阅读说明：本技术 一种利用模拟移动床色谱提取l-组氨酸的方法 (Method for extracting L-histidine by simulated moving bed chromatography ) 是由 边恩来 冯世红 张宗华 崔小红 庄会华 于 2020-12-21 设计创作，主要内容包括：本发明属于L-组氨酸的生产技术领域,公开了一种利用模拟移动床色谱提取L-组氨酸的方法,其包括发酵工序和提取工序。本方法可以实现L-组氨酸的连续化生产,具有发酵效率高、生产周期短、生产成本低、污染少、提取收率高等优点。(The invention belongs to the technical field of L-histidine production, and discloses a method for extracting L-histidine by using simulated moving bed chromatography, which comprises a fermentation process and an extraction process. The method can realize continuous production of L-histidine, and has the advantages of high fermentation efficiency, short production period, low production cost, less pollution, high extraction yield, etc.)

1. A method for extracting L-histidine by simulated moving bed chromatography is characterized by comprising a fermentation process and an extraction process.

2. The method of claim 1, wherein the fermentation process comprises the following:

1) inoculating serratia marcescens seed liquid into a fermentation culture tank containing a fermentation culture medium for fermentation, and controlling the liquid volume in the fermentation culture tank to be 50L;

2) in the whole fermentation process, feeding the dipotassium phosphate aqueous solution at the feeding flow rate of 20ml/h until the fermentation is finished; after fermentation culture is carried out for 6 hours, feeding fermentation accelerant at the feeding flow rate of 0.5L/h until fermentation is finished, and collecting histidine fermentation liquor; the total fermentation time was 52 h.

3. The method of claim 1, wherein the extraction process comprises the following: step 1) ceramic membrane filtration, step 2) simulated moving bed chromatography, step 3) primary decolorization, step 4) concentration and redissolution, step 5) secondary decolorization, step 6) refining and concentration, and step 7) crystallization and centrifugation.

4. The method as claimed in claim 2, wherein the fermentation promoter comprises glucose 100-1000g/L and calcium acetate 10-100 mg/L.

5. The method of claim 2, wherein the fermentation medium comprises: 70g/L glucose, 5g/L yeast powder, 1.5g/L betaine, 5g/L dipotassium hydrogen phosphate, 40ml/L corn steep liquor, 4g/L ammonium sulfate, 0.5g/L magnesium sulfate, 0.15mg/L biotin and vitamin B₁ 0.15mg/L。

6. The method of claim 2, wherein the aqueous solution of dipotassium phosphate has a concentration of 100 g/L.

7. The method of claim 2, wherein the extraction process comprises the following:

step 1) ceramic membrane filtration: controlling the temperature of histidine fermentation liquor to 70-80 ℃, adjusting the pH value to 5.0-5.5, filtering by a ceramic membrane, and collecting clear liquid of the ceramic membrane;

step 2) simulated moving bed chromatography: subjecting the clear solution of the ceramic membrane obtained in the step 1) to simulated moving bed chromatography filled with cation exchange resin, and eluting with ammonia water to obtain histidine eluent;

step 3), primary decoloring: removing part of pigment and small molecular impurities from the histidine eluent obtained in the step 2) through a decolorizing membrane to obtain a decolorizing solution;

step 4), concentration and redissolution: concentrating the destaining solution obtained in the step 3) by a four-effect evaporator until the content of histidine is 220-250g/L, cooling to 10 ℃ by using condensed water, carrying out crystal growth for 6h, starting a centrifugal machine for centrifugation, collecting the obtained mother solution and crude histidine, redissolving the crude histidine, adjusting the pH value to 6.5-7.0, and collecting the solution;

step 5) secondary decolorization: decoloring the solution obtained in the step 4) by using activated carbon at the decoloring temperature of 50 ℃ for 60min, filtering and collecting filtrate;

step 6) refining and concentrating: concentrating the filtrate obtained in the step 5) by a four-effect evaporator until the histidine content is 220-250g/L, cooling to 10 ℃ by using condensed water, culturing crystals for 6h, and filtering to obtain a wet finished product;

step 7), crystallization and centrifugation: centrifuging the wet finished product obtained in the step 6), and performing low-temperature vacuum drying to obtain the L-histidine finished product.

8. The method as claimed in claim 4, wherein the fermentation promoter comprises glucose 100-1000g/L, calcium acetate 10-100mg/L, and malonic acid 10-50 mg/L.

9. The method of claim 4, wherein the fermentation promoter has a composition of: 500g/L glucose, 100mg/L calcium acetate and 50mg/L malonic acid.

10. The method as claimed in claim 7, wherein the pore size of the decolorizing membrane is 300-500 mw.

Technical Field

The invention relates to the technical field of L-histidine production, in particular to a method for extracting L-histidine by using simulated moving bed chromatography.

Background

L-histidine is a basic amino acid containing an imidazole nucleus in its molecule, is a semi-essential amino acid, is not essential for adults, but is particularly important for the growth of infants and animals, and is synthesized slowly in the human body. It has multiple physiological functions, can be used as biochemical reagent and medicament, and can be used for treating heart disease, anemia, rheumatic arthritis, etc. Besides, histidine is one of the necessary raw materials for the synthesis of some medical intermediates. Due to its remarkable nutritional efficacy, it has a broad market prospect, and its role in medical research is increasingly emphasized.

At present, L-histidine is mainly extracted from pig blood meal hydrolysate in China by an ion exchange process, and can also be extracted from hydrolysate of defatted soybean, and the L-histidine is not suitable for industrialized mass production because the L-histidine has higher cost and high hydrolysis loss rate, thereby causing environmental pollution. The extraction method can realize continuous production of L-histidine, has the advantages of short production period, low production cost, less pollution, high extraction yield and the like, and has very many advantages compared with a protein hydrolysis extraction method.

Disclosure of Invention

In order to solve the problems and overcome the defects of the prior art, the invention provides the method for extracting the L-histidine by using the simulated moving bed chromatography, the method can realize the continuous production of the L-histidine, and has the advantages of short production period, low production cost, less pollution, high extraction yield and the like.

The purpose of the invention is realized by the following technical scheme.

A method for extracting L-histidine by simulated moving bed chromatography, which is characterized by comprising the following fermentation process and extraction process.

Specifically, the fermentation process comprises the following steps:

1) inoculating serratia marcescens seed liquid into a fermentation culture tank containing a fermentation culture medium, controlling the liquid volume in the fermentation culture tank to be 50L, and controlling the fermentation culture condition to be at 33 ℃, pH7.0, the rotating speed of 80rpm and the pressure of 0.05 MPa;

2) in the whole fermentation process, feeding the dipotassium phosphate aqueous solution at the feeding flow rate of 20ml/h until the fermentation is finished; after fermentation culture is carried out for 6 hours, feeding fermentation accelerant at the feeding flow rate of 0.5L/h until fermentation is finished, and collecting histidine fermentation liquor; the total fermentation time was 52 h.

Specifically, the extraction process comprises the following steps: step 1) ceramic membrane filtration, step 2) simulated moving bed chromatography, step 3) primary decolorization, step 4) concentration and redissolution, step 5) secondary decolorization, step 6) refining and concentration, and step 7) crystallization and centrifugation.

Further, the fermentation promoter comprises 1000g/L of glucose 100-100 mg/L and 10-100mg/L of calcium acetate.

Further, the fermentation medium comprises the following components: 70g/L glucose, 5g/L yeast powder, 1.5g/L betaine, 5g/L dipotassium hydrogen phosphate, 40ml/L corn steep liquor, 4g/L ammonium sulfate, 0.5g/L magnesium sulfate, 0.15mg/L biotin and vitamin B₁0.15mg/L。

Further, the concentration of the aqueous solution of dipotassium hydrogen phosphate is 100g/L

Further, the extraction process comprises the following steps:

step 1) ceramic membrane filtration: controlling the temperature of histidine fermentation liquor to 70-80 ℃, adjusting the pH value to 5.0-5.5, filtering by a ceramic membrane, and collecting clear liquid of the ceramic membrane;

step 2) simulated moving bed chromatography: subjecting the clear solution of the ceramic membrane obtained in the step 1) to simulated moving bed chromatography filled with cation exchange resin, and eluting with ammonia water to obtain an L-histidine eluent;

step 3), primary decoloring: removing part of pigment and micromolecular impurities from the L-histidine eluent obtained in the step 2) through a decolorizing membrane to obtain a decolorizing solution;

step 4), concentration and redissolution: concentrating the destaining solution obtained in the step 3) by a four-effect evaporator until the content of L-histidine is 220-one 250g/L, cooling to 10 ℃ by using condensed water, carrying out crystal growth for 6h, starting a centrifugal machine for centrifugation, collecting the obtained mother solution and crude histidine, redissolving the crude histidine, adjusting the pH value to 6.5-7.0, and collecting the solution;

step 5) secondary decolorization: decoloring the solution obtained in the step 4) by using activated carbon at the decoloring temperature of 50 ℃ for 60min, filtering and collecting filtrate;

step 6) refining and concentrating: concentrating the filtrate obtained in the step 5) by a four-effect evaporator until the histidine content is 220-250g/L, cooling to 10 ℃ by using condensed water, culturing crystals for 6h, and filtering to obtain a wet finished product;

step 7), crystallization and centrifugation: centrifuging the wet finished product obtained in the step 6), and performing low-temperature vacuum drying to obtain the L-histidine finished product.

Furthermore, the fermentation promoter comprises glucose 100-1000g/L, calcium acetate 10-100mg/L, and malonic acid 10-50 mg/L.

Preferably, the components of the fermentation promoter are: 500g/L glucose, 100mg/L calcium acetate and 50mg/L malonic acid.

Drawings

FIG. 1: influence of calcium acetate on histidine yield in fermentation broth;

FIG. 2: effect of malonic acid on histidine production in fermentation broth.

The beneficial effects achieved by the invention mainly comprise but are not limited to the following aspects:

the Serratia marcescens subjected to activation culture is adopted to produce histidine, a nitrogen source and nutrient substances required by the growth of the strain are effectively supplemented by feeding glucose and dipotassium hydrogen phosphate in the fermentation process, the growth activity of the strain is maintained, the fermentation acid production performance is greatly improved, the glucose consumption in the propagation of the strain is reduced, the sugar-acid conversion rate is increased, and the yield of histidine and the sugar-acid conversion rate are greatly improved by the method through detection.

The acid production mechanism of different strains and the tolerance degree of the acid production mechanism to the stimulating factors are greatly different and have no reference significance, and although the acid production mechanism of the glutamic acid is researched more in the prior art, for example, the amount of histidine produced by corynebacterium glutamicum can be increased by adding a proper amount of sodium citrate into a fermentation culture medium, the sodium citrate has no obvious stimulation effect on serratia marcescens.

The fermentation process of histidine is independent of TCA pathway, and the weakening of TCA pathway can improve the metabolic flow entering histidine synthesis pathway, but TCA cycle maintains normal proliferation and metabolism of somatic cells, and cannot weaken excessively. Malonic acid can be used as an inhibitor of TCA cycle, and by weakening TCA cycle, metabolic flow of histidine synthetic pathway can be improved, and further, the yield of histidine is improved.

L-histidine is an intermediate product of an HMP (high molecular weight peptide) pathway, an acetic acid byproduct is also generated in the HMP pathway, so that waste of carbon metabolic flow is caused, calcium acetate is added to generate a certain inhibition effect on the byproduct, so that more metabolic flows in the HMP pathway enter a histidine synthesis pathway, more preconditions are provided for biosynthesis of histidine, and the yield of histidine is improved.

The present invention selects to start fed-batch in the middle of fermentation because the mass synthesis of histidine occurs in the middle of fermentation, and the strain proliferation is the main in the early stage of fermentation, at which point weakening of the TCA pathway reduces the strain viability.

The invention adopts a simulated moving bed chromatographic separation technology to extract L-histidine, adopts a color reaction during chromatographic column loading and elution interception, reduces interception loss, improves chromatographic yield, and separates other impurities generated in histidine fermentation liquor by utilizing the principle that different ions pass through chromatographic resin for different time to carry out physical adsorption, thereby improving the purity of histidine.

The method can effectively remove other heteropolyacids and impurities in the fermentation liquor at one time, improve the product quality, reduce the using amount of acid and alkali, reduce the discharge amount of wastewater and realize the purposes of clean production, energy conservation and environmental protection.

The invention adopts the four-effect evaporator, ensures the sanitary requirements of materials and the environmental protection requirements in the evaporation process under the vacuum action, greatly reduces the evaporation temperature, and has the advantages of energy saving, consumption reduction, low steam consumption and low cooling water circulation.

The invention decolors twice, uses a decoloring film for one-time decoloring, does not use active carbon, reduces the using amount of the active carbon, reduces the generation amount of fixed wastes and reduces the pollution to the environment.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the following will fully describe the technical solutions in conjunction with the specific embodiments of the present application.

Example 1

A method for extracting L-histidine by simulated moving bed chromatography comprises the following steps:

1. a fermentation process:

1) the method comprises the steps of putting serratia marcescens (ATCC 31026) into an activation culture medium, maintaining the temperature of a constant-temperature incubator at 33 ℃ for constant-temperature culture for 24 hours, wherein the formula of the activation culture medium comprises 1g/L of anhydrous glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 2.5g/L of sodium chloride and 20g/L of agar strips, and thus obtaining the activation culture solution. Before the strain is transferred, the seeding tank is charged, water is added to the seeding tank to a constant volume, the constant volume is 5L, steam is directly connected to the seeding tank to heat the seeding tank to 119-122 ℃, the pressure is 0.13-0.14MPa, the temperature is kept for 30min, and sterilization treatment is carried out.

2) Inoculating the obtained activated culture solution into sterilized seed culture tank by pressure difference method, wherein the volume of the liquid is 5L, and the initial temperature of the culture condition is maintained and controlled at 33 deg.C, pH7.0, rotation speed of stirring motor 80rpm, and air volume is 70m³H, the pressure is 0.05MPa, and the continuous culture is carried out for 16 h. The components of the control feed liquid are 35g/L of glucose, 5g/L of yeast powder, 1.5g/L of betaine, 1.5g/L of dipotassium hydrogen phosphate, 30ml/L of corn steep liquor, 5g/L of ammonium sulfate, 0.4g/L of magnesium sulfate, 0.15mg/L of biotin and 10.15mg/L of vitamin B.

3) Feeding dipotassium phosphate into the fermentation at the beginning of the fermentation, wherein the concentration of the dipotassium phosphate is 100g/L, the feeding flow rate is 1.5ml/h, feeding glucose is started after 5 hours of fermentation culture, the concentration of the glucose is 500g/L, and the feeding flow rate is 0.1L/h, and continuously culturing for 16 hours until the end of the fermentation.

4) After the OD value of the seed irrigation is detected to reach 20.3, the seeds are transplanted and are inoculated into a fermentation culture tank according to the inoculation amount of 10 percent. The volume of liquid in the fermentation culture tank is 50L, the temperature of the fermentation culture condition is controlled to be 33 ℃, the pH value is 7.0, the rotating speed of a stirring motor is 80rpm, and the air volume is 400m³H and pressure of 0.05 MPa.

5) The fermentation medium components of glucose 70g/L, yeast powder 5g/L, betaine 1.5g/L, dipotassium hydrogen phosphate 5g/L, corn steep liquor 40ml/L, ammonium sulfate 4g/L, magnesium sulfate 0.5g/L, biotin 0.15mg/L and vitamin B10.15mg/L are controlled. Feeding dipotassium phosphate into the fermentation at the beginning of the fermentation, wherein the concentration of the dipotassium phosphate is 100g/L, the feeding flow rate is 20ml/h, the fermentation culture is carried out for 6h, then feeding fermentation accelerators (500 g/L of glucose, 100mg/L of calcium acetate and 50mg/L of malonic acid) is started, the feeding flow rate is 0.5L/h until the fermentation is finished, the total fermentation time is 52h, and the histidine yield in the obtained L-histidine fermentation liquor reaches 38.7 g/L;

2. an extraction process:

1) ceramic membrane filtration: controlling the temperature of the L-histidine fermentation liquor to 70-80 ℃, adjusting the pH to 5.0-5.5 by using sulfuric acid, adding 60% of water, uniformly stirring, filtering by using a ceramic membrane to remove mycoprotein and other particle impurities, and collecting a ceramic membrane clear solution;

2) simulated moving bed chromatography: subjecting the clear solution of the ceramic membrane to simulated moving bed chromatography filled with cation exchange resin, and eluting with ammonia water with a certain concentration to obtain an L-histidine eluate;

3) primary decoloring: removing partial pigment and small molecular impurities from the obtained L-histidine eluent by a decolorizing membrane to obtain decolorizing liquid, wherein the decolorizing membrane adopts a 300-500 molecular weight membrane pore diameter;

4) concentration and redissolution: concentrating the obtained decolorized solution by a four-effect evaporator until the content of L-histidine is 250g/L, cooling to 10 ℃ by using condensed water, growing crystals for 6 hours, starting a centrifugal machine for centrifugation, collecting the obtained mother solution and crude product, recycling the mother solution, purifying the crude product, then carrying out redissolution according to 60g/L, adjusting the pH value to 6.5-7.0, and collecting the obtained solution;

5) and (3) secondary decolorization: decolorizing the obtained back solution with active carbon at 50 deg.C for 60min, allowing the decolorized solution to pass through 98% of light, and collecting filtrate;

6) refining and concentrating: concentrating the obtained filtrate by a four-effect evaporator until the content of histidine is 220-fold and 250g/L, cooling to 10 ℃ by using condensate water, and filtering to obtain a wet finished product after crystal growth for 6 hours;

7) and (3) crystallization and centrifugation: centrifuging the obtained wet finished product, and performing low-temperature vacuum drying to obtain an L-histidine finished product; the purity can reach more than 97% by detection.

In conclusion, the method for extracting L-histidine by using simulated moving bed chromatography provided by the invention improves the product quality by using the technologies of ceramic membrane filtration sterilization, decolorization membrane impurity removal decolorization, simulated moving bed chromatography separation and the like, further improves the yield, increases the yield, provides a high-cost performance product for the L-histidine market, reduces the discharge of sewage, reduces the pollution to the environment, increases the social benefit, and has a great market popularization prospect.

Comparative example 1

A method for extracting L-histidine by simulated moving bed chromatography comprises the following steps:

1. a fermentation process:

1) the method comprises the steps of putting serratia marcescens (ATCC 31026) into an activation culture medium, maintaining the temperature of a constant-temperature incubator at 33 ℃ for constant-temperature culture for 24 hours, wherein the formula of the activation culture medium comprises 1g/L of anhydrous glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of yeast extract, 2.5g/L of sodium chloride and 20g/L of agar strips, and thus obtaining the activation culture solution. Before the strain is transferred, adding water into a seed tank for constant volume, wherein the constant volume is 5L, directly introducing steam for heating to 119-;

3) inoculating the obtained activated culture solution into sterilized seed culture tank by pressure difference method, wherein the volume of the liquid is 5L, and the initial temperature of the culture condition is maintained and controlled at 33 deg.C, pH7.0, rotation speed of stirring motor 80rpm, and air volume is 70m³H, the pressure is 0.05MPa, and the continuous culture is carried out for 16 h. The components of the control feed liquid are 35g/L of glucose, 5g/L of yeast powder, 1.5g/L of betaine, 1.5g/L of dipotassium hydrogen phosphate, 30ml/L of corn steep liquor, 5g/L of ammonium sulfate, 0.4g/L of magnesium sulfate, 0.15mg/L of biotin and 10.15mg/L of vitamin B.

4) Feeding dipotassium phosphate into the fermentation at the beginning of the fermentation, wherein the concentration of the dipotassium phosphate is 100g/L, the feeding flow rate is 1.5ml/h, feeding glucose is started after 5 hours of fermentation culture, the concentration of the glucose is 500g/L, and the feeding flow rate is 0.1L/h, and continuously culturing for 16 hours until the end of the fermentation. After the OD value of the seed irrigation is detected to reach 20.3, the seeds are transplanted and are inoculated into a fermentation culture tank according to the inoculation amount of 10 percent.

The volume of liquid in the fermentation culture tank is 50L, the temperature of the fermentation culture condition is controlled to be 33 ℃, the pH value is 7.0, the rotating speed of a stirring motor is 80rpm, and the air volume is 400m³H and pressure of 0.05 MPa.

4) The fermentation medium components of glucose 70g/L, yeast powder 5g/L, betaine 1.5g/L, dipotassium hydrogen phosphate 5g/L, corn steep liquor 40ml/L, ammonium sulfate 4g/L, magnesium sulfate 0.5g/L, biotin 0.15mg/L and vitamin B10.15mg/L are controlled. Feeding dipotassium phosphate into the fermentation at the beginning of the fermentation, wherein the concentration of the dipotassium phosphate is 100g/L, the feeding flow rate is 20ml/h, carrying out fermentation culture for 6h, then feeding a fermentation accelerator (500 g/L of glucose), and feeding the flow rate is 0.5L/h until the fermentation is finished, the total fermentation time is 52h, and the histidine yield reaches 22.1 g/L.

2. The extraction procedure was the same as in example 1.

Example 2

1. The process is the same as that of the comparative example 1, the fermentation promoter is optimized on the basis of the comparative example 1, calcium acetate (abscissa) with different concentrations is set to be 0, 20, 40, 60, 80, 100, 120 and 140, and the unit is mg/L, as shown in figure 1, the content of histidine (ordinate, g/L) in the fermentation broth is increased along with the increase of the calcium acetate concentration, when the calcium acetate concentration reaches 100mg/L, the histidine concentration reaches a peak value, the continuous increase of the calcium acetate concentration has no obvious influence on the yield of histidine, the sugar acid conversion rate is consistent with the trend of histidine, which indicates that the calcium acetate mainly improves the yield of histidine by improving the sugar acid conversion rate of Serratia marcescens, probably because L-histidine is an intermediate product of an HMP pathway, and an acetic acid byproduct is also generated by the HMP pathway, thereby causing waste of carbon metabolic flux, calcium acetate with proper concentration is added to generate a certain inhibition effect on the by-products, so that more metabolic flows in an HMP (HMP) pathway enter a histidine synthesis pathway, more precursor substances are provided for biosynthesis of histidine, and the yield of histidine is improved; furthermore, calcium ions are also activators of enzymes required for histidine synthesis.

2. The concentration of calcium acetate was selected to be 100mg/L, and the effect of malonic acid on histidine production in the fermentation broth was continuously evaluated. Setting different concentrations of calcium acetate, 0, 10, 20, 30, 40, 50, 60 and 70, respectively, with the unit of mg/L, as shown in FIG. 2, the content of histidine in the fermentation broth is increased along with the increase of the concentration of malonic acid, when the concentration of calcium acetate reaches 50mg/L, the concentration of histidine approaches the peak value, the continuous increase of calcium acetate has no substantial influence on the yield of histidine, the sugar acid conversion rate and the trend of histidine are basically consistent, probably because the fermentation process of histidine does not depend on the TCA pathway, the metabolic flux entering the histidine synthesis pathway can be improved by properly weakening the TCA pathway, but the normal proliferation and metabolism of somatic cells are maintained by the TCA cycle and cannot be excessively weakened, and therefore, the feeding beginning in the middle fermentation stage needs to be selected, because the mass synthesis of histidine occurs in the middle fermentation stage and the strain proliferation is mainly used in the early fermentation stage, weakening the TCA pathway at this point reduces strain viability. Proper amount of malonic acid can be used as an inhibitor of TCA cycle, and metabolic flow of histidine synthetic pathway can be improved by weakening TCA cycle, so that the saccharic acid conversion rate and the yield of histidine are improved.

While the invention has been described with reference to specific embodiments, it will be apparent to those skilled in the art that the invention is not limited thereto, and that various changes and modifications can be made without departing from the spirit and scope of the invention.