CN110693030A - Preparation method and application of macadimia nut green husk extract - Google Patents
Preparation method and application of macadimia nut green husk extract Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/004—Aftersun preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention belongs to the field of extraction of natural plant products, and particularly relates to a preparation method and application of a macadimia nut green husk extract. The preparation method comprises the steps of cellulose enzymolysis and protease enzymolysis, then the phenolic glycoside compounds with high utilization value, such as arbutin, helicid, gastrodin and the like in the green tangerine peel of the macadimia nut are extracted by an ultrasonic extraction method, and finally the green tangerine peel extract is obtained by degreasing treatment and decoloring treatment. The green tangerine peel extract obtained by the preparation method has high content of phenolic glycoside compounds such as arbutin, helicidum, gastrodin and the like, the extraction process is simple, complex extraction, purification and separation operations are not needed, and reagents, raw materials and the like used in the process are easy to obtain and have low cost. The preparation method can be applied to the preparation of health food and cosmetics containing arbutin, helicid, gastrodin and the like.
Description
Technical Field
The invention belongs to the field of extraction of natural plant products, and particularly relates to a preparation method and application of a macadimia nut green husk extract.
Background
The fruit of Macadamia nut (Macadamia integrifolia f.muell, also known as Macadamia nut) includes a green tangerine peel, a brown seed coat, and opalescent kernels. The green tangerine peel is the peel of the fruit of the macadimia nut, accounts for 45-60% of the fresh weight of the fruit, belongs to the discarded part of the fruit, only a small amount of the peel is used as a compost and for making animal feed, and a large amount of active substances in the green tangerine peel are basically not reasonably developed and utilized.
Researches show that the green tangerine orange peel of the macadimia nut contains a large amount of phenolic compounds which have the effects of bacteriostasis, antioxidation, dental caries prevention, tumor resistance and the like, and also have certain fragrant smell and whitening effect. The macadimia nut green tangerine peel polyphenol comprises arbutin, helicid, gastrodin and the like, wherein the helicid and the gastrodin have the effects of helping sleep, tranquilizing and allaying excitement, relieving mental stress and the like, and the arbutin has the effects of repairing damage after sunshine and whitening. If the glycosides can be fully utilized, the added value of the macadimia nuts can be improved, waste is turned into wealth, green tangerine peels are comprehensively utilized, and the nut industrial chain is expanded.
However, because the green tangerine peel contains a plurality of phenolic compounds and other substances, if one or more phenolic compounds are to be separated and obtained, more complicated operation processes, such as multiple extractions, column purification and separation, are required. The extraction and separation of the green husk polyphenol of the macadimia nut in the prior art are not suitable for industrial production, thereby hindering the comprehensive utilization of the green husk resource of the macadimia nut.
Disclosure of Invention
The invention mainly solves the technical problem of providing the preparation method of the extract of the green tangerine orange peel of the macadimia nut, the preparation method can be used for efficiently extracting effective substances such as arbutin, helicid, gastrodin and the like in the green tangerine orange peel of the macadimia nut, and the preparation method is simple and is suitable for industrial production.
In order to solve the technical problems, the invention provides the following technical scheme:
a preparation method of a macadimia nut green husk extract comprises the following steps:
step (1): crushing macadimia nut green peel material to obtain green peel powder;
step (2): adding water into pericarpium citri reticulatae viride powder, adding cellulase to obtain an enzymolysis system I, and carrying out a first enzymolysis reaction; after the first enzymolysis reaction is finished, adjusting the pH value to be alkaline, then adding alkaline protease to obtain an enzymolysis system II, and carrying out a second enzymolysis reaction; after the second enzymolysis reaction is finished, centrifuging to obtain a solid phase, and washing the solid phase by using an ethanol solution to obtain a precipitate after enzymolysis;
and (3): adding an ethanol solution into the precipitate after enzymolysis to obtain an extraction system, carrying out ultrasonic treatment on the extraction system, filtering to obtain a liquid phase, and concentrating the liquid phase to obtain a crude extract;
and (4): adding the crude extract into water, and stirring until the crude extract is completely dissolved to obtain a purification system; extracting the purification system by using petroleum ether, and taking a water phase; then adding activated carbon into the water phase, stirring, standing, and filtering to obtain a liquid phase to obtain a purified liquid;
and (5): concentrating the purified solution to obtain pericarpium Citri Reticulatae viride extract.
By adopting the technical scheme, the technical principle is as follows: cellulase is used to locally disrupt the cell wall of the green hide powder, and then alkaline protease is used to enzymatically hydrolyze proteins present in the cell wall. Since part of the polyphenols bind to the proteins of the cell wall by hydrogen bonding, after the proteins are broken, the part of the phenolics is released. And then, fully eluting the released phenolic substances through the washing action of an ethanol solution, and then, fully extracting the precipitates after enzymolysis by using ethanol to fully extract the polyphenol substances in the green tangerine peel cell vacuole, wherein the polyphenol substances in the green tangerine peel vacuole comprise arbutin, helicidum, gastrodin and other phenolic glycoside compounds with high utilization values. And then fully purifying the crude extract through the degreasing action of petroleum ether and the decoloring action of activated carbon. Concentrating to obtain pericarpium Citri Reticulatae viride extract rich in phenolic glycoside compounds such as arbutin, helicidum and gastrodin.
The beneficial effect of this scheme lies in:
(1) the obtained pericarpium Citri Reticulatae viride extract has high content of phenolic glycoside compounds such as arbutin, helicid and gastrodin
The inventor researches and discovers that different polyphenols are distributed in green tangerine orange peel cells to a certain extent, some polyphenols are bonded on macromolecular substances such as proteins on cell walls through hydrogen bonds, and some polyphenols are distributed in vacuoles of the cells. The inventor also finds that phenolic glycoside substances such as helicid, arbutin and gastrodin are mainly distributed in vacuole, and the substances have higher biological activity and utilization value. According to the research result, the inventor designs a novel green tangerine peel polyphenol extraction method, sufficiently excludes non-target polyphenol compounds, and the obtained green tangerine peel extract has ideal content of phenolic glycosides such as helicid, arbutin and gastrodin. The inventor uses a proper amount of cellulase to locally destroy the cell wall, so that substances such as proteins on the cell wall are exposed, but the whole cell is not disintegrated, and structures such as vacuoles in the cell wall are not destroyed. And degrading the protein by using alkaline protease so as to release phenols bound on the protein, and separating polyphenol substances distributed on cell walls from green tangerine orange peel cells by washing with ethanol, thereby increasing the content of target components in the green tangerine orange peel extract.
(2) The extraction process is simple, complex extraction, purification and separation operations are not needed, reagents, raw materials and the like used in the process are easy to obtain, and the cost is low. In the prior art, ethanol and the like are generally directly used as solvents to extract polyphenol substances, the extraction process can extract the total polyphenol in green tangerine peel, but the green tangerine peel total polyphenol components are complex, one or more polyphenol compounds in the total polyphenol are required to be separated, a very complex extraction and column purification method is required, the operation process is long, the cost is high, and the method is not suitable for industrial production.
(3) After ultrasonic extraction, besides crude extracts, green tangerine peel residues can be obtained, and the green tangerine peel residues can be continuously used for preparing fertilizers or animal feeds without toxic and side effects because organic solvents with high toxicity are not used in the steps of enzymolysis, alcohol extraction and the like.
Further, in the step (1), fresh macadimia nut green peel is taken and cleaned, and then dried in the shade, so as to obtain the macadimia nut green peel material.
By adopting the technical scheme, the green tangerine peels are cleaned, so that the influence of impurities on the extraction process is avoided; the influence of the moisture in the green tangerine peel on the extraction efficiency can be avoided by drying the green tangerine peel in the shade.
Further, in the step (2), the using amount ratio of the green skin powder to the water is 1 g: (15-20) ml;
the mass concentration of the cellulase in the enzymolysis system I is 2-4%, the duration of the first enzymolysis reaction is 20-30min, and the temperature is 40-55 ℃;
after the first enzymolysis reaction is finished, the pH value is adjusted to 9.5-10.5, the mass concentration of the alkaline protease in the enzymolysis system II is 3-5%, the time duration of the second enzymolysis reaction is 40-70min, and the temperature is 40-55 ℃.
By adopting the technical scheme, in the first enzymolysis reaction, cellulase with the mass concentration of 2-4% is used for treating green tangerine peel powder for 20-30min, so that green tangerine peel cell walls can be locally damaged but not completely disintegrated, proteins and other substances on the cell walls are exposed, vacuole and other structures in cells are kept complete, target components in the vacuole cannot be released in advance, and non-target polyphenol substances can be separated.
In the second enzymolysis reaction, 3-5% of alkaline protease is used for treating the green skin powder for 40-70min, so that the exposed protein can be fully degraded, and the release of non-target polyphenols is promoted. The pH value of 9.5-10.5 is the condition of alkaline protease reaction and can also stop the catalytic action of the cellulase. Because the enzymolysis system II is alkaline, the protein is easy to dissolve in alkaline solution, and partial protein impurities can be removed in the step.
Further, in the step (2), after the enzymolysis reaction is finished, centrifuging at 3000-; the method for washing the solid phase with ethanol solution is: adding the solid phase into an ethanol solution with the volume fraction of 80-95% to obtain a washing system, wherein the use ratio of the solid phase to the ethanol solution is 1 g: (6-15) ml; stirring the washing system for 30-60min, and then filtering to obtain a solid phase to obtain a precipitate after enzymolysis.
By adopting the technical scheme, ethanol is used as a solvent, and the washing process is assisted by stirring operation, so that the non-target polyphenols can be sufficiently eluted from the solid phase. Meanwhile, the 80-95% ethanol solution also has a certain degreasing effect, which is beneficial to improving the content of the target component in the final product.
Further, in the step (3), ethanol solution with volume fraction of 50-60% is added into the precipitate after enzymolysis to obtain an extraction system, and the dosage ratio of the precipitate after enzymolysis to the ethanol solution is 1 g: (15-20) ml.
By adopting the technical scheme, the 50-60% ethanol solution can effectively dissolve the polyphenols in the vacuole of the cells. If the volume fraction of the ethanol solution is less than 50%, the solvent may dissolve more polysaccharides, which is disadvantageous in increasing the content of the objective component in the product. If the volume fraction of the ethanol solution is more than 60%, it may result in a large amount of low-polar substances dissolved in the solvent and also may result in a decrease in the content of the objective component in the product.
Further, in the step (3), the time period of the ultrasonic treatment of the extraction system is 40-60min, and the ultrasonic power is 250-300W.
By adopting the technical scheme, phenolic glycosides such as helicid, arbutin and gastrodin can be effectively extracted. If the ultrasonic time is too long and the power is too high, the molecular motion is aggravated, a large amount of heat is generated, the molecular structure of the target component is damaged, and other impurities are continuously dissolved out, so that the extraction rate is reduced. If the ultrasonic time is too short and the power is too low, the cell structure cannot be effectively destroyed, the target component cannot be effectively dissolved out, and the extraction rate is also reduced.
Further, in the step (4), the dosage ratio of the crude extract to water is 1 g: (12-15) ml; the volume ratio of the petroleum ether to the purification system is 1:1, and the extraction process is repeated for 2-3 times.
By adopting the technical scheme, the petroleum ether extraction and purification system is used for multiple times, and fat-soluble substances in the purification system can be removed, so that the content of target components is improved.
Further, in the step (4), the mass percent of the activated carbon in the water phase is 0.5-2.5%, the stirring time is 60-90min, and the standing time is 10-20 min.
By adopting the technical scheme, the influence of pigments such as chlorophyll on the color of the green tangerine peel extract can be fully reduced, and the active carbon has a certain adsorption effect on substances such as polysaccharide and the like, so that a small amount of polysaccharide mixed into a water phase can be removed.
Further, an application of the macadimia nut green husk extract in preparing health-care food.
By adopting the technical scheme, the extract of the green tangerine orange peel of the macadimia nut is rich in phenolic glycosides such as helicid and gastrodin, the helicid and the gastrodin have the effects of helping sleep, tranquilizing and allaying excitement, relieving mental stress and the like, and the green tangerine orange peel extract is subjected to deep processing to obtain the health-care food with the function of helping sleep.
Further, an application of the macadimia nut green husk extract in preparing cosmetics.
By adopting the technical scheme, the macadimia nut green tangerine peel extract is rich in phenol glycoside substances such as arbutin and the like, the arbutin has the effects of repairing damage after sun and whitening, and the green tangerine peel extract is subjected to deep processing to obtain the cosmetic with the effects of whitening and repairing after sun.
Detailed Description
The following is further detailed by the specific embodiments, wherein:
example 1: example of preparation of extract of green husk of macadamia nut
The preparation method of the macadimia nut green husk extract comprises the following steps:
step (1): cleaning fresh green tangerine peels of macadimia nuts, drying in the shade, crushing, and sieving with a 40-mesh sieve to obtain green tangerine peel powder;
step (2): adding 20ml of water into every 1g of pericarpium Citri Reticulatae viride powder, adding cellulase (aladdin, C298989, CAS number: 9012-54-8) to obtain enzymolysis system I, and performing first enzymolysis reaction. In the enzymolysis system I, the mass concentration of cellulase is 3%, the enzymolysis reaction time is 30min, and the temperature is 50 ℃.
After the first enzymolysis reaction, the pH value is adjusted to 9.5, and then alkaline protease (Meclin, P824138, CAS number: 9014-01-1) is added to obtain an enzymolysis system II for the second enzymolysis reaction. The mass concentration of the alkaline protease in the enzymolysis system II is 5%, the time of the second enzymolysis reaction is 55min, and the temperature is 40 ℃.
Centrifuging at 3000rpm for 20min after the second enzymolysis reaction is finished, and taking a solid phase to obtain a precipitate A. And adding the precipitate A into an ethanol solution with the volume fraction of 80% to obtain a washing system. Wherein the dosage ratio of the sediment A to the ethanol solution is 1 g: 10ml, stirring and washing the system for 60min, and filtering to obtain a solid phase to obtain a precipitate B.
And (3): adding an ethanol solution with the volume fraction of 60% into the precipitate B to obtain an extraction system, wherein the dosage ratio of the precipitate B to the ethanol solution is 1 g: 20 ml. Treating the extraction system with ultrasonic wave, filtering to obtain liquid phase, and concentrating with rotary evaporator (at 50 deg.C and vacuum degree of-0.05 Mpa for 5 hr) to obtain crude extract. The ultrasonic treatment extraction system has the time length of 60min and the ultrasonic power of 250W, and the ultrasonic process is carried out at room temperature.
And (4): adding the crude extract into water, stirring until the crude extract is completely dissolved to obtain a purification system, wherein the dosage ratio of the crude extract to the water is 1 g: 12 ml. Extracting and purifying a system by using petroleum ether at 50 ℃, wherein the volume ratio of the petroleum ether to the purification system is 1:1, repeating the extraction process for 3 times, combining the water phase parts obtained by each extraction, discarding the petroleum ether phase, and obtaining the combined water phase as the purification solution A. And then adding activated carbon into the purified liquid A, wherein the mass percent of the activated carbon in the purified liquid A is 2%, stirring for 60min, standing for 10min, and then filtering to obtain a liquid phase part, thus obtaining a purified liquid B.
And (5): performing reduced pressure evaporation treatment on the purified solution B for 4h by using a rotary evaporator under the conditions that the temperature is 50 ℃ and the vacuum degree is-0.05 Mpa to obtain a concentrate A, and then evaporating the concentrate A to dryness by using a reduced pressure distillation device under the conditions that the pressure is 0.1Mpa and the temperature is 45 ℃ to obtain a dry powder green tangerine peel extract.
Example 2:
example of preparation of extract of green husk of macadamia nut
The preparation method of the macadimia nut green husk extract comprises the following steps:
step (1): cleaning fresh green tangerine peels of macadimia nuts, drying in the shade, crushing, and sieving with a 40-mesh sieve to obtain green tangerine peel powder;
step (2): adding 15ml of water into every 1g of pericarpium Citri Reticulatae viride powder, adding cellulase (aladdin, C298989, CAS number: 9012-54-8) to obtain enzymolysis system I, and performing first enzymolysis reaction. In the enzymolysis system I, the mass concentration of cellulase is 2%, the enzymolysis reaction time is 30min, and the temperature is 40 ℃.
After the first enzymolysis reaction is finished, the pH value is adjusted to 10.5, and then alkaline protease (Meclin, P824138, CAS number: 9014-01-1) is added to obtain an enzymolysis system II for the second enzymolysis reaction. The mass concentration of the alkaline protease in the enzymolysis system II is 5%, the time of the second enzymolysis reaction is 40min, and the temperature is 40 ℃.
Centrifuging at 3000rpm for 30min after the second enzymolysis reaction is finished, and taking a solid phase to obtain a precipitate A. And adding the precipitate A into an ethanol solution with the volume fraction of 95% to obtain a washing system. Wherein the dosage ratio of the sediment A to the ethanol solution is 1 g: 15ml, stirring and washing the system for 30min, and filtering to obtain a solid phase to obtain a precipitate B.
And (3): adding 50% ethanol solution into the precipitate B to obtain an extraction system, wherein the dosage ratio of the precipitate B to the ethanol solution is 1 g: 20 ml. Treating the extraction system with ultrasonic wave, filtering to obtain liquid phase, and concentrating with rotary evaporator (at 50 deg.C and vacuum degree of-0.05 Mpa for 5 hr) to obtain crude extract. The ultrasonic treatment extraction system has the time length of 60min and the ultrasonic power of 250W, and the ultrasonic process is carried out at room temperature.
And (4): adding the crude extract into water, stirring until the crude extract is completely dissolved to obtain a purification system, wherein the dosage ratio of the crude extract to the water is 1 g: 12 ml. Extracting and purifying a system by using petroleum ether at 50 ℃, wherein the volume ratio of the petroleum ether to the purification system is 1:1, repeating the extraction process for 3 times, combining the water phase parts obtained by each extraction, discarding the petroleum ether phase, and obtaining the combined water phase as the purification solution A. And then adding activated carbon into the purified liquid A, wherein the mass percent of the activated carbon in the purified liquid A is 2.5%, stirring for 60min, standing for 20min, and then filtering to obtain a liquid phase part, thus obtaining a purified liquid B.
And (5): performing reduced pressure evaporation treatment on the purified solution B for 4h by using a rotary evaporator under the conditions that the temperature is 50 ℃ and the vacuum degree is-0.05 Mpa to obtain a concentrate A, and then evaporating the concentrate A to dryness by using a reduced pressure distillation device under the conditions that the pressure is 0.1Mpa and the temperature is 45 ℃ to obtain a dry powder green tangerine peel extract.
Example 3: example of preparation of extract of green husk of macadamia nut
The preparation method of the macadimia nut green husk extract comprises the following steps:
step (1): cleaning fresh green tangerine peels of macadimia nuts, drying in the shade, crushing, and sieving with a 40-mesh sieve to obtain green tangerine peel powder;
step (2): adding 18ml of water into every 1g of pericarpium Citri Reticulatae viride powder, adding cellulase (aladdin, C298989, CAS number: 9012-54-8) to obtain enzymolysis system I, and performing first enzymolysis reaction. In the enzymolysis system I, the mass concentration of cellulase is 4%, the enzymolysis reaction time is 25min, and the temperature is 55 ℃.
After the first enzymolysis reaction is finished, the pH value is adjusted to 10.0, and then alkaline protease (Meclin, P824138, CAS number: 9014-01-1) is added to obtain an enzymolysis system II for the second enzymolysis reaction. The mass concentration of the alkaline protease in the enzymolysis system II is 4%, the time of the second enzymolysis reaction is 65min, and the temperature is 50 ℃.
Centrifuging at 3000rpm for 30min after the second enzymolysis reaction is finished, and taking a solid phase to obtain a precipitate A. And adding the precipitate A into an ethanol solution with the volume fraction of 90% to obtain a washing system. Wherein the dosage ratio of the sediment A to the ethanol solution is 1 g: 10ml, stirring and washing the system for 50min, and filtering to obtain a solid phase to obtain a precipitate B.
And (3): adding an ethanol solution with the volume fraction of 60% into the precipitate B to obtain an extraction system, wherein the dosage ratio of the precipitate B to the ethanol solution is 1 g: 18 ml. Treating the extraction system with ultrasonic wave, filtering to obtain liquid phase, and concentrating with rotary evaporator (at 50 deg.C and vacuum degree of-0.05 Mpa for 5 hr) to obtain crude extract. The ultrasonic treatment extraction system has the time length of 50min and the ultrasonic power of 300W, and the ultrasonic process is carried out at room temperature.
And (4): adding the crude extract into water, stirring until the crude extract is completely dissolved to obtain a purification system, wherein the dosage ratio of the crude extract to the water is 1 g: 15 ml. Extracting and purifying a system by using petroleum ether at 50 ℃, wherein the volume ratio of the petroleum ether to the purification system is 1:1, repeating the extraction process for 3 times, combining the water phase parts obtained by each extraction, discarding the petroleum ether phase, and obtaining the combined water phase as the purification solution A. And then adding active carbon into the purified liquid A, wherein the mass percent of the active carbon in the purified liquid A is 0.5%, stirring for 90min, standing for 20min, and then filtering to obtain a liquid phase part to obtain a purified liquid B.
And (5): performing reduced pressure evaporation treatment on the purified solution B for 4h by using a rotary evaporator under the conditions that the temperature is 50 ℃ and the vacuum degree is-0.05 Mpa to obtain a concentrate A, and then evaporating the concentrate A to dryness by using a reduced pressure distillation device under the conditions that the pressure is 0.1Mpa and the temperature is 45 ℃ to obtain a dry powder green tangerine peel extract.
Comparative example 1
This comparative example is substantially the same as example 1, except that in step (2), cellulase is not used in this example, and the details are as follows:
step (2): adding 20ml of water into every 1g of green tangerine peel powder, adjusting the pH value to 9.5, and then adding alkaline protease to obtain an enzymolysis system for enzymolysis reaction. The mass concentration of the alkaline protease in the enzymolysis system II is 5%, the enzymolysis reaction time is 55min, and the temperature is 40 ℃. Centrifuging at 3000rpm for 20min after the enzymolysis reaction is finished, and taking a solid phase to obtain a precipitate A. Adding the obtained precipitate A into an ethanol solution with the volume fraction of 80 percent to obtain a washing system. Wherein the dosage ratio of the sediment A to the ethanol solution is 1 g: 10ml, stirring and washing the system for 60min, and filtering to obtain a solid part to obtain a precipitate B.
Comparative example 2
This comparative example is substantially the same as example 1, except that in step (2), alkaline protease is not used in this example, and the details are as follows:
step (2): adding 20ml of water into 1g of pericarpium Citri Reticulatae viride powder, adding cellulase (aladdin, C298989, CAS number: 9012-54-8) to obtain an enzymolysis system, and performing enzymolysis reaction. The mass concentration of the cellulase in an enzymolysis system is 3 percent, the enzymolysis reaction time is 30min, and the temperature is 50 ℃.
After the enzymolysis reaction is finished, centrifuging at 3000rpm for 20min, and taking a solid phase to obtain a precipitate A. And adding the precipitate A into an ethanol solution with the volume fraction of 80% to obtain a washing system. Wherein the dosage ratio of the sediment A to the ethanol solution is 1 g: 10ml, stirring and washing the system for 60min, and filtering to obtain a solid phase to obtain a precipitate B.
Comparative example 3
This comparative example is substantially the same as example 1, except that in step (2), no ethanol solution is used for washing after the second enzymatic hydrolysis, as follows:
step (2): adding 20ml of water into every 1g of pericarpium Citri Reticulatae viride powder, adding cellulase (aladdin, C298989, CAS number: 9012-54-8) to obtain enzymolysis system I, and performing first enzymolysis reaction. In the enzymolysis system I, the mass concentration of cellulase is 3%, the enzymolysis reaction time is 30min, and the temperature is 50 ℃.
After the first enzymolysis reaction, the pH value is adjusted to 9.5, and then alkaline protease (Meclin, P824138, CAS number: 9014-01-1) is added to obtain an enzymolysis system II for the second enzymolysis reaction. The mass concentration of the alkaline protease in the enzymolysis system II is 5%, the time of the second enzymolysis reaction is 55min, and the temperature is 40 ℃.
Centrifuging at 3000rpm for 20min after the second enzymolysis reaction is finished, and taking a solid phase to obtain a precipitate A. Precipitate a was used directly in the subsequent ultrasonic extraction.
Comparative example 4
The comparative example is basically the same as example 1, but is different from the comparative example in that step (2) is not arranged, and ultrasonic extraction is directly performed on green tangerine peel powder, which is a conventional method in the prior art, and specifically comprises the following steps:
an ethanol solution with a volume fraction of 60% was directly added to the green sheet powder, followed by ultrasonic extraction and purification in the same manner as in steps (3) to (5) of example 1.
Comparative example 5
This comparative example is substantially the same as example 1 except that: the dosage of the cellulase is 6 percent, and the enzymolysis reaction lasts for 90 min.
Comparative example 6
This comparative example is substantially the same as example 1 except that: the dosage of the cellulase is 1 percent, and the enzymolysis reaction lasts for 10 min.
Experimental example: measurement of extraction amount
The extraction rate of the green tangerine peel extract in the examples 1-3 and the comparative examples 1-4 is calculated by the following method: the total extraction amount (mg/100g) is equal to the mass (mg) of the green tangerine peel extract/the mass (g) of the green tangerine peel powder × 100, and the calculation results are shown in table 1.
The green tangerine orange peel extracts of examples 1-3 and comparative examples 1-4 were subjected to HPLC assay. The helicid standard (purity more than 98%), arbutin standard (purity more than 98%) and gastrodin standard (purity more than 98%) used in the experiment were purchased from Ficus organisms. Precisely weighing 10mg of each of helicid, gastrodine and arbutin standard substances, respectively placing in 10mL volumetric flasks to make each 1mL contain 1mg of standard substance, adding acetonitrile to desired volume, shaking up to obtain reference solution, and keeping. 10mg of the green tangerine peel extract obtained in examples 1 to 3 and comparative examples 1 to 4 was put in a 100mL volumetric flask, and acetonitrile was added to the flask to a constant volume to prepare a test solution for future use. Accurately sucking 5 μ L of sample solution, and injecting into high performance liquid chromatograph for chromatographic determination. The chromatographic conditions are as follows: the chromatograph is an Agilent 1200 series, the chromatographic column is Eclipse XDB-C18 (4.6X 150mm,5 mu m), the column temperature is 25 ℃, and the mobile phase is acetonitrile: water 10: 90(V/V), the ultraviolet detection wavelength is 270nm, the sample injection amount is 5 mu l, and the flow rate is 1 mL/min. The results of the assay of helicid, gastrodine and arbutin in the green tangerine peel extract are shown in table 1, and are expressed in mg/100g, that is, the amount (mg) of the target component (helicid, gastrodine or arbutin) contained in each 100g of green tangerine peel powder.
The green tangerine orange peel extracts of examples 1-3 and comparative examples 1-4 were tested for total polyphenol content. The gallic acid standard (purity greater than 98%) used in the experiment was purchased from a flying organism. Using gallnutPreparing acid standard substance into standard solutions of 0, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0, 5.0, 8.0 and 10.0mg/mL, respectively taking 5mL of each standard solution with the above concentration, respectively adding 2mL of 10% forskolin phenol reagent and 2mL of 10% Na2CO3Mixing the solutions, reacting in dark for 45min, measuring absorbance at 760nm with standard solution without gallic acid as blank control, and making standard curve. 1mg of the extract of green tangerine orange peel obtained in examples 1-3 and comparative examples 1-4 was dissolved in 4mL of water (using a 10mL volumetric flask), and 2mL of 10% forskolin phenol reagent and 2mL of 10% Na were added2CO3Mixing the solutions, diluting to 10ml, reacting in dark for 45min, measuring 760nm absorbance, and calculating total polyphenol extraction amount (mg/100g) in pericarpium Citri Reticulatae viride extract of examples 1-3 and comparative examples 1-4 according to gallic acid standard curve, wherein each 100g of pericarpium Citri Reticulatae viride powder contains target component (total polyphenol).
In table 1, part of the calculation is as follows:
the ratio of helicid to total extract (R1): helicid extract/total extract × 100%
The ratio of helicid to total polyphenol (R1') -helicid extract/total polyphenol extract × 100%
The ratio of arbutin to total extract (R2) ═ arbutin extraction amount/total extraction amount × 100%
The ratio of arbutin to total polyphenol (R2') -arbutin extraction/total polyphenol extraction × 100%
The gastrodine accounts for 100% of the total extract (R3) ═ gastrodine extract/total extract
The gastrodine accounts for the total polyphenol ratio (R3') -gastrodine extract/total polyphenol extract × 100%
Table 1: measurement results of Green Tangerine Peel extract content in examples 1-3 and comparative examples 1-4
In examples 1-3, the content of three phenolic glycosides (helicid, arbutin and gastrodine) in the green tangerine peel extract is high.
Comparative example 1 does not use cellulase for enzymolysis, alkaline protease is directly added, and as the green tangerine peel cell wall is not damaged, the alkaline protease cannot fully act on the protein located at the cell wall, and the components such as non-target polyphenols and the like cannot be effectively removed, so that although the total extract and the total polyphenols are high, the extraction amount of helicid, arbutin and gastrodine in the green tangerine peel extract is not improved. Comparative example 6, in which only a small amount of cellulase was used for the enzymatic hydrolysis and the enzymatic hydrolysis time was short, similar to comparative example 1 was also found.
Comparative example 2 without degrading proteins with alkaline protease, resulting in the preparation without a step of removing undesired polyphenols, the total extract content and total polyphenol content of the present preparation scheme were high, but the content of the desired ingredient was low.
Comparative example 3 does not contain an alcohol washing step, resulting in the residue of undesired polyphenols on green tangerine peel cells, and reduced contents of helicid, arbutin and gastrodin in green tangerine peel extract.
Comparative example 4 does not use an enzymolysis step before ultrasonic extraction, non-target substances are not removed, although the content of the total extract and the content of the total polyphenol are higher, the content of helicid, arbutin and gastrodin in the green tangerine peel extract is not improved, which indicates that the method of comparative example 4 extracts more non-target components (more impurities) and causes greater interference on the subsequent separation and purification of target compounds such as helicid, arbutin and gastrodin.
In comparative example 5, the amount of cellulase used was large and the time of enzymolysis was long, the cells were sufficiently destroyed, a large amount of the cell contents were dissolved out, and the objective component was not encapsulated in the cells but was dissolved out in advance. The subsequent steps of ultrasonic extraction and the like are carried out on the sediment part, most of the target components are dissolved out and are not in the sediment, so that a large amount of impurities are extracted in the comparative example, and the target components are very few.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. The preparation method of the extract of the green tangerine orange peel of the macadimia nut is characterized by comprising the following steps:
step (1): crushing macadimia nut green peel material to obtain green peel powder;
step (2): adding water into pericarpium citri reticulatae viride powder, adding cellulase to obtain an enzymolysis system I, and carrying out a first enzymolysis reaction; after the first enzymolysis reaction is finished, adjusting the pH value to be alkaline, then adding alkaline protease to obtain an enzymolysis system II, and carrying out a second enzymolysis reaction; after the second enzymolysis reaction is finished, centrifuging to obtain a solid phase to obtain a precipitate A, and washing with an ethanol solution to obtain a precipitate B;
and (3): adding an ethanol solution into the precipitate B to obtain an extraction system, carrying out ultrasonic treatment on the extraction system, filtering to obtain a liquid phase, and concentrating to obtain a crude extract;
and (4): adding the crude extract into water, and stirring until the crude extract is completely dissolved to obtain a purification system; extracting the purification system by using petroleum ether, and taking a water phase to obtain a purified liquid A; then adding activated carbon into the purified liquid A, stirring, standing, and filtering to obtain a liquid phase to obtain a purified liquid B;
and (5): and concentrating the purified solution B to obtain a green tangerine peel extract.
2. The method of claim 1, wherein in step (1), the green husk of macadimia nut is washed and dried in the shade to obtain the material of the green husk of macadimia nut.
3. The method of claim 1, wherein in the step (2), the ratio of the green skin powder to the water is 1 g: (15-20) ml;
the mass concentration of the cellulase in the enzymolysis system I is 2-4%, the duration of the first enzymolysis reaction is 20-30min, and the temperature is 40-55 ℃;
after the first enzymolysis reaction is finished, the pH value is adjusted to 9.5-10.5, the mass concentration of the alkaline protease in the enzymolysis system II is 3-5%, the time duration of the second enzymolysis reaction is 40-70min, and the temperature is 40-55 ℃.
4. The method for preparing an extract of green tangerine peel of macadimia nut as claimed in claim 3, wherein in step (2), after the completion of the enzymatic hydrolysis reaction, centrifugation is carried out at 3000 and 4000rpm for 20-30min, and then the solid phase is taken out to obtain precipitate A;
the method for washing the precipitate A by using the ethanol solution comprises the following steps: adding the precipitate A into an ethanol solution with the volume fraction of 80-95% to obtain a washing system; the dosage ratio of the sediment A to the ethanol solution is 1 g: (6-15) ml; stirring the washing system for 30-60min, and then filtering to obtain a solid phase to obtain a precipitate B.
5. The method of claim 1, wherein in step (3), 50-60% by volume of ethanol solution is added to the precipitate B to obtain an extraction system; the dosage ratio of the precipitate B to the ethanol solution is 1 g: (15-20) ml.
6. The method for preparing extract of green tangerine orange peel of macadimia nut as claimed in claim 5, wherein in the step (3), the duration of the ultrasonic treatment of the extraction system is 40-60min, and the ultrasonic power is 250-300W.
7. The method for preparing an extract of green tangerine orange peel of macadimia nut as claimed in claim 1, wherein in the step (4), the ratio of the crude extract to water is 1 g: (12-15) ml; the volume ratio of the petroleum ether to the purification system is 1:1, and the extraction process is repeated for 2-3 times.
8. The method of claim 7, wherein in the step (4), the activated carbon is added to the purified solution A in an amount of 0.5-2.5% by weight, the stirring time is 60-90min, and the standing time is 10-20 min.
9. Use of the method of any one of claims 1-8 for preparing an extract of green tangerine orange peel of macadimia nut in the preparation of a health food.
10. The use of the method of any one of claims 1-8 for the preparation of a cosmetic composition comprising an extract of green tangerine orange peel of macadamia nut.
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| CN113230174A (en) * | 2021-05-28 | 2021-08-10 | 西南林业大学 | Macadamia nut whitening extract and preparation method thereof |
| CN113230174B (en) * | 2021-05-28 | 2022-07-08 | 西南林业大学 | Macadamia nut whitening extract and preparation method thereof |
| CN113244150A (en) * | 2021-05-31 | 2021-08-13 | 西南林业大学 | Preparation containing helicid and preparation method and application thereof |
| CN114752585A (en) * | 2022-05-20 | 2022-07-15 | 山西大学 | Walnut green husk anti-tumor active protein and preparation method and application thereof |
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