WO2021120506A1 - 一种天然食品抗氧化剂及其制备方法 - Google Patents

一种天然食品抗氧化剂及其制备方法 Download PDF

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WO2021120506A1
WO2021120506A1 PCT/CN2020/089887 CN2020089887W WO2021120506A1 WO 2021120506 A1 WO2021120506 A1 WO 2021120506A1 CN 2020089887 W CN2020089887 W CN 2020089887W WO 2021120506 A1 WO2021120506 A1 WO 2021120506A1
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red clover
crude
ethanol
polysaccharide
isoflavones
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PCT/CN2020/089887
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English (en)
French (fr)
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刘权
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兰州大学
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Priority to US17/281,920 priority Critical patent/US20220312815A1/en
Publication of WO2021120506A1 publication Critical patent/WO2021120506A1/zh

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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
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
    • 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
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/23—Removal of unwanted matter, e.g. deodorisation or detoxification by extraction with solvents
    • 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
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
    • A23L5/32—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation using phonon wave energy, e.g. sound or ultrasonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • 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

Definitions

  • the invention relates to the technical field of the food industry, in particular to a natural food antioxidant and a preparation method thereof.
  • Food antioxidants can prevent or slow down the spoilage of things, prevent the production of toxic substances in food, the loss of nutrients and changes in appearance and color, and can also avoid some peculiar smells caused by the oxidation of some animal fats and oils.
  • Commonly used artificial antioxidants are often added to food, tert-butyl hydroquinone (TBHQ), tert-butyl p-hydroxyanisole (BHA), dibutyl hydroxytoluene (BHT) and propyl gallate (PG) It is widely used. With the improvement of people's health awareness, the safety of synthetic antioxidants has attracted more and more attention. In the experiment of intraperitoneal injection of BHA, BHT and their metabolites in male young rats, it was found that the number of rat lung cells increased. Now the United States, the European Union and other countries have gradually banned the use of such synthetic antioxidants.
  • the purpose of the present invention is to provide a natural food antioxidant and a preparation method thereof to solve the above-mentioned problems in the background art.
  • a natural food antioxidant including an extract of red clover extract whose main component of the food antioxidant is crude polysaccharides and crude isoflavones, as well as refined polysaccharides and Isoflavones.
  • the refined polysaccharides and isoflavones are prepared with a mass ratio of 1:10-15:1.
  • a preparation method of natural food antioxidant including the following steps:
  • the collected red clover leaves were deactivated at 105°C for 2-60min, dried at 65°C for 1-160h, crushed, defatted with a small amount of petroleum ether for 1-10 times, evaporated off the solvent and dried, and weighed a certain quality of defatted red clover powder , Use 10-50 times 10-100% ethanol 30-70°C ultrasonic assisted extraction for 10-90min, filter and collect the supernatant, add 5-120 times solvent to extract the sample residue for 10-90min, combine the two extracts;
  • Crude polysaccharide Concentrate the extract with a vacuum rotator at 15-90°C. When it reaches one-eighth of the volume, slowly add 2-20 times the volume of absolute ethanol to the concentrated solution while stirring, and put it in the refrigerator at 4°C. Let stand overnight and centrifuge at 4800rpm for 2-20min. After centrifugation, the brown precipitate is the crude polysaccharide part. The precipitated part is washed repeatedly with absolute ethanol for 1-10 times to remove alcohol-soluble impurities. Finally, the precipitate is washed with ethanol and poured into a petri dish. The solvent evaporates to obtain brown red clover crude polysaccharide;
  • the supernatant is the isoflavone part, and the supernatant is concentrated under reduced pressure to obtain the crude red clover isoflavone;
  • Protein removal Prepare red clover crude polysaccharide with distilled water to form a 1-10mg/mL solution, add 1/4 volume of sevage solution, shake for 1-30min, transfer to a separatory funnel and let stand for 1-120min, let go of the lower layer of solution and For the protein in the middle layer, repeat this operation 1-6 times until no protein appears in the organic solvent in the lower layer;
  • Dialysis The polysaccharide solution from which the protein has been removed is concentrated at low temperature and reduced pressure, and then put into a pretreated dialysis bag, placed in a large-capacity beaker, filled with distilled water, and allowed to stand for dialysis. Change the water every 1-16h and dialyze for 1-72h. 1% AgNO 3 was used to check the residual conditions of NaCl, KCl, etc., and finally purified polysaccharides were obtained.
  • resin pretreatment soak the resin with 20-100% ethanol for 1-72h, filter, rinse repeatedly with deionized water until there is no alcohol smell, and the rinse solution is not white and turbid, and then soak it with 1-30% NaOH 1- 72h, filter, repeatedly rinse the resin with deionized water until the pH is neutral, then soak in 1-30% HCl solution for 1-72h, filter and rinse the resin to neutral, and dry at 20-90°C for use.
  • the beneficial effects of the present invention are: the natural food antioxidant proposed by the present invention and the preparation method thereof.
  • the refined red clover polysaccharide has the advantages of high activity and good water solubility. It is firstly considered for use, and secondly If you need to prepare fat-soluble components, you can consider using crude red clover polysaccharides or red clover isoflavones. The two types of components have different emphasis on activity. Crude red clover polysaccharides have high antioxidant activity to superoxide anions, but different The DPPH activity of flavonoids is better.
  • the two components can be used for the compounding scheme, especially when the mass ratio of polysaccharides to isoflavones is 1:2, the synergistic effect between the two components It is the best and has the highest activity, and its isoflavones are also sold as health products at home and abroad. Therefore, the food additives prepared by using the plant are not only green and environmentally friendly, but also very safe for the human body, but also rich in resources.
  • a natural food antioxidant including the extract of red clover extract as the main ingredient of the food antioxidant.
  • the extracts are crude polysaccharides and crude isoflavones, as well as refined polysaccharides and isoflavones. Crude polysaccharides and crude isoflavones are based on the quality of each other. The ratio is 1:2, and the refined polysaccharides and isoflavones are prepared with a mass ratio of 1:2.
  • this embodiment now proposes a method for preparing natural food antioxidants, which includes the following steps:
  • Step 1 Preparation of red clover extract:
  • the collected red clover leaves were quenched at 105°C for 10 minutes, dried at 65°C for 72 hours, crushed, defatted 4 times with a small amount of petroleum ether, evaporated off the solvent and dried, weighed 2g of defatted red clover powder, and used 25 times 40% ethanol Ultrasound assisted extraction at 50°C for 30 minutes, filter and collect the supernatant, add 20 times the solvent to extract the sample residue for 30 minutes, and combine the two extracts;
  • Step 2 Preparation of crude polysaccharides and crude isoflavones:
  • Section 1 Crude polysaccharides: Concentrate the extract with a vacuum rotator at 45°C. When it reaches one-eighth of the volume, add 4 times the volume of absolute ethanol to the concentrated solution slowly while stirring to make the solution ethanol concentration reach 80%, put it in a refrigerator at 4°C and let it stand overnight. Centrifuge at 4800rpm for 10min. After centrifugation, the brown precipitate is the crude polysaccharide part. The precipitated part is washed 3 times with absolute ethanol to remove alcohol-soluble impurities. Finally, the precipitate is washed out with ethanol and poured into In the petri dish, wait for the solvent to evaporate to obtain the brown red clover crude polysaccharide;
  • Section 2 The supernatant in step S21 is the isoflavone part, and the supernatant is concentrated under reduced pressure to obtain the crude red clover isoflavone;
  • Resin pretreatment soak the resin in 95% ethanol for 24 hours, filter, and repeatedly rinse with deionized water until there is no alcohol smell, and the rinsing liquid has no white turbidity, and then soak in 5% NaOH for 24 hours, filter, and rinse the resin with deionized water repeatedly When the pH is neutral, soak the resin with 5% HCl solution for 24 hours, filter and rinse the resin until it is neutral, and dry at 60°C for later use. Soak the pretreated resin in distilled water for more than 48 hours. The height of the wet packing resin is 13cm. 10g of crude isoflavones are dissolved in water.
  • Step 4 Refining of red clover polysaccharides:
  • Section 1 Removal of protein: Prepare red clover crude polysaccharide with distilled water to form a 2mg/mL solution, add 1/4 volume of sevage solution, shake for 20 minutes, transfer to a separatory funnel and let stand for 20 minutes, and let go of the lower layer and the middle layer. For protein, repeat this operation 3 times until no more protein appears in the lower organic solvent;
  • Section 2 Dialysis: Put the protein-removed polysaccharide solution into a pretreated dialysis bag after being concentrated under reduced pressure at low temperature, put it into a large-capacity beaker, fill up with distilled water and let stand for dialysis. Change the water every 8h and dialyze for 24h. 1% AgNO 3 was used to check the residual conditions of NaCl, KCl, etc., and finally purified polysaccharides were obtained.
  • DPPH Antioxidant activity of DPPH: DPPH belongs to a class of synthetic stable free radicals. It has characteristic absorption in the visible light region. The determination method is simple, fast and has good reproducibility;
  • the flavonoids and flavonoids and polysaccharide mixed samples prepared in Example 1 were all prepared with 70% ethanol, and the polysaccharide samples, positive control vitamin C (Vc) and vitamin E (Ve) were prepared with distilled water, and each test substance was prepared at 1.280 The stock solution of mg/mL is diluted to 7 concentration gradients for later use.
  • A0 is the absorbance of the DPPH solution plus the test solution at a concentration of zero
  • A1 is the absorbance of the test solution after the reaction with DPPH
  • A2 is the blank control, that is, the absorbance of each test solution and the blank solvent.
  • P represents refined red clover polysaccharide
  • IF represents refined red clover isoflavones
  • O 2- ⁇ is a long-lived free radical in the body.
  • O 2- ⁇ acts as an initiator to promote the generation of other free radicals, causing further harm to the body, and is closely related to many diseases Related. Therefore, the ability of the sample to remove O 2- ⁇ is usually taken as an important indicator of the activity of antioxidant substances.
  • the flavonoids and flavonoids and polysaccharide mixed samples prepared in Example 1 were all prepared with 70% ethanol, and the polysaccharide samples, positive control vitamin C (Vc) and vitamin E (Ve) were prepared with distilled water, and each test substance was prepared at 1.280 The stock solution of mg/mL is diluted to 7 concentration gradients for later use.
  • the PMS-NADH-NBT system method is used to determine the superoxide anion (O2- ⁇ ) scavenging activity.
  • the principle is to reduce phenazine methyl sulfate (PMS) with reduced coenzyme I (nicotinamide adenine dinucleotide, NADH) Produce superoxide anion, O 2- ⁇ reacts with nitrotetrazolium blue chloride (NBT) to produce a blue product, which has a strong absorption peak at 560nm.
  • PMS phenazine methyl sulfate
  • coenzyme I nicotinamide adenine dinucleotide, NADH
  • NBT nitrotetrazolium blue chloride
  • the antioxidant substance is added to the PMS-NADH-NBT system, it inhibits O 2- ⁇ reacts with NBT, and the ability of scavenging free radicals of antioxidants can be calculated by measuring absorbance.
  • P represents refined red clover polysaccharide
  • IF represents refined red clover isoflavones
  • D 1 and D 2 are the concentration when the combined inhibitory rate of flavonoids and polysaccharides is 50%;
  • Dx 1 and Dx 2 are the concentration at which the inhibitory rate of flavonoids and polysaccharides acting alone is 50%.
  • Water solubility determination method accurately weigh each test sample, 5.0mg each, in a transparent centrifuge tube filled with 1mL of distilled water at 25°C room temperature, add 5.0mg in sequence until precipitation appears, repeat the operation if it cannot be added Stop adding when completely dissolved. You can use a vortex shaker to accelerate the dissolution, then record the addition amount of each sample in 1mL of distilled water, observe and estimate the mass of the undissolved part, and get the actual amount of dissolved in 1mL of distilled water.
  • red clover as a raw material, different types of natural antioxidant components prepared have different functions and application ranges. This is a multi-faceted and multi-purpose application in the development and production of food additives, and has a broad market prospect.
  • the refined red clover polysaccharide has the advantages of high activity and good water solubility, and it is first considered for use.
  • you need to prepare fat-soluble components you can consider using crude red clover polysaccharide or red clover isoflavones.
  • the two types of ingredients have different emphasis on activity. Crude red clover polysaccharide has high antioxidant activity to superoxide anion, while the DPPH activity of isoflavones is better.
  • the two components can be used for compounding. Especially when the mass ratio of polysaccharide and isoflavone is 1:2, the synergy effect between the two components is the best and the activity is the highest. .
  • red clover itself is an excellent forage grass, and its isoflavones are also popular as health products at home and abroad. Therefore, food additives prepared from this plant are not only green and environmentally friendly, but also very safe for the human body, but also rich in resources.
  • the refined red clover polysaccharide has the advantages of high activity and good water solubility. First of all, it is considered to be used. Secondly, if fat-soluble components need to be prepared, crude red can be considered. Triloba polysaccharides or red clover isoflavones, the two types of components have different emphasis in activity. Crude red clover polysaccharides have high antioxidant activity on superoxide anions, while the DPPH activity of isoflavones is better. Considering the cost and comprehensive activity Factors such as solubility and solubility can be combined with the two components.
  • the two components have the best synergistic effect and the highest activity.
  • the isoflavones are also popular as health products.
  • the food additives prepared by using the plant are not only green and environmentally friendly, but also very safe for the human body, but also rich in resources.

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Abstract

一种天然食品抗氧化剂及其制备方法，所述抗氧化剂主要组成为红三叶提取物，成分为黄酮、多糖及它们的复配物。所述制备方法包括红三叶提取物的制备、粗多糖和粗异黄酮的制备、红三叶异黄酮的精制、红三叶多糖的精制。

Description

一种天然食品抗氧化剂及其制备方法 技术领域
本发明涉及食品工业技术领域，特别涉及一种天然食品抗氧化剂及其制备方法。
背景技术
食品抗氧化剂可以防止或减缓事物的腐败变质，防止食物中有毒物质的产生、营养物质的流失和外观色泽的变化，另外也能避免一些动物脂肪和油脂氧化带来的一些异味。常用的人工抗氧化剂常常被添加到食品中，叔丁基对苯二酚(TBHQ)、叔丁基对羟基茴香醚(BHA)、二丁基羟基甲苯(BHT)和没食子酸丙酯(PG)使用较为广泛。随着人们健康意识的提高，合成抗氧化剂的安全问题越来越引起人们的关注。在雄性幼鼠腹膜内注射BHA、BHT及其代谢物的实验中，发现鼠肺细胞数量增生，现在美国，欧盟等国家已逐渐禁止使用此类合成抗氧化剂。
开发使用天然抗氧化剂已成为当今食品科学的发展趋势，红三叶是豆科多年生牧草，在全球范围内广泛种植，资源丰富。据报道，该植物中富含异黄酮类化合物，具有较好的抗氧化能力，然而，本发明从该植物提取制备的抗氧化成分活性优于异黄酮成分，且水溶性更好，适应范围更广，具有非常好的开发应用前景。
发明内容
本发明的目的在于提供一种天然食品抗氧化剂及其制备方法，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：一种天然食品抗氧化剂，包括食品抗氧化剂主要成分为红三叶提取物的萃取物，萃取物分别为粗多糖和粗异黄酮以及精制的多糖和异黄酮。
进一步地，精制的多糖和异黄酮以质量比1：10-15：1配制。
本发明提供的另一种技术方案：一种天然食品抗氧化剂的制备方法，包括以下步骤：
S1：红三叶提取物的制备：
将采集到的红三叶叶片105℃杀青2-60min，65℃下干燥1-160h，粉碎，用少量石油醚脱脂1-10次，挥去溶剂晾干，称取一定质量脱脂红三叶粉末，用10-50倍10-100％乙醇30-70℃超声辅助提取10-90min，过滤收集上清液，样品残渣再加5-120倍溶剂提取10-90min，合并两次提取液；
S2：粗多糖和粗异黄酮的制备：
粗多糖：将提取液在15-90℃下用真空旋转仪浓缩，至八分之一体积时，向浓缩液中边搅拌边缓缓加入2-20倍体积无水乙醇，放入4℃冰箱静置过夜，4800rpm离心2-20min，离心后褐色沉淀为粗多糖部分，沉淀部分用无水乙醇反复洗涤1-10次，洗去醇溶性杂质，最后用乙醇洗出沉淀倒入培养皿，待溶剂挥发，得到褐色红三叶粗多糖；
上清为异黄酮部分，并对上清液减压浓缩，得到红三叶粗异黄酮；
S3：红三叶异黄酮的精制：
将经过预处理的树脂用蒸馏水浸泡10-90h以上，湿法装柱树脂高度在3-50cm，将粗异黄酮10g用水溶解，上完样等待吸附0.5-24h后，分别用蒸馏水、30％、50％乙醇各洗脱1-5BV，然后用70％乙醇洗脱1-5BV，最后用100％乙醇洗脱1-10BV，收集70％乙醇洗脱液，减压浓缩，得精制异黄酮；
S4：红三叶多糖的精制：
除蛋白：将红三叶粗多糖用蒸馏水配置成1-10mg/mL溶液，加入1/4体积的sevage溶液，振荡1-30min，转移至分液漏斗静置1-120min，放掉下层溶液和中层的蛋白质，如此操作反复1-6次至下层有机溶剂中不再出现蛋白质；
透析：将去除蛋白质的多糖溶液，低温减压浓缩后，装入预处理的透析 袋，放入大容量烧杯中，加满蒸馏水静置透析，每1-16h换一次水，透析1-72h，用1％AgNO 3检查NaCl、KCl等残留情况，最终得到纯化的多糖。
进一步地，树脂预处理：将树脂用20-100％乙醇浸泡1-72h，过滤，用去离子水反复冲洗至无醇味，且冲洗液无白色浑浊，再用1-30％NaOH浸泡1-72h，过滤，用去离子水反复冲洗树脂至pH呈现中性，再用1-30％HCl溶液浸泡1-72h，过滤冲洗树脂至中性，20-90℃烘干备用。
与现有技术相比，本发明的有益效果是：本发明提出的一种天然食品抗氧化剂及其制备方法，精制红三叶多糖具有活性高、水溶性好的优点，首先被考虑采用，其次，如果需要制备脂溶性组分，可以考虑采用粗红三叶多糖或红三叶异黄酮，两类成分在活性方面有不同侧重，粗红三叶多糖对超氧阴离子抗氧化活性高，而异黄酮的DPPH活性更好一些，考虑到成本，综合活性和溶解度等因素，可以采用两者成分进行复配方案，特别是多糖与异黄酮质量比在1：2时，两种成分之间协同效果最好，活性最高，其异黄酮也作为保健品畅销海内外，所以利用该植物制备的食品添加剂不仅绿色环保，对人体非常安全，而且资源丰富。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例一：
一种天然食品抗氧化剂，包括食品抗氧化剂主要成分为红三叶提取物的萃取物，萃取物分别为粗多糖和粗异黄酮以及精制的多糖和异黄酮，粗多糖和粗异黄酮相互以质量比1：2配制，精制的多糖和异黄酮以质量比1：2配制。
为了更好的展现天然食品抗氧化剂制备的流程，本实施例现提出一种天然食品抗氧化剂的制备方法，包括以下步骤：
步骤一：红三叶提取物的制备：
将采集到的红三叶叶片105℃杀青10min，65℃下干燥72h，粉碎，用少量石油醚脱脂4次，挥去溶剂晾干，称取2g脱脂红三叶粉末，用25倍40％乙醇50℃超声辅助提取30min，过滤收集上清液，样品残渣再加20倍溶剂提取30min，合并两次提取液；
步骤二：粗多糖和粗异黄酮的制备：
第一节：粗多糖：将提取液在45℃下用真空旋转仪浓缩，至八分之一体积时，向浓缩液中边搅拌边缓缓加入4倍体积无水乙醇，使溶液乙醇浓度达到80％，放入4℃冰箱静置过夜，4800rpm离心10min，离心后褐色沉淀为粗多糖部分，沉淀部分用无水乙醇反复洗涤3次，洗去醇溶性杂质，最后用乙醇洗出沉淀倒入培养皿，待溶剂挥发，得到褐色红三叶粗多糖；
第二节：步骤S21所的上清为异黄酮部分，并对上清液减压浓缩，得到红三叶粗异黄酮；
步骤三：红三叶异黄酮的精制：
树脂预处理：将树脂用95％乙醇浸泡24h，过滤，用去离子水反复冲洗至无醇味，且冲洗液无白色浑浊，再用5％NaOH浸泡24h，过滤，用去离子水反复冲洗树脂至pH呈现中性，再用5％HCl溶液浸泡24h，过滤冲洗树脂至中性，60℃烘干备用，将经过预处理的树脂用蒸馏水浸泡48h以上，湿法装柱树脂高度在13cm，将粗异黄酮10g用水溶解，上完样等待吸附2h后，分别用蒸馏水、30％、50％乙醇各洗脱1BV，然后用70％乙醇洗脱2BV，最后用100％乙醇洗脱1BV，收集70％乙醇洗脱液，减压浓缩，得精制异黄酮。
步骤四：红三叶多糖的精制：
第一节：除蛋白：将红三叶粗多糖用蒸馏水配置成2mg/mL溶液，加入1/4 体积的sevage溶液，振荡20min，转移至分液漏斗静置20min，放掉下层溶液和中层的蛋白质，如此操作反复3次至下层有机溶剂中不再出现蛋白质；
第二节：透析：将去除蛋白质的多糖溶液，低温减压浓缩后，装入预处理的透析袋，放入大容量烧杯中，加满蒸馏水静置透析，每8h换一次水，透析24h，用1％AgNO 3检查NaCl、KCl等残留情况，最终得到纯化的多糖。
实施例二：
DPPH抗氧化活性：DPPH属于人工合成的一类稳定性自由基，在可见光区有特征吸收，测定方法简便，快速且重现性良好；
将实施例一所制备的黄酮类和黄酮与多糖混合样品均由70％乙醇配制，多糖样品、阳性对照维生素C(Vc)和维生素E(Ve)用蒸馏水配制，将每个待测物质配制1.280mg/mL的原液，再稀释到7个浓度梯度待用。
在96孔酶标板上精确加入各浓度待测液75μL，再加入0.1mMDPPH70％乙醇溶液150μL，每个浓度的反应孔都设置空白对照，空白对照为75μL待测液和150μL70％乙醇，反应总体积为225μL，充分摇匀，在黑暗中室温放置30min后，测定波长为519nm处的吸光值。吸光度的下降表示自由基清除活性的增加。每个样品每个浓度进行3次重复。
清除率＝[A0-(A1-A2)]/A0
A0为DPPH溶液加浓度为零待测液的吸光值，A1为待测液与DPPH反应后的吸光值，A2为空白对照，即各待测液与空白溶剂的吸光值。
表1 DPPH抗氧化活性
Figure PCTCN2020089887-appb-000001
Figure PCTCN2020089887-appb-000002
注：P表示精制红三叶多糖，IF表示精制红三叶异黄酮，两者按照质量比混合。
小写字母表示显著性差异(P<0.05)。
从表1可知，红三叶的精制多糖活性与精制异黄酮相当。在两者混合质量比为1：2时活性最高，显著优于每个单独成分。随着多糖含量的升高，DPPH活性显著下降。
实施例三：
超氧阴离子抗氧化活性测定：
O 2-·是机体内寿命较长的一类自由基，在自由基链式反应中，O 2-·作为引发剂促使其他自由基的生成，对机体造成进一步的危害，与多种疾病密切相关。因此，通常将样品清除O 2-·的能力作为抗氧化物质活性的重要指标。
将实施例一所制备的黄酮类和黄酮与多糖混合样品均由70％乙醇配制，多糖样品、阳性对照维生素C(Vc)和维生素E(Ve)用蒸馏水配制，将每个待测物质配制1.280mg/mL的原液，再稀释到7个浓度梯度待用。
用PMS-NADH-NBT体系法测定清除超氧阴离子(O2-·)活性，其原理是，以还原型辅酶Ⅰ(烟酰胺腺嘌呤二核苷酸，NADH)还原吩嗪硫酸甲酯(PMS)产生超氧阴离子，O 2-·和氯化硝基四氮唑蓝(NBT)反应生成蓝色产物，在560nm有强吸收峰，当抗氧化物质加入到PMS-NADH-NBT体系后，抑制了O 2-·与NBT反应，通过测定吸光度可以计算出抗氧化物质的清除自由基能力。在96孔酶 标板上依次加入30μLNBT(144μmol/L)，100μL待测液(2.50-125.0μg/mL，用溶剂代替样液为空白)，30μLNADH(0.68mmol/L，用磷酸缓冲液配制)，30μL新配制的PMS(60μmol/L)30μLpH7.4磷酸缓冲液。空白管为120μL缓冲液和100μL待测液，振荡30s，5min后测定溶液在560nm处的吸光值。
表2超氧阴离子抗氧化活性
Figure PCTCN2020089887-appb-000003
注：P表示精制红三叶多糖，IF表示精制红三叶异黄酮，两者按照质量比混合。
小写字母表示显著性差异(P<0.05)。
从表2可知，红三叶的异黄酮的活性与Vc，相当，显著优于Ve，而红三叶多糖活性显著高于Vc和Ve，特别是精制多糖活性高于阳性对照(Vc和Ve)100多倍。3种复配物活性与精制多糖相当，随着多糖含量的提高，活性略有增强。
实施例四：
复配组分互作对抗氧化活性关系的评价：
利用判断两类组分抗氧化活性之间的关系方法—联合指数 (CI,combinationindex)对红三叶异黄酮和多糖的联合抗氧化作用的效果进行评价。我们这里采用Talady和Chou的中效原理联合作用指数CI。
CI＝D 1/DX 1+D 2/DX 2
D 1和D 2是黄酮和多糖联合作用抑制率为50％时的作用浓度；
Dx 1和Dx 2是黄酮和多糖单独作用时的作用抑制率为50％时的作用浓度。
根据Talady和Chou的理论，如果CI＝1，表明药物1和药物2是联合作用效果是具有叠加作用的；若CI>1时，表明药物1和药物2联合作用效果是具有拮抗作用的；若CI<1，表明药物1和药物2联合作用是具有协同作用的。
表3联合抗氧化作用指数CI
Figure PCTCN2020089887-appb-000004
注：小写字母表示显著性差异(P<0.05)。
由表3可知，多糖与异黄酮两者混合质量比为1：2时两种成分是相互协同，其中对于超氧阴离子的协同作用更强。但是对于DPPH活性，随着多糖含量增加，两种成分出现一定程度的拮抗作用。
实施例五：
不同制备物在水中的溶解性
水溶性测定方法：准确称取各测定样品，每份5.0mg，在25℃室温环境下，在加有1mL蒸馏水的透明离心管中，依次加入5.0mg，直至出现沉淀，重复操作若加入后不能完全溶解时停止添加，可以用涡旋振荡器加速溶解，然后记录1mL蒸馏水中各样品添加量，观察估算未溶解部分的质量，得出1mL蒸馏水中的实际溶解量。
表4不同制备成分在水中的溶解范围
Figure PCTCN2020089887-appb-000005
由表4可知，多糖在水中的溶解性强于异黄酮，精制后的样品溶解度均会提高。特别是精制红三叶多糖(大于25g/L)，其溶解性是精制异黄酮和粗多糖的5倍。
以红三叶为原料，所制备的不同类型天然抗氧化成分其功能和适用范围均有所差别，这为多方面和多用途应用在食品添加剂的研发和生产中，市场前景广阔。其中，精制红三叶多糖具有活性高、水溶性好的优点，首先被考虑采用。其次，如果需要制备脂溶性组分，可以考虑采用粗红三叶多糖或红三叶异黄酮。两类成分在活性方面有不同侧重，粗红三叶多糖对超氧阴离子抗氧化活性高，而异黄酮的DPPH活性更好一些。再次，如果考虑到成本，综合活性和溶解度等因素，可以采用两者成分进行复配方案，特别是多糖与异黄酮质量比在1：2时，两种成分之间协同效果最好，活性最高。
此外，红三叶本身是一种优良牧草，其异黄酮也作为保健品畅销海内外，所以利用该植物制备的食品添加剂不仅绿色环保，对人体非常安全，而且资源丰富。
综上所述：本天然食品抗氧化剂及其制备方法，精制红三叶多糖具有活性高、水溶性好的优点，首先被考虑采用，其次，如果需要制备脂溶性组分，可以考虑采用粗红三叶多糖或红三叶异黄酮，两类成分在活性方面有不同侧重，粗红三叶多糖对超氧阴离子抗氧化活性高，而异黄酮的DPPH活性更好一些，考虑到成本，综合活性和溶解度等因素，可以采用两者成分进行复配方案，特别是多糖与异黄酮质量比在1：2时，两种成分之间协同效果最好，活 性最高，其异黄酮也作为保健品畅销海内外，所以利用该植物制备的食品添加剂不仅绿色环保，对人体非常安全，而且资源丰富。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。

Claims (4)

  1. 一种天然食品抗氧化剂，其特征在于，包括食品抗氧化剂主要组分为红三叶提取物的萃取物，成分分别为粗多糖、粗异黄酮、精制多糖和精制异黄酮及精制多糖和精制异黄酮的复配物。
  2. 如权利要求1所述的一种天然食品抗氧化剂，其特征在于精制多糖和精制异黄酮的复配物质量比1：10-15：1配制。
  3. 一种如权利要求1-2任一项所述的天然食品抗氧化剂的制备方法，其特征在于，包括以下步骤：
    S1：红三叶提取物的制备：
    将采集到的红三叶叶片105℃杀青2-60min，65℃下干燥1-160h，粉碎，用少量石油醚脱脂1-10次，挥去溶剂晾干，称取一定质量脱脂红三叶粉末，用10-50倍10-100％乙醇30-70℃超声辅助提取10-90min，过滤收集上清液，样品残渣再加5-120倍溶剂提取10-90min，合并两次提取液；
    S2：粗多糖和粗异黄酮的制备
    粗多糖：将提取液在15-90℃下用真空旋转仪浓缩，至八分之一体积时，向浓缩液中边搅拌边缓缓加入2-20倍体积无水乙醇，放入4℃冰箱静置过夜，4800rpm离心2-20min，离心后褐色沉淀为粗多糖部分，沉淀部分用无水乙醇反复洗涤1-10次，洗去醇溶性杂质，最后用乙醇洗出沉淀倒入培养皿，待溶剂挥发，得到褐色红三叶粗多糖；
    上清为异黄酮部分，并对上清液减压浓缩，得到红三叶粗异黄酮；
    S3：红三叶异黄酮的精制：
    将经过预处理的树脂用蒸馏水浸泡10-90h以上，湿法装柱树脂高度在3-50cm，将粗异黄酮1-30g用水溶解，上完样等待吸附0.5-24h后，分别用蒸馏水、30％、50％乙醇各洗脱1-5BV，然后用70％乙醇洗脱1-5BV，最后用100％乙醇洗脱1-10BV，收集70％乙醇洗脱液，减压浓缩，得精制异黄酮；
    S4：红三叶多糖的精制：
    除蛋白：将红三叶粗多糖用蒸馏水配置成1-10mg/mL溶液，加入1/4体积的sevage溶液，振荡1-30min，转移至分液漏斗静置1-120min，放掉下层溶液和中层的蛋白质，如此操作反复1-6次至下层有机溶剂中不再出现蛋白质；
    透析：将去除蛋白质的多糖溶液，低温减压浓缩后，装入预处理的透析袋，放入大容量烧杯中，加满蒸馏水静置透析，每1-16h换一次水，透析1-72h，用1％的AgNO 3检查NaCl、KCl等残留情况，最终得到纯化的多糖。
  4. 如权利要求3所述的一种天然食品抗氧化剂的制备方法，其特征在于，针对步骤S3中，树脂预处理：将树脂用20-100％乙醇浸泡1-72h，过滤，用去离子水反复冲洗至无醇味，且冲洗液无白色浑浊，再用1-30％NaOH浸泡1-72h，过滤，用去离子水反复冲洗树脂至pH呈现中性，再用1-30HCl溶液浸泡1-72h，过滤冲洗树脂至中性，20-90℃烘干备用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112972321A (zh) * 2019-12-16 2021-06-18 兰州大学 一种用于化妆品的天然抗氧化剂及其制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101386613A (zh) * 2007-09-11 2009-03-18 兰州大学 从红车轴草提取异黄酮类物质的工艺
CN102302539A (zh) * 2010-07-10 2012-01-04 恩施济源药业科技开发有限公司 一种红车轴草异黄酮的生产方法
CN105434509A (zh) * 2015-12-25 2016-03-30 兰州大学 红三叶提取物制备方法及在降糖方面的应用
CN110183541A (zh) * 2019-04-23 2019-08-30 湖南华诚生物资源股份有限公司 红车轴草多糖及总异黄酮的制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101386613A (zh) * 2007-09-11 2009-03-18 兰州大学 从红车轴草提取异黄酮类物质的工艺
CN102302539A (zh) * 2010-07-10 2012-01-04 恩施济源药业科技开发有限公司 一种红车轴草异黄酮的生产方法
CN105434509A (zh) * 2015-12-25 2016-03-30 兰州大学 红三叶提取物制备方法及在降糖方面的应用
CN110183541A (zh) * 2019-04-23 2019-08-30 湖南华诚生物资源股份有限公司 红车轴草多糖及总异黄酮的制备方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112972321A (zh) * 2019-12-16 2021-06-18 兰州大学 一种用于化妆品的天然抗氧化剂及其制备方法

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