CN108088823B - Method for detecting calcium content in dairy product - Google Patents
Method for detecting calcium content in dairy product Download PDFInfo
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- CN108088823B CN108088823B CN201611048129.5A CN201611048129A CN108088823B CN 108088823 B CN108088823 B CN 108088823B CN 201611048129 A CN201611048129 A CN 201611048129A CN 108088823 B CN108088823 B CN 108088823B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Abstract
The invention discloses a method for detecting the content of calcium in a dairy product. The method comprises the following steps: diluting the dairy product by using a diluent to obtain a diluent; carrying out color reaction on the diluent and a calcium reaction reagent so as to obtain a reaction product; and measuring the reaction product by using a time-resolved fluorescence method, and comparing the reaction product with a calcium calibration solution treated in the same way to calculate the calcium content in the sample, wherein the diluent comprises: 10 to 30 mass% of hydrochloric acid and 0.5 mass% of Triton X-100. The method for detecting the calcium content in the dairy product can quickly and accurately detect the calcium content in the dairy product.
Description
Technical Field
The invention relates to the field of food. In particular, the invention relates to a method for detecting the calcium content in a dairy product.
Background
Calcium is a trace element in the human body, and although the content of calcium in the human body is very small, the effect of calcium is huge. Calcium can maintain and regulate physiological and biochemical processes in organism, regulate transmitter release, increase endocrine gland secretion, maintain cell integrity and permeability, promote cell regeneration, and increase organism resistance. The milk contains rich active calcium which is the best source of calcium for human bodies, and the proportion of calcium and phosphorus is very proper, thus being beneficial to the absorption of calcium. Therefore, the eating of milk is one of the most common ways for calcium supplement, and the determination of the calcium content of various kinds of milk helps people to select milk with the required calcium content.
However, the method for detecting the calcium content in the dairy product still needs to be improved.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a method for detecting the calcium content in the dairy product. The method can quickly and accurately determine the calcium content in the dairy product.
It should be noted that the present invention has been completed based on the following findings of the inventors:
at present, methods for detecting the content of calcium include a coordination titration method (for titrating calcium in milk by using an ethylene diamine tetraacetic acid disodium salt (EDTA) solution), GB 5413.21-2010 determination (first method flame atomic absorption spectrophotometry) of calcium, iron, zinc, sodium, potassium, magnesium, copper and manganese in infant food and dairy products in national standard of food safety) (hereinafter referred to as a national standard method), an ion selective electrode method (calcium ion meter method) and the like, wherein the accuracy of detection results of the national standard method is high. However, the detection method is long in time consumption, complicated in method and not suitable for on-line rapid detection, and in the detection method, acetylene gas, muffle furnace and other instruments and equipment are used, so that potential safety hazards exist in the operation of the detection process. Because the freshness of the raw milk needs to be ensured in the purchasing link, the raw milk needs to be shortened in the inspection process, and the requirement for rapid detection cannot be met.
Therefore, the invention provides a method for detecting the calcium content in the dairy product. According to an embodiment of the invention, the method comprises: diluting the dairy product by using a diluent to obtain a diluent; carrying out color reaction on the diluent and a calcium reaction reagent so as to obtain a reaction product; and comparing the standard calcium calibration solution (standard) with the calcium calibration solution (standard) subjected to the same treatment by using a time-resolved fluorescence method to calculate a result. Wherein the diluent comprises: 10 to 30 mass% of hydrochloric acid and 0.5 mass% of Triton X-100.
The inventor finds that the calcium content in the dairy product can be detected by performing color reaction on the dairy product diluted by the diluent and a calcium reaction reagent and detecting a color reaction product by using a time-resolved fluorescence method. Further, the inventor finds that the calcium content in the diluted sample liquid meets the requirement of subsequent detection by performing dilution treatment by using a diluent containing hydrochloric acid and Triton X-100. In addition, hydrochloric acid enables bound calcium in the dairy product to be changed into free calcium, so that subsequent color reaction with a calcium reaction reagent is facilitated, Triton X-100 can maintain the stability of the dairy product system, and the phenomenon of unstable detection results is prevented. The inventors surprisingly found that the hydrochloric acid concentration and the Triton X-100 concentration significantly affect the accuracy of the detection result. Therefore, the inventor finds that the detection result has high accuracy when the concentrations of the hydrochloric acid and the Triton X-100 are respectively 10-30 mass% and 0.5 mass% through a large amount of experiments. If the hydrochloric acid content is too high, the sample solution is precipitated, and if the Triton X-100 is too high, the property of the sample solution system is changed; if the content of the hydrochloric acid is too low, the combined calcium in the sample solution can not be completely dissociated, and if the Triton X-100 is too low, the detection result is lower and deviates from the normal value of the content of the raw milk calcium. Therefore, the method for detecting the calcium content in the dairy product can quickly and accurately detect the calcium content in the dairy product.
According to the embodiment of the invention, the method for detecting the calcium content in the dairy product can also have the following additional technical characteristics:
according to an embodiment of the invention, the diluent comprises: 20 mass% hydrochloric acid and 0.5 mass% Trion X-100. The inventor finds that the calcium content in the dairy product can be further accurately detected under the condition.
According to an embodiment of the invention, the dairy product is raw milk. Therefore, the method for detecting the calcium content in the dairy product can quickly and accurately detect the calcium content in the raw milk.
According to the embodiment of the invention, based on 100 mu L of the dairy product, the dilution ratio of the dilution treatment is 5-15 times, the volume of the diluent which is subjected to color development reaction with the calcium reactive agent is 10 mu L, and the volume of the calcium reactive agent is 800-1500 mu L. The inventor finds that the addition ratio of the diluent to the calcium reaction reagent influences the accuracy of subsequent color reaction and spectrophotometry detection results. The inventor finds that the detection result has high accuracy when the volume of the diluent is 10 mu L based on 100 mu L of the dairy product. According to a preferred embodiment of the invention, the dilution factor of the dilution treatment is 10 times based on 100 μ L of the dairy product. Therefore, the method for detecting the calcium content in the dairy product can quickly and accurately detect the calcium content in the dairy product.
According to the embodiment of the invention, the calcium reaction reagent is 0.1-0.3 g/L of azoarsine III solution. The inventor finds that calcium in the dairy product can react with the azoarsine III to generate a blue compound, and the calcium content in the sample can be calculated by comparing the blue compound with a calcium calibration solution (standard substance) which is treated in the same way through time-resolved fluorescence measurement. Therefore, the method for detecting the calcium content in the dairy product can quickly and accurately detect the calcium content in the dairy product.
According to an embodiment of the present invention, the color reaction comprises: and oscillating the diluent and the calcium reaction reagent for 30 seconds, and carrying out water bath at the temperature of 30-40 ℃ for 1-6 minutes, preferably 5 minutes. Therefore, calcium in the dairy product and the azoarsine III in the calcium reaction reagent can be subjected to full color reaction to generate a blue compound, and the calcium content in the sample can be calculated by comparing the blue compound with a calcium calibration solution (standard substance) which is treated in the same way through measurement of time-resolved fluorescence. Therefore, the method for detecting the calcium content in the dairy product can quickly and accurately detect the calcium content in the dairy product.
According to an embodiment of the invention, the detection wavelength of the spectrophotometry is 650 nm. Therefore, the method for detecting the calcium content in the dairy product can quickly and accurately detect the calcium content in the dairy product.
In another aspect of the invention, the invention provides a method for detecting the calcium content in raw milk. According to an embodiment of the invention, the method comprises: (1) putting 100 mu L of raw milk into a centrifuge tube, adding 900 mu L of diluent into the centrifuge tube, and uniformly mixing for 30s by using a vortex apparatus to obtain 10-time diluent, wherein the diluent contains 20 mass percent of hydrochloric acid and 0.5 mass percent of Trion X-100; (2) respectively taking 10 mu L of each of the diluent, the distilled water and the calcium standard solution, oscillating the diluent, the distilled water and the calcium standard solution for 30 seconds, and then reacting the diluent and the calcium reaction reagent in a water bath at 37 ℃ for 5 minutes to respectively obtain a sample solution to be detected, a blank reference solution and a standard solution to be detected, wherein the calcium reaction reagent is 0.1-0.3 g/L of azoarsine III solution; (3) respectively taking 200 mu L of sample solution to be detected, blank reference solution and standard solution to be detected, placing each micropore cup in a slot of a time-resolved fluorometer for detection, wherein the detection wavelength is 650nm, and recording the absorbance of the sample solution to be detected, the blank reference solution and the standard solution to be detected; and (4) calculating to obtain the calcium content in the raw milk based on the concentration and absorbance of the standard solution to be detected, the absorbance of the blank reference solution and the absorbance of the sample solution to be detected. Therefore, the method for detecting the calcium content in the raw milk can quickly and accurately detect the calcium content in the raw milk.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows the effect of bath temperature on the assay results according to one embodiment of the present invention; and
FIG. 2 shows the effect of the water bath time on the assay results according to one embodiment of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
In this example, the calcium content of raw milk was determined as follows:
1. instruments and reagents
1.1 instruments and consumables
Time-resolved fluorometer: model SS-330, the linear range of calcium content detection is 0-20mg/100 mL;
pipettes (0.5-10. mu.L), (20-200. mu.L), (100-1000. mu.L);
a vortex instrument;
a water bath kettle;
and (4) sampling a micropore cup.
1.2 reagents
Calcium-reactive reagent: the concentration of the azoarsine III is 0.1-0.3 g/L;
diluent agent: 20 mass% of hydrochloric acid and 0.5 mass% of Trion X-100;
calcium calibrator: the calcium content was 10mg/100 ml.
2. Step (ii) of
All reagents and samples were brought to room temperature (20. + -. 5 ℃ C.) before the assay
(1) Putting 100 mu L of raw milk into a centrifuge tube;
(2) adding 900 mu L of diluent into a centrifuge tube, and uniformly mixing for 30s by using a vortex instrument to obtain 10 times of diluent;
(3) respectively taking 10 mu L of each of the diluent, the distilled water and the calcium standard solution, shaking the diluent, the distilled water and the calcium standard solution with 1000 mu L of calcium reaction reagent for 30 seconds, and then carrying out water bath reaction at 37 ℃ for 5 minutes to respectively obtain a sample solution to be detected, a blank reference solution and a standard solution to be detected;
(4) putting 200 mu L of sample solution to be detected, blank reference solution and standard solution to be detected in micropore cups, putting each micropore cup in a clamping groove of a SS-330 instrument, measuring by using a time-resolved fluorescence method, wherein the detection wavelength is 650nm, and recording the absorbance of the sample solution to be detected, the blank reference solution and the standard solution to be detected;
(5) and calculating to obtain the calcium content in the raw milk based on the concentration and absorbance of the standard solution to be detected, the absorbance of the blank reference solution and the absorbance of the sample solution to be detected.
Example 2 analysis of accuracy
The calcium content of the same raw milk was measured according to example 1 and the national standard method, respectively, and the results are shown in Table 1. It can be seen that the deviation of the results of the two methods is not higher than 10%, which meets the national standard requirements, thereby showing that the method of example 1 can accurately detect the calcium content in raw milk. In addition, the overall time consumption of the national standard method is about 16 hours, and the overall time consumption of the method of the embodiment 1 is about 15 minutes, so that the method of the embodiment 1 has the advantages of faster detection and higher efficiency.
TABLE 1 comparison of the national Standard method and the method of example 1
Example 3
The calcium content in raw milk was measured in the same manner as in example 1 except that the hydrochloric acid concentration in the diluent was 10 mass%.
The results are shown in Table 2. It can be seen that the detection result of 10 mass% hydrochloric acid concentration in the diluent meets the national standard requirement of 10%, wherein the deviation of 1 group of data is between 5% and 10%, and is higher than that of example 1.
TABLE 2 comparison of the national Standard method and the method of example 3
Example 4
The calcium content in raw milk was measured in the same manner as in example 1 except that the concentration of hydrochloric acid in the diluent was 30% by mass.
The results are shown in Table 3. It can be seen that the detection result of 30 mass% hydrochloric acid concentration in the diluent meets 10% of the national standard, wherein 2 groups of data are between 5% and 10%, and the deviation is higher compared with example 1.
TABLE 3 comparison of the national Standard method and the method of example 4
Example 5
The calcium content of raw milk was measured according to the method of example 1 except that in step (1), the amount of the diluent was 5. mu.L.
The results are shown in Table 4. It can be seen that the detection result is higher, the deviation from the detection result of the national standard method is higher than 10%, and the accuracy is poorer.
TABLE 4 comparison of the national Standard method and the method of example 5
Example 6
The calcium content of raw milk was measured according to the method of example 1 except that the amount of the diluent used in step (1) was 20. mu.L.
The results are shown in Table 5. It can be seen that the detection results are low, wherein the deviation between the 2 groups of results and the detection results of the national standard method is higher than 10%, and the accuracy is poor.
TABLE 5 comparison of the national Standard method and the method of example 6
Example 7
The calcium content of raw milk was measured according to the method of example 1 except that the reaction amount of the calcium reactant in step (1) was 1500. mu.L.
The results are shown in Table 6. It can be seen that the deviation of the detection result from the national standard method is between 5% and 10%, and the deviation is higher compared with the example 1.
TABLE 6 comparison of the national Standard method and the method of example 7
Example 8
The calcium content of raw milk was measured according to the method of example 1 except that the reaction amount of the calcium reactant in step (1) was 800. mu.L.
The results are shown in Table 7. It can be seen that the deviation of the detection result from the national standard method is between 5% and 10%, and the deviation is higher compared with the example 1.
TABLE 7 comparison of the national Standard method and the method of example 8
Example 9
The calcium content of raw milk was determined as in example 1, except that the diluent did not contain Triton X-100.
The results are shown in Table 8. As can be seen, the detection result without Triton X-100 is low, the deviation with the detection result of the national standard method is higher than 10%, and the accuracy is poor.
TABLE 8 comparison of the national Standard method and the method of example 9
Example 10
The calcium content of raw milk was measured according to the method of example 1, except that the water bath temperature was 30-40 ℃ and the water bath reaction time was 1-6 minutes, and the results are shown in fig. 1 and 2. It can be seen that the best results are obtained with a bath temperature of 36-37 ℃ and a bath reaction time of 5-6 minutes.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (1)
1. A method for detecting the calcium content in raw milk is characterized by comprising the following steps:
(1) putting 100 mu L of raw milk into a centrifuge tube, adding 900 mu L of diluent into the centrifuge tube, and uniformly mixing for 30s by using a vortex apparatus to obtain 10-time diluent, wherein the diluent contains 20 mass percent of hydrochloric acid and 0.5 mass percent of Trion X-100;
(2) respectively taking 10 mu L of each of the diluent, the distilled water and the calcium standard solution, oscillating the diluent, the distilled water and the calcium standard solution for 30 seconds, and then reacting the diluent and the calcium reaction reagent in a water bath at 37 ℃ for 5 minutes to respectively obtain a sample solution to be detected, a blank reference solution and a standard solution to be detected, wherein the calcium reaction reagent is 0.1-0.3 g/L of azoarsine III solution;
(3) respectively taking 200 mu L of sample solution to be detected, blank reference solution and standard solution to be detected, placing each micropore cup in a slot of a time-resolved fluorometer for detection, wherein the detection wavelength is 650nm, and recording the absorbance of the sample solution to be detected, the blank reference solution and the standard solution to be detected; and
(4) and calculating to obtain the calcium content in the raw milk based on the concentration and absorbance of the standard substance to-be-detected liquid, the absorbance of the blank reference liquid and the absorbance of the sample to-be-detected liquid.
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