CN103601647B - Method for desalting and decoloring L-alanine fermentation liquor produced by genetically engineered bacteria - Google Patents

Abstract

The invention discloses a method for desalting and decolorizing fermented liquid of L-alanine produced by genetically engineered bacteria. The method is simple and convenient, and can save the cost of downstream separation and purification. The method for desalting and decolorizing the L-alanine fermentation broth produced by genetic engineering bacteria of the present invention mainly uses D101 macroporous resin to perform desalting and decolorization treatment on the L-alanine fermentation broth produced by genetic engineering bacteria.

Description

A method for desalination and decolorization of L-alanine fermentation broth produced by genetically engineered bacteria

technical field

The invention relates to a method for desalting and decolorizing fermented liquid, in particular to a method for desalting and decolorizing fermented liquid produced by genetically engineered bacteria for L-alanine.

Background technique

L-alanine, also known as α-alanine, has a molecular formula of C ₃ H ₇ NO ₂ . It is easily soluble in water, slightly soluble in ethanol, and insoluble in ether or acetone. It has a high sweetness and is optically active. One of the 20 L-type alpha amino acids that make up proteins. Although L-alanine is a non-essential amino acid for the human body, it is the amino acid with the highest content in human blood and has important physiological functions. L-alanine is the most important amino donor in the transamination reaction in vivo, and it can also be used as an excellent transport tool for nitrogen in the blood. It is closely related to glucose metabolism. In the glucose-alanine cycle, it maintains low blood ammonia levels and also produces glucose. L-alanine has a wide range of applications in the food and pharmaceutical industries. In the food industry, L-alanine can be used as preservatives and flavor seasonings for food and beverages. Adding a small amount of alanine can significantly improve the protein utilization rate in food and beverages and the nutritional value of food, and because alanine Acid has the characteristics of being directly digested and absorbed by organisms. After drinking, it can quickly recover from fatigue and invigorate the spirit; L-alanine has strong sweetness and can be used as a food sweetener. Add it to the compound sweetener A small amount of L-alanine can increase the sweetness and soften the sweetness like a natural sweetener. It is one of the raw materials for the synthesis of high-sweetness alitame; L-alanine can also improve the aftertaste and act as a sour taste corrector to improve The sour taste of organic acids makes it closer to the sour taste of natural substances; L-alanine is also used in nutritional supplements to prepare amino acid functional health wine, etc.; in the pharmaceutical industry, L-alanine is widely used in clinical infusions, Provide energy and nutrition for patients; L-alanine is also an important raw material for the production of vitamin B6, calcium pantothenate and other organic compounds.

At present, the vast majority of domestic L-alanine production is through biological fermentation. In addition to the exploration of upstream technologies such as breeding of excellent strains and improvement of fermentation process, the downstream separation and purification of L-alanine is also an important research content and one of the key technologies for the production of L-alanine by biological fermentation. There are a large number of pigment molecules and high-salt components in the microbial fermentation broth. Pigments are high-molecular organic compounds that have their own color and can color other substances. It is a metabolite produced during the fermentation process, which is related to the strain and fermentation conditions. During the pretreatment and refining process of the fermentation broth, most of the impurities and pigments have been removed, but there are still a small amount of pigments. If poor quality raw materials are used in the refining process, some pigments will also be brought in. Chromophores and auxiliary groups in various pigments generally contain a large number of various polar groups (such as -COOH, -NH ₂ , -OH, etc.). If these pigment molecules and high-salt components cannot be effectively removed, it will increase the difficulty of product separation, purification, and crystallization in subsequent downstream processes, thereby affecting the purity and quality of the product. The production cost of L-alanine is mainly composed of the upstream fermentation process and the downstream separation and purification process, among which the downstream process accounts for as high as 60% to 70% of the cost. Generally, the decolorization method commonly used for L-alanine decolorization is activated carbon decolorization. This is because activated carbon has a large specific surface area and strong adsorption capacity. However, it also has shortcomings and problems. It has certain requirements on the viscosity of the solution. When the viscosity is high, the sample liquid needs to be heated to a certain temperature to facilitate adsorption and filtration. In addition, activated carbon is not recyclable, and it is easy to adsorb products, resulting in Product loss is large. Moreover, the activated carbon decolorization method cannot effectively reduce the high salt content in the mother liquor. Therefore, how to improve the separation and purification method on the basis of saving costs and improve the quality of the finished product has become one of the key issues in the production of alanine by microbial fermentation.

D101 macroporous adsorption resin is a styrene-type non-polar copolymer with a wide range of applications. It generally has strong adsorption capacity for non-polar or weakly polar organic compounds. At present, it is widely used in the pharmaceutical industry, especially for The separation and purification effect of Chinese herbal medicines such as saponins and flavonoids is particularly good, such as: ginsenosides, notoginseng saponins, diosgenin, ginkgo flavonoids, etc. However, there are no relevant patents and reports about using D101 macroporous adsorption resin for desalting and decolorizing biological products such as L-alanine fermentation broth.

Contents of the invention

The purpose of the present invention is to solve the problems and deficiencies in the above-mentioned prior art, and provide a method for desalting and decolorizing the L-alanine fermentation broth produced by genetically engineered bacteria. The method is simple and convenient, and can save the cost of downstream separation and purification.

The present invention is achieved through the following technical solutions:

The method for desalting and decolorizing the L-alanine fermentation broth produced by genetic engineering bacteria of the present invention mainly uses D101 macroporous resin to perform desalting and decolorization treatment on the L-alanine fermentation broth produced by genetic engineering bacteria.

The method for desalting and decolorizing the L-alanine fermentation broth produced by genetically engineered bacteria of the present invention, its further scheme is that the use of D101 macroporous resin to carry out desalting and decolorizing treatment on the L-alanine fermentation broth produced by genetically engineered bacteria includes the following step:

Pretreatment of L-alanine fermentation broth produced by genetically engineered bacteria: Centrifuge the L-alanine fermentation broth produced by genetically engineered bacteria under a high-speed low-temperature centrifuge to remove impurities including bacteria, and collect the supernatant; Adjust the concentration of alanine in the supernatant to 70-80 mg/mL, and adjust the pH value to 2.0-3.0 for use;

D101 macroporous resin pretreatment: Pack D101 macroporous resin into a column, soak in ethanol for 24 hours, wash with water until the effluent is clear, then wash with 4% HCl, wash to neutral, 4% NaOH, wash to neutral The process after the property obtains the pretreatment D101 macroporous resin stand-by;

Desalination and decolorization treatment: the L-alanine fermentation broth that has been treated in the pretreatment step of the fermentation broth produced by genetically engineered bacteria enters the The pretreated D101 macroporous resin is desalted and decolorized, the pigment organic macromolecules and high-salt impurities are adsorbed on the macroporous resin, and the separated L-alanine fermentation broth is collected.

The method for desalting and decolorizing L-alanine fermentation broth produced by genetically engineered bacteria of the present invention has a further technical solution that the rotating speed of the high-speed low-temperature centrifuge is 10000-12000 r/min, and the centrifugation time is 5 minutes.

The method for the desalination and decolorization of L-alanine fermentation broth produced by genetically engineered bacteria of the present invention, its further technical scheme can also be that the D101 macroporous resin is pretreated before loading the column with 1/ 3-1/4 volume of water, then add D101 resin, the amount of resin packed into the column is 80% of the column volume after resin expansion.

The method for desalting and decolorizing the L-alanine fermentation broth produced by genetically engineered bacteria of the present invention, its further technical solution is to use 50-99% ethanol, 0.5- The flow rate of 2.0mL/min was used to elute the resin to regenerate the D101 macroporous resin.

Compared with the prior art, the present invention has the following beneficial effects:

D101 macroporous adsorption resin is used for desalination and decolorization. The adsorbed pigments and impurities are easily eluted, and 80% ethanol can be eluted. Repeated treatment with acid and alkali can realize the regeneration of the resin. D101 macroporous resin has little or no adsorption on L-alanine, and the product retention rate reaches over 94%, without causing product loss. Compared with other common resins, D101 macroporous resin can also desalinate and reduce the conductivity of the fermentation broth while decolorizing the fermentation broth. D101 macroporous resin has a high regeneration rate of over 95%, and can be reused dozens of times. Using D101 macroporous resin to desalt and decolorize L-alanine is better than activated carbon decolorization, which can save the cost of downstream separation and purification, and is suitable for industrial scale production.

Description of drawings

Figure 1 is a schematic diagram of the decolorization rate of D101 macroporous resin decolorizing L-alanine with temperature in Example 1 of the present invention.

Fig. 2 is a schematic diagram showing the decolorization rate of D101 macroporous resin for decolorization of L-alanine in Example 2 of the present invention as a function of sample loading flow rate.

Fig. 3 is a schematic diagram showing the variation of the resolution rate of the decolorization of L-alanine by the D101 macroporous resin in Example 3 of the present invention with the concentration of ethanol.

Detailed ways

In the examples, D101 macroporous adsorption resins are all commercially available products.

Example 1

(1) Pretreat the L-alanine fermentation broth, centrifuge it in a high-speed low-temperature centrifuge to remove impurities such as bacteria, and collect the supernatant;

(2) Adjust the concentration of L-alanine in the above supernatant to 75mg/mL, and adjust the pH value to 3.0;

(3) Pack D101 macroporous resin into a column, soak in ethanol for 24 hours, wash with water until the effluent is clear, then wash with 4% HCl, wash to neutral, 4% NaOH, wash to neutral pretreatment process;

(4) Control the flow rate of the fermented liquid after treatment to be 1.5mL/min, and the temperature is 35, 40, 45, 50, 55, 60°C, 65°C respectively, enter D101 macroporous resin for desalination and decolorization, and ferment to obtain alanine solution to calculate the decolorization rate. Figure 1 is the curve of temperature versus decolorization rate obtained from the dynamic decolorization test of D101 macroporous resin. It can be seen that the temperature has a great influence on the adsorption performance of D101 macroporous resin, and the decolorization performance is excellent at a temperature of about 50 °C.

(5) After the resin is saturated, use 80% ethanol at a flow rate of 1.0 mL/min to elute the resin.

In the above method, the rotating speed of the centrifuge is 11000r/min, and the centrifugation time is 5min.

Example 2

(1) Pretreat the L-alanine fermentation broth, and centrifuge it at 12000r/min for 5min in a high-speed low-temperature centrifuge to remove impurities such as bacteria, and collect the supernatant;

(2) Adjust the concentration of L-alanine in the above supernatant to 75mg/mL, and adjust the pH value to 3.0;

(3) Pack D101 macroporous resin into a column, soak in ethanol for 24 hours, wash with water until the effluent is clear, then wash with 4% HCl, wash to neutral, 4% NaOH, wash to neutral pretreatment process;

(4) Control the temperature at 30°C, enter the D101 macroporous resin at a flow rate of 0.5mL/min, 1mL/min, 1.5mL/min, 2mL/min, and 2.5mL/min for desalination and decolorization to obtain alanine fermentation broth , to calculate the decolorization rate. Figure 2 is the curve of sample loading flow rate versus decolorization rate obtained from the dynamic decolorization test of D101 macroporous resin. It can be seen that the decolorization performance is excellent when the flow rate is about 1.5mL/min.

(5) After the resin is saturated, use 80% ethanol at a flow rate of 1.0 mL/min to elute the resin.

In the above method, the rotating speed of the centrifuge is 12000r/min, and the centrifugation time is 5min.

Example 3

(1) Pretreat the L-alanine fermentation broth, and centrifuge it at 12000r/min for 5min in a high-speed low-temperature centrifuge to remove impurities such as bacteria, and collect the supernatant;

(2) Adjust the concentration of L-alanine in the above supernatant to 75mg/mL, and adjust the pH value to 3.0;

(3) Pack D101 macroporous resin into a column, soak in ethanol for 24 hours, wash with water until the effluent is clear, then wash with 4% HCl, wash to neutral, 4% NaOH, wash to neutral pretreatment process;

(4) Control the temperature at 30°C, enter the D101 macroporous resin at a flow rate of 1.5mL/min for desalination and decolorization, and obtain alanine fermentation broth;

(5) After the resin is adsorbed and saturated, the resin is eluted with the concentration of 50%, 60%, 70%, 80%, 99% ethanol, and the flow rate of 1.0mL/min, and the resolution rate is calculated. Figure 3 is the curve of ethanol concentration versus resolution rate obtained from the dynamic decolorization test of D101 macroporous resin. It can be seen that the resolution performance is excellent when the ethanol concentration is around 80%.

In the above method, the rotating speed of the centrifuge is 10000r/min, and the centrifugation time is 5min.

Claims (4)

1. genetic engineering bacterium produces a method for ALANINE fermented liquid desalination bleaching, it is characterized in that using D101 macroporous resin to produce ALANINE fermented liquid to genetic engineering bacterium carries out desalination bleaching process; Described use D101 macroporous resin carries out desalination bleaching process to genetic engineering bacterium product ALANINE fermented liquid and comprises the following steps:

Genetic engineering bacterium produces ALANINE fermentation liquor pretreatment: genetic engineering bacterium is produced ALANINE fermented liquid centrifugal to remove the impurity comprising thalline under high speed low temperature centrifugal machine, collect and obtain supernatant liquor; L-Ala concentration in supernatant liquor is adjusted to 70 ~ 80mg/mL, and pH value is adjusted to 2.0 ~ 3.0 stand-by;

D101 macroporous resin pre-treatment: by D101 macroporous resin dress post, and after carrying out alcohol immersion 24h after washing to effluent liquid clarification, then wash through 4%HCl stream, be washed to neutrality, 4%NaOH stream is washed, be washed to neutrality after process to obtain pre-treatment D101 macroporous resin stand-by;

Desalination bleaching process: genetic engineering bacterium to be produced in ALANINE fermentation liquor pretreatment step processed good ALANINE fermented liquid with flow velocity for 0.5 ~ 2.5mL/min, temperature is pass into pretreated D101 macroporous resin under 35 ~ 65 DEG C of conditions to carry out desalination bleaching, collects the ALANINE fermented liquid after being separated.

2. genetic engineering bacterium according to claim 1 produces the method for ALANINE fermented liquid desalination bleaching, and it is characterized in that the rotating speed of described high speed low temperature centrifugal machine is 10000 ~ 12000r/min, centrifugation time is 5min.

3. genetic engineering bacterium according to claim 1 produces the method for ALANINE fermented liquid desalination bleaching, when it is characterized in that described D101 macroporous resin pre-treatment before dress post prior to post in load the water of 1/3 ~ 1/4 volume, then add D101 resin, resin dress post amount is 80% of column volume after resin expands.

4. genetic engineering bacterium according to claim 1 produces the method for ALANINE fermented liquid desalination bleaching, it is characterized in that in desalination bleaching treating processes, after D101 macroporous resin adsorption is saturated, the wash-out carrying out resin with the flow velocity of 50 ~ 99% ethanol, 0.5 ~ 2.0mL/min makes D101 macroporous resin regenerate.