A method and application of preparing single-cell protein feed by liquid-solid combined fermentation of composite strains

Abstract

The present 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. The method comprises the following steps: S1. liquid fermentation: respectively preparing crassa nematocystis fermentation broth, red yeast rice fermentation broth, bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth; S2. solid-state fermentation: fully mixing the prepared fermentation broth, then adding it to a solid-state fermentation medium for solid-state fermentation to obtain material; S3. bagging and sealing the material obtained in step S2, and then stacking it for anaerobic fermentation to obtain single-cell protein feed. The present invention adopts a composite strain for fermentation, and while obtaining a large amount of bacterial cells through liquid fermentation, it can also promote the full utilization of solid-state fermentation matrix, and coordinated fermentation 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.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation and single-cell protein production, and particularly relates to a method for preparing single-cell protein feed through 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 facing a shortage of protein feed resources. In 2023, the total protein consumption of my country's livestock and poultry industry will reach 81.45 million tons, of which 37.03 million tons, or 45.5%, will be imported. Due to the large amount of protein raw materials such as soybeans and fish meal that need to be imported, production costs are relatively high. Replacing 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 attracted widespread attention as a high-quality protein substitute. SCP is rich in nutrients, with a protein content of 30%-80%. It is rich in various amino acids such as lysine, methionine, and tryptophan, as well as vitamins, minerals, trace elements, lipids, growth factors, enzymes, and other bioactive substances. These substances directly or indirectly participate in the body's vital activities and maintain normal physiological functions of animals. In addition, SCP production is not affected by seasons and climate, has abundant raw material sources, and has a short production cycle. Therefore, it has broad application prospects in feed production.

[0003] Currently, scholars have studied the preparation of single-cell proteins. For example, Chinese Patent CN118126853A discloses a strain of Kluyveromyces marxianus suitable for high-density culture and its application in single-cell protein production; Chinese Patent CN118006516A discloses a methylotrophic bacillus that can use methanol as a sole 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 method for synthesizing 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-state or liquid fermentation methods with a single strain, resulting in relatively low fermentation efficiency and amino acid abundance, insufficient utilization of nutrients in the fermentation medium, and 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 object of the invention, 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 Nervus crassa fermentation broth, Monascus fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth respectively;

[0008] S2 solid-state fermentation: The prepared crassa venosporium 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 single-cell protein feed;

[0010] In step S2, the weight portions of the Nervus crassa fermentation broth, the red yeast rice 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 red yeast rice 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 fermenting the strain through liquid fermentation, and after the fermentation is completed, transferring the fermentation liquid to a solid fermentation matrix for further fermentation. This method can shorten the fermentation cycle and improve the nutritional value of the 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 through this fermentation method does not generate wastewater and has relatively low energy consumption. In addition, this method uses a composite bacterial strain for fermentation. While obtaining a large amount of bacterial cells through liquid fermentation, it also produces a large amount of enzymes, which can promote the full utilization of the solid fermentation matrix. The coordinated fermentation of different bacterial 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, after a large number of experimental investigations, it was found that when the fermentation broth of the 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 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 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 uses the above four raw materials as culture medium matrices for solid-state fermentation of mixed strains. The raw materials are widely available and, through reasonable matching and dosage optimization, can fully exert their respective nutritional components and promote the growth of the composite strain, 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 Nervus crassa fermentation broth comprises the following steps: inoculating a cultured and activated Nervus crassa suspension into a liquid fermentation medium at an inoculum size of 8-12%, culturing the culture temperature at 28-30° C., stirring the suspension at 180-220 r / min, aerating the suspension at 1 vvm, and dissolving the oxygen at 20-30%, and culturing and fermenting the suspension for 68-72 hours to obtain the Nervus crassa fermentation broth;

[0019] The liquid fermentation culture 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 a cultured activated red yeast rice suspension into a fermentation medium at an inoculum size of 4-6%, culturing the culture temperature at 30-34° C., stirring the culture at a speed of 180-220 r / min, a ventilation volume of 0.5 vvm, and a dissolved oxygen content of 10-20%, and culturing and fermenting the culture 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 method for preparing the bacillus fermentation broth comprises the following steps: inoculating a cultured activated bacillus suspension into a fermentation medium at an inoculum size of 4-6%, culturing the culture temperature at 37-40° C., stirring speed at 180-220 r / min, ventilation volume at 0.5 vvm, dissolved oxygen content at 20-40%, and culturing and fermenting for 18-20 hours to obtain the bacillus fermentation broth;

[0023] The liquid fermentation culture 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 a cultured and activated Candida utilis suspension into a fermentation medium at an inoculum size of 4-6%, culturing the culture temperature at 28-30° C., stirring speed at 180-220 r / min, ventilation volume at 0.5 vvm, dissolved oxygen content at 20-40%, and culturing and fermenting for 18-20 hours 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 broth comprises the following steps: inoculating a cultured activated lactic acid bacteria suspension into a fermentation medium at an inoculum size of 1-3%, culturing the culture temperature at 35-40° C., stirring the suspension at 80-120 r / min, and aerating the suspension at 0.05 vvm, and culturing and fermenting the suspension for 35-40 hours to obtain the lactic acid bacteria fermentation broth;

[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 uses composite bacteria to prepare single-cell protein, which is more nutritionally comprehensive and has a higher protein content than fermentation with 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 low environmental burden.

[0034] 3. The present invention adopts liquid fermentation to prepare fermentation liquid first, and then transfers the fermentation liquid into solid fermentation matrix. This method produces a large number of bacteria through liquid fermentation. 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 N. crassa bacteria used in the present invention not only produces a large amount of bacterial cells during liquid fermentation, but also produces a large amount of cellulase, which can quickly degrade cellulose in solid materials and provide a large amount of carbon source for the production of bacterial protein. The addition of N. crassa bacteria improves the utilization rate of solid materials and saves production raw material input;

[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 Bacillus natto added in the present invention has a strong protease and amylase production capacity. It 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, providing 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 produce a large amount of single-cell protein through high-density liquid culture. After the fermentation is completed, the fermentation liquid is transferred to a solid fermentation matrix for secondary growth, further increasing the content of single-cell protein.

[0039] 8. The pentose lactic acid bacteria added in the present invention have a strong effect of degrading phytic acid phosphorus. The addition of this bacterium 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, this bacterium 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 easily deteriorating 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. However, after fermentation with the composite bacteria, the cellulose is largely decomposed, greatly improving the utilization rate of the 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 following further describes the above content of the present invention in detail through specific implementation methods in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples.

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

[0044] The Neurospora crassa described in the present invention was purchased from the China Industrial Microorganism Culture Collection Center with a strain number of CICC 40203; the Monascus is Monascus purpureus BOEN-0920, which is deposited in the Guangdong Provincial Microorganism Culture Collection Center (5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with a preservation date of April 19, 2024, and is classified as Monascus purpureus with a preservation number of GDMCC NO: 64523; the Bacillus is Bacillus natto, which was purchased from the China Industrial Microorganism Culture Collection 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 Microorganism 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 liquid-solid combined fermentation of composite bacteria (Examples 1-3 and Comparative Examples 1-8), as shown in Table 1.

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

[0048]

[0049]

[0050] The method for preparing single-cell protein feed by liquid-solid combined fermentation of a composite strain as 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 crassa nematode fermentation broth: crassa nematode was inoculated on Cha's agar medium and cultured for 5-7d, until the culture medium showed that it was covered with orange-yellow spores, and 1 ring was inoculated into a potato liquid seed medium, 28-30 ℃, 180r / min shaking culture for about 40h, 10% of the inoculum size was accessed into the liquid fermentation medium, the culture temperature was controlled at 28-30 ℃, 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 completed for about 70h to obtain crassa nematode 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: Monascus was inoculated on potato agar medium and cultured for 5-7d, until the surface of the culture medium was covered with mycelium, the culture medium was purple-red, and 1 ring was picked in a liquid potato seed medium, 30-34 ° C, 180r / min shaking culture for 40h, 5% inoculum was inoculated into the fermentation medium, 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, the dissolved oxygen was controlled at 10-20%, and the liquid fermentation was completed for about 70h 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: Bacillus natto was inoculated in LB solid medium and cultured at 35-40 ° C for 18-22h until the bacteria covered the slope and the mycelium showed wrinkles. A ring was picked in LB liquid medium and cultured at 35-40 ° C, 200r / min shaking for 40h. The inoculum size was 5% and inoculated into the fermentation medium. The temperature was controlled at 37-40 ° C, the initial stirring speed was 200r / min, the ventilation volume was 0.5vvm, and the culture was started. During the culture, the dissolved oxygen was controlled at 20-40%. The fermentation was completed about 18-20h in liquid state to obtain Bacillus fermentation broth.

[0057] The liquid fermentation medium comprises: 10% corn flour, 5% soybean cake powder, 3% cottonseed meal powder, 1% wheat bran powder, 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: Candida utilis was inoculated into YM solid medium and cultured at 25-28 ° C for 40-48h. After the yeast covered the test tube slope, 1 loop was picked and inoculated into YM liquid seed medium, 28-30 ° C, 180r / min shaking culture for 18-24h, and 5% inoculum was inoculated into the liquid fermentation medium. The temperature was controlled at 28-30 ° 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 20-40%. The fermentation was completed after about 18-20h of liquid fermentation to 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 on MRS solid medium and cultured at 37 ° C for 24-30h, until lactic acid bacteria covered the test tube slope, picked 1 loop and inoculated on MRS liquid seed medium, 36-38 ° C, 180r / min shaking culture for 18-20h, when the pH reached 4.0 and OD600 reached 6.0, 2% of the inoculum size 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, fermentation was started, and the liquid fermentation was completed 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 nematocystis fermentation broth, red yeast rice fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth were placed in a stirring tank and thoroughly mixed for 5-10min, then rice bran, corn germ meal, wheat bran and bean dregs were weighed and thoroughly stirred in a mixer for 10-20min to prepare a solid-state fermentation medium. The composite fermentation broth and the solid fermentation medium were then added to a constant temperature stirring tank, the stirring tank temperature was controlled at 30 ° C, and rapid stirring was performed for 30min. After the materials were thoroughly mixed, aerobic solid-state fermentation was continued in a constant temperature stirring tank. The stirring speed during the fermentation process was controlled at 5r / min, compressed air was introduced into the bottom of the tank, and fermentation was continued for 60-72h;

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

[0064] Effect Examples

[0065] The crude protein and amino acid contents of the single-cell proteins obtained in Examples 1-3 and Comparative Examples 1-8 were determined using Kjeldahl nitrogen determination, liquid chromatography, and an amino acid analyzer. The results are shown in Table 2. Reference Standards for Detection Methods: The crude protein content in feed was determined according to the Kjeldahl nitrogen determination method GB / T 6432, the tryptophan content in feed was determined according to GB / T 15400, and the amino acid content in feed was determined according to GB / T 18246.

[0066] Table 2

[0067]

[0068]

[0069] As can be seen from the results in Table 2, 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 nutritional value of the bacterial protein is high, which fully meets the market demand for high-quality protein resources. In contrast, in Comparative Examples 3-6, there is a lack of crassa fermentation broth, utilis fermentation broth, red yeast rice fermentation broth, and bacillus fermentation broth, and when other fermentation broths are added to keep the total amount of the composite fermentation broth unchanged, the crude protein content and total amino acid content in the single-cell protein obtained are significantly reduced compared to 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 materials, and none of them can be missing. Even if other strains are used as substitutes, the fermentation effect is not ideal; the dosage ratio of the fermentation broths 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 synergistic effect can be better exerted when the amount of the composite strain fermentation broth is within the preferred range, thereby improving the yield and abundance of the bacterial protein, and further improving the nutritional value of the 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 to give full play to their respective nutritional components and promote the growth of the composite strain, thereby reducing production costs while effectively improving the nutritional value of the single-cell protein.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing single-cell protein feed by liquid-solid combined fermentation of a composite strain, characterized in that: The following steps are involved: S1. Liquid fermentation: preparing Neurospora crassa fermentation broth, Monascus fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth respectively; S2 solid-state fermentation: The prepared Neurospora crassa fermentation broth, Monascus 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 single-cell protein feed; In step S2, the weight portions of Neurospora crassa fermentation broth, Monascus fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth are respectively: 30-80 parts of Neurospora crassa fermentation broth, 20-45 parts of Monascus fermentation broth, 3-10 parts of Bacillus fermentation broth, 80-150 parts of Candida utilis fermentation broth and 8-20 parts of lactic acid bacteria fermentation broth; 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; The bacillus is Bacillus natto.

2. The method according to claim 1, wherein In step S2, the weight portions of Neurospora crassa fermentation broth, Monascus fermentation broth, Bacillus fermentation broth, Candida utilis fermentation broth and lactic acid bacteria fermentation broth are respectively: 50 parts of Neurospora 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.

3. The method according to claim 1, wherein The preparation method of the Neurospora crassa fermentation broth comprises the following steps: inoculating a cultured and activated Neurospora crassa suspension into a liquid fermentation medium at an inoculum size of 8-12%, culturing the culture temperature at 28-30° C., stirring the speed at 180-220 r / min, aerating the air volume at 1 vvm, and dissolving oxygen at 20-30%, and culturing and fermenting for 68-72 hours to obtain the Neurospora crassa fermentation broth; 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.

4. The method according to claim 1, wherein The preparation method of the red yeast rice fermentation liquid comprises the following steps: inoculating a cultured and activated red yeast rice suspension into a liquid fermentation medium at an inoculum rate of 4-6%, culturing the culture temperature at 30-34° C., stirring the speed at 180-220 r / min, aerating the air volume at 0.5 vvm, and dissolving oxygen at 10-20%, and culturing and fermenting the culture 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.

5. The method according to claim 1, wherein The preparation method of the bacillus fermentation broth comprises the following steps: inoculating a cultured activated bacillus suspension into a liquid fermentation medium at an inoculum rate of 4-6%, culturing the culture temperature at 37-40° C., stirring the speed at 180-220 r / min, aerating the air volume at 0.5 vvm, and dissolving oxygen at 20-40%, and culturing and fermenting for 18-20 hours to obtain the bacillus fermentation broth; 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.

6. The method according to claim 1, wherein The preparation method of the Candida utilis fermentation broth comprises the following steps: inoculating a cultured and activated Candida utilis suspension into a liquid fermentation medium at an inoculum rate of 4-6%, culturing the culture temperature at 28-30° C., stirring speed at 180-220 r / min, ventilation volume at 0.5 vvm, dissolved oxygen content at 20-40%, and culturing and fermenting for 18-20 hours 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.

7. The method according to claim 1, wherein The method for preparing the lactic acid bacteria fermentation broth comprises the following steps: inoculating a cultured activated lactic acid bacteria suspension into a liquid fermentation medium at an inoculum size of 1-3%, culturing the culture temperature at 35-40° C., stirring the culture speed at 80-120 r / min, and aerating the culture at 0.05 vvm, and culturing and fermenting for 35-40 hours to obtain the lactic acid bacteria fermentation broth; 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.

8. A single-cell protein feed prepared by the method according to any one of claims 1 to 7.

9. Use of the method according to any one of claims 1 to 7 in preparing single-cell protein feed.

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