CN112921018A - Separation and purification method of momordica grosvenori proteinase - Google Patents
Separation and purification method of momordica grosvenori proteinase Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
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Abstract
The invention discloses a method for separating and purifying momordica grosvenori protease, which comprises the following steps: s1, obtaining fresh juice of fresh fructus momordicae, and separating the fresh juice by using a D101 macroporous resin column to obtain crude enzyme juice; s2, purifying by adopting a freeze-drying method or an ammonium sulfate fractional precipitation method to obtain the momordica grosvenori protease or the momordica grosvenori protease liquid, wherein the momordica grosvenori protease and the momordica grosvenori glycoside V in the momordica grosvenori juice can be well separated, and the activity of the momordica grosvenori protease can be well reserved.
Description
Technical Field
The invention relates to the technical field of extraction of momordica grosvenori, in particular to a separation and purification method of momordica grosvenori protease.
Background
Mature fruit of Momordica plant belonging to Cucurbitaceae is collected in autumn when the fruit turns from light green to dark green. It is usually in mountain slopes, forest lands, river-side wetlands and shrubs, and is distributed in Jiangxi, Hunan, Guangdong, Guangxi, Guizhou, etc. It has high nutritive value, and is rich in vitamin C, glucoside, fructose, glucose, protein, lipid, etc.
The momordica grosvenori is a famous and precious traditional Chinese medicine for both medicine and food, which is published by Ministry of health, contains momordica grosvenori glycoside which is 300 times sweeter than sucrose, does not generate heat, is a famous and precious raw material in beverage and candy industries, and is an optimal substitute for sucrose. Fructus Siraitiae Grosvenorii has high nutritive value, and has effects of clearing heat, moistening lung, relieving cough, eliminating phlegm, benefiting liver, invigorating spleen, refreshing, and promoting fluid production; modern medicine proves that the momordica grosvenori has obvious curative effect on bronchitis, hypertension, sphagitis, bronchial asthma, whooping cough, stomach heat, constipation, acute tonsillitis and other diseases, and can also play a role in preventing and treating coronary heart disease, angiosclerosis and obesity.
The momordica grosvenori is widely used in medicines and mainly used for producing momordica grosvenori sweet glycosides, wherein the momordica grosvenori sweet glycosides are low-calorie sweeteners and are main effective components in momordica grosvenori. In actual production, the mogroside V content is also used for measuring the quality of the mogroside. In addition, fresh Lo Han Guo contains protease with high hydrolysis activity to casein. Under the same condition, the activity of the momordica grosvenori proteinase is more than 10 times of that of the papain, so that the momordica grosvenori proteinase has higher research and development values.
The methods reported so far for separating momordica grosvenori protease include ammonium sulfate salting-out method and ultrafiltration membrane separation method. In practice, these methods are affected by the sticky nature of mogrosides in Lo Han Guo, making it difficult to separate the mogroside protease and the separated protease is difficult to completely remove mogroside, resulting in sweet taste in the protease, which is not favorable for application of the enzyme in food.
Therefore, the method has important significance for developing the momordica grosvenori protease by removing the momordica grosvenori sweet glycosides in the momordica grosvenori juice and separating the momordica grosvenori protease and the momordica grosvenori saponin.
Disclosure of Invention
The invention discloses a method for separating and purifying momordica grosvenori protease, which can well separate momordica grosvenori protease from momordica grosvenori glycoside V in momordica grosvenori juice and well retain the activity of the momordica grosvenori protease.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for separating and purifying momordica grosvenori protease comprises the following steps:
s1, obtaining fresh juice of fresh momordica grosvenori, separating the fresh juice through a D101 macroporous resin column, collecting separated enzyme juice which flows out and has no sweet taste, flushing the D101 macroporous resin column with water after the enzyme juice completely flows out to obtain flushing fluid, and combining the flushing fluid and the separated enzyme juice to obtain crude enzyme juice;
s2, purifying by adopting a freeze drying method or an ammonium sulfate fractional precipitation method to obtain the momordica grosvenori proteinase or the momordica grosvenori proteinase solution.
Further, the fresh fructus momordicae juice is obtained by a method selected from one of the following methods:
(1) squeezing fresh fructus Siraitiae Grosvenorii to obtain juice, or centrifuging with centrifugal dehydrator to obtain first-stage fruit juice, soaking the fruit residue with water for several times to obtain soaking solution, and mixing the soaking solution and the first-stage fruit juice to obtain fresh fruit juice;
(2) mashing or crushing fresh fructus Siraitiae Grosvenorii, soaking in water for several times to obtain soaking solution, filtering or centrifuging the soaking solution, and removing fruit residue to obtain fresh fruit juice.
Further, in step S2, the freeze-drying method specifically includes: the temperature is less than or equal to minus 50 ℃, and the vacuum degree is less than or equal to 10 Pa.
Further, in step S2, the ammonium sulfate fractionation method specifically includes: adding solid ammonium sulfate into the crude enzyme juice to ensure that the saturation degree of the crude enzyme juice is 50 to100 percent, standing, freezing and centrifuging, dissolving the precipitate by a Tris-HCl buffer solution with the concentration of 0.03-0.06 mol/L, pH being 6.5-7.5 and containing 0.5-1.5 mmol/L EDTA, and dialyzing by a Tris-HCl buffer solution after dissolving the precipitate until 1 percent of BaCl is used2 And (4) detecting no precipitate in the solution, freezing and centrifuging, and taking supernatant fluid to obtain the momordica grosvenori protease liquid.
Furthermore, the dialysis is specifically: the Tris-HCl buffer solution after dissolving the precipitate is filled into a dialysis bag to be dialyzed in pure water at 4 ℃, and the dialyzate is replaced for a plurality of times until no precipitate is detected by using 1% BaCl2 solution.
Further, the Tris-HCl buffer solution is 0.05mol/L, has a pH of 7.0, and contains 1mmol/L EDTA.
Further, in step S2, solid ammonium sulfate is added to the crude enzyme juice to adjust the saturation to 65%.
Further, in the step S1, if the separated enzyme juice separated by the D101 macroporous resin column has sweet taste, the separated enzyme juice is returned to the D101 macroporous resin column for re-separation, or is re-separated by a new D101 macroporous resin column.
The method for separating and purifying the momordica grosvenori protease can well separate the momordica grosvenori protease and the momordica grosvenori glycoside V in the momordica grosvenori juice after the D101 macroporous resin treatment, has a certain purification effect on the separated momordica grosvenori protease, and still has a small part of the momordica grosvenori glycoside V remained in the momordica grosvenori protease solution. Further, the invention further purifies the momordica grosvenori protease by using a freeze-drying method and an ammonium sulfate fractional precipitation method, wherein: by using a freeze-drying method, the recovery rate of protein is 87.613%, the recovery rate of enzyme activity is 82.587%, and the purification multiple is 0.944; using ammonium sulfate fractional precipitation; the maximum recovery rate of the momordica grosvenori protease can reach 87.681%, the maximum recovery rate of the enzyme activity can reach 93.951%, and the purification multiple can reach 1.071.
By separating and purifying the momordica grosvenori protease, the advantages of high hydrolysis activity, special structure and good stability of the momordica grosvenori protease can be better exerted, the development and application fields of the momordica grosvenori are widened, and the development and utilization values of the momordica grosvenori are improved.
Drawings
FIG. 1 is a standard graph of protein content.
FIG. 2 is a chromatogram of mogroside V standard.
FIG. 3 Lo Han Guo sample chromatogram.
FIG. 4 is a mogroside V standard curve.
FIG. 5 is a graph showing recovery of protein and recovery of enzyme activity using ammonium sulfate of different saturation levels.
Detailed Description
The present invention is further illustrated by the following specific examples, but the scope of the present invention is not limited to the following examples.
Example 1
S1, cleaning, weighing and juicing fresh momordica grosvenori, separating pomace and first-level fruit juice, soaking the pomace for a few times (5 times, 10g momordica grosvenori: 50mL of water) by using ultrapure water, discarding the pomace, taking soaking liquid, combining the first-level fruit juice and the soaking liquid to obtain fresh momordica grosvenori juice, separating by using a D101 macroporous resin column, collecting outflowing separated enzyme juice without sweet taste without applying pressure, and pouring the outflowing separated enzyme juice into the macroporous resin column to separate and purify again if the outflowing separated enzyme juice has sweet taste. And after the separated enzyme juice completely flows out, washing the D101 macroporous resin by using a small amount of ultrapure water, fully washing the momordica grosvenori protease adsorbed on the D101 macroporous resin under the condition of ensuring that the outflow washing liquid has no sweet taste, detecting the protein content of the collected washing liquid by adopting a Coomassie brilliant blue G250 method, and stopping washing the column when the result is zero. And combining the washing liquid and the separated enzyme juice to obtain crude enzyme juice.
S2, adopting a freeze-drying method, wherein the temperature is-50 ℃, and the vacuum degree is 10Pa, so as to obtain the momordica grosvenori proteinase.
Example 2
S1, cleaning, weighing and juicing fresh momordica grosvenori, separating pomace and first-level fruit juice, soaking the pomace (5 times, 10g momordica grosvenori: 50mL of water) for a few times by using ultrapure water, discarding the pomace, taking soaking liquid, combining the first-level fruit juice and the soaking liquid, separating the fresh momordica grosvenori juice by using a D101 macroporous resin column, collecting outflowing separated enzyme juice without sweet taste without applying pressure, and pouring the outflowing separated enzyme juice into the macroporous resin column to separate and purify again if the outflowing separated enzyme juice has sweet taste. And after the separated enzyme juice completely flows out, washing the D101 macroporous resin by using a small amount of ultrapure water, fully washing the momordica grosvenori protease adsorbed on the D101 macroporous resin under the condition of ensuring that the outflow washing liquid has no sweet taste, detecting the protein content of the collected washing liquid by adopting a Coomassie brilliant blue G250 method, and stopping washing the column when the result is a negative value. And combining the washing liquid and the separated enzyme juice to obtain crude enzyme juice.
S2, purifying the momordica grosvenori protease by adopting an ammonium sulfate fractional precipitation method to obtain momordica grosvenori protease liquid; the method specifically comprises the following steps: diluting 200ml of crude enzyme juice to 700ml, dividing into 7 equal parts, adding solid ammonium sulfate to reach saturation degree of 0%, 20%, 35%, 50%, 65%, 80%, 100%, standing, freeze-centrifuging (4 deg.C, 8000 r/min) for 15 min, dissolving the precipitate with 0.05mol/L Tris-HCl (pH = 7) buffer (containing 1mmol/L EDTA), dialyzing the buffer overnight to 1% BaCl2The solution can not detect precipitate, then refrigerated and centrifuged (4 ℃, 8000 r/min) for 15 min, supernatant is taken, and protein concentration and enzyme activity of the juice with ammonium sulfate saturation of 0 are respectively determined and calculated as 100 percent to obtain the recovery rate of protein and enzyme activity of each supernatant.
Experimental validation examples
First, test effect verification of separation and purification of momordica grosvenori protease by macroporous resin
1. Protein content and activity determination of momordica grosvenori protease
1.1 Coomassie brilliant blue G250 method for determining protein content before and after separation and purification of fruit juice
Detecting protein content by Coomassie brilliant blue G250 method (refer to teaching practice and methodology discussion of measuring protein content by Coomassie brilliant blue G250 method. education and teaching forum, 2020.23), simultaneously measuring protein content and enzyme activity of collected enzyme juice and fresh fruit juice of fresh fructus Siraitiae Grosvenorii, and calculating recovery rate.
FIG. 1 is a standard curve of protein content, the linear regression equation is: y =0.0148x +0.6953 (R)2= 0.9933), in the range of the protein concentration of the reaction system 0.05 mg/ml-0.5 mg/ml, the graph 1 shows that the absorbance has good linear relation with the protein content。
The protein content is detected by adopting the standard curve, and the specific result is shown in table 1:
TABLE 1 protein content determination before and after separation and purification of D101 macroporous resin
Table 1 shows that the protein recovery rate is 85.676% after separation and purification by the D101 macroporous resin, only a small part of protein is adsorbed by the macroporous resin, and the protein loss is less.
1.2 Activity assay of Momordica grosvenori proteinase separated and purified by macroporous resin
The enzyme activity of fructus Siraitiae Grosvenorii protease can be determined by comparing the activity detection methods of papain in industrial production (rejuvenation, Propharia gigantea, Wu shorea), and casein is used as substrate. Definition of units of enzyme activity: under the specified conditions, the casein is enzymatically hydrolyzed within 1min to release 1 activity unit corresponding to the absorbance of 1. mu.g/mL tyrosine at 275nm wavelength.
Table 2 shows the enzyme activity measurement data before and after separation and purification of D101 macroporous resin, where the experimental group is a, the blank group is b, and the blank group is a group of reagents and instruments that are the same in the same time period and in the same environment as the experimental group, and only the order of adding trichloroacetic acid is changed, so that the protease is denatured and inactivated, and cannot react with casein to produce a product that can have an absorbance at a wavelength of 275nm, thereby excluding any interfering factors that can have an absorbance at 275nm except the product. The test result shows that: the recovery rate of the enzyme activity reaches 92.270%, the purification multiple is 1.077, the data shows that the grosvenor momordica protease separated and purified by the D101 macroporous resin still keeps higher enzyme activity and is purified, and the D101 macroporous resin has good separation and purification effects.
TABLE 2 enzyme Activity measurement results before and after separation and purification with D101 macroporous resin
1.3 Experimental effect research of separating mogroside V by macroporous resin.
1.3.1 test methods
1.3.1.1 purification treatment of mogroside V: and (3) washing the D101 macroporous resin column after the crude enzyme juice is separated by using 95% ethanol, fully washing the mogroside adsorbed by the macroporous resin, and measuring the mogroside V content in the standard product and the eluent by using an HPLC method.
1.3.1.2 chromatographic conditions: analytical HPLC column: an AgilentZORBAXSB-C18 column; specification: 4.6mm × 150mm, 5 μm; mobile phase: methanol-water (volume ratio 15: 85); flow rate: 1 mL/min; sample introduction volume: 10 mu L of the solution; detection wavelength: 203 nm; column temperature: room temperature; the quantitative method comprises the following steps: and (4) an external standard method.
1.3.1.3 Linear relationship investigation: accurately weighing a mogroside V standard substance SGL20110.03mg prepared by a laboratory, putting the standard substance into a 10mL volumetric flask, dissolving the standard substance with a mobile phase (15% methanol aqueous solution), fixing the volume to a scale, preparing a standard sample with the concentration of 1.000mg/mL, respectively sucking a certain amount of standard sample, diluting the standard sample with the mobile phase, preparing a control solution with the mass concentration of 0.025, 0.050, 0.100, 0.250, 0.500 and 1.000mg/mL, respectively carrying out HPLC (high performance liquid chromatography) measurement, investigating a linear relation and a linear range, drawing a standard curve by taking a peak area Y (mv/min) vertical coordinate and the mass concentration X (mg/mL) of the control solution as a horizontal coordinate, and obtaining a regression equation.
1.3.1.4 reproducibility test: a sample to be tested (P2-2) was taken for HPLC and repeated 6 times.
1.3.1.5 precision test: a control sample at a concentration of 0.500mg/mL was subjected to HPLC assay for 6 replicates.
1.3.1.6 testing content of mogroside V in sample to be tested, analyzing sample introduction of reserved fresh fructus Siraitiae Grosvenorii juice and sample to be tested obtained in 1.2.1.1, introducing sample 6 times for each sample, averaging, and quantifying by external standard method.
1.3.2 HPLC chromatograms of mogroside V standard sample and sample to be detected
As can be seen from FIGS. 2 and 3, peak B is mogroside V, and the peak-off times are 6.469min and 6.391 min.
1.3.3 drawing of Standard Curve of mogroside V Standard
FIG. 4 is a plot of mogroside V standard plotted with a linear regression equation: y =4130.1X +1.0639 (R)2=0.9998), the response value of the peak area and the quality of mogroside V have good linear relation within the range of 0.00025-0.01000 mg.
1.3.4 reproducibility test
The results of the reproducibility test of the samples are shown in Table 3. RSD was 0.299% (n =6), retention time and reproducibility of peak area response values were good.
TABLE 3 precision test of mogroside V
1.3.5 precision test
The results of the precision measurement of mogroside V are shown in Table 4. RSD was 0.962% (n =6), indicating good precision. ,
TABLE 4 determination of mogroside V in fresh fruits repeatability
1.3.6 determination of mogroside V content in Momordica grosvenori samples
Table 5 shows data of HPLC detection of fresh momordica grosvenori juice and momordica grosvenori glycoside samples treated by D101 macroporous resin in this experiment, most momordica grosvenori glycoside V is adsorbed by D101 macroporous resin after being treated by D101 macroporous resin, and can be washed down by 95% ethanol, and dried to dryness to obtain 4.1957g, and the recovery rate of momordica grosvenori glycoside V measured by HPLC method reaches 84.625%.
TABLE 5 measurement results of Momordica grosvenori Swingle samples
2. Research on freeze-dried momordica grosvenori protease
Determination of specific enzyme Activity before and after lyophilization
Freeze drying the crude enzyme juice after a period of refrigeration storage. Table 6 shows the results of protein content measurement before and after drying of Momordica grosvenori proteinase, the total enzyme activity before drying is 0.402X 106U, drying to obtain crude enzyme with total enzyme activity of 0.372 × 106U, the recovery rate of the enzyme activity reaches 92.537%, the purification multiple is 0.944, and the data show that most of the enzyme activity is recovered and the loss is less after the enzyme activity is dried by a freeze dryer, so the effect of drying the momordica grosvenori protease by using the freeze dryer is good.
TABLE 6 results of enzyme activity measurement before and after drying of Momordica grosvenori proteinase
3 research on ammonium sulfate fractional precipitation of momordica grosvenori protease
3.1 assay of protein content of Momordica grosvenori protease precipitated by ammonium sulfate fractionation
The luo han guo protease solutions were precipitated with ammonium sulfate of various saturation levels and tested for protein content using the coomassie brilliant blue G250 method, the results of which are shown in table 7. The enzyme juice with the saturation of 0% is used as a contrast, when the saturation of ammonium sulfate is increased from 20% to 65%, the recovery rate of protein is also gradually increased, when the saturation of ammonium sulfate is increased from 65% to 100%, the recovery rate of protein is gradually reduced, the data show that when the saturation of ammonium sulfate is 50-100%, the recovery rate of protein of the momordica grosvenori protease solution is better, when the saturation of ammonium sulfate is 65%, the recovery rate of protein of the momordica grosvenori protease solution is the largest and reaches 87.681%, which indicates that most of momordica grosvenori protease is precipitated and recovered, and the loss of protein is the least.
TABLE 7 ammonium sulfate fractionation precipitation of the results of the assay of the protease protein content of Momordica grosvenori
3.2 determination of specific Activity of ammonium sulfate fractionated precipitation Siraitia grosvenori proteinase
The specific activity of the momordica grosvenori protease is determined by dividing the momordica grosvenori protease into an experimental group and a blank group, wherein the blank group is prepared by adding trichloroacetic acid into the same reaction reagent and instrument in the same time period and in the same environment as the experimental group, only changing the adding sequence of the trichloroacetic acid to denature and inactivate the protease, and the protease cannot react with casein to produce a product with the wavelength of 275nm and the absorbance, so that interference factors except the product with the absorbance at 275nm are eliminated.
TABLE 8 results of enzyme activity assay of ammonium sulfate fractionated precipitation of siraitia grosvenorii protease
Table 8 shows the results of the enzyme activity determination of the luo han guo protease treated with ammonium sulfate of different saturation levels, with the increase of the saturation level, the enzyme activity gradually increases within the range of 20% to 65%, the purification multiple also increases, within the range of 65% to 100%, the enzyme activity gradually decreases, and the purification multiple also decreases, the data shows that when the saturation level of ammonium sulfate is 50% to 100%, the enzyme activity loss of the luo han guo protease solution is less, wherein when the saturation level of ammonium sulfate is 65%, the enzyme activity reaches 93.951%, the recovery rate of purification multiple is 1.071, the enzyme activity loss is least, and the purification multiple is maximum.
The protein recovery rate and the enzyme activity recovery rate corresponding to different ammonium sulfate saturation degrees are shown in fig. 5, when the ammonium sulfate saturation degree is 65%, the protein recovery rate is 87.681%, the enzyme activity recovery rate is 93.951%, and the purification multiple is 1.071, which shows that the ammonium sulfate with the concentration can precipitate and recover most of the momordica grosvenori protease, can keep the higher enzyme activity of the momordica grosvenori protease, and has a certain purification effect on the momordica grosvenori protease.
Claims (8)
1. A method for separating and purifying momordica grosvenori protease is characterized by comprising the following steps:
s1, obtaining fresh juice of fresh momordica grosvenori, separating the fresh juice through a D101 macroporous resin column, collecting separated enzyme juice which flows out and has no sweet taste, flushing the D101 macroporous resin column with water after the enzyme juice completely flows out to obtain flushing fluid, and combining the flushing fluid and the separated enzyme juice to obtain crude enzyme juice;
s2, purifying by adopting a freeze drying method or an ammonium sulfate fractional precipitation method to obtain the momordica grosvenori proteinase or the momordica grosvenori proteinase solution.
2. The method for separating and purifying momordica grosvenori protease according to claim 1, wherein:
the fresh fructus Siraitiae Grosvenorii juice is obtained by one of the following methods:
(1) squeezing fresh fructus Siraitiae Grosvenorii to obtain juice, or centrifuging with centrifugal dehydrator to obtain first-stage fruit juice, soaking the fruit residue with water for several times to obtain soaking solution, and mixing the soaking solution and the first-stage fruit juice to obtain fresh fruit juice;
(2) mashing or crushing fresh fructus Siraitiae Grosvenorii, soaking in water for several times to obtain soaking solution, filtering or centrifuging the soaking solution, and removing fruit residue to obtain fresh fruit juice.
3. The method for separating and purifying momordica grosvenori protease according to claim 1, wherein:
in the step S2, the freeze-drying method specifically includes: the temperature is less than or equal to minus 50 ℃, and the vacuum degree is less than or equal to 10 Pa.
4. The method for separating and purifying momordica grosvenori protease according to claim 1, wherein:
in step S2, the ammonium sulfate fractional precipitation method specifically includes: adding solid ammonium sulfate into the crude enzyme juice to ensure that the saturation degree is 50-100%, standing, freezing and centrifuging, dissolving the precipitate by using 0.03-0.06 mol/L, pH of 6.5-7.5 and Tris-HCl buffer solution containing 0.5-1.5 mmol/L EDTA, dialyzing by using the Tris-HCl buffer solution after dissolving the precipitate until 1% BaCl is used2 And (4) detecting no precipitate in the solution, freezing and centrifuging, and taking supernatant fluid to obtain the momordica grosvenori protease liquid.
5. The method for separating and purifying momordica grosvenori protease according to claim 4, wherein:
the dialysis specifically comprises: the Tris-HCl buffer solution after dissolving the precipitate is filled into a dialysis bag to be dialyzed in pure water at 4 ℃, and the dialyzate is replaced for a plurality of times until no precipitate is detected by using 1% BaCl2 solution.
6. The method for separating and purifying momordica grosvenori protease according to claim 4, wherein:
the Tris-HCl buffer solution is 0.05mol/L, the pH value is 7.0, and the Tris-HCl buffer solution contains 1mmol/L EDTA.
7. The method for separating and purifying momordica grosvenori protease according to claim 4, wherein:
in the step S2, solid ammonium sulfate is added into the crude enzyme juice to adjust the saturation degree to 65%.
8. The method for separating and purifying momordica grosvenori protease according to claim 1, wherein:
in the step S1, if the separated enzyme juice separated by the D101 macroporous resin column has sweet taste, the separated enzyme juice is returned to the original D101 macroporous resin column for re-separation, or is re-separated by a new D101 macroporous resin column.
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