阅读说明：本技术 一种提高赖氨酸发酵效率的方法 (Method for improving lysine fermentation efficiency ) 是由 董力青 唐永强 白红兵 汲广习 关健 韩杨 于晶 李航 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,公开了一种提高赖氨酸发酵效率的方法,其包括如下步骤：将产赖氨酸的黄色短杆菌按照4-8%的接种量接入到发酵培养基中,发酵温度：0-18h为31℃,18h-结束为34℃,通风比1:0.6-0.8,搅拌转速200-400r/min,发酵总时间为50-60h；发酵过程中,通过流加葡萄糖溶液维持残糖含量在5-10g/L,通过流加硫酸铵溶液维持氨氮含量为1-2g/L,流加氨水控制pH7.0-7.2,流加消泡剂消泡。本发明方法提高了赖氨酸产酸效率和糖酸转化率。(The invention belongs to the technical field of amino acid fermentation, and discloses a method for improving lysine fermentation efficiency, which comprises the following steps: inoculating the lysine-producing brevibacterium flavum into a fermentation medium according to the inoculation amount of 4-8%, wherein the fermentation temperature is as follows: the temperature of 0-18h is 31 ℃, the temperature of 18 h-end is 34 ℃, the ventilation ratio is 1:0.6-0.8, the stirring speed is 200-; in the fermentation process, the residual sugar content is maintained at 5-10g/L by feeding glucose solution, the ammonia nitrogen content is maintained at 1-2g/L by feeding ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming. The method improves the acid production efficiency and the sugar acid conversion rate of the lysine.)

1. A method for improving the fermentation efficiency of lysine, comprising the steps of:

inoculating the lysine-producing brevibacterium flavum into a fermentation medium according to the inoculation amount of 4-8%, wherein the fermentation temperature is as follows: the temperature of 0-18h is 31 ℃, the temperature of 18 h-end is 34 ℃, the ventilation ratio is 1:0.6-0.8, the stirring speed is 200-; in the fermentation process, the residual sugar content is maintained at 5-10g/L by feeding glucose solution, the ammonia nitrogen content is maintained at 1-2g/L by feeding ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming.

2. The method of claim 1, wherein the fermentation medium comprises a mycoprotein stillage co-hydrolysate.

3. The method according to claim 1 or 2, wherein the fermentation medium comprises the following components: the thallus protein wine lees combined hydrolysate, glucose, molasses, ammonium sulfate, sodium citrate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, ferrous sulfate heptahydrate, and vitamin B ₁And biotin.

4. The method of claim 3, wherein the fermentation medium comprises the following components: 200ml/L of thallus protein and distillers' grains combined hydrolysate, 40g/L of glucose, 12g/L of molasses, 4g/L of ammonium sulfate, 5g/L of sodium citrate, 10g/L of potassium dihydrogen phosphate, 0.8g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate tetrahydrate, 0.02g/L of ferrous sulfate heptahydrate, and vitamin B ₁0.01g/L and biotin 0.5 mg/L.

5. The method of claim 4, wherein the mycoprotein distillers' grains combined hydrolysate is prepared by the following process: preparing primary hydrolysate of mycoprotein in the step (1), preparing primary hydrolysate of corn vinasse in the step (2), and performing combined enzymolysis in the step (3).

6. The method of claim 5, wherein the mycoprotein distillers' grains combined hydrolysate is prepared by the following process:

preparing primary corn vinasse hydrolysate: adding corn vinasse into 3 times of water by weight, heating to 60 ℃, performing microwave treatment, adjusting the pH of the solution to 4.5, adding cellulase and glucoamylase, and performing enzymolysis for 12-24 hours to obtain primary corn vinasse hydrolysate;

step (3) combined enzymolysis: mixing the primary hydrolysate of the mycoprotein and the primary hydrolysate of the corn vinasse according to the volume ratio of 1:1, adjusting the pH to 3.0 and the temperature to 40 ℃, adding acid protease for 5-10h, adjusting the pH to 7.0 and the temperature to 50 ℃, adding serrapeptase for 5-10h, inactivating enzyme, filtering through a filter screen, removing floccules and insoluble particles, and collecting filtrate.

7. The method of claim 6, wherein the sonication parameters are: each time of ultrasonic treatment is 6s, the interval is 3s, the total ultrasonic treatment time is 90-270s, and the ultrasonic frequency is 25 kHz.

8. The method according to claim 6, wherein the parameters of the microwave treatment are: the microwave treatment time is 10-20min, and the microwave power is 500W.

9. The method of claim 6, wherein the cellulase and glucoamylase are added in amounts of 5 and 3 million U/L, respectively.

10. The method according to claim 6, wherein the amount of the acidic protease added is 15 ten thousand U/L and the amount of the serrapeptase added is 10 ten thousand U/L.

Technical Field

The invention belongs to the technical field of amino acid fermentation, and particularly relates to a method for improving lysine fermentation efficiency.

Background

Lysine, also known as L-lysine, lysine and the like, is one of the most important essential amino acids in human and animal nutrition, and currently, the global lysine yield reaches over 240 ten thousand tons, which is second to glutamic acid and becomes the second largest amino acid variety in the global yield. The lysine residue is a new product of mycoprotein feed produced by using lysine waste liquid as a raw material through microbial fermentation, belongs to the field of treatment of biological fermentation liquid waste liquid, and is accompanied with the production of lysine. Lysine waste contains a large amount of: mycoprotein, COD, BOD, reducing sugar, lysine, alanine, aspartic acid, leucine, etc., and trace inorganic salt such as P, K, Ca, etc.

Lysine-derived products have been developed earlier abroad. A series of studies on the synthesis, isolation, and application of lysine have been conducted in countries such as the United states, France, Germany, and Japan. Especially, Japan has certain advantages in lysine production and waste liquid treatment, and the Aomoto Sorbase company is the biggest lysine production enterprise in the world. The lysine produced by the company occupies more than 60 percent of the global market, and the lysine production capacity of the gourmet European company is 3.5 ten thousand tons. The production capacity of Heetlean corporation is l ten thousand tons. The company plans to increase its lysine productivity to 5 million tons in order to increase market share. Every 10 ten thousand tons of lysine are produced, 1 ten thousand tons of theoretical mycoprotein can be recovered. Also, along with the increasing market competition, the bad lysine market conditions make the reduction of the production cost become the research direction of various large enterprises.

The mycoprotein is a by-product in the production process of amino acid, is a cell protein prepared by separating and drying amino acid fermented by amino acid producing bacteria, and is found to contain various nutrient substances such as rich protein, nucleic acid, saccharides, vitamins and the like by measuring the composition of the amino acid mycoprotein. The product with high added value can be developed by extracting the thallus eggs. At present, most amino acid manufacturers produce mycoprotein feed by using amino acid mycoprotein, so that the production cost of amino acid is reduced, the waste of available resources is avoided, certain economic benefit is obtained, and the industrial added value is still low. The applicant's prior patent technology "CN 201810634938.7, preparation process of lysine fermentation medium", comprises the following steps:

sequentially adding corn bran processed product, corn steep liquor, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate heptahydrate, magnesium sulfate heptahydrate, calcium acetate, mannitol, biotin and vitamins into water, stirring uniformly, adjusting pH to 6.8, keeping temperature at 121 ℃ for 20min, sterilizing, and cooling to obtain the final product. The corn bran treatment product is used as a carbon source in the process, so that the problems of storage and waste of the corn bran in fermentation enterprises are effectively solved, the fermentation cost is reduced, and the enterprise profits are improved; however, this medium is suitable for Corynebacterium glutamicum and is not suitable for lysine fermentation by strains such as Escherichia coli and Brevibacterium flavum. In the literature, "response surface optimization of a fermentation medium of an L-lysine-producing strain, the culture medium is optimized in 2012 by modern food science and technology on the basis of seven single-factor experiments of researching glucose, ammonium sulfate, bean cake hydrolysate, KH2PO 4.3H 2O, MgSO 4.7H 2O, FeSO 4.7H 2O and MnSO 4.H 2O, and the method is suitable for fermentation of brevibacterium flavum, but the defects of high cost and low fermentation efficiency of the fermentation culture medium still exist.

Disclosure of Invention

The invention aims to overcome the defects of low additional value of mycoprotein, high cost of a lysine fermentation culture medium, low lysine efficiency and the like in the prior art, and provides a method for improving lysine fermentation efficiency.

The invention is realized by the following technical scheme.

A method for improving the fermentation efficiency of lysine, comprising the steps of:

inoculating the lysine-producing brevibacterium flavum into a fermentation medium according to the inoculation amount of 4-8%, wherein the fermentation temperature is as follows: the temperature of 0-18h is 31 ℃, the temperature of 18 h-end is 34 ℃, the ventilation ratio is 1:0.6-0.8, the stirring speed is 200-; in the fermentation process, the residual sugar content is maintained at 5-10g/L by feeding glucose solution, the ammonia nitrogen content is maintained at 1-2g/L by feeding ammonium sulfate solution, the pH value is controlled at 7.0-7.2 by feeding ammonia water, and defoaming agent is fed for defoaming.

Further, the fermentation medium comprises mycoprotein distillers' grains combined hydrolysate.

Further, the fermentation medium comprises the following components: the thallus protein wine lees combined hydrolysate, glucose, molasses, ammonium sulfate, sodium citrate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, ferrous sulfate heptahydrate, and vitamin B ₁And biotin.

Further, the fermentation medium comprises the following components: 200ml/L of thallus protein and distillers' grains combined hydrolysate, 40g/L of glucose, 12g/L of molasses, 4g/L of ammonium sulfate, 5g/L of sodium citrate, 10g/L of potassium dihydrogen phosphate, 0.8g/L of magnesium sulfate heptahydrate, 0.02g/L of manganese sulfate tetrahydrate, 0.02g/L of ferrous sulfate heptahydrate, and vitamin B ₁0.01g/L and biotin 0.5 mg/L.

Further, the mycoprotein distillers' grains combined hydrolysate is prepared by the following process: preparing primary hydrolysate of mycoprotein in the step (1), preparing primary hydrolysate of corn vinasse in the step (2), and performing combined enzymolysis in the step (3).

Preferably, the mycoprotein distillers' grains combined hydrolysate is prepared by the following process:

preparing primary hydrolysate of mycoprotein: grinding and crushing the mycoprotein, adding the grinded mycoprotein into 10 times of hydrochloric acid solution with the concentration of 0.6M, uniformly stirring, then placing the stirred mycoprotein in a high-speed shearing machine to shear for 60-120s at the speed of 8000-;

preparing primary corn vinasse hydrolysate: adding corn vinasse into 3 times of water by weight, heating to 60 ℃, performing microwave treatment, adjusting the pH of the solution to 4.5, adding cellulase and glucoamylase, and performing enzymolysis for 12-24 hours to obtain primary corn vinasse hydrolysate;

step (3) combined enzymolysis: mixing the primary hydrolysate of the mycoprotein and the primary hydrolysate of the corn vinasse according to the volume ratio of 1:1, adjusting the pH to 3.0 and the temperature to 40 ℃, adding acid protease for 5-10h, adjusting the pH to 7.0 and the temperature to 50 ℃, adding serrapeptase for 5-10h, inactivating enzyme, filtering through a filter screen, removing floccules and insoluble particles, and collecting filtrate.

More preferably, the ultrasonic treatment parameters are: each time of ultrasonic treatment is 6s, the interval is 3s, the total ultrasonic treatment time is 90-270s, and the ultrasonic frequency is 25 kHz.

More preferably, the parameters of the microwave treatment are: the microwave treatment time is 10-20min, and the microwave power is 500W.

More preferably, the cellulase and glucoamylase are added in an amount of 5 ten thousand U/L and 3 ten thousand U/L, respectively.

More preferably, the addition amount of the acid protease is 15 ten thousand U/L, and the addition amount of the serrapeptase is 10 ten thousand U/L.

Compared with the prior art, the research starting point and the obtained beneficial effects of the invention mainly comprise but are not limited to the following aspects:

the bacterial protein hydrolysate is a better nitrogen source, the vinasse hydrolysate can be used as a nitrogen source and a carbon source, the nitrogen source and the carbon source are jointly hydrolyzed, and the proper proportion is selected, so that the nitrogen source and the carbon source used by a lysine fermentation culture medium can be replaced, and nutrient factors and inorganic mineral substances can be provided, thereby being beneficial to the fermentation of the brevibacterium flavum; however, the combined hydrolysis method of the two is difficult and a technical problem to be overcome.

The mycoprotein contains a large amount of microbial protein, but the wall breaking treatment is needed firstly; the main components of the vinasse are starch, cellulose and vegetable protein, and the cellulose is degraded firstly, so that the protein structure is looser, and the subsequent proteolysis is facilitated; thus, the initial steps of selection are performed separately and then combined for proteolysis to obtain a hydrolysate that is nutritionally balanced.

The invention adopts a microwave-assisted enzymolysis mode, can reduce the combination degree of cellulose, starch and protein, improves the leaching rate of each component, is beneficial to subsequent hydrolysis reaction, reduces the use amount of enzyme, and degrades the cellulose and the starch into reducing sugar which can be utilized by microorganisms, thereby achieving the purpose of providing a carbon source.

The invention adopts high-speed shearing and ultrasonic treatment under the condition of dilute hydrochloric acid, can accelerate the breaking of cell walls of thalli, loosen protein, change the internal structure, improve the solubility and the hydrophilicity of the protein, increase the mass transfer rate, reduce the viscosity and be beneficial to subsequent enzymolysis.

According to different characteristics of the enzymes, the invention adopts a mode of sequentially carrying out enzymolysis on the acid protease and the Serratin peptidase, both the acid protease and the Serratin peptidase are in an optimal enzymolysis reaction system, and the hydrolysis degree is obviously superior to that of the acid protease or the Serratin peptidase which is singly used.

According to the research on the lysine metabolism process distribution, the reduction of the metabolism flow of the TCA cycle is beneficial to the accumulation of lysine, and the sodium citrate can generate a certain inhibition effect on the citrate dehydrogenase which is a key enzyme in the TCA cycle, so that the accumulation of the lysine is promoted by reducing the metabolism flow of the TCA cycle; since the accumulation of lysine has already started from the early stage of fermentation, it was chosen to add a certain amount of sodium citrate to the fermentation medium.

Drawings

FIG. 1: the effect of the enzyme combination on the degree of proteolysis;

FIG. 2: influence of the addition amount of sodium citrate on the yield of lysine;

FIG. 3: influence of the addition amount of sodium citrate on the conversion rate of sugar acid.

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.