CN103601647B - Method for desalting and decolorizing L-alanine fermentation liquor produced by genetically engineered bacteria - Google Patents
Method for desalting and decolorizing L-alanine fermentation liquor produced by genetically engineered bacteria Download PDFInfo
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- CN103601647B CN103601647B CN201310611576.7A CN201310611576A CN103601647B CN 103601647 B CN103601647 B CN 103601647B CN 201310611576 A CN201310611576 A CN 201310611576A CN 103601647 B CN103601647 B CN 103601647B
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- 238000011033 desalting Methods 0.000 title abstract 4
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Abstract
The invention discloses a method for desalting and decolorizing L-alanine fermentation liquor produced by genetically engineered bacteria. The method is simple and convenient. By adopting the method, the cost of downstream separation and purification can be saved. According to the method for desalting and decolorizing the L-alanine fermentation liquor produced by the genetically engineered bacteria, D101 macroporous resin is mainly used for desalting and decolorizing the L-alanine fermentation liquor produced by the genetically engineered bacteria.
Description
Technical field
The present invention relates to a kind of method of fermented liquid desalination bleaching, produce the method for ALANINE fermented liquid desalination bleaching more specifically to a kind of genetic engineering bacterium.
Background technology
ALANINE is also known as α-aminopropionic acid, and molecular formula is C
3h
7nO
2, soluble in water, be slightly soluble in ethanol, be insoluble to ether or acetone, containing higher sweet taste, having opticity, is one of 20 kinds of L-type alpha amino acids of constitutive protein matter.Although ALANINE is human body non-essential amino acid, is the most homoamino acid of content in blood of human body, has important physiological action.ALANINE is most important amino group donor in transamination reaction in vivo, also can be used as the excellent transportation means of nitrogen in blood.There is close association with carbohydrate metabolism, in glucose alaninc cycle, keep low blood ammonia levels, also have simultaneously and produce sugar effect.ALANINE has extremely wide application at food and pharmaceutical industries.In the food industry, ALANINE can be used for sanitas, the flavoring of food-drink, add a small amount of L-Ala and just can significantly improve protein utilization in food and beverage and Nutritive value of food, and because L-Ala has the advantages that directly can be digested and assimilated by organism, can set up rapidly after drinking, inspire enthusiasm; ALANINE has stronger sugariness, can be used as sweetening agent and uses, and in compound sweetener, adding a small amount of ALANINE can make that sugariness improves, sweet taste is soft hides agent as crude sweet, is one of high sugariness alitame raw material of synthesis; ALANINE also can improve aftertaste, makes tart flavour corrigent, improves organic acid tart flavour, makes it closer to the tart flavour of crude substance; ALANINE also for accessory substance, Preparation of amino acid functional health wine etc.; ALANINE, in pharmaceutical industries, is widely used at clinical transfusion, for patient provides energy and nutrition; ALANINE still produces the important source material of vitamin B6, synthetic pantothenic acid calcium and other organic compound.
At present, the production overwhelming majority of domestic ALANINE is the mode adopting biological fermentation.It is except improving the exploration of these upstream technology to excellent species seed selection, zymotechnique, the downstream separation purification mode of ALANINE is also one of important research content and gordian technique that biological fermentation produces ALANINE.There is a large amount of pigment molecular and high salt component in microbial fermentation solution, pigment is the macromolecular organic compound itself having color and can make other mass pigmentation.It is meta-bolites produced during the fermentation, relevant with bacterial classification and fermentation condition.In fermentation liquor pretreatment and extractive process, most impurity and pigment are removed, but still have a small amount of pigment to exist, if the starting material that functional quality is poor in refinement, also can bring some pigments into.Chromophore and help group generally all to contain a fairly large number of various polar group (as-COOH ,-NH in various pigment
2,-OH etc.).If these pigment moleculars and high salt component effectively can not be removed, the difficulty of product separation, purification, crystallization in subsequent downstream process will be increased, and then affect purity and the quality of product.The production cost of ALANINE is primarily of upstream fermentation process and downstream separation purification process expense composition, and wherein, the ratio of expense shared by downstream process is up to 60% ~ 70%.The usual ALANINE decolouring mode generally adopted of decolouring is activated carbon decolorizing.This is because gac specific surface area is large, adsorptive power is strong.But there is also deficiency and problem, it has certain requirement to the viscosity of solution, needs to make sample liquid body be warmed up to certain temperature and be just beneficial to absorption and filter, gac not recoverable in addition when viscosity is higher, and easy adsorption production, cause product loss larger.And activated carbon decolorizing mode effectively can not reduce high salt component in mother liquor.Therefore on cost-saving basis, how to improve the mode of separating-purifying, the quality improving finished product has become fermentable and has produced one of key issue of L-Ala.
D101 macroporous adsorbent resin is then the nonpolar interpolymer of styrene type, the scope of application compares wide spectrum, for the organic compound not with polarity or low-pole, general high adsorption capacity, at present, be widely used in pharmaceutical industries, especially the separating-purifying of saponin, this kind of herbal medicine of flavones had better effect, such as: ginsenoside, arasaponin, dioscin, ginkgolic flavone glycoside etc.But, also there is no the Patents about desalination bleaching D101 macroporous adsorbent resin being used for similar this kind biological product of ALANINE fermented liquid and report at present.
Summary of the invention
The object of the invention is to solve above-mentioned problems of the prior art with not enough, provide a kind of genetic engineering bacterium to produce the method for ALANINE fermented liquid desalination bleaching, the method is simple and convenient, can save the cost of downstream separation purifying.
The present invention is achieved by the following technical solutions:
Genetic engineering bacterium of the present invention produces the method for ALANINE fermented liquid desalination bleaching, and it mainly uses D101 macroporous resin to carry out desalination bleaching process to genetic engineering bacterium product ALANINE fermented liquid.
Genetic engineering bacterium of the present invention produces the method for ALANINE fermented liquid desalination bleaching, and to be described use D101 macroporous resin produce ALANINE fermented liquid to genetic engineering bacterium to its further scheme carries out desalination bleaching process and comprise 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, pigment organic macromolecule and containing high salt impurity absorption on macroporous resin, collect be separated after ALANINE fermented liquid.
Genetic engineering bacterium of the present invention produces the method for ALANINE fermented liquid desalination bleaching, and further technical scheme is the rotating speed of described high speed low temperature centrifugal machine is 10000 ~ 12000r/min for it, and centrifugation time is 5min.
Genetic engineering bacterium of the present invention produces the method for ALANINE fermented liquid desalination bleaching, its when further technical scheme can also be 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.
Genetic engineering bacterium of the present invention produces the method for ALANINE fermented liquid desalination bleaching, its further technical scheme be 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.
Compared with prior art the present invention has following beneficial effect:
Utilize D101 macroporous adsorbent resin to carry out desalination bleaching, the pigment adsorbed and the easy wash-out of impurity, 80% ethanol elution, soda acid re-treatment can realize the regeneration of resin.D101 macroporous resin does not seldom even adsorb ALANINE adsorptive capacity, and product retention rate reaches more than 94%, can not cause product loss.D101 macroporous resin compared with other ordinary resin while carrying out fermented liquid decolouring, can also desalination, reduce fermented liquid specific conductivity.The regeneration rate of D101 macroporous resin is higher, reaches more than 95%, can reuse tens of times.Use D101 macroporous resin to carry out desalination bleaching to ALANINE, effect is better than activated carbon decolorizing, can save the cost of downstream separation purifying, is applicable to commercial scale production and uses.
Accompanying drawing explanation
Fig. 1 is that in the embodiment of the present invention 1, D101 macroporous resin varies with temperature schematic diagram to the percent of decolourization that ALANINE decolours.
Fig. 2 is that percent of decolourization that in the embodiment of the present invention 2, D101 macroporous resin decolours to ALANINE is with loading change in flow schematic diagram.
Fig. 3 is that in the embodiment of the present invention 3, D101 macroporous resin changes schematic diagram to the resolution factor that ALANINE decolours with alcohol concn.
Embodiment
D101 macroporous adsorbent resin is commercially available prod in an embodiment.
Embodiment 1
(1) ALANINE fermented liquid is carried out pre-treatment, centrifugal to remove the impurity such as thalline under high speed low temperature centrifugal machine, collect supernatant liquor;
(2) ALANINE concentration in above-mentioned supernatant liquor is adjusted to 75mg/mL, pH value is adjusted to 3.0;
(3) by D101 macroporous resin dress post, and after carrying out alcohol immersion 24h after washing to effluent liquid clarification, wash with 4%HCl stream successively, be washed to neutrality, 4%NaOH stream is washed, and is washed to neutral preprocessing process;
(4) the fermented liquid flow velocity after control treatment is 1.5mL/min, at temperature is respectively 35,40,45,50,55,60 DEG C, 65 DEG C, enters D101 macroporous resin and carries out desalination bleaching, obtain L-Ala fermented liquid, calculates percent of decolourization.Fig. 1 be the temperature that obtains of the dynamic Decolorant Test of D101 macroporous resin to percent of decolourization change curve, can find out, the impact of temperature on the absorption property of D101 macroporous resin is larger, and temperature is remarkable at about 50 DEG C decoloration performances.
(5) resin absorption saturated after, carry out the wash-out of resin with the flow velocity of 80% ethanol, 1.0mL/min.
In aforesaid method, centrifuge speed is 11000r/min, and centrifugation time is 5min.
Embodiment 2
(1) ALANINE fermented liquid is carried out pre-treatment, under high speed low temperature centrifugal machine, with the centrifugal 5min of 12000r/min, to remove the impurity such as thalline, collect supernatant liquor;
(2) ALANINE concentration in above-mentioned supernatant liquor is adjusted to 75mg/mL, pH value is adjusted to 3.0;
(3) by D101 macroporous resin dress post, and after carrying out alcohol immersion 24h after washing to effluent liquid clarification, wash with 4%HCl stream successively, be washed to neutrality, 4%NaOH stream is washed, and is washed to neutral preprocessing process;
(4) control temperature is at 30 DEG C, enters D101 macroporous resin and carries out desalination bleaching, obtain L-Ala fermented liquid with the flow velocity of 0.5mL/min, 1mL/min, 1.5mL/min, 2mL/min, 2.5mL/min, calculates percent of decolourization.Fig. 2 be the loading flow velocity that obtains of the dynamic Decolorant Test of D101 macroporous resin to percent of decolourization change curve, can find out, flow velocity is remarkable at about 1.5mL/min decoloration performance.
(5) resin absorption saturated after, carry out the wash-out of resin with the flow velocity of 80% ethanol, 1.0mL/min.
In aforesaid method, centrifuge speed is 12000r/min, and centrifugation time is 5min.
Embodiment 3
(1) ALANINE fermented liquid is carried out pre-treatment, under high speed low temperature centrifugal machine, with the centrifugal 5min of 12000r/min, to remove the impurity such as thalline, collect supernatant liquor;
(2) ALANINE concentration in above-mentioned supernatant liquor is adjusted to 75mg/mL, pH value is adjusted to 3.0;
(3) by D101 macroporous resin dress post, and after carrying out alcohol immersion 24h after washing to effluent liquid clarification, wash with 4%HCl stream successively, be washed to neutrality, 4%NaOH stream is washed, and is washed to neutral preprocessing process;
(4) control temperature is at 30 DEG C, enters D101 macroporous resin and carries out desalination bleaching, obtain L-Ala fermented liquid with the flow velocity of 1.5mL/min;
(5) resin absorption saturated after, with the wash-out that concentration is respectively 50%, 60%, 70%, 80%, 99% ethanol, the flow velocity of 1.0mL/min carries out resin, calculate resolution factor.Fig. 3 be the alcohol concn that obtains of the dynamic Decolorant Test of D101 macroporous resin to resolution factor change curve, can find out, alcohol concn is remarkable at about 80% analysis feature.
In aforesaid method, centrifuge speed is 10000r/min, and 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.
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| CN1099268A (en) * | 1994-03-30 | 1995-03-01 | 三门峡市化工研究院 | Process for production of amino acid oral liquid |
| CN101275149A (en) * | 2008-05-08 | 2008-10-01 | 江南大学 | Preparation for asparaginic acid protease inhibitors |
| CN103387502A (en) * | 2012-05-09 | 2013-11-13 | 上海医药工业研究院 | Method for extracting L-alanine from L-alanine fermentation liquid |
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| CN1099268A (en) * | 1994-03-30 | 1995-03-01 | 三门峡市化工研究院 | Process for production of amino acid oral liquid |
| CN101275149A (en) * | 2008-05-08 | 2008-10-01 | 江南大学 | Preparation for asparaginic acid protease inhibitors |
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