are an important parameter affecting the yields of zein, zeaxanthin (such as carotenoids), functional amino acids, polypeptides and other metabolites in the distillers' grains with alcohol. Among them, Bacillus subtilis can secrete protease to fully degrade the macromolecular proteins in the distillers' grains with alcohol into small-molecular-weight proteins, reducing the zein content, and Lactobacillus plantarum can produce a large amount of acid, which can promote animal growth, regulate the normal flora of the gastrointestinal tract, and maintain the microecological balance.

[0052] The present invention first prepares Bacillus subtilis fermentation broth and Lactobacillus plantarum fermentation broth by liquid fermentation culture, and then uses the fermentation broth to perform solid-state fermentation on the distillers' grains with alcohol. The fermented distillers' grains with alcohol can be used as a fermented feed product, that is, the present invention uses a two-stage solid-state fermentation method to prepare the fermented distillers' grains with alcohol. The total viable bacteria count and metabolite content of the fermented distillers' grains with alcohol are high. After detection, the total viable bacteria count in the fermented distillers' grains with alcohol is as high as (8.92 ± 5.02) × 10 9 CFU / g, and the zein content is reduced by 52.37%. The fermentation process of the present invention is easy to operate, the culture medium components are simple, and the solid-state culture cost is low.

[0053] The present invention provides the application of the fermented distillers' grains with alcohol obtained by the preparation method described in the above technical solution in livestock and poultry breeding.

[0054] As an alternative embodiment, the poultry in the present invention includes chickens; the chickens in the present invention include chicks.

[0055] As an alternative embodiment, the livestock in the present invention includes pigs, cows, sheep, etc.; the pigs in the present invention include weaned piglets.

[0056] The present invention provides the application of the fermented distillers' grains with alcohol obtained by the preparation method described in the above technical solution in improving the growth performance of livestock and poultry.

[0057] As an alternative embodiment, the growth performance in the present invention includes increasing body weight and / or reducing the feed-to-weight ratio; the increase in body weight includes increasing weight gain and / or average daily gain.

[0058] The present invention provides the application of the fermented distillers' grains with alcohol obtained by the preparation method described in the above technical solution in the preparation of livestock and poultry animal feed. The fermented distillers' grains with alcohol in the present invention can replace soybean meal and be added to livestock and poultry animal feed to feed livestock and poultry.

[0059] The fermented alcoholic corn distillers grains prepared by the present invention can be added to feed for feeding livestock and poultry. After feeding, it can promote the growth and development of poultry, improve the growth performance of livestock and poultry, and enhance the intestinal barrier function of poultry.

[0060] The present invention provides a feed additive, which includes the fermented alcoholic corn distillers grains obtained by the preparation method described in the above technical solution. The fermented alcoholic corn distillers grains of the present invention can be added to the feed of livestock and poultry for feeding. In a specific embodiment of the present invention, the fermented alcoholic corn distillers grains of the present invention can replace soybean meal and be added to the basal feed. The addition amount of the fermented alcoholic corn distillers grains is 5wt% - 20wt% of the mass ratio of soybean meal in the basal feed, and more preferably 10wt%.

[0061] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0062] Example 1 Isolation and Identification of Bacillus subtilis Strains

[0063] 1. Physiological and Biochemical Analysis

[0064] 20 samples were collected from dry straw in Gongzhuling City, Changchun City, Jilin Province. 5g of each sample was weighed and added to 50mL of sterile normal saline. After shaking culture at 37°C, the culture solution was diluted by a factor of 10 in gradient, and then the obtained diluted solution was spread on the separation plate of the modified LB medium and cultured at a constant temperature of 37°C for 48h to obtain 20 strains.

[0065] The single colonies of the strains obtained by separation and purification were subjected to fermentation enzyme production experiments and protease activity determination for further rescreening. The strain with the highest enzyme activity was obtained for subsequent identification experiments. Among them, the pH of the modified LB medium was 6.2 - 6.4; the components of each liter of the modified LB medium were: 10g of peptone, 3g of yeast powder, 3g of glucose, and 5g of sodium chloride.

[0066] 2. Identification of Bacillus subtilis

[0067] The strain was identified by colony morphology, physiology and biochemistry, and molecular biology. The specific identification results are as follows:

[0068] (1) Morphological identification: The colonies of the strain on the modified LB medium plate were milky white, with uneven edges, convex and wrinkled. After Gram staining, the cell morphological characteristics were observed under an oil immersion lens (100×) of an optical microscope. It was Gram-positive, the cells were short rod-shaped, with blunt ends, and the spores were oval and central. The results are as Figure 1 shown.

[0069] (2)Physiological and biochemical characteristics: The physiological and biochemical characteristics of the strain were analyzed, and the results are shown in Table 1.

[0070] Table 1 Results of physiological and biochemical experiments of the strain

[0071]

[0072] Note: "+" represents having this characteristic or a positive reaction, and "-" represents not having this characteristic or a negative reaction.

[0073] (3)16S rDNA molecular biological identification: The total DNA of the Bacillus subtilis strain was extracted. Using it as a template, PCR amplification was carried out with universal primers for bacterial 16S rDNA to obtain an amplification product with a length of about 1.4 kb. The amplification product was recovered and entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The measured 16S rDNA sequence is shown in SEQ ID NO:1, as follows:

[0074]

[0075] By using Blast for alignment and constructing a phylogenetic tree as shown in Figure 2 according to the highest similarity sequence provided by GenBank, it was found that the homology of this strain with Bacillus subtilis (GenBank accession number HQ340123.1) was 98.37%. Combining the colony morphology and physiological and biochemical identification results, the strain was identified as Bacillus subtilis ( Bacillus subtilis ), and the preservation number was CGMCC No. 32291.

[0076] Example 2 Preparation of Fermentation Bacterial Agent

[0077] The composition of LB medium was: peptone 2 wt%, yeast powder 1 wt%, glucose 1 wt%, NaCl 0.5 wt% and the balance of water; it was autoclaved at 121 °C for 20 min before use.

[0078] The composition of MRS medium was: casein peptone 2.5 wt%, beef extract 2.5 wt%, yeast powder 1.25 wt%, glucose 1.25 wt%, sodium acetate 1.25 wt%, disodium citrate 1 wt%, Tween80 0.5 wt%, K2HPO4 1 wt%, MgSO4 0.5 wt%, MnSO4·H2O 0.2 wt% and the balance of water, and it was autoclaved at 121 °C for 20 min before use.

[0079] The preparation method of the bacterial agent fermentation broth was as follows:

[0080] (1) The Bacillus subtilis strain of Example 1 was stored on a slant. One loop was scraped from the slant and inoculated into a shake flask containing 250 mL of LB medium, and cultured in a shaking incubator at a temperature of 37 °C, a rotation speed of 190 rpm, and a culture time of 24 h to obtain a seed solution; the seed solution was inoculated into a new 1000 mL of LB medium at a volume ratio (v / v) of 2% of the medium, and fermented and cultured in a shaking incubator at a rotation speed of 190 rpm, a temperature of 37 °C, and a time of 24 h to obtain a Bacillus subtilis strain fermentation broth, and the viable count of the fermentation broth was 1×10 9 CFU / mL, abbreviated as Bacillus subtilis fermentation broth.

[0081] (2) Scrape a loopful of the Lactobacillus plantarum strain preserved on the inclined plane and inoculate it into a shake flask containing 250 mL of MRS medium. Incubate it in a shaking incubator at a temperature of 30 °C, a rotation speed of 190 rpm, and for a culture time of 48 h to obtain a seed solution; inoculate the seed solution into a new 1000 mL MRS medium at a volume ratio (v / v) of 2% of the medium, and perform fermentation culture in a shaking incubator at a rotation speed of 190 rpm, a temperature of 30 °C, and for a time of 48 h to obtain a fermentation broth of the Lactobacillus plantarum strain. The viable count of the fermentation broth is 1×10 9 CFU / mL, which is simply referred to as the Lactobacillus plantarum fermentation broth.

[0082] Example 3

[0083] A method for improving the quality of fermented alcoholic corn distillers grains, the steps are as follows:

[0084] Use the alcoholic corn distillers grains as the fermentation substrate; mix the alcoholic corn distillers grains and water to obtain a mixture, and the initial water content of the mixture is 90%. Based on the weight of the mixture, inoculate the Bacillus subtilis fermentation broth prepared in Example 2 at an inoculation amount of 6 wt% of the initial mixture for solid-state aerobic fermentation at a temperature of 37 °C and an aerobic fermentation time of 3 d. The aerobic fermentation process is accompanied by ventilation; after the aerobic fermentation is completed, based on the weight of the mixture, inoculate the Lactobacillus plantarum fermentation broth prepared in Example 2 at an inoculation amount of 6 wt% of the initial mixture for solid-state anaerobic fermentation at a temperature of 30 °C and an anaerobic fermentation time of 3 d. The anaerobic fermentation is not ventilated. After the anaerobic fermentation is completed, dry the solid-state fermentation product at a drying temperature of 55 °C until the water content drops to 15 wt%, and crush it with a pulverizer to a particle size of 60 mesh and pass through a 60-mesh sieve to obtain a fermentation product.

[0085] Extraction and content determination of zein in Example 4

[0086] 1. Zein extraction

[0087] Extract zein from corn by the ethanol extraction method. The specific process is as follows: Immerse the fermentation product obtained in Example 3 in anhydrous ethanol with a volume concentration of 75%, place it in a water bath shaker at 60 °C for extraction for 3 h, draw out the supernatant, precipitate it with sterile water, and obtain zein after drying and pulverization.

[0088] 2. Zein determination

[0089] Determine the zein content by the Kjeldahl method. The test results show that the zein content of the alcoholic corn distillers grains before fermentation is 12.79 mg / 100 ml, and the zein content of the alcoholic corn distillers grains after fermentation is 6.69 mg / 100 ml. The alcohol-soluble content of the alcoholic corn distillers grains after fermentation is reduced by 52.37% compared with that before fermentation ( Figure 3), through the two-stage solid-state fermentation of the compound microbial agent, it can effectively degrade the prolamin in alcoholic corn distillers grains ( Figure 4 ), and improve the characteristics of difficult absorption and low utilization of corn gluten meal. Figure 4 Among them, D0 is the prolamin distribution of alcoholic corn distillers grains before fermentation, and D1 is the prolamin distribution of alcoholic corn distillers grains after fermentation.

[0090] Example 5 Changes in the morphology of macromolecular proteins before and after fermentation

[0091] Stain the protein by FICT staining, and the results are shown in Figure 5 , according to Figure 5 It can be seen that the morphological structure of macromolecular proteins in alcoholic corn distillers grains changes before and after fermentation. The large particle proteins are decomposed into small particle proteins, which preliminarily verifies that the fermentation of this scheme can degrade macromolecular proteins.

[0092] Example 6 Structural changes in alcoholic corn distillers grains before and after fermentation

[0093] Fourier transform infrared spectroscopy was used to determine the structure of alcoholic corn distillers grains before and after fermentation. The results showed that the secondary structure of alcoholic corn distillers grains changed after fermentation (see Figure 6 ), according to Figure 5 It can be seen that the stretching vibration of the O-H bond at wavelengths of 3293-3277 is due to the degradation of polymers such as proteins and cellulose. A change appears at a wavelength of 1631, and the stretching of the C-O bond proves that the secondary structure of the protein has changed. A strong absorption peak appears at a wavelength of 1027, which is related to the deformation of the C-H bond of the aromatic ring, indicating a large number of molecular rearrangements.

[0094] Example 7 Changes in protease activity and cellulase in alcoholic corn distillers grains before and after fermentation

[0095] Ferment according to the method of Example 3 to obtain the fermented alcoholic corn distillers grains. Measure the acid protease and cellulase activities of the alcoholic corn distillers grains before fermentation and after fermentation. The results are shown in Figure 7 , it can be seen that after fermentation, the content of acid protease increased from 1.02 U / g to 9.97 U / g, and the content of cellulase increased from 0.62 U / g to 2.46 U / G. The contents of acid protease and cellulase after fermentation both increased significantly. Acid protease promoted the degradation of macromolecular proteins in alcoholic corn distillers grains, which was consistent with the observed microscopic structural changes. Cellulase catalyzed the decomposition of cellulose in the fiber into monosaccharides.

[0096] Example 8 Application effect of solid-state fermentation of alcoholic corn distillers grains in poultry farming

[0097] For the chick feeding experiment, 225 healthy 1-day-old chicks were selected, and the experiment was carried out at the Animal Breeding Center of Jilin Agricultural University.

[0098] According to the principle that there is no significant difference in body weight, they were randomly divided into three groups: a control group, an alcoholic corn distillers grains group, and a fermented alcoholic corn distillers grains group. There were 75 chicks in each group, with 5 replicates in each group and 15 chicks in each replicate.

[0099] The control group was fed a common basal diet.

[0100] Table 1 Composition of the common basal diet

[0101]

[0102] The alcoholic corn distillers grains group was fed Diet 1, and the composition of Diet 1 was: 10% of the soybean meal in the common basal diet by mass was replaced with alcoholic corn distillers grains.

[0103] The fermented alcoholic corn distillers grains group was fed Diet 2, and the composition of Diet 2 was: 10% of the soybean meal in the common basal diet by mass was replaced with fermented alcoholic corn distillers grains.

[0104] The trial period was 28 days. After the trial ended, the growth performance, feed intake, and feed-to-weight ratio changes of the chicks in each group were measured, and the specific results are shown in Table 2 below.

[0105] Table 2 Effects of solid-state fermented alcoholic corn distillers grains on the growth performance, feed intake, and feed-to-weight ratio of poultry

[0106]

[0107] Note: * represents a significant change, P <0.05.

[0108] According to Table 2, at the end of the feeding trial, the final weight, weight gain, and average daily gain of the poultry chicks in the experimental groups were significantly increased, and the feed-to-weight ratio was decreased, indicating that adding the fermented alcoholic corn distillers grains of Example 2 to the common basal diet could significantly improve the growth performance of the chicks.

[0109] Application effect of solid-state fermented alcoholic corn distillers grains in livestock farming in Example 9

[0110] For the weaned piglet feeding trial, 180 weaned piglets were selected, and the trial was carried out in the animal breeding center of Jilin Agricultural University.

[0111] According to the principle that there is no significant difference in body weight, they were randomly divided into three groups: a control group, an alcoholic corn distillers grains group, and a fermented alcoholic corn distillers grains group. There were 60 weaned piglets in each group, with 5 replicates in each group and 12 weaned piglets in each replicate.

[0112] The control group was fed a common basal diet.

[0113] The group fed with alcoholic corn distillers grains was fed diet 1, and the composition of diet 1 was as follows: 10% of the soybean meal in the common basal diet by mass was replaced with alcoholic corn distillers grains.

[0114] The group fed with fermented alcoholic corn distillers grains was fed diet 2, and the composition of diet 2 was as follows: 10% of the soybean meal in the common basal diet by mass was replaced with fermented alcoholic corn distillers grains.

[0115] The daily management of the weaned piglets in the three groups of experiments was the same. The experimental period was 90 days. After the experiment ended, the growth performance, feed intake, and feed-to-weight ratio changes of the weaned piglets in each group were measured, and the specific results are shown in Table 3 below.

[0116] Table 3 Effects of solid-state fermented alcoholic corn distillers grains on the growth performance, feed intake, and feed-to-weight ratio of weaned piglets

[0117]

[0118] Note: * represents a significant change, P <0.05.

[0119] According to Table 3, at the end of the feeding experiment, the final weight, weight gain, and average daily gain of the weaned piglets of the experimental group of poultry were significantly increased, and the feed-to-weight ratio was decreased, indicating that adding the fermented alcoholic corn distillers grains of Example 2 to the common basal diet could significantly improve the growth performance of weaned piglets.

[0120] In summary, the Bacillus subtilis provided by the present invention can secrete protease and cellulase, degrade the macromolecular proteins in the alcoholic corn distillers grains into small molecular weight proteins during the fermentation process, and reduce the content of prolamin. The Bacillus subtilis and Lactobacillus plantarum of the present invention are compounded to ferment the alcoholic corn distillers grains, and the fermented alcoholic corn distillers grains can improve the growth performance of chicks.

[0121] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Bacillus subtilis strain ( Bacillus subtilis ), characterized in that The preservation number is CGMCC No. 32291.

2. A composite bacterial agent, characterized in that, It includes Lactobacillus plantarum and the Bacillus subtilis described in claim 1.

3. The composite bacterial agent according to claim 2, characterized in that, The application form of the Bacillus subtilis includes a fermentation broth, and the viable count of the fermentation broth is ≥ 10 9 CFU / mL; the application form of the Lactobacillus plantarum includes a fermentation broth, and the viable count of the fermentation broth is ≥ 10 9 CFU / mL.

4. Use of the compound microbial agent described in claim 2 or 3 in fermenting alcoholic corn distillers grains and / or reducing the prolamin content of alcoholic corn distillers grains.

5. A preparation method of fermented alcoholic corn distillers grains, characterized in that, It includes: Inoculate the Bacillus subtilis in the compound microbial agent described in claim 2 or 3 into the raw material of alcoholic corn distillers grains for solid-state aerobic fermentation culture, and then inoculate the Lactobacillus plantarum in the compound microbial agent described in claim 2 or 3 for solid-state anaerobic fermentation culture.

6. The preparation method according to claim 5, characterized in that, The temperature of the solid-state aerobic fermentation culture is 28 - 35 °C, and the time is 3 - 5 d; the temperature of the solid-state anaerobic fermentation culture is 30 - 40 °C, and the time is 3 - 5 d.

7. According to the preparation method described in claim 5, characterized in that, Mix the raw material of alcoholic corn distillers grains with water to obtain a mixture; the initial water content of the mixture is 60 wt% - 90 wt%.

8. The preparation method according to claim 5, characterized in that, The inoculation amount of the Bacillus subtilis is 4 wt% - 10 wt% of the raw material of alcoholic corn distillers grains; the inoculation amount of the Lactobacillus plantarum is 4 wt% - 10 wt% of the raw material of alcoholic corn distillers grains.

9. Use of the fermented alcoholic corn distillers grains obtained by the preparation method described in any one of claims 5 - 8 in livestock and poultry breeding.

10. The application according to claim 9, characterized in that, The livestock and poultry breeding includes improving the growth performance of livestock and poultry.

11. Use of the fermented alcoholic corn distillers grains obtained by the preparation method described in any one of claims 5 - 8 in preparing livestock and poultry animal feed.

12. A feed additive, characterized in that, It includes the fermented alcoholic corn distillers grains obtained by the preparation method described in any one of claims 5 - 8.

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