阅读说明：本技术 一种发酵生产异柠檬酸的方法 (Method for producing isocitric acid by fermentation ) 是由 任轶锴 吴松海 马骏 于 2021-03-15 设计创作，主要内容包括：本发明提供了一种发酵生产异柠檬酸的方法,包括以下步骤：1)采用特殊的异柠檬酸发酵培养基原料制得培养基液体；2)将培养基液体进行灭菌处理；3)将无菌的培养基在发酵罐中冷却到30℃以下,接入异柠檬酸菌种,调节培养温度26℃-30℃,所述发酵罐通入净化空气进行有氧培养,发酵培养120h-160h得到发酵液,发酵过程使用氨水调节发酵液的pH至4.0-6.0,4)发酵液经过滤去除菌体,然后进行沉淀、酸溶、结晶、干燥,得到异柠檬酸产品。本发明通过优化有机复合氮源和发酵条件,控制发酵过程中pH,提高发酵单位,发酵结束后,异柠檬酸的含量达到125 g/L。与优化前相比,异柠檬酸产量提高了38.1%,异柠檬酸和柠檬酸的比例达到了8.2:1,易于后期提取及纯化。(The invention provides a method for producing isocitric acid by fermentation, which comprises the following steps: 1) preparing a culture medium liquid by adopting a special isocitric acid fermentation culture medium raw material; 2) sterilizing the culture medium liquid; 3) cooling a sterile culture medium to below 30 ℃ in a fermentation tank, inoculating an isocitric acid strain, adjusting the culture temperature to 26-30 ℃, introducing purified air into the fermentation tank for aerobic culture, performing fermentation culture for 120-160 h to obtain fermentation liquor, adjusting the pH of the fermentation liquor to 4.0-6.0 by using ammonia water in the fermentation process, 4) filtering the fermentation liquor to remove thalli, and then performing precipitation, acid dissolution, crystallization and drying to obtain an isocitric acid product. According to the invention, by optimizing the organic compound nitrogen source and the fermentation conditions, the pH value in the fermentation process is controlled, the fermentation unit is increased, and after the fermentation is finished, the content of the isocitric acid reaches 125 g/L. Compared with the prior art, the yield of the isocitric acid is improved by 38.1 percent, the ratio of the isocitric acid to the citric acid reaches 8.2:1, and the post-extraction and purification are easy.)

1. A method for producing isocitric acid by fermentation is characterized by comprising the following steps:

1) preparing a culture medium: preparing a culture medium liquid by adopting an optimized isocitric acid fermentation culture medium raw material containing enzymatic vegetable protein powder;

2) and (3) sterilization: sterilizing the culture medium liquid in a sterilizing device; killing microorganisms in the culture medium liquid without loss of nutrients;

3) inoculating and culturing: cooling the culture medium liquid after the aseptic treatment to below 30 ℃ in a fermentation tank, adding a proper amount of isocitric acid producing bacteria, adjusting the culture temperature to 26-30 ℃, introducing purified air into the fermentation tank for aerobic culture, fermenting for 60-120h, supplementing sterilized 40% glycerol solution at a constant speed, wherein the supplementing amount is 8-15% of the volume of the fermentation liquid, adjusting the pH of the fermentation liquid to 4.0-6.0 by using ammonia water in the fermentation process, and collecting the fermentation liquid after fermentation culture for 120-160 h;

4) filtering the fermentation liquor to remove thalli, and precipitating, acid dissolving, crystallizing and drying to obtain an isocitric acid product.

2. The fermentative production method of isocitric acid according to claim 1, characterized in that: the optimized isocitric acid fermentation medium containing the enzymatic vegetable protein powder comprises the following components: 0.1-0.5 g/L of yeast extract, 0.1-0.3 g/L of enzymolysis vegetable protein powder, 0.05-0.3 g/L of monopotassium phosphate, 0.05-0.3 g/L of magnesium sulfate heptahydrate, 40-60 g/L of glycerol and 0.02-0.06 g/L of itaconic acid, and the pH is adjusted to 6.0-7.0 before sterilization.

3. The fermentative production method of isocitric acid according to claim 1, characterized in that: the amount of the glycerol added in the step (3) is 32 g to 48g per liter of fermentation liquor.

4. The fermentative production method of isocitric acid according to claim 1, characterized in that: the time for supplementing the glycerol in the step (3) is 60-120 h.

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to a method for producing isocitric acid by fermentation.

Background

Citric acid is the first large acid in organic acid, 90% is obtained by microbial fermentation, and isocitric acid is a by-product which is difficult to remove in the citric acid fermentation process. The biosynthesis pathways of the two are roughly that sugars generate pyruvic acid through glycolysis and pentose phosphate pathways, the pyruvic acid generates acetyl CoA through oxidative decarboxylation and enters tricarboxylic acid circulation to generate citric acid, and the citric acid generates isocitric acid through the isomerization of aconitase. Research in the last two decades shows that isocitrate is increasingly used as a marker for drug chiral modeling, biological fluid anticoagulation and orange juice quality detection, and monopotassium isocitrate is used for several biochemical analyses (detection of aconitate hydratase, NAD-isocitrate dehydrogenase, NADP-isocitrate dehydrogenase, isocitrate lyase). With the increase of application scenes, the scale production of the isocitric acid which is generated as a byproduct in the past is more important.

However, the existing isocitric acid fermentation culture medium has many problems in the industrial production process, such as difficulty in complete metabolism of oil and fat, high extraction difficulty of citric acid content in fermentation liquor, low conversion rate of fermentation substrate and the like, and the quality and production cost of products are restricted. According to the invention, by optimizing the organic compound nitrogen source and the fermentation conditions, the yield of the isocitric acid is effectively improved, and the isocitric acid is easier to extract and purify in the later period.

Disclosure of Invention

The invention aims to provide a method for producing isocitric acid by fermentation, which uses enzymolysis plant protein powder and yeast extract compound organic nitrogen source as nitrogen source, and improves the utilization rate of the nitrogen source. The method uses glycerol as substrate instead of conventional heavy oil such as vegetable oil or liquid paraffin, and has the advantages of high substrate conversion rate and high substrate conversion rate.

The method has the advantages that 32 g-48 g of glycerol is supplemented into each liter of fermentation liquor in the fermentation period of 60 h-120 h, and the yield of a single tank batch is improved.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for producing isocitric acid by fermentation specifically comprises the following steps:

(1) preparing a culture medium: preparing a culture medium liquid by adopting an optimized isocitric acid fermentation culture medium raw material containing enzymatic vegetable protein powder;

(2) and (3) sterilization: sterilizing the culture medium liquid in a sterilizing device; killing microorganisms in the culture medium liquid without loss of nutrients;

(3) inoculating and culturing: cooling the sterile culture medium liquid to below 30 ℃ in a fermentation tank, inoculating an isocitric acid strain, adjusting the culture temperature to 26-30 ℃, introducing purified air into the fermentation tank for aerobic culture, fermenting for 60-120h, supplementing sterilized 40% glycerol solution at constant speed, wherein the supplement amount is 32-48 g/L of the volume of the fermentation liquid, performing fermentation culture for 120-160 h to obtain the fermentation liquid, and adjusting the pH of the fermentation liquid to 4.0-6.0 by using ammonia water in the fermentation process;

(4) filtering the fermentation liquor to remove thalli, precipitating, acid-dissolving, crystallizing and drying to obtain an isocitric acid product.

Preferably, in the step (1), the optimized isocitric acid fermentation medium containing the enzymatic hydrolyzed plant protein powder is as follows: 0.1-0.5 g/L of yeast extract, 0.1-0.3 g/L of enzymolysis vegetable protein powder, 0.05-0.3 g/L of monopotassium phosphate, 0.05-0.3 g/L of magnesium sulfate heptahydrate, 40-60 g/L of glycerol and 0.02-0.06 g/L of itaconic acid, and the pH is adjusted to 6.0-7.0 before sterilization.

Preferably, in step (3), the amount of glycerol to be supplemented is 32 g to 48g per liter of the fermentation broth.

Preferably, in the step (3), the glycerol is supplemented for 60-120 h.

Compared with the prior art, the invention has the beneficial effects that:

(1) the method has the advantages that the substrate in the isocitric acid fermentation production process is the traditional heavy oil such as vegetable oil or liquid paraffin, and the like, so that the problems of incomplete substrate utilization or conversion, difficult equipment cleaning, large extraction difficulty and the like exist;

(2) the method of the invention uses glycerol as a substrate instead of glycerol, thereby promoting the conversion rate of the substrate and improving the conversion rate of the substrate.

The specific implementation mode is as follows:

the technical solution of the present invention is described in detail by examples below. The following examples are intended to further illustrate the content of the invention, but do not limit the scope of protection of the invention.

The original protein powder and the equipment are commercially available, and the enzymatic hydrolysis plant protein powder is produced by Hebei Baoding flavor group food science and technology company Limited.

EXAMPLE 1 preparation of Isocitric acid by fermentation of Shake flasks

(1) Preparing a culture medium: preparing a culture medium liquid by adopting an optimized isocitric acid fermentation culture medium raw material containing enzymatic vegetable protein powder; the optimized isocitric acid fermentation medium containing the enzymatic vegetable protein powder comprises the following components: 0.3g/L of yeast extract, 0.2 g/L of enzymolysis vegetable protein powder, 0.1 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 60g/L of glycerol and 0.02 g/L of itaconic acid, and adjusting the pH to 6.5 before sterilization; mixing, fixing the volume, subpackaging, and bottling by 40mL for 250mL fermentation;

(2) and (3) sterilization: sterilizing the culture medium liquid in a sterilizing device; killing microorganisms in the culture medium liquid without loss of nutrients;

(3) inoculating and culturing: cooling the culture medium liquid after the killing treatment to be below 28 ℃ in a fermentation tank, inoculating an isocitric acid seed liquid under aseptic operation, inoculating 5% of the isocitric acid seed liquid, placing the culture medium liquid in a thermostatic chamber at 26-28 ℃ and a shaking flask machine at 220 rpm/min for fermentation and culture, fermenting for 60 hours, supplementing sterilized 40% glycerol solution, wherein the supplementing amount is 0.032 g/mL of the volume of the fermentation liquid, and performing fermentation and culture for 140 hours to obtain the fermentation liquid.

The yield of the isocitric acid prepared by the method is improved by 25 percent, and the ratio of the isocitric acid to the citric acid is improved by 56 percent.

The isocitrate producing strain is CICC 32287.

EXAMPLE 2 preparation of Isocitric acid by fermentation of Shake flasks

(1) Preparing a culture medium: preparing a culture medium liquid by adopting an optimized isocitric acid fermentation culture medium raw material containing enzymatic vegetable protein powder; the optimized isocitric acid fermentation medium containing the enzymatic vegetable protein powder comprises the following components: 0.4g/L of yeast extract, 0.2 g/L of enzymolysis vegetable protein powder, 0.2 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 40g/L of glycerol and 0.04 g/L of itaconic acid, and adjusting the pH to 6.5 before sterilization; mixing, fixing the volume, subpackaging, and bottling by 40mL for 250mL fermentation;

(2) and (3) sterilization: sterilizing the culture medium liquid in a sterilization device; killing bacteria in the culture medium liquid without losing nutrition;

(3) inoculating and culturing: cooling the culture medium liquid after the killing treatment to be below 28 ℃ in a fermentation tank, inoculating an isocitric acid seed liquid under aseptic operation, inoculating 5% of the seed liquid, placing the culture medium liquid in a thermostatic chamber at 26-28 ℃ and a shaking flask machine at 220rpm for fermentation culture, fermenting for 70h, supplementing sterilized 40% of glycerol solution, wherein the supplementing amount is 0.048g/ml of the volume of the fermentation liquid, and performing fermentation culture for 140h to obtain the fermentation liquid.

The yield of the isocitric acid prepared by the method is improved by 28 percent, and the ratio of the isocitric acid to the citric acid is improved by 87 percent.

The isocitrate producing strain is CICC 32287.

Example 310L fermentor preparation of isocitric acid

(1) Preparing a culture medium: preparing a culture medium liquid by adopting an optimized isocitric acid fermentation culture medium raw material containing enzymatic vegetable protein powder; the optimized isocitric acid fermentation medium containing the enzymatic vegetable protein powder comprises the following components: 0.4g/L of yeast extract, 0.2 g/L of enzymolysis vegetable protein powder, 0.2 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 40g/L of glycerol and 0.04 g/L of itaconic acid, and adjusting the pH to 6.5 before sterilization;

(2) and (3) sterilization: sterilizing the culture medium liquid in a sterilization device; killing bacteria in the culture medium liquid without losing nutrition;

(3) inoculating and culturing: cooling the culture medium liquid after the killing treatment to be below 28 ℃ in a fermentation tank, inoculating an isocitric acid strain, adjusting the culture temperature to be 26-28 ℃, introducing purified air into the fermentation tank for aerobic culture, fermenting for 60-120h, supplementing sterilized 40% glycerol solution at constant speed, wherein the supplementing amount is 40g/L of the volume of the fermentation liquid, performing fermentation culture for 140h to obtain fermentation liquid, and adjusting the pH of the fermentation liquid to be 4.5-5.5 by using ammonia water in the fermentation process;

(4) filtering the fermentation liquor to remove thalli, precipitating, acid-dissolving, crystallizing and drying to obtain an isocitric acid product.

The yield of the isocitric acid prepared by the method is improved by 32 percent compared with that of the isocitric acid prepared by the same process, and the proportion of the isocitric acid to the citric acid is improved by 96 percent.

The isocitrate producing strain is CICC 32287.

Example 410L fermentor preparation of isocitric acid

(1) Preparing a culture medium: preparing a culture medium liquid by adopting an optimized isocitric acid fermentation culture medium raw material containing enzymatic vegetable protein powder; the optimized isocitric acid fermentation medium containing the enzymatic vegetable protein powder comprises the following components: 0.5 g/L of yeast extract, 0.3g/L of enzymolysis vegetable protein powder, 0.1 g/L of monopotassium phosphate, 0.1 g/L of magnesium sulfate heptahydrate, 60g/L of glycerol and 0.06 g/L of itaconic acid, and adjusting the pH to 6.5 before sterilization;

(2) and (3) sterilization: sterilizing the culture medium liquid in a fermenter; killing microorganisms in the culture medium liquid without loss of nutrients;

(3) inoculating and culturing: cooling the culture medium liquid after the killing treatment to be below 28 ℃ in a fermentation tank, inoculating an isocitric acid strain, adjusting the culture temperature to be 26-28 ℃, introducing purified air into the fermentation tank for aerobic culture, fermenting for 60-120h, supplementing sterilized 40% glycerol solution at constant speed, wherein the supplement amount is 48 g/L of the volume of the fermentation liquid, fermenting for 160h to obtain the fermentation liquid, and adjusting the pH of the fermentation liquid to be 5.0-6.0 by using ammonia water in the fermentation process;

(4) filtering the fermentation liquor to remove thalli, precipitating, acid-dissolving, crystallizing and drying to obtain an isocitric acid product.

Compared with the traditional method, the method for preparing the isocitric acid has the advantages that the yield of the isocitric acid prepared by the method is improved by 38 percent, and the proportion of the isocitric acid to the citric acid is improved by 105 percent under the same process condition.

The isocitrate producing strain is CICC 32287.