阅读说明：本技术 一种坚果炒制品的复配添加剂中tbhq检测方法 (Detection method for TBHQ in compound additive of fried nut product ) 是由 李功 周燕霞 石凯文 张艳 马士飞 杨雅芹 李俐 于 2019-12-10 设计创作，主要内容包括：本发明公开了一种坚果炒制品的复配添加剂中TBHQ检测方法,属于食品检测技术领域。本申请的坚果炒制品的复配添加剂中TBHQ检测方法,包括依次的样品前处理、标准品制备、平衡色谱条件和样品检测的步骤,所述标准品制备步骤中,TBHQ标准品工作液浓度梯度为50mg/L、20mg/L、10mg/L、5mg/L、1mg/L；可以更全面的覆盖坚果炒货产品中的TBHQ含量。所述平衡色谱条件为使用高效液相色谱仪,包括C<Sub>18</Sub>色谱柱,柱长250mm,内径4.6mm粒径5um,或等效色谱柱及紫外检测器,流动相为0.5％甲酸水溶液：甲醇＝50:50,柱温35℃,进样量10ul,检测波长280nm,该色谱条件更适宜于坚果炒货行业复配添加剂中TBHQ的检测。(The invention discloses a method for detecting TBHQ in a compound additive of a fried nut product, and belongs to the technical field of food detection. The method for detecting TBHQ in the compound additive of the fried nut product comprises the steps of sample pretreatment, standard product preparation, equilibrium chromatographic conditions and sample detection in sequence, wherein in the step of standard product preparation, the concentration gradient of a TBHQ standard product working solution is 50mg/L, 20mg/L, 10mg/L, 5mg/L and 1 mg/L; the TBHQ content in the nut roasted product can be more completely covered. The equilibrium chromatographic condition is to use a high performance liquid chromatograph comprising C 18 A chromatographic column with the length of 250mm and the inner diameter of 4.6mm and the particle size of 5um, or an equivalent chromatographic column and an ultraviolet detector, and a mobile phase is 0.5 percent formic acid water solution: the methanol is 50:50, the column temperature is 35 ℃, the sample injection amount is 10ul, the detection wavelength is 280nm, and the chromatographic condition is more suitable for nutsAnd (3) detecting TBHQ in the compound additive in the roasted seeds and nuts industry.)

1. Nut stir-frying deviceThe detection method of TBHQ in the compound additive of the product is characterized by comprising the steps of sample pretreatment, standard preparation, equilibrium chromatographic condition and sample detection in sequence, wherein in the step of standard preparation, the concentration gradient of a TBHQ standard working solution is 50mg/L, 20mg/L, 10mg/L, 5mg/L and 1 mg/L; the equilibrium chromatographic condition is to use a high performance liquid chromatograph comprising C₁₈A chromatographic column with the length of 250mm and the inner diameter of 4.6mm and the particle size of 5um, or an equivalent chromatographic column and an ultraviolet detector, and a mobile phase is 0.5 percent formic acid water solution: the methanol is 50:50, the column temperature is 35 ℃, the sample injection amount is 10ul, and the detection wavelength is 280 nm.

2. The method for detecting TBHQ in the compound additive of the fried nut product as claimed in claim 1,

the calculation formula of the sample detection is as follows:

xi is AxV/m; in the formula:

xi: the antioxidant content of the sample in milligrams per kilogram;

a: the TBHQ solution concentration, in micrograms per milliliter, was obtained from the standard curve;

v: the final volume of the sample solution in milliliters;

m: weighing the mass of the sample oil, wherein the unit is gram;

the result is to retain three significant digits or two digits after the decimal point.

3. The method for detecting TBHQ in the compound additive of the fried nut product as claimed in claim 2, wherein in the pretreatment of the sample, the shells of the nuts to be detected are removed by a huller to obtain nut kernels, the nut kernels are picked to obtain the nut kernels without impurities, the nut kernels are smashed into uniform granular samples and then placed into a ground reagent bottle, and petroleum ether is added into the uniform granular samples to be uniformly mixed and soaked to obtain soaking liquid; filtering the soak solution by qualitative filter paper, collecting the filtrate by using a round-bottomed flask, and separating solid impurities to obtain the required filtrate; connecting the round-bottom flask containing the filtrate with a rotary evaporator, placing the round-bottom flask in a water bath kettle, and performing rotary evaporation, wherein petroleum ether in the flask is completely volatilized, and the operation is stopped only when the oil sample is remained; weighing the grease sample, and placing the grease sample in a centrifuge tube after weighing; adding an acetonitrile saturated normal hexane solution into a centrifugal tube filled with a grease sample, uniformly mixing in a vortex mode, and then carrying out ultrasonic treatment; adding an acetonitrile solution saturated by normal hexane into the centrifugal tube after ultrasonic treatment, uniformly mixing by vortex, and then centrifuging; taking out acetonitrile extracting solution from the centrifuged solution for later use, adding n-hexane saturated acetonitrile solution into a centrifugal tube, uniformly mixing by vortex, and centrifuging; taking out acetonitrile extracting solution from the solution after the second centrifugation, mixing the acetonitrile extracting solution with the first acetonitrile extracting solution, repeatedly adding n-hexane saturated acetonitrile solution into the centrifugal tube, uniformly mixing in a vortex mode, and then centrifuging; and taking out acetonitrile extracting solution from the solution after the third centrifugation, and mixing the acetonitrile extracting solution with the acetonitrile extracting solution obtained in the previous two times to obtain a final target product for use on a computer.

4. The method for detecting TBHQ in the compound additive of the fried nut product as claimed in claim 2, wherein the purity requirement of the TBHQ standard product in the standard product preparation step is more than or equal to 98%, the TBHQ standard product is accurately weighed and placed in a volumetric flask, and then acetonitrile solvent is added to the volumetric flask to be constant volume to a scale, so as to prepare a stock solution, and the stock solution is stored in a dark place; sucking the stock solution to a certain amount, placing the stock solution in a brown volumetric flask, adding an acetonitrile solvent to the volume to a certain scale, preparing an intermediate solution, and storing the intermediate solution in a dark place; respectively sucking 50ml, 20ml, 10ml, 5ml and 1ml of the intermediate solution, placing in a brown volumetric flask, adding acetonitrile solvent to a constant volume to scale, and preparing into working solutions of 50mg/L, 20mg/L, 10mg/L, 5mg/L and 1mg/L for use on a machine.

5. The method for detecting TBHQ in the compound additive of the fried nut product as claimed in claim 3, wherein specific operating parameters in the sample pretreatment are as follows: removing shell of nut with a huller to obtain nut kernel of at least 200g, picking to obtain impurity-free shelled melon seed, and crushing into uniform granular sample of about 5 mm; putting the uniform granular sample into a 250ml ground reagent bottle, adding petroleum ether with a boiling range of 30-60 ℃ in an amount which is 2-3 times that of the sample, and fully and uniformly mixing and soaking for about 12 hours to prepare a soaking solution; filtering the soak solution by qualitative filter paper, collecting the filtrate by using a round-bottom flask, and separating solid impurities to obtain the required filtrate; connecting the round-bottom flask containing the filtrate with a rotary evaporator, placing the flask in a water bath kettle at 60 ℃, performing rotary evaporation for 3-10 minutes, volatilizing all petroleum ether in the flask, and stopping only residual grease sample; weighing a grease sample by using an electronic balance, accurately weighing 1g to 0.01g, and placing the grease sample in a 50ml centrifuge tube; adding 5ml of acetonitrile saturated normal hexane solution into a centrifugal tube filled with a grease sample, uniformly mixing in a vortex mode for 1min, and carrying out ultrasonic treatment for 2 h; adding 5ml of n-hexane saturated acetonitrile solution into the centrifugal tube after ultrasonic treatment, uniformly mixing in a vortex manner for 2min, and centrifuging at 3000r/min for 5 min; taking out acetonitrile extracting solution from the centrifuged solution for later use, adding 5ml of n-hexane saturated acetonitrile solution into a centrifugal tube, uniformly mixing for 2min by vortex, and centrifuging for 5min at 3000 r/min; and taking out acetonitrile extracting solution from the solution after the second centrifugation, mixing the acetonitrile extracting solution with the first acetonitrile extracting solution, repeatedly adding 5ml of n-hexane saturated acetonitrile solution into the centrifugal tube, uniformly mixing for 2min in a vortex mode, and centrifuging for 5min at 3000 r/min.

6. The method for detecting TBHQ in the compound additive of the fried nut product as claimed in any one of claims 1 to 5, wherein the specific operating parameters in the preparation of the standard product are as follows: the purity requirement of the TBHQ standard product is more than or equal to 98 percent; accurately weighing 0.1g of TBHQ standard product to 0.1mg, placing in a 100ml brown volumetric flask, adding acetonitrile solvent to constant volume to scale, making into 1000mg/L stock solution, and storing at 0-4 deg.C in dark place; sucking 10ml of the stock solution, placing in a 100ml brown volumetric flask, adding acetonitrile solvent to a constant volume to a scale to prepare 100mg/L intermediate solution, and storing at 0-4 ℃ in a dark place.

Technical Field

The invention belongs to the technical field of detection, and particularly relates to a method for detecting TBHQ in a compound additive of a fried nut product.

Background

The compound additive is a formula mixture containing two or more than two food additives. According to the definition of national food safety standard 'Compound food additive general rule' issued by Ministry of health of 7/6/2011, the compound food additive is prepared by physically and uniformly mixing two or more kinds of single-variety food additives with or without auxiliary materials in order to improve the food quality and facilitate the food processing.

TBHQ (tert-butylhydroquinone) is an edible antioxidant which is allowed to be added in a small amount by national regulations, and compared with BHT and BHA, TBHQ has safer and nontoxic performance because of the small addition amount. There was an incident of exposure to media of TBHQ from the german cherry bloc in a famous fast food chain at one time in the country. According to the FDA regulations in the united states, the limit of use of TBHQ in fats and oils or the like is 0.02% of the fat in fats and oils or foods. [1] Foreign studies have shown that TBHQ shows effects on the health of experimental animals, such as development of gastric cancer and damage to DNA, with increasing doses. [2] TBHQ has a disadvantage over other antioxidants in that it is used in a narrow range of industries, both food and pharmaceutical and cosmetic, but is expensive and widely used as an antioxidant.

At present, an antioxidant is added into a compound additive, THBQ is an antioxidant with a large using amount, so that the supervision on TBHQ is stricter, but the detection on TBHQ by the existing standard is not perfect, a detection method aiming at the nut roasting industry is not provided, and the detection is easy to be abnormal due to the fact that a sample is not suitable in the detection process.

Through retrieval, the Chinese patent application publication number: CN105699535A, publication date: 2016.6.22 discloses a method for detecting tert-butylhydroquinone in food. The detection method comprises the following steps: (1) separating tert-butyl hydroquinone in the food to be detected by using a high performance liquid chromatography; (2) under the condition of flow injection, mixing the effluent liquid in the step (1) with a chemiluminescence reagent to obtain a system, carrying out chemiluminescence reaction, and obtaining the concentration of the tert-butylhydroquinone in the food according to the chemiluminescence intensity value of the system; the chemiluminescence reagent is a mixed solution of a luminol-potassium permanganate system and an alkaline pH regulator. The detection method aims at the detection of the THBQ in the liquid fluid oil, and the accuracy of the THBQ detection method is difficult to guarantee when the THBQ detection method is applied to the detection of the THBQ in the solid fried nut products.

Disclosure of Invention

1. Problems to be solved

Aiming at the problem that TBHQ in the compound additive in the existing nut roasted product industry has no specific detection method, the invention provides a method for detecting TBHQ in the compound additive of a nut roasted product. The pretreatment part can be used for extracting grease in the nut roasted products more pertinently, the standard product preparation part can be used for more comprehensively covering the TBHQ content in the nut roasted products, and the TBHQ content in the compound additive of the nut roasted products can be accurately detected.

2. Technical scheme

In order to solve the above problems, the present invention adopts the following technical solutions.

A method for detecting TBHQ in a compound additive of a fried nut product comprises the steps of sample pretreatment, standard substance preparation, equilibrium chromatographic condition and sample detection in sequence, wherein in the step of standard substance preparation, the concentration gradient of a TBHQ standard substance working solution is 50mg/L, 20mg/L, 10mg/L, 5mg/L and 1 mg/L; the TBHQ content in the nut roasted product can be more completely covered. The equilibrium chromatographic condition is to use a high performance liquid chromatograph comprising C₁₈Chromatographic column with length of 250mm and inner diameter of 4.6mm and particle size of 5um, or equivalent chromatographic column and ultraviolet detector, mobile phase0.5% aqueous formic acid: the methanol is 50:50, the column temperature is 35 ℃, the sample loading is 10ul, the detection wavelength is 280nm, and the chromatographic condition is more suitable for detecting TBHQ in the compound additive in the nut roasting industry.

Further, the calculation formula of the sample detection is as follows:

xi is AxV/m; in the formula:

xi: the antioxidant content of the sample in milligrams per kilogram;

a: the TBHQ solution concentration, in micrograms per milliliter, was obtained from the standard curve;

v: the final volume of the sample solution in milliliters;

m: weighing the mass of the sample oil, wherein the unit is gram;

the result is to retain three significant digits or two digits after the decimal point.

Further, in the sample pretreatment, the shells of nuts to be detected are removed by a huller to prepare nut kernels, the nut kernels are picked to obtain the nut kernels without impurities, the nut kernels are crushed into uniform granular samples and then are placed into a ground reagent bottle, and petroleum ether is added into the uniform granular samples to be uniformly mixed and soaked to prepare soaking liquid; filtering the soak solution by qualitative filter paper, collecting the filtrate by using a round-bottomed flask, and separating solid impurities to obtain the required filtrate; connecting the round-bottom flask containing the filtrate with a rotary evaporator, placing the round-bottom flask in a water bath kettle, and performing rotary evaporation, wherein petroleum ether in the flask is completely volatilized, and the operation is stopped only when the oil sample is remained; weighing the grease sample, and placing the grease sample in a centrifuge tube after weighing; adding an acetonitrile saturated normal hexane solution into a centrifugal tube filled with a grease sample, uniformly mixing in a vortex mode, and then carrying out ultrasonic treatment; adding an acetonitrile solution saturated by normal hexane into the centrifugal tube after ultrasonic treatment, uniformly mixing by vortex, and then centrifuging; taking out acetonitrile extracting solution from the centrifuged solution for later use, adding n-hexane saturated acetonitrile solution into a centrifugal tube, uniformly mixing by vortex, and centrifuging; taking out acetonitrile extracting solution from the solution after the second centrifugation, mixing the acetonitrile extracting solution with the first acetonitrile extracting solution, repeatedly adding n-hexane saturated acetonitrile solution into the centrifugal tube, uniformly mixing in a vortex mode, and then centrifuging; and taking out acetonitrile extracting solution from the solution after the third centrifugation, and mixing the acetonitrile extracting solution with the acetonitrile extracting solution obtained in the previous two times to obtain a final target product for use on a computer.

Further, in the standard preparation step, the purity requirement of the TBHQ standard substance is more than or equal to 98%, the TBHQ standard substance is accurately weighed and placed in a volumetric flask, and then acetonitrile solvent is added to the volumetric flask to a constant volume to a scale, so that a stock solution is prepared and stored in a dark place; sucking the stock solution to a certain amount, placing the stock solution in a brown volumetric flask, adding an acetonitrile solvent to the volume to a certain scale, preparing an intermediate solution, and storing the intermediate solution in a dark place; respectively sucking 50ml, 20ml, 10ml, 5ml and 1ml of the intermediate solution, placing in a brown volumetric flask, adding acetonitrile solvent to a constant volume to scale, and preparing into working solutions of 50mg/L, 20mg/L, 10mg/L, 5mg/L and 1mg/L for use on a machine.

Further, in the sample pretreatment, specific operating parameters are as follows: removing shell of nut with a huller to obtain nut kernel of at least 200g, picking to obtain impurity-free shelled melon seed, and crushing into uniform granular sample of about 5 mm; putting the uniform granular sample into a 250ml ground reagent bottle, adding petroleum ether with a boiling range of 30-60 ℃ in an amount which is 2-3 times that of the sample, and fully and uniformly mixing and soaking for about 12 hours to prepare a soaking solution; filtering the soak solution by qualitative filter paper, collecting the filtrate by using a round-bottom flask, and separating solid impurities to obtain the required filtrate; connecting the round-bottom flask containing the filtrate with a rotary evaporator, placing the flask in a water bath kettle at 60 ℃, performing rotary evaporation for 3-10 minutes, volatilizing all petroleum ether in the flask, and stopping only residual grease sample; weighing a grease sample by using an electronic balance, accurately weighing 1g to 0.01g, and placing the grease sample in a 50ml centrifuge tube; adding 5ml of acetonitrile saturated normal hexane solution into a centrifugal tube filled with a grease sample, uniformly mixing in a vortex mode for 1min, and carrying out ultrasonic treatment for 2 h; adding 5ml of n-hexane saturated acetonitrile solution into the centrifugal tube after ultrasonic treatment, uniformly mixing in a vortex manner for 2min, and centrifuging at 3000r/min for 5 min; taking out acetonitrile extracting solution from the centrifuged solution for later use, adding 5ml of n-hexane saturated acetonitrile solution into a centrifugal tube, uniformly mixing for 2min by vortex, and centrifuging for 5min at 3000 r/min; and taking out acetonitrile extracting solution from the solution after the second centrifugation, mixing the acetonitrile extracting solution with the first acetonitrile extracting solution, repeatedly adding 5ml of n-hexane saturated acetonitrile solution into the centrifugal tube, uniformly mixing for 2min in a vortex mode, and centrifuging for 5min at 3000 r/min.

Further, in the preparation of the standard product, the specific operating parameters are as follows: the purity requirement of the TBHQ standard product is more than or equal to 98 percent; accurately weighing 0.1g of TBHQ standard product to 0.1mg, placing in a 100ml brown volumetric flask, adding acetonitrile solvent to constant volume to scale, making into 1000mg/L stock solution, and storing at 0-4 deg.C in dark place; sucking 10ml of the stock solution, placing in a 100ml brown volumetric flask, adding acetonitrile solvent to a constant volume to a scale to prepare 100mg/L intermediate solution, and storing at 0-4 ℃ in a dark place.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) in the sample pretreatment step, the sample is crushed, a required target object is extracted by using a reagent, and a final product is prepared by reacting a standard reagent with the extracted target object, so that the method can better extract grease, is convenient for subsequent detection, and the detection result can be closer to the national standard limit requirement;

(2) according to the preparation step of the standard product, the concentration gradient of the working solution of the TBHQ standard product is 50mg/L, 20mg/L, 10mg/L, 5mg/L and 1mg/L, the equivalent content of the TBHQ in the roasted nut product is about 10mg/L, and the working solution of the TBHQ standard product with the concentration gradient can more comprehensively cover the roasted nut product;

(3) according to the step of balancing chromatographic conditions, parameters are referred to special conditions in daily detection, and the chromatographic conditions are more suitable for detecting TBHQ in the sample due to the fact that the complex additive added into the roasted nuts is complex, so that impurity interference is distinguished, and detection accuracy is improved.

Detailed Description

The invention is further described with reference to specific examples.