CN116655962A - Preparation method of gamma-polyglutamic acid - Google Patents
Preparation method of gamma-polyglutamic acid Download PDFInfo
- Publication number
- CN116655962A CN116655962A CN202310791369.8A CN202310791369A CN116655962A CN 116655962 A CN116655962 A CN 116655962A CN 202310791369 A CN202310791369 A CN 202310791369A CN 116655962 A CN116655962 A CN 116655962A
- Authority
- CN
- China
- Prior art keywords
- gamma
- polyglutamic acid
- acid
- degradation
- ultrasonic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 230000015556 catabolic process Effects 0.000 claims abstract description 33
- 238000006731 degradation reaction Methods 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 25
- 230000004151 fermentation Effects 0.000 claims abstract description 25
- 239000002253 acid Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 21
- 238000000502 dialysis Methods 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 7
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 235000013305 food Nutrition 0.000 claims abstract description 5
- 239000002537 cosmetic Substances 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims abstract description 4
- 239000000047 product Substances 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 238000009210 therapy by ultrasound Methods 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 8
- 241001052560 Thallis Species 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 238000012869 ethanol precipitation Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000000049 pigment Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 6
- 239000007857 degradation product Substances 0.000 claims description 3
- 230000000593 degrading effect Effects 0.000 claims description 2
- 238000006386 neutralization reaction Methods 0.000 claims description 2
- 230000003472 neutralizing effect Effects 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 3
- 230000014759 maintenance of location Effects 0.000 abstract description 3
- 230000003647 oxidation Effects 0.000 abstract description 3
- 238000007254 oxidation reaction Methods 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
- 229920000642 polymer Polymers 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 108010020346 Polyglutamic Acid Proteins 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 5
- -1 agriculture Substances 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 239000005457 ice water Substances 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 229960002989 glutamic acid Drugs 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229930182847 D-glutamic acid Natural products 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-Glutamic acid Natural products OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 238000002792 antioxidant assay Methods 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008394 flocculating agent Substances 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000012667 polymer degradation Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 229910000406 trisodium phosphate Inorganic materials 0.000 description 1
- 235000019801 trisodium phosphate Nutrition 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/28—Treatment by wave energy or particle radiation
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2377/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers
- C08J2377/04—Polyamides derived from alpha-amino carboxylic acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/62—Plastics recycling; Rubber recycling
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Cosmetics (AREA)
- Polyamides (AREA)
Abstract
The invention discloses a preparation method of gamma-polyglutamic acid, and particularly belongs to the field of degradation of high molecular polymers. The method comprises the following steps: adding water into the ethanol precipitated gamma-polyglutamic acid fermentation liquor for re-dissolving, carrying out ultrasonic-assisted acid degradation and high-temperature acid degradation, and then obtaining gamma-polyglutamic acid with different weight average molecular weight ranges through centrifugation, dialysis and drying. The method has simple process and high degradation efficiency, the prepared product has high uniformity and polydispersity coefficient of 1.0-1.5, good moisture retention and oxidation resistance, and wide application range, and can be used in the fields of cosmetics, foods, health care products, medicines and the like.
Description
Technical Field
The invention relates to a preparation method of gamma-polyglutamic acid, and in particular belongs to the field of high polymer degradation.
Background
Gamma-polyglutamic acid (gamma-PGA) is an anionic polypeptide polymer formed by connecting an L-glutamic acid monomer and a D-glutamic acid monomer through an amide bond, and the polymerization degree is generally between 200 and 7000. Gamma-polyglutamic acid is generally produced by microbial fermentation and is nontoxic. Because the structure is special, the novel environment-friendly biological product contains a large amount of hydrogen bonds and free carboxyl groups, has good water solubility, moisture retention, adsorptivity, film forming property and the like, can be used as a water-retaining agent, a flocculating agent, a drug carrier and the like to be applied to the fields of foods, cosmetics, agriculture, medicines and the like, and has wide application prospect.
The molecular weight of the gamma-polyglutamic acid produced by the microorganism is large, the solution viscosity is also large, the rheology is difficult to control, and the gamma-polyglutamic acid is difficult to modify by chemical reagents, so that the application of the polyglutamic acid is limited. The application directions of the polyglutamic acid are different, and the degradation of the polyglutamic acid is a key step in the application. There are reports of degradation of gamma-polyglutamic acid such as Chen Xiong (preliminary study of poly-gamma-glutamic acid degradation characteristics, food research and development, 2009) and studies on the degradation mechanism of polyglutamic acid under heat, acid, alkali and ultrasonic conditions. However, the degradation research is aimed at developing gamma-polyglutamic acid after extraction and purification, and is not suitable for industrial production. And the fermentation liquor is subjected to pretreatment steps such as separation and purification, and the like, so that the method is complex in operation, high in energy consumption and not friendly to the environment, can reduce the molecular weight of gamma-polyglutamic acid and widen the molecular weight distribution to different degrees, increases the workload of subsequent separation and purification, and reduces the overall efficiency, and the quality of the product is low.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides the preparation method of the gamma-polyglutamic acid, which has the advantages of simple process, high concentration of molecular weight distribution of the product, and control of the dispersion coefficient between 1.0 and 1.5, and the method has the advantages of simple steps, easy operation, high degradation efficiency, small polydispersity coefficient of the produced product, good moisture retention and oxidation resistance and high application value.
A first object of the present invention is to provide a method for preparing gamma-polyglutamic acid, the method comprising the steps of:
(1) And (3) re-dissolving: adding water into the gamma-polyglutamic acid fermentation broth obtained by ethanol precipitation for redissolution until the mass concentration of the fermentation broth is 1-10%;
(2) Ultrasonic assisted acid degradation: adding hydrochloric acid to regulate the pH value of the fermentation liquor obtained in the step (1), and carrying out ultrasonic treatment for a period of time at a certain frequency and power; the ultrasonic frequency is 20-60 kHz, the ultrasonic power is 80-160W, and the ultrasonic time is 0.5-1 h;
(3) High-temperature acid degradation: heating and stirring the acid solution obtained in the step (2), preserving heat and degrading to obtain degraded gamma-polyglutamic acid solution, cooling and neutralizing;
(4) And (3) centrifuging: centrifuging the feed liquid obtained in the step (3) to remove thalli and foreign proteins, and taking a supernatant;
(5) And (3) dialysis: dialyzing the supernatant obtained in the step (4) to remove small molecular salts, sugar and pigment;
(6) And (3) drying: and (5) performing vacuum freeze drying on the dialysate obtained in the step (5) to obtain a polyglutamic acid finished product.
In one embodiment of the present invention, the pH of the fermentation broth after reconstitution in step (2) is 1 to 3.
In one embodiment of the present invention, the temperature of the heating and stirring in the step (3) is 70 to 90 ℃, preferably 80 to 90 ℃.
In one embodiment of the present invention, the holding time in step (3) is 0.5 to 12 hours.
In one embodiment of the invention, the neutralization is carried out in step (3) by adjusting the pH to 6-7 with a base.
In one embodiment of the present invention, the centrifugation speed in step (4) is 6000-10000r/min, the centrifugation time is 5-15min, and the centrifugation temperature is 20 ℃.
In one embodiment of the invention, the degradation products are dialyzed in step (5) using an 8kDa-1.5kDa dialysis bag and the contents of the dialysis bag are removed.
A second object of the present invention is to provide a gamma-polyglutamic acid prepared according to the above method.
In one embodiment of the present invention, the gamma-polyglutamic acid has a number average molecular weight of 65000Da to 75000Da, a weight average molecular weight of 80000Da to 100000Da, and a polydispersity PDI of 1.2 to 1.5.
The third object of the present invention is to provide a use of the above-mentioned gamma-polyglutamic acid in foods, cosmetics, agriculture and medicine.
The invention has the beneficial effects that:
(1) The invention reduces the step of separating and purifying the original fermentation liquor in advance, and separates and purifies the original fermentation liquor after direct degradation, and has the advantages of simple step, easy operation, high treatment efficiency, less foreign substances introduced into a separation system, less residues and high safety.
(2) The invention reduces the step of adding alcohol reagent into degradation products to precipitate, reduces the use of organic solvents, and has easy operation, high safety and high treatment efficiency.
(3) The invention adopts a mode of combining ultrasonic technology and high-temperature acid degradation, improves degradation efficiency, and saves energy and time. The ultrasonic wave in a certain range can play a role in dispersing, so that the concentration of the product is higher, the three-dimensional structure of gamma-polyglutamic acid can be opened, the reaction contact area is increased, and the degradation rate is accelerated.
(4) The product prepared by the invention has small polydisperse coefficient, good uniformity and application value, and can omit the subsequent process steps of membrane separation to obtain different molecular weights.
(5) The medium and small molecular polyglutamic acid prepared by the method has the characteristics of safety, no stimulation and the like, and can effectively meet the diversified requirements of markets on polyglutamic acid with different molecular weights.
Detailed Description
The following further illustrates the invention in connection with examples, but the embodiments of the invention are not limited to the scope indicated by the examples.
In the following examples, the gamma-polyglutamic acid broth was obtained from Shandong Jin Yang pharmaceutical Co., ltd, and all other chemical reagents were analytically pure.
1. Molecular weight measurement method: dissolving the prepared gamma-polyglutamic acid finished product with pure water to prepare an aqueous solution with the concentration of 1mg/mL, filtering the aqueous solution with a 0.22 mu m membrane, and determining the molecular weight distribution of gamma-PGA by adopting a Gel Permeation Chromatography (GPC) method under the following chromatographic conditions: chromatographic column TSKgel G3000SWXL (7.8 mm. Times.300 mm), mobile phase 0.1M Na2SO4-0.1M Na3PO4 (pH 6.7), flow rate 0.5mL/min, column temperature 30 ℃,2487 ultraviolet detector UV 220nm.
2. The dispersion coefficient testing method comprises the following steps: polydisperse coefficient (PDI) =mw/Mn.
Examples 1-3:
the preparation process of gamma-polyglutamic acid with different molecular weight includes the following steps:
(1) And (3) re-dissolving: adding purified water into the gamma-polyglutamic acid fermentation broth obtained by ethanol precipitation, stirring at room temperature, and redissolving the solution until the mass concentration of the fermentation broth is 8%;
(2) Ultrasonic assisted acid degradation: adding hydrochloric acid to adjust the pH of the fermentation liquor obtained in the step (1) to 1, and respectively carrying out ultrasonic treatment for 0.5h, 1h and 2h under the conditions of ultrasonic frequency of 53kHz and ultrasonic power of 80W;
(3) High-temperature acid degradation: heating and stirring the acid solution obtained in the step (2) at 90 ℃, preserving heat for 1h to degrade to obtain degraded gamma-polyglutamic acid solution, rapidly cooling in ice water bath, and adding sodium hydroxide to adjust the pH to 7;
(4) And (3) centrifuging: centrifuging the feed liquid obtained in the step (3) for 10min at the temperature of 20 ℃ at 8000r/min, removing thalli and foreign proteins, and taking a supernatant;
(5) And (3) dialysis: dialyzing the supernatant obtained in the step (4) by using an 8kDa dialysis bag to remove small molecular salts, sugar and pigment;
(6) And (3) drying: and (5) performing vacuum freeze drying on the dialysate obtained in the step (5) for 48 hours to obtain a polyglutamic acid finished product.
Examples 4-6:
the preparation process of gamma-polyglutamic acid with different molecular weight includes the following steps:
(1) And (3) re-dissolving: adding purified water into the gamma-polyglutamic acid fermentation broth obtained by ethanol precipitation, stirring at room temperature, and redissolving the solution until the mass concentration of the fermentation broth is 8%;
(2) Ultrasonic assisted acid degradation: adding hydrochloric acid to adjust the pH of the fermentation liquor obtained in the step (1) to 1, and respectively carrying out ultrasonic treatment for 0.5h, 1h and 2h under the conditions of ultrasonic frequency of 53kHz and ultrasonic power of 160W;
(3) High-temperature acid degradation: heating and stirring the acid solution obtained in the step (2) at 90 ℃, preserving heat for 1h to degrade to obtain degraded gamma-polyglutamic acid solution, rapidly cooling in ice water bath, and adding sodium hydroxide to adjust the pH to 7;
(4) And (3) centrifuging: centrifuging the feed liquid obtained in the step (3) for 10min at the temperature of 20 ℃ at 8000r/min, removing thalli and foreign proteins, and taking a supernatant;
(5) And (3) dialysis: dialyzing the supernatant obtained in the step (4) by using an 8kDa dialysis bag to remove small molecular salts, sugar and pigment;
(6) And (3) drying: and (5) performing vacuum freeze drying on the dialysate obtained in the step (5) for 48 hours to obtain a polyglutamic acid finished product.
Examples 7 to 9:
the preparation process of gamma-polyglutamic acid with different molecular weight includes the following steps:
(1) And (3) re-dissolving: adding purified water into the gamma-polyglutamic acid fermentation broth obtained by ethanol precipitation, stirring at room temperature, and redissolving the solution until the mass concentration of the fermentation broth is 8%;
(2) Ultrasonic assisted acid degradation: adding hydrochloric acid to adjust the pH of the fermentation liquor obtained in the step (1) to 1, and respectively carrying out ultrasonic treatment for 0.5h, 1h and 2h under the conditions of ultrasonic frequency of 53kHz and ultrasonic power of 200W;
(3) High-temperature acid degradation: heating and stirring the acid solution obtained in the step (2) at 90 ℃, preserving heat for 1h to degrade to obtain degraded gamma-polyglutamic acid solution, rapidly cooling in ice water bath, and adding sodium hydroxide to adjust the pH to 7;
(4) And (3) centrifuging: centrifuging the feed liquid obtained in the step (3) for 10min at the temperature of 20 ℃ at 8000r/min, removing thalli and foreign proteins, and taking a supernatant;
(5) And (3) dialysis: dialyzing the supernatant obtained in the step (4) by using an 8kDa dialysis bag to remove small molecular salts, sugar and pigment;
(6) And (3) drying: and (5) performing vacuum freeze drying on the dialysate obtained in the step (5) for 48 hours to obtain a polyglutamic acid finished product.
Comparative example 1:
the preparation process of gamma-polyglutamic acid with different molecular weight includes the following steps:
(1) And (3) re-dissolving: adding purified water into the gamma-polyglutamic acid fermentation broth obtained by ethanol precipitation, stirring at room temperature, and redissolving the solution until the mass concentration of the fermentation broth is 8%;
(2) High-temperature acid degradation: adding hydrochloric acid to regulate the pH of the fermentation liquor obtained in the step (1) to 1, heating and stirring at 90 ℃, preserving heat for 1h to degrade to obtain degraded gamma-polyglutamic acid solution, rapidly cooling in ice water bath, and adding sodium hydroxide to regulate the pH to 7;
(3) And (3) centrifuging: centrifuging the feed liquid obtained in the step (2) for 10min at the temperature of 20 ℃ at 8000r/min, removing thalli and foreign proteins, and taking a supernatant;
(4) And (3) dialysis: dialyzing the supernatant obtained in the step (3) by using an 8kDa dialysis bag to remove small molecular salts, sugar and pigment;
(5) And (3) drying: and (3) performing vacuum freeze drying on the dialysate obtained in the step (4) for 48 hours to obtain a polyglutamic acid finished product.
Comparative example 2:
gamma-polyglutamic acid without any treatment.
The number average molecular weight (Mn), weight average molecular weight (Mw) and Polydispersity (PDI) of the samples prepared in examples 1, 2 and 3 and comparative examples 1 and 2 are shown in Table 1.
TABLE 1
It can be seen from Table 1 that the degradation rate in the range of 80W to 160W of ultrasonic power is substantially faster than that without ultrasonic treatment at the same degradation temperature, degradation time and pH, and the degradation rate at 200W of ultrasonic power is not faster than that without ultrasonic treatment. When the ultrasonic power is the same, the polydispersion coefficient of gamma-polyglutamic acid is smaller than that of gamma-polyglutamic acid which is not treated by ultrasonic within 0.5h-1h, and the molecular weight distribution is more concentrated within 1.0-1.5. When the ultrasonic time is the same, the gamma-polyglutamic acid with the ultrasonic power of 80W-160W has smaller polydisperse coefficient. In conclusion, the ultrasonic treatment is controlled within the range of 80W-160W and 0.5h-1h, and the prepared gamma-polyglutamic acid has better dispersion coefficient and better degradation effect than single high-temperature acid.
The antioxidant activity assay used in this control and example was carried out as follows: the specific determination method takes the ABTS free radical clearance as an index and comprises the following steps: the sample was prepared into 0.2% (w/w) aqueous solution with pure water, 400. Mu.L of the sample solution was added to 400. Mu.L of ABTS+ -working solution, and the mixture was mixed, reacted at room temperature under a dark condition for 6 minutes, and absorbance at 734nm was measured.
The sample to ABTS radical clearance calculation equation is as follows:
ABTS radical clearance (%) = (Ac-As)/(Ac-Ab) ×100%
Wherein Ac, as and Ab are absorbance values of a control group, an experimental group and a blank group respectively. The test results are shown in Table 2.
TABLE 2
As can be seen from table 2, the antioxidant effect of examples 1, 2 after ultrasonic treatment was better than that of comparative example 1 and comparative example 3 without degradation treatment at the same high temperature acidolysis time, and the antioxidant effect of example 1 was the best. Therefore, the oxidation resistance of the gamma-polyglutamic acid after the ultrasonic wave and high-temperature acid degradation treatment is better than that of the gamma-polyglutamic acid produced by the single high-temperature acid degradation treatment to a certain extent.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for preparing gamma-polyglutamic acid, comprising the steps of:
(1) And (3) re-dissolving: adding water into the gamma-polyglutamic acid fermentation broth obtained by ethanol precipitation for redissolution until the mass concentration of the fermentation broth is 1-10%;
(2) Ultrasonic assisted acid degradation: adding hydrochloric acid to regulate the pH value of the fermentation liquor obtained in the step (1), and carrying out ultrasonic treatment for a period of time at a certain frequency and power; the ultrasonic frequency is 20-60 kHz, the ultrasonic power is 80-160W, and the ultrasonic time is 0.5-1 h;
(3) High-temperature acid degradation: heating and stirring the acid solution obtained in the step (2), preserving heat and degrading to obtain degraded gamma-polyglutamic acid solution, cooling and neutralizing;
(4) And (3) centrifuging: centrifuging the feed liquid obtained in the step (3) to remove thalli and foreign proteins, and taking a supernatant;
(5) And (3) dialysis: dialyzing the supernatant obtained in the step (4) to remove small molecular salts, sugar and pigment;
(6) And (3) drying: and (3) performing vacuum freeze drying on the dialysate obtained in the step (5) to obtain a gamma-polyglutamic acid finished product.
2. The method according to claim 1, wherein the pH of the fermentation broth after reconstitution in step (2) is 1 to 3.
3. The method according to claim 1 or 2, wherein the temperature of the heating and stirring in the step (3) is 70 to 90 ℃.
4. A method according to any one of claims 1 to 3, wherein the holding time in step (3) is from 0.5 to 12 hours.
5. The method according to any one of claims 1 to 4, wherein neutralization is carried out in step (3) by adjusting the pH to 6 to 7 with a base.
6. The method according to any one of claims 1 to 5, wherein the centrifugation speed in step (4) is 6000 to 10000r/min and the centrifugation time is 5 to 15min.
7. The method according to any one of claims 1 to 6, wherein in step (5) the degradation products are dialyzed against an 8kDa-1.5kDa dialysis bag and the contents of the dialysis bag are taken.
8. A gamma-polyglutamic acid produced according to the method of any one of claims 1-7.
9. The gamma-polyglutamic acid according to claim 8, characterized in that the gamma-polyglutamic acid has a number average molecular weight of 65000Da to 75000Da, a weight average molecular weight of 80000Da to 100000Da and a polydispersity PDI of 1.2 to 1.5.
10. A use of the gamma-polyglutamic acid according to claim 8 or 9 in foods, cosmetics, agriculture and medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310791369.8A CN116655962A (en) | 2023-06-30 | 2023-06-30 | Preparation method of gamma-polyglutamic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310791369.8A CN116655962A (en) | 2023-06-30 | 2023-06-30 | Preparation method of gamma-polyglutamic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116655962A true CN116655962A (en) | 2023-08-29 |
Family
ID=87726235
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310791369.8A Pending CN116655962A (en) | 2023-06-30 | 2023-06-30 | Preparation method of gamma-polyglutamic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116655962A (en) |
-
2023
- 2023-06-30 CN CN202310791369.8A patent/CN116655962A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108264574B (en) | Ozone degradation method of polysaccharide | |
| CN108559763B (en) | Preparation method for improving polymerization degree of chitosan oligosaccharide and sugar content of 3-6 | |
| CN111718972B (en) | Preparation method of chitosan oligosaccharide with specific polymerization degree | |
| WO2021081999A1 (en) | Low-molecular-weight chondroitin sulfate and preparation method therefor | |
| WO2010013174A2 (en) | Process for the co-production of chitin, its derivatives and polymers containing glucose, mannose and/or galactose, by the fermentation of the yeast pichia pastoris | |
| CN103266146B (en) | Two enzyme immobilization coupling multistage membrane sepn prepare the method for medicinal dextran and fructose | |
| US11952436B2 (en) | Ultrasound and pressure assisted method for extracting pectin rich in RG-I | |
| CN102228125A (en) | Preparation method of algal active peptide | |
| WO2011034341A2 (en) | Method for manufacturing low molecular weight hyaluronic acid | |
| CN105154496B (en) | A method of specified molecular weight water soluble chitosan is prepared using enzyme process | |
| CN111393536B (en) | Degradation method of konjac glucomannan | |
| CN116655962A (en) | Preparation method of gamma-polyglutamic acid | |
| CN110982862B (en) | Method for large-scale preparation of high-purity unsaturated hyaluronic acid disaccharide | |
| WO2023137759A1 (en) | Hyperbranched polylysine powder with low polydispersity index and production method therefor | |
| CN111893151A (en) | Method for continuously producing low molecular weight hyaluronic acid, low molecular weight hyaluronic acid obtained by method and application of low molecular weight hyaluronic acid | |
| JPH0632742A (en) | Production of calcium ion solubilization agent | |
| LU502460B1 (en) | Preparation of porphyra haitanensis polysaccharide with low molecular weight | |
| CN113502312B (en) | Method for extracting functional glycopeptides from scallop viscera degreasing residues | |
| CN114591499B (en) | Preparation method and application of poly (R) -3-hydroxybutyrate | |
| CN116925257B (en) | Method for preparing low-methoxy orange peel pectin by regional temperature control and electrochemical cathode and anode co-operation | |
| CN113430238B (en) | Method for producing resistant dextrin by adding sucrose/fructo-oligosaccharide | |
| CN216295782U (en) | Cosmetic-grade polyglutamic acid solution circulating dialysis concentration device | |
| CN112266944A (en) | Industrial production method for obtaining chlorella protein peptide by acid-enzyme method | |
| CN111004295A (en) | Preparation method for efficiently synthesizing N-acetylglucosamine | |
| CN111197065A (en) | Method for producing algin hydrolysate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |