WO2006099798A1 - A method for using bamboo leaf extract as acrylamide inhibitor for heat processing food - Google Patents

A method for using bamboo leaf extract as acrylamide inhibitor for heat processing food Download PDF

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WO2006099798A1
WO2006099798A1 PCT/CN2006/000431 CN2006000431W WO2006099798A1 WO 2006099798 A1 WO2006099798 A1 WO 2006099798A1 CN 2006000431 W CN2006000431 W CN 2006000431W WO 2006099798 A1 WO2006099798 A1 WO 2006099798A1
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extract
acrylamide
bamboo leaf
food
leaf extract
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English (en)
French (fr)
Inventor
Ying Zhang
Xiaoqin Wu
Yu Zhang
Genyi Zhang
Dingding Luo
Yi Dong
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ZHEJIANG UNIVERSITY (HANGZHOU) INNOESSEN BIO-TECHNOLOGY Co Ltd
Zhejiang University ZJU
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ZHEJIANG UNIVERSITY (HANGZHOU) INNOESSEN BIO-TECHNOLOGY Co Ltd
Zhejiang University ZJU
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Priority claimed from CNB2005100494017A external-priority patent/CN100384349C/zh
Priority claimed from CNB2005100494021A external-priority patent/CN1313036C/zh
Application filed by ZHEJIANG UNIVERSITY (HANGZHOU) INNOESSEN BIO-TECHNOLOGY Co Ltd, Zhejiang University ZJU filed Critical ZHEJIANG UNIVERSITY (HANGZHOU) INNOESSEN BIO-TECHNOLOGY Co Ltd
Priority to US11/909,265 priority Critical patent/US8206766B2/en
Publication of WO2006099798A1 publication Critical patent/WO2006099798A1/zh
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A23B2/733—Compounds of undetermined constitution obtained from animals or plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B4/00—Preservation of meat, sausages, fish or fish products
    • A23B4/10—Coating with a protective layer; Compositions or apparatus therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B4/00—Preservation of meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by group A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
    • A23B7/16—Coating with a protective layer; Compositions or apparatus therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F5/00—Coffee; Coffee substitutes; Preparations thereof
    • A23F5/10—Treating roasted coffee; Preparations produced thereby
    • A23F5/14—Treating roasted coffee; Preparations produced thereby using additives, e.g. milk or sugar; Coating
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/30—Removing undesirable substances, e.g. bitter substances
    • A23L11/34—Removing undesirable substances, e.g. bitter substances using chemical treatment, adsorption or absorption
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A23L19/10—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops
    • A23L19/12—Products from fruits or vegetables; Preparation or treatment thereof of tuberous or like starch containing root crops of potatoes
    • A23L19/18—Roasted or fried products, e.g. snacks or chips
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
    • A24B15/30—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances
    • A24B15/302—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances by natural substances obtained from animals or plants

Definitions

  • the invention relates to the field of hot processed food safety; in particular to the application of natural bamboo leaf extracts containing flavonoids and phenolic acids as main chemical constituents as acrylamide inhibitors in hot processed foods. Background technique
  • Food safety is a major problem related to life health and national economy and people's livelihood. Its decisive factor is food hazard. Therefore, solving food safety problems should start with food hazards, that is, three kinds of food hazards: physical hazards, chemical hazards and microbial hazards. Physical hazards are easy to monitor and prevent, microbial hazards need to be controlled in the food processing process, and chemical hazards (such as pesticides, veterinary drug residues, etc.) are added in the general sense due to human factors in the growth of food raw materials. The source implements control. The most embarrassing thing is that certain chemical hazards are not derived from the external environment, but are naturally formed during food processing, such as acrylamide in hot processed foods. .
  • Acrylamide is a recognized neurotoxin and carcinogen [JIFSAN/NCFST. Workshop "Acrylamide in food, scientific issues, uncertainties, and research strategies' " 28- 30th October 2002. Rosemont, USA] , animal experiments show long-term exposure In the environment of acrylamide, not only the pathological changes of the nervous system but also various cancers may be caused. Further research indicates that this chemical hazard is not present in food materials, but is formed during food processing [Mottram, DS, et al. Acrylamide is formed in the Mai Hard reaction. Nature, 2002, 419: 448- 449; Stadler, RH, et al. Acrylamide from Maillard reaction products. Na ture, 2002, 419: 449-450].
  • Acrylamide is a white crystal that is soluble in water, ethanol, methanol, dimethyl ether, acetone, and is insoluble in non-polar solvents such as heptane and benzene. Its -unsaturated amino system is very easy and nucleophilic (eg The thiol group of cysteine in the protein is chemically affected by the Michael Addition, which affects the normal function of the protein and causes disease.
  • the Maillard reaction is a complex chemical reaction of reducing sugars and amino acids or free amino groups in proteins under high temperature conditions. It is one of the important ways to produce flavors of hot processed foods.
  • the Maillard reaction mainly has three reaction stages. The first stage is a Schiff base with an "ON" bond formed by a few groups of a reducing sugar and an amino group of an amino acid, which is rearranged to form an Amadori or Heyns product; the second stage is Amadori. Or Heyns products are degraded into various flavor compounds and intermediates by different routes; the final stage is the formation of Maillard reaction brownish yellow matter.
  • the mechanism by which asparagine participates in the Maillard reaction to form acrylamide is called the asparagine pathway.
  • the initiation of the asparagine pathway is the initial stage of the Maillard reaction.
  • Schiff base intermediate in the homeostasis with the 7-glycosyl amino acid
  • two different reaction routes can lead to the production of acrylamide:
  • One continues the Maillard reaction Schiff base is rearranged by Amadori to form Amadori product, which is then dehydrated and deaminated to form a carbonyl-containing product.
  • Asparagine in the presence of these carbonyl-containing molecules can be decarboxylated and deaminated by Strecker degradation mechanism.
  • this reaction mechanism can be called the Strecker pathway; the other is the intramolecular cyclization of Schiff base to form oxazolone, which forms the decarboxylated Amadori product, the "C-N” bond of this product It is broken at high temperature to form acrylamide.
  • This reaction mechanism can be called glycoside [glycoside] pathway [Zhang Genyi. Formation mechanism and risk analysis of acrylamide in hot processed food. Journal of Wuxi University of Light Industry, 2003, 22 (4) : 91 -99].
  • acrylamide can be reduced or inhibited by changing or rationally controlling the processing properties of the food or adding other components.
  • PCT patent, W0 2004/028278 A2 Jung, MY et al. Method for J.
  • Amino acids include cysteine, lysine, glycine, histidine, alanine, methionine, glutamic acid, aspartic acid, proline, phenylalanine, proline and triterpenes [Elder, VA et al. Method for reducing acrylamide formation in thermally processed foods. PCT patent, WO 2004/075655 A2]. Although these methods may theoretically have different degrees of inhibition on the production of acrylamide, it is difficult to meet practical needs in terms of practicality of the method, requirements for color and flavor of food, and food safety. New ways to reduce the formation of acrylamide and maintain the original flavor and texture of the food are yet to be explored.
  • Flavonoids are an important food function factor. They are widely found in medicinal plants, vegetables and fruits. They have strong biological antioxidant activity and have obvious control effects on cardiovascular and cerebrovascular diseases, tumors and diabetes.
  • the use of phytoflavonoids to inhibit the formation of acrylamide in food systems may be a relatively useful method, as it combines both food safety and functional foods that are important for life and health.
  • bamboo leaf extract (bamboo leaf antioxidant) recently developed by the inventors was approved by the Ministry of Health in April 2004 to be included in the "Hygienic Standard for the Use of Food Additives of the People's Republic of China" (GB-2760).
  • the bamboo leaf antioxidant (A0B), as described in Patent Application No. 200310107871. 5, is a natural phenolic part extracted from bamboo leaves, with flavonoids and phenolic acids as the main chemical components, including four main components.
  • the bamboo leaf carbon flavonoids and three phenolic acids are valerin, isoorientin, vitexin, isovite and chlorogenic acid, ferulic acid, caffeic acid. Its molecular structure is as follows:
  • a bamboo leaf extract for the preparation of a acrylamide inhibitor in a hot processed food.
  • the inhibitor contains from 1 to 99% by weight (preferably from 34 to 95% by weight) of bamboo leaf extract.
  • the inhibitor further comprises Ginkgo biloba extract, tea extract, rosemary extract, apple polyphenol extract, hawthorn extract, onion extract, licorice extract, puerarin extract, At least one of grape seed extract and burdock extract.
  • the inhibitor contains 34 to 95% by weight of bamboo leaf extract, and 5 to 66% by weight of the natural extract (e.g., plant extract or animal extract).
  • the natural extract e.g., plant extract or animal extract.
  • the bamboo leaf extract is an aqueous extract or an alcohol extract of bamboo leaves. More preferably, the flavonoid content is 4-50% (aluminum nitrate-sodium nitrite colorimetric method, using rutin as a standard), and the total phenol content is 10-80% (Fulin reagent reduction colorimetric method, to Hydroxybenzoic acid is a standard).
  • a method of thermally processing a food product comprising the steps of:
  • the inhibitor further comprises Ginkgo biloba extract, tea extract, rosemary extract, apple polyphenol extract, hawthorn extract, onion extract, licorice extract, puerarin extract, At least one of grape seed extract and burdock extract.
  • the food to be processed comprises French fries, potato chips, wafers, biscuits, cakes, breads obtained by frying, baking, grilling, baking, microwave heating, puffing, burning. , cereal breakfast, fried dough sticks, flatbread, instant noodles, hamburger, fried chicken nuggets, coffee, cocoa, tobacco, cigarettes.
  • each kilogram of the food to be processed in step (a) is mixed with 0.003 to 1-5 grams of the acrylamide inhibitor. In another preferred embodiment, each kilogram of the food to be processed in step (a) is mixed with 0.01 to 1 gram of the acrylamide inhibitor. In another preferred embodiment, each kilogram of the food to be processed is mixed with 0.11 g of the acrylamide inhibitor in step (a). In another preferred embodiment, in step (a), the mixing step is carried out by means of wrapping, soaking or spraying.
  • the acrylamide inhibitor is added to the package to form a mixed package, and the mixed package is used to uniformly wrap the raw material to be processed.
  • 0.001 1-5 g of the acrylamide inhibitor is added per kg of the package.
  • 0.1-1 g of the acrylamide inhibitor is added per kg of the package.
  • the acrylamide inhibitor is added to an aqueous solution or a low alcohol solution to prepare an infusion solution, and the soaking liquid is used to soak the food to be processed.
  • 0.001 1-5 g of the acrylamide inhibitor is added to make an infusion solution per liter of the aqueous solution or the low alcohol solution.
  • 0.1 to 1-1 g of the acrylamide inhibitor is added per liter of the aqueous solution or the low alcohol solution to prepare a soaking liquid.
  • the acrylamide inhibitor is added to an aqueous solution or a low alcohol solution to prepare a spray liquid, and the spray liquid is used to uniformly spray the surface of the food to be processed.
  • 0.005 to 1-5 g of the acrylamide inhibitor is added to prepare a spray liquid per liter of the aqueous solution or the low alcohol solution.
  • 0.1 to 1-1 g of the acrylamide inhibitor is added per liter of the aqueous solution or the low alcohol solution to prepare a spray liquid.
  • an acrylamide inhibitor composition for use in a thermally processed food comprising 1 - 99% by weight of bamboo leaf extract and 1 - 99% by weight of Ginkgo biloba extract, tea extract, rosemary At least one of an extract, an apple polyphenol extract, a hawthorn extract, an onion extract, a licorice extract, a pueraria extract, a grape seed extract, and a burdock extract.
  • the acrylamide inhibitor provided by the present invention can effectively inhibit acrylamide produced in the thermal processing of foods.
  • Figure 1 is a GC spectrum of an acrylamide standard
  • Figure 2 is a GC chromatogram of acrylamide produced by the asparagine pathway in a blank control group
  • Figure 3 is a GC spectrum of acrylamide in test group 1 (with bamboo leaf extract (A0B) content of 10 mg/kg);
  • Figure 4 is a GC chromatogram of acrylamide in test group 2 (with bamboo leaf extract (A0B) content of 150 mg/kg);
  • Figure 5 is a GC spectrum of acrylamide produced by frying a group of potato chips after frying;
  • Figure 6 is a GC spectrum of acrylamide after deep-frying of group B potato chips (potato chips soaked in an aqueous solution containing lg/L bamboo leaf extract);
  • Figure 7 is a GC spectrum of acrylamide after deep-frying of Group C potato chips (potato chips soaked in an aqueous solution containing lg/L tea extract);
  • Figure 8 is a LC-MS/MS spectrum of acrylamide and 13 C-labeled acrylamide standards
  • Figure 9 is a LC-MS/MS spectrum of the acrylamide obtained by frying the outer surface of the group A chicken wings (with no added ingredients in the fried chicken);
  • Figure 10 is a IX-MS/MS spectrum of acrylamide after frying on the outer surface of group B chicken wings (containing 94 g/kg bamboo leaf extract in fried chicken);
  • Figure 11 is a LC-MS/MS spectrum of acrylamide after frying on the outer surface of group C chicken wings (containing 94 g/kg tea extract in fried chicken);
  • Fig. 12 is a LC-MS/MS spectrum of acrylamide after being fried in the outer surface of the group D chicken wings (containing 4.9 g/kg rutin in fried chicken). detailed description
  • a composition comprising a bamboo leaf extract can effectively inhibit the toxic acrylamide produced during the thermal processing of food.
  • other extracts e.g., ginkgo extract, tea extract, etc.
  • the acrylamide inhibitor composition of the present invention may be coated, immersed or sprayed on the food to be processed during the food thermal processing.
  • bamboo leaf extract, acrylamide inhibitor containing bamboo leaf extract, and a compound containing bamboo leaf extract can be used as an acrylamide inhibitor in the preparation of a hot processed food.
  • the acrylamide inhibitor containing the bamboo leaf extract and the compound containing the bamboo leaf extract are used interchangeably, and the composition contains the bamboo leaf extract and the ginkgo extract, the tea extract, and the rosemary extract. At least one of an extract of apple, apple polyphenol extract, hawthorn extract, onion extract, licorice extract, puerarin extract, grape seed extract and burdock extract.
  • the bamboo leaf extract referred to in the present invention is a natural bamboo leaf extract obtained from the leaves of Gramineae (frs M' ⁇ e), Bambusoideae, and the Phyllostachys Sieb. Et Zucc variety, and the production process thereof is applied.
  • the two previous invention patents (patent numbers ZL 98104563. 4 and ZL 98104564. 2, respectively) have been addressed.
  • the bamboo leaf extract referred to in this patent may be obtained by using the above-mentioned patented process, or may be further purified by using high-tech and combination methods such as adsorption-desorption and membrane separation.
  • bamboo leaf extract [including bamboo leaf antioxidant (A0B)] products.
  • the bamboo leaf extract has a yellow or brownish yellow powder (also in the form of an extract), and its main components include Orientin, Homoorientin, Vitexin and Isovitexin is a representative flavonoid compound and a phenolic acid compound represented by chlorogenic acid, ferulic acid, and caffeic acid.
  • the total flavonoid content is generally 4-50% (aluminum nitrate-sodium nitrite colorimetric method, using rutin as a standard), wherein the total phenol content is generally 10-80% (Fulin reagent reduction colorimetric method, For light benzoic acid as a standard).
  • the reaction is carried out in an equimolar ratio of asparagine and glucose, and the reaction system may be an aqueous solution or a mixture of wetted bodies, and heated at a temperature of 120 ° C. -40 min, thereby producing acrylamide, and measuring the amount of acrylamide produced.
  • the reaction system is used as a blank control, and based on this, the acrylamide inhibitor containing the bamboo leaf extract in the added range declared by the present invention is added, and the amount of acrylamide produced is measured under the same conditions, and the blank is compared with the blank control. For comparison, the inhibition rate of acrylamide inhibition by acrylamide inhibitor containing bamboo leaf extract was calculated.
  • the determination of acrylamide was carried out by GC or LC-MS/MS.
  • the first is to directly add the acrylamide inhibitor containing the bamboo leaf extract to the food raw material or the package to make it act in the thermal processing process.
  • the asparagine pathway blocks the reaction chain that produces acrylamide, thereby reducing or inhibiting the production of acrylamide during hot processing.
  • the acrylamide inhibitor containing bamboo leaf extract is proportioned into an aqueous solution or a small amount of ethanol or The cooking wine is made into a low alcohol solution for soaking the food material or evenly spraying on the surface of the food material.
  • the actual heat processing system without adding the acrylamide inhibitor containing the bamboo leaf extract is used as a blank control, and the test group is added.
  • the acrylamide inhibitor containing the bamboo leaf extract in the range of the invention was added, the amount of acrylamide was measured by heating under the same conditions, and compared with the above blank control, the acrylamide inhibitor containing the bamboo leaf extract was calculated.
  • the determination of acrylamide was carried out by GC or LC-MS/MS.
  • the sample must be derivatized to improve the degree of volatilization before gas chromatography.
  • KBr and KBr0 3 are used to generate bromine molecules by redox reaction, and bromine molecules are reacted with acrylamide to form monobromo or dibromopropionamide.
  • N3 ⁇ 4S 2 0 3 is added to terminate the derivatization reaction and reduce the excess desert to Br.
  • Capillary voltage 3. 50kV ; cone voltage: 50V; source temperature: 100 ° C ; solvent removal temperature: 350 ° C ; cone gas flow rate: 45 L / h; desolvation gas flow rate: 400 L / h;
  • MRM parameters acrylamide standard 72>55, 13 C 3 -acrylamide internal standard 75>58;
  • Adding natural antioxidants such as flavonoids and phenolic acids to foods as acrylamide inhibitors can also play a dual role in nutritional enhancement and prolonging the shelf life of foods while ensuring food safety.
  • the bamboo leaf extract is the bamboo leaf antioxidant (product code A0B) produced by Hangzhou Zhejiang Biotechnology Co., Ltd., and the appearance is a brownish yellow powder, the total flavonoid content is 32.5%, and the total phenol content is 56.7%.
  • Figure 2 is a GC chromatogram of acrylamide produced by the asparagine pathway in the blank control group;
  • Figure 3 is a GC chromatogram of acrylamide produced by the test group 1 (A0B concentration of 10 mg/kg);
  • Figure 4 is the experimental group 2 (A0B concentration is 150 mg) /k g ) produces a GC map of acrylamide.
  • the amount of acrylamide produced by the peak area was 6460.31 g/kg, 410.10 g/kg and 190.66 g/kg, respectively.
  • the inhibition rate of A0B on the acrylamide produced by the simulated reaction system was 93. 6% and 97.0% (as shown in Table 1).
  • Potato chips Fresh potatoes are washed, peeled and cut into thin slices of approximately 1 awake. The sliced potato chips are selected and rinsed twice with running water and blotted dry with absorbent paper.
  • Tea extract A water-soluble tea polyphenol preparation for the Department of Tea Science, Zhejiang University, with a content of 98%.
  • the bamboo leaf extract was formulated into an aqueous solution having a mass content of lg/L, i.e., 1 gram of bamboo leaf extract was added per liter of water, and then the potato pieces were immersed in an aqueous solution of bamboo leaf extract for an immersion time of 1 min.
  • This group was set as the bamboo leaf extract dosage group B.
  • the tea extract is formulated into an aqueous solution having a mass content of lg/L, that is, 1 gram of the water-soluble tea polyphenol preparation is added per liter of water, and then the potato pieces are immersed in the aqueous solution of the tea extract, and the immersion time is lmir! .
  • This group is set to the tea extract dose group (.
  • each of the B and C groups is 5. 5min, each of the B and C groups is 5. 5min. .
  • the dried potato chips A, B and C are respectively fried in the oil pan.
  • the oil used is commercially available peanut oil, and the oil temperature is controlled at 140-160°. Between C, the frying time is about 3 minutes, and the surface of the potato chips is golden brown or brownish yellow, which is taken out, drained, and tested; new oil is activated after each group is fried.
  • the potato chips were subjected to the above pretreatment and the content of acrylamide was determined by GC.
  • Figure 5 is a GC spectrum of acrylamide produced by frying a group of potato chips
  • Figure 6 is a batch of potato chips (potato chips soaked with an aqueous solution containing lg/L bamboo leaf extract) to produce acrylamide by frying.
  • GC map is a GC chromatogram of a group C potato chips (potato chips soaked in an aqueous solution containing lg/L tea extract) by frying to produce acrylamide.
  • the inhibition rates of acrylamide produced by the bamboo leaf extract and the tea extract on the fried potato chips were 95.7% and 73. 4%, respectively (as shown in Table 3).
  • bamboo leaf extract dose (Group B) 24. 11 + 2. 46 95. 7 + 0. 3 Tea extract dose (group c) 147. 85 ⁇ 14.33 73. 4 ⁇ 2. 8 It can be seen from Table 3 that bamboo leaf extract and tea polyphenol have significant inhibitory effects on acrylamide produced by sleeve fried potato chips. And the inhibition effect of bamboo leaf extract is better than that of tea polyphenol.
  • Example 3 a test group in which a flavonol glycoside compound (rutin) was added, and the concentration of the bamboo leaf extract (A0B) soaking solution was increased to 5 g/L, that is, 5 g per liter of water was added. A0B.
  • the source of the bamboo leaf extract was the same as that of Example 1.
  • the source of the tea extract was the same as in Example 3.
  • Rutin is a standard purchased from Sigma and its purity is ⁇ 95%.
  • the oil used was the same as in Example 3, and the oil temperature was controlled between 140 and 160 ° C.
  • the method of sample pretreatment was the same as that in Example 3.
  • the content of acrylamide was determined by LC-MS/MS.
  • the inhibition rates of bamboo leaf extract, tea extract and rutin on the production of acrylamide on fried potato chips were calculated according to the internal standard method. 40.0%, 37.7% and 39.7% (as shown in Table 4).
  • bamboo leaf extract, tea extract and rutin all have a certain inhibitory effect on acrylamide produced by fried potato chips, but compared with Example 3, the inhibitory effects are significantly decreased, indicating that inhibition There is not a simple linear relationship between the effect and the amount of additive. It is indicated that the inhibitory effect of flavonoids on acrylamide in fried potato chips does not increase with the addition amount, but there is an optimum addition range, which varies with the food system and processing conditions.
  • Coffee green beans are commercially available products, and bamboo leaf extract (A0B) is of the same source as in Example 1.
  • the immersion time is 1 min, and the immersion time is 1 min, and the immersion time is 1 min, and the immersion time is 1 min, and the immersion time is 1 min.
  • the coffee beans are for the bamboo leaf extract dose group 8.
  • the coffee beans in the B and B groups were baked and the baking temperature was controlled between 190 and 200 ° C.
  • the roasted coffee is pulverized and sufficiently extracted with hot water, and spray-dried to obtain a coffee product.
  • the content of acrylamide was determined by LC-MS/MS after pretreatment of the coffee product, and the sample pretreatment method was the same as in Example 3. According to the internal standard method, the inhibition rate of the bamboo leaf extract on the production of acrylamide in roasted coffee was 85.0% (as shown in Table 6).
  • the bamboo leaf extract is produced by Hangzhou Zhejufu Biotechnology Co., Ltd., the total flavonoid content is 16.5%, the total phenol content is 33.7%; the source of tea extract and rutin is the same as in Example 4; The oil source was the same as in Example 3; the fried chicken was a commercially available product.
  • the sample pretreatment method was the same as in Example 3. After the sample was pretreated, the content of acrylamide was determined by LC-MS/MS. (4) Results
  • Figure 9 is a LC-MS/MS spectrum of A group of chicken wings (no added ingredients in fried chicken) with flour on the outer surface and flour to produce acrylamide;
  • Figure 10 is a LC-MS/MS spectrum of B group of chicken wings (containing 94 g/kg bamboo leaf extract in fried chicken) with flour on the outer surface and flour to produce acrylamide;
  • Figure 11 is an LC-MS/MS spectrum of the acrylamide obtained by frying the outer surface of the group C chicken wings (containing 94 g/kg tea extract in fried chicken) after frying;
  • Figure 12 is an LC-MS/MS spectrum of the group A chicken wings (containing 94 g/kg rutin in fried chicken) wrapped with flour on the outer surface and fried to produce acrylamide.
  • the inhibition rates of the bamboo leaf extract, the water-soluble tea polyphenols and the rutin on the acrylamide produced by the fried chicken wings were 38.3%, 32.5% and 22.5% respectively. Show).
  • bamboo leaf extract, tea extract and rutin have a certain inhibitory effect on acrylamide produced by fried chicken wings.
  • the bamboo leaf extract was obtained in the same manner as in Example 1. In order to find out the inhibitory effect of the non-dose bamboo leaf extract and the encapsulation on the production of acrylamide in fried chicken wings, six different additions of bamboo leaf extract were set in the actual frying system. The sample pretreatment method was the same. Example 3, after pretreatment of the sample, the content of acrylamide was determined by LC-MS/MS.
  • the source and content of the bamboo leaf extract were the same as in Example 1, and the sources and contents of the tea extract and rutin were the same as in Example 4.
  • Blank control group A (the flour is not treated);
  • the fermented dough of each group is kneaded into strips, cut into small pieces, and then pulled into a strip shape and then fried in a frying pan. After the golden color and bulking, the fritters are removed from the oil pan and the oil is drained.
  • the sample pretreatment method was the same as in Example 3. After the sample was pretreated, the content of acrylamide was determined by LC-MS/MS.
  • the bamboo leaf extract was obtained in the same manner as in Example 1. In order to determine the inhibitory effect of different doses of bamboo leaf extract on the production of acrylamide from fritters, six different levels of addition were set in the actual frying system.
  • the sample pretreatment method was the same as in Example 3. The sample was pretreated and its acrylamide content was determined by LC-MS/MS.
  • Test group 1 0.002 184.12 + 10.23 8.5 ⁇ 1.0 Test group 2 0.01 123.94 + 8.66 38.4 ⁇ 7.0 Test group 3 0.1 67.70 + 4.86 66.4 ⁇ 5.5 Test group 4 1 34.41 ⁇ 2.98 82.9 ⁇ 6.9 Test group 5 2.5 74.52 Soil 9.44 63.0 + 5.8 Test group 6 4.9 141.00 soil 11.01 29.9 + 5.3 It can be seen from Table 10 that when the amount of bamboo leaf extract (AOB) added in the fritters is in the range of 002-4. 9g/kg, the acrylamide produced by the fritters after mixing with the raw materials has different degrees of inhibition. The inhibition rate is increased with the addition amount of A0B being 0. 002 lg/kg, and decreasing with the addition amount, but decreasing with the addition amount when the addition amount is 1-4. 9g/L, which indicates that there is one Optimal range of additions.
  • AOB bamboo leaf extract
  • bamboo Leaf Extract The product produced by Hangzhou Zheqiangfu Biotechnology Co., Ltd. (product code is EOB-S03), the appearance is dark brown concentrate, the total flavonoid content is 4.5%, and the solid content is 25. 2%.
  • Tea extract a water-soluble tea polyphenol preparation for the Department of Tea Science, Zhejiang University, with a content of 98%;
  • Rutin The purity of the standard purchased from Sigma is 95%.
  • blank control group A is a cigarette made of common tobacco.
  • the bamboo leaf extract is prepared in an aqueous solution of 4. 9 g / L, that is, in an aqueous solution containing 4.9 g of the bamboo leaf extract per liter of the aqueous solution, and then the solution is uniformly sprayed to the cut tobacco at a ratio of 10 mL / kg.
  • the surface, made of cigarettes was test group B.
  • the tea extract is prepared to have an aqueous solution of 4.9 g/L, that is, a tea extract of 4.9 g on a dry basis per liter of the aqueous solution, and then the solution is uniformly sprayed to the surface of the tobacco at a ratio of 10 mL/kg.
  • the finished cigarette is the test group (.
  • the rutin is prepared in an aqueous solution of 4.9 g/L, that is, 4.9 g of rutin is added per liter of the aqueous solution, and then the solution is uniformly sprayed onto the surface of the shredded tobacco at a ratio of 10 mL/kg.
  • the cigarette was test group D.
  • the smoking test of cigarettes was carried out under standard conditions using a smoking machine, and the tar phase and the gas phase were separately collected to determine the content of acrylamide in the tar phase.
  • the tar phase sample was pretreated in the same manner as in Example 3. The sample was pretreated and its acrylamide content was determined by LC-MS/MS.
  • the relative inhibition rates of bamboo leaf extract, tea extract and rutin on acrylamide production in tobacco were 67.8%, 55.0% and 65.3%, respectively (as shown in Table 11).
  • bamboo leaf extract, tea extract and rutin have different degrees of inhibition on acrylamide produced during tobacco combustion.
  • the bamboo leaf extract was obtained in the same manner as in Example 11.
  • blank control group A is a cigarette made of common tobacco.
  • the bamboo leaf extract is formulated into a low alcohol solution having a concentration of 10 g/L, that is, in a low alcohol solution per liter (low alcohol in this embodiment)
  • the solution refers to an alcohol solution having a volume fraction of 20%) containing 10 grams of bamboo leaf extract on a dry basis, and then uniformly spraying the solution to the surface of the tobacco at a ratio of 10 mL/kg, and the prepared cigarette is the test group D. .
  • the bamboo leaf extract was adjusted to a low alcohol solution having a concentration of lg/L, that is, 1 gram of bamboo leaf extract per liter of the solution, and then the solution was uniformly sprayed to the surface of the tobacco at a ratio of 10 mL/kg.
  • the made cigarette was test group C.
  • the bamboo leaf extract is adjusted to a concentration of 0. lg / L of the low alcohol solution, that is, in each liter of the solution, the bamboo leaf extract is 0.1 g, and then the solution is 10 mL / kg.
  • the cigarette was uniformly sprayed onto the surface of the tobacco, and the prepared cigarette was the test group 8.
  • the smoking test of cigarettes was carried out under standard conditions using a smoking machine, and the tar phase and the gas phase were separately collected to determine the content of acrylamide in the tar phase.
  • the tar phase sample was pretreated in the same manner as in Example 3.
  • the sample was pretreated and its acrylamide content was determined by LC-MS/MS.
  • the relative inhibition rate of bamboo leaf extract to acrylamide produced by tobacco was calculated based on the peak area (as shown in Table 3).
  • the bamboo leaf extract, the rosemary extract and the licorice extract were mixed at a mass ratio of 3:1:1 to obtain a compound, at which time the bamboo leaf extract accounted for 60% of the total weight of the compound.
  • test group 1 Take 20 L of blank control group, test group 1, test group 2 - into 20 mL colorimetric tube - add 0. 6mL10% (v / v) H 2 S0 4 - add water to volume of lOmL - 4 ° C refrigerator placed Pre-cooling for 20 min ⁇ Add 1. 5 g KBr powder to dissolve fully. Add lmL 0. lmol/L KBr0 3 - mix thoroughly in a refrigerator and let stand for 30 min - remove and add 0.
  • the total phenolic content is 56.7%
  • the bamboo leaf extract is the bamboo leaf antioxidant (product code A0B) produced by Hangzhou Zhejiang Biotechnology Co., Ltd., the appearance is a brownish yellow powder
  • the total flavonoid content is 32.5%
  • the total phenol content is 56.7% .
  • Four kinds of bamboo leaf carbon glycosides The ratio of flavonoids - isohumuloside, valerin, isovite and vitexin is 2. 75: 1. 05: 1. 15: 1 [Yu Zhang et al.
  • the amount of acrylamide produced by the blank control group, test group 1 and test group 2 calculated according to the peak area was 5421. 12 g/kg 421. 44 g/kg and 148. 40 g/kg, respectively, including bamboo leaf extract.
  • the inhibitory rate of the acrylamide produced by the simulated reaction system was 92.2% and 97.3%, respectively (as shown in Table 13).
  • the compound containing the bamboo leaf extract has a very significant inhibitory effect on the acrylamide produced by the simulated reaction system.
  • the bamboo leaf extract and the tea extract are mixed at a mass ratio of 19:1 to obtain a compound, at which time the bamboo leaf extract accounts for 95% of the total weight of the compound.
  • Fresh potatoes are washed, peeled and cut into thin slices of about 1 thickness.
  • the cut potato chips are picked, rinsed twice with running water, and blotted dry with absorbent paper.
  • Tea extract a water-soluble tea polyphenol preparation for the Department of Tea Science, Zhejiang University, with a content of 98%
  • Blank control group A (potato chips are not treated with any solution).
  • the aging solution is made up to a concentration of 0. 001 g / L, that is, dissolved in 0.001 g of the compound, and then the potato pieces are immersed in an aqueous solution of bamboo leaf extract, and the immersion time is lmi.
  • the potato chips are the same as the test group 13, according to the method of impregnation of the test group B, when the compound is made into an aqueous solution having a mass concentration of 0.01 g/L, the obtained potato chips are the test group C;
  • the obtained potato chips are the test group E when the aqueous solution is made into an aqueous solution having a mass concentration of 0.1 g/L, and the obtained potato pieces are made into an aqueous solution having a mass concentration of 0.5 g/L.
  • the compound is made into an aqueous solution having a mass concentration of lg/L
  • the obtained potato pieces are the test group F; when the compound is made into an aqueous solution having a mass concentration of 2.5 g/L, the obtained potato pieces are obtained.
  • the test group G when the compound was made into an aqueous solution having a mass concentration of 4.9 g/L, the obtained potato chips were the test group 11
  • the dried potato chips of all the above groups are separately fried in the oil pan.
  • the oil used is commercially available peanut oil, and the oil temperature is controlled between 140-160 ° C. , frying time is about 3min, fry until the surface of the potato chips is golden yellow or yellow brown, remove, drain, test; start new oil after each group is fried.
  • the inhibition rate of the acrylamide produced on the fried potato chips by the compound containing the bamboo leaf extract was calculated from the peak area (as shown in Table 14).
  • the concentration of the soaking solution containing the bamboo leaf extract (E0B-C01) is in the range of 0.0000 to 1.9 g/L
  • the potato chips obtained by the soaking are fried to produce propylene.
  • the amide has different degrees of inhibition, and the inhibition rate increases with the concentration of the soaking solution when the concentration of the solution soaking solution is 0.001-lg/L, and the concentration of the soaking solution in the compound is 1-1-4. At 9g/L, it decreases with the concentration of the soaking solution, which indicates that there is an optimum addition range. Therefore, the acrylamide produced by the compound containing the bamboo leaf extract on the fried potato chips has different degrees of inhibition after being soaked in different concentrations of the soaking solution.
  • the bamboo leaf extract, the ginkgo extract, and the hawthorn extract were mixed at a mass ratio of 1:1:1 to obtain a compound, at which time the bamboo leaf extract accounted for 34% of the total weight of the compound.
  • a low alcohol solution is used as the solvent for the soaking solution, which means an alcohol solution having a volume fraction of 20%.
  • Blank control group A is a cigarette made of ordinary tobacco.
  • the compound was formulated into a low alcohol solution having a concentration of 10 g/L, that is, 10 gram of the compound on a dry basis per liter of the low alcohol solution, and then the solution was uniformly sprayed to the cut tobacco at a ratio of 10 mL/kg.
  • the surface, made of cigarettes was test group D.
  • the compound was adjusted to a low alcohol solution having a concentration of lg/L, that is, 1 gram of the compound per liter of the solution, and then uniformly sprayed to the surface of the tobacco at a ratio of 10 mL/kg.
  • the cigarette is test group C.
  • the solution of the solution is adjusted to a concentration of 0. lg / L of the low alcohol solution, that is, in each liter of the solution containing 0.1% of the compound on a dry basis, and then the solution is evenly sprayed at a ratio of 10 mL / kg To the surface of the cut tobacco, the made cigarette was the test group B.
  • the smoking test of cigarettes was carried out under standard conditions using a smoking machine, and the tar phase and the gas phase were separately collected to determine the content of acrylamide in the tar phase.
  • the tar phase sample was pretreated in the same manner as in Example 2.
  • the sample was pretreated and its acrylamide content was determined by LC-MS/MS.
  • the relative inhibition rate of the acrylamide produced by the tobacco leaf extract was calculated based on the peak area (as shown in Table 15).
  • the bamboo leaf extract, the apple polyphenol extract and the onion extract were mixed at a mass ratio of 1.63 : 1 :1 to obtain a compound, at which time the bamboo leaf extract accounted for 45% of the total weight of the compound.
  • the bamboo leaf extract is a product produced by Hangzhou Zheqiangfu Biotechnology Co., Ltd., and the total flavonoid content is 16.5%, and the total phenolic ⁇ is 33.7%.
  • the apple polyphenol extract and onion extract are all from Zhejiang University. The Natural Products Laboratory of the School of Systems Engineering and Food Science produces 30% ethanol extract dry powder.
  • Blank control group A using ordinary fried chicken stock to evenly wrap chicken wings, of which fried chicken is commercially available.
  • the gram of chicken feed per kg of fried chicken is added to the test group B.
  • the mixture is made into a mixed package, and the chicken wings wrapped with the mixed package are tested group C; when each kilogram of fried chicken is added, 0.1 gram of the compound is added to make a mixed package, and the mixed package is used. 5 ⁇
  • the wrapping material, the chicken wings wrapped with the mixed wrapping material is the test group E; when 1 kg of the compound is added to each kilogram of fried chicken material to make a mixed package, the chicken wings wrapped with the mixed wrapping material are the test group F;
  • the gram of compound is added to each kilogram of fried chicken. 4. 9 grams of compound is added to each kilogram of fried chicken.
  • a mixed package was prepared, and the chicken wings wrapped with the mixed package were the test group H.
  • the inhibition rate of acrylamide produced by fried chicken wings was calculated according to the internal standard method (see Table 16).
  • the bamboo leaf extract and the grape seed extract were mixed at a mass ratio of 3:1 to obtain a compound, at which time the bamboo leaf extract accounted for 75% of the total weight of the compound.
  • the source of the bamboo leaf extract (A0B) was the same as in Example 1.
  • the grape seed extract was a commercially available product.
  • the test group B was prepared by adding 0.0001 g of the compound to each kilogram of flour; similarly, 0.01 g of the compound was added to the test group C per kilogram of flour; 0 was added to each kilogram of flour.
  • a gram of the compound was prepared into test group D; 0. 5 grams of the compound was added to each test of the test group E; 1 gram of compound per kilogram of flour was added to make the test group F
  • the test group H was prepared by adding 2. 5 g of the compound to the reference control group G per kilogram of flour; and adding 4.9 g of the compound per kg of flour to prepare the test group H.
  • the above 8 groups were used to make flour for fritters, and an appropriate amount of soda and baking powder were added, respectively, and then water and dough were added. After standing for 12 h, it was once again mixed with the noodles and then allowed to stand until fried.
  • the fermented dough of each group is kneaded into strips, cut into small pieces, and then pulled into a strip shape and then fried in a frying pan. After the golden color and bulking, the fritters are removed from the oil pan and the oil is drained.
  • the sample pretreatment method was the same as in Example 2. After pretreatment of the sample, the content of acrylamide was determined by LC-MS/MS.
  • bamboo leaf extract and Ginkgo biloba extract, tea extract, rosemary extract, apple polyphenol extract, hawthorn extract, onion extract, licorice extract, puerarin extract, grape seed extract, burdock extract The mixture was mixed with a mass ratio of 30:1:1:1:1:1:1:1:1:1 to obtain a compound, at which time the bamboo leaf extract accounted for 75% of the total weight of the compound.
  • the source of bamboo leaf extract (A0B) is the same as that of Example 1; the tea extract is the water-soluble tea polyphenol preparation provided by the Tea Science Department of Zhejiang University, the content is 98%; the apple polyphenol extract and onion extract are all Zhejiang University Self-made by the Natural Products Laboratory of the School of Biosystems Engineering and Food Science, all of which are 30% ethanol extract dry powder; Ginkgo biloba extract, rosemary extract, hawthorn extract, licorice extract, puerarin extract, grape seed extract, Burdock extract is a commercially available product.
  • the test group B was prepared by adding 0.0001 g of the compound to each kilogram of flour; similarly, 0.01 g of the compound was added to the test group C per kilogram of flour; 0 was added to each kilogram of flour.
  • a gram of the compound was prepared into test group D; 0. 5 grams of the compound was added to each test of the test group E; 1 gram of compound per kilogram of flour was added to make the test group F The gram of compound is added to each kilogram of flour. 4. 9 grams of compounding is added to each kilogram of flour.
  • the test group H was prepared.
  • the above 8 groups were used to make flour for fritters, and an appropriate amount of soda and baking powder were added, respectively, and then water and dough were added. Allow to stand for 12 h and then face again, then let stand until fried.
  • the fermented dough of each group is kneaded into strips, cut into small pieces, and then pulled into a strip shape and then fried in a frying pan. After the golden color and bulking, the fritters are removed from the oil pan and the oil is drained.
  • the sample pretreatment method was the same as in Example 2. After pretreatment of the sample, the content of acrylamide was determined by LC-MS/MS.
  • the inhibition rate of the acrylamide produced by the fritters was calculated according to the internal standard method (the mass content was 75%) (see Table 18).

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Description

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 技术领域
本发明涉及热加工食品安全领域；尤其是涉及以黄酮和酚酸类化合物为主要化学成分 的天然竹叶提取物， 在热加工食品中作为丙烯酰胺抑制剂的应用。 背景技术
食品安全是关系生命健康和国计民生的重大问题，它的决定因素是食品危害，所以解 决食品安全问题应从食品危害入手， 也就是从物理危害、 化学危害和微生物危害这三种食 品危害入手。 物理危害容易监测和防犯， 微生物危害需在食品加工过程中加以控制， 而化 学危害 (例如农药、 兽药残留等)从一般意义上来讲， 是在食品原料生长中由于人为因素而 加入的， 可从源头实施控制。 最令人棘手的是， 某些化学危害物并非来源于外部环境， 而 是在食品加工过程中自然形成的， 如热加工食品中的丙烯酰胺 (acrylamide)。.
2002年 4月， 瑞典斯德哥尔摩大学的 Margareta T5rnqvist首次在油炸或焙烤的马 铃薯和谷物类食品中发现了具有神经毒性的潜在致癌物丙烯酰胺 [Tareke, E. et al. Analysis of Acrylamide, a carcinogen Formed in Heated Foodstuffs. J. Agri. Food Chem. , 2002, 50 : 4998-5006]， 她在报道中指出 "淀粉类食品经过 120° C以上的高温加 工后， 其中所含的丙烯酸胺会大大超出安全标准， 长期食用者可导致癌症"的观点一度引 起了人们的恐慌。 这一发现促使瑞典国家食品管理局（Swedish National Food Administration, SNFA)对随机抽取的一百多种食品进行了检验和分析， 并在其官方网站 上公布了检验结果。 2002年 5月 17日， 英国食品标准局（Food Standards Agency, FSA) 公布了类似的结果， 随后， 挪威、 美国、 澳大利亚、 新西兰、 加拿大等国的负责食品安全 的政府机构对丙烯酰胺在食品中的含量也进行了测定并公布了结果， 瑞典科学家的发现被 广泛证实。 与此同时， 许多国际组织和研究机构对丙烯酰胺在食品中的形成机理、 毒理学 、 风险评估等各方面展开了研究。 2005年 3月 2日， 世界卫生组织 (WHO)和联合国粮农组 织 (FA0)属下的食品添加剂联合专家委员会在日内瓦发表声明， 含有致癌毒素——丙烯酰 胺化合物 (简称 "丙毒"）的食品会严重危害人的健康， 特别是 "洋快餐"的多种食物中均 含有大量丙毒。 丙烯酰胺是一种公认的神经毒素和致癌物 [JIFSAN/NCFST. Workshop "Acrylamide in food, scientific issues, uncertainties, and research strategies' " 28- 30th October 2002. Rosemont, USA] , 动物实验表明， 长期暴露在丙 烯酰胺的环境中， 不仅会引发神经系统的病变， 而且可能导致各种癌变。 进一步研究表明 这一化学危害物并不存在于食品原料中， 而是在食品加工过程中形成的 [Mottram, D. S.， et al. Acrylamide is formed in the Mai Hard reaction. Nature, 2002, 419 : 448-449； Stadler, R. H. , et al. Acrylamide from Maillard reaction products. Na ture, 2002, 419 : 449-450]。
丙烯酰胺是一种白色晶体，溶于水、 乙醇、 甲醇、 二甲醚、 丙酮，而不溶于非极性溶 剂如庚烷和苯， 它的 ， -不饱和氨基系统非常容易和亲核物质 (例如蛋白质中半胱氨酸 的巯基)通过 Michael Addition发生化学反应， 从而影响蛋白质的正常功能而致病。
各国政府对丙烯酰胺都有一定的限量标准， 如饮用水中规定不超过 0. 5 g/L。 比照 这个标准， 每 kg油炸薯片中应至多含有 0. 5 g的丙烯酸胺， 而实际情况是， 炸薯片中丙 烯酰胺的含量高达 1480 g/kg, 是正常安全标准的 2960倍。 其它一些淀粉类食品， 像烤 面包片、 饼干等经高温处理的食品中丙烯酸胺的含量也大大超出安全标准。 在我国广泛消 费的大众化食品中有着类似热加工方式 (加工温度≥120° C)的有以油条、 烧饼为代表的传 统早餐食品， 以油炸方便面、谷物早餐为代表的引进速食品， 以咖啡、可可为代表的饮品， 还有以烟草和香烟为代表的副食品。 可令人遗憾的是， 自 2002年热加工食品中丙烯酰胺 的发现距今两年多的时间里， 我国在这方面的监测、 监控和危害评估几近空白。
对丙烯酰胺产生机理的研究表明， 丙烯酰胺是由游离的天门冬酰胺通过美拉德反应 (Maillard Reaction)而形成的， 如今这一机理基本得到确认。 美拉德反应是由还原性糖 和氨基酸或蛋白质中的自由氨基在高温条件下所发生的一系列复杂的化学反应， 它是热加 工食品风味产生的重要途径之一。 美拉德反应主要有三个反应阶段， 第一阶段是由还原糖 的幾基和氨基酸的氨基形成的具有 "ON"键的 Schiff碱，经过重排而生成 Amadori或 Heyns 产物;第二阶段是 Amadori或 Heyns产物通过不同途径降解而成多种风味化合物和中间体； 最后阶段是美拉德反应棕黄色物质的形成。
由天门冬酰胺参与美拉德反应而形成丙烯酰胺的机理被称作天门冬酰胺途径。天门冬 酰胺途径的开始是美拉德反应的初始阶段，当 Schiff碱中间物 (与 7^糖基氨基酸处于动态 平衡之中）形成以后， 两条不同的反应路线都可导致丙烯酰胺的产生： 一条继续美拉德反 应， Schiff碱经过 Amadori重排生成 Amadori产物，继而脱水、脱氨生成含有羰基的产物， 天门冬酰胺在这些含有羰基的分子存在下可以通过 Strecker 降解机制在脱羧、 脱氨后生 成丙烯酰胺， 这一反应机理可以称作 Strecker途径； 而另一条则是由 Schiff碱经过分子 内环化反应生成唑垸酮， 进而形成脱羧 Amadori 产物， 这一产物的 "C- N"键在高温下被 断裂而生成丙烯酰胺， 这一反应机理可叫做 糖苷 ^glycoside)途径 [张根义. 热加工食 品中丙烯酰胺的形成机理和风险分析. 无锡轻工大学学报， 2003， 22 (4) : 91-99]。
Yaylayan等和 Becalski等的工作进一步证实了天门冬酰胺是形成丙烯酰胺的关键前体物 质 [Yaylayan, V. A.， et al. Why asparagine needs carbohydrates to generate acryl amide. J. Agric. Food Chem. , 2003, 51 : 1753—1757 ; Becalski, A. , et al. Acrylamide in foods : occurrence, sources, and modeling. J. Agric. Food Chem. , 2003, 51 : 802-808]； Elmore等用土豆、 小麦和黑麦的热加工模式系统也证实了食品中丙烯酰胺 的形成机理和前体物质 [Elmore,. J. S. , et al. Measurement of acrylamide and its precursors in potato, wheat and rye model systems. J. Agric. Food Chem. , 2003, 51 : 4782-4787]。
根据丙烯酰胺形成的上述理论，如果去除食品原料中游离的天门冬酰胺或者抑制美拉 德反应的进行， 热加工时丙烯酰胺的形成就会受到抑制。 目前的实验研究表明， 在热加工 过程中降低或抑制丙烯酰胺产生的途径主要有两种， 一是通过改变热加工的条件 (包括热 加工方式、 时间、 温度)来实现； 二是通过改变食品的加工属性来实现。 首先， 丙烯酰胺 的产生因热加工的时间和温度而异， 且受热加工方式的影响， 通过控制这些热加工过程中 的关键条件， 从而达到降低或抑制丙烯酰胺产生之目的。 例如， 通过水煮的方法， 控制加 热温度在 45- 78° C以及加热时间在 4min以上，可达到抑制丙烯酰胺生成的目的 [Lindsay,
R. C. and Jang, S. Method for suppressing acrylamide formation. US patent, US2004/0224066 Al] ; 通过控制油炸和加热过程以及先前的洗涤过程中的关键环节， 如完 全去皮、充分洗涤、及时翻炸和沥干油分等措施来降低丙烯酰胺的生成 [Barry, D. L. et al. Method for reducing acrylamide formation in thermally processed foods. PCT patent, W02004/075656 A2]， 其机理是通过降低油脂的热解度来抑制油脂-甘油-丙烯醛-丙烯酸- 两烯酰胺途径 [Tricoit, J. et al. Method for preventing acrylamide formation during heat treatment of food. US patent, US2004/0115321 Al ; Tricoit, J. et al. Method for preventing acrylamide formation during heat-treatment of food. EU patent, 03292813. 7] 其次， 可通过改变或合理控制食品的加工属性或添加其它组分来降低或抑 制丙烯酰胺的产生， 如富含盐类 (包括钙、 镁、 铜、 铝、 铁盐）的食品， 由于其二价或三价 金属阳离子的存在而使得它们在热加工过程中产生的丙烯酰胺很少 [Elder, V. A. et al. Method for reducing acrylamide formation in thermally processed foods. PCT patent, W0 2004/075657 A2; Elder, V. A. et al. Method for reducing acrylamide formation in thermally processed foods. US patent, US 2004/0085045 Al] ; 通过酸处理降低食 品原料的 pH值，使亲核性强的氨基 (- NH2)质子化为亲核性弱的胺类物质 (-N¾+)，可以抑制 两烯酷胺的产生 [Baaxdseth, P. et al. Reduction of acrylamide formation. PCT patent, W0 2004/028278 A2 ; Jung, M. Y. et al. Method for the reduction of acrylamide formation. PCT patent, W0 2004/060078 Al ; Jung, M. Y. et al. A novel technique for limitation of acrylamide formation in fried and baked corn chips and in french fries. J. Food Sci. , 2003, 68 : 1287-1290]； 通过降低丙烯酰胺前体物质在食品原料 中的含量 (包括以微生物糖代谢的方式消耗还原糖或添加天门冬酰胺酶使天门冬酰胺转化 为天冬氨酸）来抑制丙烯酰胺的产生 [Awad, A. C. Reduction of acrylamide formation in cooked starchy foods. US patent, US 2004/0086597 Al ; Elder, V. A. et al. Method for reducing acrylamide formation in thermally processed foods. PCT patent, W0 2004/026042 Al] ; 另外， 还可以利用竞争性抑制作用的原理， 添加一些其它氨基酸， 使 其与还原糖反应， 从而限制天门冬酰胺与还原糖的反应， 这些氨基酸包括半胱氨酸、 赖氨 酸、 甘氨酸、 组氨酸、 丙氨酸、 甲硫氨酸、 谷氨酸、 天冬氨酸、 脯氨酸、 苯丙氨酸、 缬氨 酸禾口精氨酸 [Elder, V. A. et al. Method for reducing acrylamide formation in thermally processed foods. PCT patent, W0 2004/075655 A2]。 尽管这些方法从理论 上可能对丙烯酰胺的产生具有不同程度的抑制作用， 但从方法的实用性、 食品对色香味的 要求以及食用安全性的角度来讲， 很难满足实际的需要， 所以还有待探索新的方法来降低 丙烯酰胺的形成， 并保持食品原有的风味和质构。 最近， 国内学者采用钙离子和阿魏酸作 用于由天门冬酰胺和葡萄糖组成的模拟反应体系， 在钙离子和阿魏酸的最适添加范围以及 最适反应温度和时间内发现其对丙烯酰胺的抑制率可达 80%以上 [欧仕益等.用于高温加工 食品的丙烯酰胺抑制剂及其应用工艺方法. 公开号 CN 1561866A] , 但实际体系与模拟体 系往往存在显著的差异， 其在实际体系中的应用效果还有待检验。 同时， 由天门冬酰胺途 径产生的丙烯酰胺随热加工条件变化的规律也有待进一步研究。
芬兰赫尔辛基大学食品技术研究所 2004年最近公告的 PCT专利称， 在制作炸薯条的 过程中， 使用黄酮类化合物可大大减少薯条中的丙烯酰胺含量。 其在制作薯条过程中添加 了 0. 05-0. 15%的植物提取物，该提取物由绿茶提取物 (45%)、苹果浓缩汁 (45%)和洋葱浓缩 汁 (10%)组成， 富含大量的黄酮类化合物。 结果发现， 在实际油炸过程中产生的丙烯酰胺 减少了 50% [Kurppa, L. A process and composition for prevention of reducing the formation of acrylamide in foods. PCT patent, WO 2004/032647 Al]。
黄酮类化合物是一种重要的食物功能因子， 广泛存在于药用植物和蔬菜、水果中， 具 有很强的生物抗氧化活性， 对心脑血管疾病、 肿瘤、 糖尿病等具有明显的防治效果。 用植 物黄酮来抑制食品体系中丙烯酰胺的形成可能是目前比较适用的方法， 因为这一方法融合 了食品安全和功能性食品这两个对于保障生命健康都十分重要的方面。
就其抗氧化性质而言，植物黄酮类提取物中的许多品种，如茶叶提取物、甘草提取物、 迷迭香提取物等在世界范围内被广泛用作食品抗氧化剂。本发明者近期研发的一种竹叶提 取物 (竹叶抗氧化物）已于 2004年 4月被卫生部批准列入了 《中华人民共和国食品添加剂 使用卫生标准》 （GB- 2760)。 如专利申请号为 200310107871. 5所述的竹叶抗氧化物 (A0B)， 是一种从竹叶中提取的天然酚性部位， 以黄酮和酚酸类化合物为主要化学成分， 包括四种 主要的竹叶碳苷黄酮和三种酚酸， 分别是荭草苷、异荭草苷、牡荆苷、异牡荆苷和绿原酸、 阿魏酸、 咖啡酸。 其分子结构式如下：
Figure imgf000005_0001
(I) 荭草苷 (II) 异荭草苷
Figure imgf000005_0002
(III) 牡荆苷 (IV) 异牡荆苷
Figure imgf000005_0003
(V) 绿原酸
COOH H3CO 、COOH
(VI) 咖啡酸 （VII) 阿魏酸 竹叶碳苷黄酮的结构特点是黄酮母核在 6位或 8位以 C-C键与葡萄糖相连， 由于 C-C 键合的强大键能， 这类化合物具有极高的结构稳定性， 遇酸完全不水解， 并能抗热解和酶 解， 同时具有良好的亲水性， 适用于多种食品体系， 具有氧苷黄酮 (0- glycosyl flavone) 无法比拟的优越性， 尤其是应用于高温处理的热加工食品。 尽管人类对黄酮类化合物的研 究已经有 100多年的历史， 但大量的研究工作主要集中在黄酮苷元 (如槲皮素等)和氧苷黄 酮 (如芦丁等)上。 自 20世纪 90年代起， 国际上对碳苷黄酮的结构与功能性的研究幵始起 步， 属国际前沿领域。 迄今用碳苷黄酮抑制高温处理的食品中丙烯酰胺形成的研究未见公 开报道。
鉴于竹叶"药食两用"的独特背景和竹叶提取物的优良禀赋， 它在食品工业中的应用 前景将十分广阔。 发明内容
本发明的目的是提供一种竹叶提取物的新用途，即在热加工食品中作为丙烯酰胺抑制 剂的应用。 在本发明的第一方面，提供了一种竹叶提取物的用途，它被用于制备热加工食品中丙 烯酰胺抑制剂。
在另一优选例中， 所述抑制剂中含有 1- 99wt% (较佳地 34- 95wt%)竹叶提取物。
在另一优选例中， 所述抑制剂中还含有银杏提取物、茶叶提取物、迷迭香提取物、苹 果多酚提取物、 山楂提取物、 洋葱提取物、 甘草提取物、 葛根提取物、 葡萄籽提取物和牛 蛭提取物中的至少一种。
在另一优选例中，所述抑制剂中含有 34- 95wt%竹叶提取物， 以及 5- 66wt%的所述天然 提取物 (如植物提取物或动物提取物）。
在另一优选例中，所述的竹叶提取物是竹叶的水提取物或醇提取物。更佳地其黄酮含 量为 4-50% (硝酸铝-亚硝酸钠比色法， 以芦丁为标准品）， 总酚含量为 10- 80% (福林试剂还 原比色法测定， 以对羟基苯甲酸为标准品）。
在本发明的第二方面， 提供了一种热加工食品的方法， 包括步骤：
(a)将待加工的食品与丙烯酰胺抑制剂混合， 形成混有抑制剂的食品， 其中所述抑制 剂含有竹叶提取物；
(b)在 120°C或以上温度热加工所述的混有抑制剂的食品， 形成热加工食品。
在另一优选例中， 所述抑制剂中还含有银杏提取物、茶叶提取物、迷迭香提取物、苹 果多酚提取物、 山楂提取物、 洋葱提取物、 甘草提取物、 葛根提取物、 葡萄籽提取物和牛 蛭提取物中的至少一种。
在另一优选例中， 所述的待加工食品包括以油炸、焙烤、烧烤、 烘烤、 微波加热、 膨 化、 燃烧方式得到的炸薯条、 炸薯片、 薄脆饼、 饼干、 蛋糕、 面包、 谷物早餐、 油条、 大 饼、 方便面、 汉堡包、 炸鸡块、 咖啡、 可可、 烟草、 香烟。
在另一优选例中， 步骤 (a)中每千克待加工食品与 0. 001-5克丙烯酰胺抑制剂混合。 在另一优选例中， 步骤 (a)中每千克待加工食品与 0. 01-2克丙烯酰胺抑制剂混合。 在另一优选例中， 步骤 (a)中每千克待加工食品与 0. 1-1克丙烯酰胺抑制剂混合。 在另一优选例中， 在步骤 (a)中， 混合步骤采用包裹、 浸泡或喷洒的方式进行。
在另一优选例中，包裹料中添加所述的丙烯酰胺抑制剂制成混合包裹料，再用所述混 合包裹料均匀包裹待加工食品原料。 在另一优选例中， 每千克包裹料中添加 0. 001-5克所述丙烯酰胺抑制剂。 在另一优选例中， 每千克包裹料中添加 0. 1-1克所述丙烯酰胺抑制剂。
在另一优选例中，在水溶液或低醇溶液中添加所述的丙烯酰胺抑制剂制成浸泡液，再 用所述浸泡液浸泡待加工食品。
在另一优选例中，在每升水溶液或低醇溶液中添加 0. 001-5克所述丙烯酰胺抑制剂制 成浸泡液。
在另一优选例中，每升水溶液或低醇溶液中添加 0. 1-1克所述丙烯酰胺抑制剂制成浸 泡液。
在另一优选例中，在水溶液或低醇溶液中添加所述的丙烯酰胺抑制剂制成喷洒液， 再 用所述喷洒液均匀喷洒待加工食品的表面。
在另一优选例中，在每升水溶液或低醇溶液中添加 0. 001-5克所述丙烯酰胺抑制剂制 成喷洒液。
在另一优选例中，每升水溶液或低醇溶液中添加 0. 1-1克所述丙烯酰胺抑制剂制成喷 洒液。
在本发明的第三方面，提供了一种用于热加工食品中的丙烯酰胺抑制剂组合物，它含 有 l_99wt%竹叶提取物以及 1- 99wt%银杏提取物、 茶叶提取物、 迷迭香提取物、 苹果多酚 提取物、 山楂提取物、 洋葱提取物、 甘草提取物、 葛根提取物、 葡萄籽提取物和牛蛭提取 物中的至少一种。
本发明提供的丙烯酰胺抑制剂能有效抑制食品热加工中所产生的丙烯酰胺。 附图说明
图 1为丙烯酰胺标准品的 GC图谱；
图 2为空白对照组由天门冬酰胺途径产生丙烯酰胺的 GC图谱；
图 3为试验组 1 (竹叶提取物 (A0B)含量为 lOmg/kg)丙烯酰胺的 GC图谱；
图 4为试验组 2 (竹叶提取物 (A0B)含量为 150mg/kg)丙烯酰胺的 GC图谱； 图 5为 A组土豆片经油炸后产生丙烯酰胺的 GC图谱；
图 6为 B组土豆片 (用含有 lg/L竹叶提取物的水溶液浸泡后的土豆片)经油炸后丙烯 酰胺的 GC图谱；
图 7为 C组土豆片（用含有 lg/L茶叶提取物的水溶液浸泡后的土豆片)经油炸后丙烯 酰胺的 GC图谱；
图 8为丙烯酰胺以及 13C标记的丙烯酰胺标准品的 LC-MS/MS图谱；
图 9为 A组鸡翅 (炸鸡料中无任何添加成分)外表面裹上面粉后经油炸产生丙烯酰胺的 LC - MS/MS图谱；
图 10为 B组鸡翅 (炸鸡料中含有 4. 9g/kg竹叶提取物)外表面裹上面粉后经油炸后丙 烯酰胺的 IX- MS/MS图谱；
图 11为 C组鸡翅 (炸鸡料中含有 4. 9g/kg茶叶提取物)外表面裹上面粉后经油炸后丙 烯酰胺的 LC- MS/MS图谱；
图 12为 D组鸡翅 (炸鸡料中含有 4. 9g/kg芦丁)外表面裹上面粉后经油炸后丙烯酰胺 的 LC-MS/MS图谱。 具体实施方式
发明人经过广泛而深入的研究，发现包含有竹叶提取物的组合物可以有效地抑制食品 热加工过程中所产生的有毒物质丙烯酰胺。 此外， 在上述的丙烯酰胺抑制剂组合物中还可 以包括其他提取物 (如银杏提取物、 茶叶提取物等）。 在食品热加工过程中， 可以本发明的 丙烯酰胺抑制剂组合物包裹、 浸泡或喷洒于待加工的食品。 如本文所用，竹叶提取物、含竹叶提取物的丙烯酰胺抑制剂以及含竹叶提取物的复配 物， 都可在制备热加工食品时用作丙烯酰胺抑制剂。 其中含竹叶提取物的丙烯酰胺抑制剂 和含竹叶提取物的复配物可互换使用， 所述的组合物中含有竹叶提取物以及银杏提取物、 茶叶提取物、 迷迭香提取物、 苹果多酚提取物、 山楂提取物、 洋葱提取物、 甘草提取物、 葛根提取物、 葡萄籽提取物和牛蛭提取物中的至少一种。
本发明所指的竹叶提取物是从禾本科（frs M'^e)、 Bambusoideae、 刚竹属 {Phyllostachys Sieb. Et Zucc)品种的叶子中得到的天然竹叶提取物， 其生产工艺在申 请者以前的两项发明专利（专利号分别为 ZL 98104563. 4和 ZL 98104564. 2)中已经涉及。 需要声明的是， 本专利所指的竹叶提取物既可以是采用上述专利工艺得到的产品， 也可以 是在此基础上进一步运用吸附-解吸和膜分离等高新技术及其组合方法精制得到的竹叶提 取物 [包括竹叶抗氧化物 (A0B) ]制品。
竹叶提取物的外观为黄色或棕黄色粉末 (也可以浸膏的形式存在），其主要成分包括以 荭草苷（Orientin)、异荭草苷 (Homoorientin)、牡荆苷（Vitexin)和异牡荆苷（Isovitexin) 为代表的黄酮类化合物和以绿原酸（Chlorogenic acid)、 阿魏酸（Ferulic acid)和咖啡酸 (Caffeic acid)为代表的酚酸类化合物。 其总黄酮含量一般在 4-50% (硝酸铝-亚硝酸钠比 色法， 以芦丁为标准品）， 其中总酚含量一般在 10- 80% (福林试剂还原比色法测定， 以对轻 基苯甲酸为标准品）。
在由天门冬酰胺途径产生丙烯酰胺的模拟反应体系中，以天门冬酰胺和葡萄糖按等摩 尔浓度比例进行反应， 反应体系可以是水溶液或混合物润湿体，在 120° C的温度条件下 加热 10-40min， 从而产生丙烯酰胺， 并测定丙烯酰胺的生成量。 同时， 以此反应体系为空 白对照， 并在此基础上添加本发明声明的添加范围内的含竹叶提取物的丙烯酰胺抑制剂， 按同样条件加热并测定丙烯酰胺的生成量， 与空白对照作比较， 计算含竹叶提取物的丙烯 酰胺抑制剂对丙烯酰胺形成的抑制率。 丙烯酰胺的测定采用 GC或 LC- MS/MS进行。
在食品原料经过热加工而产生丙烯酰胺的实际反应体系中，一是将含竹叶提取物的丙 烯酰胺抑制剂按比例直接添加至食品原料或包裹料中， 使其在热加工过程中作用于天门冬 酰胺途径， 阻断产生丙烯酰胺的反应链， 从而降低或抑制热加工过程中丙烯酰胺的产生； 二是将含竹叶提取物的丙烯酰胺抑制剂按比例配成水溶液或加入少量乙醇或料酒制成低 醇溶液， 用来浸泡食品原料或均匀喷洒在食品原料的表面， 同时， 以不添加含竹叶提取物 的丙烯酰胺抑制剂的实际热加工体系为空白对照， 将试验组添加本发明声明的添加范围内 的含竹叶提取物的丙烯酰胺抑制剂后， 按同样条件加热并测定丙烯酰胺的生成量， 与上述 空白对照作比较， 计算含竹叶提取物的丙烯酰胺抑制剂对丙烯酰胺生成的抑制率。 丙烯酰 胺的测定采用 GC或 LC-MS/MS进行。
(1)气相色谱法 (GC)分析的实验条件如下：
仪器名称： Fuli GC9790气相色谱仪； 检测器： ECD (电子捕获）； 色谱柱： HP- 5 (30 mX 0. 32醒， 25 m)； 进样器： SLIP (不分流毛细管）；
流动相以及流速： 氮气（lmL/min) ; 进样量： 1 L;
柱箱初温： 100°C; 检测口温度： 250°C; 进样口温度： 250。C;
升温程序： 100°C Imin ― 10°C/min 140°C 15min ― 30°C /min 240°C 7min。
与此同时， 气相色谱法测定前须对样品进行衍生化处理提高其挥发程度， 采用 KBr 和 KBr03通过氧化还原反应产生溴分子，使溴分子与丙烯酰胺反应生成一溴或二溴丙酰胺， 最后加入 N¾S203终止衍生化反应并将多余的漠还原为 Br"。
丙烯酰胺标准品的 GC图谱如图 1所示。
(2)液相色谱-两级质谱联用（LC-MS/MS)分析的实验条件如下：
仪器名称： Micromass公司液相色谱 -两级质谱联用仪；
LC条件：
色谱柱： Atlantis (1. 5 X 210mm, 5 m)；
流动相： 甲醇（0. 1%甲酸）： 水（0. 1%甲酸) = 2 ： 98；
流速： lmL/min; 柱温： 20°C; 进样量： 10 L。
MS条件：
毛细管电压： 3. 50kV; 锥孔电压： 50V; 源温： 100°C; 脱溶剂温度： 350°C; 锥孔气 流速： 45L/h; 脱溶剂气流速： 400L/h;
MRM参数： 丙烯酰胺标样 72>55， 13C3-丙烯酰胺内标 75>58；
碰撞能量 6eV。
丙烯酰胺以及 13C标记的丙烯酰胺标准品的 LC- MS/MS图谱如图 8所示。 本发明的主要优点在于：
1、 釆用外源性植物化学素 (黄酮和酚酸等)在所述浓度范围内处理食品物料， 在有效 抑制热加工过程中丙毒形成的同时， 对终端产品的色、 香、 味、 型、 质地等感官品质无显 著的不良影响， 易于为消费者接受。
2、 在食品中添加植物黄酮和酚酸等天然抗氧化物作为丙烯酰胺抑制剂， 在保障食品 安全性的同时， 还可起到营养强化和延长食品保质期的双重作用， 一举数得。
3、 原料来源广泛， 成本低。 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， 否则所有的百分比和份数按重量计。
实施例 1
竹叶提取物对模拟反应体系产生丙烯酰胺的抑制作用
取 0. lmol/L的 天门冬酰胺和 D-葡萄糖各 10mL混合一置于 lOOraL锥形瓶中一设空 白对照组； 还在上述空白对照组的基础上加入竹叶提取物， 制成最终体系中竹叶提取物浓 度分别为 10mg/kg的试验组 1和 150mg/kg的试验组 2—将空白对照组、 试验组 1、 试验组
2分别在 120°C水浴中加热 15min—直接取得到的反应溶液后进行衍生化一 GC分析。
其中衍生化反应具体过程如下：
各取 20 L 的空白对照组、 试验组 1、 试验组 2→入 20mL 比色管中—加入 0. 6mL 10 (v/v) H2S04—加水定容至 lOmL— 4°C冰箱放置 20min后预冷一加入 1. 5 g KBr粉末充 分溶解一加入 1 mL 0. 1 mol/L KBr03—充分混匀一冰箱中静置衍生 30min—取出后加入 0. lmL lmol/L Na2S203—充分混匀—取出后加入 5mL重蒸后或者 HPLC纯的乙酸乙酯充分萃 取一取乙酸乙酯相并用无水 NasSO,,脱水—待用。
其中竹叶提取物为杭州浙大力夫生物科技有限公司生产的竹叶抗氧化物 (产品代码为 A0B)，外观为棕黄色粉末，总黄酮含量 32. 5%,总酚含量 56. 7%。其中四种竹叶碳苷黄酮—— 异荭草苷、 荭草苷、 异牡荆苷和牡荆苷的含量比例为： 2. 75： 1. 05： 1. 15 ： 1 [Yu Zhang et al.， Determination of flavone C- glucosides in antioxidant of bamboo leaves (AOB) fortified foods by reversed-phase high-performance liquid chromatography with ultraviolet diode array detection, Journal of Chromatography A, 2005, 1065： 177-185]
图 2为空白对照组由天门冬酰胺途径产生丙烯酰胺的 GC图谱； 图 3为试验组 1 (A0B 浓度为 10mg/kg)产生丙烯酰胺的 GC图谱； 图 4为试验组 2 (A0B浓度为 150mg/kg)产生丙 烯酰胺的 GC图谱。 根据峰面积计算并折合后得丙烯酰胺的生成量分别为 6460. 31 g/kg, 410. 10 g/kg 和 190. 66 g/kg , A0B对模拟反应体系产生的丙烯酰胺的抑制率分别为 93. 6%和 97. 0% (如表 1所示）。
Figure imgf000010_0001
由表 1可见， A0B对模拟反应体系产生的丙烯酰胺具有极显著的抑制作用。
实施例 2
不同剂量的竹叶提取物对模拟反应体系产生丙烯酰胺的抑制作用
竹叶提取物 (A0B)来源同实施例 1。 为了探明其抑制丙烯酰胺的最适作用剂量范围， 在模拟体系中分别设置了 4个不同浓度的添加剂量。 模拟体系反应过程、 取样以及衍生化 过程同实施例 1， 采用 GC法测定丙烯酰胺含量。
表 2不同剂量的竹叶提取物 (A0B)对模拟反应体系产生的丙烯酰胺的抑制率 ( ? =6)
Figure imgf000010_0002
由表 2可见， 在模拟反应体系中当 A0B添加量在 1- 9mg/kg的范围内对模拟反应体系 产生的丙烯酰胺具有不同程度的抑制作用， 其抑制率具有显著的线性相关性。
实施例 3
竹叶提取物对油炸土豆片产生丙烯酰胺的抑制作用
(1)土豆片 新鲜土豆经清洗后去皮并切成厚度大约为 1醒的薄片，切好的土豆片经挑选后用流水 漂洗两次， 并用吸水纸吸干。
(2)植物提取物
竹叶提取物： 为杭州浙大力夫生物科技有限公司生产的产品（产品代码为 EOB- C01)， 总黄酮含量为 40. 7%， 总酚含量为 79. 8%;
茶叶提取物： 为浙江大学茶学系提供的水溶性茶多酚制剂， 含量为 98%。
(3)实验分组
本实验共设三个组， 分别为空白对照组 A (土豆片不用任何溶液处理）、 竹叶提取物剂 量组 B (土豆片用竹叶提取物水溶液浸泡处理)和茶叶提取物剂量组 C (土豆片用茶叶提取物 水溶液浸泡处理）。
将竹叶提取物配制成质量含量为 lg/L的水溶液， 即在每升水中添加 1克的竹叶提取 物， 然后将土豆片浸没在竹叶提取物水溶液中， 浸渍时间为 lmin。该组设为竹叶提取物剂 量组 B。
将茶叶提取物配制成质量含量为 lg/L的水溶液， 即在每升水中添加 1克的水溶性茶 多酚制剂， 然后将土豆片浸没在茶叶提取物水溶液中， 浸渍时间为 lmir！。该组设为茶叶提 取物剂量组 (。
将八、 B、 C三个组中的土豆片分别放入功率为 750W的家用微波炉中， 在中火档加热 干燥， 干燥时间为 A组 3. 5 min, B、 C组各为 5. 5min。
(4)油炸
设定各组土豆片的量均为 50- 60g， 将干燥后的 A、 B、 C三组土豆片分别在油锅中炸 制， 所用油为市售花生油， 油温控制在 140- 160° C之间, 油炸时间在 3min左右， 炸至土 豆片表面呈金黄色或棕黄色， 捞出， 沥油， 检测； 每炸完一组后启用新油。
(5)取样及样品预处理
取适量油炸土豆片样品并用研钵碾碎一称取 1. 5g样品一加入浓度为 1 g/mL的内标 500 L—静置 10min→两次加入 20mL重蒸石油醚脱脂并充分振荡 lOmin—两次加入 8mL 2mol/L NaCl超声振荡提取 20min→15000rpm离心 15min—三次加入 15mL重蒸乙酸乙酯充 分萃取一合并萃取液旋转蒸发→N2吹干一1. 5mL蒸馏水重溶一 6cc HLB柱固相萃取纯化一进 样分析。
(6)结果检测
土豆片经上述预处理后用 GC测定丙烯酰胺的含量。
图 5为 A组土豆片经油炸后产生丙烯酰胺的 GC图谱；图 6为 B组土豆片 (用含有 lg/L 竹叶提取物的水溶液浸泡后的土豆片)经油炸产生丙烯酰胺的 GC图谱； 图 7为 C组土豆片 (用含有 lg/L茶叶提取物的水溶液浸泡后的土豆片)经油炸产生丙烯酰胺的 GC图谱。根据 峰面积计算得竹叶提取物和茶叶提取物对油炸土豆片产生的丙烯酰胺的抑制率分别为 95. 7%和 73· 4% (如表 3所示）。
表 3竹叶提取物对油炸土豆片产生丙烯酰胺的抑脾（73 =6)
组 别 丙烯酰胺生成量（ g/kg) 抑制率 (%) 空白对照 (A组） 556. 12 + 84. 35
竹叶提取物剂量 (B组） 24. 11 + 2. 46 95. 7 + 0. 3 茶叶提取物剂量 (c组) 147. 85 士 14. 33 73. 4 ± 2. 8 由表 3可见，竹叶提取物和茶多酚对袖炸土豆片产生的丙烯酰胺均具有显著的抑制作 用， 且竹叶提取物的抑制效果好于茶多酚。
实施例 4
竹叶提取物对油炸土豆片产生丙烯酰胺的抑制作用
与实施例 3 不同的是增加了一黄酮醇糖苷化合物 (芦丁）的试验组， 并将竹叶提取物 (A0B)浸泡液的浓度增大到 5g/L, 即在每升水中添加 5克的 A0B。 竹叶提取物的来源同实 施例 1， 茶叶提取物的来源同实施例 3。 芦丁为购自 Sigma公司的标准品， 其纯度≥95%。 所用油同实施例 3， 油温控制在 140-160° C之间。 样品预处理的方法同实施例 3， 采用 LC- MS/MS测定丙烯酰胺含量，根据内标法计算得竹叶提取物、茶叶提取物和芦丁对油炸土 豆片产生丙烯酰胺的抑制率分别为 40. 0%、 37. 7%和 39. 7% (如表 4所示）。
表 4竹叶提取物 (A0B)对油炸土豆片产生的丙烯酰胺的抑制率 (/7 =6)
Figure imgf000012_0001
由表 4可见，竹叶提取物、茶叶提取物和芦丁对油炸土豆片产生的丙烯酰胺均具有一 定的抑制作用， 但与实施例 3相比， 抑制效果均显著下降， 这表明， 抑制效果与添加剂量 之间并非呈简单的线性关系。说明黄酮类化合物对油炸土豆片中丙烯酰胺的抑制效果并非 随着添加量的增加而增加， 而是存在一个最适添加量区间， 并且随食品体系和加工条件的 变化而变化。
实施例 5
不同剂量的竹叶提取物对油炸土豆片产生丙烯酰胺形成的抑制作用
竹叶提取物 (A0B)来源同实施例 1。 为了探明不同剂量的 A0B对油炸土豆片中丙烯酰 胺形成的抑制作用，分别设置了 6个不同浓度的浸泡液，油炸后的土豆片样品经预处理后， 用 LC- MS/MS测定丙烯酰胺的含量， 样品预处理方法同实施例 3。
表 5不同剂量的竹叶提取物对油炸土豆片产生丙烯酰胺的抑制作用 n =6)
Figure imgf000012_0002
由表 5可见，在油炸土豆片中当竹叶提取物 (A0B)浸泡液浓度在 0. 002-4. 9g/L的范围 内时， 浸泡土豆片后对其油炸产生丙烯酰胺具有不同程度的抑制作用， 其抑制率在浸泡液 浓度为 0. 002-lg/L时随着浸泡液浓度的增加而增加， 而在浸泡液浓度为 1-4. 9g/L时却随 着浸泡液浓度的增加而减少， 这说明存在一个最适添加量区间。
实施例 6
竹叶提取物对焙烤咖啡产生丙烯酰胺的抑制作用
(1)原料来源
咖啡生豆为市售产品， 竹叶提取物 (A0B)来源同实施例 1。
(2)实验分组
本实验共设两个组， 分别为空白对照组 A (咖啡豆不用任何溶液处理）、竹叶提取物剂 量组 B (咖啡豆用竹叶提取物水溶液浸泡处理）。
将竹叶提取物 (A0B)制成质量含量为 0. lg/L的水溶液，即每升水中添加 0. 1克的 A0B， 然后将咖啡生豆浸没在 A0B水溶液中，浸渍时间为 lmin,所得的咖啡豆为竹叶提取物剂量 组8。
(3)咖啡制作
将 、 B两组中的咖啡豆进行焙烤，焙烤温度控制在 190- 200° C之间。将焙烤后的咖 啡粉碎， 并用热水充分萃取， 经喷雾干燥后得到咖啡产品。
咖啡产品经样品预处理后用 LC- MS/MS测定其丙烯酰胺的含量， 样品预处理方法同实 施例 3。 根据内标法计算得竹叶提取物对焙烤咖啡产生丙烯酰胺的抑制率为 85. 0% (如表 6 所示)。
表 6竹叶提取物 (A0B)对焙烤咖啡产生丙烯酰胺的抑制率 6)
Figure imgf000013_0001
由表 6可见，釆用 0. lg/L的竹叶提取物 (A0B)水溶液浸泡咖啡生豆，进而对咖啡焙烤 过程中丙烯酰胺的产生具有非常显著的抑制作用， 说明在该竹叶提取物添加剂量下对丙烯 酰胺的抑制率已接近最佳水平。
实施例 7
竹叶提取物对炸鸡翅产生丙烯酰胺的抑制作用
竹叶提取物为杭州浙大力夫生物科技有限公司生产的产品， 总黄酮含量为 16. 5%， 总 酚含量为 33. 7%； 茶叶提取物和芦丁的来源与实施例 4相同； 煎炸用油来源同实施例 3; 炸鸡料为市售产品。
(1)分组
本实验共设四个组， 分别为空白对照组 A (炸鸡翅所用包裹料不添加任何试样）、 竹叶 提取物剂量组 B、 茶叶提取物剂量组 C和芦丁剂量组 D。 添加方法和剂量均为在每千克包 裹料中分别均匀混入 4. 9克试样。 然后制作面糊： 加水调成稀薄糊状， 并打入鸡蛋一个， 拌匀。
(2)油炸
用各组对应调好的炸鸡料裹匀鸡翅， 然后将鸡翅挂上面糊， 立即上油锅炸制， 油温控 制在 140- 160° C之间， 炸至金黄色出锅， 每次炸完一组后启用新油。
(3)检测
样品预处理方法同实施例 3， 经样品预处理后用 LC-MS/MS测定其丙烯酰胺的含量。 (4)结果
图 9为 A组鸡翅 (炸鸡料中无任何添加成分)外表面裹上面粉后经油炸产生丙烯酰胺的 LC-MS/MS图谱；
图 10为 B组鸡翅 (炸鸡料中含有 4. 9g/kg竹叶提取物)外表面裹上面粉后经油炸产生 丙烯酰胺的 LC- MS/MS图谱；
图 11为 C组鸡翅 (炸鸡料中含有 4. 9g/kg茶叶提取物)外表面裹上面粉后经油炸产生 丙烯酰胺的 LC- MS/MS图谱；
图 12为 D组鸡翅 (炸鸡料中含有 4. 9g/kg芦丁)外表面裹上面粉后经油炸产生丙烯酰 胺的 LC- MS/MS图谱。
根据内标法计算得竹叶提取物、水溶性茶多酚和芦丁对炸鸡翅产生的丙烯酰胺的抑制 率分别为 38. 3%, 32. 5%和 22. 5% (如表 7所示）。
Figure imgf000014_0001
由表 7可见，竹叶提取物、茶叶提取物和芦丁对炸鸡翅产生的丙烯酰胺均具有一定的 抑制作用。
实施例 8
不同剂量的竹叶提取物对炸鸡翅产生丙烯酰胺的抑制作用
竹叶提取物来源同实施例 1。为了探明不 剂量的竹叶提取物与包裹料混合后对炸鸡 翅产生丙烯酰胺的抑制作用，在实际油炸体系中分别设置了 6个不同添加量的竹叶提取物， 样品预处理方法同实施例 3， 经样品预处理后用 LC- MS/MS测定其丙烯酰胺的含量。
表 8不同剂量的竹叶提取物 (A0B)对炸鸡翅产生丙烯酰胺的抑制作用（ =6)
Figure imgf000014_0002
由表 8可见， 在炸鸡翅中当竹叶提取物 (A0B)添加量在 0. 002- 4. 9g/kg的范围内时， 与包裹料混合后对炸鸡翅产生丙烯酰胺具有不同程度的抑制作用， 其抑制率在 A0B添加量 为 0. 002- lg/kg时随着添加量的增加而增加， 而在添加量为 l-4. 9g/L时却随着添加量的 增加而减少， 这说明存在一个最适添加量区间。
实施例 9
竹叶提取物对油条产生丙烯酰胺的抑制作用 (1)原料来源
竹叶提取物的来源和含量与实施例 1相同，茶叶提取物和芦丁的来源和含量与实施例 4相同。
(2)试验分组
本实验共设四个组， 分别为：
空白对照组 A (面粉不作任何处理）；
在每千克面粉中加入 2.5g竹叶提取物， 制成 B组；
在每千克面粉中加入 2.5g茶叶提取物， 制成 C组；
在每千克面粉中加入 2.5g芦丁， 制成 D组。
将上述 4组用于制作油条的面粉， 加入适量苏打和发酵粉， 然后加水和成面团。静置
12 h后再和面一次， 然后静置直至油炸。
(3)油炸
将各组发酵好的面团搓成条状，切成小团，然后拉成长条状以后入油锅炸制， 待金黄 色且膨松后将油条捞出油锅并沥干油分即成。 样品预处理方法同实施例 3， 经样品预处理 后用 LC- MS/MS测定其丙烯酰胺的含量。
根据内标法计算得竹叶提取物、茶叶提取物和芦丁对油条产生丙烯酰胺的抑制率分别 为 67.5%、 64.9%和 53.7% (如表 9所示）。
表 9竹叶提取物 (A0B)对油条产生丙烯酰胺的抑制率 ( 6)
Figure imgf000015_0001
由表 9可见，竹叶提取物、茶多酚和芦丁对油条产生的丙烯酰胺均具有一定的抑制作 用。
实施例 10
不同剂量的竹叶提取物对油条产生丙烯酰胺的抑制作用
竹叶提取物来源同实施例 1。为了探明不同剂量的竹叶提取物对油条产生丙烯酰胺的 抑制作用， 在实际油炸体系中分别设置了 6个不同的添加量水平。 样品预处理方法同实施 例 3， 样品经预处理后用 LC- MS/MS测定其丙烯酰胺的含量。
表 10不同剂量的竹叶提取物 (A0B)对油条产生丙烯酰胺的抑制 用 (^6) 组别 添加的竹叶提取物（g/kg) 丙烯酰胺生成量（ g/kg) 抑制率 (%) 空白对照组 0 201.23 土 12.40
试验组 1 0.002 184.12 + 10.23 8.5 士 1.0 试验组 2 0.01 123.94 + 8.66 38.4 ± 7.0 试验组 3 0.1 67.70 + 4.86 66.4 ± 5.5 试验组 4 1 34.41 士 2.98 82.9 ± 6.9 试验组 5 2.5 74.52 土 9.44 63.0 + 5.8 试验组 6 4.9 141.00 土 11.01 29.9 + 5.3 由表 10可见，在油条中当竹叶提取物 (AOB)添加量在 0. 002-4. 9g/kg的范围内时，与 原料混合后对油条产生丙烯酰胺具有不同程度的抑制作用， 其抑制率在 A0B添加量为 0. 002 lg/kg时随着添加量的增加而增加，而在添加量为 1-4. 9g/L时却随着添加量的增加 而减少， 这说明存在一个最适添加量区间。
实施例 11
竹叶提取物对香烟燃烧过程中产生丙烯酰胺的抑制作用
(1)试样来源
竹叶提取物： 为杭州浙大力夫生物科技有限公司生产的产品 (产品代码为 EOB- S03)， 外观为深棕色浓缩液， 总黄酮含量为 4. 5%， 固形物含量为 25. 2%。
茶叶提取物： 为浙江大学茶学系提供的水溶性茶多酚制剂， 含量为 98%;
芦丁： 为购自 Sigma公司的标准品， 其纯度为 95%。
(2)试验分组
本实验共设 4个组- 空白对照组 A为普通烟丝制成的卷烟。
将竹叶提取物配制成 4. 9g/L的水溶液， 即在每升水溶液中含有以干基计 4. 9克的竹 叶提取物，然后将此溶液以 10mL/kg的比例均匀喷洒至烟丝表面，制成的卷烟为试验组 B。
将茶叶提取物配制成 4. 9g/L的水溶液， 即在每升水溶液中含有以干基计 4. 9克的茶 叶提取物，然后将此溶液 '以 10mL/kg的比例均匀喷洒至烟丝表面，制成的卷烟为试验组 (。
将芦丁配制成 4. 9g/L的水溶液， 即在每升水溶液中含有以干基计 4. 9克的芦丁， 然 后将此溶液以 lOmL/kg的比例均匀喷洒至烟丝表面， 制成的卷烟为试验组 D。
(3)卷烟燃烧
采用吸烟机在标准条件下进行卷烟的燃烧试验，并分别收集焦油相和气相，测定焦油 相中丙烯酰胺的含量。 焦油相样品的预处理方法同实施例 3， 样品经预处理后用 LC- MS/MS 测定其丙烯酰胺的含量。
根据峰面积计算竹叶提取物、茶叶提取物和芦丁对烟草产生丙烯酰胺的相对抑制率分 别为 67. 8%、 55. 0%和 65. 3% (如表 11所示）。
表 11竹叶提取物对烟草燃烧过程中产生的丙烯酰胺的抑制率 0τ=6)
Figure imgf000016_0001
由表 11可见， 竹叶提取物、 茶叶提取物和芦丁对烟草燃烧过程中产生的丙烯酰胺均 具有不同程度的抑制作用。
实施例 12
不同剂量的竹叶提取物对香烟燃烧过程中产生丙烯酰胺的抑制作用
竹叶提取物来源同实施例 11。
本实验共设 4个组： 空白对照组 A为普通烟丝制成的卷烟。
将竹叶提取物配制成浓度为 10g/L的低醇溶液， 即在每升低醇溶液 (本实施例中低醇 溶液是指体积分数为 20%的酒精溶液）中含有以干基计 10克的竹叶提取物， 然后将此溶液 以 10mL/kg的比例均匀喷洒至烟丝表面， 制成的卷烟为试验组 D。
将竹叶提取物调整为浓度 lg/L的低醇溶液， 即在每升溶液含以干基计 1克的竹叶提 取物， 然后将此溶液以 10mL/kg的比例均匀喷洒至烟丝表面， 制成的卷烟为试验组 C。
将竹叶提取物调整为浓度 0. lg/L的低醇溶液， 即在每升溶液中含以干基计为 0. 1克 的竹叶提取物， 然后将此溶液以 10mL/kg的比例均匀喷洒至烟丝表面， 制成的卷烟为试验 组8。
(3)卷烟燃烧
采用吸烟机在标准条件下进行卷烟的燃烧试验，并分别收集焦油相和气相，测定焦油 相中丙烯酰胺的含量。 焦油相样品的预处理方法同实施例 3， 样品经预处理后用 LC- MS/MS 法测定其丙烯酰胺的含量。根据峰面积计算竹叶提取物对烟草产生丙烯酰胺的相对抑制率 (如表 3所示）。
Figure imgf000017_0001
由表 12可见，当竹叶提取物 (EOB- S03)的低醇溶液浓度在 0. 1- 10g/L的范围内时， 以 lOmL/kg的比例均勾喷洒至烟丝表面， 采用吸烟机在标准条件下进行卷烟的燃烧试验， 对 其燃烧过程焦油相中产生丙烯酰胺具有不同程度的抑制作用， 其抑制率在竹叶提取物的低 醇溶液浓度为 0. l-10g/L (即烟丝中竹叶提取物添加量为 0. 001- 0. lg/kg)时随着喷洒液浓 度的增加而增加。
实施例 13
含竹叶提取物的复配物对模拟反应体系产生丙烯酰胺的抑制作用
将竹叶提取物、迷迭香提取物和甘草提取物按 3 : 1 : 1的质量比混合得到复配物，此时 竹叶提取物占复配物总重的 60%。
取 0. lmol/L的 L-天门冬酰胺和 D-葡萄糖各 10mL混合—置于 lOOmL锥形瓶中—设空 白对照组； 还在上述空白对照组的基础上加入复配物， 制成最终体系中复配物浓度分别为 10rag/kg的试验组 1和 150mg/kg的试验组 2—将空白对照组、 试验组 1、 试验组 2分别在 120°C水浴中加热 15min→直接取得到的反应溶液后进行衍生化一 GC分析。
其中衍生化反应具体过程如下：
各取 20 L 的空白对照组、 试验组 1、 试验组 2—入 20mL 比色管中—加入 0. 6mL10% (v/v) H2S04—加水定容至 lOmL— 4°C冰箱放置 20min预冷→加入 1. 5g KBr粉末 充分溶解一加入 lmL 0. lmol/L KBr03 —充分混匀一冰箱中静置衍生 30min—取出后加入 0. IraL lmol/L Na2S203—充分混匀—取出后加入 5mL重蒸后或者 HPLC纯的乙酸乙酯充分萃 取一取乙酸乙酯相并用无水 Na2S04脱水—待用。
其中竹叶提取物为杭州浙大力夫生物科技有限公司生产的竹叶抗氧化物 (产品代码为 A0B) , 外观为棕黄色粉末， 总黄酮含量为 32. 5%， 总酚含量为 56. 7%。 其中四种竹叶碳苷 黄酮——异荭草苷、 荭草苷、 异牡荆苷和牡荆苷的含量比例为 2. 75： 1. 05： 1. 15： 1 [Yu Zhang et al. , Determination of f lavone C— glucosides in antioxidant of bamboo leaves (AOB) fortified foods by reversed - phase high - performance liquid chromatography with ultraviolet diode array detection, Journal of Chromatography A, 2005 1065 : 177-185]。 迷迭香提取物和甘草提取物均为市售产品。
根据峰面积计算并折合后得空白对照组、试验组 1和试验组 2的丙烯酰胺生成量分别 为 5421. 12 g/kg 421. 44 g/kg和 148. 40 g/kg，含竹叶提取物的复配物对模拟反应体 系产生的丙烯酰胺的抑制率分别为 92. 2%和 97. 3% (如表 13所示）。
表 13含竹叶提取物的复配物对模拟反应体系产生丙烯酰胺的抑制率 (/7=6)
Figure imgf000018_0001
由表 13可见， 含竹叶提取物的复配物对模拟反应体系产生的丙烯酰胺具有极显著的 抑制作用。
实施例 14
含竹叶提取物的复配物对油炸土豆片产生丙烯酰胺的抑制作用
将竹叶提取物和茶叶提取物按 19 : 1的质量比混合得到复配物， 此时竹叶提取物占复 配物总重的 95%
(1)土豆片
新鲜土豆经清洗后去皮并切成厚度大约为 1 的薄片，切好的土豆片经挑选后用流水 漂洗两次， 并用吸水纸吸干。
(2)植物提取物
竹叶提取物： 为杭州浙大力夫生物科技有限公司生产的产品 (产品代码为 E0B- C01) , 总黄酮含量为 40. 7%, 总酚含量为 79. 8%；
茶叶提取物： 为浙江大学茶学系提供的水溶性茶多酚制剂， 含量为 98%
(3)实验分组
本实验共设 8个组：
空白对照组 A (土豆片不用任何溶液处理）。
将复配物制成质量浓度为 0. 001g/L的水溶液， 即在每升水中溶入 0. 001克复配物， 然后将土豆片浸没在竹叶提取物水溶液中， 浸渍时间为 lmi 所得的土豆片为试验组13 同理， 依照试验组 B的浸渍方法， 当将复配物制成质量浓度为 0. 01g/L的水溶液时， 所得的土豆片为试验组 C; 当将复配物制成质量浓度为 0. lg/L的水溶液时，所得的土豆片 为试验组 D; 当将复配物制成质量浓度为 0. 5g/L的水溶液时， 所得的土豆片为试验组 E; 当将复配物制成质量浓度为 lg/L的水溶液时，所得的土豆片为试验组 F; 当将复配物制成 质量浓度为 2. 5g/L 的水溶液时， 所得的土豆片为试验组 G; 当将复配物制成质量浓度为 4. 9g/L的水溶液时， 所得的土豆片为试验组11
将上述所有组中的土豆片分别放入功率为 750W的家用微波炉中，在中火档加热干燥， 干燥时间： A组 3. 5 rain, 其余组均为 5. 5 min (4)油炸
设定各组土豆片的量均为 50-60g, 将干燥后的上述所有组的土豆片分别在油锅中炸 制， 所用油为市售花生油， 油温控制在 140- 160° C之间， 油炸时间在 3min左右， 炸至土 豆片表面呈金黄色或黄棕色， 捞出， 沥油， 检测； 每炸完一组后启用新油。
(5)取样及样品预处理
取适量油炸土豆片样品并用研钵碾碎一称取 1. 5g样品→加入浓度为 1 g/mL的内标 500 L—静置 lOrain—两次加入 20mL重蒸石油醚脱脂、并充分振荡 lOmin—两次加入 8mL 2M NaCl超声振荡提取 20min— 15000rpm离心 15min—三次加入 15mL重蒸乙酸乙酯充分萃取 —合并萃取液旋转蒸发→N2吹干→1. 5mL蒸馏水重溶一 6cc HLB柱固相萃取纯化一进样分 析。
(6)结果检测
土豆片经上述预处理后， 用 LC- MS/MS测定丙烯酰胺的含量。
根据峰面积计算得含竹叶提取物的复配物对油炸土豆片产生丙烯酰胺的抑制率 (如表 14所示）。
表 14含竹叶提取物的复配物对油炸土豆片产生丙烯酰胺的抑制率 0=6)
Figure imgf000019_0001
由表 14可见， 当含竹叶提取物 (E0B-C01)复配物的浸泡液浓度在 0. 001-4. 9g/L的范 围内时， 浸泡所得到的土豆片对其油炸产生丙烯酰胺具有不同程度的抑制作用， 其抑制率 在复配物浸泡液浓度为 0. 001-lg/L时随着浸泡液浓度的增加而增加， 而在复配物浸泡液 浓度为 1- 4. 9g/L时却随着浸泡液浓度的增加而减少， 这说明存在一个最适添加量区间。 因此， 含竹叶提取物的复配物对油炸土豆片产生的丙烯酰胺在经不同浓度浸泡液浸泡后具 有不同程度的抑制作用。
实施例 15
含竹叶提取物的复配物对香烟燃烧过程中产生丙烯酰胺的抑制作用
将竹叶提取物、银杏提取物、 山楂提取物按 1 : 1 : 1的质量比混合得到复配物， 此时竹 叶提取物占复配物总重的 34%。
(1)试样来源
竹叶提取物： 为杭州浙大力夫生物科技有限公司生产的产品（产品代码为 E0B-S03)， 外观为深棕色浓缩液， 总黄酮含量为 4. 5%， 固形物含量为 25. 2%; 银杏提取物和山楂提取 物均为市售产品。 本实施例中采用低醇溶液作为浸泡液溶剂， 是指体积分数为 20%的酒精 溶液。 (2)试验分组
本实验共设 4个组：
空白对照组 A为普通烟丝制成的卷烟。
将复配物配制成浓度为 10g/L的低醇溶液， 即在每升低醇溶液中含有以干基计 10克 的复配物，然后将此溶液以 10mL/kg的比例均匀喷洒至烟丝表面，制成的卷烟为试验组 D。
将复配物调整为浓度 lg/L的低醇溶液， 即在每升溶液含以干基计 1克的复配物， 然 后将此溶液以 10mL/kg的比例均匀喷洒至烟丝表面， 制成的卷烟为试验组 C。
将复配物调整为浓度 0. lg/L的低醇溶液， 即在每升溶液中含以干基计为 0. 1克的复 配物， 然后将此溶液以 10mL/kg的比例均匀喷洒至烟丝表面， 制成的卷烟为试验组 B。
(3)卷烟燃烧
采用吸烟机在标准条件下进行卷烟的燃烧试验，并分别收集焦油相和气相，测定焦油 相中丙烯酰胺的含量。 焦油相样品的预处理方法同实施例 2， 样品经预处理后用 LC- MS/MS 法测定其丙烯酰胺的含量。根据峰面积计算含竹叶提取物的复配物对烟草产生丙烯酰胺的 相对抑制率 (如表 15所示）。
表 15含竹叶提取物的复配物对烟草燃烧过程中产生的丙烯酰胺的抑制率 0τ=6)
Figure imgf000020_0001
由表 15可见，当含竹叶提取物 (E0B- S03)复配物的低醇溶液浓度在 0. l-10g/L的范围 内时， 以 lOmL/kg的比例均匀喷洒至烟丝表面， 采用吸烟机在标准条件下进行卷烟的燃烧 试验， 对其燃烧过程焦油相中产生丙烯酰胺具有不同程度的抑制作用， 其抑制率在复配物 的低醇溶液浓度为 0. 1- 10g/L (即烟丝中复配物添加量为 0. 001-0. lg/L)时随着喷洒液浓度 的增加而增加。
实施例 16
含竹叶提取物的复配物对炸鸡翅产生丙烯酰胺的抑制作用
将竹叶提取物、苹果多酚提取物和洋葱提取物按 1. 63 : 1 : 1的质量比混合得到复配物， 此时竹叶提取物占复配物总重的 45%。
竹叶提取物为杭州浙大力夫生物科技有限公司生产的产品， 总黄酮含量为 16. 5%， 总 酚^ "量为 33. 7%。 苹果多酚提取物和洋葱提取物均为浙江大学生物系统工程与食品科学学 院天然产物实验室自制， 均为 30%的乙醇提取物干粉。
(1)实验分组
本实验共设 8个组：
空白对照组 A， 使用普通的炸鸡料用来均匀包裹鸡翅， 其中炸鸡料为市售品。
在每千克炸鸡料中加入 0. 001克的复配物制成混合包裹料，用此混合包裹料包裹的鸡 翅为试验组 B; 同理， 在每千克炸鸡料中加入 0. 01克的复配物制成混合包裹料， 用此混合 包裹料包裹的鸡翅为试验组 C; 当每千克炸鸡料中加入 0. 1克的复配物制成混合包裹料， 用此混合包裹料包裹的鸡翅为试验组 D; 当每千克炸鸡料中加入 0. 5克的复配物制成混合 包裹料， 用此混合包裹料包裹的鸡翅为试验组 E; 当每千克炸鸡料中加入 1克的复配物制 成混合包裹料， 用此混合包裹料包裹的鸡翅为试验组 F; 当每千克炸鸡料中加入 2. 5克的 复配物制成混合包裹料， 用此混合包裹料包裹的鸡翅为试验组 G; 当每千克炸鸡料中加入 4. 9克的复配物制成混合包裹料， 用此混合包裹料包裹的鸡翅为试验组 H。
(¾油炸
将面粉加水调成稀薄糊状， 并打入鸡蛋一个， 拌匀制成面糊。 将上述各组鸡翅（已经 裹有相应的炸鸡料)再分别挂上面糊， 立即上油锅炸制， 等到金黄色即出锅， 每次炸完一 组后将油弃去。样品预处理方法同实施例 2,经样品预处理后用 LC- MS/MS测定其丙烯酰胺 的含量。
根据内标法计算得含竹叶提取物的复配物对炸鸡翅产生丙烯酰胺的抑制率 (如表 16 所示）。
表 16含竹叶提取物的复配物对炸鸡翅产生的丙烯酰胺的抑制率 ( ^6)
Figure imgf000021_0001
由表 16可见， 在炸鸡翅过程中当含竹叶提取物的复配物添加量在 0. 001-4. 9g/kg的 范围内时， 与原料混合后对炸鸡翅产生丙烯酰胺具有不同程度的抑制作用， 其抑制率在复 配物添加量为 0. 001-lg/kg时随着添加量的增加而增加， 而在添加量为 1-4. 9g/kg时却随 着添加量的增加而减少， 这说明存在一个最适添加量区间。 因此， 含竹叶提取物的复配物 对炸鸡翅产生的丙烯酰胺具有不同程度的抑制作用。
实施例 17
含竹叶提取物的复配物对油条产生丙烯酰胺的抑制作用
将竹叶提取物和葡萄籽提取物按 3 : 1的质量比混合得到复配物，此时竹叶提取物占复 配物总重的 75%。
竹叶提取物 (A0B)的来源同实施例 1， 葡萄籽提取物为市售产品。
试验分组
本实验共设 8个组：
空白对照组 A， 面粉不作任何处理。
在每千克面粉中加入 0. 001克的复配物制成试验组 B;同理，在每千克面粉中加入 0. 01 克的复配物制成试验组 C; 在每千克面粉中加入 0. 1克的复配物制成试验组 D; 在每千克 面粉中加入 0. 5克的复配物制成试验组 E; 在每千克面粉中加入 1克的复配物制成试验组 F;在每千克面粉中加入 2. 5克的复配物制成参比对照组 G;在每千克面粉中加入 4. 9克的 复配物制成试验组 H。 将上述 8组用于制作油条的面粉， 分别加入适量苏打和发酵粉， 然后加水和成面团。 静置 12 h后再和面一次， 然后静置直至油炸。
(3)油炸
将各组发酵好的面团搓成条状， 切成小团， 然后拉成长条状以后入油锅炸制， 待金黄 色且膨松后将油条捞出油锅并沥干油分即成。 样品预处理方法同实施例 2， 经样品预处理 后用 LC- MS/MS测定其丙烯酰胺的含量。
根据内标法计算得含竹叶提取物的复配物对油条产生丙烯酰胺的抑制率 (如表 17 所 表 17含竹叶提取物的复配物对油条产生的丙烯酰胺的抑制率 (Λ=6)
Figure imgf000022_0001
由表 17可见， 在制作油条过程中当含竹叶提取物的复配物添加量在 0. 001-4. 9g/kg 的范围内时， 与原料混合后对炸油条产生丙烯酰胺具有不同程度的抑制作用， 其抑制率在 含竹叶提取物的复配物添加量为 0. 001- lg/kg时随着添加量的增加而增加， 而在添加量为 1 - 4. 9g/kg时却随着添加量的增加而减少， 这说明存在一个最适添加量区间。 因此， 含竹 叶提取物的复配物对炸油条产生的丙烯酰胺具有不同程度的抑制作用。
实施例 18
含竹叶提取物的复配物对油条产生丙烯酰胺的抑制作用
将竹叶提取物和银杏提取物、 茶叶提取物、 迷迭香提取物、苹果多酚提取物、 山楂提 取物、 洋葱提取物、 甘草提取物、 葛根提取物、 葡萄籽提取物、 牛蛭提取物按 30 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1的质量比混合得到复配物，此时竹叶提取物占复配物总重的 75%。
竹叶提取物 (A0B)的来源同实施例 1 ; 茶叶提取物为浙江大学茶学系提供的水溶性茶 多酚制剂， 含量为 98%; 苹果多酚提取物和洋葱提取物均为浙江大学生物系统工程与食品 科学学院天然产物实验室自制， 均为 30%的乙醇提取物干粉； 银杏提取物、迷迭香提取物、 山楂提取物、 甘草提取物、 葛根提取物、 葡萄籽提取物、 牛蛭提取物为市售产品。
试验分组
本实验共设 8个组：
空白对照组 A， 面粉不作任何处理。
在每千克面粉中加入 0. 001克的复配物制成试验组 B;同理，在每千克面粉中加入 0. 01 克的复配物制成试验组 C; 在每千克面粉中加入 0. 1克的复配物制成试验组 D; 在每千克 面粉中加入 0. 5克的复配物制成试验组 E; 在每千克面粉中加入 1克的复配物制成试验组 F;在每千克面粉中加入 2. 5克的复配物制成试验组 G;在每千克面粉中加入 4. 9克的复配 物制成试验组 H。
将上述 8组用于制作油条的面粉， 分别加入适量苏打和发酵粉， 然后加水和成面团。 静置 12 h后再和面一次， 然后静置直至油炸。
(3)油炸
将各组发酵好的面团搓成条状， 切成小团， 然后拉成长条状以后入油锅炸制， 待金黄 色且膨松后将油条捞出油锅并沥干油分即成。 样品预处理方法同实施例 2， 经样品预处理 后用 LC- MS/MS测定其丙烯酰胺的含量。
根据内标法计算得含竹叶提取物的复配物 (质量含量为 75%)对油条产生丙烯酰胺的抑 制率 (如表 18所示）。
表 18含竹叶提取物的复配物对油条产生的丙烯酰胺的抑制率 0?=6)
Figure imgf000023_0001
由表 18可见， 在制作油条过程中当含竹叶提取物的复配物添加量在 0. 001-4. 9g/kg 的范围内时， 与原料混合后对炸油条产生丙烯酰胺具有不同程度的抑制作用， 其抑制率在 含竹叶提取物的复配物添加量为 0. 001- lg/kg时随着添加量的增加而增加， 而在添加量为 1 - 4. 9g/kg时却随着添加量的增加而减少， 这说明存在一个最适添加量区间。 因此， 含竹 叶提取物的复配物对炸油条产生的丙烯酰胺具有不同程度的抑制作用。 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

权 利 要 求
1.一种竹叶提取物的用途， 其特征在于， 用于制备热加工食品中丙烯酰胺抑制 剂。
2.如权利要求 1所述的用途， 其特征在于， 所述抑制剂中含有 1- 99^%竹叶提 取物。
3.如权利要求 1所述的用途， 其特征在于， 所述抑制剂中还含有银杏提取物、 茶叶提取物、 迷迭香提取物、 苹果多酚提取物、 山楂提取物、 洋葱提取物、 甘草提 取物、 葛根提取物、 葡萄籽提取物和牛蛭提取物中的至少一种。
4.一种热加工食品的方法， 其特征在于， 包括步骤-
(a)将待加工的食品与丙烯酰胺抑制剂混合， 形成混有抑制剂的食品， 其中所 述抑制剂含有竹叶提取物；
(b)在 120°C或以上温度热加工所述的混有抑制剂的食品， 形成热加工食品。
5.如权利要求 4所述的方法， 其特征在于， 所述的待加工食品包括以油炸、焙 烤、 烧烤、 烘烤、 微波加热、 膨化、 燃烧方式得到的炸薯条、 炸薯片、 薄脆饼、 饼 干、 蛋糕、 面包、 谷物早餐、 油条、 大饼、 方便面、 汉堡包、 炸鸡块、 咖啡、 可可、 烟草、 香烟。
6.如权利要求 4所述的方法， 其特征在于， 步骤 (a)中每千克待加工食品与 0. 001-5克丙烯酰胺抑制剂混合。
7.如权利要求 4所述的方法， 其特征在于， 步骤 (a)中每千克待加工食品与
0. 01-2克丙烯酰胺抑制剂混合。
8.如权利要求 4所述的方法， 其特征在于， 步骤 (a)中每千克待加工食品与 0. 1-1克丙烯酰胺抑制剂混合。
9.如权利要求 4所述的方法， 其特征在于， 在步骤 (a)中， 混合步骤采用包裹、 浸泡或喷洒的方式进行。
10.—种用于热加工食品中的丙烯酰胺抑制剂组合物， 其特征在于， 它含有 1-99^%竹叶提取物以及 1- 9 t%银杏提取物、 茶叶提取物、 迷迭香提取物、 苹果多 酚提取物、 山楂提取物、 洋葱提取物、 甘草提取物、 葛根提取物、 葡萄籽提取物和 牛蛭提取物中的至少一种。
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