WO2015176670A1 - 一种酶法制备得到的脂溶性竹叶抗氧化物及其用途 - Google Patents

一种酶法制备得到的脂溶性竹叶抗氧化物及其用途 Download PDF

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WO2015176670A1
WO2015176670A1 PCT/CN2015/079475 CN2015079475W WO2015176670A1 WO 2015176670 A1 WO2015176670 A1 WO 2015176670A1 CN 2015079475 W CN2015079475 W CN 2015079475W WO 2015176670 A1 WO2015176670 A1 WO 2015176670A1
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bamboo leaf
fat
group
antioxidant
soluble
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PCT/CN2015/079475
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English (en)
French (fr)
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张英
刘零怡
金成�
黄骆镰
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杭州凝竹生物科技有限公司
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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
    • C11B5/00—Preserving by using additives, e.g. anti-oxidants
    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides

Definitions

  • the invention relates to the field of food additives, in particular to enzymatically catalyzing the esterification of water-soluble bamboo leaf antioxidants (AOB-w), and the product fat-soluble bamboo leaf antioxidants (eAOB-o) in the food industry It is mainly used as a lipid antioxidant and can simultaneously inhibit the formation of acrylamide (AA), advanced glycation end products (AGEs) and heterocyclic amines (HAAs) associated with Maillard reaction during high temperature processing, especially suitable for High-temperature oils (such as frying oil, baking oil, and release oil) and high-fat foods (such as fried potato products and cookies) that require hot processing.
  • AOB-w water-soluble bamboo leaf antioxidants
  • eAOB-o product fat-soluble bamboo leaf antioxidants
  • AA acrylamide
  • AGEs advanced glycation end products
  • HAAs heterocyclic amines
  • Oxidation of oils and fats is an important factor leading to deterioration of oil quality.
  • the oil is easily affected by light, temperature, oxidation, microorganisms, moisture, etc., and is oxidized to form short-chain aldehydes, ketones, acids, etc., which produce a sulphate, causing oxidative rancidity.
  • the oxidation products of fats and oils have an adverse effect on the flavor, color and composition of edible fats and oils, reducing the nutritional quality of fats and oils and shortening the shelf life.
  • the human body's intake of rancid oil will also destroy the structure and function of cell membranes, enzymes and proteins, leading to cardiovascular disease, accelerated body aging and even cancer, which seriously endangers human health.
  • antioxidants directly to fats and oils is the most effective way to prevent or delay the oxidative deterioration of oils and prolong the shelf life of foods.
  • Synthetic food antioxidants although good in anti-oxidation effect and low in price, have been widely used in the food industry, but they all have certain safety hazards, and their use has been gradually limited.
  • BHA butylated hydroxyanisole
  • the United States has removed it from the GRAS (Generally Recognized Safety) list. In Japan, BHA is only approved for palm oil and Palm kernel oil.
  • BHT 2,6-di-tert-butyl-4-methylphenol
  • TBHQ tert-butyl hydroquinone
  • RE Natural antioxidants
  • RE show good results in antioxidants, but they are limited in fat solubility, expensive, and have a special odor, which limits their oil to a certain extent. Large-scale applications in industry.
  • Natural antioxidants are mainly derived from plants, mostly polyhydroxy substances, which have good hydrophilicity and weak lipophilicity, which cannot be fully mixed with fats and oils, thereby affecting their antioxidant effects.
  • Molecular modification of natural antioxidants is an important way to improve their fat solubility.
  • the means of esterification include chemical methods and enzymatic methods.
  • the antioxidants obtained by chemical modification include vitamin C palmitate (AP), fat-soluble tea polyphenol (OTP) and fat-soluble bamboo leaf antioxidant (cAOB-o), which are vitamin C and tea, respectively.
  • the esterification product of phenol, bamboo leaf antioxidant (AOB-w) and fatty acid can be added to fats and oils or foods as a fat-soluble antioxidant and/or a nutrient enhancer.
  • Structural modification of natural antioxidants by chemical methods can satisfy the need for fat solubility to some extent, as disclosed by the inventors in the prior art for the preparation of fat-soluble bamboo leaf antioxidants by oxyacylation (CN 103005007 A).
  • the chemical acylation selectivity is low, the reaction conditions are severe, the process is difficult to control, and often accompanied by the formation of a large amount of by-products, which brings difficulties to the subsequent separation and purification work, and the production process generates a large amount of waste liquid. Improper handling can pollute the environment. Enzymatic acylation can achieve directional modification of the substrate, and the reaction conditions are mild and the by-products are small.
  • CN 102747117A discloses a method for synthesizing fat-soluble tea polyphenol by enzymatic method, using organic solvent as a reaction medium, using lipase to catalyze the synthesis of fat-soluble tea polyphenols from tea polyphenols and fatty acid vinyl esters, and other chemical synthesis methods (CN) 101255151 has certain advantages compared to CN 101270108).
  • CN 102787146A discloses a microwave-assisted lipase-catalyzed synthesis of modified epigallocatechin gallate (EGCG). This method can significantly accelerate the reaction rate and increase the reaction yield and shorten on the basis of CN 102747117A. Reaction time.
  • CN 103131741A discloses a biological modification method of active plant polyphenols, which comprises reacting monomeric compounds of plant polyphenols with C2-C18 fatty acids or fatty acid vinyl esters under the catalysis of immobilized lipase using an organic solvent as a medium. A fatty acid ester of a plant polyphenol is obtained.
  • antioxidant activity is the key.
  • the proportion of active phenolic hydroxyl groups in natural phenolic compounds is inevitably lowered due to the incorporation of fatty acids, and the antioxidant activity (such as DPPH ⁇ clearing activity, etc.) depending on the number of active phenolic hydroxyl groups is inevitable. The ground will be lowered.
  • the antioxidant activity of antioxidants in lipid matrix should be a comprehensive characterization of effective phenolic hydroxyl, fat solubility, substrate dispersion and other factors. Therefore, an antioxidant evaluation system close to real lipid matrix is selected for screening. High performance fat soluble antioxidants are critical.
  • the ionic liquid has the characteristics of non-combustible, good thermal stability, wide range of soluble substances, and strong designability. It is gradually becoming a green, environmentally friendly new solvent in the food industry, and has broad application prospects.
  • the solvent-free system can effectively solve the solvent residue problem and simplify the separation and purification steps, which has obvious advantages. However, since the solvent-free system requires the substrate to be in a liquid state, its wide application is limited to some extent.
  • Maillard reaction products include not only coloring substances and antioxidant substances, but also some associated harmful substances that affect human health. Since these Maillard-associated hazards are not artificially added, but are produced by components of food raw materials (such as amino acids, reducing sugars) during thermal processing, they are called "endogenous chemical contaminants.” Typical Maillard-associated hazards include Acrylamide (AA), Heterocyclic Aromatic Amines (HAAs), Nitrosamine, Polycyclic Aromatic Hydrocarbons, and Advanced Protein Glycosylation Advanced Glycation End-products (AGEs), etc. In recent years, with the deepening of the understanding of Maillard's reaction, the research on these associated hazards has gradually become a hot spot.
  • AA Acrylamide
  • HAAs Heterocyclic Aromatic Amines
  • AGEs Advanced Protein Glycosylation Advanced Glycation End-products
  • Acrylamide is a food rich in carbohydrates and amino acids produced by Maillard reaction under high temperature conditions. Fried foods are high-exposure areas, especially fried potato products.
  • the acrylamide production pathway of fried foods mainly includes the asparagine pathway and the acrolein pathway. Asparagine and reducing sugar form during Maillard reaction Acrylamide (ie, asparagine pathway) is the main pathway for the production of acrylamide, contributing about 70% to 80% of the total amount of acrylamide in fried potato products.
  • the reaction mainly occurs in the aqueous phase; Oxidative decomposition produces acrolein, which in turn forms acrylamide (ie, acrolein pathway), which accounts for about 20% to 25% of the total amount of acrylamide produced in the fried potato product.
  • the reaction mainly occurs in the oil phase.
  • Naturally derived phenolic compounds such as tea polyphenols, apple polyphenols, and bamboo leaf antioxidants, all exhibit good acrylamide inhibitory activity, mainly through the asparagine pathway
  • Advanced glycation end products refer to the free amino groups of macromolecules such as proteins, amino acids, lipids or nucleic acids and the aldehyde groups of reducing sugars (glucose, fructose, pentose, etc.) under non-enzymatic conditions.
  • AGEs there are more than 20 kinds of AGEs, and representative compounds include carboxymethyl lysine [N ⁇ - (carboxymethyl)-lysine, CML], carboxyethyl lysine [N ⁇ - (carboxyethyl)-lysine, CEL], pentosidine, pyrraline, crosslines, imidazolysine, and versperlysine.
  • AGEs are cross-linkable, and their amino groups and carboxyl groups can be stably and prolongedly combined with macromolecules such as proteins by covalent bonds to form a crosslinked structure with a very large molecular weight.
  • the crosslinked structure is stable to the enzyme, is not easily degraded, and is irreversible.
  • the formation and accumulation of AGEs in the body is closely related to many diseases, especially the complications of diabetes.
  • Heterocyclic amines are mutagenic, carcinogenic substances formed during high-temperature cooking of protein-rich foods. Barbecue-like meat products are known to be highly exposed foods of HAAs.
  • HAAs compounds can be divided into two major classes: Amino imidazo azaren (AIA) and Amino-carboline congener, which can be subdivided into ⁇ -porphyrins. ⁇ -porphyrin, ⁇ -carboline and ⁇ -carboline heterocyclic amine.
  • the ⁇ -carboline heterocyclic amine mainly contains Harman and Norharman.
  • Norharman and Harman In addition to the typical barbecue meat products, the researchers also detected high levels of Norharman and Harman, especially coffee, in coffee, chocolate, soy sauce and other food systems. It has been reported that the Norharman content in coffee can be as high as 9.34 ⁇ g / g, Harman content Up to 1.67 ⁇ g/g, indicating that drinking coffee and its products are one of the main ways for humans to consume ⁇ -carboline heterocyclic amines. However, there have been no reports on the detection and inhibition of Norharman and Harman in potato products. Norharman and Harman are non-polar heterocyclic amines and are not themselves mutagenic. However, animal experiments have shown that they can bind to different sites in the brain and liver tissues of mice, thereby affecting the physiological behavior of animals.
  • Norharman and Harman can enhance the genotoxicity of other heterocyclic amines; when combined with non-mutagenic compounds such as aniline or toluidine, they can produce processogenic activity and form DNA adducts; Organize and inhibit some key enzymes.
  • Other studies have shown that Norharman and Harman may be associated with some human diseases, such as Parkinson's disease, cancer and muscle pain syndrome.
  • Antioxidant of bamboo leaves is a natural, safe and cost-effective food antioxidant created by the inventor. In 2004, it was listed as "Food Additive Sanitation in the People's Republic of China”.
  • Standards (GB 2760), currently approved applications include: basically non-aqueous fats and oils, cooked nuts and seeds, fried noodles, ready-to-eat cereals, baked goods, cured meat products, marinated meat products Class, (smoked, roasted, roasted) meat, fried meat, Western ham, meat enema, fermented meat products, aquatic products and products, fruit and vegetable juice (meat) drinks, tea drinks and puffed foods, etc.
  • Six categories (GB 2760-2014).
  • Naturally derived water-soluble bamboo leaf antioxidant is a medium to large polarity composition with strong hydrophilicity and weak lipophilicity, which greatly limits its oil industry and high oil foods.
  • AOB-w inhibits the formation of acrylamide in hot processed foods mainly through the asparagine pathway, and its inhibition of the acrolein pathway is not obvious.
  • Fried foods such as potato chips, French fries, instant noodles, fried chicken, fried dough sticks, twists, etc.
  • high-fat baked foods such as cookies, egg yolks, etc.
  • Maillard reactions acrylamide and advanced sugar
  • a fat-soluble bamboo leaf antioxidant comprising the following characteristic components: p-coumaric acid, and one or more a product of an acyl modification of a 5,7-dihydroxy-4H-1-benzopyran-4-one derivative; wherein the 5,7-dihydroxy-4H-1-benzopyran-4- a ketone derivative is a substituted 5,7-dihydroxy-4H-1-benzopyran-4-one;
  • the substitution means that the benzopyran ring is substituted by the following: any one hydrogen atom on the benzene ring portion of the benzopyran ring is substituted by one glyco group, and the 2-position of the benzopyran ring is 1-3 Phenyl substitution of a hydroxy substituent;
  • acyl modification means that the hydroxyl group on the glycosyl group is modified by a C8-C18 acyl modification group; and the 5,7-dihydroxy-4H-1-benzopyran-4-one derivative
  • the molar ratio to the acyl modifying group is 1:0.5 to 2.
  • the molar ratio of the 5,7-dihydroxy-4H-1-benzopyran-4-one derivative to the acyl modification group is from 1:0.8 to 1.2.
  • the lipophilic bamboo leaf antioxidant has a p-coumaric acid content of ⁇ 0.5%, preferably 0.51% to 0.80%.
  • the fat-soluble bamboo leaf antioxidant has a total phenolic content of ⁇ 20%, preferably 20.1% to 28.0%.
  • the lipophilic bamboo leaf antioxidant has an ethyl acetate solubility of ⁇ 3 g / 100 mL, preferably 3.5 g / 100 mL - 6.3 g / 100 mL (with fat-soluble bamboo leaf antioxidants in The mass of dissolution in 100 mL of ethyl acetate was calculated).
  • the fat-soluble bamboo leaf antioxidant is a pale yellow to yellowish brown solid.
  • the fat-soluble bamboo leaf antioxidant is a pale yellow to yellowish brown powder or granule. Or flake solid.
  • the fat-soluble bamboo leaf antioxidant has the property of inhibiting the formation of acrylamide (AA) and/or advanced glycation end products (AGEs) during high temperature processing.
  • the advanced glycation end product is carboxymethyl lysine (CML) and/or carboxyethyl lysine (CEL).
  • the fat-soluble bamboo leaf antioxidant has an inhibition rate of acrylamide formation of ⁇ 20%, preferably ⁇ 25%, more preferably ⁇ 27%.
  • the fat-soluble bamboo leaf antioxidant has an inhibition rate of acrylamide to acrylamide to ⁇ 50%, preferably ⁇ 70%, more preferably ⁇ 80%.
  • the fat-soluble bamboo leaf antioxidant can completely inhibit the acrylamide formed by the acrolein pathway.
  • the product of the 5,7-dihydroxy-4H-1-benzopyran-4-one derivative modified with an acyl group is selected from the group consisting of A.1, A.2, A. 3. A.4, B.1, B.2, B.3, B.4, C.1, C.2, C.3, C.4;
  • R group shown is a C8-C18 acyl group.
  • the C8-C18 acyl group refers to a group formed by decarboxylation of a C8-C18 fatty acid.
  • the fatty acid is selected from the group consisting of caprylic acid, capric acid, and lauric acid. , myristic acid, brown Palmitic acid, oleic acid, linoleic acid, stearic acid, or a combination thereof.
  • the fat-soluble bamboo leaf antioxidant is prepared by the following method:
  • the water-soluble bamboo leaf antioxidant (AOB-w) is reacted with the C8-C18 fatty acid to carry out monohydroxy acylation to obtain a fat-soluble bamboo leaf antioxidant;
  • the method comprises the steps of: acylating the water-soluble bamboo leaf antioxidant with the C8-C18 fatty acid at 40 ° C to 80 ° C under enzyme catalysis to obtain a fat-soluble bamboo leaf antioxidant.
  • a method for preparing a fat-soluble bamboo leaf antioxidant comprising the steps of: reacting a water-soluble bamboo leaf antioxidant with a C8-C18 fatty acid under an enzyme catalysis to carry out a monohydroxyl group; To obtain a fat-soluble bamboo leaf antioxidant;
  • the method comprises the steps of: acylating the water-soluble bamboo leaf antioxidant with the C8-C18 fatty acid at 40 ° C to 80 ° C under enzyme catalysis to obtain a fat-soluble bamboo leaf antioxidant.
  • the fat-soluble bamboo leaf antioxidant is a fat-soluble bamboo leaf antioxidant as described in the first aspect of the invention.
  • reaction time of the step is from 4 h to 48 h; preferably from 6 h to 24 h.
  • the mass ratio of the water-soluble bamboo leaf antioxidant to the fatty acid is 1:1 to 30; preferably 1:5 to 15.
  • the yield of the method is from 40 to 100%, preferably from 50 to 100%, more preferably from 60 to 100%.
  • the method further comprises the step of concentrating the extract prior to performing the extraction.
  • the step is carried out in the absence of a solvent, or the step is carried out in an organic solvent or an ionic solvent;
  • the organic solvent is selected from the group consisting of tert-amyl alcohol (preferably water-saturated tert-amyl alcohol), acetone, ethyl acetate, tert-butanol, or a combination thereof;
  • the ionic solvent is a salt composed of a cation and an anion, wherein the cation is an imidazole which is unsubstituted or substituted by a C1-C8 alkyl group, and the anion is selected from the group consisting of BF 4 - , PF 6 - , TFSI - , or a combination thereof.
  • the ratio of the AOB-w to the organic solvent is 1 g: 25 to 250 mL.
  • the enzyme is selected from the group consisting of Candida antarctica lipase, subtilisin, Rhizomucor miehei lipase, Pseudomonas lipase, and Thermomyces lanuginosus An enzyme, or a combination thereof; preferably a Candida antarctica lipase;
  • the C8-C18 fatty acid is selected from the group consisting of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, stearic acid, or a combination thereof.
  • the fatty acid is a C8-C18 fatty acid.
  • the fatty acid is a C8-C12 fatty acid.
  • the weight ratio of the AOB-w to the enzyme is from 1:0.5 to 5; preferably from 1:1-4.
  • a food additive comprising the fat-soluble bamboo leaf antioxidant according to the first aspect of the invention, and optionally a food acceptable carrier.
  • the food additive is a food additive formed by dissolving a fat-soluble bamboo leaf antioxidant as described in the first aspect of the invention in a medium long-chain fatty acid.
  • the food additive is a medium long-chain fatty acid ester solution containing a mass fraction of 1% to 20% of a fat-soluble bamboo leaf antioxidant; wherein the medium-long chain fatty acid is selected from the group consisting of Group: glyceryl octanoate, glyceryl laurate, glyceryl myristate, glyceryl palmitate, glyceryl stearate, glyceryl oleate, glycerol linoleate, or a combination thereof.
  • the fat-soluble bamboo leaf antioxidant is used to inhibit the formation of acrylamide, carboxymethyl lysine, and/or carboxyethyl lysine during hot processing of food.
  • the fat-soluble bamboo leaf antioxidant is used to inhibit the formation of acrylamide, and/or heterocyclic amines during hot processing of food.
  • the antioxidant refers to a decrease in one or more indicators selected from the group consisting of: peroxide value, anisidine value, thiobarbituric acid value, acid value, polar compound Content, free fatty acid content, malondialdehyde content.
  • a food processing method comprising the steps of: adding a fat-soluble bamboo leaf anti-antibody according to the first aspect of the invention to a food material before, during or after the processing An oxide, or a food additive as described in the third aspect of the invention.
  • the method comprises the steps of: dissolving the fat-soluble bamboo leaf antioxidant or food additive in a fat or oil for processing a food; preferably, the oil is selected from the group consisting of : frying oil, baking oil, release oil, or a combination thereof.
  • the fat-soluble bamboo leaf antioxidant or food additive is added in an amount of 0.01 to 0.05% by weight based on the mass of the oil-soluble bamboo leaf antioxidant.
  • the food additive is a food additive formed by dissolving a fat-soluble bamboo leaf antioxidant as described in the first aspect of the invention in a medium long-chain fatty acid.
  • the food processing method further comprises: adding a water-soluble bamboo leaf antioxidant (AOB-w) before, during or after the processing.
  • AOB-w water-soluble bamboo leaf antioxidant
  • the food processing method comprises: adding a fat-soluble bamboo leaf antioxidant according to the first aspect of the invention to a fat or oil, or adding a food additive according to the third aspect of the invention;
  • the food material is treated with water-soluble bamboo leaf antioxidant.
  • a food product which, during processing, is added with a fat-soluble bamboo leaf antioxidant according to the first aspect of the invention or a fat according to the first aspect of the invention Food component of soluble bamboo leaf antioxidants.
  • the fat-soluble bamboo leaf antioxidant inhibits acrylamide formation in the hot processed food.
  • the fat-soluble bamboo leaf antioxidant according to the first aspect of the invention is preferably added in an amount of from 100 to 500 ppm, preferably from 100 to 300 ppm, in the fat or oil.
  • the acrylamide inhibition rate is ⁇ 20%, preferably 25% to 30%, in the food, and is converted into acrylamide formed by the oxidation pathway of the oil (ie, the acrolein pathway).
  • the inhibition rate is 60% or more, more preferably 85% to 95%.
  • the food product is selected from the group consisting of fried foods, baked foods, puffed foods, edible oils, and food processing oils.
  • the content of acrylamide in the food product is less than 200 ⁇ g/kg.
  • the fried food when the fried food is French fries, the fried food has an acrylamide content of less than 200 ⁇ g/kg.
  • the fried food when the fried food is a traditional Chinese food fritter, the fried food has an acrylamide content of less than 30 ⁇ g/kg.
  • the content of the fat-soluble bamboo leaf antioxidant according to the first aspect of the invention is from 10 to 150 ppm.
  • Figure 1 UPLC detection of bamboo leaf glucoside flavonoids
  • Figure 2 is a mass spectrometric detection of bamboo leaf glucoside flavonoids
  • Figure 3 is a schematic view showing the structure of bamboo leaf glucoside flavonoids
  • Figure 5 Inhibition of acrylamide (AA) formation in cookies by eAOB-o and OTP;
  • Figure 13 A combination of AOB-w and eAOB-o for the inhibition of acrylamide formation in fritters and French fries;
  • Figure 14 AOB-w combined with eAOB-o for the inhibition of acrylamide and ⁇ -carboline heterocyclic amines in fried compound potato chips.
  • the present inventors have unexpectedly discovered that the water-soluble bamboo leaf antioxidant is modified by an enzymatic method, and the acylation selectively occurs on the sugar hydroxyl group of the bamboo leaf carbon flavonoid, while the benzene ring is on the benzene ring.
  • the phenolic hydroxyl group does not undergo acylation, thereby improving the fat solubility of the product while maintaining good antioxidant performance, and simultaneously inhibiting acrylamide (especially the acrylamide produced by the acrolein pathway) during the hot processing of food. produce.
  • the inventors completed the present invention.
  • enzyme-catalyzed acylation a fat-soluble bamboo leaf anti-defect prepared using the method of the invention (ie, enzyme-catalyzed acylation). Oxide.
  • water-soluble bamboo leaf antioxidant is used interchangeably with "AOB-w”, both of which are fat-soluble bamboo leaf antioxidants prepared by the method of the invention (ie, enzyme-catalyzed acylation). Substrate.
  • a preferred water-soluble bamboo leaf antioxidant has the following characteristics: a yellow-brown powder containing 46.53% of total phenol, 2.35% of isophorin and 5.87% of water.
  • C8-C18 fatty acid refers to a carboxylic acid having 8 to 18 carbon atoms such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, stearic acid, or the like. acid.
  • acyl group of C8-C18 refers to a group formed by the removal of an -OH group of a carboxylic acid having 8 to 18 carbon atoms, in the form of Shown wherein R is a C7-C17 aliphatic chain.
  • glycosyl refers to a group formed by the loss of a hydrogen atom from any one of the hydroxyl groups (-OH) of glucose.
  • the present invention provides an enzymatically modified fat-soluble bamboo leaf antioxidant (eAOB-o).
  • the fat-soluble bamboo leaf antioxidant comprises the following characteristic components: p-coumaric acid, and one or more 5,7-dihydroxy-4H-1-benzopyran-4-one derivatives are modified with an acyl group a product of the 5,7-dihydroxy-4H-1-benzopyran-4-one derivative substituted with 5,7-dihydroxy-4H-1-benzopyran-4- a ketone, wherein the substitution means that the benzopyran ring is substituted by: any one of the hydrogen atoms on the benzene ring portion of the benzopyran ring is substituted by a monosaccharide group, and the two positions of the benzopyran ring are 1-3 hydroxy substituted phenyl substitutions;
  • acylation modification means that the sugar hydroxyl group of the flavonoid carbon glycoside is modified by a C8-C18 fatty acid donor; and the 5,7-dihydroxy-4H-1-benzopyran-4-one
  • the molar ratio of the derivative to the acyl donor is from 1:0.5 to 2, preferably from 1:0.8 to 1.2.
  • the 5,7-dihydroxy-4H-1-benzopyran-4-one derivative comprises two or more of the following structural compounds:
  • the eAOB-o provided by the present invention has a total phenolic content of ⁇ 20%, preferably 20.1% to 28.0%.
  • the eAOB-o provided by the present invention has a p-coumaric acid content of ⁇ 0.5%, preferably 0.51% to 0.8%.
  • the fat-soluble bamboo leaf antioxidant has a pale yellow to yellowish brown powder, granules or flakes with a slight ester taste.
  • the fat-soluble bamboo leaf antioxidant can be directly dissolved in edible fats and oils, medium long-chain fatty acids and non-polar solvents.
  • the lipophilic bamboo leaf antioxidant has an ethyl acetate solubility of ⁇ 3 g / 100 mL, preferably 3.5 g / 100 mL - 6.3 g / 100 mL.
  • the fat-soluble bamboo leaf antioxidant of the invention has significantly superior to the commercially available oil antioxidant products (rosemary extract RE, fat-soluble tea polyphenol OTP, hydroxyanisole BHA, 2,6-di-tert-butyl pair)
  • the oxidative and thermal stability of cresol BHT, etc. is comparable to the synthetic antioxidant tert-butyl hydroquinone (TBHQ).
  • TBHQ synthetic antioxidant tert-butyl hydroquinone
  • the fat-soluble bamboo leaf antioxidant of the present invention can effectively improve the oxidation stability and frying stability of the oil, and the oil of the fat-soluble bamboo leaf antioxidant of the present invention is added to the protective factor (PF).
  • Indicator values such as peroxide value (PV), thiobarbituric acid value (TBARS), acid value (AV), anisidine value (p-AnV), free fatty acid (FFA) and polar compound (PC) content It has a large improvement and the improvement effect is better than the existing oil antioxidant.
  • the fat-soluble bamboo leaf antioxidant of the invention can simultaneously inhibit the formation of the acrylamide (AA) and advanced glycation end products (AGEs) associated with the Maillard reaction in the high temperature processing, and is particularly suitable for high temperature oil. (such as frying oil, baking oil, release oil, etc.) and high-fat foods (such as fried potato products and cookies) that require hot processing.
  • the fat-soluble bamboo leaf antioxidant has an inhibition rate of acrylamide formation of the finished product of ⁇ 20%, preferably ⁇ 25%, more preferably ⁇ 27%.
  • the fat-soluble bamboo leaf antioxidant of the present invention can also simultaneously inhibit the formation of AA and heterocyclic amines (Harman and Norharman) during high temperature processing.
  • the liposoluble bamboo leaf antioxidant has an inhibition ratio of acrylamide of 75.86%, a Harman inhibition rate of 45.70%, and a Norharman inhibition rate of 35.36%.
  • the fat-soluble bamboo leaf antioxidant of the present invention can particularly effectively block acrylamide (i.e., acrylamide produced by the acrolein pathway) produced by the oil oxidation process.
  • acrylamide pathway In hot processed foods, there are two main routes of formation of acrylamide: the "asparagine pathway” and the “acrolein pathway”.
  • the amine ie asparagine pathway
  • acrylamide pathway is the main route of acrylamide production (about 70% to 80% of the total amount of acrylamide in the final product)
  • the reaction mainly occurs in the aqueous phase; the oil is oxidatively decomposed at high temperature to produce acrolein.
  • acrylamide i.e., acrolein pathway
  • acrolein pathway is formed, which contributes about 20 to 30% of the total amount of acrylamide in the product, and the reaction mainly occurs in the oil phase.
  • the fat-soluble bamboo leaf antioxidant eAOB-o of the present invention has a good inhibitory effect on acrylamide produced in the oil phase, in some preferred embodiments of the present invention, the fat-soluble bamboo leaf antioxidant pair
  • the inhibition rate of the acrolein pathway to acrylamide is ⁇ 50%, preferably ⁇ 70%, more preferably ⁇ 80%. In a particularly preferred embodiment, the inhibition rate (acrylamide produced by the acrolein route) can reach nearly 100%.
  • the present invention also provides a method for enzymatically preparing eAOB-o.
  • the method adopts enzymatic catalysis, and directly esterifies AOB-w with a fatty acid in an organic solvent, an ionic solvent as a medium or a solventless reaction system.
  • the preparation process can be used with little or no organic solvent, green, environmentally friendly and environmentally friendly.
  • the method includes the steps of:
  • the water-soluble bamboo leaf antioxidant is reacted with the C8-C18 fatty acid to carry out monohydroxyacylation to obtain a fat-soluble bamboo leaf antioxidant;
  • the method comprises the steps of: acylating the water-soluble bamboo leaf antioxidant with the C8-C18 fatty acid at 40 ° C to 80 ° C under enzyme catalysis to obtain a fat-soluble bamboo leaf antioxidant.
  • reaction time of the step is from 4 h to 48 h.
  • the mass ratio of the water-soluble bamboo leaf extract to the fatty acid in the step is 1:1 to 30.
  • the yield of the process is relatively high and can be from 40% to 100%, preferably from 50% to 100%, more preferably from 60% to 100%.
  • Step 1 In the solventless system, organic solvent system or ionic solvent system, under the catalysis of enzyme, AOB-w and fatty acid are acylated at 40 °C ⁇ 80 °C for 4h ⁇ 48h (mass ratio of AOB-w to fatty acid) It is 1:1 ⁇ 30).
  • Step 2 After the completion of the above reaction, the reaction mixture was filtered and concentrated to give a concentrate (cr.).
  • Step 3 The concentrate obtained above was extracted with acetonitrile-n-heptane solution (2:5, v/v), and the lower phase was concentrated and extracted with ethyl acetate-water (3:5, v/v).
  • the concentrate is concentrated and dried to obtain eAOB-o;
  • the concentrate obtained in step 2) can also be extracted with ethyl acetate-water (3:5, v/v), and the upper phase is concentrated.
  • the extract was extracted with 88% to 92% aqueous ethanol solution - petroleum ether (2:5, v/v), and the phase was concentrated and dried to obtain the target eAOB-o.
  • any one of C8-C18 fatty acids namely caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid (linoleic acid) or stearic acid, may be used.
  • the fatty acid has a chain length of from C8 to C12.
  • the organic solvent used in the reaction system may be tert-amyl alcohol (water-saturated tert-amyl alcohol), acetone, ethyl acetate, tert-butanol; the ionic solvent is a hydrophobic ionic liquid, and the cation is a substituent chain length of not more than 8 carbons.
  • the imidazole of the atom, the anion is BF 4 - , PF 6 - or TFSI - .
  • the catalytic enzyme in the present invention may be selected from Candida antarctica lipase, subtilisin, Rhizomucor miehei lipase, Pseudomonas lipase, Thermomyces lanuginosa lipase or the like. a mixture of enzyme preparations; preferably Candida antarctica lipase.
  • the organic solvent described in the step 1) is tert-amyl alcohol or water-saturated tert-amyl alcohol, acetone, ethyl acetate, tert-butanol, etc., the ratio of the substrate AOB-w to the organic solvent is 1g: 25 ⁇ 250mL;
  • the ionic solvent described in the step 1) is a hydrophobic ionic liquid
  • the cation is an imidazole having a substituent chain length of not more than 8 carbon atoms
  • the anion is BF 4 - , PF 6 - or TFSI - .
  • the process parameters of the present invention are preferably as follows:
  • AOB-w Water-soluble bamboo leaf antioxidant
  • the eAOB-o provided by the invention can be directly dissolved in edible oils and fats and/or medium and long-chain fatty acids, and is insoluble or insoluble in water.
  • the edible fats and oils include (but are not limited to): palm oil, peanut oil, corn oil, rapeseed oil, lard, butter, butter, fish oil, camellia oil, pecan oil, linseed oil, olive oil , sunflower oil, perilla oil, etc.
  • the recommended addition amount is 0.01% to 0.05% of the oil content.
  • eAOB-o can also be added to medium and long-chain fatty acid solutions (such as glyceryl octanoate, glyceryl laurate, glyceryl myristate, palmitate, in a certain proportion (mass fraction 1% to 20%).
  • medium and long-chain fatty acid solutions such as glyceryl octanoate, glyceryl laurate, glyceryl myristate, palmitate, in a certain proportion (mass fraction 1% to 20%).
  • glyceryl stearate, glyceryl oleate, glyceryl linoleate, etc. it is used as a liquid preparation.
  • the invention has the advantages that the eAOB-o of the invention can be directly dissolved in edible oils, medium and long-chain fatty acids and non-polar solvents, exhibiting good anti-lipid peroxidation performance and heat stability.
  • Sex The effect of eAOB-o on the antioxidant stability of palm oil at 130 °C was studied by Rancimat. The results showed that the addition of 0.03% eAOB-o could increase the service life of palm oil by 1.83 times, which was significantly better than that of chemistry.
  • a lipophilic bamboo leaf antioxidant (cAOB-o) prepared by acylation.
  • the frying oil stability test proves that the anti-oxidation performance and thermal stability of eAOB-o at 180 °C are significantly better than the similar products (RE, OTP, BHA and BHT) and cAOB-o.
  • the most widely used synthetic antioxidant, tert-butyl hydroquinone (TBHQ) is comparable.
  • TBHQ tert-butyl hydroquinone
  • adding the eAOB-o of the present invention to the cookie formulation in a certain ratio, while prolonging the shelf life of the product, can significantly reduce acrylamide (AA) and AGEs (carboxymethyl lysine CML and carboxylate) in the product.
  • the content of ethyl lysine CEL); adding the eAOB-o of the present invention to the frying oil of the fried potato chips in a certain ratio can significantly reduce the acrylamide (AA) in the product while prolonging the shelf life of the product.
  • the content of heterocyclic amines (Harman and Norharman); exhibits the dual effect of anti-lipid oxidation and simultaneous inhibition of multiple Maillard-related associated hazards.
  • the eAOB-o provided by the invention has good anti-lipid peroxidation performance and thermal stability, and can be directly dissolved in edible oils, medium-long-chain fatty acids and non-polar solvents, and adopts an oil oxidation stability tester.
  • Rancimat studied the effect of eAOB-o on the antioxidant stability of palm oil at 130 °C. The results showed that the addition of 0.03% eAOB-o could increase the service life of palm oil by 1.83 times, and the fat-soluble bamboo prepared by chemical acylation method.
  • Leaf antioxidants (cAOB-o) increased significantly.
  • the frying oil stability test proves that the anti-oxidation performance and thermal stability of eAOB-o at 180 °C are significantly better than the similar products (RE, OTP, BHA and BHT) and cAOB-o.
  • the most widely used synthetic antioxidant, tert-butyl hydroquinone (TBHQ) is comparable.
  • the eAOB-o provided by the present invention has excellent lipophilic properties and has good solubility in fats and oils, so that it can be directly added to oils and used as a lipid antioxidant.
  • the eAOB-o of the present invention can also inhibit the formation of acrylamide caused by oxidation of oil during high-temperature processing, while significantly reducing the content of AGEs and heterocyclic amines in high oil, high sugar, high protein foods.
  • the enzymatic catalysis of the present invention can realize the targeted esterification of the antioxidant active ingredient in AOB-w, and the characteristic component is bamboo leaf carbon flavonoids (valalin, isohumulosin, vitexin, and isophora)
  • the position of fatty acid linkage mainly occurs on the sugar hydroxyl group, and the number of effective phenolic hydroxyl groups which have a greater influence on the antioxidant activity is not reduced, thereby increasing the fat solubility of eAOB-o without significantly reducing the resistance thereof. Oxidative activity.
  • the enzymatic catalysis of the present invention has an unparalleled advantage in the preparation process, product performance, and environmental friendliness.
  • the eAOB-o of the present invention has both outstanding oxidation resistance, thermal stability, and the ability to simultaneously suppress Maillard reaction to form a variety of associated hazards, which are in the food industry (especially those that require high temperature treatment such as frying) Food, baked goods, etc. have extremely broad applications. In addition to producing safe, high-quality, long-life assured food, it can significantly extend the life of frying oil and reduce production costs.
  • AOB-w was purchased from Hangzhou Youmet Technology Co., Ltd., and its appearance was yellow-brown powder, containing 56.53% of total phenol, 3.78% of isophoraside and 5.87% of water.
  • AOB-w was purchased from Hangzhou Youmet Technology Co., Ltd., and its appearance was yellow-brown powder, containing 56.53% of total phenol, 3.78% of isophoraside and 5.87% of water.
  • CAB Candida antarctic lipase
  • the average molecular weight of AOB-w is calculated from its eight characteristic components.
  • the invention selects the content of chlorogenic acid, caffeic acid, isohumuloside, valerin, p-coumaric acid, vitexin, isovite, and ferulic acid in AOB-w to be 0.73%, 0.40%, 3.78, respectively. %, 1.08%, 0.79%, 1.40%, 0.35% and 0.07%, the average relative molecular weight of AOB-w was calculated to be 410.7. The same below
  • the upper liquid was collected and concentrated to dryness to obtain 1.2 g of the target eAOB-o after purification, which was a pale yellow powder, the total phenol content was 20.45%, the p-coumaric acid content was 0.55%, and the ethyl acetate solubility was 4.82 g/ 100g.
  • the upper liquid was collected and concentrated to dryness to obtain 1.6 g of the target eAOB-o after refining, which was a pale yellow powder, the total phenol content was 20.13%, the p-coumaric acid content was 0.51%, and the ethyl acetate solubility was 5.70 g/ 100g.
  • AOB-w 1g as the reaction substrate, add 25g of lauric acid, stir evenly at 60°C, and completely dissolve AOB-w in 5mL 1-ethyl-3-methylimidazolium hexafluorophosphate [Emim][PF 6 ]; Adding 2.1 g (Novozym 435, purchased from Novozymes, with a viability of about 10000 PLU/g) of Candida antarctica, and catalyzing the reaction at 65 ° C for 6 h. After completion of the reaction, the mixture was filtered and concentrated under reduced pressure to give a crude eAOB-o (1.2 g).
  • the crude product was extracted twice with 100 mL of acetonitrile-n-heptane (2:5, v/v) solution, and the lower phase was collected, and the solvent was recovered. After extracting twice with 100 mL of ethyl acetate-water solution (3:5, v/v), the upper layer liquid was collected, and concentrated to dryness to give 0.8 g of the target eAOB-o as a yellow powder with a total phenol content of 20.13%.
  • the p-coumaric acid content was 0.56%, and the ethyl acetate solubility was 4.67 g/100 g.
  • the crude product was dissolved in acetonitrile / n-heptane (2:5, v / v) and extracted twice at 60 ° C.
  • the acetonitrile layer was collected and concentrated to dryness ethyl acetate / n-heptane (3:5, v/v
  • the extract was extracted twice, and the ethyl acetate layer was collected and combined, and concentrated to dryness to give an acylated product.
  • the acylation products were identified and separated and characterized by ultra-high performance liquid chromatography (UPLC), semi-preparative high performance liquid chromatography (Semi-Pre HPLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR). The identification results are shown in Figures 1 and 2.
  • Figure 1 shows the UPLC detection spectrum of four kinds of bamboo leaf carboflavonol laurate after 72h reaction. It can be seen that under the catalysis of immobilized enzyme CALB, after 72h reaction, the bamboo leaf carbon flavonoid laurate Unreacted bamboo leaf carbon glycoside remains.
  • Figure 2 is a mass spectrometric detection of bamboo leaf carboflavonol laurate.
  • the relative molecular weights of isovilin, vitexin, isoorientin and valerin were 432.38, 432.38, 448.37 and 448.37, respectively.
  • the mass spectrometric detection of the bamboo leaf carboflavonol laurate prepared by four enzymatic methods can be seen (Fig.
  • Vitexin laurate 1 H (CDC1 3 ): ⁇ (ppm) 7.19 (s, 2H, H2', H6'), 6.69 (s, 1H, H3', H5') 6.14-6.38 (d, 2H, H3), 6.07 (s, 1H, H6), 4.96 (s, 1H, H1"), 4.33 (s, 1H, H6a"), 4.28 (s, 1H, H5"), 4.09 (s, 1H, H6b” ), 3.77 (s, 1H, H2"), 3.48 (s, 1H, H3"), 3.41 (s, 1H, H4"), 2.33 (s, 2H, CH 2 ⁇ fatty acid carbon chain), 1.62 (s, 2H, CH 2 ⁇ fatty acid carbon chain), 1.26-1.31 (m, 16H, CH 2 fatty acid carbon chain) and 0.87 (s, 3H, CH 3 fatty acid carbon chain).
  • CALB catalyzes the acylation of the carbon flavonoids of bamboo leaves on the primary hydroxyl group of the glycosyl group, mainly because the primary hydroxyl group of the glycosyl group is more active and sterically hindered relative to the hydroxyl group at other positions. smaller.
  • the structural formula of the bamboo leaf carbon flavonoid laurate is shown in Fig. 3.
  • the eAOB-o sample was dissolved in methanol, using acetonitrile-1% acetic acid aqueous solution as mobile phase, and used as a liquid chromatography column with C18 as a packing material (Luna C18 ODS column, column length 250 mm, inner diameter 4.6 mm, containing C18 filler, pellet 5 ⁇ m or equivalent) and UV detector or diode array detector, the coumarinic acid in the sample is separated and determined by reversed-phase high performance liquid chromatography, which is qualitative compared with the retention time of the standard, and the peak area is externally labeled. Quantitative.
  • Standard stock solution configuration accurately weigh 10 mg of coumarin standard (accurate to 0.0001 g), dissolve with methanol and dilute to 10 mL, mix and store in the refrigerator. 1 mL of this solution contained 1.0 mg of p-coumaric acid.
  • Detection wavelength 330 nm.
  • Flow rate 1.0 mL/min.
  • Sample detection accurately weigh 100 mg of eAOB-o sample (accurate to 0.0001 g), dissolve it with methanol solution and dilute to 100 mL, and filter through a microporous membrane (0.45 ⁇ m) to obtain a sample solution. Accurately absorb 10 ⁇ L of the sample solution, perform chromatographic analysis under the specified chromatographic conditions, and quantify the retention time, and quantify the peak area by external standard method.
  • the p-coumaric acid content is represented by w 1 and its value is expressed in %, which is calculated by the following formula:
  • c 1 the concentration of p-coumaric acid in the test liquid calculated according to the standard curve, in milligrams per milliliter (mg/mL);
  • V 1 the volume of the sample for the volume of the sample, in milliliters (mL);
  • m 1 the sample size for the sample, in milligrams (mg).
  • Example 1 The measurement results showed that the content of p-coumaric acid in eAOB-o prepared in Example 1, Example 2, Example 3 and Example 4 was 0.58%, 0.53%, 0.55% and 0.56%, respectively.
  • the eAOB-o sample was dissolved in methanol (chromatographically pure) using an aqueous solution of acetonitrile (chromatographically pure)-1% acetic acid (premium pure) as the mobile phase, and an ultra-high performance liquid chromatography column and UV detector for C18 as a packing or The diode array detector analyzes the fingerprint of the eAOB-o sample.
  • Detection wavelength 260 nm.
  • Column temperature 40 ° C.
  • Mobile phase A. acetonitrile; B. 1% aqueous acetic acid (v/v).
  • Gradient elution conditions were as follows: 0 to 13 min, 12% A; 13 min to 16 min, 12% A to 40% A; 16 min to 19 min, 40% A; 19 min to 21 min, 40% A to 12% A.
  • Flow rate 0.28 mL/min.
  • Injection volume 2 ⁇ L.
  • Example 2 2.55 mg of the eAOB-o sample prepared in Example 1 was weighed, dissolved in methanol (chromatographically pure) and made up to 5 mL, and filtered through a microporous membrane (0.22 ⁇ m) to obtain a sample solution. Accurately pipet 1 ⁇ L of the sample solution and perform chromatographic analysis under the above chromatographic conditions.
  • Example 4 The UPLC fingerprint analysis results of the eAOB-o of Example 1 are shown in Fig. 4.
  • the eAOB-o samples obtained in Example 2, Example 3 and Example 4 were analyzed and tested, and the fingerprint of the obtained UPLC was identical to the eAOB-o obtained in Example 1.
  • Sample pretreatment Weigh (1.00 ⁇ 0.01) g of food (cookie or potato chips, etc.) powder in a 50 mL centrifuge tube, add 400 ⁇ L of 13 C 3 -AA internal standard (1 ⁇ g / mL), and mix well. 9.6 mL of a formic acid aqueous solution (0.3%) was added to the sample, and after vortexing for 1 min, it was centrifuged at 15,000 rpm for 5 min at 0 °C. The centrifuge tube was taken out, the grease layer floating on the surface of the sample was discarded, and the clear liquid was collected. Further, 10 mL of a formic acid aqueous solution (0.3%) was added to the sample, and the operation was repeated once in the above extraction step.
  • the two obtained clear liquids (about 20 mL) were combined, vortexed and mixed, and 2 mL of the clear liquid was accurately taken up and purified by a well-balanced Oasis MCX solid phase extraction column (3 cc, 60 mg).
  • the first 16 drops of the effluent were discarded, and the remaining effluent was collected, and the volume was adjusted to 2 mL with ultrapure water.
  • the mixture was passed through a 0.22 ⁇ m PTFE microporous membrane, and the filtrate was measured by UPLC-MS/MS.
  • Mass spectrometry conditions electrospray ionization positive (ESI+) mode.
  • Capillary voltage 3.5 kV; drying gas temperature: 200 ° C; drying gas flow rate: 16 L/min; atomizing gas pressure: 45 psi; sheath gas temperature: 350 ° C; sheath gas flow rate: 12 L/min.
  • Monitoring method MRM mode.
  • Quantitative analysis the standard concentration/internal standard concentration is plotted on the abscissa, the standard peak area/internal standard peak area is plotted on the ordinate, and the standard curve is drawn.
  • the linear regression equation of the curve is obtained, and the linear range is 2 to 160 ng/mL (2, 5). , 10, 20, 40, 80 and 160 ng/mL), correlation coefficient 0.9999.
  • Sample pretreatment Weigh (1.00 ⁇ 0.01) g sample powder in a 50 mL centrifuge tube, add 5 mL of n-hexane, shake vigorously for 3 min, centrifuge at 13,000 rpm for 5 min, discard the solvent, and repeat the above steps twice. After degreasing three times (5+5+5 mL) with 15 mL of n-hexane, the sample was dried with nitrogen until the powder was recovered. 10 mL of Na 2 B 4 O 7 buffer (0.2 mol/L, pH 9.2) and 5 mL of NaBH 4 (1 mol/L, prepared with 0.1 mol/L NaOH) were added to the sample, and reduced at 4 ° C overnight (about 8 h).
  • the column was a Waters Acquity UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 ⁇ m).
  • the mobile phase A was 5 mmol/L aqueous solution of nonafluoropentanoic acid
  • the mobile phase B was acetonitrile
  • the mobile phase gradient was shown in Table 1. Flow rate: 0.2 mL/min; injection amount: 7.5 ⁇ L, column temperature: 50 °C.
  • Mass spectrometry conditions ESI + mode.
  • Capillary voltage 3.0 kV; cone voltage: 20 eV; ion source temperature: 130 ° C; solvent removal temperature: 400 ° C.
  • Monitoring method MRM mode.
  • the standard concentration/internal standard concentration is the abscissa
  • the standard peak area/internal standard peak area is the ordinate
  • the standard curve is drawn.
  • the linear regression equation of the curve is obtained, and the linear range is 3 ⁇ 300ng/mL (3, 10). , 50, 100, 150, 200 and 300 ng / mL), the correlation coefficient is between 0.9997 and 0.9998.
  • Chromatographic conditions The column was a Waters UPLC BEH C18 column (100 mm x 2.1 mm i.d., 1.7). ⁇ m); mobile phase A is 0.3% formic acid aqueous solution, mobile phase B is acetonitrile; mobile phase gradient is shown in Table 2; flow rate: 300 ⁇ L/min; column temperature: 40 ° C; injection amount: 5 ⁇ L.
  • Mass spectrometry conditions ESI + mode.
  • Capillary voltage 4.0 kV; cone voltage: 30 V; ion source temperature: 120 ° C; solvent removal temperature: 400 ° C; monitoring mode: MRM mode.
  • the characteristic ion information and mass spectrometry parameters of Harman and Norharman and the internal standard are shown in Table 3.
  • the standard concentration/internal standard concentration is the abscissa
  • the standard peak area/internal standard peak area is the ordinate
  • the standard curve is drawn.
  • the linear regression equation of the curve is obtained, and the linear range is 1 ⁇ 25ng/mL (1, 2) , 5, 10 and 25 ng / mL), the correlation coefficient is between 0.9926 and 0.9998.
  • the production of cookies is based on the recommended method of the American Association of Cereal Chemistry Law AACC 10-54.
  • the basic formula is shown in Table 4.
  • eAOB-o (sample prepared in Example 1, the same below) was added in an amount of 0.1 g/kg, 0.2 g/kg, 0.3 g/kg, 0.4 g/kg, and 0.5 g/kg (based on the dough).
  • the blank control group was the group without the addition of antioxidants.
  • the dough was made into a cookie having a diameter of 5 cm and a thickness of 3 mm, and baked at 205 ° C for 11 minutes. The baked cookies were cooled at room temperature (25 ° C) for 30 min and then used.
  • UPLC-MS/MS was used to measure acrylamide (AA), and carboxymethyl lysine (CML) and carboxyethyl lysine were simultaneously measured.
  • CEL acrylamide
  • the inhibition effect of eAOB-o on the formation of CML and CEL during cookie baking is shown in Fig. 6.
  • the CML and CEL contents measured in the blank control group without the addition of antioxidant were 79.58 ⁇ 1.93 mg/kg and 11.97 ⁇ 0.26 mg/kg, respectively.
  • the inhibition rates of eAOB-o on CML were 36.83%, 53.72%, 65.42%, 43.13% and 25.96%, respectively, and the inhibition rates for CEL were 34.71%, 56.74%, 69.00%, respectively. 31.61% and 13.83%.
  • the maximum inhibition rates for CML and CEL were found at the level of eAOB-o added by three tenths of a third (0.3 g/kg).
  • Example 10 Effect of AOB-o obtained by different preparation methods on oxidation stability of palm oil and lard
  • the air flow rate is set to 20L/h.
  • the test was carried out in accordance with the method specified in GB/T 21121-2007, and the difference in the antioxidant properties of eAOB-o and cAOB-o was compared by the size of the protective factor (PF).
  • the formula for calculating PF is as follows:
  • the test results are shown in Fig. 7.
  • the cAOB-o and eAOB-o can significantly enhance the antioxidant stability of palm oil in the range of 0.01%-0.05% of the additive amount.
  • the antioxidant stability of eAOB-o enhanced palm oil was significantly better than that of cAOB-o (p ⁇ 0.01).
  • the amount of cAOB-o increased from 0.01% to 0.03%, the oxidative stability gradually increased, and the PF values of palm oil with 0.01%, 0.02% and 0.03% cAOB-o were 1.24 ⁇ respectively.
  • the acrylamide content was determined after the samples of the French fries and the fritters were cooled, and the formation inhibition rate was calculated as compared with the blank control.
  • the inhibition rate of fried potato fries acrylamide after adding 0.02% eAOB-o can reach 27.8%, which is significantly higher than other commonly used antioxidants (OTP 12.2%, TBHQ 9.7% and RE 8.4%);
  • the addition of 0.01% eAOB-o inhibited the formation of acrylamide in fried French fries by 23.1%, which was also significantly higher than other commonly used lipid antioxidants (OTP 14.5%, TBHQ 8.3% and RE 9.2%).
  • OTP 14.5%, TBHQ 8.3% and RE 9.2% The above results indicate that eAOB-o almost completely blocks the formation of acrylamide caused by the acrolein pathway.
  • Example 16 Combination of eAOB-o and AOB-w inhibits acrylamide formation in French fries and fritters
  • French fries aqueous phase treatment 0.1% AOB-w and asparaginase aqueous solution are prepared, and the frozen fries are thawed, soaked for 45s, drained, and to be fried;
  • French fries oil phase treatment 3 kg of palm oil was weighed, 0.02% eAOB-o was added, and the mixture was uniformly mixed, and the same treatment as in Example 15 was carried out.
  • Oil stick water phase treatment adding 0.05% AOB-w to the fritter base formula to make fritters;
  • Oil fritter phase treatment 3 kg of palm oil was weighed, 0.01% eAOB-o was added, and the mixture was uniformly mixed, and the same treatment as in Example 15 was carried out.
  • the acrylamide value in the sample was measured to calculate the inhibition ratio of acrylamide formation.
  • AOB-w water-soluble bamboo leaf antioxidant
  • eAOB-o fat-soluble bamboo leaf antioxidant
  • Example 17 Combination of eAOB-o and AOB-w inhibits the formation of acrylamide and heterocyclic amines in fried potato chips
  • Preparation of compound potato chips 5 parts of potato snow powder and 3 parts of flour are thoroughly dry-mixed with eggbeater, and 6 parts of distilled water (both in mass ratio) are added, and the dough is thoroughly mixed.
  • the dough was repeatedly pressed into a uniform dough with a thickness of 1 mm using a hand-held pressing machine, and then a potato chip embryo having a diameter of 4 cm and a thickness of 1 mm was pressed out using a biscuit mold (single embryo weight was about 1.37 ⁇ 0.06 g) and stored at -20 ° C. In the refrigerator, take it out before frying, and leave it at room temperature for 15-20 min.
  • Aqueous phase treatment of composite potato chips 0.02% of AOB-w is completely dissolved in water, gradually added to the pre-mixed dry powder, thoroughly mixed to form a dough, and pressed into a potato chip blank.
  • Compound potato chip oil phase treatment Measure 2.5L palm oil, add 0.01% eAOB-o after the oil temperature rises to a certain extent, mix evenly, and add no antioxidant in the blank group.
  • the potato chips were fried at 170 ° C; the amount of potato chips per batch was 30 (about 41 g) and fried for 3.0 min.
  • the potato chips were dried and pulverized. After cooling, the acrylamide content, Harman and Norharman content in the samples were determined, and the inhibition rate was calculated compared with the blank control. As can be seen from Figure 14, the combination of eAOB-o and AOB-w has significant inhibitory effects on acrylamide, Harman and Norharman, the inhibition ratio of acrylamide is 75.86%, the Harman inhibition rate is 45.70%, and the Norharman inhibition rate is 35.36. %.

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Abstract

一种酶法制备得到的脂溶性竹叶抗氧化物（eAOB-o）及其用途，所述eAOB-o包括对香豆酸和一种或多种5，7-二羟基-4H-1-苯并吡喃-4-酮衍生物被酰基修饰的产物；其中，所述的5，7-二羟基-4H-1-苯并吡喃-4-酮衍生物为取代的5，7-二羟基-4H-1-苯并吡喃-4-酮；所述的取代指苯并吡喃环被以下取代：苯并吡喃环的苯环部分上的任意一个氢原子被一个糖基所取代，且苯并吡喃环的2位被具有1-3个羟基取代基的苯基取代；其中，所述的酰基修饰指所述糖基上的羟基被C8-C18的酰基修饰基所修饰；且所述5，7-二羟基-4H-1-苯并吡喃-4-酮衍生物与酰基修饰基的摩尔比为1:0.5-2。

Description

一种酶法制备得到的脂溶性竹叶抗氧化物及其用途 技术领域
本发明涉及食品添加剂领域，具体是指以酶法催化的方式实现水溶性竹叶抗氧化物(AOB-w)的酯化，产物脂溶性竹叶抗氧化物(eAOB-o)在食品工业中主要作为油脂抗氧化剂，并可同步抑制高温加工过程中美拉德反应伴生危害物丙烯酰胺(AA)、晚期糖基化终末产物(AGEs)和杂环胺(HAAs)的形成，尤为适合于高温用油(如煎炸油、焙烤油、脱膜油)以及需经热加工处理的高脂食品(如油炸薯类制品和曲奇)。
背景技术
油脂氧化是导致油脂品质劣变的一个重要因素。在储藏过程中，油脂容易受到光照、温度、氧化、微生物、水分等的影响，被氧化形成短链醛、酮、酸等，产生哈喇味，造成氧化酸败。油脂氧化产物会对食用油脂的风味、色泽以及组成产生不良的影响，降低油脂的营养品质和缩短货架期。人体摄入酸败的油脂还会破坏细胞膜、酶和蛋白质的结构与功能，导致心血管疾病、加速机体老化甚至致癌，严重危害人体健康。直接在油脂中添加抗氧化剂是阻止或延缓油脂氧化变质、延长食品货架寿命最为行之有效的方法。合成的食品抗氧化剂虽然抗氧化效果良好且价格低廉，曾在食品工业中应用广泛，但均存在一定的安全隐患，其使用已逐渐受限。如美国国家卫生研究院的报告认为丁基羟基茴香醚(BHA)是一种人类可疑的致癌物，美国已将其从GRAS(公认安全)名单中删除，在日本BHA只准用于棕榈油和棕榈仁油。又如在日本、瑞典、澳洲等国，2,6-二叔丁基-4-甲基苯酚(BHT)被明令禁止添加于食品中，在美国也只被允许添加于某些食品。尽管特丁基对苯二酚(TBHQ)是合成品中安全性最高、性能最好的一种，但由于缺少足够的长期安全性试验数据，致突变性不能排除，故在EEC所属的国家(欧洲国家和日本等)未批准其使用。天然抗氧化剂如迷迭香提取物(Rosemary extract,RE)在油脂抗氧化方面表现出良好的效果，但是脂溶性有限、且价格较昂贵，并具有特殊的气味，一定程度上限制了其在油脂工业中的大规模应用。
天然抗氧化剂主要来源于植物，大多为多羟基物质，亲水性良好，而亲脂性较弱，无法充分与油脂混合，进而影响其抗氧化作用的发挥。对天然抗氧化剂进行分子修饰是改善其脂溶性的重要途径，酯化的手段包括化学法和酶法。目前通过化学法修饰得到的油脂抗氧化剂有维生素C棕榈酸酯(AP)、脂溶性茶多酚(OTP)及脂溶性竹叶抗氧化物(cAOB-o)，它们分别是维生素C、茶多酚、竹叶抗氧化物(AOB-w)与脂肪酸的酯化产物，可作为脂溶性抗氧化剂和/或营养强化剂添加于油脂或食品中。
利用化学法对天然抗氧化剂进行结构修饰能在一定程度上满足脂溶性的需求，如发明人前期公开的一种利用氧酰化法制备脂溶性竹叶抗氧化物的方法(CN 103005007A)。然而，化学酰化选择性较低，反应条件剧烈，过程不易控制，常伴随大量副产物的生成，给后续分离纯化工作带来困难，同时制造过程产生大量的废液， 如处理不当会污染环境。而酶法酰化可实现对底物的定向修饰，且反应条件温和，副产物少。如CN 102747117A公开了一种酶法合成脂溶性茶多酚的方法，以有机溶剂为反应介质，利用脂肪酶催化茶多酚与脂肪酸乙烯酯合成脂溶性茶多酚，与其他化学合成法(CN 101255151和CN 101270108)相比具有一定优势。又如CN 102787146A公布了一种微波辅助脂肪酶催化合成改性表没食子儿茶素没食子酸酯(EGCG)的方法，该方法在CN 102747117A基础上，可显著加快反应速度，提高反应产率，缩短反应时间。然而，脂肪酸乙烯酯中目前仅有月桂酸乙烯酯被允许在胶基糖果作为基础剂物质及其配料使用(GB 2760)，其他食品领域均未被批准使用。同时，与脂肪酸相比，脂肪酸乙烯酯的沸点、闪点均大大降低，过程中往往需添加稳定剂，且价格较昂贵，具有显著的局限性。CN 103131741A公开了一种活性植物多酚类物质的生物修饰法，以有机溶剂为介质，在固定化脂肪酶催化下，使植物多酚的单体化合物与C2-C18的脂肪酸或脂肪酸乙烯酯反应得到植物多酚的脂肪酸酯。以上均为竹叶碳苷黄酮脂溶性的改善提供了技术指导，然而，它们均只在有机溶剂体系中尝试对茶多酚或EGCG进行酶法酰化，且主要针对转化率的优化。天然来源的抗氧化剂往往是多组分的复杂体系，其分子修饰的难度较单体物质要大，酯化过程对产品性能的影响也具有不确定性。如何实现天然抗氧化剂的酯化修饰并确保其抗氧化活性是问题的关键所在。
对于抗氧化剂而言，抗氧化活性是其关键。在酰化反应中，天然酚类化合物中活性酚羟基所占的比例由于脂肪酸的接入不可避免地会降低，依赖于活性酚羟基数目的抗氧化活性(如DPPH·清除活性等)亦不可避免地会降低。抗氧化剂在脂类基质中的抗氧化活性应该是有效活性酚羟基、脂溶性、底物分散性等诸多因素的综合表征，因此选择一种接近于真实脂类基质中的抗氧化评价体系对于筛选高性能脂溶性抗氧化剂是至关重要的。
就酰化反应的体系而言，常采用有机溶剂，但其具有易挥发、易燃的特点，对过程管理及生产环境会带来一定影响，且产品中溶剂残留的问题也尚未得到很好解决。离子型液体具有不可燃、热稳定性好、可溶解的物质范围广、以及可设计性强等特点，正逐渐成为食品工业中一种绿色、环保的新型溶剂，应用前景广阔。无溶剂体系则能够有效解决溶剂残留问题，并且简化分离纯化步骤，具有明显的优势，但是由于无溶剂体系要求底物必须为液体状态，在一定程度上限制了其广泛应用。
食品经历高温加工往往伴随着美拉德反应的发生。美拉德反应产物不仅包括呈色呈味物质和抗氧化物质，往往还包括一些伴生的对人体健康有影响的危害物。由于这些美拉德反应伴生危害物并非人为添加，而是由食品原料中的组分(如氨基酸、还原糖)在热加工过程中产生的，所以被称为“内源性化学污染物”。典型的美拉德反应伴生危害物包括丙烯酰胺(Acrylamide，AA)、杂环胺(Heterocyclic Aromatic Amines，HAAs)、亚硝胺(Nitrosamine)、杂环芳烃(Polycyclic Aromatic Hydrocarbons)和蛋白晚期糖基化终末产物(Advanced Glycation End-products，AGEs)等。近年来，随着对美拉德反应认识的深入，对这些伴生危害物的研究逐渐成为热点。
丙烯酰胺是富含碳水化合物和氨基酸的食品在高温条件下经美拉德反应产生，油炸食品是其高暴露领域，其中尤以油炸马铃薯制品最为突出。油炸食品的丙烯酰胺生成途径主要有天冬酰胺途径和丙烯醛途径。天冬酰胺与还原糖在美拉德反应过程中形 成丙烯酰胺(即天冬酰胺途径)是丙烯酰胺产生的主要途径，约贡献了油炸薯类制品中丙烯酰胺总量的70％～80％，反应主要发生在水相中；油脂在高温下氧化分解产生丙烯醛、进而形成丙烯酰胺(即丙烯醛途径)，约占油炸薯类制品丙烯酰胺生成总量的20％～25％，反应主要发生在油相中。天然来源的酚类化合物，如茶多酚、苹果多酚、竹叶抗氧化物等均表现出良好的丙烯酰胺抑制活性，主要是通过天冬酰胺途径实现的。
晚期糖基化终末产物(AGEs)是指在非酶促条件下，蛋白质、氨基酸、脂类或核酸等大分子物质的游离氨基与还原糖(葡萄糖、果糖、戊糖等)的醛基经过缩合、重排、裂解、氧化修饰后产生的一类稳定的终末产物，即通过美拉德反应形成的一类蛋白糖基化后的交联产物。目前已经发现的AGEs物质有20多种，代表性化合物包括羧甲基赖氨酸[Nε-(carboxymethyl)-lysine,CML]、羧乙基赖氨酸[Nε-(carboxyethyl)-lysine,CEL]、戊糖苷素(pentosidine)、吡咯素(pyrraline)、交联素(crosslines)、咪唑赖氨酸(imidazolysine)和versperlysine等。AGEs具有交联性，其结构上的氨基和羧基可通过共价键与蛋白质等大分子物质发生稳定及长时间的结合，形成分子量极大的交联结构。而且，此交联结构对酶稳定，不易被降解，具有不可逆性。AGEs在体内的形成和积聚与许多疾病有着密切关系，尤其是糖尿病的各种并发症。
杂环胺(HAAs)是富含蛋白质的食品在高温烹调加工过程中形成的具有致突变、致癌性的物质，已知烧烤类肉制品是HAAs的高暴露食品。HAAs化合物从化学结构上可以分为氨基咪唑氮杂芳烃(Amino imidazo azaren，AIA)和氨基咔啉杂环胺(Amino-carboline congener)二大类，后者又可细分为α-咔啉、β-咔啉、γ-咔啉和δ-咔啉类杂环胺。β-咔啉类杂环胺主要包含Harman和Norharman。除了典型的烧烤肉制品外，研究人员还在咖啡、巧克力、酱油等食品体系中检出了高含量的Norharman和Harman，尤其是咖啡，有报道咖啡中Norharman含量可高达9.34μg/g、Harman含量高达1.67μg/g，表明饮用咖啡及其制品是人体摄入β-咔啉类杂环胺的主要途径之一。但迄今未见有关薯类制品中Norharman和Harman的含量检测及抑制方面的报道。Norharman和Harman属于非极性杂环胺，本身不具有致突变性。但是动物试验表明，它们能与小鼠大脑和肝脏组织的不同位点结合，从而影响动物的生理行为。作为辅助致突物，Norharman和Harman能够强化其他杂环胺的基因毒性；当与苯胺或甲苯胺等非致突变化合物一起作用时，能产生致突活性并形成DNA加合物；能毒害部分神经组织、并抑制一些关键的酶。另有研究表明，Norharman和Harman可能与人类的一些疾病有关，如帕金森症、癌症和肌肉疼痛综合症等。
发明人的最新研究发现，在油炸薯类制品中含有高含量的Norharman和Harman。对中国大陆市售的106种膨化休闲食品(其中大部分为油炸薯类制品)的面上调查结果显示，全部106种膨化休闲食品中均检出Norharman和Harman，且含量分别为8.27±6.25μg/kg和4.31±4.07μg/kg，达到甚至超过了公认杂环胺高暴露的烧烤肉制品中杂环胺的含量水平，表明油炸薯类制品中的杂环胺风险不容忽视。
竹叶抗氧化物(Antioxidant of bamboo leaves，AOB)是发明人创制的一种天然、安全、经济高效的食品抗氧化剂，2004年列入《中华人民共和国食品添加剂使用卫生 标准》(GB 2760)，目前已批准的应用领域包括：基本不含水的脂肪和油、熟制坚果与籽类、油炸面制品、即食谷物、焙烤食品、腌腊肉制品类、酱卤肉制品类、(熏、烧、烤)肉类、油炸肉类、西式火腿、肉灌肠类、发酵肉制品类、水产品及其制品、果蔬汁(肉)饮料、茶饮料类和膨化食品等十六类(GB 2760-2014)。天然来源的水溶性竹叶抗氧化物(AOB-w)是一种中等偏大极性的组合物，亲水性强，亲油性弱，很大程度上限制了其在油脂行业和高油食品领域中的应用。大量的研究和应用试验表明，AOB-w对热加工食品中丙烯酰胺的形成抑制也主要是通过天冬酰胺途径实现的，其对丙烯醛途径的抑制不明显。油炸食品(如薯片、薯条、方便面、炸鸡、油条、麻花等)和高脂焙烤类食品(如曲奇、蛋黄派等)是美拉德反应伴生危害物(丙烯酰胺和晚期糖基化终末产物)的高暴露和高风险领域，如何抑制和延缓煎炸油在高温下的氧化变质、控制食品中内源性化学污染物的产生，迄今尚无好的解决方案。
综上所述，本领域尚缺乏一种能够抑制和延缓煎炸油在高温下的氧化变质、控制食品中内源性化学污染物产生的方法。
发明内容
本发明的第一方面，提供了一种脂溶性竹叶抗氧化物(eAOB-o)，所述脂溶性竹叶抗氧化物包括以下特征性成分：对香豆酸、和一种或多种5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物被酰基修饰的产物；其中，所述的5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物为取代的5,7-二羟基-4H-1-苯并吡喃-4-酮；
所述的取代指苯并吡喃环被以下取代：苯并吡喃环的苯环部分上的任意一个氢原子被一个糖基所取代，且苯并吡喃环的2位被具有1-3个羟基取代基的苯基取代；
其中，所述的酰基修饰指所述糖基上的羟基被C8-C18的酰基修饰基所修饰；且所述5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物与酰基修饰基的摩尔比为1:0.5～2。
在另一优选例中，所述5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物与酰基修饰基的摩尔比为1:0.8～1.2。
在另一优选例中，所述的脂溶性竹叶抗氧化物的对香豆酸含量为≥0.5％，较佳地为0.51％～0.80％。
在另一优选例中，所述的脂溶性竹叶抗氧化物的总酚含量为≥20％，较佳地为20.1％～28.0％。
在另一优选例中，所述的脂溶性竹叶抗氧化物的乙酸乙酯溶解度≥3g/100mL，较佳地为3.5g/100mL～6.3g/100mL(以脂溶性竹叶抗氧化物在100mL乙酸乙酯中的溶解质量计算)。
在另一优选例中，所述的脂溶性竹叶抗氧化物是淡黄色至黄棕色的固体，较佳地，所述的脂溶性竹叶抗氧化物是淡黄色至黄棕色的粉末、颗粒或片状固体。
在另一优选例中，所述的脂溶性竹叶抗氧化物具有以下性能：可抑制高温加工过程中丙烯酰胺(AA)和/或晚期糖基化终末产物(AGEs)的形成。
在另一优选例中，所述的晚期糖基化终末产物为羧甲基赖氨酸(CML)和/或羧乙基赖氨酸(CEL)。
在另一优选例中，所述的脂溶性竹叶抗氧化物对丙烯酰胺形成的抑制率为≥20％，较佳地为≥25％，更佳地为≥27％。
在另一优选例中，所述的脂溶性竹叶抗氧化物对丙烯醛途径形成丙烯酰胺的抑制率为≥50％，较佳地为≥70％，更佳地为≥80％。
在另一优选例中，所述的脂溶性竹叶抗氧化物可以完全抑制丙烯醛途径形成的丙烯酰胺。
在另一优选例中，所述5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物被酰基修饰的产物选自下组：A.1、A.2、A.3、A.4、B.1、B.2、B.3、B.4、C.1、C.2、C.3、C.4；
Figure PCTCN2015079475-appb-000001
其中，所示的R基团是C8-C18的酰基。
在另一优选例中，所述的C8-C18的酰基指C8-C18的脂肪酸脱去羟基后形成的基团，较佳地，所述的脂肪酸选自下组：辛酸、癸酸、月桂酸、肉豆蔻酸、棕 榈酸、油酸、亚油酸、硬脂酸，或其组合。
在另一优选例中，所述的脂溶性竹叶抗氧化物是通过以下方法制备的：
在酶催化下，使水溶性竹叶抗氧化物(AOB-w)与C8-C18的脂肪酸反应，进行单羟基酰化，得到脂溶性竹叶抗氧化物；
较佳地，所述的方法包括步骤：在酶催化下，用水溶性竹叶抗氧化物与C8-C18的脂肪酸在40℃～80℃下进行酰化反应，得到脂溶性竹叶抗氧化物。
本发明的第二方面，提供了一种脂溶性竹叶抗氧化物的制备方法，包括步骤：在酶催化下，使水溶性竹叶抗氧化物与C8-C18的脂肪酸反应，进行单羟基酰化，得到脂溶性竹叶抗氧化物；
较佳地，所述的方法包括步骤：在酶催化下，用水溶性竹叶抗氧化物与C8-C18的脂肪酸在40℃～80℃下进行酰化反应，得到脂溶性竹叶抗氧化物。
在另一优选例中，所述的脂溶性竹叶抗氧化物是如本发明第一方面所述的脂溶性竹叶抗氧化物。
在另一优选例中，所述步骤的反应时间为4h～48h；较佳地为6h～24h。
在另一优选例中，所述步骤中，水溶性竹叶抗氧化物与脂肪酸的质量比为1：1～30；较佳地为1:5～15。
在另一优选例中，所述方法的收率为40～100％，较佳地为50～100％，更佳地为60～100％。
在另一优选例中，所述方法还包括步骤：反应完成后，用乙腈-正庚烷溶液对反应液进行萃取，得到含有脂溶性竹叶抗氧化物的下相；较佳地，所述的乙腈-正庚烷溶液的体积比为乙腈:正庚烷＝2:5。
在另一优选例中，所述方法还包括步骤：对所述萃取得到的下相用乙酸乙酯-水溶液进行二次萃取，得到含有脂溶性竹叶抗氧化物的上相；较佳地，所述的乙酸乙酯-水溶液的体积比为乙酸乙酯：水＝3:5。
在另一优选例中，所述方法还包括步骤：在进行萃取前，对待萃取液进行浓缩。
在另一优选例中，所述方法还包括步骤：反应完成后，用乙腈-正庚烷溶液对反应液进行萃取，得到含有脂溶性竹叶抗氧化物的下相；较佳地，所述的乙腈-正庚烷溶液的体积比为乙腈:正庚烷＝2:5。
在另一优选例中，所述方法还包括步骤：对上述萃取得到的下相用乙醇水溶液-石油醚进行二次萃取，得到含有脂溶性竹叶抗氧化物的下相；较佳地，所述的乙醇水溶液-石油醚的体积比为乙醇水溶液：石油醚＝2:5。
在另一优选例中，所述的步骤在无溶剂条件下进行，或所述的步骤在有机溶剂或离子型溶剂中进行；
较佳地，所述的有机溶剂选自下组：叔戊醇(优选为水饱和叔戊醇)、丙酮、乙酸乙酯、叔丁醇，或其组合；
所述的离子型溶剂为阳离子和阴离子组成的盐，其中，所述的阳离子为未取代或被C1-C8的烷基取代的咪唑，且所述的阴离子选自下组：BF4 -、PF6 -、TFSI-，或其组合。
在另一优选例中，所述AOB-w与有机溶剂的用量比为1g：25～250mL。
在另一优选例中，所述的酶选自下组：南极假丝酵母脂肪酶、枯草杆菌蛋白酶、米黑根毛霉脂肪酶、假单胞菌脂肪酶、疏棉状嗜热丝孢菌脂肪酶，或其组合；优选南极假丝酵母脂肪酶；
所述的C8-C18的脂肪酸选自下组：辛酸、癸酸、月桂酸、肉豆蔻酸、棕榈酸、油酸、亚油酸、硬脂酸，或其组合。
在另一优选例中，当所述反应在有机溶剂或离子型溶剂中进行时，所述的脂肪酸为C8-C18的脂肪酸。
在另一优选例中，当所述反应在无溶剂条件下进行时，所述的脂肪酸为C8-C12的脂肪酸。
在另一优选例中，所述AOB-w与所述酶的用量重量比为1：0.5-5；较佳地为1：1-4。
本发明的第三方面，提供了一种食品添加剂，所述的食品添加剂含有如本发明第一方面所述的脂溶性竹叶抗氧化物，和任选的食品上可接受的载体。
在另一优选例中，所述的食品添加剂为如本发明第一方面所述的脂溶性竹叶抗氧化物溶于中长链脂肪酸形成的食品添加剂。
在另一优选例中，所述的食品添加剂为含质量分数1％～20％的脂溶性竹叶抗氧化物的中长链脂肪酸酯溶液；其中，所述的中长链脂肪酸选自下组：辛葵酸甘油酯、月桂酸甘油酯、肉豆蔻酸甘油酯、棕榈酸甘油酯、硬脂酸甘油酯、油酸甘油酯、亚油酸甘油酯，或其组合。
本发明的第四方面，提供了一种如本发明第一方面所述的脂溶性竹叶抗氧化物的用途，用于选自下组的一种或多种用途：
(a)作为高温食品加工的抗氧化剂；
(b)作为高温食品加工的丙烯酰胺抑制剂；
(c)用于制备高温食品加工的抗氧化剂；
(d)用于制备高温食品加工的丙烯酰胺抑制剂；
(e)在高温食品加工工艺中用于抑制丙烯酰胺和/或晚期糖基化终末产物和/或杂环胺的生成；
(f)作为油脂抗氧化剂。
在另一优选例中，所述的脂溶性竹叶抗氧化物用于抑制食品热加工过程中丙烯酰胺、羧甲基赖氨酸、和/或羧乙基赖氨酸的生成。
在另一优选例中，所述的脂溶性竹叶抗氧化物用于抑制食品热加工过程中丙烯酰胺、和/或杂环胺的生成。
在另一优选例中，所述抗氧化指油脂或食品中选自下组的一个或多个指标降低：过氧化值、茴香胺值、硫代巴比妥酸值、酸价、极性化合物含量、游离脂肪酸含量、丙二醛含量。
本发明的第五方面，提供了一种食品加工方法，所述方法包括步骤：在加工过程之前、之中或之后，在食品原料中添加如本发明第一方面所述的脂溶性竹叶抗氧化物，或添加如本发明第三方面所述的食品添加剂。
在另一优选例中，所述的方法包括步骤：将所述的脂溶性竹叶抗氧化物或食品添加剂溶于油脂中，用于加工食品；较佳地，所述的油脂选自下组：煎炸油、焙烤油、脱膜油，或其组合。
在另一优选例中，所述的脂溶性竹叶抗氧化物或食品添加剂的添加量为油品质量分数的0.01～0.05wt％，以脂溶性竹叶抗氧化物的质量计。
在另一优选例中，所述的食品添加剂为如本发明第一方面所述的脂溶性竹叶抗氧化物溶于中长链脂肪酸形成的食品添加剂。
在另一优选例中，所述的食品加工方法还包括：在加工过程之前、之中或之后，添加水溶性竹叶抗氧化物(AOB-w)。
在另一优选例中，所述的食品加工方法包括：在油脂中添加如本发明第一方面所述的脂溶性竹叶抗氧化物，或添加如本发明第三方面所述的食品添加剂；并用水溶性竹叶抗氧化物处理食品原料。
本发明的第六方面，提供了一种食品，所述的食品在加工过程中添加如本发明第一方面所述的脂溶性竹叶抗氧化物或含有如本发明第一方面所述的脂溶性竹叶抗氧化物的食品组分。
在另一优选例中，所述的脂溶性竹叶抗氧化物使得热加工食品中的丙烯酰胺生成被抑制。
在另一优选例中，所述的食品中，如本发明第一方面所述的脂溶性竹叶抗氧化物在油脂中建议添加量为100～500ppm，优选100～300ppm。
在另一优选例中，所述的食品中，丙烯酰胺抑制率≥20％，较佳的为25％～30％；折合成对油脂氧化途径(即丙烯醛途径)形成的丙烯酰胺而言，其抑制率为60％以上，更佳地为85％～95％。
在另一优选例中，所述的食品选自下组：油炸食品、烘烤食品、膨化食品、食用油、食品加工用油。
在另一优选例中，所述食品中，丙烯酰胺的含量低于200μg/kg。
在另一优选例中，所述的油炸食品为薯条时，所述油炸食品的丙烯酰胺的含量低于200μg/kg。
在另一优选例中，所述的油炸食品为传统中式食品油条时，所述油炸食品的丙烯酰胺的含量低于30μg/kg。
在另一优选例中，所述的食品中，如本发明第一方面所述的脂溶性竹叶抗氧化物的含量为10～150ppm。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
附图1竹叶碳苷黄酮月桂酸酯的UPLC检测；
附图2竹叶碳苷黄酮月桂酸酯的质谱检测；
附图3竹叶碳苷黄酮月桂酸酯的结构示意图；
附图4 eAOB-o的UPLC指纹图谱；
附图5 eAOB-o和OTP对曲奇中丙烯酰胺(AA)形成的抑制作用；
附图6 eAOB-o和OTP对曲奇中CML(黑色棒)和CEL(灰色棒)的抑制作用；
附图7 eAOB-o及cAOB-o对棕榈油和猪油氧化稳定性的影响；
附图8 eAOB-o与常用油脂抗氧化剂对棕榈油煎炸稳定性的影响；
附图9 eAOB-o和cAOB-o对棕榈油煎炸稳定性的影响；
附图10 eAOB-o与常用油脂抗氧化剂对油炸薯条抗氧化的影响比较；
附图11 eAOB-o与常用油脂抗氧化剂对油条抗氧化作用的影响比较；
附图12 eAOB-o与常用油脂抗氧化剂对薯条和油条丙烯酰胺的抑制作用比较；
附图13 AOB-w与eAOB-o联合使用对油条和薯条中丙烯酰胺形成的抑制作用；
附图14 AOB-w与eAOB-o联合使用对油炸复合薯片中丙烯酰胺和β-咔啉类杂环胺的抑制作用。
具体实施方式
本发明人经过长期而深入的研究，意外地发现，通过酶法对水溶性竹叶抗氧化物进行修饰，酰化作用选择性地发生在竹叶碳苷黄酮的糖羟基上，而苯环上的酚羟基则不发生酰化，从而使得产物在脂溶性改善的同时保持较好的抗氧化性能，且能够同时抑制食品热加工过程中丙烯酰胺(特别是丙烯醛途径所产生的丙烯酰胺)的生产。基于上述发现，发明人完成了本发明。
术语
如本文所用，术语“酶法修饰的脂溶性竹叶抗氧化物”与“eAOB-o”可互换使用，均指使用本发明方法(即酶催化酰化)制备得到的脂溶性竹叶抗氧化物。
如本文所用，术语“水溶性竹叶抗氧化物”与“AOB-w”可互换使用，均为使用本发明方法(即酶催化酰化)制备得到的脂溶性竹叶抗氧化物的反应底物。一种优选的水溶性竹叶抗氧化物具有以下特性：黄棕色粉末，含总酚46.53％、异荭草苷2.35％和水分5.87％。
如本文所用，术语“5,7-二羟基-4H-1-苯并吡喃-4-酮”指具有如下式所示结构的化合物：
Figure PCTCN2015079475-appb-000002
术语“C8-C18的脂肪酸”指具有8-18个碳原子的羧酸，如辛酸、癸酸、月桂酸、肉豆蔻酸、棕榈酸、油酸、亚油酸、硬脂酸，或类似的酸。
术语“C8-C18的酰基”指具有8-18个碳原子的羧酸脱去-OH基团形成的基团， 形如
Figure PCTCN2015079475-appb-000003
所示，其中，R为C7-C17的脂链。
术语“糖基”指葡萄糖的任意一个羟基(-OH)失去氢原子形成的基团。
酶法修饰的脂溶性竹叶抗氧化物
为了解决上述技术问题，本发明提供了一种酶法修饰的脂溶性竹叶抗氧化物(eAOB-o)。所述脂溶性竹叶抗氧化物包括以下特征性成分：对香豆酸、和一种或多种5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物被酰基修饰的产物；其中，所述的5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物为取代的5,7-二羟基-4H-1-苯并吡喃-4-酮，其中，所述的取代指苯并吡喃环被以下取代：苯并吡喃环的苯环部分上的任意一个氢原子被单糖基所取代，且苯并吡喃环的2位被具有1-3个羟基取代的苯基取代；
其中，所述的酰化修饰指所述黄酮碳苷的糖羟基被C8-C18的脂肪酸供体所修饰；且所述5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物与酰基供体的摩尔比为1:0.5～2，较佳地为1:0.8～1.2。
更佳地，所述的5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物包括以下结构化合物中的两种或以上：
Figure PCTCN2015079475-appb-000004
Figure PCTCN2015079475-appb-000005
本发明提供的eAOB-o的总酚含量≥20％，较佳地为20.1％～28.0％。
本发明提供的eAOB-o的对香豆酸含量≥0.5％，较佳地为0.51％～0.8％。
在另一优选例中，所述的脂溶性竹叶抗氧化物的外观为淡黄色至黄棕色的粉末、颗粒或片状，略带酯味。
所述的脂溶性竹叶抗氧化物能直接溶于食用油脂、中长链脂肪酸和非极性溶剂中。在另一优选例中，所述的脂溶性竹叶抗氧化物的乙酸乙酯溶解度≥3g/100mL，较佳地为3.5g/100mL～6.3g/100mL。
本发明的脂溶性竹叶抗氧化物具有显著优于市售同类油脂抗氧化剂产品(迷迭香提取物RE、脂溶性茶多酚OTP、羟基茴香醚BHA、2,6-二叔丁基对甲酚BHT等)的抗氧化性和热稳定性，可与合成抗氧化剂特丁基对苯二酚(TBHQ)媲美。具体地，本发明的脂溶性竹叶抗氧化物能够有效地改善油品的抗氧化稳定性和煎炸稳定性，添加本发明的脂溶性竹叶抗氧化物的油品在保护因子(PF)、过氧化值(PV)、硫代巴比妥酸值(TBARS)、酸价(AV)、茴香胺值(p-AnV)、游离脂肪酸(FFA)和极性化合物(PC)含量等指标水平上均有较大改善，且改善效果优于现有的油脂抗氧化剂。
本发明的脂溶性竹叶抗氧化物还可同步抑制高温加工过程中美拉德反应伴生危害物丙烯酰胺(AA)和晚期糖基化终末产物(AGEs)的形成，尤为适合于高温用油(如煎炸油、焙烤油、脱膜油等)以及需经热加工处理的高脂食品(如油炸薯类制品和曲奇等)。在另一优选例中，所述的脂溶性竹叶抗氧化物对制成品丙烯酰胺形成的抑制率≥20％，较佳地为≥25％，更佳地为≥27％。
本发明的脂溶性竹叶抗氧化物还可同步抑制高温加工过程中AA和杂环胺(Harman和Norharman)的形成。在另一优选例中，所述的脂溶性竹叶抗氧化物对丙烯酰胺抑制率为75.86％，对Harman抑制率为45.70％，对Norharman抑制率为35.36％。
在另一优选例中，本发明所述的脂溶性竹叶抗氧化物可以特别有效地阻断经由油脂氧化过程产生的丙烯酰胺(即丙烯醛途径产生的丙烯酰胺)。
热加工食品中，丙烯酰胺的形成途径主要有以下两条：“天冬酰胺途径”和“丙烯醛途径”。在油炸薯类制品为例，天冬酰胺与还原糖在美拉德反应过程中形成丙烯酰 胺(即天冬酰胺途径)是丙烯酰胺产生的主要途径(约贡献最终产品中丙烯酰胺总量的70％～80％)，反应主要发生在水相中；油脂在高温下氧化分解产生丙烯醛、进而形成丙烯酰胺(即丙烯醛途径)，约贡献了制品中丙烯酰胺总量的20～30％，反应主要发生在油相中。目前，一系列天然来源的酚类化合物，如茶多酚、苹果多酚、竹叶抗氧化物等对于水相中丙烯酰胺的产生均表现出良好的抑制活性(主要通过天冬酰胺途径实现)，但对于油脂中产生的丙烯酰胺却未见报道。而本发明的脂溶性竹叶抗氧化物eAOB-o对于油相中产生的丙烯酰胺具有很好的抑制效果，在本发明的一些优选实施例中，所述的脂溶性竹叶抗氧化物对丙烯醛途径形成丙烯酰胺的抑制率≥50％，较佳地为≥70％，更佳地为≥80％。在特别优选的实施例中，其抑制率(丙烯醛途径所产生的丙烯酰胺)可达到将近100％。
酶法制备脂溶性竹叶抗氧化物(eAOB-o)的方法
本发明还提供了一种酶法制备eAOB-o的方法。所述方法采用酶法催化，在有机溶剂、离子型溶剂为介质或无溶剂反应体系中，将AOB-w与脂肪酸直接酯化而成。该制备过程可少用或不用有机溶剂，绿色、环保、环境友好。所述方法包括步骤：
在酶的催化下，使水溶性竹叶抗氧化物与C8-C18的脂肪酸反应，进行单羟基酰化，得到脂溶性竹叶抗氧化物；
较佳地，所述的方法包括步骤：在酶催化下，用水溶性竹叶抗氧化物与C8-C18的脂肪酸在40℃～80℃下进行酰化反应，得到脂溶性竹叶抗氧化物。
在另一优选例中，所述步骤的反应时间为4h～48h。
在另一优选例中，所述步骤中，水溶性竹叶提取物与脂肪酸的质量比为1：1～30。
所述方法的收率较高，可以达到40％～100％，较佳地为50％～100％，更佳地为60％～100％。
在一个优选例中，所述制备过程的具体步骤如下：
步骤1：在无溶剂体系、有机溶剂体系或离子型溶剂体系中，在酶的催化下，AOB-w与脂肪酸在40℃～80℃下酰化4h～48h(AOB-w与脂肪酸的质量比为1：1～30)。
步骤2：上述反应结束后，反应液经过滤、浓缩后得到浓缩液(粗品)。
步骤3：将以上获得的浓缩液用乙腈-正庚烷溶液(2:5，v/v)进行萃取，下相经浓缩后再用乙酸乙酯-水(3:5，v/v)萃取，取上相经浓缩、干燥后即得到eAOB-o；也可将步骤2)中获得的浓缩液先用乙酸乙酯-水(3:5，v/v)萃取，上相经浓缩后再用88％～92％的乙醇水溶液-石油醚(2:5，v/v)萃取，取下相浓缩、干燥后即得目标物eAOB-o。
当采用有机溶剂或离子型溶剂体系时，可选C8-C18脂肪酸中的任何一种，即辛酸、癸酸、月桂酸、肉豆蔻酸、棕榈酸、油酸(亚油酸)或硬脂酸；而当采用无溶剂体系时，优选脂肪酸的链长为C8～C12。
反应体系采用的有机溶剂可以是叔戊醇(水饱和叔戊醇)、丙酮、乙酸乙酯、 叔丁醇；离子型溶剂为疏水性离子型液体，阳离子为取代基链长不超过8个碳原子的咪唑类，阴离子为BF4 -、PF6 -或TFSI-。
本发明中的催化用酶可选用南极假丝酵母脂肪酶、枯草杆菌蛋白酶、米黑根毛霉脂肪酶、假单胞菌脂肪酶、疏棉状嗜热丝孢菌脂肪酶或者为所述几种酶制剂的混合物；优选南极假丝酵母脂肪酶。
作为本发明的脂溶性竹叶抗氧化物(eAOB-o)的制备方法的进一步描述：
步骤1)中所述的有机溶剂为叔戊醇或水饱和叔戊醇、丙酮、乙酸乙酯、叔丁醇等，底物AOB-w与有机溶剂的用量比为1g：25～250mL；
步骤1)中所述的离子型溶剂为疏水性离子型液体，阳离子为取代基链长不超过8个碳原子的咪唑类，阴离子为BF4 -、PF6 -或TFSI-。
本发明的工艺参数优选如下：
水溶性竹叶抗氧化物(AOB-w)：脂肪酸的质量比为1:5～15，反应时间为6h～24h。
酶法修饰的脂溶性竹叶抗氧化物在食品加工中的应用
本发明提供的eAOB-o能直接溶于食用油脂和/或中、长链脂肪酸，难溶或不溶于水。其中，所述的食用油脂包括(但并不限于)：棕榈油、花生油、玉米油、菜籽油、猪油、牛油、黄油、鱼油、山茶油、山核桃油、亚麻籽油、橄榄油、葵花籽油、紫苏油等。
建议添加量为油品质量分数的0.01％～0.05％。也可将eAOB-o以一定比例(质量分数1％～20％)加到中、长链脂肪酸溶液(如辛葵酸甘油酯、月桂酸甘油酯、肉豆蔻酸甘油酯、棕榈酸甘油酯、硬脂酸甘油酯、油酸甘油酯、亚油酸甘油酯等)中，作为液态制剂使用。
本发明与现有技术相比，其优点在于：本发明的eAOB-o能够直接溶于食用油脂、中长链脂肪酸和非极性溶剂中，表现出良好的抗脂质过氧化性能和热稳定性。采用油脂氧化稳定性测定仪(Rancimat)研究130℃下eAOB-o对棕榈油抗氧化稳定性的影响，结果表明添加0.03％eAOB-o能使棕榈油的使用寿命延长1.83倍，明显优于化学酰化制备的脂溶性竹叶抗氧化物(cAOB-o)。煎炸油稳定性试验证明，eAOB-o在180℃的高温下抗油脂氧化的性能及其热稳定性显著优于市售同类产品(RE、OTP、BHA和BHT)及cAOB-o，可与目前工业界应用最为广泛的合成抗氧化剂特丁基对苯二酚(TBHQ)媲美。将本发明的eAOB-o以一定比例添加于煎炸油中，能够显著降低薯片、薯条、油条中的过氧化物值和硫代巴比妥酸值，并显著降低制品中丙烯酰胺的含量；将本发明的eAOB-o以一定比例添加于曲奇的配方中，在延长产品货架寿命的同时，能显著降低产品中丙烯酰胺(AA)和AGEs(羧甲基赖氨酸CML和羧乙基赖氨酸CEL)的含量；将本发明的eAOB-o以一定比例添加于油炸薯片的煎炸用油中，在延长产品货架寿命的同时，能显著降低产品中丙烯酰胺(AA)和杂环胺(Harman和Norharman)的含量；表现出抗脂质氧化和同步抑制多种美拉德反应伴生危害物的双重效果。
与现有技术相比，本发明的主要优点在于：
(1)本发明提供的的eAOB-o具有良好的抗脂质过氧化性能和热稳定性，能够直接溶于食用油脂、中长链脂肪酸和非极性溶剂中，采用油脂氧化稳定性测定仪(Rancimat)研究130℃下eAOB-o对棕榈油抗氧化稳定性的影响，结果表明，添加0.03％eAOB-o能使棕榈油的使用寿命延长1.83倍，与化学酰化方法制备的脂溶性竹叶抗氧化物(cAOB-o)相比明显上升。煎炸油稳定性试验证明，eAOB-o在180℃的高温下抗油脂氧化的性能及其热稳定性显著优于市售同类产品(RE、OTP、BHA和BHT)及cAOB-o，可与目前工业界应用最为广泛的合成抗氧化剂特丁基对苯二酚(TBHQ)媲美。
(2)本发明提供的eAOB-o具有优良的亲脂性能，在油脂中具有很好的溶解度，使其能直接添加于油品中，作为油脂抗氧化剂使用。
(3)本发明的eAOB-o还能够在高温加工过程中抑制油脂氧化导致的丙烯酰胺形成，同时显著降低高油、高糖、高蛋白食品中的AGEs和杂环胺含量。
(4)本发明的酶法催化可实现AOB-w中抗氧化活性成分的定向酯化，以特征性成分竹叶碳苷黄酮(荭草苷、异荭草苷、牡荆苷、异牡荆苷)为例，脂肪酸连接的位置主要发生在糖羟基上，而对抗氧化活性具有较大影响的有效酚羟基的数目没有减少，从而在增加eAOB-o的脂溶性的同时不会显著降低其抗氧化活性。同时，糖基虽然发生了修饰，但并不影响糖基与黄酮母核之间的-C＝C-键合，依然保持了碳苷黄酮高的热稳定性和抗酸解、酶解的特性。
(5)本发明的酶法催化与化学酰化法相比，在制备过程、产品性能、环境友好等方面具有前者无可比拟的优势。
(6)本发明的eAOB-o同时拥有突出的抗氧化性、热稳定性和同步抑制美拉德反应多种伴生危害物形成的能力，其在食品工业(尤其是需要高温处理的如油炸食品、焙烤食品等)中具有极为广阔的用途。除了能生产出安全、优质、保质期长的放心食品以外，还能显著延长煎炸油的使用寿命，降底生产成本。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1 酶法制备eAOB-o(无溶剂体系)
AOB-w购于杭州尤美特科技有限公司，外观为黄棕色粉末，含总酚56.53％、异荭草苷3.78％和水分5.87％。称取AOB-w 0.02mol作为反应底物，加入月桂酸0.40mol，60℃下搅拌均匀，使AOB-w完全溶解于月桂酸中；加入南极假丝酵母脂肪酶(CALB)15g(Novozym 435，购自诺维信公司，活力约为10000PLU/g，下同)，水0.5mL，65℃下催化反应12h。反应结束后，经过滤、减压浓缩，得eAOB-o粗品1.1g；将粗品用100mL乙腈-正庚烷(2:5，v/v)溶液萃取两次，收集下相，回收溶剂后再用100mL乙酸乙酯-水溶液(3:5，v/v)萃取两次，收集上层液体，减压 浓缩至干后即得到0.9g目标物eAOB-o，呈淡黄色粉末，总酚含量21.89％，对香豆酸含量为0.58％，乙酸乙酯溶解度为4.19g/100g。
【注：AOB-w平均相对分子质量以其八种特征性成分含量计算而来。本发明选用AOB-w中绿原酸、咖啡酸、异荭草苷、荭草苷、对香豆酸、牡荆苷、异牡荆苷、阿魏酸含量分别为0.73％、0.40％、3.78％、1.08％、0.79％、1.40％、0.35％和0.07％，AOB-w平均相对分子量计算为410.7。下同】
实施例2 酶法制备eAOB-o(有机溶剂体系)
同上称取AOB-w(来源同实施例1，下同)0.02mmol作为反应底物，加入100mL叔戊醇(尽可能使原料浸没于叔戊醇中，可采用超声波助溶)，加入肉豆蔻酸0.20mmol搅拌均匀，加入米黑根毛霉脂肪酶(RM IM，购自诺维信，活力约为275IUN/g)2.4g，55℃下催化反应30h。反应结束后，经过滤、减压浓缩、得eAOB-o粗品1.1g；用100mL乙酸乙酯-水(3:5，v/v)萃取粗品两次，收集上层液体，回收溶剂后再用100mL 88％乙醇-石油醚(2:5，v/v)萃取两次，收集下层液体，减压浓缩至干后得到0.7g目标物eAOB-o，呈淡黄色粉末，总酚含量20.87％，对香豆酸含量为0.53％，乙酸乙酯溶解度为4.31g/100g。
实施例3 酶法制备eAOB-o(有机溶剂体系)
同上称取AOB-w 1g作为反应底物，加入100mL丙酮(尽可能使原料浸没于丙酮中，可采用超声波助溶)，加入棕榈酸15g，45℃下疏棉状嗜热丝孢菌脂肪酶1.8g(TL IM，购自诺维信，活力约为250IUN/g)催化反应48h。反应结束后，经过滤、旋转、浓缩收集eAOB-o粗品1.7g。用125mL乙腈-正庚烷(2:5，v/v)萃取eAOB-o粗品两次，收集下层液体，浓缩至干后用125mL乙酸乙酯-水(3:5，v/v)萃取两次，收集上层液体，浓缩至干后得到精制后的1.2g目标物eAOB-o,呈淡黄色粉末，总酚含量20.45％，对香豆酸含量为0.55％，乙酸乙酯溶解度为4.82g/100g。
实施例4 酶法制备eAOB-o(有机溶剂体系)
同上称取AOB-w 1g作为反应底物，加入50mL丙酮(尽可能使原料浸没于丙酮中，可采用超声波助溶)，加入月桂酸15g，45℃下加入CALB 2g催化反应72h。反应结束后，经过滤、旋转、浓缩收集eAOB-o粗品2.2g。用125mL乙腈-正庚烷(2:5，v/v)萃取eAOB-o粗品两次，收集下层液体，浓缩至干后用125mL乙酸乙酯-水(3:5，v/v)萃取两次，收集上层液体，浓缩至干后得到精制后的1.6g目标物eAOB-o,呈淡黄色粉末，总酚含量20.13％，对香豆酸含量为0.51％，乙酸乙酯溶解度为5.70g/100g。
实施例5 酶法制备eAOB-o(离子型溶剂体系)
同上称取AOB-w 1g作为反应底物，加入月桂酸25g，60℃下搅拌均匀，使AOB-w完全溶解于5mL 1-乙基-3-甲基咪唑六氟磷酸盐[Emim][PF6]；加入南极假 丝酵母脂肪酶用量2.0g(Novozym 435，购自诺维信公司，活力约为10000PLU/g)，65℃下催化反应6h。反应结束后，经过滤、减压浓缩，得eAOB-o粗品1.2g；将粗品用100mL乙腈-正庚烷(2:5，v/v)溶液萃取两次，收集下相，回收溶剂后再用100mL乙酸乙酯-水溶液(3:5，v/v)萃取两次，收集上层液体，减压浓缩至干后即得到0.8g目标物eAOB-o，呈黄色粉末，总酚含量20.13％，对香豆酸含量为0.56％，乙酸乙酯溶解度为4.67g/100g。
实施例6 特征性碳苷黄酮的酰化产物及其结构特点
竹叶碳苷黄酮(异牡荆苷、牡荆苷、异荭草苷和荭草苷标准品购自南京景竹生物科技有限公司)各0.02mmol、月桂酸0.40mmol、固定化酶CALB 0.015g、水0.005mL混合均匀后于60℃反应72h。反应自加入固定化CALB酶起开始计时，反应结束后，离心将脂肪酶分离，终止反应。去除溶剂后得到酰化产物粗品。以乙腈/正庚烷(2:5，v/v)溶解粗品，60℃下萃取2次，收集并合并乙腈层，浓缩至干以乙酸乙酯/正庚烷(3:5，v/v)萃取2次，收集并合并乙酸乙酯层，浓缩至干后得酰化产物终产物。采用对酰化产物进行鉴别，采用超高效液相色谱(UPLC)、半制备高效液相色谱(Semi-Pre HPLC)、质谱(MS)以及核磁共振(NMR)等酰化产物进行分离及结构鉴定，鉴定结果如附图1、2中所示。
附图1所示为反应72h后四种竹叶碳苷黄酮月桂酸酯的UPLC检测图谱，可以看到，在固定化酶CALB催化作用下，经72h反应后竹叶碳苷黄酮月桂酸酯中仍残留有未反应完的竹叶碳苷黄酮。
附图2为竹叶碳苷黄酮月桂酸酯的质谱检测。异牡荆苷、牡荆苷、异荭草苷和荭草苷的相对分子量分别为432.38、432.38、448.37和448.37。对四种酶法制备的竹叶碳苷黄酮月桂酸酯的质谱检测可以看到(附图2)，异牡荆苷月桂酸酯、牡荆苷月桂酸酯、异荭草苷月桂酸酯和荭草苷月桂酸酯的分子量分别为618.15、618.17、629.15和629.19，说明在无溶剂体系中固定化酶CALB催化竹叶碳苷黄酮月桂酸酯为单酯。
采用半制备(Semi-Pre HPLC)对竹叶碳苷黄酮月桂酸酯进一步纯化后，进行1H-NMR和13C-NMR分析。
异牡荆苷月桂酸酯1H(CDC13)：δ(ppm)7.19(s，2H，H2′，H6′)，6.68(s，1H，H3′，H5′)，6.13-6.18(d，2H，H3)，6.11(s，1H，H8)，4.96(s，1H，H1")，4.34(s，1H，H6a")，4.27(s，1H，H5")，4.09(s，1H，H6b")，3.79(s，1H，H2")，3.49(s，1H，H3")，3.40(s，1H，H4")，2.32(s，2H，CH2α脂肪酸碳链)，1.64(s，2H，CH2β脂肪酸碳链)，1.26-1.31(m，16H，脂肪酸碳链)和0.88(s，3H，CH3脂肪酸碳链)。
异牡荆苷月桂酸酯13C(CDC13)：δ(ppm)196.82(C4)，173.14(C＝O)，168.23(C2)，166.43(C7)，162.67(C5)，160.85(C9)，157.46(C4′)，130.93(C1′)，127.44(C2′，C6′)，116.14(C3′，C5′)，105.81(C6)，102.69(C10)，102.52(C3)，94.43(C8)，81.52(C5")，78.41(C3")，76.64(C1")，71.75(C4")，70.87(C2")，61.33(C6")，33.94(脂肪酸碳链)，31.87(脂肪酸碳链)，29.64(脂肪酸碳链)，29.61(脂肪酸碳链)，29.60(脂肪酸碳链)，29.33(脂肪酸碳链)，29.32(脂肪酸碳链)，29.04(脂肪酸碳链)，25.03(脂肪酸碳链)， 22.68(脂肪酸碳链)，14.14(CH3，脂肪酸碳链)。
牡荆苷月桂酸酯1H(CDC13)：δ(ppm)7.19(s，2H，H2′，H6′)，6.69(s，1H，H3′，H5′)6.14-6.38(d，2H，H3)，6.07(s，1H，H6)，4.96(s，1H，H1")，4.33(s，1H，H6a")，4.28(s，1H，H5")，4.09(s，1H，H6b")，3.77(s，1H，H2")，3.48(s，1H，H3")，3.41(s，1H，H4")，2.33(s，2H，CH2α脂肪酸碳链)，1.62(s，2H，CH2β脂肪酸碳链)，1.26-1.31(m，16H，CH2脂肪酸碳链)和0.87(s，3H，CH3脂肪酸碳链)。
牡荆苷月桂酸酯13C(CDC13)：δ(ppm)196.81(C4)，173.12(C＝O)，168.21(C2)，163.43(C7)，161.04(C5)，158.68(C9)，157.41(C4′)，130.90(C1′)，127.44(C2′)，127.43(C6′)，116.13(C3′，C5′)，103.53(C8)，102.63(C10)，102.47(C3)，94.88(C6)，81.51(C5")，78.41(C3")，76.93(C1")，71.72(C4")，70.78(C2")，63.14(C6")，33.93(脂肪酸碳链)，31.92(脂肪酸碳链)，29.63(脂肪酸碳链)，29.62(脂肪酸碳链)，29.60(脂肪酸碳链)，29.32(脂肪酸碳链)，29.31(脂肪酸碳链)，29.04(脂肪酸碳链)，25.02(脂肪酸碳链)，22.70(脂肪酸碳链)，14.13(CH3，脂肪酸碳链)。
异荭草苷月桂酸酯1H(CDC13)：δ(ppm)6.93(s，1H，H2′)，6.75(s，1H，H6′)，6.71(s，1H，H5′)，6.13-6.36(d，1H，H3)，6.13(s，1H，H8)，5.30(s，1H，H5，H7)，5.11(s，1H，H2)，4.97(s，1H，H1")，4.32(s，1H，H6a")，4.29(s，1H，H5")，4.10(s，1H，H6b")，3.81(s，1H，H2")，3.46(s，1H，H3")，3.42(s，1H，H4")，2.31(s，2H，CH2α脂肪酸碳链)，1.64(s，2H，CH2β脂肪酸碳链)，1.26-1.29(m，16H，CH2脂肪酸碳链)和0.88(s，3H，CH3脂肪酸碳链)。
异荭草苷月桂酸酯13C(CDC13)：δ(ppm)196.79(C4)，173.13(C＝O)，168.42(C2)，166.44(C7)，162.57(C5)，160.83(C9)，146.20(C4′)，145.91(C3′)，131.09(C1′)，120.03(C6′)，116.23(C5′)，114.18(C2′)，105.79(C6)，102.55(C10)，102.42(C3)，94.43(C8)，81.52(C5")，78.44(C3")，76.56(C1")，71.67(C4")，70.76(C2")，63.11(C6")，33.92(脂肪酸碳链)，31.88(脂肪酸碳链)，29.64(脂肪酸碳链)，29.59(脂肪酸碳链)，29.62(脂肪酸碳链)，29.31(脂肪酸碳链)，29.32(脂肪酸碳链)，29.02(脂肪酸碳链)，25.03(脂肪酸碳链)，22.70(脂肪酸碳链)，14.13(CH3，脂肪酸碳链)。
荭草苷月桂酸酯1H(CDC13)：δ(ppm)6.92(s，1H，H2′)，6.77(s，1H，H6′)，6.71(s，1H，H5′)，6.13-6.36(d，1H，H3)，6.07(s，1H，H6)，5.51(s，1H，H2)，4.99(s，1H，H1")，4.32(s，1H，H6a")，4.25(s，1H，H5")，4.08(s，1H，H6b")，3.78(s，1H，H2")，3.48(s，1H，H3")，3.40(s，1H，H4")，2.33(s，2H，CH2α脂肪酸碳链)，1.62(s，2H，CH2β脂肪酸碳链)，1.26-1.31(m，16H，CH2脂肪酸碳链)和0.88(s，3H，CH3脂肪酸碳链)。
荭草苷月桂酸酯13C(CDC13)：δ(ppm)196.83(C4)，173.10(C＝O)，168.43(C2)，163.40(C7)，161.07(C5)，158.71(C9)，146.19(C4′)，145.88(C3′)，131.10(C1′)，120.02(C6′)，116.23(C5′)，114.20(C2′)，103.54(C8)，102.61(C10)，102.39(C3)，94.93(C6)，81.53(C5")，78.43(C3")，76.86(C1")，71.68(C4")，70.81(C2")，63.11(C6")，33.91(脂肪酸碳链)，31.90(脂肪酸碳链)，29.64(脂肪酸碳链)，29.61(脂肪酸碳链)，29.60(脂肪酸碳链)，29.30(脂肪酸碳链)，29.33(脂肪酸碳链)，29.01(脂肪酸碳链)， 25.02(脂肪酸碳链)，22.69(脂肪酸碳链)，14.12(CH3，脂肪酸碳链)。
从以上结构解析数据可知，CALB催化竹叶碳苷黄酮酰化的位点是在糖基的伯羟基上，这主要是因为糖基的伯羟基相对于其他位置的羟基更活泼，且空间位阻更小。竹叶碳苷黄酮月桂酸酯结构式如附图3所示。
实施例7 eAOB-o中对香豆酸含量测定及UPLC指纹图谱分析
(1)对香豆酸含量测定
eAOB-o试样经甲醇溶解，以乙腈-1％乙酸水溶液为流动相，用以C18为填料的液相色谱柱(Luna C18 ODS柱，柱长250mm，内径4.6mm，内装C18填充物，粒径5μm或是相当者)和紫外检测器或二极管阵列检测器，对试样中的对香豆酸进行反相高效液相色谱分离和测定，与标准品保留时间比较定性，峰面积外标法定量。
标准储备液的配置：准确称取对香豆酸标准品10mg(精确至0.0001g)，用甲醇溶解并定容至10mL，混匀，置冰箱中保存。此溶液1mL含对香豆酸1.0mg。
准确吸取一定量的对香豆酸标准溶液，分别稀释8、10、16、32、64、128和256倍，分别进样10μL，在下述规定色谱条件下，进行色谱分析，根据对香豆酸标准溶液的不同进样量及相应谱峰面积，以色谱峰面积为纵坐标、对香豆酸浓度为横坐标，绘制标准曲线。
色谱柱：Luna C18 ODS柱，柱长250mm，内径4.6mm，内装C18填充物，粒径5μm或是相当者。
参考色谱条件如下：
检测波长：330nm。柱温：40℃。流动相：A.乙腈；B.1％乙酸水溶液(v/v)。梯度洗脱条件：0～15min时，A 15％，B 85％；15～25min时，A 15％～40％，B 85％～0％；25～34min时，A 40％，B 60％；34～40min时，A 40％～15％，B 60％～85％。流量：1.0mL/min。进样量：10μL。
样品检测：准确称取eAOB-o试样100mg(精确至0.0001g)，用甲醇溶液溶解并定容至100mL，经微孔滤膜(0.45μm)过滤，即得试样溶液。准确吸取试样溶液10μL，在规定色谱条件下，进行色谱分析，以保留时间定性，峰面积外标法定量。
对香豆酸含量以w1表示，其数值以％表示，按下式计算：
Figure PCTCN2015079475-appb-000006
式中：
w1——试样中对香豆酸的含量，％；
c1——依据标准曲线计算出的被测液中的对香豆酸浓度，单位为毫克每毫升(mg/mL)；
V1——供试样定容体积，单位为毫升(mL)；
m1——供试样取样量，单位为毫克(mg)。
测定结果表明实施例1、实施例2、实施例3和实施例4制备的eAOB-o中对香豆酸含量分别为0.58％、0.53％、0.55％和0.56％。
(2)UPLC指纹图谱分析
eAOB-o试样用甲醇(色谱纯)溶解，以乙腈(色谱纯)-1％乙酸(优级纯)水溶液为流动相，用以C18为填料的超高效液相色谱柱和紫外检测器或二级管阵列检测器分析eAOB-o试样的指纹图谱。
色谱柱：ACQUITY UPLC HSS T3柱，柱长100mm，内径2.1mm，粒径1.8μm或是相当者。
参考色谱条件如下：
检测波长：260nm。柱温：40℃。流动相：A.乙腈；B.1％乙酸水溶液(v/v)。梯度洗脱条件如下：0～13min，12％A；13min～16min，12％A～40％A；16min～19min，40％A；19min～21min，40％A～12％A。流速：0.28mL/min。进样量：2μL。
称取实施例1制备的eAOB-o样品2.55mg，用甲醇(色谱纯)溶解并定容至5mL，经微孔滤膜(0.22μm)过滤，即得试样溶液。准确吸取试样溶液1μL，在上述色谱条件下，进行色谱分析。
实施例1的eAOB-o的UPLC指纹图谱分析结果见附图4。实施例2、实施例3和实施例4所得的eAOB-o试样经分析测试，所得UPLC的指纹图谱与实施例1所得的eAOB-o一致。
实施例8 食品试样中丙烯酰胺(AA)、AGEs(CML和CEL)和HAAs(Harman和Nonharman)含量的测定
(1)丙烯酰胺的测定
样品预处理：称取(1.00±0.01)g食品(曲奇或薯片等)粉末于50mL离心管中，加入400μL 13C3-AA内标(1μg/mL)，混合均匀。向样品中加入9.6mL甲酸水溶液(0.3％)，涡旋提取1min后，在0℃下，于15,000rpm离心5min。取出离心管，弃去浮在样品表面的油脂层，收集澄清液。再向样品中加入10mL甲酸水溶液(0.3％)，按以上提取步骤重复操作1次。将两次得到的澄清液(约20mL)合并，漩涡振荡混匀后，准确吸取2mL澄清液，利用事先平衡好的Oasis MCX固相萃取柱(3cc，60mg)净化。前16滴流出液弃去，收集剩余流出液，用超纯水定容至2mL，充分混匀后通过0.22μm PTFE微孔滤膜，滤液供UPLC-MS/MS测定。
色谱条件：色谱柱采用Phenomenex Synergi MAX-RP柱(150×2.0mm,4μm)。流动相A为0.3％甲酸水溶液，流动相B为乙腈，V(A):V(B)＝99:1，等梯度洗脱。流速：0.3mL/min；进样量：5.0μL；柱温：40℃；样品室温度：10℃。
质谱条件：电喷雾正离子(electrospray ionization positive，ESI+)模式。毛细管电压：3.5kV；干燥气温度：200℃；干燥气流速：16L/min；雾化气压力：45psi；鞘气温度：350℃；鞘气流速：12L/min。监测方式：MRM模式。AA：72>55(碰撞能量8eV)；13C3-AA：75>58(碰撞能量9eV)。
定量分析：以标样浓度/内标浓度为横坐标，标样峰面积/内标峰面积为纵坐标，绘制标准曲线，求曲线线性回归方程，线性范围为2～160ng/mL(2、5、10、20、40、80和160ng/mL)，相关系数0.9999。
(2)CML和CEL的同步测定
样品预处理：称取(1.00±0.01)g试样粉末于50mL离心管中，加入5mL正己烷，剧烈振荡3min后，于13,000rpm离心5min，弃去溶剂，重复以上步骤2次。经15mL正己烷脱脂3次(5+5+5mL)后，将样品利用氮气吹干至恢复粉末状。向样品中加入10mL Na2B4O7缓冲液(0.2mol/L，pH 9.2)和5mL NaBH4(1mol/L，以0.1mol/L NaOH配制)，于4℃还原过夜(约8h)。接着，加入10mL氯仿/甲醇(2:1，v/v)，于13,000rpm离心10min后，弃去上清液；向得到的沉淀物中加入20mL HCl(6mol/L)，置于110℃烘箱中，水解24h。水解完成后，将水解样品取出并冷却至室温(25℃)，用超纯水定容至50mL，然后用定性滤纸过滤并收集滤液。准确量取1mL滤液，加入0.8μg/mL d4-CML和0.8μg/mL d4-CEL内标各200μL，充分混匀后利用事先平衡好的C18固相萃取柱净化，供UPLC-MS/MS测定。
色谱条件：色谱柱采用Waters Acquity UPLC BEH C18柱(50mm×2.1mm，1.7μm)。流动相A为5mmol/L九氟戊酸水溶液，流动相B为乙腈，流动相梯度见表1。流速：0.2mL/min；进样量：7.5μL，柱温：50℃。
表1 UPLC-MS/MS检测CML、CEL的梯度洗脱表
Figure PCTCN2015079475-appb-000007
质谱条件：ESI+模式。毛细管电压：3.0kV；锥孔电压：20eV；离子源温度：130℃；脱溶剂温度：400℃。监测方式：MRM模式。CML：205>84(碰撞能量16eV)；d4-CML：209>88(碰撞能量18eV)；CEL：219>84(碰撞能量16eV)；d4-CEL：223>88(碰撞能量18eV)。
定量分析：以标样浓度/内标浓度为横坐标，标样峰面积/内标峰面积为纵坐标，绘制标准曲线，求曲线线性回归方程，线性范围为3～300ng/mL(3、10、50、100、150、200和300ng/mL)，相关系数在0.9997～0.9998之间。
(3)Harman和Norharman的同步测定
样品预处理：称取(1.00±0.01)试样粉末于50mL离心管中，加入5μg/mL的4,7,8-TriMeIQx内标工作液8μL。加入10mL甲醇/5％盐酸(v/v＝20/80)溶液，在涡旋振荡器上充分混合后，置入超声清洗机中，在室温下超声提取10分钟。取出离心管，0℃下离心(10000r/min，10min)。将PCX固相萃取柱预先用3mL甲醇、3mL 0.5％甲酸溶液活化。从上述离心管中移取5mL上清液至萃取柱中，弃去流出液；然后依次用3mL水、3mL 0.5％甲酸-甲醇淋洗，最后用3mL 5％氨水-甲醇溶液(v/v)洗脱。控制流速约为每3秒一滴，收集洗脱液并在35℃水浴下用氮气吹干，加入复溶液(5％甲酸-乙腈v/v＝95:5)，定容至2mL，过0.22μm PTFE滤膜，供UPLC-MS/MS测定。
色谱条件：色谱柱采用Waters UPLC BEH C18柱(100mm×2.1mm i.d.,1.7 μm)；流动相A为0.3％甲酸水溶液，流动相B为乙腈；流动相梯度见表2；流速：300μL/min；柱温：40℃；进样量：5μL。
表2 UPLC-MS/MS检测杂环胺的梯度洗脱表
Figure PCTCN2015079475-appb-000008
质谱条件：ESI+模式。毛细管电压：4.0kV；锥孔电压：30V；离子源温度：120℃；脱溶剂温度：400℃；监测方式：MRM模式。Harman和Norharman及内标的特征离子信息和质谱参数详见表3。
表3 Harman和Norharman及内标的特征离子信息和质谱参数
Figure PCTCN2015079475-appb-000009
*为定量离子对；4,7,8-TriMeIQx为内标
定量分析：以标样浓度/内标浓度为横坐标，标样峰面积/内标峰面积为纵坐标，绘制标准曲线，求曲线线性回归方程，线性范围为1～25ng/mL(1、2、5、10和25ng/mL)，相关系数在0.9926～0.9998之间。
实施例9 eAOB-o对曲奇中AA和CML、CEL的同步抑制作用
曲奇的制作参照美国谷物化学法协会AACC 10-54的推荐方法，基础配方如表4所示。按照0.1g/kg、0.2g/kg、0.3g/kg、0.4g/kg和0.5g/kg(以面团为基数)的添加剂量将eAOB-o(实施例1制备的试样，下同)溶解后加入配方面团中。空白对照组为不添加抗氧化剂组。面团用模具制成直径5cm、厚度3mm的曲奇坯后，在205℃下焙烤11min。焙烤后的曲奇在室温(25℃)下冷却30min后备用，采用UPLC-MS/MS测定丙烯酰胺(AA)，并同步测定羧甲基赖氨酸(CML)和羧乙基赖氨酸(CEL)。
表4 曲奇的基础配方
Figure PCTCN2015079475-appb-000010
Figure PCTCN2015079475-appb-000011
结果表明，在上述五个不同添加剂量下，eAOB-o对AA的抑制率分别为15.98％、36.32％、53.87％、48.01％和35.65％，最大抑制率出现在万分之三的添加水平(0.3g/kg)(见附图5)。
eAOB-o对曲奇焙烤过程中CML和CEL形成的抑制作用如附图6所示。不添加抗氧化剂的空白对照组中测得的CML和CEL含量分别为79.58±1.93mg/kg和11.97±0.26mg/kg。在上述五个不同添加剂量下，eAOB-o对CML的抑制率分别为36.83％、53.72％、65.42％、43.13％和25.96％，对CEL的抑制率分别为34.71％、56.74％、69.00％、31.61％和13.83％。对CML和CEL的最大抑制率均出现在万分之三(0.3g/kg)的eAOB-o添加量水平上。
实施例10 不同制备方法获得的AOB-o对棕榈油和猪油氧化稳定性的影响
称取20g不含外加抗氧化剂的棕榈油和猪油(猪板油购自当地市场后低温熬制得到)，分别按质量分数0、0.01％、0.02％、0.03％、0.04％、0.05％添加量加入eAOB-o试样，同时与化学法制备得到的脂溶性竹叶抗氧化物(cAOB-o，自制，总酚含量≥20％，专利号CN 103005007A)进行比较，超声波处理10min助溶。称取3g左右添加抗氧化剂后的油品，用油脂氧化酸败仪(Rancimat法)比较在130℃下eAOB-o和cAOB-o对棕榈油抗氧化性能的影响，空气流速设定为20L/h，测试按照GB/T 21121-2007规定的方法进行，通过保护因子(PF)的大小比较eAOB-o和cAOB-o抗氧化性能的差异。PF的计算公式如下：
Figure PCTCN2015079475-appb-000012
试验结果如附图7所示，cAOB-o和eAOB-o在0.01％-0.05％的添加剂量范围内，均能显著增强棕榈油的抗氧化稳定性。在同等添加浓度下(0.01％-0.05％五个水平)，eAOB-o增强棕榈油抗氧化稳定性均极显著优于cAOB-o(p<0.01)。对棕榈油而言，当cAOB-o添加量从0.01％增加至0.03％时，其氧化稳定性逐渐增强，添加0.01％、0.02％和0.03％cAOB-o的棕榈油的PF值分别为1.24±0.02、1.36±0.04和1.58±0.03，而当添加量继续增加，棕榈油的氧化稳定性反而下降；eAOB-o添加量从0.01％增加至0.04％时，棕榈油氧化稳定性不断增强，添加0.01％、0.02％、0.03％和0.04％eAOB-o的棕榈油的PF值分别为1.70±0.09、1.73±0.11、1.83±0.08和1.94±0.10。与cAOB-o结果类似，eAOB-o对猪油氧化稳定性的增强作用显著 高于cAOB-o。0.02％添加量的cAOB-o和eAOB-o对猪油氧化稳定性的增强作用最明显，其PF值分别为3.74±0.09和3.98±0.12。在相同添加量下，eAOB-o对棕榈油和猪油氧化稳定性的增强作用显著高于cAOB-o。
实施例11 eAOB-o与常用油脂抗氧化剂对棕榈油煎炸稳定性的影响比较
分别配置1kg分别含0.02％添加量的eAOB-o、RE(购自广州方道食品添加剂有限公司，鼠尾草酸≥20％，下同)、OTP(购自杭州普丽美地生物科技有限公司，儿茶素含量≥20％，下同)、BHT(食品级，购自上海津颂食品有限公司，有效含量≥99％，下同)、BHA(食品级，购自上海驰为食品配料实业有限公司，有效含量≥99％，下同)和TBHQ(食品级，购自东莞广益食品添加剂实业有限公司，有效含量≥99％，下同)的棕榈油试样，于180℃下持续加热36h，每隔6h取样一次，分别测定油品的过氧化值(PV)、硫代巴比妥酸值(TBARS)、酸价(AV)、茴香胺值(p-AnV)、游离脂肪酸(FFA)、极性化合物(PC)含量，测定加热前后的脂肪酸组成，并以不加任何抗氧化剂的棕榈油做空白对照。结果表明(见附图8)，0.02％的eAOB-o对棕榈油煎炸稳定性的改善明显优于同等剂量的BHT、BHA和OTP；180℃下持续加热36h后，0.02％eAOB-o与0.02％RE对油品过氧化值和茴香胺值的影响虽无明显差异，但eAOB-o表现出能显著降低油品的硫代巴比妥酸值(TBARS)、酸价(AV)和游离脂肪酸(FFA)含量。
eAOB-o与常用油脂抗氧化剂对煎炸棕榈油脂肪酸组成的影响见表5。eAOB-o抑制多不饱和脂肪酸(PUFA)降解的能力显著高于其他抗氧化剂(P<0.05)。经过36h持续加热，添加0.02％eAOB-o棕榈油中的PUFA含量为8.34％，MUFA含量为43.53％。以上结果表明，eAOB-o能够显著抑制在煎炸条件下的棕榈油中PUFA氧化。
表5 eAOB-o与常用油脂抗氧化剂对煎炸棕榈油脂肪酸组成的影响
Figure PCTCN2015079475-appb-000013
Figure PCTCN2015079475-appb-000014
实施例12 eAOB-o与cAOB-o对棕榈油煎炸稳定性影响的比较
分别配置1kg含0.02％eAOB-o和0.02％cAOB-o(自制，总酚含量≥20％，专利号CN 103005007A)的棕榈油，于180℃下持续加热36h，每隔6h取样一次，分别测定油品的PV、TBARS、AV、p-AnV、FFA和PC值，同时用不加任何抗氧化剂的纯棕榈油做空白对照。附图9显示出eAOB-o的各项指标均优于cAOB-o。
实施例13 eAOB-o与常用油脂抗氧化剂对油炸薯条中脂质氧化的抑制
分别取3kg纯棕榈油(不含外加抗氧化剂)，添加质量分数为0.02％的eAOB-o和OTP、RE和TBHQ，分别于(180±2)℃下用来煎炸薯条(市售冷冻薯条)。每批薯条用量为200g，煎炸时间为45s。待薯条冷却后粉碎，用一定量的石油醚(沸程30-60℃)浸没12h提取薯条中的脂质，测定其PV和TBARS值。从附图10可以看出，较目前常用的油脂抗氧化剂而言，eAOB-o能更为有效地控制油炸薯条的PV值；从TBARS测定结果可以看出，与空白对照和OTP组相比，eAOB-o降低薯条中TBARS的作用也更为显著。
实施例14 eAOB-o与常用油脂抗氧化剂对油条中脂质氧化的抑制
分别称取3kg纯棕榈油，添加质量分数0.01％的eAOB-o、OTP、RE和TBHQ，于180℃下炸制油条，每批加入油条胚为400g、煎炸110s。待油条冷却后粉碎，用一定量的石油醚(30℃～60℃)浸没12h提取油条中的脂质，测定PV值和TBARS值。从附图11可见，较目前常用的天然油脂抗氧化剂(OTP和RE)而言，eAOB-o的抗氧化作用更为显著，且TBARS值低于TBHQ。
实施例15 eAOB-o对薯条和油条中丙烯酰胺的抑制作用
称取3kg纯棕榈油，分别添加质量分数为0.02％的eAOB-o、OTP、RE和TBHQ试样，于180℃下炸制薯条；每批薯条用量为200g，煎炸6.5min。
称取3kg纯棕榈油，分别添加质量分数0.01％的eAOB-o、OTP、RE和TBHQ，于180℃下炸制油条，每批加入油条胚为400g、煎炸110s。
待薯条和油条样品冷却后测定丙烯酰胺含量，并与空白对照相比计算其形成抑制率。从附图12可见，添加0.02％eAOB-o后对油炸薯条丙烯酰胺的抑制率可达27.8％，显著高于其他常用抗氧化剂(OTP为12.2％，TBHQ 9.7％和RE 8.4％)；添加0.01％eAOB-o后对油炸薯条中丙烯酰胺生成的抑制率可以达到23.1％，亦显著高于其他常用油脂抗氧化剂(OTP 14.5％，TBHQ 8.3％和RE 9.2％)。以上结果说明，eAOB-o几乎完全阻断了丙烯醛途径导致的丙烯酰胺形成。
实施例16 eAOB-o和AOB-w组合抑制薯条和油条中的丙烯酰胺形成
薯条水相处理：配置0.1％AOB-w和天冬酰胺酶水溶液，将速冻薯条解冻后，浸泡45s后沥干，待炸；
薯条油相处理：称取3kg棕榈油，加入0.02％eAOB-o，混合均匀，处理同实施例15。
油条水相处理：在油条基础配方中添加0.05％AOB-w制成油条胚；
油条油相处理：称取3kg棕榈油，加入0.01％eAOB-o，混合均匀，处理同实施例15。
待薯条和油条样品冷却后，测定样品中的丙烯酰胺值，计算丙烯酰胺生成抑制率。从附图13可以看出，水溶性竹叶抗氧化物(AOB-w)和脂溶性竹叶抗氧化物(eAOB-o)的协同作用，最大程度地阻断了油炸薯条和油条中丙烯酰胺的形成，抑制率均在90％以上。
实施例17 eAOB-o和AOB-w组合抑制油炸薯片中丙烯酰胺和杂环胺的形成
复合薯片的制作：将5份马铃薯雪花全粉和3份的面粉用打蛋机充分干混后，加入6份的蒸馏水(均为质量比)，充分混合制成面团。使用手持式压面机将面团反复压制成厚1mm的均匀面皮，然后使用饼干模具压出直径4cm、厚度1mm的薯片胚(单胚重量约为1.37±0.06g)，保存于-20℃的冰箱中，油炸前提前取出，室温下放置15～20min，待炸。
复合薯片水相处理：将0.02％的AOB-w完全溶解于水中，逐步加到预混的干粉料中，充分混匀，制成面团，压制成薯片坯。
复合薯片油相处理：量取2.5L棕榈油，待油温升至一定程度后加入0.01％eAOB-o，混合均匀，空白组则不加抗氧化剂。于170℃下炸制薯片；每批薯片用量为30片(约41g)，煎炸3.0min。
油炸结束后，将薯片控干、粉碎，冷却后测定样品中丙烯酰胺含量、Harman和Norharman含量，并与空白对照相比计算其抑制率。由附图14可见，eAOB-o和AOB-w组合后对于丙烯酰胺、Harman和Norharman均有显著的抑制效果，丙烯酰胺抑制率为75.86％，Harman抑制率为45.70％，而Norharman抑制率为35.36％。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种脂溶性竹叶抗氧化物(eAOB-o)，其特征在于，所述脂溶性竹叶抗氧化物包括以下特征性成分：对香豆酸、和一种或多种5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物被酰基修饰的产物；其中，所述的5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物为取代的5,7-二羟基-4H-1-苯并吡喃-4-酮；
    所述的取代指苯并吡喃环被以下取代：苯并吡喃环的苯环部分上的任意一个氢原子被一个糖基所取代，且苯并吡喃环的2位被具有1-3个羟基取代基的苯基取代；
    其中，所述的酰基修饰指所述糖基上的羟基被C8-C18的酰基修饰基所修饰；且所述5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物与酰基修饰基的摩尔比为1:0.5-2。
  2. 如权利要求1所述的脂溶性竹叶抗氧化物，其特征在于，所述5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物与酰基修饰基的摩尔比为1:0.8-1.2。
  3. 如权利要求1所述的脂溶性竹叶抗氧化物，其特征在于，所述的酰基修饰指所述糖基上的伯羟基被C8-C18的酰基修饰基所修饰。
  4. 如权利要求1所述的脂溶性竹叶抗氧化物，其特征在于，所述5,7-二羟基-4H-1-苯并吡喃-4-酮衍生物被酰基修饰的产物选自下组：A.1、A.2、A.3、A.4、B.1、B.2、B.3、B.4、C.1、C.2、C.3、C.4；
    Figure PCTCN2015079475-appb-100001
    Figure PCTCN2015079475-appb-100002
    其中，所示的R基团是C8-C18的酰基。
  5. 如权利要求1所述的脂溶性竹叶抗氧化物，其特征在于，所述的脂溶性竹叶抗氧化物是通过以下方法制备的：
    在酶催化下，使水溶性竹叶抗氧化物(AOB-w)与C8-C18的脂肪酸反应，进行单羟基酰化，得到脂溶性竹叶抗氧化物；
    较佳地，所述的方法包括步骤：在酶催化下，用水溶性竹叶抗氧化物与C8-C18的脂肪酸在40℃～80℃下进行酰化反应，得到脂溶性竹叶抗氧化物。
  6. 一种脂溶性竹叶抗氧化物的制备方法，其特征在于，包括步骤：在酶催化下，使水溶性竹叶抗氧化物与C8-C18的脂肪酸反应，进行单羟基酰化，得到脂溶性竹叶抗氧化物；
    较佳地，所述的方法包括步骤：在酶催化下，用水溶性竹叶抗氧化物与C8-C18的脂肪酸在40℃～80℃下进行酰化反应，得到脂溶性竹叶抗氧化物。
  7. 如权利要求6所述的制备方法，其特征在于，所述的步骤在无溶剂条件下进行，或所述的步骤在有机溶剂或离子型溶剂中进行；
    较佳地，所述的有机溶剂选自下组：叔戊醇(优选为水饱和叔戊醇)、丙酮、乙酸乙酯、叔丁醇，或其组合；
    所述的离子型溶剂为阳离子和阴离子组成的盐，其中，所述的阳离子为未取代或被C1-C8的烷基取代的咪唑，且所述的阴离子选自下组：BF4 -、PF6 -、TFSI-，或其组合。
  8. 如权利要求6所述的制备方法，其特征在于，所述的酶选自下组：南极假丝酵母脂肪酶、枯草杆菌蛋白酶、米黑根毛霉脂肪酶、假单胞菌脂肪酶、疏棉状嗜热丝孢菌脂肪酶，或其组合；优选南极假丝酵母脂肪酶；和/或
    所述的C8-C18的脂肪酸选自下组：辛酸、癸酸、月桂酸、肉豆蔻酸、棕榈酸、油酸、亚油酸、硬脂酸，或其组合。
  9. 一种食品添加剂，其特征在于，所述的食品添加剂含有如权利要求1-5任一所述的脂溶性竹叶抗氧化物，和任选的食品上可接受的载体。
  10. 如权利要求9所述的食品添加剂，其特征在于，所述的食品添加剂为如权利要求1-5任一所述的脂溶性竹叶抗氧化物溶于中长链脂肪酸形成的食品添加 剂。
  11. 如权利要求9所述的食品添加剂，其特征在于，所述的食品添加剂为含质量分数1～20％的脂溶性竹叶抗氧化物的中长链脂肪酸酯溶液；其中，所述的中长链脂肪酸选自下组：辛葵酸甘油酯、月桂酸甘油酯、肉豆蔻酸甘油酯、棕榈酸甘油酯、硬脂酸甘油酯、油酸甘油酯、亚油酸甘油酯，或其组合。
  12. 一种如权利要求1-5任一所述的脂溶性竹叶抗氧化物的用途，其特征在于，用于选自下组的一种或多种用途：
    (a)作为高温食品加工的抗氧化剂；
    (b)作为高温食品加工的丙烯酰胺抑制剂；
    (c)用于制备高温食品加工的抗氧化剂；
    (d)用于制备高温食品加工的丙烯酰胺抑制剂；
    (e)在高温食品加工工艺中用于抑制丙烯酰胺和/或晚期糖基化终末产物和/或杂环胺的生成；
    (f)作为油脂抗氧化剂。
  13. 一种食品加工方法，其特征在于，包括步骤：在加工过程之前、之中或之后，在食品原料中添加如权利要求1-5任一所述的脂溶性竹叶抗氧化物，或添加如权利要求9所述的食品添加剂。
  14. 如权利要求13所述的方法，其特征在于，所述的食品加工方法还包括：在加工过程之前、之中或之后，添加水溶性竹叶抗氧化物(AOB-w)。
  15. 一种食品，其特征在于，所述的食品在加工过程中添加如权利要求1-5任一所述的脂溶性竹叶抗氧化物或含有如权利要求1-5任一所述的脂溶性竹叶抗氧化物的食品组分。
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CN112451434A (zh) * 2020-11-09 2021-03-09 刘德康 一种玫瑰花提取物保湿面霜及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1528197A (zh) * 2003-10-08 2004-09-15 ƽ 竹叶抗氧化物及其用途
CN1663477A (zh) * 2005-03-21 2005-09-07 浙江大学 应用竹叶提取物作为热加工食品中丙烯酰胺抑制剂的方法
CN103005007A (zh) * 2012-12-25 2013-04-03 浙江大学 脂溶性竹叶抗氧化物及其制备方法
CN103045674A (zh) * 2012-12-10 2013-04-17 禇慧林 黄酮碳苷类6"羟基-酯衍生物的制备方法和用途
CN103131741A (zh) * 2013-02-27 2013-06-05 成都伟晖生物科技有限公司 一种活性植物多酚类物质分子的生物修饰方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1911101A (zh) * 2006-08-25 2007-02-14 胡林福 一种从竹叶中提取竹叶抗氧化物的生产方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1528197A (zh) * 2003-10-08 2004-09-15 ƽ 竹叶抗氧化物及其用途
CN1663477A (zh) * 2005-03-21 2005-09-07 浙江大学 应用竹叶提取物作为热加工食品中丙烯酰胺抑制剂的方法
CN103045674A (zh) * 2012-12-10 2013-04-17 禇慧林 黄酮碳苷类6"羟基-酯衍生物的制备方法和用途
CN103005007A (zh) * 2012-12-25 2013-04-03 浙江大学 脂溶性竹叶抗氧化物及其制备方法
CN103131741A (zh) * 2013-02-27 2013-06-05 成都伟晖生物科技有限公司 一种活性植物多酚类物质分子的生物修饰方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113698989A (zh) * 2021-07-28 2021-11-26 暨南大学 一种适用高含水方便面的煎炸油组合物及其制备方法
CN113698989B (zh) * 2021-07-28 2023-06-13 暨南大学 一种适用高含水方便面的煎炸油组合物及其制备方法

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