A microbial protein raw material and preparation method thereof

Abstract

The present invention relates to the field of microbial application technology, and in particular to a microbial protein raw material and a preparation method thereof, comprising the steps of raw material pretreatment, enzymolysis, heating to inactivate enzymes, microbial fermentation, centrifugal separation, freeze drying, etc. In the present invention, modified nanoliposomes, modified biotin and sodium alginate play a role through a synergistic effect, the modified nanoliposomes encapsulate nutrients such as glucose, vitamins, minerals and modified biotin to form vesicles, the modified biotin further enhances the encapsulation ability of the vesicles, the cell penetrating peptide groups on the surface of the vesicles specifically bind to the receptors on the surface of the cells to induce endocytosis so that the vesicles are swallowed into the cells, thereby improving the bioavailability of the nutrients, the modified biotin enhances the ability of microorganisms to synthesize proteins by activating the intracellular signal transduction mechanism, the buffering effect of sodium alginate stabilizes the pH value of the system, and together with the modified biotin enhances the antioxidant capacity of microbial cells.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, in particular to a microbial protein raw material and a preparation method thereof. Background Art

[0002] With the global population growth and increasing environmental pressure, traditional protein sources can no longer meet the human demand for sustainable protein. As an emerging protein source, microbial protein raw materials have the characteristics of high production efficiency and environmental friendliness, and therefore have received widespread attention. Microbial protein raw materials refer to protein-rich biomass raw materials produced by fermentation processes using microorganisms such as yeast, bacteria, and fungi. These microorganisms can grow rapidly under specific conditions, and then convert simple nutrients such as carbon sources and nitrogen sources into high-quality proteins and are widely used in many fields. For example, they can be used in the food industry as meat substitutes, nutritional supplements, etc., and can also be used as feed for livestock, poultry, aquaculture and other animals. Because they can produce certain specific proteins or amino acids, they are used in the pharmaceutical industry, and as functional ingredients in the cosmetics industry. At the same time, they can also be used to produce biodegradable materials and other bioplastic products. Compared with traditional protein preparation processes, the preparation of microbial protein raw materials has significant advantages, such as high production efficiency, environmental friendliness, high nutritional value, controllable production process, and a wide range of raw material sources. Commonly used raw materials include agricultural by-products such as corn stalks, rice husks, and wheat bran, industrial waste such as bagasse and sawdust, starch raw materials such as corn, cassava, and potatoes, sugar raw materials such as sucrose and glucose, and petrochemical by-products such as methanol and ethanol. With the continuous advancement of technology, the quality requirements for microbial protein raw materials are also significantly improved. How to reasonably and effectively improve the absorption of nutrients such as carbon sources and nitrogen sources by microorganisms requires further improvement of the preparation process. Therefore, by adding chemical additives such as modified nanoliposomes and modified biotin in the process of preparing microbial protein raw materials, the absorption of nutrients such as carbon sources and nitrogen sources by microorganisms is significantly improved, and the ability of microorganisms to synthesize high-quality proteins is improved, and finally a high-quality protein raw material is obtained. The high-quality protein raw material is then applied to food, feed and other fields to achieve large-scale production, thereby promoting the healthy and orderly development of the industry. Summary of the invention

[0003] (1) Technical issues to be resolved

[0004] The object of the present invention is to provide a microbial protein raw material and a preparation method thereof. By further improving the preparation process, that is, adding chemical additives such as modified nanoliposomes and modified biotin during the preparation of the microbial protein raw material, the absorption of nutrients such as carbon sources and nitrogen sources by microorganisms is significantly improved, and the ability of microorganisms to synthesize high-quality protein is improved, thereby finally obtaining a high-quality protein raw material.

[0005] (2) Technical solution

[0006] To achieve the above object, the present invention provides a method for preparing a microbial protein raw material, characterized in that it comprises the following steps:

[0007] S11. Raw material pretreatment: clean corn, cassava and rice husk, remove impurities, and crush them into particles of 60-80 mesh. Add the crushed corn, cassava and rice husk into a container in the following proportions, with the mass ratio of corn, cassava and rice husk being (3-5): (2-4): (1-3). Add purified water and soak for 18-24 hours to obtain an enzymatic substrate.

[0008] S12. Enzyme hydrolysis: adding the complex enzyme solution and sodium alginate to the enzymatic substrate under stirring, wherein the mass ratio of the enzymatic substrate, the complex enzyme solution and the sodium alginate is (5-12):(0.5-1):(1-2), the reaction temperature is controlled at 50-60°C, and the enzymatic product is obtained after reacting for 8-12 hours;

[0009] S13. Inactivate the enzyme by heating: Under stirring, treat the enzymatic hydrolysate at 80-100°C for 15-25 min, cool to room temperature, add modified nanoliposomes and modified biotin, and continue stirring for 2-4 h to obtain a fermentation substrate. The modified nanoliposomes have a nanometer size of 80-100 nm and a specific surface area of ​​60-80 m 2 / g, and the surface of the modified nanoliposomes contains cell-penetrating peptide groups;

[0010] S14. Microbial fermentation: inoculating the mixed microbial liquid into the fermentation substrate under stirring for fermentation at a temperature of 35 to 50° C. and obtaining a fermentation product after 60 to 72 hours of fermentation;

[0011] S15. Centrifugation: The fermentation product was centrifuged at a speed of 6000 to 8000 rpm for 15 to 20 min to separate the supernatant and the precipitate, and the precipitate was washed three times with purified water and then freeze-dried;

[0012] S16. Freeze drying: freeze-dry the precipitate at a temperature of -10 to 0°C for 20 to 25 hours to obtain the microbial protein raw material.

[0013] Furthermore, the preparation method of the modified nanoliposome comprises:

[0014] S21. Dissolve phospholipid, cholesterol and polyethylene glycol-lipid in a mixed solution of methanol and chloroform under stirring, wherein the molar ratio of phospholipid, cholesterol and polyethylene glycol-lipid is (60-75):(15-20):(5-10), stir for 1-2 hours, perform rotary evaporation at a temperature of 40-60°C, evaporate for 2-4 hours, vacuum dry at a temperature of 40-50°C, and dry for 2-4 hours to obtain an intermediate substance;

[0015] S22. The intermediate substance is added to a PBS buffer for hydration and placed in an oscillator. After oscillating at a temperature of 50 to 60° C. for 0.5 to 1 h, the liquid nitrogen freeze-water bath thaw cycle is performed 5 times to obtain a nanoliposome solution.

[0016] S23. Slowly add N-succinimidyl-3-(2-pyridyldithio) propionate to the nanoliposome solution under stirring, continue stirring for 4 to 6 hours to obtain an activated nanoliposome solution, dissolve the cell penetrating peptide in PBS buffer and stir for 1 to 2 hours until fully dissolved, add Traut's reagent and continue stirring for 1 to 2 hours to obtain a cell penetrating peptide solution;

[0017] S24. slowly dropwise adding the cell penetrating peptide solution into the activated nanoliposome solution under stirring, reacting at room temperature for 4-6 hours, and purifying the reaction product using an ultrafiltration centrifuge tube with a molecular weight of 10 kDa to obtain a modified nanoliposome solution;

[0018] S25. The modified nanoliposome solution is freeze-dried at a drying temperature of -40 to -60°C for a drying time of 36 to 48 hours to obtain the modified nanoliposome, which is then ground into powder for later use.

[0019] Furthermore, the preparation method of the modified biotin comprises:

[0020] S31. Dissolve biotin in PBS buffer under stirring, add NF-κB transcription factor after stirring at room temperature for 1-2 hours, the mass ratio of biotin to NF-κB transcription factor is (15-20): (1-2), continue stirring for 4-6 hours to obtain a modified biotin solution;

[0021] S32. The modified biotin solution is freeze-dried at a drying temperature of -40 to -60°C for 36 to 48 hours to obtain the modified biotin, which is then ground into powder for later use.

[0022] Furthermore, the modified biotin surface is coupled to the NF-κB transcription factor, and the specific surface area is 150-180 m 2 / g.

[0023] Furthermore, the mass ratio of the modified nanoliposome to the modified biotin is (1-2): (0.5-1).

[0024] Furthermore, the complex enzyme solution is a compound of α-amylase, glucoamylase and cellulase, and the weight ratio of α-amylase, glucoamylase and cellulase is (1-2): (0.5-1): (0.5-1).

[0025] Furthermore, the mixed microbial solution is a compound of Candida utilis solution, Serratia marcescens solution and Bacillus thermophilus solution, and the mass ratio of Candida utilis solution, Serratia marcescens solution and Bacillus thermophilus solution is (0.5~1): (0.5~0.8): (0.2~0.5).

[0026] Furthermore, the microbial protein raw material is prepared by the method for preparing a microbial protein raw material.

[0027] The mechanism of action of the above raw material components is as follows:

[0028] Microbial protein raw materials are prepared from corn, cassava and rice husks. These raw materials are mainly used as carbon and energy sources to provide necessary nutrition for the growth and protein synthesis of microorganisms. Corn and cassava are rich in starch and are high-quality carbon sources. The glucose produced after starch decomposition can provide energy for microorganisms. At the same time, corn contains a certain amount of protein as a nitrogen source supplement, and also contains a variety of vitamins and minerals, which are conducive to the growth of microorganisms. Rice husks are rich in cellulose and can be used as a carrier for microbial growth, providing an attachment surface and improving aeration. In the enzymatic hydrolysis process, α-amylase, glucoamylase and cellulase play a very important role. α-amylase and glucoamylase decompose the starch in corn and cassava into sugars with smaller molecular weights such as maltose and glucose. Glucoamylase further decomposes the maltose produced by α-amylase hydrolysis into glucose, providing nutrients for microbial growth. Cellulase decomposes the cellulose in rice husks into cellobiose and glucose, while increasing the aeration of the enzymatic hydrolysis environment, reducing viscosity, and promoting the growth and reproduction of microorganisms.

[0029] Fermentation with Candida utilis, Serratia marcescens and Bacillus thermophilus can significantly improve the yield and quality of protein. Candida utilis is a unicellular fungus that is widely used in microbial protein production. Candida utilis can reproduce rapidly under suitable conditions, can efficiently utilize carbon sources such as glucose and xylose, and convert them into protein. The protein content in the cell is high, reaching 50-60% of the dry weight, and it has strong tolerance and strong adaptability to pH and temperature changes. Serratia marcescens is a Gram-negative bacterium that can decompose complex organic matter such as cellulose and hemicellulose, has nitrogen fixation ability, can increase the nitrogen content in the environment, and thus improve the utilization of nitrogen sources. Bacillus thermophilus is a thermostable bacterium that can continue to ferment under high temperature conditions. It can grow rapidly under suitable conditions and accumulate biomass. The protein content in the cell can reach 60-70% of the dry weight, and it can form heat-resistant spores, which is easy to store and transport.

[0030] In the process of preparing microbial protein raw materials, modified nanoliposomes, modified biotin and sodium alginate play a role through synergistic effect. Nanoliposomes are a vesicle-like nanoscale carrier material formed by wrapping a phospholipid bilayer. Its core is a hydrophilic cavity that can encapsulate hydrophilic substances. Its structure is similar to that of a cell membrane and has excellent biological activity. Cell-penetrating peptide groups are introduced on the surface of nanoliposomes through chemical modification to obtain modified nanoliposomes. During the process of heating and inactivating enzymes, the modified nanoliposomes will wrap up nutrients such as glucose, vitamins, minerals and modified biotin to form modified nanoliposome vesicles. The modified biotin further enhances the encapsulation ability of the modified nanoliposome vesicles. During the microbial fermentation process, the cells on the surface of the modified nanoliposome vesicles The penetrating peptide group specifically binds to specific receptors on the surface of microbial cells to trigger endocytosis, so that the modified nanoliposome vesicles are engulfed into the cells. In the intracellular environment, the modified nanoliposome vesicles are gradually dissociated, and nutrients are gradually released for absorption and utilization by microbial cells, thereby improving their bioavailability. Biotin is a water-soluble vitamin. The modified biotin is obtained by coupling NF-κB transcription factor on the surface of biotin through chemical modification. The modified biotin significantly enhances the ability of microbial cells to synthesize protein by activating the intracellular signal transduction mechanism. At the same time, the modified nanoliposome vesicles provide a protective layer for the modified biotin, reducing the contact of the modified biotin with intracellular hydrolases, prolonging its half-life, and thus prolonging the time for the modified biotin to promote protein synthesis. However, many intermediate products will be produced during the enzymatic hydrolysis, temperature inactivation of enzymes and microbial fermentation, which will lead to unstable pH value of the system. Fluctuation of pH value will lead to adverse consequences such as reduced enzyme activity, inhibited microbial growth, reduced product stability and increased pollution risk. The buffering effect of sodium alginate can maintain the stability of pH value of the system. Sodium alginate is a linear polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) connected by 1,4-glycosidic bonds. At the same time, sodium alginate and modified biotin jointly enhance the antioxidant capacity of microbial cells. Modified biotin, as a cofactor of antioxidant enzymes, enhances the activity of antioxidant enzymes. Sodium alginate reduces oxidative damage of microbial cells by capturing free radicals and maintains the function of microbial cells.

[0031] (3) Beneficial effects

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The modified nanoliposomes, modified biotin and sodium alginate work through a synergistic effect. During the process of heating and inactivating enzymes, the modified nanoliposomes encapsulate nutrients such as glucose, vitamins, minerals and modified biotin to form vesicles, and the modified biotin further enhances the encapsulation ability of the vesicles.

[0034] 2. During the microbial fermentation process, the cell-penetrating peptide groups on the surface of the vesicles specifically bind to specific receptors on the surface of microbial cells, triggering endocytosis, so that the modified nanoliposome vesicles are engulfed into the cells, thereby improving bioavailability;

[0035] 3. Modified biotin enhances the ability of microbial cells to synthesize proteins by activating the intracellular signal transduction mechanism. At the same time, the vesicles provide a protective layer for the modified biotin, reducing the contact between the modified biotin and the intracellular hydrolases and prolonging the time for the modified biotin to promote protein synthesis.

[0036] 4. The buffering effect of sodium alginate stabilizes the pH value of the system, and together with modified biotin, it enhances the antioxidant capacity of microbial cells. Modified biotin acts as a cofactor of antioxidant enzymes to enhance the activity of antioxidant enzymes, while sodium alginate reduces oxidative damage to microbial cells by capturing free radicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the SEM image of the modified nanoliposomes of Example 1 of the present invention. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The experimental equipment and preparations of the embodiments described below are as follows: electronic balance (Sartorius, Germany), magnetic stirrer (Meiyingpu, Shanghai), high-speed centrifuge (Guangzhou Jidi), freeze dryer (Shanghai Pudong Freeze Drying), vacuum drying oven (Shanghai Jiecheng), high-speed pulverizer (Shandong Tianfang Machinery), pH meter (Shanghai Yidian), rotary evaporator (Henan Boda), oscillator (Shanghai Analyzer), scanning electron microscope (Zeiss, Germany), specific surface area analyzer (Beijing Best), automatic nitrogen analyzer (Shanghai Heguan), amino acid automatic analyzer (Qingdao Haitai), UV-visible spectrophotometer (Agilent Technologies); α-amylase, glucoamylase and cellulase (Shanghai Yuanye), Candida utilis (Shandong Xinxiong), Serratia marcescens (Shanghai Bohu), Thermophilic Bacillus (Shanghai Yushao); chemicals and reagents were purchased from Sigma-Aldrich.

[0040] Example 1: This example discloses a method for preparing a microbial protein raw material, which is characterized by comprising the following steps:

[0041] S11. Raw material pretreatment: clean corn, cassava and rice husk, remove impurities, crush into 60-80 mesh particles, add crushed corn, cassava and rice husk into a container in proportion, the mass ratio of corn, cassava and rice husk is 4:3:2, and soak in purified water for 18-24 hours to obtain an enzymatic substrate;

[0042] S12. Enzymolysis: adding the complex enzyme solution and sodium alginate to the enzymolysis substrate under stirring, the mass ratio of the enzymolysis substrate, the complex enzyme solution and the sodium alginate is 8:0.8:1, the reaction temperature is controlled at 50-60°C, and the enzymolysis product is obtained after reacting for 8-12 hours;

[0043] S13. Inactivate the enzyme by heating: Under stirring, treat the enzymatic hydrolysate at 80-100°C for 15-25 min, cool to room temperature, add modified nanoliposomes and modified biotin, and continue stirring for 2-4 h to obtain a fermentation substrate. The modified nanoliposomes have a nanometer size of 80-100 nm and a specific surface area of ​​60-80 m 2 / g, and the surface of the modified nanoliposomes contains cell-penetrating peptide groups;

[0044] S14. Microbial fermentation: inoculating the mixed microbial liquid into the fermentation substrate under stirring for fermentation at a temperature of 35 to 50° C. and obtaining a fermentation product after 60 to 72 hours of fermentation;

[0045] S15. Centrifugation: The fermentation product was centrifuged at a speed of 6000 to 8000 rpm for 15 to 20 min to separate the supernatant and the precipitate, and the precipitate was washed three times with purified water and then freeze-dried;

[0046] S16. Freeze drying: freeze-dry the precipitate at a temperature of -10 to 0°C for 20 to 25 hours to obtain the microbial protein raw material.

[0047] The preparation method of the modified nanoliposome comprises:

[0048] S21. Dissolve phospholipid, cholesterol and polyethylene glycol-lipid in a mixed solution of methanol and chloroform under stirring, wherein the molar ratio of phospholipid, cholesterol and polyethylene glycol-lipid is 70:18:8, stir for 1-2 hours, perform rotary evaporation at a temperature of 40-60°C, evaporate for 2-4 hours, perform vacuum drying at a temperature of 40-50°C, and dry for 2-4 hours to obtain an intermediate substance;

[0049] S22. The intermediate substance is added to a PBS buffer for hydration and placed in an oscillator. After oscillating at a temperature of 50 to 60° C. for 0.5 to 1 h, the liquid nitrogen freeze-water bath thaw cycle is performed 5 times to obtain a nanoliposome solution.

[0050] S23. Slowly add N-succinimidyl-3-(2-pyridyldithio) propionate to the nanoliposome solution under stirring, continue stirring for 4 to 6 hours to obtain an activated nanoliposome solution, dissolve the cell penetrating peptide in PBS buffer and stir for 1 to 2 hours until fully dissolved, add Traut's reagent and continue stirring for 1 to 2 hours to obtain a cell penetrating peptide solution;

[0051] S24. slowly dropwise adding the cell penetrating peptide solution into the activated nanoliposome solution under stirring, reacting at room temperature for 4-6 hours, and purifying the reaction product using an ultrafiltration centrifuge tube with a molecular weight of 10 kDa to obtain a modified nanoliposome solution;

[0052] S25. The modified nanoliposome solution is freeze-dried at a drying temperature of -40 to -60°C for a drying time of 36 to 48 hours to obtain the modified nanoliposome, which is then ground into powder for later use.

[0053] The preparation method of the modified biotin comprises:

[0054] S31. Dissolve biotin in PBS buffer under stirring, add NF-κB transcription factor after stirring at room temperature for 1-2 hours, the mass ratio of biotin to NF-κB transcription factor is 18:1, continue stirring for 4-6 hours to obtain a modified biotin solution;

[0055] S32. The modified biotin solution is freeze-dried at a drying temperature of -40 to -60°C for 36 to 48 hours to obtain the modified biotin, which is then ground into powder for later use.

[0056] The modified biotin surface is coupled to the NF-κB transcription factor, and the specific surface area is 150-180 m 2 / g.

[0057] The mass ratio of the modified nanoliposome to the modified biotin is 1.5:0.8.

[0058] The complex enzyme solution is a compound of alpha-amylase, glucoamylase and cellulase, wherein the weight ratio of alpha-amylase, glucoamylase and cellulase is 1.5:0.8:0.8.

[0059] The mixed microbial liquid is a compound of Candida utilis liquid, Serratia marcescens liquid and thermophilic Bacillus liquid, and the mass ratio of the Candida utilis liquid, Serratia marcescens liquid and thermophilic Bacillus liquid is 1:0.5:0.5.

[0060] In the preparation process of microbial protein raw materials, modified nanoliposomes, modified biotin and sodium alginate play a role through synergistic effects. Nanoliposomes are a vesicle-like nanoscale carrier material formed by a phospholipid bilayer. Its core is a hydrophilic cavity that can encapsulate hydrophilic substances. It also has a hydrophobic tail that can encapsulate fat-soluble substances. Its structure is similar to that of a cell membrane and has excellent biological activity. Cell-penetrating peptide groups are introduced into the surface of nanoliposomes through chemical modification to obtain modified nanoliposomes, such as Figure 1As shown in the SEM image of the modified nanoliposomes, the modified nanoliposomes have uniform particle size and spherical morphology, and have good biocompatibility. During the process of heating and inactivating enzymes, the modified nanoliposomes will encapsulate nutrients such as glucose, vitamins, minerals and modified biotin through physical adsorption and chemical action to form modified nanoliposome vesicles. The modified biotin further enhances the encapsulation ability of the modified nanoliposome vesicles. The biotin molecule contains hydrophilic carboxyl (-COOH) and urea (-NH-CO-NH-), as well as hydrophobic tetrahydroimidazole ring. The unique chemical structure enables the biotin molecule to The modified nanoliposomes interact with the phospholipid bilayer, and the carboxyl group (-COOH) and urea group (-NH-CO-NH-) of the biotin molecule can further react with the hydroxyl group (-OH) in glucose and vitamins to form an ester bond (-COO-), allowing more nutrients to enter the core structure of the modified nanoliposomes, thereby promoting the efficient encapsulation of nutrients by the modified nanoliposomes. During the microbial fermentation process, the cell-penetrating peptide groups on the surface of the modified nanoliposome vesicles specifically bind to specific receptors on the surface of microbial cells, triggering calcium protein, actin and mesh protein-mediated The cell membrane invaginates to form coated pits, allowing the modified nanoliposome vesicles to be engulfed into the cell. In the intracellular environment, the modified nanoliposome vesicles are gradually dissociated, and nutrients such as glucose, vitamins, minerals and modified biotin are gradually released for absorption and utilization by microbial cells, thereby improving their bioavailability. Biotin is a water-soluble vitamin. The modified biotin is obtained by chemically modifying the surface of biotin to couple NF-κB transcription factors. In the intracellular environment, the modified biotin binds to Toll-like receptors and cytokine receptors to trigger a signal cascade reaction to activate IκB kinase. The enzyme complex then activates the NF-κB transcription factor, which significantly improves the ability of microbial cells to synthesize protein by increasing mRNA levels, improving translation efficiency, enhancing energy metabolism and enhancing amino acid metabolism. Biotin molecules, as cofactors of carboxylases, participate in fatty acid synthesis, amino acid metabolism and gluconeogenesis, providing more precursor substances and energy for protein synthesis. At the same time, the modified nanoliposome vesicles provide a protective layer for the modified biotin, reducing the contact of the modified biotin with intracellular hydrolases, prolonging its half-life, and thus prolonging the time for the modified biotin to promote protein synthesis. However, many intermediates will be produced during the enzymatic hydrolysis, temperature inactivation and microbial fermentation, which will lead to unstable pH value of the system. Fluctuation of pH value will lead to adverse consequences such as reduced enzyme activity, inhibited microbial growth, reduced product stability and increased pollution risk. The buffering effect of sodium alginate can maintain the stability of pH value of the system. Sodium alginate is a linear polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G) connected by 1,4-glycosidic bonds. The uronic acid unit contains a carboxyl group (-COOH) that can accept or release protons (H+ ), forming a buffer system. When the pH value decreases, the carboxylate (-COO - ) can accept H + The formation of carboxyl groups (-COOH) prevents the pH from falling further. When the pH rises, -COOH can release H + , preventing the pH value from rising further, thereby maintaining the stability of the system's pH value. At the same time, sodium alginate and modified biotin jointly enhance the antioxidant capacity of microbial cells. Modified biotin, as a cofactor of antioxidant enzymes, enhances the activity of antioxidant enzymes and improves the active defense ability of cells. Sodium alginate reduces oxidative damage to microbial cells by capturing the formed free radicals, forming a complementary mechanism with modified biotin. At the same time, sodium alginate maintains the optimal activity of antioxidant enzymes by stabilizing the pH value of the system, thereby maintaining microbial cell functions and extending the life of microorganisms.

[0061] Example 2: This example discloses a method for preparing a microbial protein raw material, which is characterized by comprising the following steps:

[0062] S11. Raw material pretreatment: clean corn, cassava and rice husk, remove impurities, crush into 60-80 mesh particles, add crushed corn, cassava and rice husk into a container in proportion, the mass ratio of corn, cassava and rice husk is 3:2:1, and soak in purified water for 18-24 hours to obtain an enzymatic substrate;

[0063] S12. Enzymolysis: adding the complex enzyme solution and sodium alginate to the enzymolysis substrate under stirring, the mass ratio of the enzymolysis substrate, the complex enzyme solution and the sodium alginate is 5:0.5:1, the reaction temperature is controlled at 50-60°C, and the enzymolysis product is obtained after reacting for 8-12 hours;

[0064] S13. Inactivate the enzyme by heating: Under stirring, treat the enzymatic hydrolysate at 80-100°C for 15-25 min, cool to room temperature, add modified nanoliposomes and modified biotin, and continue stirring for 2-4 h to obtain a fermentation substrate. The modified nanoliposomes have a nanometer size of 80-100 nm and a specific surface area of ​​60-80 m 2 / g, and the surface of the modified nanoliposomes contains cell-penetrating peptide groups;

[0065] S14. Microbial fermentation: inoculating the mixed microbial liquid into the fermentation substrate under stirring for fermentation at a temperature of 35 to 50° C. and obtaining a fermentation product after 60 to 72 hours of fermentation;

[0066] S15. Centrifugation: The fermentation product was centrifuged at a speed of 6000 to 8000 rpm for 15 to 20 min to separate the supernatant and the precipitate, and the precipitate was washed three times with purified water and then freeze-dried;

[0067] S16. Freeze drying: freeze-dry the precipitate at a temperature of -10 to 0°C for 20 to 25 hours to obtain the microbial protein raw material.

[0068] The preparation method of the modified nanoliposome comprises:

[0069] S21. Dissolve phospholipid, cholesterol and polyethylene glycol-lipid in a mixed solution of methanol and chloroform under stirring, wherein the molar ratio of phospholipid, cholesterol and polyethylene glycol-lipid is 60:15:5, stir for 1-2 hours, perform rotary evaporation at a temperature of 40-60°C, evaporate for 2-4 hours, perform vacuum drying at a temperature of 40-50°C, and dry for 2-4 hours to obtain an intermediate substance;

[0070] S22. The intermediate substance is added to a PBS buffer for hydration and placed in an oscillator. After oscillating at a temperature of 50 to 60° C. for 0.5 to 1 h, the liquid nitrogen freeze-water bath thaw cycle is performed 5 times to obtain a nanoliposome solution.

[0071] S23. Slowly add N-succinimidyl-3-(2-pyridyldithio) propionate to the nanoliposome solution under stirring, continue stirring for 4 to 6 hours to obtain an activated nanoliposome solution, dissolve the cell penetrating peptide in PBS buffer and stir for 1 to 2 hours until fully dissolved, add Traut's reagent and continue stirring for 1 to 2 hours to obtain a cell penetrating peptide solution;

[0072] S24. slowly dropwise adding the cell penetrating peptide solution into the activated nanoliposome solution under stirring, reacting at room temperature for 4-6 hours, and purifying the reaction product using an ultrafiltration centrifuge tube with a molecular weight of 10 kDa to obtain a modified nanoliposome solution;

[0073] S25. The modified nanoliposome solution is freeze-dried at a drying temperature of -40 to -60°C for a drying time of 36 to 48 hours to obtain the modified nanoliposome, which is then ground into powder for later use.

[0074] The preparation method of the modified biotin comprises:

[0075] S31. Dissolve biotin in PBS buffer under stirring, add NF-κB transcription factor after stirring at room temperature for 1-2 hours, the mass ratio of biotin to NF-κB transcription factor is 15:1, continue stirring for 4-6 hours to obtain a modified biotin solution;

[0076] S32. The modified biotin solution is freeze-dried at a drying temperature of -40 to -60°C for 36 to 48 hours to obtain the modified biotin, which is then ground into powder for later use.

[0077] The modified biotin surface is coupled to the NF-κB transcription factor, and the specific surface area is 150-180 m 2 / g.

[0078] The mass ratio of the modified nanoliposome to the modified biotin is 1:0.5.

[0079] The complex enzyme solution is a compound of alpha-amylase, glucoamylase and cellulase, wherein the weight ratio of alpha-amylase, glucoamylase and cellulase is 1:0.5:0.5.

[0080] The mixed microbial liquid is a compound of Candida utilis liquid, Serratia marcescens liquid and thermophilic Bacillus liquid, and the mass ratio of the Candida utilis liquid, Serratia marcescens liquid and thermophilic Bacillus liquid is 0.5:0.5:0.2.

[0081] Example 3: This example discloses a method for preparing a microbial protein raw material, which is characterized by comprising the following steps:

[0082] S11. Raw material pretreatment: clean corn, cassava and rice husk, remove impurities, crush into 60-80 mesh particles, add crushed corn, cassava and rice husk into a container in proportion, the mass ratio of corn, cassava and rice husk is 5:4:3, and soak in purified water for 18-24 hours to obtain an enzymatic substrate;

[0083] S12. Enzymolysis: adding the complex enzyme solution and sodium alginate to the enzymolysis substrate under stirring, the mass ratio of the enzymolysis substrate, the complex enzyme solution and the sodium alginate is 12:1:2, the reaction temperature is controlled at 50-60°C, and the enzymolysis product is obtained after reacting for 8-12 hours;

[0084] S13. Inactivate the enzyme by heating: Under stirring, treat the enzymatic hydrolysate at 80-100°C for 15-25 min, cool to room temperature, add modified nanoliposomes and modified biotin, and continue stirring for 2-4 h to obtain a fermentation substrate. The modified nanoliposomes have a nanometer size of 80-100 nm and a specific surface area of ​​60-80 m 2 / g, and the surface of the modified nanoliposomes contains cell-penetrating peptide groups;

[0085] S14. Microbial fermentation: inoculating the mixed microbial liquid into the fermentation substrate under stirring for fermentation at a temperature of 35 to 50° C. and obtaining a fermentation product after 60 to 72 hours of fermentation;

[0086] S15. Centrifugation: The fermentation product was centrifuged at a speed of 6000 to 8000 rpm for 15 to 20 min to separate the supernatant and the precipitate, and the precipitate was washed three times with purified water and then freeze-dried;

[0087] S16. Freeze drying: freeze-dry the precipitate at a temperature of -10 to 0°C for 20 to 25 hours to obtain the microbial protein raw material.

[0088] The preparation method of the modified nanoliposome comprises:

[0089] S21. Dissolve phospholipid, cholesterol and polyethylene glycol-lipid in a mixed solution of methanol and chloroform under stirring, wherein the molar ratio of phospholipid, cholesterol and polyethylene glycol-lipid is 75:20:10, stir for 1-2 hours, perform rotary evaporation at a temperature of 40-60°C, evaporate for 2-4 hours, perform vacuum drying at a temperature of 40-50°C, and dry for 2-4 hours to obtain an intermediate substance;

[0090] S22. The intermediate substance is added to a PBS buffer for hydration and placed in an oscillator. After oscillating at a temperature of 50 to 60° C. for 0.5 to 1 h, the liquid nitrogen freeze-water bath thaw cycle is performed 5 times to obtain a nanoliposome solution.

[0091] S23. Slowly add N-succinimidyl-3-(2-pyridyldithio) propionate to the nanoliposome solution under stirring, continue stirring for 4 to 6 hours to obtain an activated nanoliposome solution, dissolve the cell penetrating peptide in PBS buffer and stir for 1 to 2 hours until fully dissolved, add Traut's reagent and continue stirring for 1 to 2 hours to obtain a cell penetrating peptide solution;

[0092] S24. slowly dropwise adding the cell penetrating peptide solution into the activated nanoliposome solution under stirring, reacting at room temperature for 4-6 hours, and purifying the reaction product using an ultrafiltration centrifuge tube with a molecular weight of 10 kDa to obtain a modified nanoliposome solution;

[0093] S25. The modified nanoliposome solution is freeze-dried at a drying temperature of -40 to -60°C for a drying time of 36 to 48 hours to obtain the modified nanoliposome, which is then ground into powder for later use.

[0094] The preparation method of the modified biotin comprises:

[0095] S31. Dissolve biotin in PBS buffer under stirring, add NF-κB transcription factor after stirring at room temperature for 1-2 hours, the mass ratio of biotin to NF-κB transcription factor is 20:2, continue stirring for 4-6 hours to obtain a modified biotin solution;

[0096] S32. The modified biotin solution is freeze-dried at a drying temperature of -40 to -60°C for 36 to 48 hours to obtain the modified biotin, which is then ground into powder for later use.

[0097] The modified biotin surface is coupled to the NF-κB transcription factor, and the specific surface area is 150-180 m 2 / g.

[0098] The mass ratio of the modified nanoliposome to the modified biotin is 2:1.

[0099] The composite enzyme solution is a compound of alpha-amylase, glucoamylase and cellulase, wherein the weight ratio of alpha-amylase, glucoamylase and cellulase is 2:1:1.

[0100] The mixed microbial liquid is a compound of Candida utilis liquid, Serratia marcescens liquid and thermophilic Bacillus liquid, and the mass ratio of the Candida utilis liquid, Serratia marcescens liquid and thermophilic Bacillus liquid is 1:0.8:0.5.

[0101] Control group 1: This example differs from Example 1 in that the modified nanoliposome is not included. The preparation method of the modified biotin in this example is the same as that in Example 1. The preparation method of a microbial protein raw material in this example is the same as that in Example 1.

[0102] Control group 2: This example differs from Example 1 in that modified biotin is not contained. The preparation method of the modified nanoliposomes in this example is consistent with that in Example 1. The preparation method of a microbial protein raw material in this example is consistent with that in Example 1.

[0103] Control group 3: This example differs from Example 1 in that sodium alginate is not contained. The preparation methods of the modified nanoliposomes and modified biotin in this example are the same as those in Example 1. The preparation method of a microbial protein raw material in this example is the same as that in Example 1.

[0104] Effect evaluation: Detection of nutritional components of microbial protein raw materials: Three microbial protein raw materials prepared by each experimental group were randomly selected to determine the contents of crude protein, total amino acids, bacterial protein and acid-soluble protein. The method for determining crude protein was in accordance with GB / T 6432-2018 Determination of crude protein in feed, the method for determining total amino acids was in accordance with GB 5009.124-2016 Determination of amino acids in food, the method for determining bacterial protein was in accordance with GB 5009.5-2016 Determination of protein in food, and the method for determining acid-soluble protein was in accordance with GB / T 22492-2008 Determination of acid-soluble protein in soybean peptide powder. The average value was taken and the determination results were recorded.

[0105]

[0106] Table 1 shows the statistical results of the determination of the nutritional components of the microbial protein raw materials in each experimental group. The crude protein, total amino acids, bacterial protein and acid-soluble protein contents are important indicators for evaluating the nutritional value and application potential of the microbial protein raw materials. As can be seen from Table 1, there are certain differences in the nutritional content of the microbial protein raw materials in each experimental group. By comparing the nutritional components of the microbial protein raw materials prepared in Examples 1 to 3 with those prepared in Control Groups 1 to 3, it can be found that, overall, the nutritional value of the microbial protein raw materials prepared in Examples 1 to 3 is better, and the crude protein, total amino acids, bacterial protein and acid-soluble protein contents of the microbial protein raw materials prepared in Example 1 are significantly the highest, which are 34.24%, 28.53%, 3.70% and 20.86% respectively. A higher crude protein content means better nutritional supply for animal growth and production. Total amino acids include essential amino acids and non-essential amino acids. A higher bacterial protein content indicates higher nutritional value and higher feed conversion rate, and a higher acid-soluble protein content indicates better digestion and absorption performance. By comparing the microbial protein raw material prepared in Example 1 with control groups 1 to 3, it can be found that the content of each indicator is very different, which shows that when adding modified nanoliposomes, modified biotin and sodium alginate at the same time during the preparation of the microbial protein raw material, the nutritional value of the microbial protein raw material can be significantly improved.

[0107] Through the above limited experiments, a microbial protein raw material of Example 1 of the present invention has significant effects. By optimizing the preparation method of the microbial protein raw material, chemical additives such as modified nanoliposomes and modified biotin are added during the preparation of the microbial protein raw material, the absorption of nutrients such as carbon sources and nitrogen sources by microorganisms is significantly improved, and the ability of microorganisms to synthesize high-quality protein is improved. Finally, a high-quality protein raw material is obtained. The obtained microbial protein raw material has high nutritional value and great application potential. The high-quality protein raw material can be applied to food, feed and other fields to achieve large-scale production, thereby promoting the healthy and orderly development of the industry.

[0108] Finally, it should be noted that: Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a microbial protein raw material, characterized in that: The following steps are involved: S11. Raw material pretreatment: clean corn, cassava and rice husk, remove impurities, and crush them into particles of 60-80 mesh. Add the crushed corn, cassava and rice husk into a container in the following proportions, with the mass ratio of corn, cassava and rice husk being (3-5): (2-4): (1-3). Add purified water and soak for 18-24 hours to obtain an enzymatic substrate. S12. Enzyme hydrolysis: adding the complex enzyme solution and sodium alginate to the enzymatic substrate under stirring, wherein the mass ratio of the enzymatic substrate, the complex enzyme solution and the sodium alginate is (5-12):(0.5-1):(1-2), the reaction temperature is controlled at 50-60°C, and the enzymatic product is obtained after reacting for 8-12 hours; S13. Inactivate the enzyme by heating: Under stirring, treat the enzymatic hydrolysate at 80-100°C for 15-25 min, cool to room temperature, add modified nanoliposomes and modified biotin, and continue stirring for 2-4 h to obtain a fermentation substrate. The modified nanoliposomes have a nanometer size of 80-100 nm and a specific surface area of ​​60-80 m 2 / g, and the surface of the modified nanoliposome contains a cell penetrating peptide group, the modified biotin surface is coupled to the NF-κB transcription factor, and the specific surface area is 150-180m 2 / g, the mass ratio of the modified nanoliposome to the modified biotin is (1-2): (0.5-1); S14. Microbial fermentation: inoculating the mixed microbial liquid into the fermentation substrate under stirring for fermentation at a temperature of 35 to 50° C. and obtaining a fermentation product after 60 to 72 hours of fermentation; S15. Centrifugation: The fermentation product was centrifuged at a speed of 6000 to 8000 rpm for 15 to 20 min to separate the supernatant and the precipitate, and the precipitate was washed three times with purified water and then freeze-dried; S16. Freeze drying: freeze drying the precipitate at a temperature of -10 to 0°C for 20 to 25 hours to obtain a microbial protein raw material; The preparation method of the modified nanoliposome comprises: S21. Dissolve phospholipid, cholesterol and polyethylene glycol-lipid in a mixed solution of methanol and chloroform under stirring, wherein the molar ratio of phospholipid, cholesterol and polyethylene glycol-lipid is (60-75):(15-20):(5-10), stir for 1-2 hours, perform rotary evaporation at a temperature of 40-60°C, evaporate for 2-4 hours, vacuum dry at a temperature of 40-50°C, and dry for 2-4 hours to obtain an intermediate substance; S22. The intermediate substance is added to a PBS buffer for hydration and placed in an oscillator. After oscillating at a temperature of 50 to 60° C. for 0.5 to 1 h, the liquid nitrogen freeze-water bath thaw cycle is performed 5 times to obtain a nanoliposome solution. S23. Slowly add N-succinimidyl-3-(2-pyridyldithio) propionate to the nanoliposome solution under stirring, continue stirring for 4 to 6 hours to obtain an activated nanoliposome solution, dissolve the cell penetrating peptide in PBS buffer and stir for 1 to 2 hours until fully dissolved, add Traut's reagent and continue stirring for 1 to 2 hours to obtain a cell penetrating peptide solution; S24. slowly dropwise adding the cell penetrating peptide solution into the activated nanoliposome solution under stirring, reacting at room temperature for 4-6 hours, and purifying the reaction product using an ultrafiltration centrifuge tube with a molecular weight of 10 kDa to obtain a modified nanoliposome solution; S25. The modified nanoliposome solution is freeze-dried at a drying temperature of -40 to -60 ° C for a drying time of 36 to 48 h to obtain the modified nanoliposomes and ground into a powder for later use; The preparation method of the modified biotin comprises: S31. Dissolve biotin in PBS buffer under stirring, add NF-κB transcription factor after stirring at room temperature for 1-2 hours, the mass ratio of biotin to NF-κB transcription factor is (15-20): (1-2), continue stirring for 4-6 hours to obtain a modified biotin solution; S32. The modified biotin solution is freeze-dried at a drying temperature of -40 to -60 ° C for a drying time of 36 to 48 h to obtain modified biotin and ground into a powder for standby use; The complex enzyme solution is a compound of α-amylase, glucoamylase and cellulase, wherein the weight ratio of α-amylase, glucoamylase and cellulase is (1-2): (0.5-1): (0.5-1); The microbial mixed bacterial liquid is a compound of Candida utilis liquid, Serratia marcescens liquid and Bacillus thermophilus liquid, and the mass ratio of Candida utilis liquid, Serratia marcescens liquid and Bacillus thermophilus liquid is (0.5-1): (0.5-0.8): (0.2-0.5); The microbial protein raw material is prepared by the method for preparing a microbial protein raw material.

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