阅读说明：本技术 一种降低l-组氨酸发酵液粘度的方法 (Method for reducing viscosity of L-histidine fermentation liquor ) 是由 李闯 曹华杰 李静 于 2020-12-31 设计创作，主要内容包括：本发明涉及一种降低L-组氨酸发酵液粘度的方法,属于发酵工程技术领域。本发明在发酵培养基中添加高浓度氯化钾,所述氯化钾浓度为5-15g/L,然后在发酵过程中补入0.1-1.0g/L灭菌胰蛋白酶溶液进行混合发酵。本发明在发酵过程中解决料液粘稠问题,通过添加高浓度氯化钾提高菌体渗透液,补加胰蛋白酶不但可以降低发酵液中胞外蛋白多糖的含量,降低粘度改善溶解氧而且水解蛋白后生成多种氨基酸又可以被菌体再次利用,有效提高L-组氨酸发酵单位,由原来30g/L提高到45g/L。(The invention relates to a method for reducing viscosity of L-histidine fermentation liquor, belonging to the technical field of fermentation engineering. According to the invention, high-concentration potassium chloride is added into a fermentation culture medium, wherein the concentration of the potassium chloride is 5-15g/L, and then 0.1-1.0g/L of sterile trypsin solution is supplemented in the fermentation process for mixed fermentation. The invention solves the problem of feed liquid viscosity in the fermentation process, improves the thallus penetrating fluid by adding high-concentration potassium chloride, and adds trypsin, so that the content of extracellular proteoglycan in the fermentation liquid can be reduced, the viscosity is reduced, the dissolved oxygen is improved, a plurality of amino acids are generated after protein hydrolysis, the amino acids can be reused by the thallus, the L-histidine fermentation unit is effectively improved, and the L-histidine fermentation unit is improved to 45g/L from the original 30 g/L.)

1. A method for reducing the viscosity of L-histidine fermentation liquor, which adopts serratia marcescens to ferment and produce L-histidine and is characterized in that: adding 5-15g/L potassium chloride into fermentation culture medium, adding sterile trypsin solution during fermentation culture to make the final concentration of trypsin be 0.1-1.0g/L, and mixing and fermenting.

2. The method for reducing the viscosity of an L-histidine fermentation broth as claimed in claim 1, wherein: the timing of supplementing the sterile trypsin solution is when the fermentation culture is carried out to the middle stage.

3. The method for reducing the viscosity of an L-histidine fermentation broth as claimed in claim 2, wherein: the timing of addition of the sterile trypsin solution was when the fermentation culture proceeded for 15 h.

4. The method for reducing the viscosity of an L-histidine fermentation broth as claimed in claim 1, wherein: the final concentration of the potassium chloride is 10g/L, and the final concentration of the trypsin is 0.5 g/L.

Technical Field

The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for reducing viscosity of an L-histidine fermentation solution.

Background

L-Histidine (L-Histindine) is a basic amino acid with an imidazole nucleus in its molecule, chemically known as L-a-amino-p-imidazole propionic acid. L-histidine has various physiological functions, is widely used in the industries of medicine, feed and food, and particularly has an increasingly important role in medical research. At present, L-histidine, L-tryptophan, L-arginine and L-serine are amino acid varieties which are urgently needed in the market, and are one of four amino acids which influence the national goal of realizing all localization of amino acid infusion raw materials in China.

At present, the domestic industrial production of L-histidine is mainly extracted from pig blood meal hydrolysate, and a factory for producing L-histidine by adopting a microbial fermentation method on a large scale does not exist in China. The yield of histidine extracted from the pig blood powder is low, the extraction difficulty is high, the process is complex, the production cost is high, and the environmental cost is extremely high. The method for producing the L-histidine by adopting the microbial fermentation method has the advantages of mild production control conditions, low production cost, no pollution to the environment and small extraction difficulty, and the development of a new control process for improving the L-histidine fermentation unit has great significance for improving the international competitiveness of China.

The L-histidine fermentation is carried out by utilizing the serratia, as the bacterial concentration increases, the thalli can generate proteoglycan byproducts and secrete the proteoglycan byproducts to the outside of cells, the fermentation liquor is increasingly viscous, the oxygen mass transfer is influenced, the utilization rate of dissolved oxygen is lower and lower, the growth rate of the L-histidine is slowed down, the fermentation period is prolonged, the sugar-acid conversion rate is lower, and the viscous fermentation liquor brings about a large fullness to the post-extraction, which is the bottleneck of the L-histidine fermentation produced by the serratia.

Disclosure of Invention

In order to solve various problems caused by the viscosity of fermentation liquor, the invention aims to provide a method for reducing the viscosity of an L-histidine fermentation liquor.

In order to achieve the purpose, the invention adopts the specific scheme that:

a method for reducing viscosity of L-histidine fermentation liquor comprises producing L-histidine by fermentation of Serratia marcescens, adding potassium chloride with final concentration of 5-15g/L into fermentation culture medium, simultaneously supplementing sterile trypsin solution during fermentation culture to make the final concentration of trypsin be 0.1-1.0g/L, and performing mixed fermentation.

Further, the timing of addition of the sterile trypsin solution is when the fermentation culture proceeds to the middle stage. Further, when the fermentation culture was carried out for 15 hours.

Further, the final concentration of the potassium chloride is 10g/L, and the final concentration of the trypsin is 0.5 g/L.

Has the advantages that:

the invention adopts serratia marcescens to ferment and produce L-histidine, adds high-concentration potassium chloride and replenishes sterile trypsin solution in the fermentation process, solves the problem of material liquid viscosity, improves thallus penetrating fluid by adding high-concentration potassium chloride, replenishes trypsin, can reduce the content of extracellular proteoglycan in fermentation liquor, reduces viscosity, improves dissolved oxygen, generates various amino acids after hydrolyzing protein, can be reused by thallus, effectively improves L-histidine fermentation unit, and is improved to 45g/L from the original 30 g/L. The unit yield is improved, the difficulty of post extraction is reduced, and the production cost is low.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1

A method for reducing viscosity of L-histidine fermentation liquor adopts serratia marcescens as a strain, firstly, the strain is activated on a slant, then the strain is inoculated in a seed culture medium, the inoculation amount is a test tube slant, and the strain is cultured for 12 hours at 34 ℃. Inoculating the mixture into a 50L automatic control fermentation tank containing a fermentation culture medium according to 15 percent of inoculation amount, controlling the fermentation temperature to be 34 ℃, introducing sterile air, properly adjusting the air volume, the rotating speed and the tank pressure, controlling the dissolved oxygen to be 25 +/-5 percent, automatically adding 25 percent ammonia water in a flowing manner to control the pH to be 7.0, adding a defoaming agent in a flowing manner to defoam, supplementing sterile 0.5g/L trypsin solution for 15 hours of fermentation to perform fermentation, controlling zero residual sugar, and finishing the fermentation until 45 hours.

The fermentation medium per liter contains the following components in percentage by weight: glucose 80g, (NH)₄)₂SO₄10g, 30mL of corn steep liquor, 20mL of cane molasses and KH₂PO₄ 2g，MgSO₄·7H₂O 0.5g，KCl 10g，MnSO₄·H₂O 0.05g，FeSO₄·7H₂O 0.05g，VB₁ 0.2mg，VH0.2mg。

The seed culture medium (g/L) contains the following components: glucose 80, (NH)₄)₂SO₄10, 30mL of corn steep liquor, 20mL of cane molasses and KH₂PO₄ 2，MgSO₄·7H₂O 0.5，MnSO₄·H₂O 0.05，FeSO₄·7H₂O 0.05，VB₁0.2mg，VH 0.2mg。

Control 1, control 2 and control 3 were set up in a similar manner as described above. Wherein, 10g/L KCl is not added into the fermentation culture medium in the control group 1, and sterile trypsin solution is not supplemented in the middle stage of fermentation, and the rest is the same as that in the embodiment 1; in the control group 2, 10g/L of KCl is added into the fermentation medium, sterile trypsin solution is not supplemented in the middle stage of fermentation, and the rest is the same as that in the example 1; control 3 was the same as example 1 except that 10g/L KCl was not added to the fermentation medium and 0.5g/L sterile trypsin solution was added at the time of 15 hours of fermentation. The L-histidine content of the fermentation broth is shown in Table 1 below, and the viscosity is shown in Table 2 below.

Table 1: the content of L-histidine is compared with the table when the tank is placed.

Group of	Experimental group	Control group 1	Control group 2	Control group 3
					Yield (g/L)	45	32	37	40
Rate of increase	40.63%		15.62%	25%

Table 2: and (5) detecting the viscosity of the fermentation liquor when the fermentation tank is placed.

Group of	Experimental group	Control group 1	Control group 2	Control group 3
					Viscosity (mPa/S)	2438	4358	3706	3089
Rate of decrease	44.06%		14.96%	29.12%

Example 2

The adopted bacterial strain is serratia marcescens, and the culture method comprises the following steps: inoculating the seeds into a seed culture medium, wherein the inoculation amount is a test tube inclined plane, and culturing for 12h at 34 ℃. Inoculating the mixture into a 50L automatic control fermentation tank containing a fermentation culture medium according to 15 percent of inoculation amount, controlling the fermentation temperature to be 34 ℃, introducing sterile air, properly adjusting the air volume, the rotating speed and the tank pressure, controlling the dissolved oxygen to be 25 +/-5 percent, automatically adding 25 percent ammonia water in a flowing manner to control the pH to be 7.0, adding a defoaming agent in a flowing manner to defoam, supplementing sterile 0.1g/L trypsin solution for 15 hours of fermentation to perform fermentation, controlling zero residual sugar, and finishing the fermentation until 45 hours.

Fermentation Medium (g/L) [ glucose 80, (NH)₄)₂SO₄10, 30mL of corn steep liquor, 20mL of cane molasses and KH₂PO₄2，MgSO₄·7H₂O 0.5，KCl 5，MnSO₄·H₂O 0.05，FeSO₄·7H₂O 0.05，VB₁0.2mg，VH0.2mg]。

Seed Medium (g/L) [ glucose 80, (NH)₄)₂SO₄10, 30mL of corn steep liquor, 20mL of cane molasses and KH₂PO₄2，MgSO₄·7H₂O 0.5，MnSO₄·H₂O 0.05，FeSO₄·7H₂O 0.05，VB₁0.2mg，VH0.2mg] 。

Three sets of controls were set up as in example 1, with the results shown in tables 3 and 4 below.

Table 3: the content of L-histidine is compared with the table when the tank is placed.

Group of	Experimental group	Control group 1	Control group 2	Control group 3
					Yield (g/L)	43	31	34	38
Rate of increase	46.67%		9.68%	22.58%

Table 4: and (5) detecting the viscosity of the fermentation liquor when the fermentation tank is placed.

Group of	Experimental group	Control group 1	Control group 2	Control group 3
					Viscosity (mPa/S)	3520	4420	4031	3923
Rate of decrease	20.36%		9.12%	11.24%

Example 3

This example was carried out in substantially the same manner as example 1 except that the concentration of KCl in the fermentation medium was 15g/L and that trypsin was added in an amount of 1.0g/L up to 15 hours of fermentation. The control group was set in the same manner as in example, and the results are shown in tables 5 and 6 below.

Table 5: the content of L-histidine is compared with the table when the tank is placed.

Group of	Experimental group	Control group 1	Control group 2	Control group 3
					Yield (g/L)	46	32	38	41
Rate of increase	43.75%		18.75%	28.13%

Table 6: and (5) detecting the viscosity of the fermentation liquor when the fermentation tank is placed.

Group of	Experimental group	Control group 1	Control group 2	Control group 3
					Viscosity (mPa/S)	2106	4386	3560	3012
Rate of decrease	51.98%		18.84%	31.33%

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.