CN102174194B - Method for extracting gamma-polydiaminobutyric acid and polylysine from fermentation liquor - Google Patents
Method for extracting gamma-polydiaminobutyric acid and polylysine from fermentation liquor Download PDFInfo
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- Polyamides (AREA)
Abstract
The invention discloses a method for extracting gamma-poly (diaminobutyric acid) and polylysine from fermentation liquor, which comprises the steps of pretreatment, resin adsorption and desorption, decoloration, concentration and drying of the fermentation liquor, and then drying of the fermentation liquor to obtain the finished products of the poly (lysine) and the poly (diaminobutyric acid). The raw materials and instruments adopted by the invention are easy to obtain, and the cost is low; the extraction and purification process is simple and feasible; the final product has high yield and high purity. And is beneficial to environmental protection and suitable for industrial mass production.
Description
Technical field
The invention belongs to the separation engineering technical field, be specifically related to a kind of γ-gather method of DAB and polylysine of from fermented liquid, extracting.
Background technology
γ-gather DAB by L-2, the homopolymer that the gamma-amino of 4-DAB and α-carboxyl are polymerized.At present result of study shows, γ-gather DAB has and the similar character of polylysine at thermostability, aspect such as water-soluble, it should be noted that γ-gather DAB has extremely strong yeast and suppresses ability, and has certain inhibition G
+Bacterium, G
-Bacterium and mould vigor are the good biological preservatives of a kind of potential.Monomer whose DAB (2,4-diaminobutyricacid, english abbreviation DABA) also claims 2, and 4-fourth two propylhomoserins are a kind of secondary nonprotein amino acid, belong to C4 compound, and its structural formula is (NH
2) CH
2CH
2CH (NH
2) COOH, belong to basic aminoacids.It extensively is present in animal, plant and the microbe body, has special physiological properties.It then has more wide application prospect as medicine synthetic precursor.Also there is not at present both at home and abroad relevant γ-the gather report that DAB production is extracted.
Polylysine gathers DAB
(ε-PL) is the homotype polymkeric substance of L-Methionin to polylysine, and it forms peptide bond α-carboxyl and epsilon-amino and is formed by connecting, and general ε-PL is made up of 20-40 L-lysine residue.ε-PL is a kind of has a broad antifungal spectrum (comprising that gram-positive microorganism, Gram-negative bacteria, yeast, mould all have good inhibition effect), biological safety high (ε-PL degradable be human body must amino acid L-Methionin), thermostability strong (120 ℃ are heated 20min and still keep anti-microbial activity), the wide novel nourishing type food preservatives of the pH scope of application.Production efficiency is low in ε-PL production process, production concentration is low, product separates problems such as difficulty, is perplexing the suitability for industrialized production of ε-PL.The research of ε-PL at present mainly is in laboratory study and the stage; Only have Japanese Chisso company to realize the large-scale production of ε-PL, only there are produced in small quantities in domestic Nanjing Shinekingbiotech, Ltd., Yinxiang Biological Engineering Co., Ltd., Zhejiang Prov and Zhengzhou Bai Nafo biotechnology limited-liability company.
This seminar finds that when screening has the bacterial strain of polylysine ability a strain can produce polylysine simultaneously and gather the bacterial strain Streptomyces albulus PD-1 (CCTCC NO:M2011043) of DAB.This bacterial strain is preserved in Wuhan China typical culture collection center (being called for short CCTCC) at present; The address: Wuhan Wuhan University, postcode: 430072, the numbering of registering on the books is CCTCC NO:M2011043; Preservation date is: on February 22nd, 2011, see Chinese patent 2011100499868 for details.Because two kinds of products all have good physical and chemical performance and wide application prospect, therefore the separation and purification to two kinds of products has crucial meaning.
Summary of the invention
Technical problem to be solved by this invention provides a kind of γ-gather method of DAB and polylysine of from fermented liquid, extracting.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
Fermented liquid of the present invention is to utilize little streptomyces albus (Streptomyces albulus PD-1, CCTCC NO:M2011043), and fermentation obtains.
The preparation of fermentation liquid method specifically can be referring to Chinese patent 2011100499868.Be about to bacterial strain CCTCC NO:M2011043 and be inoculated in the no bacteria fermentation culture medium that contains carbon source, nitrogenous source, inorganic salt and water, under the condition of proper growth, carry out aerobic cultivation.
Wherein, said fermention medium comprises following component: carbon source 20～100g/L, and nitrogenous source 2～40g/L, inorganic salt 0.1～20g/L, all the other are water, pH6.0～7.0.Described carbon source is one or more in glucose, wood sugar, fructose, lactic acid, glycerine and the cellulose hydrolysis liquid glucose; Nitrogenous source is Carnis Bovis seu Bubali cream, peptone, yeast extract paste, steeping water, soybean cake powder, cottonseed meal, urea, (NH
4)
2SO
4And NH
4Among the Cl one or more; Inorganic salt are one or more in sylvite, cobalt salt, sodium salt, phosphoric acid salt, dihydrogen phosphate and the hydrochloride.
Wherein, the condition of described proper growth is: culture temperature is 28～37 ℃, and incubation time is 48～150 hours, wherein, when fermented liquid pH value is reduced to 4.0～4.5, keep the pH value 4.0～4.5 until fermentation ends.
Generally can make polylysine content through above-mentioned fermentation process is about 30g/L, and gathering DAB content is the fermented liquid about 12g/L.
From above-mentioned fermented liquid, extract γ-gather the method for DAB and polylysine, comprise the steps:
1) fermentation liquor pretreatment: add acid and regulate fermented liquid pH value to 2.0～6.0,50～110 ℃ insulation 10～30 minutes, be cooled to room temperature,, add alkali and regulate pH value to 6.0～10.0 of filtrating, obtain pretreatment fluid through filtering filtrating;
2) resin absorption and parsing: pretreatment fluid is pumped in the ion exchange resin adsorption column, saturated until resin absorption; Wash assorted then with deionized water; Resolve with hydrochloric acid or glacial acetic acid aqueous solution again, collect polylysine desorbed solution respectively and gather the DAB desorbed solution with different RTs;
3) decolouring: with the polylysine desorbed solution of collecting with gather the DAB desorbed solution and carry out activated carbon decolorizing respectively, filter respectively and collect destainer;
4) concentrate: it is 15%～30% that the polylysine destainer that step 3) is obtained is concentrated into the polylysine weight percentage; The DAB destainer that gathers that obtains of step 3) is concentrated into that to gather the DAB weight percentage be 8%～20%;
5) drying: the polylysine liquid concentrator that step 4) is obtained with gather the DAB liquid concentrator and carry out drying treatment respectively, obtain polylysine and gather the DAB finished product.
In the step 1), described filtration can be adopted Plate Filtration.
Step 2) in, the resin of described ion exchange resin adsorption column filling be weakly acidic cation-exchange resin (for example: 110,112; 122,151) or large porous strong acid type ion exchange resin (for example: D001, D011; D031) a kind of in, the adsorption column aspect ratio is 2～30: 1.The absorption flow velocity is 0.2～15BV/h, preferred 1～5BV/h; Washing assorted flow velocity is 1～20BV/h, preferred 2～10BV/h; The total consumption of deionized water is 1～10 times of column volume, preferred 3～8 times of column volumes; The parsing flow velocity is 0.1～10BV/h, preferred 0.2～3BV/h.
Step 2) in, described concentration of hydrochloric acid is 0.2～4mol/L, and described glacial acetic acid aqueous solution concentration is 0.2～3mol/L.
Step 2) in; Resolving; Utilize method for detecting specificity to follow the tracks of and detect polylysine and gather the concentration of DAB in desorbed solution, thereby collect polylysine respectively and gather DAB, described method for detecting specificity is Itzhaki method or GPC.Concrete detection method is following:
The Itzhaki method:
Getting the 2mL desorbed solution mixes with the tropeolin-D of 2mL 10mmol/L; Vibration is fully reacted 30min, centrifugal 10min under the 8000r/min condition; Getting supernatant suitably dilutes; With the phosphoric acid buffer is blank, under 465nm, surveys its absorbance A 465, calculates in the desorbed solution polylysine or gathers DAB content through typical curve.【Itzhaki?R?F.Colorimetric?Method?for?Estimating?Polylysine?and?Polyargine.Analytical?Biochemistry[J]1972，50：569～574.】
HPLC:
Use GPC measures polylysine simultaneously or gathers the content and the molecular weight of DAB.Chromatographic condition: T SK-gelG3000PWXL chromatographic column (7.8mm * 300mm); Moving phase is 0.2mol/L Na
2SO4 (acetate is transferred pH to 4.0); Detect wavelength 210nm; 35 ℃ of column temperatures; Sample size 20 μ l, flow velocity 0.5ml/min.[all pretty Xu Hong Wang Jun etc. kitasatosporia PL 6-3 produces the separation and purification and the structural characterization [J] of polylysine. chemical industry journal, 2006,57 (8): 1957-1961.]
In the step 3), the add-on of gac is 0.5～5.0% of a desorbed solution weight.
In the step 3), the activated carbon decolorizing process temperature is 60～100 ℃, and stirring bleaching time is 5～120 minutes, is cooled to room temperature then, filters.
In the step 4), described concentrated employing freeze concentration or evaporation concentration.
In the step 5), said drying is spraying drying or decompression lyophilize.
Through the said extracted method, the yield of gained γ-gather DAB product is not less than 80%, and purity is not less than 96%; The yield of polylysine product is not less than 85%, and purity is not less than 95%.
The characteristic of products obtained therefrom of the present invention: γ-gather DAB has and the similar character of polylysine at thermostability, aspect such as water-soluble, in addition, gathers DAB and has extremely strong yeast and suppress ability, and have certain inhibition G
+Bacterium, G
-Bacterium and mould vigor are the good biological preservatives of a kind of potential.Can reach the unrivaled effect of other sanitas with another product polylysine is composite.
The γ of the present invention preparation-gather DAB and polylysine also can be used as bioabsorbable polymer material, microcapsule technology material, pharmaceutical carrier, biochip and macromolecule water absorbent material etc.
Beneficial effect: extraction process of the present invention is that the method for utilization resin isolation is extracted from fermented liquid and made with extra care γ-gather DAB and polylysine.Raw material, instrument that the present invention adopts are drawn materials easily, and cost is low; Extraction and purification process is simple, feasible; The finished product yield is high, and purity is high.
Description of drawings
Fig. 1 is fermented liquid liquid phase figure.
The polylysine liquid phase figure of Fig. 2 for finally obtaining through the inventive method.
Fig. 3 gathers DAB liquid phase figure for what finally obtain through the inventive method.
Wherein, No. 1 peak is a polylysine, and No. 2 peaks are for gathering DAB.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
The free fermentation of embodiment 1:5L jar S.albulus PD-1 produces polylysine and gathers DAB
Slant medium: glucose 10g/L, Carnis Bovis seu Bubali cream 5g/L, K
2HPO
41g/L, MgSO
47H
2O 0.5g/L, agar 20g/L, pH 7.0.
Seed culture medium: glucose 10g/L, Carnis Bovis seu Bubali cream 5g/L, yeast extract paste 5g/L, K
2HPO
41g/L, MgSO
47H
2O0.5g/L utilizes 6mol/LNaOH solution to transfer pH 7.0
Fermention medium: glucose 50g/L, yeast extract paste 5g/L, (NH
4)
2SO
45g/L, Na
2HPO
412H
2O 1.58g/L, K
2HPO
47H
2O 0.82g/L, MgSO
47H
2O 0.1g/L utilizes 6mol/L NaOH solution to transfer pH 7.0.
Little streptomyces albus S.albulus PD-1 (CCTCC NO:M2011043) is cultivated 24h under seed culture medium, 28 ℃, the shaking table condition of 200r/min; The 300mL seed liquor is inoculated in prepackage by the seed dose of 10% (v/v) cultivation that dissociates in the fermentor tank of the fermention medium after the 2.7L sterilization is arranged, culture condition: 28 ℃, 400r/min; Air flow 3L/min; The pH value of fermented liquid when the pH value is reduced to 4.2 left and right sides, is opened the pH automatic control device along with the carrying out of fermentation descends gradually; The ammoniacal liquor control fermented liquid pH value of utilizing 10% (v/v) is between 4.0-4.2; Fermentation 150h, polylysine concentration reaches 32.25g/L, gathers DAB concentration and reaches 13.32g/L.
The employed fermented liquid of following examples all is to ferment with the method for this enforcement to obtain.
Embodiment 2:
In souring tank, using 3mol/L hydrochloric acid to regulate fermented liquid pH value is 2.0, is heated to 60 ℃ then, is incubated 20 minutes, and acidizing fluid is cooled to room temperature, through Plate Filtration, gets clear filtrate after the multiple filter.Using the 6mol/L sodium hydroxide solution to transfer clear filtrate pH value is 9.0, obtains pretreatment fluid.
It is to adsorb in 20: 1 weak-type Zeo-karb (110) adsorption column that pretreatment fluid is pumped into aspect ratio, and adsorption process control flow velocity is 4mL/min (about 2BV/h), reaches state of saturation up to resin absorption.Use the deionized water wash saturated resin, the control flow velocity is 6mL/min (about 3BV/h), and wash volumes is 3 column volumes.3mol/L hydrochloric acid with preparation is in advance resolved, and control flow velocity is 1mL/min (about 0.5BV/h), and under different RTs, joint Itzhaki method or HPLC are collected respectively and gathered DAB and polylysine with the acquisition desorbed solution.The gac that in containing the desorbed solution that gathers DAB, adds 4.0% (w/w); The gac that in the desorbed solution that contains polylysine, adds 4.0% (w/w), respectively heat temperature raising to 60 ℃ stirs decolouring 10 minutes; Be cooled to room temperature then, cross and filter destainer.
With the polylysine destainer with gather the DAB destainer and be pressed into the freeze concentration device respectively and circulate concentratedly, be concentrated into and gather DAB content and when 15% (w/w), stop to concentrate, polylysine content stops to concentrate when 25% (w/w).
Spray-dried respectively promptly the getting of liquid concentrator gathered DAB and the pure article of polylysine.It is 82% that gained gathers the DAB yield, and purity is 96.8%; The polylysine yield is 86%, and purity is 96.3%.
Embodiment 3:
In souring tank, using 2mol/L hydrochloric acid to transfer fermented liquid pH value is 5.0, heats this acidizing fluid to 100 ℃ then, be incubated 10 minutes, and acidizing fluid is cooled to room temperature, through Plate Filtration, gets clear filtrate after multiple the filter.Add 6mol/L sodium hydroxide to clear filtrate, transferring clear filtrate pH value is 7.2 must pretreatment fluid.
Pretreatment fluid pumped in large porous strong acid type ion exchange resin (D001) adsorption column (aspect ratio is 10: 1) adsorb, it is 4mL/min (about 4BV/h) that adsorption process should be controlled flow velocity, reaches state of saturation up to resin absorption.Deionized water with 5 times of column volumes washs saturated resin under the condition of flow velocity 5mL/min (about 5BV/h).1mol/L aqueous acetic acid with preparation is in advance resolved, and control resolution speed 1mL/min (about 1BV/h) under different RTs, engages Itzhaki method or HPLC and collects respectively and gather DAB and polylysine with the acquisition desorbed solution.The gac that in containing the desorbed solution that gathers DAB, adds 2.0% (w/w); The gac that in the desorbed solution that contains polylysine, adds 2.0% (w/w), respectively heat temperature raising to 100 ℃ stirs decolouring 60 minutes; Be cooled to room temperature then, cross and filter destainer.
With the polylysine destainer with gather the DAB destainer and be pressed into evaporation concentration device respectively and circulate concentratedly, be concentrated into and gather DAB content and when 10% (w/w), stop to concentrate, polylysine content stops to concentrate when 20% (w/w).
Liquid concentrator promptly got through lyophilize respectively gather DAB and the pure article of polylysine.It is 85% that gained gathers the DAB recovery, and purity is 96.5%; The polylysine recovery is 88%, and purity is 96.8%.
The ion exchange resin that uses in the inventive method is through well known to a person skilled in the art the process of pickling, alkali cleaning and pickling, and is reusable.
Adopt the polylysine that HPLC obtains fermented liquid and separation and purification and gather DAB and carry out test result shown in Fig. 1～3.
Claims (9)
1. from fermented liquid, extract γ-gather the method for DAB and polylysine, it is characterized in that it comprises the steps:
1) fermentation liquor pretreatment: add acid and regulate fermented liquid pH value to 2.0～6.0,50～110 ℃ insulation 10～30 minutes, be cooled to room temperature,, add alkali and regulate pH value to 6.0～10.0 of filtrating, obtain pretreatment fluid through filtering filtrating;
2) resin absorption and parsing: pretreatment fluid is pumped in the ion exchange resin adsorption column, saturated until resin absorption; Wash assorted then with deionized water; Resolve with hydrochloric acid or glacial acetic acid aqueous solution again, collect polylysine desorbed solution respectively and gather the DAB desorbed solution with different RTs;
3) decolouring: with the polylysine desorbed solution of collecting with gather the DAB desorbed solution and carry out activated carbon decolorizing respectively, filter respectively and collect destainer;
4) concentrate: it is 15%～30% that the polylysine destainer that step 3) is obtained is concentrated into the polylysine weight percentage; The DAB destainer that gathers that obtains of step 3) is concentrated into that to gather the DAB weight percentage be 8%～20%;
5) drying: the polylysine liquid concentrator that step 4) is obtained with gather the DAB liquid concentrator and carry out drying treatment respectively, obtain polylysine and gather the DAB finished product;
Step 2) in, the resin of described ion exchange resin adsorption column filling is weakly acidic cation-exchange resin or large porous strong acid type ion exchange resin.
2. the γ-gather the method for DAB and polylysine of from fermented liquid, extracting according to claim 1 is characterized in that step 2) in, described ion exchange resin adsorption column aspect ratio is 2～30: 1.
3. the γ-gather the method for DAB and polylysine of from fermented liquid, extracting according to claim 1 is characterized in that step 2) in, the absorption flow velocity is 0.2～15BV/h; Washing assorted flow velocity is 2～20BV/h, and the total consumption of deionized water is 1～10 times of column volume; The parsing flow velocity is 0.1～10BV/h.
4. the γ-gather the method for DAB and polylysine of from fermented liquid, extracting according to claim 1 is characterized in that step 2) in, described concentration of hydrochloric acid is 0.2～4mol/L, described glacial acetic acid aqueous solution concentration is 0.2～3mol/L.
5. γ-the gather method of DAB and polylysine of from fermented liquid, extracting according to claim 1; It is characterized in that: said step 2); Resolving; Utilize method for detecting specificity to follow the tracks of and detect polylysine and gather the concentration of DAB in desorbed solution, described method for detecting specificity is Itzhaki method or HPLC.
6. the γ-gather the method for DAB and polylysine of from fermented liquid, extracting according to claim 1 is characterized in that in the step 3) that the add-on of gac is 0.5～5.0% of a desorbed solution weight.
7. γ-the gather method of DAB and polylysine of from fermented liquid, extracting according to claim 1; It is characterized in that in the step 3) that the activated carbon decolorizing process temperature is 60～100 ℃, stirring bleaching time is 5～120 minutes; Be cooled to room temperature then, filter.
8. the γ-gather the method for DAB and polylysine of from fermented liquid, extracting according to claim 1 is characterized in that in the step 4) described concentrated employing freeze concentration or evaporation concentration.
9. the γ-gather the method for DAB and polylysine of from fermented liquid, extracting according to claim 1 is characterized in that in the step 5), and said drying is spraying drying or decompression lyophilize.
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