CN108752625A - The method for preparing different molecular weight polyglutamic acid - Google Patents
The method for preparing different molecular weight polyglutamic acid Download PDFInfo
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Abstract
The invention discloses a kind of methods preparing different molecular weight polyglutamic acid, are related to chemistry and daily chemical products field.The preparation method is added into macromolecular polyglutamic acid solution by acid treatment step sour to be obtained the acid solution of 0.5-4mmol/L and/or the acid solution of 80-120mmol/L and alkali treatment alkali is added into above-mentioned acid solution, adjust its pH to 6-8, macromolecular polyglutamic acid can be made to the corresponding middle molecule polyglutamic acid of the acid solution small molecule polyglutamic acid corresponding with the acid solution by 80-120mmol/L by 0.5-4mmol/L, the small molecular polyglutamic acid being prepared has safety, reliably, it is without side-effects, the features such as non-stimulated, diversified demand of the market to different molecular weight polyglutamic acid can effectively be met.
Description
Technical field
The present invention relates to chemistry and daily chemical products fields, in particular to the side for preparing different molecular weight polyglutamic acid
Method.
Background technology
Polyglutamic acid (γ-PGA) is all natural as one kind, multi-functional, the boiomacromolecule product of bioerodible,
The polyglutamic acid of different relative molecular weights can be applicable to different fields.And there are many various, such as chemistry conjunctions for the approach in its source
Cheng Fa, extraction method synthesis and microbial fermentation synthetic method, wherein in the form of microbial fermentation synthesizes main source, because its
His approach complex process, difficulty is big, and cost is higher.
Since most of present polyglutamic acid is all microbial fermentation, molecular weight of product range narrow distribution, and with
The development of science and technology, the progress in epoch, polyglutamic acid will be used wider and wider, and the industry being related to is also more and more, especially
In cosmetic applications, the polyglutamic acid of single molecular weight can not meet multiple demands, go by fermentation condition
The process is more complicated for molecular size range, and the size of regulatory molecule amount is gone by screening different bacterial strains, and the screening period is too
Long, difficulty is big.
In consideration of it, special propose the present invention.
Invention content
The purpose of the present invention is to provide a kind of method preparing different molecular weight polyglutamic acid, using the preparation method,
Macromolecular polyglutamic acid can be prepared into middle molecule or small molecule polyglutamic acid, which has step simply and operation side
Just the features such as.
The invention is realized in this way：
A method of preparing different molecular weight polyglutamic acid comprising：
Acid treatment step：Acid is added into macromolecular polyglutamic acid solution, obtains containing the acid that acid concentration is 0.5-4mmol/L
Solution and/or containing acid concentration be 80-120mmol/L acid solution；
Alkali treatment：Alkali is added into above-mentioned acid solution, adjusts its pH to 6-8.
The addition of strong acid can promote the decomposition of macromolecular polyglutamic acid, and filling into for alkali can be terminated decomposing, and pH is controlled
System is in safe range.
Further, in some embodiments of the present invention, a concentration of 1%- of the macromolecular polyglutamic acid solution
5%.Degree of decomposition of macromolecular polyglutamic acid is easy to control within the scope of this.
Further, in some embodiments of the present invention, in acid treatment step, acid used is hydrochloric acid or dense sulphur
The strong acid such as acid.
Further, in some embodiments of the present invention, before acid treatment step, the preparation method is also wrapped
It includes：Macromolecular polyglutamic acid solution preparation process：
The macromolecular polyglutamic acid solution preparation process includes：Macromolecular polyglutamic acid raw material is mixed with water, is made
The macromolecular polyglutamic acid solution.
Further, in some embodiments of the present invention, before macromolecular polyglutamic acid solution preparation process, institute
Stating preparation method further includes：Macromolecular polyglutamic acid material preparation step；
The macromolecular polyglutamic acid material preparation step includes：
The bacterium for producing polyglutamic acid is seeded in fermentation medium and carries out fermented and cultured；
The fermented liquid supernatant collected is concentrated by ultrafiltration, it is macromolecular polyglutamic acid raw material to take the filtrate of concentration.
Further, in some embodiments of the present invention, the fermentation medium includes：Yeast powder 0.5%-3%,
Ammonium chloride 1%-2.5%, sodium glutamate 5%-10%, disodium hydrogen phosphate 0.1%-0.3%, potassium dihydrogen phosphate 0.02%-
0.15%, sodium chloride 1%-2%, glucose 2%-5%.
Further, in some embodiments of the present invention, the condition of the fermented and cultured is as follows：
30 DEG C -40 DEG C, stir speed (S.S.) 100rpm-700rpm, ventilatory capacity 50VVM-400VVM of cultivation temperature, fermentation time
24hr-64hr。
Further, in some embodiments of the present invention, poly- paddy is produced by described with the ratio of inoculum concentration 1%-10%
The bacterium of propylhomoserin is seeded to the fermentation medium.
Further, in some embodiments of the present invention, it is described production polyglutamic acid bacterium be bafillus natto or
Bacillus subtilis etc..
Further, in some embodiments of the present invention, after residing acid treatment step, the alkali treatment
Before, the preparation method further includes：
Sterilize 5-10min under the conditions of the acid solution is placed in 115-121 DEG C, is then cooled to the acid solution rapidly
Room temperature.
The invention has the advantages that：
The method provided by the invention for preparing different molecular weight polyglutamic acid is by acid treatment step i.e. toward the poly- paddy of macromolecular
Acid is added in propylhomoserin solution, obtains the acid solution of 0.5-4mmol/L and/or the acid solution of 80-120mmol/L and alkali treatment
Alkali is added into above-mentioned acid solution, adjusts its pH to 6-8, macromolecular polyglutamic acid can be made to the acid by 0.5-4mmol/L
The corresponding middle molecule polyglutamic acid of solution small molecule polyglutamic acid corresponding with the acid solution by 80-120mmol/L；The preparation side
Method is simple, and the time is short, need not carry out other complicated process means.
Wherein, macromolecular polyglutamic acid molecular weight has height moisturizing more than 1,000,000 dalton in cosmetic industry,
The effect of thickening, moreover it is possible to as brightening agent.Middle molecule polyglutamic acid molecular weight is mainly protected in 600,000-80 ten thousand dalton or so
Wet effect.Small molecule polyglutamic acid molecular weight has moisturizing in 10,000 dalton or so in cosmetics, crease-resistant, can penetrate skin
Skin arrives at skin corium, keeps skin delicacy smooth.
The small molecular polyglutamic acid being prepared has the features such as safe and reliable, without side-effects, non-stimulated, Neng Gouyou
Effect meets diversified demand of the market to different molecular weight polyglutamic acid.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the agarose gel electrophoresis figure of the polyglutamic acid solution of different molecular weight made from 1-4 of the embodiment of the present invention；
Fig. 2 is the embodiment of the present invention 1, the agarose gel electrophoresis of the polyglutamic acid solution of different molecular weight made from 5-7
Figure；
Fig. 3 is that the present invention divides the polyglutamic acid solution of different molecular weight made from embodiment by GFC chromatographs
The result of analysis；
Fig. 4 is GFC chromatography result of the present invention to the polyglutamic acid standard items of different molecular weight.
Specific implementation mode
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
Required raw material and equipment and various chemical reagent have as follows：
Strain：Bacillus subtilis (being provided by Guangzhou no added doctrine cosmetics Co., Ltd) or bafillus natto
(being provided by Guangzhou no added doctrine cosmetics Co., Ltd).
Raw material：Nacl, peptone, yeast powder, sodium glutamate, glucose, disodium hydrogen phosphate, potassium dihydrogen phosphate；
Instrument and equipment：Airlift fermentor, vertical pressure steam sterilization pan (Shanghai Bo Xun Industrial Co., Ltd.s), electrophoresis
Instrument (6 1 bio tech ltd of Beijing), the liquid-transfering gun and pipette tips of 1ml ranges, the liquid-transfering gun and pipette tips of 0.2ml ranges,
The liquid-transfering gun and pipette tips of 0.01ml, the EP pipes of 1.5ml, 200ml shaking flasks.
Chemical reagent：Concentrated hydrochloric acid (12mol/L), NaOH (10mol/L), Tirs-base, methylene blue, ethyl alcohol, agar
Sugar, boric acid, standard protein mark, deionized water.
The method provided in this embodiment for preparing different molecular weight polyglutamic acid, includes the following steps：
One, prepared by the fermentation of macromolecular polyglutamic acid
1, fermentation tank culture bacillus subtilis
Can also be the bacterial strain of the production polyglutamic acid such as bafillus natto in other examples.
1.1, prepared by seed liquor
The bacillus subtilis with production polyglutamic acid ability frozen is seeded in seed liquid culture medium (LB culture mediums),
Bacillus subtilis seed liquor is made in shaking flask culture 16h, 37 DEG C, shaking speed 195rpm of cultivation temperature.
Seed liquid culture medium used includes：Peptone 1%, yeast powder 0.5%, sodium chloride 1%.
1.2, fermentation tank culture
Bacillus subtilis seed liquor is seeded in the ratio of inoculum concentration 8% in the fermentation tank equipped with 50L fermentation mediums
Carry out fermented and cultured, condition of culture：37 DEG C, stir speed (S.S.) 100rpm-700rpm, ventilatory capacity 50VVM-400VVM of cultivation temperature,
Fermentation time r for 24 hours.
Wherein, fermentation medium used includes：Yeast powder 1.5%, ammonium chloride 1%, sodium glutamate 8%, disodium hydrogen phosphate
0.1%, potassium dihydrogen phosphate 0.05%, sodium chloride 1% and glucose 5%.
2, continuous flow tube centrifuge is used to collect fermented liquid supernatant after fermentation.
3, fermented liquid supernatant is concentrated by ultrafiltration using doughnut membrane filtration system.
4, the filtrate concentrated -20 DEG C of refrigerators are placed in as macromolecular polyglutamic acid raw material to preserve, it is spare.
Two, the polyglutamic acid of small molecular is prepared into using macromolecular polyglutamic acid as raw material
1,2% or so macromolecular polyglutamic acid solution is made into after the macromolecular polyglutamic acid raw material of preservation thawing,
2, by strong acid, that is, hydrochloric acid be added above-mentioned macromolecular polyglutamic acid solution be configured to respectively it is hydrochloric a concentration of
The acid solution B of the acid solution A of 0.5mmol/L and hydrochloric a concentration of 80mmol/L.
3, acid solution is put into vertical pressure autoclave and is sterilized 121 DEG C, 5min.
4, acid solution is taken out into then rapid cooling after subject to sterilization.
5, after dropping to room temperature, above-mentioned acid solution A and acid solution B is added in NaOH solution, adjusts pH value, make it on 6.8 left sides
The right side, so it is poly- in the polyglutamic acid and small molecule polyglutamic acid, that is, acid solution B being made in middle molecule polyglutamic acid, that is, acid solution A
Glutamic acid.
Experimental example 1
Using agarose gel electrophoresis to the identification experiment of different molecular weight polyglutamic acid
1, the Ago-Gel for preparing 1.0%, to the 15 μ L of polyglutamic acid sample introduction of each acid concentration, Sample Buffer solution
TRIS- borate buffer solutions 6.8, solvent：TRIS- borate buffer solutions pH is 8.5, voltage 150mv, electric current 50mA, the time
60min, 0.5 methylene blue solution of coloring agent (3% ethyl alcohol) dye 15min, and decolourize 10min.
2, small molecule with standard protein mark as a contrast, middle molecule is produced with Taiwan Wei Dan enterprise stocks Co., Ltd
Middle molecule polyglutamic acid (since molecular weight is excessive, is not suitable for conventional standard protein mark as a contrast) as reference, and
It is analyzed by electrophoresis pattern.
3, electrophoresis result is analyzed, as depicted in figs. 1 and 2：The polyglutamic acid molecular weight that 0.5mmol acid solutions obtain
In the range of 600kD-800kD (Fig. 1), belong to middle molecule polyglutamic acid molecule, the obtained polyglutamic of 80mmol acid solutions
Acid molecule amount in the range of 10kD-15kD (Fig. 2), belongs to small molecule polyglutamic acid, as a result meets expection.
Embodiment 2
The method provided in this embodiment for preparing different molecular weight polyglutamic acid is substantially the same manner as Example 1, unlike,
In the present embodiment：Hydrochloric acid addition macromolecular polyglutamic acid solution is configured to the acid solution of hydrochloric a concentration of 1mmol/L respectively.
Obtained polyglutamic acid molecular weight electrophoresis detection the result is shown in Figure 1, can be seen that, poly- paddy made from the present embodiment according to Fig. 1 results
Propylhomoserin molecular weight is in the range of 600kD-800kD.
Embodiment 3
The method provided in this embodiment for preparing different molecular weight polyglutamic acid is substantially the same manner as Example 1, unlike,
In the present embodiment：Hydrochloric acid addition macromolecular polyglutamic acid solution is configured to the acid solution of hydrochloric a concentration of 2mmol/L respectively.
Obtained polyglutamic acid molecular weight electrophoresis detection the result is shown in Figure 1, can be seen that, poly- paddy made from the present embodiment according to Fig. 1 results
Propylhomoserin molecular weight is in the range of 600kD-800kD.
Embodiment 4
The method provided in this embodiment for preparing different molecular weight polyglutamic acid is substantially the same manner as Example 1, unlike,
In the present embodiment：Hydrochloric acid addition macromolecular polyglutamic acid solution is configured to the acid solution of hydrochloric a concentration of 4mmol/L respectively.
Obtained polyglutamic acid molecular weight electrophoresis detection the result is shown in Figure 1.It can be seen that according to Fig. 1 results, poly- paddy made from the present embodiment
Propylhomoserin molecular weight is in the range of 600kD-800kD.
Embodiment 5
The method provided in this embodiment for preparing different molecular weight polyglutamic acid is substantially the same manner as Example 1, unlike,
In the present embodiment：The acid that hydrochloric acid addition macromolecular polyglutamic acid solution is configured to hydrochloric a concentration of 60mmol/L respectively is molten
Liquid.Obtained polyglutamic acid molecular weight electrophoresis detection result is shown in Fig. 2.Result can be seen that according to fig. 2, gather made from the present embodiment
Glutamate molecule amount is in the range of 10kD-17kD.
Embodiment 6
The method provided in this embodiment for preparing different molecular weight polyglutamic acid is substantially the same manner as Example 1, unlike,
In the present embodiment：The acid that hydrochloric acid addition macromolecular polyglutamic acid solution is configured to hydrochloric a concentration of 100mmol/L respectively is molten
Liquid.Obtained polyglutamic acid molecular weight electrophoresis detection result is shown in Fig. 2.Result can be seen that according to fig. 2, gather made from the present embodiment
Glutamate molecule amount is most of in the range of 10kD or less.
Embodiment 7
The method provided in this embodiment for preparing different molecular weight polyglutamic acid is substantially the same manner as Example 1, unlike,
In the present embodiment：The acid that hydrochloric acid addition macromolecular polyglutamic acid solution is configured to hydrochloric a concentration of 120mmol/L respectively is molten
Liquid.Obtained polyglutamic acid molecular weight electrophoresis detection result is shown in Fig. 2.- B results can be seen that according to fig. 2, made from embodiment 7
Polyglutamic acid molecular weight is substantially all less than 10kD.
Experimental example 2
The identification experiment of different molecular weight polyglutamic acid solution is analyzed using GFC chromatographs
Each molecule is analyzed by GFC chromatographs, takes each molecular weight polyisoprene glutamic acid solution (fermenation raw liquid, 0.5mmol
Acid solution is provided by embodiment 1,100mmol acid solutions are provided by embodiment 6) in phosphate is added, it is 6.8 to be configured to pH
The phosphate buffer solution of 0.1mol/L, 20 μ L of sample introduction, mobile phase are the phosphate buffer solution of 0.1mol/L, chromatographic column BIQ-
5 columns of BIQ SIL SEC 40Q of RID productions, flow velocity 1ml/min, detector wavelength 212nm.The GFC of polyglutamic acid is analyzed
Collection of illustrative plates is as shown in Figure 3.
By the small molecule polyglutamic acid of standard items (purchasing in Taiwan Wei Dan enterprise stocks Co., Ltd), middle molecule polyglutamic
Acid and macromolecular polyglutamic acid are dissolved, and 2% solution are respectively prepared, it is molten that 0.1mol/L phosphoric acid buffers are made with phosphate
Liquid, while also 20 μ L of sample introduction, are analyzed, the collection of illustrative plates of standard items is as shown in Figure 4 with GFC chromatographs.
The self-produced polyglutamic acid molecule of our company can be calculated by the comparison of the retention time of the polyglutamic acid of Fig. 3 and Fig. 4
The polyglutamic acid molecular weight that amount is handled about more than 1,000,000 dalton, by 0.5mmol acid in ten thousand dalton of 50-70 or so,
The small molecule of 100mmol acid processing is about below 10000 dalton.Experimental result is met the requirements.
To sum up, the method provided in an embodiment of the present invention for preparing different molecular weight polyglutamic acid, has the advantages that：
1, macromolecular polyglutamic acid controllable rapidly can be prepared into middle molecule (600,000-80 ten thousand dalton) or small molecule
(10,000 dalton or so) polyglutamic acid disclosure satisfy that the various diversified demands of our company.
2, the polyglutamic acid safety after converting, it is non-stimulated, it is without side-effects, it can be safe to use.
3, the polyglutamic acid that can obtain various molecular weight simultaneously, meets the R and D of product.
4, the polyglutamic acid method for preparing small molecular is simple, and the time is short, need not carry out other complicated process means.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method preparing different molecular weight polyglutamic acid, which is characterized in that it includes：
Acid treatment step：Acid is added into macromolecular polyglutamic acid solution, obtains containing the acid solution that acid concentration is 0.5-4mmol/L
And/or containing the acid solution that acid concentration is 80-120mmol/L；
Alkali treatment：Alkali is added into above-mentioned acid solution, adjusts its pH to 6-8.
2. according to the method described in claim 1, it is characterized in that, a concentration of 1%- of the macromolecular polyglutamic acid solution
5%.
3. method according to claim 1 or 2, which is characterized in that in acid treatment step, acid used is hydrochloric acid or sulphur
Acid.
4. according to the method described in claim 2, it is characterized in that, before acid treatment step, the method further includes：Big point
Sub- polyglutamic acid solution preparation process：
The macromolecular polyglutamic acid solution preparation process includes：Macromolecular polyglutamic acid raw material is mixed with water, is made described
Macromolecular polyglutamic acid solution.
5. according to the method described in claim 4, it is characterized in that, before macromolecular polyglutamic acid solution preparation process, institute
The method of stating further includes：Macromolecular polyglutamic acid material preparation step；
The macromolecular polyglutamic acid material preparation step includes：
The bacterium for producing polyglutamic acid is seeded in fermentation medium and carries out fermented and cultured；
The fermented liquid supernatant collected is concentrated by ultrafiltration, it is macromolecular polyglutamic acid raw material to take the filtrate of concentration.
6. according to the method described in claim 5, it is characterized in that, the fermentation medium includes：Yeast powder 0.5%-3%,
Ammonium chloride 1%-2.5%, sodium glutamate 5%-10%, disodium hydrogen phosphate 0.1%-0.3%, potassium dihydrogen phosphate 0.02%-
0.15%, sodium chloride 1%-2%, glucose 2%-5%.
7. according to the method described in claim 5, it is characterized in that, the condition of the fermented and cultured is as follows：
30 DEG C -40 DEG C, stir speed (S.S.) 100rpm-700rpm, ventilatory capacity 50VVM-400VVM of cultivation temperature, fermentation time r- for 24 hours
64hr。
8. according to the method described in claim 5, it is characterized in that, with the ratio of inoculum concentration 1%-10% by the production polyglutamic
The bacterium of acid is seeded to the fermentation medium.
9. according to claim 5-8 any one of them methods, which is characterized in that the bacterium of the production polyglutamic acid is natto gemma
Bacillus or bacillus subtilis.
10. according to the method described in claim 9, it is characterized in that, after residing acid treatment step, the alkali treatment
Before, the method further includes：
Sterilize 5-10min under the conditions of the acid solution is placed in 115-121 DEG C, and the acid solution is then cooled to rapidly room
Temperature.
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Cited By (1)
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