Method for preparing microbial protein from crop straw and application of microbial protein

Abstract

The invention belongs to the technical field of microbial protein preparation, and particularly relates to a method for preparing microbial protein from crop straw and application of the microbial protein. According to the method, the crop straw is subjected to biomass conversion pretreatment, so that the accessibility and hydrolysis efficiency of cellulose are improved; on the basis, the pretreated biomass is subjected to enzymolysis through cellulase, cellulose and hemicellulose components in the biomass are converted into glucose and xylose, and the glucose and the xylose are utilized by saccharomycetes in the subsequent saccharomycetes fermentation process to synthesize the microbial protein. The microbial protein obtained through the method has the advantages of being rich in nutrition and high in essential amino acid content, and has wide application prospects in the fields of food and feed. Therefore, after the crop straw is subjected to chemical or physical-biological treatment, a new way is provided for high-value utilization of the crop straw on the basis of obtaining the nutrient-rich microbial protein.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial protein preparation, and specifically relates to a method for preparing microbial protein by utilizing crop straw and application thereof. Background Art

[0002] Crop straw, such as wheat, corn, rice, cotton straw, etc., is a major by-product of agricultural production. The annual production of straw of major crops such as corn, rice, wheat, rapeseed, soybean, cotton, etc. is as high as nearly 1 billion tons. However, most crop straw is directly returned to the fields or burned, resulting in resource waste and environmental pollution, and seriously hindering the development of sustainable agriculture.

[0003] Crop straw is an ideal lignocellulosic raw material, mainly composed of cellulose, hemicellulose and lignin. Converting straw cellulose and hemicellulose components into high-value products such as microbial protein is a feasible way to promote the utilization of crop straw resources as feed. Although there are some studies that use fungal solid fermentation to convert crop straw into protein, which can solve the problem of straw being directly returned to the field or burned to a certain extent, this conversion mode usually requires a long cultivation time, and the operation process is difficult to control, the conversion rate is low, and the content of certain specific amino acids is low or unbalanced. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing microbial protein using crop straw and its application, wherein the method can efficiently convert crop straw into microbial protein, the conversion rate of lignocellulose-derived sugar-synthesized protein is high, and the protein is rich in essential amino acids.

[0005] The present invention provides a method for preparing microbial protein using crop straw, comprising the following steps:

[0006] 1) pre-treating crop straw to obtain pre-treated slurry;

[0007] The pretreatment method includes dilute alkali pretreatment or dilute acid pretreatment; the pretreatment temperature is 100-140°C, the pressure is 0.1-2.5Mpa, and the time is 10-50min;

[0008] 2) adjusting the pH value of the pretreated slurry to 7.0 and then drying to obtain pretreated biomass;

[0009] 3) mixing the pretreated biomass with cellulase and performing enzymatic hydrolysis in a solution with a pH value of 4.6 to 5.0 to obtain a crop straw hydrolyzate;

[0010] 4) inoculating yeast into a protein fermentation medium for fermentation to obtain a fermentation liquid, wherein the fermentation liquid contains the microbial protein; the protein fermentation medium includes the crop straw hydrolyzate, and the volume of the crop straw hydrolyzate is 10% to 20% of the volume of the protein fermentation medium.

[0011] Preferably, the water content of the pretreated biomass is 10 wt.% to 20 wt.%.

[0012] Preferably, the cellulase comprises, by weight percentage, 10% to 30% of exo-β-1,4-glucanase, 10% to 30% of endo-β-1,4-glucanase, 10% to 30% of β-glucosidase and 50% to 70% of cellobiohydrolase.

[0013] Preferably, in the step 3), based on the cellulose in the pretreated biomass, the amount of the cellulase used is 0.02% to 0.1% of the mass of the cellulose.

[0014] Preferably, the temperature of the enzymatic hydrolysis is 45-55°C, the stirring speed is 250r / min, and the time is 96h.

[0015] Preferably, the yeast comprises Candida utilis, Pichia pastoris, Rhodosporidium toruloides or Yarrowia lipolytica.

[0016] Preferably, the protein fermentation medium comprises components in the following concentrations: 10% to 20% v / v crop straw hydrolyzate, 13.56 g / LNa2HPO4·12H2O, 6 g / LKH2PO4, 1 g / LNaCl, 2 g / LNH4Cl, 0.492 g / LMgSO4·7H2O, 0.111 g / L CaCl2, 10 mL / L Hoagland's nutrient solution and a nitrogen source; the carbon-nitrogen ratio of the protein fermentation medium is 1:4 to 1:18.

[0017] Preferably, the fermentation temperature is 28-30° C., the rotation speed is 200-250 r / min, and the fermentation time is 3-6 days.

[0018] Preferably, the OD of the yeast after inoculation into the protein fermentation medium is 600 The value is 1 to 2.

[0019] The present invention also provides the use of the microbial protein prepared by the method described in the above technical scheme in feed and / or feed additives.

[0020] Beneficial effects:

[0021] The present invention provides a method for preparing microbial protein from crop straw, wherein the method pre-treats the crop straw (including dilute alkali pre-treatment or dilute acid pre-treatment) and treats the straw by physical or chemical means to improve the accessibility and hydrolysis efficiency of cellulose; on this basis, the pre-treated biomass is enzymatically hydrolyzed by cellulase to convert the cellulose and hemicellulose components in the biomass into glucose and xylose, which are used by yeast to synthesize microbial protein in the subsequent yeast fermentation process. The microbial protein obtained in this way has the advantages of short synthesis time and high essential amino acid content, and has a wide range of application prospects in the field of food and feed. It can be seen that the present invention provides a new way for the high-value utilization of crop straw on the basis of obtaining nutrient-rich microbial protein after chemical or physical-biological treatment of crop straw. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 This is a process flow chart for preparing microbial protein using crop straw in Example 1;

[0024] Figure 2 is the concentration of microbial protein synthesized by the hydrolyzate of different parts of cotton stalks transformed by Candida utilis ACCC20060 in Examples 1-2;

[0025] Figure 3 This is the concentration of microbial protein produced by the seven yeasts transforming the straw hydrolysate in Example 3. DETAILED DESCRIPTION

[0026] The present invention provides a method for preparing microbial protein using crop straw, comprising the following steps:

[0027] 1) pre-treating crop straw to obtain pre-treated slurry;

[0028] The pretreatment method includes dilute alkali pretreatment or dilute acid pretreatment; the pretreatment temperature is 100-140°C, the pressure is 0.1-2.5Mpa, and the time is 10-50min;

[0029] 2) adjusting the pH value of the pretreated slurry to 7.0 and then drying to obtain pretreated biomass;

[0030] 3) mixing the pretreated biomass with cellulase and performing enzymatic hydrolysis in a solution with a pH value of 4.6 to 5.0 to obtain a crop straw hydrolyzate;

[0031] 4) inoculating yeast into a protein fermentation medium for fermentation to obtain a fermentation liquid, wherein the fermentation liquid contains the microbial protein; the protein fermentation medium includes the crop straw hydrolyzate, and the volume of the crop straw hydrolyzate is 10% to 20% of the volume of the protein fermentation medium.

[0032] As an embodiment, the present invention washes and crushes crop straw to obtain washed crop straw particles. As an embodiment, the particle size of the crop straw particles is 40 to 100 meshes. The present invention does not specifically limit the crushing method, and the conventional crushing method in the art can be used. The present invention does not specifically limit the washing method, as long as the dust removal effect of the crop straw is achieved. The present invention can remove impurities on the surface of the crop straw through the washing and crushing, further improving the efficiency of subsequent treatment.

[0033] As an embodiment, the crop straw of the present invention may be one or more of cotton straw, wheat straw, corn straw and wheat straw; as another embodiment, the crop straw may be cotton straw; as another embodiment, the crop straw may be different parts of the crop straw; as in a specific embodiment, the crop straw is cotton straw, and is one or more of cotton roots, dehulled cotton stalks, fruit branches, cotton stalk skin, cotton husks and cotton leaves.

[0034] After obtaining the washed crop straw particles, the present invention pre-treats the washed crop straw particles to obtain a pre-treated slurry. The pre-treatment method of the present invention can be a dilute alkali pre-treatment or a dilute acid pre-treatment; the pre-treatment temperature is 100-140°C, the pressure is 0.1-2.5Mpa, and the time is 10-50min. As an embodiment, the step of the dilute alkali pre-treatment is: the washed crop straw particles are immersed in a dilute alkali solution and treated for 10-50min at 100-140°C and 0.1-2.5Mpa; as another embodiment, the dilute alkali solution is a NaOH solution with a mass concentration of 2%. As an embodiment, the step of the dilute acid pre-treatment is: the washed crop straw particles are immersed in a dilute acid solution and treated for 10-50min at 100-140°C and 0.1-2.5Mpa; as another embodiment, the dilute acid solution is a sulfuric acid solution with a mass concentration of 1%. As an embodiment, the pretreatment temperature of the present invention is 110-120°C, the pressure is 0.1-0.4Mpa, and the time is 40min; in a specific embodiment, the pretreatment can be carried out in an autoclave or a high pressure sterilizer to ensure the pressure during the pretreatment.

[0035] After obtaining the pretreated slurry, the present invention adjusts the pH value of the pretreated slurry to 7.0 and then dries it to obtain pretreated biomass. As an embodiment, the present invention adjusts the pH value with concentrated hydrochloric acid. The reason why the present invention adjusts the pH value of the pretreated slurry to 7.0 is that microorganisms grow and metabolize best at neutral pH. As an embodiment, the present invention also includes a step of filtering the pretreated slurry after adjusting the pH value with a filtering device before drying to remove liquid from the pretreated slurry. As an embodiment, the drying method is drying; as another embodiment, the drying temperature is 60°C, and the time is based on the water content in the pretreated biomass reaching 10wt.% to 20wt.%.

[0036] After obtaining the pretreated biomass, the present invention mixes the pretreated biomass with cellulase and then performs enzymatic hydrolysis in a solution with a pH value of 4.6 to 5.0 to obtain a crop straw hydrolysis slurry. As an embodiment, the cellulase of the present invention includes 10% to 30% of exo-β-1,4-glucanase, 10% to 30% of endo-β-1,4-glucanase, 10% to 30% of β-glucosidase and 50% to 70% of cellobiohydrolase in terms of mass percentage; as another embodiment, the mass percentage of the exo-β-1,4-glucanase in the cellulase of the present invention is 15% to 20%; the mass percentage of the endo-β-1,4-glucanase is 15% to 20%; the mass percentage of the β-glucosidase is 15% to 20%; the mass percentage of the cellobiohydrolase is 55% to 65%. As an embodiment, based on the cellulose in the pretreated biomass, the amount of the cellulase in the present invention is 0.02% to 0.1% of the mass of the cellulose; as another embodiment, the amount of the cellulase is 0.05% to 0.08% of the mass of the cellulose. As an embodiment, the solution with a pH value of 4.6 to 5.0 can be a citric acid buffer; as another embodiment, the mass ratio of the dry matter in the pretreated biomass to the citric acid buffer is 1:5 to 1:10. As an embodiment, the temperature of the enzymatic hydrolysis of the present invention is 45 to 55°C; as another embodiment, the temperature of the enzymatic hydrolysis is 50°C. As an embodiment, the pH value of the enzymatic hydrolysis of the present invention is 4.8. As an embodiment, the stirring speed of the enzymatic hydrolysis is 150r / min; as another embodiment, the stirring speed of the enzymatic hydrolysis is 250r / min. As an embodiment, the time of the enzymatic hydrolysis can be 72h; as another embodiment, the time of the enzymatic hydrolysis is 96h. The enzymatic hydrolysis of the present invention can hydrolyze the cellulose and hemicellulose components in the crop straw to generate glucose and xylose, thereby providing raw materials for the subsequent synthesis of microbial protein.

[0037] After obtaining the crop straw hydrolysis slurry, as an embodiment, the present invention centrifuges the crop straw hydrolysis slurry to obtain a crop straw hydrolyzate. As an embodiment, the centrifuge speed of the present invention is 10000r / min and the time is 10min.

[0038] After obtaining the crop straw hydrolyzate, the present invention inoculates yeast into a protein fermentation medium for fermentation to obtain a fermentation liquid, wherein the fermentation liquid contains the microbial protein; the protein fermentation medium includes the crop straw hydrolyzate, and the volume of the crop straw hydrolyzate is 10% to 20% of the volume of the protein fermentation medium. As an embodiment, the yeast of the present invention includes Candida utilis, Pichia pastoris, Rhodosporidium toruloides or Yarrowia lipolytica; in a specific embodiment, the Candida utilis is Candida utilis ACCC20060; the Pichia pastoris is Pichia pastoris X-33, Pichia pastoris GS115 and Pichia pastoris KM17; the Rhodosporidium toruloides is Rhodosporidium toruloides NP11. The advantage of using yeast instead of other strains for the fermentation of the present invention is that the growth rate is fast and the sugar released from the straw can be quickly utilized. As an embodiment, the protein fermentation medium includes the following components: 10% to 20% v / v crop straw hydrolyzate, 13.56g / LNa2HPO4·12H2O, 6g / LKH2PO4, 1g / LNaCl, 2g / LNH4Cl, 0.492g / LMgSO4·7H2O, 0.111g / L CaCl2, 10mL / L Hoagland's nutrient solution and nitrogen source; the carbon-nitrogen ratio of the protein fermentation medium is 1:4 to 1:18; as an embodiment, the carbon-nitrogen ratio of the protein fermentation medium can be 1:18. The present invention adds the crop straw hydrolyzate of the volume percentage content to the protein fermentation medium to have the effect of rapidly converting the straw hydrolyzate. As an embodiment, the nitrogen source of the present invention is ammonium sulfate, and the amount of the ammonium sulfate used is based on the carbon-nitrogen ratio of 1:4 to 1:18 achieved in the protein fermentation culture after being added to the protein fermentation medium. As an embodiment, the fermentation temperature of the present invention is 28-30°C; as another embodiment, the fermentation temperature is 30°C. As an embodiment, the fermentation speed of the present invention is 200-250r / min; as another embodiment, the fermentation speed is 250r / min. As an embodiment, the fermentation time of the present invention is 3-6 days; as another embodiment, the fermentation time is 3 days. As an embodiment, the OD of the yeast after inoculation into the protein fermentation medium is 600 As another embodiment, the OD value of the yeast after inoculation into the protein fermentation medium is 1 to 5. 600The value is 1 to 2.

[0039] After obtaining the fermentation liquid, as an embodiment, the present invention centrifuges the fermentation liquid to obtain bacterial cells. The present invention has no special limitation on the centrifugation method, and the conventional centrifugation method in the art can be used.

[0040] After obtaining the bacterial cells, as an embodiment, the present invention washes and freeze-dries the bacterial cells to obtain freeze-dried bacterial cells. The present invention has no special limitation on the washing and freeze-drying methods, and conventional washing and freeze-drying methods in the art can be used.

[0041] After obtaining the freeze-dried cells, the present invention extracts the protein in the freeze-dried cells. As an embodiment, the protein extraction method refers to GB / T6432-1994 "Method for Determination of Crude Protein in Feed", and the crude protein in the freeze-dried cells is determined using a DigiPREPTKN DRK-K616 Kjeldahl nitrogen analyzer.

[0042] The microbial protein prepared by the method of the present invention is rich in nutrition, contains higher quality protein, and contains more essential amino acids, and can be used as high-quality protein feed or feed additive for breeding of livestock, poultry, aquatic products and other animals. The present invention also provides the use of the microbial protein prepared by the method described in the above technical solution in feed and / or feed additive.

[0043] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] The strains used in the following examples, Candida utilis (ACCC20060) was purchased from the China Agricultural Microbiological Culture Collection Center, Pichia pastoris X-33, GS115 and KM71 were purchased from Biyuntian Biotechnology Co., Ltd. (product numbers: D0414, D0412 and D0413, respectively), Yarrowia lipolytica (ATCC 8661), Rhodosporidium toruloides (ATCC10788) and Saccharomyces cerevisiae (ATCC26603) were purchased from the China Agricultural Microbiological Culture Collection Center.

[0045] Example 1

[0046] A method for preparing microbial protein using crop straw, the process flow chart is as follows Figure 1 As shown, the steps are as follows:

[0047] 1. Preparation and pretreatment of cotton straw raw materials

[0048] Cotton straw (specifically, cotton roots) is dusted and crushed into cotton straw particles of 40 to 100 meshes; the moisture content in the cotton straw is determined by a moisture meter, and the pretreated biomass is actually charged into a 2% sodium hydroxide solution with a solid content of 10% (w / w), and treated in an autoclave at 120° C. for 40 minutes to obtain a pretreated slurry; after the pretreated slurry is cooled to room temperature, its pH is adjusted to 7.0 using concentrated hydrochloric acid, the liquid is filtered out using gauze, and the solid matter is dried in an oven at 60° C. to a moisture content of 10% to 20%, and the content of different lignocellulose components is determined using the technical report NREL / TP-510-42622 of the National Renewable Energy Laboratory of the United States, and the amount of cellulase mixture added is determined.

[0049] 2. Enzymatic hydrolysis of cotton straw

[0050] A cellulase mixture was added, consisting of exo-β-1,4-glucanase (~15%, w / w), endo-β-1,4-glucanase (~15%, w / w), β-glucosidase (~15%, w / w) and cellobiohydrolase (~55%, w / w). The Cellic CTec3 HS cellulase mixture used in the experiment was purchased from Novozymes (China) Biotechnology Co., Ltd. The nominal enzyme activity of cellulase in hydrolyzing β-(1,4)-glucosidic bonds and other β-glucans in cellulose was 1000 BHU-2-HS / g. The amount of cellulase mixture added was 0.1% (w / w, cellulase mixture / cellulose). Enzyme hydrolysis was carried out at 50°C, the pH value was maintained at 4.8, the stirring speed was 250r / min, and it lasted for 96 hours to obtain a hydrolyzed slurry. After the hydrolysis was completed, the hydrolyzate was separated by centrifuging the enzyme hydrolyzed slurry at a speed of 10000 r / min for 10 min to obtain the cotton root hydrolyzate.

[0051] 3. Bacteria activation, expansion culture and fermentation

[0052] Preparation of yeast seed liquid: Streak the yeast Candida utilis ACCC20060 on YPD plates, culture at 30℃ for 2 days, pick a single clone and inoculate it in YPD liquid medium, culture at 30℃, 200r / min, and culture for 24 hours. Collect the cells by centrifugation at 8000r / min, rinse the cells with saline for 3 times, resuspend the cells with saline and measure their OD with a spectrophotometer 600 According to the initial inoculation OD 600 The value is 1-2 and inoculated into the cotton stalk hydrolyzate obtained in step 2 containing 10% to 20%, and the liquid co-fermentation is carried out at 30° C. and 250 r / min for 3-6 days.

[0053] The protein fermentation culture medium comprises: 10% (v / v) cotton root hydrolyzate, 13.56 g / L Na2HPO4·12H2O, 6 g / L KH2PO4, 1 g / L NaCl, 2 g / L NH4Cl, 0.492 g / LMgSO4·7H2O, 0.111 g / L CaCl2 and 10 mL / L Hoagland's nutrient solution, and ammonium sulfate is added as a nitrogen source at a carbon-nitrogen ratio of 1:18 according to the measured carbon content.

[0054] 4. Determination of crude protein content

[0055] After the fermentation is finished, the cells are collected by centrifugation, washed with distilled water, and freeze-dried using a freeze-drying device. The protein content is determined by referring to GB / T6432-1994 "Determination of crude protein in feed", and the crude protein is quantified using a DigiPREPTKNDRK-K616 Kjeldahl nitrogen analyzer. The crude protein content of the fermentation product is calculated according to crude protein (%) = total solid nitrogen content (%) × 6.25. The results are as follows: Figure 2 As shown, the five-pointed star indicates the experimental group with higher concentration of protein synthesized by straw hydrolysate.

[0056] Example 2

[0057] The microbial protein was synthesized by the method in Example 1, with the only difference being that the cotton roots in step 1 were replaced with cotton leaves, cotton husks, cotton stalk skins, cotton fruit branches or dehulled cotton stalks.

[0058] The crude protein content synthesized by Candida utilis from hydrolyzates of different parts of cotton straw was determined according to the method for determining the crude protein content in step 4 of Example 1. The results are as follows: Figure 2 shown.

[0059] Depend on Figure 2 It can be concluded that after fermentation, the crude protein synthesized by Candida utilis from the hydrolyzate of cotton leaves, cotton husks, cotton stalk skin, cotton fruit branches, dehulled cotton stalks and cotton roots had protein contents of 1.15g / L, 4.12g / L, 5.21g / L, 5.14g / L, 2.11g / L and 5.45g / L respectively, with the highest reaching 5.45g / L.

[0060] Example 3

[0061] To further verify the feasibility of this technical solution, other commonly used protein-producing yeasts, such as Pichia pastoris X-33 (P. pastoris X33), GS115 (P. pastoris GS115), KM71 (P. pastoris KM71), Saccharomyces cerevisiae ATCC26603 (S. cerevisiae ATCC26603), Candida utilis ACCC20060 (C. utilis ACCC20060), Yarrowia lipolytica Y1095 (Y. lipolytica NRRLY1095) and Rhodosporidium toruloides NP11 (R. toruloides NP11), were used to transform cotton straw hydrolysate to produce protein. The steps are as follows:

[0062] 1. Preparation and pretreatment of cotton straw raw materials

[0063] Cotton straw is dusted and crushed into cotton straw particles of 40-100 mesh; the cotton straw particles are immersed in a sodium hydroxide solution with a mass fraction of 2%, and the cotton straw (biomass) is added in an amount of 10% (w / w, cotton straw / reaction system), and then put into a 250 ml flask, and treated in an autoclave at 120° C. for 40 minutes to obtain a pretreated slurry; after the pretreated slurry is cooled to room temperature, the pH value thereof is adjusted to 7.0 using concentrated hydrochloric acid, the liquid is filtered out using gauze, and the solid matter is dried in an oven at 60° C., and the content of different lignocellulose components is determined using the technical report NREL / TP-510-42622 of the National Renewable Energy Laboratory of the United States, and the amount of cellulase mixture added is determined.

[0064] 2. The steps of enzymatic hydrolysis of cotton straw are the same as those in Example 1.

[0065] 3. Bacteria activation, expansion culture and fermentation

[0066] The steps in Example 1 are used to activate and expand the strains, except that Pichia pastoris X-33, GS115, KM17, Saccharomyces cerevisiae ATCC26603, Candida utilis ACCC20060 and Rhodosporidium toruloides NP11 are activated, expanded and fermented respectively;

[0067] The fermentation conditions were the same as those in Example 1, except that the fermentation time was 72 h.

[0068] 4. Determination of crude protein content

[0069] The crude protein content was determined by the method of step 4 in Example 1. Figure 3 shown.

[0070] Depend on Figure 3It can be concluded that the protein concentrations of crude proteins obtained by fermentation of Pichia pastoris X-33, GS115, KM17, Saccharomyces cerevisiae ATCC26603, Yarrowia lipolytica Y1095, Candida utilis ACCC20060 and Rhodosporidium toruloides NP11 were 8.29 g / L, 4.39 g / L, 4.21 g / L, 7.25 g / L, 7.41 g / L, 5.16 g / L and 7.73 g / L, respectively;

[0071] 5. Calculation of sugar-protein conversion rate

[0072] According to the crude protein concentration obtained by fermentation, the conversion rate of straw sugar to protein can be calculated, according to protein yield (%) = obtained crude protein concentration (g / L) / initial total concentration of straw sugar (g / L).

[0073] The crude protein concentration produced by Pichia pastoris X-33 reached the highest 8.29g / L, and the yield of protein synthesized from lignocellulose-derived sugars was 0.33g / g. Secondly, the crude protein concentration produced by Rhodosporidium toruloides NP11 was 7.73g / L, which was higher than the protein concentration produced by Candida utilis ACCC20060.

[0074] The above results indicate that straw-derived sugars can be effectively converted into microbial proteins using this straw-synthesized microbial protein technology route and based on dominant microorganisms.

[0075] 5. Determination of amino acid content

[0076] The determination was performed using a Waters e2695 amino acid analyzer in accordance with the National Standard of the People's Republic of China GB / T18246-2000 "Determination of Amino Acids in Feed". The crude protein obtained by fermentation of Saccharomyces cerevisiae ATCC26603 and Rhodosporidium toruloides NP11 was used as the object to determine the content of different amino acids in the protein. The results of amino acid determination are shown in Table 1.

[0077] Table 1 Amino acid content of microbial proteins in two yeasts

[0078] Amino Acids Saccharomyces cerevisiae ATCC26603 (mg / g) Rhodosporidium toruloides NP11 (mg / g) Aspartic acid 67.01 34.22 Threonine 32.03 19.59 Serine 27.97 20.39 Glutamate 82.01 53.98 Glycine 29.33 18.95 Alanine 47.16 26.64 Cysteine 13.36 13.88 Valine 36.94 21.17 Methionine 17.02 8.33 Isoleucine 29.98 14.92 Leucine 51.96 31.92 Tyrosine 29.35 14.55 Phenylalanine 38.06 40.56 Histidine 17.84 11.56 Lysine 47.32 28.41 Arginine 41.63 25.16 Proline 25.40 20.06

[0079] It can be concluded from Table 1 that the lysine content of SCP produced by Saccharomyces cerevisiae ATCC26603 is 47.32 mg / g, which exceeds the standard of 36 mg / g stipulated in the Food Safety and Standards Regulations of 2011 (the fourth edition issued on November 9, 2017) and is higher than the cottonseed content, and has great potential for feed application.

[0080] It can be concluded from the above examples that the method of the present invention can efficiently convert crop straw into microbial protein, which not only has a high conversion rate but also contains rich essential amino acids.

[0081] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing microbial protein using crop straw, characterized in that: The steps include: 1) pre-treating crop straw to obtain pre-treated slurry; The pretreatment method includes dilute alkali pretreatment or dilute acid pretreatment; the pretreatment temperature is 100-140°C, the pressure is 0.1-2.5Mpa, and the time is 10-50min; 2) adjusting the pH value of the pretreated slurry to 7.0 and then drying to obtain pretreated biomass; 3) mixing the pretreated biomass with cellulase and performing enzymatic hydrolysis in a solution with a pH value of 4.6 to 5.0 to obtain a crop straw hydrolyzate; 4) inoculating yeast into a protein fermentation medium for fermentation to obtain a fermentation liquid, wherein the fermentation liquid contains the microbial protein; the protein fermentation medium includes the crop straw hydrolyzate, and the volume of the crop straw hydrolyzate is 10% to 20% of the volume of the protein fermentation medium.

2. The method according to claim 1, characterized in that The water content of the pretreated biomass is 10 wt.% to 20 wt.%.

3. The method according to claim 1, characterized in that Calculated by mass percentage, the cellulase comprises 10% to 30% of exo-β-1,4-glucanase, 10% to 30% of endo-β-1,4-glucanase, 10% to 30% of β-glucosidase and 50% to 70% of cellobiohydrolase.

4. The method according to claim 1 or 3, characterized in that: Based on the cellulose in the pretreated biomass, the dosage of the cellulase is 0.02% to 0.1% of the mass of the cellulose.

5. The method according to claim 1, characterized in that The temperature of the enzymatic hydrolysis is 45-55° C., the stirring speed is 250 r / min, and the time is 96 h.

6. The method according to claim 1, characterized in that The yeast includes Candida utilis, Pichia pastoris, Rhodosporidium toruloides or Yarrowia lipolytica.

7. The method according to claim 1, characterized in that The protein fermentation medium comprises components of the following concentrations: 10% to 20% v / v crop straw hydrolyzate, 13.56 g / L Na2HPO4·12H2O, 6 g / L KH2PO4, 1 g / L NaCl, 2 g / L NH4Cl, 0.492 g / LMgSO4·7H2O, 0.111 g / L CaCl2, 10 mL / L Hoagland's nutrient solution and a nitrogen source; the carbon-nitrogen ratio of the protein fermentation medium is 1:4 to 1:

18.

8. The method according to claim 1, characterized in that The fermentation temperature is 28-30° C., the rotation speed is 200-250 r / min, and the fermentation time is 3-6 days.

9. The method according to claim 1 or 8, characterized in that: The OD after the yeast is inoculated into the protein fermentation medium 600 The value is 1 to 2.

10. Use of the microbial protein prepared by the method according to any one of claims 1 to 9 in feed and / or feed additives.

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