CN114858943B

CN114858943B - Method for detecting content of animal-derived natural pigment in food

Info

Publication number: CN114858943B
Application number: CN202210575402.9A
Authority: CN
Inventors: 孟鹏; 潘城; 黄永辉; 黄姝玲; 蔡小明; 李颖
Original assignee: Fujian Institute Of Product Quality Inspection Fujian Defective Product Recall Technology Center
Current assignee: Fujian Institute Of Product Quality Inspection Fujian Defective Product Recall Technology Center
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2023-05-30
Anticipated expiration: 2042-05-25
Also published as: CN114858943A

Abstract

The invention discloses a method for detecting the content of animal-derived natural pigment in food, wherein the animal-derived natural pigment is lac red pigment. The method comprises the following steps: (1) preparing a standard stock solution and a standard working solution; (2) preparing a sample solution to be tested; (3) high performance liquid chromatography conditions; (4) establishing a standard working curve; (5) analysis of results. The method establishes a solid phase extraction-high performance liquid chromatography method of natural pigment lac red pigment of animal origin in food. The method fills the blank in the field of detecting the content of the natural pigment lac in the multi-class food, can effectively reduce the interference of the food matrix components, has high sensitivity and good repeatability, and is suitable for the daily detection and supervision requirements of the lac pigment in the food.

Description

Method for detecting content of animal-derived natural pigment in food

Technical Field

The invention relates to the field of foods, in particular to a method for detecting the content of animal-derived natural pigment, and specifically relates to a solid-phase extraction-high performance liquid chromatography detection method of natural pigment lac-red pigment in foods.

Background

Shellac, also called shellac, red gum, shikonin, etc. is a product obtained by secretion of shellac on a host tree after feeding, mainly contains shellac wax, shellac resin, shellac pigment, etc., has good film forming property and acid resistance, and is used as a film coating agent or a coating material in industries such as food, medicine, etc. The lac pigment is obtained by extracting lac, is a typical natural pigment of insect source, has strong coloring capability and bright color, can be used as food coloring agent, textile dye, cell dye and the like, and has higher economic value. The lac pigment mainly contains A, B, C, D, E active components with definite structures, and the content of each component fluctuates with the change of the production place and seasons. In general, the lac A, B has the highest content, accounting for 80-90% of the total content, and the rest components accounting for 10-20%. The maximum usage amount of lac pigment in food is 0.5 g/kg, and the lac pigment can be used in jam, cocoa products, chocolate and chocolate products (including cocoa butter substitute chocolate and products), candies, baked food stuffing and surface hanging paste (limited flavor pie stuffing), compound seasonings, fruit and vegetable juice (paste) beverages, carbonated beverages, flavored beverages (limited fruit flavored beverages) and prepared wine. Natural pigments have higher safety and stability than synthetic pigments with potential risks of carcinogenesis, teratogenesis, etc., but may also be harmful to human health if overdose, oversrange are used.

In addition, black tea and brown sugar are native to China, have long history of application, respectively contain tea polyphenol, tea pigment, alkaloid, vitamin, amino acid and other components, have pharmacological effects of resisting inflammation, protecting cardiovascular and cerebrovascular diseases, resisting oxidation, promoting growth and the like, are accepted as green and healthy food, and are deeply favored by masses. Color is one of factors influencing consumers to purchase black tea and brown sugar, and black tea and brown sugar with bright and gorgeous color and good eye effect are often selected more. Because of the large daily usage, the disorder of counterfeiting of black tea and brown sugar by using artificial synthetic pigments exists in the past, but with the increasingly strict supervision of related departments, illegal merchants begin to try to replace the artificial synthetic pigments by natural pigments to be added into the black tea and brown sugar with relatively poor quality, in an attempt to change the aged tea into 'new tea' and the secondary sugar into 'good sugar', thereby obtaining higher profit. The food additive use standard in China clearly prescribes that black tea and brown sugar cannot be added with any additive ingredients, including natural pigments. At present, the detection method of the lac pigment in the black tea and the brown sugar is blank at home and abroad, so that the establishment of the accurate, rapid and sensitive detection method of the lac pigment in the black tea and the brown sugar has important significance for meeting the supervision requirement.

At present, the pigment detection method mainly comprises a high performance liquid chromatography method, a high performance liquid chromatography-tandem mass spectrometry method and the like, the research of the method is concentrated on the aspect of artificially synthesizing pigment, and the detection of natural pigment lac red is still reported. The method only researches single-component single matrix, and is easy to be influenced by metal ions in complex food matrix to generate fluorescence quenching phenomenon; the method only researches a single-substrate juice sample, selects a low-band 286nm as detection wavelength, and targets are easily interfered by various foods and complex substrates; in addition, the method is completely used for testing, and the peak type of each component of the lac red pigment is found to have poor sensitivity and recovery rate, the standard adding recovery rate of the component A, B, C, E is respectively about 10%, 0%, 6% and 20%, and the detection requirements of various foods are difficult to meet.

The invention establishes the combination of solid phase extraction and high performance liquid chromatography to simultaneously determine the content of the lac A, B, C, D, E in the food. The method can realize the complete separation of each component of the lac red pigment on a common liquid-phase instrument for 7min, has good anti-interference capability, high sensitivity and accuracy and good repeatability, can meet the daily detection requirement of the natural pigment lac red in various foods, and provides a method basis for food production enterprises, regulatory departments and import and export detection departments.

Disclosure of Invention

The invention aims to provide a solid-phase extraction-high performance liquid chromatography detection method for the content of natural pigment lac in food, which is used for overcoming and solving the defect that the detection method for the content of lac in various foods which is not satisfied with the use standard of food additives in China at present is not provided, and filling the blank of the supervision field of the detection methods for the lac in black tea and brown sugar, so that the method has extremely strong applicability and practicability in the related field, and can completely satisfy the limit requirement of the related laws and regulations in China and forbidden detection limit requirement.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the method is a separation detection method capable of realizing natural pigment lac red pigment in food, and comprises the following steps:

the invention relates to a method for detecting the content of animal-derived natural pigment in food, the method comprises the following steps:

(1) Preparing standard stock solution and standard working solution, wherein the standard stock solution is 1000 mg/L, and the standard working solution is a series of standard working solutions with the concentration of 0.05mg/L, 0.1 mg/L, 0.5 mg/L, 1.0 mg/L, 5.0 mg/L, 10.0 mg/L and 100.0 mg/L prepared from the standard stock solution of lac red pigment by adopting a progressive dilution method;

(2) Preparing a sample solution to be tested: if the food sample is solid samples such as candy, baked food stuffing, chocolate, jam, flavoring, black tea, brown sugar and the like, placing the sample in a 50mL colorimetric tube, adding 30 mL of 0.02% ammonia water solution, mixing uniformly, performing ultrasonic treatment at 60 ℃ for 30 min, diluting and fixing the volume to 50mL by using the 0.02% ammonia water solution, mixing uniformly, centrifuging 12000 r/min for 3 min, taking supernatant 10 mL, adding 2 drops of acetic acid, mixing uniformly, centrifuging 12000 r/min for 3 min, and passing through a column; if the food sample is fruit and vegetable juice, carbonated beverage, flavored beverage, wine preparation and other liquid samples, placing the sample in a 50mL colorimetric tube, diluting the sample with pure water to constant volume to 50mL, mixing, performing ultrasonic treatment at room temperature for 10min, collecting supernatant 10 mL, adding 2 drops of acetic acid, mixing, centrifuging at 12000 r/min for 3 min, and standing; activating the PWAX column with 3mL methanol/acetic acid (99/1, v/v) solution and 3mL pure water/acetic acid (99/1, v/v) solution, transferring the 10 mL sample solution to the PWAX column (black tea and brown sugar sample are transferred to self-filling polyamide column), keeping the flow rate of effluent liquid to be 1-2 drops per minute, eluting with 3mL pure water/acetic acid (99/1, v/v) solution and 3mL methanol/acetic acid (99/1, v/v) solution after the upper column liquid completely flows out, discarding the eluent, eluting with 3mL methanol/1.6% ammonia water (70/30, v/v) solution, blowing the eluent liquid nitrogen to near dryness, adding pure water to constant volume to 1.0 mL, mixing, filtering with 0.45 μm water phase filter membrane, and measuring the filtrate by liquid phase chromatography.

(3) The instrument is a high performance liquid chromatograph, and the detector is a photodiode array detector; the chromatographic column is a C18 column, the column temperature is 35 ℃, and the detection wavelength is 484 nm; mobile phase A is methanol, mobile phase B is 0.02mol/L ammonium acetate solution (containing 0.009% phosphoric acid); flow rate: 1 mL/min; sample injection amount: 20. mu L; gradient elution procedure: 0-1.0 min,2% A;1.0 to 3.0min,2 percent A to 20 percent A;3.0 to 6.5min,20 percent of A to 55 percent of A; 6.5-7.1 min,55% A-2%A; 7.1 to 10.0min,2 percent of A.

(4) Drawing a standard curve: injecting a series of mixed standard working solutions of the lac pigment into a high performance liquid chromatograph, performing gradient elution and detection under the chromatographic condition of the step (3), qualitatively determining the retention time, and drawing a standard curve according to the corresponding relation between the peak area measured by the concentration and the concentration.

(5) Analysis of results: and (3) pouring the filtrate obtained in the step (2) into a high performance liquid chromatograph, performing gradient elution and detection under the chromatographic condition of the step (3), measuring the peak area of the target object in the filtrate, qualitatively determining the retention time, quantifying according to the standard curve manufactured in the step (4), and calculating the content of the lac pigment in the sample to be measured.

Compared with the prior art, the invention has the following outstanding advantages:

1. the invention fills the blank in the field of detecting the content of natural pigment lac in multi-category foods, the researched food matrix not only completely covers the food matrix range of jam, cocoa products, chocolate and chocolate products (including cocoa butter substitute chocolate and products) specified in the use standard of food additives in China, candies, baked food stuffing and surface hanging pulp (only limited flavor stuffing), composite seasonings, fruit and vegetable juice (pulp) beverage, carbonated beverage, flavored beverage (only limited fruit flavored beverage), wine preparation and the like, but also covers the food matrix range of brown sugar, black tea and the like with outstanding safety problems in the food field, can completely meet the current legal regulation in China and the food matrix range required by the burst safety detection of foods, has wide applicability, and the prior art only aims at a single food matrix of fruit juice, and has obvious technical limitation. The method can effectively reduce the interference of food matrix components, has high sensitivity and good reproducibility, and is suitable for daily detection and supervision requirements of lac pigment in food.

2. The invention relates to a detection technology of five components of lac dye A, B, C, D, E, wherein the existing detection technology is only used for lac dye A or only used for four components of lac dye A, B, C, E, and the lac dye comprises A, B, C, D, E five components, and the detection technology of the five components of the lac dye A, B, C, D, E is necessary to be developed from the aspects of the rigor of academic and methodology and the applicability of the method although the proportion of the component D is not high.

3. The invention selects the high-band sub-strong absorption peak 484 nm as the quantitative wavelength, which is different from the prior art that selects the low-band 286nm as the quantitative wavelength, and the accurate quantification of lac pigment is seriously affected by the interference of various food matrix samples including fruit juice at the low wavelength 286nm, and the 484 nm as the quantitative wavelength can completely meet the detection requirements of all food matrixes specified by the food additive standard of China without passing through a solid-phase extraction column, so that the interference phenomenon of the sample matrixes disappears.

4. According to the invention, by selecting a proper type C18 column (a C18 short column with the length of 50 mm) as a stationary phase, various mobile phase compositions and proportions are continuously explored and optimized, the chromatographic separation effect of the ultra-high performance liquid phase instrument can be realized in a conventional common liquid phase instrument, and compared with a common liquid phase system, the complete separation of each component of the lac red pigment can be realized in 7min, so that the method is simple and rapid, and better peak type and sensitivity are obtained. This is particularly important in screening large batches of samples, can greatly reduce solvent consumption, does not need to frequently prepare mobile phase by operators, does not need to operate a plurality of instruments, waits for the completion of the operation of large batches of sample tasks, greatly reduces the cost of consumables, time, instruments and labor, and saves more than 1/2 of the cost of chromatographic short columns by using a conventional liquid chromatograph and a more conventional liquid chromatographic column, so that the invention is easier to popularize and popularize.

5. The invention adopts methanol to prepare the standard stock solution of the lac haematochrome, pure water is adopted to prepare the series of standard working solutions, and the invention is different from the prior art that adopts ultrapure water to prepare the standard stock solution, and adopts acetonitrile/water (1/1, V/V) to dilute the stock solution into the series of standard working solutions. Experiments show that the standard substance cannot be dissolved completely when 1000 mg/L standard stock solution is prepared by adopting ultrapure water according to the prior art, and meanwhile, the peak type of each component of lac red is poor and the sensitivity is reduced when acetonitrile/water (1/1, V/V) is adopted to dilute the stock solution into a series of standard working solutions. The invention adopts pure methanol solvent to prepare standard stock solution, which has good solubility to the mauve red pigment standard substance, and adopts pure water to prepare series standard working solution to obtain good peak type and sensitivity of the target substance, and meanwhile, the solvent is pure water, and the preparation of the standard solution is not required to prepare a complex solvent system such as acetonitrile/water (1/1, V/V), thus the operation is simple and easy. However, it should be noted that when the standard stock solution is stored in a refrigerator at 0 ℃ to 4 ℃, a small amount of crystallization occurs, but the phenomenon does not occur at room temperature, which may be caused by the reduced temperature and the reduced solubility of the standard in the methanol solvent. Therefore, when preparing a series of standard solutions, the standard stock solution taken out of the refrigerator needs to be treated by ultrasonic treatment for 10min, cooled to room temperature, and then diluted with pure water to form a series of standard working solutions.

6. The invention explores the multi-dimensional deep level of the multi-class food pretreatment technology, finally optimizes the optimal extraction process of various lac red pigment components in the multi-class food, and has satisfactory recovery rate effect and primary purification effect. And the ultrasonic-assisted extraction treatment is carried out on the liquid food matrix sample by adopting pure water, the labeling recovery rate of each component of the lac red pigment in the liquid matrix sample is 90.1% -102.6%, and the labeling recovery effect is good. Because the lac pigment is stable under the acidic condition, the adsorption capacity is stronger, the desorption is easy under the alkaline condition, and the experiment further examines the standard recovery rate of each component of the lac pigment in the solid matrix sample under the condition of ammonia water solutions with different proportions (0%, 0.02%, 0.04%, 0.4% and 0.8%). The result shows that under the condition of 0% ammonia water, the standard adding recovery rate of each component of the lac red pigment in the chocolate and the candy is 90.5% -103.2%, the standard adding recovery rate of the lac red pigment A, B in the yolk pie sandwich, the jam, the seasonings, the brown sugar and the black tea is 76.1% -80.5%, and the standard adding recovery rate of C, D, E is 90.6% -102.5%. After the proportion of the ammonia water is increased to 0.02%, the labeling recovery rate of the lac dye A, B is increased to 83.4% -90.1%, but the C, D, E chromatographic peak disappears. Therefore, the invention selects 0.02% ammonia water solution as the extraction solvent of the solid matrix sample. The experiment further examines the effect of the recovery rate of each component of lac red pigment at 60 ℃. The result shows that the standard adding recovery rate of each component of the lac pigment is improved to 91.6% -103.4%, which further shows that the lac pigment has good thermal stability. In addition, the experiment further examines the influence of precipitant precipitation and acidification precipitation on the primary purification effect of the sample on the column solution and the labeling recovery rate of the lac red pigment. The results show that after the zinc acetate solution 2mL, the potassium ferrocyanide solution and the acetic acid solution 2 drops are respectively added, the purifying effect can be obviously improved, and the upper column solution is clear without turbidity; however, after 2mL of zinc acetate solution and potassium ferrocyanide solution are added, the labeling recovery rate of lac red A, B is 10.8% -11.9%, and the C, D, E chromatographic peak disappears; and after the addition of 2 drops of acetic acid solution, the recovery rate of each component of the lac pigment is 92.5% -107.4%, and the recovery rate effect is good. Therefore, the invention finally adopts pure water solvent to carry out ultrasonic treatment on the liquid matrix sample at room temperature, and heating the solid matrix sample by using 0.02% ammonia water solution, performing ultrasonic extraction at 60 ℃, and simultaneously adding 2 drops of acetic acid into the upper column liquid for acidification, precipitation and purification treatment. Under the optimized extraction conditions, the recovery rate effect and the primary purification effect of each component of the lac red pigment in each sample matrix are satisfactory.

7. The invention adopts the solid phase extraction technology to realize the functions of concentrating, enriching and purifying the lac red pigment on various food substrate samples, and effectively ensures the requirements of the anti-interference capability and the sensitivity of the method. Unlike available HLB solid phase extraction column, the present invention has enriched and purified components and recovered red pigment in HLB, PWAX, PWCX, PWA-2, PEP-2, PSA and other 7 solid phase extraction columns, and has excellent PWAX solid phase extraction column for all food substrates, the method has the advantages that the method completely meets the methodological requirements, meanwhile, the HLB column passing experiment is completely carried out according to the prior art, the standard adding recovery rate of each component A, B, C, E of the lac is about 10%, 0%, 6% and 20%, and the detection requirements of multi-category complex food matrixes can not be met. In addition, as the substrate components of the brown sugar and black tea samples are complex, the interference is serious, the self-filling polyamide solid phase extraction column with stronger anti-interference capability is selected, the anti-interference capability and the recovery rate effect completely meet the methodological requirements, meanwhile, the cost of the self-filling polyamide solid phase extraction column is lower than that of a commercial solid phase extraction column, the screening operation cost of a large number of samples is lower, and the filling material of the polyamide solid phase extraction column is simple and easy to obtain, so that the method is easier to popularize.

8. The analysis instrument used in the invention is a photodiode array detector for the high performance liquid chromatograph, and is easier to popularize and apply compared with the high-cost instruments such as the liquid chromatography, the ultra-high performance liquid chromatograph and the like, and meanwhile, the invention can overcome the defects that the fluorescence spectrometer cannot identify multiple components of lac, the fluorescence of the lac pigment is easily influenced by metal ions in complex food matrixes to generate quenching phenomenon, and the stability and reproducibility are poor.

Drawings

FIG. 1 is a high performance liquid chromatography separation diagram of a standard solution of a method for detecting the content of animal-derived natural pigment in food according to the invention.

Detailed Description

The invention is described in further detail below with reference to the drawings of the specification of the specific embodiments. The scope of the invention is not limited to the following examples.

Example 1

The present embodiment is implemented on the premise of the above technology of the present invention, and detailed embodiments and specific operation procedures are now given to illustrate the present invention:

1. reagents and materials

Unless otherwise indicated, all reagents were analytically pure and water was primary water as specified in GB/T6682.

1.1 methanol: chromatographic purity.

1.2 Ammonia Water

1.3 ammonium acetate

1.4 acetic acid

1.5 PWAX solid phase extraction column: 60 mg/3 mL

1.6 Polyamide solid phase extraction column: self-filling column, 500 mg/6 mL

1.7 standard: lac red pigment standard (CAS No.60687-93-6, purity 100%).

1.8 preparation of standard stock solution: accurately weighing the mauve red pigment standard 10 mg, dissolving with methanol, fixing the volume to 10 mL, preparing into standard stock solution with the concentration of 1000 mg/L, and storing in a refrigerator at 0-4 ℃.

1.9 Preparing a standard working solution: at the time of use, the standard stock solution (1.8) is taken out of the refrigerator, is cooled to room temperature after ultrasonic treatment for 10min, and is diluted into a series of standard working solutions by pure water.

Apparatus and device

2.1 high performance liquid chromatograph: a photodiode array detector is provided.

2.2 electronic balance: the sensing amount was 0.0001 and g.

2.3 ultrasonic cleaner.

2.4 centrifuge: the rotating speed is not lower than 15000 r/min.

2.5 solid phase extraction device.

2.6 Nitrogen blow dryer.

2.7 vortex mixer.

2.8 with plug colorimetric tube: 50. 50 and (3) mL.

Method

3.1 conditions for high Performance liquid chromatography

a) Chromatographic column: c18 column (4.6X105 mm,3.0 μm);

b) Mobile phase: the mobile phase A is methanol, and the mobile phase B is pure water; gradient elution procedure: 0-1.0 min,2% A;1.0 to 3.0min,2 percent A to 20 percent A;3.0 to 6.5min,20 percent of A to 55 percent of A; 6.5-7.1 min,55% A-2%A; 7.1 to 10.0min,2 percent of A;

c) Flow rate: 1.0 mL/min;

d) Column temperature: 35 ℃;

e) Sample injection amount: 20. mu L;

f) Detection conditions of the photodiode array detector: detection wavelength 484 nm

3.2 Standard curve drawing

The standard working solutions (1.9) were each used, and the chromatographic measurements were performed under the chromatographic conditions of 3.1. And (3) carrying out linear regression on the corresponding mass concentration (X, mg/L) by using the peak area (Y) of each analyte to draw a standard curve, thereby obtaining a linear regression equation.

Sample testing procedure

4.1 sample pretreatment

4.1.1 Solid sample (candy, roasted food stuffing, chocolate, jam, flavoring, black tea, brown sugar, etc.)

Weighing 5 g sample to 0.01 g, placing in a 50mL colorimetric tube, adding 30 mL of 0.02% ammonia water solution, mixing, performing ultrasonic treatment at 60deg.C for 30 min, diluting with 0.02% ammonia water solution to constant volume of 50mL, mixing, centrifuging at 12000 r/min for 3 min, collecting supernatant at 10 mL, adding 2 drops of acetic acid, mixing, centrifuging at 12000 r/min for 3 min, and standing.

4.1.2 Liquid sample (fruit and vegetable juice, carbonated beverage, flavor beverage, mixed liquor, etc.)

Weighing 5. 5 g sample to 0.01 g, placing in a 50mL colorimetric tube, diluting with pure water to constant volume of 50mL, mixing, performing ultrasonic treatment at room temperature for 10min, collecting supernatant 10 mL, adding 2 drops of acetic acid, mixing, centrifuging at 12000 r/min for 3 min, and standing.

4.1.3 Post passing

Activating the PWAX column with 3mL methanol/acetic acid (99/1, v/v) solution and 3mL pure water/acetic acid (99/1, v/v) solution, transferring the 10 mL sample solution to the PWAX column (black tea and brown sugar sample are transferred to self-filling polyamide column), keeping the flow rate of effluent liquid to be 1-2 drops per minute, eluting with 3mL pure water/acetic acid (99/1, v/v) solution and 3mL methanol/acetic acid (99/1, v/v) solution after the upper column liquid completely flows out, discarding the eluent, eluting with 3mL methanol/1.6% ammonia water (70/30, v/v) solution, blowing the eluent liquid nitrogen to near dryness, adding pure water to constant volume to 1.0 mL, mixing, filtering with 0.45 μm water phase filter membrane, and collecting filtrate for analysis.

4.2 test solution measurement

The prepared sample solutions were chromatographed under the same chromatographic conditions, and were characterized by retention time and quantified by an external standard method.

Representation of analysis results

The content of lac pigment in the sample is calculated according to the following formula (1):

………………… （1）

wherein:

Xthe content of lac pigment in the sample, mg/kg;

c-the concentration of a component of lac-red pigment in the sample solution, measured in micrograms per milliliter (mg/L), in a standard working curve;

Vthe volume of the extracting solution is fixed, units are milliliters (mL);

V ₂ -final volumetric volume in milliliters (mL);

m-mass of the sample in grams (g);

V ₁ the volume is divided into milliliters (mL).

Expressed as the arithmetic mean of two independent measurements obtained under repetitive conditions, the results retain three significant digits.

Methodology investigation, including linearity, limit of detection, limit of quantification, recovery, precision

6.1 linearity, limit of detection, limit of quantification: preparing a series of standard working solutions with the mass concentration of the lac pigment between 0.05 and 100.0 and mg/L, and carrying out linear regression on the mass concentration (unit mg/L) according to the peak area, wherein a linear equation, a correlation coefficient, a linear range, a detection limit and a quantitative limit are shown in Table 1. The result shows that the components of the lac pigment have good linear relation in the linear range, and the correlation coefficient r is more than 0.999. The detection limit and the quantitative limit of the method are obtained by the signal to noise ratio (S/N) =3 and (S/N) =10, the detection limit range of each component of the lac red pigment is 0.016-1.6 mg/kg, the quantitative limit range is 0.052-5.4 mg/kg, and the quantitative limit of the method can completely meet the limit requirement of the national food additive use standard on the lac red pigment in foods.

TABLE 1 Linear equation, correlation coefficient, linear range, detection limit and quantitative limit for lac-red pigment

Table 1 Linear equations, correlation coefficients (r), linear ranges, limits of detection (LODs) and limits ofquantification （LOQs）of laccaic acids

The relative content of each component in the lac red pigment is determined by adopting an area normalization method, and the relative contents of five components of the lac red A, B, C, D, E are 58.56%, 22.67%, 12.97%, 0.87% and 4.92% respectively.

6.2 recovery and precision: under optimized test conditions, blank food substrate samples were taken for standard recovery tests at standard levels of 0.1, 0.6, 1.0, 5.0, 6.0, 10.0 and 50.0 mg/kg, each level was repeated 3 times, and the results are shown in Table 2. As shown in Table 2, the standard recovery rate of the method is 85.0% -107.7%, and the RSDs are 0.2% -4.3%. The result shows that the method has good accuracy and precision, and is suitable for daily analysis and detection of natural pigment lac in food.

TABLE 2 recovery rate of lac dye from food and relative standard deviation [ ]n＝3）

Table 2 Recoveries and relative standard deviations (RSDs) of laccaic acids infoods (n=3）

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Finally, it is noted that the above embodiments are merely for illustrating the technical aspects of the present invention, not for limiting the same, and although the present invention has been described in detail with reference to the embodiments, it will be understood by those of ordinary skill in the art, modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be covered by the claims.

Claims

1. A method for detecting the content of animal-derived natural pigment in food is characterized in that the animal-derived natural pigment is lac pigment; the detection method comprises the following steps:

(2) Preparing a sample solution to be tested: if the food sample is a solid sample of candy, roasted food stuffing, chocolate, jam, flavoring, black tea and brown sugar, placing the sample in a 50mL colorimetric tube, adding 30 mL of 0.02% ammonia water solution, mixing uniformly, performing ultrasonic treatment at 60 ℃ for 30 min, diluting and fixing the volume to 50mL by using the 0.02% ammonia water solution, mixing uniformly, centrifuging 12000 r/min for 3 min, taking supernatant 10 mL, adding 2 drops of acetic acid, mixing uniformly, centrifuging 12000 r/min for 3 min, and passing through a column; if the food sample is fruit and vegetable juice, carbonated beverage, flavored beverage, and liquor liquid sample, placing the sample in a 50mL colorimetric tube, diluting with pure water to constant volume of 50mL, mixing, performing ultrasonic treatment at room temperature for 10min, collecting supernatant 10 mL, adding 2 drops of acetic acid, mixing, centrifuging at 12000 r/min for 3 min, and standing; activating a PWAX small column by using 99/1, v/v 3mL methanol/acetic acid solution and 99/1, v/v 3mL pure water/acetic acid solution successively, transferring the 10 mL sample solution to the PWAX small column, transferring black tea and brown sugar samples to a self-filling polyamide small column, keeping the flow rate of effluent liquid to be 1-2 drops per minute, eluting by using 99/1, v/v 3mL pure water/acetic acid solution and 99/1, v/v 3mL methanol/acetic acid solution after the upper column liquid completely flows out, discarding the eluent, eluting by using 70/30, v/v 3mL methanol/1.6% ammonia water solution, blowing the eluent liquid nitrogen to near dryness, adding pure water to a constant volume of 1.0 mL, uniformly mixing, filtering by using a 0.45 mu m aqueous phase filter membrane, and measuring the filtrate by liquid chromatography;

(3) The instrument is a high performance liquid chromatograph, and the detector is a photodiode array detector; the chromatographic column is a C18 column, the column temperature is 35 ℃, and the detection wavelength is 484 nm; mobile phase A is methanol, mobile phase B is 0.02mol/L ammonium acetate solution containing 0.009% phosphoric acid; flow rate: 1 mL/min; sample injection amount: 20. mu L; gradient elution procedure: 0-1.0 min,2% A;1.0 to 3.0min,2 percent A to 20 percent A;3.0 to 6.5min,20 percent of A to 55 percent of A; 6.5-7.1 min,55% A-2%A; 7.1 to 10.0min,2 percent of A;

(4) Drawing a standard curve: injecting a series of mixed standard working solutions of the lac pigment into a high performance liquid chromatograph, performing gradient elution and detection under the chromatographic condition of the step (3), qualitatively determining the retention time, and drawing a standard curve according to the corresponding relation between the peak area measured by the concentration and the concentration;

2. The method for detecting the content of animal-derived natural pigment in food according to claim 1, wherein the standard stock solution in step (1) is prepared by the following specific steps: accurately weighing a mauve pigment standard 10 mg, dissolving with methanol, and fixing the volume to 10 mL to prepare a standard stock solution with the concentration of 1000 mg/L; (1) The preparation of the standard working solution comprises the following specific steps: taking out the stock solution from the refrigerator at the time of use, cooling to room temperature after ultrasonic treatment for 10min, and preparing a series of standard working solutions with the concentration of 0.05mg/L, 0.1 mg/L, 0.5 mg/L, 1.0 mg/L, 5.0 mg/L, 10.0 mg/L and 100.0 mg/L by using pure water by adopting a progressive dilution method;

and (5) placing the standard stock solution in a refrigerator and preserving at the temperature of 0-4 ℃.

3. The method for detecting the content of animal-derived natural pigment in food according to claim 1, wherein the preparation of the sample solution to be detected in the step (2) specifically comprises the steps of: if the food sample is a solid sample of candy, baked food stuffing, chocolate, jam and flavoring, weighing 5 g samples to 0.01 g accurately, placing the samples in a 50mL colorimetric tube, adding 30 mL of 0.02% ammonia water solution, mixing uniformly, performing ultrasonic treatment at 60 ℃ for 30 min, diluting with 0.02% ammonia water solution to constant volume of 50mL, mixing uniformly, performing centrifugal treatment for 12000 r/min for 3 min, taking supernatant for 10 mL, adding 2 drops of acetic acid, mixing uniformly, performing centrifugal treatment for 12000 r/min for 3 min, and passing through a column; if the food sample is fruit and vegetable juice, carbonated beverage, flavored beverage, and wine liquid sample, weighing 5. 5 g sample to 0.01 g, placing the sample in a 50mL colorimetric tube, diluting with pure water to constant volume of 50mL, mixing, performing ultrasonic treatment at room temperature for 10min, collecting supernatant 10 mL, adding 2 drops of acetic acid, mixing, centrifuging 12000 r/min for 3 min, and standing; activating a PWAX small column by using 99/1, v/v 3mL methanol/acetic acid solution and 99/1, v/v 3mL pure water/acetic acid solution successively, transferring the 10 mL sample solution to the PWAX small column, keeping the flow rate of effluent liquid to be 1-2 drops per minute, eluting by using 99/1, v/v 3mL pure water/acetic acid solution and 99/1, v/v 3mL methanol/acetic acid solution after the upper column liquid completely flows out, discarding the eluent, eluting by using 70/30, v/v 3mL methanol/1.6% ammonia water solution, blowing the eluting liquid nitrogen to be nearly dry, adding pure water to fix the volume to 1.0 mL, uniformly mixing, filtering by using a 0.45 mu m water phase filter membrane, and measuring the filtrate by liquid chromatography;

if the food sample is tea and brown sugar solid sample, weighing 5 g sample to 0.01 g accurately, placing the sample in a 50mL colorimetric tube, adding 30 mL of 0.02% ammonia water solution, mixing uniformly, performing ultrasonic treatment at 60 ℃ for 30 min, diluting and fixing the volume to 50mL by using 0.02% ammonia water solution, mixing uniformly, centrifuging 12000 r/min for 3 min, taking supernatant 10 mL, adding 2 drops of acetic acid, mixing uniformly, centrifuging 12000 r/min for 3 min, and passing through a column; eluting the PA column with 99/1, v/v 3mL methanol/acetic acid solution and 3 mL1% acetic acid solution successively, transferring the 10 mL sample solution to the PA column, keeping the flow rate of effluent liquid at 1-2 drops per minute, eluting with 3 mL1% acetic acid solution and 99/1, v/v 3mL methanol/acetic acid solution after the column liquid completely flows out, discarding the eluent, eluting with 70/30, v/v 3mL methanol/1.6% ammonia water solution, blowing the eluting liquid nitrogen to near dryness, adding pure water to constant volume of 1.0 mL, mixing uniformly, filtering with 0.45 μm water phase filter membrane, and filtering the filtrate for analysis.

4. The method for detecting the content of animal-derived natural pigment in food according to claim 1, wherein in the step (3), the specification of the C18 chromatographic column is 4.6 mm ×50mm inner diameter, and the particle size is 3 μm.

5. The method for detecting the content of animal-derived natural pigment in food according to claim 1, wherein the standard curve coefficient in the step (4) is not less than 0.999.

6. The method for detecting the content of the natural pigment of the animal source in the food according to claim 1, wherein the food is jam, cocoa products, chocolate and chocolate products, candies, baked food flavor pie fillings, surface coating, compound seasonings, fruit and vegetable juice beverages, carbonated beverages, fruit flavor beverages, wine, black tea and brown sugar.