CN106244645B - Method for producing fructo-oligosaccharide by recycling aspergillus oryzae thallus - Google Patents
Method for producing fructo-oligosaccharide by recycling aspergillus oryzae thallus Download PDFInfo
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Abstract
The invention relates to the technical field of microbial fermentation, in particular to a method for producing fructo-oligosaccharide by recycling aspergillus oryzae thalli, which comprises the following steps: mixing the cultured aspergillus oryzae suspension with a sucrose solution for saccharification to obtain fermentation liquor containing fructo-oligosaccharide; treating the fermentation liquor with a microfiltration membrane, recovering Aspergillus oryzae thallus, and filtering to obtain fructo-oligosaccharide clear solution; treating the aspergillus oryzae thallus recovered for the first time by glutaraldehyde, and washing and recovering the aspergillus oryzae thallus through a microfiltration membrane; adjusting the concentration of the aspergillus oryzae suspension, refluxing to a saccharification tank for carrying out the next batch of saccharification, and carrying out post-treatment on a fructo-oligosaccharide clear solution to obtain fructo-oligosaccharide syrup; and (4) after the second batch and the subsequent batch of saccharification are finished, the recovered thalli are not treated by glutaraldehyde any more, and the thalli are used for the next batch of saccharification after the concentration of the bacterial suspension is adjusted. The glutaraldehyde treatment and the microfiltration membrane are adopted to recover the thalli, so that the recycling of the thalli is realized, the thalli can be used for at least 8 times, the times of strain culture are reduced, the energy consumption and the raw material consumption in the process of strain culture are reduced, and the production cost is reduced.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a method for producing fructo-oligosaccharide by recycling aspergillus oryzae thalli.
Background
FOS (Fructooligosaccharides) refers to functional oligosaccharide with polymerization degree of 2-9 formed by connecting fructosyl with β -2,1 glycosidic bond, and belongs to food ingredientStructurally, fructooligosaccharides can be classified into cane-fruit type and fruit-fruit type. There are two main methods for industrially producing fructooligosaccharides: an enzyme hydrolysis method using inulin as raw material and an enzyme conversion method using sucrose as raw material. The fructo-oligosaccharide obtained by the former has a large polymerization degree range (DP)2-DP9) Belongs to fruit-fruit type; the polymerization degree range of the fructo-oligosaccharide obtained by the latter is small (DP)3-DP6) It belongs to the sugarcane-fruit type. Research shows that the animal or human body can improve intestinal flora, relieve constipation, improve lipid metabolism, inhibit intestinal putrefaction, promote absorption of mineral elements such as calcium and magnesium, and enhance immunity by eating fructo-oligosaccharide. The fructo-oligosaccharide has good physical and chemical properties and physiological effects, so that the fructo-oligosaccharide can be widely applied to the industries of food, health care products, feed and the like.
Industrial methods for producing fructo-oligosaccharide from sucrose include submerged fermentation with microorganism liquid, free enzyme method and immobilized enzyme method. Although the post-treatment of the free enzyme method is simple, the enzyme can be used only once and the cost of the enzyme preparation is higher; although the immobilized enzyme method can realize the repeated utilization of the enzyme, the technical requirement of the immobilization process of the enzyme is higher, and the content of fructo-oligosaccharide obtained by converting the enzyme into the sucrose after immobilization is often lower than that of free enzyme; although the microbial liquid submerged fermentation method is a traditional method, the production process is mature and the production amount is large, so the method is still widely used for producing fructo-oligosaccharide.
Patent CN 103045489B discloses Aspergillus niger and a method for producing fructo-oligosaccharide by whole-cell catalysis. The method is improved on the basis of the traditional microorganism liquid submerged fermentation method. The whole cells are added into the sucrose solution to catalyze and synthesize the fructo-oligosaccharide, and the fructo-oligosaccharide can be concentrated into the final product syrup by simple filtration without decolorization and ion exchange, which is the advantage of the method. This advantage is obtained thanks to the previous treatment of the transformed aspergillus niger biomass: and (3) fermenting the obtained aspergillus niger whole-cell solution, filtering by using sterile filter cloth, washing, filtering again and drying to obtain aspergillus niger whole-cell suspension with conversion activity. The process is complicated in operation, special drying equipment is needed, and the FOS content in the syrup obtained by the method is not high.
Patent CN 105524847A discloses aureobasidium pullulans AP172-1B and a method for producing fructo-oligosaccharide by using the same, and the method also belongs to a microbial fermentation method. The purity of the fructo-oligosaccharide finally obtained by the method is not lower than 93 percent, which is the advantage of the method. However, the method has a long period for producing fructo-oligosaccharide: the Aureobasidium pullulans is inoculated into a fermentation tank to obtain fermentation liquor within 60-72h, the fermentation liquor is heated to 80 ℃, is cooled and then is placed for 48-60h, the enzyme reaction needs 10-15h after decolorization, and the culture time of the strain is added for 24-36h, so that the total use time reaches 142-183 h. In addition, the fermentation liquor is heated to 80 ℃, is cooled and then is placed at 4 ℃, and a centrifuge is used for collecting supernatant, so that the energy consumption of the steps is large; the fructosyl transferase is added for reaction at the later stage, and the cost of the enzyme is higher.
Disclosure of Invention
The invention improves the traditional microorganism liquid submerged fermentation method and provides a method for producing fructo-oligosaccharide by recycling aspergillus oryzae thalli. The method is improved on the basis of the traditional microorganism liquid submerged fermentation method, and microfiltration equipment is introduced after saccharification is finished so as to realize the reutilization of microorganism thalli; after the microfiltration is finished, introducing ultrafiltration equipment, further refining the feed liquid and recovering macromolecular substances such as protein (derived from culture medium) with high added value. The traditional process adopts plate-frame filtration in the filtration link, and the working procedure has high labor intensity, open operation system (feed liquid is easy to be polluted) and longer working hours; after the microfiltration equipment is adopted, the automatic operation can be realized, the labor intensity is reduced, the labor cost is saved, and the feed liquid can be prevented from being polluted due to the airtight operation system.
A method for producing fructo-oligosaccharide by circularly utilizing Aspergillus oryzae thallus comprises the following steps:
(1) mixing the cultured aspergillus oryzae suspension with a sucrose solution for saccharification to obtain fermentation liquor containing fructo-oligosaccharide;
(2) filtering the fermentation liquor with a microfiltration membrane, recovering Aspergillus oryzae thallus, and filtering to obtain fructo-oligosaccharide clear solution;
(3) treating the aspergillus oryzae thalli recovered for the first time by glutaraldehyde, and then washing and recovering the aspergillus oryzae thalli through a microfiltration membrane for the next batch of saccharification; after the subsequent saccharification is finished, the recovered thalli are not treated by glutaraldehyde any more, and the concentration of the recovered bacterial suspension is adjusted to the concentration of the bacterial suspension in the step (1), namely, the recovered bacterial suspension is used for the next batch of saccharification;
(4) and (4) adjusting the concentration of the aspergillus oryzae suspension recovered in the step (3) to the concentration of the bacterial suspension in the step (1), refluxing to a saccharification tank for carrying out saccharification of the next batch, and carrying out post-treatment on a fructo-oligosaccharide clear solution to obtain fructo-oligosaccharide syrup.
In the method, the microfiltration membranes in the steps (2) and (3) are preferably inorganic ceramic membranes, and the interception aperture is 0.1-1 μm.
In the method, the preferable microfiltration temperature is 30-42 ℃, and the operation pressure is 0.05-0.2 MPa.
In the method, preferably, when the first-recovered Aspergillus oryzae thallus is treated by glutaraldehyde in the step (3), the thallus concentration is adjusted to be 5-15 g/L, and the final concentration after adding the glutaraldehyde is 5-15 g/L.
In the method, preferably, when the first-time recovered aspergillus oryzae thalli is treated by glutaraldehyde in the step (3), the reaction temperature is 10-15 ℃, the stirring speed is 30-60 rpm, and the reaction time is 6-10 hours.
In the method, the mass ratio of the bacteria to the glutaraldehyde in the step (3) is preferably 1: 0.8-1: 1.2, and the mass ratio of the bacteria to the glutaraldehyde is preferably 1:1.
In the method, the enzyme activity recovery rate is 88-92% after washing and recycling through a microfiltration membrane in the step (3) is preferred.
The method, preferably the post-treatment step in the step (4), comprises ultrafiltration, activated carbon decolorization, ion exchange desalination and vacuum concentration.
In the method, the recovery is preferably not carried out in the step (3) until the recovery rate of the enzyme activity is less than or equal to 60 percent.
Advantageous effects
1. The use of the microfiltration membrane successfully solves the problems of easy pollution, high labor intensity, production limitation and the like of fructo-oligosaccharide liquid caused by the plate-and-frame filtration in the production of fructo-oligosaccharide in the traditional process;
2. in the traditional microbial liquid submerged fermentation method, microbial thalli can be used only once; the process adopts the microfiltration membrane to recover the thalli, realizes the recycling of the thalli (at least 8 times of use), reduces the times of strain culture, and reduces the energy consumption and the raw material consumption in the process of strain culture, thereby reducing the production cost and reducing the discharge amount of waste thalli;
3. the concentration of the first recovered thallus is adjusted and is treated by glutaraldehyde, so that the stability of intracellular fructosyltransferase can be improved, and the use frequency of the recovered thallus is increased;
4. the feed solution is refined by ultrafiltration, during which large molecules such as proteins can be recovered from the feed solution, and these proteins can be further processed for addition to animal feed. The recycling of the proteins can improve the added value of the product;
5. the fructo-oligosaccharide prepared by the invention has high quality, and the content (accounting for the total solid) of the fructo-oligosaccharide is more than or equal to 56 percent.
Detailed Description
The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.
The enzyme activity determination method in the embodiment refers to GB/T23528-2009 fructo-oligosaccharide; when the recovery rate of the enzyme activity is lower than 60 percent, the subsequent saccharification is too long, so the recovery of the thalli is stopped, and the thalli are inactivated and discharged.
Example 1
(1) Mixing 1 ton of cultured aspergillus oryzae suspension with 5 ton of sugar-concentrated 50% sucrose solution as a dry basis for saccharification, controlling the saccharification temperature to be 40-42 ℃ and the pH value to be 6.0-6.5, and saccharifying for 30 hours to obtain the fermentation liquor containing fructo-oligosaccharide.
(2) Filtering the fermentation liquor by a ceramic membrane to obtain a fructo-oligosaccharide clear liquid, and recovering Aspergillus oryzae thallus, wherein the aperture of the ceramic membrane is 0.1-0.4 mu m, the microfiltration temperature is 30-33 ℃, the operation pressure is 0.15-0.2 Mpa, and the microfiltration speed is 4.4m3/h。
(3) Adding water into the first recovered thallus, adjusting the thallus concentration to 5 g/L, adding glutaraldehyde to make the final concentration reach 5 g/L, reacting for 6h at 13-15 ℃ and 30rpm, fully washing and recovering the thallus through a ceramic membrane, and the enzyme activity recovery rate reaches 88%.
(4) Adjusting the concentration of the recovered strain suspension after microfiltration to the concentration of the aspergillus oryzae suspension in the step (1), refluxing to a saccharification tank, starting the next batch of saccharification after a sucrose solution is supplemented, wherein the saccharification conditions are the same as those in the step (1), the saccharification time of the batch and the subsequent batch is determined according to the detection result, and when the FOS content in the saccharification liquid reaches more than 56%, the saccharification of the corresponding batch can be finished; introducing the clear liquid obtained by microfiltration into an ultrafiltration membrane to obtain an ultrafiltration fructo-oligosaccharide liquid, and recovering macromolecular substances such as protein, wherein the ultrafiltration membrane has a molecular weight cutoff range of 27000-30000D, an operating pressure of 0.2-0.35 Mpa and an ultrafiltration temperature of 30-33 ℃; decolorizing the fructo-oligosaccharide solution obtained by ultrafiltration with active carbon, desalting by ion exchange, and vacuum concentrating to obtain refined fructo-oligosaccharide syrup; the FOS content (in total solid content) in the syrup is 57.0% by high performance liquid chromatography detection.
(5) The recovery rate of enzyme activity of the cultured thalli is 55 percent after the thalli is recycled for 8 times.
Example 2
(1) Mixing 1.5 tons of cultured aspergillus oryzae suspension with 7.5 tons of sucrose solution with sugar concentration of 51 percent in dry basis, saccharifying at the temperature of 40-42 ℃ and pH controlled between 6.0-6.5, and saccharifying for 32 hours to obtain fermentation liquor containing fructo-oligosaccharide.
(2) Filtering the fermentation liquor by a ceramic membrane to obtain a fructo-oligosaccharide clear liquid, and recovering Aspergillus oryzae thallus, wherein the aperture of the ceramic membrane is 0.3-0.7 mu m, the microfiltration temperature is 34-37 ℃, the operation pressure is 0.05-0.1 Mpa, and the microfiltration speed is 4.1m3/h。
(3) Adding water into the first recovered thallus, adjusting the thallus concentration to 15 g/L, adding glutaraldehyde to make the final concentration to 15 g/L, reacting for 10h at 10-12 ℃ and 60rpm, fully washing and recovering the thallus through a ceramic membrane, wherein the recovery rate of enzyme activity reaches 90%.
(4) Adjusting the concentration of the recovered strain suspension after microfiltration to the concentration of the aspergillus oryzae suspension in the step (1), refluxing to a saccharification tank, starting the next batch of saccharification after a sucrose solution is supplemented, wherein the saccharification conditions are the same as those in the step (1), the saccharification time of the batch and the subsequent batch is determined according to the detection result, and when the FOS content in the saccharification liquid reaches more than 56%, the saccharification of the corresponding batch can be finished; introducing the clear liquid obtained by microfiltration into an ultrafiltration membrane to obtain an ultrafiltration fructo-oligosaccharide liquid, and recovering macromolecular substances such as protein, wherein the ultrafiltration membrane has a molecular weight cutoff range of 24000-27000D, an operating pressure of 0.35-0.5 Mpa and an ultrafiltration temperature of 34-37 ℃; decolorizing the fructo-oligosaccharide solution obtained by ultrafiltration with active carbon, desalting by ion exchange, and vacuum concentrating to obtain refined fructo-oligosaccharide syrup; the FOS content (in total solid content) in the syrup is 57.3% by high performance liquid chromatography detection.
(5) The recovery rate of enzyme activity of the cultured thalli is 58 percent after the thalli is recycled for 8 times.
Example 3
(1) Mixing 3 tons of cultured aspergillus oryzae suspension with a sucrose solution with a dry base of 15 tons of sugar concentration and 53 percent for saccharification, controlling the saccharification temperature to be 40-42 ℃ and the pH value to be 6.0-6.5, and saccharifying for 36 hours to obtain the fermentation liquor containing fructo-oligosaccharide.
(2) Filtering the fermentation liquor by a ceramic membrane to obtain a fructo-oligosaccharide clear liquid, and recovering Aspergillus oryzae thallus, wherein the aperture of the ceramic membrane is 0.7-1 μm, the microfiltration temperature is 38-42 ℃, the operation pressure is 0.1-0.15 Mpa, and the microfiltration speed is 5.2m3/h。
(3) Adding water into the first recovered thallus, adjusting the thallus concentration to be 7 g/L, adding glutaraldehyde to make the thallus concentration to be 7 g/L, reacting for 8 hours at 12-14 ℃ and 45rpm, fully washing and recovering the thallus through a ceramic membrane, wherein the enzyme activity recovery rate reaches 91%.
(4) Adjusting the concentration of the recovered strain suspension after microfiltration to the concentration of the aspergillus oryzae suspension in the step (1), refluxing to a saccharification tank, starting the next batch of saccharification after a sucrose solution is supplemented, wherein the saccharification conditions are the same as those in the step (1), the saccharification time of the batch and the subsequent batch is determined according to the detection result, and when the FOS content in the saccharification liquid reaches more than 56%, the saccharification of the corresponding batch can be finished; introducing the clear liquid obtained by microfiltration into an ultrafiltration membrane to obtain an ultrafiltration fructo-oligosaccharide solution, and recovering macromolecular substances such as protein, wherein the ultrafiltration membrane has a molecular weight cutoff range of 21000-24000D, an operating pressure of 0.5-0.6 Mpa and an ultrafiltration temperature of 37-40 ℃; decolorizing the fructo-oligosaccharide solution obtained by ultrafiltration with active carbon, desalting by ion exchange, and vacuum concentrating to obtain refined fructo-oligosaccharide syrup; the FOS content (in total solid content) in the syrup is 58.1% by high performance liquid detection.
(5) The recovery rate of enzyme activity of the cultured thalli is 60 percent after the thalli is recycled for 8 times.
Example 4
(1) Mixing 2 tons of cultured aspergillus oryzae suspension with a sucrose solution with a dry basis of 10 tons of sugar concentration and 54 percent for saccharification, controlling the saccharification temperature to be 40-42 ℃, controlling the saccharification pH to be 6.0-6.5, and saccharifying for 35 hours to obtain the fermentation liquor containing fructo-oligosaccharide.
(2) Filtering the fermentation liquor by a ceramic membrane to obtain a fructo-oligosaccharide clear liquid, and recovering Aspergillus oryzae thallus, wherein the aperture of the ceramic membrane is 0.7-1 μm, the microfiltration temperature is 36-40 ℃, the operation pressure is 0.15-0.2 Mpa, and the microfiltration speed is 5.5m3/h。
(3) Adding water into the first recovered thallus, adjusting the thallus concentration to 10 g/L, adding glutaraldehyde to make the final concentration to 10 g/L, reacting for 7h at 13-15 ℃ and 40rpm, fully washing and recovering the thallus through a ceramic membrane, wherein the recovery rate of enzyme activity reaches 91%.
(4) Adjusting the concentration of the recovered strain suspension after microfiltration to the concentration of the aspergillus oryzae suspension in the step (1), refluxing to a saccharification tank, starting the next batch of saccharification after a sucrose solution is supplemented, wherein the saccharification conditions are the same as those in the step (1), the saccharification time of the batch and the subsequent batch is determined according to the detection result, and when the FOS content in the saccharification liquid reaches more than 56%, the saccharification of the corresponding batch can be finished; introducing the clear liquid obtained by microfiltration into an ultrafiltration membrane to obtain an ultrafiltration fructo-oligosaccharide liquid, and recovering macromolecular substances such as protein, wherein the ultrafiltration membrane has a molecular weight cutoff range of 18000-21000D, an operating pressure of 0.6-0.7 Mpa and an ultrafiltration temperature of 34-37 ℃; decolorizing the fructo-oligosaccharide solution obtained by ultrafiltration with active carbon, desalting by ion exchange, and vacuum concentrating to obtain refined fructo-oligosaccharide syrup; the FOS content (in total solid content) in the syrup is 58.3% by high performance liquid detection.
(5) The recovery rate of enzyme activity of the cultured thalli is 62 percent after the thalli is recycled for 8 times.
Example 5
(1) Mixing 2.5 tons of cultured aspergillus oryzae suspension with a sucrose solution with a dry base of 12.5 tons of sugar concentration and 55 percent for saccharification, controlling the saccharification temperature to be 40-42 ℃ and the pH value to be 6.0-6.5, and saccharifying for 34 hours to obtain the fermentation liquor containing fructo-oligosaccharide.
(2) Passing the fermentation broth through a ceramic membraneFiltering to obtain fructo-oligosaccharide clear solution, and recovering Aspergillus oryzae thallus, wherein the aperture of the ceramic membrane is 0.5-0.8 μm, the microfiltration temperature is 35-38 deg.C, the operation pressure is 0.15-0.2 Mpa, and the microfiltration speed is 5.2m3/h。
(3) Adding water into the first recovered thallus, adjusting the thallus concentration to be 13 g/L, adding glutaraldehyde to make the final concentration reach 13 g/L, reacting for 9 hours at the temperature of 10-12 ℃ and at the speed of 55rpm, fully washing and recovering the thallus through a ceramic membrane, and the enzyme activity recovery rate reaches 92%.
(4) Adjusting the concentration of the recovered strain suspension after microfiltration to the concentration of the aspergillus oryzae suspension in the step (1), refluxing to a saccharification tank, starting the next batch of saccharification after a sucrose solution is supplemented, wherein the saccharification conditions are the same as those in the step (1), the saccharification time of the batch and the subsequent batch is determined according to the detection result, and when the FOS content in the saccharification liquid reaches more than 56%, the saccharification of the corresponding batch can be finished; introducing the clear liquid obtained by microfiltration into an ultrafiltration membrane to obtain an ultrafiltration fructo-oligosaccharide liquid, and recovering macromolecular substances such as protein, wherein the ultrafiltration membrane has a molecular weight cutoff range of 15000-18000D, an operating pressure of 0.7-0.8 Mpa and an ultrafiltration temperature of 30-34 ℃; decolorizing the fructo-oligosaccharide solution obtained by ultrafiltration with active carbon, desalting by ion exchange, and vacuum concentrating to obtain refined fructo-oligosaccharide syrup; the FOS content (in total solid content) in the syrup is 57.9% by high performance liquid detection.
(5) The recovery rate of enzyme activity is 61% after the cultured thalli is recycled for 8 times.
Comparative example 1
Compared with the example 5, the water is added into the thallus recovered for the first time, the thallus concentration is adjusted to be 13 g/L, the glutaraldehyde is added to make the final concentration to be 5 g/L, the other operations are the same as the example 5, the recovery rate of the enzyme activity is 84%, and the recovery rate of the enzyme activity after the finally cultured thallus is recycled for 6 times is 57%.
Comparative example 2
Compared with the example 5, the water is added into the thallus recovered for the first time, the thallus concentration is adjusted to be 13 g/L, the glutaraldehyde is added to make the final concentration be 50 g/L, the rest operations are the same as the example 5, because the addition amount of the glutaraldehyde is larger, the intracellular fructosyltransferase is greatly limited, the cross-linking between thalli is too dense, the glutaraldehyde is treated and then washed by a ceramic membrane and recovered, the recovery rate of the enzyme activity is only 64 percent, and therefore, the circulating saccharification is not further carried out.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (7)
1. A method for producing fructo-oligosaccharide by circularly utilizing aspergillus oryzae thalli is characterized by comprising the following steps:
(1) mixing the cultured aspergillus oryzae suspension with a sucrose solution for saccharification to obtain fermentation liquor containing fructo-oligosaccharide;
(2) filtering the fermentation liquor by a microfiltration membrane, recovering aspergillus oryzae thallus, and filtering to obtain a fructo-oligosaccharide clear solution;
(3) treating the aspergillus oryzae thalli recovered for the first time by glutaraldehyde, and then washing and recovering the aspergillus oryzae thalli through a microfiltration membrane for the next batch of saccharification; after the subsequent saccharification is finished, the recovered thalli are not treated by glutaraldehyde any more, and the concentration of the recovered bacterial suspension is adjusted to the concentration of the bacterial suspension in the step (1), namely, the recovered bacterial suspension is used for the next batch of saccharification;
(4) adjusting the concentration of the aspergillus oryzae suspension recovered in the step (3) to the concentration of the bacterial suspension in the step (1), refluxing to a saccharification tank for carrying out saccharification of the next batch, and carrying out post-treatment on a fructo-oligosaccharide clear liquid to obtain fructo-oligosaccharide syrup;
the microfiltration membranes in the steps (2) and (3) are inorganic ceramic membranes, and the interception aperture is 0.1-1 mu m;
when the first-time recovered aspergillus oryzae thalli is treated by glutaraldehyde in the step (3), adjusting the concentration of the thalli to be 5-15 g/L, and adjusting the final concentration of added glutaraldehyde to be 5-15 g/L;
the mass ratio of the thalli to the glutaraldehyde in the step (3) is 1: 0.8-1.2.
2. The method according to claim 1, wherein the microfiltration temperature is 30 to 42 ℃ and the operation pressure is 0.05 to 0.2 MPa.
3. The method according to claim 1, wherein the step (3) of treating the Aspergillus oryzae mycelia recovered for the first time with glutaraldehyde is carried out at a reaction temperature of 10 to 15 ℃, a stirring rotation speed of 30 to 60rpm, and a reaction time of 6 to 10 hours.
4. The method according to claim 1, wherein the recovery rate of the enzyme activity after washing and recovering through the microfiltration membrane in the step (3) is 88 to 92%.
5. The method according to claim 1, wherein the post-treatment step in step (4) comprises ultrafiltration, activated carbon decolorization, ion exchange desalination, and vacuum concentration.
6. The method according to claim 1, wherein the Aspergillus oryzae cells are recycled for at least 8 times.
7. The method according to claim 1, wherein in step (3), the enzyme activity is not recovered until the recovery rate is less than or equal to 60%.
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