阅读说明：本技术 一种醋酸杆菌和丁酸梭菌级联发酵生产丁酸的方法 (Method for producing butyric acid by cascade fermentation of acetobacter and clostridium butyricum ) 是由 曹华伟 吕向云 李冰 杨永 娄百勇 远万里 于 2021-01-15 设计创作，主要内容包括：本发明公开了一种醋酸杆菌和丁酸梭菌级联发酵生产丁酸的方法,发酵工艺为三级,包括一级种子培养、二级种子培养和发酵培养,丁酸梭菌摇瓶种子培养,接种于一级种子培养基中进行一级种子培养,然后接种于二级种子培养基中进行二级种子培养,最后接种于发酵罐培养基和醋酸杆菌中进行发酵培养,发酵培养分为两个阶段,第一阶段培养温度为30-35℃,发酵pH降至4.5-5.5时,开始升温至35-37℃,进行第二阶段培养,控制pH为6.0。本发明通过级联发酵,醋酸杆菌利用部分葡萄糖和淀粉生产出丁酸的合成原料乙酸,以此来减少原料中乙酸钙的成本,增加丁酸生产效率,提高发酵效率,提升发酵效能。(The invention discloses a method for producing butyric acid by cascade fermentation of clostridium acetobutylicum and clostridium butyricum, which comprises three stages of fermentation processes, wherein the fermentation processes comprise first-stage seed culture, second-stage seed culture and fermentation culture, the clostridium butyricum shake flask seed culture is inoculated in a first-stage seed culture medium for first-stage seed culture, then is inoculated in a second-stage seed culture medium for second-stage seed culture, and finally is inoculated in a fermentation tank culture medium and the bacillus acetobacter for fermentation culture, the fermentation culture is divided into two stages, the culture temperature of the first stage is 30-35 ℃, when the fermentation pH is reduced to 4.5-5.5, the temperature is initially increased to 35-37 ℃, the second-stage culture is carried out, and the pH is controlled to be 6.0. According to the invention, through cascade fermentation, acetobacter utilizes part of glucose and starch to produce acetic acid which is a synthetic raw material of butyric acid, so that the cost of calcium acetate in the raw material is reduced, the production efficiency of butyric acid is increased, the fermentation efficiency is improved, and the fermentation efficiency is improved.)

1. A method for producing butyric acid by cascade fermentation of acetobacter and clostridium butyricum comprises the following steps: the fermentation method is three-stage, and comprises first-stage seed culture, second-stage seed culture and fermentation culture, and is characterized in that clostridium butyricum shake flask seed culture is inoculated in a first-stage seed culture medium for first-stage seed culture, then is inoculated in a second-stage seed culture medium for second-stage seed culture, and finally is inoculated in a fermentation tank culture medium and acetobacter for fermentation culture, the fermentation culture is divided into two stages, the first stage culture temperature is 30-35 ℃, when the fermentation pH is reduced to 4.5-5.5, the temperature is raised to 35-37 ℃, the second stage culture is carried out, and the pH is controlled to be 6.0.

2. The method of claim 1, wherein the composition of the primary seed medium comprises: 0.5 to 0.6 percent of glucose, 1 to 2 percent of yeast extract powder, 0.5 to 1.0 percent of peptone and 0.05 to 0.2 percent of sodium chloride.

3. The method of claim 1, wherein the secondary seed medium comprises: 1 to 2 percent of yeast extract powder, 0.5 to 1 percent of peptone, 0.1 to 0.2 percent of soluble starch, 0.4 to 0.6 percent of glucose, 0.01 to 0.2 percent of manganese chloride, 0.05 to 0.06 percent of sodium chloride and 0.3 to 0.4 percent of sodium acetate.

4. The method of producing butyric acid by the cascade fermentation of acetobacter and clostridium butyricum according to claim 1, wherein the composition of the fermenter medium comprises: 10 to 20 percent of glucose, 0.1 to 0.2 percent of ammonium sulfate, 0.4 to 0.6 percent of peptone, 0.1 to 0.2 percent of yeast powder, 0.1 to 0.2 percent of monopotassium phosphate, 0.2 to 0.4 percent of calcium carbonate, 0.07 to 0.8 percent of magnesium sulfate, 0.01 to 0.03 percent of manganese sulfate and 0.1 percent of defoaming agent.

5. The method for producing butyric acid by the cascade fermentation of acetobacter and clostridium butyricum according to claim 1, wherein the conditions of the primary seed culture are as follows: the culture temperature is 37 ℃, the rotation speed is 200rpm, and the culture time is 24-48 hours.

6. The method for producing butyric acid by the cascade fermentation of acetobacter and clostridium butyricum according to claim 1, wherein the conditions of the secondary seed culture are as follows: the culture temperature was 37 ℃, the rotation speed was 200rpm, and the culture time was 24 hours.

7. The method for producing butyric acid by the cascade fermentation of acetobacter and clostridium butyricum according to claim 1, wherein the second stage culture conditions of the fermentation culture are: the pressure is 0.01Mpa, the rotating speed is 200rpm, and the culture time is 48 hours.

Technical Field

The invention belongs to the technical field of butyric acid fermentation, and particularly relates to a method for producing butyric acid by cascade fermentation of acetobacter and clostridium butyricum.

Background

Butyric acid is a product of microbial fermentation of carbohydrates in the rumen of ruminants and the colon of omnivores, has a strong bactericidal effect, and is considered as a potential substitute of antibiotics. The combination of butyric acid and antibiotics can improve the curative effect, and a large number of experiments show that the butyric acid has no toxic or side effect and is widely applied to medicines, functional health-care foods and additives of animal feeds. At present, butyric acid fermentation culture still mostly stays at a laboratory stage, the product concentration is basically about 10-30g/L, and the conditions of low fermentation efficiency and high cost exist in industrial production.

At present, the fermentation production of butyric acid is anaerobic culture, and thalli cannot grow or even die in the presence of oxygen in a fermentation environment; in addition, the cell body is strictly required to have an optimum pH for growth, and if the pH deviates from the optimum pH, the target product may not be obtained. The fermentation process is three-stage fermentation, and the first-stage seed culture process comprises the following steps: the temperature is 37 ℃, the anaerobic reaction is carried out, the rotating speed is 200rpm/min, the ph is natural, and the culture time is 20-24 h; secondary seed culture: the temperature is 37 ℃, the rotating speed is 200rpm/min, the culture period is 10-24h, the ORP is controlled to be-300 to-500 mv, and the pH is controlled to be 5.90; fermentation culture: the temperature is 37 ℃, the rotation speed is 150-; and (4) detecting the content of butyric acid after fermentation is finished, and adding a proper amount of sodium salt or calcium salt according to the concentration of the butyric acid to obtain the butyrate. The existing butyric acid fermentation technology has more byproducts, lower production efficiency and higher production cost, and is not suitable for modern industrial production.

Disclosure of Invention

The invention aims to provide a method for producing butyric acid by cascade fermentation of acetobacter and clostridium butyricum, which has the advantages of reasonable fermentation conditions, low cost and stable product quality and is suitable for industrial production.

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

a method for producing butyric acid by cascade fermentation of acetobacter and clostridium butyricum comprises the following steps: the fermentation method is three-stage, and comprises primary seed culture, secondary seed culture and fermentation culture, wherein clostridium butyricum shake flask seed culture is carried out for 12-16 hours, the clostridium butyricum shake flask seed culture is inoculated into a primary seed culture medium for primary seed culture, the inoculum size is 0.25%, then the clostridium butyricum shake flask seed culture is inoculated into a secondary seed culture medium for secondary seed culture, the inoculum size is 10%, finally the clostridium butyricum shake flask seed culture is inoculated into a fermentation tank culture medium and bacillus aceticus for fermentation culture, the inoculum size is 20%, the fermentation culture is divided into two stages, the culture temperature of the first stage is 30-35 ℃, when the fermentation pH is reduced to 4.5-5.5, the temperature is raised to 35-37 ℃, the second stage culture is carried out, and the pH.

Further, the composition of the primary seed culture medium comprises: 0.5 to 0.6 percent of glucose, 1 to 2 percent of yeast extract powder, 0.5 to 1.0 percent of peptone and 0.05 to 0.2 percent of sodium chloride.

Further, the composition of the secondary seed culture medium comprises: 1 to 2 percent of yeast extract powder, 0.5 to 1 percent of peptone, 0.1 to 0.2 percent of soluble starch, 0.4 to 0.6 percent of glucose, 0.01 to 0.2 percent of manganese chloride, 0.05 to 0.06 percent of sodium chloride and 0.3 to 0.4 percent of sodium acetate.

Further, the composition of the fermenter medium comprises: 10 to 20 percent of glucose, 0.1 to 0.2 percent of ammonium sulfate, 0.4 to 0.6 percent of peptone, 0.1 to 0.2 percent of yeast powder, 0.1 to 0.2 percent of monopotassium phosphate, 0.2 to 0.4 percent of calcium carbonate, 0.07 to 0.8 percent of magnesium sulfate, 0.01 to 0.03 percent of manganese sulfate and 0.1 percent of defoaming agent.

Further, the conditions of the primary seed culture are as follows: the culture temperature is 37 ℃, the rotation speed is 200rpm, and the culture time is 24-48 hours.

Further, the conditions of the secondary seed culture are as follows: the culture temperature was 37 ℃, the rotation speed was 200rpm, and the culture time was 24 hours.

Further, the second stage culture conditions of the fermentation culture are as follows: the pressure is 0.01Mpa, the rotating speed is 200rpm, and the culture time is 48 hours.

The invention has the advantages that:

according to the method for producing butyric acid by the acetic acid bacillus and the clostridium butyricum through cascade fermentation, the acetic acid bacillus utilizes part of glucose and starch to produce acetic acid serving as a synthetic raw material of butyric acid, so that the cost of calcium acetate in the raw material is reduced, the production efficiency of butyric acid is increased, the fermentation efficiency is improved, and the fermentation efficiency is improved. The content of butyric acid in the normal process tank is 45-50g/L, the content of acetic acid is 5g/L, and the content of lactic acid is 2-3g/L, the content of butyric acid in the fermentation tank is 44-48g/L, the content of acetic acid is 7g/L, and the content of lactic acid is 1-2g/L, which are detected by HPLC, the experimental result shows that the content of butyric acid in the fermentation tank after the improvement of the process is basically consistent with that of the initial process, the content of acetic acid is increased by 40%, the content of lactic acid is reduced by about 50%, but the cost of a single tank is reduced by 3000-5000 yuan (calculated by 15 tons of liquid in a 30.

The fermentation tank provided by the invention has the advantages that calcium carbonate is added into the culture medium components of the fermentation tank, 5% of calcium acetate is reduced, and the cost of each ton of fermentation liquid culture medium is reduced by 300 yuan. The invention has reasonable fermentation condition, low cost and stable product quality, and is suitable for industrial production.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the specific examples described herein are only for explaining the present invention and are not intended to limit the present invention.

Example 1

A method for producing butyric acid by cascade fermentation of acetobacter and clostridium butyricum comprises the following steps: the fermentation method is three-stage, and comprises primary seed culture, secondary seed culture and fermentation culture. The clostridium butyricum is cultured for 12 hours in a shake flask and is inoculated in a first-level seed culture medium for first-level seed culture, the inoculation amount is 0.25 percent, and the first-level seed culture medium comprises the following components: 0.5% of glucose, 1% of yeast extract powder, 0.5% of peptone and 0.05% of sodium chloride. The conditions for primary seed culture are as follows: the culture temperature was 37 ℃, the rotation speed was 200rpm, and the culture time was 24 hours.

Then inoculating the strain in a secondary seed culture medium for secondary seed culture, wherein the inoculation amount is 10 percent, and the secondary seed culture medium comprises the following components: 1% of yeast extract powder, 0.5% of peptone, 0.1% of soluble starch, 0.4% of glucose, 0.01% of manganese chloride, 0.05% of sodium chloride and 0.3% of sodium acetate. The conditions for secondary seed culture are as follows: the culture temperature was 37 ℃, the rotation speed was 200rpm, and the culture time was 24 hours.

Finally inoculating the strain into a fermentation tank culture medium and acetobacter for fermentation culture, wherein the inoculation amount is 20%, and the fermentation tank culture medium comprises the following components: 10% of glucose, 0.1% of ammonium sulfate, 0.4% of peptone, 0.1% of yeast powder, 0.1% of monopotassium phosphate, 0.2% of calcium carbonate, 0.07% of magnesium sulfate, 0.01% of manganese sulfate and 0.1% of defoaming agent. The fermentation culture is divided into two stages, the first stage culture temperature is 30 ℃, when the fermentation pH is reduced to 4.5, the temperature is raised to 35 ℃, the second stage culture is carried out, the pH is controlled to be 6.0, the pressure is 0.01Mpa, the rotating speed is 200rpm, and the culture time is 48 hours.

Example 2

A method for producing butyric acid by cascade fermentation of acetobacter and clostridium butyricum comprises the following steps: the fermentation method is three-stage, and comprises primary seed culture, secondary seed culture and fermentation culture. The clostridium butyricum is cultured for 16 hours in a shake flask and is inoculated in a first-level seed culture medium for first-level seed culture, the inoculation amount is 0.25 percent, and the first-level seed culture medium comprises the following components: 0.6 percent of glucose, 2 percent of yeast extract powder, 1.0 percent of peptone and 0.2 percent of sodium chloride. The conditions for primary seed culture are as follows: the culture temperature was 37 ℃, the rotation speed was 200rpm, and the culture time was 48 hours.

Then inoculating the strain in a secondary seed culture medium for secondary seed culture, wherein the inoculation amount is 10 percent, and the secondary seed culture medium comprises the following components: 2% of yeast extract powder, 1% of peptone, 0.2% of soluble starch, 0.6% of glucose, 0.2% of manganese chloride, 0.06% of sodium chloride and 0.4% of sodium acetate. The conditions for secondary seed culture are as follows: the culture temperature was 37 ℃, the rotation speed was 200rpm, and the culture time was 24 hours.

Finally inoculating the strain into a fermentation tank culture medium and acetobacter for fermentation culture, wherein the inoculation amount is 20%, and the fermentation tank culture medium comprises the following components: 20% of glucose, 0.2% of ammonium sulfate, 0.6% of peptone, 0.2% of yeast powder, 0.2% of monopotassium phosphate, 0.4% of calcium carbonate, 0.8% of magnesium sulfate, 0.03% of manganese sulfate and 0.1% of defoaming agent. The fermentation culture is divided into two stages, the first stage culture temperature is 35 ℃, when the fermentation pH is reduced to 5.5, the temperature is raised to 37 ℃, the second stage culture is carried out, the pH is controlled to be 6.0, the pressure is 0.01Mpa, the rotating speed is 200rpm, and the culture time is 48 hours.