CN101580587B - Novel technology for extracting polyglutamic acid - Google Patents

Abstract

The invention discloses novel technology for extracting polyglutamic acid, which comprises the following steps: adopting dilute acid to adjust the pH value of a zymotic fluid to between 3.0 and 5.0 toreduce the viscosity of the zymotic fluid first; then centrifugating the zymotic fluid at a high speed to remove thalli; adding enzyme into centrifugated clear solution further to hydrolyze protein,centrifugating to remove undissolved substances at a high speed; performing acid regulation and using an ultrafiltration membrane to dialysis and concentrate so as to remove small molecular impuritiessuch as pigment, salt and the like; using ethanol to precipitate colloid residue, finally using the ethanol to extract a colloid by adding salt; performing solid-liquid separation; and performing vacuum low-temperature or vacuum freeze drying on the solid colloid to obtain a finished product of polyglutamic acid. The technology has the advantages of simple process, low production cost, and good product quality.

Description

A kind of polyglutamic acid extraction process

Technical field the invention belongs to the polyglutamic acid production technical field, relates to a kind of polyglutamic acid extraction process.

The background technology gamma-polyglutamic acid-is the outer polypeptide of a kind of born of the same parents that is produced by multiple bacillus, and it is the main ingredient of certain micro-organisms pod membrane, is a kind of water-soluble and biodegradable polymer substance.It is to be formed by connecting by the γ amido linkage by D-type or L-type L-glutamic acid.Gamma-polyglutamic acid-is found in nineteen thirty-seven the earliest, and the researchist has found gamma-polyglutamic acid-in the cell pod membrane of Bacillus anthracis and in the cell pod membrane of amylomyces, and proves that it is one of main component of certain micro-organisms pod membrane.In subtilis and bacillus natto, also find gamma-polyglutamic acid-afterwards.Gamma-polyglutamic acid-is a kind of peptide molecule that is formed with the form condensation of peptide bond with γ-carboxyl and alpha-amino group by D-L-glutamic acid (D-GLu) and L-L-glutamic acid (L-GLu) monomer.Have the higher side chain carboxyl group of a large amount of activity on the molecular chain of gamma-polyglutamic acid-, have high moisture retention and water-absorbent (1: 3500, m/v).Be easy to some medicines in conjunction with generating stabilized complex, be the biodegradable pharmaceutical macromolecular material of a class ideal.The gamma-polyglutamic acid-environmentally safe is the green bio product.The nineties in 20th century, some developed countries such as Japan, the U.S. were very active to its preparation and the research of using, and carried out the research that utilizes liquid submerged fermentation to produce gamma-polyglutamic acid-.The gamma-polyglutamic acid-research and development of Japan are positioned at prostatitis, the world, and Ajincomoto Co., Inc has successfully developed the biological production technology of gamma-polyglutamic acid-.At present, the method for extraction mainly contains: organic solvent precipitation method, chemical precipitation method or the membrane sepn precipitator method obtain gamma-polyglutamic acid-.But the method that prior art is extracted exists production cost height, extraction process complicated technology problem.

Summary of the invention the purpose of this invention is to provide the polyglutamic acid extraction process that a kind of technology is simple, production cost is low.

The present invention operates by following processing step:

(1) detects gamma-polyglutamic acid-content, pH, soluble protein content in the fermented liquid;

(2) open stirring, slowly transfer fermented liquid pH to 3.0-5.0 with 1-15% dilute hydrochloric acid;

(3) carrying out thalline with the pump delivery fermented liquid to supercentrifuge separates, separating clear liquid goes in the enzymatic vessel, open the sodium hydroxide solution that stirs with 1-20% and transfer fermented liquid pH to 6.0-8.0, opening steam slowly heats up, the fermented liquid temperature is remained between 30 ℃-50 ℃,, add proteolytic enzyme in the ratio of 0.1%-10% according to soluble protein content in the fermented liquid, open and stir insulation 5-7 hour, make protein hydrolysis thorough;

(4) remove behind the albumen clear liquid once more high speed centrifugation remove insolubles, slowly transfer centrifugal stillness of night pH to 3.0-5.0 behind the enzymolysis with 1-15% dilute hydrochloric acid, dialyse concentrated to ultra-filtration membrane with pump delivery;

(5) through ultrafiltration and concentration, remove pigment and salt, other amino acid small molecular weight impurity, ultrafiltration and concentration liquid goes in extractor;

(6) open stirring and slowly transfer ultrafiltration and concentration liquid pH to 6.0-8.0 or 2.0-4.0 with the alkali lye of 1-20%, slowly stream adds the ethanol of 60-90 degree, makes fermented liquid form milky suspension liquid, has grey or xanchromatic glue slag to occur simultaneously;

(7) suspension liquid and glue slag go to whizzer with pump, and the centrifugal slag that removes photoresist that removes obtains pure emulsion;

(8) emulsion goes in the second extraction jar, opens an amount of solid salt of the slow adding of stirring and separates out colloid, separates out fully until colloid, stops stirring the sedimentation colloid 1-5 hour, shifts top ethanol;

(9) open stirring and repeatedly added an amount of 80-100 degree alcohol immersion colloid 1-3 hour, reduce colloid viscosity, go to whizzer with pump and carry out solid-liquid separation, the colloid after the separation carries out vacuum lyophilization or vacuum dehydrating at lower temperature, makes the gamma-polyglutamic acid-finished product.

Gamma-polyglutamic acid-extraction process of the present invention adopts multiple effective means to remove impurity in the fermented liquid, has promoted end product quality, has following outstanding advantage:

1, transfer fermented liquid pH to 3.0-5.0 to reduce fermentation broth viscosity with diluted acid, the high speed centrifugation fermented liquid is removed thalline, the simple efficient large-scale production that is fit to of technology;

2, centrifugal clear liquid adds protein hydrolysis and is further purified feed liquid;

3, the centrifugal clear liquid acid adjustment behind the enzymolysis is dialysed through ultrafiltration and concentration, removes pigment, salt, other hydrolysis amino acid small molecular weight impurity, has effectively saved steam, ethanol consumption simultaneously;

4, add salt and cooperate the extraction using alcohol colloid, compare with independent use ethanol to extract and saved the ethanol consumption;

5, lyophilize or vacuum dehydrating at lower temperature have guaranteed quality product;

Embodiment is an example with tank body volume 100L fermentor tank, adopts the present invention to extract gamma-polyglutamic acid-technology, and step is as follows:

(1) detects fermentation sol content 4.6g/100ml, pH6.65, soluble protein content 0.54g/L, volume 50L;

(2) open stirring, slowly transfer fermented liquid pH to 3.0 with 10% dilute hydrochloric acid;

(3) carry out 13000r/min high-speed separation thalline with the pump delivery fermented liquid to supercentrifuge, separating clear liquid goes in the enzymatic vessel, open the sodium hydroxide lye that stirs with 10% and transfer fermented liquid pH to 6.6, opening steam slowly heats up, make the fermented liquid temperature remain on 40 ℃, add 1.4g proteolytic enzyme according to fermented liquid soluble protein content, open and stirred insulation effect 6 hours, make the enzyme effect thorough;

(4) carry out 13000r/min high-speed separation insolubles with pump delivery enzymatic hydrolysis and fermentation liquid to supercentrifuge, centrifugal clear liquid is slowly transferred pH to 3.0 with 1-15% dilute hydrochloric acid behind the enzymolysis, dialyses concentrated to ultra-filtration membrane with pump delivery;

(5) concentrate through ultrafiltration dialysis, remove wherein pigment and salt. the total free aminoacids small molecular weight impurity, concentrate back material liquid volume 40L, ultrafiltration and concentration liquid goes in extractor;

(6) open stirring and slowly transfer ultrafiltration and concentration liquid pH6.0 with 10% sodium hydroxide lye, slowly stream adds the ethanol of 80 degree, and making fermented liquid form milky suspension liquid has the glue slag of grey or yellow-gray to occur simultaneously again;

(7) suspension liquid and glue slag go to whizzer with pump, and the centrifugal slag that removes photoresist that removes of 4000r/min obtains pure emulsion;

(8) emulsion goes in the second extraction jar, opens the slow solid food level salt that adds of stirring and separates out colloid, separates out fully until colloid, stops stirring the sedimentation colloid 1 hour, shifts top ethanol;

(9) open stirring and repeatedly added an amount of 95 degree alcohol immersion colloids 3 hours, go to whizzer 500r/min with pump and carry out solid-liquid separation, the colloid after the separation carries out lyophilize or vacuum-drying, makes 1.35Kg finished product gamma-polyglutamic acid-.

Claims (1)

1. polyglutamic acid extraction process is characterized in that by following processing step operation:

(1) detects gamma-polyglutamic acid-content, pH, soluble protein content in the fermented liquid;

(2) open stirring, slowly transfer fermented liquid pH to 3.0-5.0 with 1-15% dilute hydrochloric acid;

(3) carrying out thalline with the pump delivery fermented liquid to supercentrifuge separates, separating clear liquid goes in the enzymatic vessel, open the sodium hydroxide solution that stirs with 1-20% and transfer fermented liquid pH to 6.0-8.0, opening steam slowly heats up, the fermented liquid temperature is remained between 30 ℃-50 ℃,, add proteolytic enzyme in the ratio of 0.1%-10% according to soluble protein content in the fermented liquid, open and stir the insulation effect certain hour, make protein hydrolysis thorough;

(4) remove behind the albumen clear liquid once more high speed centrifugation remove insolubles, slowly transfer centrifugal clear liquid pH to 3.0-5.0 behind the enzymolysis with 1-15% dilute hydrochloric acid, dialyse concentrated to ultra-filtration membrane with pump delivery;

(5) through ultrafiltration and concentration, remove pigment and salt, other amino acid small molecular weight impurity, ultrafiltration and concentration liquid goes in extractor;

(6) open stirring and slowly transfer ultrafiltration and concentration liquid pH to 6.0-8.0 or 2.0-4.0 with the alkali lye of 1-20%, slowly stream adds the ethanol of 60-90 degree, and making fermented liquid form milky suspension liquid has grey or xanchromatic glue slag to occur simultaneously;

(7) suspension liquid and glue slag go to whizzer with pump, and the centrifugal slag that removes photoresist that removes obtains pure emulsion;

(8) emulsion goes in the second extraction jar, opens an amount of solid salt of the slow adding of stirring and separates out colloid, separates out fully until colloid, stops stirring the sedimentation colloid 1-5 hour, shifts top ethanol;

(9) open stirring and repeatedly added an amount of 80-100 degree alcohol immersion colloid 1-3 hour, reduce colloid viscosity, go to whizzer with pump and carry out solid-liquid separation, the colloid after the separation carries out vacuum lyophilization or vacuum dehydrating at lower temperature, makes the gamma-polyglutamic acid-finished product.