Production of bacterial protein biological feed by means of two-step fermentation of corn husk

Abstract

A method for preparing a bacterial protein biological feed. Aspergillus niger is inoculated into a first fermentation substrate for first fermentation to obtain seed liquor; a composite strain is inoculated into a second fermentation substrate for second fermentation to obtain a bacterial protein biological feed. The first fermentation substrate comprises corn husk, bran, soybean meal and wheat middlings. The second fermentation substrate comprises corn husk, molasses, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, acetic acid and seed liquor; and the composite strain comprises Saccharomyces cerevisiae and Candida utilis. Further provided is the use of the bacterial protein biological feed in the preparation of an animal feed and in the improvement of milk production and milk quality of animals.

Description

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

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 19, 2022, with application number CN202211628999.5 and invention name “Two-step fermentation of corn husks to produce bacterial protein biological feed”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention belongs to the technical field of biological fermentation feed preparation, and particularly relates to a two-step method for producing bacterial protein biological feed by fermenting corn husks. Background Art

[0003] With the development of animal husbandry and aquaculture, my country's protein feed resource shortage is becoming increasingly severe. Large quantities of protein feeds such as soybean meal and fish meal must be imported annually, significantly increasing feed costs and impacting food security. Therefore, the feed industry urgently needs unconventional protein feeds to replace conventional protein feeds. Currently, protein feed alternatives typically use inexpensive industrial and agricultural byproducts as raw materials. Through fermentation with various microorganisms, these raw materials are degraded and their protein content is increased.

[0004] Corn husks are one of the largest byproducts of corn-based fermentation. Currently, the most common method is to wash, dry, and sell them as ruminant feed. However, this process suffers from low nutritional value, poor palatability, and poor feeding efficiency. To address this issue, researchers have conducted research on microbial fermentation of corn husks. 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, which 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 fungi 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 base liquid; mixing tropical yeast seed liquid and natto spore liquid to obtain a mixed seed liquid, inoculating the mixed seed liquid into the base liquid, fermenting and culturing for 24 hours, and then inoculating thermophilic Streptococcus seed liquid, 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 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.

[0005] Although the research on microbial fermentation of corn husks has achieved certain results, there are still some problems, such as high cost, poor strain safety, high fiber content and low protein content in the feed. Moreover, the tropical yeast Candida 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.

[0006] Summary of the Invention

[0007] The present invention aims to provide a two-step method for producing bacterial protein biofeed from corn husk fermentation. The method maximizes fiber degradation and increases protein content in fermented feed while maintaining cost-effectiveness, providing a method for producing high-quality protein feed for the feed industry. Furthermore, the strain used is safe.

[0008] The present invention provides a method for preparing bacterial protein biological feed, which comprises the following steps:

[0009] Inoculating the Aspergillus niger bacterial agent into the first fermentation base material to perform the first fermentation to obtain koji seed;

[0010] Inoculating the composite bacterial agent into the second fermentation base material to carry out the second fermentation to obtain bacterial protein biological feed;

[0011] The first fermentation base 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-grade flour;

[0012] The second fermentation base comprises the following raw materials in parts by weight: 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;

[0013] The composite bacterial agent comprises Saccharomyces cerevisiae and Candida utilis.

[0014] Preferably, the inoculation amount of Aspergillus niger is 5-10% of the mass of the first fermentation base;

[0015] The effective viable bacteria count of Aspergillus niger is 1.0×10 9 ~2.0×10 9 CFU / mL.

[0016] Preferably, the Aspergillus niger includes Aspergillus niger CJH-JXSFZh-B703, with a deposit number of CGMCC No.22439.

[0017] Preferably, the first fermentation time is 24 to 48 hours and the temperature is 30 to 35°C.

[0018] Preferably, the inoculation amount of the composite bacterial agent is 5% to 10% of the mass of the corn bran in the second fermentation base;

[0019] The effective viable bacterial count of the Saccharomyces cerevisiae is independently 1.5×10 8 CFU / mL;

[0020] The effective viable count of the Candida utilis was 1.8×10 8 CFU / mL;

[0021] The ratio of the effective live bacteria counts of saccharomyces cerevisiae and Candida utilis in the composite bacterial agent is 1.5-10.5:1.8-12.6.

[0022] Preferably, the Saccharomyces cerevisiae includes Saccharomyces cerevisiae CICC 32236; the Candida utilis includes Candida utilis CGMCC 2.2878.

[0023] Preferably, the fermentation time is 48 to 72 hours and the temperature is 30 to 32°C.

[0024] The present invention provides a bacterial protein biological feed, which is prepared by using the preparation method described in the above technical solution.

[0025] Preferably, the effective viable bacteria count of Saccharomyces cerevisiae in the bacterial protein biological feed is 1.4×10 9 CFU / kg~1.4×10 11 CFU / kg, the effective viable count of Candida utilis is preferably 1.9×10 9 CFU / kg~1.9×10 11 CFU / kg.

[0026] The present invention provides the use of the bacterial protein biological feed described in the above technical solution in the preparation of animal feed.

[0027] 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. Beneficial effects:

[0028] The present invention provides a method for preparing a bacterial protein biological feed, comprising the steps of inoculating Aspergillus niger into a first fermentation base material for a first fermentation to obtain a koji seed; inoculating a composite bacterial agent into a second fermentation base material for fermentation to obtain a bacterial protein biological feed, and defining the composition of the first fermentation base material, the second fermentation base material, and the composite bacterial agent. The fermentation base material, which is mainly composed of corn husks, is treated by step-by-step fermentation, and the fiber in the fermented feed can be degraded to the maximum extent while taking into account the cost, and its protein content can be increased. The strain used is highly safe, and a method for preparing a high-quality protein feed is provided for the feed industry. The obtained bacterial protein biological feed can not only provide high-quality protein, but also provide a variety of probiotics and enzymes. While ensuring protein supply, it also plays a certain role in reducing or eliminating the use of antibiotics and ensuring the green development of the aquaculture industry, and has broad application prospects. In addition, the present invention subjects the by-products of corn starch processing to secondary processing, which not only provides a high-quality and low-cost antibiotic-free bacterial protein microbial feed for the animal husbandry industry, but also solves the problem of insufficient utilization of corn husks, a by-product of corn processing, and increases their added value; the solid-state fermentation method adopted has the characteristics of simple equipment structure, low investment, low energy consumption, and simple operation; all fermentation products are products, there is no other waste, and there is less pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0030] FIG1 is a diagram showing the state of mycelium in the first fermentation product;

[0031] FIG2 is an experimental flow chart of Example 1, wherein the second fermentation base material in FIG2 refers to the dry corn husk, molasses, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride and magnesium sulfate in the second fermentation base material.

[0032] Biological deposit information

[0033] Aspergillus niger CJH-JWSFZh-B703, classified as Aspergillus niger, was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on June 8, 2021. The deposit address is No. 2, Yard 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

[0034] Unless otherwise specified, the raw materials used in the present invention are all purchased by those skilled in the art.

[0035] The present invention provides a method for preparing bacterial protein biological feed, comprising the following steps:

[0036] Inoculating the Aspergillus niger bacterial agent into the first fermentation base material to perform the first fermentation to obtain koji seed;

[0037] Inoculating the composite bacterial agent into the second fermentation base material to carry out the second fermentation to obtain bacterial protein biological feed;

[0038] The first fermentation base 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-grade flour;

[0039] The second fermentation base comprises the following raw materials in parts by weight: 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;

[0040] The composite bacterial agent comprises Saccharomyces cerevisiae and Candida utilis.

[0041] In the present invention, a niger Aspergillus niger agent is inoculated into a first fermentation base material for a first fermentation to obtain a koji. The niger Aspergillus niger of the present invention is preferably niger Aspergillus niger CJH-JXSFZh-B703; the deposit number of the niger Aspergillus niger CJH-JXSFZh-B703 is CGMCC No. 22439; the number of spores of the niger Aspergillus niger in the koji is preferably 6.52×10 9 CFU / 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 all act on corn husks. 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 husks, Aspergillus niger can grow well by utilizing some nutrients in the corn husks, and degrade cellulose into small molecules such as glucose for bacterial growth. The treated corn husks are rich in bacterial proteins, amino acids, vitamins, minerals and other elements. It has great utilization value as a feed.

[0042] The invention inoculates Aspergillus niger into a first fermentation base material before the first fermentation, and preferably performs slant culture on the Aspergillus niger in a malt agar medium; after the slant culture is completed, the Aspergillus niger obtained by the slant culture is picked out and inoculated into a malt liquid medium for shake flask culture to obtain an Aspergillus niger fungus agent.

[0043] In the present invention, the temperature of the slant culture is preferably 30°C; during the slant culture, the inoculum size of Aspergillus niger is preferably 1 loop. The method for preparing the malt extract agar medium of the present invention preferably comprises the following steps: mixing malt extract, agar, and water to obtain a malt extract agar medium; the amount of the malt extract used is preferably 1.0 L; the amount of the agar used is preferably 15.0 g; the amount of water used is preferably an amount used to adjust the Brix of the malt extract to 12° Brix; the malt extract agar medium preferably has a natural pH, and the malt extract agar medium is preferably a malt extract agar medium sterilized at 121°C for 20 minutes.

[0044] 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; and the time of the shake flask culture is preferably 24h. In the present invention, the malt wort liquid culture medium is preferably a malt saccharification 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 sterilizing at 115°C for 30 minutes. The spore count of the Aspergillus niger fungus 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.

[0045] After obtaining the Aspergillus niger agent, the present invention preferably adjusts the Aspergillus niger agent so that the spore count is 1.0×10 9 ~2.0×10 9 The present invention does not limit the method of the adjustment, and any method known to those skilled in the art can be used. Preferably, sampling is performed every 2 hours to monitor the number of Aspergillus niger spores in the Aspergillus niger inoculum in real time, and the number of Aspergillus niger spores is calculated using a hemocytometer; the sampling preferably refers to taking Aspergillus niger seed liquid.

[0046] 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-grade flour.

[0047] The first fermented base of the present invention comprises 84-92 parts by mass of corn husks, preferably 86-90 parts, and more preferably 86 parts. The corn husks of the present invention preferably comprise dried corn husks, more preferably dried corn husks. The corn husks preferably comprise coarse corn husks and / or fine corn husks, more preferably coarse corn husks and fine corn husks. The mass ratio of coarse corn husks to fine corn husks is preferably 66-82:10-20, more preferably 66:20, 72:10, or 80:10. The coarse corn husks preferably have a particle size of 5-10 mesh, and the fine corn husks preferably have a pass rate of at least 85% through a 20-mesh sieve. That is, the fine corn husks are preferably the undersize obtained after crushing corn husks through a 20-mesh sieve. However, fine corn husks with particle sizes slightly larger than 20 mesh may exist during the crushing process. The purpose of selecting corn husks of different mesh sizes is to control the gaps between the corn husks to avoid excessive coarse or fine corn husks, which can lead to excessive gaps, making it difficult to control the moisture content, or causing excessive viscosity. The moisture content of the dried corn husks of the present invention is preferably 9 wt.%. The source of the corn husks is not particularly limited in the present invention, and can be purchased by a person skilled in the art. In a specific embodiment of the present invention, the corn husks were purchased from Inner Mongolia Fufeng Biotechnology Co., Ltd. The moisture content of the first fermentation base of the present invention is preferably controlled to be 35 wt.% to 40 wt.%.

[0048] Based on the mass parts of corn bran, the first fermentation base material of the present invention includes 4 to 6 parts of bran, preferably includes 5 to 6 parts, and most preferably includes 6 parts.

[0049] Based on the mass fraction of corn husks, the first fermentation base material of the present invention includes 2 to 4 parts of soybean meal, preferably includes 2.5 to 4 parts, and most preferably includes 4 parts.

[0050] Based on the mass parts of corn husks, the first fermentation base material of the present invention includes 2 to 4 parts of secondary flour, preferably includes 2.5 to 5 parts, and most preferably includes 4 to 5 parts.

[0051] Before inoculation, the present invention preferably performs a first steaming, a first stirring, water addition, a second stirring, a second steaming, and a third stirring on the first fermentation base material to obtain a 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 10 minutes; the time of the third stirring is preferably 20 minutes. When adding water according to the present invention, the amount of water used 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.

[0052] 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, more preferably 5%.

[0053] After inoculation with the Aspergillus niger inoculum, the present invention performs a further inoculation and a first fermentation on the steamed material inoculated with the Aspergillus niger inoculum to produce a koji seed, also referred to as a first fermentation product. The temperature for the further inoculation is preferably 30°C; the duration of the further inoculation is preferably 48 hours. In the present invention, the duration of the first fermentation is preferably 24-48 hours, more preferably 28-45 hours, more preferably 30-42 hours, and most preferably 33-37 hours. The indoor temperature during the first fermentation is preferably 25-30°C, meaning that during the first fermentation, the ambient temperature is preferably 25-30°C. The fermentation product temperature during the first fermentation is preferably 30-35°C. The relative humidity of the first fermentation is preferably ≥95%. The fermentation product temperature herein refers to the temperature of the steamed material inoculated with the Aspergillus niger inoculum during the first fermentation. In the present invention, if the fermentation product temperature exceeds 35°C and continuous ventilation cannot reduce the temperature to below 35°C, turning or stirring is preferably performed to reduce the temperature. The present invention does not limit the methods of turning and stirring; methods known to those skilled in the art can be used.

[0054] After obtaining the koji seed, the present invention inoculates the composite microbial agent into the second fermentation base material for a second fermentation to obtain bacterial protein biological feed. In the present invention, the composite microbial agent includes saccharomyces cerevisiae and Candida utilis; the effective viable cell count of the saccharomyces cerevisiae is preferably 1.5×10 8 CFU / mL; the effective viable count of Candida utilis is preferably 1.8×10 8CFU / mL; the ratio of the effective viable counts of Saccharomyces cerevisiae and Candida utilis in the composite bacterial agent is preferably 1.5-10.5:1.8-12.6. The present invention preferably mixes Saccharomyces cerevisiae and Candida utilis in a volume ratio to obtain the composite bacterial agent; the volume ratio of Saccharomyces cerevisiae and Candida utilis is preferably 1-7:1-7, more preferably 1:1-2, 1-2:1, 1.5:1-2, 7:3 or 3:7.

[0055] In the present invention, the Saccharomyces cerevisiae strain is preferably Saccharomyces cerevisiae strain with accession number CICC 32236, a strain of the China Industrial Culture Collection (CICC). The CICC maintains a dedicated website at http: / / www.china-cicc.org, where the public can directly order strains. The website address for Saccharomyces cerevisiae strain CICC 32236 is http: / / www.china-cicc.org / search / ?classtype=0&keyword=32236, where the public can obtain the strain from the CICC. The present invention does not specifically limit the source of the Saccharomyces cerevisiae strain; it can be obtained from conventional sources by those skilled in the art. In specific embodiments of the present invention, the Saccharomyces cerevisiae strain is preferably purchased from the China Industrial Culture Collection (CICC).

[0056] The Candida utilis described herein is preferably the Candida utilis strain with the accession number CGMCC 2.2878, a strain from the China General Microbiological Culture Collection Center (CGMCC). CGMCC maintains a dedicated website at https: / / cgmcc.net / english / , where the public can directly order the strain. The website address for Candida utilis CGMCC2.2878 is https: / / cgmcc.net / english / detail?id=66762, and the public can obtain this strain from CGMCC. The source of the Candida utilis strain is not particularly limited in the present invention; it can be obtained through conventional commercial means by those skilled in the art. In specific embodiments of the present invention, the Candida utilis strain is preferably purchased from the China General Microbiological Culture Collection Center. The brewer's yeast used in this invention is rich in nutrients such as protein, amino acids, and small peptides. Fermentation with the yeast can improve the palatability and nutritional value of feed, increasing animal feed intake and digestibility. Candida utilis itself has a high protein content, accounting for 32% to 75% of its dry weight, and is rich in nutrients such as fat, vitamins, and nucleic acids. It can utilize a wide range of carbon sources, including both pentose and hexose sugars. Furthermore, fermentation of the two together significantly increases true protein content.

[0057] The present invention also preferably provides a method for preparing the composite bacterial agent, comprising the following steps:

[0058] Saccharomyces cerevisiae and Candida utilis were inoculated on YPD agar medium for slant culture.

[0059] The slant cultured Saccharomyces cerevisiae and Candida utilis were inoculated into YPD liquid medium and cultured in a shaking table to obtain Saccharomyces cerevisiae seed liquid and Candida utilis seed liquid;

[0060] 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.

[0061] In the present invention, Saccharomyces cerevisiae and Candida utilis are preferably inoculated separately onto YPD agar medium for slant culture. The temperature of the slant culture is preferably 30°C. During the slant culture of Saccharomyces cerevisiae, preferably, a single colony of Saccharomyces cerevisiae is streaked onto the YPD agar medium; during the slant culture of Candida utilis, preferably, a single colony of Candida utilis is streaked onto the YPD agar medium. The YPD agar medium preferably comprises the following components by mass: 10g peptone, 5g yeast extract powder, 10g glucose, 20g agar powder, and 1kg distilled water. The pH of the YPD agar medium is preferably natural. The YPD agar medium is preferably sterilized at 115°C for 30 minutes.

[0062] The present invention preferably inoculates the slant cultured Saccharomyces cerevisiae and Candida utilis 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 shaking flask culture time 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.

[0063] After obtaining the saccharomyces cerevisiae seed solution and the Candida utilis seed solution, the present invention further 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 bacteria count of Candida utilis seed solution is 1.8×10 8CFU / mL. The present invention does not impose any restrictions on the manner of adjustment, and methods familiar to those skilled in the art may be employed. In a specific embodiment of the present invention, the adjustment method preferably comprises sampling every 2 hours, monitoring the number of viable Saccharomyces cerevisiae cells in a Saccharomyces cerevisiae seed solution or the number of viable Candida utilis cells in a Candida utilis seed solution in real time, and calculating the number of viable Saccharomyces cerevisiae cells or the number of viable Candida utilis cells using a hemocytometer. The sampling preferably comprises sampling the Saccharomyces cerevisiae seed solution or the Candida utilis seed solution.

[0064] The second fermentation base of the present invention comprises the following raw materials in parts by weight: 25-30 parts corn husks, 3-6 parts molasses, 2-4 parts ammonium sulfate, 0.1-0.2 parts potassium dihydrogen phosphate, 0.1-0.2 parts magnesium sulfate, 0.10-0.15 parts sodium chloride, 0.10-0.15 parts acetic acid, and 10-35 parts of the koji. In the present invention, the water content of the second fermentation base is preferably controlled to 70 wt.%, so that the texture of the second fermentation base is slightly sticky.

[0065] In parts by mass, the second fermentation base of the present invention comprises 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 husks is preferably 5 to 10 mesh, and the fine corn husks are preferably corn husks with a pass rate of 85% or more through a 20-mesh sieve, that is, the fine corn husks are preferably the undersize obtained by crushing corn husks and passing them 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 9 wt.%; the present invention does not specifically limit the source of the corn husks, and those conventionally purchased by those skilled in the art can be used.

[0066] The second fermentation base of the present invention comprises 3 to 6 parts of molasses, preferably 3.2 to 3.8 parts, based on the mass fraction of corn husks. In the present invention, the source of the molasses is not particularly limited, and can be obtained by conventional purchase by a person skilled in the art. In a specific embodiment of the present invention, the molasses is preferably purchased from Inner Mongolia Fufeng Biotechnology Co., Ltd.

[0067] Based on the mass parts of corn husks, the second fermentation base material of the present invention includes 2 to 4 parts of ammonium sulfate, preferably 2 to 3 parts.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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 acetic acid, preferably 0.1 parts.

[0072] Based on the mass parts of corn husks, the second fermentation base material of the present invention includes 10 to 35 parts of the koji, preferably includes 10 to 10.5 parts, and more preferably includes 10.5 parts.

[0073] Before inoculating the composite bacterial agent, the present invention preferably further comprises: steaming the corn husks, molasses, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, and sodium chloride in the second fermentation base and adding water to obtain a steamed material; the steaming temperature is preferably 115° C. to 121° C., more preferably 121° C.; the steaming pressure is preferably 0.1 MPa to 0.2 MPa, more preferably 0.2 MPa; and the steaming time is 20 minutes. When adding water in the present invention, the amount of water used is preferably 70% of the total mass of the water and corn husks.

[0074] After obtaining the steamed material, the present invention preferably further comprises mixing the steamed material with koji and performing enzymatic hydrolysis to obtain an enzymatic hydrolysis base material; the enzymatic hydrolysis time is preferably 6 hours, and the enzymatic hydrolysis temperature is preferably 25° C. to 30° C. During the enzymatic hydrolysis of the present invention, tap water is preferably added to the steamed material; the amount of tap water used is preferably the amount of water that causes the steamed material to become slightly viscous after adding tap water.

[0075] After obtaining the enzymatic hydrolysis base, the present invention preferably mixes the enzymatic hydrolysis substrate with acetic acid, and inoculates the resulting mixture with a composite bacterial agent for a second fermentation. The duration of the second fermentation 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 feeding are: adding 1% ammonium sulfate and 1% molasses before stirring at the 24th hour of the second fermentation; and 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 base. The present invention uses 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 in the second fermentation base can be fully degraded, and the resulting components after degradation have a higher nutritional content after fermentation.

[0076] 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 bran in the second fermentation base material.

[0077] 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 11 CFU / kg; the effective viable bacteria 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.

[0078] The present invention uses Aspergillus niger to perform a first fermentation on the first fermentation base to obtain a koji seed, and then uses a composite bacterial liquid containing brewer's yeast and utilis to ferment the second fermentation substrate. It can be seen that the process adopted by the present invention is a multi-strain mixed step-by-step fermentation, and the microorganisms cooperate with each other to greatly promote the rapid degradation and fermentation of corn husks, with the following characteristics: 1. Multi-bacteria symbiosis, rich in cellulase, protease, and amylase, to achieve enzyme complementation; 2. Energy conservation, simplified process equipment, and more perfect biochemical reactions; 3. Rich substrates, which can provide nutrients in stages; 4. Nutrients such as monosaccharides generated by enzymatic action are immediately utilized by other microorganisms, while maintaining the concentration of degradation products, eliminating the inhibitory effect of enzymatic hydrolysis products on enzymes; 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. Moreover, the method of the present invention can maximize the degradation of fiber in the fermented feed while taking into account cost and increase its protein content.

[0079] 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, which will not be repeated here. The raw material combination used in the bacterial protein biological feed provided by the present invention is low in cost and widely available; the bacterial protein biological feed described in the present invention can hydrolyze soybean meal in the raw materials into small peptide substances, degrade the cellulose and hemicellulose in corn husks into soluble sugars, and convert them into bacterial protein. In addition, the aromatic smell produced after fermentation of multiple strains 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 both beneficial microorganisms in the rumen of dairy cows. 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 production 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.

[0080] Based on the above advantages, the present invention provides the bacterial protein bio-feed described in the above technical solution or the use of the bacterial protein bio-feed described in the above technical solution in preparing animal feed. The animals described in the present invention preferably include mammals, more preferably include cattle, and more preferably include dairy cows.

[0081] Based on the aforementioned advantages, the present invention provides the use of the specific protein feed described in the above technical solution for improving milk production and quality in animals. The corn husk protein biofeed produced by the present invention contains multiple yeasts and is rich in protein, B vitamins, amino acids, and other substances. The protein content is approximately 30% to 40%, making it widely used as a protein supplement for animal feed. This feed can promote animal growth and development, shorten the feeding period, and improve product yield and quality, such as milk production and quality.

[0082] The present invention also provides the use of Aspergillus niger, Saccharomyces cerevisiae and Candida utilis in preparing bacterial protein biological feed. Aspergillus niger, Saccharomyces cerevisiae and Candida utilis of the present invention have been described in detail above and will not be repeated here.

[0083] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0084] Preparation Example

[0085] Culture medium preparation:

[0086] The malt extract agar medium is a 12° Brix malt extract agar medium, specifically 1.0 L of malt extract, 15.0 g of agar and the balance of water, with natural pH, and sterilized at 121° C. for 20 min.

[0087] The wort liquid medium is malt saccharification liquid with an apparent sugar content of 12°Brix;

[0088] YPD agar medium is composed of 10 g of peptone, 5 g of yeast extract powder, 10 g of glucose, 20 g of agar powder, and 1 L of distilled water, with a pH of 7.0-7.4; sterilize at 15°C for 30 min.

[0089] YPD liquid medium consists 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.

[0090] Aspergillus niger CJH-JXSFZh-B703 was cultured on a slant in malt agar medium. One loop of a single colony of Aspergillus niger was streaked onto the malt agar medium and cultured at 30°C. After the slant culture, one loop of the slant of Aspergillus niger CJH-JXSFZh-B703 was picked and inoculated into 100 mL of malt wort liquid medium in a 250 mL Erlenmeyer flask. The culture was cultured at 30°C, 180 rpm, for 24 h to prepare a seed solution of Aspergillus niger CJH-JXSFZh-B703. The concentration of the seed solution was adjusted to obtain an effective viable count of 1.0 × 10 9 CFU / mL, get the Aspergillus niger agent and set aside;

[0091] Saccharomyces cerevisiae CICC 32236 was cultured on a slant in YPD agar medium. One loop of a single colony of Saccharomyces cerevisiae was streaked onto the YPD agar medium and cultured at 30°C. After the slant culture, one loop of the slant of Saccharomyces cerevisiae CICC 32236 was inoculated into 100 mL of YPD liquid medium in a 250 mL Erlenmeyer flask and cultured at 30°C, 180 rpm, for 11 h to prepare a seed solution of Saccharomyces cerevisiae CICC 32236. The concentration of the seed solution was adjusted to obtain an effective viable count of 1.5 × 10 8 CFU / mL, spare;

[0092] Candida utilis CGMCC 2.2878 was cultured on a slant in YPD agar medium. One loopful of a single Saccharomyces cerevisiae colony was streaked onto the YPD agar medium and cultured at 30°C. After the slant culture, one loopful of the Candida utilis CGMCC 2.2878 slant was inoculated into 100 mL of YPD liquid medium in a 250 mL Erlenmeyer flask. The culture was constant temperature at 30°C and 180 rpm for 11 h to prepare a seed solution of Candida utilis CGMCC 2.2878. The concentration of the seed solution was adjusted to obtain an effective viable count of 1.8 × 10 8 CFU / mL, spare;

[0093] After the shake flask culture was completed, the obtained Saccharomyces cerevisiae CICC 32236 seed liquid and Candida utilis CGMCC 2.2878 seed liquid were mixed in equal volumes to obtain a composite bacterial solution, which was recorded as a composite bacterial agent and set aside.

[0094] The following examples all use the Aspergillus niger fungus agent or composite fungus agent prepared herein.

[0095] Example 1

[0096] Dried corn husks were obtained from Inner Mongolia Fufeng Biotechnology Co., Ltd., with a moisture content of 9 wt.%. Bran was obtained from Inner Mongolia Shenggu Brewing Food Co., Ltd., soybean meal was obtained from Inner Mongolia Shenggu Brewing Food Co., Ltd., and second-grade flour was obtained from Inner Mongolia Shenggu Brewing Food Co., Ltd. The specific experimental steps are shown in FIG2 , wherein the second fermentation base in FIG2 refers to the dried corn husks, molasses, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride, and magnesium sulfate in the second fermentation base:

[0097] (1) Preparation of fermentation substrate

[0098] The first fermentation base material (fermentation substrate 1) is composed of 86 parts of dry corn husks, 6 parts of bran, 4 parts of soybean meal and 4 parts of second-grade flour, among which the dry corn husks are composed of coarse corn husks and fine corn, and the mass ratio of coarse corn husks to fine corn is 66:20; converted into specific mass: 172kg dry corn husks, 12kg bran, 8kg soybean meal, 8kg second-grade flour, among which the coarse corn husks are 132kg and the fine corn husks are 40kg.

[0099] 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). The dry corn husks are composed of coarse corn husks and fine corn, and the mass ratio of coarse corn husks to fine corn is 14.5:5; the specific mass converted is: 290 kg of coarse corn husks and 100 kg of fine corn husks, 60 kg of molasses, 40 kg of ammonium sulfate, 4 kg of potassium dihydrogen phosphate, 2 kg of sodium chloride, 4 kg of magnesium sulfate, 2.5 L of acetic acid and 210 kg of koji seeds.

[0100] (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.% to 40wt.%, and the initial pH value is 7.0±0.2.

[0101] (3) After the sterilization is completed, the Aspergillus niger agent is directly mixed with the steamed material, the material is transported 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 48h. The temperature of the base material after the expanded inoculation is about 40°C. Here, "the temperature of the base material is about 40°C" specifically refers to the temperature of the material in the Quchi just after the expanded culture. The inoculated base material is sent into the Quchi by a cart, and the material layer is tilted. The material layer was stacked obliquely, with a 25 cm layer near the air inlet and a 30 cm layer on the other side. The upper layer was scraped flat and the first fermentation began. The temperature in the fermentation room was controlled at 25°C to 30°C, and the relative humidity was ≥95wt.%. The product temperature was controlled at 30°C to 35°C by intermittent ventilation. If the product temperature was higher than 35°C and continuous ventilation could not cool it down to below 35°C, the pile was turned or stirred to cool it down. After cooling it down to below 35°C, the first fermentation was continued for 5h to 10h. A large number of mycelia were observed to be very strong, and a large number of spores were also observed, as shown in Figure 1. The fermentation was completed and the koji was obtained. The number of Aspergillus niger spores in the koji reached 6.52×10 9 CFU / kg.

[0102] (4) Sterilization: The dried corn husks, molasses, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride and magnesium sulfate in the second fermentation base material are placed in a sterilizer, and then sterilized at 121°C and 0.2 MPa steam for 20 minutes, and 1400 kg of tap water is added to obtain the steamed material.

[0103] (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. The enzymatic hydrolysis reaction 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.

[0104] (6) After the enzymatic hydrolysis, 2.5 L of acetic acid and composite bacterial agent were added. The inoculation amount of the composite bacterial agent was 5% of the mass of the corn bran in the second fermentation base, which was converted into a specific volume of: 50 L of brewer's yeast seed liquid and 50 L of 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 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 were added before stirring; at the end of the fermentation, corn bran bacterial protein biofeed fermented by the step-by-step method was obtained, which was recorded as bacterial protein biofeed 1. The content of viable Candida utilis in bacterial protein biofeed 1 was 1.9×10 11 CFU / kg, the content of live yeast Saccharomyces cerevisiae is 1.4×10 11 CFU / kg.

[0105] Example 2

[0106] The steps are the same as those in Example 1, with the only difference being that when performing step (3), the inoculation amount of the Aspergillus niger agent is ensured to be 10% of the mass of the first fermentation base material, and the corn husk mycelial protein biological feed obtained after the fermentation is recorded as mycelial protein biological feed 2.

[0107] Example 3

[0108] The steps are the same as those in Example 1, with the only difference being that when performing step (1), the first fermentation base material is replaced with fermentation substrate 3, and the other steps remain unchanged. After the fermentation is completed, a corn husk mycelial protein biofeed fermented by a multi-step method is obtained, which is recorded as mycelial protein biofeed 3.

[0109] 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.

[0110] Example 4

[0111] The steps are the same as those in Example 1, with the only difference being that when performing step (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.

[0112] Among them, fermentation substrate 4 consists 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.

[0113] Example 5

[0114] The steps are the same as those in Example 1, with the only difference being that when performing step (6), the composite bacterial agent is replaced with composite bacterial agent 2, and the other steps remain unchanged. The corn husk mycelial protein biofeed obtained after the fermentation is recorded as mycelial protein biofeed 5.

[0115] The composite bacterial solution 2 was prepared by mixing the fermentation broth of Saccharomyces cerevisiae and the fermentation broth of Candida utilis in the preparation example at a volume ratio of 7:3.

[0116] Example 6

[0117] The steps are the same as those in Example 1, with the only difference being that when performing step (6), the composite bacterial solution is replaced with composite bacterial solution 3, and the other steps remain unchanged. The corn husk mycelial protein biofeed obtained after the fermentation is recorded as mycelial protein biofeed 6.

[0118] The composite bacterial solution 3 was obtained by mixing the fermentation broth of Saccharomyces cerevisiae and the fermentation broth of Candida utilis in the preparation example at a volume ratio of 3:7.

[0119] Example 7

[0120] The steps are the same as those in Example 1, with the only difference being that when performing step (1), the second fermentation base material 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.

[0121] Fermentation substrate 5 consisted of 19.5 parts of corn husks, 10.5 parts of the 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. The corn husks consisted of 14.5 parts of coarse corn husks and 5 parts of fine corn husks, which translated into a specific mass of 290 kg of coarse corn husks, 100 kg of fine corn husks, 210 kg of koji, 100 kg of molasses, 80 kg of ammonium sulfate, 4 kg of potassium dihydrogen phosphate, 2 kg of sodium chloride, 4 kg of magnesium sulfate, and 2.5 L of acetic acid.

[0122] Example 8

[0123] The steps are the same as those in Example 1, with the only difference being that when performing step (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.

[0124] 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 part of potassium dihydrogen phosphate, 0.2 part of sodium chloride, 0.2 part of magnesium sulfate and 0.125 part 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.

[0125] Application Example 1

[0126] The bacterial protein biological feeds of Examples 1 to 8 were fed to lactating dairy cows to detect the effect of the bacterial protein biological feeds of the present invention on the milk production of the dairy cows. The specific steps are as follows:

[0127] 27 Holstein black and white dairy cows were selected and randomly divided into 9 groups, with 3 cows in each group, namely: 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. Among them, experimental group 0 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, converted to a specific mass of 290kg coarse corn husks and 100kg fine corn husks, experimental group 1 was fed with bacterial protein bio-feed 1; experimental group 2 was fed with bacterial protein bio-feed 2, experimental group 3 was fed with bacterial protein bio-feed 3, experimental group 4 was fed with bacterial protein bio-feed 4, experimental group 5 was fed with bacterial protein bio-feed 5, experimental group 6 was fed with bacterial protein bio-feed 6, experimental group 7 was fed with bacterial protein bio-feed 7, and experimental group 8 was fed with bacterial protein bio-feed 8. Cows in each experimental group were tethered and fed by a dedicated person at 7:00 AM and 7:00 PM daily. Water and food were freely available. Milking took place at 4:30 AM and 3:30 PM daily, and milk production was recorded. The experimental period lasted 30 days.

[0128] Milk production determination: 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.

[0129] 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 the test groups 1 to 8 were significantly increased compared with those of the test group 0, indicating that the bacterial protein biological feed of Examples 1 to 8 can increase the milk production of dairy cows.

[0130] Comparative Example 1

[0131] The steps are the same as those in Example 1, with the only difference being that when performing step (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.

[0132] Fermentation substrate 7 consisted of 19.5 parts of dry corn husks, 10.5 parts of the koji seeds prepared in Example 1, 3 parts of molasses, 6 parts of ammonium sulfate, 0.1 part of potassium dihydrogen phosphate, 0.2 part of sodium chloride, 0.2 part of magnesium sulfate, and 0.125 part of acetic acid. The corn husks consisted of 14.5 parts of coarse corn husks and 5 parts of fine corn husks, which translated into a specific mass of 290 kg of coarse corn husks, 100 kg of fine corn husks, 210 kg of the koji seeds, 60 kg of molasses, 120 kg of ammonium sulfate, 2 kg of potassium dihydrogen phosphate, 4 kg of sodium chloride, 4 kg of magnesium sulfate, and 2.5 L of acetic acid.

[0133] Comparative Example 2

[0134] The steps are the same as those in Example 1, with the only difference being that when performing step (1), the first fermentation base material is replaced with fermentation substrate 8, and the corn husk mycelial protein biofeed obtained after fermentation is recorded as mycelial protein biofeed 10.

[0135] Among them, fermentation substrate 8 consists 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: corn husks are 290 kg of coarse corn husks, 100 kg of fine corn husks, 210 kg of koji, 80 kg of molasses, 20 kg of ammonium sulfate, 12 kg of potassium dihydrogen phosphate, 6 kg of sodium chloride, 4 kg of magnesium sulfate and 2.5 L of acetic acid.

[0136] Comparative Example 3

[0137] The steps are the same as those in Example 1, with the only difference being that when performing step (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.

[0138] Among them, the fermentation substrate 9 consists 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 part of potassium dihydrogen phosphate, 0.3 part of sodium chloride, 0.2 part of magnesium sulfate and 0.125 part 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.

[0139] Comparative Example 4

[0140] The steps are the same as those in Example 1, with the only difference being that when performing step (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.

[0141] Among them, the fermentation substrate 10 consists 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: 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.

[0142] Test Example 1

[0143] The physicochemical and sanitary indexes of the bacterial protein biofeeds prepared in Examples 1 to 8 and Comparative Examples 1 to 4 were measured. The measuring methods of the physicochemical and sanitary indexes are as follows:

[0144] Physical and chemical indicators: crude protein content is determined in accordance with GB / T 6432-2018, crude fiber content is determined in accordance with GB / T 6434-2006, moisture content is determined in accordance with GB / T 6435-2014, crude ash content is determined in accordance with GB / T 6438-1992, crude fat content is determined in accordance with GB / T 6433-2006, and starch content is determined in accordance with GB / T 20194-2018;

[0145] 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 ... The content of vomitoxin is determined in accordance with GB / T19540-2004, the content of vomitoxin is determined in accordance with GB / T30956-2014, the content of T-2 toxin is determined in accordance with GB / T28718-2012, the content of fumonisin is determined in accordance with NY / T1970-2010, the content of cyanide is determined in accordance with GB / T13084-2006, the content of free gossypol is determined in accordance with GB / T13086-1991, the content of isothiocyanate is determined in accordance with GB / T13087-1991, the content of oxazolidinethione is determined in accordance with GB / T13089-1991, the content of hexachlorobenzene is determined in accordance with GB / T34270-2017, the content of polychlorinated biphenyls is determined in accordance with GB / T 8381.8-2005, and the detection of Salmonella is determined in accordance with GB / T 13091-2002. The specific process is common knowledge and will not be repeated here.

[0146] The true protein determination method is as follows:

[0147] (1) Experimental steps

[0148] Accurately weigh approximately 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 a boil. 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 let it sit for at least 1 hour. Filter with qualitative filter paper, then wash the precipitate 5 or 6 times with 60-80°C hot water. Check the filter paper with 5 drops of barium chloride solution and 1 drop of hydrochloric acid solution. Repeat the previous process of checking the filter paper until no white barium sulfate precipitate is formed. Dry the precipitate and filter paper in a 65°C oven for 2 hours, then transfer everything to a Kjeldahl flask and determine nitrogen using the semi-micro Kjeldahl method.

[0149] (2) Calculation formula

[0150] Where:

[0151] V2: The volume of hydrochloric acid standard titrant consumed in titrating the sample, in milliliters (mL);

[0152] V1: The volume of the hydrochloric acid standard titrant solution consumed in the titration blank, in milliliters (mL);

[0153] C: concentration of hydrochloric acid standard titration solution, in moles per liter (mol / L);

[0154] m: sample mass, in grams (g);

[0155] V: total volume of sample digestion solution, in milliliters (mL);

[0156] V': volume of digestate for distillation, in milliliters (mL);

[0157] 6.25: Average conversion factor of nitrogen to crude protein;

[0158] Yeast detection method is as follows:

[0159] 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 cfu / mL bacterial suspension, and then draw 1mL of yeast concentration of 10 -1 cfu / mL of bacterial suspension was added to 9 mL of sterile water, shaken thoroughly, and diluted to a yeast concentration of 10 -2 cfu / mL bacterial suspension. Pipette 10 μL of yeast with a concentration of 10 -2 The bacterial suspension with a concentration of cfu / mL was dropped onto a 0.0025 mm plate. 2Count the area, then drop an equal volume of methylene blue staining solution, mix it evenly, cover it with a coverslip, be sure not to produce 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.

[0160] The calculation formula is:

[0161] Yeast cell number / g = number of yeast cells in 80 cells / 80×400×10 6 × dilution factor

[0162] The calculation formula is: yeast cell number / g = number of yeast cells in 80 small grids / 80×400×10 6 × dilution factor.

[0163] The test results are shown in Table 1, where % represents mass percentage.

[0164] Table 1 Results of bacterial protein biological feed detection indicators

[0165] 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 this may be that the inorganic salt content ratio is improperly distributed, which inhibits the growth and reproduction of yeast. As can be seen from Table 1, the number of yeast in Comparative Example 1 is significantly lower than that of the examples. 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 this may be that the content of inorganic nitrogen source available to yeast during the fermentation process is low, which cannot meet the needs of its own growth and reproduction. As can be seen from Table 1, the number of yeast in Comparative Example 2 is significantly lower than that of Examples 1 to 8.

[0166] 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 nitrogen source content needs to be further improved.

[0167] 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 and biotin, which inhibit the growth of yeast. Therefore, the crude protein and true protein contents of Comparative Example 2 are lower, and the number of yeast is smaller.

[0168] By comparing the number of yeasts, it was found that the number of yeasts in Examples 1 to 8 was higher than that in Comparative Examples 1 to 4. As shown in Table 1, a comprehensive comparison of the experimental data of Examples 1 to 8 and Comparative Examples 1 to 4 shows that by comparing the crude protein content, true protein content, and crude fiber content, it was found that the crude protein content and true protein content in Examples 1 to 8 were significantly higher than those in Comparative Examples 1 to 4, and met the standards for protein feed; the crude fiber content was significantly lower than that in the comparative example, meeting the standard for 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 was not detected; the content of mycotoxins met the requirements of health indicators; the content of natural toxins and organic pollutants was not detected; the probiotic content of the bacterial protein biological feed prepared in Examples 1 to 8 of the present invention was significantly increased, that is, the number of cerevisiae and utilis increased, the number of Aspergillus niger decreased, and the feeding effect was obvious, and the milk production of dairy cows increased.

[0169] In summary, the corn husk protein fermented feed provided by 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. It has broad application prospects.

[0170] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection 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 the Aspergillus niger bacterial agent into the first fermentation base material to perform the first fermentation to obtain koji seed; Inoculating the composite bacterial agent into the second fermentation base material to carry out the second fermentation to obtain bacterial protein biological feed; The first fermentation base 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-grade flour; The second fermentation base comprises the following raw materials in parts by weight: 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; The composite bacterial agent comprises Saccharomyces cerevisiae and Candida utilis.

2. The preparation method according to claim 1, characterized in that The inoculation amount of Aspergillus niger is 5-10% of the mass of the first fermentation base material; The effective viable bacteria count of Aspergillus niger is 1.0×10 9 ~2.0×10 9 CFU / mL.

3. The preparation method according to claim 1 or 2, characterized in that The Aspergillus niger includes Aspergillus niger CJH-JXSFZh-B703, and its preservation number is CGMCC No.22439.

4. The preparation method according to claim 1, characterized in that The first fermentation lasts for 24 to 48 hours at a temperature of 30 to 35°C.

5. The preparation method according to claim 1, characterized in that The inoculation amount of the composite bacterial agent is 5% to 10% of the mass of the corn husk in the second fermentation base; The effective viable bacteria count of the brewer's yeast is 1.5×10 8 CFU / mL; The effective viable count of the Candida utilis is 1.8×10 8 CFU / mL; The ratio of the effective live bacteria counts of saccharomyces cerevisiae and Candida utilis in the composite bacterial agent is 1.5-10.5:1.8-12.

6.

6. The preparation method according to claim 1 or 5, characterized in that The cerevisiae yeast includes cerevisiae CICC 32236; the Candida utilis includes Candida utilis CGMCC 2.2878.

7. The preparation method according to claim 1, characterized in that The second fermentation lasts for 48 to 72 hours at a temperature of 30 to 32°C.

8. A bacterial protein biological feed, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 7.

9. The bacterial protein biological feed according to claim 8, characterized in that The effective viable bacteria count of Saccharomyces cerevisiae in the bacterial protein biological feed is 1.4×10 9 CFU / kg~1.4×10 11 CFU / kg, the effective viable count of Candida utilis was 1.9×10 9 CFU / kg~1.9×10 11 CFU / kg.

10. Use of the bacterial protein biological feed according to claim 8 or 9 in the preparation of animal feed.

11. Use of the bacterial protein biological feed according to claim 8 or 9 in improving animal milk production and milk quality.

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