Bacillus subtilis for improving available protein in alcoholic corn distillers' grains and preparation and application of complex microbial inoculant of bacillus subtilis

Abstract

The invention belongs to the technical field of microbial engineering, and particularly relates to bacillus subtilis for improving available protein in alcoholic corn distillers' grains and preparation and application of a complex microbial inoculant of the bacillus subtilis. The invention provides a strain of bacillus subtilis, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.32291. The bacillus subtilis can secrete protease and cellulase, macromolecular protein in the alcoholic corn vinasse is degraded into small-molecular-weight protein in the fermentation process, and the content of prolamin is reduced. The invention further provides a complex microbial inoculant which comprises lactobacillus plantarum and the bacillus subtilis in the technical scheme. The complex microbial inoculant can improve the viable count and the content of corresponding beneficial metabolites in the fermented alcoholic corn vinasse, enrich the nutrition of the alcoholic corn vinasse, and improve the utilization rate of the alcoholic corn vinasse.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial engineering, and particularly relates to a Bacillus subtilis for enhancing 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 come 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 will be generated, causing environmental pollution and resource waste. 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 to improve 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 resource waste. At the same time, after hydrolysis, alcoholic corn distillers grains lack essential amino acids for the body, restricting their 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 enhancing 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] To solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a strain of Bacillus subtilis ( Bacillus subtilis ), and the preservation number is CGMCC No. 32291.

[0008] The present invention provides a compound bacterium agent, which includes Lactobacillus plantarum and the Bacillus subtilis described in the above technical solution.

[0009] 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.

[0010] The present invention provides the application of the compound bacterial agent described in the above technical solution in fermenting alcoholic corn distillers grains and / or reducing the prolamin content of alcoholic corn distillers grains.

[0011] The present invention provides a preparation method of fermented alcoholic corn distillers grains, including: inoculating the Bacillus subtilis in the compound bacterial 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 the Lactobacillus plantarum in the compound bacterial agent described in the above technical solution for solid-state anaerobic fermentation culture.

[0012] Preferably, the temperature of the solid-state aerobic fermentation culture is 28~35°C, and the time is 3~5d; the temperature of the solid-state anaerobic fermentation culture is 30~40°C, and the time is 3~5d.

[0013] Preferably, the initial water content of the raw material of alcoholic corn distillers grains is 60wt%~100wt%.

[0014] 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.

[0015] 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 two or more of the following 1)~4), 1) Livestock and poultry breeding; 2) Improving the growth performance of livestock and poultry; 3) Preparing livestock and poultry animal feed.

[0016] The present invention provides a feed additive, including the fermented alcoholic corn distillers grains obtained by the preparation method described in the above technical solution.

[0017] The beneficial effects of the present invention: The present invention provides a strain of Bacillus subtilis, and the preservation number is CGMCC No.32291. The Bacillus subtilis described in the present invention can secrete protease and cellulase, and the Bacillus subtilis can fully degrade the macromolecular proteins in the alcoholic corn distillers grains into small molecular weight proteins during the fermentation process, reducing the prolamin content.

[0018] The present invention also provides a compound bacterium agent, which includes Lactobacillus plantarum and the Bacillus subtilis described in the above technical solution. The 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 in the animal gastrointestinal tract, promoting animal growth, and maintaining the microecological balance. The compound bacterium agent of the present invention can increase the viable bacteria count and the content of corresponding beneficial metabolites in the fermented alcoholic corn distillers grains, enrich the nutrition of the alcoholic corn distillers grains, and improve the utilization rate of the 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

[0019] Figure 1 It is a cell morphology diagram of Bacillus subtilis under an oil immersion objective of an optical microscope; 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 ); Figure 3 It is the change in the content of prolamin in alcoholic corn distillers grains before and after fermentation; Figure 4 It is the change in the molecular weight of prolamin in alcoholic corn distillers grains before and after fermentation; Figure 5 It is the change in the protein of alcoholic corn distillers grains before and after fermentation, Figure 5 The left side is the content of prolamin in alcoholic corn distillers grains before fermentation, and the right side is the content of prolamin in alcoholic corn distillers grains after fermentation; Figure 6 It is the change in the structure of alcoholic corn distillers grains before and after fermentation, Figure 6 In it, the red line represents the alcoholic corn distillers grains before fermentation, and the blue line is the alcoholic corn distillers grains after fermentation.

[0020] Figure 7 It is the change in the activity of acid protease and cellulase before and after fermentation.

[0021] Biological Deposit Description Bacillus subtilis ( Bacillus subtilis ), classified and named as Bacillus subtilis ( Bacillus subtilis ), was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 21, 2024, with the deposit number CGMCC No.32291, and the address of the deposit unit: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Embodiments

[0022] The present invention provides a strain of Bacillus subtilis ( Bacillus subtilis ), and the preservation number is CGMCC No. 32291.

[0023] The Bacillus subtilis described in the present invention is isolated from dry straw in Gongzhuling City, Changchun City, Jilin Province. The colony of this strain is milky white, with an irregular edge, convex and wrinkled; after Gram staining, the cell morphological characteristics are observed under an oil immersion lens (100×) of an optical microscope, indicating that the strain is a Gram-positive bacterium, the cells are rod-shaped, with blunt ends and an oval central spore. Using the 16S rDNA of the Bacillus subtilis for Blast alignment, a phylogenetic tree as shown in Figure 2 is constructed according to the highest similarity sequence provided by GenBank, and it is found that the strain is Bacillus subtilis ( Bacillus subtilis ).

[0024] The Bacillus subtilis described in the present invention has the function of secreting protease and cellulase, and through mouse pathogenicity and toxicity tests, it is proved that the strain has no toxic and side effects, can be used as a feed additive for livestock and poultry breeding, improve growth performance, improve the intestinal barrier function of livestock and poultry, effectively improve the quality of livestock and poultry products, and has a wide market application prospect.

[0025] 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 described in 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.

[0026] As an alternative embodiment, the application form of the Bacillus subtilis described in the present invention includes a fermentation broth, and the viable count of the fermentation broth is ≥10 9 CFU / mL, more preferably 10 9 CFU / mL. The preparation method of the fermentation broth described in the present invention includes: inoculating the seed broth of the Bacillus subtilis into a fermentation medium for fermentation culture to obtain the fermentation broth.

[0027] As an alternative embodiment, the method for preparing the seed liquid of Bacillus subtilis of the present invention includes: inoculating the Bacillus subtilis into a seed medium for seed culture to obtain a Bacillus subtilis seed liquid. The present invention has no special limitation on the inoculation method, and conventional methods can be used. The present invention has no strict requirement for the inoculation amount of the seed culture. In specific embodiments of the present invention, picking a loop of bacterial cells with an inoculation loop and putting it into the seed medium for conventional seed culture operations 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 specific embodiments 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 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 medium. In specific embodiments of the present invention, the inoculation amount can be 1%, 2% or 3% of the volume of the medium. The temperature of the fermentation culture in the present invention is 30-37 °C, or can also be 35-36 °C; in specific embodiments 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 medium and fermentation 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.

[0028] 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 into a fermentation medium for fermentation culture to obtain a fermentation broth.

[0029] 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 it 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.

[0030] 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.

[0031] The seed culture medium and the 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% casein peptone, 2.5 wt% beef extract, 1.25 wt% yeast powder, 1.25 wt% glucose, 1.25 wt% sodium acetate, 1 wt% disodium citrate, 0.5 wt% Tween 80, K 2 HPO 4 1 wt%, MgSO 4 0.5 wt%, MnSO 4 ·H 2O 0.2 wt% and the balance of water. Before use, the MRS culture medium described in the present invention is preferably autoclaved at 121 °C for 20 min. The present invention has no special limitation on the source of the Lactobacillus plantarum, and conventional strains can be used. In a specific embodiment of the present invention, Lactobacillus plantarum numbered CICC 21790 is used for effect verification. The Lactobacillus plantarum of the present invention is a lactic acid bacterium that 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, Lactobacillus plantarum helps to reduce the antibacterial acidity of the fermentation and inhibit the proliferation of certain pathogenic microorganisms.

[0032] The present invention provides the application of the above-mentioned composite bacterium agent in fermenting alcoholic corn distillers grains and / or reducing the prolamin content of alcoholic corn distillers grains.

[0033] The composite bacterium agent described in 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 level of aquaculture productivity.

[0034] The present invention provides a preparation method of fermented alcoholic corn distillers grains, including: inoculating Bacillus subtilis in the composite 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 under anaerobic conditions.

[0035] As an optional implementation manner, 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% to 100 wt%, more preferably 90 wt%. The function of adding water is to promote the growth and reproduction of Bacillus subtilis and Lactobacillus plantarum.

[0036] As an optional implementation manner, 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 described in the present invention has been discussed above and will not be elaborated here. As an optional implementation manner, the inoculation amount of the Bacillus subtilis fermentation broth of the present invention is 4 wt% to 10 wt% of the initial mixture, more preferably 6 wt%. The temperature of the solid-state aerobic fermentation culture of the present invention is 28 to 37 °C, more preferably 37 °C; the time is 3 to 5 d, or can also be 3 to 4 d, more preferably 3 d.

[0037] As an optional implementation manner, after the solid-state aerobic fermentation culture is completed, the present invention inoculates Lactobacillus plantarum into the product obtained from the solid-state aerobic fermentation culture 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.

[0038] 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, and more preferably 6wt%.

[0039] 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, and can also be 3 - 4 days.

[0040] After the solid-state anaerobic fermentation culture is completed, a two-stage solid fermentation product is obtained in the present invention. As an alternative embodiment, the two-stage solid fermentation product is subjected to drying treatment and crushing treatment 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, and 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 fermentation 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 dried product is crushed and passed through a 60-mesh sieve to obtain the undersize. The obtained undersize is the finished product of fermented alcoholic corn distillers grains. The crushing in the present invention is preferably carried out using a crusher.

[0041] The present invention separately conducts seed culture and fermentation culture on Bacillus subtilis and Lactobacillus plantarum, and performs two-stage solid fermentation on the obtained Bacillus subtilis fermentation broth and Lactobacillus plantarum fermentation broth, creating a process for producing fermented alcoholic corn distillers grains for feed by two-stage solid fermentation, which can increase the number of viable fermenting bacteria and the yield of corresponding beneficial metabolites in the fermented alcoholic corn distillers grains product. When performing two-stage solid fermentation, the complex bacteria in the present invention are an important parameter affecting the yield of metabolites such as zein, zeaxanthin (such as carotenoids), functional amino acids, and polypeptides in alcoholic corn distillers grains. Among them, Bacillus subtilis can secrete protease to fully degrade the macromolecular proteins in alcoholic corn distillers grains 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.

[0042] The present invention first prepares Bacillus subtilis fermentation broth and Lactobacillus plantarum fermentation broth by liquid fermentation culture, and then uses the fermentation broth for solid-state fermentation of alcoholic corn distillers grains. The fermented alcoholic corn distillers grains 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 alcoholic corn distillers grains. The total viable count and metabolite content of the fermented alcoholic corn distillers grains are high. After detection, the total viable count in the fermented alcoholic corn distillers grains 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 composition is simple, and the solid-state culture cost is low.

[0043] 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 livestock and poultry breeding.

[0044] As an alternative embodiment, the poultry in the present invention includes chickens; the chickens in the present invention include chicks.

[0045] As an alternative embodiment, the livestock in the present invention includes pigs, cows, sheep, etc.; the pigs in the present invention include weaned piglets.

[0046] 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 improving the growth performance of livestock and poultry.

[0047] 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.

[0048] 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 the preparation of livestock and poultry animal feed. The fermented alcoholic corn distillers grains in the present invention can replace soybean meal and be added to livestock and poultry animal feed to feed livestock and poultry.

[0049] The fermented alcoholic corn distillers grains prepared by the present invention can be added to feed to feed livestock and poultry. After feeding, it can promote the growth and development of poultry, improve the growth performance of livestock and poultry, and improve the intestinal barrier function of poultry.

[0050] 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 in the present invention can be added to livestock and poultry animal feed to feed livestock and poultry. In a specific embodiment of the present invention, the fermented alcoholic corn distillers grains 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%.

[0051] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] Example 1 Isolation and Identification of Bacillus subtilis Strains 1. Physiological and Biochemical Analysis Twenty samples were collected from dry straw in Gongzhuling City, Changchun City, Jilin Province. 5 g of each sample was weighed and added to 50 mL of sterile normal saline. After shaking culture at 37 °C, the culture solution was diluted by 10-fold gradient, and then the obtained dilution was spread on the separation plate of the modified LB medium and cultured at a constant temperature of 37 °C for 48 h to obtain 20 strains.

[0053] The single colonies of the strains obtained by separation and purification were subjected to fermentation enzyme production experiments, determination of protease activity, and further rescreening to obtain the strain with the highest enzyme activity 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: 10 g of peptone, 3 g of yeast powder, 3 g of glucose, and 5 g of sodium chloride.

[0054] 2. Identification of Bacillus subtilis The strain was identified by colony morphology, physiology and biochemistry, and molecular biology. The specific identification results are as follows: (1) Morphological identification: The colonies of the strain on the modified LB medium plate were milky white, with irregular 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.

[0055] (2) Physiological and biochemical characteristics: The physiological and biochemical characteristics of the strain were analyzed, and the results are shown in Table 1.

[0056] Table 1 Results of Physiological and Biochemical Experiments of the Strain

[0057] Note: "+" represents having this characteristic or the reaction is positive, and "-" represents not having this characteristic or the reaction is negative.

[0058] (3) 16S rDNA molecular biology identification: The total DNA of the Bacillus subtilis strain was extracted and used as a template. Using the universal primers for bacterial 16S rDNA, PCR amplification was carried out 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, specifically as follows:

[0059] By using Blast for alignment and constructing a phylogenetic tree as shown based on the highest similarity sequence provided by GenBank, it was found that the homology of this strain with Figure 2 (GenBank accession number HQ340123.1) was 98.37%. Combining the colony morphology and the results of physiological and biochemical identification, the strain was identified as Bacillus subtilis Bacillus subtilis ( Bacillus subtilis ), and its preservation number was CGMCC No. 32291.

[0060] Example 2 Preparation of Fermentation Bacterial Agent The composition of the 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.

[0061] The composition of the 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%, K 2 HPO 4 1 wt%, MgSO 4 0.5 wt%, MnSO 4 ·H 2 O 0.2 wt%, and the balance of water; it was autoclaved at 121 °C for 20 min before use.

[0062] The preparation method of the bacterial agent fermentation broth was as follows: (1) The Bacillus subtilis strain of Example 1 was preserved 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 liquid; the seed liquid 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. The viable count of the fermentation broth was 1×10 9 CFU / mL, which was simply called Bacillus subtilis fermentation broth.

[0063] (2)Scrape a loopful of Lactobacillus plantarum species preserved on the inclined plane and inoculate it into a shaking flask containing 250 mL of MRS medium. Cultivate it in a shaking incubator at a temperature of 30 °C, a rotation speed of 190 rpm, and a cultivation 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 cultivation in a shaking incubator at a rotation speed of 190 rpm, a temperature of 30 °C, and a time of 48 h to obtain a fermentation broth of Lactobacillus plantarum strain. The viable count of the fermentation broth is 1×10 9 CFU / mL, which is simply called Lactobacillus plantarum fermentation broth.

[0064] Example 3 A method for fermenting alcoholic corn distillers grains to improve their quality, the steps are as follows: Use alcoholic corn distillers grains as the fermentation substrate; mix 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.

[0065] Example 4 Extraction and content determination of zein 1. Zein extraction Extract zein from corn by the ethanol extraction method. The specific process is as follows: Immerse the fermentation product obtained in Example 3 in absolute 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.

[0066] 2. Zein determination Determine the zein content by the Kjeldahl method. The test results show that the zein content of alcoholic corn distillers grains before fermentation is 12.79 mg / 100 ml, and the zein content of alcoholic corn distillers grains after fermentation is 6.69 mg / 100 ml. The alcohol-soluble content of alcoholic corn distillers grains after fermentation is reduced by 52.37% compared with that before fermentation ( Figure 3 ), and through two-stage solid-state fermentation of the composite microbial agent, the zein in alcoholic corn distillers grains can be effectively degraded ( Figure 4 ), improving the characteristics of difficult absorption and low utilization of corn protein powder.Figure 4 Among them, D0 is the gliadin distribution of the distillers dried grains with solubles before fermentation, and D1 is the gliadin distribution of the distillers dried grains with solubles after fermentation.

[0067] Example 5 Changes in the morphology of macromolecular proteins before and after fermentation The proteins were stained 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 the distillers dried grains with solubles changes before and after fermentation. The large particle proteins are decomposed into small particle proteins, preliminarily verifying that the macromolecular proteins can be degraded by fermentation with this scheme.

[0068] Example 6 Structural changes in the distillers dried grains with solubles before and after fermentation The structure of the distillers dried grains with solubles before and after fermentation was determined by Fourier transform infrared spectroscopy. The results show that the secondary structure of the distillers dried grains with solubles changes after fermentation (see Figure 6 ). According to Figure 5 It can be seen that the stretching vibration of the O-H bond at the wavelength of 3293 - 3277 is due to the degradation of polymers such as proteins and cellulose. The change at the wavelength of 1631, the stretching of the C-O bond, proves the change of the protein secondary structure. A strong absorption peak appears at the wavelength of 1027, which is related to the deformation of the aromatic ring C-H bond, indicating a large number of molecular rearrangements.

[0069] Example 7 Changes in the protease activity and cellulase of the distillers dried grains with solubles before and after fermentation Fermentation was carried out according to the method of Example 3 to obtain the distillers dried grains with solubles after fermentation. The acid protease and cellulase activities of the distillers dried grains with solubles before and after fermentation were measured. The results are shown in Figure 7 , it can be seen that after fermentation, the content of acid protease increases from 1.02 U / g to 9.97 U / g, and the content of cellulase increases from 0.62 U / g to 2.46 U / G. The contents of acid protease and cellulase after fermentation both increase significantly. The acid protease promotes the degradation of macromolecular proteins in the distillers dried grains with solubles, which is consistent with the observed microscopic structural changes. The cellulase catalyzes the decomposition of cellulose in the fiber into monosaccharides.

[0070] Example 8 Application effect of solid-state fermented distillers dried grains with solubles in poultry farming 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.

[0071] According to the principle of insignificant basic differences in body weight, they were randomly divided into three groups: the control group, the distillers dried grains with solubles group, and the fermented distillers dried grains with solubles group, with 75 chicks in each group. Each group was set with 5 replicates, and each replicate had 15 chicks.

[0072] The control group was fed with a common basic diet.

[0073] Table 1 Composition of the common basal diet

[0074] The alcoholic corn distillers grains group was fed diet 1, and the composition of diet 1 was: replacing 10% of the soybean meal in the common basal diet by alcoholic corn distillers grains.

[0075] The fermented alcoholic corn distillers grains group was fed diet 2, and the composition of diet 2 was: replacing 10% of the soybean meal in the common basal diet by fermented alcoholic corn distillers grains.

[0076] The experimental period was 28 d. After the experiment ended, the growth performance, feed intake and feed to weight ratio changes of each group of chicks were measured, and the specific results are shown in Table 2 below.

[0077] Table 2 Effects of solid-state fermented alcoholic corn distillers grains on the growth performance, feed intake and feed to weight ratio of poultry

[0078] Note: * represents significant change, P <0.05.

[0079] As can be seen from Table 2, at the end of the feeding experiment, the final weight, weight gain and average daily weight 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 on the basis of the common basal diet could significantly improve the growth performance of chicks.

[0080] Application effect of solid-state fermented alcoholic corn distillers grains in livestock farming in Example 9 For the weaned piglet feeding experiment, 180 weaned piglets were selected, and the experiment was carried out in the animal breeding center of Jilin Agricultural University.

[0081] According to the principle that the body weights were not significantly different basically, they were randomly divided into three groups: the control group, the alcoholic corn distillers grains group and the fermented alcoholic corn distillers grains group, with 60 weaned piglets in each group, 5 replicates were set in each group, and 12 weaned piglets in each replicate.

[0082] The control group was fed the common basal diet.

[0083] The alcoholic corn distillers grains group was fed diet 1, and the composition of diet 1 was: replacing 10% of the soybean meal in the common basal diet by alcoholic corn distillers grains.

[0084] The fermented alcoholic corn distillers grains group was fed diet 2, and the composition of diet 2 was: replacing 10% of the soybean meal in the common basal diet by fermented alcoholic corn distillers grains.

[0085] The daily management of weaned piglets in the three groups of experiments was the same. The experimental period was 90 days. After the experiment, the growth performance, feed intake, and feed-to-weight ratio of weaned piglets in each group were measured, and the specific results are shown in Table 3 below.

[0086] 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

[0087] Note: * represents a significant change, P <0.05.

[0088] According to Table 3, at the end of the feeding experiment, the final weight, weight gain, and average daily gain of the weaned piglets in 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.

[0089] In summary, the Bacillus subtilis provided by the present invention can secrete protease and cellulase, degrade macromolecular proteins in alcoholic corn distillers grains into small molecular weight proteins during the fermentation process, and reduce the content of prolamin. The compound of the Bacillus subtilis and Lactobacillus plantarum of the present invention ferments the alcoholic corn distillers grains, and the fermented alcoholic corn distillers grains can improve the growth performance of chicks.

[0090] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

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

2. A composite bacterial agent, characterized in that: The invention comprises Lactobacillus plantarum and the Bacillus subtilis according to 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, wherein the number of viable bacteria in the fermentation broth is ≥10 9 CFU / mL; the application form of the plant lactobacillus includes a fermentation broth, and the number of viable bacteria in the fermentation broth is ≥10 9 CFU / mL.

4. Use of the composite bacterial agent according to claim 2 or 3 in fermenting alcoholic corn distiller's grains and / or reducing the alcohol-soluble protein content of alcoholic corn distiller's grains.

5. A method for preparing fermented alcoholic corn distiller's grains, characterized in that: include: The Bacillus subtilis in the composite bacterial agent described in claim 2 or 3 is inoculated into the alcoholic corn distiller's grains raw material for solid-state aerobic fermentation culture, and then the Lactobacillus plantarum in the composite bacterial agent described in claim 2 or 3 is inoculated 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 days; the temperature of the solid-state anaerobic fermentation culture is 30-40° C., and the time is 3-5 days.

7. The preparation method according to claim 5, characterized in that: The initial water content of the alcoholic corn distiller's grains raw material is 60wt%-100wt%.

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

9. Use of the fermented alcoholic corn distiller's grains obtained by the preparation method according to any one of claims 5 to 8 in any one or more of the following 1) to 4), 1) Livestock and poultry farming; 2) Improve the growth performance of livestock and poultry; 3) Preparation of livestock and poultry feed.

10. A feed additive, characterized in that: The invention comprises fermented alcoholic corn distiller's grains obtained by the preparation method according to any one of claims 5 to 8.

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