Crocin extraction method based on enzymolysis and microwave ultrasound

Abstract

The invention discloses a crocin extraction method based on enzymolysis and microwave ultrasound, and relates to the technical field of crocin extraction, and the crocin extraction method comprises the following steps: taking fresh saffron crocus petals, removing impurities in the fresh saffron crocus petals, and putting the dried saffron crocus petals into an ultrafine grinder for grinding. According to the method, the extraction effect of crocin is improved by virtue of enzymolysis and microwave synergism, cellulase and xylanase are added, cell wall skeletons are destroyed under mild conditions, release of crocin is promoted, even if the enzyme dosage is reduced, the extraction rate is still higher than that of a traditional method, microwave heating is rapid and uniform, local overheating is avoided by virtue of a pulse mode, and the extraction efficiency is greatly improved. And through the synergistic effect of the two components, the target component is efficiently released, the impurity extraction is reduced, the product purity is improved, the heating time is greatly shortened, and the overall extraction efficiency and the product quality are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crocin extraction, in particular to a crocin extraction method based on enzymolysis and microwave ultrasonic. BACKGROUND

[0002] Crocus, a Chinese herbal medicine, is a plant of the Iridaceae family, also known as safflower and safflower. It contains chemical components such as crocin and crocetin. The core value of crocus lies in the crocin in the crocus style. Crocus grows in the coastal areas of the Middle East to the foothills of the Himalayas. Due to its extremely low yield, time-consuming and laborious harvesting, it is listed as a rare and precious Chinese herbal medicine, making it expensive. It is known as "plant gold". Crocus contains rich active ingredients such as crocin, crocin and crocetin, which also make it have great potential in the cosmetics and pharmaceutical fields.

[0003] Crocetin is usually extracted by ethanol or water extraction. Patent "Safflower extract, its preparation method and its application" with application number "202110619330.9" is disclosed. The patent mentions that in the safflower extraction process, there is high solvent residue (≥500ppm) or low extraction rate, low active substance extraction rate and long extraction time, which is not conducive to industrial production. Patent "Crocus fermentation and its preparation method and application" with application number "202111634342.5" is disclosed. The patent uses Saccharomyces cerevisiae and Monascus fermentation to ferment crocus stigma, but the fermentation condition is difficult to control, the product composition is uncertain, and other risks exist. At the same time, the yield of crocus stigma is extremely low, which cannot meet the actual market demand, and further increases the difficulty of technical development and commercialization. Therefore, we propose a crocin extraction method based on enzymolysis and microwave ultrasonic. Enzymolysis, microwave, macroporous resin are used for enrichment, separation and purification to improve the content of crocin in crocus. SUMMARY

[0004] The purpose of the present application is to provide a crocin extraction method based on enzymolysis and microwave ultrasonic to solve the problems raised in the background art.

[0005] To achieve the above purpose, the present application provides the following technical scheme: a crocin extraction method based on enzymolysis and microwave ultrasonic, comprising the following steps:

[0006] S1: Take fresh crocus petals, remove impurities, dry at 25℃ for 6h to remove surface moisture, then put in constant temperature and humidity conditions at 35℃ for 12h, and then put the dried crocus petals into a super micro grinder;

[0007] S2: Citric acid buffer solution was added to the crushed saffron powder at a ratio of 1:20, the mixture was transferred to the enzyme hydrolysis reactor and stirred for 30 min, nitrogen was filled into the reactor at a concentration of 0.5% to 5%, cellulase was added, pre-reaction was carried out at 35°C for 30 min, and stirring was carried out, xylanase was added, and enzyme hydrolysis was carried out at 40°C for 2 h with stirring;

[0008] S3: The enzyme-hydrolyzed liquid was transferred to a microwave reaction kettle:

[0009] A1: The first stage, power control 300W, temperature rise to 50°C, last for 5 min;

[0010] A2: The second stage, power control 500W, pulse mode, temperature 55°C, maintain 15 min;

[0011] A3: The third stage, turn off the microwave, start the built-in cooling coil, and cool to below 40°C within 10 min;

[0012] S4: The microwave-processed mixture was quickly transferred to an ice water bath, cooled to below 10°C, stirred for 5 min, filtered using a 800-1000 mesh sieve, then centrifugal filtration was carried out at 4°C, the supernatant was taken, the supernatant was eluted on D101 macroporous resin, impurities were eluted with pure water until colorless and odorless, 10 column volumes were eluted, then 4-8 column volumes of 50% ethanol aqueous solution were used for elution, and the eluate was collected;

[0013] S5: The eluate was vacuum concentrated at low temperature, ethanol was recovered, the concentrated extract was freeze-dried into powder, and high-purity crocin was obtained.

[0014] Preferably, the saffron petals are put into a supermicro grinder and crushed until the particle size is ≤10μm.

[0015] Preferably, the concentration of the citric acid buffer solution is 0.1M, the pH is 5.0, and the concentration of nitrogen is controlled at 0.5% to 5%.

[0016] Preferably, the concentration of the cellulase is 0.5% to 1.0% w / w, and the concentration of the xylanase is 0.3% to 0.6% w / w.

[0017] Preferably, the stirring rate of the pre-reaction is 150rpm / min, the stirring rate of the vibration enzyme hydrolysis is 100rpm / min, and the stirring time is 30 min.

[0018] Preferably, the frequency of the pulse in the second stage is 10s on / 5s off.

[0019] Preferably, the flow rate of the supernatant on the D101 macroporous resin is 1.0 BV / H, the elution flow rate of 10 column volumes is 2.0 BV / H, and the flow rate of the ethanol aqueous solution elution is 1.0 BV / H.

[0020] Preferably, the eluent is concentrated under reduced pressure and low temperature, the temperature T is ≤40℃, and the pressure ≥0.1kpa.

[0021] Preferably, the supernatant is loaded on the D101 macroporous resin by wet loading, and saturated adsorption is 45min.

[0022] Preferably, the enzyme solution is transferred to a microwave reaction kettle, and the liquid layer thickness is controlled to be ≤10cm.

[0023] Compared with the prior art, the beneficial effects of the present application are:

[0024] Through the method, the extraction effect of crocin is improved by enzyme hydrolysis and microwave cooperation, cellulase and xylanase are added, the cell wall skeleton is destroyed under mild conditions, and the release of crocin is promoted. Even if the enzyme dosage is reduced, the extraction rate is still higher than that of the traditional method. The microwave heating is rapid and uniform, the pulse mode avoids local overheating, accelerates the diffusion of crocin and reduces the degradation, and the synergistic effect of the two not only efficiently releases the target components, but also reduces the extraction of impurities, so that the product purity is improved. At the same time, the heating time is greatly shortened, and the overall extraction efficiency and product quality are improved. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The flow chart of the crocin extraction method of the present application. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0027] Embodiment: please refer to Figure 1 The present application provides a technical solution: a crocin extraction method based on enzyme hydrolysis and microwave ultrasonic, comprising the following steps:

[0028] S1: Take fresh crocus petals, remove impurities, dry at 25℃ for 6h to remove surface moisture, then put into constant temperature and humidity conditions at 35℃ for 12h, and then put the dried crocus petals into a super micro grinder for grinding;

[0029] S2: Citric acid buffer solution is added to the crushed saffron powder at a ratio of 1:20, the mixture is put into an enzymatic hydrolysis reactor and stirred for 30 min, nitrogen gas is filled into the reactor, the concentration is controlled between 0.5% and 5%, cellulase is added, pre-reaction is carried out at 35°C for 30 min, and stirring is carried out, xylanase is added, and enzymatic hydrolysis is carried out at 40°C for 2 h, and stirring is carried out;

[0030] S3: The enzyme-hydrolyzed liquid is transferred to a microwave reaction kettle:

[0031] A1: In the first stage, the power is controlled at 300 W, the temperature is raised to 50°C, and the process is continued for 5 min;

[0032] A2: In the second stage, the power is controlled at 500 W, the pulse mode is used, the temperature is 55°C, and the process is maintained for 15 min;

[0033] A3: In the third stage, the microwave is turned off, the built-in cooling coil is started, and the temperature is reduced to below 40°C within 10 min;

[0034] S4: The microwave-treated mixture is quickly transferred to an ice water bath, cooled to below 10°C, stirred for 5 min, filtered using a 800-1000 mesh sieve, then centrifuged and filtered at 4°C, the supernatant is taken, the supernatant is passed through a D101 macroporous resin, impurities are eluted with pure water until no color and odor are present, 10 column volumes are eluted, then 4-8 column volumes of 50% ethanol aqueous solution are used for elution, and the eluate is collected;

[0035] S5: The eluate is vacuum-concentrated at low temperature, ethanol is recovered, the concentrated extract is freeze-dried into powder, and high-purity crocin is obtained.

[0036] As a further limitation of the application, the saffron petals are put into a supermicro grinder and crushed until the particle size is ≤10 μm.

[0037] The concentration of the citric acid buffer solution is 0.1 M, the pH is 5.0, and the concentration of the nitrogen gas is controlled between 0.5% and 5%.

[0038] The concentration of the cellulase is 0.5%-1.0% w / w, and the concentration of the xylanase is 0.3%-0.6% w / w.

[0039] The stirring rate of the pre-reaction is 150 rpm / min, the stirring rate of the vibration enzymolysis is 100 rpm / min, and the stirring time is 30 min.

[0040] The frequency of the pulse in the second stage is 10 s on / 5 s off.

[0041] The flow rate of the supernatant on the D101 macroporous resin is 1.0 BV / H, the elution flow rate of 10 column volumes is 2.0 BV / H, and the flow rate of the ethanol aqueous solution elution is 1.0 BV / H.

[0042] The eluent is concentrated under reduced pressure and low temperature, and the temperature T≤40℃, and the pressure≥0.1kpa.

[0043] The supernatant is subjected to D101 macroporous resin, and wet column is used, and saturated adsorption is 45 min.

[0044] The enzymatic solution is transferred to a microwave reaction kettle, and the liquid layer thickness is controlled to be≤10 cm.

[0045] The specific implementation of the embodiment is:

[0046] Experiment 1

[0047] Take the fresh safflower petals 2.50 kg (water content≤8%, crocin content≥15%), remove impurities, get 2.45 kg, loss 2%, placed in 35℃ constant temperature low humidity environment drying 12h, the dried safflower petals are put into a super micro grinder (rotating speed 20000rp / min, 10min) and sieved (sieve 100-200 mesh), get powder (particle size≤10μm);

[0048] The above powder is added into the citric acid buffer solution (pH 5.0, concentration 0.1M) according to the mass ratio 1:20, and is put into the enzymatic reaction tank, and after stirring for 30 min, cellulase (1.0% w / w) is added, and the temperature is controlled at 35℃ for pre-reaction for 30 min, and then the cellulose skeleton is destroyed by stirring (150rpm / min, 30min), and then xylanase (0.6% w / w) is added, and the enzymatic hydrolysis is carried out at 40℃ for 2h, and then stirring (100rpm / min, 30min) and nitrogen gas are used to maintain anaerobic environment (N2 concentration 0.5%-5%);

[0049] The enzymatic solution is transferred to a microwave reaction kettle (liquid layer thickness≤10cm), the first stage power is controlled to be 300W, the temperature is raised to 50℃, and the process is continued for 5min, the second time power is controlled to be 500W, the pulse mode (open 10s / close 5s), the temperature is 55℃, and the process is maintained for 15min, the third time microwave is turned off, the built-in cooling coil is started, and the temperature is reduced to below 40℃ within 10min;

[0050] The microwave treated mixture was quickly transferred to an ice water bath, cooled to below 10°C, stirred for 5 min, high speed centrifuged (8000 r / min, 15 min) and filtered (800-1000 mesh), the supernatant was collected, the filtered precipitate was added to 10% volume of 30% ethanol (v / v), vortexed and mixed, then high speed centrifuged (8000 r / min, 15 min) and filtered (800-1000 mesh) again to collect the supernatant, the precipitate was discarded, and the two collected supernatants were combined;

[0051] The supernatant was loaded onto a D101 macroporous resin column using a wet loading method. After loading, the column was saturated for 45 min, then impurities were eluted with pure water until the eluate was colorless and odorless, which took about 10 column volumes. The loading flow rate was 2 BV / h. Then, 50% ethanol aqueous solution was used to elute the column for 4-8 column volumes at a flow rate of 1 BV / h. The eluate was collected and concentrated under reduced pressure and low temperature vacuum. Ethanol was recovered (temperature ≤40°C, pressure ≥0.1 kpa). A powder of 96 g was obtained by freeze-drying the concentrated extract. The water content was 8%, the extraction yield was 94%, and the crocin content was 98% HPLC.

[0052] Experiment 2

[0053] Fresh saffron petals weighing 2.50 kg (water content ≤8%, crocin content ≥15%) were obtained by removing impurities. The petals were placed in a constant temperature and low humidity environment at 35°C for 12 h of drying. The dried saffron petals were ground in a super micro grinder (20000 rpm / min, 10 min) and sieved (100-200 mesh) to obtain a powder (particle size ≤10 μm).

[0054] The above powder was added to a citric acid buffer (pH 5.0, concentration 0.1 M) at a mass ratio of 1:20, and then was put into an enzyme reaction tank. After stirring for 30 min, cellulase (0.86% w / w) was added, and the temperature was controlled at 35°C for pre-reaction for 30 min. The cellulose framework was destroyed by stirring (150 rpm / min, 30 min). Then, xylanase (0.48% w / w) was added, and the enzyme hydrolysis was carried out at 40°C for 2 h with shaking (100 rpm / min, 30 min) and under anaerobic conditions (N2 concentration 0.5%-5%).

[0055] The enzyme hydrolysate was transferred to a microwave reactor (liquid layer thickness ≤10 cm). In the first stage, the power was controlled at 300 W, and the temperature was raised to 50°C for 5 min. In the second stage, the power was controlled at 500 W in pulse mode (on for 10 s and off for 5 s), and the temperature was maintained at 55°C for 15 min. In the third stage, the microwave was turned off, and the built-in cooling coil was started to cool the temperature to below 40°C within 10 min.

[0056] The microwave treated mixture was quickly transferred to an ice water bath, cooled to below 10°C, stirred for 5 min, filtered (800-1000 mesh), the supernatant was collected, the filtered precipitate was added to 10% by volume of 30% ethanol (v / v), vortexed and mixed, then filtered again (800-1000 mesh) to collect the supernatant, and the precipitate was discarded, and the two collected supernatants were combined;

[0057] The supernatant was loaded onto a D101 macroporous resin column using a wet loading method. After loading, the column was saturated for 45 min, then impurities were eluted with purified water until the eluate was colorless and odorless, which took about 10 column volumes. The loading flow rate was 2 BV / h. Then, 50% ethanol aqueous solution was used to elute the column for 4-8 column volumes at a flow rate of 1 BV / h. The eluate was collected and concentrated under reduced pressure and low temperature vacuum, and ethanol was recovered (temperature T≤40°C, pressure ≥0.1 kpa). This resulted in 76 g of concentrated extract powder after freeze-drying, with a water content of 8% and an extraction yield of 92%. The crocin content was 96% HPLC.

[0058] Experiment 3

[0059] Fresh safflower petals weighing 2.50 kg (water content ≤8%, crocin content ≥15%) were obtained by removing impurities. The petals were placed in a constant temperature and low humidity environment at 35°C for 12 h of drying. The dried safflower petals were ground in a super micro grinder (rotating speed 20000 rpm / min, 10 min) and sieved (100-200 mesh). The powder (particle size ≤10 μm) was obtained.

[0060] The powder was added to citric acid buffer (pH 5.0, concentration 0.1 M) at a mass ratio of 1:20, and was placed in an enzyme hydrolysis reactor. After stirring for 30 min, cellulase (0.56% w / w) was added, and the temperature was controlled at 35°C for pre-reaction for 30 min. The cellulose framework was destroyed by stirring (150 rpm / min, 30 min). Then, xylanase (0.4% w / w) was added, and the enzyme hydrolysis was carried out at 40°C for 2 h. The stirring (100 rpm / min, 30 min) was performed, and the anaerobic environment was maintained by nitrogen gas (N2 concentration 0.5%-5%).

[0061] The enzyme hydrolysis solution was transferred to a microwave reactor (liquid layer thickness ≤10 cm). The power was controlled at 300 W, and the temperature was raised to 50°C for 5 min. Then, the power was controlled at 500 W in pulse mode (on for 10 s and off for 5 s), and the temperature was maintained at 55°C for 15 min. Finally, the microwave was turned off, and the built-in cooling coil was started to cool the temperature to below 40°C within 10 min.

[0062] The microwave treated mixture was quickly transferred to an ice water bath, cooled to below 10°C, stirred for 5 min, filtered (800-1000 mesh), the supernatant was collected, the filtered precipitate was added to 10% by volume of 30% ethanol (v / v), vortexed and mixed, then filtered again (800-1000 mesh) to collect the supernatant, the precipitate was discarded, and the two collected supernatants were combined;

[0063] The supernatant was loaded onto a D101 macroporous resin column using a wet loading method. After loading, the column was saturated for 45 min, then impurities were eluted with purified water until the eluate was colorless and odorless, which took about 10 column volumes. The flow rate was 2 BV / h. Then, 50% ethanol aqueous solution was used to elute for 4-8 column volumes at a flow rate of 1 BV / h, and the eluate was collected. The eluate was concentrated under reduced pressure and low temperature vacuum, and ethanol was recovered (temperature T≤40°C, pressure≥0.1 kpa). A powder of 68 g was obtained by freeze-drying the concentrated extract, with a water content of 5% and an extraction yield of 87%. The crocin content was 94% HPLC.

[0064] Control experiment

[0065] Fresh saffron petals weighing 2.50 kg (water content≤8%, crocin content≥15%) were obtained by removing impurities. The petals were placed in a constant temperature and low humidity environment at 35°C for 12 h of drying. The dried saffron petals were ground in a super micro grinder (rotating speed 20000 rpm / min, 10 min) and sieved (100-200 mesh). The powder (particle size≤10 μm) was obtained.

[0066] The powder was added to a citric acid buffer solution (pH 5.0, concentration 0.1 M) at a mass ratio of 1:20, and was placed in an enzyme hydrolysis reactor. After stirring for 30 min, cellulase (0.57% w / w) was added, and the temperature was controlled at 35°C for pre-reaction for 30 min. The cellulose framework was destroyed by stirring (150 rpm / min, 30 min). Then, xylanase (0.31% w / w) was added, and the enzyme hydrolysis was carried out at 40°C for 2 h under shaking (100 rpm / min, 30 min) and anaerobic conditions (N2 concentration 0.5%-5%).

[0067] The enzyme hydrolysate was transferred to a microwave reactor (liquid layer thickness≤10 cm). In the first stage, the power was controlled at 300 W, and the temperature was raised to 50°C for 5 min. In the second stage, the power was controlled at 500 W in pulse mode (on for 10 s / off for 5 s), and the temperature was maintained at 55°C for 15 min. In the third stage, the microwave was turned off, and the built-in cooling coil was started to cool the temperature to below 40°C within 10 min.

[0068] The microwave-treated mixture was quickly transferred to an ice water bath, cooled to below 10°C, stirred for 5 min, filtered (800-1000 mesh), the supernatant was collected, the filtered precipitate was added to 10% volume of 30% ethanol (v / v), vortexed and mixed, then filtered again (800-1000 mesh) to collect the supernatant, and the precipitate was discarded, and the two collected supernatants were combined;

[0069] The supernatant was loaded onto a D101 macroporous resin column using a wet loading method. After loading, the column was saturated for 45 min, then impurities were eluted with purified water until the eluate was colorless and odorless, which took about 10 column volumes. The flow rate was 2 BV / h. Then, 50% ethanol aqueous solution was used to elute for 4-8 column volumes at a flow rate of 1 BV / h, and the eluate was collected. The eluate was concentrated under reduced pressure and low temperature vacuum, and ethanol was recovered (temperature T≤40°C, pressure≥0.1 kpa). This resulted in 86 g of concentrated extract powder, which was freeze-dried, with a water content of 8%, an extraction rate of 82%, and a crocin content of 88% HPLC.

[0070] By adding cellulase and xylanase, the cellulose and hemicellulose framework in the cell wall of crocus petals were destroyed, making the cell contents, including crocin, more easily released into the extraction solution. This method significantly improved the extraction rate of crocin, with an extraction rate of 94% in Example 1. Even in Examples 2 and 3, where the enzyme dosage was reduced, the extraction rates remained at 92% and 87%, respectively, which was higher than that of traditional extraction methods. Microwave heating can quickly and uniformly raise the system temperature, promote molecular motion, and accelerate the diffusion of crocin from the cell matrix to the extraction solution. At the same time, the pulse mode (10s on / 5s off) helps to avoid local overheating and degradation of crocin, maintaining the extraction efficiency. Through the synergistic effect of enzyme hydrolysis and microwave treatment, crocin can be more effectively released, and the extraction of impurities can be reduced. The crocin powder obtained in Example 1 had a content of up to 98% (HPLC), and the contents of Examples 2 and 3 reached 96% and 94%, respectively, which was significantly higher than the purity of products obtained by non-optimized extraction methods. The enzyme hydrolysis process was carried out under relatively mild conditions (e.g., 35°C pre-reaction for 30 min, 40°C shaking enzyme hydrolysis for 2 h), which can more quickly destroy the cell wall and reduce the risk of thermal degradation of crocin compared to high-temperature long-time extraction. Microwave can quickly raise the system temperature to the required level and maintain it constant, greatly shortening the heating time during the extraction process and improving the overall extraction efficiency.

[0071] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting; it is not intended to exclude myriad other embodiments of the present application that other inventors can develop based on the same general inventive concepts embodied by the described embodiments. That is, although the present application is described in terms of particular embodiments and illustrative figures, it should be apparent that the scope of the present application is not limited to these specific embodiments.

[0072] While the embodiments of the application have been shown and described herein, it will be understood by those of ordinary skill in the art that various changes, modifications, alternatives, and variations can be made to the embodiments without departing from the spirit and scope of the application, which is defined by the claims and their equivalents.

Claims

1. A method for extraction of crocin based on enzymatic hydrolysis and microwave-ultrasound, characterized by, It comprises the following steps: S1: Take fresh saffron petals, remove impurities, dry at 25℃ for 6h to remove surface moisture, then put into a constant temperature and humidity condition at 35℃ for 12h, and then put the dried saffron petals into an ultrafine grinder for grinding; S2: Add citric acid buffer solution to the ground saffron powder at a ratio of 1:20, put the mixture into an enzymatic reaction tank and stir for 30min, fill nitrogen gas into the reaction tank at a concentration of 0.5%-5%, add cellulase, pre-react at 35℃ for 30min, and stir, then add xylanase, and oscillate for 2h at 40℃ and stir; S3: Transfer the enzymatic liquid to a microwave reaction kettle for three-stage temperature control, A1: first stage, power control 300W, temperature rises to 50℃, lasts for 5min; A2: second stage, power control 500W, pulse mode, temperature 55℃, maintains for 15min; A3: third stage, turn off the microwave, start the built-in cooling coil, and reduce the temperature to below 40℃ within 10min; S4: quickly transfer the microwave-treated mixture to an ice water bath, cool to below 10℃, stir for 5min, filter with a 800-1000 mesh sieve, then centrifugal filter at 4℃, take the supernatant, pass the supernatant through D101 macroporous resin, elute impurities with pure water until colorless and odorless, elute for 10 column volumes, then elute with 50% ethanol aqueous solution for 4-8 column volumes, and collect the eluate; S5: vacuum low-temperature concentrate the eluate, recover ethanol, freeze-dry the concentrated extract into powder, and obtain high-purity saffron.

2. The method according to claim 1, wherein the method is characterized by: The saffron petals are ground in an ultrafine grinder until the particle size is ≤10μm.

3. The method according to claim 1, wherein the method is characterized by: The citric acid buffer solution has a concentration of 0.1M and a pH of 5.0, and the nitrogen gas has a concentration of 0.5%-5%.

4. The method according to claim 1, wherein the method is characterized by: The concentration of cellulase is 0.5%-1.0% w / w, and the concentration of xylanase is 0.3%-0.6% w / w.

5. The method of claim 1, wherein the method is characterized by: The stirring rate of pre-reaction is 150rpm / min, and the stirring rate of vibration enzymolysis is 100rpm / min and stirring for 30min.

6. The method according to claim 1, wherein the method is characterized by: The pulse frequency of the second stage is 10s on / 5s off.

7. The method according to claim 1, wherein the method is characterized by: The flow rate of D101 macroporous resin for the supernatant is 1.0BV / H, the flow rate for eluting 10 column volumes is 2.0BV / H, and the flow rate of ethanol aqueous solution for elution is 1.0BV / H.

8. The method according to claim 1, wherein the method is characterized by: The eluate is vacuum low-temperature concentrated at a temperature of ≤40℃ and a pressure of ≥0.1kpa.

9. The method according to claim 1, wherein the method is characterized by: The D101 macroporous resin for the supernatant is loaded by wet method, and saturated adsorption lasts for 45min.

10. The method of claim 1, wherein the method is a method of extracting crocin by enzymatic hydrolysis and microwave ultrasonic. The enzymatic liquid is transferred to a microwave reaction kettle, and the liquid layer thickness is controlled to be ≤10cm.

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