WO2018133600A1 - 一种高活性甘蔗花色苷的制备方法 - Google Patents

一种高活性甘蔗花色苷的制备方法 Download PDF

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WO2018133600A1
WO2018133600A1 PCT/CN2017/116390 CN2017116390W WO2018133600A1 WO 2018133600 A1 WO2018133600 A1 WO 2018133600A1 CN 2017116390 W CN2017116390 W CN 2017116390W WO 2018133600 A1 WO2018133600 A1 WO 2018133600A1
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anthocyanin
solution
sugar cane
sugarcane
anthocyanins
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French (fr)
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赵振刚
闫怀锋
刘瑞海
扶雄
于淑娟
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华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans

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  • the invention relates to food coloring, in particular to a method for preparing high-activity sugar cane anthocyanin, which is the method for extracting and purifying anthocyanins from sugar cane; belonging to the technical field of food additives and health foods.
  • Synthetic food coloring is made by using aniline dye separated from coal tar as raw material, so it is also called coal tar pigment or aniline pigment, such as synthetic amaranth, carmine and lemon yellow. These synthetic pigments are harmful to the human body because they are easy to induce poisoning, diarrhea and even cancer, so they cannot be used or used as much as possible.
  • Anthocyanin is a general term for glycosyl-derived compounds in which anthocyanins are combined with various monosaccharides and disaccharides by glycosidic bonds, and is an important water-soluble natural pigment. Studies have shown that anthocyanins have multiple functions such as anti-oxidation, anti-mutation, protection of the liver, prevention of cardiovascular diseases, scavenging free radicals, and improving human microcirculation. In recent years, a large number of studies have been carried out on various plant anthocyanins at home and abroad, but the market for anthocyanin pigments is far from saturated, and there is still much room for development.
  • Sugarcane bagasse is the residue of sugarcane stems after sugarcane is broken and extracted from cane juice, and is the main by-product of the sugar industry. Its components are mainly cellulose, hemicellulose, lignin, protein, starch and pectin. At present, the use of bagasse is mainly pulping and papermaking, preparation of activated carbon, hydrolysis and fermentation of ethanol, production of livestock and poultry feed and compost, etc., while bagasse is rich in anthocyanins (mainly cyanidin-3-O). - Glucosin) is an ideal natural food coloring resource.
  • the traditional anthocyanin extraction methods are solvent extraction, microwave and ultrasonic assisted extraction.
  • the purification methods include paper chromatography, thin layer chromatography, column chromatography, high performance liquid chromatography and high speed countercurrent chromatography.
  • the anthocyanins obtained by the above extraction method not only contain a large amount of impurities, but also affect the biological activity of anthocyanins.
  • the preparation of anthocyanins by enzymatic method has been carried out at home and abroad, and some achievements have been made.
  • due to the high specificity of the enzyme a single enzyme preparation can not effectively decompose plant tissues to release anthocyanins, and different plant tissues are different.
  • the object of the present invention is to solve the problems of low extraction efficiency and poor antioxidation activity in the process of extracting anthocyanins from sugar cane.
  • the present invention extracts anthocyanins from sugar cane skin or bagasse as raw materials, and provides a high activity color of sugarcane waste.
  • the method of glycosides turns wastes from sugar production into waste, improves the utilization rate of sugar cane, prolongs the production chain of sugar cane, and greatly increases the added value of sugar cane.
  • a method for preparing a highly active sugar cane anthocyanin comprises the following steps:
  • the cellulase, hemicellulase and pectinase are mixed to prepare a compound enzyme; the compound enzyme is sterilized, mixed, added to the reaction solution, and stirred at 40-50 ° C for 1 to 5 hours to remove the extract.
  • the enzyme is centrifuged for 1 to 10 minutes, and concentrated under vacuum to obtain crude extract of anthocyanins of sugar cane; the mass ratio of the cellulase, hemicellulase and pectinase is (1-20): (1-20): (1) ⁇ 5); the amount of the complex enzyme added is 5-15 mg/kg bagasse;
  • step 2) The crude extract of sugar cane anthocyanin obtained in step 1) is diluted with deionized water to 3-10 g/L, the pH is adjusted to 1.0-3.0 with acid, and then the dilution is subjected to the first stage chromatography of the macroporous adsorption resin. Column adsorption, anthocyanins are exchanged from the solution to the resin, washed with water to remove salts, sugars and other impurities; eluted with an eluent to obtain an impurity after the impurity removal, the eluate is concentrated in vacuo, and the concentrate is removed. After the mixed anthocyanin solution, the concentrated solution is diluted to 520 nm and the absorbance is 0.3-1.2;
  • the dilute solution of the anthocyanin obtained in the step 2) is adjusted to a pH of 1.0 to 3.0 with an acid, adsorbed through a second column packed with a dextran gel resin, eluted with acidified methanol, and ultraviolet-visible spectroscopy
  • the photometer monitors the elution process, collects the eluate at 520 nm, and concentrates the eluate in vacuo, and the concentrate is an anthocyanin purification solution;
  • the anthocyanin purified solution obtained in the step 3) is freeze-dried to obtain a powdery anthocyanin purified product.
  • the first pore-absorbing resin of the step 2) comprises an AB-8, D101, NKA-9, DA201 or DM-130 type resin.
  • the dextran gel-containing resin of step 3 comprises Sephadex LH-20, Sephadex G-25, Sephadex G-50 or Sephadex G-100 type resin.
  • the eluent is an acidified methanol solution having a pH of 1 to 3, wherein the methanol volume content is 90 to 100%.
  • the vacuum obtained by the vacuum concentration in step 2) is used for elution of the next macroporous adsorption resin.
  • the eluate obtained by vacuum-concentrating the eluate in step 3 is used for elution of the next glucan gel resin.
  • the ultraviolet-visible spectrophotometer of step 3 monitors a wavelength of 500 to 560 nm.
  • the acid described in step 2) and step 3) is hydrochloric acid, sulfuric acid, acetic acid or citric acid.
  • the water for removing the salt by washing with water as described in step 2) is deionized water, purified water or ultrapure water.
  • the cellulase is composed of endoglucanase, exoglucanase and cellobiase, pectinase from pectin methylesterase, polygalacturonase and pectin lyase composition.
  • the invention uses the sugarcane waste to prepare anthocyanins as high-purity and high-activity sugarcane anthocyanins, and the obtained anthocyanin purified products include cyanidin 3-o-glucoside, cyanidin- 3,5-O-diglucoside (cyanidin 3,5-di-O-glucoside), cyanidin-3-O-feruloyl-5-O-glucoside (cyanidin3 ⁇ O ⁇ feruloylglucoside ⁇ 5 ⁇ O-glucoside), peonidin 3 ⁇ O-glucoside, cyanidin ⁇ 3 ⁇ O ⁇ (malonyl)-glucoside , cyanidin-3-O-(succinyl)-glucoside, cyanidin-3 ⁇ O-caffeoyl glucoside-5-O-propanoid Cyanodin3 ⁇ O-caffeoylglucoside ⁇ 5 ⁇ O ⁇ malonylglucoside, cyanidin 3 ⁇ (6′′ ⁇ acetoyl glu
  • the anthocyanin purified product obtained by the invention has a content of 47.33-60.75 mg/100 g of bagasse.
  • the sugar cane anthocyanins of the present invention have important applications in the preparation of functional foods and medicines for preventing and mitigating antioxidants.
  • the cellulase of the present invention is composed of endoglucanase, exoglucanase and cellobiase, and the pectinase is composed of pectin methylesterase, polygalacturonase and pectin lyase. .
  • the invention finds that cellulase, hemicellulase and pectinase are compounded into a complex enzyme, and the bagasse tissue can be decomposed gently and efficiently by the corresponding ratio of the three types of enzymes, thereby accelerating anthocyanins. The release, while ensuring the biological activity of anthocyanins.
  • the invention acts on cellulose, hemicellulose and pectin of the cell wall of bagasse, and promotes the rapid release of anthocyanins in cells and adsorbed on cellulose and cell walls, and at the same time ensures its biological activity.
  • cellulase hydrolyzes cellulose in bagasse and destroys its chain structure.
  • Hemicellulose xylan is bound to the surface of cellulose microfibers and interconnected to form a hard cell network structure. Hemicellulase can be hydrolyzed.
  • Hemicellulose which reduces the connection with cellulose, synergizes with cellulase to increase the effect on cellulose, releases anthocyanins between cells and attached to cellulose, and an appropriate amount of pectinase can hydrolyze polysaccharides in cell membranes. Change the permeability of the cells and release the anthocyanin substances in the cells.
  • the model of the macroporous adsorption resin is AB-8
  • the eluent is acidified methanol (pH 1.0)
  • the elution flow rate is 2 to 10 BV/h.
  • the model of the dextran gel is Sephadex G-20, the elution flow rate is 30-100 mL/h, and the model of the ultraviolet-visible spectrophotometer is Backman DU700, and the monitoring wavelength is 500-550 nm.
  • the complex enzyme can extract the anthocyanins in the bagasse in a gentle and effective manner, and the yield is increased by more than 20%, and the anthocyanin has a strong antioxidant activity.
  • Macroporous adsorption resin-dextran gel resin is used for the purification of anthocyanins.
  • the resin can be continuously injected without repeated regeneration, and the anthocyanins in the eluent can be monitored in real time. This method is simple and operates. Convenient and rapid separation, the resulting anthocyanin purified material retains its original structure and biological activity.
  • Figure 1 is a graph showing the total ion current of MS of high content of sugarcane anthocyanins obtained in Example 1.
  • Example 2 is a MS/MS total ion chromatogram of a high content of sugarcane anthocyanins obtained in Example 1.
  • the composite enzyme is prepared; the complex enzyme is composed of cellulase, hemicellulase and pectinase, wherein the mass ratio of cellulase, hemicellulase and pectinase is 5:3:1.
  • the above complex enzyme was mixed, sterilized, added to an extraction tank, and the amount of 8 mg/kg bagasse was added, and the mixture was stirred and immersed at 35 ° C for 2 h, and the extract was sterilized for 10 min, centrifuged at 3000 r/min, and concentrated in vacuo to obtain Sugar cane anthocyanin crude extract A 1 .
  • the macroporous resin AB-8 and the Sephadex LH-20 were pretreated and packed, and the obtained concentrate was diluted to 4.2 g/L by using a glass chromatography column (7.5 mm ⁇ 240 mm).
  • the sample was loaded with 1000 mL of diluent, and the flow rate was controlled at 4 BV/h. When the absorbance of the effluent at 520 nm reached one tenth of the dilution, the adsorption reached saturation.
  • step 3 Adjust the pH of the dilution obtained in step 2 to 1.0 with hydrochloric acid, load 0.40 BV, elute with acidified methanol (pH 1.0), elute at a flow rate of 60 mL/h, collect the eluate every 10 min, and at 520 nm. The absorbance was measured, and the eluent with the absorbance greater than zero was combined to obtain anthocyanin purified A 2 , and the eluate was concentrated in vacuo at a temperature of 40 ° C and a rotation speed of 80 r / min to make the solid matter reach 70%.
  • step 4 The concentrated liquid in step 3 is freeze-dried to obtain a dark red powder which is an anthocyanin.
  • the composite enzyme is prepared; the complex enzyme is composed of cellulase, hemicellulase and pectinase, wherein the mass ratio of cellulase, hemicellulase and pectinase is 16:8:3.
  • the above complex enzyme was mixed, sterilized, added to an extraction tank, and the amount of 8 mg/kg bagasse was added, and the mixture was stirred and incubated at 40 ° C for 4 hours, and the extract was inactivated for 10 min, centrifuged at 3000 r/min, and concentrated in vacuo to obtain Sugar cane anthocyanin crude extract B 1 .
  • the macroporous resin D101 and the Sephadex G-25 were pretreated and packed, and the obtained condensed solution was diluted to 6.4 g/L by a glass chromatography column (7.5 mm ⁇ 240 mm), and diluted to 800 mL.
  • the loading flow rate is controlled to 3BV/h, and when the absorbance of the effluent at 520 nm reaches one tenth of the diluent, the adsorption reaches saturation.
  • step 3 Adjust the pH of the dilution obtained in step 2 to 1.0 with hydrochloric acid, load 0.52 BV, elute with acidified methanol (pH 1.0), elute at a flow rate of 65 mL/h, collect the eluate every 10 min, and at 520 nm. The absorbance was measured, and the eluent with the absorbance greater than zero was combined to obtain anthocyanin purified B 2 , and the eluate was concentrated in vacuo at a temperature of 35 ° C and a rotation speed of 80 r/min to make the solid matter reach 70%.
  • step 4 The concentrated liquid in step 3 is freeze-dried to obtain a dark red powder which is an anthocyanin.
  • the composite enzyme is prepared; the complex enzyme is composed of cellulase, hemicellulase and pectinase, wherein the mass ratio of cellulase, hemicellulase and pectinase is 3:2:1.
  • the above complex enzyme was mixed, sterilized, added to an extraction tank, and the amount of 14 mg/kg bagasse was added, and the mixture was stirred and immersed for 5 hours at 30 ° C, and the extract was inactivated for 10 min, centrifuged at 3000 r/min, and concentrated in vacuo to obtain Sugar cane anthocyanin crude extract C 1 .
  • the macroporous resin NKA-9 and the Sephadex G-50 were pretreated and packed, and the obtained concentrate was diluted to 5.2 g/L by using a glass chromatography column (7.5 mm ⁇ 240 mm).
  • the sample was loaded with 900 mL of diluent, and the flow rate was controlled to 4 BV/h.
  • the absorbance of the effluent at 520 nm reached one tenth of the dilution, the adsorption reached saturation.
  • step 3 Adjust the pH of the dilution obtained in step 2 to 1.0 with hydrochloric acid, load 0.65 BV, elute with acidified methanol (pH 1.0), elute at a flow rate of 80 mL/h, collect the eluate every 10 min, and at 523 nm. The absorbance was measured, and the eluent with the absorbance greater than zero was combined to obtain the anthocyanin purified C 2 , and the eluate was concentrated in vacuo at a temperature of 35 ° C and a rotation speed of 80 r/min to make the solid matter reach 70%.
  • step 4 The concentrated liquid in step 3 is freeze-dried to obtain a dark red powder which is an anthocyanin.
  • the total content of anthocyanins in sugar cane peel was determined by pH differential method, and the results were expressed as cyanidin-3-glucoside/100g sample with cyanidin-3-O-glucoside as standard.
  • pH difference method take the sugarcane skin anthocyanin purified to a certain volume, centrifuge at 3000r/min for 10min, take 4mL supernatant in two 20mL centrifuge tubes, and then add 6mL KCl-HCl with pH1.0 respectively.
  • the buffer and CH3COONa-HCl buffer with a pH of 4.5 were mixed and equilibrated for 60 min.
  • distilled water as a blank control, take 1 mL, and measure the absorbance A at ⁇ max (maximum absorption wavelength of an anthocyanin of sugar cane skin, scanned) and 700 nm, respectively, using an ultraviolet-visible spectrophotometer, and calculate according to the following formula:
  • ⁇ max Take the appropriate amount of the anthocyanin extract of the sugarcane skin after centrifugation, and use the DU-730 UV-Vis spectrophotometer to scan the absorption spectrum in the range of 200-800 nm to obtain the sugarcane skin color. The maximum absorption wavelength of the glycoside in the visible region.
  • Anthocyanin purifier Anthocyanin content (mg/100g) A 2 60.75 ⁇ 2.78 B 2 47.33 ⁇ 1.06 C 2 51.95 ⁇ 1.29
  • the content of the anthocyanin purified product obtained in the embodiment of the present invention is at least 47.33 mg/100 g of bagasse, and the highest is 60.75 mg/100 g of bagasse.
  • the total amount of anthocyanins in the various parts of sugar cane obtained by conventional hydroalcohol extraction is 36.6 mg/100 g.
  • Sugarcane dry weight Li, X., et al., Determination and comparison of flavonoids and anthocyanins in Chinese sugarcane tips, stems, roots and leaves. J Sep Sci, 2010. 33(9): p. 1216-23).
  • the enzymatic hydrolysis method of the present invention increases the content of anthocyanins by 22.67 to 39.75%, and the effect is remarkable.
  • Prior art He Xiong et al., Extraction process and stability of anthocyanins from sugar cane skin. Food Industry Science and Technology, 2011 (12): 371-373, page 376) refers to the scraping of sugarcane epidermis, supplemented by hydroalcohol Ultrasonic leaching, the content of anthocyanins in sugar cane is 288.06mg/100g, which is expressed by the content of anthocyanins in sugarcane skin, and in the present invention, the content of anthocyanins in bagasse is calculated.
  • the content comparison is different, so the results are quite different, and the method does not take into account the practical application of separating sugar cane skin requires a lot of manpower and material resources, not only greatly increase production costs, but also difficult to achieve industrialization, the resulting anthocyanins
  • the content does not have practical application value.
  • the complex enzyme method destroys the tissue structure of bagasse by synergistic action between various enzymes, and releases the anthocyanins inside and outside the cell as much as possible.
  • the specificity of the enzyme ensures the natural state of anthocyanins, macroporous adsorption resin and
  • the combination of the glucan gel resin removes a large amount of impurities such as polyphenol flavonoids, and greatly reduces the degradation of the anthocyanin substance by the impurities.
  • the anthocyanin purified obtained in the examples was analyzed using an ultrahigh liquid phase-mass spectrometry method.
  • the obtained product A 2 was dissolved in methanol, and each was set to a solution of 10 ⁇ g/ml. After passing through a 0.45 ⁇ m filter, it was analyzed by UPLC-MS/MS.
  • Chromatographic conditions injection volume 5 ⁇ L; mobile phase A is chromatographic acetonitrile, mobile phase B is 0.1% TFA ultrapure water; gradient conditions: 0 min, 95% A; 2.3 min, 80% A; 3.4 min, 75% A; 6.8 min, 60%; 7.9 min, 95% A; 9 min, 95% A.
  • Mass spectrometry conditions ion source: ESI, 50 m/z to 1500 m/z in positive ion mode, capillary pressure 3500 V, dry gas: N 2 , flow rate: 4.0 l/min, nebulizer pressure: 0.3 bar, dryer temperature: 180 °C.
  • Example 2 The test results of Example 1 are shown in Table 2. There are only two to five species of anthocyanins obtained by the existing extraction and purification methods.
  • the method for extracting and purifying an anthocyanin of the sugarcane peel of the invention not only improves the yield of anthocyanins, but also ensures the naturalness and stability of the anthocyanins. The main reason is that the compound enzymatic method of the invention is mild and specific, and the extracted sugar cane is obtained.
  • the anthocyanin structure was preserved to the greatest extent.
  • the combination of macroporous adsorption resin and dextran gel resin can remove a large amount of impurities and avoid the degradation of anthocyanins by other impurities.
  • a and b are ion fragments of anthocyanin primary mass spectrometry (MS) and ion fragments of secondary mass spectrometry (MS/MS), respectively.
  • MS total ion current map and the MS/MS total ion current map are shown in Figures 1 and 2, respectively.
  • Example 1 Ten anthocyanins were obtained in Example 1: cyanidin 3-O-glucoside, cyanidin-3,5-O-diglucoside (cyanidin 3,5- di ⁇ O-glucoside), cyanidin-3 ⁇ O ⁇ feruloylglucoside ⁇ 5 ⁇ O-glucoside, paeoniflorin-3O-glucoside peonidin3 ⁇ O ⁇ glucoside), cyanidin ⁇ 3 ⁇ O ⁇ (malonyl)-glucoside, cyanidin-3 ⁇ O ⁇ succinyl-glucoside Cyanodin-3 ⁇ O ⁇ (succinyl)-glucoside, cyanidin-3 ⁇ O-caffeoylglucoside-5-O-malonyl-glucoside (cyanidin3 ⁇ O ⁇ caffeoylglucoside ⁇ 5 ⁇ O ⁇ Malonylglucoside), cyanidin 3 ⁇ (6′′-acetoyl glucoside), malvidin-3 ⁇ O ⁇ glucoside, corn Inulin-7-me
  • the purified product A 2 in the above Example 1 was diluted 100 times, 125 times, 167 times, 250 times and 500 times, respectively, to obtain 20, 16, 12, 8.0, 4.0 mg (as bagasse powder) / mL sample solution. .
  • Each group of experiments was carried out in parallel 3 times. 2 mL of each gradient sample solution was added, 2 mL of 0.2 mM DPPH ethanol solution was added, and the mixture was thoroughly mixed. After standing for 30 minutes in the dark, it was centrifuged for 15 minutes to separate the precipitate, and the rotation speed was 4800 r/min. .
  • the mixture was adjusted to volume with an equal volume of absolute ethanol and deionized water, and the supernatant after centrifugation of the sample was taken to measure the absorbance A sample at 517 nm.
  • the absorbance of the absolute volume of anhydrous ethanol and DPPH ethanol solution (2 mL) measured at 517 nm was A0, and the absorbance at 517 nm measured by mixing the bagasse extract sample liquid with the absolute ethanol solution was A control.
  • DPPH ⁇ radical scavenging rate of sugarcane bagasse anthocyanin/% [1-(A sample-A control)/A0] ⁇ 100, and IC50 value was calculated at the same time.
  • the scavenging rate of DPPH ⁇ free radicals by the enzymatic extraction of sugarcane anthocyanins reached 72.58%, which was 15.8% higher than that of the traditional hydroalcohol extraction method. It may be that the enzymatic hydrolysis exposed more H. And the method is mild and does not destroy this electronic structure, so its ability to resist DPPH ⁇ free radicals is stronger.
  • the scavenging rate of ABTS free radicals by the enzymatic method of sugarcane anthocyanin reached 59.32%, which was 26.3% higher than that of the traditional hydroalcoholic extraction method. It may be that the anthocyanins with more charged groups were extracted by the present invention. The substance is such that its activity against ABTS groups is significantly enhanced.
  • the purified product A 2 in the above Example 1 was diluted into a solution of 1 mg (in terms of cane skin powder) / mL, and 20 ⁇ L of each was taken in a 96-well plate, each group was paralleled by three sets of experiments, and then 7 mM of FL was separately added.
  • the ORAC value of the sugarcane anthocyanin extracted by the complex enzymatic method reached 909.50 ⁇ mol Trolox/g, which was 60% higher than the traditional hydroalcohol extraction method. It may be that the enzymatic hydrolysis method is mild, and H ⁇ in the anthocyanin is preserved to the utmost extent, so that it has a strong scavenging ability to the peroxidation radical ROO ⁇ produced in the method.
  • the present invention obtains a high content of sugar cane anthocyanins, prolongs the production chain of sugar cane, and greatly increases the added value of sugar cane.

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Abstract

本发明公开了一种高活性甘蔗花色苷的制备方法。该方法包括:1)取榨汁后的甘蔗渣，晾干，破碎，加入蒸馏水；将纤维素酶，半纤维素酶，果胶酶混合配制复合酶，灭菌，加入到反应液中，浸提，灭酶，离心，真空浓缩，得甘蔗花色苷粗提液；2)将粗提液经装有大孔吸附树脂第一段色谱柱进行除盐，提取剂洗脱后，真空浓缩，得花色苷溶液；3)用酸调节后，再经装有葡聚糖凝胶树脂的第二段色谱柱，收集洗脱液，真空浓缩，浓缩液为花色苷纯化液；4)冷冻干燥，得花色苷纯化物。本发明以甘蔗皮或甘蔗渣为原料提取花色苷，将制糖生产后的废弃物变废为宝，提高了甘蔗的利用率，延长了甘蔗的生产链，大大提高了甘蔗的附加价值。

Description

一种高活性甘蔗花色苷的制备方法 技术领域
本发明涉及食用色素，特别是涉及一种高活性甘蔗花色苷的制备方法，该方法是从甘蔗中萃取和纯化花色苷；属于食品添加剂及保健食品技术领域。
背景技术
人工合成食用色素，是用煤焦油中分离出来的苯胺染料为原料制成的，故又称煤焦油色素或苯胺色素，如合成苋菜红、胭脂红及柠檬黄等等。这些人工合成的色素因易诱发中毒、泻泄甚至癌症，对人体有害，故不能多用或尽量不用。花色苷(Anthocyanin)是由花青素与各种单糖、二糖以糖苷键结合而成的糖基衍生化合物的总称，是一种重要的水溶性天然色素。研究表明，花色苷具有抗氧化，抗突变，保护肝脏，预防心血管疾病，清除自由基，改善人体微循环等多重功效。近年来，国内外对各种植物花色苷类物质进行了大量研究，但花色苷类色素的市场远未饱和，仍有很大的发展空间。
甘蔗渣是甘蔗经破碎和提取蔗汁后的甘蔗茎的残渣，是制糖工业的主要副产物。其成分主要为纤维素，半纤维素，木质素，蛋白，淀粉和果胶类物质等。目前对甘蔗渣的利用主要是制浆造纸，制备活性炭，水解发酵乙醇，生产畜禽饲料和堆肥等，而甘蔗渣中富含丰富的花色苷类物质(主要为矢车菊素-3-O-葡萄糖苷)，是一种理想的天然食用色素资源。
传统的花色苷提取方法是溶剂萃取法，微波和超声辅助提取法，纯化方法主要有纸层析，薄层层析，柱层析，高效液相色谱法以及高速逆流色谱法等。上述提取方法得到的花色苷不仅含有大量的杂质，而且影响花色苷的生物活性。目前国内外已经开始通过酶法制备花色苷，取得了一些成果，但由于酶具有高度专一性，单一的酶制剂并不能有效的分解植物组织释放花色苷，并且不同的植物组织构成不同，所需的酶的种类和组成也会有所差异，这些差异必定对酶的实用性提出更高要求。传统纯化方法中柱层析法应用最为广泛，但主要采用单一的大孔吸附树脂，得到的花色苷杂质成分高，产品活性较低，其它方法不仅得到的花色苷含量低，而且设备昂贵，严重影响其在食品，医药等领域的高值化应用。
发明内容
本发明的目的在于针对目前甘蔗花色苷提取过程中提取效率低和抗氧化活性差等问题，本发明以甘蔗皮或甘蔗渣为原料提取花色苷，提供了一种用甘蔗废弃物制备高活性花色苷的方法，将制糖生产后的废弃物变废为宝，提高了甘蔗的利用率，延长了甘蔗的生产 链，大大提高了甘蔗的附加价值。
本发明解决技术问题所采用的技术方案是：
一种高活性甘蔗花色苷的制备方法，包括以下步骤：
1)取榨汁后的甘蔗渣，晾干，破碎，加入蒸馏水，用缓冲液调节pH3.0～5.0；
将纤维素酶，半纤维素酶，果胶酶混合配制复合酶；将复合酶灭菌，混匀，加入到反应液中，在40～50℃下搅拌浸提1～5h，将提取液灭酶1～10min，离心，真空浓缩，得到甘蔗花色苷粗提液；所述纤维素酶、半纤维素酶和果胶酶的质量比为(1～20):(1～20):(1～5)；所述复合酶的添加量为5～15mg/kg甘蔗渣；
2)将步骤1)得到的甘蔗花色苷粗提液用去离子水稀释至3～10g/L，用酸调节pH至1.0～3.0，然后将稀释液经装有大孔吸附树脂第一段色谱柱吸附，花色苷从溶液中交换到树脂上，用水洗除去盐、糖和其它杂质；用洗脱剂洗脱，得到除杂后的洗脱液，将洗脱液真空浓缩，浓缩液为除杂后的花色苷溶液，将浓缩液稀释至520nm处吸光值为0.3～1.2；
3)将步骤2)中得到的花色苷稀溶液用酸调整pH到1.0～3.0，经装有葡聚糖凝胶树脂的第二段色谱柱进行吸附，用酸化甲醇洗脱，紫外-可见光分光光度计监测洗脱过程，收集520nm处的洗脱液，将洗脱液真空浓缩，浓缩液为花色苷纯化液；
4)将步骤3)中得到的花色苷纯化液冷冻干燥，得到粉末状的花色苷纯化物。
为进一步实现本发明目的，优选地，步骤2)所述第大孔吸附树脂包括AB-8、D101、NKA-9、DA201或DM-130型号树脂。
优选地步骤3)所述装有葡聚糖凝胶的树脂包括Sephadex LH-20、Sephadex G-25、Sephadex G-50或Sephadex G-100型号树脂。
优选地，所述洗脱剂为酸化甲醇溶液，pH为1～3，其中甲醇体积含量为90～100％。
优选地，步骤2)所述真空浓缩所得蒸出液用于下次大孔吸附树脂的洗脱。
优选地，步骤3)所述将洗脱液真空浓缩所得蒸出液用于下次葡聚糖凝胶树脂的洗脱。
优选地，步骤3)所述紫外-可见光分光光度计监测波长为500～560nm。
优选地，步骤2)和步骤3)所述的酸为盐酸、硫酸、醋酸或柠檬酸。
优选地，步骤2)所述的用水洗除去盐的水为去离子水、纯净水或超纯水。
优选地，所述纤维素酶由葡聚糖内切酶、葡聚糖外切酶和纤维二糖酶组成，果胶酶由果胶甲酯酶、聚半乳糖醛酸酶和果胶裂解酶组成。
本发明用甘蔗废弃物制备花色苷为高纯度和高活性甘蔗花色苷，所得花色苷纯化物包括矢车菊素-3-O-葡萄糖苷(cyanidin 3‐O‐glucoside)，矢车菊素‐3，5‐O‐二葡萄糖苷(cyanidin 3，5‐di‐O‐glucoside)，矢车菊素‐3‐O‐阿魏酰‐5‐O‐葡萄糖苷(cyanidin3‐O‐feruloylglucoside‐5‐O‐glucoside)，芍药色素‐3‐O‐葡萄糖苷(peonidin3‐O‐glucoside)，矢车菊素‐3‐O‐丙二酰‐萄糖苷(cyanidin‐3‐O‐(malonyl)‐glucoside)，矢车菊素‐3‐O‐丁二酰‐萄糖苷(cyanidin‐3‐O‐(succinyl)‐glucoside)，矢车菊素‐3‐O‐咖啡酰葡萄糖苷‐5‐O‐丙二酰‐葡萄糖苷(cyanidin3‐O‐caffeoylglucoside‐5‐O‐malonylglucoside)，矢车菊素‐3‐(6‐乙酰)葡萄糖苷(cyanidin 3‐(6″‐acetoyl)glucoside)，锦葵色素‐3‐O‐葡萄糖苷(malvidin3‐O‐glucoside)，矢车菊素‐7甲氧基‐3‐O‐(2‐没食子酰)‐葡萄糖苷(7‐O‐Methyl‐cyanidin‐3‐O‐(2″galloyl)‐galactoside)共10中花色苷的混合物。
本发明所得到的花色苷纯化物含量为47.33-60.75mg/100g甘蔗渣。本发明所述的甘蔗花色苷在制备预防和减缓抗氧化的功能食品和药品中具有重要应用。
本发明所述纤维素酶由葡聚糖内切酶，葡聚糖外切酶和纤维二糖酶组成，果胶酶由果胶甲酯酶，聚半乳糖醛酸酶和果胶裂解酶组成。本发明根据甘蔗渣成分特点，发现纤维素酶，半纤维素酶和果胶酶配制成复合酶，通过三类酶的相应配比，能温和高效地将甘蔗渣组织分解，加速花色苷类物质的释放，同时保证了花色苷的生物活性。本发明作用于甘蔗渣细胞壁的纤维素，半纤维素和果胶，促使细胞内和吸附在纤维素及细胞壁上的花色苷类物质快速释放，同时保证了其生物活性。其中，纤维素酶水解甘蔗渣中的纤维素，破坏其链状结构，半纤维素木聚糖结合在纤维素微纤维的表面，并相互连接构成坚硬的细胞网络结构，半纤维素酶可以水解半纤维素，降低其和纤维素的连接，从而协同纤维素酶增加对纤维素的作用，释放细胞间和附着在纤维素上的花色苷，适量的果胶酶可以水解细胞膜的多糖类，改变细胞的通透性，释放细胞内的花色苷物质。
作为优选技术方案，选用大孔吸附树脂的型号为AB-8，洗脱剂为酸化甲醇(pH1.0)，洗脱流速为2～10BV/h。
作为优选技术方案，选用葡聚糖凝胶的型号为Sephadex G-20，洗脱流速为30～100mL/h；紫外-可见光分光光度计的型号为：Backman DU700，监测波长为500～550nm。
相对于现有技术，本发明的有益效果是：
1)以甘蔗渣为原料提取花色苷，将制糖生产后的废弃物变废为宝，提高了甘蔗的利用率，延长了甘蔗的生产链，大大提高了甘蔗的附加价值。
2)本发明中复合酶可温和并有效地提取甘蔗渣中的花色苷，产量增加20％以上，同时保证了花色苷具有较强的抗氧化活性。
3)大孔吸附树脂-葡聚糖凝胶树脂联用进行花色苷的纯化，树脂可连续进样，不需反 复再生，并且可以实时监测洗脱液中的花色苷，此方法设备简单，操作方便，分离迅速，得到的花色苷纯化物保持了原始的结构和生物学活性。
附图说明
图1为实施例1所得产物高含量甘蔗皮花色苷的MS总离子流图。
图2为实施例1所得产物高含量甘蔗皮花色苷的MS/MS总离子流图。
具体实施方式
为更好地理解本发明，下面结合附图和实施例对本发明进一步的说明，但本发明的实施方式不限如此。
实施例1
1.从甘蔗糖厂收集刚榨汁后的甘蔗渣，晾干后破碎，取500g甘蔗渣粉，加入5L的蒸馏水，用缓冲液调节pH至4.0。配制复合酶；复合酶由纤维素酶、半纤维素酶和果胶酶组成，其中，纤维素酶、半纤维素酶和果胶酶的质量比为5:3:1。将上述复合酶混匀，灭菌，加入到提取罐中，加入量为8mg/kg甘蔗渣，在35℃下搅拌浸提2h，将提取液灭酶10min，3000r/min离心，真空浓缩，得到甘蔗花色苷粗提液A 1。
2.大孔树脂AB-8和葡聚糖凝胶Sephadex LH-20经预处理后装柱，采用玻璃层析柱(7.5mm×240mm)，将得到的浓缩液稀释至4.2g/L，取1000mL稀释液上样，上样流速控制4BV/h，流出液在520nm处的吸光值达到稀释液十分之一时，吸附达到饱和。用纯水以1.5BV/h流速冲洗5BV，洗去杂质；然后用酸化甲醇(pH1.0)洗脱，收集洗脱液；将洗脱液进行真空浓缩，温度40℃，转速80r/min，得到除杂后的花色苷浓缩液。用酸化甲醇稀释浓缩液，使最终稀释液在520nm的吸光值为0.6。
3.用盐酸将步骤2得到的稀释液pH调整为1.0，上样0.40BV，用酸化甲醇(pH1.0)洗脱，洗脱流速为60mL/h，每10min收集洗脱液，并在520nm处测量吸光值，合并吸光值大于零的洗脱液，得到花色苷纯化物A 2，将洗脱液进行真空浓缩，温度40℃，转速80r/min，使固形物达到70％。
4.将步骤3中浓缩液冷冻干燥，得到暗红色粉末即为花色苷。
实施例2
1.从甘蔗糖厂收集刚榨汁后的甘蔗渣，晾干后破碎，取750g甘蔗渣粉，加入7.5L的蒸馏水，用缓冲液调节pH至4.0。配制复合酶；复合酶由纤维素酶、半纤维素酶和果胶酶组成，其中，纤维素酶、半纤维素酶和果胶酶的质量比为16:8:3。将上述复合酶混匀，灭菌，加入到提取罐中，加入量为8mg/kg甘蔗渣，在40℃下搅拌浸提4h，将提取液灭酶10min， 3000r/min离心，真空浓缩，得到甘蔗花色苷粗提液B 1。
2.大孔树脂D101和葡聚糖凝胶Sephadex G-25经预处理后装柱，采用玻璃层析柱(7.5mm×240mm)，将得到的缩液稀释至6.4g/L，取800mL稀释液上样，上样流速控制3BV/h，流出液在520nm处的吸光值达到稀释液十分之一时，吸附达到饱和。用纯水以2.0BV/h流速冲洗6BV，洗去杂质；然后用酸化甲醇(pH1.0)洗脱，收集洗脱液；将洗脱液进行真空浓缩，温度40℃，转速80r/min，得到除杂后的花色苷浓缩液。用酸化甲醇稀释浓缩液，使最终稀释液在519nm的吸光值为0.6。
3.用盐酸将步骤2得到的稀释液pH调整为1.0，上样0.52BV，用酸化甲醇(pH1.0)洗脱，洗脱流速为65mL/h，每10min收集洗脱液，并在520nm处测量吸光值，合并吸光值大于零的洗脱液，得到花色苷纯化物B 2，将洗脱液进行真空浓缩，温度35℃，转速80r/min，使固形物达到70％。
4.将步骤3中浓缩液冷冻干燥，得到暗红色粉末即为花色苷。
实施例3
1.从甘蔗糖厂收集刚榨汁后的甘蔗渣，晾干后破碎，取839g甘蔗渣粉，加入8.39L的蒸馏水，用缓冲液调节pH至4.0。配制复合酶；复合酶由纤维素酶、半纤维素酶和果胶酶组成，其中，纤维素酶、半纤维素酶和果胶酶的质量比为3:2:1。将上述复合酶混匀，灭菌，加入到提取罐中，加入量为14mg/kg甘蔗渣，在30℃下搅拌浸提5h，将提取液灭酶10min，3000r/min离心，真空浓缩，得到甘蔗花色苷粗提液C 1。
2.大孔树脂NKA-9和葡聚糖凝胶Sephadex G-50经预处理后装柱，采用玻璃层析柱(7.5mm×240mm)，将得到的浓缩液稀释至5.2g/L，取900mL稀释液上样，上样流速控制4BV/h，流出液在520nm处的吸光值达到稀释液十分之一时，吸附达到饱和。用纯水以1.5BV/h流速冲洗5BV，洗去杂质；然后用酸化甲醇(pH1.0)洗脱，收集洗脱液；将洗脱液进行真空浓缩，温度40℃，转速80r/min，得到除杂后的花色苷浓缩液。用酸化甲醇稀释浓缩液，使最终稀释液在520nm的吸光值为0.6。
3.用盐酸将步骤2得到的稀释液pH调整为1.0，上样0.65BV，用酸化甲醇(pH1.0)洗脱，洗脱流速为80mL/h，每10min收集洗脱液，并在523nm处测量吸光值，合并吸光值大于零的洗脱液，得到花色苷纯化物C 2，将洗脱液进行真空浓缩，温度35℃，转速80r/min，使固形物达到70％。
4.将步骤3中浓缩液冷冻干燥，得到暗红色粉末即为花色苷。
上述实施例所得高含量甘蔗花色苷的理化性质的测定：
1).花色苷含量的测定
采用pH示差法测定甘蔗皮花色苷总含量，并以矢车菊-3-O-葡萄糖苷为标准，检测结果表示为mg矢车菊-3-葡萄糖苷/100g样品。
pH示差法的具体操作：取甘蔗皮花色苷纯化物定容到一定体积，3000r/min离心10min，分别取4mL上清液于两个20mL离心管中，再分别加入6mLpH1.0的KCl-HCl缓冲液和pH值为4.5的CH3COONa-HCl缓冲液，混匀，平衡60min。用蒸馏水做空白对照，取1mL，使用紫外-可见分光光度计在λmax(甘蔗皮花色苷的最大吸收波长，经扫描确定)和700nm处分别测定吸光度A，按下列公式计算：
花色苷含量(mg/100g)＝A/εL×Mr×DF×V/Wt
其中，吸光度A＝(A max—A 700)pH 1.0—(A max—A 700)pH 4.5
式中：A—吸光度；ε—矢车菊-3-葡萄糖苷的消光系数，29600；L—光程，1cm；Mr—矢车菊-3-葡萄糖苷的相对分子量，449.2；DF—稀释倍数；V：最终体积,mL；Wt:原料质量，g；A max：最大吸收波长处的吸光值；A 700：700nm处的吸光值；pH 1.0：在pH值为1.0的溶液中测量吸光度；pH 4.5：在pH值为1.0的溶液中测量吸光度
λ max的确定：取离心后的甘蔗皮花色苷提取液适量，以溶剂为空白，用DU-730紫外-可见分光光度计在波长为200-800nm的范围内扫描其吸收光谱，得到甘蔗皮花色苷在可见光区的最大吸收波长。
pH1.0KCl-HCl缓冲液的配制：取125mL 0.2mol/L的KCl溶液与375mL 0.2mol/L的HCl溶液均匀混合，即得。
pH4.5CH 3COONa-HCl缓冲液的配制：取400mL 1mol/L的CH 3COONa溶液、240mL 1mol/L的HCl溶液和360mL H 2O均匀混合，即得。
表1 不同实施例中甘蔗花色苷含量
花色苷纯化物 花色苷含量(mg/100g)
A 2 60.75±2.78
B 2 47.33±1.06
C 2 51.95±1.29
本发明实施例所得到的花色苷纯化物含量最低为47.33mg/100g甘蔗渣，最高为60.75mg/100g甘蔗渣，传统水醇浸提得到的甘蔗各部分中花色苷总量为36.6mg/100g甘蔗干重(Li,X.,et al.,Determination and comparison of flavonoids and anthocyanins  in Chinese sugarcane tips,stems,roots and leaves.J Sep Sci,2010.33(9):p.1216-23)。相比传统方法本发明中酶解法得到花色苷的含量增加了22.67～39.75％，效果显著。现有技术(何雄等，甘蔗皮花色苷的提取工艺及稳定性初探.食品工业科技，2011(12)：第371-373，376页)提及将甘蔗表皮刮下，通过水醇辅以超声浸提，得到甘蔗花色苷的含量达到288.06mg/100g，此方法是以甘蔗皮中花色苷的含量来表示，而本发明中以甘蔗渣中花色苷的含量来计算得出，两者的含量比较基础不同，故结果有较大差异，而且该方法没有考虑到实际应用中分离甘蔗皮需要投入大量人力和物力，不但极大增加生产成本，而且难以实现产业化，由此得到的花色苷含量不具有实际应用价值。复合酶法通过各种酶之间的协同作用，破坏甘蔗渣的组织结构，使细胞内外花色苷尽可能多的释放出来，酶的专一性确保了花色苷的天然状态，大孔吸附树脂和葡聚糖凝胶树脂联用除去了大量多酚黄酮等杂质，极大地减弱了杂质对花色苷物质地降解作用。
2).UPLC-MS和MS/MS测定甘蔗花色苷成分
使用超高液相-质谱联用方法对实施例所得花色苷纯化物进行分析。将所得产物A 2溶于甲醇，分别配置成10μg/ml的溶液。过0.45μm的滤膜后，用UPLC-MS/MS分析。
色谱条件：进样体积5μL；流动相A为色谱乙腈，流动相B为0.1％TFA的超纯水；梯度条件：0min，95％A；2.3min，80％A；3.4min，75％A；6.8min，60％；7.9min，95％A；9min，95％A。
质谱条件：离子源：ESI，正离子模式下50m/z到1500m/z，毛细管电压3500V，干燥气体：N 2，流速：4.0l/min，雾化器压力:0.3bar，干燥器温度:180℃。
实施例1的测试结果如表2所示。现有的提取纯化方法得到的甘蔗花色苷种类只有2～5种。说明本发明中甘蔗皮花色苷的提取纯化方法不仅提高了花色苷的产量，而且保证了花色苷的天然和稳定性，主要原因为本发明复合酶法温和且具有专一性，提取得到的甘蔗花色苷结构得到了最大程度的保存，大孔吸附树脂和葡聚糖凝胶树脂的联用可以去除大量的杂质，避免了其它杂质对花色苷的降解作用。
表2 所得甘蔗花色苷一级质谱和二级质谱分析结果
Figure PCTCN2017116390-appb-000001
Figure PCTCN2017116390-appb-000002
注： a和 b分别为花色苷一级质谱(MS)的离子碎片和二级质谱(MS/MS)的离子碎片。
其MS总离子流图和MS/MS总离子流图分别如图1和图2所示。
实施例1中得到10种花色苷：矢车菊素-3-O-葡萄糖苷(cyanidin 3‐O‐glucoside)，矢车菊素‐3，5‐O‐二葡萄糖苷(cyanidin 3，5‐di‐O‐glucoside)，矢车菊素‐3‐O‐阿魏酰‐5‐O‐葡萄糖苷(cyanidin3‐O‐feruloylglucoside‐5‐O‐glucoside)，芍药色素‐3‐O‐葡萄糖苷(peonidin3‐O‐glucoside)，矢车菊素‐3‐O‐丙二酰‐萄糖苷(cyanidin‐3‐O‐(malonyl)‐glucoside)，矢车菊素‐3‐O‐丁二酰‐萄糖苷(cyanidin‐3‐O‐(succinyl)‐glucoside)，矢车菊素‐3‐O‐咖啡酰葡萄糖苷‐5‐O‐丙二酰‐葡萄糖苷(cyanidin3‐O‐caffeoylglucoside‐5‐O‐malonylglucoside)，矢车菊素‐3‐(6‐乙酰)葡萄糖苷(cyanidin 3‐(6″‐acetoyl)glucoside)，锦葵色素‐3‐O‐葡萄糖苷(malvidin3‐O‐glucoside)，矢车菊素‐7甲氧基‐3‐O‐(2‐没食子酰)‐葡萄糖苷(7‐O‐Methyl‐cyanidin‐3‐O‐(2″galloyl)‐galactoside)。
3)甘蔗皮多酚DPPH自由基清除力测定
取上述实施例1中的纯化物A 2分别稀释100倍、125倍、167倍、250倍及500倍，得到20、16、12、8.0、4.0mg(以蔗渣粉计)/mL的样品溶液。每组试验平行3次进行，取各梯度样品液2mL，分别加入0.2mM的DPPH乙醇溶液2mL，充分混匀，在暗处静置30分钟后离心15分钟以分离沉淀物，转速为4800r/min。然后，用无水乙醇和去离子水等体积混合调零，取样品离心后的上清液测定其在517nm处的吸光度A样品。其中，无水乙醇与DPPH乙醇溶液等体积混合(2mL)在517nm处测得的吸光度为A0，而甘蔗渣提取物样品液与无水乙醇液混匀测得的517nm处吸光值为A对照。
甘蔗渣花色苷的DPPH·自由基清除率/％＝[1-(A样品-A对照)/A0]×100，同时计算IC50值。
复合酶法提取得到的甘蔗花色苷对DPPH·自由基的清除率达到了72.58％，和传统水醇浸提方法相比提高了15.8％，可能是酶解较大程度地暴露了更多的H，·且方法比较温和不会 破坏这种电子结构，所以其抗DPPH·自由基能力更强。
4)甘蔗皮多酚ABTS+清除力测定
首先取适量的14mM的ABTS+原溶液与等体积的4.9mM的过硫酸钾混匀，然后在常温下暗处保藏15小时制成储备液。然后在30℃室温内将储备液用50％的乙醇溶液稀释至其吸光度达到0.7±0.02(波长为734nm)，以备用。每组实验平行3次进行，取上述实施例1中的纯化物A 2用去离子水稀释至25、20、15、10、5mg(以蔗渣粉计)/mL，各取0.1mL。然后加入2.9mL的ABTS+溶液振荡混合，室温下静置25分钟。待反应完成后测得的吸光度为Ai(734nm)，其中，样品利用去离子水取代，其他条件相同，在734nm处测得的吸光度是Aj。
将各吸光度代入公式可求出样品的ABTS+清除百分率/％＝(Aj-Ai)/Aj×100，同时算出IC50值。
复合酶法得到的甘蔗花色苷对ABTS自由基的清除率达到了59.32％，和传统水醇浸提方法相比提高了26.3％，可能是本发明提取出了更多具有带电基团的花色苷类物质，从而使其抗ABTS基团相关的活性明显增强。
5)甘蔗皮多酚ORAC自由基吸收能力测定
取上述实施例1中的纯化物A 2稀释成1mg(以蔗皮粉计)/mL的溶液，各取20μL于96孔板中，每组以三组实验为平行，再分别加入7mM的FL荧光素溶液和磷酸盐缓冲溶液(pH7.4)各20μL，37℃处静置15分钟，然后在各微孔中迅速用多道移液器加入140μL的12mM的AAPH自由基溶液，同时将该孔板至于37℃环境中的酶标仪，然后在2小时内连续测定每隔2分钟各微孔的荧光强度，其中485nm为激发波长，538nm为发射波长，求出样品的荧光曲线面积AUC。另外一组不添加APPH的FL荧光剂作为空白对照，为自然衰减面积AUC空白，则样品的荧光净面积为AUC-AUC空白。
同时，以Trolox物质作为标准抗氧化剂作出标准曲线，然后由样品的荧光净面积和Trolox的标准曲线就可以求出甘蔗皮提取物的ORAC值(结果以μM Trolox/g蔗皮粉计)
复合酶法提取的甘蔗花色苷的ORAC值达到了909.50μmol Trolox/g，相比传统水醇浸提方法提高了60％。可能是酶解方法温和，花色苷中的H·得到最大程度的保存，所以其对该方法中产生的过氧化自由基ROO·有很强的清除能力。
从上述结果可以看出，本发明得到高含量的甘蔗花色苷，延长了甘蔗的生产链，极大提高了甘蔗的附加价值。

Claims (10)

  1. 一种高活性甘蔗花色苷的制备方法，其特征在于包括以下步骤：
    1)取榨汁后的甘蔗渣，晾干，破碎，加入蒸馏水，用缓冲液调节pH3.0～5.0；
    将纤维素酶，半纤维素酶，果胶酶混合配制复合酶；将复合酶灭菌，混匀，加入到反应液中，在40～50℃下搅拌浸提1～5h，将提取液灭酶1～10min，离心，真空浓缩，得到甘蔗花色苷粗提液；所述纤维素酶、半纤维素酶和果胶酶的质量比为(1～20):(1～20):(1～5)；所述复合酶的添加量为5～15mg/kg甘蔗渣；
    2)将步骤1)得到的甘蔗花色苷粗提液用去离子水稀释至3～10g/L，用酸调节pH至1.0～3.0，然后将稀释液经装有大孔吸附树脂第一段色谱柱吸附，花色苷从溶液中交换到树脂上，用水洗除去盐、糖和其它杂质；用洗脱剂洗脱，得到除杂后的洗脱液，将洗脱液真空浓缩，浓缩液为除杂后的花色苷溶液，将浓缩液稀释至520nm处吸光值为0.3～1.2；
    3)将步骤2)中得到的花色苷稀溶液用酸调整pH到1.0～3.0，经装有葡聚糖凝胶树脂的第二段色谱柱进行吸附，用酸化甲醇洗脱，紫外-可见光分光光度计监测洗脱过程，收集520nm处的洗脱液，将洗脱液真空浓缩，浓缩液为花色苷纯化液；
    4)将步骤3)中得到的花色苷纯化液冷冻干燥，得到粉末状的花色苷纯化物。
  2. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，步骤2)所述第大孔吸附树脂包括AB-8、D101、NKA-9、DA201或DM-130型号树脂。
  3. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，步骤3)所述装有葡聚糖凝胶的树脂包括Sephadex LH-20、Sephadex G-25、Sephadex G-50或Sephadex G-100型号树脂。
  4. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，所述洗脱剂为酸化甲醇溶液，pH为1～3，其中甲醇体积含量为90～100％。
  5. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，步骤2)所述真空浓缩所得蒸出液用于下次大孔吸附树脂的洗脱。
  6. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，步骤3)所述将洗脱液真空浓缩所得蒸出液用于下次葡聚糖凝胶树脂的洗脱。
  7. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，步骤3)所述紫外-可见光分光光度计监测波长为500～560nm。
  8. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，步骤2)和步骤3)所述的酸为盐酸、硫酸、醋酸或柠檬酸。
  9. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，步骤2)所述的用水洗除去盐的水为去离子水、纯净水或超纯水。
  10. 根据权利要求1所述的高活性甘蔗花色苷的制备方法，其特征在于，所述纤维素酶由葡聚糖内切酶、葡聚糖外切酶和纤维二糖酶组成；所述果胶酶由果胶甲酯酶、聚半乳糖醛酸酶和果胶裂解酶组成。
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