CN114686544A - Method for spontaneously regulating hydrolysis to generate alpha-glucan with specific molecular weight by utilizing three-water-phase system and application of method - Google Patents
Method for spontaneously regulating hydrolysis to generate alpha-glucan with specific molecular weight by utilizing three-water-phase system and application of method Download PDFInfo
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Abstract
The invention discloses a method for spontaneously regulating hydrolysis to generate alpha-glucan with a specific molecular weight by using a three-water phase system and application thereof, belonging to the technical field of enzymatic reaction regulation. The invention utilizes glucan which is a polymer capable of forming a phase body and the molecular weight of the glucan is also a key factor influencing the phase forming condition, and the final target molecular weight of the enzymolysis reaction falls on a phase separation point by adjusting the composition of other high polymer phase bodies, thereby achieving the purpose of stopping the reaction spontaneously. Meanwhile, the enzyme protein is rapidly recovered from the reaction phase by utilizing the high partition coefficient of the alpha-glucanase in part of the polymer phase, the enzymolysis reaction is stopped, and the enzyme protein is recovered again through concentrated salt solution after the low molecular weight alpha-glucan is separated.
Description
Technical Field
The invention belongs to the technical field of enzymatic reaction regulation and control, and particularly relates to a method for spontaneously regulating and controlling hydrolysis to generate alpha-glucan with specific molecular weight by using a three-water-phase system and application thereof
Background
The current process for producing low molecular weight alpha-glucans in the food or pharmaceutical industry consists of the following steps: fermentation-ethanol precipitation-hydrolysis-separation and purification-freeze-drying. The hydrolysis mode generally comprises acidolysis and enzymolysis, and various improved schemes are combined with other processes in hydrolysis, for example, CN101205257 obtains high molecular weight α -glucan through fermentation, then acidolysis is performed to a specified molecular weight, and the finished product is obtained through the steps of multi-stage membrane separation, ion exchange resin desalination and activated carbon decolorization. CN103993052 is prepared by one step through directly controlling the molecular weight of alpha-glucan in the fermentation process by using hydrolytic enzyme. CN105671104 is that penicillium echinosporii and acetobacter which secrete hydrolytic enzyme are respectively fixed on carrageenan matrix microspheres, and the aim of regulating enzymatic reaction is achieved by controlling the contact of the enzyme and a reaction substrate. Generally, acidolysis has good control capability on the molecular weight distribution range of hydrolysate, but has more triggered side reactions, particularly generates a large amount of monosaccharide, is easy to increase the color value of downstream products, and aggravates the downstream purification difficulty; the enzymolysis has specificity, few byproducts, different enzymological properties among isozymes, difficult control of reaction process and wider molecular weight distribution of final products. The immobilized fermentation is easy to cause the problems of reduction of the growth speed of thalli and reduction of the utilization rate of nutrients. The principle of the method is that the hydrogen bonds of polymer long chain molecules interact with water molecules in the environment to form liquid phases which are mutually geometrically saturated but have obviously different structures. According to factors such as polymer molecular weight, density, interfacial tension, potential difference and the like, proteins, nucleic acids, viruses, cell particles and the like have corresponding distribution behaviors in different aqueous phase systems, and the theoretical basis of aqueous phase extraction and purification is formed. CN103571806 proposes two aqueous phase extraction of dextran sucrase, wherein the target object is synthetase, the catalytic direction is opposite, and exogenous α -glucan needs to be introduced, so that the control of the final α -glucan molecular weight cannot be realized. Because the alpha-glucan is taken as a common constituent component of aqueous phase extraction, the alpha-glucan is not considered to participate in the aqueous phase extraction and reaction system of corresponding alpha-glucanase generally, and no method for quickly stopping the reaction and recovering free alpha-glucanase in the process of hydrolyzing the alpha-glucan in the aqueous phase extraction system is seen at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for generating alpha-glucan with specific molecular weight by spontaneously regulating and controlling hydrolysis by using a three-water phase system; the invention is based on the principle of aqueous phase extraction, and the alpha-glucan of the invention is not only a polymer forming a phase body, but also a substrate of enzymatic reaction. The molecular weight of the alpha-glucan is a key factor influencing the phase forming condition, and the final target molecular weight of the enzymolysis reaction falls on a phase separation point by adjusting the composition of other high polymer phase bodies. Meanwhile, the high distribution coefficient of the alpha-glucanase in a part of polymer phases is utilized to realize the rapid recovery of the enzyme protein from the reaction phase, stop the enzymolysis reaction, and recover the enzyme protein again through a specific concentrated salt solution which is compatible with the alpha-glucanase protein after the separation of the low molecular weight alpha-glucan.
Another object of the present invention is to provide the application of the above method for spontaneously controlling hydrolysis to produce alpha-glucan with a specific molecular weight by using a three-aqueous phase system.
The purpose of the invention is realized by the following technical scheme:
a method for spontaneously regulating and controlling hydrolysis to generate alpha-glucan with a specific molecular weight by using a three-water phase system comprises the following steps:
mixing a mixed solution of polypropylene glycol, polyethylene glycol (PEG4000) and alpha-glucan by shaking, standing for layering, dividing the mixed solution into three layers from top to bottom, wherein the upper layer is a polypropylene glycol phase, the middle phase is a polyethylene glycol phase, and the lower layer is an alpha-glucan phase, adding alpha-glucanase to the position, close to the interface of the two phases, of the middle phase and the lower phase, slightly shaking, keeping the temperature, observing that the middle phase and the lower phase are mixed, centrifuging, separating the liquid into the upper phase and the lower phase, taking the lower phase, adding alcohol, centrifuging, and taking a precipitate, namely the target low-molecular-weight alpha-glucan; adding a magnesium sulfate solution into the upper phase liquid, shaking, standing and layering to obtain two phases, wherein the upper phase is a polypropylene glycol phase, and the lower phase is a salt solution phase, taking the lower phase, and performing ultrafiltration, desalination and concentration to obtain an alpha-glucanase recovery liquid.
In the mixed solution of polypropylene glycol, polyethylene glycol (PEG4000) and alpha-glucan, the mass ratio of the polypropylene glycol, the polyethylene glycol (PEG4000) and the alpha-glucan is preferably 6.5-9: 4-8: 12-17.
In the mixed solution of the polypropylene glycol, the polyethylene glycol (PEG4000) and the alpha-glucan, the alpha-glucan is prepared by single batch fermentation of leuconostoc mesenteroides with the preservation number of GDMCC1.473 through a shake flask; the fermentation process for producing the alpha-glucan by single batch fermentation in the shake flask is preferably as follows: the liquid loading of a 5L fermentation tank is 2.7L, the inoculation amount is 10% (v/v), the initial pH is 5.5, the culture temperature is 25 ℃, and fermentation is carried out under the condition of 100 r/min. After fermentation for 12h, adding alpha-glucanase with the final concentration of 0.005U/mL, and simultaneously supplementing a sucrose flowing liquid at a flowing speed of 7 g/(h.L), and adding 280g of sucrose in total; then, two-stage pH regulation fermentation is carried out, the natural pH fermentation is carried out in the growth period of the thalli, the pH is regulated to 5.5 after 10 hours, and the fermentation is carried out in 20 hours; vacuum evaporation and concentration are carried out, and then heating sterilization is carried out to obtain the alpha-glucan.
The solvent of the mixed solution of the polypropylene glycol, the polyethylene glycol (PEG4000) and the alpha-glucan is preferably acetic acid-sodium acetate buffer solution; more preferably 0.2mol/L, pH ═ 5.4 in acetic acid-sodium acetate buffer.
The final addition amount of the alpha-glucanase is increased by 0.0005-0.0015U/mL when the addition amount of the alpha-glucanase is increased by 1% of the total reaction system.
The alpha-glucanase is derived from at least one of a bacterial source and a commercial source; when the alpha-glucanase is derived from bacteria, the alpha-glucanase is preferably derived from a strain with a preservation number of CCTCC No: m2013096 engineering bacteria of Pichia pastoris.
The enzyme activity of the alpha-glucanase is preferably 0.5-1.5U/mL; more preferably 1U/mL.
The temperature of the heat preservation is preferably 40-50 ℃; more preferably 45 deg.c.
The centrifugation conditions are preferably as follows: centrifuging at 4000-6000 rpm for 4-6 min; more preferably, centrifugation is carried out at 5000rpm for 5 min.
The alcohol is preferably 80-95% (v/v) alcohol; more preferably 92% (v/v) alcohol.
The final concentration of the alcohol in the lower phase and the alcohol is preferably 70-90% (v/v); more preferably 80% (v/v).
The conditions of centrifugation after adding alcohol are preferably as follows: centrifuging at 4000-6000 rpm for 4-6 min; more preferably, centrifugation is carried out at 5000rpm for 5 min.
The mass fraction of the magnesium sulfate in the magnesium sulfate solution is preferably 25-36% (w/w).
The adding amount of the magnesium sulfate in the system formed by the upper phase liquid and the magnesium sulfate solution is preferably 14-15% (w/w) of the system.
The method for spontaneously regulating and controlling hydrolysis to generate the alpha-glucan with the specific molecular weight by utilizing a three-water phase system is applied to food and medicines.
Compared with the prior art, the invention has the following advantages and effects:
(1) the finally obtained low molecular weight alpha-glucan is relatively concentrated in distribution, the reaction is easy to regulate and control, the condition is mild, and the waste strong acid solution does not need to be additionally treated; the enzyme preparation used in the reaction can be simply recovered, and the economic benefit is improved.
(2) The alpha-glucan of the invention is a polymer which can form a phase body, the molecular weight of the alpha-glucan is also a key factor influencing the phase forming condition, and the final target molecular weight of the enzymolysis reaction falls on a phase separation point by adjusting the composition of other high polymer phase bodies, thereby achieving the purpose of spontaneous stopping the reaction. Meanwhile, the enzyme protein is rapidly recovered from the reaction phase by utilizing the high partition coefficient of the alpha-glucanase in part of the polymer phase, the enzymolysis reaction is stopped, and the enzyme protein is recovered again through concentrated salt solution after the low molecular weight alpha-glucan is separated.
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FIG. 1 is a graph showing the results of comparing the liquid phase patterns of conventional enzymatic hydrolysis of alpha-glucanase with that of a three-aqueous phase process for producing alpha-glucan having a weight average molecular weight of about 100 kDa; wherein the peak between 9 and 10min represents fructose; peaks between 5 and 8min represent alpha-glucan.
FIG. 2 is a graph showing the results of comparing the liquid phase patterns of conventional enzymatic hydrolysis of alpha-glucanase with that of alpha-glucan having a weight average molecular weight of about 500kDa prepared by a three-aqueous phase method.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1: method for spontaneously regulating and controlling hydrolysis to generate alpha-glucan with weight average molecular weight of about 100kDa by utilizing three-aqueous phase system
In the present example, reagents such as polyethylene glycol, polypropylene glycol, potassium dihydrogen phosphate, magnesium sulfate, acetic acid, and sodium acetate were used as analytical reagents.
The alpha-glucanase is a pichia pastoris engineering bacterium from China center for type culture collection, and the collection number is CCTCC No: m2013096; the preservation number is CCTCC NO: the pichia pastoris engineering bacteria of M2013096 and the preparation method of alpha-glucanase are disclosed in patent 2013102877278. The alpha-glucan is prepared by shaking a flask and performing single batch fermentation on Leuconostoc mesenteroides (Leuconostoc mesenteroides purchased from Guangdong province culture Collection) with the preservation number of GDMCC1.473, and the specific fermentation process comprises the steps of filling 2.7L of 5L fermentation tank, inoculating 10% (v/v), initially pH6.5, culturing at 25 ℃ and fermenting at 100 r/min. After 12h of fermentation, 0.01U/mL final concentration of alpha-glucanase was added, and at the same time, a sucrose addition solution was added at a rate of 7 g/(h.L), and a total of 280g sucrose was added.
Then two-stage pH regulation fermentation is carried out, the pH of the thallus in the growing period is naturally fermented, the pH is regulated to 5.5 after 10 hours, and the fermentation is stopped after 20 hours. Concentrating by vacuum evaporation, and then heating and sterilizing to obtain the alpha-glucan for later use, wherein the molecular weight of the alpha-glucan is more than 1000 kDa.
Taking 0.2mol/L, pH ═ 5.4 acetic acid-sodium acetate buffer solution as a solvent, preparing 100g of mixed solution of polypropylene glycol, polyethylene glycol (PEG4000) and alpha-glucan, wherein the mass fractions of the polypropylene glycol, the polyethylene glycol (PEG4000) and the alpha-glucan are 9%, 4% and 17% in sequence, shaking, mixing, standing and layering. Dividing the mixture into three layers from top to bottom, wherein the upper layer is a polypropylene glycol phase, the middle phase is a polyethylene glycol phase, the lower layer is an alpha-glucan phase, 1mL of alpha-glucanase with the final concentration of 1U/mL is added to the lower layer close to the interface of the two phases, after slight oscillation, the temperature is kept at 45 ℃, after the middle and lower phases are observed to be mixed, the mixture is centrifuged at 5000rpm for 5min, the liquid is divided into the upper and lower phases at the moment, the lower phase is taken, alcohol is added until the final concentration is 80% (v/v), the mixture is centrifuged at 5000rpm for 5min, the precipitate is re-dissolved by distilled water, the precipitate is detected by HPLC by using a ligand exchange chromatographic column Shodex SUR-803, the alpha-glucan with the target low molecular weight of 100kDa is determined after external standard method calibration, and the alpha-glucan is stored at 4 ℃.
Adding concentrated magnesium sulfate solution into the upper phase liquid until the mass fraction of magnesium sulfate reaches 14%, shaking, standing for layering, distributing polypropylene glycol phase and salt solution phase from top to bottom, taking the lower phase, performing ultrafiltration desalination on the lower phase by Vivaflow 200PES 10000 by about 4 times of volume, and concentrating by one time of volume to obtain the alpha-glucanase recovery liquid.
The conversion rate of sucrose in the whole process is about 85%, the recovery rate of alpha-glucan ethanol precipitation is about 90%, and the recovery rate of alpha-glucanase is about 70%.
In addition, a process sample for producing the alpha-glucan by direct enzymolysis is prepared, namely the alpha-glucan is prepared by a common enzymolysis method, and the alpha-glucan process sample is obtained by fermentation culture by the following method: the liquid loading of the 5L fermentation tank is 2.7L, the inoculation amount is 10% (v/v), the initial pH is 6.5, the culture temperature is 25 ℃, and the fermentation is carried out under the condition of 100 r/min. After 16h of fermentation, the alpha-glucanase is added until the final concentration is 0.1U/mL, and simultaneously, the sucrose flowing liquid is supplemented at a certain flowing speed of 8g/(h & L), and the fermentation is stopped when the residual sugar concentration of the fermentation liquor is 1 to 2 percent before and after 26h according to HPLC monitoring. When HPLC detects that the hydrolysis degree of the alpha-glucan is close to the target molecular weight, the pH value is adjusted to be neutral, the hydrolysis reaction is stopped after heating for 20min at 85 ℃, alcohol is added until the final concentration is 80% (v/v), centrifugation is carried out for 5min at 5000rpm, and precipitates are taken, namely the alpha-glucan prepared by the common enzymolysis method. Through detection, the number average molecular weight (Mn) of the alpha-glucan prepared by the common enzymolysis method is 36kDa, the weight average molecular weight (Mw) is 63kDa, and the distribution coefficient is 1.7. The number average molecular weight (Mn) of an alpha-glucan sample obtained by the three-aqueous phase system spontaneous regulation process flow is 89kDa, the weight average molecular weight (Mw) is 102kDa, the distribution coefficient is 1.2, and the molecular weight distribution condition is obviously narrower than that of a common enzymolysis method.
Wherein the enzymology is defined as that the enzyme activity is one unit of enzyme activity, namely that the hydrolase reacts at 45 ℃ for 1min to hydrolyze to generate 1 mu mol of glucose. Sucrose conversion (%) ([ α -glucan end product weight/(sucrose weight/2) ] × 100%; the recovery rate (%) of ethanol precipitation was ═ 100% of [ weight of α -glucan after freeze-drying/total glucan content in solution after aqueous phase extraction reaction ] ×; the α -glucanase recovery (%) was ═ 100% x [ original concentration × addition volume/(concentration after concentration × volume after concentration) ].
Example 2: method for spontaneously regulating and controlling hydrolysis to generate alpha-glucan with weight average molecular weight of about 500kDa by utilizing three-aqueous phase system
The reagents used in this example, such as polyethylene glycol, polypropylene glycol, potassium dihydrogen phosphate, sodium chloride, ammonium sulfate, sodium sulfate, magnesium sulfate, acetic acid, and sodium acetate, were all analytical grade.
The glucanase is a pichia pastoris engineering bacterium from China center for type culture collection, and the collection number is CCTCC No: m2013096; the preservation number is CCTCC NO: 2013096 the preparation method of Pichia pastoris engineering bacteria and alpha-glucanase is disclosed in patent 2013102877278. Alpha-glucan is obtained from Leuconostoc mesenteroides with the collection number GDMCC1.473 (Leuconostoc mesenteroides from the Collection of microorganisms of Guangdong province). The specific fermentation process comprises the following steps: the liquid loading of a 5L fermentation tank is 2.7L, the inoculation amount is 10% (v/v), the initial pH is 5.5, the culture temperature is 25 ℃, and fermentation is carried out under the condition of 100 r/min. After 12h of fermentation, 0.005U/mL final concentration of alpha-glucanase was added, and at the same time, a sucrose addition solution was added at a rate of 7 g/(h.L), and a total of 280g sucrose was added.
Then two-stage pH regulation fermentation is carried out, the natural pH fermentation is carried out in the growth period of the thalli, the pH is regulated to 5.5 after 10 hours, and the fermentation is stopped after 20 hours. Vacuum evaporating and concentrating, and then heating and sterilizing to obtain the alpha-glucan for later use, wherein the molecular weight of the alpha-glucan is larger than 2000 kDa.
Taking acetic acid-sodium acetate buffer solution with the concentration of 0.2mol/L, pH-5.4 as a solvent, preparing 100g of mixed solution of polypropylene glycol, polyethylene glycol (PEG4000) and alpha-glucan, wherein the mass fractions of the polypropylene glycol, the polyethylene glycol and the alpha-glucan are 6.5%, 8% and 12% in sequence, shaking, mixing, standing and layering. Dividing the mixture into three layers from top to bottom, wherein the upper layer is a polypropylene glycol phase, the middle phase is a polyethylene glycol phase, the lower layer is an alpha-glucan phase, 1mL of alpha-glucanase with the final concentration of 1U/mL is added to the lower layer close to the interface of the two phases, after slight oscillation, the temperature is kept at 45 ℃, after the middle and lower phases are observed to be mixed, the mixture is centrifuged at 5000rpm for 5min, the liquid is divided into the upper and lower phases at the moment, the lower phase is taken, alcohol is added until the final concentration is 80% (v/v), the mixture is centrifuged at 5000rpm for 5min, the precipitate is re-dissolved by distilled water, the precipitate is detected by HPLC by using a ligand exchange chromatographic column Shodex SUR-803, the alpha-glucan with the target low molecular weight of 500kDa is determined after external standard method calibration, and the alpha-glucan is stored at 4 ℃.
Adding concentrated magnesium sulfate solution into the upper phase liquid until the mass fraction of magnesium sulfate reaches 14%, shaking, standing for layering, distributing polypropylene glycol phase and salt solution phase from top to bottom, taking the lower phase, performing ultrafiltration desalination by Vivaflow 200PES 10000 by about 4 times of volume, and concentrating by 2 times of volume to obtain the alpha-glucanase recovery liquid.
The conversion rate of sucrose in the whole process is about 90%, the recovery rate of alpha-glucan ethanol precipitation is about 92%, and the recovery rate of alpha-glucanase is about 50%.
In addition, a process sample for producing the alpha-glucan by direct enzymolysis is prepared, namely the alpha-glucan is prepared by a common enzymolysis method, and the alpha-glucan process sample is obtained by fermentation culture by the following method: the liquid loading of the 5L fermentation tank is 2.7L, the inoculation amount is 10% (v/v), the initial pH is 6.5, the culture temperature is 25 ℃, and the fermentation is carried out under the condition of 100 r/min. After 16h of fermentation, the alpha-glucanase is added to the final concentration of 0.02U/mL, meanwhile, the sucrose flowing liquid is supplemented at a certain flowing speed of 7g/(h & L), and the fermentation is stopped before and after 26h when the residual sugar concentration of the fermentation liquor is 1-2% according to HPLC monitoring. When HPLC detects that the hydrolysis degree of the alpha-glucan is close to the target molecular weight, heating at 85 ℃ to stop the reaction, adding alcohol until the final concentration is 80% (v/v), centrifuging at 5000rpm for 5min, and taking the precipitate, namely the alpha-glucan prepared by the common enzymolysis method. Through detection, the number average molecular weight (Mn) of the alpha-glucan prepared by the common enzymolysis method is 385kDa, the weight average molecular weight (Mw) is 585kDa, and the distribution coefficient is 1.6. The number average molecular weight (Mn) of an alpha-glucan sample obtained by the three-aqueous phase system spontaneous regulation process flow is 350kDa, the weight average molecular weight (Mw) is 508kDa, the distribution coefficient is 1.4, and the molecular weight distribution condition is obviously narrower than that of a common enzymolysis method.
Wherein the enzymology is defined as that the enzyme activity is one unit of enzyme activity, namely that the hydrolase reacts at 45 ℃ for 1min to hydrolyze to generate 1 mu mol of glucose. Sucrose conversion (%) ([ α -glucan end product weight/(sucrose weight/2) ] × 100%; the recovery rate (%) of ethanol precipitation was ═ 100% of [ weight of α -glucan after freeze-drying/total glucan content in solution after aqueous phase extraction reaction ] ×; the α -glucanase recovery (%) was ═ 100% x [ original concentration × addition volume/(concentration after concentration × volume after concentration) ].
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 (10)
1. A method for spontaneously regulating and controlling hydrolysis to generate alpha-glucan with a specific molecular weight by using a three-water phase system is characterized by comprising the following steps of:
taking a mixed solution of polypropylene glycol, polyethylene glycol PEG4000 and alpha-glucan, shaking and mixing, standing and layering, dividing the mixed solution into three layers from top to bottom, wherein the upper phase is a polypropylene glycol phase, the middle phase is a polyethylene glycol phase, and the lower phase is an alpha-glucan phase, adding alpha-glucanase to the position, close to the interface of the two phases, of the middle phase and the lower phase, slightly shaking, keeping the temperature, observing that the middle phase and the lower phase are mixed, centrifuging, separating the liquid into the upper phase and the lower phase, taking the lower phase, adding alcohol, centrifuging, and taking a precipitate, namely the target low-molecular-weight alpha-glucan; adding a magnesium sulfate solution into the upper phase liquid, shaking, standing and layering to obtain two phases, wherein the upper phase is a polypropylene glycol phase, and the lower phase is a salt solution phase, taking the lower phase, and performing ultrafiltration, desalination and concentration to obtain an alpha-glucanase recovery liquid.
2. The method according to claim 1, wherein in the mixed solution of polypropylene glycol, polyethylene glycol PEG4000 and alpha-glucan, the mass ratio of polypropylene glycol, polyethylene glycol PEG4000 and alpha-glucan is 6.5-9: 4-8: 12-17;
in the mixed solution of the polypropylene glycol, the polyethylene glycol PEG4000 and the alpha-glucan, the alpha-glucan is prepared by single batch fermentation of leuconostoc mesenteroides with the preservation number of GDMCC1.473 through a shake flask.
3. The method according to claim 1, wherein the solvent of the mixture of polypropylene glycol, polyethylene glycol and α -glucan is acetic acid-sodium acetate buffer.
4. The method of claim 1, wherein the amount of the α -glucanase added is increased by 0.0005 to 0.0015U/mL per 1% of the α -glucan added to the total reaction system.
5. The method of claim 1, wherein the alpha-glucanase is derived from at least one of a bacterial source and a commercial source; when the alpha-glucanase is derived from bacteria, the alpha-glucanase is derived from a culture medium with a preservation number of CCTCC No: m2013096 engineering bacteria of Pichia pastoris.
6. The method according to claim 1, wherein the enzyme activity of the alpha-glucanase is 0.5-1.5U/mL;
the temperature of the heat preservation is 40-50 ℃;
the centrifugation conditions are as follows: centrifuging at 4000-6000 rpm for 4-6 min;
the alcohol is 80-95% v/v alcohol.
7. The method of claim 1, wherein the final concentration of alcohol in the lower phase and alcohol is 70% to 90% v/v;
the centrifugation conditions after adding the alcohol are as follows: centrifuging at 4000-6000 rpm for 4-6 min.
8. The method of claim 1,
the mass fraction of the magnesium sulfate in the magnesium sulfate solution is 25-36% w/w.
9. The method of claim 1,
the adding amount of magnesium sulfate in the system formed by the upper phase liquid and the magnesium sulfate solution is 14-15% w/w of the system.
10. Use of a method according to any of claims 1-9 for the spontaneous controlled hydrolysis of a three-aqueous phase system to produce α -glucan of a specific molecular weight in food and pharmaceutical products.
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