Production of bacterial protein biofeed by two-step fermentation of corn husk

Abstract

This invention belongs to the field of bio-fermentation feed technology, specifically involving a two-step fermentation method for producing microbial protein bio-feed from corn husks. The invention provides a method for preparing microbial protein bio-feed, specifically: inoculating a first fermentation substrate with Aspergillus niger inoculum for a first fermentation to obtain a starter culture; and inoculating a second fermentation substrate with a compound microbial agent for a second fermentation to obtain the microbial protein bio-feed. The preparation method described in this invention can maximize the degradation of cellulose and hemicellulose in corn husk raw materials while considering cost, and increase its protein content through secondary fermentation, providing the feed industry with a method for preparing high-quality protein feed.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation feed preparation, and specifically relates to a two-step method for fermenting corn husk to produce bacterial protein biological feed. Background Art

[0002] With the development of animal husbandry and breeding, the shortage of protein feed resources in my country is becoming more and more serious. Every year, a large amount of protein feed such as soybean meal and fish meal needs to be imported from abroad, which has led to a substantial increase in feed costs and affected food security. Therefore, the feed industry urgently needs to develop and apply unconventional protein feed to replace conventional protein feed. At present, in the field of protein feed replacement, cheap industrial and agricultural by-products are generally used as raw materials. Through fermentation by a variety of microorganisms, the indigestible substances in the raw materials are degraded and their protein content is increased.

[0003] Corn husk is one of the largest byproducts of fermentation enterprises using corn as raw material. Currently, the most common treatment method is to wash and dry it and then sell it as ruminant feed. It has the disadvantages of low nutritional value, poor palatability and poor feeding effect. To solve this problem, some researchers have conducted relevant research on microbial fermentation of corn husk. For example, Chinese patent publication number CN112868906A discloses a method for preparing corn husk dietary fiber feed, which includes preparing a composite microecological preparation, a composite enzyme preparation and a fermentation substrate. The invention converts corn husks that are difficult for animals to digest and absorb into a high-quality feed raw material product containing nutrients such as protein, prebiotics, soluble dietary fiber and insoluble dietary fiber through the synergistic action of bacteria and enzymes, while increasing the added value of corn husks; Chinese patent publication number CN110934223A discloses a process for preparing protein feed by solid-state microbial fermentation of corn husks, which includes mixing wet corn husks, wheat bran, shell powder, calcium phosphate and water, stirring, sterilizing, and naturally cooling to room temperature to obtain a basic feed solution; mixing tropical Candida seed solution and natto spore solution to obtain a mixed seed solution, inoculating the mixed seed solution into the basic feed solution, fermenting and culturing for 24 hours, and then inoculating thermophilic Streptococcus seed solution, and continuing to ferment and culture for 48 to 60 hours; stopping the fermentation, and drying the fermentation product at 60 to 70°C to a constant weight to obtain a protein feed. The invention solves the problems of high energy consumption and high pollution in the corn husk drying process in the prior art, and proposes a process for preparing protein feed by solid-state microbial fermentation of corn husk.

[0004] Although the relevant research on microbial fermentation of corn husks has achieved certain results, there are still some problems, such as high cost, strain safety, high fiber content and low protein content in feed. Moreover, the tropical Candida albicans and Bacillus natto in the above-mentioned invention patent are not included in the "Feed Additive Variety Catalog", and subsequent production cannot be carried out. Summary of the invention

[0005] The purpose of the present invention is to provide a two-step method for fermenting corn husk to produce bacterial protein biological feed. The preparation method of the present invention can maximize the degradation of fiber in the fermented feed while taking into account the cost, and increase its protein content, providing a preparation method for high-quality protein feed for the feed industry, and the strain used is safe.

[0006] The present invention provides a method for preparing bacterial protein biological feed, which comprises the following steps:

[0007] Inoculating the black Aspergillus fungus agent into the first fermentation base material for first fermentation to obtain koji seed;

[0008] Inoculating the composite bacterial agent into the second fermentation base material for second fermentation to obtain bacterial protein biological feed;

[0009] The first fermentation base material comprises the following raw materials in parts by weight: 84 to 92 parts of corn husks, 4 to 6 parts of bran, 2 to 4 parts of soybean meal and 2 to 5 parts of second flour;

[0010] The second fermentation base material includes the following raw materials in parts by mass: 19.5 to 30 parts of corn husks, 3 to 6 parts of molasses, 2 to 4 parts of ammonium sulfate, 0.1 to 0.2 parts of potassium dihydrogen phosphate, 0.1 to 0.2 parts of magnesium sulfate, 0.10 to 0.20 parts of sodium chloride, 0.10 to 0.15 parts of acetic acid and 10 to 35 parts of the koji.

[0011] Preferably, when inoculating the Aspergillus niger agent, the inoculation amount of the Aspergillus niger agent is 5-10% of the mass of the first fermentation base material.

[0012] Preferably, the first fermentation takes 24 to 48 hours at a temperature of 30 to 35°C.

[0013] Preferably, the composite bacterial agent comprises a saccharomyces cerevisiae seed solution and a utilis candida seed solution; the number of saccharomyces cerevisiae live bacteria in the saccharomyces cerevisiae seed solution is 1.4×10 11 CFU / kg; the number of viable Candida utilis in the Candida utilis seed solution is 1.9×10 11 CFU / kg; the volume ratio of the saccharomyces cerevisiae seed liquid to the Candida utilis seed liquid in the composite bacterial agent is 1-7:1-7.

[0014] Preferably, when inoculating the composite bacterial agent, the inoculation amount of the composite bacterial agent is 5% to 10% of the mass of the corn husk in the second fermentation base material.

[0015] Preferably, the fermentation time is 48 to 72 hours and the temperature is 30 to 32°C.

[0016] The present invention provides a bacterial protein biological feed prepared by the preparation method described in the above technical scheme.

[0017] The present invention provides the use of the bacterial protein biological feed described in the above technical solution in the preparation of animal feed.

[0018] The present invention provides the use of the bacterial protein biological feed described in the above technical solution in improving the milk production and milk quality of animals.

[0019] The invention provides the use of Aspergillus niger, Saccharomyces cerevisiae and Candida utilis in preparing bacterial protein biological feed.

[0020] Beneficial effects:

[0021] The present invention provides a method for preparing bacterial protein biological feed, specifically: inoculating Aspergillus niger in a first fermentation base material for a first fermentation to obtain a koji seed; inoculating a composite bacterial agent in a second fermentation base material for fermentation to obtain a bacterial protein biological feed. The preparation method of the present invention utilizes step-by-step fermentation to treat a fermentation base material mainly composed of corn husks, which can maximize the degradation of fiber in the fermented feed while taking into account the cost, and increase its protein content. The strain used is highly safe, and a method for preparing high-quality protein feed is provided for the feed industry. The corn husk bacterial protein fermented feed of the present invention can not only provide high-quality protein, but also provide a variety of probiotics and enzymes. While ensuring protein supply, it also has a certain effect on reducing or eliminating the use of antibiotics and ensuring the green development of the aquaculture industry, and has broad application prospects.

[0022] Moreover, the present invention subjects the by-products of corn starch processing to secondary processing, which not only provides a high-quality and low-cost non-antibiotic bacterial protein microbial feed for animal husbandry, but also solves the problem of insufficient utilization of corn husks, a by-product of corn processing, and improves its added value; the solid-state fermentation method adopted has the characteristics of simple equipment structure, low investment, low energy consumption, simple operation, etc.; all fermentation products are products, there is no other waste, and there is less pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0024] Figure 1 is a diagram of the state of mycelium in the first fermentation product;

[0025] Figure 2 It is the experimental flow chart of Example 1, wherein Figure 2 The second fermentation base material refers to the dry corn husk, molasses, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride and magnesium sulfate in the second fermentation base material.

[0026] Biological deposit information

[0027] Aspergillus niger CJH-JWSFZh-B703 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on June 8, 2021. The deposit address is No. 2, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the deposit number is CGMCC No. 22439. DETAILED DESCRIPTION

[0028] Unless otherwise specified, the raw materials used in the present invention can be purchased conventionally by those skilled in the art.

[0029] The present invention provides a method for preparing bacterial protein biological feed, comprising the following steps:

[0030] Inoculating Aspergillus niger into the first fermentation base material for first fermentation to obtain koji seed;

[0031] Inoculating the composite bacterial agent into the second fermentation base material for second fermentation to obtain bacterial protein biological feed;

[0032] The first fermentation base material comprises the following raw materials in parts by weight: 84 to 92 parts of corn husks, 4 to 6 parts of bran, 2 to 4 parts of soybean meal and 2 to 5 parts of second flour;

[0033] The second fermentation base material includes the following raw materials in parts by mass: 19.5 to 30 parts of corn husks, 3 to 6 parts of molasses, 2 to 4 parts of ammonium sulfate, 0.1 to 0.2 parts of potassium dihydrogen phosphate, 0.1 to 0.2 parts of magnesium sulfate, 0.10 to 0.20 parts of sodium chloride, 0.10 to 0.15 parts of acetic acid and 10 to 35 parts of the koji.

[0034] In the present invention, the Aspergillus niger agent is inoculated into the first fermentation base material for the first fermentation to obtain a koji seed. The Aspergillus niger strain of the present invention is preferably Aspergillus niger CJH-JXSFZh-B703; the Aspergillus niger CJH-JXSFZh-B703 has a deposit number of CGMCC No. 22439; the number of Aspergillus niger spores in the koji seed is preferably 6.52×10 9CFU / kg. The Aspergillus niger used in the present invention is a safe strain that is allowed to be used as a microbial feed additive in the "Catalogue of Feed Additives"; and the enzyme system produced by Aspergillus niger itself is rich, and more than ten enzymes that can be used as feed enzyme preparations allowed by the Ministry of Agriculture can be derived from Aspergillus niger, among which cellulase, protease, amylase, and pectinase can act on corn husk. Moreover, Aspergillus niger contains enzymes with a maximum enzyme activity of 39.52U / mL. Aspergillus niger has a strong ability to degrade cellulose. When inoculated into corn husk, Aspergillus niger can grow well by utilizing some nutrients in the corn husk, and degrade cellulose into small molecules such as glucose for bacterial growth. The treated corn husk is rich in bacterial protein, amino acids, vitamins, minerals and other elements. It is used as feed and has a great utilization value.

[0035] The present invention also preferably provides a method for preparing the Aspergillus niger agent, comprising the following steps:

[0036] Aspergillus niger was cultured on slant in malt extract agar medium;

[0037] After the slant culture is completed, the Aspergillus niger obtained from the slant culture is picked up and inoculated into a malt juice liquid culture medium for shake flask culture to obtain an Aspergillus niger fungus agent.

[0038] The temperature of the slant culture of the present invention is preferably 30°C; during the slant culture, the inoculation amount of Aspergillus niger is preferably 1 ring. The preparation method of the malt agar medium of the present invention preferably comprises the following steps: after mixing malt juice, agar and water, a malt agar medium is obtained; the amount of the malt juice is preferably 1.0L; the amount of the agar is preferably 15.0g; the amount of water is preferably the amount used when the Brix of the malt juice is adjusted to 12°Brix; the malt agar medium is preferably a natural pH, and the malt agar medium is preferably a malt agar medium obtained by sterilization at 121°C for 20 minutes.

[0039] The inoculation amount of the shake flask culture of the present invention is preferably 1 ring; the temperature of the shake flask culture is preferably a constant temperature of 30°C; the rotation speed of the shake flask culture is preferably 180r / min; the time of the shake flask culture is preferably 24h. In the present invention, the malt wort liquid culture medium is preferably a maltose liquid with an apparent sugar content of 12°Brix; the malt wort liquid culture medium is preferably a malt wort liquid culture medium obtained by sterilization at 115°C for 30min. The spore count of the Aspergillus niger agent of the present invention is preferably ≥10 9 CFU / mL, more preferably 1.0×10 9 ~2.0×10 9 CFU / mL, the most preferred is 1.0×10 9 CFU / mL.

[0040] After obtaining the Aspergillus niger agent, the present invention also preferably adjusts the Aspergillus niger agent so that the spore count is 1.0×10 9 ~2.0×10 9 CFU / mL. The present invention has no limitation on the adjustment method, and a method familiar to those skilled in the art can be used; in a specific embodiment of the present invention, the adjustment method is preferably to take samples every 2 hours, monitor the number of Aspergillus niger spores in the Aspergillus niger inoculant in real time, and calculate the number of Aspergillus niger spores by a hemocytometer; the sampling preferably refers to taking Aspergillus niger seed liquid.

[0041] In the present invention, the first fermentation base material comprises the following raw materials in parts by mass: 84 to 92 parts of corn husks, 4 to 6 parts of bran, 2 to 4 parts of soybean meal and 2 to 4 parts of second flour.

[0042] In parts by mass, the first fermented base material of the present invention comprises 84 to 92 parts of corn husks, preferably 86 to 90 parts, and more preferably 86 parts. The corn husks of the present invention preferably comprise dry corn husks, more preferably dry corn husks; the types of the corn husks preferably include coarse corn husks and / or fine corn husks, more preferably coarse corn husks and fine corn husks; the mass ratio of the coarse corn husks to the fine corn husks is preferably 66 to 82:10 to 20, more preferably 66:20, 72:10 or 80:10; the particle size of the coarse corn husks is preferably 5 to 10 meshes, and the fine corn husks are preferably corn husks with a 20-mesh sieve pass rate of more than 85%, that is, the fine corn husks are preferably corn husks obtained by crushing corn husks and passing through a 20-mesh sieve, but fine corn husks with a particle size slightly larger than 20 meshes may exist during the crushing process. The purpose of selecting corn husks of different meshes is to control the gaps between the corn husks to avoid excessive gaps due to too much coarse corn husks or too much fine corn husks, which makes it difficult to control the moisture content or is too sticky. The moisture content of the dry corn husk of the present invention is preferably 9wt.%; the source of the corn husk is not particularly limited in the present invention, and the corn husk can be purchased by a person skilled in the art; in the specific embodiment of the present invention, the corn husk is purchased from Inner Mongolia Fufeng Biotechnology Co., Ltd. The moisture content of the first fermentation base material of the present invention is preferably controlled at 35wt.% to 40wt.%.

[0043] Based on the mass fraction of corn husk, the first fermentation base material of the present invention comprises 4 to 6 parts of bran, preferably 5 to 6 parts, and most preferably 6 parts.

[0044] Based on the mass percentage of corn husk, the first fermentation base material of the present invention includes 2 to 4 parts of soybean meal, preferably 2.5 to 4 parts, and most preferably 4 parts.

[0045] Based on the mass percentage of corn husk, the first fermentation base material of the present invention includes 2 to 4 parts of secondary flour, preferably 2.5 to 5 parts, and most preferably 4 to 5 parts.

[0046] Before inoculating the Aspergillus niger agent, the present invention preferably performs the first steaming, the first stirring, the adding of water, the second stirring, the second steaming and the third stirring on the first fermentation base material to obtain the steamed material; the temperature of the first steamed material and the second steamed material is preferably 115°C to 120°C, more preferably 120°C; the air pressure of the first steamed material and the second steamed material is preferably 0.1MPa to 0.2MPa, more preferably 0.2MPa; the time of the first stirring and the second stirring is preferably 10min; the time of the third stirring is preferably 20min. When adding water according to the present invention, the amount of water is 60% to 80% of the mass of the first fermentation base material, more preferably 80%. Steaming under the conditions set by the present invention can eliminate other harmful bacteria in the first fermentation base material, and can also expand the fiber structure and promote hydrolysis. The water content of the steamed material of the present invention is preferably 40wt.% to 60wt.%, more preferably 43wt.% to 58wt.%, and more preferably 45wt.% to 55wt.%; the initial pH value of the steamed material is preferably 7.0±0.2, that is, the pH value of the steamed material is 7.0±0.2.

[0047] When the Aspergillus niger is inoculated in the present invention, the inoculation amount of Aspergillus niger is preferably 5% to 10% of the mass of the steamed material, and more preferably 5%.

[0048] After inoculating the Aspergillus niger agent, the present invention performs expanded inoculation and first fermentation on the steamed material inoculated with the Aspergillus niger agent to obtain a koji seed, also known as a first fermentation product. The temperature of the expanded inoculation of the present invention is preferably 30°C; the time of the expanded inoculation is preferably 48h. In the present invention, the time of the first fermentation is preferably 24-48h, further preferably 28-45h, more preferably 30-42h, and most preferably 33-37h; the indoor temperature of the first fermentation is preferably 25°C-30°C, that is, during the first fermentation, the temperature in the environment is preferably 25°C-30°C; during the first fermentation, the fermentation product temperature is preferably 30-35°C; the relative humidity of the first fermentation is preferably ≥95%; the fermentation product temperature of the present invention preferably refers to the temperature of the steamed material inoculated with the Aspergillus niger agent during the first fermentation. When the fermentation product temperature exceeds 35°C and continuous ventilation cannot be cooled to below 35°C, the present invention preferably performs compost turning or stirring to cool down. The present invention does not have any restrictions on the methods of compost turning and stirring, and the methods well known to those skilled in the art can be used.

[0049] After obtaining the koji seed, the present invention inoculates the composite bacterial agent into the second fermentation base material for second fermentation to obtain bacterial protein biological feed. In the present invention, the composite bacterial agent preferably includes a fermentation broth of saccharomyces cerevisiae and a fermentation broth of Candida utilis; the number of live saccharomyces cerevisiae in the fermentation broth of saccharomyces cerevisiae is preferably 1.4×10 11 CFU / kg; the number of viable Candida utilis in the Candida utilis fermentation broth is preferably 1.9×1011 The volume ratio of the fermentation broth of Saccharomyces cerevisiae to the fermentation broth of Candida utilis in the composite bacterial agent of the present invention is 1-7:1-7, more preferably 1:1-2, 1-2:1, 1.5:1-2, 7:3 or 3:7.

[0050] In the present invention, the brewer's yeast (Saccharomyces cerevisiae) is preferably a brewer's yeast with a deposit number of CICC32236, and the brewer's yeast (Saccharomyces cerevisiae) CICC32236 is a strain of the China Industrial Microorganism Culture Collection Administration Center (CICC). CICC has a special website, the website address is: http: / / www.china-cicc.org, and the public can directly order strains online. The website address of brewer's yeast (Saccharomyces cerevisiae) CICC32236 is http: / / www.china-cicc.org / search / ? classtype=0&keyword=32236, and the public can obtain the strain from CICC. The present invention does not specifically limit the source of the brewer's yeast, and the yeast can be obtained by conventional purchase by a technician in the field; in a specific embodiment of the present invention, the brewer's yeast is preferably purchased from the China Industrial Microorganism Culture Collection Administration Center.

[0051] The Candida utilis of the present invention is preferably a Candida utilis with a deposit number of CGMCC2.2878. The Candida utilis CGMCC2.2878 is a strain of the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration. CGMCC has a special website, the website address is: https: / / cgmcc.net / english / , and the public can directly order strains online. The website address of Candida utilis CGMCC2.2878 is https: / / cgmcc.net / english / detail?id=66762, and the public can obtain the strain from CGMCC. The present invention does not specifically limit the source of the Candida utilis, and the conventional purchase by a technician in the field can be used; in a specific embodiment of the present invention, the Candida utilis is preferably purchased from the China General Microbiological Culture Collection Administration Center. The brewer's yeast used in the present invention is rich in nutrients such as protein, amino acids and small peptides. After fermentation by brewer's yeast, the palatability and nutritional value of feed can be improved, and the animal's feed intake and digestion utilization rate can be increased. The protein content of Candida utilis itself is relatively high, accounting for 32% to 75% of its own dry weight. It is rich in nutrients such as fat, vitamins, nucleic acids, and has a wide range of carbon source utilization, and can utilize both pentose and hexose. And the true protein content is greatly increased after the two are fermented together.

[0052] The present invention also preferably provides a method for preparing the composite bacterial agent, comprising the following steps:

[0053] Saccharomyces cerevisiae and Candida utilis were inoculated on YPD agar medium for slant culture.

[0054] The slant cultured Saccharomyces cerevisiae and Candida utilis were inoculated into YPD liquid medium for shaking culture to obtain Saccharomyces cerevisiae seed liquid and Candida utilis seed liquid;

[0055] The brewer's yeast seed liquid and the Candida utilis seed liquid are mixed in a volume ratio of 1-7:1-7 to obtain a composite bacterial agent.

[0056] The present invention preferably inoculates Saccharomyces cerevisiae and Candida utilis on YPD agar medium for slant culture. The temperature of the slant culture of the present invention is preferably 30°C; when the Saccharomyces cerevisiae slant culture is performed, preferably one ring of a single colony of Saccharomyces cerevisiae is streaked on YPD agar medium; when the Candida utilis slant culture is performed, preferably one ring of a single colony of Candida utilis is streaked on YPD agar medium. The YPD agar medium of the present invention preferably includes the following components by mass: 10g of peptone, 5g of yeast extract powder, 10g of glucose, 20g of agar powder and 1kg of distilled water; the pH of the YPD agar medium is preferably natural; the YPD agar medium is preferably a YPD agar medium sterilized at 115°C for 30min.

[0057] The present invention preferably inoculates the Saccharomyces cerevisiae and Candida utilis obtained by slant culture into YPD liquid culture medium for shaking culture to obtain Saccharomyces cerevisiae seed solution and Candida utilis seed solution. The inoculation amount of the shaking culture in the present invention is preferably 1 ring; the temperature of the shaking flask culture is preferably a constant temperature of 30°C; the rotation speed of the shaking flask culture is preferably 180r / min; and the time of the shaking flask culture is preferably 11h. In the present invention, the YPD liquid culture medium preferably includes the following components by mass: 10g of peptone, 5g of yeast extract powder, 10g of glucose and 1L of distilled water; the pH of the YPD liquid culture medium is 7.0-7.4; the YPD liquid culture medium is preferably a YPD liquid culture medium obtained by sterilization at 115°C for 30min.

[0058] After obtaining the saccharomyces cerevisiae seed solution and the Candida utilis seed solution, the present invention also preferably adjusts the saccharomyces cerevisiae seed solution and the Candida utilis seed solution respectively so that the effective viable bacteria count of the saccharomyces cerevisiae seed solution is 1.5×10 8 CFU / mL, so that the effective viable count of Candida utilis seed solution is 1.8×10 8 CFU / mL. The present invention has no limitation on the method of adjustment, and the method known to those skilled in the art can be adopted; in a specific embodiment of the present invention, the method of adjustment is preferably to take samples every 2 hours, monitor the number of live yeasts in the saccharomyces cerevisiae seed liquid or the number of live Candida utilis in the Candida utilis seed liquid in real time, and calculate the number of live yeasts or the number of live Candida utilis by a hemocytometer; the sampling preferably refers to taking the saccharomyces cerevisiae seed liquid or the Candida utilis seed liquid.

[0059] The second fermentation base material of the present invention comprises the following raw materials in parts by weight: 25-30 parts of corn husks, 3-6 parts of molasses, 2-4 parts of ammonium sulfate, 0.1-0.2 parts of potassium dihydrogen phosphate, 0.1-0.2 parts of magnesium sulfate, 0.10-0.15 parts of sodium chloride, 0.10-0.15 parts of acetic acid and 10-35 parts of the koji. In the present invention, the water content of the second fermentation base material is preferably controlled to 70wt.%, so that the texture of the second fermentation base material is slightly sticky.

[0060] In parts by mass, the second fermentation base material of the present invention includes 25 to 30 parts of corn husks, preferably 26 to 28 parts. The corn husks of the present invention are dry corn husks; the types of corn husks preferably include coarse corn husks and fine corn husks; the mass ratio of coarse corn husks to fine corn husks is preferably 66 to 82:10 to 20; the particle size of the coarse corn is preferably 5 mesh to 10 mesh, and the fine corn husks are preferably corn husks with a pass rate of more than 85% on a 20-mesh sieve, that is, the fine corn husks are preferably the sieve-down obtained after the corn husks are crushed and passed through a 20-mesh sieve, but fine corn husks with a particle size slightly larger than 20 mesh may exist during the crushing process. The moisture content of the corn husks of the present invention is preferably 9wt.%; the present invention does not specifically limit the source of the corn husks, and the corn husks conventionally purchased by those skilled in the art can be used.

[0061] Based on the mass percentage of corn husk, the second fermentation base material of the present invention includes 3 to 6 parts of molasses, preferably 3.2 to 3.8 parts. In the present invention, there is no special limitation on the source of the honey, and it can be purchased by a person skilled in the art; in a specific embodiment of the present invention, the honey is preferably purchased from Inner Mongolia Fufeng Biotechnology Co., Ltd.

[0062] Based on the mass fraction of corn husk, the second fermentation base material of the present invention comprises 2 to 4 parts of ammonium sulfate, preferably 2 to 3 parts.

[0063] Based on the mass fraction of corn husk, the second fermentation base material of the present invention includes 0.1 to 0.2 parts of potassium dihydrogen phosphate, preferably 0.12 to 0.19 parts.

[0064] Based on the mass percentage of corn husk, the second fermentation base material of the present invention includes 0.1 to 0.2 parts of magnesium sulfate, preferably 0.13 to 0.18 parts of magnesium sulfate.

[0065] Based on the mass fraction of corn husk, the second fermentation base material of the present invention includes 0.10 to 0.15 parts of sodium chloride, preferably 0.1 parts.

[0066] Based on the mass fraction of corn husk, the second fermentation base material of the present invention comprises 0.10 to 0.15 parts of acetic acid, preferably 0.1 parts.

[0067] Based on the mass percentage of corn husk, the second fermentation base material of the present invention includes 10 to 35 parts of the koji, preferably 10 to 10.5 parts, and more preferably 10.5 parts.

[0068] Before inoculating the composite bacterial agent, the present invention preferably further comprises: steaming the corn husk, molasses, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate and sodium chloride in the second fermentation base material and adding water to obtain steamed material; the temperature of the steaming treatment is preferably 115°C to 121°C, more preferably 121°C; the air pressure of the steaming treatment is preferably 0.1MPa to 0.2MPa, more preferably 0.2MPa; the time of the steaming treatment is 20min. When adding water in the present invention, the amount of water is preferably 70% of the total mass of water and corn husk.

[0069] After obtaining the steamed material, the present invention preferably further comprises mixing the steamed material with the koji, performing enzymolysis, and obtaining an enzymolysis base material; the enzymolysis time is preferably 6 hours, and the enzymolysis temperature is preferably 25° C. to 30° C. During the enzymolysis of the present invention, the present invention preferably adds tap water to the steamed material; the amount of tap water used is preferably the amount of water when the steamed material becomes slightly sticky after adding tap water.

[0070] After obtaining the enzymatic substrate, the present invention preferably mixes the enzymatic substrate with acetic acid, and inoculates the mixture obtained after mixing the enzymatic substrate with acetic acid for the second fermentation. The time of the second fermentation of the present invention is preferably 48 to 72 hours, more preferably 48 hours; the temperature of the second fermentation is preferably 30 to 32°C. The present invention performs feeding during the second fermentation process; the specific steps of the feeding are: adding 1% ammonium sulfate and 1% molasses before stirring at the 24th hour of the second fermentation; adding 1% ammonium sulfate and 1% molasses before stirring at the 48th hour of the second fermentation; the amount of ammonium sulfate and molasses is preferably based on the weight of the second fermentation substrate. The present invention adopts a step-by-step feeding method to supplement the carbon source, trace elements and inorganic salts required for bacterial growth and metabolism, so that the crude fiber in the corn husk of the second fermentation substrate can be fully degraded, and the components obtained after degradation can contain higher nutrition after fermentation.

[0071] When the composite bacterial agent is inoculated in the present invention, the inoculation amount of the composite bacterial agent is preferably 5% of the mass of the corn husk in the second fermentation base material.

[0072] The effective viable bacteria count of Saccharomyces cerevisiae in the bacterial protein biological feed of the present invention is preferably 1.4×10 9 CFU / kg~1.4×10 11 CFU / kg, more preferably 1.4×10 10 CFU / kg~1.4×10 11 CFU / kg, the most preferred is 1.4×10 11CFU / kg; the effective viable count of Candida utilis in the bacterial protein biological feed is preferably 1.9×10 9 CFU / kg~1.9×10 11 CFU / kg, more preferably 1.9×10 10 CFU / kg~1.9×10 11 CFU / kg, the most preferred is 1.9×10 11 CFU / kg.

[0073] The present invention uses Aspergillus niger to perform the first fermentation on the first fermentation substrate to obtain the koji, and then uses a composite bacterial liquid containing saccharomyces cerevisiae and Candida utilis to ferment the second fermentation substrate. It can be seen that the process adopted by the present invention is multi-strain mixed step-by-step fermentation, and the microorganisms cooperate with each other, which greatly promotes the rapid degradation and fermentation of corn husks, and has the following characteristics: 1. Multi-bacteria symbiosis, rich in cellulase, protease, and amylase, and realizes enzyme system complementation; 2. Energy saving, simplified process equipment, and more perfect biochemical reactions; 3. Rich substrates, nutrients can be provided in stages; 4. The monosaccharides and other nutrients generated by the enzymatic action are immediately used by other microorganisms, while maintaining the concentration of the degradation product, eliminating the inhibitory effect of the enzymatic hydrolysis product on the enzyme; 5. Through the mutualistic symbiosis and partial symbiotic relationship between microorganisms, soybean meal can be hydrolyzed to generate small peptide substances, and corn husks can be fermented into a biological fermentation feed product with the characteristics of nutrition, immunity, anti-infection, and easy absorption. And the method described in the present invention can maximize the degradation of fiber in the fermented feed while taking into account the cost, and increase its protein content.

[0074] The present invention also provides a bacterial protein biological feed prepared by the method described in the above technical solution. The present invention has already discussed the preparation method in detail in the previous text, and will not be repeated here. The raw material combination used in the bacterial protein biological feed provided by the present invention has low cost and wide sources; the bacterial protein biological feed described in the present invention can hydrolyze soybean meal in the raw material into small peptide substances, degrade cellulose and hemicellulose in corn husk into soluble sugars, and convert them into bacterial protein. In addition, after fermentation of multiple strains, an aromatic odor is produced, which can also increase palatability. At the same time, the feed contains a large number of beneficial microorganisms and enzymes, among which Aspergillus niger and yeast are beneficial microorganisms in the rumen of dairy cows, and the various cellulose and hemicellulose hydrolases produced help the rumen to degrade crude fiber in the diet, thereby improving feed utilization. Moreover, the feed contains a variety of yeasts, rich in protein, B vitamins, amino acids and other substances, and can be widely used as a protein supplement for animal feed. It can promote the growth and development of animals, shorten the feeding period, and improve product output and quality, such as milk output and milk quality; and the feed contains Aspergillus niger, which can produce a variety of hydrolases and saccharifying enzymes such as cellulase and hemicellulase, which can improve the utilization rate of roughage and save feeding costs.

[0075] According to the above advantages, the present invention provides the bacterial protein biological feed described in the above technical solution or the use of the bacterial protein biological feed described in the above technical solution in preparing animal feed. The animal described in the present invention preferably includes mammals, further preferably includes cattle, and more preferably includes dairy cows.

[0076] According to the above advantages, the present invention provides the application of the specific protein feed described in the above technical solution in improving the milk production and milk quality of animals. The corn husk bacterial protein biological feed produced by the present invention contains a variety of yeasts, rich in protein, B vitamins and amino acids and other substances, wherein the protein content is about 30% to 40%, and can be widely used as a protein supplement for animal feed. The feed can promote the growth and development of animals, shorten the feeding period, and improve product yield and quality, such as milk yield and milk quality.

[0077] The present invention also provides the use of Aspergillus niger, Saccharomyces cerevisiae and Candida utilis in preparing bacterial protein biological feed. The Aspergillus niger, Saccharomyces cerevisiae and Candida utilis of the present invention have been described in detail above and will not be repeated here.

[0078] In order to further illustrate the present invention, the technical solution provided by the present invention is 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.

[0079] Preparation Example

[0080] Medium preparation:

[0081] The malt extract agar medium is a malt extract agar medium of 12° Brix, specifically 1.0 L of malt extract, 15.0 g of agar and the balance of water, natural pH, and sterilized at 121° C. for 20 min.

[0082] The wort liquid medium is maltose liquid with an apparent sugar content of 12°Brix;

[0083] YPD agar medium is peptone 10g, yeast extract powder 5g, glucose 10g, agar powder 20g and distilled water 1L, pH is 7.0-7.4; sterilize at 15℃ for 30min;

[0084] YPD liquid culture medium is composed of 10 g of peptone, 5 g of yeast extract powder, 10 g of glucose and 1 L of distilled water, with a pH of 7.0-7.4; sterilize at 15°C for 30 min.

[0085] Aspergillus niger CJH-JXSFZh-B703 was cultured on a malt agar medium, and a ring of Aspergillus niger colonies was streaked on the malt agar medium, and the culture was carried out at 30°C. After the slant culture, a ring of Aspergillus niger CJH-JXSFZh-B703 slant was picked and inoculated into 100 mL of malt liquid medium in a 250 mL triangular flask, and the culture was carried out at a constant temperature of 30°C and 180 r / min for 24 hours to prepare Aspergillus niger CJH-JXSFZh-B703 seed liquid, and the concentration of the seed liquid was adjusted so that the effective viable count was 1.0×10 9 CFU / mL, get the Aspergillus niger agent, and keep it for later use;

[0086] Saccharomyces cerevisiae CICC32236 was cultured on a slant in YPD agar medium, and a single colony of Saccharomyces cerevisiae was streaked on the YPD agar medium and cultured at 30°C. After the slant culture, a slant of Saccharomyces cerevisiae CICC32236 was picked and inoculated into 100 mL of YPD liquid medium in a 250 mL triangular flask, and cultured at 30°C and 180 r / min for 11 h to prepare a seed solution of Saccharomyces cerevisiae CICC32236. The concentration of the seed solution was adjusted to make the effective viable count of 1.5×10 8 CFU / mL, reserve;

[0087] Candida utilis CGMCC2.2878 was cultured on a slant in YPD agar medium, and a single colony of Saccharomyces cerevisiae was streaked on the YPD agar medium and cultured at 30°C. After the slant culture, a slant of Candida utilis CGMCC2.2878 was picked and inoculated into 100 mL of YPD liquid medium in a 250 mL triangular flask, and cultured at 30°C and 180 r / min for 11 h to prepare a seed solution of Candida utilis CGMCC2.2878. The concentration of the seed solution was adjusted so that the effective viable count was 1.8×10 8 CFU / mL, reserve;

[0088] After the shake flask culture was completed, the obtained Saccharomyces cerevisiae CICC32236 seed solution and the Candida utilis CGMCC2.2878 seed solution were mixed in equal volumes to obtain a composite bacterial solution, which was recorded as a composite bacterial agent and set aside.

[0089] The following examples all use the Aspergillus niger fungus agent or the composite fungus agent prepared herein.

[0090] Example 1

[0091] The dried corn husk was obtained from Inner Mongolia Fufeng Biotechnology Co., Ltd., and the moisture content of the dried corn husk was 9wt.%, the bran was obtained from Inner Mongolia Shenggu Brewing Food Co., Ltd., the soybean meal was obtained from Inner Mongolia Shenggu Brewing Food Co., Ltd., and the second flour was obtained from Inner Mongolia Shenggu Brewing Food Co., Ltd.; the specific experimental steps are as follows Figure 2 As shown, Figure 2 The second fermentation base material refers to the dry corn husk, molasses, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride and magnesium sulfate in the second fermentation base material:

[0092] (1) Preparation of fermentation substrate

[0093] The first fermentation substrate (fermentation substrate 1) is composed of 86 parts of corn husks, 6 parts of bran, 4 parts of soybean meal and 4 parts of second-rate flour, wherein the dry corn husks are composed of 66 parts of coarse corn husks and 20 parts of fine corn husks; converted into specific mass: 172kg of dry corn husks, 12kg of bran, 8kg of soybean meal, 8kg of second-rate flour, wherein the coarse corn husks are 132kg and the fine corn husks are 40kg.

[0094] The second fermentation substrate (fermentation substrate 2) is composed of 19.5 parts of dry corn husks, 3 parts of molasses, 2 parts of ammonium sulfate, 0.2 parts of potassium dihydrogen phosphate, 0.1 parts of sodium chloride, 0.2 parts of magnesium sulfate, 0.125 parts of acetic acid and 10.5 parts of koji seeds, wherein the dry corn husks, molasses, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride and magnesium sulfate are obtained after sterilization. The specific steps are detailed in step (4); wherein the dry corn husks are composed of 14.5wt.% of coarse corn husks and 5wt.% of fine corn husks; converted into specific masses: 290kg of coarse corn husks and 100kg of fine corn husks, 60kg of molasses, 40kg of ammonium sulfate, 4kg of potassium dihydrogen phosphate, 2kg of sodium chloride, 4kg of magnesium sulfate, 2.5L of acetic acid and 210kg of koji seeds.

[0095] (2) Sterilization: The first fermentation base material is loaded into the sterilizer. After all the materials are added, 120°C, 0.2MPa steam is introduced and stirred for 10 minutes. After the pressure is released, tap water accounting for 80% of the mass of the first fermentation base material, i.e. 160 kg, is added and stirred for 10 minutes. Finally, 120°C, 0.2MPa steam is introduced and stirred for 20 minutes to obtain the steamed material. The water content is controlled at 35wt.%~40wt.%, and the initial pH value is 7.0±0.2.

[0096] (3) After the sterilization is completed, the Aspergillus niger agent is directly mixed with the steamed material, the material is conveyed by an auger, and the Aspergillus niger agent is started to be inoculated, ensuring that the inoculation amount of the Aspergillus niger agent is 5% of the mass of the first fermentation base material. First, a car of about 50 kg of the first fermentation base material inoculated with the Aspergillus niger agent is inoculated, and then the inoculation is expanded to ensure that the Aspergillus niger is evenly connected to the fermentation base material. The culture conditions for the expanded inoculation are 30° C. and 48 h. The base material temperature after the expanded inoculation is about 40° C. Here, "the base material temperature of about 40° C." specifically refers to the temperature of the material in the Quchi just after the expanded culture. The inoculated bottom material is sent into the Quchi by a cart. The material layer is stacked at an angle, with a 25cm layer near the air inlet and a 30cm layer on the other side. The upper layer is scraped flat and the first fermentation begins. The temperature in the fermentation room is controlled at 25℃~30℃, and the relative humidity is ≥95wt.%. Intermittent ventilation is used to control the product temperature at 30℃~35℃. If the product temperature is higher than 35℃ and continuous ventilation cannot cool it down to below 35℃, turn the pile or stir it to cool it down. After cooling down to below 35℃, continue the first fermentation for 5h~10h. Observe that there are a large number of mycelium and they are very strong. At the same time, there are also a large number of spores. Figure 1 As shown, the result is as follows, and the koji is obtained. The number of Aspergillus niger spores in the koji reaches 6.52×10 9 CFU / kg.

[0097] (4) Sterilization: The dried corn husk, molasses, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride and magnesium sulfate in the second fermentation base material are placed in a sterilizing pot, and then sterilized at 121° C. and 0.2 MPa steam for 20 min. 1400 kg of tap water is added to obtain the steamed material.

[0098] (5) After sterilization, the material in the steamer is transported to the yeast fermentation tank by a cart, and mixed with the 210 kg of koji prepared above. Appropriate ventilation and cooling are carried out to ensure that the temperature of the corn husk is not lower than 60°C. Tap water is added to adjust the moisture content to a slightly sticky state. Enzymatic hydrolysis is carried out at 30°C for 6 hours. When liquid seeps out from the bottom, it is promptly shoveled to the surface of the material layer.

[0099] (6) After the enzymatic hydrolysis, 2.5 L of acetic acid and a composite bacterial agent were added. The inoculation amount of the composite bacterial agent was 5% of the mass of the corn husk in the second fermentation base material, which was converted into a specific volume of: 50 L of saccharomyces cerevisiae seed liquid and 50 L of Candida utilis seed liquid were stirred, and the second fermentation was started, which was recorded as yeast fermentation. The fermentation temperature was 30° C. and the fermentation time was 3 days. At the 24th hour of fermentation, 1 wt.% of ammonium sulfate and 1% of molasses of the total mass of the second fermentation base material were added before stirring; at the 48th hour of fermentation, 1% of ammonium sulfate and 1% of molasses of the total mass of the second fermentation base material were added before stirring; at the end of the fermentation, a corn husk mycelial protein biological feed fermented by the step-by-step method was obtained, which was recorded as mycelial protein biological feed 1. The content of live Candida utilis in mycelial protein biological feed 1 was 1.9×10 11CFU / kg, the content of live yeast of Saccharomyces cerevisiae is 1.4×10 11 CFU / kg.

[0100] Example 2

[0101] The steps are the same as those in Example 1, with the only difference being that, according to the method in step (3) of Example 1, the inoculation amount of the Aspergillus niger agent is adjusted to 10%, and the corn husk mycelial protein biological feed obtained after the fermentation is recorded as mycelial protein biological feed 2.

[0102] Example 3

[0103] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (1) in Example 1, the first fermentation base material is replaced with fermentation substrate 3, and the other steps remain unchanged. After the fermentation is completed, corn husk mycelial protein biological feed fermented by a multi-step method is obtained, which is recorded as mycelial protein biological feed 3.

[0104] Among them, fermentation substrate 3 consists of 82 parts of coarse corn husks, 10 parts of fine corn husks, 4 parts of bran, 2 parts of soybean meal, and 2 parts of second-grade flour.

[0105] Example 4

[0106] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (1) in Example 1, the first fermentation base material is replaced with fermentation substrate 4, and the other steps remain unchanged. After the fermentation is completed, corn husk mycelial protein biological feed fermented by a multi-step method is obtained, which is recorded as mycelial protein biological feed 4.

[0107] Among them, fermentation substrate 4 is composed of 74 parts of coarse corn husks, 10 parts of fine corn husks, 6 parts of bran, 5 parts of soybean meal, and 5 parts of second-grade flour.

[0108] Example 5

[0109] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (6) in Example 1, the composite bacterial agent is replaced with composite bacterial agent 2, and the other steps remain unchanged. The corn husk mycelial protein biological feed obtained after the fermentation is recorded as mycelial protein biological feed 5.

[0110] The composite bacterial solution 2 is obtained by mixing the fermentation solution of Saccharomyces cerevisiae and the fermentation solution of Candida utilis in the preparation example at a volume ratio of 7:3.

[0111] Example 6

[0112] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (6) in Example 1, the composite bacterial solution is replaced with composite bacterial solution 3, and the other steps remain unchanged. The corn husk mycelial protein biological feed obtained after the fermentation is recorded as mycelial protein biological feed 6.

[0113] The composite bacterial solution 3 is obtained by mixing the fermentation solution of Saccharomyces cerevisiae and the fermentation solution of Candida utilis in the preparation example at a volume ratio of 3:7.

[0114] Example 7

[0115] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (1) in Example 1, the second fermentation base is replaced with fermentation substrate 5, and the other steps remain unchanged. The corn husk mycelial protein biological feed obtained after the fermentation is recorded as mycelial protein biological feed 7.

[0116] Among them, fermentation substrate 5 is composed of 19.5 parts of corn husks, 10.5 parts of koji prepared in Example 1, 5 parts of molasses, 4 parts of ammonium sulfate, 0.2 parts of potassium dihydrogen phosphate, 0.1 parts of sodium chloride, 0.2 parts of magnesium sulfate and 0.125 parts of acetic acid. Among them, the corn husks are 14.5 parts of coarse corn husks and 5 parts of fine corn husks, which are converted into specific masses: 290kg of coarse corn husks, 100kg of fine corn husks, 210kg of koji, 100kg of molasses, 80kg of ammonium sulfate, 4kg of potassium dihydrogen phosphate, 2kg of sodium chloride, 4kg of magnesium sulfate and 2.5L of acetic acid.

[0117] Example 8

[0118] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (1) in Example 1, the second fermentation base material is replaced with fermentation substrate 6, and the other steps remain unchanged. The corn husk mycelial protein biological feed obtained after the fermentation is recorded as mycelial protein biological feed 8.

[0119] Among them, fermentation substrate 6 is composed of 19.5 parts of corn husks, 10.5 parts of koji prepared in Example 1, 6 parts of molasses, 2 parts of ammonium sulfate, 0.1 parts of potassium dihydrogen phosphate, 0.2 parts of sodium chloride, 0.2 parts of magnesium sulfate and 0.125 parts of acetic acid, wherein the corn husks are 14.5 parts of coarse corn husks and 5 parts of fine corn husks, which is converted into a specific mass of: 290kg coarse corn husks, 100kg fine corn husks, 210kg koji, 120kg molasses, 40kg ammonium sulfate, 2kg potassium dihydrogen phosphate, 4kg sodium chloride, 4kg magnesium sulfate and 2.5L acetic acid.

[0120] Application Example 1

[0121] Feeding the bacterial protein biological feeds of Examples 1 to 8 to lactating dairy cows to detect the effect of the bacterial protein biological feeds of the present invention on the milk production of dairy cows, the specific steps are as follows:

[0122] 27 Holstein black and white dairy cows were selected and randomly divided into 9 groups, each with 3 cows, namely: No. 0 experimental group, No. 1 experimental group, No. 2 experimental group, No. 3 experimental group, No. 4 experimental group, No. 5 experimental group, No. 6 experimental group, No. 7 experimental group, and No. 8 experimental group. Among them, the 0 experimental group was fed with wet corn husks, with a humidity of 35-45wt.%, specifically 14.5 parts of coarse corn husks and 5 parts of fine corn husks, which were converted into a specific mass of 290kg coarse corn husks and 100kg fine corn husks, the No. 1 experimental group was fed with bacterial protein biological feed 1; the No. 2 experimental group was fed with bacterial protein biological feed 2, the No. 3 experimental group was fed with bacterial protein biological feed 3, the No. 4 experimental group was fed with bacterial protein biological feed 4, the No. 5 experimental group was fed with bacterial protein biological feed 5, the No. 6 experimental group was fed with bacterial protein biological feed 6, the No. 7 experimental group was fed with bacterial protein biological feed 7, and the No. 8 experimental group was fed with bacterial protein biological feed 8. The cows in each experimental group were fed by a tethered person at 7:00 a.m. and 7:00 p.m. every day. They were allowed to drink water and eat freely. Milking was performed at 4:30 a.m. and 3:30 p.m. every day, and the milk production was recorded. The experimental period was 30 days.

[0123] Milk production measurement: At the beginning of feeding, the milk production of each cow in experimental groups 0 to 8 was measured every day, and the average daily milk production and total milk production of each group of cows were calculated.

[0124] The results showed that at the end of the experiment, the daily milk production of experimental group 0, experimental group 1, experimental group 2, experimental group 3, experimental group 4, experimental group 5, experimental group 6, experimental group 7, and experimental group 8 were 24.8kg±0.15kg, 32.9kg±0.27kg, 27.8kg±0.12kg, 29.9kg±0.45kg, 30.8kg±0.32kg, 30.5kg±0.22kg, 30.2kg±0.28kg, 28.8kg±0.19kg, 32.5kg±0. 0.37kg; the total milk production was 745kg±2.35kg, 987kg±2.68kg, 834kg±6.54kg, 897kg±3.97kg, 926kg±1.23kg, 915kg±2.34kg, 907kg±5.62kg, 864kg±3.16kg, 975kg±5.67kg respectively; the average daily milk production and total milk production of test groups 1 to 8 were significantly increased compared with those of test group 0, indicating that the bacterial protein biological feed of Examples 1 to 8 can increase the milk production of dairy cows.

[0125] Comparative Example 1

[0126] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (1) in Example 1, the second fermentation base material is replaced with fermentation substrate 7, and the other steps remain unchanged. The corn husk mycelial protein biological feed obtained after the fermentation is recorded as mycelial protein biological feed 9.

[0127] Wherein, fermentation substrate 7 is composed of 19.5 parts of dry corn husks, 10.5 parts of koji seeds prepared in Example 1, 3 parts of molasses, 6 parts of ammonium sulfate, 0.1 parts of potassium dihydrogen phosphate, 0.2 parts of sodium chloride, 0.2 parts of magnesium sulfate and 0.125 parts of acetic acid. Wherein, the corn husks are 14.5 parts of coarse corn husks and 5 parts of fine corn husks, which are converted into specific masses: 290kg of coarse corn husks, 100kg of fine corn husks, 210kg of koji seeds, 60kg of molasses, 120kg of ammonium sulfate, 2kg of potassium dihydrogen phosphate, 4kg of sodium chloride, 4kg of magnesium sulfate and 2.5L of acetic acid.

[0128] Comparative Example 2

[0129] The steps are the same as those in Example 1, with the only difference being that: according to the method in step (1) of Example 1, the first fermentation base material is replaced with fermentation substrate 8, and the corn husk mycelial protein biological feed obtained after fermentation is recorded as mycelial protein biological feed 10.

[0130] Among them, the fermentation substrate 8 is composed of 19.5 parts of dry corn husks, 10.5 parts of koji prepared in Example 1, 4 parts of molasses, 1 part of ammonium sulfate, 0.6 parts of potassium dihydrogen phosphate, 0.3 parts of sodium chloride, 0.2 parts of magnesium sulfate and 0.125 parts of acetic acid, of which 14.5 parts of coarse corn husks and 5 parts of fine corn husks are converted into specific mass: the corn husks are 290kg of coarse corn husks, 100kg of fine corn husks, 210kg of koji, 80kg of molasses, 20kg of ammonium sulfate, 12kg of potassium dihydrogen phosphate, 6kg of sodium chloride, 4kg of magnesium sulfate and 2.5L of acetic acid.

[0131] Comparative Example 3

[0132] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (1) in Example 1, the first fermentation base material is replaced with fermentation substrate 9, and the other steps remain unchanged. After the fermentation is completed, corn husk mycelial protein biological feed fermented by a multi-step method is obtained, which is recorded as mycelial protein biological feed 11.

[0133] Among them, the fermentation substrate 9 is composed of 19.5 parts of dry corn husks, 10.5 parts of koji prepared in Example 1, 1 part of molasses, 5 parts of ammonium sulfate, 0.5 parts of potassium dihydrogen phosphate, 0.3 parts of sodium chloride, 0.2 parts of magnesium sulfate and 0.125 parts of acetic acid, of which 14.5 parts of coarse corn husks and 5 parts of fine corn husks are converted into specific mass: the corn husks are 290kg of coarse corn husks, 100kg of fine corn husks, 210kg of koji, 20kg of molasses, 100kg of ammonium sulfate, 10kg of potassium dihydrogen phosphate, 6kg of sodium chloride, 4kg of magnesium sulfate and 2.5L of acetic acid.

[0134] Comparative Example 4

[0135] The steps are the same as those in Example 1, with the only difference being that: according to the method of step (1) in Example 1, the first fermentation base material is replaced with fermentation substrate 10, and the other steps remain unchanged. After the fermentation is completed, corn husk mycelial protein biological feed fermented by a multi-step method is obtained, which is recorded as mycelial protein biological feed 12.

[0136] Among them, the fermentation substrate 10 is composed of 19.5 parts of dry corn husks, 10.5 parts of koji prepared in Example 1, 3 parts of molasses, 1 part of ammonium sulfate, 0.4 parts of potassium dihydrogen phosphate, 0.3 parts of sodium chloride, 0.1 parts of magnesium sulfate and 0.125 parts of acetic acid, of which 14.5 parts of coarse corn husks and 5 parts of fine corn husks are converted into specific mass: the corn husks are 290kg of coarse corn husks, 100kg of fine corn husks, 210kg of koji, 60kg of molasses, 20kg of ammonium sulfate, 8kg of potassium dihydrogen phosphate, 6kg of sodium chloride, 2kg of magnesium sulfate and 2.5L of acetic acid.

[0137] The physical and chemical indicators and sanitary indicators of each bacterial protein biological feed prepared in Examples 1 to 8 and Comparative Examples 1 to 4 were measured. The methods for measuring the physical and chemical indicators and sanitary indicators are as follows:

[0138] Physical and chemical indicators: crude protein content is determined in accordance with GB / T6432-2018, crude fiber content is determined in accordance with GB / T6434-2006, moisture content is determined in accordance with GB / T6435-2014, crude ash content is determined in accordance with GB / T6438-1992, crude fat content is determined in accordance with GB / T6433-2006, starch content is determined in accordance with GB / T20194-2018;

[0139] Hygiene indicators: total arsenic content is determined in accordance with GB / T13079-2006, lead content is determined in accordance with GB / T13080-2004, mercury content is determined in accordance with GB / T13081-2006, cadmium content is determined in accordance with GB / T13082-1991, chromium content is determined in accordance with GB / T13088-2006, fluorine content is determined in accordance with GB / T13083-2002, nitrite content is determined in accordance with GB / T13085-2005, aflatoxin B1 content is determined in accordance with GB / T17480-1998, ochratoxin A content is determined in accordance with GB / T19539-2004, zearalenone content is determined in accordance with GB / T19540-2004, and vomitoxin content is determined in accordance with GB / T30956-2014, T-2 toxin content determination complies with GB / T28718-2012, fumonisin content determination complies with NY / T1970-2010, cyanide content determination complies with GB / T13084-2006, free gossypol content determination complies with GB / T13086-1991, isothiocyanate content determination complies with GB / T13087-1991, oxazolidinethione content determination complies with GB / T13089-1991, hexachlorobenzene content determination complies with GB / T34270-2017, polychlorinated biphenyl content determination complies with GB / T8381.8-2005, and Salmonella detection complies with GB / T13091-2002; the specific process is common knowledge and will not be repeated here.

[0140] The true protein determination method is as follows:

[0141] (1) Experimental procedures

[0142] Accurately weigh about 1g (accurate to 0.0001g) of the sample to be tested and place it in a 200ml beaker, add 50ml of water, and heat to boiling. Then add 20ml of copper sulfate solution and 20ml of 2.5% (m / m) sodium hydroxide aqueous solution, stir thoroughly with a glass rod, and leave for more than 1h. Filter with qualitative filter paper, then wash the precipitate with 60-80℃ hot water 5 or 6 times, check the filter paper with 5 drops of barium chloride solution and 1 drop of hydrochloric acid solution, and repeat the previous process of checking the filter paper until no white barium sulfate precipitate is generated. Dry the precipitate and filter paper in a 65℃ oven for 2h, then transfer them all to a Kjeldahl flask and determine nitrogen according to the semi-micro Kjeldahl method.

[0143] (2) Calculation formula

[0144]

[0145] Where:

[0146] V2: The volume of the hydrochloric acid standard titration solution consumed by the titration sample, in milliliters (mL);

[0147] V1: The volume of the hydrochloric acid standard titration solution consumed in the titration blank, in milliliters (mL);

[0148] C: concentration of hydrochloric acid standard titration solution, in moles per liter (mol / L);

[0149] m: sample mass, in grams (g);

[0150] V: total volume of sample digestion solution, in milliliters (mL);

[0151] V': volume of digestion liquid for distillation, in milliliters (mL);

[0152] 6.25: Average coefficient for conversion of nitrogen into crude protein;

[0153] The yeast detection method is as follows:

[0154] Accurately weigh 1g of bacterial protein biological feed, put it into 9mL of sterile water and vortex for 15 minutes to make a yeast concentration of 10 -1 1mL of yeast suspension was then drawn to a concentration of 10 -1 Add the bacterial suspension to 9 mL of sterile water, shake thoroughly, and dilute to a yeast concentration of 10 -2 Pipette 10 μL of yeast suspension with a concentration of 10 -2 The concentration of bacterial suspension was dropped onto the blood cell plate 0.0025mm 2 Then add an equal volume of methylene blue staining solution into the counting area, mix it evenly, cover it with a coverslip, and be sure not to create bubbles. Stain for about 3 to 5 minutes, and within 5 minutes after staining, transfer it to a microscope to determine the yeast activity and count the number of live yeast cells.

[0155] The calculation formula is:

[0156] Yeast cell number / g = number of yeast cells in 80 cells / 80×400×10 6 × Dilution factor

[0157] The calculation formula is: number of yeast cells / g = number of yeast cells in 80 small grids / 80×400×10 6 × dilution factor.

[0158] The test results are shown in Table 1, where % represents mass percentage.

[0159] Table 1 Results of bacterial protein biological feed detection indicators

[0160]

[0161]

[0162]

[0163]

[0164] By comparing the data of Example 1 with Comparative Example 1, it can be seen that when the inorganic salt content ratio is changed, the crude protein content is lower than the standard value, the true protein content is significantly lower than that of Example 1, and the crude fiber content is higher than the standard value. The reason for the analysis may be that the inorganic salt content ratio is improperly distributed, which inhibits the growth and reproduction of yeast. It can be seen from Table 1 that the number of yeasts in Comparative Example 1 is significantly lower than that of the embodiment. By comparing the data of Example 2 with Comparative Example 2, it can be seen that when the inorganic nitrogen content is reduced, the crude protein content is lower than the standard value, the true protein content is significantly lower than that of Example 2, and the crude fiber content is higher than the standard value. The reason for the analysis may be that the content of inorganic nitrogen sources available to yeast during the fermentation process is low and cannot meet the needs of its own growth and reproduction. It can be seen from Table 1 that the number of yeasts in Comparative Example 2 is significantly lower than that of Examples 1 to 8.

[0165] By comparing the data of Example 3 with that of Comparative Example 3, it can be seen that when the content of inorganic nitrogen source is increased, the crude protein content, true protein content and crude fiber content are all close to the standard values. The reason for this may be that increasing the content of inorganic nitrogen source can promote the growth and reproduction of yeast, but the increase in the nitrogen source content needs to be further improved.

[0166] By comparing the data of Example 6 with that of Comparative Example 2, it can be seen that when the molasses concentration is increased, the crude protein content and true protein content of Comparative Example 2 are significantly lower than those of Example 6; the crude fiber content is higher than the standard value. The reason for this may be that the molasses concentration is relatively high, containing a large amount of sugars, minerals, biotin, etc., which inhibit the growth of yeast, so the crude protein and true protein contents of Comparative Example 2 are lower, and the number of yeast is relatively small.

[0167] By comparing the number of yeasts, it is found that the number of yeasts in Examples 1 to 8 is higher than that in Comparative Examples 1 to 4. As shown in Table 1, the experimental data of Examples 1 to 8 and Comparative Examples 1 to 4 are comprehensively compared, and by comparing the crude protein content, true protein content and crude fiber content, it is found that the crude protein content and true protein content in Examples 1 to 8 are significantly higher than those in Comparative Examples 1 to 4, and meet the standards of protein feed; the crude fiber content is significantly lower than that in the comparative example, meeting the standard of crude fiber content ≤ 18% in protein feed. The content of inorganic pollutants in Examples 1 to 8 of the present invention and Comparative Examples 1 to 4 is not detected; the content of mycotoxins meets the requirements of health indicators; the content of natural toxins and organic pollutants is not detected; the content of probiotics in the bacterial protein biological feed prepared in Examples 1 to 8 of the present invention is significantly increased, that is, the number of saccharomyces cerevisiae and Candida utilis increases, the number of Aspergillus niger decreases, and the feeding effect is obvious, and the milk production of dairy cows increases.

[0168] In summary, the corn husk mycelium protein fermented feed of the present invention can not only provide high-quality protein, but also provide a variety of probiotics and enzymes. While ensuring the supply of protein, it also plays a certain role in reducing or eliminating the use of antibiotics and ensuring the green development of the breeding industry, and has broad application prospects.

[0169] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing bacterial protein biological feed, characterized in that: The preparation method comprises the following steps: inoculating a black Aspergillus fungus agent into a first fermentation base material for first fermentation to obtain a koji seed; Inoculating the composite bacterial agent into the second fermentation base material for second fermentation to obtain bacterial protein biological feed; The first fermentation base material comprises the following raw materials in parts by weight: 84-92 parts of corn husks, 4-6 parts of bran, 2-4 parts of soybean meal and 2-5 parts of second flour; The second fermentation base material comprises the following raw materials in parts by weight: 19.5 parts of corn husks, 3-6 parts of molasses, 2-4 parts of ammonium sulfate, 0.1-0.2 parts of potassium dihydrogen phosphate, 0.1-0.2 parts of magnesium sulfate, 0.10-0.20 parts of sodium chloride, 0.10-0.15 parts of acetic acid and 10.5 parts of the koji; The types of corn husks include coarse corn husks and / or fine corn husks; the particle size of the coarse corn husks is 5-10 meshes; the fine corn husks are the sieve-down obtained after the corn husks are crushed and passed through a 20-mesh sieve; The first fermentation time is 24-48 hours and the temperature is 30-35°C; The second fermentation time is 48-72 hours at a temperature of 30-32°C; The composite bacterial agent comprises a saccharomyces cerevisiae seed solution and a utilis candida seed solution; the number of saccharomyces cerevisiae live bacteria in the saccharomyces cerevisiae seed solution is 1.4×10 11 CFU / kg; the number of viable Candida utilis in the Candida utilis seed solution is 1.9×10 11 CFU / kg; the volume ratio of the saccharomyces cerevisiae seed solution to the Candida utilis seed solution in the composite bacterial agent is 1-7:1-7; The deposit number of the Aspergillus niger is CGMCC 22439; the deposit number of the Saccharomyces cerevisiae is CICC 32236; the deposit number of the Candida utilis is CGMCC 2.2878; When inoculating the Aspergillus niger agent, the inoculation amount of the Aspergillus niger agent is 5-10% of the mass of the first fermentation base material; When the composite bacterial agent is inoculated, the inoculation amount of the composite bacterial agent is 5% to 10% of the mass of the corn husk in the second fermentation base material.

2. A bacterial protein biological feed prepared by the preparation method according to claim 1.

3. Use of the bacterial protein biological feed according to claim 2 in the preparation of animal feed.

4. Use of the bacterial protein biological feed according to claim 2 in increasing animal milk production.

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