WO2017088211A1 - 一种半固态水酶法制备菜籽油的方法 - Google Patents

一种半固态水酶法制备菜籽油的方法 Download PDF

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WO2017088211A1
WO2017088211A1 PCT/CN2015/096664 CN2015096664W WO2017088211A1 WO 2017088211 A1 WO2017088211 A1 WO 2017088211A1 CN 2015096664 W CN2015096664 W CN 2015096664W WO 2017088211 A1 WO2017088211 A1 WO 2017088211A1
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oil
rapeseed
rapeseed oil
water
added
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French (fr)
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刘元法
曹培让
李进伟
张亮
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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
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
    • A23D9/04—Working-up
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
    • 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
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • 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)
    • A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
    • 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
    • C11B1/02—Pretreatment
    • C11B1/04—Pretreatment of vegetable raw material
    • 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
    • C11B1/06—Production of fats or fatty oils from raw materials by pressing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/25—Threonine endopeptidases (3.4.25)
    • C12Y304/25001—Proteasome endopeptidase complex (3.4.25.1)
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine

Definitions

  • the invention relates to a method for preparing rapeseed oil by semi-solid water enzymatic method, belonging to the field of functional foods and health care products.
  • Rapeseed oil is one of the world's four major vegetable oil varieties, and its output is second only to palm oil and soybean oil. Rapeseed oil is a liquid obtained by seed rapeseed of the cruciferous plant Brassica (ie, rape), and is generally yellow or brown. Rapeseed oil contains higher oleic acid and linoleic acid.
  • Rapeseed oil is the main vegetable oil variety in China. It has large reserves and many varieties. The quality of rapeseed oil is related to the health of Chinese people. With the development of the oil industry, the oil and fat composition of vegetable oils and fats, such as unsaturated fatty acids, and some biologically active ingredients containing special physiological effects on the human body have gradually received attention. In particular, although the trace components are low in content, they are related to certain nutrients and health effects of fats and oils. Therefore, current researchers, while cultivating high-yield oil crops, are moving toward improving certain nutrient functional components in oil products.
  • phytosterols which have structural properties similar to those of cholesterol, compete with cholesterol molecules in the intestine to form components of chylomicrons, while co-crystallizing effects with cholesterol increase cholesterol excretion.
  • VE which is rich in fats and oils, can inhibit LDL oxidation by virtue of its affinity with lipoproteins in the prevention and treatment of cardiovascular and cerebrovascular diseases. Plant polyphenols in oils and fats can also effectively reduce the risk of cardiovascular disease.
  • the amount of trace active ingredients such as tocopherol, sterols, phenols and beta carotene in rapeseed oil can affect the quality of the oil.
  • the cold pressing method is prepared by pressing at less than 60 ° C; the hot pressing method is obtained by steaming and frying at 120 ° C; the leaching method is extracted with a solvent such as n-hexane.
  • the aqueous enzymatic method is prepared by hydrolyzing an oil body with a protease and then separating the protein and the oil by centrifugation.
  • the rapeseed oil produced by the water enzymatic method has the best quality.
  • the traditional water enzymatic method for preparing rapeseed oil is a mass ratio of rapeseed solid to water of 1:4-6, adding a mixed complex enzyme, and the reaction is carried out in a liquid state.
  • this traditional method has serious shortages in the preparation of rapeseed oil, such as emulsification is relatively serious, and the yield of clear oil is not high.
  • Technical treatments that require further demulsification result in wasted energy and time.
  • the traditional water enzymatic method due to the high water content, the phenolic substance is highly polar, and is easily dissolved in water to cause the loss of trace nutrients.
  • the present invention provides a semi-solid aqueous enzymatic method for preparing rapeseed oil, which is:
  • the enzymatic hydrolysis process is carried out by gradient temperature control, maintained at 40 ° C for 20-40 min, the temperature is raised from 40 ° C to 60 ° C in 20-40 min, and maintained at 60 ° C for 20-40 min.
  • the gradient temperature control fully exerts the effect of the enzyme and significantly increases the oil yield.
  • the pulverization is carried out to obtain an oil having a particle size of 40 to 80 mesh.
  • the agitation mixing at 20-40 ° C is agitation at 10-20 rpm.
  • the separation of the pomace and the hair oil is carried out at 3500 rpm.
  • the rapeseed oil is obtained by centrifugation at 6500 rpm.
  • the pectinase, cellulase, and alkaline protease have an enzyme activity of 1.5 AU/g, 2.4 AU/g, and 1.5 AU/g, respectively.
  • AU is Anson unit.
  • the step (4) specifically: pulverizing the cooked embryos after low temperature preparation to obtain an oil material having a particle size of 40 to 80 meshes; and mixing the oil materials with water in a mass ratio of 1:1.
  • Obtain a semi-solid paste; add 0.5% to 2% (w/w) pectinase: cellulase: alkaline protease 1:1:1 to the rapeseed paste, at 40- Mixing at 10 °C for 1-2 h at 60 ° C; then separating the rapeseed paste with a two-phase centrifuge at 3500 rpm to obtain pomace and hair oil; finally, the oil is 0.25 L/kg oil.
  • the amount of water added was added to distilled water, centrifuged at 40 ° C, centrifuged at 6500 rpm to obtain rapeseed oil, and finally vacuum dried to obtain the final aqueous enzymatic rapeseed oil without any refining steps.
  • the invention also provides an application of the rapeseed oil prepared by the above method, which is to add the rapeseed oil to the feed.
  • the feed contains 1% cholesterol, 0.2 sodium cholate, 10% lard, 10% rapeseed oil, 10% egg yolk powder, 68.85. % basic feed.
  • the invention adopts semi-solid water enzymatic method to prepare rapeseed oil, the process is simple, and the enzymatic hydrolysis is carried out by pectinase, cellulase and alkaline protease, and the gradient temperature control strategy is adopted, and the yield of clear oil can reach 93.5%. .
  • the invention has simple process and mild conditions, does not need to use organic solvent, is safe and non-toxic; the prepared rapeseed oil has high quality, and the micronutrient active ingredient retains more, tocopherol, sterol, phenol, ⁇ - Carotene content can reach 672ppm, 5869ppm, 154ppm, 5.8ppm or more respectively, which is significantly higher than traditional water enzymatic method and low-moisture water enzymatic method.
  • the rapeseed oil prepared by the invention is added to the animal feed, which can significantly inhibit the increase of body weight of the hyperlipidemic rats, reduce the accumulation of fat, and significantly reduce the total triglyceride (TG) and total cholesterol in the serum. And LDL-C content, and increase the serum HDL-C content; and the rapeseed oil prepared by the water enzymatic method can improve liver fatty degeneration in hyperlipidemic rats.
  • the rapeseed oil prepared by the present invention can be used for the purpose of preventing, alleviating or treating a drug or food which is associated with a disease or a discomfort associated with elevated blood fat.
  • the hyperlipidemia described therein is hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia or low high density lipoproteinemia.
  • the hyperlipidemia described therein is hypercholesterolemia, hypertriglyceridemia, mixed hyperlipidemia or low-density lipoproteinemia; wherein the disease or physical discomfort associated with elevated blood lipids Symptoms are atherosclerosis, coronary heart disease, stroke or myocardial infarction.
  • Figure 1 Morphology of liver tissue of normal mice
  • Figure 2 Morphological map of liver tissue of mice in the high fat group
  • Figure 3 Morphological map of liver tissue of refining mice
  • Figure 4 Morphological map of liver tissue of mice in the cold-press group
  • Figure 5 Morphological map of liver tissue of mice in the hot pressed group
  • Figure 6 Morphological map of liver tissue of mice in the hydroenzyme group
  • Figure 7 Morphological map of liver tissue of mice in the leaching group.
  • Example 1 Preparation of rapeseed oil by semi-solid aqueous enzymatic method
  • the rapeseed obtained in the previous step is subjected to rolling, and the rapeseed embryo is obtained, and the rapeseed embryo is subjected to low temperature preparation to have a moisture content of 4.5 wt%, and the cooked embryo obtained at a low temperature is obtained;
  • the temperature is 60 ° C, 60 ° C for 40 min, the gradient temperature control fully exerts the effect of the enzyme, significantly improving the oil yield; then 3500 rpm centrifugation, the rapeseed paste is separated to obtain pomace and hair oil; finally the oil Water was added according to the added amount of 0.2 L/kg of oil, and the rapeseed oil was obtained by centrifugation at 6500 rpm at 40 ° C, and finally dried under vacuum to obtain a final aqueous enzymatic rapeseed oil.
  • Example 2 Preparation of rapeseed oil by semi-solid aqueous enzymatic method
  • the gradient temperature control fully exerted the effect of the enzyme, significantly improved the oil yield; then centrifuged at 3500 rpm to separate the rapeseed paste to obtain pomace and hair oil; finally, the oil was 0.4L/ The water content of the kg oil was added to water, and the rapeseed oil was obtained by centrifugation at 6500 rpm at 40 ° C, and finally dried under vacuum to obtain the final aqueous enzymatic rapeseed oil.
  • Example 3 Comparison of trace components in semi-solid water enzymatic method and traditional water enzymatic method and low-moisture water enzymatic method for rapeseed oil
  • the rapeseed oil was prepared by the traditional aqueous enzymatic method, the low-moisture water enzymatic method and the semi-solid aqueous enzymatic method of the present invention 1-2, respectively, and the tocopherol, sterol, phenol and ⁇ -carotene in the oil were respectively determined. Table 1.
  • the rapeseed oil prepared by the semi-solid water enzymatic method of the present invention has a significantly higher content of trace active ingredients than the conventional aqueous enzymatic method and low moisture aqueous enzymatic method.
  • the ratio of the oil to the water in the step (4) was changed by the method of the first embodiment of the present invention, and the other steps were identical.
  • the results are shown in Table 2.
  • the results show that the 1:1 ratio of the semi-solid aqueous enzymatic method of the present invention can significantly increase the rate of clear oil.
  • the clear oil yield is the total oil quality in the non-emulsified oil/vegetable granules obtained by centrifugation.
  • Fifty-five male SD rats (170 ⁇ 10g) were acclimatized for 7 days in the experimental environment, and then the rats were randomly divided into 7 groups: normal control group, high fat lard group, refined rapeseed oil group, cold mustard Seed oil group, hot pressed rapeseed oil group, semi-solid water enzyme rapeseed oil group (the rapeseed oil prepared by the method of the invention) and the rapeseed oil group, 8 in each group, except for the normal group for normal feed
  • the high-fat lard group was given high-fat diet (1% cholesterol + 0.2 sodium cholate + 20% lard + 10% egg yolk powder + 68.85% basic feed), and the other 5 groups were prepared with 10% different processing methods.
  • the oil replaces 10% lard and the other conditions remain unchanged.
  • the rats were weighed for 12 hours after fasting.
  • the heart was pierced and blood was taken, and the supernatant of the blood was centrifuged, and the biochemical indexes such as TC, TG, HDL-C and LDL-C in the serum were determined by a medical kit.
  • the liver tissue (about 1 cm ⁇ 1 cm) was placed in a 4% paraformaldehyde fixative solution for tissue fixation, paraffin section and H/E tissue staining to observe the morphology of the liver tissue.
  • the results are shown in Table 3.
  • the results show that the rapeseed oil prepared by the semi-solid aqueous enzymatic method of the present invention can be significantly suppressed from the table.
  • the body weight and liver-to-body ratio of high-fat rats were increased.
  • liver to body ratio liver weight / weight
  • Table 4 shows the levels of TCH and TG in the serum of the rats in the hydroenzymatic group compared with the normal group, compared with the rats given the high fat lard feed and other rapeseed oils obtained by different processing methods. The lowest, in which the serum TCH, TG content in the high fat lard, decreased by 28% and 12%, respectively.
  • LDL-C commonly known as bad cholesterol
  • Table 5 shows that LDL-C increased by 300% in the high-fat group compared with the normal control group (P ⁇ 0.05); LDL-C in the serum of the water-enzymatic group decreased compared with the high-fat lard group. The lowest is up to 36%. The difference between the HDL-C levels is not significant.
  • the LDL-C level and the HDL-C level were the lowest in the leaching group.
  • FIG. 1-7 A micrograph of the histopathological observation of the rat is shown in Figures 1-7. It can be seen that the liver cells of the normal control group are arranged neatly, the liver cells are abundant, and the boundary is clear. Compared with the liver tissue of the rapeseed oil fed by other methods, the water enzymatic method is most close to the normal tissue morphology, ie Hepatocytes maintain a radially intact cell arrangement and structure. However, in the high fat lard group, hepatocytes in the cytoplasm of the liver showed vesicles of different sizes.
  • the fat droplets in the liver cells were mostly larger, and the intercellular gap was not clear; the relative fat droplets in the cold-pressing group, the hot-pressing group and the water-enzyme group were less.

Abstract

一种半固态水酶法制备菜籽油的方法，以半固态水酶法，以果胶酶、纤维素酶、碱性蛋白酶复配进行酶解，从菜籽中得到菜籽油。

Description

一种半固态水酶法制备菜籽油的方法 技术领域
本发明涉及一种半固态水酶法制备菜籽油的方法，属于功能食品和保健品领域。
背景技术
菜籽油是世界四大植物油品种之一，产量仅次于棕榈油和豆油。菜籽油是以十字花科植物芸苔(即油菜)的种子油菜籽制取所得的液体，一般呈黄色或棕色。菜籽油中含有较高的油酸和亚油酸。
菜籽油是我国主要的植物油品种，储量大，品种多，菜籽油其品质质量关系着国人的身体健康。随着油脂工业的发展，植物油脂中油脂组成如不饱和脂肪酸以及含有的一些对人体有特殊生理作用的生物活性成分逐渐受到了人们的重视。尤其是微量成分虽然含量很低，但与油脂所具有的某些营养和保健功效有关。因此现在的研究者，在培育高产油量作物的同时，正朝着提高油品中某些特殊营养功能成分的方向发展。例如，植物甾醇由于具有和胆固醇相似的结构特性，能够在肠道中与胆固醇分子竞争构成乳糜微粒的成分，同时与胆固醇的共结晶效应增加了胆固醇的排遗。油脂中富含的VE能够在心脑血管疾病防治中通过与脂蛋白特有的亲和力，能够抑制LDL氧化。油脂中的植物多酚类也能够有效地减少心血管疾病风险。
菜籽油中富含的微量活性成分如生育酚、甾醇、酚类和β胡萝卜素等含量会影响到油的品质。目前菜籽油的制取方法主要有四种，冷榨法是在低于60℃下压榨制得；热榨法是经过120℃蒸炒后压榨制得；浸出法是用溶剂如正己烷萃取得到；水酶法是用蛋白酶水解油小体，然后通过离心分离蛋白质和油而制得。在这四种方法中，水酶法制得的菜籽油品质最好。因为其制备条件温和，微量营养活性成分如生育酚、酚类、β胡萝卜素等保留较多。传统的水酶法制备菜籽油的过程是菜籽固体和水的质量比1:4-6，加入混合复合酶，反应以液态方式进行。但此种传统的方法在制备菜籽油时，存在严重不足，譬如乳化现象比较严重，清油得率不高。需要进一步破乳的技术处理，这样造成能源和时间的浪费。同时，传统水酶法中由于水的含量很高，酚类物质极性强，易溶于水中造成微量营养成分地流失。
发明内容
为了克服以上问题，本发明提供了一种半固态水酶法制备菜籽油的方法，所述方法是：
(1)将油菜籽清理干净，去除碎屑、瘪籽、杂质；
(2)对除去杂质的菜籽进行水分调制，使其水分含量为8-10wt％；
(3)对上一步得到的菜籽进行轧胚，得到菜籽胚片，对菜籽胚片进行低温调制(50℃-70℃)，使其水分含量为3.5-4.5wt％，得到低温调制后的熟胚；
(4)将低温调制后的熟胚粉碎过筛，得到油料；将油料与水按1:1的质量比例混合得到半固态的糊状物；按总加酶量0.5％～2％(w/w)添加果胶酶：纤维素酶：碱性蛋白酶＝1:1:1的酶至菜籽糊状物，在40-60℃下搅拌混合1-2h；然后离心，将菜籽糊状物分离得到果渣和毛油；最后将毛油按0.2-0.4L/kg油的加水量加入水，在40-60℃下离心分离得到菜籽清油，最后经过真空干燥得到最终的水酶法菜籽油。
在本发明的一种实施方式中，酶解过程采用梯度控温的方法，40℃保持20-40min，温度在20-40min内从40℃升温到60℃，60℃下保持20-40min。梯度控温充分发挥了酶的作用，显著提高了出油率。
在本发明的一种实施方式中，所述粉碎过筛得到粒度40～80目的油料。
在本发明的一种实施方式中，所述20-40℃下搅拌混合是在10-20rpm下搅拌。
在本发明的一种实施方式中，所述果渣和毛油的分离是在3500rpm下离心。
在本发明的一种实施方式中，所述菜籽清油是在6500rpm下离心得到的。
在本发明的一种实施方式中，所述果胶酶、纤维素酶、碱性蛋白酶的酶活分别为1.5AU/g、2.4AU/g、1.5AU/g。AU即为Anson unit。
在本发明的一种实施方式中，所述步骤(4)具体是：将低温调制后的熟胚粉碎过筛，得到粒度40～80目的油料；将油料与水按1:1的质量比例混合得到半固态的糊状物；按加酶量0.5％～2％(w/w)果胶酶：纤维素酶：碱性蛋白酶＝1:1:1添加至菜籽糊状物，在40-60℃下用混合机在10-20rpm下混合1-2h；然后用两相离心机在3500rpm下离心将菜籽糊状物分离得到果渣和毛油；最后将毛油按0.25L/kg油的加水量加入蒸馏水，在40℃下，用立式离心机离心，6500rpm分离得到菜籽清油，最后经过真空干燥得到最终的水酶法菜籽油，无需任何精炼步骤。
本发明还提供了一种上述方法制备得到的菜籽油的应用，是将所述菜籽油添加到饲料中。
在本发明的一种实施方式中，所述菜籽油添加到饲料中后，饲料中含有1％胆固醇、0.2胆酸钠、10％猪油、10％菜籽油、10％蛋黄粉、68.85％基础饲料。
本发明的有益效果：
(1)本发明采用半固态水酶法制备菜籽油，工艺简单，以果胶酶、纤维素酶、碱性蛋白酶复配进行酶解，联合梯度控温策略，清油得率可达93.5％。
(2)本发明工艺简单、条件温和，不需要使用到有机溶剂，安全、无毒；制备得到的菜籽油品质高、微量营养活性成分保留较多，生育酚、甾醇、酚类、β-胡萝卜素含量分别可达672ppm、5869ppm、154ppm、5.8ppm以上，较传统水酶法和低水分水酶法提高显著。
(3)本发明制备的菜籽油添加到动物饲料中，能够明显抑制高血脂大鼠体重的增加、减少脂肪的富集生成，明显降低血清中总甘三脂(TG)、总胆固醇(TCH)和LDL-C含量，并升高血清中HDL-C的含量；并且以本水酶法制得的菜籽油能够改善高血脂大鼠肝脏脂肪变性。
(4)本发明制备的菜籽油，可以用于预防、缓解或治疗与血脂升高有关的疾病或者身体不适症的药物或食品中的应用。其中所述的高脂血症为高胆固醇血症、高甘油三酯血症、混合型高脂血症或者低高密度脂蛋白血症。其中所述的高脂血症为高胆固醇血症、高甘油三脂血症、混合型高脂血症或者低高密度脂蛋白血症；其中所述的与血脂升高有关的疾病或者身体不适症为动脉粥样硬化、冠心病、中风或心肌梗塞。
附图说明
图1：正常组小鼠的肝脏组织的形态图；
图2：高脂组小鼠的肝脏组织的形态图；
图3：精炼组小鼠的肝脏组织的形态图；
图4：冷榨组小鼠的肝脏组织的形态图；
图5：热榨组小鼠的肝脏组织的形态图；
图6：水酶组小鼠的肝脏组织的形态图；
图7：浸出组小鼠的肝脏组织的形态图。
具体实施方式
实施例1：半固态水酶法制备菜籽油
采用以下方法制备：
(1)将油菜籽清理干净，去除碎屑、瘪籽、杂质；
(2)对除去杂质的菜籽进行调制，使其水分含量为8wt％；
(3)对上一步得到的菜籽进行轧胚，得到菜籽胚片，对菜籽胚片进行低温调制，使其水分含量为4.5wt％，得到低温调制后的熟胚；
(4)将低温调制后的熟胚粉碎过筛，得到粒径40目油料；将油料与水按1:1的质量比例混合得到半固态的糊状物；按加酶量0.5％(w/w)果胶酶：纤维素酶：碱性蛋白酶＝1:1:1添加至菜籽糊状物，在10rpm下搅拌混合，先40℃保持40min，温度在40min内从40℃升 温到60℃，60℃下保持40min，梯度控温充分发挥了酶的作用，显著提高了出油率；然后3500rpm离心，将菜籽糊状物分离得到果渣和毛油；最后将毛油按0.2L/kg油的加水量加入水，在40℃下6500rpm离心分离得到菜籽清油，最后经过真空干燥得到最终的水酶法菜籽油。
实施例2：半固态水酶法制备菜籽油
采用以下方法制备：
(1)将油菜籽清理干净，去除碎屑、瘪籽、杂质；
(2)对除去杂质的菜籽进行调制，使其水分含量为10wt％；
(3)对上一步得到的菜籽进行轧胚，得到菜籽胚片，对菜籽胚片进行低温调制，使其水分含量为3.5wt％，得到低温调制后的熟胚；
(4)将低温调制后的熟胚粉碎过筛，得到粒径80目油料；将油料与水按1:1的质量比例混合得到半固态的糊状物；按加酶量2％(w/w)果胶酶：纤维素酶：碱性蛋白酶＝1:1:1添加至菜籽糊状物，在20rpm下搅拌混合，先40℃保持20min，温度在20min内从40℃升温到60℃，60℃下保持20min，梯度控温充分发挥了酶的作用，显著提高了出油率；然后3500rpm离心，将菜籽糊状物分离得到果渣和毛油；最后将毛油按0.4L/kg油的加水量加入水，在40℃下6500rpm离心分离得到菜籽清油，最后经过真空干燥得到最终的水酶法菜籽油。
实施例3：半固态水酶法和传统水酶法、低水分水酶法菜籽油微量成分含量比较
分别采用传统水酶法、低水分水酶法和本发明实施例1-2的半固态水酶法制备菜籽油，分别测定油中生育酚、甾醇、酚类和β-胡萝卜素，结果如表1。
传统水酶法：油料破碎后加水，固液比1:4～6，加入果胶酶、纤维素酶、蛋白质酶等，酶在水相中进行水解，使油脂从油料固体粒子中渗出。由于水的比例较高，蛋白溶液容易与油形成乳化液，清油含量得率低，大约只有50％，给后续分离提取带来困难。
低水分水酶法，料液比1:0.1～0.4进行预处理后，采用有机溶剂进行油脂浸出，提油率(制取得到的总油(包括清油和乳化的油)/菜籽颗粒里的总油质量)虽然提高，但有机溶剂的脱除和残留仍然影响消费者的健康。
表1不同水酶法制备得到的菜籽油微量成分含量比较
Figure PCTCN2015096664-appb-000001
Figure PCTCN2015096664-appb-000002
由表1可知，本发明的半固态水酶法制得的菜籽油，微量活性成分含量显著高于传统水酶法和低水分水酶法。
实施例4：提取工艺对菜籽油品质的影响
采用本发明实施例1的方法，改变步骤(4)中油料与水的比例，其他步骤一致，结果如表2所示。结果表明，采用本发明的半固态水酶法的1:1可以显著提高清油的率。清油得率是离心得到的非乳化油/菜籽颗粒里的总油质量。
表2固液比对清油得率的影响
Figure PCTCN2015096664-appb-000003
实施例5：菜籽油对高血脂大鼠影响
取正常SD(170±10g)雄性大鼠56只在实验环境下适应7天，然后将大鼠随机分为7组：正常对照组、高脂猪油组、精炼菜籽油组、冷榨菜籽油组、热榨菜籽油组、半固态水酶菜籽油组(使用本发明方法制备得到的菜籽油)和浸出菜籽油组，每组8只，除了正常组给予正常饲料外，高脂猪油组给予高脂饲料(1％胆固醇+0.2胆酸钠+20％猪油+10％蛋黄粉+68.85％基础饲料)，其他5组用10％不同加工方式制得的菜籽油代替10％猪油，其他条件不变。给予高脂饲料12周后，禁食12小时候后，称量大鼠体重。再以水合氯醛动物麻醉后，心脏刺穿取血，离心取的血液上清液，分别以医用试剂盒测定血清中TC、TG、HDL-C、LDL-C等生化指标。取肝脏组织(1㎝×1cm左右大小)置于4％的多聚甲醛固定溶液中进行组织固定，石蜡切片和H/E组织染色的制作，以观察肝脏组织的形态。
(1)对体重、体质比的影响
结果如表3所示，结果表明从表中显示，本发明的半固态水酶法制得的菜籽油能明显抑 制高脂大鼠体重、肝体比的增加。
表3菜籽油对高血脂大鼠体重、体脂比的影响(x±s，n＝8)
Figure PCTCN2015096664-appb-000004
肝体比：肝脏重量/体重
(2)对血液生化指标的影响
表4菜籽油对高脂大鼠血清中TC、TG含量的影响(x±s，n＝8)
Figure PCTCN2015096664-appb-000005
由表4显示与正常组相比，和给予高脂猪油饲料，以及其它给予不同加工方式制取的菜籽油的大鼠相比较，以水酶法组大鼠血清中的TCH和TG水平为最低，其中血清中TCH、TG含量于高脂猪油比较，分别降低达到28％和12％。
表5菜籽油对高脂大鼠血清中LDL-C、HDL-C含量的影响(x±s，n＝8)
Figure PCTCN2015096664-appb-000006
当机体LDL-C(俗称坏胆固醇)含量过高，可沉积于心脑血管壁上，导致动脉粥样硬化。表5表明与正常对照组相比，高脂组的LDL-C增加300％(P<0.05)；以水酶法组大鼠血清中的LDL－C相较于高脂猪油组则降低水平为最低，可达36％。HDL-C水平两者之间差异不明显。其他不同加工方式所得菜籽油组中，浸出组的LDL-C水平最高和HDL-C水平最低。
(3)对病理组织学的影响
大鼠病理组织学观察的显微镜图如附图1-7所示。可知，正常对照组大鼠肝脏细胞呈放射状排列整齐，肝细胞质丰富，界限清晰；与其它方法制备所得菜籽油喂养的动物肝脏组织相比，本水酶法组最为明显接近正常组织形态，即肝细胞保持放射状完整的细胞排列和结构。然而高脂猪油组大鼠肝细胞胞浆内可见大小不等的脂滴空泡。精炼组和正己烷浸出组，肝脏细胞内脂肪液滴较多偏大，细胞间间隙不清楚；冷榨组、热榨组和水酶组相对脂肪液滴较少偏小。
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 (10)

  1. 一种半固态水酶法制备菜籽油的方法，其特征在于，所述方法是：
    (1)将油菜籽清理干净，去除碎屑、瘪籽、杂质；
    (2)对除去杂质的菜籽进行水分调制，使其水分含量为8-10wt％；
    (3)对上一步得到的菜籽进行轧胚，得到菜籽胚片，对菜籽胚片于50℃-70℃进行调制，使其水分含量为3.5-4.5wt％，得到熟胚；
    (4)将熟胚粉碎过筛，得到油料；将油料与水按1:1的质量比例混合得到半固态的糊状物；按总加酶量0.5％～2％w/w添加质量比果胶酶：纤维素酶：碱性蛋白酶＝1:1:1的酶至菜籽糊状物，在40-60℃下搅拌混合1-2h；然后离心，将菜籽糊状物分离得到果渣和毛油；最后将毛油按0.2-0.4L/kg油的加水量加入水，在40-60℃下离心分离得到菜籽清油，最后经过真空干燥得到最终的水酶法菜籽油。
  2. 根据权利要求1所述的方法，其特征在于，所述酶解过程采用梯度控温的方法，40℃保持20-40min，温度在20-40min内从40℃升温到60℃，60℃下保持20-40min。
  3. 根据权利要求1所述的方法，其特征在于，所述粉碎过筛得到粒度40～80目的油料。
  4. 根据权利要求1所述的方法，其特征在于，所述40-60℃下搅拌混合是在10-20rpm下搅拌。
  5. 根据权利要求1所述的方法，其特征在于，所述果渣和毛油的分离是在3500rpm下离心。
  6. 根据权利要求1所述的方法，其特征在于，所述菜籽清油是在6500rpm下离心得到的。
  7. 根据权利要求1所述的方法，其特征在于，所述步骤(4)具体是：
    将熟胚粉碎过筛，得到粒度40～80目的油料；将油料与水按1:1的质量比例混合得到半固态的糊状物；按加酶量0.5％～2％w/w果胶酶：纤维素酶：碱性蛋白酶＝1:1:1添加至菜籽糊状物，在40-60℃下用混合机在10-20rpm下混合1-2h；然后用两相离心机在3500rpm下离心将菜籽糊状物分离得到果渣和毛油；最后将毛油按0.25L/kg油的加水量加入蒸馏水，在40℃下，用立式离心机离心，6500rpm分离得到菜籽清油，最后经过真空干燥得到最终的水酶法菜籽油，无需任何精炼步骤。
  8. 根据权利要求1-7任一所述方法得到的菜籽油。
  9. 权利要求8所述菜籽油在食品或者饲料方面的应用。
  10. 权利要求8所述菜籽油在降低血脂、降低胆固醇、降低甘三酯或者降低体重方面的应用。
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