Threonine fermentation medium prepared by using mycoprotein as raw material
阅读说明：本技术 利用菌体蛋白作为原料制备的苏氨酸发酵培养基 (Threonine fermentation medium prepared by using mycoprotein as raw material ) 是由 孙钦波 赵凤良 赵春晓 边建军 张婷婷 于 2019-12-22 设计创作，主要内容包括：本发明属于生物技术领域,公开了用菌体蛋白作为原料制备的苏氨酸发酵培养基,其包括如下组分：蔗糖,葡萄糖,菌体蛋白和大豆蛋白联合水解液,硫酸铵,磷酸二氢钾,磷酸氢二钾,七水硫酸镁,七水硫酸亚铁,一水硫酸锰,V<Sub>B1</Sub>,V<Sub>H</Sub>。本发明利用菌体蛋白和大豆蛋白水解液来制备发酵培养基,能够提高苏氨酸发酵产量和糖酸转化率,并且成本相对低廉。(The invention belongs to the technical field of biology, and discloses a threonine fermentation medium prepared by using mycoprotein as a raw material, which comprises the following components: sucrose, glucose, mycoprotein and soybean protein combined hydrolysate, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, V B1 ，V H . The invention utilizes the mycoprotein and the soybean protein hydrolysate to prepare the fermentation medium, can improve the threonine fermentation yield and the saccharic acid conversion rate, and has relatively low cost.)
1. The threonine fermentation medium prepared by using the mycoprotein as the raw material comprises the following components:
sucrose, glucose, mycoprotein and soybean protein combined hydrolysate, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, VB1，VH。
2. Threonine fermentation medium according to claim 1, characterized in that it comprises the following components:
60g/L of sucrose, 30g/L of glucose, 200g/L of thallus protein and soybean protein combined hydrolysate, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, VB12mg/L，VH50μg/L。
3. The threonine fermentation medium according to claim 1 or 2, wherein the mycoprotein and soy protein combined hydrolysate is prepared by the following process:
taking mycoprotein and soybean protein according to the weight ratio of 2-4:1, and then mixing the mycoprotein and the soybean protein according to the weight ratio of 1 g: adding citric acid aqueous solution in a proportion of 5-10ml, stirring uniformly, then placing in a high-speed shearing machine for shearing, standing for 30-90min, then treating with ultrasonic waves, standing for 2h, then adjusting the pH to 3.0 and the temperature to 40 ℃, then adding acid protease, performing enzymolysis for 4-6h, then adjusting the pH to 7.0 and the temperature to 50 ℃, then adding Serratin, performing enzymolysis for 4-6h, and finally inactivating enzyme to obtain hydrolysate.
4. A threonine fermentation medium according to claim 3, wherein the concentration of the aqueous citric acid solution is 0.4-0.8M.
5. Threonine fermentation medium according to claim 4, characterized in that the concentration of aqueous citric acid is 0.6M.
6. The threonine fermentation medium as claimed in claim 4, wherein the shear rate of the high-speed shearing machine is 8000-10000rpm, and the shear time is 60-90 s.
7. Threonine fermentation medium according to claim 4, characterized in that the sonication time is 30-90s and the sonication frequency is 25 kHz.
8. Threonine fermentation medium according to claim 4, characterized in that the acidic protease is added in an amount of 2000U/g dry matter.
9. A threonine fermentation medium according to claim 4, wherein serrapeptidase is added in an amount of 1000U/g dry matter.
10. Use of a threonine fermentation medium according to claims 1 to 9 for the fermentative preparation of threonine in a microorganism.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a threonine fermentation medium prepared by using mycoprotein as a raw material.
Background
The mycoprotein is a byproduct in the process of producing amino acid by microbial fermentation, and is rich in protein and other nutrient substances. Researches show that the microbial fermentation is not greatly influenced by adopting the mycoprotein hydrolysate to replace a soybean meal hydrolysate as a nitrogen source for culturing microorganisms, and the applicant finds that the yield of threonine produced by microbial fermentation is improved by adding a certain proportion of the soybean meal hydrolysate in the mycoprotein hydrolysate by accident in actual industrial production, but the subsequent purification of threonine is not facilitated by considering that the soybean meal hydrolysate contains certain impurities such as pigment, so that the soybean protein hydrolysate can be adopted for replacement, the mycoprotein and the soybean protein can be subjected to combined hydrolysis by considering the simplicity and controllability of operation, but the structures and components of the two proteins have large differences, the hydrolysis process suitable for the mycoprotein cannot be suitable for the soybean protein, and vice versa, the prior art does not describe the process parameters of the combined hydrolysis of the two proteins in detail, how to carry out the joint hydrolysis to mycoprotein and soy protein to simplify the procedure, reduce the energy consumption, make the nutritive value maximize, thus promote the fermentation efficiency of threonine more effectively, it is the technical problem that needs to solve in the industrial production process.
Document 1, "study on peptide molecular distribution in soy protein hydrolysate, the chinese food and oil institute in 2001" study on the distribution of molecular weight of oligopeptide mixture of soy protein hydrolysate by using an experimental method combining ultrafiltration and Gel Filtration Chromatography (GFC). Research results show that small peptides with the molecular weight of less than 1000D are mainly used in the enzymatic hydrolysate of the papain and the Asl.398 protease, namely 63.9 percent of the enzymatic hydrolysate of the papain with the molecular weight of less than 1000D, 4.64 percent of the enzymatic hydrolysate of the papain with the molecular weight of 1000-2000D, 8.21 percent of the enzymatic hydrolysate of 2000-4000D, 8.20 percent of the enzymatic hydrolysate of the papain with the molecular weight of more than 4000-10000D, 15 percent of the enzymatic hydrolysate of the papain with the molecular weight of more than 10000D, 72.1 percent of the enzymatic hydrolysate of the protease of the As1.398 with the molecular weight of less than 1000D, 6.42 percent of the enzymatic hydrolysate of 1000D-2000D, 2.5 percent of the enzymatic hydrolysate of 2000D-4000D, 3.92 percent of. However, papain alone or asl.398 protease alone has poor hydrolysis efficiency on mycoprotein and is not suitable for co-hydrolysis.
Disclosure of Invention
In order to simultaneously carry out combined hydrolysis on mycoprotein and soybean protein and prepare a culture medium by using a hydrolysate as a nitrogen source so as to improve the fermentation efficiency and overcome the defects of the prior art, the invention provides a threonine fermentation culture medium prepared by using mycoprotein as a raw material.
The invention is realized by the following scheme.
The threonine fermentation medium prepared by using the mycoprotein as the raw material comprises the following components:
sucrose, glucose, mycoprotein and soybean protein combined hydrolysate, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, VB1，VH。
Further, the threonine fermentation medium comprises the following components:
60g/L of sucrose, 30g/L of glucose, 200g/L of thallus protein and soybean protein combined hydrolysate, 5g/L of ammonium sulfate, 0.5g/L of monopotassium phosphate, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate, 10mg/L of ferrous sulfate heptahydrate, 10mg/L of manganese sulfate monohydrate, VB12mg/L，VH50μg/L。
Further, the mycoprotein and soybean protein combined hydrolysate is prepared by the following process:
taking mycoprotein and soybean protein according to the weight ratio of 2-4:1, and then mixing the mycoprotein and the soybean protein according to the weight ratio of 1 g: adding a citric acid aqueous solution in a proportion of 5-10ml, uniformly stirring, then placing in a high-speed shearing machine for shearing, standing for 30-90min, then treating with ultrasonic waves, standing for 2h, then adjusting the pH to 3.0 and the temperature to 40 ℃, then adding acid protease, performing enzymolysis for 4-6h, then adjusting the pH to 7.0 and the temperature to 50 ℃, then adding Serratin, performing enzymolysis for 4-6h, and finally inactivating enzyme to obtain a hydrolysate.
Preferably, the concentration of the aqueous citric acid solution is 0.4-0.8M.
Preferably, the concentration of the aqueous citric acid solution is 0.6M.
Preferably, the shearing speed of the high-speed shearing machine is 8000-10000rpm, and the shearing time is 60-90 s.
Preferably, the ultrasonic treatment time is 30-90s, and the ultrasonic frequency is 25 kHz.
Preferably, the amount of the acidic protease added is 2000U/g dry matter.
Preferably, the amount of serrapeptase added is 1000U/g dry matter.
The invention also claims the application of the threonine fermentation medium in preparing threonine by microbial fermentation.
The starting point and the beneficial effects obtained by the invention mainly comprise but are not limited to the following aspects:
because the mycoprotein and the soybean protein have great difference in the three-dimensional structure composition of the protein, a conventional system suitable for hydrolyzing a single protein is not suitable for jointly hydrolyzing the two proteins, and a system for jointly hydrolyzing the two proteins needs to be developed.
The invention carries out physical auxiliary preliminary hydrolysis on two proteins under the weak acid condition, not only can hydrolyze partial proteins, but also is beneficial to subsequent enzyme hydrolysis.
The invention adopts citric acid to replace hydrochloric acid, has mild hydrolysis conditions, does not damage amino acid components, and improves the hydrolysis degree to a certain extent.
The invention adopts high-speed shearing and ultrasonic treatment to carry out treatment under the weak acid condition, thereby not only breaking the cell wall of mycoprotein, but also accelerating the mass transfer process, reducing the viscosity of the system and being beneficial to hydrolysis; and the protein chain can be damaged or loosened, so that the internal structure of the protein is changed, the solubility and the hydrophilicity of the protein are improved, and further hydrolysis is facilitated.
The invention adopts a mode of sequentially carrying out enzymolysis on the acid protease and the serrapeptase, both the acid protease and the serrapeptase are in an optimal reaction system, and the hydrolysis degree is respectively improved by 32 percent or 51 percent compared with the hydrolysis degree when the acid protease or the serrapeptase is singly used.
Compared with a pure acid method and a pure alkali method, the method provided by the invention has the advantages that the two proteins are hydrolyzed, the hydrolysis efficiency is high, the reaction condition is mild, the possibility of generating toxic substances is low, the molecular weight of the proteins is gradually reduced along with the proceeding of enzyme reaction, the proteins are converted into peptides or amino acids, the physical properties and the functional properties of the proteins are obviously changed, and the method has more advantages than the proteins and the amino acids in nutrition.
The method utilizes the hydrolysate to prepare the fermentation medium, can improve the fermentation yield of threonine and the conversion rate of saccharic acid, and has relatively low cost.
Drawings
FIG. 1: the effect of different acids on the degree of hydrolysis;
FIG. 2: the effect of different enzymes and combinations thereof on the degree of hydrolysis.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
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