阅读说明：本技术 氨基酸高效绿色生产提取工艺 (Efficient green production and extraction process of amino acid ) 是由 赵兰坤 赵凤良 王小平 魏冉冉 刘世周 程士清 于 2020-05-21 设计创作，主要内容包括：本发明属于生物工程技术领域,氨基酸高效绿色生产提取工艺,其包括：收L-丙氨酸发酵液,离心,收集上清液；将上清液通过陶瓷膜进行微滤,收集滤过液；往滤过液中添加活性炭脱色,板框过滤,收集脱色液；然后通过强酸性阳离子交换树脂,L-丙氨酸被交换到树脂上,随后用氨水将树脂上的L-丙氨酸完全洗脱下来,通过浓缩结晶、离心、干燥获得成品。本发明工艺能够提高发酵生产强度,进而降低发酵丙氨酸的生产成本。(The invention belongs to the technical field of bioengineering, and discloses an efficient and green production and extraction process of amino acid, which comprises the following steps: collecting L-alanine fermentation liquor, centrifuging, and collecting supernatant; microfiltering the supernatant through a ceramic membrane, and collecting filtrate; adding activated carbon into the filtrate for decolorization, filtering with a plate frame, and collecting the decolorized solution; then passing through strong acid cation exchange resin, L-alanine is exchanged on the resin, then ammonia water is used for completely eluting the L-alanine on the resin, and the finished product is obtained by concentration, crystallization, centrifugation and drying. The process can improve the fermentation production intensity and further reduce the production cost of fermentation alanine.)

1. An efficient green process for extracting amino acid from it includes

Collecting L-alanine fermentation liquor, centrifuging, and collecting supernatant; microfiltering the supernatant through a ceramic membrane, and collecting filtrate; adding activated carbon into the filtrate for decolorization, filtering with a plate frame, and collecting the decolorized solution; then passing through 732 type strong acid cation exchange resin, L-alanine is exchanged onto the resin, then completely eluting L-alanine on the resin with ammonia water, and obtaining the finished product by concentration crystallization, centrifugation and drying.

2. The process of claim 1, wherein the L-alanine fermentation broth is prepared by:

1) inoculating the Escherichia coli seed liquid for producing L-alanine into a fermentation tank containing a basic fermentation culture medium, starting fermentation for 45h, pumping the fermentation liquid in the fermentation tank into a microfiltration membrane, retaining cells, returning the cells to the fermentation tank, dialyzing the fermentation liquid containing alanine out of the microfiltration membrane, and collecting the fermentation liquid in a collection tank;

2) activating the culture medium to enter a fermentation tank, starting the second batch of fermentation, wherein the fermentation time is 35h, the fermentation liquid in the fermentation tank is pumped into a microfiltration membrane, the cells are intercepted and returned to the fermentation tank, and the fermentation liquid containing alanine is permeated and separated out from the microfiltration membrane and collected into a collection tank;

3) activating the culture medium to enter a fermentation tank, starting the third batch of fermentation, wherein the fermentation time is 35h, the fermentation liquid in the fermentation tank is pumped into a microfiltration membrane, the cells are intercepted and returned to the fermentation tank, and the fermentation liquid containing alanine is permeated and separated out from the microfiltration membrane and collected into a collection tank;

4) activating the culture medium to enter a fermentation tank, starting fermentation for the fourth batch for 25h, pumping the fermentation liquid in the fermentation tank into a microfiltration membrane, retaining cells to return to the fermentation tank, and dialyzing out the fermentation liquid containing alanine from the microfiltration membrane to be collected in a collection tank;

in the whole fermentation process, when the glucose concentration in the fermentation liquor is lower than 5g/L, feeding the glucose solution to maintain the glucose concentration in the fermentation liquor at 5-10 g/L, and automatically feeding ammonia water to maintain the pH value at 7.0 +/-0.3.

3. The process of claim 1, wherein the centrifugation is: centrifuging for 3-5min at 4000-.

4. The process according to claim 1, wherein the ceramic membrane has a molecular weight cut-off of 1-2 ten thousand Da.

5. The process according to claim 1, characterized in that the decolourization is: the adding amount of the activated carbon is 1 g: 100ml of filtrate, 60 ℃ of decolorization temperature and 60min of decolorization time.

6. The process of claim 2, wherein the basal fermentation medium comprises: 80g/L glucose, 20g/L corn steep liquor, 5g/L ammonium chloride, 2.5g/L dipotassium phosphate, 0.1g/L magnesium sulfate heptahydrate, 0.1g/L calcium chloride, 10mg/L manganese sulfate monohydrate and 100 mu g/L biotin.

7. The process of claim 2, wherein the activation medium comprises the following components: adding a composition containing two or more of aspartic acid, magnesium carbonate and lactose into the basic fermentation medium.

8. The process according to claim 2 or 7, wherein the activation medium has the composition: basal fermentation medium + aspartic acid + magnesium carbonate + lactose.

9. The process according to claim 2 or 7, wherein the activation medium has the composition: basic fermentation medium + aspartic acid 1g/L + magnesium carbonate 0.5g/L + lactose 2 g/L.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an efficient and green production and extraction process of amino acid.

Background

L-alanine colorless to white crystalline powder, soluble in water, ethanol, and insoluble in ether and acetone. The flavoring agent is mainly used for biochemical research, tissue culture, liver function measurement and flavoring agent, can increase the flavoring effect of the flavoring agent, and can also be used as sour taste correcting agent to improve the sour taste of organic acid. The application of alanine in the field of medicine is discovered, and clinical researches show that the alanine can prevent kidney stones, assist the metabolism of glucose, help to alleviate hypoglycemia and improve body energy. Alanine is also widely used as a starting material for the synthesis of novel sweeteners and certain chiral pharmaceutical intermediates.

Currently, alanine is usually produced industrially by a chemical method, but the method has low conversion rate and a large amount of byproducts, and is easy to generate industrial three wastes, thereby polluting the environment. In contrast, the renewable resource microbial fermentation method is a green production method with more development potential. Microbial fermentation is the primary method for producing alanine.

CN110982857A discloses a fermentation production method of L-alanine, which adopts a one-time feeding fermentation process, puts sugar source, nitrogen source, inorganic salt and the like required by fermentation into a fermentation tank at one time, after sterilization and cooling, transfers seeds into the fermentation tank, and produces the L-alanine by fermentation culture. By optimizing fermentation conditions, such as increasing sugar feeding amount, controlling culture air volume, ensuring the oxygen amount required by culture, controlling culture temperature, pH value and the like in the fermentation process, the fermentation acid production rate of L-alanine is increased, and the fermentation period is shortened.

CN109468254A adopts a genetic engineering method to modify the strain, modifies the screened mutant strain FMME-A232 which grows to produce acid under high-concentration glucose, strengthens the transfer path of glucose, and uses pEtac plasmid to express glucose transporter EI protein and EIIAClc protein in series to obtain escherichia coli recombinant bacteria, wherein the escherichia coli recombinant bacteria can improve the consumption rate of glucose, so that the fermentation period of the strain is reduced from 55h to 45h, and the production efficiency of L-alanine is improved.

CN109055451A discloses a biological fermentation method of L-alanine, which comprises the following steps: the L-alanine strain is cultured by adopting a seed culture medium, and yeast extract powder and ammonium sulfate are adopted as nitrogen sources in the seed culture medium. According to the invention, the fermentation process in the seed culture stage is optimized, the activity of the seeds is improved, the production intensity of the L-alanine is effectively improved, and the raw material cost is not additionally increased. By optimizing the fermentation process in the seed culture stage, including optimizing the culture medium and the fermentation mode, the production intensity of the L-alanine is improved, the production cost of biologically preparing the L-alanine is reduced, and the fermentation period is shortened to 25 h.

Disclosure of Invention

The invention aims to improve the fermentation production intensity and further reduce the production cost of fermentation alanine.

The invention is realized by the following technical scheme.

An efficient green process for extracting amino acid from it includes

Collecting L-alanine fermentation liquor, centrifuging, and collecting supernatant; microfiltering the supernatant through a ceramic membrane, and collecting filtrate; adding activated carbon into the filtrate for decolorization, filtering with a plate frame, and collecting the decolorized solution; then passing through 732 type strong acid cation exchange resin, L-alanine is exchanged onto the resin, then completely eluting L-alanine on the resin with ammonia water, and obtaining the finished product by concentration crystallization, centrifugation and drying.

Specifically, the preparation method of the L-alanine fermentation liquor comprises the following steps:

1) inoculating the Escherichia coli seed liquid for producing L-alanine into a fermentation tank containing a basic fermentation culture medium, starting fermentation for 45h, pumping the fermentation liquid in the fermentation tank into a microfiltration membrane, retaining cells, returning the cells to the fermentation tank, dialyzing the fermentation liquid containing alanine out of the microfiltration membrane, and collecting the fermentation liquid in a collection tank;

2) activating the culture medium to enter a fermentation tank, starting the second batch of fermentation, wherein the fermentation time is 35h, the fermentation liquid in the fermentation tank is pumped into a microfiltration membrane, the cells are intercepted and returned to the fermentation tank, and the fermentation liquid containing alanine is permeated and separated out from the microfiltration membrane and collected into a collection tank;

3) activating the culture medium to enter a fermentation tank, starting the third batch of fermentation, wherein the fermentation time is 35h, the fermentation liquid in the fermentation tank is pumped into a microfiltration membrane, the cells are intercepted and returned to the fermentation tank, and the fermentation liquid containing alanine is permeated and separated out from the microfiltration membrane and collected into a collection tank;

4) activating the culture medium to enter a fermentation tank, starting fermentation for the fourth batch for 25h, pumping the fermentation liquid in the fermentation tank into a microfiltration membrane, retaining cells to return to the fermentation tank, and dialyzing out the fermentation liquid containing alanine from the microfiltration membrane to be collected in a collection tank;

in the whole fermentation process, when the glucose concentration in the fermentation liquor is lower than 5g/L, feeding the glucose solution to maintain the glucose concentration in the fermentation liquor at 5-10 g/L, and automatically feeding ammonia water to maintain the pH value at 7.0 +/-0.3.

Preferably, the centrifugation is: centrifuge for 5min at 4000rpm with a disk centrifuge.

Preferably, the ceramic membrane has a molecular weight cut-off of 1 ten thousand Da,

preferably, the decolorization is: the adding amount of the activated carbon is 1 g: 100ml of filtrate, 60 ℃ of decolorization temperature and 60min of decolorization time.

Further, the basic fermentation medium comprises the following components: 80g/L glucose, 20g/L corn steep liquor, 5g/L ammonium chloride, 2.5g/L dipotassium phosphate, 0.1g/L magnesium sulfate heptahydrate, 0.1g/L calcium chloride, 10mg/L manganese sulfate monohydrate and 100 mu g/L biotin.

Further, the components of the activation medium are: adding composition containing two or more of aspartic acid, magnesium carbonate and lactose into basic fermentation medium

Preferably, the components of the activation medium are: basal fermentation medium + aspartic acid + magnesium carbonate + lactose.

More preferably, the components of the activation medium are: basic fermentation medium + aspartic acid 1g/L + magnesium carbonate 0.5g/L + lactose 2 g/L.

Compared with the fermentation extraction process in the prior art, the method for producing alanine by batch fermentation has the advantages and positive effects that:

1. in the production process, a cyclic batch fermentation technology of cell recycling is used, the biomass is large in the next batch of fermentation, high-density fermentation is formed, the fermentation period is shortened due to the high-density fermentation, and the production intensity is improved.

2. As the thallus cells are recovered and recycled, the circular batch fermentation only needs one seed culture process, thereby saving a large amount of time, materials and energy.

3. The method can be continuously carried out for a long time by adopting the circular batch fermentation, thereby reducing the process of cleaning and sterilizing the fermentation tank between batches and reducing the non-production time.

4. The fermentation process is coupled with the extraction process, so that metabolites are separated in time, the feedback inhibition of the products is reduced, the cell activity is kept, and the production efficiency is improved.

5. According to the invention, the culture medium is optimized, and aspartic acid, magnesium carbonate and lactose are added, so that the three have strong synergistic effect, the growth promotion effect on escherichia coli is obvious, the continuous growth of the biomass of the strain is maintained, and the acid production activity of the strain is ensured.

Drawings

FIG. 1: effect of different media on the viability of the strain.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.