CN112359073A - Method for preparing high-purity conjugated linoleic acid isomer by double-enzyme method resolution - Google Patents
Method for preparing high-purity conjugated linoleic acid isomer by double-enzyme method resolution Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
- C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
Abstract
The invention discloses a method for preparing a high-purity conjugated linoleic acid isomer by resolution of a double-enzyme method, which comprises the following steps of (1) mixing a conjugated linoleic acid mixed isomer, alcohol and enzyme liquid for esterification reaction, and then layering to obtain an upper phase product; (2) adding alkali liquor, alcohol and a hydrophobic organic solvent into the upper phase product, layering to obtain an upper phase which is a solvent phase rich in the conjugated linoleate of the c9, t11 isomer, and performing reduced pressure rotary evaporation to obtain the conjugated linoleate rich in the c9, t11 isomer; after the solution of the lower layer is taken out and regulated to be acidic, a hydrophobic organic solvent and soluble salt are added to form three phases, and the upper phase is conjugated linoleic acid rich in t10 and c12 isomers; (3) and (3) adding a soluble salt solution, lipase and a hydrophilic solvent into the conjugated linoleate obtained in the step (2), carrying out enzyme catalytic hydrolysis reaction, and then layering to obtain conjugated linoleic acid rich in c9, t11 isomer in an upper phase. The method has mild reaction conditions and high resolution efficiency, and can realize the reutilization of salt and enzyme.
Description
Technical Field
The invention belongs to the technical field of bioengineering and food, relates to a separation and application technology of enzyme, and particularly relates to a method for separating conjugated linoleic acid isomer by using lipase.
Background
Conjugated linoleic acid is a functional fatty acid having various physiological activities, and is considered as a new resource food in the publication No. 12 of the ministry of health in china. Conjugated linoleic acid has the effects of resisting tumors, atherosclerosis, hypertension, fat deposition and the like, is a substance which is beneficial to preventing diseases and safely reducing human body fat, and is often used for food and health care products.
The different isomers of conjugated linoleic acid exist mainly in the forms of c9, t11, t10 and c12 isomers, have different physiological effects, the c9 and t11 isomers have antitumor and anticancer activities, and the t10 and c12 isomers have important effects on reducing the accumulation of body fat. At present, the conjugated linoleic acid is mainly produced by a chemical isomerization method, a mixed isomer is obtained, and the separation process of the isomer is complex. To avoid the side effect of the t10, c12 isomer, the preparation of the c9, t11 isomer in high purity is the focus of current research. However, due to the similarity of physical and chemical properties between isomers, it is difficult to separate them by conventional separation methods such as direct extraction and distillation. The urea is separated to a certain extent by adopting a urea inclusion method and a low-temperature crystallization method, but the overall purity is not high, the operation is extremely complex, and the loss is huge. In recent years, an enzyme with high selectivity is utilized to esterify one isomer or hydrolyze the ester of the isomer, the other isomer is retained in the form of acid or ester, and finally, the two acids are separated to form different acids and esters, which are widely regarded. Multiple enzymatic resolution is advantageous to obtain higher purity isomers, for example NAGAO, Toshihiro attempted to obtain 93.1% pure conjugated linoleic acid by using two esterifications. However, the separation and purification process needs multiple times of molecular distillation, acid-base treatment and extraction, and the like, and has extremely complex process and extremely high cost. More importantly, the process requires multiple high-temperature heating treatments, and conjugated linoleic acid is extremely easy to oxidize, so that more harmful byproducts are finally enriched, and a large amount of energy is consumed. In a traditional oil-water or micro-water splitting system, the catalytic selectivity of the enzyme is low, the separation of product acid and ester requires a series of complex processes such as alkali washing, acid washing or high-temperature and ultra-low-pressure molecular distillation, and the problems of serious acid and alkali consumption, large environmental pollution, difficult enzyme recycling and the like are solved. Therefore, it is necessary to develop a high-efficiency and controllable continuous catalysis and separation system.
Disclosure of Invention
The invention aims to provide a method for efficiently and quickly splitting conjugated linoleic acid isomers by an enzyme method, aiming at the problems of high cost, complex reaction operation, difficult continuous production, low isomer selectivity and the like in the process of splitting conjugated linoleic acid by the enzyme method at present.
The purpose of the invention is realized by the following technical scheme:
a method for splitting conjugated linoleic acid isomer by a two-enzyme method comprises the following steps:
(1) mixing the conjugated linoleic acid mixed isomer, short-chain alcohol and enzyme solution of lipase or phospholipase, stirring for esterification reaction, synthesizing conjugated linoleate rich in single isomer, centrifuging or standing for layering to obtain an upper phase product;
(2) adding an ethanol-containing alkali liquor and a hydrophobic organic solvent into the upper-phase product, centrifuging or standing for layering, wherein the upper-layer extraction phase is a solvent phase rich in the conjugated linoleate of the c9, t11 isomer, and performing reduced pressure rotary evaporation to obtain the conjugated linoleate rich in the c9, t11 isomer; adding acid into the lower layer solution to adjust the solution to be acidic, and then adding a hydrophobic organic solvent and soluble salt to form three phases, wherein the upper phase is conjugated linoleic acid rich in t10 and c12 isomers; the middle phase is an ethanol-rich phase, and the lower layer is a soluble salt solution of a salt-rich phase;
(3) adding a soluble salt solution, lipase and a hydrophilic solvent into the conjugated linoleate obtained in the step (2), carrying out an enzymatic hydrolysis reaction under a stirring condition, centrifuging or standing after the reaction is finished to divide the conjugated linoleate into three layers, and obtaining conjugated linoleic acid rich in c9, t11 isomer in an upper phase.
The soluble salt solution in the steps (2) and (3) of the invention can be recycled.
Preferably, the molar ratio of the short-chain alcohol to the conjugated linoleic acid mixed isomer in the step (1) is 1: 0.5-3; the short-chain alcohol in the step (1) is one or more than two of methanol, ethanol, isopropanol, n-butanol, isoamyl alcohol and n-octanol.
Preferably, the esterification reaction condition in the step (1) is that the reaction time is 0.5-48h and the reaction temperature is 15-50 ℃; the enzyme adding amount is 80-200U/g, relative to the mass of the conjugated linoleic acid isomer mixture.
Preferably, the alkali liquor in the step (2) is one or more than two of ammonia water-ethanol solution, sodium hydroxide-ethanol solution and potassium hydroxide-ethanol solution with the concentration of 0.1-10 mol/L; wherein the concentration of ethanol is 10-50%.
Preferably, the hydrophobic organic solvent in step (2) is one or more of n-hexane, ethyl acetate, isooctane and isopropyl ether.
Preferably, the acid in step (2) is one or two of hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid.
Preferably, the soluble salt in steps (2) and (3) is one or more of sodium sulfate, ammonium sulfate, dipotassium hydrogen phosphate, sodium carbonate and potassium dihydrogen phosphate.
Preferably, the hydrophilic solvent in step (3) is one or more of polyethylene glycol, polypropylene glycol, dextran, n-propanol, isopropanol, n-butanol, isobutanol, ethylene glycol and acetone; or the hydrophilic solvent is [ BMIM]Br、[BMIM]BF4、[EMIM]ETSO4、[OMIM]One or more of Cl.
Preferably, the Lipase in the step (1) is Lipase AYS, Lipase AY30, CALB, Novozyme 435, the phospholipase is PLA2, PLC, PLD; the Lipase in the step (3) is Lipase AYS, Lipase AY30, CALB and Novozyme 435.
Preferably, the concentration of the soluble salt solution is 10% to 40%.
Preferably, the mass ratio of the hydrophilic solvent, the lipase, the conjugated linoleate and the soluble salt solution in the step (3) is 0.2-0.8, 0.005-0.5 and 0.05-0.8.
Preferably, the pH of the system in the step (3) is 5-9; the reaction conditions are that the time is 0.5-24h and the temperature is 25-55 ℃.
According to the method, a proper amount of water is needed for an enzyme in the esterification reaction in the step (1) to improve the reaction activity of the enzyme, alkali liquor is added in the step (2) to adjust the pH value of the enzyme to be an alkaline environment of 8-10, acid is added to adjust the pH value of the enzyme to be an acidic environment of 3-5, and the enzyme is extracted by a hydrophobic solvent to obtain an isomer rich in c9 and t11 and an isomer rich in t10 and c12 respectively, and can be used for further resolution.
According to the invention, ethanol with a certain volume fraction is required to be added in the alkali liquor prepared in the step (2), the extraction efficiency is influenced by no addition of ethanol, and the recovered conjugated linoleate product is reduced.
Compared with the prior art, the invention has the beneficial effects that:
the invention overcomes the problems of high cost, complex reaction operation, difficult continuous production, low reaction efficiency and the like in the process of splitting the conjugated linoleic acid isomer by the enzyme method at present, and provides a method for efficiently and quickly splitting the conjugated linoleic acid isomer by the enzyme method. The method has the specific characteristics that (1) the reaction steps are simple, special equipment and high energy consumption are not needed, and the high-purity c9 and t11 conjugated linoleic acid isomer can be obtained; (2) by utilizing the multi-enzyme reaction process, the enzyme, the unreacted ethanol and partial salt can be synchronously recovered, which is beneficial to the purification and recovery of the enzyme and the product, greatly reduces the environmental pollution, and reduces the material consumption, thereby reducing the cost. The method has the advantages of low energy consumption, high utilization rate of raw materials, high reaction efficiency and the like, solves the problem of the resolution of the conjugated linoleic acid isomer by the existing enzyme method, effectively obtains the c9 and t11 isomers with high purity, and has the resolution efficiency of more than 98 percent at most.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Lipase AYS and Lipase AY30(Candida rugosa lipases) used in this example were obtained from Tianye, Japan, conjugated linoleic acid mixed isomer was obtained from Australian biology, Inc., Qingdao, CALB, PLA2, PLC, PLD from Gaorhenson, Beijing, and Novozyme 435 were obtained from Novozymes.
Example 1
Step (1): 10g of conjugated linoleic acid isomer mixture is taken, 2.1ml of absolute ethyl alcohol (molar ratio is 1:1) and 4ml of Lipase AYS enzyme solution with pH value of 7 are added, the enzyme adding amount is 180U/g, the mixture is uniformly mixed in a 50ml conical flask, and the mixture is reacted for 4 hours at the temperature of 40 ℃ and the rpm of 300 in a constant-temperature water bath kettle.
Step (2): after the reaction is finished, centrifuging for 5 minutes at 3000 r/L for layering, and adding 150ml of 0.5mol/L sodium hydroxide-30% ethanol solution and 200ml of n-hexane into the upper phase for extraction. Standing for layering, and performing rotary evaporation on the upper layer solution to obtain 3.23g of conjugated linoleic acid ethyl ester. Adding sulfuric acid into the lower layer solution to adjust the lower layer solution to acidity, adding 200ml of n-hexane for extraction and a proper amount of sodium sulfate to enable the concentration of a salt solution of the lower layer solution to reach 23% (w/v), obtaining three phases, wherein the upper phase is an n-hexane phase, carrying out rotary evaporation to obtain 2.52g of conjugated linoleic acid rich in t10, c12 isomers, the middle phase is an ethanol-rich phase, and the lower layer solution is a salt solution containing sodium sulfate.
And (3): 0.01g of AY30 enzyme and 0.3g of polyethylene glycol 400 are added into 0.975g of the obtained salt solution, and 0.08g of the conjugated linoleic acid ethyl ester in the step (2) is added into the mixed solution to construct a heterogeneous catalytic system. The reaction was carried out for 2h at 37 ℃ on a constant temperature shaker at 200 rpm. Finally, the resolution efficiency of the c9 and t11 isomers reaches 98.01 percent, and the reaction hydrolysis efficiency is 30 percent.
Example 2
Step (1): taking 10g of conjugated linoleic acid isomer mixture, adding 1.4ml of methanol (molar ratio is 1:1) and 4ml of Lipase AYS enzyme solution with pH value of 6, adding the enzyme amount of 160U/g, uniformly mixing in a 50ml conical flask, and reacting for 4 hours at 300rpm and 40 ℃ in a constant-temperature water bath kettle.
Step (2): after the reaction is finished, centrifuging for 5 minutes at 3000 r/L, taking the upper phase, adding 30ml of 2mol/L ammonia water-30% ethanol solution, and adding 200ml of isopropyl ether for extraction. Standing for layering, and performing rotary evaporation on the upper layer solution to obtain 2.8g of conjugated methyl linoleate. Adding sulfuric acid into the lower layer solution to adjust the solution to acidity, adding 200ml of n-hexane for extraction and a proper amount of ammonium sulfate to enable the concentration of the salt solution to reach 26% (w/v), obtaining three phases, wherein the upper phase is an n-hexane phase, carrying out rotary evaporation to obtain 3.4g of conjugated linoleic acid rich in t10, c12 isomers, the middle phase is an ethanol-rich phase, and the lower layer solution is a salt solution containing ammonium sulfate.
And (3): 0.01g of AYS enzyme and 0.3g of polyethylene glycol 400 are added into 0.9g of the obtained salt solution, and 0.08g of conjugated methyl linoleate obtained in the step (2) is added into the mixed solution to construct a heterogeneous catalytic system. The reaction was carried out for 2h at 37 ℃ on a constant temperature shaker at 200 rpm. The resolution efficiency of the final c9, t11 isomer reaches 97.86%.
Example 3
Step (1): taking 10g of conjugated linoleic acid isomer mixture, adding 2.8ml of isopropanol (molar ratio is 1:1) and 4ml of Lipase AYS enzyme solution with pH value of 7, adding the enzyme amount to 200U/g, uniformly mixing in a 50ml conical flask, and reacting for 4 hours at 300rpm and 40 ℃ in a constant-temperature water bath kettle.
Step (2): after the reaction, the mixture was centrifuged at 3000 rpm for 5 minutes, and 70ml of a 1mol/L ammonia-10% ethanol solution was added to the above solution, followed by extraction with 200ml of isooctane. Standing for layering, and performing rotary evaporation on the upper layer solution to obtain 2.14g of conjugate isopropyl linoleate. Adding sulfuric acid into the lower layer solution to adjust the solution to acidity, adding 200ml of n-hexane for extraction and a proper amount of ammonium sulfate to enable the concentration of the salt solution to reach 26% (w/v), obtaining three phases, wherein the upper phase is an n-hexane phase, carrying out rotary evaporation to obtain 3.6g of conjugated linoleic acid rich in t10, c12 isomers, the middle phase is an ethanol-rich phase, and the lower layer solution is a salt solution containing ammonium sulfate.
And (3): 0.03g of AY30 enzyme and 0.3g of [ BMIM ] were added to 0.9g of the salt solution obtained above]BF4And (3) adding 0.08g of the conjugate isopropyl linoleate obtained in the step (2) into the mixed solution to construct a heterogeneous catalytic system. The reaction was carried out for 4h at 37 ℃ on a constant temperature shaker at 200 rpm. The resolution efficiency of the final c9, t11 isomer reaches 98.92%, and the reaction hydrolysis efficiency is 48%.
Example 4
Step (1): taking 10g of conjugated linoleic acid isomer mixture, adding 3.3ml of n-butanol (molar ratio is 1:1) and 4ml of Lipase AYS enzyme solution with pH of 7, adding the enzyme amount of 140U/g, uniformly mixing in a 50ml conical flask, and reacting for 4 hours at 300rpm and 40 ℃ in a constant-temperature water bath kettle.
Step (2): after the reaction, the mixture was centrifuged at 3000 rpm for 5 minutes, 150ml of 0.5mol/L potassium hydroxide-50% ethanol solution was added to the above solution, and 200ml of ethyl acetate was added thereto for extraction. Standing for layering, and rotary-steaming the upper layer solution to obtain 4.28g of n-butyl conjugated linoleic acid. Adding sulfuric acid into the lower layer solution to adjust the lower layer solution to acidity, adding 200ml of n-hexane for extraction and a proper amount of potassium sulfate to enable the concentration of a salt solution of the lower layer solution to reach 23% (w/v), obtaining three phases, wherein the upper phase is an n-hexane phase, carrying out rotary evaporation to obtain 1.8g of conjugated linoleic acid rich in t10 and c12 isomers, the middle phase is an ethanol-rich phase, and the lower layer solution is a salt solution containing potassium sulfate.
And (3): 0.03g of AYS enzyme and 0.285g of isopropanol are added into 0.9g of the obtained salt solution, and 0.08g of conjugated butyl linoleate obtained in the step (2) is added into the mixed solution to construct a heterogeneous catalytic system. The reaction was carried out for 4h at 37 ℃ on a constant temperature shaker at 200 rpm. The resolution efficiency of the final c9, t11 isomer reaches 95.80 percent, and the reaction hydrolysis efficiency is 40 percent.
Example 5
Step (1): taking 10g of conjugated linoleic acid isomer mixture, adding 4.2ml of absolute ethyl alcohol (molar ratio is 1:2) and 2ml of Lipase AYS enzyme solution with pH value of 6, adding the enzyme amount of 180U/g, uniformly mixing in a 50ml conical flask, and reacting for 8 hours at 300rpm and 40 ℃ in a constant-temperature water bath kettle.
Step (2): after the reaction, the mixture was centrifuged at 3000 rpm for 5 minutes, and 70ml of 0.5mol/L NaOH-30% ethanol solution was added to the above solution, followed by extraction with 200ml of ethyl acetate. Standing for layering, and performing rotary evaporation on the upper layer solution to obtain 3.6g of conjugated linoleic acid ethyl ester. Adding sulfuric acid into the lower layer solution to adjust the lower layer solution to acidity, adding 200ml of n-hexane for extraction and a proper amount of sodium sulfate to enable the concentration of a salt solution of the lower layer solution to reach 20% (w/v), obtaining three phases, wherein the upper phase is an n-hexane phase, carrying out rotary evaporation to obtain 2.0g of conjugated linoleic acid rich in t10, c12 isomers, the middle phase is an ethanol-rich phase, and the lower layer solution is a salt solution containing sodium sulfate.
And (3): 0.01g of AYS enzyme and 0.3g of PEG400 are added into 0.9g of the obtained salt solution, and then 0.08g of conjugated linoleic acid ethyl ester in the step (2) is added into the mixed solution to construct a heterogeneous catalytic system. The reaction was carried out for 4h at 37 ℃ on a constant temperature shaker at 200 rpm. The resolution efficiency of the final c9, t11 isomer reaches 98.65%, and the reaction hydrolysis efficiency is 56.5%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A method for preparing high-purity conjugated linoleic acid isomer by resolution of a two-enzyme method is characterized by comprising the following steps:
(1) mixing the conjugated linoleic acid mixed isomer, short-chain alcohol and enzyme solution of lipase or phospholipase, stirring for esterification reaction, synthesizing conjugated linoleate rich in single isomer, centrifuging or standing for layering to obtain an upper phase product;
(2) adding an ethanol-containing alkali liquor and a hydrophobic organic solvent into the upper-phase product, centrifuging or standing for layering, wherein the upper-layer extraction phase is a solvent phase rich in the conjugated linoleate of the c9, t11 isomer, and performing reduced pressure rotary evaporation to obtain the conjugated linoleate rich in the c9, t11 isomer; adding acid into the lower layer solution to adjust the solution to be acidic, and then adding a hydrophobic organic solvent and soluble salt to form three phases, wherein the upper phase is conjugated linoleic acid rich in t10 and c12 isomers; the middle phase is an ethanol-rich phase, and the lower layer is a soluble salt solution of a salt-rich phase;
(3) adding a soluble salt solution, lipase and a hydrophilic solvent into the conjugated linoleate obtained in the step (2), carrying out an enzymatic hydrolysis reaction under a stirring condition, centrifuging or standing after the reaction is finished to divide the conjugated linoleate into three layers, and obtaining conjugated linoleic acid rich in c9, t11 isomer in an upper phase.
2. The method according to claim 1, wherein the molar ratio of the short-chain alcohol to the conjugated linoleic acid mixed isomer in the step (1) is 1: 0.5-3; the short-chain alcohol in the step (1) is one or more than two of methanol, ethanol, isopropanol, n-butanol, isoamyl alcohol and n-octanol.
3. The method according to claim 2, wherein the esterification reaction in step (1) is carried out under the conditions of 0.5-48h of reaction time and 15-50 ℃ of reaction temperature; the enzyme adding amount is 80-200U/g, relative to the mass of the conjugated linoleic acid isomer mixture.
4. The method according to claim 3, wherein the alkali solution in the step (2) is one or more of an ammonia-ethanol solution, a sodium hydroxide-ethanol solution and a potassium hydroxide-ethanol solution with a concentration of 0.1-10 mol/L; wherein the concentration of ethanol is 10-50%;
the hydrophobic organic solvent in the step (2) is one or more than two of n-hexane, ethyl acetate, isooctane and isopropyl ether;
and (3) the acid in the step (2) is one or two of hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid.
5. The method according to claim 1 or 2 or 3 or 4, wherein the soluble salt in steps (2) and (3) is one or more of sodium sulfate, ammonium sulfate, dipotassium hydrogen phosphate, sodium carbonate and potassium dihydrogen phosphate;
the hydrophilic solvent in the step (3) is one or more than two of polyethylene glycol, polypropylene glycol, dextran, normal propyl alcohol, isopropyl alcohol, normal butyl alcohol, isobutyl alcohol, ethylene glycol and acetone; or the hydrophilic solvent is [ BMIM]Br、[BMIM]BF4、[EMIM]ETSO4、[OMIM]One or more of Cl.
6. The method of claim 1 or 2 or 3 or 4, wherein the Lipase of step (1) is Lipase AYS, Lipase AY30, CALB, Novozyme 435, the phospholipase is PLA2, PLC, PLD; the Lipase in the step (3) is Lipase AYS, Lipase AY30, CALB and Novozyme 435.
7. The method of claim 5, wherein the concentration of the soluble salt solution is 10% to 40%.
8. The method according to claim 1, 2, 3 or 4, wherein the mass ratio of the hydrophilic solvent, the lipase, the conjugated linoleate and the soluble salt solution in the step (3) is 0.2-0.8, 0.005-0.5 and 0.05-0.8.
9. The method according to claim 8, wherein the system in step (3) has a pH of 5 to 9; the reaction conditions are that the time is 0.5-24h and the temperature is 25-55 ℃.
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