CN116904529A

CN116904529A - Method for continuously catalyzing and preparing GABA (gamma-amino acid) by gel bead immobilized lactobacillus brevis cells

Info

Publication number: CN116904529A
Application number: CN202211423996.8A
Authority: CN
Inventors: 郭小雷; 王暐; 翁成光; 刘丽霞; 李韶雄; 陈子昂
Original assignee: Fujian Kehong Biological Engineering Co ltd
Current assignee: Fujian Kehong Biological Engineering Co ltd
Priority date: 2022-11-15
Filing date: 2022-11-15
Publication date: 2023-10-20

Abstract

The invention discloses a method for continuously catalyzing and preparing GABA by gel bead immobilized lactobacillus brevis cells. It comprises the following steps: the pretreatment of adsorption and then crosslinking is carried out on lactobacillus brevis by chitosan and glutaraldehyde; then mixing the mixture with sodium alginate solution, dripping the mixture into calcium chloride solution, and collecting formed rubber balls; respectively performing primary film coating treatment on the rubber ball in a chitosan solution and a sodium alginate solution to obtain a coated rubber ball; mixing the coated rubber ball with sodium citrate solution or EDTA solution for reaction to obtain immobilized cells; carrying out conversion reaction on the immobilized cell catalytic L-glutamic acid reaction solution to obtain a conversion solution; removing GABA from the conversion solution through electrodialysis, enriching GABA, transferring the residual conversion solution into a conversion tank, adding new L-glutamic acid, and continuously carrying out immobilized cell catalytic reaction to obtain GABA. The invention reduces the energy consumption of the post concentration crystallization and the wastewater treatment capacity, and improves the yield of unit volume in unit time and the conversion rate of the L-glutamic acid.

Description

Method for continuously catalyzing and preparing GABA (gamma-amino acid) by gel bead immobilized lactobacillus brevis cells

Technical Field

The invention belongs to the technical field of biology, and relates to a method for continuously catalyzing gel bead immobilized lactobacillus brevis cells to prepare GABA.

Background

Gamma-aminobutyric acid (gamma-aminobutyric acid, GABA for short) is a new food raw material approved by the national commission of Wei Jian, is an inhibitory neurotransmitter naturally existing in human bodies, has the function of assisting sleep, is mainly reflected in shortening the falling-into-sleep time and prolonging the deep sleep time, and is mainly used as a functional dietary supplement in the market. In addition, the method has certain application in agriculture, feed and medicine fields, and has wide market prospect.

The preparation method of GABA includes chemical synthesis method and biological synthesis method, in which most of chemical synthesis methods use pyrrolidone or N-methyl pyrrolidone as substrate, and the substrate itself has toxicity, and the use of the substrate in food industry has safety risk; while biosynthesis methods include enzyme catalysis and direct fermentation. The enzyme catalysis method has the characteristics of high product concentration, short period, easy product separation and high purity, and has obvious cost advantage. The enzymes used in the enzyme catalysis method are all glutamate decarboxylases, and the microorganisms are of a plurality of sources, and the microorganisms allowed to be applied to food raw materials in the present country are only lactobacillus johnsonii and lactobacillus brevis. The concentration of the fermentation thalli of the lactobacillus johnsonii and the lactobacillus brevis is greatly different from that of the modeling strains such as escherichia coli, bacillus subtilis and the like, and the low GABA yield of unit bacterium concentration leads to high GABA production cost. The application of the immobilized cells or immobilized enzyme technology can greatly improve the GABA yield of unit bacteria concentration or unit enzyme activity, and domestic scientific researchers do a great deal of work on how to improve the GABA yield of unit bacteria concentration. The method comprises the steps of mixing lactobacillus plantarum with sodium alginate uniformly in CN 101838672B, then dripping the mixture into 0.2mol/L calcium chloride solution to prepare calcium alginate gel, and catalyzing to prepare GABA. The method is the most basic immobilized cell process by an embedding method, and has the defects that the access channels of the prepared gel bead substrate and the prepared gel bead product are blocked, so that the catalytic activity of the enzyme cannot be expressed, the catalytic efficiency is reduced, 3g/L of L-glutamic acid is used as the substrate, the reaction is carried out for 24 hours, and the average conversion rate is only 91.2%. In CN 107345233B, 732 strong acid cation exchange resin and immobilized enterococcus faecium cell column are coupled to form a continuous conversion system, and the immobilized cell method is a calcium alginate embedding method, so that the acid consumption is saved on the premise of controlling the pH of the catalytic reaction, and the 732 resin is utilized to realize the prepurification process of GABA to a certain extent. However, the process of preparing GABA by using L-Glu as a substrate in a catalysis way can control the pH value by supplementing the substrate L-Glu without the need of additional acid supplementing solution; the use of 732 resin necessarily involves elution and regeneration of the resin, producing a large amount of acid-base waste liquid, increasing environmental treatment pressure. In CN 107287253B, the D101 macroporous adsorption resin column and the immobilized enterococcus faecium cell column are coupled to form a continuous conversion system, and the same problems as in CN 107345233B exist. In CN 108642039A, modified silicon dioxide particles are used as a carrier, immobilized enzyme for preparing glutamic acid decarboxylase is used for catalyzing and preparing GABA, and after the conversion reaction is finished, the carrier is directly centrifugally separated, but the conversion rate of glutamic acid is lower and is only 78%; meanwhile, the preparation cost of the immobilized carrier is high, and the immobilized carrier is not suitable for large-scale production.

Disclosure of Invention

The invention aims to provide a method for continuously catalyzing gel bead immobilized lactobacillus brevis cells to prepare GABA.

The invention provides a method for continuously catalyzing and preparing GABA by gel bead immobilized lactobacillus brevis cells, which comprises the following steps:

1) The pretreatment of adsorption and then crosslinking is carried out on lactobacillus brevis by chitosan and glutaraldehyde;

2) Mixing the lactobacillus brevis pretreated in the step 1) with sodium alginate solution, dripping into calcium chloride solution to form rubber balls, and collecting the rubber balls;

3) In chitosan solution, performing first film coating treatment on the rubber ball to obtain the rubber ball subjected to first film coating;

4) Performing second film coating treatment on the rubber ball subjected to the first film coating in the sodium alginate solution to obtain a rubber ball subjected to the second film coating;

5) Mixing the rubber ball subjected to the second film coating with a sodium citrate solution or an EDTA solution for reaction to obtain relatively hollow microcapsule gel beads, namely immobilized cells;

6) Carrying out conversion reaction on the immobilized cell catalytic L-glutamic acid reaction solution to obtain a conversion solution; removing GABA from the conversion solution through electrodialysis, enriching GABA, transferring the residual conversion solution into a conversion tank, adding new L-glutamic acid, and continuously carrying out the immobilized cell catalytic reaction to obtain GABA.

In the invention, the units of the mass volume percent concentration (%) are expressed in g/100ml.

In the above method, in step 1), the final mass volume concentration of the chitosan and glutaraldehyde may be 0.1 to 0.5%;

the initial mass volume concentration of the wet weight of the thallus of the lactobacillus brevis can be 5-20%, and can be 10% in particular;

the mass ratio of the chitosan to the glutaraldehyde is 0.2-0.5% and 2-5% of the wet bacterial weight of the lactobacillus brevis respectively.

In the above method, the pretreatment process is as follows: mixing the lactobacillus brevis collected by centrifugation with normal saline uniformly, and adding the prepared chitosan solution with the mass volume concentration of 0.6-1.0%; then dripping glutaraldehyde solution with mass volume concentration of 2-4% to make the final mass volume concentration of chitosan and glutaraldehyde be 0.1-0.5%, regulating pH value of system to be 5.0-7.0, adopting sodium hydroxide solution to control pH value;

the step 1) also comprises the steps of centrifugally collecting the pretreated lactobacillus brevis thalli, and washing the thalli with normal saline (specifically washing twice) for later use.

In the above method, in step 2), the mass-volume percentage concentration of the sodium alginate before mixing may be 2.0-5.0%, and specifically may be 2.0%;

the mass and volume percentage concentration of the calcium chloride before mixing is 1.0-5.0%, and can be 1.5%;

in the step 2), the volume mass ratio of the sodium alginate solution, the calcium chloride solution and the thallus wet weight of the lactobacillus brevis is 5ml (15-50 ml) and 1g.

In the above method, in step 3), the initial mass-volume percentage concentration of the chitosan is 0.5-2.0%, specifically 1.0%, and the pH is 3.0-5.5, specifically 3.5;

in the above method, in step 4), the initial mass volume percentage concentration of the sodium alginate may be 1.0 to 3.0%, and specifically may be 2.0%.

In the above method, in step 5), the initial mass-volume percentage concentration of sodium citrate may be 1.0-5.0%;

the mass volume percentage concentration of the EDTA solution can be 0.5-2.0%, and can be specifically 2.0%; the pH of the reaction system is adjusted to 6.0 to 10.0 by sodium hydroxide solution, and can be specifically 10.0.

In the above method, the immobilized cell catalysis is performed in a conversion tank, and the electrodialysis is performed in an electrodialysis device;

the immobilized cells catalyze electrodialysis coupling mode is that the conversion tank is connected with electrodialysis equipment; wherein, the bottom of the transformation tank is provided with a screen for separating the immobilized cells from the transformation liquid;

the immobilized cell continuous catalytic reaction specifically comprises the following steps: transferring the conversion liquid into the electrodialysis equipment to carry out the first electrodialysis treatment after the reaction of the first batch of L-glutamic acid reaction liquid is completed, transferring GABA to enrich, and simultaneously adding the second batch of L-glutamic acid reaction liquid into the conversion tank to start the reaction; after the first electrodialysis treatment is finished, transferring the second batch of L-glutamic acid reaction solution into a transfer tank, transferring the feed liquid after the first electrodialysis treatment into the transfer tank, supplementing substrate L-glutamic acid to 145g/L in batches, and performing electrodialysis treatment on the second batch of L-glutamic acid reaction solution; and stopping collecting until the GABA concentration in the electrodialysis GABA concentration chamber reaches 500g/L, and collecting after pure water is replaced.

In the invention, the immobilized cell catalyzed electrodialysis coupling reaction can be specifically carried out for 40 batches of reaction.

In the method, the pH of the initial system of the conversion reaction is 4.0-4.5, and the concentration of the initial substrate L-glutamic acid is 60g/L;

and in the conversion reaction process, tracking and detecting the residual L-glutamic acid in the conversion tank, supplementing the concentration of the L-glutamic acid in the conversion tank to 50g/L when the concentration is lower than 10g/L, and stopping supplementing when the total concentration value is 145g/L, until the reaction is finished when the glutamic acid content in the conversion liquid is lower than 0.2 g/L.

In the method, the operation voltage of the electrodialysis is 25-50V, the electrodialysis is a two-chamber bipolar membrane dissociation tank, the pH of the conversion end point of the conversion tank is 5.0-6.0, and the electrodialysis treatment is stopped when the pH of the stock solution of the L-glutamic acid reaction solution is reached to 4.5.

The invention has the following beneficial effects:

1. compared with the whole cell catalysis and direct fermentation methods, the immobilized cell conversion solution has the advantages of less impurities and easy separation and purification of products; the conversion liquid is recycled after electrodialysis treatment, so that the energy consumption of post concentration crystallization and the wastewater treatment capacity are greatly reduced.

2. The immobilized cells are subjected to continuous catalytic reaction, so that the yield of unit volume in unit time is improved;

3. and supplementing the residual reaction liquid after electrodialysis treatment with L-glutamic acid, and continuing the next conversion reaction, so that the conversion rate of the L-glutamic acid is improved to more than 99.5%.

Drawings

FIG. 1 is a schematic diagram of a unit flow scheme for coupling conversion reactions with electrodialysis in accordance with the present invention.

The individual labels in the figures are as follows:

1 a conversion tank; 2, transferring the tank; 3 electrodialysis apparatus.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In the following examples, lactobacillus brevis is a known strain, and the preparation method of Lactobacillus brevis is as follows:

(1) Aseptically inoculating the glycerol storage tube to a slant culture medium for streaking, and culturing at 29 ℃ for 3 days;

(2) Scraping the monocyclopedia on an inclined plane until the seed culture medium reaches 75/250ml, and standing and culturing for 16-20h at 29 ℃;

(3) Inoculating the seed into 75/250mL seed culture medium according to the inoculation amount of 3%, and standing and culturing for 6-9h at 29 ℃;

(4) 1% of the inoculation amount is inoculated to 1.5L/5L of fermentation medium, 60-100rpm and 29 ℃, and bacterial cells are collected by centrifugation for about 24 hours for standby.

The composition of the slant/seed medium and the preparation conditions are shown in table 1.

TABLE 1

The composition of the fermentation medium and the preparation conditions are shown in Table 2.

TABLE 2

In the examples described below, the unit of mass volume concentration (%) is g/100ml.

Example 1

The preparation of the immobilized cells comprises the following specific steps:

weighing lactobacillus brevis with the wet weight of 10g, adding 40ml of physiological saline for resuspension; taking 40ml of prepared chitosan solution with the mass volume concentration of 0.6%, dropwise adding the chitosan solution into the bacterial liquid, and uniformly stirring the solution to obtain bacterial flocculation suspension; then 10ml of glutaraldehyde solution with the mass volume concentration of 3.0% is added dropwise, during which 2M NaOH solution is used for controlling pH=6.0, and stirring is carried out for 60min; the pretreated cells were collected by centrifugation and washed twice with 0.3L of physiological saline.

The collected thalli are evenly mixed with 50ml of sodium alginate solution with 2.0 percent, and the mixture is dropwise added into calcium chloride solution with the mass volume concentration of 1.5 percent by a syringe (the adding proportion is 5ml sodium alginate solution and 15ml calcium chloride solution in each gram of thalli), and the mixture is slowly stirred during the period to form the rubber ball. After the dripping is completed, stirring is continuously maintained for 0.5h, and the rubber balls are filtered and collected by a sand core funnel.

Adding the gel balls into the prepared chitosan solution (pH 3.5) with the mass and volume percentage concentration of 1.0%, and slowly stirring for 2 hours; and after the sand core funnel is separated, adding the rubber ball into sodium alginate solution with the mass and volume percentage concentration of 2.0%, and slowly stirring for 1h to perform the second film coating treatment.

After the sand core funnel separation is carried out again, the gel balls are added into EDTA solution (pH 10.0) with the mass and volume percentage concentration of 1.0 percent, and after the mixture is slowly stirred for 1 hour, the gel balls are collected, namely the immobilized cells are ready for use.

The enzyme activity recovery rate of the immobilized cells was 91.2% compared to the free Lactobacillus brevis.

Example 2

As shown in fig. 1, the continuous catalytic preparation of GABA by electrodialysis coupling was carried out as follows:

all the immobilized cells prepared in example 1 of the present invention were added to a transformation tank, 900ml of water, 60. 60g L-glutamic acid and 100mg of PLP were added, the pH was adjusted to 4.2 with 2M NaOH, the reaction was started at 37℃to detect glutamic acid residues, the concentration was supplemented to 50g/L when the concentration was lower than 10g/L, and the reaction was stopped when the total concentration value was 145g/L, and ended when the glutamic acid content was 0.1g/L, the total reaction time was 40min, and the end-point pH was 5.3.

Discharging the conversion liquid from the bottom of the conversion tank, pumping the conversion liquid to an electrodialysis treatment feed liquid chamber, and simultaneously adding the next batch of reaction liquid into the conversion tank for continuous reaction; opening the electrodialysis equipment, maintaining the voltage at 35V, and closing the electrodialysis equipment when the pH value is slowly reduced to 4.5, wherein the treatment time is 30min; after the second reaction is finished, pumping the reaction liquid into a buffer tank, pumping the electrodialysis treated feed liquid back into a conversion tank again, adding substrates except PLP, and continuing the third conversion reaction. The continuous reaction was continued for 5 batches, and the GABA content in the GABA concentration chamber of electrodialysis was 490g/L, which was replaced with pure water, to prepare the next round of continuous reaction.

After repeating 8 rounds of continuous catalytic reactions of 5 batches per round, the conversion reaction time was prolonged to 75min, the enzyme activity retention was 80.2%, the space-time yield of the total 40 batches of catalytic reaction GABA was 108 g/L.h, and the molar conversion of L-glutamic acid was 99.5%.

According to the experimental results, the immobilized cell catalysis and the electrodialysis concentration GABA collecting process are coupled, the pH value is not additionally regulated by adding acid or alkali in the whole process, the continuous catalysis preparation of GABA by the immobilized cell is realized, and the L-glutamic acid conversion rate and the GABA yield are improved.

Claims

1. A method for continuously catalyzing and preparing GABA by gel bead immobilized lactobacillus brevis cells comprises the following steps:

2. A method as claimed in claim 1, characterized in that: in the step 1), the final mass volume concentration of the chitosan and the glutaraldehyde is 0.1-0.5%;

the initial mass volume concentration of the wet weight of the thallus of the lactobacillus brevis is 5-20%;

3. A method according to claim 1 or 2, characterized in that: the pretreatment process is as follows: mixing the lactobacillus brevis collected by centrifugation with normal saline uniformly, and adding the prepared chitosan solution with the mass volume concentration of 0.6-1.0%; then dripping glutaraldehyde solution with the mass volume concentration of 2-4% to ensure that the final mass volume concentration of the chitosan and glutaraldehyde is 0.1-0.5%, and regulating the pH value of the system to be 5.0-7.0;

the step 1) also comprises centrifugally collecting the pretreated lactobacillus brevis thallus, and washing the thallus with normal saline for later use.

4. A method according to any one of claims 1-3, characterized in that: in the step 2), the mass and volume percentage concentration of the sodium alginate is 2.0-5.0% before mixing;

the mass and volume percentage concentration of the calcium chloride before mixing is 1.0-5.0%;

5. The method according to any one of claims 1-4, wherein: in the step 3), the initial mass volume percentage concentration of the chitosan is 0.5-2.0%, and the pH value of the chitosan is 3.0-5.5.

6. The method according to any one of claims 1-5, wherein: in the step 4), the initial mass volume percentage concentration of the sodium alginate is 1.0-3.0%.

7. The method according to any one of claims 1-6, wherein: in the step 5), the initial mass volume percentage concentration of the sodium citrate is 1.0-5.0%; the initial mass volume percentage concentration of the EDTA solution is 0.5-2.0%; and regulating the pH value of the reaction system to 6.0-10.0 by using sodium hydroxide solution.

8. The method according to any one of claims 1-7, wherein: the immobilized cell catalysis is performed in a conversion tank, and the electrodialysis is performed in an electrodialysis device;

the immobilized cell continuous catalytic reaction comprises the following steps that when the reaction of a first batch of L-glutamic acid reaction liquid is completed, the conversion liquid is transferred into electrodialysis equipment to carry out first electrodialysis treatment, GABA is migrated out for enrichment, and a second batch of L-glutamic acid reaction liquid is added into the conversion tank to start the reaction; after the first electrodialysis treatment is finished, transferring the second batch of L-glutamic acid reaction solution into a transfer tank, transferring the feed liquid after the first electrodialysis treatment into the transfer tank, supplementing substrate L-glutamic acid to 145g/L in batches, and performing electrodialysis treatment on the second batch of L-glutamic acid reaction solution; and stopping collecting until the GABA concentration in the electrodialysis GABA concentration chamber reaches 500g/L, and collecting after pure water is replaced.

9. The method according to any one of claims 1-8, wherein: the pH value of the initial system of the conversion reaction is 4.0-4.5, and the concentration of the initial substrate L-glutamic acid is 60g/L;

10. The method according to any one of claims 1-6, wherein: the operation voltage of the electrodialysis is 25-50V, the electrodialysis is a two-chamber bipolar membrane dissociation tank, the pH of the conversion end point of the conversion tank is 5.0-6.0, and the electrodialysis is stopped when the pH of the stock solution of the L-glutamic acid conversion solution is 4.5 after the electrodialysis treatment.