Method for preparing microbial protein by using crop stalks and application thereof

Abstract

The present application belongs to the technical field of microbial protein preparation, and particularly relates to a method for preparing microbial protein by using crop stalks and application thereof. The method improves the accessibility of cellulose and the hydrolysis efficiency by carrying out biomass conversion pretreatment on the crop stalks; on this basis, the pretreated biomass is subjected to enzymolysis by cellulase, so that the cellulose and hemicellulose components in the biomass are converted into glucose and xylose; and the glucose and xylose are utilized by yeast in the subsequent yeast fermentation process to synthesize microbial protein. The microbial protein obtained in this way has the advantages of rich nutrition and high content of essential amino acids, and has a wide application prospect in the fields of food and feed. It can be seen that the present application provides a new way for high-value utilization of crop stalks on the basis of obtaining rich-nutrition microbial protein through chemical or physical-biological treatment of the crop stalks.

Description

Technical Field

[0001] This invention belongs to the field of microbial protein preparation technology, specifically relating to a method for preparing microbial protein using crop straw and its application. Background Technology

[0002] Crop straw, such as wheat, corn, rice, and cotton straw, is a major byproduct of agricultural production. The annual straw production from major crops like corn, rice, wheat, rapeseed, soybeans, and cotton reaches nearly 1 billion tons. However, most crop straw is directly returned to the field or burned, causing resource waste and environmental pollution, and seriously hindering the development of sustainable agriculture.

[0003] Crop straw is an ideal source of lignocellulose, mainly composed of cellulose, hemicellulose, and lignin. Converting the cellulose and hemicellulose components of straw into high-value products such as microbial protein is a feasible way to promote the utilization of crop straw resources as animal feed. Although some studies have shown that using fungal solid-state fermentation to convert crop straw into protein can address the problem of straw being directly returned to the field or burned to some extent, this conversion method usually requires a long cultivation time, the process is difficult to control, the conversion rate is low, and certain amino acids are often insufficient or unbalanced. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing microbial protein from crop straw and its application. The method can efficiently convert crop straw into microbial protein, with a high conversion rate of lignocellulose-derived sugars to synthesized protein, and it contains abundant essential amino acids.

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

[0006] 1) Pre-treat 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℃, the pressure is 0.1-2.5 MPa, and the time is 10-50 min.

[0008] 2) After adjusting the pH value of the pretreated slurry to 7.0, it is dried to obtain pretreated biomass;

[0009] 3) The pretreated biomass is mixed with cellulase and then enzymatically hydrolyzed in a solution with a pH of 4.6 to 5.0 to obtain crop straw hydrolysate;

[0010] 4) Inoculate yeast into a protein fermentation medium for fermentation to obtain a fermentation broth containing the microbial protein; the protein fermentation medium includes the crop straw hydrolysate, the volume of which is 10% to 20% of the volume of the protein fermentation medium.

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

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

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

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

[0015] Preferably, the yeast includes Candida utilis, Pichia pastoris, Rhodotorula rubrum, or Yersinia lipolytica.

[0016] Preferably, the protein fermentation medium comprises the following components at the following concentrations: 10%–20% v / v crop straw hydrolysate, 13.56 g / L Na₂HPO₄·12H₂O, 6 g / L KH₂PO₄, 1 g / L NaCl, 2 g / L NH₄Cl, 0.492 g / L MgSO₄·7H₂O, 0.111 g / L CaCl₂, 10 mL / L Hoagland's nutrient solution, and a nitrogen source; the carbon-to-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 after the yeast is inoculated into the protein fermentation medium is... 600 The value is 1 to 2.

[0019] The present invention also provides the application of microbial proteins prepared by the method described above in feed and / or feed additives.

[0020] Beneficial effects:

[0021] This invention provides a method for preparing microbial protein from crop straw. The method involves pretreating the crop straw (including dilute alkali or dilute acid pretreatment) using physical or chemical means to improve the accessibility and hydrolysis efficiency of cellulose. Following this, cellulase is used to enzymatically hydrolyze the pretreated biomass, converting the cellulose and hemicellulose components into glucose and xylose. These glucose and xylose are then utilized by yeast during subsequent fermentation to synthesize microbial protein. The microbial protein obtained in this way has the advantages of short synthesis time and high essential amino acid content, showing broad application prospects in the food and feed industries. Therefore, this invention provides a new pathway for the high-value utilization of crop straw by obtaining nutrient-rich microbial protein through chemical or physical-biological treatment. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 This is a process flow diagram of preparing microbial protein from crop straw in Example 1;

[0024] Figure 2 The concentrations of microbial proteins synthesized from hydrolysates of different parts of cotton stalks by *Candida utilis* ACCC20060 in Examples 1 and 2.

[0025] Figure 3 The concentration of microbial protein produced by the conversion of straw hydrolysate by the seven types of yeast in Example 3. Detailed Implementation

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

[0027] 1) Pre-treat 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℃, the pressure is 0.1-2.5 MPa, and the time is 10-50 min.

[0029] 2) After adjusting the pH value of the pretreated slurry to 7.0, it is dried to obtain pretreated biomass;

[0030] 3) The pretreated biomass is mixed with cellulase and then enzymatically hydrolyzed in a solution with a pH of 4.6 to 5.0 to obtain crop straw hydrolysate;

[0031] 4) Inoculate yeast into a protein fermentation medium for fermentation to obtain a fermentation broth containing the microbial protein; the protein fermentation medium includes the crop straw hydrolysate, the volume of which is 10% to 20% of the volume of the protein fermentation medium.

[0032] In one embodiment, the present invention washes and crushes crop straw to obtain washed crop straw particles. In another embodiment, the particle size of the crop straw particles is 40-100 mesh. The present invention does not specifically limit the crushing method; conventional crushing methods in the art are acceptable. The present invention also does not specifically limit the washing method, as long as it achieves the effect of removing dust from the crop straw. The present invention, through washing and crushing, can remove impurities from the surface of the crop straw, further improving the efficiency of subsequent processing.

[0033] In one embodiment, the crop straw of the present invention can be one or more of cotton straw, wheat straw, corn straw, and wheat straw; in another embodiment, the crop straw can be cotton straw; in yet another embodiment, the crop straw can be different parts of the crop straw; for example, in a specific embodiment, the crop straw is cotton straw, and is one or more of cotton root, peeled cotton stalk, fruit branch, cotton stalk bark, cotton hull, and cotton leaf.

[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 dilute alkali pre-treatment or dilute acid pre-treatment; the pre-treatment temperature is 100–140℃, the pressure is 0.1–2.5 MPa, and the time is 10–50 min. As one embodiment, the dilute alkali pre-treatment step is: immersing the washed crop straw particles in a dilute alkali solution and treating them at 100–140℃ and 0.1–2.5 MPa for 10–50 min; as another embodiment, the dilute alkali solution is a 2% (w / w) NaOH solution. As one embodiment, the dilute acid pre-treatment step is: immersing the washed crop straw particles in a dilute acid solution and treating them at 100–140℃ and 0.1–2.5 MPa for 10–50 min; as another embodiment, the dilute acid solution is a 1% (w / w) sulfuric acid solution. In one embodiment, the pretreatment temperature of the present invention is 110-120°C, the pressure is 0.1-0.4 MPa, and the time is 40 min; 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 one embodiment, the present invention adjusts the pH value using concentrated hydrochloric acid. The reason for adjusting the pH value of the pretreated slurry to 7.0 is that microorganisms grow and metabolize optimally at neutral pH. As one embodiment, before drying, the present invention further includes a step of filtering the pH-adjusted pretreated slurry using a filtration device to remove liquid from the pretreated slurry. As one embodiment, the drying method is oven drying; as another embodiment, the drying temperature is 60°C, and the drying time is based on the moisture content of the pretreated biomass reaching 10wt.% to 20wt.%.

[0036] After obtaining the pretreated biomass, the present invention mixes the pretreated biomass with cellulase and performs enzymatic hydrolysis in a solution with a pH of 4.6–5.0 to obtain crop straw hydrolysate. As one embodiment, the cellulase of the present invention, by mass percentage, comprises 10%–30% exo-β-1,4-glucanase, 10%–30% endo-β-1,4-glucanase, 10%–30% β-glucosidase, and 50%–70% cellobiose hydrolase; as another embodiment, the cellulase of the present invention comprises 15%–20% exo-β-1,4-glucanase, 15%–20% endo-β-1,4-glucanase, 15%–20% β-glucosidase, and 55%–65% cellobiose hydrolase. In one embodiment, based on the cellulose in the pretreated biomass, the amount of cellulase used in this invention is 0.02% to 0.1% of the cellulose mass; in another embodiment, the amount of cellulase used is 0.05% to 0.08% of the cellulose mass. In one embodiment, the solution with a pH of 4.6 to 5.0 can be a citrate buffer; in another embodiment, the mass ratio of the dry matter in the pretreated biomass to the citrate buffer is 1:5 to 1:10. In one embodiment, the enzyme hydrolysis temperature is 45 to 55°C; in another embodiment, the enzyme hydrolysis temperature is 50°C. In one embodiment, the pH value for enzyme hydrolysis is 4.8. In one embodiment, the stirring speed for enzyme hydrolysis is 150 r / min; in another embodiment, the stirring speed for enzyme hydrolysis is 250 r / min. In one embodiment, the enzyme hydrolysis time can be 72 h; in another embodiment, the enzyme hydrolysis time is 96 h. The enzymatic hydrolysis described in this invention can hydrolyze the cellulose and hemicellulose components in crop straw into glucose and xylose, providing raw materials for the subsequent synthesis of microbial proteins.

[0037] After obtaining the hydrolyzed crop straw slurry, as one embodiment, the present invention centrifuges the hydrolyzed crop straw slurry to obtain a hydrolysate of crop straw. As one embodiment, the centrifugation speed is 10000 r / min; the centrifugation time is 10 min.

[0038] After obtaining the crop straw hydrolysate, the present invention inoculates yeast into a protein fermentation medium for fermentation to obtain a fermentation broth containing the microbial protein. The protein fermentation medium includes the crop straw hydrolysate, and the volume of the crop straw hydrolysate is 10% to 20% of the volume of the protein fermentation medium. As one embodiment, the yeast of the present invention includes *Candida utilis*, *Pichia pastoris*, *Rhodotorula glutinis*, 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; and the *Rhodotorula glutinis* is *Rhodotorula glutinis* NP11. The advantage of using yeast instead of other strains for fermentation in the present invention is its faster growth rate, allowing for rapid utilization of the sugars released from the straw. In one embodiment, the protein fermentation medium comprises the following components at the following concentrations: 10%–20% v / v crop straw hydrolysate, 13.56 g / L Na₂HPO₄·12H₂O, 6 g / L KH₂PO₄, 1 g / L NaCl, 2 g / L NH₄Cl, 0.492 g / L MgSO₄·7H₂O, 0.111 g / L CaCl₂, 10 mL / L Hoagland's nutrient solution, and a nitrogen source; the carbon-to-nitrogen ratio of the protein fermentation medium is 1:4–1:18; in another embodiment, the carbon-to-nitrogen ratio of the protein fermentation medium can be 1:18. This invention, by adding the volume percentage of crop straw hydrolysate to the protein fermentation medium, achieves a rapid conversion of the straw hydrolysate. In another embodiment, the nitrogen source is ammonium sulfate, and the amount of ammonium sulfate added is such that the carbon-to-nitrogen ratio of the protein fermentation culture reaches 1:4–1:18 after addition. In one embodiment, the fermentation temperature of the present invention is 28–30°C; in another embodiment, the fermentation temperature is 30°C. In one embodiment, the fermentation speed of the present invention is 200–250 r / min; in another embodiment, the fermentation speed is 250 r / min. In one embodiment, the fermentation time of the present invention is 3–6 days; in another embodiment, the fermentation time is 3 days. In one embodiment, the OD of the yeast inoculated into the protein fermentation medium… 600 The value is 1 to 5; as another embodiment, the OD value after the yeast is inoculated into the protein fermentation medium is... 600The value is 1 to 2.

[0039] After obtaining the fermentation broth, as one embodiment, the present invention centrifuges the fermentation broth to obtain bacterial cells. The present invention does not have a particular limitation on the centrifugation method; conventional centrifugation methods in the art are acceptable.

[0040] After obtaining the bacterial cells, as one embodiment, the present invention rinses and freeze-dries the bacterial cells to obtain freeze-dried bacterial cells. The present invention does not have any particular limitation on the rinsing and freeze-drying methods; conventional rinsing and freeze-drying methods in the art can be used.

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

[0042] The microbial protein prepared by the method described in this invention is rich in nutrients, contains higher quality protein, and contains more essential amino acids. It can be used as a high-quality protein feed or feed additive for the breeding of livestock, poultry, aquatic animals, and other animals. This invention also provides the application of the microbial protein prepared by the method described above in feed and / or feed additives.

[0043] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0044] The strains used in the following examples were: Candida utilis (ACCC20060) purchased from the China Agricultural Microbiological Culture Collection Center; Pichia pastoris X-33, GS115 and KM71 purchased from Beyotime Biotechnology Co., Ltd. (product numbers: D0414, D0412 and D0413, respectively); and Yersinia lipolyticosa (ATCC 8661), Rhodotorula glutinis (ATCC10788) and Saccharomyces cerevisiae (ATCC26603) 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 diagram is as follows: Figure 1 As shown, the steps are as follows:

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

[0048] Cotton stalks (specifically cotton roots) were dusted and pulverized into 40-100 mesh cotton stalk particles. The moisture content of the cotton stalks was measured using a moisture analyzer. The pretreated biomass was then added to 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 cooled to room temperature, its pH was adjusted to 7.0 using concentrated hydrochloric acid. The liquid was filtered out using gauze, and the solid material was dried in a 60°C oven to a moisture content of 10%-20%. The content of different lignocellulose components was determined using the National Renewable Energy Laboratory (NREL) technical report NREL / TP-510-42622 to determine the amount of cellulase mixture to be added.

[0049] 2. Enzymatic hydrolysis of cotton stalks

[0050] A cellulase mixture, consisting of exo-β-1,4-glucanase (~15%, w / w), endo-β-1,4-glucanase (~15%, w / w), β-glucosidase (~15%, w / w), and cellobiase (~55%, w / w), was added. The Cellic CTec3 HS cellulase mixture used in the experiment was purchased from Novozymes (China) Biotechnology Co., Ltd., and its nominal enzyme activity for 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). Enzymatic hydrolysis was carried out at 50°C, pH maintained at 4.8, and stirring at 250 rpm for 96 hours to obtain a hydrolyzed slurry. After hydrolysis, the hydrolysis products were separated by centrifuging the enzyme hydrolysate at 10,000 r / min for 10 min to obtain cotton root hydrolysate.

[0051] 3. Activation, scale-up culture and fermentation of microbial strains

[0052] Yeast seed culture preparation: Streak *Candida utilis* ACCC20060 onto YPD plates and incubate at 30°C for 2 days. Single colonies are then picked and inoculated into YPD liquid medium and incubated at 30°C and 200 rpm for 24 hours. Cells are collected by centrifugation at 8000 rpm, washed three times with physiological saline, resuspended in physiological saline, and their OD values ​​are measured using a spectrophotometer. 600 Value. According to the initial inoculation OD 600 The inoculum with a value of 1-2 is inoculated into a solution containing 10%-20% of the cotton stalk hydrolysate obtained in step 2, and liquid co-fermented at 30℃ and 250r / min for 3-6 days.

[0053] The protein fermentation medium consisted of: 10% (v / v) cotton root hydrolysate, 13.56 g / L Na2HPO4·12H2O, 6 g / L KH2PO4, 1 g / L NaCl, 2 g / L NH4Cl, 0.492 g / L MgSO4·7H2O, 0.111 g / L CaCl2, and 10 mL / L Hoagland's nutrient solution. Ammonium sulfate was added as a nitrogen source at a carbon-to-nitrogen ratio of 1:18 based on the measured carbon content.

[0054] 4. Crude protein content determination

[0055] After fermentation, the bacterial cells were collected by centrifugation, rinsed with distilled water, and then freeze-dried using a freeze-drying device. Protein content was determined according to GB / T6432-1994 "Determination of Crude Protein in Feed," using a DigiPREPTKNDRK-K616 Kjeldahl nitrogen analyzer. The crude protein content of the fermentation product was calculated as follows: Crude Protein (%) = Total Solid Nitrogen Content (%) × 6.25. The results are shown below. Figure 2 As shown, the pentagrams represent the experimental groups with higher concentrations of protein synthesized from straw hydrolysate.

[0056] Example 2

[0057] Microbial proteins were synthesized using the method described in Example 1, with the only difference being that the cotton roots in step 1 were replaced with cotton leaves, cotton hulls, cotton stalk bark, cotton fruit branches, or peeled cotton stalks.

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

[0059] Depend on Figure 2 It can be concluded that, after fermentation, the crude protein content of *Candida utilis* synthesized from the hydrolysates of cotton leaves, cotton bolls, cotton stalk bark, cotton fruit branches, peeled cotton stalks, and cotton roots was 1.15 g / L, 4.12 g / L, 5.21 g / L, 5.14 g / L, 2.11 g / L, and 5.45 g / L, respectively, with the highest reaching 5.45 g / L.

[0060] Example 3

[0061] To further verify the feasibility of this technical solution, other commonly used protein-producing yeasts were used to convert cotton straw hydrolysate into protein: Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71, Saccharomyces cerevisiae ATCC26603, Candida utilis ACCC20060, Yersinia lipolytica NRRLY1095, and Rhodotorula toruloides NP11. The steps are as follows:

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

[0063] Cotton stalks were dusted and pulverized into 40-100 mesh cotton stalk particles. The cotton stalk particles were then immersed in a 2% sodium hydroxide solution, with a cotton stalk (biomass) addition rate of 10% (w / w, cotton stalk / reaction system). The mixture was then placed in a 250 mL flask and autoclaved at 120°C for 40 min to obtain a pretreated slurry. After the pretreated slurry cooled to room temperature, its pH was adjusted to 7.0 using concentrated hydrochloric acid. The liquid was filtered out using gauze, and the solid material was dried in a 60°C oven. The content of different lignocellulose components was determined using the National Renewable Energy Laboratory (NREL) technical report NREL / TP-510-42622 to determine the amount of cellulase mixture to be added.

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

[0065] 3. Activation, scale-up culture and fermentation of microbial strains

[0066] The steps in Example 1 were used to activate and expand the culture of the strains, except that Pichia pastoris X-33, GS115, KM17, Saccharomyces cerevisiae ATCC26603, Candida utilis ACCC20060 and Rhodotorula glutinis NP11 were activated, expanded and fermented respectively.

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

[0068] 4. Crude protein content determination

[0069] The crude protein content was determined using the method in step 4 of Example 1, and the results are as follows: Figure 3 As shown.

[0070] Depend on Figure 3The crude protein concentrations obtained from fermentation of Pichia pastoris X-33, GS115, KM17, Saccharomyces cerevisiae ATCC26603, Yersinia lipolytica Y1095, Candida utilis ACCC20060, and Rhodotorula glutinis 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] Based on the crude protein concentration obtained from fermentation, the conversion rate of straw sugar to protein can be calculated as follows: protein yield (%) = crude protein concentration obtained (g / L) / initial total straw sugar concentration (g / L).

[0073] The highest crude protein concentration was achieved by Pichia pastoris X-33, reaching 8.29 g / L, with a protein yield of 0.33 g / g from lignocellulose-derived sugars. Secondly, Rhodotorula glutinis NP11 produced a crude protein concentration of 7.73 g / L, higher than that of Candida utilis ACCC20060.

[0074] The above results indicate that using this straw-based microbial protein synthesis technology and leveraging dominant microorganisms can effectively convert straw-derived sugars into microbial proteins.

[0075] 5. Determination of amino acid content

[0076] The amino acid content in feed was determined using a Waters e2695 amino acid analyzer, in accordance with the People's Republic of China National Standard GB / T18246-2000, "Determination of Amino Acids in Feed". The crude protein obtained from fermentation of *Saccharomyces cerevisiae* ATCC26603 and *Rhodotorula glutinis* NP11 was used as the model, and the results are shown in Table 1.

[0077] Table 1. Amino acid content of microbial proteins in two types of yeast

[0078] amino acids Saccharomyces cerevisiae ATCC26603 (mg / g) Rhodotorula glutinis NP11 (mg / g) Aspartic acid 67.01 34.22 threonine 32.03 19.59 Serine 27.97 20.39 glutamic acid 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] Table 1 shows that the lysine content of SCP produced by Saccharomyces cerevisiae ATCC26603 is 47.32 mg / g, which exceeds the standard of 36 mg / g for cottonseed in the 2011 Food Safety and Standards Regulations (the fourth edition released on November 9, 2017), indicating that it has great potential for feed application.

[0080] From the above examples, it can be concluded 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 abundant essential amino acids.

[0081] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, 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, Includes the following steps: 1) Pre-treat crop straw to obtain pre-treated slurry; The pretreatment method includes dilute alkali pretreatment or dilute acid pretreatment; the pretreatment temperature is 100~140℃, the pressure is 0.1~2.5Mpa, and the time is 10~50min; 2) After adjusting the pH value of the pretreated slurry to 7.0, it is dried to obtain pretreated biomass; 3) The pretreated biomass is mixed with cellulase and then enzymatically hydrolyzed in a solution with a pH of 4.6-5.0 to obtain crop straw hydrolysate; 4) Yeast is inoculated into a protein fermentation medium for fermentation to obtain a fermentation broth containing the microbial protein; the protein fermentation medium includes the crop straw hydrolysate, the volume of which is 10% to 20% of the volume of the protein fermentation medium; The cellulase comprises 10%–30% exo-β-1,4-glucanase, 10%–30% endo-β-1,4-glucanase, 10%–30% β-glucosidase, and 50%–70% cellobiase; The enzymatic hydrolysis was performed at a temperature of 45-55℃, a stirring speed of 250 r / min, and a time of 96 h.

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

3. The method according to claim 1, characterized in that, The amount of cellulase used is 0.02% to 0.1% of the mass of the cellulase in the pretreated biomass.

4. The method according to claim 1, characterized in that, The yeasts include Candida utilis, Pichia pastoris, Rhodotorula rubrum, or Yersinia lipolytica.

5. The method according to claim 1, characterized in that, The protein fermentation medium comprises the following components at the following concentrations: 10%–20% v / v crop straw hydrolysate, 13.56 g / L Na₂H₂O. P The protein fermentation medium contains O4·12H2O, 6 g / L KH2PO4, 1 g / L NaCl, 2 g / L NH4Cl, 0.492 g / L MgSO4·7H2O, 0.111 g / L CaCl2, 10 mL / L Hogrange nutrient solution, and a nitrogen source; the carbon-to-nitrogen ratio of the protein fermentation medium is 1:4 to 1:

18.

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

7. The method according to claim 1 or 6, characterized in that, OD after the yeast is inoculated into the protein fermentation medium 600 The value is 1 to 2.

8. The use of the microbial protein prepared by the method according to any one of claims 1 to 7 in feed and / or feed additives.

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