阅读说明：本技术 利用玉米皮水解产物制备苏氨酸发酵培养基的工艺 (Process for preparing threonine fermentation medium by using corn bran hydrolysate ) 是由 白红兵 董力青 王振义 韩杨 李航 于晶 于 2019-12-01 设计创作，主要内容包括：本发明属于氨基酸发酵技术领域,公开了利用玉米皮水解产物制备苏氨酸发酵培养基的工艺,其包括如下步骤：步骤1)取发酵培养基原料,蔗糖,葡萄糖,玉米皮水解产物,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>；步骤2)将各原料搅拌均匀后,调节pH为6-7,灭菌,自然冷却,即得发酵培养基。本发明利用了玉米皮废弃作为原料进行水解,利用水解产物制备了苏氨酸发酵培养基,实现了废弃物的再利用,提高了工业附加值。(The invention belongs to the technical field of amino acid fermentation, and discloses a process for preparing a threonine fermentation medium by using a corn bran hydrolysate, which comprises the following steps: step 1) taking fermentation medium raw materials, sucrose, glucose, corn bran hydrolysate, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, and V B1 ，V H (ii) a And 2) uniformly stirring all the raw materials, adjusting the pH value to 6-7, sterilizing, and naturally cooling to obtain the fermentation medium. The invention utilizes the corn bran waste as the raw material to hydrolyze, and utilizes the hydrolysate to prepare the threonine fermentation medium, thereby realizing the reutilization of the waste and improving the industrial added value.)

1. The process for preparing the threonine fermentation medium by utilizing the corn bran hydrolysate comprises the following steps:

step 1) taking fermentation medium raw materials: sucrose, glucose, corn bran hydrolysate, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, V_B1，V_H；

And 2) uniformly stirring all the raw materials, adjusting the pH value to 6-7, sterilizing, and naturally cooling to obtain the fermentation medium.

2. The process according to claim 1, characterized in that it comprises the following steps:

step 1) taking fermentation medium raw materials, and preparing according to the following concentration: 60g/L of sucrose, 10g/L of glucose, 300ml/L of corn bran 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, V_B12mg/L，V_H50μg/L；

And 2) uniformly stirring all the raw materials, adjusting the pH value to 7, sterilizing, and naturally cooling to obtain the fermentation medium.

3. The process of claim 1, wherein the corn bran hydrolysate is prepared by the steps of: step 1) corn bran pretreatment, step 2) ultrasonic-assisted hydrochloric acid hydrolysis, step 3) microbial hydrolysis, step 4) thallus hydrolysis, and step 5) concentration and steam treatment.

4. The process of claim 3, wherein the corn bran hydrolysate is prepared by the steps of:

step 1) corn husk pretreatment: washing corn bran with water for 2 times, drying, crushing, sieving with a 50-100 mesh sieve, and degreasing with anhydrous ether to obtain pretreated corn bran;

step 2) ultrasonic-assisted hydrochloric acid hydrolysis: adding the pretreated corn bran into 0.4-0.6M diluted hydrochloric acid, wherein the addition amount is 1 g: 3-10ml, ultrasonic treating, heating to 80-90 ℃, standing for 2-5h, cooling to 55 ℃, and then dropwise adding ammonia water, wherein the pH value is controlled to be 6.5-7.0;

step 3) microbial hydrolysis: inoculating heat-resistant bacillus seed solution according to the inoculum size of 2-5%, culturing for 36-48h, inoculating clostridium thermocellum seed solution according to the inoculum size of 2-5%, continuously culturing for 96h, centrifuging by a high-speed disc centrifuge, and collecting precipitate and upper layer liquid;

step 4) hydrolysis of the thalli: the precipitate was stirred at 1 g: adding 0.5-0.8M hydrochloric acid solution at a ratio of 3-10ml, performing microwave-assisted hydrolysis at 100 deg.C, adding neutral protease, hydrolyzing for 12-24 hr, filtering with filter screen to remove insoluble substances, and collecting supernatant;

step 5) concentration and steam treatment: mixing the supernatant liquid and the supernatant liquid according to the volume ratio of 2-4:1, evaporating and concentrating to one third, then carrying out steam treatment at 121 ℃ for 5-20min, and naturally cooling to room temperature to obtain a hydrolysate.

5. The process according to claim 4, characterized in that the conditions of the ultrasonication are: the ultrasonic time is 6s, the interval time is 3s, the total ultrasonic time is 180-270s, and the ultrasonic frequency is 20-25 kHz.

6. The process of claim 4, wherein the conditions of the high speed disc centrifuge centrifugation are: centrifuging at 4000-.

7. The process according to claim 4, wherein the parameters of the microwave-assisted hydrolysis are: the time is 1h, and the microwave power is 440W.

8. The process according to claim 4, wherein the hydrolysis parameters of the neutral protease are: the hydrolysis time is 12-24h, the hydrolysis temperature is 40 ℃, the pH is 7.5, and the enzyme adding amount is 400U/ml.

9. The process of claim 4, wherein the filter mesh has a pore size of 50-100 mesh.

10. A fermentation medium prepared according to the process of claims 1-9.

Technical Field

The invention belongs to the technical field of amino acid fermentation, and particularly relates to a process for preparing a threonine fermentation medium by using a corn bran hydrolysate.

Background

The main production route of threonine is microbial fermentation, in recent years, the yield is increased and increased by 20% every year, and the addition amount of threonine is further increased for improving the product quality of the feed industry. The major market for high quality threonine products is also the pharmaceutical industry, mainly for infusion of various amino acids. At present, the main threonine production enterprises in the world are Japan monosodium glutamate company, Germany Texas company, American ADM company, Japan Co-and fermentation industry company and the like, the yield of the major companies accounts for about 90% of the global share, the capacity reaches about 10 ten thousand tons, the domestic threonine production is in the development stage, as early as 2014, domestic manufacturers have 8 to 9 families, most of the threonine supply domestic requirements, and part of the companies export amino acids abroad, the domestic production level of the threonine still has a certain gap with foreign countries, and the total price of the product is slightly higher than the foreign price; the technical problem to be solved by production enterprises is to reduce the fermentation cost of threonine.

Corn (Corn), an annual herbaceous plant of the genus Zea of the family Gramineae, is an important food and feed crop and is the crop with the highest overall yield worldwide, second only to rice and wheat in terms of planting area and overall yield. Corn is native to Mexico and Peru in Central America, is transferred to China in the 16 th century, and has over 400 years of cultivation history to date. At present, the plants are planted all over the country, particularly in northeast, northwest and southwest provinces. Hard corn is generally planted in northeast regions, and horse-tooth corn suitable for grinding is mostly planted in northeast regions, and the quality of the horse-tooth corn is inferior to that of the hard corn. Corn contains rich protein, fat, vitamins, trace elements, cellulose and the like, and has great potential for developing high-nutrition and high-biological function food.

Corn bran is a byproduct produced by corn deep processing enterprises. The corn is prepared by soaking corn particles, then performing starch production, washing, squeezing water, drying and the like. The main components are fiber, starch, protein, etc. The corn coat has high content of protein and starch, and is mainly used for the feed industry; corn Fiber (Corn Fiber) in Corn peel is dried after Corn steep liquor is added to obtain a product, namely the steep liquor Fiber, the protein content can reach more than 16% (dry basis), and the Corn steep liquor Fiber is mainly used for producing feed.

In recent years, researchers have processed corn bran into dietary fiber in order to improve the application value of the corn bran, and the hydrolysis of cellulose is mainly inorganic acid and enzyme hydrolysis at present. The inorganic acid hydrolysis has high efficiency, low temperature and less by-products, but has the problems of difficult acid recovery, high equipment corrosion, high cost, difficult product separation, serious environmental pollution and the like. The enzymolysis method has mild conditions and few byproducts, and is a common method. For example, in the literature, "the research on extracting water-soluble dietary fiber from corn husk residue by complex enzyme method, 2016 (snow and horse) years" of Maxue et al: the preparation method comprises the steps of taking corn bran residues as raw materials, carrying out enzymolysis on insoluble dietary fibers to modify and convert the insoluble dietary fibers into water-soluble dietary fibers, carrying out composite enzymolysis on the corn bran residues by using cellulase and hemicellulase, and researching a preparation process for preparing the high-quality water-soluble dietary fibers by modifying the water-insoluble dietary fibers by adopting a single-factor experiment and an orthogonal experiment method. The applicant's previous patent technology ' a lysine fermentation culture medium ', which treats corn bran by degreasing, amylase and neutral protease hydrolysis processes to obtain dietary fiber and corn bran treated matter, wherein the corn bran treated matter is applied to the lysine fermentation culture medium, thereby achieving two purposes; however, the enzyme has the problem of high price, which affects the large-scale production.

In order to reduce the technical problem of high enzyme cost in the enzymolysis method, researchers try to degrade corn bran by using bacterial strains, such as a document 'Aspergillus niger solid state fermentation and enzymolysis of corn bran, Huhuidodong and the like' in 2011, the corn bran residue obtained after starch is extracted from corn is used as a main raw material, and a two-step method of producing enzyme by an Aspergillus niger solid state fermentation method and then carrying out enzymolysis is adopted to degrade cellulose substances in the corn bran. By the Plackeu-Bunnan method and the response surface design, the fermentation conditions are optimized, the degradation rates of neutral detergent fiber and acidic detergent fiber are 46.09% and 48.82%, and the mass fraction of reducing sugar reaches 9.02%. The method has low cellulose degradation rate, the obtained product has high cellulose content and more impurities, and the product cannot be used as a high-value product and has low industrial use value; it is possible that the enzymatic activity and type of cellulase produced by Aspergillus niger does not completely degrade cellulose.

Disclosure of Invention

In order to overcome the technical problem of higher cost of a threonine fermentation culture medium in the prior art, the invention provides a process for preparing the threonine fermentation culture medium by using a corn bran hydrolysate.

The invention is realized by the following technical scheme.

The process for preparing the threonine fermentation medium by utilizing the corn bran hydrolysate comprises the following steps:

step 1) taking fermentation medium raw materials, sucrose, glucose, corn bran hydrolysate, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, and V_B1，V_H；

And 2) uniformly stirring all the raw materials, adjusting the pH value to 6-7, sterilizing, and naturally cooling to obtain the fermentation medium.

Further, the process comprises the following steps:

step 1) taking fermentation medium raw materials, and preparing according to the following concentration: 60g/L of sucrose, 10g/L of glucose, 300ml/L of corn bran 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, V_B12mg/L，V_H50μg/L；

And 2) uniformly stirring all the raw materials, adjusting the pH value to 7, sterilizing, and naturally cooling to obtain the fermentation medium.

Further, the corn bran hydrolysate is prepared by the following process:

step 1) corn husk pretreatment: washing corn bran with water for 2 times, drying, crushing, sieving with a 50-100 mesh sieve, and degreasing with anhydrous ether to obtain pretreated corn bran;

step 2) ultrasonic-assisted hydrochloric acid hydrolysis: adding the pretreated corn bran into 0.4-0.6M diluted hydrochloric acid, wherein the addition amount is 1 g: 3-10ml, ultrasonic treating, heating to 80-90 ℃, standing for 2-5h, cooling to 55 ℃, and then dropwise adding ammonia water, wherein the pH value is controlled to be 6.5-7.0;

step 3) microbial hydrolysis: inoculating heat-resistant bacillus seed solution according to the inoculum size of 2-5%, culturing for 36-48h, inoculating clostridium thermocellum seed solution according to the inoculum size of 2-5%, continuously culturing for 96h, centrifuging by a high-speed disc centrifuge, and collecting precipitate and upper layer liquid;

step 4) hydrolysis of the thalli: the precipitate was stirred at 1 g: adding 0.5-0.8M hydrochloric acid solution at a ratio of 3-10ml, performing microwave-assisted hydrolysis at 100 deg.C, adding neutral protease, hydrolyzing for 12-24 hr, filtering with filter screen to remove insoluble substances, and collecting supernatant;

step 5) concentration and steam treatment: mixing the supernatant liquid and the supernatant liquid according to the volume ratio of 2-4:1, evaporating and concentrating to one third, then carrying out steam treatment at 121 ℃ for 5-20min, and naturally cooling to room temperature to obtain a hydrolysate.

Preferably, the ultrasonic treatment conditions are: the ultrasonic time is 6s, the interval time is 3s, the total ultrasonic time is 180-270s, and the ultrasonic frequency is 20-25 kHz.

Preferably, the conditions for the high speed disc centrifuge centrifugation are: centrifuging at 4000-.

Preferably, the parameters of the microwave-assisted hydrolysis are: the time is 1h, and the microwave power is 440W.

Preferably, the hydrolysis parameters of the neutral protease are: the hydrolysis time is 12-24h, the hydrolysis temperature is 40 ℃, the pH is 7.5, and the enzyme adding amount is 400U/ml.

Preferably, the aperture of the filter screen is 50-100 meshes.

Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:

the protein in the corn bran forms a protein net in a special three-dimensional structure, the protein net wraps starch particles and cellulose components, and the protein net is prehydrolyzed by ultrasonic-assisted dilute acid to loosen the protein and reduce the binding tightness, so that the subsequent bacteria utilization is facilitated;

the ammonia water reacts with the surplus hydrochloric acid to produce ammonium chloride, so that the pH can be increased to adapt to the growth of the strain, and a certain inorganic nitrogen source can be provided for the strain;

inoculating heat-resistant bacillus, namely inoculating clostridium thermocellum after the protein and the cellulose are completely separated, and utilizing the nitrogen source generated by the cellulose and the hydrolyzed protein to highly produce C1, Cx glucanase and β -1,4 glycosidase, so as to hydrolyze the cellulose and hemicellulose into glucose reducing sugar;

the heat-resistant bacillus and the clostridium thermocellum can be symbiotic with each other and act on the corn bran in a synergistic manner, so that the corn bran is completely degraded;

the method comprises the following steps of physically breaking the wall of a microorganism treatment product of the corn bran, performing microwave-assisted dilute acid hydrolysis, filtering to remove insoluble substances to obtain a hydrolysate containing polypeptide, amino acid and reducing sugar, wherein the hydrolysate can be applied to threonine fermentation, the cost is reduced, and the fermentation efficiency is improved.

Drawings

FIG. 1: influence of different strain selection on NDF and ADF degradation rates;

FIG. 2: influence of strain treatment time on reducing sugar content.

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.