A method for coproduction of cellulase and microbial protein based on an integrated microorganism-fiber microcarrier-surfactant suspension system

Abstract

The application discloses a method for co-production of cellulase and bacterial protein based on an integrated microorganism-fiber microcarrier-surfactant suspension system, and belongs to the technical field of cellulase and bacterial protein production. First, the microorganism is inoculated on a culture medium to obtain a seed culture solution, then fiber microcarriers are prepared through micro-bubble continuous flotation, and then the fiber microcarriers are hydrolyzed by acid to form a fiber microcarrier hydrolysis mixed solution, which is dispersed in the microorganism and surfactant to prepare an integrated microorganism-fiber microcarrier-surfactant suspension system, microcarrier suspension fermentation is carried out, and after centrifugation and drying, the supernatant is cellulase, and the solid is bacterial protein. The method can effectively improve the yield of cellulase and bacterial protein, realize partial replacement of exogenous cellulase in the process of lignocellulose saccharification, and thus effectively reduce the preparation cost of lignocellulose saccharification.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cellulase and bacterial protein production, and specifically relates to a method for co-producing cellulase and bacterial protein based on an integrated microorganism-fiber microcarrier-surfactant suspension system. BACKGROUND

[0002] China is the world's largest country in animal husbandry and the first country in demand for feed grains. China is also the world's largest grain importer, with 91.08 million tons of soybeans and 20.62 million tons of corn imported in 2022. The large amount of grain imports and low implicit self-sufficiency rate means that these imported grains are mainly used for feed grain consumption. According to the current feed conversion level in China, the corn energy feed gap will exceed 88 million tons in 2035, and the grain consumption will be 867 million tons, with a soybean protein feed gap of over 124 million tons. Developing diversified feed resources, especially protein alternative feed resources, fully tapping the potential of microbial resources, replacing traditional feed production methods, and researching and establishing corresponding standard and specification systems to form a relatively complete soybean and corn reduction substitution technology system and open up new ways for China's feed raw material supply are of great significance to ensuring food security in China.

[0003] Fiber feed includes forage, crop straw, woody feed and other cellulose-rich feed. It can be converted into sugar by cellulase and then into microbial protein by microorganisms, thereby replacing part of the protein feed. However, the crude fiber feed has poor palatability, which leads to the decrease of animal feed intake and metabolic energy, and affects the growth rate of animals, which is not conducive to the healthy growth of animals. At the same time, the cost of cellulase preparation is too high, which is also a stumbling block restricting the rapid development of the entire industry. Microbial fermentation can not only effectively degrade anti-nutritional factors and increase the content of nutrients and functional substances in feed, but also be beneficial to the health of animal intestinal tract and the enhancement of immunity. Some microorganisms also have the function of producing cellulase. Aspergillus niger is a kind of strain that can be used as enzyme preparation and strain preparation in the list of feed additives in China. It is an important industrial strain in fermentation industry and shows many advantages in the field of feed additives: 1. Improving the nutritional value and digestion and absorption rate of feed: Aspergillus niger contains almost a complete set of decomposition enzymes for botanical raw materials, such as amylase, glucoamylase, lipase, protease, cellulase, xylanase, glucanase, mannanase, etc. It can decompose macromolecular sugars in feed into monosaccharides and oligosaccharides, and generate various organic acids, vitamins, biological enzymes and growth factors, which greatly improves the nutritional level and digestion and absorption rate of fermented feed; 2. Detoxification of feed: through the life activities of microorganisms, Aspergillus niger can degrade and remove toxic and harmful substances contained in feed, thereby greatly improving the safety of feed; 3. Improving the disease resistance of animals: Aspergillus niger can directly participate in the barrier function of animal intestinal tract, promote the proliferation of beneficial microorganisms in animal intestinal tract, and prevent the colonization and growth and reproduction of pathogenic microorganisms through biological competition mechanism, thereby restoring and maintaining the microecological balance of animal intestinal tract, and improving the immunity and disease resistance of animals; 4. Improving the palatability of feed and the appetite of animals: Aspergillus niger can produce various enzymes during fermentation, which can efficiently convert indigestible crude fiber into small molecular compounds, thereby increasing the palatability. The organic acids, alcohols, esters and aldehydes produced during fermentation are beneficial to increasing the appetite of animals; 5. Wide applicability: Aspergillus niger can ferment all kinds of feed such as energy feed, protein feed and roughage. The fermented feed is suitable for livestock animals such as pigs, cattle and sheep, poultry animals such as chickens, ducks and geese, and various animals such as fish, shrimp, insects and so on; 6. Economic, environmental and stable: Aspergillus niger is safe and non-toxic, and can produce enzymes and proteins, and also can detoxify and degrade crude fiber, which has the function of one strain with multiple uses; Aspergillus niger has high spore content, which is convenient for product storage, has strong adaptability to environment and high fermentation regeneration capacity, so Aspergillus niger as a feed additive has good stability and economic practicality.

[0004] Microcarrier suspension fermentation is a fermentation method that combines solid-state fermentation and liquid fermentation. It uses microcarriers as a support for microbial growth, allowing microorganisms to attach and grow in a liquid environment. This fermentation method combines the advantages of both, with the advantages of improving cell density, improving product quality, easy to scale up production, high flexibility, easy to automate control, and easy to separate and purify products. It is a very promising fermentation technology. Natural wood fibers have good voids, and through homogenization and classification processing, small size fiber carriers with uniform particle size can be obtained. Culturing cellulase-producing strains can achieve slow-release degradation and utilization of fibers, which is beneficial to the continuous proliferation of microorganisms. Ultrafine grinding technology uses high-speed shearing or air flow grinding and other collaborative methods, with the characteristics of short time, high work efficiency; not easy to produce heat, high stability of ingredient quality control; fine particle size, uniform distribution, easy for the next stage of microcarrier fermentation; saves raw materials, high utilization rate, one-step grinding, reduces intermediate links; environmentally friendly, safe, low energy consumption. Microbubble separation fiber particle technology is a method that uses microbubbles generated by a microbubble generator to adsorb and float small size fiber particles in water. These microbubbles can effectively adsorb fiber particles in water, and then bring them to the water surface by buoyancy, achieving the classification and separation of different size fiber particles, and then separating the fiber microcarriers. The floated fiber microcarriers can effectively reduce the crude ash value, which is beneficial to the subsequent microbial fermentation for enzyme and protein production.

[0005] Surfactants play an important role in fermentation processes. Their main functions include increasing enzyme activity, improving cell permeability, and acting as fermentation accelerators. Surfactants can change the fluidity of cell membranes, affecting enzyme secretion and activity. In addition, surfactants can also improve oxygen solubility by changing the surface tension of the fermentation broth, thereby increasing enzyme activity. Surfactants can also improve cell permeability, which is crucial for improving fermentation efficiency. By increasing cell membrane permeability, surfactants can help nutrients enter cells more effectively, while also helping metabolic products be more easily expelled from cells. Surfactants are widely used as fermentation accelerators in the fermentation industry. They can promote fermentation processes through various mechanisms, such as reducing surface tension, improving mass transfer efficiency, enhancing cell membrane fluidity and stability, etc. Studies have shown that surfactants have important applications in glutamic acid fermentation, molasses fermentation glutamic acid, xanthan gum fermentation, corn straw silage feed fermentation, and alcohol fermentation, etc. Sodium tripolyphosphate and sodium hexametaphosphate are two commonly used food additives and important inorganic surfactants, with multiple regulatory functions, including as quality improvement and stabilizing agents, pH regulators, metal ion chelating agents, dispersants, etc.

[0006] The present application takes agroforestry lignocellulose as raw material, forms fiber slurry liquid by ultrafine grinding, forms uniform slurry liquid by micro-bubble continuous flotation, and finally realizes the purpose of improving the yield of cellulase and bacterial protein by dispersing lignocellulose micro-particles in a microbial-surfactant system for micro-carrier suspension fermentation, which has high application value in reducing the cost of cellulase preparation, increasing the efficiency of fiber conversion and utilization, and high-value utilization of lignocellulose. SUMMARY

[0007] The technical problem to be solved by the present application is to provide a method for co-production of cellulase and bacterial protein based on an integrated microbial-fiber micro-carrier-surfactant suspension system, which is controllable, easy to operate and environmentally friendly, realizes partial replacement of exogenous cellulase in the lignocellulose saccharification process, thereby effectively reducing the cost of lignocellulose preparation, and has good application prospect.

[0008] In order to solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0009] A method for co-production of cellulase and bacterial protein based on an integrated microbial-fiber micro-carrier-surfactant suspension system, wherein the seed culture solution is obtained by inoculating microorganisms into the culture medium; the fiber micro-carrier is prepared by micro-bubble continuous flotation, and the fiber micro-carrier hydrolysis mixed solution is formed by acid hydrolysis; the fiber micro-carrier hydrolysis solution and the surfactant are mixed and the pH is adjusted by an alkaline nitrogen-containing solution to obtain a fiber micro-carrier-surfactant mixed solution; then the seed culture solution and the fiber micro-carrier-surfactant mixed solution are mixed to obtain a microbial-fiber micro-carrier-surfactant suspension, and micro-carrier suspension fermentation is carried out, and after centrifugation and drying, the supernatant is cellulase and the solid is bacterial protein.

[0010] The microorganism is Aspergillus niger; the fiber micro-carrier includes bamboo shoot shell, corn straw, shrub, and lignocellulose micro-carriers prepared from poplar wood; the surfactant is selected from one or more of sodium tripolyphosphate and sodium hexametaphosphate; the mixing ratio of the microorganism, the fiber micro-carrier and the surfactant is 10 9 ~10 10 cfu: 50~500 g: 1 g.

[0011] The mass ratio of lignocellulose to water during flotation is 1:7~11; the gas flow during flotation is 1~8 m 3 / h; the flotation grading times are 1 grade and 2 grades.

[0012] The acid type of acid hydrolysis is sulfuric acid, hydrochloric acid or citric acid, the acid concentration is 0.5~2%, and the solid-liquid mass ratio of fiber micro-carrier to acid is 1:5~20.

[0013] The hydrolysis temperature is 100-130 DEG C, and the hydrolysis time is 30-120 min.

[0014] The formula of the seed culture solution is: 1-2% glucose, 1-2% peptone, 0.5-1% yeast extract, 1-2% agar, 0.2-0.3% potassium dihydrogen phosphate, 0.7-0.8% sodium dihydrogen phosphate, 0.01%-0.02% sodium chloride, 0.02-0.05% magnesium sulfate and 0.005-0.01% calcium chloride.

[0015] The culture condition of the seed culture solution is that the seed culture solution is cultured at 30-32 DEG C for 2-3 days.

[0016] The alkaline nitrogen-containing solution is ammonia solution, ammonium carbonate solution or ammonium bicarbonate solution; and the pH is adjusted to 5-6.5.

[0017] The fermentation temperature is 28-32 DEG C, the fermentation time is 6 days, and the fermentation rotation speed is 10-30 r / min; and the total aeration amount of the fermentation is 0.1-0.5 V / V·min.

[0018] The centrifugal rotation speed is 5000-12000 r / min, the centrifugal time is 5-15 min, and the drying temperature is 50-80 DEG C.

[0019] Compared with the prior art, the present application has the following advantages:

[0020] (1) The present application utilizes abundant and low-cost agricultural and forestry fiber raw materials, which is beneficial to the high-value utilization of agricultural and forestry biomass resources.

[0021] (2) The preparation process of the present application is controllable and easy to implement, the material is environmentally friendly, the process parameters have good stability, the wood fiber particles are prepared by using ultrafine grinding, the fiber microcarriers are prepared by using micro-bubble continuous flotation, the fiber microcarrier hydrolysis mixed solution is formed by acid hydrolysis, and the integrated microbial-fiber microcarrier-surfactant suspension system is prepared by dispersing the microbial-fiber microcarrier-surfactant suspension system in the microorganisms and surfactants, so that the microcarrier suspension fermentation can be carried out, the yield of cellulase and bacterial protein can be effectively improved, the partial replacement of exogenous cellulase in the wood fiber saccharification process can be realized, and thus the preparation cost of fiber saccharification can be effectively reduced.

[0022] (3) The main raw materials used in the present application meet the requirements of feed raw materials and additives, and the present application has good application prospect in the fields of agricultural and forestry biomass resource development and utilization, feed processing industry and the like. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The process flow chart for producing cellulase and bacterial protein by using the microbial-fiber microcarrier-surfactant suspension of the present application is shown in the figure.

[0024] Figure 2 Schematic diagram of microbubble continuous flotation-reaction device and microcarrier fermentation device for preparing fiber microcarriers in Example 1 of the present application;

[0025] Figure 3 Particle size distribution diagram of fiber microcarriers prepared under final optimization conditions in Example 1 of the present application;

[0026] Figure 4 Comparison diagram of effects of Example 10 (CICC 41481 Aspergillus niger) suspension microcarrier fermentation and hydrolysate conventional liquid fermentation on filter paper enzyme activity (FPA);

[0027] Figure 5 Comparison diagram of effects of Example 10 (CICC 41481 Aspergillus niger) suspension microcarrier fermentation and hydrolysate conventional liquid fermentation on carboxymethyl cellulase activity (CMCA);

[0028] Figure 6 Comparison diagram of effects of Example 10 (CICC 40273 Aspergillus niger) suspension microcarrier fermentation and hydrolysate conventional liquid fermentation on filter paper enzyme activity (FPA);

[0029] Figure 7 Comparison diagram of effects of Example 10 (CICC 40273 Aspergillus niger) suspension microcarrier fermentation and hydrolysate conventional liquid fermentation on carboxymethyl cellulase activity (CMCA);

[0030] Figure 8 Comparison diagram of effects of Example 10 (CICC 41481 Aspergillus niger) suspension microcarrier fermentation and hydrolysate conventional liquid fermentation on true protein yield of mycelium;

[0031] Figure 9 Comparison diagram of effects of Example 10 (CICC 40273 Aspergillus niger) suspension microcarrier fermentation and hydrolysate conventional liquid fermentation on true protein yield of mycelium. DETAILED DESCRIPTION

[0032] The present application will be further illustrated below in combination with specific examples. The examples are implemented on the premise of the technical solutions of the present application, and it should be understood that the examples are only used to illustrate the present application and not used to limit the scope of the present application.

[0033] In the following examples, the microorganism is Aspergillus niger, numbered CICC 41481 and CICC 40273, both purchased from China Industrial Microbial Culture Collection Center.

[0034] The true protein content in the following examples was determined according to the standard "T / NAIA 060-2021", and the cellulase content was determined according to the standard "QBT 2583-2003" "Cellulase preparation" of the People's Republic of China Light Industry Standard, the filter paper enzyme activity (FPA) appendix A method and the methyl cellulase activity (CMCA) appendix B method.

[0035] Example 1

[0036] Process research on preparation of fibrous microcarriers by continuous flotation of microbubbles

[0037] Fibrous microcarriers are separated by microbubble (microporous aeration) flotation. This method requires low equipment and can produce a large number of bubbles with low energy consumption. Microbubbles can effectively adsorb fibrous particles in water, and then bring them to the water surface by buoyancy, achieving the separation of fibrous particles of different sizes. This method can effectively float small size (particle size less than 100 μm) fibrous microcarriers, and is environmentally friendly, low in energy consumption, easy to operate and high in stability.

[0038] The following takes corn straw as an example of fibrous raw material. Corn straw is a natural porous material that can be suspended in liquid under certain size and liquid system, and can be used as a carrier for microbial fermentation and a slow-release carbon source for microorganisms in fermentation.

[0039] (1) Effect of solid-liquid ratio (1st flotation)

[0040] After shearing and vibration grinding, corn straw lignocellulose particles 100 g (calculated on a dry basis) were obtained by sieving through a 0.15 mm sieve. Different proportions of water were added, and 1st flotation was carried out (gas flow rate was 4 m 3 / h). The effect of solid-liquid ratio on separation is shown in Table 1.

[0041] Table 1 Effect of solid-liquid ratio on separation

[0042]

[0043] The results in Table 1 show that when the solid-liquid mass ratio of fiber to water is 1:10 and 1:11, the particle size range of the float is 31-104 μm and 39-110 μm, respectively, and the PDI is 0.391 and 0.386, respectively. The uniformity of particle size distribution is significantly better than that of other groups. At this time, the mass fraction of the float is 21% and 20%, respectively. Considering the reduction of water consumption as much as possible, the solid-liquid mass ratio of 1:10 is selected as the best 1st flotation process condition.

[0044] (2) Selection of gas flow rate (1st flotation)

[0045] Corn stalk lignocellulose particles 100 g (dry basis) prepared by ultrafine grinding were added with water at a mass ratio of 1:10, and floated at different gas flow rates. The effect of gas flow rate on separation efficiency is shown in Table 2.

[0046] Table 2 Effect of gas flow rate on separation efficiency

[0047]

[0048] The results in Table 2 show that when the gas flow rate is 4 m 3 / h, the mass fraction of float is 21%, the particle size range of float is 31-104 μm, and the PDI is 0.391. Considering the uniformity of particle size and working time, the gas flow rate of 4 m 3 / h is selected as the optimal first-stage flotation process condition.

[0049] (3) Combination effect of flotation process (first-stage-second-stage combined flotation)

[0050] Corn stalk lignocellulose particles 100 g (dry basis) prepared by ultrafine grinding were added with water at a mass ratio of 1:10, and then floated at different classification times and methods (the gas flow rate is 4 m 3 / h). The effect of flotation process combination on separation efficiency is shown in Table 3.

[0051] Table 3 Effect of flotation process combination on separation efficiency

[0052]

[0053] The results in Table 3 show that the mass fraction of float of the first-stage-second-stage combined method is 25%, the particle size range of float is 8-87 μm Figure 3 , and the PDI is 0.383. Considering the uniformity of particle size and working time, the first-stage-second-stage combined method is selected as the optimal flotation process condition.

[0054] Based on the above single-factor investigation conditions and parameter results, the final optimized conditions are as follows: the first-stage-second-stage combined method is selected for flotation, the solid-liquid mass ratio is 1:10, and the gas flow rate is 4 m 3 / h. After flotation, the crude ash content of the fiber microcarrier is reduced from 9.7% to 5.6% (referring to the determination of crude ash content in feed according to national standard GB / T 6438-2007).

[0055] Example 2

[0056] Preparation method of CICC 41481 Aspergillus niger-bamboo shoot shell fiber microcarrier-sodium tripolyphosphate suspension

[0057] CICC 41481 Aspergillus niger was inoculated on YPD plate medium and cultured at 32℃ for 2d, diluted with sterile water to a spore concentration of about 10 6 6 CICC 41481 Aspergillus niger culture solution also includes the following formula: 1.5% glucose, 1.5% peptone, 1% yeast extract, 1.5% agar, 0.2% potassium dihydrogen phosphate, 0.8% sodium dihydrogen phosphate, 0.01% sodium chloride, 0.02% magnesium sulfate, 0.005% calcium chloride. 10 g bamboo shoot shell fiber microcarriers were mixed with 100 mL 0.5% dilute sulfuric acid and heated at 110℃ for 60 min to obtain a bamboo shoot shell fiber microcarrier hydrolysis mixture with a mass fraction of about 9.5%, then 0.1 g sodium tripolyphosphate was added to the above-mentioned bamboo shoot shell fiber microcarrier hydrolysis mixture and mixed well, and ammonia water was added to adjust the pH value to 6.5 to obtain a bamboo shoot shell fiber microcarrier-sodium tripolyphosphate mixture with a mass fraction of about 10%; then 10 8 8 CICC 41481 Aspergillus niger culture solution containing a total of about 10 5 5 CICC 41481 Aspergillus niger-bamboo shoot shell fiber microcarrier-sodium tripolyphosphate suspension containing about 5x10

[0058] Example 3

[0059] Preparation method of CICC 40273 Aspergillus niger-corn straw fiber microcarrier-sodium hexametaphosphate suspension

[0060] CICC 40273 Aspergillus niger was inoculated on YPD plate medium and cultured at 32℃ for 2d, diluted with sterile water to a spore concentration of about 10 6 6 CICC 40273 Aspergillus niger culture solution also includes the following formula: 1% glucose, 1% peptone, 0.5% yeast extract, 1% agar, 0.3% potassium dihydrogen phosphate, 0.7% sodium dihydrogen phosphate, 0.02% sodium chloride, 0.04% magnesium sulfate, 0.01% calcium chloride. 100 g corn straw fiber microcarriers were mixed with 800 mL 1% hydrochloric acid and heated at 120℃ for 40 min to obtain a corn straw fiber microcarrier hydrolysis mixture with a mass fraction of about 11.9%, then 2 g sodium hexametaphosphate was added to the above-mentioned corn straw fiber microcarrier hydrolysis mixture and mixed well, and ammonium carbonate solution was added to adjust the pH value to 6 to obtain a corn straw fiber microcarrier-sodium hexametaphosphate mixture with a mass fraction of about 12.8%; then 10 9CICC 40273 Aspergillus niger culture solution containing about 5 x 10 5 CICC 40273 Aspergillus niger-corn straw fiber microcarrier-sodium hexametaphosphate suspension containing about 5 x 10

[0061] Example 4

[0062] Preparation method of CICC 41481 Aspergillus niger-sinowood fiber microcarrier-sodium tripolyphosphate suspension

[0063] CICC 41481 Aspergillus niger strain was inoculated on YPD plate culture medium and cultured at 30 °C for 3 d, diluted with sterile water to a spore concentration of about 10 6 CICC 41481 Aspergillus niger culture solution containing about 5 x 10 9 CICC 41481 Aspergillus niger culture solution containing about 5 x 10 5 CICC 41481 Aspergillus niger-sinowood fiber microcarrier-sodium tripolyphosphate suspension containing about 5 x 10

[0064] Example 5

[0065] Preparation method of CICC 40273 Aspergillus niger-poplar wood fiber microcarrier-sodium hexametaphosphate / sodium tripolyphosphate suspension

[0066] CICC 40273 Aspergillus niger strain was inoculated on YPD plate culture medium and cultured at 30 °C for 3 d, diluted with sterile water to a spore concentration of about 10 6CICC 40273 Aspergillus niger culture solution also includes the following formula: 1% glucose, 1% peptone, 0.5% yeast extract, 1% agar, 0.2% potassium dihydrogen phosphate, 0.7% sodium dihydrogen phosphate, 0.01% sodium chloride, 0.02% magnesium sulfate, 0.005% calcium chloride. 20 g poplar fiber microcarriers were mixed with 120 mL of 1% sulfuric acid and heated at 130 ℃ for 30 min to obtain a fiber microcarrier hydrolysis mixture with a mass fraction of about 14.9%, then 0.02 g of sodium hexametaphosphate and 0.02 g of sodium tripolyphosphate were added to the above fiber microcarrier hydrolysis mixture and mixed well, and then adjusted to pH 6 with ammonia water to obtain a fiber microcarrier-surfactant hydrolysis mixture with a mass fraction of about 14.7%; then 10 g of CICC 40273 Aspergillus niger spores were added to the above fiber microcarrier-surfactant mixture and mixed well to obtain a CICC 40273 Aspergillus niger-fiber microcarrier-surfactant suspension containing about 5×10 8 cfu / mL of spores. 5 5 cfu / mL of spores.

[0067] Example 6

[0068] Method for producing cellulase and mycelial protein based on CICC 41481 Aspergillus niger-bamboo shoot shell fiber microcarrier-sodium tripolyphosphate suspension fermentation

[0069] Take 100 mL of CICC 41481 Aspergillus niger-bamboo shoot shell fiber microcarrier-sodium tripolyphosphate suspension containing about 5×10 5 cfu / mL of spores obtained in Example 2 and place it in a micro-stirring and air circulation fermentation tank with an initial pH of 6.5, continuously ferment at 30 ℃ for 6 d, the fermentation stirring speed is 10 r / min, and the total aeration amount of sterile air is 0.1 V / V·min; after the fermentation is completed, the solid is separated out by centrifugation at a speed of 8000 r / min for 10 min, the obtained solid is dried at 60 ℃, then crushed into powder to obtain 1.31 g of mycelial protein powder, the true protein content is 34.57%, and the equivalent mycelial true protein mass is 0.45 g; the obtained supernatant is the enzyme solution, and the enzyme activity (FPA) is 63.38 U / mL and the carboxymethyl cellulase activity (CMCA) is 76.12 U / mL.

[0070] Example 7

[0071] Method for producing cellulase and mycelial protein based on CICC 40273 Aspergillus niger-corn straw fiber microcarrier-sodium hexametaphosphate suspension fermentation

[0072] Take 100 mL of CICC 40273 Aspergillus niger-corn straw fiber microcarrier-sodium hexametaphosphate suspension containing about 5×10 5A CICC 40273 Aspergillus niger suspension with cfu / mL spores, corn straw fiber microcarriers, and sodium hexametaphosphate was placed in a fermenter equipped with micro-stirring and air circulation. The initial pH was 5, and fermentation was carried out at 32 °C for 6 days with a stirring speed of 20 r / min and a total sterile air ventilation of 0.2 V / V·min. After fermentation, the solids were separated by centrifugation at 12000 r / min for 5 min. The obtained solids were dried at 70 °C and pulverized into powder to obtain 1.37 g of mycelial protein powder with a true protein content of 35.96%, which is equivalent to 0.49 g of true mycelial protein. The obtained supernatant was the enzyme solution, and the filter paper enzyme activity (FPA) was measured to be 62.06 U / mL and the carboxymethyl cellulase activity (CMCA) was measured to be 65.05 U / mL.

[0073] Example 8

[0074] A method for producing cellulase and microbial protein by fermentation of CICC 41481 Aspergillus niger-Caragana korshinskii fiber microcarrier-sodium tripolyphosphate suspension.

[0075] Take 100 mL of the solution obtained in Example 4 containing approximately 5 × 10 5 A CICC 41481 Aspergillus niger suspension with CFU / mL spores, containing *Caragana korshinskii* fiber microcarriers and sodium tripolyphosphate, was placed in a fermenter equipped with micro-stirring and air circulation. The initial pH was 6, and fermentation was carried out at 28°C for 6 days with a stirring speed of 30 r / min and a total sterile air ventilation of 0.5 V / V·min. After fermentation, the solids were separated by centrifugation at 5000 r / min for 15 min. The obtained solids were dried at 80°C and pulverized to obtain 0.93 g of bacterial protein powder with a true protein content of 34.06%, which translates to 0.32 g of true bacterial protein. The resulting supernatant was the enzyme solution, and the filter paper enzyme activity (FPA) was measured to be 41.55 U / mL and the carboxymethyl cellulase activity (CMCA) was 53.10 U / mL.

[0076] Example 9

[0077] A method for producing cellulase and microbial protein by fermentation of CICC 40273 Aspergillus niger-poplar fiber microcarrier-sodium hexametaphosphate / sodium tripolyphosphate suspension.

[0078] Take 100 mL of the solution obtained in Example 5 containing approximately 5 × 10 5CICC 40273 Aspergillus niger - poplar fiber microcarrier - sodium hexametaphosphate / sodium tripolyphosphate suspension, placed in a micro-stirring and air circulation fermenter, initial pH value of 6, continuous fermentation at 30 °C for 6 d, fermentation stirring speed of 20 r / min, total aeration of sterile air of 0.3 V / V·min; after the fermentation is completed, the solid is separated out by centrifugation at a speed of 10000 r / min for 10 min, the obtained solid is dried at 50 °C, then pulverized into powder, 1.27 g of mycelial protein powder is obtained, the true protein content is 36.11%, the mass of the true mycelial protein is 0.46 g; the obtained supernatant is the enzyme liquid, the filter paper activity (FPA) is 63.23 U / mL and the carboxymethyl cellulase activity (CMCA) is 66.24 U / mL.

[0079] Example 10

[0080] Comparison of the effects of microbial - fiber microcarrier - surfactant suspension fermentation and conventional liquid fermentation on cellulase production and mycelial protein

[0081] According to the similar conditions and methods of Examples 1-9, microbial - fiber microcarrier - surfactant suspensions are prepared, respectively, CICC 41481 / CICC 40273 Aspergillus niger - bamboo shoot shell / corn straw / shrub / poplar - fiber microcarrier - sodium tripolyphosphate suspension (referred to as: bamboo shoot shell / corn straw / shrub / poplar suspension, the same below), and the corresponding bamboo shoot shell / corn straw / shrub / poplar hydrolysate is prepared for conventional liquid fermentation (referred to as hydrolysate, the same below). The Aspergillus niger culture solution formula used in the above suspension and hydrolysate is uniformly 2% glucose, 2% peptone, 1% yeast extract, 2% agar, 0.3% potassium dihydrogen phosphate, 0.8% sodium dihydrogen phosphate, 0.02% sodium chloride, 0.05% magnesium sulfate, and 0.01% calcium chloride. The fiber microcarrier preparation conditions in the above suspension are: after the bamboo shoot shell / corn straw / shrub / poplar is ultra-micro pulverized, the fiber microcarrier with a particle size distribution range of 20-100 μm and a PDI range of 0.3-0.4 is obtained by 1st-2nd combined flotation (solid-liquid mass ratio 1:10, gas flow rate of 4 m 3 / h). The acid hydrolysis conditions used in the above suspension and hydrolysate are uniformly 10 g of each fiber sample, solid-liquid mass ratio of 1:12, 1% dilute sulfuric acid at 120 °C for 60 min. The above suspension contains 0.5% sodium tripolyphosphate, and the pH value is 6.5. The above hydrolysate is adjusted to a pH value of 6.5 with ammonia water. The above suspension and hydrolysate are uniformly contained in the starting fermentation liquid at about 5×10 5cfu / mL Aspergillus spores. The fermentation was continuously carried out for 6 days at 28℃, the stirring speed was 30 r / min, and the total sterile air flux was 0.5 V / V·min; wherein, the fermentation broth was taken for measuring the corresponding enzyme activity on the 2nd, 3rd, 4th, 5th and 6th day of fermentation, respectively. After the fermentation was completed, the solid was separated by centrifugation at a speed of 10000 r / min for 10 min, the obtained solid was dried at 80℃, then was crushed into powder, the corresponding mycelial protein powder was obtained, and the true protein mass of the mycelium was measured; the obtained supernatant was the enzyme activity, the filter paper enzyme activity (FPA) and carboxymethyl cellulase activity (CMCA) were measured.

[0082] The results show that the cellulase production and mycelial protein production efficiency of the microcarrier fermentation mode of the suspension group are higher than those of the conventional liquid fermentation mode of the hydrolysate control group. Among them, the CICC 41481 Aspergillus is the fermentation strain, the filter paper enzyme activity (FPA) of the bamboo shoot shell / corn straw / saskatoon / yang wood suspension group is increased by 17.58%, 25.33%, 17.09% and 18.52% (the maximum value is taken in different fermentation days, the same below) respectively compared with the hydrolysate control group, and the carboxymethyl cellulase (CMCA) is increased by 20.15%, 22.07%, 16.66% and 20.93% (the maximum value is taken in different fermentation days, the same below) respectively. Figure 4 Figure 5 Figure 6 Figure 7 The enzyme production period of the suspension group is shortened by 1d compared with the hydrolysate control group. The CICC 41481 Aspergillus is the fermentation strain, the true protein mass of the bamboo shoot shell / corn straw / saskatoon / yang wood suspension group is increased by 14.94%, 18.09%, 10.31% and 17.33% (the maximum value is taken in different fermentation days, the same below) respectively compared with the hydrolysate control group; the CICC 40273 Aspergillus is the fermentation strain, the true protein mass of the bamboo shoot shell / corn straw / saskatoon / yang wood suspension group is increased by 15.31%, 20.19%, 13.43% and 17.96% (the maximum value is taken in different fermentation days, the same below) respectively compared with the hydrolysate control group. Figure 8 Figure 9

[0083] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​​​​​

Claims

1. A method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system, characterized in that, Microorganisms are cultured and inoculated onto a culture medium to obtain a seed culture solution; fiber microcarriers are prepared by continuous microbubble flotation, and then hydrolyzed with acid to form a fiber microcarrier hydrolysate mixture; the fiber microcarrier hydrolysate is mixed with a surfactant and the pH is adjusted with an alkaline nitrogen-containing solution to obtain a fiber microcarrier-surfactant mixture. The seed culture medium and the fiber microcarrier-surfactant mixture are then mixed to obtain a microbial-fiber microcarrier-surfactant suspension. This suspension is then subjected to microcarrier suspension fermentation. After centrifugation and drying, the supernatant contains cellulase, and the solid contains bacterial protein. The surfactant is selected from one or more of sodium tripolyphosphate and sodium hexametaphosphate. The mass ratio of lignocellulose to water during flotation is 1:7~11. The gas flow rate during flotation is 1~8 m³ / s. 3 / h; the number of flotation stages is 1 stage and 2 stages.

2. The method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system according to claim 1, characterized in that: The microorganism is Aspergillus niger; the fibrous microcarrier includes fibrous microcarriers prepared from lignocellulose derived from bamboo shoot shells, corn stalks, Caragana korshinskii, and poplar wood; the mixing ratio of the microorganism, fibrous microcarrier, and surfactant is 10:

1. 9 ~10 10 CFU: 50~500 g: 1 g.

3. The method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system according to claim 1, characterized in that: The acid used in the acid hydrolysis is sulfuric acid, hydrochloric acid, or citric acid, with an acid concentration of 0.5-2%, and the solid-liquid mass ratio of the fiber microcarrier to the acid is 1:5-20.

4. The method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system according to claim 1, characterized in that: The hydrolysis temperature is 100~130 ℃, and the hydrolysis time is 30~120 min.

5. The method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system according to claim 1, characterized in that: The seed culture medium is formulated as follows: 1-2% glucose, 1-2% peptone, 0.5-1% yeast extract, 1-2% agar, 0.2-0.3% potassium dihydrogen phosphate, 0.7-0.8% sodium dihydrogen phosphate, 0.01%-0.02% sodium chloride, 0.02-0.05% magnesium sulfate, and 0.005-0.01% calcium chloride.

6. The method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system according to claim 1, characterized in that: The seed culture medium is cultured at 30-32 ℃ for 2-3 days.

7. The method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system according to claim 1, characterized in that: The alkaline nitrogen-containing solution is an ammonia solution, an ammonium carbonate solution, or an ammonium bicarbonate solution; the pH is adjusted to 5-6.

5.

8. The method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system according to claim 1, characterized in that: The fermentation temperature is 28~32 ℃, the fermentation time is 6 days, and the fermentation speed is 10~30 r / min; the total fermentation aeration rate is 0.1~0.5 V / V·min.

9. The method for co-producing cellulase and bacterial protein based on an integrated microbial-fiber microcarrier-surfactant suspension system according to claim 1, characterized in that: The centrifugation speed is 5000~12000 r / min, the centrifugation time is 5~15 min, and the drying temperature is 50~80 ℃.

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