CN111965270B - Method for detecting 8 synthetic colorants in glutinous rice food - Google Patents
Method for detecting 8 synthetic colorants in glutinous rice food Download PDFInfo
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Abstract
The invention relates to the technical field of food detection, and discloses a method for detecting 8 synthetic colorants in glutinous rice food, which comprises the following steps: 1) preparing a standard solution of lemon yellow, sunset yellow, carmine, amaranth, erythrosine, allura red, indigo and brilliant blue, and drawing a standard curve; 2) carrying out enzymolysis on the sample by using medium-temperature amylase to obtain an enzymolysis liquid; 3) extracting with polyamide adsorption method to obtain eluate containing synthetic colorant, and filtering with filter membrane; 4) detecting by using a high performance liquid chromatograph-ultraviolet or diode array ultraviolet detector; 5) the content of 8 synthetic colorants in the sample was calculated. The invention adopts the medium temperature amylase with specific enzyme activity and carries out enzymolysis on the starch in the glutinous rice food at a specific temperature, so that the sample is fully dissolved and dispersed in water, the content of 8 synthetic colorants in the glutinous rice food can be simultaneously and accurately detected, the recovery rate range is 71.83-82.40%, and the recovery rate range of the treatment by adopting the national standard method is 0-34.6%.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a method for detecting a synthetic colorant in glutinous rice food.
Background
Glutinous rice foods such as 'Qingyuan', 'Qingmingfu', 'moxa dumpling' and the like are traditional festival foods in China, have over 1000 years of history, and are popular with modern people due to special taste, rich nutrition and natural faint scent. The food has color mainly caused by natural pigment such as folium Artemisiae Argyi juice, fructus Hordei vulgaris juice, etc.; however, in recent years, illegal vendors use artificial pigments to replace natural pigments to dye the food, which not only seriously affects the confidence of consumers in the food, but also seriously attacks the traditional food industry.
At present, China has approved a plurality of synthetic colorants and specified the use range and dosage thereof in detail, wherein the most commonly used artificially synthesized colorants are lemon yellow, sunset yellow, carmine, amaranth, erythrosine, allura red, indigo, brilliant blue and the like, and the detection method of the synthetic colorants in the food specified in the reported national food safety standard GB 5009.35-2016 is to add distilled water into a sample, warm and dissolve the sample, add a citric acid solution into the sample solution to adjust the pH to 6.0, heat the sample solution to 60 ℃, add a little water into polyamide powder to prepare porridge, add the porridge into the sample solution, stir and adsorb the mixture, elute the eluent by using ethanol-ammonia water-water mixed solution to obtain the eluent, heat and evaporate the eluent, move a volumetric flask, fix the volume, filter membrane filtration to obtain filtrate, detect the filtrate by using high performance liquid chromatography, and quantify the external standard method.
However, the above-mentioned technical method has the following technical problems:
the pretreatment operation of the sample by the method is suitable for most food substrates, but the detection effect is often poor for special food substrates such as 'green rice cakes', 'Qingming rice cakes' and 'moxa dumplings' which are prepared by taking glutinous rice flour as a main raw material, and the food is a traditional food which is popular with people in China; the main raw material of the food is glutinous rice flour which contains high amylopectin, so that the glutinous rice food is high in viscosity and colloidal, when the coloring agent is detected, the glutinous rice food cannot be dissolved and dispersed by heating after distilled water is added, a sample still becomes a dough under the action of the glutinous rice starch, except that a small part of the sample surface contacted with water is slightly dissolved out, the interior of the sample cannot be contacted with the water, so that the sample after polyamide adsorption cannot pass through a G3 sand core funnel, the suction filtration is difficult, the coloring agent detection is difficult, the actual content in the sample cannot be detected, the detection result is low, and even false negative is caused.
Disclosure of Invention
The invention provides the method for detecting the 8 synthetic colorants in the glutinous rice food, so that the content of the 8 synthetic colorants in the glutinous rice food can be simultaneously and accurately detected, and the method is simple and easy to operate; the detection method provided by the invention is verified through a recovery rate experiment, and the result shows that the recovery rate can reach 71.83-82.40%.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a detection method for 8 synthetic colorants in glutinous rice food comprises the following steps:
1) preparing a mixed standard series working solution of substances to be detected, namely lemon yellow, sunset yellow, carmine, amaranth, erythrosine, allura red, indigo and brilliant blue, respectively injecting the mixed standard series working solution into a high performance liquid chromatograph-ultraviolet detector for analysis to obtain a chromatogram of the mixed standard series working solution, and respectively drawing a relation curve graph according to the concentration of each substance to be detected standard solution and the value of the chromatographic peak area for measuring the corresponding concentration to obtain a fitting equation of a standard curve;
2) weighing a glutinous rice food sample in a beaker, adding distilled water, oscillating under the water bath condition of 55-65 ℃, adding a medium-temperature amylase with the enzyme activity of 2000-7000U/g for enzymolysis, wherein the mass ratio of the medium-temperature amylase to the sample is 0.05-0.15: 5, and the enzymolysis time is 5-10 hours, so as to obtain an enzymolysis solution A;
3) adjusting the pH value of the enzymatic hydrolysate A obtained in the step 2) to 5.5-6.5 by using a citric acid solution, and maintaining the heating temperature at 55-65 ℃, wherein the concentration range of the citric acid solution is 150-200 g/L to obtain an enzymatic hydrolysate B;
adding a little water into polyamide powder to be blended into porridge, pouring the polyamide powder and the sample with the mass ratio of 0.8-1.2: 5 into the enzymolysis liquid B, uniformly stirring for 1-3 min, performing suction filtration, washing for 3-5 times by using a citric acid solution with the temperature of 55-65 ℃ and the pH value of 5.5-6.5 in the suction filtration process, and eluting for 3-5 times by using an ethanol-ammonia water-water mixed solution to obtain an eluent; collecting the eluent in a beaker, heating and evaporating the eluent, then moving the eluent into a volumetric flask, fixing the volume, and filtering the eluent by a filter membrane to obtain filtrate;
4) injecting the filtrate obtained in the step 3) into a high performance liquid chromatograph-ultraviolet detector for detection, respectively measuring the values of chromatographic peak areas of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo blue and brilliant blue in the sample, and calculating the obtained value of the chromatographic peak area through the fitting equation of the corresponding standard curve in the step 1) to obtain the concentration c of each component to be detected;
5) the contents of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo and brilliant blue in the sample are respectively obtained according to the following conversion formulas:
in the formula:
x is the content of the substance to be detected in the sample, and the unit is gram per kilogram (g/kg);
c, calculating the concentration of each substance to be detected by a fitting equation of a standard curve, wherein the unit is milligram per liter (mg/L);
v-volumetric volume of sample solution in milliliters (mL);
m is sample weighing, and the unit is gram (g);
1000-unit conversion factor.
The medium-temperature amylase in the step 2) is alpha-amylase suitable for being used under the conditions of 55-65 ℃.
The oscillation speed in the step 2) is 100-200 r/min.
The specific preparation method of the standard series working solution mixed by lemon yellow, sunset yellow, carmine, amaranth, erythrosine, allura red, indigo and brilliant blue in the step 1) is as follows:
accurately transferring 200 mu L of standard stock solutions of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo blue and brilliant blue with the concentration of 1000mg/L into a 5mL volumetric flask to obtain a mixed standard substance use solution of 40 mg/L;
precisely transferring 10, 20, 50, 100, 200 and 500 mu L of the mixed standard substance use solution, respectively placing the mixed standard substance use solution into 62 mL volumetric flasks, metering the volume to the scale with water, and shaking up to obtain the mixed standard solution with the concentration gradients of 0.2, 0.4, 1.0, 2.0, 4.0 and 10.0 mg/L;
respectively injecting the mixed standard solutions with different concentrations into a high performance liquid chromatograph-ultraviolet detector for analysis, and measuring the value of the chromatographic peak area with corresponding concentration, wherein the chromatographic conditions are as follows:
a) a chromatographic column: c18 column, 5 μm, 4.6mm × 250 mm;
b) mobile phase: phase A: methanol, phase B: 20 mmol/L ammonium acetate aqueous solution, wherein the volume ratio of the phase A to the phase B is shown in the following table 1:
TABLE 1 mobile phase gradient elution conditions
c) Sample introduction amount: 10 mu L of the solution;
d) flow rate: 1.0 mL/min;
e) column temperature: 30-40 ℃;
f) wavelength of the detector: 430nm, 500nm and 600 nm;
and (3) drawing a standard curve by taking the concentration of each standard solution of the substance to be measured as an abscissa and the value of the corresponding chromatographic peak area as an ordinate, and respectively drawing a relation curve graph of the concentrations of the standard solutions of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo blue and brilliant blue and the value of the chromatographic peak area to obtain a fitting equation of the standard curve.
The volume ratio of the ethanol-ammonia water-water mixed solution in the step 3) is that the ratio of ethanol to ammonia water to water is =7:2: 1.
Compared with the prior art, the invention has the advantages that:
according to the invention, the moderate temperature amylase with specific enzyme activity is adopted, and the starch in the glutinous rice food is subjected to enzymolysis at a specific temperature, so that the compound wrapped in the viscous agglomerated sample is fully released and dissolved, and the sample after polyamide adsorption is easy to pass through a G3 sand core funnel and suction filtration, so that the pretreatment process is quick, convenient and simple, the technical problem that the sample is difficult to treat according to the national standard method is effectively solved, and meanwhile, the contents of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo and brilliant blue in the glutinous rice food can be accurately detected, and the method is simple and easy to operate; the detection method provided by the invention is verified through a recovery rate experiment, and the result shows that the recovery rate range of the detection method is 71.83-82.40%, and the recovery rate range of the glutinous rice food processed by the national standard method is 0-34.6%; particularly, for the detection of three colorants of allura red, erythrosine and indigo, the recovery rates detected by the method are 73.40%, 77.58% and 71.83% respectively, while the recovery rate is 0% when the national standard method is adopted to detect no obvious chromatographic peak on the same sample.
Drawings
FIG. 1 is a chromatogram of a mixed standard solution of 10 mg/L concentration in example 1 of the present invention, in which the detection wavelengths of (a), (b), and (c) are 430nm, 500nm, and 600nm, respectively;
FIG. 2a is a graph showing the relationship between standard solutions of tartrazine in the present invention;
FIG. 2b is a graph showing the relationship between standard solutions of amaranth according to the present invention;
FIG. 2c is a graph showing the relationship between a standard solution of carmine according to the present invention;
FIG. 2d is a graph showing the relationship between standard solutions of sunset yellow in the present invention;
FIG. 2e is a graph of a standard solution of allura red according to the present invention;
FIG. 2f is a graph of a relationship between a standard solution of erythrosine in the present invention;
FIG. 2g is a graph of a standard solution of indigo according to the invention;
FIG. 2h is a graph showing the relationship between a brilliant blue standard solution according to the present invention;
FIG. 3 is a chromatogram of each substance to be detected of the cyan cluster sample A in example 1 of the present invention, in which the detection wavelengths (a), (b), and (c) are 430nm, 500nm, and 600nm, respectively;
FIG. 4 is a chromatogram of each test substance of the cyan group sample A in comparative example 1 of the present invention, in which the detection wavelengths of (a), (b), and (c) are 430nm, 500nm, and 600nm, respectively;
FIG. 5 is a chromatogram of each of the test substances of the supernatant drugs of example 2 of the present invention, wherein the detection wavelengths of (a), (b), and (c) are 430nm, 500nm, and 600nm, respectively;
FIG. 6 is a chromatogram of each of test substances of the example of the present invention, which is obtained from the drug of "Qingming rice" in comparative example 2, wherein the detection wavelengths of (a), (b), and (c) are 430nm, 500nm, and 600nm, respectively;
fig. 7 is a chromatogram of each substance to be detected of the dumpling samples in example 3 of the present invention, in which the detection wavelengths (a), (b), and (c) are 430nm, 500nm, and 600nm, respectively;
FIG. 8 is a chromatogram of each substance to be measured of a sample of the moxa dumpling in comparative example 3 of the present invention, wherein the detection wavelengths of (a), (b), and (c) are 430nm, 500nm, and 600nm, respectively;
FIG. 9 is a chromatogram of each substance to be detected of the cyan cluster sample B in example 4 of the present invention, in which the detection wavelengths (a), (B), and (c) are 430nm, 500nm, and 600nm, respectively;
FIG. 10 is a chromatogram of each test substance of the cyan group sample C in example 5 of the present invention, in which the detection wavelengths (a), (b), and (C) are 430nm, 500nm, and 600nm, respectively;
FIG. 11 is a chromatogram of each test substance of the cyan cluster sample C in example 6 of the present invention, in which the detection wavelengths (a), (b), and (C) are 430nm, 500nm, and 600nm, respectively.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Reagent: methanol, chromatographically pure, merck; medium temperature amylase, biochemical reagent, storage condition 2-8 deg.C, chemical reagent of national medicine group limited; water, deionized water purified by Milli-Q system; polyamide powder, 200 mesh, national chemical group, ltd; lemon yellow, amaranth, carmine, sunset yellow, allura red, erythrosine, indigo and brilliant blue standard stock solutions, the concentrations of which are all 1000mg/L, China institute of metrology science; ammonium acetate, reagent pure, national pharmaceutical group chemical reagents ltd;
materials: c18 column (5 μm, 4.6 mm. times.250 mm); high performance liquid chromatography-ultraviolet or diode array detectors; shaking the water bath, G3 sand core funnel, 0.22 μm aqueous phase microporous filter membrane.
Preparation of a standard solution:
accurately transferring 200 mu L of standard stock solutions of lemon yellow, amaranth, carmine, sunset yellow, allura red, erythrosine, indigo blue and brilliant blue with the concentration of 1000mg/L into a 5mL volumetric flask, and adding water to a constant volume to a scale mark to obtain a mixed standard substance use solution of 40 mg/L.
Precisely transferring 10, 20, 50, 100, 200 and 500 mu L of 40mg/L mixed standard substance using solution, respectively placing the solution into 62 mL volumetric flasks, fixing the volume to the scale mark with water, and shaking uniformly to obtain mixed standard solutions with concentration gradients of 0.2, 0.4, 1.0, 2.0, 4.0 and 10.0 mg/L; respectively injecting the mixed standard solutions with different concentrations into a high performance liquid chromatograph-ultraviolet detector for analysis, and measuring corresponding chromatograms, wherein as shown in fig. 1, the chromatograms of the mixed standard solutions with the concentration of 10 mg/L are subjected to the chromatograms under the conditions of detection wavelengths of 430nm, 500nm and 600nm, so as to obtain values of chromatographic peak areas of the standard solutions with corresponding concentrations of the substances to be detected, wherein the chromatographic conditions are as follows:
a) a chromatographic column: c18 column, 5 μm, 4.6mm × 250 mm;
b) mobile phase: phase A: methanol, phase B: 20 mmol/L ammonium acetate aqueous solution, wherein the volume ratio of the phase A to the phase B is shown in the following table 1:
TABLE 1 mobile phase gradient elution conditions
c) Sample introduction amount: 10 mu L of the solution;
d) flow rate: 1.0 mL/min;
e) column temperature: 30-40 ℃;
f) wavelength of the detector: 430nm, 500nm and 600 nm;
and (3) taking the concentration of the standard solution of each substance to be detected as an abscissa and the value of the corresponding chromatographic peak area as an ordinate to draw a standard curve, and respectively drawing a relation curve graph of the concentrations of the standard solutions of lemon yellow, amaranth, carmine, sunset yellow, allura red, erythrosine, indigo blue and brilliant blue and the value of the chromatographic peak area, as shown in fig. 2a-2h, so as to obtain a fitting equation and a correlation coefficient of the standard curve, and the result is shown in table 2.
TABLE 28 fitting equation and correlation coefficient of standard curve for substances to be tested
Example 1:
A) weighing 5g of the cyan cluster sample A in a beaker, adding 40mL of distilled water, oscillating under the water bath condition at the temperature of 60 ℃, wherein the oscillation speed is 150 r/min, adding 100mg of medium-temperature amylase with the enzyme activity of 2000U/g for enzymolysis, and the enzymolysis time is 5h to obtain an enzymolysis liquid A;
B) adjusting the pH value of the enzymolysis solution A to 6.0 by using a citric acid solution with the concentration of 200g/L, and maintaining the heating temperature at 60 ℃ to obtain an enzymolysis solution B;
C) adding a little water into 1G of polyamide powder to prepare porridge, pouring into the enzymolysis liquid B, stirring and uniformly mixing for 1min, pouring into a G3 sand core funnel, performing suction filtration by using a vacuum pump, leaching for 3 times by using citric acid aqueous solution with the temperature of 60 ℃ and the pH value of 6.0 in the suction filtration process, wherein 10mL of the citric acid aqueous solution is used for each time, and eluting for 3 times by using ethanol-ammonia water-water mixed solution with the volume ratio of 7:2:1, wherein 15 mL of the ethanol-ammonia water-water mixed solution is used for each time to obtain eluent;
D) collecting eluate in a 150 mL beaker, adding glass beads, heating and boiling on an electric hot plate, evaporating until the residual volume is about 5mL, transferring to a 50mL volumetric flask, adding water to a constant volume to a scale mark, and filtering through a 0.22 μm water phase microporous membrane to obtain filtrate;
E) injecting the filtrate into a high performance liquid chromatograph-ultraviolet detector for detection, obtaining chromatograms of various substances to be detected of the cyan group sample A under the conditions that the detection wavelengths are 430nm, 500nm and 600nm respectively, and respectively measuring the values of chromatographic peak areas of lemon yellow, amaranth, carmine, sunset yellow, allura red, erythrosine, indigo blue and brilliant blue in the sample as shown in fig. 3, wherein the chromatographic conditions are as follows:
a) a chromatographic column: c18 column, 5 μm, 4.6mm × 250 mm;
b) mobile phase: phase A: methanol, phase B: 20 mmol/L ammonium acetate aqueous solution, wherein the volume ratio of the phase A and the phase B is shown in the following table 3:
TABLE 3 mobile phase gradient elution conditions
c) Sample introduction amount: 10 mu L of the solution;
d) flow rate: 1.0 mL/min;
e) column temperature: 30-40 ℃;
f) wavelength of the detector: 430nm, 500nm and 600 nm;
calculating the value of the chromatographic peak area of the substance to be measured through a fitting equation of a corresponding standard curve to obtain the concentration c of each substance to be measured; the contents of lemon yellow, amaranth, carmine, sunset yellow, allura red, erythrosine, indigo and brilliant blue in the sample are respectively obtained according to the following conversion formulas:
in the formula:
x is the content of the component to be measured in grams per kilogram (g/kg) in the sample;
c, calculating the concentration of each component to be measured through a fitting equation of a standard curve, wherein the unit is milligram per liter (mg/L);
v-volumetric volume of sample solution in milliliters (mL);
m is sample weighing, and the unit is gram (g);
1000-unit conversion factor.
Comparative example 1:
comparative example 1 differs from example 1 in that: 100mg of medium-temperature amylase with the enzyme activity of 2000U/g is not added for enzymolysis, and the rest is the same as that in the embodiment 1; and obtaining chromatograms of the substances to be detected of the cyan group sample A with the detection wavelengths of 430nm, 500nm and 600nm respectively, as shown in figure 4.
And (4) experimental conclusion: example 1 and comparative example 1 were each performed on an actual cyan cluster sample a, and the obtained comparison table of the detection results of the substances to be detected of the cyan cluster sample a is shown in table 4; from the experimental data, the blue cluster sample A treated in comparative example 1 did not have a distinct peak in the synthesized colorant, while the blue cluster sample A treated in example 1 had peaks in the colors lemon yellow and brilliant blue, indicating that the synthesized colorant in the sample was more completely released and could be detected by the instrument for qualitative and quantitative analysis when the blue cluster sample was treated in accordance with the method of example 1.
Table 4 comparison table of test results of substances to be tested of the cyan group sample a in example 1 and comparative example 1
Example 2:
A) weighing 5g of the kumquat sample, adding 40mL of distilled water into a beaker, oscillating under the water bath condition at the temperature of 65 ℃, wherein the oscillation speed is 100 r/min, adding 150mg of medium-temperature amylase with the enzyme activity of 5000U/g for enzymolysis, and the enzymolysis time is 10 hours to obtain an enzymolysis liquid A;
B) adjusting the pH value of the enzymolysis solution A to 5.5 by using citric acid solution with the concentration of 150g/L, and maintaining the heating temperature at 65 ℃ to obtain enzymolysis solution B;
C) adding a little water into 1G of polyamide powder to prepare porridge, pouring into the enzymolysis liquid B, stirring and uniformly mixing for 1min, pouring into a G3 sand core funnel, carrying out suction filtration by using a vacuum pump, leaching for 4 times with a citric acid aqueous solution with the temperature of 65 ℃ and the pH value of 5.5 in the suction filtration process, eluting for 10mL each time for 4 times with an ethanol-ammonia water-water mixed solution with the volume ratio of 8:1:1 to obtain an eluent, and eluting for 10mL each time;
the remaining samples were analyzed in the same manner as in example 1 to obtain chromatograms of the respective analytes of the kumquat samples at detection wavelengths of 430nm, 500nm and 600nm, respectively, as shown in FIG. 5.
Comparative example 2:
comparative example 2 differs from example 2 in that: the chromatogram of each substance to be detected of the supernatant cake samples with the detection wavelengths of 430nm, 500nm and 600nm is obtained without adding 150mg of medium temperature amylase with the enzyme activity of 5000U/g for enzymolysis, as shown in fig. 6, and the rest is the same as that in example 2.
And (4) experimental conclusion: in example 2 and comparative example 2, the actual qingming kui samples were processed to obtain comparison tables of the test results of the substances to be tested of the qingming kui samples, as shown in table 5; according to experimental data, the sample of the domino drugs treated in the comparative example 2 has the chromatographic peaks of lemon yellow and brilliant blue, and the calculated content of the lemon yellow in 5g of the domino drugs is 3.22mg/kg, and the calculated content of the brilliant blue is 0.61 mg/kg; the sample of the cyan cake treated in example 2 also has measured the chromatographic peaks of lemon yellow and brilliant blue, and the calculated content of lemon yellow in 5g of the sample of the Qingming cake is 8.64mg/kg, and the content of brilliant blue is 1.84 mg/kg; it is shown that the synthetic colorant in the qingming rice cake samples treated according to the method of example 2 was released more completely, and the result of the test was 3 times that of comparative example 2.
TABLE 5 comparison table of the test results of the test substances of the domino drugs in example 2 and comparative example 2
Example 3:
A) weighing 5g of a moxa dumpling sample, placing the sample in a beaker, adding 40mL of distilled water, oscillating the sample in a water bath at the temperature of 55 ℃, wherein the oscillation speed is 200r/min, adding 50mg of medium-temperature amylase with the enzyme activity of 7000U/g for enzymolysis, and the enzymolysis time is 8 hours to obtain an enzymolysis liquid A;
B) adjusting the pH value of the enzymolysis solution A to 6.5 by using citric acid solution with the concentration of 180g/L, and maintaining the heating temperature to 55 ℃ to obtain enzymolysis solution B;
C) adding a little water into 1G of polyamide powder to prepare porridge, pouring into the enzymolysis liquid B, stirring and uniformly mixing for 1min, pouring into a G3 sand core funnel, carrying out suction filtration by using a vacuum pump, leaching with a citric acid aqueous solution with the temperature of 55 ℃ and the pH value of 6.5 for 5 times, wherein 8mL of the citric acid aqueous solution is used in the suction filtration process, and eluting with an ethanol-ammonia water-water mixed solution with the volume ratio of 7:2:1 for 5 times, wherein 10mL of the ethanol-ammonia water-water mixed solution is used to obtain an eluent;
the rest is the same as example 1, and chromatograms of each substance to be detected of the boiled dumpling samples with detection wavelengths of 430nm, 500nm and 600nm are obtained, as shown in fig. 7.
Comparative example 3:
comparative example 3 differs from example 3 in that: 50mg of medium-temperature amylase with the enzyme activity of 7000U/g is not added for enzymolysis, and chromatograms of various substances to be detected of the moxa dumpling samples with the detection wavelengths of 430nm, 500nm and 600nm are obtained, as shown in figure 8, and the rest is the same as that in embodiment 3.
And (4) experimental conclusion: example 3 and comparative example 3 were performed on actual moxa dumpling samples, and the obtained comparison table of the detection results of the substances to be detected of the moxa dumpling samples is shown in table 6; from experimental data, the chromatographic peaks of lemon yellow and brilliant blue are obtained in the moxa dumpling sample treated in the comparative example 3, and the content of lemon yellow and the content of brilliant blue in 5g of the moxa dumpling sample are calculated to be 1.32 mg/kg and 0.62 mg/kg respectively; the sample of the cyan cake treated in example 3 also has measured the chromatographic peaks of lemon yellow and brilliant blue, and the calculated content of lemon yellow in 5g of the sample of the Qingming cake is 13.06 mg/kg, and the content of brilliant blue is 3.18 mg/kg; it is shown that the synthetic colorant in the sample was more completely released when the moxa dumpling sample was processed according to the method of example 3, the lemon yellow test result of example 3 was 10 times the lemon yellow test result of comparative example 3, and the brilliant blue test result of example 3 was 5 times the brilliant blue test result of comparative example 3.
Table 6 comparison table of the test results of the test substances of the moxa dumpling samples in example 3 and comparative example 3
In order to prove that the scheme of the invention can accurately detect the content of the synthetic colorant in the glutinous rice food, the content is further verified by a labeling experiment.
Example 4:
the chromatogram of each substance to be detected of the cyan group sample B under the conditions of detection wavelengths of 430nm, 500nm and 600nm, respectively, was obtained as shown in fig. 9, with the "cyan group sample a" in example 1 being changed to "cyan group sample B", and the rest being the same as in example 1.
Example 5:
weighing the cyan ball sample B in the example 4, accurately adding 25 muL of lemon yellow with the concentration of 1000mg/L, 25 muL of sunset yellow with the concentration of 1000mg/L, 25 muL of carmine with the concentration of 1000mg/L, 25 muL of amaranth with the concentration of 1000mg/L, 25 muL of allura red with the concentration of 1000mg/L, 25 muL of erythrosine with the concentration of 1000mg/L, 25 muL of indigo with the concentration of 1000mg/L and 25 muL of brilliant blue with the concentration of 1000mg/L into a beaker to obtain a mixed standard substance stock solution, standing at normal temperature for 24 hours to enable the mixed standard substance stock solution to be completely absorbed by the cyan ball sample B, and marking the sample obtained after adding the label as a cyan ball sample C;
changing the cyan group sample A in the embodiment 1 into the cyan group sample C, and obtaining chromatograms of the substances to be detected of the cyan group sample C under the conditions that the detection wavelengths are 430nm, 500nm and 600nm respectively as shown in the attached figure 10, wherein the rest of the chromatograms are the same as the embodiment 1;
the recovery and relative standard deviation were calculated by the formula: recovery (%) = (measured addition/theoretical addition) 100%; relative Standard Deviation (RSD) = Standard Deviation (SD)/arithmetic mean of calculated results (X) × 100%; the resulting normalized recovery data are shown in table 7.
TABLE 78 data sheet for standard recovery rate of substance to be measured
And (4) experimental conclusion: example 4, an actual cyan cluster sample B is processed and detected, and no obvious chromatographic peak of the synthetic colorant is obtained, which indicates that the content of the synthetic colorant in the cyan cluster sample B is low or even 0, and the obtained value is taken as the background value of the substance to be detected and is marked as 0; example 5 the cyan cluster sample B was treated and tested after addition of the mixed standard stock solution, and the calculated recovery ranged from 71.83% to 82.40%.
Example 6:
example 6 differs from example 5 in that: and (3) detecting the substance to be detected in the cyan cluster sample B without adding 100mg of medium-temperature amylase with the enzyme activity of 2000U/g for enzymolysis, wherein the rest is the same as in example 5, the obtained chromatogram is shown in figure 11, and the obtained data table of the standard recovery rate is shown in table 8.
TABLE 88 results of recovery of test substances with spiked
And (4) experimental conclusion: example 6 the green cluster sample B is added with the mixed standard stock solution and then processed and detected, and the calculated recovery rate range is 0-34.6%; the results of the labeling experiments of example 5 and example 6 show that: adding a standard substance of a synthetic colorant into glutinous rice food, and detecting according to the method disclosed by the invention that the recovery rate of the obtained lemon yellow is 2.38 times that of the lemon yellow obtained by the national standard method; the recovery rate of amaranth detected by the method is 49.1 times of that of the national standard method; the recovery rates of the carmine and the sunset yellow detected by the method are respectively 10.02 times and 10.85 times of those of the traditional method; the recovery rates of the allura red, erythrosine and indigo obtained by detection of the method are 73.40%, 77.58% and 71.83%, while the recovery rates obtained by detection of the national standard method are 0% without detection of obvious chromatographic peaks of the allura red, erythrosine and indigo; the recovery rate of brilliant blue detected by the method is 4.41 times of that of the national standard method; the method of the invention is adopted to process the glutinous rice food, so that the synthetic colorant in the sample is more completely released, and can be detected by an instrument for qualitative and quantitative analysis.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention are within the scope of the present invention, and the scope of the present invention is subject to the claims.
Claims (4)
1. A detection method for 8 synthetic colorants in glutinous rice food is characterized by comprising the following steps:
1) preparing a mixed standard series working solution of substances to be detected, namely lemon yellow, sunset yellow, carmine, amaranth, erythrosine, allura red, indigo and brilliant blue, respectively injecting the mixed standard series working solution into a high performance liquid chromatograph-ultraviolet detector for analysis to obtain a chromatogram of the mixed standard series working solution, and respectively drawing a relation curve graph according to the concentration of each substance to be detected standard solution and the value of the chromatographic peak area for measuring the corresponding concentration to obtain a fitting equation of a standard curve;
2) weighing a sample of glutinous rice food in a beaker, adding distilled water, oscillating under the water bath condition of 55-65 ℃, adding a medium-temperature amylase with the enzyme activity of 2000-7000U/g for enzymolysis, wherein the mass ratio of the medium-temperature amylase to the sample is 0.05-0.15: 5, the enzymolysis time is 5-10 h, and obtaining an enzymolysis solution A, wherein the medium-temperature amylase is alpha-amylase applicable to the condition of 55-65 ℃;
3) adjusting the pH value of the enzymatic hydrolysate A obtained in the step 2) to 5.5-6.5 by using a citric acid solution, and maintaining the heating temperature at 55-65 ℃, wherein the concentration range of the citric acid solution is 150-200 g/L to obtain an enzymatic hydrolysate B;
adding a little water into polyamide powder to be blended into porridge, pouring the polyamide powder and the sample with the mass ratio of 0.8-1.2: 5 into the enzymolysis liquid B, uniformly stirring for 1-3 min, performing suction filtration, washing for 3-5 times by using a citric acid solution with the temperature of 55-65 ℃ and the pH value of 5.5-6.5 in the suction filtration process, and eluting for 3-5 times by using an ethanol-ammonia water-water mixed solution to obtain an eluent; collecting the eluent in a beaker, heating and evaporating the eluent, then transferring the eluent into a volumetric flask, fixing the volume, and filtering the eluent by a filter membrane to obtain filtrate;
4) injecting the filtrate obtained in the step 3) into a high performance liquid chromatograph-ultraviolet detector for detection, respectively measuring the values of chromatographic peak areas of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo blue and brilliant blue in the sample, and calculating the obtained value of the chromatographic peak area through the fitting equation of the corresponding standard curve in the step 1) to obtain the concentration c of each component to be detected;
5) the contents of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo and brilliant blue in the sample are respectively obtained according to the following conversion formulas:
in the formula:
x is the content of the substance to be detected in the sample, and the unit is gram per kilogram (g/kg);
c, calculating the concentration of each substance to be detected by a fitting equation of a standard curve, wherein the unit is milligram per liter (mg/L);
v is the volume of the sample solution to volume in milliliters (mL);
m is sample weighing, and the unit is gram (g);
1000-unit conversion factor;
wherein, the detection conditions of the high performance liquid chromatograph-ultraviolet detector are as follows:
a) a chromatographic column: c18 column, 5 μm, 4.6mm × 250 mm;
b) mobile phase: phase A: methanol, phase B: 20 mmol/L ammonium acetate aqueous solution, wherein the volume ratio of the phase A to the phase B is shown in the following table 1:
TABLE 1 mobile phase gradient elution conditions
c) Sample introduction amount: 10 mu L of the solution;
d) flow rate: 1.0 mL/min;
e) column temperature: 30-40 ℃;
f) wavelength of the detector: 430nm, 500nm and 600 nm.
2. The method for detecting 8 synthetic colorants in glutinous rice food according to claim 1, wherein the oscillation speed in step 2) is 100-200 r/min.
3. The method for detecting 8 synthetic colorants in glutinous rice food according to claim 1, wherein the specific preparation method of the mixed standard series working solution of lemon yellow, sunset yellow, carmine, amaranth, erythrosine, allura red, indigo blue and brilliant blue in step 1) is as follows:
accurately transferring 200 mu L of standard stock solutions of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo blue and brilliant blue with the concentration of 1000mg/L into a 5mL volumetric flask respectively to obtain a mixed standard use solution of 40 mg/L;
precisely transferring 10, 20, 50, 100, 200 and 500 mu L of the mixed standard substance use solution, respectively placing the mixed standard substance use solution into 62 mL volumetric flasks, metering the volume to the scale with water, and shaking up to obtain the mixed standard solution with the concentration gradients of 0.2, 0.4, 1.0, 2.0, 4.0 and 10.0 mg/L;
respectively injecting the mixed standard solutions with different concentrations into a high performance liquid chromatograph-ultraviolet detector for analysis, and measuring the values of chromatographic peak areas with corresponding concentrations;
and (3) drawing a standard curve by taking the concentration of each standard solution of the substance to be measured as an abscissa and the value of the corresponding chromatographic peak area as an ordinate, and respectively drawing a relation curve graph of the concentrations of the standard solutions of lemon yellow, sunset yellow, carmine, amaranth, allura red, erythrosine, indigo blue and brilliant blue and the value of the chromatographic peak area to obtain a fitting equation of the standard curve.
4. The method for detecting 8 synthetic colorants in glutinous rice food as claimed in claim 1, wherein the volume ratio of ethanol-ammonia water-water mixed solution in step 3) is ethanol-ammonia water: water =7:2: 1.
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