CN112778182A - Green extraction method of high-purity beta-carotene - Google Patents
Green extraction method of high-purity beta-carotene Download PDFInfo
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- C07C403/00—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone
- C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
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Abstract
The invention discloses a green extraction method of high-purity beta-carotene, which comprises the following steps: (1) pretreatment: crushing fresh carrots by using a stirrer to obtain carrot paste; (2) carrying out enzymolysis; (3) static pressurizing and primary extracting; (4) static pressurizing and secondary extracting; (5) freezing and concentrating; (6) freeze drying; (7) crushing and sieving to obtain the high-yield and high-purity beta-carotene. The yield and the purity of the beta-carotene extracted by the method are effectively improved, toxic chemical reagents are not contained in the extraction process, the extraction effect is obvious and safe, and the method is green and environment-friendly.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to a green extraction method of high-yield and high-purity beta-carotene.
Background
Beta-carotene is a natural pigment, antioxidant and precursor of high-effective vitamin A, and is a safe and harmless food additive. The extraction of various bioactive compounds from natural plants has been carried out in many industries. The traditional extraction method of dipping and Soxhlet extraction has the defects of large solvent consumption, long extraction time, low extraction rate and the like, which greatly limits the yield and application of the beta-carotene, and because the beta-carotene is extremely unstable and has low solubility in water, is sensitive to light, oxygen and heat and is easy to degrade and isomerize, the difficulty of extracting the beta-carotene is increased.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a green extraction method of high-yield and high-purity beta-carotene, which comprises the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: uniformly mixing cellulase, carrot paste and a cocrystal solvent, carrying out enzymolysis at 35-40 ℃, and then filtering an enzymolysis product to obtain a filtrate A and a filter residue A;
(3) static pressurized primary extraction: carrying out static pressure extraction on the filter residue A, wherein the extract is a mixture of ethanol and water, and filtering after extraction to obtain an extract B and a filter residue B;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C;
(5) freezing and concentrating: combining the extract B and the extract C, putting the mixture into a freezing device, cooling to-5 to-3 ℃, observing for 1 time every 30 seconds after 5min of freezing until snowflake-shaped and flocculent small ice crystals appear in the extract, namely reaching the freezing point, continuing to freeze for 15-25 min after reaching the freezing point, and then separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
Further, the preparation method of the eutectic solvent comprises the following steps: choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1: 0.7-1.3, and dissolving in water, wherein the total mass percentage of the choline chloride and the citric acid is 10% -16%; evaporating at 45-55 ℃ by using a rotary evaporator, putting the obtained liquid into a dryer containing silica gel, and drying for 2-3 days until the weight of the liquid is constant to obtain the eutectic solvent.
Further, before enzymolysis, cyclocarya paliurus leaf-radix puerariae extract is mixed into the eutectic solvent, and the preparation method of the cyclocarya paliurus leaf-radix puerariae extract comprises the following steps: respectively cutting fresh cyclocarya paliurus leaves and fresh radix puerariae, mixing, adding the mixture into deionized water with the mass 6-8 times of the total mass of the mixture, extracting in a water bath at the temperature of 80 +/-5 ℃ for more than 20 hours at constant temperature, cooling in air to normal temperature after extraction is finished, filtering, decompressing and concentrating filtrate to 1/4 of the volume before concentration, and obtaining the cyclocarya paliurus leaf-radix puerariae extract.
Further, the mixing mass ratio of the fresh cyclocarya paliurus leaves to the fresh radix puerariae is as follows: the kudzu root =1: 2-5.
Furthermore, the mass ratio of the cyclocarya paliurus leaf-radix puerariae extract to the eutectic solvent is 0.6-1: 10.
Further, in the enzymolysis step, the mixing mass ratio of the cellulase, the carrot mud and the cocrystallization solvent is as follows: carrot paste: the ratio of the eutectic solvent to the eutectic solvent is 1: 15-25: 70-90, the enzymolysis temperature is 35-40 ℃, and the enzymolysis time is 2-3 h.
Further, in the extraction steps in the steps (4) and (5), the volume ratio of ethanol/water in the extraction liquid is =1: 0.5-1.5; the conditions of the two extractions are as follows: the pressure is 8-12 MPa, the temperature is 70-80 ℃, the ratio of material to liquid is 1: 15-25 g/mL, and the extraction time is 20-30 min.
The beneficial effects of the invention include: the eutectic solvent mainly comprises a binary system and a ternary system consisting of hydrogen bond donors (polyhydric alcohols, urea and carboxylic acid) and hydrogen bond acceptors (quaternary ammonium salts such as choline chloride and the like), and the most remarkable physical property of the formed eutectic solvent is the reduction of the melting point of the solvent. Experiments show that the eutectic solvent has the effect of promoting enzymolysis as a novel green reagent capable of replacing ionic liquid, the yield and the purity of the beta-carotene extracted by the composite cellulase are effectively improved, toxic chemical reagents are not contained in the extraction process, the extraction effect is obvious and safe, and the green environment is protected.
Drawings
FIG. 1 shows the results of the respective experimental groupsβ-graph of carotene yield comparison;
FIG. 2 shows the results of the respective experimental groupsβComparison of carotene purity.
Detailed Description
The following is a detailed description with reference to examples:
example 1
A green extraction method of high-yield high-purity beta-carotene comprises the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: cellulase, carrot paste and a cocrystallization solvent are mixed according to the mass ratio of the cellulase: carrot paste: uniformly mixing eutectic solvent =1:15:70, carrying out enzymolysis at 40 ℃ for 2 h, and then filtering the zymolyte to obtain filtrate A and filter residue A;
(3) static pressurized primary extraction: performing static pressure extraction on the filter residue A, wherein an extraction liquid is a mixture of ethanol and water, and the volume ratio of ethanol to water in the extraction liquid is =1: 0.5; filtering after extraction to obtain extract B and filter residue B; the extraction conditions were all: the pressure is 8 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:15g/mL, and the extraction time is 20 min;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C; the extraction conditions were all: the pressure is 8 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:15g/mL, and the extraction time is 20 min;
(5) freezing and concentrating: mixing the extract B and the extract C, placing into a freezing device, cooling to-5 deg.C, freezing for 5min, observing for 1 time every 30 s until small ice crystals appear in the extract, i.e. reaching freezing point, continuing to freeze for 15 min after reaching the freezing point, and separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
The preparation method of the eutectic solvent comprises the following steps: choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1:0.7, and dissolving in water, wherein the total mass percentage of the choline chloride and the citric acid is 10%; the solution was evaporated at 50 ℃ for 20 hours using a rotary evaporator, and the obtained liquid was put into a desiccator containing silica gel and dried for 2 days until the weight thereof became constant, to obtain a eutectic solvent as described in the present example.
Example 2
A green extraction method of high-yield high-purity beta-carotene comprises the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: cellulase, carrot paste and a cocrystallization solvent are mixed according to the mass ratio of the cellulase: carrot paste: uniformly mixing eutectic solvent =1:20:80, carrying out enzymolysis at 40 ℃ for 2 h, and filtering the zymolyte to obtain filtrate A and filter residue A;
(3) static pressurized primary extraction: performing static pressure extraction on the filter residue A, wherein an extraction liquid is a mixture of ethanol and water, and the volume ratio of ethanol to water in the extraction liquid is =1: 1; filtering after extraction to obtain extract B and filter residue B; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(5) freezing and concentrating: mixing the extract B and the extract C, placing into a freezing device, cooling to-4 deg.C, freezing for 5min, observing for 1 time every 30 s until small ice crystals appear in the extract, i.e. reaching freezing point, continuing to freeze for 20min after reaching the freezing point, and separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
The preparation method of the eutectic solvent comprises the following steps: choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1:1, and dissolving in water, wherein the total mass percentage of choline chloride and citric acid is 12%; the solution was evaporated at 50 ℃ for 20 hours using a rotary evaporator, and the obtained liquid was put into a desiccator containing silica gel and dried for 2 days until the weight thereof became constant, to obtain a eutectic solvent as described in the present example.
Example 3
A green extraction method of high-yield high-purity beta-carotene comprises the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: cellulase, carrot paste and a cocrystallization solvent are mixed according to the mass ratio of the cellulase: carrot paste: uniformly mixing eutectic solvent =1:25:90, carrying out enzymolysis at 40 ℃ for 3 h, and filtering the zymolyte to obtain filtrate A and filter residue A;
(3) static pressurized primary extraction: performing static pressure extraction on the filter residue A, wherein an extraction liquid is a mixture of ethanol and water, and the volume ratio of ethanol to water in the extraction liquid is =1: 1.5; filtering after extraction to obtain extract B and filter residue B; the extraction conditions were all: the pressure is 12 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:25g/mL, and the extraction time is 30 min;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C; the extraction conditions were all: the pressure is 12 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:25g/mL, and the extraction time is 30 min;
(5) freezing and concentrating: mixing the extract B and the extract C, placing into a freezing device, cooling to-3 deg.C, freezing for 5min, observing for 1 time every 30 s until small ice crystals appear in the extract, i.e. reaching freezing point, continuing to freeze for 25 min after reaching the freezing point, and separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
The preparation method of the eutectic solvent comprises the following steps: choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1:1.3, and dissolving in water, wherein the total mass percentage of the choline chloride and the citric acid is 16%; the solution was evaporated at 50 ℃ for 20 hours using a rotary evaporator, and the obtained liquid was put into a desiccator containing silica gel and dried for 3 days until the weight thereof became constant, to obtain a eutectic solvent as described in the present example.
Comparative example 1
This comparative example is a green extraction process of beta-carotene, with the same extraction procedure and parameters as in example 2, except that in the co-crystal solvent preparation process of this comparative example, the molar ratio of choline chloride to citric acid choline chloride: citric acid =1: 3. Other parameters, steps and example 2 were exactly the same.
Comparative example 2
This comparative example is a green extraction process of beta-carotene, with the same extraction procedure and parameters as in example 2, except that in the eutectic solvent preparation process of this comparative example, evaporation was performed at 30 ℃ for 20h using a rotary evaporator. Other parameters, steps and example 2 were exactly the same.
Comparative example 3
This comparative example is a green extraction method of beta-carotene, the extraction procedure and parameters are the same as those of example 2, and the only difference is that the extraction pressure in steps (3) and (4) of this comparative example is 6 MPa. Other parameters, steps and example 2 were exactly the same.
Comparative example 4
This comparative example is a green extraction process of beta-carotene, with the same extraction procedure and parameters as example 2, except that in step (5) of this comparative example, the freezing temperature was 4 ℃. Other parameters, steps and example 2 were exactly the same.
Comparative example 5
A green extraction method of high-yield high-purity beta-carotene comprises the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: cellulase, carrot paste and a cocrystallization solvent are mixed according to the mass ratio of the cellulase: carrot paste: uniformly mixing eutectic solvent =1:20:80, carrying out enzymolysis at 40 ℃ for 2 h, and filtering the zymolyte to obtain filtrate A and filter residue A;
(3) static pressurized primary extraction: performing static pressure extraction on the filter residue A, wherein an extraction liquid is a mixture of ethanol and water, and the volume ratio of ethanol to water in the extraction liquid is =1: 1; filtering after extraction to obtain extract B and filter residue B; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(5) freezing and concentrating: mixing the extract B and the extract C, placing into a freezing device, cooling to-4 deg.C, freezing for 5min, observing for 1 time every 30 s until small ice crystals appear in the extract, i.e. reaching freezing point, continuing to freeze for 20min after reaching the freezing point, and separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
The preparation method of the eutectic solvent comprises the following steps:
1) choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1:1, and dissolving in water, wherein the total mass percentage of choline chloride and citric acid is 12%; evaporating at 50 + -5 deg.C for 20h with a rotary evaporator, and drying the obtained liquid in a drier containing silica gel for 2 days until the weight is constant to obtain solvent A;
2) respectively cutting fresh cyclocarya paliurus leaves and fresh radix puerariae, and mixing, wherein the mass ratio of the fresh cyclocarya paliurus leaves to the fresh radix puerariae is as follows: kudzu root =1: 2. Adding the mixture into deionized water 6 times of the total mass of the mixture, extracting in 80 + -5 deg.C water bath at constant temperature for 20 hr, air cooling to room temperature after extraction, filtering, and concentrating the filtrate under reduced pressure to 1/4 volume before concentration to obtain cyclocarya paliurus leaf-radix Puerariae extract;
3) and mixing the cyclocarya paliurus leaf-radix puerariae extract into the solvent A to obtain the eutectic solvent of the comparative example. The mass ratio of the cyclocarya paliurus leaf-radix puerariae extract to the solvent A is that the cyclocarya paliurus leaf-radix puerariae extract/the solvent A =0.6: 10.
Comparative example 6
A green extraction method of high-yield high-purity beta-carotene comprises the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: cellulase, carrot paste and a cocrystallization solvent are mixed according to the mass ratio of the cellulase: carrot paste: uniformly mixing eutectic solvent =1:20:80, carrying out enzymolysis at 40 ℃ for 2 h, and filtering the zymolyte to obtain filtrate A and filter residue A;
(3) static pressurized primary extraction: performing static pressure extraction on the filter residue A, wherein an extraction liquid is a mixture of ethanol and water, and the volume ratio of ethanol to water in the extraction liquid is =1: 1; filtering after extraction to obtain extract B and filter residue B; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(5) freezing and concentrating: mixing the extract B and the extract C, placing into a freezing device, cooling to-4 deg.C, freezing for 5min, observing for 1 time every 30 s until small ice crystals appear in the extract, i.e. reaching freezing point, continuing to freeze for 20min after reaching the freezing point, and separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
The preparation method of the eutectic solvent comprises the following steps:
1) choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1:1, and dissolving in water, wherein the total mass percentage of choline chloride and citric acid is 12%; evaporating at 50 + -5 deg.C for 20h with a rotary evaporator, and drying the obtained liquid in a drier containing silica gel for 2 days until the weight is constant to obtain solvent A;
2) respectively cutting fresh cyclocarya paliurus leaves and fresh radix puerariae, and mixing, wherein the mass ratio of the fresh cyclocarya paliurus leaves to the fresh radix puerariae is as follows: kudzu root =1: 5. Adding the mixture into deionized water 8 times of the total mass of the mixture, extracting in 80 + -5 deg.C water bath at constant temperature for 20 hr, air cooling to room temperature after extraction, filtering, and concentrating the filtrate under reduced pressure to 1/4 volume before concentration to obtain cyclocarya paliurus leaf-radix Puerariae extract;
3) and mixing the cyclocarya paliurus leaf-radix puerariae extract into the solvent A to obtain the eutectic solvent of the comparative example. The mass ratio of the cyclocarya paliurus leaf-radix puerariae extract to the solvent A is 1: 10.
Comparative example 7
A green extraction method of high-yield high-purity beta-carotene comprises the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: cellulase, carrot paste and a cocrystallization solvent are mixed according to the mass ratio of the cellulase: carrot paste: uniformly mixing eutectic solvent =1:20:80, carrying out enzymolysis at 40 ℃ for 2 h, and filtering the zymolyte to obtain filtrate A and filter residue A;
(3) static pressurized primary extraction: performing static pressure extraction on the filter residue A, wherein an extraction liquid is a mixture of ethanol and water, and the volume ratio of ethanol to water in the extraction liquid is =1: 1; filtering after extraction to obtain extract B and filter residue B; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(5) freezing and concentrating: mixing the extract B and the extract C, placing into a freezing device, cooling to-4 deg.C, freezing for 5min, observing for 1 time every 30 s until small ice crystals appear in the extract, i.e. reaching freezing point, continuing to freeze for 20min after reaching the freezing point, and separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
The preparation method of the eutectic solvent comprises the following steps:
1) choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1:1, and dissolving in water, wherein the total mass percentage of choline chloride and citric acid is 12%; evaporating at 50 + -5 deg.C for 20h with a rotary evaporator, and drying the obtained liquid in a drier containing silica gel for 2 days until the weight is constant to obtain solvent A;
2) cutting fresh cyclocarya paliurus leaves, adding the cut fresh cyclocarya paliurus leaves into deionized water with the mass 8 times of that of the fresh cyclocarya paliurus leaves, carrying out water bath constant-temperature extraction at the temperature of 80 +/-5 ℃ for 20 hours, cooling the obtained product to the normal temperature after the extraction is finished, filtering, decompressing and concentrating the filtrate to 1/4 of the volume of the obtained product before concentration, and obtaining an extract of the cyclocarya pali;
3) and (3) mixing the cyclocarya paliurus leaf extract into the solvent A to obtain the eutectic solvent of the comparative example. The mass ratio of the cyclocarya paliurus leaf extract to the solvent A is 1: 10.
Comparative example 8
A green extraction method of high-yield high-purity beta-carotene comprises the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: cellulase, carrot paste and a cocrystallization solvent are mixed according to the mass ratio of the cellulase: carrot paste: uniformly mixing eutectic solvent =1:20:80, carrying out enzymolysis at 40 ℃ for 2 h, and filtering the zymolyte to obtain filtrate A and filter residue A;
(3) static pressurized primary extraction: performing static pressure extraction on the filter residue A, wherein an extraction liquid is a mixture of ethanol and water, and the volume ratio of ethanol to water in the extraction liquid is =1: 1; filtering after extraction to obtain extract B and filter residue B; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C; the extraction conditions were all: the pressure is 10 MPa, the temperature is 70 ℃, the ratio of material to liquid is 1:20g/mL, and the extraction time is 20 min;
(5) freezing and concentrating: mixing the extract B and the extract C, placing into a freezing device, cooling to-4 deg.C, freezing for 5min, observing for 1 time every 30 s until small ice crystals appear in the extract, i.e. reaching freezing point, continuing to freeze for 20min after reaching the freezing point, and separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
The preparation method of the eutectic solvent comprises the following steps:
1) choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1:1, and dissolving in water, wherein the total mass percentage of choline chloride and citric acid is 12%; evaporating at 50 + -5 deg.C for 20h with a rotary evaporator, and drying the obtained liquid in a drier containing silica gel for 2 days until the weight is constant to obtain solvent A;
2) cutting fresh radix Puerariae, adding into deionized water 8 times of the fresh radix Puerariae, extracting at 80 + -5 deg.C with water bath at constant temperature for 20 hr, air cooling to room temperature, filtering, and concentrating the filtrate under reduced pressure to 1/4 of the volume before concentration to obtain radix Puerariae extract;
3) and mixing the kudzu root extract into the solvent A to obtain the eutectic solvent of the comparative example. The mass ratio of the kudzu root extract/the solvent A =1:10 is mixed in the solvent A.
Example 4
Extracted for examples 1 to 3 and comparative examples 1 to 8βCarotene was evaluated for yield. The evaluation method comprises the following steps: the same batch of purchased carrots are used for the extraction experiments of the examples 1 to 3 and the comparative examples 1 to 8, and the weight of the input carrot raw material is recorded as m0Is to be extractedβWeighing the dry ground carotene and recording its mass as m1。βThe ratio m of carotene to carrot material weight1/m0That is, the yield, the results are shown in FIG. 1.
As shown in figure 1, the eutectic solvent prepared by the method of the invention is obtained after subsequent enzymolysis, pressure extraction and freeze dryingβCarotene has a higher yield. Comparing comparative example 1 and comparative example 2, it can be seen that the right method for preparing the eutectic solvent isβThe improvement of the extraction yield of the carotene has the promotion effect, the cellulose enzymolysis effect of the eutectic solvent treatment is better,βyield of caroteneAnd higher. And of comparative example 4βCarotene is lowest, which may be the temperature during freeze concentration not reaching below the freezing point of water, and no effective concentration, resulting in a decrease in yield. As can be seen from comparison of example 2 with comparative examples 5 and 6, addition of the cyclocarya paliurus leaf-puerariae radix extract to the eutectic solvent can better promoteβ-extraction of carotene, expressed asβThe highest yields of carotene are obtained. And the effect of the compound extract is obviously better than that of the single extract.
Example 5
Extracted for examples 1-3 and comparative examples 1-8 by using an ultraviolet spectrophotometerβCarotene toβ-carotene purity identification. The identification method comprises the following steps: is to be extractedβCarotene was dissolved in 0.01 mol/L PBS solution (pH 5), and the sample was extracted with a mixture of 2 mL ethanol and 3 mL n-hexane (1 mL). After shaking the mixture thoroughly, the hexane phase was removed. The extraction was repeated twice and the hexane phases were combined. After appropriate dilution with n-hexane, the absorbance was measured at 450 nm with an ultraviolet-visible spectrophotometer. High purity in commercially known purityβCarotene as standard, to be testedβThe purity of carotene is expressed as: OD1/OD0Wherein OD1And OD0Respectively to be tested and commercially availableβCarotene absorbance, results are shown in figure 2.
As can be seen from FIG. 2, the samples extracted in examples 1 to 3βThe purity of the carotene is higher, and the carotene reaches the high purity of the commercial productsβAround 90% of carotene, extracted from comparative example 3βThe lowest purity of carotene, which may be due to too low extraction pressure in the pressure extraction step, causes boiling and overheating of the extractβ-the thermal degradation of carotene takes place. Comparative examples 1 to 2βThe low purity of the carotene may be due to the fact that the cellulase hydrolysis effect is not high due to incorrect configuration of the eutectic solvent,βcarotene is difficult to dissolve. Comparative example 2, comparative example 5 and comparative example 6 it can be seen that cyclocarya paliurus leaf-puerariae radix extract was added to a eutectic solvent to obtainβThe carotene has higher purity, the subsequent treatment process can be reduced or simplified, and the application prospect is wide.
The technical solutions provided by the present invention are described in detail above, and for those skilled in the art, the ideas according to the embodiments of the present invention may be changed in the specific implementation manners and the application ranges, and in summary, the content of the present description should not be construed as limiting the present invention.
Claims (7)
1. A green extraction method of high-yield high-purity beta-carotene is characterized by comprising the following steps:
(1) pretreatment: cutting off two ends of fresh carrot, cleaning, and crushing with a stirrer to obtain carrot paste;
(2) enzymolysis: uniformly mixing cellulase, carrot paste and a cocrystal solvent, carrying out enzymolysis at 35-40 ℃, and then filtering an enzymolysis product to obtain a filtrate A and a filter residue A;
(3) static pressurized primary extraction: carrying out static pressure extraction on the filter residue A, wherein the extract is a mixture of ethanol and water, and filtering after extraction to obtain an extract B and a filter residue B;
(4) static pressurized secondary extraction: adding fresh extract liquor into the filter residue B, carrying out static pressurized secondary extraction, and filtering after extraction is finished to obtain extract liquor C;
(5) freezing and concentrating: combining the extract B and the extract C, putting the mixture into a freezing device, cooling to-5 to-3 ℃, observing for 1 time every 30 seconds after 5min of freezing until snowflake-shaped and flocculent small ice crystals appear in the extract, namely reaching the freezing point, continuing to freeze for 15-25 min after reaching the freezing point, and then separating ice blocks to obtain concentrated solution;
(6) and (3) freeze drying: pre-freezing the concentrated solution at-80 ℃ for 24 h, and then freezing and drying to obtain a beta-carotene crude product;
(7) crushing and sieving: and (3) crushing the beta-carotene crude product in a crusher, and sieving the crushed product with a 100-mesh sieve to obtain the high-yield and high-purity beta-carotene.
2. The green extraction method of beta-carotene with high yield and high purity according to claim 1, wherein the preparation method of the eutectic solvent comprises the following steps: choline chloride and citric acid are mixed in a molar ratio of choline chloride: mixing citric acid =1: 0.7-1.3, and dissolving in water, wherein the total mass percentage of the choline chloride and the citric acid is 10% -16%; evaporating at 45-55 ℃ by using a rotary evaporator, putting the obtained liquid into a dryer containing silica gel, and drying for 2-3 days until the weight of the liquid is constant to obtain the eutectic solvent.
3. The green extraction method of beta-carotene with high yield and high purity according to claim 2, characterized in that before enzymolysis, cyclocarya paliurus leaf-radix puerariae extract is mixed into the eutectic solvent, and the preparation method of the cyclocarya paliurus leaf-radix puerariae extract comprises the following steps: respectively cutting fresh cyclocarya paliurus leaves and fresh radix puerariae, mixing, adding the mixture into deionized water with the mass 6-8 times of the total mass of the mixture, extracting in a water bath at the temperature of 80 +/-5 ℃ for more than 20 hours at constant temperature, cooling in air to normal temperature after extraction is finished, filtering, decompressing and concentrating filtrate to 1/4 of the volume before concentration, and obtaining the cyclocarya paliurus leaf-radix puerariae extract.
4. The green extraction method of beta-carotene with high yield and high purity according to claim 3, wherein the fresh cyclocarya paliurus leaves and the fresh radix puerariae are mixed according to the mass ratio of cyclocarya paliurus leaves: the kudzu root =1: 2-5.
5. The green extraction method of beta-carotene with high yield and high purity according to claim 3, wherein the mass ratio of cyclocarya paliurus leaf-radix puerariae extract/eutectic solvent mixed in the eutectic solvent is = 0.6-1: 10.
6. The green extraction method of beta-carotene with high yield and high purity according to claim 1, wherein in the enzymolysis step, the mixing mass ratio of the cellulase, the carrot mud and the co-crystal solvent is as follows: carrot paste: the ratio of the eutectic solvent to the eutectic solvent is 1: 15-25: 70-90, the enzymolysis temperature is 35-40 ℃, and the enzymolysis time is 2-3 h.
7. The green extraction method of β -carotene with high yield and high purity according to claim 1, wherein in the extraction steps (4) and (5), the volume ratio of ethanol/water in the extract is =1: 0.5-1.5; the conditions of the two extractions are as follows: the pressure is 8-12 MPa, the temperature is 70-80 ℃, the ratio of material to liquid is 1: 15-25 g/mL, and the extraction time is 20-30 min.
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