WO2018130091A1 - 一种结合酶与超声处理由带内种皮核桃仁中提取多酚物质的方法 - Google Patents

一种结合酶与超声处理由带内种皮核桃仁中提取多酚物质的方法 Download PDF

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WO2018130091A1
WO2018130091A1 PCT/CN2017/119777 CN2017119777W WO2018130091A1 WO 2018130091 A1 WO2018130091 A1 WO 2018130091A1 CN 2017119777 W CN2017119777 W CN 2017119777W WO 2018130091 A1 WO2018130091 A1 WO 2018130091A1
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walnut
polyphenol
walnut kernel
seed coat
kernel
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PCT/CN2017/119777
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French (fr)
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张文斌
蒋将
李笑笑
杨瑞金
华霄
赵伟
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • 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 relates to a method for extracting polyphenol substances from the in-band seed coat walnut kernel by combining enzyme and ultrasonic treatment, and belongs to the technical field of oil crop processing.
  • Walnut (Juglans regia L.), also known as walnut, is a walnut, is the first of the "four major dried fruits", is a good economic crop with both medicine and food.
  • Walnut kernel is rich in nutrients such as oil, protein and vitamins. It has many functions such as stomach, blood, lungs, kidney and brain. It is a good tonic, nutritious and delicious, so it has The reputation of “Long live the child”, “longevity fruit”, “music fruit” and “beauty fruit”. Modern research has shown that walnut seed's antioxidant, anti-aging and prevention of cardiovascular diseases are mainly attributed to the rich oils, proteins and polyphenols in walnut kernels.
  • Walnut kernels have high oil content, generally 40-65%, some up to 80%, walnut oil fatty acid composition, linoleic acid accounts for about 61%, oleic acid accounts for about 23%, linolenic acid accounts for about 7.0%, and contains ⁇ .
  • the -3 and omega-6 series of polyunsaturated fatty acids make it a significant advantage in lowering blood lipids and serum cholesterol, and are a good choice for medicinal oils or nutritional oils for the elderly and infants.
  • walnut oil is favored by consumers like olive oil, and the prospects are very broad.
  • Walnut protein has 18 kinds of amino acids, and the 8 essential amino acids required by the human body are reasonable.
  • the content of glutamic acid, aspartic acid and arginine which have important functions on the human body is high, and it is a full-price nutrient protein. Its digestibility rate is 87.2%.
  • the main components of walnut kernels are cellulose, pectin, tannins and pigments. There is a pectin between the skin and the kernel of the walnut kernel, which makes the bond between the kernel and the skin relatively firm and difficult to separate. Studies have shown that the tannins in walnut kernels are mainly found in the epidermis (more than 90% of the total), which is the most important cause of bitterness and browning.
  • lye immersion is the main pretreatment method.
  • the pectin layer between the skin and the kernel of the walnut is first dissolved, and then the whole skin is peeled off, which can be washed away by the water flow.
  • the mechanism of the lye immersion method is that the pectin will be hydrolyzed by the action of the alkali solution, and a part of the pectin is depolymerized, and the solubility in the water is improved; on the other hand, the pectic acid is converted into a water-soluble solution by the action of the alkali. Salts such as sodium pectate.
  • the lye soaking can remove the pectin more fully and remove the walnut skin.
  • the flaws of the law are also very obvious.
  • a large number of beneficial polyphenols in the walnut skin are destroyed by the lye or wasted in the peeling process;
  • some of the walnut kernels are irregular in shape, and the walnut skin in the creases with wrinkles is often not completely removed;
  • the hot lye soaking time has been shortened (about 10 min)
  • the processed walnut kernels still suffer from protein denaturation and loss.
  • Colaric et al. Cold M, Veberic R, Solar A, Hudina M, Stampar F. Phenolic Acids, Syringaldehyde, and Juglone in Fruits of Different Cultivars of Juglans regia L.
  • the contents of the three inner seeds were 1003.24, 317.90, and 128.98 mg/100 g, respectively, which were about 20 to 30 times that of walnut kernels.
  • Wan Zhengmin et al. Wang Zhengmin, Hao Yanbin, Yang Chunmei, Qi Jianxun, Zhao Liqin, Wang Kejian. High-pressure liquid chromatography analysis of polyphenols in walnut seed coat. Food Industry Science and Technology, 2007, 28(7): 212-213, 224.
  • High performance liquid chromatography (HPLC) was used to analyze polyphenols in walnut seed coats.
  • the method steps are as follows: (1) immersing the walnut to be treated in liquid nitrogen for freezing; (2) placing the treated walnut in the air until a white frost appears on the surface of the walnut; (3) white will appear
  • the frosted walnut kernel is placed in an aqueous solution of vitamin C for rehydration treatment; (4) the rehydrated walnut is subjected to ultrasonication and shaking treatment in sequence, and then washed to obtain the walnut kernel for removing the inner seed coat and the removed Inner seed coat.
  • the invention essentially utilizes the principle of different thermal expansion and contraction coefficients of materials having different structural compositions, thereby achieving separation of the inner seed coat and the walnut kernel.
  • the inner seed coat is separated by the combination of lye immersion and high pressure rinsing, but since the raw material of the walnut kernel often contains a lot of granules, the high pressure impact causes considerable loss of walnut kernel, and the polyphenol in the seed coat is also seriously lost.
  • Kang Lili et al. optimized the peeling method of walnut kernels (Kang Lili, Tang Junhu, Jing Siqun. Optimization of peeling method of walnut kernel. Food and Fermentation Industry, 2013, 36(8): 127-131), found to be dried by hot air The baking-microwave method can remove the walnut kernels.
  • the optimum processing conditions are: baking temperature 130 ° C, baking time 16 min, microwave power 769 W, microwave time 51 s, walnut peeling rate 98.01%, walnut oil peroxide value 1.65 Meq/kg. Due to the high proportion of linolenic acid in walnut oil, this high-strength heat treatment not only increases the peroxide value of the oil, but may also cause other adverse effects.
  • Another idea is to extract the polyphenols from the inner seed coat and then develop and use the walnut kernels.
  • Osborne and Campbell et al. Osborne, TB; Campbell, GF Conglutin and vitellin. J. Am. Chem. Soc., 1896, 18: 609-623.
  • the inner seed coat of walnuts achieves the purpose of removing tannins, and also increases the globulin dissolution rate of defatted walnut powder from about 3.5% to 20%.
  • pure hot water soaking polyphenol extraction efficiency is not high and protein and other losses are relatively high.
  • the present invention proposes a method for extracting polyphenol substances from walnut kernels by combining enzymes and ultrasonic treatment. Under the conditions, combined with the action of ethanol solution and pectinase and / or cellulase complex, the mass transfer process is enhanced by ultrasonic treatment to achieve efficient extraction of polyphenols from intact walnut kernels.
  • the obtained polyphenol extract can be further separated and refined, and used as an antioxidant for dietary supplements, beverages, fruit and vegetable products and snack foods; the walnut kernel extracted after polyphenol can be pulverized to obtain high quality walnut oil and low fat walnut powder, taking into consideration
  • the main components and active ingredients in walnut kernel have important reference value for the comprehensive utilization of walnut.
  • a first object of the present invention is to provide a method for extracting polyphenolic substances from in-band seed coat walnut kernels by combining enzymes and sonication, using walnut kernels with inner seed coat as raw material, in pectinase and/or With the aid of cellulase, combined with ultrasonic treatment, the polyphenols in the inner seed coat of walnut kernels were extracted efficiently by ethanol solution, and the crude polyphenol extract and the dephenolized walnut kernel product were separated by filtration.
  • the method is implemented by the following technical solutions:
  • Preparation of walnut kernels with seed coat shelling and cleaning of walnuts, and obtaining walnut kernels with inner seed coat without crushing treatment;
  • the walnut kernel comprises an inner seed coat walnut kernel obtained by a walnut sheller or a manual shell peeling, or a commercially available inner seeded walnut kernel, or other conventional shelling.
  • the walnut kernel product obtained by the separation is operated.
  • the in-band seed coat walnut kernel can reduce the consumption of the solution after the subsequent combined dephenolization treatment, and reduce the dissolution of protein, etc., which is beneficial to improve the purity of the crude polyphenol extract and the quality of the dephenolized walnut kernel product. .
  • the step (2) is carried out according to a ratio of the mass of the inner seed coat walnut kernel to the volume of the ethanol aqueous solution in terms of g: 1:3 to 1:15.
  • the obtained in-plant seed coat walnut kernel is put into an aqueous ethanol solution.
  • the volume concentration of the aqueous ethanol solution is 0 to 20%
  • the pH of the mixture is adjusted to 3.5 to 6.5 with food grade citric acid or hydrochloric acid, followed by adding pectinase to the mixture at a temperature of 10 to 60 ° C and / or cellulase complex
  • the amount of enzyme is 0.01 ⁇ 10.0U / g walnut kernel, thereby obtaining a polyphenol extraction system.
  • the ratio of the mass of the inner seed coat walnut kernel to the volume of the aqueous ethanol solution in terms of g in the step (2) is 1:3 to 1:15.
  • the ratio of the mass of the inner seed coat walnut kernel to the volume of the aqueous ethanol solution in terms of g in the step (2) is 1:5 to 1:10.
  • the volume concentration of the aqueous ethanol solution in the step (2) is 0 to 20%, and when the volume concentration is higher than 20%, the cellulase and pectinase are deactivated.
  • the volume concentration of the aqueous ethanol solution in the step (2) is 5 to 15%; when the volume concentration of the aqueous ethanol solution is 0%, the extraction time needs to be extended by more than 2 times.
  • the pH of the mixture of the inner seed coat walnut kernel and the aqueous solution containing ethanol is adjusted to 3.5 to 6.5 in the step (2) using food grade citric acid or hydrochloric acid.
  • citric acid or hydrochloric acid is used because the two are economical and have no adverse effect on the flavor of the product; alternatively, the pH is adjusted by using a mixture of citric acid and hydrochloric acid. Adjusting the pH to 3.5 to 6.5 is to meet the optimum pH of the enzyme. The pH value is either high or low, which is not conducive to the efficient action of the enzyme.
  • the pH is adjusted to 4.8 to 6.0 in the step (2).
  • the complex enzyme in the step (2) is a pectinase and/or a cellulose complex enzyme containing a polygalacturonase activity, that is, a pectinase or a cellulose complex enzyme. , or a complex of pectinase and cellulose complex enzyme; when the enzyme used is a complex of pectinase and cellulose complex enzyme, the ratio of the two is optional; polygalacturonase activity refers to The ability to hydrolyze the pectin polygalacturonic acid backbone per unit time.
  • Pectinase and cellulose complex enzymes are well known to those skilled in the art and are currently marketed products.
  • Pectinase is, for example, SMASH produced and sold by Novozymes, and cellulase such as Amano enzyme preparation.
  • the Cellulase AP3 produced and sold by the company, Cellic CTec2 produced and sold by Novozymes.
  • the amount of the enzyme in the step (2) is 0.01 to 10.0 U/g of walnut kernel by weight of the walnut kernel; when the amount of the enzyme is less than 0.01 U/g of the walnut kernel, It causes a decrease in extraction efficiency and an extension of extraction time; if it is higher than 10.0 U/g walnut kernel, it will cause excessive cost and even peeling of the inner seed coat.
  • the polyphenol extraction system obtained in the step (2) is subjected to ultrasonic treatment for 10 to 120 minutes under the condition of ultrasonic power of 0.2 to 20 W/g of walnut kernel, and the extraction is carried out. Phenolic substances.
  • the effect of ultrasonic physics-assisted extraction is mainly to strengthen the effect of the enzyme on the seed coat related substrate of the walnut kernel, and realize the efficient release of the polyphenols.
  • the inventors determined that the ultrasonic power of 0.2-20 W/g walnut kernel and the sonication time of 10 to 120 min are suitable. In the case where the power used is high, the time can be appropriately shortened, but the medium power combined with the appropriate extended time is better for the extraction of polyphenols.
  • the separation and recovery is a solid-liquid separation of the polyphenol extraction system after the ultrasonic treatment in the step (3), and the dephenolized walnut kernel and the walnut polyphenol extract are respectively recovered.
  • the solid-liquid separation includes, but is not limited to, filtration, sedimentation centrifugation.
  • the resulting walnut polyphenol extract contains a quantity of ethanol, optionally after separation and recovery, including a distillation recovery treatment step.
  • the steps (2) to (4) are repeated 1 to 4 times to achieve a better extraction effect.
  • the step (4) further comprises refining: the walnut polyphenol extract obtained in the step (4) is subjected to vacuum rotary evaporation to remove ethanol, followed by concentration, and then vacuum freeze-drying. In addition to moisture, a crude extract of walnut polyphenols is obtained.
  • the vacuum concentration is carried out at -1 to -20 kPa, 20 to 50 °C.
  • the obtained crude polyphenol polyphenol extract may be further separated by macroporous adsorption resin and/or industrial chromatography to prepare products such as ellagic acid, hydrolyzed tannin, and walnut.
  • the invention also claims the use of the process in the preparation of polyphenol-containing products.
  • the polyphenol-containing product refers to a product comprising a walnut polyphenol extract.
  • the polyphenol-containing product refers to a food, a drug, or a health care product containing at least one of flavonoids, phenolic acids, ellagic acids, hydrolyzed tannins, or walnuts.
  • the present invention mainly has the following beneficial effects:
  • the crude extract of walnut polyphenol prepared by the method of the invention is dried after freeze-drying
  • the total weight accounts for more than 2% of the original walnut kernels, and the content of polyphenols in the crude extract of walnut polyphenols is more than 15%.
  • the method proposed by the present invention can provide a useful reference for the extraction of other food function factors.
  • Figure 1 is a process flow of the method of the present invention
  • A is a total particle flow diagram
  • B is a UV absorption (280 nm) diagram.
  • % for describing the concentration is a weight percentage unless otherwise specified.
  • UPLC detection method UPLC-PDA-MS/MS of walnut polyphenol crude extract adopts Waters UPLC system, chromatographic conditions are as follows: GeminiC18 column; column temperature 35 ° C; mobile phase A: pure acetonitrile; mobile phase B: 1 % aqueous formic acid; flow rate 0.3 mL / min; gradient elution: 0 ⁇ 0.1 min / 2% ⁇ 2% A; 0.1 ⁇ 18min / 2% ⁇ 30% A; 18 ⁇ 20min / 30% ⁇ 50% A; ⁇ 25min/50% ⁇ 100%A; 25 ⁇ 27min/100% ⁇ 100%A.
  • the mass spectrum was in negative ion scan mode and the data was analyzed by Masslynxv4.1 software.
  • the polyphenol extraction system is placed in an ultrasonic cleaning machine (such as Kunshan Shumei KQ-J2000GKDE high-power constant temperature ultrasonic cleaner), and the ultrasonic power is adjusted to 250W according to the ratio of ultrasonic power 0.25W/g walnut, ultrasonic treatment for 120min, and then gauze filtration respectively Recycling dephenolized walnut kernel and walnut polyphenol crude extract.
  • an ultrasonic cleaning machine such as Kunshan Shumei KQ-J2000GKDE high-power constant temperature ultrasonic cleaner
  • the ultrasonic power is adjusted to 250W according to the ratio of ultrasonic power 0.25W/g walnut, ultrasonic treatment for 120min, and then gauze filtration respectively Recycling dephenolized walnut kernel and walnut polyphenol crude extract.
  • the obtained dephenolized walnut kernel was again leached under the same conditions as above, and the crude extract of the dephenolized walnut kernel and the walnut polyphenol was again recovered.
  • the obtained crude extracts were combined twice, and the ethanol was removed by vacuum rotary evaporation, and then concentrated to a suitable concentration, and then lyophilized by vacuum freeze-drying to obtain 21.6 g of crude powder of walnut polyphenol. According to UPLC analysis, the effective substances such as phenolic acid and flavonoids accounted for 17.0%.
  • the commercially available Xinjiang paper-skinned walnuts are peeled off to obtain 200g of walnut kernels (about 2% of the crushed kernels), and the ratio of the mass of the walnut kernel to the volume ratio of the ethanol aqueous solution in terms of mL is 1:15.
  • Mix 3L of 20% ethanol solution adjust the pH of the system to 6.0 with 1M citric acid, increase the temperature of the system to 40 °C, and add Novozymes CellicCTec2 to the ratio of 5U/g walnut.
  • the mixed system was placed in an ultrasonic cleaner, and the ultrasonic power was adjusted to 2000 W according to the ratio of ultrasonic power of 10 W/g walnut kernel, sonicated for 20 min, and then filtered by a Buchner funnel to separately recover the walnut kernel and the extract.
  • the extract was evaporated to dryness by vacuum evaporation, concentrated to a suitable concentration, and then subjected to vacuum freeze-drying to remove water to obtain 4.76 g of crude powder of walnut polyphenol. According to UPLC analysis, the effective substances such as phenolic acid and flavonoids accounted for 15.4%.
  • the commercially available inner-planted walnut kernel (about 10% of the crushed kernel) is 500 g and the volume concentration of 5 L is 10%.
  • the ethanol solution was mixed, the pH of the system was adjusted to 4.5 with 1 M citric acid, the temperature of the system was raised to 50 ° C, and the complex enzymes SMASH and Max of Novozymes were added at a ratio of 0.1 U/g and 0.2 U/g of walnut.
  • the mixed system was placed in an ultrasonic cleaner, and the ultrasonic power was adjusted to 1000 W according to the ratio of ultrasonic power of 2 W/g walnut kernel, sonicated for 30 min, and then the gauze was separately filtered to recover the walnut kernel and the extract.
  • the walnut kernels were repeatedly leached twice under the same conditions as above to recover the dephenolized walnut kernels and the extract.
  • the obtained extracts were combined three times, and ethanol was removed by vacuum rotary evaporation, and concentrated to an appropriate concentration, followed by vacuum freeze-drying to remove water to obtain 11.4 g of a crude powder of walnut polyphenol.
  • UPLC analysis showed that the effective substances such as phenolic acid and flavonoids accounted for 17.6%.
  • the commercially available in-plant seed coat walnut kernel 50 g and 0.15 L volume concentration is 0%.
  • the ethanol solution ie pure water
  • the pH of the system is adjusted to 3.5 with 1 M citric acid
  • the temperature of the system is raised to 60 ° C
  • the pectinase SMASH of Novozymes is added at a ratio of 10 U/g walnut.
  • the system was placed in an ultrasonic cleaner, and the ultrasonic power was adjusted to 1000 W according to the ratio of ultrasonic power of 20 W/g walnut kernel, sonicated for 10 min, and then the gauze was separately filtered to recover the walnut kernel and the extract.
  • the walnut kernel was repeatedly leached twice under the same conditions as above to recover the dephenolized walnut kernel and the extract.
  • the obtained extracts were combined three times, and ethanol was removed by vacuum rotary evaporation, and concentrated to an appropriate concentration, and then subjected to vacuum freeze-drying to remove water to obtain 1.22 g of a crude powder of walnut polyphenol.
  • UPLC analysis showed that the effective substances such as phenolic acid and flavonoids accounted for 15.2%.
  • the commercially available Xinjiang paper-skinned walnuts are peeled off to obtain 200g of walnut kernels (about 2% of crushed kernels), and the ratio of the mass of walnut kernels in g to the ratio of the volume of ethanol aqueous solution in mL is 1:8.
  • the mixed system was placed in an ultrasonic cleaner, the ultrasonic power was adjusted to 40 W according to the ratio of ultrasonic power of 0.2 W/g walnut, sonicated for 120 min, and then filtered by a Buchner funnel to recover the walnut kernel and the extract respectively.
  • the extract was evaporated to dryness by vacuum evaporation, concentrated to a suitable concentration, and then subjected to vacuum freeze-drying to remove water to obtain 4.68 g of a crude powder of walnut polyphenol. According to UPLC analysis, the effective substances such as phenolic acid and flavonoids accounted for 15.5%.
  • the specific embodiment is the same as Example 1, except that the ultrasonic extraction treatment is not used, and the yield of the crude polyphenol extract is 1.52%.
  • the crude extract of walnut polyphenols and walnut kernels were tested. The results are shown in the following table:
  • the specific embodiment is the same as that of the first embodiment, except that the composite enzymatic treatment is not used, and the yield of the crude polyphenol extract is 1.76%.
  • the crude extract of walnut polyphenols and walnut kernels were tested. The results are shown in the following table:

Abstract

一种结合酶与超声处理由带内种皮核桃仁中提取多酚物质的方法，所述方法包括得到带种皮的核桃仁；然后以体积比1:3～1:15将带内种皮核桃仁投入到乙醇水溶液中，然后用食品级柠檬酸或盐酸将混合物的pH调整为3.5～6.5，然后加入果胶酶和/或纤维素酶复合物，得到多酚提取体系。根据所述方法制备得到的核桃多酚粗提物，经冷冻干燥后其干物质总重量占原带皮核桃仁的2％以上，核桃多酚粗提物中多酚类有效物含量则达到15％以上。此外，还可采用大孔吸附树脂和工业色谱对得到的核桃多酚粗提物进行进一步分离，制备鞣花酸、水解单宁、胡桃醌等产品。

Description

一种结合酶与超声处理由带内种皮核桃仁中提取多酚物质的方法 技术领域
本发明涉及一种结合酶与超声处理由带内种皮核桃仁中提取多酚物质的方法，属于油料作物加工技术领域。
背景技术
核桃(Juglans regiaL.)又名胡桃、弟桃，是胡桃科胡桃属植物，位居“四大干果”之首，是一种药食兼备的优良经济作物。核桃仁富含油脂、蛋白质及维生素等营养成份，并具有健胃、补血、润肺、益肾和补脑等多种功效，是一种很好的滋补品，营养丰富而味美，故又有“万岁子”、“长寿果”、“益智果”、“美容果”的美誉。现代研究表明核桃仁的抗氧化、防衰老和预防心血管疾病等多重功效主要归功于核桃仁中丰富的油脂、蛋白质和多酚类物质。核桃仁含油量很高，一般为40～65％，有的高达80％，核桃油脂肪酸组成中亚油酸约占61％，油酸约占23％，亚麻酸约占7.0％，同时含有ω-3和ω-6系列多不饱和脂肪酸使得其在降低血脂和血清胆固醇方面具有显著的优势，是药用油或老人和婴幼儿营养油的良好选择。在国际市场上，核桃油同橄榄油一样都倍受消费者青睐，前景非常广阔。核桃蛋白18种氨基酸种类齐全，且人体所需的8种必需氨基酸含量合理，对人体有重要功能的谷氨酸、天冬氨酸、精氨酸含量较高，作为一种全价的营养蛋白，其可消化率达87.2％。核桃仁皮的主要成分是纤维素、果胶质、单宁和色素等。在核桃仁的皮与仁之间存在着果胶质，使得仁与皮之间结合的比较牢固，难于分开。研究表明，核桃仁中的单宁主要存在于表皮中(占总量的90％以上)，是产生苦涩味及导致果实褐变的最主要原因。
目前，在核桃的开发利用中，碱液浸泡是主要的前处理手段。通过在一定温度下的碱液浸泡，核桃的皮与仁之间的果胶层先被溶解，然后整皮脱落，可被水流冲洗去除。碱液浸泡法的机理是：果胶质在碱液作用下会发生水解，一部分果胶被解聚，其在水中的溶解度得以提高；另一方面，果胶酸在碱的作用下转变成水溶性好的果胶酸钠等盐类。所以碱液浸泡可以较充分地去除果胶质，脱除核桃皮。然而该法的缺陷也非常明显。首先，核桃皮中大量有益的多酚类物质被碱液破坏或在脱皮过程中浪费掉；其次，部分核桃仁的形状不规则，带有皱褶的夹缝中的核桃皮往往无法彻底脱除；再次，尽管热碱液浸泡时间目前已经有所缩短(约10min)，经过处理的核桃仁还是会发生蛋白质变性和流失等问题。吴世兰等(吴世兰，秦礼康，蒋成刚，张帅.核桃仁碱液去皮过程中营养功能成分动态变化.中国油脂,2013,38(2):84-87)对核桃仁碱液去皮过程中营养功能成分的变化进行了研究。结果表明：碱液去皮后，核桃仁的粗 蛋白、粗脂肪含量分别减少了24.7％、4.1％；必需氨基酸评分(AAS)从1.82降低到1.28；核桃仁内种皮总酚和总黄酮含量分别下降81.7％和72.2％；核桃仁蛋白的溶解性也明显降低。去皮核桃仁在后续烘干过程中颜色变成暗黄色，且核桃仁油的酸值(KOH)和过氧化值增加(分别增加0.43mg/g、1.78mmol/kg)。碱液去皮工艺废水COD为17500～21100mg/L，远远超过了国家二级污水排放标准。
尽管有研究对碱液尝试过回收利用，但收益与成本比例并不高。宋浩等对核桃仁去衣液中黄酮的回收开展了研究(宋浩，赵声兰，陈朝银，李汝荣，张天财，武万兴，史丹丹.核桃仁去衣液中核桃黄酮的回收及抗氧化活性研究.食品工业科技,2015,36(6):94-98,103.)，采用AB-8型大孔树脂分离和纯化核桃黄酮，结果表明AB-8树脂在优化的工艺条件下可回收81.73％～86.51％的核桃黄酮，产品纯度61.35％～74.16％。此法不仅周期较长，而且碱液中不稳定的其他多酚类物质也被浪费掉。
因此，改进核桃内种皮的处理方式，是实现核桃仁高效开发利用的主要着眼点之一。荣瑞芬等(荣瑞芬，历重先，刘雪峥，裴东，陈佳，王怡婷.核桃内种皮营养与功能成分初步分析研究.食品科学,2008,29(11):541-543.)对核桃内种皮营养与功能成分进行了分析研究,发现核桃内种皮中总酚和黄酮类分别为1.038g/100g和0.744g/100g，分别是仁中含量的12.51和1.78倍，表明核桃内种皮具有较高的营养和保健价值。Colaric等(Colaric M,Veberic R,Solar A,Hudina M,Stampar F.Phenolic Acids,Syringaldehyde,and Juglone in Fruits of Different Cultivars of Juglans regia L.Journal of Agricultural and Food Chemistry,2005,53(16):6390-6396.)采用高效液相色谱分析了核桃及其内种皮中的酚酸(绿原酸、咖啡酸、香豆酸、阿魏酸、芥子酸、鞣花酸、丁香酸)、丁香醛及胡桃醌等酚类物质，结果表明丁香酸、胡桃醌、鞣花酸是核桃和内种皮中含量较高的物质，核桃中三者含量分别为33.83、11.75、5.90mg/100g，而核桃内种皮中三者含量则分别为1003.24、317.90、128.98mg/100g，均为核桃仁中20至30倍左右。万政敏等(万政敏,郝艳宾,杨春梅,齐建勋,赵丽芹,王克建.核桃仁种皮中的多酚类物质高压液相色谱分析.食品工业科技，2007，28(7):212-213,224.)采用高效液相色谱对核桃内种皮中的多酚类物质进行了分析研究。在核桃内种皮中检测到没食子酸、绿原酸、咖啡酸、对羟基苯甲酸、香豆酸和阿魏酸等17种酚酸类物质，含量最高的没食子酸达到146.2mg/100g干样，而在无种皮的核桃仁中只检测到7种酚酸类物质，其中没食子酸的含量仅为5.3mg/100g干样，与前述研究结论基本一致。
其中一种思路是将核桃内种皮与核桃仁分离开再分别加以利用。周晔等(周晔；王伟；裴东.一种核桃内种皮的脱除方法.CN201210552825.5)公开了一种核桃内种皮的脱除方法。该方 法步骤如下：(1)将待处理的核桃浸渍于液氮中进行冷冻；(2)将处理后的核桃置于空气中，直至所述核桃的表面出现白霜；(3)将出现白霜的核桃仁置于维生素C的水溶液中进行复水处理；(4)将经复水后的核桃依次进行超声和震荡处理，然后经冲刷，得到脱除内种皮的核桃仁以及脱除的内种皮。该发明本质上是利用不同结构组成的物质热胀冷缩系数不同的原理，进而实现内种皮与核桃仁的分离。尽管该法相对环保、处理难度适中，但使用液氮耗费较大，且该法回收的内种皮中多酚流失严重(仅保留约28％的多酚物质)。王群学等(王群学，赵西周，贺跃东，梁少华，张富强.核桃仁连续脱皮的方法.CN201510261863.9)公布了一种核桃仁连续脱皮的方法。利用碱液浸泡和高压冲洗相结合对内种皮进行分离，但由于带皮核桃仁原料往往含有不少碎粒，高压冲击造成了很可观的核桃仁损失，且种皮中多酚也严重流失。康玮丽等对核桃仁去皮方法进行了优化研究(康玮丽，唐军虎，敬思群.核桃仁去皮方法优化.食品与发酵工业,2013,36(8):127-131)，发现采用热风烘烤-微波联用法可去除核桃仁皮，优化工艺条件为：烘烤温度130℃，烘烤时间16min，微波功率769W，微波时间51s，核桃仁脱皮率为98.01％，核桃油过氧化值为1.65meq/kg。由于核桃油中亚麻酸比例较高，因此这一高强度热处理不仅会增加油脂过氧化值，还可能造成其它不利影响。
另一种思路是先对内种皮中多酚类物质进行提取，然后再开发利用核桃仁。早在1896年，Osborne和Campbell等(Osborne,T.B.；Campbell,G.F.Conglutin and vitellin.J.Am.Chem.Soc.,1896,18:609-623.)研究发现，用热水处理英国核桃可除去核桃内种皮，达到去除单宁的目的，同时也将脱脂核桃粉的球蛋白溶出率从原来的3.5％左右提高到20％。然而，单纯的热水浸泡，多酚提取效率不高且蛋白等损失也比较高。国内有些研究者以纯核桃仁内种皮为原料，研究了多酚的提取工艺。如魏静和郝利平(魏静，郝利平.核桃仁种皮中多酚的提取工艺研究.山西农业大学学报,2010,30(2):150-153.)研究了核桃仁种皮中多酚的提取工艺。通过对乙醇浓度、料液比、提取时间、提取温度等因素的探讨，确定较佳工艺条件为：60％乙醇、料液比1:8、提取时间30min、提取温度55℃，实现多酚的粗提得率571mg/g。但此粗提物的多酚类物质含量未见报道。张春梅等也以核桃内种皮为原料开展了多酚提取工艺的研究(张春梅，陈朝银，赵声兰，黄再强，樊启猛.核桃内种皮多酚提取工艺及其体外抗氧化活性的初步研究.中国酿造,2014,33(7):130-134.)，确定核桃内种皮多酚的提取工艺条件为：乙醇体积分数45％、液固比60:1(mL/g)、提取温度70℃、提取时间60min。在此条件下，多酚得率为250.5mg/g。王莉等(王莉，赵新亮，董文，贺志龙，李佳，马亚茹.核桃仁种皮中类黄酮提取工艺的比较.食品与发酵科技,2013,49(4):90-92.)也以核桃仁种皮为原料，比较了其中类黄酮的提取工艺，得出提取类黄酮的优化条件为：水浴温度65℃，乙醇浓度60％，提取时间 为1.5h，固液比为1∶30。上述研究对多酚类物质的提取有参考价值，但由于需要先分离出核桃仁内种皮，在实际应用时受到很大限制。
其他一些新型提取技术也被应用于核桃仁加工上。正如郜海燕等(郜海燕,李兴飞,陈杭君,房祥军.山核桃多酚物质提取及抗氧化研究进展.食品科学,2011,32(05):336-341)指出的，微波萃取技术和超临界萃取技术在山核桃油的提取上已经达到可应用的程度，但由于多酚类物质含量相对较低以及微波处理导致的局部温升等不利因素的限制，这些技术应用于核桃多酚物质提取还不成熟。
发明内容
考虑到核桃仁中多酚主要分布在内种皮上，而内种皮的物理分离还难以工业化实现，本发明提出一种结合酶与超声处理由核桃仁中提取多酚物质的方法，在适宜的条件下，结合乙醇溶液和果胶酶和/或纤维素酶复合物的作用，利用超声处理强化传质过程，实现对完整核桃仁的多酚物质的高效提取。所得多酚提取液可进一步分离精制，作为抗氧化剂用于膳食补充剂、饮料、果蔬产品及休闲食品；提取多酚后的核桃仁可粉碎制取高品质核桃油和低脂核桃粉，兼顾了核桃仁中主要的组分和活性成分，对核桃综合利用具有重要参考价值。
本发明的第一个目的是提供一种结合酶与超声处理由带内种皮核桃仁中提取多酚物质的方法，是以带内种皮的核桃仁为原料，在果胶酶和/或纤维素酶的辅助下，结合超声处理，采用乙醇溶液将核桃仁内种皮中的多酚类物质高效提取出来，经过滤分离得到多酚粗提物和脱酚核桃仁产品。
在本发明的一种实施方式中，所述方法通过下述技术方案实现：
(1)制备带种皮的核桃仁：对核桃进行脱壳和清理除杂，不经破碎处理即可得到带内种皮核桃仁；(2)制备多酚提取体系：将步骤(1)得到的带内种皮核桃仁投入到乙醇水溶液中，加入果胶酶和/或纤维素酶复合物进行多酚提取处理：(3)超声处理：在0.2～20W/g核桃仁的条件下对步骤(2)得到的多酚提取体系进行超声处理；(4)分离回收。
在本发明的一种实施方式中，所述的核桃仁包括由核桃剥壳机或手工剥壳获得的带内种皮核桃仁，或市售的带内种皮核桃仁，或其它常规剥壳操作分离获得的核桃仁产品。
带内种皮核桃仁不经过破碎处理可以降低后继联合脱酚处理的溶液消耗，同时降低蛋白等的溶出，既有利于提高多酚粗提物的纯度，也对脱酚核桃仁产品的品质有利。
在本发明的一种实施方式中，所述步骤(2)按照以g计带内种皮核桃仁的质量与以mL计乙醇水溶液的体积之比1:3～1:15的比例，将步骤(1)得到的带内种皮核桃仁投入到乙醇水溶液中。其中，乙醇水溶液的体积浓度是0～20％，然后用食品级柠檬酸或盐酸将混合物的 pH调整为3.5～6.5，接着在温度10～60℃条件下向所述混合物中加入果胶酶和/或纤维素酶复合物，酶用量为0.01～10.0U/g核桃仁，从而得到多酚提取体系。
在本发明的一种实施方式中，所述步骤(2)中以g计带内种皮核桃仁的质量与以mL计乙醇水溶液的体积之比为1:3～1:15。
在本发明的一种实施方式中，所述步骤(2)中以g计带内种皮核桃仁的质量与以mL计乙醇水溶液的体积之比为1:5～1:10。
在本发明的一种实施方式中，所述步骤(2)中乙醇水溶液的体积浓度是0～20％，当其体积浓度高于20％，会造成纤维素酶和果胶酶的失活。
在本发明的一种实施方式中，所述步骤(2)中乙醇水溶液体积浓度为5～15％；当乙醇水溶液的体积浓度为0％时，提取时间需要延长2倍以上。
在本发明的一种实施方式中，所述步骤(2)中使用食品级柠檬酸或盐酸将带内种皮核桃仁与含有乙醇的水溶液的混合物的pH调整为3.5～6.5。之所以使用柠檬酸或盐酸，是由于二者经济性好，且对产品风味无不良影响；可选地，采用柠檬酸与盐酸混合物调整pH。调整pH为3.5～6.5，是为了满足酶的最适pH的要求，pH值或高或低都不利于酶的高效作用。
在本发明的一种实施方式中，所述步骤(2)中调整pH为4.8～6.0。
在本发明的一种实施方式中，所述步骤(2)中的复合酶是含有聚半乳糖醛酸酶活力的果胶酶和/或纤维素复合酶，即果胶酶或纤维素复合酶，或果胶酶与纤维素复合酶的复合物；当使用的酶是果胶酶与纤维素复合酶的复合物时，二者的比例是任选的；聚半乳糖醛酸酶活力是指单位时间内水解果胶聚半乳糖醛酸主链的能力。果胶酶和纤维素复合酶都是本技术领域的技术人员熟知的，也是目前市场上销售的产品，果胶酶例如是由诺维信公司生产销售的SMASH，纤维素酶例如由天野酶制剂有限公司生产销售的CellulaseAP3、由诺维信公司生产销售的Cellic CTec2。
在本发明的一种实施方式中，所述步骤(2)中酶的用量是以核桃仁的重量计0.01～10.0U/g核桃仁；当酶的用量低于0.01U/g核桃仁，会造成提取效率的下降和提取时间的延长；如果高于10.0U/g核桃仁，则会造成成本过高，甚至内种皮的脱落。
在本发明的一种实施方式中，所述步骤(3)在超声功率0.2～20W/g核桃仁的条件下，对步骤(2)得到的多酚提取体系进行超声处理10～120min，提取多酚类物质。
本发明中，超声物理场辅助提取的作用主要是强化酶对核桃仁内种皮相关底物的作用，实现对多酚类物质的高效释放。发明人经过多次试验，确定超声功率0.2～20W/g核桃仁、超声处理时间10～120min是合适的。在所用功率较高的情况下，时间可以适当缩短，但中等功 率结合适当延长的时间对多酚类的提取效果更好。
在本发明的一种实施方式中，所述分离回收是对步骤(3)超声处理后的多酚提取体系进行固液分离，分别回收脱酚核桃仁和核桃多酚浸提液。
在本发明的一种实施方式中，所述固液分离包括但不限于过滤、沉降式离心。
在本发明的一种实施方式中，所得的核桃多酚浸提液含有一定量乙醇，在分离回收后可选地，包括蒸馏回收处理步骤。
在本发明的一种实施方式中，所述步骤(2)～(4)重复1～4次以达到更好的提取效果。
在本发明的一种实施方式中，所述步骤(4)后还包括精制：即将步骤(4)得到的核桃多酚浸提液经过真空旋转蒸发除去乙醇，接着浓缩，然后经真空冷冻干燥脱除水分，得到核桃多酚粗提物。
在本发明的一种实施方式中，所述真空浓缩在-1～-20kPa，20～50℃下进行。
在本发明的一种实施方式中，还可采用大孔吸附树脂和/或工业色谱对得到的核桃多酚粗提物进行进一步分离，制备鞣花酸、水解单宁、胡桃醌等产品。
本发明还要求保护所述方法在制备含多酚的产品中的应用。
在本发明的一种实施方式中，所述含多酚的产品是指含核桃多酚提取物的产品。
在本发明的一种实施方式中，所述含多酚的产品是指含黄酮、酚酸、鞣花酸、水解单宁或胡桃醌中至少一种的食品、药品或保健品。
与现有技术相比，本发明主要具有以下有益效果：
(1)从带内种皮核桃仁中直接提取种皮中的多酚类物质，脱酚效果与热碱液浸泡脱除法相当，所提取的多酚物质可开发利用，显著提升了本发明的经济价值；
(2)无需从核桃仁表面脱除内种皮，使得本发明技术成果应用便利性大幅改进；脱酚核桃仁的开发利用中，残留的内种皮对油脂提取、蛋白粉制备基本无不良影响。
(3)充分发挥生物酶处理和超声处理的作用特性，使得核桃多酚的提取效果和效率得到大幅的改进；根据本发明方法制备得到的核桃多酚粗提物，经冷冻干燥后其干物质总重量占原带皮核桃仁的2％以上，核桃多酚粗提物中多酚类有效物含量则达到15％以上。
(4)本发明提出的方法可为其他食品功能因子的提取提供有益参考。
附图说明
图1为本发明方法的工艺流程；
图2为本发明的核桃多酚粗提物的UPLC洗脱曲线；其中，A为总粒子流图；B为紫外吸收(280nm)图。
具体实施方式
下面结合实施例对本发明做进一步的说明，但不限于实施例。
在本发明中，如无特殊说明，用于描述浓度的“％”均为重量百分比。
UPLC检测方法：核桃多酚粗提物的UPLC-PDA-MS/MS采用Waters公司的UPLC系统，色谱条件如下：GeminiC18色谱柱；柱温35℃；流动相A：纯乙腈；流动相B：1％的甲酸水溶液；流速0.3mL/min；梯度洗脱：0～0.1min/2％～2％A；0.1～18min/2％～30％A；18～20min/30％～50％A；20～25min/50％～100％A；25～27min/100％～100％A。质谱采用负离子扫描模式，数据通过Masslynxv4.1软件进行分析。
实施例1 本发明的核桃多酚粗提物的提取
称取市售的带内种皮核桃仁(碎仁率5％左右)1000g，按照以g计核桃仁的质量与以mL计乙醇水溶液的体积之比1:4的比例，将其与4L体积浓度为5％的乙醇溶液混合，以1M的盐酸调节体系pH至4.0，将体系温度升至55℃，按0.2U/g核桃仁的比例加入诺维信公司的果胶酶SMASH，将得到的多酚提取体系置于超声清洗机(如昆山舒美KQ-J2000GKDE高功率恒温超声波清洗器)中，按照超声功率0.25W/g核桃仁的比例调节超声功率250W，超声处理120min，而后纱布过滤分别回收脱酚核桃仁和核桃多酚粗提液。将得到的脱酚核桃仁采用与上述相同的条件再次浸提，再次回收脱酚核桃仁和核桃多酚粗提液。合并两次所得粗提液，真空旋转蒸发除去乙醇后浓缩至适当浓度，而后经真空冷冻干燥脱除水分，得到21.6g核桃多酚粗提物粉末。经UPLC分析，其酚酸和黄酮等有效物占比17.0％。
Figure PCTCN2017119777-appb-000001
实施例2 本发明的核桃多酚粗提物的提取
将市售新疆纸皮核桃剥壳，得到核桃仁200g(碎仁率2％左右)，按照以g计核桃仁的质量与以mL计乙醇水溶液的体积之比1:15的比例，将其与3L的体积浓度为20％的乙醇溶液混合，以1M的柠檬酸调节体系pH至6.0，将体系温度升至40℃，按5U/g核桃仁的比例加入诺维信公司的纤维素酶CellicCTec2，将混合体系放置于超声清洗机中，按照超声功率10W/g核桃仁的比例调节超声功率2000W，超声处理20min，而后布氏漏斗过滤，分别回收 核桃仁和浸提液。将浸提液真空旋转蒸发除去乙醇后浓缩至适当浓度，而后经真空冷冻干燥脱除水分，得到4.76g核桃多酚粗提物粉末。经UPLC分析，其酚酸和黄酮等有效物占比15.4％。
Figure PCTCN2017119777-appb-000002
实施例3 本发明的核桃多酚粗提物的提取
按照以g计核桃仁的质量与以mL计乙醇水溶液的体积之比1:10的比例，将市售带内种皮核桃仁(碎仁率10％左右)500g与5L体积浓度为10％的乙醇溶液混合，以1M的柠檬酸调节体系pH至4.5，将体系温度升至50℃，分别按0.1U/g、0.2U/g核桃仁的比例加入诺维信公司的复合酶SMASH和Max，将混合体系置于超声清洗机中，按照超声功率2W/g核桃仁的比例调节超声功率1000W，超声处理30min，而后纱布过滤分别回收核桃仁和浸提液。将核桃仁采用与上述相同的条件重复浸提2次，回收脱酚核桃仁和浸提液。合并三次所得浸提液，真空旋转蒸发除去乙醇后浓缩至适当浓度，而后经真空冷冻干燥脱除水分，得到11.4g核桃多酚粗提物粉末。UPLC分析显示其酚酸和黄酮等有效物占比17.6％。
Figure PCTCN2017119777-appb-000003
实施例4 本发明的核桃多酚粗提物的提取
按照以g计核桃仁的质量与以mL计乙醇水溶液的体积之比1:3的比例，将市售带内种皮核桃仁(碎仁率10％左右)50g与0.15L体积浓度为0％的乙醇溶液(即纯水)混合，以1M的柠檬酸调节体系pH至3.5，将体系温度升至60℃，按10U/g核桃仁的比例加入诺维信公司的果胶酶SMASH，将混合体系置于超声清洗机中，按照超声功率20W/g核桃仁的比例调节超声功率1000W，超声处理10min，而后纱布过滤分别回收核桃仁和浸提液。将核桃仁采用与 上述相同的条件重复浸提2次，回收脱酚核桃仁和浸提液。合并三次所得浸提液，真空旋转蒸发除去乙醇后浓缩至适当浓度，而后经真空冷冻干燥脱除水分，得到1.22g核桃多酚粗提物粉末。UPLC分析显示其酚酸和黄酮等有效物占比15.2％。
Figure PCTCN2017119777-appb-000004
实施例5 本发明的核桃多酚粗提物的提取
将市售新疆纸皮核桃剥壳，得到核桃仁200g(碎仁率2％左右)，按照以g计核桃仁的质量与以mL计乙醇水溶液的体积之比1:8的比例，将其与1.6L的体积浓度为15％的乙醇溶液混合，以1M的柠檬酸调节体系pH至6.5，将体系温度升至10℃，按0.01U/g核桃仁的比例加入诺维信公司的纤维素酶CellicCTec2，将混合体系放置于超声清洗机中，按照超声功率0.2W/g核桃仁的比例调节超声功率40W，超声处理120min，而后布氏漏斗过滤，分别回收核桃仁和浸提液。将浸提液真空旋转蒸发除去乙醇后浓缩至适当浓度，而后经真空冷冻干燥脱除水分，得到4.68g核桃多酚粗提物粉末。经UPLC分析，其酚酸和黄酮等有效物占比15.5％。
Figure PCTCN2017119777-appb-000005
对照例1
具体实施方式同实施例1，区别在于，其核桃仁经过预先粉碎处理，平均粒径68μm，进行酶与超声结合处理，按0.8U/g核桃仁的比例加入诺维信公司的果胶酶SMASH，按超声功率0.25W/g核桃仁的比例调节超声功率250W，超声处理180min，而后离心(5000g，15min)，上清液回收得到核桃多酚粗提液，真空浓缩并冻干后得到48.7g核桃多酚粗提物；离心所得沉淀干燥后得到脱酚核桃仁，对脱酚核桃仁和核桃粗提物进行分析，结果如下表所示：
Figure PCTCN2017119777-appb-000006
对照例2
具体实施方式同实施例1，区别在于，不采用超声提取处理，多酚粗提物得率1.52％。对处理后的核桃多酚粗提物含量和核桃仁进行检测，结果如下表所示：
Figure PCTCN2017119777-appb-000007
对照例3
具体实施方式同实施例1，区别在于，不采用复合酶解处理，多酚粗提物得率1.76％。对处理后的核桃多酚粗提物含量和核桃仁进行检测，结果如下表所示：
Figure PCTCN2017119777-appb-000008
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (25)

  1. 一种提取多酚物质的方法，其特征在于，是以带内种皮的核桃仁为原料，在果胶酶和/或纤维素酶的辅助下，结合超声处理，采用乙醇溶液将核桃仁内种皮中的多酚类物质高效提取出来，经过滤分离得到多酚粗提物和脱酚核桃仁产品。
  2. 根据权利要求1所述的方法，其特征在于，包括如下步骤：
    (1)制备带种皮的核桃仁：对核桃进行脱壳和清理除杂，不经破碎处理即可得到带内种皮核桃仁；(2)制备多酚提取体系：将步骤(1)得到的带内种皮核桃仁投入到乙醇水溶液中，加入果胶酶和/或纤维素酶复合物进行多酚提取处理：(3)超声处理：在0.2～20W/g核桃仁的条件下对步骤(2)得到的多酚提取体系进行超声处理；(4)分离回收。
  3. 根据权利要求2所述的方法，其特征在于，所述的核桃仁包括由核桃剥壳机或手工剥壳获得的带内种皮核桃仁，或市售的带内种皮核桃仁，或其它常规剥壳操作分离获得的核桃仁产品。
  4. 根据权利要求2所述的方法，其特征在于，所述步骤(2)按照每g带内种皮核桃仁与3～15mL乙醇水溶液的比例混合，然后用柠檬酸或盐酸或二者的混合物将pH调整为3.5～6.5，在10～60℃条件下加入果胶酶和/或纤维素酶复合物，酶用量为0.01～10.0U/g核桃仁。
  5. 根据权利要求4所述的方法，其特征在于，按g/mL计带内种皮核桃仁与乙醇水溶液的质量体积比为1:5～1:10。
  6. 根据权利要求4所述的方法，其特征在于，乙醇水溶液的体积浓度是0～20％。
  7. 根据权利要求6所述的方法，其特征在于，乙醇水溶液的体积浓度是5～15％。
  8. 根据权利要求4所述的方法，其特征在于，调整pH为4.8～6.0。
  9. 根据权利要求4所述的方法，其特征在于，所述复合酶是含有聚半乳糖醛酸酶活力的果胶酶和/或纤维素复合酶。
  10. 根据权利要求9所述的方法，其特征在于，复合酶的用量是以核桃仁的重量计0.01～10.0U/g核桃仁。
  11. 根据权利要求2所述的方法，其特征在于，在超声功率0.2～20W/g核桃仁的条件下，对步骤(2)得到的多酚提取体系进行超声处理10～120min，提取多酚类物质。
  12. 根据权利要求2所述的方法，其特征在于，所述分离回收是对步骤(3)超声处理后的多酚提取体系进行固液分离，分别回收脱酚核桃仁和核桃多酚浸提液。
  13. 根据权利要求12所述的方法，其特征在于，所述固液分离包括但不限于过滤、沉降式离心。
  14. 根据权利要求12或13所述的方法，其特征在于，在分离回收后进行蒸馏回收处理。
  15. 根据权利要求2所述的方法，其特征在于，将步骤(2)～(4)重复1～4次。
  16. 根据权利要求2或15所述的方法，其特征在于，步骤(4)后还包括精制；所述精制是即将步骤(4)得到的核桃多酚浸提液经过真空旋转蒸发除去乙醇，浓缩，并经真空冷冻干燥脱除水分，得到核桃多酚粗提物。
  17. 根据权利要求16所述的方法，其特征在于，所述真空浓缩在-1～-20kPa，20～50℃下进行。
  18. 根据权利要求17所述的方法，其特征在于，对核桃多酚粗提物采用大孔吸附树脂和/或工业色谱进行进一步分离。
  19. 根据权利要求2所述的方法，其特征在于，所述方法包括以下步骤：
    (1)得到带内种皮核桃仁：
    对核桃进行脱壳和清理除杂，然后不经破碎处理即得到带内种皮核桃仁；
    (2)制备多酚提取体系：
    将步骤(1)得到的带内种皮核桃仁投入到乙醇水溶液中，调节得到的混合物的pH，接着向混合物中加入果胶酶和/或纤维素酶复合物，从而得到多酚提取体系；
    (3)超声处理：
    对步骤(2)得到的多酚提取体系进行超声处理；超声处理使用的超声功率在0.2～20W/g核桃仁，超声处理时间为10～120min；
    (4)分离回收：
    对步骤(3)超声处理后的多酚提取体系进行固液分离，分别回收脱酚核桃仁和核桃多酚浸提液；所述的固液分离是采用过滤或沉降式离心方式；
    (5)精制：
    步骤(4)得到的核桃多酚浸提液经过真空旋转蒸发除去乙醇，接着浓缩，然后经真空冷冻干燥脱除水分，得到核桃多酚粗提物。
  20. 根据权利要求19所述的方法，其特征在于在步骤(2)中，以g计带内种皮核桃仁的质量与以mL计乙醇水溶液的体积之比为1:3～1:15，所述乙醇水溶液的体积浓度为0～20％。
  21. 根据权利要求19所述的方法，其特征在于在步骤(2)中，使用柠檬酸或盐酸将所述混合物的pH调整为3.5～6.5。
  22. 根据权利要求19所述的方法，其特征在于步骤(2)中，在温度10～60℃条件下向所述混合物中加入酶，所述酶是含有聚半乳糖醛酸酶活力的果胶酶和/或纤维素酶，酶的用量为0.01～10.0U/g核桃仁。
  23. 应用权利要求1～13、15、17～20任一所述方法制备的含多酚的产品。
  24. 根据权利要求23所述的产品，其特征在于，所述含多酚的产品是指含核桃多酚提取 物的产品。
  25. 根据权利要求23所述的产品，其特征在于，所述含多酚的产品是指含黄酮、酚酸、鞣花酸、水解单宁或胡桃醌中至少一种的食品、药品或保健品。
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