阅读说明：本技术 一种利用核黄素废液发酵生产赤霉素a3的方法 (Method for producing gibberellin A by fermentation of riboflavin waste liquid3Method (2) ) 是由 陆建卫 祝金山 吴烨飞 沈波 陆春锋 冯佩杰 江红杰 俞海燕 于 2020-07-30 设计创作，主要内容包括：本发明公开了一种利用核黄素废液发酵生产赤霉素A<Sub>3</Sub>的方法,属于微生物发酵技术领域,其技术方案要点是发酵培养,将种子液接种至由处理后的核黄素废液和必需营养物质配制成的发酵培养基中发酵,利用核黄素废液发酵生产赤霉素A<Sub>3</Sub>,既解决核黄素生产过程中的废液处理问题,同时也达到降低赤霉素A<Sub>3</Sub>的生产成本的效果。(The invention discloses a method for producing gibberellin A by fermentation of riboflavin waste liquid 3 The method belongs to the technical field of microbial fermentation, and the key point of the technical scheme is fermentation culture, the seed liquid is inoculated into a fermentation culture medium prepared by treated riboflavin waste liquid and essential nutrient substances for fermentation, and the gibberellin A is produced by utilizing the fermentation of the riboflavin waste liquid 3 Not only solves the problem of waste liquid treatment in the production process of riboflavin, but also achieves the aim of reducing gibberellin A 3 The production cost of (a).)

1. Method for producing gibberellin A by fermentation of riboflavin waste liquid₃The method is characterized by comprising the following preparation steps: and (4) fermentation culture, namely inoculating the seed liquid into a fermentation culture medium prepared from the treated riboflavin waste liquid and essential nutrients for fermentation.

2. The method for producing gibberellin A according to claim 1, wherein the method comprises fermentation of riboflavin waste solution₃The method of (1), wherein the method of treating the riboflavin waste liquid is as follows：

Putting the riboflavin waste liquid into a fermentation tank, preserving the heat for 30-60min to inactivate mycelium under the condition that the temperature is 70 +/-5 ℃, centrifuging for 10-30min under the condition of 5000 plus-8000 rpm to obtain a light phase containing the mycelium, and adjusting the pH of the obtained light phase to 5.5-6.5 by using a pH regulator to obtain the treated riboflavin waste liquid.

3. The method for producing gibberellin A according to claim 2, wherein the fermentation of the riboflavin waste solution is performed₃The method of (2), characterized by: the pH regulator is 20-30% sodium hydroxide solution.

4. The method for producing gibberellin A according to claim 1, wherein the method comprises fermentation of riboflavin waste solution₃The method of (2), characterized by: the riboflavin waste liquid is waste liquid generated in an extraction stage in the process of producing riboflavin by fermentation.

5. The method for producing gibberellin A according to claim 1, wherein the method comprises fermentation of riboflavin waste solution₃The method of (2), characterized by: the method comprises seed culture before fermentation culture, wherein the seed culture comprises two-stage culture:

first-order seed culture: picking the red-eared mold mutant strain into a shake flask filled with seed culture solution, and culturing for 40-60h under the conditions of 180-280rmp and 30 +/-1 ℃ to obtain first-level seed solution;

secondary seed culture: inoculating the first-stage seed solution to a second-stage seed tank at a volume ratio of 0.5-1.0%, and culturing at 30 + -1 deg.C, dissolved oxygen of 20-70%, and natural pH for 40-60h to obtain second-stage seed solution.

6. The method for producing gibberellin A according to claim 5, wherein the fermentation of the riboflavin waste solution is performed₃The method of (2), characterized by: in the fermentation culture, the secondary seed liquid is inoculated in a fermentation culture medium according to the volume proportion of 5-10%, and cultured for 7-8 days under the conditions of 30 +/-1 ℃, dissolved oxygen of 20-60% and natural pH to obtain fermentation liquid.

7. According to claim 5The method for producing gibberellin A by fermenting riboflavin waste liquid₃The method is characterized in that the culture solution of the two-stage culture comprises the following components: 20-30g/L of corn starch, 5-10g/L of cane sugar and 20-30g/L, KH of peanut powder₂PO₄1-2g/L、MgSO₄1-2g/L。

8. The method for producing gibberellin A according to claim 1, wherein the method comprises fermentation of riboflavin waste solution₃The method of (2), characterized by: the fermentation culture medium comprises 400-600g/L of riboflavin waste liquid, 50-120g/L of starch and 5-10g/L, KH of peanut powder₂PO₄0.2-0.5g/L、K₂SO₄0.2-0.5g/L、MgSO₄0.1-0.2g/L。

Technical Field

The invention relates to the technical field of microbial fermentation, in particular to a method for producing gibberellin A by fermentation of riboflavin waste liquid₃The method of (1).

Background

Gibberellin A₃Is an endogenous plant regulating substance, has obvious effects on regulating and controlling the elongation of stems of plants, seed germination, bolting and fruit setting and the like, and is widely applied to the field of planting and production of economic crops. Industrial gibberellin A₃The red-eared mold is usually produced by liquid fermentation, and a fermentation culture medium of the red-eared mold mainly comprises organic nitrogen carbon sources such as corn starch, peanut cake powder and the like, so that the production cost is high. How to optimize the culture medium and reduce the production cost is that all gibberellin A₃A problem that the manufacturing enterprise must face.

Riboflavin also known as vitamin B₂The vitamin is a water-soluble vitamin necessary for human and animals, has obvious effect on inhibiting digestive tract diseases and nervous system diseases (such as Parkinson's disease, migraine, multiple sclerosis and the like), and is widely applied to the fields of clinical treatment, feed industry, cosmetic industry, food industry and the like. Riboflavin is mainly produced by a microbial fermentation method, a large amount of waste liquid is generated in the extraction stage, and with the increasing environmental protection requirement, how to treat the waste liquid can meet the emission requirement, which is a problem to be solved urgently in riboflavin production enterprises. The riboflavin waste liquid contains carbon nitrogen source and inorganic substance which are not used up in partial fermentationSalt and residual mycelium cause high viscosity of the riboflavin waste liquid, great treatment difficulty and high production cost of the riboflavin.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for producing gibberellin A by fermentation of riboflavin waste liquid₃The method for producing gibberellin A by fermentation of riboflavin waste liquid₃Not only solves the problem of waste liquid treatment in the production process of riboflavin, but also achieves the aim of reducing gibberellin A₃The production cost of (a).

The technical purpose of the invention is realized by the following technical scheme:

method for producing gibberellin A by fermentation of riboflavin waste liquid₃The method comprises the following preparation steps: and (4) fermentation culture, namely inoculating the seed liquid into a fermentation culture medium prepared from the treated riboflavin waste liquid and essential nutrients for fermentation.

The invention is further set as the method for treating the riboflavin waste liquid comprises the steps of placing the riboflavin waste liquid in a fermentation tank, carrying out heat preservation for 30-60min to inactivate mycelium under the condition that the temperature is 70 +/-5 ℃, then carrying out centrifugation for 10-30min under the condition of 5000 plus-8000 rpm to obtain a light phase containing the mycelium, and adjusting the pH of the obtained light phase to 5.5-6.5 by using a pH regulator to obtain the treated riboflavin waste liquid.

By adopting the technical scheme, the treated riboflavin waste liquid is used as gibberellin A₃The fermentation culture solution is beneficial to reducing the treatment cost of the riboflavin waste liquid, can fully utilize organic nitrogen sources and carbon sources in the riboflavin waste liquid, and reduces the amount of other nutrient substances in the fermentation culture medium, thereby effectively reducing the gibberellin A content₃The experiment proves that when the riboflavin waste liquid is used as the component of the fermentation culture medium, the gibberellin A finally obtained is₃The yield of the method is higher than that of the method for producing the gibberellin A by normal fermentation₃The yield of (a).

In addition, after the riboflavin waste liquid is treated, the step of adjusting the pH of the fermentation medium is not needed to be added in the fermentation process, so that the gibberellin fermentation can be carried out under the condition of natural pH when other components are added into the fermentation medium, and convenience is brought to the operation.

The invention is further provided that the pH regulator is a 20-30% sodium hydroxide solution.

By adopting the technical scheme, after the pH regulator adopts the sodium hydroxide solution, the treatment on the riboflavin waste liquid can be achieved, and meanwhile, the nitrogen content in the riboflavin waste liquid can not be influenced, so that the later fermentation is guaranteed.

The invention is further configured that the riboflavin waste liquid is waste liquid generated in an extraction stage in the process of producing riboflavin by fermentation.

The invention is further configured that the fermentation culture comprises seed culture before the fermentation culture, and the seed culture comprises two stages of culture:

first-order seed culture: taking the red-eared mold mutant strain to a shake flask filled with a seed culture solution, and culturing for 40-60h under the conditions of 250rmp and 30 +/-1 ℃ to obtain a primary seed solution; a mutant strain of Gibberella fujikuroi of the present application (A)Fusarium fujikuroi GA-347) Purchased from China center for type culture Collection, wherein, the mutant of Gibberella fujikuroi: (Fusarium fujikuroi GA-347) Deposited in China center for type culture Collection, address: wuhan university, Wuhan, China, zip code 430072, preservation number: CCTCC NO: m2019378, date of deposit 2019, 5 months and 20 days;

secondary seed culture: inoculating the first-stage seed solution to a second-stage seed tank at a volume ratio of 0.5-1.0%, and culturing at 30 + -1 deg.C, dissolved oxygen of 20-70%, and natural pH for 40-60h to obtain second-stage seed solution.

The invention is further set that in the fermentation culture, the secondary seed liquid is inoculated in the fermentation culture medium according to the volume proportion of 5-10%, and the fermentation culture medium is cultured for 7-8 days under the conditions of 30 +/-1 ℃, dissolved oxygen of 20-60% and natural pH value, so as to obtain the fermentation liquid.

The invention is further configured that the culture solution for the two-stage culture comprises the following components: 20-30g/L of corn starch, 5-10g/L of cane sugar and 20-30g/L, KH of peanut powder₂PO₄1-2g/L、MgSO₄1-2g/L。

The invention is further set that the fermentation medium comprises 600g/L of riboflavin waste liquid 400-₂PO₄0.2-0.5g/L、K₂SO₄0.2-0.5g/L、MgSO₄0.1-0.2g/L。

In conclusion, the invention has the following beneficial effects:

1. the application utilizes the riboflavin waste liquid as the production of gibberellin A₃The main components of the fermentation culture medium not only reduce the discharge amount of the riboflavin waste liquid, save the energy consumed by sewage treatment and reduce the production cost of the riboflavin, but also utilize the riboflavin waste liquid to ferment and produce the gibberellin A₃Later, the gibberellin A is effectively reduced₃The production cost of (2);

2. because residual sugar contained in the riboflavin waste liquid can meet the demand of the carbon source in the early growth process of the single colony, the hydrolysis step of gibberellin A3 fermentation culture medium starch and rice flour is omitted, and the gibberellin A is simplified₃The production process saves the cost;

3. the yield of the gibberellin A3 in the application is 2458mg/L at most, while the gibberellin A in the existing production₃The yield of (5) was 2123 mg/L.

Drawings

FIG. 1 is a high performance liquid chromatogram of a standard;

FIG. 2 is a high performance liquid chromatogram of example 1 of the present invention;

FIG. 3 is a high performance liquid chromatogram of the comparative example.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

The riboflavin waste liquid used in the application is the riboflavin waste liquid obtained in industrial production, wherein the riboflavin waste liquid is the riboflavin waste liquid obtained when the residual carbon content is about 1% when the riboflavin production is stopped.

The concentration of the standard sample after 50-fold dilution was 65. mu.g/mL.

The standard chromatogram is shown in FIG. 1, gibberellin A₃The integration results of (a) are shown in table 1.

TABLE 1 Table of integration results of standard gibberellins

Peak #	Retention time	Area of	Height	Area%	Theoretical plate #
						1	6.033	1395930	126308	100.000	7017.251
Total of	——	1395930	126308	100.000	——

The yield of the comparative example was calculated by the following formula: yield of control = (control peak area/standard peak area) × concentration after dilution of standard × dilution factor.

Since the peak area ratio is proportional to the yield ratio, therefore: the yield calculation formula for example 1 is: example yield = (example sample peak area/control peak area) × control yield.