阅读说明：本技术 一种利用柠檬酸发酵尾气制备苹果酸的方法 (Method for preparing malic acid by using citric acid fermentation tail gas ) 是由 穆晓玲 秦晴 唐浩 刘宗亮 戴嘉 于 2020-12-31 设计创作，主要内容包括：本发明提供了一种利用柠檬酸发酵尾气制备苹果酸的方法,所述方法包括：以柠檬酸发酵尾气代替无菌空气通入苹果酸发酵培养基中进行发酵,得到所述苹果酸。本发明提供的制备方法操作简单易行,在既不影响柠檬酸发酵的同时可以实现柠檬酸发酵尾气的利用,节约了资源,保护了环境；并且利用柠檬酸发酵尾气可以促进嗜热菌发酵制备苹果酸,提高了苹果酸的产量和总酸转化率,缩短了发酵周期。(The invention provides a method for preparing malic acid by using citric acid fermentation tail gas, which comprises the following steps: and introducing the citric acid fermentation tail gas instead of sterile air into a malic acid fermentation culture medium for fermentation to obtain the malic acid. The preparation method provided by the invention is simple and easy to operate, the citric acid fermentation tail gas can be utilized while the citric acid fermentation is not influenced, the resources are saved, and the environment is protected; moreover, the citric acid fermentation tail gas can promote thermophilic bacteria to ferment and prepare malic acid, so that the yield of the malic acid and the total acid conversion rate are improved, and the fermentation period is shortened.)

1. A method for preparing malic acid by using citric acid fermentation tail gas is characterized by comprising the following steps: and introducing the citric acid fermentation tail gas instead of sterile air into a malic acid fermentation culture medium for fermentation to obtain the malic acid.

2. The method according to claim 1, characterized in that it comprises: after fermentation is started for 16-20h, replacing sterile air with citric acid fermentation tail gas and introducing the citric acid fermentation tail gas into a malic acid fermentation medium.

3. The method according to claim 1 or 2, characterized in that the method further comprises: and (4) introducing sterile air into the fermentation medium within 16-20h of fermentation.

4. The method of claim 3, wherein the dissolved oxygen in the malic acid fermentation medium is 20-30% within 16-20h of fermentation.

5. The method of claim 3, wherein the ventilation ratio of the malic acid fermentation tank is 1 (0.2-0.3) V/V-m within 16-20h of fermentation.

6. The method according to any one of claims 1 to 5, wherein the temperature of the fermentation is 40-50 ℃.

7. The process according to any one of claims 1 to 6, wherein the pressure of the fermentation is 0.02 to 0.05 MPa.

8. The method according to any one of claims 1 to 7, wherein the preparation method employs thermophilic bacteria as the species for fermenting malic acid.

9. The method of any one of claims 1-8, wherein the citric acid fermentation tail gas is produced from citric acid fermentation;

preferably, the ventilation ratio of citric acid fermentation is 1 (0.2-0.3) V/V.m, the fermentation temperature is 35-40 ℃, and the pressure of the fermentation tank is 0.08-0.15 MPa.

10. The method according to any one of claims 1-9, characterized in that the method comprises:

(1) inoculating a seed culture solution of thermophilic bacteria into a malic acid fermentation culture medium, starting fermentation at 40-50 ℃, introducing sterile air into the malic acid culture medium within 16-20h, and controlling the dissolved oxygen in the malic acid culture solution to be 20-30% for fermentation;

(2) after fermenting for 16-20h, introducing citric acid fermentation tail gas generated by citric acid fermentation into a malic acid culture medium instead of sterile air, and continuing to ferment until the fermentation is finished to obtain a fermentation liquid containing malic acid.

Technical Field

The invention belongs to the technical field of fermentation, and relates to a method for preparing malic acid by using citric acid fermentation tail gas.

Background

The synthesis route of malic acid in the thallus is generally three: (1) in the pyruvate carboxylation branch circuit, oxygen is not needed in the process, carbon dioxide needs to be fixed, and the theoretical conversion rate of malic acid to glucose is 148.8%; (2) TCA circulation, oxygen participation is needed in the process, and the theoretical conversion rate of malic acid to glucose is 74%; (3) the TCA cycle is added with a glyoxylic acid branch, and oxygen is required to participate in the process. From three approaches, in order to improve the conversion rate of malic acid, it is preferable to use all of the cells as pyruvate carboxylation branches (anaerobic metabolic pathways), but ATP (adenosine triphosphate), NADH (reduced coenzyme I), and the like required for these pathways generally require TCA cycles (aerobic metabolic pathways) to provide them, and therefore, malic acid fermentation is generally maintained in a micro-aerobic state, but it is difficult to find a micro-aerobic state in the actual control process, and it is necessary to balance the ratio of the aerobic metabolic pathway and the anaerobic metabolic pathway, which is difficult to grasp.

Therefore, a new method for fermenting malic acid is desired.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the method for preparing malic acid by using the citric acid fermentation tail gas, the method provided by the invention is simple and easy to operate, the citric acid fermentation tail gas can be utilized while the citric acid fermentation is not influenced, the resources are saved, and the environment is protected; moreover, the citric acid fermentation tail gas can promote thermophilic bacteria to ferment and prepare malic acid, so that the yield of the malic acid and the total acid conversion rate are improved, and the fermentation period is shortened.

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

in a first aspect, the present invention provides a method for preparing malic acid by using citric acid fermentation tail gas, wherein the method comprises: and introducing the citric acid fermentation tail gas instead of sterile air into a malic acid fermentation culture medium for fermentation to obtain the malic acid.

The proportion of oxygen and carbon dioxide in the citric acid fermentation tail gas is probably more suitable for micro oxygen consumption fermentation of malic acid than sterile air, the total acid conversion rate can be improved by utilizing the citric acid fermentation tail gas, meanwhile, water vapor in the citric acid fermentation tail gas can properly supplement water lost in the fermentation process of a malic acid fermentation culture medium, and citric acid in the citric acid fermentation tail gas can enter TCA (ternary content addressable memory) circulation to further promote fermentation, so that the citric acid fermentation tail gas is introduced into the malic acid fermentation culture medium instead of the sterile air, the fermentation can be promoted, the total acid conversion rate is improved, and the yield of the malic acid is improved; and the citric acid bacteria in the citric acid fermentation tail gas are difficult to survive in the environment of malic acid fermentation, and have no influence on the malic acid fermentation.

The method for preparing malic acid by using the citric acid fermentation tail gas provided by the invention can realize the waste utilization of the citric acid tail gas, can promote the malic acid fermentation, saves resources and protects the environment.

As a preferable technical solution of the present invention, the preparation method comprises: after 16-20h, such as 17h, 18h, 19h and the like, of starting fermentation, sterile air is replaced by citric acid fermentation tail gas and then is introduced into the malic acid fermentation medium.

When thermophilic bacteria are inoculated to a fermentation culture medium to start fermentation, the thermophilic bacteria need to grow and propagate within a certain time, and the required air flow is larger than that in the acid production metabolism stage, so that the strains in the malic acid fermentation culture medium are mainly propagated within a period of starting fermentation, citric acid fermentation tail gas is preferably introduced for fermentation for 16-20h, the thermophilic bacteria start to enter the acid production metabolism stage, the required air flow is reduced, and the citric acid fermentation tail gas can meet the requirement of dissolving oxygen in the malic acid fermentation, and can play a role in promoting the malic acid fermentation by the citric acid fermentation tail gas.

In the invention, the flow rate of the citric acid fermentation tail gas is actually determined by the tank pressure of the malic acid fermentation tank, and a certain pressure difference is obtained.

The method of the invention also comprises the following steps: sterile air is introduced into the fermentation medium within 16-20h after the fermentation is started.

In a preferred embodiment of the present invention, the dissolved oxygen content in the malic acid fermentation medium is 20-30%, for example, 22%, 25%, 28%, etc., within 16-20h from the start of fermentation.

The dissolved oxygen in the culture solution of malic acid fermentation needs to be controlled to be 20-30% within 16-20h from the beginning of the fermentation, and the control method can be the conventional method at present, for example, the dissolved oxygen in the culture solution is controlled by controlling the stirring rate under the stirring state.

The ventilation ratio is 1 (0.2-0.3) V/V.m within 16-20h of the initial fermentation.

The temperature of the fermentation according to the invention is 40-50 deg.C, for example 45 deg.C.

The pressure of the fermentation is 0.02-0.05 MPa.

In the invention, the preparation method adopts thermophilic bacteria as the strains for fermenting malic acid.

The strain for fermenting and culturing the malic acid adopted by the invention is myceliophthora thermophila, and is purchased from Tianjin industrial microorganism research institute of Chinese academy of sciences.

The citric acid fermentation tail gas is generated by citric acid fermentation.

The citric acid fermentation tail gas utilized by the invention is generated in the citric acid fermentation process, any citric acid fermentation tail gas generated by the existing citric acid fermentation method can be used in the invention, and in order to avoid the problem that the cost is easily increased due to the fact that the citric acid fermentation tail gas needs to be stored and applied, a citric acid fermentation device can be simultaneously arranged for fermenting the citric acid when the method is practically applied, and the tail gas pipe for citric acid fermentation can be directly connected with the gas inlet pipe of the malic acid fermentation tank.

In order to avoid the influence of the citric acid fermentation period and ensure the continuous supply of the citric acid fermentation tail gas in the malic acid fermentation process, the air inlet of one malic acid fermentation tank can be simultaneously connected with more than two citric acid fermentation tank tail gas pipes.

Further, in order to facilitate switching, each tail gas pipe is provided with a switch, and each path of tail gas can be independently turned on or turned off, so that when one tank of citric acid fermentation needs to be placed, other citric acid fermentation tail gases can be switched.

Meanwhile, in order to ensure that the tail gas pipe is in a sterile state, each tail gas pipe can be sterilized independently.

In the practical application process, the tank pressure for producing citric acid by fermentation is maintained at 0.08-0.15MPa, the tank pressure for producing citric acid by fermentation can be maintained by adjusting the tank pressure for producing malic acid by fermentation, and the corresponding air inflow can meet the requirement of malic acid fermentation.

The invention directly arranges a citric acid fermentation device to carry out citric acid fermentation to generate citric acid fermentation tail gas, wherein the ventilation ratio of the citric acid fermentation is 1 (0.2-0.3) V/V.m, the fermentation temperature is 35-40 ℃, and the pressure of a fermentation tank is 0.08-0.15 MPa.

As a specific embodiment of the present invention, the preparation method comprises:

(1) inoculating a seed culture solution of thermophilic bacteria into a malic acid fermentation culture medium, starting fermentation at 40-50 ℃, introducing sterile air into the malic acid culture medium within 16-20h, and controlling the dissolved oxygen in the malic acid culture solution to be 20-30% for fermentation;

(2) after fermenting for 16-20h, introducing citric acid fermentation tail gas generated by citric acid fermentation into a malic acid culture medium instead of sterile air, and continuing to ferment until the fermentation is finished to obtain a fermentation liquid containing malic acid.

Compared with the prior art, the method has the following advantages that the citric acid fermentation tail gas is used for replacing sterile air:

(1) the proportion of oxygen and carbon dioxide contained in the citric acid fermentation tail gas is more suitable for micro oxygen consumption fermentation of malic acid than sterile air, so that the total acid conversion rate can be improved, and the malic acid yield can be improved;

(2) the citric acid fermentation tail gas also contains a small amount of water vapor which can supplement water lost due to overhigh temperature of thermophilic bacteria fermentation;

(3) a small amount of citric acid contained in the citric acid fermentation tail gas can directly enter TCA cycle, so that the preparation of malic acid by thermophilic bacteria fermentation is promoted, the yield of malic acid is further improved, and the fermentation period is shortened;

(4) a small amount of citric acid bacteria contained in the citric acid tail gas are difficult to survive under the fermentation temperature and humidity of thermophilic bacteria, and the preparation of malic acid by thermophilic bacteria fermentation is not influenced;

(5) the malic acid fermentation by using the citric acid fermentation tail gas belongs to waste gas utilization, saves resources and protects the environment;

(6) the malic acid prepared by the preparation method provided by the invention has higher yield and higher total acid conversion rate, wherein the malic acid yield can reach more than 195g/L, the total acid conversion rate is more than 90%, and the fermentation period is within 120 h.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Performance test method

The fermentation broth provided in the following examples was tested for performance using the following test methods:

(1) determination of malic acid and succinic acid: 1mL of fermentation broth was added to 1mL of 3M H₂SO₄Hydrolyzing, centrifuging, collecting supernatant, and determining malic acid and succinic acid content by Agilent 1200 high performance liquid chromatograph, 5M H₂SO₄The flow rate is 0.5mL/min, the sample volume is 20 mu L, the malic acid peak time is 10.745min, and the succinic acid peak time is 12.953 min;

(2) total acid conversion: the following calculation formula is used for calculation:

wherein M is_AThe malic acid yield is kg; m_BThe yield of the succinic acid is kg; m_CKg for the amount of sugar added.

Example 1

The present example provides a method for producing citric acid fermentation tail gas from citric acid fermentation.

Mixing corn flour according to 25% of flour-pulp ratio, adding high-temperature resistant alpha-amylase according to the amount of 50U/g starch, liquefying at 90 ℃ until iodine test reaction does not change color, taking 10% of liquefied liquid according to volume ratio as return material, centrifuging the remaining liquefied liquid or filtering plate pressure to obtain clear liquid of corn liquefaction. Mixing the returned material and the corn liquefied clear liquid in a fermentation tank, and then sterilizing for 10min at 110 ℃ to obtain a fermentation culture medium, wherein the total sugar after the seed culture is about 17 percent, and the pH is natural.

Inoculating Aspergillus niger liquid into a fermentation medium according to the inoculation amount of 10%, fermenting at 37 deg.C with the ventilation ratio of 1: 0.25V/V.m, stirring at rotation speed of 55%, introducing fermentation tail gas into a malic acid fermentation tank of corresponding culture period, and maintaining the pressure of citric acid fermentation tank at 0.1MPa by controlling the pressure of malic acid fermentation tank. The citric acid fermentation period is 62 hours, the acid production is 15.8 percent, and the conversion rate is about 92.9 percent.

Example 2

The embodiment provides a method for preparing malic acid by using citric acid fermentation tail gas.

In this embodiment, the vent pipe of the citric acid fermentation tank in embodiment 1 is connected to the air inlet of the malic acid fermentation tank in this embodiment, and the malic acid fermentation tank is provided with an air inlet pipe for sterile air;

the method comprises the following steps:

(1) activating seeds and preparing a seed culture solution:

myceliophthora thermovorans (purchased from Tianjin Industrial microbiology institute of Chinese academy of sciences) were inoculated in a solid slant culture medium under aseptic conditions, incubated at a constant temperature of 45 ℃ for 7 days, spores were washed with 0.8% NaCl and 0.1% Tween-80 and counted, and 2.5X 10 cells were cultured⁷Transferring the spores into a 250mL triangular flask (containing 100mL seed culture medium), and culturing at 45 ℃ and 150rpm for 24h to obtain a seed culture solution;

(2) fermentation culture:

the fermentation medium (per liter) consists of the following components: 230g of glucose, 180g of soybean meal and K₂HPO₄ 0.15g、KH₂PO₄ 0.15g、MgSO₄·7H₂0.1g of O, 0.05g of malic acid, 2mL of 0.1g/L biotin, 0.08g of NaCl, and ZnSO₄·7H₂O 0.1g、FeSO₄·7H₂0.15g of O and tap water as a solvent to prepare a fermentation medium;

inoculating the seed culture solution to a fermentation culture medium according to the inoculation amount of 6%, fermenting at 45 ℃ and 0.03MPa, introducing sterile air from the beginning of fermentation to within 18h, wherein the ventilation ratio is 1: 0.3V/V.m, and fermenting by controlling the stirring speed to control the dissolved oxygen amount in the malic acid culture solution to be 25%;

after fermenting for 18 hours, closing the sterile air inlet, opening the citric acid fermentation tail gas inlet, introducing the citric acid fermentation tail gas into a malic acid culture medium, controlling the gas inlet rate of the citric acid fermentation tail gas by controlling the tank pressure of the citric acid fermentation tank in the embodiment 1 to be 0.1MPa, and continuing to ferment until the fermentation is finished to obtain fermentation liquor containing malic acid;

in the embodiment, the fermentation period is 120h, the malic acid content is 195g/L, the succinic acid content is 25g/L, and the total acid conversion rate is 90.2%.

Example 3

The embodiment provides a method for preparing malic acid by using citric acid fermentation tail gas.

The difference from example 2 is that in this example, citric acid fermentation off-gas is introduced from the beginning of the fermentation.

In the embodiment, the fermentation period is 135h, the malic acid content is 189g/L, the succinic acid content is 20g/L, and the total acid conversion rate is 85.9%.

Example 4

The embodiment provides a method for preparing malic acid by using citric acid fermentation tail gas.

The difference from example 2 is that in this example, after 25h of fermentation, citric acid fermentation off-gas was passed into malic acid medium.

In the embodiment, the fermentation period is 120h, the malic acid content is 185g/L, the succinic acid content is 27g/L, and the total acid conversion rate is 86.9%.

Comparative example 1

The present comparative example provides a conventional method for the preparation of malic acid.

(1) Activating seeds and preparing a seed culture solution:

myceliophthora thermovorans (purchased from Tianjin Industrial microbiology institute of Chinese academy of sciences) were inoculated in a solid slant culture medium under aseptic conditions, incubated at a constant temperature of 45 ℃ for 7 days, spores were washed with 0.8% NaCl and 0.1% Tween-80 and counted, and 2.5X 10 cells were cultured⁷Spores were transferred into 250mL flasks (containing 100mL seed medium),culturing at 45 deg.C and 150rpm for 24 hr to obtain seed culture solution;

(2) fermentation culture:

the fermentation medium (per liter) consists of the following components: 230g of glucose, 180g of soybean meal and K₂HPO₄ 0.15g、KH₂PO₄ 0.15g、MgSO₄·7H₂0.1g of O, 0.05g of malic acid, 2mL of 0.1g/L biotin, 0.08g of NaCl, and ZnSO₄·7H₂O 0.1g、FeSO₄·7H₂0.15g of O and tap water as a solvent to prepare a fermentation medium;

inoculating the seed culture solution to a fermentation culture medium according to the inoculation amount of 6%, fermenting at 45 ℃ and 0.03MPa, introducing sterile air from the beginning of fermentation to within 18h, wherein the ventilation ratio is 1:0.3VV/m, and fermenting by controlling the stirring speed to control the dissolved oxygen amount in the malic acid culture solution to be 25%;

after fermenting for 18h, controlling the ventilation ratio to be 1:0.15VV/m, changing the stirring speed to control the dissolved oxygen amount to be 10%, and continuing fermenting until the fermentation is finished to obtain the fermentation liquor containing malic acid.

In the comparative example, the fermentation period was 122h, the malic acid content was 185g/L, the succinic acid content was 29g/L, and the total acid conversion was 85.06%.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.