Method for preparing single-cell protein feed through liquid-solid combined fermentation of compound strains and application of single-cell protein feed

Abstract

The invention belongs to the technical field of biological fermentation and single-cell protein production, and particularly relates to a method for preparing a single-cell protein feed through compound strain liquid-solid combined fermentation. The method comprises the following steps: S1, liquid fermentation: respectively preparing a neurospora crassa fermentation broth, a monascus fermentation broth, a bacillus fermentation broth, a candida utilis fermentation broth and a lactic acid bacteria fermentation broth; s2, solid-state fermentation: fully mixing the prepared fermentation liquor, and then adding the mixture into a solid-state fermentation culture medium for solid-state fermentation to obtain a material; and S3, bagging the material obtained in the step S2, sealing, stacking, and carrying out anaerobic fermentation to obtain the single-cell protein feed. According to the invention, a compound strain is adopted for fermentation, full utilization of a solid-state fermentation substrate can be promoted while a large number of thalli are obtained through liquid-state fermentation, the abundance of mycoprotein can be increased through coordinated fermentation, the content of restrictive amino acids in livestock and poultry can be increased, and the nutritional value of the mycoprotein can be further increased.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation and single-cell protein production, and specifically relates to a method for preparing single-cell protein feed by liquid-solid combined fermentation of a composite strain and application thereof. Background Art

[0002] With the continuous improvement of living standards, people's demand for protein is also increasing, which has promoted the rapid development of the livestock and poultry industry. However, my country is short of protein feed resources. In 2023, the total protein consumed by my country's breeding industry will be 81.45 million tons, of which 37.03 million tons will be imported, accounting for 45.5% of the total. Due to the need to import a large amount of protein raw materials such as soybeans and fish meal, the production cost is relatively high. How to replace protein raw materials such as soybean meal and fish meal has become an important means for feed companies to reduce costs and increase efficiency. Single-cell protein (SCP) has received widespread attention as a high-quality protein substitute. Single-cell protein contains rich nutrients, with a protein content of 30%-80%, and is rich in lysine, methionine, tryptophan and other amino acids, vitamins, minerals, trace elements, lipids, growth factors, enzymes and other biologically active substances. These substances can directly or indirectly participate in the life activities of the body and maintain the normal physiological functions of animals. In addition, the production of single-cell protein is not affected by seasons and climate, the source of raw materials is rich, and the production cycle is short, so it has broad application prospects in feed production.

[0003] At present, scholars have studied the preparation of single-cell protein, such as Chinese patent CN118126853A discloses a strain of Kluyveromyces marxianus suitable for high-density culture and its application in single-cell protein, Chinese patent CN118006516A discloses a methylotrophic bacillus that can use methanol as the only carbon source to produce high-yield bacterial protein and its application, Chinese patent CN117384770A discloses a method for continuous flow production of single-cell protein, Chinese patent CN115305219A discloses a microbial fermentation synthesis of single-cell protein and its preparation method and application, and Chinese patent CN103749957A discloses a method for preparing cyanobacterial single-cell protein feed. However, most of these methods use solid or liquid methods to ferment with a single strain, so the fermentation efficiency and amino acid abundance are relatively low, the nutrients in the fermentation medium are not fully utilized, and it also causes certain environmental pollution. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing single-cell protein feed by liquid-solid combined fermentation of a composite strain.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a method for preparing single-cell protein feed by liquid-solid combined fermentation of a composite strain, comprising the following steps:

[0007] S1. Liquid fermentation: preparing respectively Neurospora crassa fermentation broth, Monascus fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth;

[0008] S2 solid-state fermentation: The prepared spore spore fermentation broth, red yeast rice fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth were fully mixed and then added to the solid-state fermentation medium for solid-state fermentation to obtain a material;

[0009] S3. The material obtained in step S2 is bagged, sealed, and then stacked for anaerobic fermentation to obtain a single-cell protein feed;

[0010] In step S2, the weight portions of the Nervus crassa fermentation broth, the Monascus fermentation broth, the Bacillus fermentation broth, the Candida utilis fermentation broth and the lactic acid bacteria fermentation broth are respectively: 30-80 parts of the Nervus crassa fermentation broth, 20-45 parts of the Monascus fermentation broth, 3-10 parts of the Bacillus fermentation broth, 80-150 parts of the Candida utilis fermentation broth and 8-20 parts of the lactic acid bacteria fermentation broth.

[0011] The present invention uses liquid-solid combined fermentation, first ferments the strain through liquid fermentation, and after the fermentation is completed, transfers the fermentation liquid to a solid fermentation matrix for further fermentation. The method can shorten the fermentation cycle and improve the nutritional value of bacterial protein. At the same time, it has the characteristics of easy scale-up of liquid fermentation and high yield of solid fermentation. At the same time, the production of bacterial protein by this fermentation method does not generate wastewater and has relatively low energy consumption. In addition, the method uses a composite strain for fermentation, and a large amount of enzymes can be generated while obtaining a large amount of bacterial cells through liquid fermentation, which can promote the full utilization of the solid fermentation matrix. The coordinated fermentation of different strains can increase the abundance of bacterial protein, increase the content of livestock and poultry limiting amino acids, and further improve the nutritional value of bacterial protein.

[0012] Moreover, through a large number of experimental investigations, it was found that when the fermentation broth of different strains was used in the above-mentioned preferred range for solid-state fermentation, the strains cooperated with each other to synergistically improve the fermentation efficiency and single-cell protein yield while avoiding excessive degradation of the culture medium matrix, thereby ensuring the good growth and metabolism of each strain and reducing vicious competition during the fermentation process.

[0013] Preferably, in step S2, the weight parts of the Nervus crassa fermentation broth, the Monascus fermentation broth, the Bacillus fermentation broth, the Candida utilis fermentation broth and the lactic acid bacteria fermentation broth are respectively: 50 parts of Nervus crassa fermentation broth, 30 parts of Monascus fermentation broth, 8 parts of Bacillus fermentation broth, 120 parts of Candida utilis fermentation broth and 15 parts of lactic acid bacteria fermentation broth.

[0014] Through experimental research, it was found that when the strains described in the present invention are compounded in the above-mentioned optimal ratio for solid-state fermentation, the substrate utilization efficiency can be effectively improved, the overall fermentation effect can be enhanced, thereby significantly increasing the bacterial protein content and the total amount of amino acids, and improving the nutritional value of the bacterial protein.

[0015] Preferably, in step S2, the solid-state fermentation medium comprises the following components in parts by weight: 500-800 parts of rice bran, 100-200 parts of corn germ meal, 200-500 parts of wheat bran and 150-250 parts of bean dregs.

[0016] More preferably, the solid-state fermentation medium comprises the following components in parts by weight: 600 parts of rice bran, 150 parts of corn germ meal, 400 parts of wheat bran and 200 parts of bean dregs.

[0017] The present invention adopts the above four raw materials as culture medium matrix to carry out solid-state fermentation of mixed strains. The sources of each raw material are wide, and after reasonable matching and dosage optimization, they can give full play to their respective nutrients and promote the growth of composite strains, thereby reducing production costs while effectively improving the nutritional value of single-cell protein and meeting the market demand for high-quality protein resources.

[0018] Preferably, the method for preparing the fermentation broth of Nervus crassa comprises the following steps: inoculating the cultured activated Nervus crassa bacterial suspension with an inoculation amount of 8-12% into a liquid fermentation medium for culturing, controlling the culture temperature to 28-30° C., the stirring speed to 180-220 r / min, the ventilation volume to 1 vvm, the dissolved oxygen content to 20-30%, and culturing and fermenting for 68-72 hours to obtain the fermentation broth of Nervus crassa;

[0019] The liquid fermentation medium comprises the following components: 3-8% microcrystalline cellulose, 2-5% soybean cake powder, 0.5-2% rice bran, 0.1-0.8% peptone, 0.05-0.4% magnesium sulfate, 0.01-0.03% manganese sulfate, 0.001-0.006% cobalt chloride, 0.001-0.004% ferrous sulfate, 0.005-0.025% zinc sulfate and the balance water.

[0020] Preferably, the method for preparing the red yeast rice fermentation liquid comprises the following steps: inoculating the activated red yeast rice suspension into the fermentation medium at an inoculum amount of 4-6% for culturing, controlling the culture temperature to 30-34° C., the stirring speed to 180-220 r / min, the ventilation volume to 0.5 vvm, the dissolved oxygen content to 10-20%, and culturing and fermenting for 68-72 hours to obtain the red yeast rice fermentation liquid;

[0021] The liquid fermentation medium comprises the following components: 5-10% indica rice flour, 8-15% soy peptone, 1-4% glucose, 0.5-2% corn steep liquor, 0.1-0.3% potassium dihydrogen phosphate, 0.1-0.3% dipotassium hydrogen phosphate, 0.01-0.04% manganese sulfate, 0.01-0.03% zinc sulfate and the balance water.

[0022] Preferably, the preparation method of the bacillus fermentation liquid comprises the following steps: inoculating the cultured activated bacillus suspension into the fermentation medium at an inoculum amount of 4-6% for culturing, controlling the culture temperature to 37-40°C, the stirring speed to 180-220r / min, the ventilation volume to 0.5vvm, the dissolved oxygen content to 20-40%, and culturing and fermenting for 18-20h to obtain the bacillus fermentation liquid;

[0023] The liquid fermentation medium comprises the following components: 8-15% corn flour, 3-8% soybean cake flour, 2-5% cottonseed meal flour, 0.5-2% wheat bran flour, 0.05-0.25% potassium dihydrogen phosphate, 0.05-0.25% dipotassium hydrogen phosphate, 0.01-0.08% magnesium sulfate, 0.001-0.003% manganese sulfate and the balance water.

[0024] Preferably, the method for preparing the Candida utilis fermentation broth comprises the following steps: inoculating the cultured activated Candida utilis suspension into a fermentation medium at an inoculation rate of 4-6% for culturing, controlling the culture temperature to 28-30°C, the stirring speed to 180-220r / min, the ventilation volume to 0.5vvm, the dissolved oxygen content to 20-40%, and culturing and fermenting for 18-20h to obtain the Candida utilis fermentation broth;

[0025] The liquid fermentation medium comprises the following components: 3-7% corn starch, 8-15% soybean cake powder, 1-5% yeast extract, 0.5-2% peptone, 0.05-0.2% potassium dihydrogen phosphate, 0.01-0.04% manganese sulfate, 0.03-0.08% magnesium sulfate and the balance water.

[0026] Preferably, the method for preparing the lactic acid bacteria fermentation liquid comprises the following steps: inoculating the cultured activated lactic acid bacteria suspension into the fermentation medium at an inoculum amount of 1-3% for culturing, controlling the culture temperature to 35-40°C, the stirring speed to 80-120r / min, the ventilation volume to 0.05vvm, and culturing and fermenting for 35-40h to obtain the lactic acid bacteria fermentation liquid;

[0027] The liquid fermentation medium comprises the following components: 8-14% glucose, 0.5-2% yeast powder, 1-4% peptone, 3-8% corn steep liquor, 0.05-0.3% potassium dihydrogen phosphate, 0.05-0.3% dipotassium hydrogen phosphate, 0.01-0.07% sodium acetate, 2-8% light calcium carbonate and the balance water.

[0028] Preferably, in step S2, the specific conditions of solid-state fermentation are: temperature of 28-32° C., stirring speed of 3-7 r / min, and fermentation time of 60-72 h.

[0029] In a second aspect, the present invention provides a single-cell protein feed prepared by the method described above.

[0030] In a third aspect, the present invention provides application of the method in preparing single-cell protein feed.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention adopts composite bacteria to prepare single-cell protein, which has more comprehensive nutrition and higher protein content than fermentation of a single strain;

[0033] 2. The present invention adopts a liquid-solid combined fermentation method to prepare single-cell protein. This production method does not generate wastewater during the production process and has a small load on the environment;

[0034] 3. The present invention adopts liquid fermentation to prepare fermentation liquid, and then transfers the fermentation liquid into a solid fermentation matrix. This method produces a large number of bacteria through liquid fermentation, and the fermentation liquid is inoculated into the solid matrix for fermentation. The inoculated fermentation liquid has an advantage, so the fermentation matrix does not need to be strictly disinfected and sterilized, which greatly saves energy consumption;

[0035] 4. The crassa spores selected in the present invention not only produce a large amount of bacterial cells but also produce a large amount of cellulase through liquid fermentation, which can quickly degrade cellulose in solid materials to provide a large amount of carbon source for the production of bacterial proteins. The addition of crassa spores improves the utilization rate of solid materials and saves input of production raw materials;

[0036] 5. The Monascus described in the present invention can produce a large amount of protease, saccharifying enzyme, liquefying enzyme, bacterial protein, etc. through liquid fermentation culture. The addition of Monascus can further promote the decomposition of materials and improve the utilization rate of solid matrix;

[0037] 6. The natto Bacillus subtilis added in the present invention has a strong protease and amylase production capacity, and produces a large amount of protease and amylase through liquid fermentation, which promotes the decomposition of materials. At the same time, during the anaerobic fermentation stage, Bacillus subtilis quickly utilizes the oxygen in the substrate to provide anaerobic conditions for anaerobic fermentation;

[0038] 7. The Candida utilis added in the present invention has a high single-cell protein production capacity. The bacteria can obtain a large amount of single-cell protein through liquid high-density culture. After the fermentation is completed, the fermentation liquid is transferred to the solid fermentation matrix for secondary growth, which further increases the content of single-cell protein.

[0039] 8. The pentose lactic acid bacteria added in the present invention has a strong effect of degrading phytic acid phosphorus. The addition of the bacteria can effectively degrade the phytic acid phosphorus in plant raw materials, improve the utilization rate of raw materials, reduce the addition of inorganic phosphorus in feed, and reduce phosphorus pollution to the environment. At the same time, the bacteria can produce a large amount of L-lactic acid under anaerobic fermentation conditions, thereby improving the palatability and nutritional value of single-cell protein.

[0040] 9. The rice bran and bean dregs used in the present invention are both nutritious and perishable materials. After being prepared with single-cell protein, the shelf life of the materials is greatly extended; the added wheat bran and corn germ have a high content of broken fiber, so the utilization rate is relatively low when used directly, but after being fermented with composite bacteria, cellulose is decomposed in large quantities, which greatly improves the utilization rate of raw materials;

[0041] 10. The single-cell protein prepared by the method of the present invention has relatively high contents of lysine, methionine and tryptophan, has high utilization value, and meets the market demand for high-quality protein resources. DETAILED DESCRIPTION

[0042] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments.

[0043] Unless otherwise specified, the reagents used in the examples are conventional reagents in the art and can be purchased through commercial channels. The experimental operations not specifically described in the examples are conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common knowledge they master.

[0044] The Neurospora crassa described in the present invention was purchased from the China Industrial Microbiological Culture Collection Administration Center, with a strain number of CICC 40203; the Monascus is Monascus purpureus BOEN-0920, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center (5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with a preservation date of April 19, 2024, and is classified and named Monascus purpureus, with a preservation number of GDMCC NO: 64523; the Bacillus is Bacillus natto, purchased from the China Industrial Microbiological Culture Collection Administration Center, with a strain number of CICC 20643; the utilis is purchased from the North Na Chuanglian Biotechnology Research Institute, with a strain number of BNCC 186221; the lactic acid bacteria is Lactobacillus pentosus, which is deposited in the Guangdong Provincial Microbiological Culture Collection Center, with a preservation number of GDMCC NO: 63561.

[0045] Examples 1-3, Comparative Examples 1-8

[0046] The present invention provides a formula for preparing single-cell protein feed by composite bacteria liquid-solid combined fermentation (Examples 1-3 and Comparative Examples 1-8), as shown in Table 1.

[0047] Table 1: Formula table for preparing single-cell protein feed by the combined fermentation of composite bacteria liquid-solid (parts by weight)

[0048]

[0049]

[0050] The method for preparing single-cell protein feed by liquid-solid combined fermentation of a composite strain described in Examples 1-3 and Comparative Examples 1-8 (a certain component is missing and the method does not contain the step of preparing the component) comprises the following steps:

[0051] S1. Liquid fermentation:

[0052] a. Preparation of fermentation broth of crassa ventriculi: crassa ventriculi was inoculated into Chadwick agar medium and cultured for 5-7 days. When the medium showed that it was covered with orange-yellow spores, 1 ring was inoculated into potato liquid seed medium, cultured at 28-30°C and 180r / min with shaking for about 40h, and 10% of the inoculum was accessed into the liquid fermentation medium, the culture temperature was controlled to be 28-30°C, the initial stirring speed was 200r / min, the ventilation volume was 1vvm, and the culture was started. During the culture process, the dissolved oxygen was controlled at 20-30%, and the liquid fermentation was ended after about 70h of fermentation to obtain crassa ventriculi fermentation broth;

[0053] The liquid fermentation medium comprises: 5% microcrystalline cellulose, 3% soybean cake powder, 1% rice bran, 0.5% peptone, 0.2% magnesium sulfate, 0.01% manganese sulfate, 0.005% cobalt chloride, 0.03% ferrous sulfate, 0.01% zinc sulfate and the balance water.

[0054] b. Preparation of red yeast rice fermentation broth: The red yeast rice was inoculated into a potato agar medium and cultured for 5-7 days. When the surface of the medium was covered with mycelium and the medium was purple-red, 1 ring was picked up in a potato liquid seed medium, and cultured at 30-34 ° C, 180r / min shaking for 40h, and inoculated into the fermentation medium at a rate of 5%, the temperature was controlled at 30-34 ° C, the initial stirring speed was 200r / min, the ventilation volume was 0.5vvm, and the culture was started. During the culture process, the dissolved oxygen was controlled at 10-20%, and the liquid fermentation was ended after about 70h of fermentation to obtain red yeast rice fermentation broth;

[0055] The liquid fermentation medium comprises: 8% indica rice flour, 10% soy peptone, 2% glucose, 1% corn steep liquor, 0.2% potassium dihydrogen phosphate, 0.2% dipotassium hydrogen phosphate, 0.02% manganese sulfate, 0.015% zinc sulfate and the balance water.

[0056] c. Preparation of Bacillus fermentation broth: Inoculate Bacillus natto in LB solid medium, culture at 35-40°C for 18-22h until the bacteria cover the slope and the hyphae show wrinkles, pick 1 ring in LB liquid medium, 35-40°C, 200r / min shaking culture for 40h, inoculate 5% of the inoculum into the fermentation medium, control the temperature to 37-40°C, the initial stirring speed is 200r / min, the ventilation volume is 0.5vvm, start cultivation, control the dissolved oxygen at 20-40% during the culture process, ferment for about 18-20h to end the liquid fermentation, and obtain Bacillus fermentation broth;

[0057] The liquid fermentation medium comprises: 10% corn flour, 5% soybean cake flour, 3% cottonseed meal flour, 1% wheat bran flour, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.001% manganese sulfate and the balance water.

[0058] d. Preparation of Candida utilis fermentation broth: inoculate Candida utilis in YM solid medium, culture at 25-28°C for 40-48h, wait for the yeast to cover the test tube slope, pick 1 loop and inoculate in YM liquid seed medium, culture at 28-30°C, 180r / min shaking for 18-24h, inoculate into liquid fermentation medium at 5% inoculum, control the temperature at 28-30°C, the initial stirring speed is 200r / min, the ventilation volume is 0.5vvm, start culture, control the dissolved oxygen at 20-40% during the culture process, ferment for about 18-20h to end the liquid fermentation, and obtain Candida utilis fermentation broth;

[0059] The liquid fermentation medium comprises: 5% corn starch, 10% soybean cake powder, 2% yeast extract, 1% peptone, 0.1% potassium dihydrogen phosphate, 0.025% manganese sulfate, 0.05% magnesium sulfate and the balance water.

[0060] e. Preparation of lactic acid fermentation broth: Lactobacillus pentosus was inoculated into MRS solid medium and cultured at 37 ° C for 24-30h. When lactic acid bacteria covered the test tube slope, 1 loop was picked and inoculated into MRS liquid seed medium, 36-38 ° C, 180r / min shaking culture for 18-20h. When the pH reached 4.0 and the OD600 reached 6.0, 2% of the inoculum was inoculated into the liquid fermentation medium, the temperature was controlled at 35-40 ° C, the initial stirring speed was 100r / min, the ventilation volume was 0.05vvm, and fermentation was started. The liquid fermentation was ended for 35-40h to obtain lactic acid fermentation broth;

[0061] The fermentation medium comprises: 10% glucose, 1% yeast powder, 2% peptone, 6% corn steep liquor, 0.1% potassium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, 0.05% sodium acetate, 4% light calcium carbonate and the balance water.

[0062] S2. Solid-state fermentation: The prepared spore fermentation liquid of crassa, red yeast rice fermentation liquid, Bacillus fermentation liquid, Candida utilis fermentation liquid and lactic acid bacteria fermentation liquid were placed in a stirring tank and mixed thoroughly for 5-10 minutes, and then rice bran, corn germ meal, wheat bran and bean dregs were weighed and mixed thoroughly in a mixer for 10-20 minutes to prepare a solid-state fermentation medium, and then the composite fermentation liquid and the solid fermentation medium were added to a constant temperature stirring tank, the temperature of the stirring tank was controlled to 30°C, and the mixture was stirred rapidly for 30 minutes. After the materials were fully mixed, aerobic solid-state fermentation was continued in a constant temperature stirring tank. The stirring speed was controlled to be 5r / min during the fermentation process, and compressed air was introduced into the bottom of the tank, and the fermentation was continued for 60-72 hours;

[0063] S3. After the aerobic fermentation is completed, the materials are packed into breathing bags, which are sealed with a sealing machine, and the materials are piled together for anaerobic fermentation. The fermentation is completed after 3-5 days to obtain the single-cell protein feed.

[0064] Effect example

[0065] The crude protein and amino acid contents in the single cell proteins obtained in Examples 1-3 and Comparative Examples 1-8 were detected by Kjeldahl nitrogen determination, liquid chromatography and amino acid determination instrument, and the results are shown in Table 2. Detection method reference standard: The crude protein in feed was determined according to the Kjeldahl nitrogen determination method GB / T 6432, the tryptophan in feed was determined according to GB / T 15400, and the amino acid in feed was determined according to GB / T 18246.

[0066] Table 2

[0067]

[0068]

[0069] From the results in Table 2, it can be seen that the crude protein content, total amino acid content, and limiting amino acid content such as lysine, threonine, and tryptophan in the single-cell protein feed prepared by the technical solution of the present invention are relatively high, and the bacterial protein has high nutritional value, which fully meets the market demand for high-quality protein resources. While in Comparative Examples 3-6, there is a lack of crassa fermentation broth, Candida utilis fermentation broth, Monascus fermentation broth, and Bacillus fermentation broth, respectively, and when other fermentation broths are added to keep the total amount of composite fermentation broth unchanged, the crude protein content and total amino acid content in the prepared single-cell protein are significantly reduced compared with Examples 1-3, indicating that the strains used in the present invention can cooperate with each other, thereby synergistically improving the fermentation efficiency and the yield and quality of single-cell protein, and effectively improving the utilization rate of raw materials, thereby reducing the input cost of raw material production, and none of them can be missing, and even if other strains are used as substitutes, the fermentation effect is not ideal; the dosage ratio of the fermentation broth of different strains described in Comparative Examples 1-2 exceeds When the scope is limited by the present invention, the crude protein content and the total amount of amino acids in the prepared single-cell protein are also reduced, indicating that the use amount of the composite strain fermentation broth can better exert the synergistic effect within the preferred range, thereby improving the yield and abundance of bacterial protein, and further improving the nutritional value of bacterial protein; while in comparative examples 7-8, wheat bran and corn germ meal fermentation raw materials are respectively lacking, and other raw materials are used to replace them so that the total amount of fermentation substrate remains unchanged, the crude protein content and the total amount of amino acids in the single-cell protein are significantly reduced, indicating that the raw materials selected by the present invention are reasonably matched, and can give full play to their respective nutrients and promote the growth of the composite strain, thereby reducing production costs while effectively improving the nutritional value of single-cell protein.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing single-cell protein feed by liquid-solid combined fermentation of composite strains, characterized in that: The following steps are involved: S1. Liquid fermentation: preparing respectively Neurospora crassa fermentation broth, Monascus fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth; S2 solid-state fermentation: The prepared spore spore fermentation broth, red yeast rice fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth were fully mixed and then added to the solid-state fermentation medium for solid-state fermentation to obtain a material; S3. The material obtained in step S2 is bagged, sealed, and then stacked for anaerobic fermentation to obtain a single-cell protein feed; In step S2, the weight portions of the Nervus crassa fermentation broth, the Monascus fermentation broth, the Bacillus fermentation broth, the Candida utilis fermentation broth and the lactic acid bacteria fermentation broth are respectively: 30-80 parts of the Nervus crassa fermentation broth, 20-45 parts of the Monascus fermentation broth, 3-10 parts of the Bacillus fermentation broth, 80-150 parts of the Candida utilis fermentation broth and 8-20 parts of the lactic acid bacteria fermentation broth.

2. The method according to claim 1, characterized in that In step S2, the weight portions of the Nervus crassa fermentation broth, the Monascus fermentation broth, the Bacillus fermentation broth, the Candida utilis fermentation broth and the lactic acid bacteria fermentation broth are respectively: 50 parts of the Nervus crassa fermentation broth, 30 parts of the Monascus fermentation broth, 8 parts of the Bacillus fermentation broth, 120 parts of the Candida utilis fermentation broth and 15 parts of the lactic acid bacteria fermentation broth.

3. The method according to claim 1, characterized in that In step S2, the solid-state fermentation medium comprises the following components in parts by weight: 500-800 parts of rice bran, 100-200 parts of corn germ meal, 200-500 parts of wheat bran and 150-250 parts of bean dregs.

4. The method according to claim 1, characterized in that The preparation method of the crassa pulverulospora fermentation liquid comprises the following steps: inoculating the cultured activated crassa pulverulospora suspension into a liquid fermentation medium at an inoculation rate of 8-12% for culturing, controlling the culture temperature to be 28-30° C., the stirring speed to be 180-220 r / min, the ventilation volume to be 1 vvm, the dissolved oxygen content to be 20-30%, and culturing and fermenting for 68-72 hours to obtain the crassa pulverulospora fermentation liquid; The liquid fermentation medium comprises the following components: 3-8% microcrystalline cellulose, 2-5% soybean cake powder, 0.5-2% rice bran, 0.1-0.8% peptone, 0.05-0.4% magnesium sulfate, 0.01-0.03% manganese sulfate, 0.001-0.006% cobalt chloride, 0.001-0.004% ferrous sulfate, 0.005-0.025% zinc sulfate and the balance water.

5. The method according to claim 1, characterized in that The preparation method of the red yeast rice fermentation liquid comprises the following steps: inoculating a cultured activated red yeast rice suspension into a liquid fermentation medium at an inoculation rate of 4-6% for culturing, controlling the culture temperature to be 30-34° C., the stirring speed to be 180-220 r / min, the ventilation volume to be 0.5 vvm, the dissolved oxygen content to be 10-20%, and culturing and fermenting for 68-72 hours to obtain the red yeast rice fermentation liquid; The liquid fermentation medium comprises the following components: 5-10% indica rice flour, 8-15% soy peptone, 1-4% glucose, 0.5-2% corn steep liquor, 0.1-0.3% potassium dihydrogen phosphate, 0.1-0.3% dipotassium hydrogen phosphate, 0.01-0.04% manganese sulfate, 0.01-0.03% zinc sulfate and the balance water.

6. The method according to claim 1, characterized in that The preparation method of the bacillus fermentation liquid comprises the following steps: inoculating the cultured activated bacillus suspension into a liquid fermentation medium at an inoculation rate of 4-6% for culturing, controlling the culture temperature to be 37-40°C, the stirring speed to be 180-220r / min, the ventilation volume to be 0.5vvm, the dissolved oxygen content to be 20-40%, and culturing and fermenting for 18-20h to obtain the bacillus fermentation liquid; The liquid fermentation medium comprises the following components: 8-15% corn flour, 3-8% soybean cake flour, 2-5% cottonseed meal flour, 0.5-2% wheat bran flour, 0.05-0.25% potassium dihydrogen phosphate, 0.05-0.25% dipotassium hydrogen phosphate, 0.01-0.08% magnesium sulfate, 0.001-0.003% manganese sulfate and the balance water.

7. The method according to claim 1, characterized in that The preparation method of the Candida utilis fermentation broth comprises the following steps: inoculating a cultured activated Candida utilis bacterial suspension into a liquid fermentation medium at an inoculation rate of 4-6% for culturing, controlling the culture temperature to be 28-30°C, the stirring speed to be 180-220r / min, the ventilation volume to be 0.5vvm, the dissolved oxygen content to be 20-40%, and culturing and fermenting for 18-20h to obtain the Candida utilis fermentation broth; The liquid fermentation medium comprises the following components: 3-7% corn starch, 8-15% soybean cake powder, 1-5% yeast extract, 0.5-2% peptone, 0.05-0.2% potassium dihydrogen phosphate, 0.01-0.04% manganese sulfate, 0.03-0.08% magnesium sulfate and the balance water.

8. The method according to claim 1, characterized in that The preparation method of the lactic acid bacteria fermentation liquid comprises the following steps: inoculating the cultured activated lactic acid bacteria suspension into a liquid fermentation medium at an inoculum amount of 1-3% for culturing, controlling the culture temperature to be 35-40°C, the stirring speed to be 80-120r / min, the ventilation volume to be 0.05vvm, and culturing and fermenting for 35-40h to obtain the lactic acid bacteria fermentation liquid; The liquid fermentation medium comprises the following components: 8-14% glucose, 0.5-2% yeast powder, 1-4% peptone, 3-8% corn steep liquor, 0.05-0.3% potassium dihydrogen phosphate, 0.05-0.3% dipotassium hydrogen phosphate, 0.01-0.07% sodium acetate, 2-8% light calcium carbonate and the balance water.

9. A single cell protein feed prepared by the method according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 in preparing single cell protein feed.

Images