阅读说明：本技术 一种澳洲坚果叶片提取物的制备方法及其应用 (Preparation method and application of macadamia nut leaf extract ) 是由 徐斌 施蕊 万举河 卢淼 马宁 李彪 于 2019-10-31 设计创作，主要内容包括：本发明属于植物天然产物提取领域,具体涉及一种澳洲坚果叶片提取物的制备方法及其应用。本制备方法包括超声辅助的水提醇沉的步骤,并且利用凝胶柱层析将酚酸类物质除去,再经过脱盐处理,可获得多糖含量较高,不含有酚酸类物质的澳洲坚果叶片提取物。该提取方法可以为澳洲坚果叶片的综合利用创造条件,大量的叶片资源可以不再被弃用,可以被用于制备含有植物多糖的食品或者保健品。(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 leaf extract. The preparation method comprises the steps of water extraction and alcohol precipitation assisted by ultrasound, phenolic acid substances are removed by utilizing gel column chromatography, and the macadimia nut leaf extract with high polysaccharide content and no phenolic acid substances can be obtained through desalination treatment. The extraction method can create conditions for comprehensive utilization of macadimia nut leaves, and a large amount of leaf resources can not be discarded any more, and can be used for preparing food or health care products containing plant polysaccharides.)

1. A preparation method of a macadamia nut leaf extract is characterized by comprising the following steps:

step (1): pretreating macadimia nut leaves to obtain leaf powder;

step (2): soaking the leaf powder in an ethanol solution, and then filtering to obtain a solid phase to obtain degreased leaf powder; adding the degreased leaf powder into water to obtain an aqueous extraction system, carrying out ultrasonic treatment on the aqueous extraction system, and then centrifuging to obtain a liquid phase to obtain an extracting solution; adding active carbon into the extract, stirring, standing, filtering, and collecting liquid phase to obtain decolorized extract; concentrating the decolorized extract, adding ethanol solution, standing, centrifuging, and collecting solid phase to obtain precipitate;

and (3): dissolving the precipitate in water, filtering to obtain liquid phase to obtain solution A; adjusting the pH value of the solution A to be alkaline, then loading the solution A to a DEAE sepharose gel chromatographic column, standing, eluting the chromatographic column by water, and removing eluent; eluting the chromatographic column with sodium chloride solution, and retaining the eluent to obtain polysaccharide liquid; concentrating the polysaccharide solution to obtain an extract;

and (4): dissolving the extract in water, filtering to obtain liquid phase to obtain solution B; loading the solution B into a sephadex chromatographic column, standing, eluting the chromatographic column with water, and retaining an eluent to obtain a desalted polysaccharide solution;

and (5): concentrating the desalted polysaccharide solution to obtain the leaf extract.

2. The method of claim 1, wherein in step (1), the macadimia nut leaves are washed, dried in the shade, and ground to obtain a leaf powder.

3. The method of claim 1, wherein in the step (2), the leaf powder is soaked in 90-98% ethanol solution at 30-40 ℃ for 2-3h, and the ratio of the leaf powder to the ethanol solution is 1 g: (5-8) ml, filtering to obtain a solid phase, and volatilizing ethanol to obtain defatted leaf powder.

4. The method of claim 3, wherein in the step (2), the ratio of the defatted leaf powder to water in the aqueous extraction system is 1 g: (20-30) ml; the power of ultrasonic treatment is 200-300W, the time duration is 60-90min, and the temperature of a water extraction system is maintained at 70-80 ℃; after the ultrasonic treatment is finished, centrifuging at 3000-.

5. The method of claim 4, wherein in the step (2), the activated carbon is present in the extract in an amount of 2-5% by weight, and the stirring time is 20-30min and the standing time is 10-20 min.

6. The method of claim 5, wherein in step (2), a 95% ethanol solution is added to the concentrated decolorized extract, and the volume ratio of the concentrated decolorized extract to the 95% ethanol solution is 1: (4-6), standing at 4-6 ℃ for 24-36h, centrifuging at 3000-4000rpm for 20-30min, and taking a solid phase to obtain a precipitate.

7. The method of claim 1, wherein in step (3), the pH of solution A is adjusted to 7.8-8.5, and then the solution A is loaded on a DEAE sepharose chromatography column, and after standing for 1.5-2h, the column is eluted with 6-8 times of column volume of water, and the eluate is discarded; then eluting the chromatographic column by using 2-3 times of the column volume of 0.1-0.5mol/L sodium chloride solution, and reserving the eluent to obtain polysaccharide liquid.

8. The method of claim 7, wherein in the step (3), the precipitate is dissolved in water, and the ratio of the precipitate to the water is (10-20) mg: 1 ml; the sample loading speed of the solution A is 20-30 ml/min; eluting the chromatographic column with water at a flow rate of 9-15 ml/min; the flow rate of elution of the column with sodium chloride solution is 7-12 ml/min.

9. The method of claim 1, wherein in step (4), the solution B is applied to a Sephadex column, the Sephadex column is allowed to stand for 1-2h, and then the column is eluted with 3-4 column volumes of water at a flow rate of 0.7-1.2ml/min, and the eluate is retained to obtain a desalted polysaccharide solution.

10. Use of the method of any one of claims 1 to 9 for the preparation of a macadamia nut leaf extract for the preparation of a food product.

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 leaf extract.

Background

Macadamia nut (Macadamia integrifolia f. muell, also known as Macadamia nut) is a tree-grown nut native to Macadamia. The macadimia nut kernel can be used for food production and oil extraction, the green tangerine peel can be used for fertilizer production, and the shell can be used for preparing products such as an adsorbent. However, the leaf resources of the macadimia nuts occupying a large amount of biomass are not effectively utilized all the time, and generally in a macadimia nut plantation, branches and leaves are discarded in situ after being trimmed and are allowed to decay to become fertilizer for fruit trees, so that the environment is polluted, and the resources are wasted.

Chinese patent "a processing method of macadimia nut leaf green tea" (CN106035882A) proposes a method for preparing tea leaves by comprehensively utilizing macadimia nut leaves, combines the preparation processes of green tea and oolong tea, and carries out deep processing on the macadimia nut leaves to obtain the macadimia nut leaf green tea. However, the prior art has the following defects: the leaves of the macadimia nuts contain a large amount of polysaccharide components, and the leaves are directly made into tea leaves, so that the polysaccharide components cannot be effectively utilized by a human body; the components of the leaves of the macadimia nuts are greatly different from the components of the tea leaves, the content of phenolic acid compounds in the leaves of the macadimia nuts is high, and the phenolic acid compounds have the effects of sterilization, inflammation resistance and the like.

Disclosure of Invention

The invention mainly solves the technical problem of providing the preparation method of the macadimia nut leaf extract, the leaf extract obtained by the preparation scheme has high polysaccharide content and low content of polyphenol impurities such as phenolic acids and the like, and the edible safety of the leaf extract is improved.

In order to solve the technical problems, the invention provides the following technical scheme:

a method for preparing macadamia nut leaf extract comprises the following steps:

step (1): pretreating macadimia nut leaves to obtain leaf powder;

step (2): soaking the leaf powder in an ethanol solution, and then filtering to obtain a solid phase to obtain degreased leaf powder; adding the degreased leaf powder into water to obtain an aqueous extraction system, carrying out ultrasonic treatment on the aqueous extraction system, and then centrifuging to obtain a liquid phase to obtain an extracting solution; adding active carbon into the extract, stirring, standing, filtering, and collecting liquid phase to obtain decolorized extract; concentrating the decolorized extract, adding ethanol solution, standing, centrifuging, and collecting solid phase to obtain precipitate;

and (3): dissolving the precipitate in water, filtering to obtain liquid phase to obtain solution A; adjusting the pH value of the solution A to be alkaline, then loading the solution A to a DEAE sepharose gel chromatographic column, standing, eluting the chromatographic column by water, and removing eluent; eluting the chromatographic column with sodium chloride solution, and retaining the eluent to obtain polysaccharide liquid; concentrating the polysaccharide solution to obtain an extract;

and (4): dissolving the extract in water, filtering to obtain liquid phase to obtain solution B; loading the solution B into a sephadex chromatographic column, standing, eluting the chromatographic column with water, and retaining an eluent to obtain a desalted polysaccharide solution;

and (5): concentrating the desalted polysaccharide solution to obtain the leaf extract.

By adopting the technical scheme, the technical principle is as follows: after macadimia nut leaves are crushed into leaf powder, the powder is soaked in an ethanol solution, and the degreasing treatment is carried out on the leaf powder, so that the influence of lipid substances on the subsequent polysaccharide extraction is avoided. Then, the water-soluble substances in the leaf powder are extracted by using an ultrasonic-assisted water extraction method. After extraction is finished, the extract is decolorized by using activated carbon, so that the influence of pigments such as chlorophyll on the quality of a final product is avoided. In order to remove water-soluble impurities, the decolored extract is subjected to alcohol precipitation treatment, and target substances such as polysaccharide and the like are further enriched. In order to eliminate the influence of phenolic acid substances on a final product, a DEAE (diethylaminoethyl) Sepharose chromatography step is added in the scheme, under an alkaline condition, the phenolic acid substances cannot be adsorbed on DEAE Sepharose gel (DEAE-Sepharose, diethylaminoethyl-Sepharose), the DEAE Sepharose chromatography column is eluted by water, the phenolic acid substances can be eluted, and the polysaccharide substances are also adsorbed on the chromatography column. Then, the column was eluted with a sodium chloride solution, and the polysaccharide substance was desorbed from DEAE Sepharose and eluted. Desalting by Sephadex (Sephadex) chromatography, concentrating, and drying to obtain leaf extract. The leaf extract has high polysaccharide content and contains no phenolic acid.

In this embodiment, the phenolic acid compound refers to an aromatic carboxylic acid compound having a plurality of phenolic hydroxyl groups substituted on one benzene ring, and the structure is unstable and easily oxidized and deteriorated. Plant polysaccharides, also known as plant polysaccharides, are carbohydrates produced by plant cell metabolism with a degree of polymerization of more than 10.

The beneficial effect of this scheme lies in:

(1) the obtained leaf extract has high polysaccharide content, and the plant polysaccharide has various biological activities, such as immunoregulation, anti-tumor, and blood sugar lowering effects. The extraction method can create conditions for comprehensive utilization of macadimia nut leaves, and a large amount of leaf resources can not be discarded any more, and can be used for preparing food or health care products containing plant polysaccharides.

(2) Effectively removes phenolic acid substances in the polysaccharide, reduces hepatotoxicity possibly caused by taking the leaf extract and reduces the probability of drug resistance generated by pathogenic microorganisms. Because the components of the leaves of the macadimia nuts are different from those of common tea leaves, and a large amount of phenolic acid substances are contained in the leaves, the leaves can cause certain stimulation to the digestive tract of a human body and even can generate certain toxic effects when being eaten directly. Therefore, in order to prepare related food or health care products by using macadamia nut leaf resources, the phenolic acid substances are required to be sufficiently removed so as to improve the safety of the leaf extract. The inventor researches and discovers that phenolic acid substances in leaves of the macadimia nuts are not adsorbed to DEAE sepharose gel under alkaline conditions, and the phenolic acid substances and polysaccharide can be fully separated by the characteristic. In the prior art, the phenolic acid substances and the polysaccharide are separated, the polysaccharide needs to be purified and separated in a multi-stage manner by using macroporous adsorption resin, so that the phenolic acid substances can be fully removed, the operation is complex, and the period is long.

(3) Phenolic acid substances (such as chlorogenic acid and the like) are easily oxidized, so that the color of the leaf extract is browned, and the quality of a final product is influenced. The preparation method removes phenolic acid substances, thereby avoiding the influence of the substances on the color and luster of the product.

(4) The preparation method is simple and safe to operate, the production period is short, the comprehensive utilization rate of the macadimia nut leaves is improved, the purity of the obtained polysaccharide is high, and the preparation method is suitable for industrial production.

Further, in the step (1), fresh macadamia nut leaves are cleaned, dried in the shade and crushed to obtain leaf powder.

By adopting the technical scheme, the leaves are cleaned, so that the influence of impurities on the extraction process is avoided; the influence of moisture in the leaves on the extraction efficiency can be avoided by drying the leaves in the shade; the leaves are crushed, so that the effective contact area of the materials and the extracting solution is increased.

Further, in the step (2), 90-98% ethanol solution is used for soaking the leaf powder for 2-3h at the temperature of 30-40 ℃, and the dosage ratio of the leaf powder to the ethanol solution is 1 g: (5-8) ml, filtering to obtain a solid phase, and volatilizing ethanol to obtain defatted leaf powder.

By adopting the technical scheme, part of fat-soluble substances in the leaf powder can be removed, the influence of the part of fat-soluble substances on the subsequent extraction step is reduced, and the purity of the target component is improved.

Further, in the step (2), in the aqueous extraction system, the ratio of the amount of the defatted leaf powder to the amount of water is 1 g: (20-30) ml; the power of ultrasonic treatment is 200-300W, the time duration is 60-90min, and the temperature of a water extraction system is maintained at 70-80 ℃; after the ultrasonic treatment is finished, centrifuging at 3000-.

By adopting the technical scheme, the water-soluble substances including the polysaccharide 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 (2), the mass percentage of the active carbon in the extracting solution is 2-5%, the stirring time is 20-30min, 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 leaf extract can be fully reduced, the polysaccharide substance can be prevented from being adsorbed by the activated carbon by controlling the stirring time to be 20-30min, and the material loss is avoided.

Further, in the step (2), a 95% ethanol solution is added to the concentrated decolorized extract, and the volume ratio of the concentrated decolorized extract to the 95% ethanol solution is 1: (4-6), standing at 4-6 ℃ for 24-36h, centrifuging at 3000-4000rpm for 20-30min, and taking a solid phase to obtain a precipitate.

By adopting the technical scheme, 95% ethanol is used for precipitating the polysaccharide, so that the purity of the polysaccharide substance in the final product can be improved.

Further, in the step (3), the pH value of the solution A is adjusted to 7.8-8.5, then the solution A is loaded on a DEAE sepharose gel chromatographic column, the DEAE sepharose gel chromatographic column is kept stand for 1.5-2h and then is eluted by 6-8 times of the volume of the column, and the eluent is discarded; then eluting the chromatographic column by using 2-3 times of the column volume of 0.1-0.5mol/L sodium chloride solution, and reserving the eluent to obtain polysaccharide liquid.

By adopting the technical scheme, phenolic acid substances in the polysaccharide can be removed. The inventor researches and discovers that phenolic acid substances in the leaves of the macadamia nut are not adsorbed to DEAE sepharose when the pH value is 7.8-8.5, but polysaccharide substances can be well adsorbed to the DEAE sepharose. The phenolic acid substances are eluted by water, and then the polysaccharide is eluted by a salt solution, so that the phenolic acid substances and the polysaccharide can be effectively separated. The pH value lower than 7.8 can cause trace amount of phenolic acid substances to be adsorbed on the gel, and a better separation effect cannot be achieved; and the excessive pH value can easily hydrolyze part of polysaccharide, destroy the active structure of the polysaccharide and reduce the yield of the polysaccharide.

Further, in the step (3), when the precipitate is dissolved in water, the ratio of the amount of the precipitate to the amount of water is (10-20) mg: 1 ml; the sample loading speed of the solution A is 20-30 ml/min; eluting the chromatographic column with water at a flow rate of 9-15 ml/min; the flow rate of elution of the column with sodium chloride solution is 7-12 ml/min.

By adopting the technical scheme, the phenol and acid substances can be fully eluted by water, and the polysaccharide substances can be fully collected by the sodium chloride solution.

Further, in the step (4), the solution B is loaded on a sephadex chromatographic column, the sephadex chromatographic column is kept stand for 1 to 2 hours, then the chromatographic column is eluted by water with the volume of 3 to 4 times of the column volume, the flow rate of the water is 0.7 to 1.2ml/min, and the eluent is reserved to obtain the desalted polysaccharide solution.

By adopting the technical scheme, salt impurities brought by the elution of the sodium chloride solution in the previous step can be removed, and the purity of the final product is improved.

Further, a preparation method of the macadamia nut leaf extract is applied to preparation of food.

By adopting the technical scheme, the leaf extract obtained by the method contains a large amount of polysaccharide substances, and the polysaccharide substances have the health-care effects of regulating immunity, resisting tumors, reducing blood sugar and the like. The leaf extract obtained by the method removes phenolic acid impurities, so that the leaf extract is safer. The leaf extract is further processed to obtain food or health food with immunoregulation function.

Detailed Description

The following is further detailed by the specific embodiments, wherein: