Compound bacteria, biological leavening agent containing compound bacteria, fermented brewer's grains and application of compound bacteria, biological leavening agent and fermented brewer's grains

Abstract

The invention relates to the technical field of animal feed, and discloses compound bacteria, a biological fermentation agent containing the compound bacteria, fermented brewer's grains and application of the compound bacteria, the biological fermentation agent and the fermented brewer's grains. The compound bacterium comprises aspergillus niger, bacillus subtilis and a pichia pastoris modified bacterium, the preservation number of the aspergillus niger is GDMCC (Graphics Digital Microorganisms for China Center for Culture Collection) No.64995, the preservation number of the bacillus subtilis is GDMCC No.64996, and the pichia pastoris modified bacterium is a gene modified bacterium P.pastoris GS115 / pGAPZ-NBLF-N. The invention further discloses a preparation method of the compound bacterium for preparing the compound bacterium, and the compound bacterium has the advantages that the preservation number of the aspergillus niger is GDMCC No.64995, the preservation number of the bacillus subtilis is GDMCC No.64995, and the preservation number of the bacillus subtilis is GDMCC No.64996. The biological leavening agent comprises compound bacteria and bran, and the fermented brewer's grains obtained through treatment of the biological leavening agent can improve the protein content, reduce cellulose, enhance the nutritional quality of animal feed, increase the resistance of animals and improve economic benefits.

Description

Technical Field

[0001] The invention relates to the technical field of animal feed, in particular to a composite bacteria, a biological fermentation agent containing the composite bacteria, fermented beer grains and applications thereof. Background Art

[0002] Sorghum, wheat, corn and other grains are used as raw materials. The solids are produced after solid fermentation and distillation in the traditional liquor brewing process. It is an important by-product produced in large quantities and in large concentrations in the production process of beer and liquor. Fresh lees have a high water content and are rich in nutrients. They are easily contaminated by microorganisms and rot and deteriorate. Drying lees to reduce their moisture content can extend their storage time, but it requires a lot of heat energy to achieve this, which does not meet the requirements of low-carbon development.

[0003] At present, most liquor companies mainly use spent grains and distiller's grains as animal (mainly cattle and pig) feed and sell them directly to prevent the proliferation of microorganisms in them, which will reduce their nutritional value and pollute the environment. The economic added value generated by the direct sale of distiller's grains as feed is low, and the distiller's grain resources have not been fully developed and utilized. In addition, compared with areas with more developed breeding industries, it is more difficult to sell spent grains in coastal areas. Therefore, finding a suitable method to develop and utilize distiller's grains in a high-value manner according to local conditions is an urgent problem that the beer and liquor industry needs to solve.

[0004] The main components of brewer's grains are malt husks, insoluble protein, hemicellulose, fat and a small amount of starch. It is an unconventional feed raw material with high yield and nutritional value. Studies have found that the protein content in brewer's grains is about 26%-30%, containing 17 kinds of amino acids, of which methionine and lysine content are 3.23% and 2.15% respectively, serine and alanine content in non-essential amino acids are 4.09% and 4.29% respectively, rumen bypass protein and rumen bypass starch content are 53.79% and 58.54% respectively, neutral detergent fiber content is 45.08%, and acid detergent fiber content is 25.85%. Due to its unique aroma, it can greatly increase the animal's feed intake. At the same time, the non-degradable fiber content in brewer's grains is relatively low compared to other feed raw materials, which increases the palatability of the feed, thereby increasing the animal's dry matter intake. Brewer's grains also contain 51.97% neutral detergent fiber and 18.15% acid detergent fiber, which can reduce the time that chyme stays in the digestive tract, increase the food circulation rate, and thus increase the dry matter intake of animals. Brewer's grains also contain arabinoxylan and β-glucan, which can promote the growth of probiotics such as bifidobacteria, enterococci, and lactic acid bacteria, thereby regulating the structure and function of intestinal flora. In addition, brewer's grains are also rich in vitamins, minerals, and a variety of trace elements, which are nutrients that other grain raw materials cannot match.

[0005] Based on the above research, brewer's grains can be used as a substitute for protein feed raw materials. At present, only a few animal food manufacturers have used brewer's grains as raw materials for related product development. The main reason is that when brewer's grains are used as animal feed, the lysine content in brewer's grain protein is low and unstable, and it is easily degraded by microorganisms. Therefore, when brewer's grain protein feed is used as animal feed, it is necessary to add proteins with high lysine content such as methionine. In addition, the cellulose in brewer's grain protein will affect the appetite of animals and should not be used as animal feed for a long time. If it is used directly as animal feed for a long time, it will cause some intestinal diseases, such as soft stools and diarrhea. Since brewer's grains contain a large amount of tannins and crude fiber substances, if the intake is not controlled during feeding, it is easy to cause oral diseases in animals. There is also a big problem when brewer's grains are used as animal feed: brewer's grains are easy to rot and deteriorate; brewer's grains will produce some gas during the fermentation process, and if they are not ventilated and stored in time, they will rot and deteriorate. Moreover, the spoiled brewer's grains will produce a large amount of toxic substances. Therefore, in actual operation, it needs to be deeply processed. Summary of the invention

[0006] The purpose of the present invention is to overcome the problem that the current brewer's grains are directly sold as animal feed, long-term use by animals will cause some intestinal diseases and oral diseases, and the brewer's grains are easily contaminated by microorganisms and rot and deteriorate to produce a large amount of toxic substances. A composite bacteria, a biological fermentation agent containing the composite bacteria, fermented brewer's grains and their applications are provided. The composite bacteria are applied to the biological fermentation treatment of fermented brewer's grains, which can increase the protein content, reduce cellulose, enhance the nutritional quality of animal feed, and also increase the resistance of animals, thereby reducing the use of antibiotics and improving economic benefits.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a composite bacteria, which comprises Aspergillus niger, Bacillus subtilis and Pichia pastoris modified bacteria, the Aspergillus niger (Aspergillus niger) has a preservation number of GDMCC No. 64995, the Bacillus subtilis (Bacillus subtilis) has a preservation number of GDMCC No. 64996, and the Pichia pastoris modified bacteria is a genetically modified bacteria P. pastoris GS115 / pGAPZ-NBLF-N.

[0008] Preferably, the weight ratio of Aspergillus niger, Bacillus subtilis and Pichia pastoris modified bacteria in the composite bacteria is 1-2:1-2:1.

[0009] The second aspect of the present invention provides a biological fermentation agent, which contains the composite bacteria and bran as described above.

[0010] Preferably, the content of the composite bacteria in the biological fermentation agent is 1.45×109 CFU / ml-2.11×109 CFU / ml, and the added amount of bran accounts for 10-15wt% of the weight of the biological fermentation agent.

[0011] A third aspect of the present invention provides fermented brewer's grains, which are obtained by mixing the above-mentioned biological fermentation agent with brewer's grains for fermentation and culturing.

[0012] Preferably, the mass ratio of the fermented beer grains to the biological fermentation agent is 10:1-2.

[0013] Preferably, the fermentation culture conditions include: a temperature of 32-33° C. and a time of 3-4 days.

[0014] The fourth aspect of the present invention provides the use of the aforementioned composite bacteria, biological fermentation agent, and fermented brewer's grains in the preparation of animal feed.

[0015] A fifth aspect of the present invention provides an animal feed, which contains the fermented brewer's grains as described above and fresh chicken, fresh duck, beef powder, broccoli, carrots, potatoes, goat milk powder, eggs, pears, seaweed powder, butter and additives;

[0016] Preferably, the raw materials in the animal feed include: based on 1 part by weight of the additives, the amount of fresh chicken is 22-28 parts by weight, the amount of fresh duck is 18-22 parts by weight, the amount of fermented brewer's grains is 13-17 parts by weight, the amount of beef powder is 8-12 parts by weight, the amount of broccoli is 4-8 parts by weight, the amount of carrots is 3-7 parts by weight, the amount of potatoes is 3-5 parts by weight, the amount of goat milk powder is 3-5 parts by weight, the amount of eggs is 2-4 parts by weight, the amount of pears is 2-4 parts by weight, the amount of seaweed powder is 1-3 parts by weight, and the amount of butter is 1-3 parts by weight.

[0017] Preferably, the additive ingredients of the animal feed include DL-methionine, vitamin A, vitamin D 3 , Vitamin B 1 , Vitamin B 2 , Vitamin B 6 , Vitamin B 12 , Vitamin C, Folic Acid, Niacin, D-Calcium Pantothenate, DHA, Anthocyanins, Honeysuckle Extract, Probiotics (Saccharomyces cerevisiae), Antioxidants, Fructooligosaccharides, Manno-oligosaccharides, Chondroitin Sulfate, Astaxanthin and Green Tea Powder.

[0018] Through the above technical solution, the beneficial effects of the present invention are:

[0019] The composite bacteria provided by the present invention can effectively increase the protein content of fermented brewer's grains and reduce the cellulose content. The modified Pichia pastoris can not only directly secrete milk protein, but also cooperate with Bacillus subtilis and Aspergillus niger to promote brewer's grain fermentation, increase the formation of functional components such as nicotinic acid and acetoin, and cover up the poor taste caused by the fermentation of Bacillus subtilis, thereby enhancing the nutritional quality of animal feed, increasing animal resistance, reducing the incidence of oral diseases, reducing costs, and improving economic benefits. The composite bacteria are further combined with bran, and the bran mainly plays a loosening role. Since the strains in the composite bacteria are all aerobic bacteria and the brewer's grains are prone to agglomeration during fermentation, sufficient oxygen can improve the fermentation effect of the brewer's grains, and the crude protein content in the brewer's grain fermentation obtained by fermentation is significantly increased compared with that before fermentation, and various properties are improved.

[0020] Biological Deposit

[0021] Aspergillus niger 1015 is deposited in Guangdong Microbiological Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code 510070, the abbreviation of the depository is GDMCC), the deposit number is GDMCC No.64995, and the deposit date is August 8, 2024.

[0022] Bacillus subtilis 1M is deposited in Guangdong Microbiological Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code 510070, the deposit unit abbreviation is GDMCC), the deposit number is GDMCC No.64996, and the deposit date is August 8, 2024. DETAILED DESCRIPTION

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] The first aspect of the present invention provides a composite bacteria, which comprises Aspergillus niger, Bacillus subtilis and Pichia pastoris modified bacteria, the Aspergillus niger (Aspergillus niger) has a preservation number of GDMCC No. 64995, the Bacillus subtilis (Bacillus subtilis) has a preservation number of GDMCC No. 64996, and the Pichia pastoris modified bacteria is a genetically modified bacteria P. pastoris GS115 / pGAPZ-NBLF-N.

[0025] According to the present invention, preferably, the weight ratio of Aspergillus niger, Bacillus subtilis and Pichia pastoris modified bacteria in the composite bacteria is 1-2:1-2:1.

[0026] The Aspergillus niger is Aspergillus niger 1015, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code 510070, the abbreviation of the depository unit is GDMCC), the deposit number is GDMCC No.64995, and the deposit date is August 8, 2024.

[0027] The Bacillus subtilis is Bacillus subtilis 1M, which is deposited in the Guangdong Microbial Culture Collection Center (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code 510070, the abbreviation of the depository unit is GDMCC), the deposit number is GDMCC No. 64996, and the deposit date is August 8, 2024.

[0028] The preparation process of the modified Pichia pastoris is as follows: the N-lobe (BLF-N) of bovine lactoferrin is cloned into Pichia pastoris GS115, and the heterologous and efficient expression of BLF-N is achieved through codon optimization and fermentation condition optimization.

[0029] In the present invention, Aspergillus niger strains (Aspergillus niger H1, Aspergillus niger H2, Aspergillus niger H3, Aspergillus niger 1015) and Bacillus subtilis strains (Bacillus subtilis 1M, Bacillus subtilis 3M, Bacillus subtilis 1-X-1, Bacillus subtilis 1-X-8, Bacillus subtilis 2-2, Bacillus subtilis 2-X-4, Bacillus subtilis 2-X-5, Bacillus subtilis 7-X-4, Bacillus subtilis 7-F-5, Bacillus subtilis 7-F-6, Bacillus subtilis G1.17-X-5, Bacillus subtilis G1.17-X-6) are activated, and then a spot test is carried out, and the strains are stained with a Congo red solution, decolorized with a sodium chloride solution, washed with distilled water to compare cellulose degradation capabilities, and degradation circles are observed. The Hc value is measured and calculated, and a strain with a strong cellulose degradation capability is selected according to the Hc value.

[0030] There is no particular limitation on the culture medium used in the present invention. Preferably, the activation culture medium and fermentation culture medium for Bacillus subtilis, Aspergillus niger and Pichia pastoris are as follows:

[0031] The Aspergillus niger activation medium uses PDA liquid medium;

[0032] The components of the fermentation medium for Aspergillus niger enzyme production are: sodium carboxymethyl cellulose (CMC-Na) 4-6 g / L, beef extract 4-6 g / L, sodium chloride 4-6 g / L, and peptone 8-12 g / L;

[0033] The components of the Bacillus subtilis activation culture medium are: beef extract 2-4 g / L, peptone 8-10 g / L, sodium chloride 4-6 g / L;

[0034] The components of the fermentation medium for Bacillus subtilis enzyme production are: 9-11 g / L konjac flour, 5-7 g / L peptone, 0.8-1.2 g / L sodium chloride, 0.4-0.6 g / L magnesium sulfate, and 0.4-0.6 g / L potassium dihydrogen phosphate;

[0035] Growth medium for Pichia pastoris: 0.5% yeast extract, 1.0% peptone, 1.0% sodium chloride; YPD: 1.0% yeast extract, 2.0% peptone, 2.0% glucose;

[0036] The components of the selective medium MD (solid) for the modified Pichia pastoris are: glucose 18.0-22.0 g / L, agar 18.0-22.0 g / L, amino-free yeast nitrogen base (YNB) 13-14 g / L, biotin 0.3-0.5 mg / L;

[0037] The components of YPDS medium (for verifying positive transformants of Pichia pastoris) are: 0.8-1.2% yeast extract, 1.8-2.2% tryptone, 1.8-2.2% glucose, 3.5-4.5×10 -5 %, potassium phosphate buffer 80-120mmol / L, pH 6.0-7.0.

[0038] The fermentation medium of the modified Pichia pastoris contains 0.8-1.2% yeast extract, 1.8-2.2% tryptone, 1.8-2.2% glucose, and KH 2 PO 4 0.4-0.6%, MgSO 4 7H 2 O 0.04-0.06%, pH 6.0-7.0.

[0039] The invention studies the influence of mixed fermentation and continuous fermentation of Bacillus subtilis, Aspergillus niger and Pichia pastoris on the degradation rate of anti-nutrient factors.

[0040] According to the present invention, whether mixed fermentation or step-by-step continuous fermentation, the degradation rate of anti-nutritional factors can be significantly improved. Preferably, the fermentation method of the composite bacteria is mixed fermentation of Bacillus subtilis, Aspergillus niger and Pichia pastoris.

[0041] The present invention studies the mixed fermentation of Bacillus subtilis, Aspergillus niger and Pichia pastoris, and observes the influence of different proportions of mixed bacteria on fermentation. The content of composite bacteria is 1.45×10 9 CFU / ml-2.11×10 9 CFU / ml, 12 groups of composite bacteria with different proportions of Bacillus subtilis, Aspergillus niger and Pichia pastoris were inoculated to observe the degradation rate of nutritional factors.

[0042] According to the present invention, preferably, the weight ratio of Aspergillus niger, Bacillus subtilis and Pichia pastoris modified bacteria in the composite bacteria is 1-2:1-2:1.

[0043] The second aspect of the present invention provides a biological fermentation agent, which is prepared by mixing the composite bacteria as mentioned above with bran.

[0044] The inventors found that different contents of composite bacteria and bran in the biological fermentation agent can affect the contents of reducing sugar, crude protein, crude fat, lysine and crude fiber in the fermentation product. The present invention sets a biological fermentation agent with different composite bacteria inoculation amounts and bran addition amounts to ferment brewer's grains, and observes the changes in the contents of reducing sugar, crude protein, crude fat, lysine and crude fiber in the fermentation product.

[0045] According to the present invention, the biological fermentation agent can be a solid fermentation agent or a liquid fermentation agent, preferably a liquid fermentation agent. Further preferably, the content of the composite bacteria in the biological fermentation agent is 1.45×10 9 CFU / ml-2.11×10 9 CFU / ml, the amount of bran added accounts for 10-15wt% of the weight of the biological fermentation agent. Specifically, ultrapure water can be used as a solvent to mix with the composite bacteria and bran to form a liquid fermentation agent.

[0046] A third aspect of the present invention provides a fermented brewer's grains, wherein the fermented brewer's grains are obtained by inoculating the aforementioned biological fermentation agent into the brewer's grains for fermentation and culture.

[0047] The inventors have found that different mass ratios of brewer's grains to biological fermentation agents will also affect the content of reducing sugars, crude protein, crude fat, lysine, and crude fiber in the fermentation products. The present invention sets different mass ratios of brewer's grains to biological fermentation agents for fermentation, and observes the changes in the content of reducing sugars, crude protein, crude fat, lysine, and crude fiber in the fermentation products.

[0048] According to the present invention, preferably, the mass ratio of the brewer's grains to the biological fermentation agent is 10:1-2.

[0049] In the present invention, there is no particular limitation on the fermentation conditions of the fermented brewer's grains. According to the present invention, preferably, the fermentation conditions include: a temperature of 32-33° C. and a time of 3-4 days.

[0050] The third aspect of the present invention provides the use of the aforementioned composite bacteria, the aforementioned biological fermentation agent, and the aforementioned fermented brewer's grains in the preparation of animal feed.

[0051] The fourth aspect of the present invention provides a method for preparing animal feed, wherein the fermented brewer's grains and beef powder, broccoli, carrots, potatoes, goat milk powder, eggs, pears, seaweed powder, butter and additives as described above are crushed and mixed according to the formula ratio to make them fully mixed, the mixed raw materials are extruded and dried, and freeze-dried particles of fresh chicken and fresh duck meat with a diameter of about 0.2-0.4 cm are added. The freeze-dried particles of fresh chicken and fresh duck meat are homemade in the inventor's laboratory.

[0052] According to the present invention, preferably, the animal feed contains the following raw material composition: based on 1 weight part of additives, the amount of fresh chicken is 22-28 weight parts, the amount of fresh duck is 18-22 weight parts, the amount of fermented brewer's grains is 13-17 weight parts, the amount of beef powder is 8-12 weight parts, the amount of broccoli is 4-8 weight parts, the amount of carrots is 3-7 weight parts, the amount of potatoes is 3-5 weight parts, the amount of goat milk powder is 3-5 weight parts, the amount of eggs is 2-4 weight parts, the amount of pears is 2-4 weight parts, the amount of seaweed powder is 1-3 weight parts, and the amount of butter is 1-3 weight parts.

[0053] According to the present invention, preferably, the additive ingredients of the animal feed include DL-methionine, vitamin A, vitamin D 3 , Vitamin B 1 , Vitamin B 2 , Vitamin B 6 , Vitamin B 12 , Vitamin C, Folic Acid, Niacin, D-Calcium Pantothenate, DHA, Anthocyanins, Honeysuckle Extract, Probiotics (Saccharomyces cerevisiae), Antioxidants, Fructooligosaccharides, Manno-oligosaccharides, Chondroitin Sulfate, Astaxanthin and Green Tea Powder.

[0054] The present invention will be described in detail below through examples.

[0055] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0056] Aspergillus niger strains and Bacillus subtilis strains were screened by the laboratory. Pichia pastoris GS115 (host bacteria) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., Escherichia coli TOP10F' strain was preserved by Professor Huang He's laboratory of Nanjing Normal University, Pichia pastoris GS115, yeast constitutive expression vector pPIC9K, legacy mycin, and pGAPZαA plasmid were purchased from Shanghai Sangon Biotechnology Co., Ltd., plasmid mini-extraction kit, DNA fragment purification kit, and gel recovery kit were purchased from Qiagen, nucleic acid and protein molecular weight standards, Pyrobest DNA polymerase, T4 DNA ligase, restriction endonucleases EcoR I, Not I, and Avr II were purchased from NEB, NBLF-N gene and all primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd., amino-free yeast nitrogen source (YNB) was purchased from MacLean Reagent Co., Ltd., and brewer's grains and bran were purchased from Hubei Jiuxing Feed Co., Ltd.

[0057] The following culture medium was used in the following examples:

[0058] Aspergillus niger activation medium: PDA liquid medium;

[0059] Aspergillus niger enzyme fermentation medium: sodium carboxymethyl cellulose (CMC-Na) 5g / L, beef extract 5g / L, sodium chloride 5g / L, peptone 10g / L.

[0060] Bacillus subtilis activation medium: beef extract 3g / L, peptone 10g / L, sodium chloride 5g / L;

[0061] Bacillus subtilis enzyme production fermentation medium: konjac flour 10g / L, peptone 6g / L, sodium chloride 1g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 0.5g / L.

[0062] Growth medium for Pichia pastoris: 0.5% yeast extract, 1.0% peptone, 1.0% sodium chloride; YPD: 1.0% yeast extract, 2.0% peptone, 2.0% glucose;

[0063] The components of the selective medium MD (solid) for the modified Pichia pastoris are: glucose 20.0 g / L, agar 20.0 g / L, amino-free yeast nitrogen base (YNB) 13.4 g / L, biotin 0.4 mg / L;

[0064] YPDS medium (for verifying positive transformants of Pichia pastoris): 1% yeast extract, 2% tryptone, 2% glucose, 4.0×10 -5 %, potassium phosphate buffer 100 mmol / L, pH 6.0.

[0065] Fermentation medium for Pichia pastoris: 1% yeast extract, 2% tryptone, 2% glucose, KH 2 PO 4 0.5%, MgSO 4 7H 2 O 0.05%, pH 6.0.

[0066] Example 1

[0067] This example illustrates the genetic modification method of Pichia pastoris modified bacteria

[0068] 1. Synthesis of target gene

[0069] The BLF-N coding sequence was designed and synthesized according to the BLF-N sequence (NCBI database gene accession number JX294418.1) and Pichia pastoris codon preference, an EcoR I restriction site was introduced at the 5' end, and a Sal I restriction site was introduced at the 3' end. After gene synthesis, the recombinant plasmid pPIC9K-NBLF-N was constructed by ligating the gene into the pPIC9K vector.

[0070] 2. Construction of recombinant plasmid

[0071] pPIC9K-NBLF-N was transformed into E. coli TOP10F', and the recombinant plasmid was extracted after amplification and culture; the pPIC9K-NBLF-N plasmid was double-digested with EcoRI and Not I, and the target fragment was recovered and connected to the pGAPZαA vector that was also double-digested to construct pGAPZ-NBLF-N; pGAPZ-NBLF-N was transformed into E. coli TOP10F' competent cells, and LB agar plates (containing 25ug / ml Zeocin) were coated, and finally a single positive colony was picked and inoculated in LB liquid culture medium for culture, and the recombinant plasmid was extracted and amplified using pGAP Forward primer and 3'-AOX1 primer, and double-digestion identification was performed. The primers used are shown in Table 1.

[0072] Table 1

[0073]

[0074] 3. Electroporation of recombinant vector into Pichia pastoris

[0075] pGAPZ-NBLF-N was linearized with restriction endonuclease SacⅠ and then electroporated into P. pastoris GS115 competent cells (Pichia pastoris GS115 was purchased from Beijing Biolab Technology Co., Ltd.). The preparation method of Pichia pastoris competent cells was referred to the "Pichia Pastoris Expression Operation Manual" of Invitrogen. The electroporated cells were incubated at 30°C for 1-2h, coated on MD plates, and cultured at 30°C for 3d until larger colonies grew.

[0076] 4. Identification of positive transformants of recombinant yeast

[0077] The His obtained from MD plate + Ten single clones were selected from the transformants, inoculated into YPD medium for overnight culture, and the genome was extracted by the "boil-freeze-boil" method. PCR was performed using Pgap as a primer to identify whether pGAPZ-NBLF-N was integrated into the yeast genome.

[0078] 5. Induced expression and identification

[0079] The positive transformants were selected and inoculated into YPDS medium. The culture was carried out at 30°C and 250 rpm for 18 h until the OD 600 4-5; centrifuge at 4000×g for 10 min at room temperature, discard the supernatant, and resuspend the cells in BMMY medium to OD 600 1; then, the expression was induced at 28°C and 250r / min, and the total induction time was 96h; the cultured cells were collected, and the expression of proteins in the cell supernatant and precipitate was detected by SDS-PAGE, and the positive transformant was the genetically modified bacteria P. pastoris GS115 / pGAPZ-NBLF-N. The genetically modified Pichia pastoris was used for subsequent example studies.

[0080] Example 2

[0081] This example compares the Hc values ​​of cellulose and mannan degradation zones to screen strains.

[0082] The present invention uses the following Aspergillus niger and Bacillus subtilis (1)-(3) as test strains:

[0083] (1) Aspergillus niger strains: Aspergillus niger H1, Aspergillus niger H2, Aspergillus niger H3, Aspergillus niger 1015;

[0084] (2) Bacillus subtilis strains: Bacillus subtilis 1M, Bacillus subtilis 3M, Bacillus subtilis 1-X-1, Bacillus subtilis 1-X-8, Bacillus subtilis 2-2, Bacillus subtilis 2-X-4, Bacillus subtilis 2-X-5, Bacillus subtilis 7-X-4, Bacillus subtilis 7-F-5, Bacillus subtilis 7-F-6, Bacillus subtilis G1.17-X-5, Bacillus subtilis G1.17-X-6;

[0085] The Aspergillus niger strain and the Bacillus subtilis strain described in the present invention are both screened by the laboratory.

[0086] The above 16 strains were activated twice with the corresponding culture medium, and then spotted on sodium carboxymethyl cellulose medium and konjac mannan medium plates with sterilized toothpicks and cultured at 33°C for 48h.

[0087] The sodium carboxymethyl cellulose plate and the konjac mannan medium plate were stained with 0.5% Congo red solution for 30 minutes, the Congo red dye solution was discarded, 1% sodium chloride solution was added, and the decolorization treatment was performed for 10 minutes. The sodium chloride solution was discarded, and the degradation zone was observed after washing twice with distilled water. The Hc value of the degradation zone was measured and calculated, and the selected strain was determined according to the Hc value; the results are shown in Table 2:

[0088] Table 2

[0089] strain Strain diameter (d) Degradation zone diameter (D) Hc value (D / d) Aspergillus niger 1015 6.5±0.8 14.2±0.6 2.19 Bacillus subtilis 1M 4.4±0.6 10.0±0.7 2.27

[0090] The results showed that the Aspergillus niger 1015 strain described in Table 2 had good cellulose degradation ability, and its cellulose degradation circle Hc value reached 2.19, while the mannan degradation circle Hc value of Bacillus subtilis 1M reached 2.27, which was higher than that of other strains.

[0091] Therefore, Aspergillus niger 1015 and Bacillus subtilis 1M were selected for subsequent example studies.

[0092] The deposit number of Aspergillus niger 1015 is GDMCC No.64995, and the deposit number of Bacillus subtilis 1M is GDMCC No.64996.

[0093] Example 3

[0094] This example illustrates the effect of different mixed bacteria ratios on the degradation rate of anti-nutrient factors.

[0095] Taking brewer's grains as substrate, the bio-fermentation agents with different mixed bacteria ratios shown in Table 3 were inoculated into the brewer's grains for fermentation culture (the mass ratio of brewer's grains to the bio-fermentation agent was 10:1.5, wherein the amount of bran in the bio-fermentation agent accounted for 10wt% of the weight of the bio-fermentation agent), the fermentation temperature was 32°C, the fermentation time was 3 days, the fermented brewer's grains were dried, crushed, and sieved to obtain brewer's grain fermentation product.

[0096] The experiment studied the effect of different mixed bacteria ratios on fermentation. The total content of composite bacteria in the biological fermentation agent was 1.65×10 9 CFU / ml, the specific proportions of different strains are shown in Table 3, and the results of cellulose content detection in brewer's grains fermentation are shown in Table 3.

[0097] Table 3

[0098] combination Aspergillus niger Bacillus subtilis Pichia pastoris Cellulose content (%) 1 1 1 1.5 9.1 2 1 1.5 1 8.9 3 1.5 1 1 7.9 4 1 1 2 9.2 5 1 2 1 8.6 6 2 1 1 7.8 7 1 1.5 1.5 8.2 8 1.5 1 1.5 8.4 9 1.5 1.5 1 7.5 10 1 2 2 7.9 11 2 1 2 7.6 12 2 2 1 7.3

[0099] The results in Table 3 show that no matter what the inoculation ratio is, the degradation rate of anti-nutritional factors can be significantly improved. Among them, the mixed fermentation combination 12, that is, the composite bacteria ratio of Bacillus subtilis, Aspergillus niger, and Pichia pastoris is 2:2:1, has the highest degradation rate of anti-nutritional factors and a cellulose content of 7.3%. Therefore, combination 12 was selected for subsequent fermentation.

[0100] Example 4

[0101] This example illustrates the effect of fermenting brewer's grains with a biological fermentation agent on the contents of reducing sugars, crude protein, crude fat, lysine, and crude fiber in the brewer's grains.

[0102] Reducing sugar determination of fermentation products: The experimental group was set up in such a way that the brewer's grains sample fermented by the combination of the 12 methods in Example 3 was removed and placed in a 60°C oven for drying for 12 hours; 1 g of the dried sample was taken, 10 mL of boiling water was added, mixed and placed at 37°C, 220 rpm shaker for 15 minutes, 2 mL was taken out and mixed and placed in a centrifuge tube, centrifuged at 3000 rpm for 10 minutes, 0.1 mL of the supernatant was taken and placed in a test tube, 0.1 mL of deionized water was added, and 0.15 mL of DNS was added, mixed and placed in a boiling water bath for 5 minutes, cooled and fixed to 2.5 mL, mixed and placed in a 96-well ELISA plate, and its OD was determined 540 The reducing sugar content of the sample was calculated based on the reducing sugar standard curve; the results are shown in Table 4.

[0103] Control group: No biological fermentation agent was added to the unfermented brewer's grains for fermentation, and the cultured samples were cultured in culture containers. The cultured samples were processed and calculated according to the processing method of the fermented brewer's grains of the experimental group. The results are shown in Table 4.

[0104] Table 4

[0105] Nutritional value index Experimental Group Control group Crude Protein 34.8％ 25.13％ Crude fat 9.1％ 7.13％ Reducing sugar content 22.6mg / mL 12.5 mg / mL Lysine 0.934％ 0.528％ Crude Fiber 7.3％ 13.81％

[0106] The results in Table 4 show that the reducing sugar yield is 22.6 mg / mL, which is 80.80% higher than that of the control group.

[0107] The crude protein content of the fermentation product was determined according to GB / T 6432-2018. The results are shown in Table 4. Compared with the control group, the crude protein content of the experimental group increased by 38.47%.

[0108] The crude fat content of the fermented beer grains was determined according to GB / T 6433-2006. The results are shown in Table 4. Compared with the control group, the crude fat content of the experimental group increased by 27.63%.

[0109] The lysine content of the fermented beer grains product was determined according to GB / T 18246-2019. The results are shown in Table 4. Compared with the control group, the lysine content of the experimental group increased by 0.406%.

[0110] The crude fiber content of the fermented beer grains was determined according to GB / T 6434-2006. The results are shown in Table 4. Compared with the control group, the crude fiber content of the experimental group was reduced by 47.14%.

[0111] According to this embodiment, it can be concluded that the fermented brewer's grains provided by the present invention have higher nutritional value than the unfermented brewer's grains, and the reduction of crude fiber content can reduce the incidence of oral diseases in animals.

[0112] Example 5

[0113] To explain the effect of different inoculation amounts of composite bacteria in biological fermentation agents on the contents of reducing sugar, crude protein, crude fat, lysine and crude fiber in fermentation products;

[0114] In this example, brewer's grains fermentation was carried out according to the method described in Example 3, and the content of reducing sugar, crude protein, crude fat, lysine and crude fiber in the fermentation product was detected by the method of Example 4; the difference was that the content of composite bacteria in the biological fermentation agent used in the fermentation product was 1.35×10 9 CFU / ml, 1.45×10 9 CFU / ml, 2.11×10 9 CFU / ml, 2.22×10 9 CFU / ml, the results are shown in Table 5:

[0115] Table 5

[0116]

[0117] The results in Table 5 show that when the inoculation amount of the composite bacteria is between 1.45×109CFU / ml and 2.11×109CFU / ml, the fermented brewer's grains have a good fermentation effect, can significantly improve the nutritional value of the fermented brewer's grains, and reduce the crude fiber content.

[0118] Example 6

[0119] The embodiment illustrates the influence of different addition amounts of bran in the biological fermentation agent on the reducing sugar, crude protein, crude fat, lysine and crude fiber content of the fermentation product; this embodiment is tested according to the method described in Example 4, except that the addition amounts of bran in the biological fermentation agent used for the fermentation product are 5, 10, 15 and 20 wt %, respectively. The results are shown in Table 6;

[0120] Table 6

[0121]

[0122] The results in Table 6 show that when the bran addition amount is 10-15wt%g, the fermentation of brewer's grains has a better fermentation effect, can significantly improve the nutritional value of the fermented brewer's grains, and reduce the crude fiber content.

[0123] Example 7

[0124] The example illustrates the influence of different mass ratios of brewer's grains and biological fermentation agent on the reducing sugar, crude protein, crude fat, lysine and crude fiber content of the fermentation product; this comparative example is operated according to the method described in Example 4, except that the mass ratios of brewer's grains and biological fermentation agent used in the fermentation product are 10:0.5, 10:1, 10:1.5, 10:2 and 10:2.5 respectively; the results are shown in Table 7.

[0125] Table 7

[0126]

[0127] The results in Table 7 show that when the mass ratio of brewer's grains to biological fermentation agent is 10:1-2, fermented brewer's grains have a good fermentation effect, can significantly improve the nutritional value of fermented brewer's grains, and reduce the crude fiber content.

[0128] Example 8

[0129] This example is used to illustrate the preparation process of the biological fermented feed. The preparation environment of the present invention is not particularly limited and is carried out in a normal temperature laboratory. The raw materials are divided into four groups according to the weight ratio of fresh chicken, fresh duck and fermented beer grains. 1 、w 2 、w 3 、w 4 express:

[0130] w1 : 20 parts by weight of fresh chicken, 15 parts by weight of fresh duck, 10 parts by weight of fermented beer grains, 5 parts by weight of beef powder, 2 parts by weight of broccoli, 2 parts by weight of carrots, 2 parts by weight of potatoes, 2 parts by weight of goat milk powder, 1 part by weight of eggs, 1 part by weight of pears, 0.5 parts by weight of seaweed powder, 0.5 parts by weight of butter, 1 part by weight of additives

[0131] w 2 : 22 parts by weight of fresh chicken, 18 parts by weight of fresh duck, 13 parts by weight of fermented brewer's grains, 8 parts by weight of beef powder, 4 parts by weight of broccoli, 3 parts by weight of carrots, 3 parts by weight of potatoes, 3 parts by weight of goat milk powder, 2 parts by weight of eggs, 2 parts by weight of pears, 1 part by weight of seaweed powder, 1 part by weight of butter, and 1 part by weight of additives;

[0132] w 3 : 25 parts by weight of fresh chicken, 20 parts by weight of fresh duck, 14 parts by weight of fermented brewer's grains, 10 parts by weight of beef powder, 6 parts by weight of broccoli, 5 parts by weight of carrots, 4 parts by weight of potatoes, 4 parts by weight of goat milk powder, 3 parts by weight of eggs, 3 parts by weight of pears, 2 parts by weight of seaweed powder, 2 parts by weight of butter, and 1 part by weight of additives;

[0133] w 4 : 28 parts by weight of fresh chicken, 22 parts by weight of fresh duck, 15 parts by weight of fermented beer grains, 12 parts by weight of beef powder, 8 parts by weight of broccoli, 7 parts by weight of carrots, 5 parts by weight of potatoes, 5 parts by weight of goat milk powder, 4 parts by weight of eggs, 4 parts by weight of pears, 3 parts by weight of seaweed powder, 3 parts by weight of butter, 1 part by weight of additives:

[0134] The above additives consist of 0.06% DL-methionine, 0.24% vitamin A, and 0.1% vitamin D. 3 0.04%, Vitamin B 1 1.5%, Vitamin B 2 1%, Vitamin B 6 1%, Vitamin B 12 1%, vitamin C 5%, folic acid 1.2%, niacin 2.5%, D-pantothenate calcium 0.1%, DHA 0.6%, anthocyanin 0.3%, honeysuckle extract 6%, probiotics (brewer's yeast) 15%, antioxidant 3%, fructooligosaccharide 0.5%, manno-oligosaccharide 0.7%, chondroitin sulfate 0.06%, astaxanthin 0.7%, green tea powder 59.5%.

[0135] The process of preparing animal feed is:

[0136] S1: Raw material preparation: fresh chicken (freeze-dried), fresh duck meat (freeze-dried), fermented brewer's grains, beef powder, broccoli, carrots, potatoes, goat milk powder, eggs, pears, seaweed powder, butter, additives.

[0137] S2: Raw material processing: Mix the prepared fermented beer grains, beef powder, broccoli, carrots, potatoes, goat milk powder, eggs, pears, seaweed powder, butter and additives, crush them to make them fully mixed and evenly mixed, and pass them through a 10-16 mesh sieve to prepare for subsequent processing.

[0138] S3: The mixed raw materials are formed by an extruder to form animal feed pellets of different shapes and sizes. Subsequently, the formed animal feed pellets are sent to a drying device to remove excess moisture at a drying temperature of 60°C to ensure that the moisture content is below 10wt%, thereby ensuring the dryness of the animal feed and increasing the storage time.

[0139] S4: Cooling and screening: The dried animal feed pellets need to be cooled to prevent the loss of nutrients due to high temperature. Then, unqualified pellets are removed through a 35-40 mesh sieve to ensure that each animal feed meets the quality standards. The animal feed pellets are dark brown in color, rough on the surface, with fine residue attached, and have a natural meaty aroma.

[0140] S5: Finally, add freeze-dried fresh chicken and freeze-dried fresh duck meat particles with a diameter of 0.3 cm.

[0141] Four groups of animal feed products (w 1 、w 2 、w 3 and w 4 ) to carry out subsequent examples and comparative examples.

[0142] Example 9

[0143] This example illustrates the quality standard test of brewer's grains bio-fermented feed, and the test sample used is taken from the finished animal feed prepared by the animal feed preparation process described in Example 8. The quality standard index of the animal feed refers to the national standard GB / T 31216-2014, as shown in Table 8; the lysine content in the feed is determined according to the method specified in the national standard GB / T 18246-2019; the water-soluble chloride (as Cl -The content of water-soluble chloride in feed shall be determined according to the method specified in national standard GB / T 6439-2023; the content of crude protein shall be determined according to the method specified in national standard GB / T 6432-2018; the content of total phosphorus shall be determined according to the method specified in national standard GB / T 6437-2018; the content of calcium shall be determined according to the method specified in national standard GB / T 6436-2018; the content of crude ash shall be determined according to the method specified in national standard GB / T 6438-2007; the content of crude fat shall be determined according to the method specified in national standard GB / T 6433-2006; the content of crude fiber shall be determined according to the method specified in national standard GB / T 6434-2022; the content of water shall be determined according to the method specified in national standard GB / T 6435-2014.

[0144] Table 8

[0145]

[0146]

[0147] As shown in the results in Table 8, all indicators of the animal feed quality standard set by the present invention are better than the national standard. Therefore, the animal feed quality standard set by the present invention is used as the detection standard for subsequent examples.

[0148] The results of the present embodiment are shown in Table 9:

[0149] Table 9

[0150]

[0151] The results are shown in Table 9. When the raw materials of the animal feed include 22-28 parts by weight of fresh chicken, 18-22 parts by weight of fresh duck, 13-17 parts by weight of fermented brewer's grains, 8-12 parts by weight of beef powder, 4-8 parts by weight of broccoli, 3-7 parts by weight of carrots, 3-5 parts by weight of potatoes, 3-5 parts by weight of goat milk powder, 2-4 parts by weight of eggs, 2-4 parts by weight of pears, 1-3 parts by weight of seaweed powder, 1-3 parts by weight of butter, and 1 part by weight of additives, the quality test results of the brewer's grains bio-fermentation animal feed of the present invention all meet the set quality standards of the animal feed.

[0152] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A composite bacteria, characterized in that: The composite bacteria comprises Aspergillus niger, Bacillus subtilis and Pichia pastoris modified bacteria, the Aspergillus niger (Aspergillus niger) has a preservation number of GDMCC No. 64995, the Bacillus subtilis (Bacillus subtilis) has a preservation number of GDMCC No. 64996, and the Pichia pastoris modified bacteria is a genetically modified bacteria P. pastoris GS115 / pGAPZ-NBLF-N.

2. The composite bacteria according to claim 1, characterized in that: The weight ratio of Aspergillus niger, Bacillus subtilis and Pichia pastoris modified bacteria in the composite bacteria is 1-2:1-2:

1.

3. A biological fermentation agent, characterized in that: The biological fermentation agent contains the composite bacteria as claimed in any one of claims 1 or 2 and bran; Preferably, the content of the composite bacteria in the biological fermentation agent is 1.45×10 9 CFU / ml-2.11×10 9 CFU / ml, and the added amount of bran accounts for 10-15wt% of the weight of the biological fermentation agent.

4. A fermented brewer's grains, characterized in that: The fermented brewer's grains are obtained by mixing the biological fermentation agent described in claim 3 with brewer's grains for fermentation and culturing.

5. The fermented brewer's grains according to claim 4, characterized in that The mass ratio of the brewer's grains to the biological fermentation agent is 10:1-2.

6. The fermented brewer's grains according to claim 4 or 5, wherein The fermentation culture conditions include: a temperature of 32-33° C. and a time of 3-4 days.

7. Use of the composite bacteria according to claim 1 or 2, the biological fermentation agent according to claim 3, and the fermented brewer's grains according to any one of claims 4 to 6 in preparing animal feed.

8. An animal feed, characterized in that: The animal feed contains the fermented brewer's grains according to any one of claims 4 to 6.

9. The animal feed according to claim 8, characterized in that The animal feed also contains fresh chicken, fresh duck, beef meal, broccoli, carrots, potatoes, goat milk powder, eggs, pears, seaweed powder, butter and additives; Preferably, the animal feed contains the following raw material composition: based on 1 weight part of additives, the amount of fresh chicken is 22-28 weight parts, the amount of fresh duck is 18-22 weight parts, the amount of fermented brewer's grains is 13-17 weight parts, the amount of beef powder is 8-12 weight parts, the amount of broccoli is 4-8 weight parts, the amount of carrots is 3-7 weight parts, the amount of potatoes is 3-5 weight parts, the amount of goat milk powder is 3-5 weight parts, the amount of eggs is 2-4 weight parts, the amount of pears is 2-4 weight parts, the amount of seaweed powder is 1-3 weight parts, and the amount of butter is 1-3 weight parts.

10. The animal feed according to claim 8 or 9, characterized in that The animal feed additive contains DL-methionine, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B6, vitamin B 12 , Vitamin C, Folic Acid, Niacin, D-Calcium Pantothenate, DHA, Anthocyanins, Honeysuckle Extract, Probiotics, Antioxidants, Fructooligosaccharides, Manno-oligosaccharides, Chondroitin Sulfate, Astaxanthin and Green Tea Powder.

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