Method for ultrasonic-enhanced extraction of multicoloured leaf extract by using ethanol and ethyl acetate cosolvent

Abstract

The invention relates to a method for ultrasonic-enhanced extraction of a multicoloured leaf extract by using an ethanol and ethyl acetate cosolvent, and belongs to the technical field of chemical engineering and plant extraction. (2) synergistically degrading cellulose by using multi-component glycosidase; (3) cosolvent extraction; 4, chloroplast pigment is efficiently separated and purified, chlorophyll and other impurities in an extracting solution are effectively separated and removed through silica gel column chromatography, and therefore the high purity of the multicoloured leaf extract is achieved; according to the invention, ethanol and ethyl acetate are used as cosolvents to extract the multicoloured leaf extract, so that the extraction efficiency and the purity of the extract can be improved.

Description

Technical Field

[0001] The invention relates to a method for ultrasonically strengthening the extraction of a pandan leaf extract by using ethanol and ethyl acetate as a co-solvent, and belongs to the technical fields of chemical engineering and plant extraction. Background Art

[0002] Pandanus amaryllifolius, also known as pandan leaf, is scientifically named Pandanus amaryllifolius Roxb. Its origin may be the Maluku Islands in Indonesia. Its cultivation has spread to Singapore, Malaysia, Sri Lanka, India, China, Thailand and other countries. Hainan is the origin and dominant production area of ​​pandan leaf cultivation in my country. The main planting areas are Wanning, Qionghai, Danzhou, Lingshui, Baoting, Ding'an and other places. Among them, Wanning is the main distribution area and traditional utilization area, with a planting area of ​​about 5,000 mu. At present, there is a rich culture of pandan leaf utilization in southern my country, which is mainly based on Nanyang culture and folk comprehensive utilization mode, and is widely used in special catering, ornamental gardening, leisure tourism and other industries.

[0003] Pandan leaf extract is widely used in the food industry, mainly due to its unique aroma and therapeutic value. For example, in Singapore and Malaysia, pandan leaves are widely used in making cakes, bread, ice cream, candy and other foods because of their natural aroma. In addition, pandan leaves are also used to add a fresh and sweet taste to food, especially in Nyonya dishes. However, since traditional methods of extracting pandan leaf extracts mostly use water or organic solvents, these methods often have problems such as low extraction efficiency and low purity of the extract.

[0004] Therefore, a new method for extracting Paniculata leaf extract using ethanol and ethyl acetate as co-solvents was explored in order to improve the extraction efficiency and the purity of the extract. Summary of the invention

[0005] The invention provides a method for ultrasonically enhanced extraction of a Panax notoginseng leaf extract by co-dissolving ethanol and ethyl acetate. The method not only improves the extraction efficiency, but also significantly improves the purity of the extract.

[0006] In order to achieve the above object, the present invention adopts the following technical solution, which specifically includes the following steps:

[0007] A method for ultrasonically enhancing the extraction of a Pandan leaf extract using ethanol and ethyl acetate as a co-solvent, comprising the following steps:

[0008] Step (i) pretreatment: soak fresh Pandan leaves or Pandan powder in an appropriate amount of distilled water to remove surface impurities;

[0009] Step (ii) synergistically degrading cellulose with multi-component glycosidases, placing the pandan leaves or pandan powder treated in step (i) in a solution of a composite enzyme preparation of cellulase and multiple glycosidase components to generate an enzymatic hydrolyzate and unenzymatically hydrolyzed solid residues;

[0010] Step (iii) co-solvent ultrasonic enhanced extraction: placing the solid residue material in a mixed solvent consisting of anhydrous ethanol and ethyl acetate binary system and a buffer solution, extracting the effective components therein through multiple rounds of heating extraction steps, and then ultrasonically enhancing the mixture at 40-60° C., filtering the mixture with a centrifuge to separate the solid residue and the liquid filtrate, and concentrating the liquid filtrate under reduced pressure to remove the solvent and obtain a preliminary extract;

[0011] Step (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract of Pandanus tinctorius leaves is treated by vacuum concentration technology to reduce its water content, and then the chlorophyll and other impurities in the extract are effectively separated and removed by silica gel column chromatography, thereby achieving high purity of the Pandanus tinctorius leaf extract.

[0012] Furthermore, the multi-component glycosidase used in step (ii) includes one or more of cellulase, cellobiase, xylanase, β-glucosidase, polyphenol oxidase and fructase.

[0013] Furthermore, in the step (ii), the volume ratio of the pandan leaves or pandan powder to the composite enzyme preparation solution is 1:2-1:20, and the enzymatic hydrolysis temperature is in the range of 40-75°C.

[0014] Furthermore, the concentration of the complex enzyme preparation solution in step (ii) is 0.1%-1.5%.

[0015] Furthermore, the composition ratio of the mixed solvent in the step (iii) is 70% to 75% by weight of ethanol, 20% to 30% by weight of ethyl acetate, and the buffer solution is 0.8% by weight of glycolic acid-sodium glycolate buffer solution.

[0016] Furthermore, in the step (iii), the heating extraction reaction temperature is controlled at 65-73° C., and the water bath heating time is 5 hours.

[0017] Furthermore, in the step (iii), the volume ratio of the solid residue material to the mixed solvent is 1:1, and the mixture is stirred evenly and then allowed to stand for a certain period of time.

[0018] The pandan leaf extract prepared by the above method is in the form of concentrated liquid / paste / powder. The pandan paste can be used for tobacco, baking, beverages, cold drinks, etc.; the pandan concentrate can be used for tobacco, beverages, cold drinks, baking, etc.; the pandan extract can be used for baking, cold drinks, instant drinks, etc.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention processes Pandan leaves and Pandan powder, and uses ethanol and ethyl acetate as co-solvents to extract Pandan leaf extract, which greatly improves the extraction efficiency and the purity of the extract.

[0021] 2. The binary co-solvents ethanol and ethyl acetate have better dissociation performance and separation effect. During separation, the binary mixed solvent has higher separation performance than a single solvent, which can better solve the contradiction between selectivity and solubility and improve separation efficiency and purity.

[0022] 3. Ultrasonic enhanced extraction is used to make the effective ingredients in the cells more easily dissolved in the solvent, so that the best extraction effect can be achieved in a shorter time, shortening the extraction time and improving productivity.

[0023] 4. Using multi-component glycosidase for decomposition and hydrolysis can increase the hydrolysis process. DETAILED DESCRIPTION

[0024] The present invention is further described with reference to the embodiments:

[0025] The present invention adopts ethanol / ethyl acetate co-solvent as an extractant to extract Paniculata. The ethanol / ethyl acetate co-solvent has better dissociation performance and separation effect. Ethanol can dissolve many organic substances and some inorganic substances, while ethyl acetate, as an organic solvent, has good solubility to certain specific organic substances, and glycolic acid-sodium glycolate buffer solution can maintain the pH stability of the system. Therefore, when they are used as co-solvents, the solubility range of the solvent can be widened to meet more diverse dissolution requirements. During separation, the binary mixed solvent has higher separation performance than a single solvent, can better solve the contradiction between selectivity and solubility, and improve separation efficiency and purity. In addition, the extraction is carried out in an ultrasonic enhanced manner, so that the effective components in the cell are more easily dissolved in the solvent, and the best extraction effect can be achieved in a relatively short time, the extraction time is shortened, and productivity is improved.

[0026] Embodiment 1:

[0027] (i) Pretreatment: Take 100 g of Pandan leaves, soak them in 500 g of distilled water, let them stand for 24 hours, then take them out and remove surface impurities.

[0028] (II) Multi-component glycosidase synergistically degrades cellulose: soak the soaked Pandan leaves in a solution of a composite enzyme preparation containing cellulase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 1%, the temperature is controlled at 40°C, the reaction time is 2 hours, the multi-component glycosidase includes cellulase, cellobiase, xylanase, glucan sucrase, and fructase, wherein the volume ratio of cellulase, cellobiase, xylanase, glucan sucrase, and fructase is 1:1:1:1:1:1. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:2; then, the mixture is filtered using a centrifuge to separate the enzymatic solution generated by the action of the glycosidase and the solid residue material that has not been enzymatically hydrolyzed. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0029] (III) Co-solvent ultrasonic enhanced extraction: The solid residue obtained in step (II) is mixed with 500g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the composition of the co-solvent is 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution is 0.8% by mass of glycolic acid-sodium glycolate buffer solution, and the heating extraction reaction temperature is controlled at 60°C, and the water bath heating time is used for 5 hours, and the mixture is stirred evenly and then allowed to stand for 2 hours. The residue is filtered, the filtrate is collected and vacuum concentrated to obtain a preliminary extract.

[0030] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated to 250 g by vacuum concentration to remove some of the water and chlorophyll and other impurities were removed by silica gel column chromatography to finally obtain 195 g of a 99% pure concentrated solution. The main chemical components in the concentrated solution were chlorophyll, neophytadiene, and linoleic acid, accounting for 23%, 18%, and 20%, respectively.

[0031] Embodiment 2:

[0032] (i) Pretreatment: Take 300 g of Pandan leaves, soak them in 900 g of distilled water, and take them out after standing for 24 hours.

[0033] (ii) Multi-component glycosidase synergistically degrades cellulose: soak the soaked Pandan leaves in a solution of a composite enzyme preparation containing cellulase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 0.1%, the temperature is controlled at 40°C, the reaction time is 2 hours, the multi-component glycosidase includes cellulase, cellobiase, xylanase, β-glucosidase, polyphenol oxidase, wherein the volume ratio of cellulase, cellobiase, xylanase, β-glucosidase, polyphenol oxidase added to the composite enzyme preparation solution is 1:1:1:1:1. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:10; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0034] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 900g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 73°C, and the water bath heating time was 5 hours. After stirring evenly, 20kHz ultrasonic waves were used at 60°C for 2 hours, and the ultrasonic wave was stopped and allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0035] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated to 350 g by vacuum concentration to remove some water and chlorophyll and other impurities by silica gel column chromatography, and then concentrated to a paste to obtain 80 g of a 99% pure Banlan concentrated paste. The main chemical components contained in the concentrated paste are phytol, neophytadiene, and linoleic acid, accounting for 25%, 20%, and 21% respectively.

[0036] Embodiment 3:

[0037] (i) Pretreatment: Take 500 g of Pandan leaves, soak them in 1000 g of distilled water, and take them out after standing for 24 hours.

[0038] (II) Synergistic degradation of cellulose by multi-component glycosidase: The soaked Pandan leaves are soaked in a solution of a composite enzyme preparation containing cellulase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 1.5%, the temperature is controlled at 40°C, and the reaction time is 2 hours. The multi-component glycosidase includes cellulase, xylanase, β-glucosidase, polyphenol oxidase, and fructase, wherein the volume ratio of cellulase, xylanase, β-glucosidase, polyphenol oxidase, and fructase added to the composite enzyme preparation solution is 1:1:1:1:1. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:15; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0039] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 1000g of ethanol-ethyl acetate co-solvent (volume ratio of 7:3) at a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 40°C, and the water bath was used for heating for 5 hours. After stirring evenly, it was allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0040] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated to 250 g by vacuum concentration to remove some water and chlorophyll and other impurities by silica gel column chromatography, and dried to finally obtain 50 g of colorful powder with a purity of 98%. The main chemical components contained in the powder are phytol, neophytadiene, and linoleic acid, accounting for 22%, 19%, and 18%, respectively.

[0041] Embodiment 4:

[0042] (i) Pretreatment: Take 100 g of Pandan leaves, soak them in 500 g of distilled water, and take them out after standing for 24 hours.

[0043] (II) Multi-component glycosidase synergistically degrades cellulose: soak the soaked Pandan leaves in a solution of a composite enzyme preparation containing xylanase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 1.5%, the temperature is controlled at 70°C, the reaction time is 2 hours, the multi-component glycosidase includes cellobiase, xylanase, β-glucosidase, polyphenol oxidase, and fructase, wherein the volume ratio of cellobiase, xylanase, β-glucosidase, polyphenol oxidase, and fructase added to the composite enzyme preparation solution is 1:1:1:1:1. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:20; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0044] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 500g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 55°C, and the water bath heating time was 5 hours. After stirring evenly, 20kHz ultrasonic waves were used at 50°C for 2 hours, and the ultrasonic wave was stopped and allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0045] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated to 230 g by vacuum concentration to remove some water and chlorophyll and other impurities were removed by silica gel column chromatography, and finally 180 g of a 99% pure concentrated solution was obtained. The main chemical components of the concentrated solution were phytol, neophytadiene, and linoleic acid, accounting for 24%, 18%, and 19%, respectively. After replacing cellulase with xylanase, the content of the main chemical components in the concentrated solution obtained in Case 1 did not change much.

[0046] Embodiment 5:

[0047] (i) Pretreatment: Take 300 g of Pandan leaves, soak them in 900 g of distilled water, and take them out after standing for 24 hours.

[0048] (II) Synergistic degradation of cellulose by multi-component glycosidase: The soaked Pandan leaves are soaked in a solution of a composite enzyme preparation containing xylanase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 1.0%, the temperature is controlled at 68°C, the reaction time is 2 hours, the multi-component glycosidase includes cellulase, cellobiase, β-glucosidase, polyphenol oxidase, and fructase, wherein the volume ratio of cellulase, cellobiase, β-glucosidase, polyphenol oxidase, and fructase added to the composite enzyme preparation solution is 1:1:1:1:1. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:18; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0049] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 900g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 60°C, and the water bath was used for heating for 5 hours. After stirring evenly, it was allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0050] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated by vacuum concentration to remove part of the water, concentrated to 300g, and impurities such as chlorophyll were removed by silica gel column chromatography, and then concentrated to a paste, and finally 60g of a colorful concentrated paste with a purity of 98% was obtained. The main chemical components contained in the concentrated paste are phytol, neophytadiene, and linoleic acid, accounting for 20%, 18%, and 20%, respectively. After replacing cellulase with xylanase, the content of phytol in the concentrated solution obtained in Case 2 decreased, while the content of neophytadiene and linoleic acid remained basically unchanged.

[0051] Embodiment 6:

[0052] (i) Pretreatment: Take 500 g of Pandan leaves, soak them in 1000 g of distilled water, and take them out after standing for 24 hours.

[0053] (ii) Multi-component glycosidase synergistically degrades cellulose: soak the Pandan leaves after soaking in a solution of a composite enzyme preparation containing xylanase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 1.5%, the temperature is controlled at 65°C, the reaction time is 2 hours, the multi-component glycosidase includes cellulase and cellobiase, wherein the volume ratio of cellulase and cellobiase added to the composite enzyme preparation solution is 1:1. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:5; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0054] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 1000g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 60°C, and the water bath heating time was 5 hours. After stirring evenly, 40kHz ultrasonic wave was used at 50°C for 2 hours, and the ultrasonic wave was stopped and allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0055] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated to 200 g by vacuum concentration to remove some of the water and chlorophyll and other impurities were removed by silica gel column chromatography. The final purity was 40 g of colorful powder with a purity of 99%. The main chemical components contained in the powder were phytol, neophytadiene, and linoleic acid, accounting for 24%, 23%, and 22%, respectively. After replacing cellulase with xylanase, the content of phytol in the concentrated solution obtained in Case 3 did not change much, while the content of neophytadiene and linoleic acid increased.

[0056] Embodiment 7:

[0057] (i) Pretreatment: Take 100 g of Pandan leaves, soak them in 800 g of distilled water, and take them out after standing for 24 hours.

[0058] (II) Synergistic degradation of cellulose by multi-component glycosidase: The soaked Pandan leaves are soaked in a solution of a composite enzyme preparation containing 0.1% xylanase, 1% cellulase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 1%, the temperature is controlled at 70°C, the reaction time is 2 hours, the multi-component glycosidase includes cellulase, β-glucosidase, polyphenol oxidase, and fructase, wherein the volume ratio of cellulase, β-glucosidase, polyphenol oxidase, and fructase added to the composite enzyme preparation solution is 1:1:1:1. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:2; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0059] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 800g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 40°C, and the water bath was used for heating for 5 hours. After stirring evenly, it was allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0060] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated to 300 g by vacuum concentration to remove some water and chlorophyll and other impurities were removed by silica gel column chromatography to finally obtain 150 g of 99% pure Banlan concentrate. The main chemical components contained in the concentrate were phytol, neophytadiene, and linoleic acid, accounting for 22%, 18%, and 21% respectively.

[0061] Embodiment 8:

[0062] (i) Pretreatment: Take 200 g of Pandan leaves, soak them in 1200 g of distilled water, and take them out after standing for 24 hours.

[0063] (II) Multi-component glycosidase synergistically degrades cellulose: soak the soaked Pandan leaves in a solution containing a composite enzyme preparation with a concentration of 0.2% xylanase, 1% cellulase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 0.2%, the temperature is controlled at 70°C, the reaction time is 2 hours, the multi-component glycosidase includes cellobiase, β-glucosidase, polyphenol oxidase, and fructase, wherein the volume ratio of cellobiase, β-glucosidase, polyphenol oxidase, and fructase added to the composite enzyme preparation solution is 1:1:1:1. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:12; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0064] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 1200g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 45°C, and the water bath heating time was 5 hours. After stirring evenly, 40kHz ultrasonic waves were used at 50°C for 2 hours, and the ultrasonic wave was stopped and allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0065] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated to 250 g by vacuum concentration to remove some water and chlorophyll and other impurities by silica gel column chromatography, and then concentrated to a paste to obtain 60 g of a 98% pure Banlan concentrated paste. The main chemical components contained in the concentrated paste are phytol, neophytadiene, and linoleic acid, accounting for 24%, 21%, and 22% respectively.

[0066] Embodiment 9:

[0067] (i) Pretreatment: Take 100 g of Pandan leaves, soak them in 1000 g of distilled water, and take them out after standing for 24 hours.

[0068] (II) Multi-component glycosidase synergistically degrades cellulose: soak the Pandan leaves in a solution containing a composite enzyme preparation with a concentration of 2% xylanase, 1% cellulase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 1.2%, the temperature is controlled at 65°C, the reaction time is 2 hours, the multi-component glycosidase includes cellulase, cellobiase, xylanase, β-glucosidase, polyphenol oxidase, and fructase, wherein the volume ratio of cellulase, cellobiase, xylanase, β-glucosidase, polyphenol oxidase, and fructase added to the composite enzyme preparation solution is 1:1:1:1:1:1, and the concentration of the composite enzyme preparation solution is 1.5%. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:20; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0069] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 1000g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 48°C, and the water bath was used for heating for 5 hours. After stirring evenly, it was allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0070] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated to 100 g by vacuum concentration to remove some water and chlorophyll and other impurities by silica gel column chromatography, and dried to finally obtain 20 g of colorful powder with a purity of 99%. The main chemical components contained in the powder are phytol, neophytadiene, and linoleic acid, accounting for 21%, 18%, and 20%, respectively.

[0071] Embodiment 10:

[0072] (i) Pretreatment: Take 100 g of Pandan leaves, soak them in 1000 g of distilled water, and take them out after standing for 24 hours.

[0073] (II) Multi-component glycosidase synergistically degrades cellulose: soak the Pandan leaves after soaking in a solution of a composite enzyme preparation containing 0.4% xylanase, 1% cellulase and multiple glycosidase components, the concentration of the composite enzyme preparation solution is 0.4%, the temperature is controlled at 65°C, the reaction time is 2 hours, the multi-component glycosidase includes cellulase, cellobiase, xylanase, β-glucosidase, polyphenol oxidase, and fructase, wherein the volume ratio of cellulase, cellobiase, xylanase, β-glucosidase, polyphenol oxidase, and fructase added to the composite enzyme preparation solution is 1:1:1:1:1:1, and the concentration of the composite enzyme preparation solution is 1.5%. The volume ratio of the Pandan leaves to the composite enzyme preparation solution is 1:15; then, the mixture is filtered using a centrifuge to separate the solid residue and the liquid filtrate. Then, the obtained filtrate is concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0074] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 1000g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, wherein the co-solvent composition was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution. The heating extraction reaction temperature was controlled at 55°C, and the water bath heating time was 5 hours. After stirring evenly, 40kHz ultrasonic waves were used at 50°C for 2 hours, and the ultrasonic wave was stopped and allowed to stand for 2 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0075] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated by vacuum concentration to remove part of the water, concentrated to 100g and removed impurities such as chlorophyll by silica gel column chromatography, 250g of cyclodextrin was added, dispersed evenly by a homogenizer, and spray dried to finally obtain 40g of colorful powder with a purity of 99%. The main chemical components contained in the powder are phytol, neophytadiene, and linoleic acid, accounting for 23%, 22%, and 20% respectively. Compared with Case 9, the content of phytol and linoleic acid in the colorful powder remains basically unchanged, and the content of neophytadiene has increased.

[0076] Comparative Example 1

[0077] (ii) Pretreatment: Take 100 g of Pandan leaves, soak them in 1000 g of distilled water, and take them out after standing for 24 hours.

[0078] (ii) Cellulase decomposition: The soaked Paniculata leaves were soaked in a solution containing 1% cellulase, the temperature was controlled at 37°C, and the reaction time was 2 hours. Subsequently, the mixture was filtered using a centrifuge to separate the solid residue and the liquid filtrate. Next, the obtained filtrate was concentrated under reduced pressure to remove the solvent and obtain a preliminary extract.

[0079] (III) Co-solvent ultrasonic enhanced extraction: The enzymatic residue was mixed with 1000g of ethanol-ethyl acetate co-solvent in a ratio of 1:1, the composition of the co-solvent system was 70% to 75% by mass of ethanol, 20% to 30% by mass of ethyl acetate, and the buffer solution was 0.8% by mass of glycolic acid-sodium glycolate buffer solution, the heating extraction reaction temperature was controlled at 73°C, and the water bath heating time was 5 hours. The residue was filtered, the filtrate was collected and vacuum concentrated to obtain a preliminary extract.

[0080] (IV) Efficient separation and purification of chloroplast pigments: The preliminary extract was concentrated by vacuum concentration to remove part of the water, concentrated to 100g and removed impurities such as chlorophyll by silica gel column chromatography, 250g of cyclodextrin was added, dispersed evenly by a homogenizer, and spray dried to finally obtain 40g of colorful powder with a purity of 99%. The main chemical components contained in the powder are phytol, neophytadiene, and linoleic acid, accounting for 10%, 8%, and 13% respectively.

[0081] The experimental data of Examples 1 to 10 and the comparative example are shown in Table 1:

[0082]

[0083] Table 1

[0084] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for ultrasonically enhancing the extraction of Pandanus variegata leaf extract using ethanol and ethyl acetate as a co-solvent, characterized in that: The steps include: Step (i) pretreatment: soak fresh Pandan leaves or Pandan powder in an appropriate amount of distilled water to remove surface impurities; Step (ii) synergistically degrading cellulose with multi-component glycosidases, placing the pandan leaves or pandan powder treated in step (i) in a solution of a composite enzyme preparation of cellulase and multiple glycosidase components to generate an enzymatic hydrolyzate and unenzymatically hydrolyzed solid residues; Step (iii) co-solvent ultrasonic enhanced extraction: placing the solid residue material in a mixed solvent consisting of anhydrous ethanol and ethyl acetate binary system and a buffer solution, extracting the effective components therein through multiple rounds of heating extraction steps, and then ultrasonically enhancing the mixture at 40 to 60° C., filtering the mixture with a centrifuge to separate the solid residue and the liquid filtrate, and concentrating the liquid filtrate under reduced pressure to remove the solvent and obtain a preliminary extract; Step (iv) Highly efficient separation and purification of chloroplast pigments: The preliminary extract described in step (iii) is treated by vacuum concentration technology to reduce its water content, and then the chlorophyll and other impurities in the extract are effectively separated and removed by silica gel column chromatography, thereby achieving high purity of the Pandan leaf extract.

2. The method for ultrasonic enhanced extraction of Pandanus pandanus leaf extract using ethanol and ethyl acetate co-solvent as claimed in claim 1, characterized in that: The multi-component glycosidase in step (ii) includes one or more of cellulase, cellobiase, xylanase, β-glucosidase, polyphenol oxidase and fructase.

3. The method for ultrasonic enhanced extraction of Pandanus pandanus leaf extract using ethanol and ethyl acetate co-solvent as claimed in claim 1, characterized in that: In the step (ii), the volume ratio of the pandan leaves or pandan powder to the composite enzyme preparation solution is 1:2-1:20, and the enzymatic hydrolysis temperature is in the range of 40-70°C.

4. The method for ultrasonic enhanced extraction of Pandanus pandanus leaf extract using ethanol and ethyl acetate co-solvent as claimed in claim 1, characterized in that: The concentration of the complex enzyme preparation solution in step (ii) is 0.1%-1.5%.

5. The method for ultrasonic enhanced extraction of Pandanus pandanus leaf extract using ethanol and ethyl acetate co-solvent as claimed in claim 1, characterized in that: The composition of the mixed solvent in the step (iii) is that the mass proportion of ethanol is 70% to 75%, the mass proportion of ethyl acetate is 20% to 30%, and the mass proportion of the buffer solution is 0.8% glycolic acid-sodium glycolate buffer solution.

6. The method for ultrasonic enhanced extraction of Pandanus pandanus leaf extract using ethanol and ethyl acetate as co-solvent according to claim 1, characterized in that: In the step (iii), the heating extraction reaction temperature is controlled at 65-73° C., and the water bath heating time is 5 hours.

7. The method for ultrasonic enhanced extraction of Pandanus pandanus leaf extract using ethanol and ethyl acetate as co-solvents as claimed in claim 1, characterized in that: In the step (iii), the volume ratio of the solid residue material to the mixed solvent is 1:1, and the mixture is stirred evenly and then allowed to stand for a certain period of time.

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