CN110885864A

CN110885864A - Process for preparing threonine fermentation medium by using corn bran hydrolysate

Info

Publication number: CN110885864A
Application number: CN201911209468.0A
Authority: CN
Inventors: 白红兵; 董力青; 王振义; 韩杨; 李航; 于晶
Original assignee: Qigihar Longjiang Fufeng Biotechnology Co Ltd
Current assignee: Qigihar Longjiang Fufeng Biotechnology Co Ltd
Priority date: 2019-12-01
Filing date: 2019-12-01
Publication date: 2020-03-17

Abstract

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.

Description

Process for preparing threonine fermentation medium by using corn bran hydrolysate

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.

Example 1

A corn bran hydrolysate prepared by the following process:

washing corn bran with water for 2 times, drying, crushing, sieving with a 100-mesh sieve, and degreasing with anhydrous ether to obtain pretreated corn bran.

The pretreated corn bran is added into 0.5M dilute hydrochloric acid, and the addition amount is 1 g: 4ml, carrying out ultrasonic treatment, wherein the ultrasonic time is 6s, the interval time is 3s, the total ultrasonic time is 180s, the ultrasonic frequency is 20kHz, then heating to 85 ℃, standing for 3h, cooling to 55 ℃, then dropwise adding ammonia water, and controlling the pH to be 6.8;

inoculating Bacillus thermotolerans XJT9503 seed liquid (density 1 × 10) in an inoculation amount of 5%⁹cfu/ml), culturing for 48h, and inoculating Clostridium thermocellum ATCC27405 seed solution (density 1 × 10) according to an inoculation amount of 5%⁹cfu/ml), continuously culturing for 96h, centrifuging for 5min at 4000rpm by a high-speed disc centrifuge, and collecting precipitate and upper-layer liquid;

the precipitate was stirred at 1 g: adding 0.6M hydrochloric acid solution at a ratio of 5ml, performing microwave-assisted hydrolysis at 100 ℃ for 1h with microwave power of 440W, adding AS.1398 neutral protease for hydrolysis for 12h, wherein the hydrolysis temperature is 40 ℃, the pH value is 7.5, the enzyme addition amount is 400U/ml, filtering with a filter screen with a pore size of 50 meshes to remove insoluble substances after hydrolysis is completed, and collecting supernatant;

mixing the supernatant liquid and the supernatant liquid according to the volume ratio of 2:1, evaporating and concentrating to one third, then carrying out steam treatment at 121 ℃ for 10min, and naturally cooling to room temperature to obtain a hydrolysate.

Example 2

A corn bran hydrolysate prepared by the following process:

washing the corn bran with water for 2 times, drying, crushing, sieving with a 80-mesh sieve, and degreasing with anhydrous ether to obtain the pretreated corn bran.

The pretreated corn bran is added into 0.5M dilute hydrochloric acid, and the addition amount is 1 g: 5ml, carrying out ultrasonic treatment, wherein the ultrasonic time is 6s, the interval time is 3s, the total ultrasonic time is 270s, the ultrasonic frequency is 25kHz, then heating to 85 ℃, standing for 5h, cooling to 55 ℃, then dropwise adding ammonia water, and controlling the pH to be 7.0;

inoculating heat-resistant Bacillus strain XJT9503 seed liquid (density of 3 × 10) in an amount of 3%⁹cfu/ml), culturing for 48h, and inoculating Clostridium thermocellum ATCC27405 seed solution (density 3X 10) according to 3% of inoculation amount⁹cfu/ml), continuously culturing for 96h, centrifuging for 3min at 5000rpm by a high-speed disc centrifuge, and collecting precipitate and upper-layer liquid;

the precipitate was stirred at 1 g: adding 0.6M hydrochloric acid solution at a ratio of 5ml, performing microwave-assisted hydrolysis at 100 ℃ for 1h with microwave power of 440W, adding AS.1398 neutral protease for hydrolysis for 18h, wherein the hydrolysis temperature is 40 ℃, the pH value is 7.5, the enzyme addition amount is 400U/ml, filtering with a filter screen with a pore size of 50 meshes to remove insoluble substances after hydrolysis is completed, and collecting supernatant;

mixing the supernatant liquid and the supernatant liquid according to the volume ratio of 3:1, evaporating and concentrating to one third, then carrying out steam treatment at 121 ℃ for 10min, and naturally cooling to room temperature to obtain a hydrolysate.

Example 3

1. The influence of the strain type and the compatibility on the hydrolysis of the corn bran.

The detection method comprises the following steps: the content of Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) is measured by a paradigm method; protein content determination: kjeldahl method.

Setting a control group;

control group 1: culturing for 144h by using heat-resistant bacillus only;

control group 2: culturing with Clostridium thermocellum only for 144 h;

control group 3: inoculating and culturing the two simultaneously for 144 h;

control group 4: culturing Aspergillus niger for 144 h;

experimental groups: inventive example 1.

As shown in figure 1, the cellulose degradation rate is low because heat-resistant bacillus is weak in cellulase production capacity and cannot effectively degrade cellulose, the cellulose degradation rate is reduced because heat-resistant bacillus is weak in protease production capacity and cannot completely degrade protein to release cellulose, and Aspergillus niger adopted in the prior art also has a similar problem, an experimental group is inoculated with heat-resistant bacillus firstly, can produce neutral protease by using starch as a carbon source and ammonium chloride as a nitrogen source under a high-temperature condition, hydrolyzes protein to release cellulose, and is favorable for hydrolyzing protein and cellulose under the high-temperature condition, and is inoculated with Clostridium thermocellum after the protein and the cellulose are completely separated, and a nitrogen source generated by the cellulose and the hydrolyzed protein is utilized to highly produce C1, Cx glucanase and β -1,4 glycosidase, so that the cellulose and hemicellulose can be hydrolyzed into glucose, the content of reducing sugar in a treatment solution is increased along with the increase of the treatment time, as shown in figure 2h, the total treatment time is 144h, the content of the degradable cellulose is highest, the reducing sugar is increased, and the degradation time is reduced, and the reducing sugar is maintained.

2. The content of the main components of the corn bran after pretreatment is shown in the table 1:

TABLE 1

Components	Protein	Starch	Total cellulose
				Pretreated corn bran	10.6％	28.3％	57.9％

The detection of the main components in the hydrolysate after evaporation concentration is shown in table 2.

TABLE 2

Components	Reducing sugar g/L	Protein content g/L
			Example 1	56.1	64.9

As shown in Table 2, the corn bran hydrolysate has high content of reducing sugar and available protein and proper carbon-nitrogen ratio, and can be applied to a culture medium for preparing threonine by microbial fermentation through fine adjustment.

Example 4

Preparing a culture medium by using hydrolysate:

the hydrolysate of the invention is adopted to replace part of components of the threonine fermentation medium:

glucose and yeast powder in the culture medium are replaced by calculating the content of reducing sugar and protein in the hydrolysate.

Control medium:

60g/L of sucrose, 30g/L of glucose, 20g/L of yeast powder, 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。

Improving a culture medium:

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。

The fermentation process by utilizing the fermentation medium comprises the following steps:

inoculating the seed liquid of Escherichia coli engineering bacteria TRFC into a fermentation tank containing a fermentation medium according to the inoculation amount of 2% for fermentation, and inoculating the seed liquid to the fermentation tank with the inoculation density OD₆₀₀At the temperature of 0.5 ℃, the temperature of 36 ℃, the stirring speed of 300rpm, controlling the dissolved oxygen amount to be 20% by aeration and stirring, defoaming by using a foam killer, stopping fermentation for 40 hours, and collecting threonine fermentation liquor; in the fermentation process: controlling the sugar content to be 2-3% by feeding 50% of sucrose solution until the fermentation is finished;

the pH was controlled to 7.0 by feeding 20% ammonia until the end of the fermentation.

Through detecting the yield of threonine in fermentation liquor, the acid yield of the improved culture medium is higher and reaches 125.8g/L, while the yield of the contrast culture medium is 116.2g/L, so that the hydrolysate prepared by the method is more beneficial to fermentation, and the fermentation cost can be reduced.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

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；

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

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:

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.