WO2018214458A1 - 一种富含可溶性多酚以及黄酮苷元的番石榴叶及制备方法与应用 - Google Patents

一种富含可溶性多酚以及黄酮苷元的番石榴叶及制备方法与应用 Download PDF

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WO2018214458A1
WO2018214458A1 PCT/CN2017/113883 CN2017113883W WO2018214458A1 WO 2018214458 A1 WO2018214458 A1 WO 2018214458A1 CN 2017113883 W CN2017113883 W CN 2017113883W WO 2018214458 A1 WO2018214458 A1 WO 2018214458A1
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guava
mass
guava leaf
cellulase
hemicellulase
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French (fr)
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吴振强
王露
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华南理工大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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  • the invention belongs to the field of foods, in particular to a guava leaf rich in soluble polyphenols and flavonoid aglycones, and a preparation method and application thereof.
  • guava leaves As a kind of medicine and food, guava leaves have many years of use history, and have various effects such as anti-oxidation, inhibition of DNA damage, blood sugar lowering, anti-inflammatory, antibacterial, blood pressure lowering and heart protection. Numerous studies have shown that the main bioactive functional components in guava leaves include polyphenols, which can eliminate body damage caused by damage to the antioxidant defense system caused by excess oxygen or nitrogen free radicals in the body. Since plant polyphenols are mainly present in plants in three forms (free state, conjugated state, and bound state), bound polyphenols are usually combined with polysaccharides on plant cell walls, and proteins are chemically bonded. It is extracted, resulting in low utilization rate of phenolic leaf polyphenols active substances.
  • the primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a method for preparing guava leaves rich in soluble polyphenols and flavonoid aglycones.
  • Another object of the present invention is to provide a guava leaf product obtained by the above production method.
  • a further object of the present invention is to provide the use of the guava leaf product described.
  • a preparation method of guava leaves rich in soluble polyphenols and flavonoid aglycones comprising the following steps:
  • the drying conditions described in the step (1) are preferably dried to a constant weight at 50 to 80 ° C; more preferably dried at 60 ° C to a constant weight.
  • the sieving described in step (1) is preferably selected through a sieve having a pore size of 4 mesh.
  • the amount of water used in the step (2) is preferably such that the guava leaves finally obtained in the step (1) are dispersed to facilitate the enzymatic hydrolysis reaction; preferably the quality of the guava leaf finally obtained in the step (1). 4 times.
  • the pH in the step (2) is from 4.5 to 6.0; preferably from 5 to 5.5.
  • the temperature of the enzymatic reaction described in the step (2) is 45 to 55 ° C; preferably 50 ° C.
  • the time of the enzymatic reaction described in the step (2) is preferably 5 to 8 hours per enzyme reaction; more preferably 6 hours for each enzyme reaction.
  • the enzyme described in the step (2) is at least one of a cellulase, a hemicellulase, a ⁇ -glucosidase, and a xylanase; preferably a cellulase, a hemicellulase, and a ⁇ -glucoside A combination of enzymes is used.
  • the cellulase is preferably a cellulase having an enzyme activity of 8000 U/g.
  • the hemicellulase is preferably a hemicellulase having an enzyme activity of 8000 U/g.
  • the ⁇ -glucosidase is preferably a ⁇ -glucosidase having an enzyme activity of 8000 U/g.
  • the xylanase is preferably a xylanase having an enzyme activity of 8000 U/g.
  • the mass ratio of the cellulase is preferably equivalent to 0.5% of the mass of the guava leaf portion.
  • the mass amount of the hemicellulase is preferably equivalent to 0.5% of the mass of the guava leaf portion.
  • the mass amount of the ⁇ -glucosidase is preferably equivalent to 0.5% of the mass of the guava leaf part.
  • the mass amount of the xylanase is preferably equivalent to 0.5% of the mass of the guava leaf part.
  • step 1) The specific process of the enzymatic reaction is preferably as shown in steps 1), 2) or 3), most preferably step 1):
  • reaction conditions of the first enzymatic hydrolysis, the second enzymatic hydrolysis and the third enzymatic hydrolysis are preferably respectively reacted at 50 ° C for 6 h;
  • the inactivation condition is preferably 80 ° C for 10 min;
  • the mass of the cellulase is preferably equal to 0.5% of the mass of the guava leaf part
  • the mass of the hemicellulase is preferably equal to 0.5% of the mass of the guava leaf part
  • the mass amount of the ⁇ -glucosidase is preferably equivalent to 0.5% of the mass of the guava leaf part.
  • the drying temperature described in the step (3) is preferably 50 to 70 ° C; more preferably 60 ° C.
  • the drying time described in the step (3) is preferably at least 12 h; more preferably 16 h.
  • a guava leaf product rich in soluble polyphenols and flavonoid aglycones is obtained by the above preparation method.
  • the guava leaf product rich in soluble polyphenols and flavonoid aglycones is applied in the field of food and/or health care products; it can be directly eaten; and can be further processed into various foods, such as soluble polyphenols. And flavonoid agave guava leaf tea beverage, guava leaf biscuit rich in soluble polyphenols and flavonoid aglycones, nutritious meals and the like.
  • the preparation method provided by the invention is to release the insoluble, insoluble, bound polyphenols in the guava leaves by various enzymatic hydrolysis, and convert them into easy-to-extract, soluble polyphenols; and the macromolecular function of the guava leaves
  • the components are degraded into aglycone and kaempferol with higher absorption capacity and higher functional activity.
  • the enzymatic hydrolysis reaction time is short, the conditions are mild, and the efficiency is high, which can be used for the processing and enhancement of medicinal plants.
  • the preparation method enhances the anti-oxidation ability of guava leaf products, inhibits DNA damage ability, lowers blood sugar, cholesterol, and prevents cardiovascular and cerebrovascular diseases.
  • Figure 1 is a graph showing the results of determination of total soluble polyphenols and insoluble polyphenols in guava leaves in different examples.
  • Figure 2 is a graph showing the results of determination of total soluble flavonoids and insoluble flavonoids in guava leaves in different examples.
  • step (1) a plurality of enzymatic hydrolysis reactions: then xylanase (8000 U / g) and the guava leaves finally obtained in step (1) are uniformly mixed in a triangular flask, and after enzyme treatment for 6 hours in a water bath at 50 ° C, Placed in an oven at 80 ° C for 10 min (inactivated xylanase), cooled to room temperature; then added cellulase, mixed evenly, enzymatically hydrolyzed for 6 h in a water bath at 50 ° C, placed in an oven at 80 ° C for 10 min (inactivated cellulase) ), cooled to room temperature; continue to add hemicellulase, mix well, enzymatic hydrolysis in a water bath at 50 ° C for 6 h, placed in an oven at 80 ° C for 10 min (inactivated hemicellulase); wherein, xylanase, fiber
  • step (1) a variety of enzymatic hydrolysis reactions: then the ⁇ -glucosidase and the guava leaves finally obtained in step (1) are uniformly mixed in a triangular flask, and subjected to enzymatic treatment for 6 hours in a water bath at 50 ° C, at 80 ° C.
  • guava leaf products prepared in Examples 1 to 4 and the untreated guava leaves were pulverized by a mill and passed through a 40-mesh sieve for extraction and detection of the following components:
  • Soluble polyphenol extraction 1.0 g of the guava leaf products prepared in Examples 1 to 4 were respectively taken in a 50 mL colorimetric tube, and 25 mL of a 50% (v/v) methanol solution was added and immersed in a 45 ° C water bath for 1 hour. The filter was filtered through a 0.45 ⁇ m filter paper, and the filtrate was subjected to rotary evaporation on a vacuum rotary evaporator at 37 ° C for 30 min to remove methanol to obtain a concentrate. 40 mL of distilled water was added to the concentrate, followed by degreasing with 10 mL of hexane, followed by extraction with 70 mL of ethyl acetate.
  • Detection of polyphenol content 100 ⁇ L of the above extracted soluble polyphenols and insoluble bound polyphenol extract were separately taken and diluted to a suitable concentration. Take 1mL of diluted sample solution or gallic acid standard solution (10-100 ⁇ g/mL), add 0.5mL of phenylephrine reagent in sequence, mix for 3-8min, then add 1.5mL of 20% (w/v) Na 2 CO 3 solution. Add water to a volume of 10 mL, mix well and shake for 30 min. The absorbance at 760 nm was measured using a blank reagent as a control.
  • the mobile phase used was: A-0.1% (v/v) aqueous formic acid, B-acetonitrile solution, flow rate of 0.8 mL/min, and injection volume of 10 ⁇ L.
  • Detection conditions gradient elution - 0 min, 85% A + 15% B, 5 min, 85% A + 15% B, 10 min, 80% A + 20% B, 20 min, 65% A + 35% B, 30 min, 50 %A+50%B, 31min, 20%A+80%B, 40min, 20%A+80%B, 45min, 85%A+15%B, 50min, 85%A+15%B (all volumes) ratio).
  • the analysis time is 50 min.
  • ⁇ L of the above extracted soluble polyphenol and the insoluble bound polyphenol extract were respectively taken and diluted to a suitable concentration. Take 100 ⁇ L of the diluted sample solution or vitamin C standard solution (5-30 ⁇ g/mL), add 400 ⁇ L of DPPH-methanol reagent, and let stand at 30 ° C for 30 min in the dark. A negative control was used for water, and VC was used as a positive control to measure the absorbance at 510 nm.
  • the DPPH radical scavenging ability of the sample is expressed by VC, that is, the sample per g of guava leaves corresponds to the number of mmol/L of VC.
  • the content of flavonoid aglycones was determined by high performance liquid chromatography.
  • the quercetin and kaempferol were the highest in the order of addition of various enzymes in the order of Example 1, which were 248.95 mg/100 g DM and 11.35 mg/100 g DM, respectively, which was 1.97 times and 1.82 times higher than that of the untreated group. .
  • the total antioxidant activity of guava leaf soluble polyphenol extract and the inhibition of DNA damage after various enzyme treatments in Examples 1 and 2 were significantly improved, and Example 1 was subjected to various enzymes (0.5% cellulase, 0.5% and half).
  • the guava leaves treated with cellulase and 0.5% ⁇ -glucosidase had the highest biological activity.
  • the DPPH and ABTS + radical scavenging capacity is equivalent to 74.29 mmol VC/g DM and 77.41 mmol VC/g DM.
  • the inhibition rate of DNA damage reached 81.23%.
  • the guava leaves treated by the method of Example 3 had the lowest activity.
  • the test data is shown in Table 1.

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Abstract

一种富含可溶性多酚以及黄酮苷元的番石榴叶及制备方法与应用。通过将清洗干净的番石榴叶沥干、烘干、揉碎、过筛，得到番石榴叶部位；再将其与水混合后，调节好pH值，再加入酶，进行酶解反应；结束酶解反应的体系烘干，得到富含可溶性多酚以及黄酮苷元的番石榴叶产品。通过该制备方法得到的番石榴叶茶可溶性多酚含量得到了极大的提高，并将番石榴叶的黄酮糖苷成分降解为功能活性更强的黄酮苷元成分，提高槲皮素、山奈酚等苷元含量，提升了番石榴叶抗氧化以及抗DNA损伤作用。从而，该富含可溶性多酚以及黄酮苷元番石榴叶在食品领域和/或保健品领域中应用的潜力大。

Description

一种富含可溶性多酚以及黄酮苷元的番石榴叶及制备方法与应用 技术领域
本发明属于食品领域，特别涉及一种富含可溶性多酚以及黄酮苷元的番石榴叶及制备方法与应用。
背景技术
番石榴叶作为一种药食两用的物质，有着多年的使用历史，具有抗氧化、抑制DNA损伤，降血糖、抗炎、抑菌、降血压，保护心脏等多种疗效。不少研究表明番石榴叶中主要的生物活性功能成分包括多酚类物质，这些活性物质能够消除由体内过剩氧或者氮自由基引起的抗氧化防御系统受损引起的机体伤害。由于植物多酚类物质主要以三种形式(自由态，共轭态以及绑定态)存在于植物体内，而绑定态多酚通常与植物细胞壁上的多糖，蛋白质以化学键形式结合，很难被提取，造成番石榴叶多酚类活性物质利用率低。
因此，有必要促进番石榴叶的可溶性多酚和黄酮苷元的释放，从而充分利用番石榴叶多酚类活性物质。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法。
本发明的另一目的在于提供通过上述制备方法得到的番石榴叶产品。
本发明的再一目的在于提供所述的番石榴叶产品的应用。
本发明的目的通过下述技术方案实现：一种富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法，包括如下步骤：
(1)将清洗干净的番石榴叶沥干、烘干、揉碎，将揉碎的番石榴叶过筛，去除番石榴叶茎干部位，获得大小基本一致的番石榴叶部位；
(2)将步骤(1)最终得到的番石榴叶部位与水混合后，调节好pH值，再加入酶，进行酶解反应；
(3)将步骤(2)进行酶解反应后的体系烘干，得到富含可溶性多酚以及黄酮苷元的番石榴叶产品。
步骤(1)中所述的烘干的条件优选为于50～80℃烘干至恒重；更优选为于60℃烘干至恒重。
步骤(1)中所述的过筛优先选过孔径为4目的筛。
步骤(2)中所述的水的用量为将步骤(1)最终得到的番石榴叶分散为宜，以有利于进行酶解反应；优选为相当于步骤(1)最终得到的番石榴叶质量的4倍。
步骤(2)中所述的pH值为4.5～6.0；优选为5～5.5。
步骤(2)中所述的酶解反应的温度为45～55℃；优选为50℃。
步骤(2)中所述的酶解反应的时间优选为按每一种酶反应5～8h计；更优选为每一种酶反应6h计。
步骤(2)中所述的酶为纤维素酶、半纤维素酶、β-葡萄糖苷酶和木聚糖酶中的至少一种；优选为纤维素酶、半纤维素酶和β-葡萄糖苷酶的组合使用。
所述的纤维素酶优选为酶活力是8000U/g的纤维素酶。
所述的半纤维素酶优选为酶活力是8000U/g的半纤维素酶。
所述的β-葡萄糖苷酶优选为酶活力是8000U/g的β-葡萄糖苷酶。
所述的木聚糖酶优选为酶活力是8000U/g的木聚糖酶。
所述的纤维素酶的质量用量优选为相当于番石榴叶部位质量的0.5％。
所述的半纤维素酶的质量用量优选为相当于番石榴叶部位质量的0.5％。
所述的β-葡萄糖苷酶的质量用量优选为相当于番石榴叶部位质量的0.5％。
所述的木聚糖酶的质量用量优选为相当于番石榴叶部位质量的0.5％。
所述的酶解反应的具体过程优选如步骤1)、2)或3)所示，最优选为步骤1)：
1)先加入纤维素酶进行第一次酶解，灭活纤维素酶；再加入半纤维素酶进行第二次酶解，灭活半纤维素酶；最后加入β-葡萄糖苷酶进行第三次酶解，灭活β-葡萄糖苷酶；
2)先加入半纤维素酶进行第一次酶解，灭活半纤维素酶；再加入纤维素酶进行第二次酶解，灭活纤维素酶；最后加入β-葡萄糖苷酶进行第三次酶解，灭活β-葡萄糖苷酶；
3)先加入β-葡萄糖苷酶进行第一次酶解，灭活β-葡萄糖苷酶；再加入纤维素酶进行第二次酶解，灭活纤维素酶；最后加入半纤维素酶进行第三次酶解，灭活半纤维素酶。
步骤1)、2)和3)中，
所述的第一次酶解、第二次酶解和第三次酶解的反应条件分别优选为于50℃反应6h；
所述的灭活的条件优选为80℃处理10min；
所述的纤维素酶的质量用量优选为相当于番石榴叶部位质量的0.5％；
所述的半纤维素酶的质量用量优选为相当于番石榴叶部位质量的0.5％；
所述的β-葡萄糖苷酶的质量用量优选为相当于番石榴叶部位质量的0.5％。
步骤(3)中所述的烘干的温度优选为50～70℃；更优选为60℃。
步骤(3)中所述的烘干的时间优选至少12h；更优选为16h。
一种富含可溶性多酚以及黄酮苷元的番石榴叶产品，通过上述制备方法得到。
所述的富含可溶性多酚以及黄酮苷元的番石榴叶产品在食品领域和/或保健品领域中进行应用；其可直接食用；也可进一步加工成各类食品，如富含可溶性多酚及黄酮苷元的番石榴叶茶饮料、富含可溶性多酚及黄酮苷元的番石榴叶饼干，营养餐条等。
本发明相对于现有技术具有如下的优点及效果：
本发明提供的制备方法，是通过多种酶水解将番石榴叶中不易提取的，不可溶的绑定态多酚释放，转变为易提取，可溶性多酚；并将番石榴叶的大分子功能成分降解为吸收能力更强，功能活性更高的小分子槲皮素及山奈酚等苷元含量；而且酶解反应时间短，条件温和，效率高，可以用于药用植物加工增效。该制备方法提升番石榴叶产品抗氧化能力，抑制DNA损伤能力，降低血糖，胆固醇，预防心脑血管疾病等作用。
附图说明
图1是不同实施例中番石榴叶总可溶性多酚与不可溶性多酚含量的测定结果图。
图2是不同实施例中番石榴叶总可溶性黄酮与不可溶性黄酮含量的测定结果图。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
(1)番石榴叶基质的制备：将清洗干净的番石榴叶放入60℃烘箱烘干16h，揉碎过孔径为4目的筛，过筛的番石榴叶即为酶促水解基质；在酶促反应基质中加入水，水的用量(用柠檬酸调pH＝5.5)是总重量的80％；
(2)酶水解反应：接着将纤维素酶(8000U/g，下同)和步骤(1)最终得到的番石榴叶混合均匀置于三角瓶中，在50℃水浴下，酶处理6h后，置于80℃烘箱10min(灭活纤维素酶)，冷却至室温；然后加入半纤维素酶(8000U/g，下同)，混合均匀，在50℃水浴下，酶解6h，置于80℃烘箱10min(灭活半纤 维素酶)，冷却至室温；继续再加入β-葡萄糖苷酶(8000U/g，下同)，混合均匀，在50℃水浴下，酶解6h，置于80℃烘箱10min(灭活β-葡萄糖苷酶)；其中，纤维素酶、半纤维素酶、β-葡萄糖苷酶的质量用量分别是相当于步骤(1)得到的过筛的番石榴叶(即酶促水解基质)质量的0.5％。
(3)酶水解后番石榴叶产品处理：将多种酶水解后的番石榴叶置于烘箱60℃烘干16h，得到富含可溶性多酚与黄酮苷元的番石榴叶产品。
实施例2
(1)番石榴叶基质的制备：基本与实施例1步骤(1)相同，区别在于，用柠檬酸调pH＝5.5。
(2)多酶水解反应：接着将半纤维素酶和步骤(1)最终得到的番石榴叶混合均匀置于三角瓶中，在50℃水浴下，酶处理6h后，置于80℃烘箱10min(灭活半纤维素酶)，冷却至室温；然后加入纤维素酶，混合均匀，在50℃水浴下，酶解6h，置于80℃烘箱10min(灭活纤维素酶)，冷却至室温；继续再加入β-葡萄糖苷酶，混合均匀，在50℃水浴下，酶解6h，置于80℃烘箱10min(灭活β-葡萄糖苷酶)；其中，半纤维素酶、纤维素酶、β-葡萄糖苷酶的质量用量分别是相当于步骤(1)得到的过筛的番石榴叶(即酶促水解基质)质量的0.5％。
(3)酶水解后番石榴叶产品处理：将多种酶水解后的番石榴叶置于烘箱60℃烘干16h，得到富含可溶性多酚与黄酮苷元的番石榴叶产品。
实施例3
(1)番石榴叶基质的制备：与实施例2步骤(1)相同。
(2)多种酶水解反应：接着将木聚糖酶(8000U/g)和步骤(1)最终得到的番石榴叶混合均匀置于三角瓶中，在50℃水浴下，酶处理6h后，置于80℃烘箱10min(灭活木聚糖酶)，冷却至室温；然后加入纤维素酶，混合均匀，在50℃水浴下，酶解6h，置于80℃烘箱10min(灭活纤维素酶)，冷却至室温；继续再加入半纤维素酶，混合均匀，在50℃水浴下，酶解6h，置于80℃烘箱10min(灭活半纤维素酶)；其中，木聚糖酶、纤维素酶、半纤维素酶的质量用量分别是相当于步骤(1)得到的过筛的番石榴叶(即酶促水解基质)质量的0.5％。
(3)酶水解后番石榴叶产品处理：将多种酶水解后的番石榴叶置于烘箱60℃烘干16h，得到富含可溶性多酚与黄酮苷元的番石榴叶产品。
实施例4
(1)番石榴叶基质的制备：与实施例2步骤(1)相同。
(2)多种酶水解反应：接着将β-葡萄糖苷酶和步骤(1)最终得到的番石榴叶混合均匀置于三角瓶中，在50℃水浴下，酶处理6h后，置于80℃烘箱10min(灭活β-葡萄糖苷酶)，冷却至室温；然后加入纤维素酶，混合均匀，在50℃水浴下，酶解6h，置于80℃烘箱10min(灭活纤维素酶)，冷却至室温；继续再加入半纤维素酶，混合均匀，在50℃水浴下，酶解6h，置于80℃烘箱10min(灭活半纤维素酶)；其中，β-葡萄糖苷酶、纤维素酶、半纤维素酶的质量用量分别是相当于步骤(1)得到的过筛的番石榴叶(即酶促水解基质)质量的0.5％。
(3)酶水解后番石榴叶产品处理：将多种酶水解后的番石榴叶置于烘箱60℃烘干16h，得到富含可溶性多酚与黄酮苷元的番石榴叶产品。
效果实施例
一、检测方法
将实施例1～4制备的番石榴叶产品以及未经处理的番石榴叶用磨粉机粉碎，通过40目筛，用于如下成分的提取与检测：
①可溶性多酚提取：分别取1.0g实施例1～4制备的番石榴叶产品于50mL比色管中，加入25mL 50％(v/v)甲醇溶液，在45℃水浴浸提1h后，用0.45μm滤纸过滤，滤液通过真空旋转蒸发仪在37℃下，旋蒸30min，去除甲醇，获得浓缩液，向浓缩液中加入40mL蒸馏水，然后加入10mL己烷脱脂，再用70mL乙酸乙酯萃取3次，合并萃取液，在35℃下，真空旋干，去除乙酸乙酯。最后加入5mL 50％(v/v)甲醇溶解，即为可溶性多酚提取液。置于-20℃保存，用于多酚含量分析以及HPLC定量分析。
②不可溶绑定态多酚提取：将步骤①中提取可溶性多酚后剩余的番石榴叶残渣加入40mL蒸馏水去除有机溶剂，滤干，于60℃烘干至恒重，记录残渣的重量。加入40mL 4M NaOH溶液，室温下提取4h，然后用浓盐酸(浓度为37％)调整pH至2左右，加入70mL乙酸乙酯萃取3次，合并萃取液，在35℃下，真空旋干，去除乙酸乙酯，最后加入5mL 50％甲醇溶解，即为不可溶绑定态多酚提取液。置于-20℃保存，用于多酚含量分析以及HPLC定量分析。
③多酚含量的检测：分别吸取100μL上述提取的可溶性多酚与不可溶性绑定态多酚提取液，稀释至合适浓度。取1mL稀释样液或者没食子酸标准液(10-100μg/mL)，依次加入0.5mL福林酚试剂混匀，反应3-8min，再加入1.5mL20％(w/v)Na2CO3溶液，加水定容至10mL，充分振荡混匀，静置30min。以空白试剂做对照，测定760nm下的吸光值。
④黄酮含量的的测定：分别吸取100μL上述提取的可溶性多酚与不可溶性绑定态多酚提取液，稀释至合适浓度。取1mL稀释样液或者芦丁标准液 (10-100μg/mL)，依次加入0.3mL 5％(w/v)NaNO2溶液混匀，静置5min。加入0.3mL 10％(w/v)AlCl3溶液混匀，静置6min。再加入2mL 4％(w/v)NaOH溶液混匀，加70％(v/v)乙醇溶液定容至10mL，充分振荡，静置10min。以空白试剂做对照，测定510nm下的吸光值。
⑤黄酮苷元(槲皮素与山萘酚)检测：分别吸取将上述提取的可溶性多酚与不可溶性绑定态多酚提取液用0.45μm滤纸过滤，取清液过0.22μm有机微孔滤膜，滤液进行HPLC分析。具体分析条件为：紫外检测器(Waters 2998)的高效液相色谱系统(Waters 2695)，检测波长350nm，柱温30℃，C18色谱柱。所用的流动相为：A-0.1％(v/v)甲酸水溶液，B-乙腈溶液，流速为0.8mL/min，进样量10μL。检测条件：梯度洗脱—0min、85％A+15％B，5min、85％A+15％B，10min、80％A+20％B，20min、65％A+35％B，30min、50％A+50％B，31min、20％A+80％B，40min、20％A+80％B，45min、85％A+15％B，50min、85％A+15％B(均为体积比)。分析时间为50min。
⑤抗氧化能力检测：
a：DPPH自由基清除能力
分别吸取100μL上述提取的可溶性多酚与不可溶性绑定态多酚提取液，稀释至合适浓度。取100μL稀释样液或者维生素C标准液(5-30μg/mL)，加入400μL DPPH-甲醇试剂，于30℃，黑暗处静置30min。以水做阴性对照，VC作为阳性对照，测定510nm下的吸光值。样品的DPPH自由基清除能力用VC表示，即每g番石榴叶样品相当于VC的mmol/L数。
b：ABTS+自由基清除能力
分别吸取100μL上述提取的可溶性多酚与不可溶性绑定态多酚提取液，稀释至合适浓度。取50μL稀释样液或者维生素C标准液(5-30μg/mL)，加入400μL ABTS+(7mM ABTS与2.45mM K2S2O8以2:1体积比混合，黑暗静置16h)试剂，于30℃，黑暗处静置30min。以水做阴性对照，VC作为阳性对照，测定510nm下的吸光值。样品的ABTS+自由基清除能力用VC表示，即每g番石榴叶样品相当于VC的mmol/L数。
⑥抗氧化能力检测：
分别取上述2μL稀释可溶性多酚与不可溶性绑定态多酚提取液样液(2mg/mL)以及槲皮素标准液(2mg/mL)，加入5μL pMD 18-T质粒DNA(200ng/μL)，10μL Fenton试剂(50mM VC、80mM FeCl3、以及30mM H2O2)，用移液枪混匀，在37℃下黑暗静置30min。以PBS缓冲液做空白对照，槲皮素作为阳性对照，然后将混合液加样于1％琼脂糖凝胶电泳，电泳后的DNA胶于紫外条件下观察，计算螺旋状DNA占总DNA的比例。DNA损伤抑制率计算公式如 下：
Figure PCTCN2017113883-appb-000001
二、检测结果
结果如图1和2所示，发现按照实施例1和2顺序添加酶处理后的番石榴叶产品可溶性多酚含量均明显增加，可溶性黄酮含量也明显增加。而实施例1酶反应顺序按照纤维素酶、半纤维素酶和β-葡萄糖苷酶处理后番石榴叶可溶性多酚含量最高(实施例1处理过的番石榴叶相对于未处理组可溶性多酚含量提高94.74％，可溶性黄酮提高89.48％，而不可溶的绑定态多酚含量明显降低。这说明多种酶处理可以促进番石榴叶可溶性多酚释放)。而用实施例3顺序添加木聚糖酶、纤维素酶、半纤维素酶多种酶后多酚释放效率最差；然而按照实施例4先用β-葡萄糖苷酶处理，再用处理过的番石榴叶相对于未处理组获得可溶性多酚效率较实施例1，2要差，但好于实施例3。
采用高效液相色谱法检测黄酮苷元(槲皮素及山奈酚)含量。采用实施例1顺序添加多种酶共同水解后，槲皮素及山奈酚的含量最高，分别为248.95mg/100g DM，11.35mg/100g DM，相对于未处理组，分别提高1.97倍，1.82倍。而实施例1、2多种酶处理后番石榴叶可溶性多酚提取液总抗氧化活性以及对DNA损伤抑制作用明显提高，其中实施例1经过多种酶(0.5％纤维素酶，0.5％半纤维素酶，以及0.5％β-葡萄糖苷酶)处理后的番石榴叶生物活性最高。DPPH、ABTS+自由基清除能力相当于74.29mmol VC/g DM、77.41mmol VC/g DM。对DNA损伤的抑制率达到了81.23％。而实施例3方法处理后的番石榴叶活性最低。检测数据见表1。
表1
Figure PCTCN2017113883-appb-000002
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法，其特征在于包括如下步骤：
    (1)将清洗干净的番石榴叶沥干、烘干、揉碎、过筛，去除番石榴叶茎干部位，获得大小基本一致的番石榴叶部位；
    (2)将步骤(1)最终得到的番石榴叶部位与水混合后，调节好pH值，再加入酶，进行酶解反应；
    (3)将步骤(2)进行酶解反应后的体系烘干，得到富含可溶性多酚以及黄酮苷元的番石榴叶产品。
  2. 根据权利要求1所述富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法，其特征在于：
    步骤(2)中所述的酶为纤维素酶、半纤维素酶、β-葡萄糖苷酶和木聚糖酶中的至少一种。
  3. 根据权利要求2所述富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法，其特征在于：
    步骤(2)中所述的酶为纤维素酶、半纤维素酶和β-葡萄糖苷酶的组合。
  4. 根据权利要求2所述富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法，其特征在于：
    所述的纤维素酶为酶活力是8000U/g的纤维素酶；
    所述的半纤维素酶为酶活力是8000U/g的半纤维素酶；
    所述的β-葡萄糖苷酶为酶活力是8000U/g的β-葡萄糖苷酶；
    所述的木聚糖酶为酶活力是8000U/g的木聚糖酶；
    所述的纤维素酶的质量用量相当于番石榴叶部位质量的0.5％；
    所述的半纤维素酶的质量用量相当于番石榴叶部位质量的0.5％；
    所述的β-葡萄糖苷酶的质量用量相当于番石榴叶部位质量的0.5％；
    所述的木聚糖酶的质量用量相当于番石榴叶部位质量的0.5％。
  5. 根据权利要求1所述富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法，其特征在于：
    步骤(1)中所述的烘干的条件为于50～80℃烘干至恒重；
    步骤(3)中所述的烘干的温度为50～70℃。
  6. 根据权利要求1所述富含可溶性多酚以及黄酮苷元的番石榴叶的制备方 法，其特征在于：
    步骤(2)中所述的pH值为4.5～6.0；
    步骤(2)中所述的酶解反应的温度为45～55℃；
    步骤(2)中所述的酶解反应的时间按每一种酶反应5～8h计。
  7. 根据权利要求1所述富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法，其特征在于：
    所述的酶解反应的具体过程如步骤1)、2)或3)所示：
    1)先加入纤维素酶进行第一次酶解，灭活纤维素酶；再加入半纤维素酶进行第二次酶解，灭活半纤维素酶；最后加入β-葡萄糖苷酶进行第三次酶解，灭活β-葡萄糖苷酶；
    2)先加入半纤维素酶进行第一次酶解，灭活半纤维素酶；再加入纤维素酶进行第二次酶解，灭活纤维素酶；最后加入β-葡萄糖苷酶进行第三次酶解，灭活β-葡萄糖苷酶；
    3)先加入β-葡萄糖苷酶进行第一次酶解，灭活β-葡萄糖苷酶；再加入纤维素酶进行第二次酶解，灭活纤维素酶；最后加入半纤维素酶进行第三次酶解，灭活半纤维素酶。
  8. 根据权利要求7所述富含可溶性多酚以及黄酮苷元的番石榴叶的制备方法，其特征在于：
    步骤1)、2)和3)中，
    所述的第一次酶解、第二次酶解和第三次酶解的反应条件分别为于50℃反应6h；
    所述的灭活的条件为80℃处理10min；
    所述的纤维素酶的质量用量为相当于番石榴叶部位质量的0.5％；
    所述的半纤维素酶的质量用量为相当于番石榴叶部位质量的0.5％；
    所述的β-葡萄糖苷酶的质量用量为相当于番石榴叶部位质量的0.5％。
  9. 一种富含可溶性多酚以及黄酮苷元的番石榴叶产品，其特征在于：通过权利要求1～8任一项所述的制备方法得到。
  10. 权利要求9所述的富含可溶性多酚以及黄酮苷元的番石榴叶产品在食品领域和/或保健品领域中的应用。
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