Method for detecting peroxide value of euphausia superba oil

Abstract

The invention relates to the technical field of inspection and detection, in particular to a method for detecting the peroxide value of euphausia superba oil. Excessive DPPP reacts with hydroperoxide in the euphausia superba oil, the fluorescence intensity after the reaction is measured, the content of the hydroperoxide in the euphausia superba oil is calculated according to a corresponding standard curve of the fluorescence intensity and the concentration of the hydroperoxide, and therefore the peroxide value of the euphausia superba oil is measured. The method for detecting the peroxide value of the shrimp sauce by measuring the fluorescence intensity is not influenced by the color of the shrimp sauce; according to the method disclosed by the invention, the DPPP is used as a raw material, the use amount of the shrimp oil is optimized, an emulsification phenomenon is avoided in a reaction process, and the reaction temperature, time and the like of the DPPP and the hydroperoxide are optimized, so that the method is relatively low in detection limit and quantitation limit and relatively high in detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of inspection and detection technology, and in particular to a method for detecting the peroxide value of Antarctic krill oil. Background Art

[0002] Antarctic krill oil is one of the high value-added products developed with Antarctic krill as raw materials. It is rich in energy factors, including n-3 polyunsaturated fatty acids (n-3PUFA), phospholipids, astaxanthin, tocopherol, vitamin A and other physiologically active ingredients. It has many benefits for human health, including preventing cardiovascular and cerebrovascular diseases, fighting cancer, preventing inflammation and promoting growth and development. However, the n-3PUFA in Antarctic krill oil is prone to oxidation, which not only reduces the nutritional value of the oil, but also produces oxidation products that are harmful to human health. Peroxide value is one of the important indicators reflecting the degree of oil oxidation.

[0003] At present, the determination of the peroxide value of oils and fats is mainly based on "GB5009.227-2016 Determination of Peroxide Value in Food". The two methods of this national standard (titration and potentiometric titration) both use peroxide to react with potassium iodide to generate iodine, and use sodium thiosulfate standard solution to titrate the precipitated iodine, and calculate the peroxide value in the food based on the consumption of sodium thiosulfate standard solution. In the first method (titration), starch indicator is used. The reaction of iodine and starch shows blue. When the titration endpoint is reached by titration with sodium thiosulfate standard solution, the blue color disappears. The second method (potentiometric titration) determines the titration endpoint by measuring the potential change. During the titration process, the solution is titrated until the potential value is stable.

[0004] The current patent applications for the determination of the peroxide value of Antarctic krill oil are as follows: 1. Application No. 201611202858.1, invention name "Method for detecting the peroxide value of Antarctic krill oil", discloses a method for detecting the peroxide value of Antarctic krill oil, the steps of which are as follows: taking a certain amount of Antarctic krill oil m and dissolving it in an organic solvent, then adding an excess of potassium iodide solution for reaction, then continuously dripping a sodium thiosulfate solution with a concentration of C into the solution and detecting the potential value of the solution until the potential value of the solution stabilizes, and discretely recording the titration volume V and the potential value E of the solution at this time during the titration process; using the second-order derivative method to draw a relationship curve between the titration volume V and the potential value E, finding the titration volume Vx at the potential jump point, and calculating the peroxide value according to the formula. However, when the peroxide value is determined by titration, the disappearance of the blue color produced by the reaction of starch and iodine is required as the titration endpoint, but the deep red color of Antarctic krill oil will seriously interfere with the judgment of the titration endpoint; and Antarctic krill oil has a high phospholipid content, and emulsification is prone to occur during the titration process using sodium thiosulfate solution, which is not conducive to the full reaction of elemental iodine and sodium thiosulfate. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the method for determining the peroxide value of Antarctic krill oil mainly adopts the iodine titration method ("Determination of Peroxide Value in Foods GB5009.227-2016"), and there are two problems when the method determines the peroxide value of Antarctic krill oil: 1. Antarctic krill oil contains a large amount of astaxanthin and is dark red. When the traditional iodine titration method is used to determine the peroxide value, the titration end point is determined according to the color development of starch and iodine. The dark red Antarctic krill oil seriously interferes with the determination of the titration end point; 2. The phospholipid content of Antarctic krill oil is as high as 40% or more. During the titration process using sodium thiosulfate solution, emulsification is likely to occur, which is not conducive to the full reaction of elemental iodine and sodium thiosulfate.

[0006] In view of the problems of the prior art, the present invention provides a method for detecting the peroxide value of Antarctic krill oil. When the peroxide value of shrimp oil is detected by measuring the fluorescence intensity, it is not affected by the color of the shrimp oil; and the amount of shrimp oil used is optimized, and emulsification does not occur during the reaction process. At the same time, the reaction temperature and time of DPPP and hydroperoxide are optimized, so that the detection limit and quantitative limit of the method are low and the detection sensitivity is high.

[0007] To achieve the above object, the present invention is implemented by the following technical scheme: a method for detecting the peroxide value of Antarctic krill oil, using excess DPPP to react with hydroperoxides in Antarctic krill oil, measuring the fluorescence intensity after the reaction, and calculating the hydroperoxide content in the krill oil according to the corresponding standard curve of fluorescence intensity and hydroperoxide concentration, thereby determining the peroxide value of Antarctic krill oil.

[0008] The principle of the reaction of the present invention is that 2-phenyl-1-pyrene phosphine (DPPP) itself does not produce fluorescence, but it can specifically bind to hydroperoxides and then produce strong fluorescent substances, and the fluorescence intensity is proportional to the content of hydroperoxides. Therefore, excess DPPP is used to react with hydroperoxides in Antarctic krill oil, the fluorescence intensity after the reaction is measured, and the hydroperoxide content in the shrimp oil is calculated according to the corresponding standard curve of fluorescence intensity and hydroperoxide concentration, so as to determine the peroxide value of Antarctic krill oil.

[0009] Furthermore, the method comprises the following steps: taking 100 μL of chloroform / methanol solution containing 2-5 mg of shrimp oil into a graduated test tube, adding 40-60 μL of 20 mM BHT (2,6-di-tert-butyl-p-cresol) solution and 20-35 μL of 3 mM DPPP solution, and then making the volume to 10 mL with chloroform / methanol, incubating in a water bath at 60-70° C. for 145-220 min, and terminating the reaction by ice bathing after the incubation time is up; and measuring the fluorescence intensity under 351 nm excitation light, 380 nm emission light, and 2.5 nm slit width.

[0010] Furthermore, 0.2-0.5 g of Antarctic krill oil was taken and the volume was adjusted to 10 mL with chloroform / methanol to prepare a chloroform / methanol solution containing Antarctic krill oil.

[0011] Furthermore, the solvent of the BHT and DPPP solutions is chloroform / methanol.

[0012] Furthermore, the volume ratio of chloroform to methanol in the above chloroform / methanol is 4:1 to 1:4.

[0013] Furthermore, during the incubation period, vortexing was performed for 10 seconds every 6 minutes for the first hour and 10 seconds every 30 minutes for the next 1.5 hours. Vortexing can promote a full reaction.

[0014] Furthermore, after the incubation time is up, the reaction is terminated by placing the mixture in an ice bath for 10 to 15 minutes.

[0015] Furthermore, the reaction was placed at room temperature for 3 to 5 min after the incubation and vortexed for 10 s before measurement.

[0016] The beneficial effects of the present invention are:

[0017] Pigments such as astaxanthin in shrimp oil do not produce fluorescence. Therefore, when this method uses the method of measuring fluorescence intensity to detect the peroxide value of shrimp oil, it is not affected by the color of the shrimp oil. This method uses a small amount of shrimp oil, and emulsification will not occur during the reaction. In addition, this method optimizes the amount of shrimp oil used, as well as the reaction temperature and time of DPPP and hydroperoxide, so that the detection limit and quantification limit of this method are low, and the detection sensitivity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the verification result diagram of the solvent ratio;

[0019] Figure 2 This is the verification result diagram of the reaction temperature;

[0020] Figure 3 This is the verification result diagram of reaction time;

[0021] Figure 4 This is the verification result diagram of the amount of DPPP added;

[0022] Figure 5 This is the verification result diagram of sample usage;

[0023] Figure 6 This is a comparison chart of the test results when the peroxide value of five fish oil samples was tested using two methods. DETAILED DESCRIPTION

[0024] The following description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0025] The following examples can be understood as a part of the local structure or method of the present invention alone, and can also be understood as the combination of the examples to explain the connotation of the larger structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are obtained from commercial sales.

[0026] Embodiment 1:

[0027] A method for detecting the peroxide value of Antarctic krill oil comprises the following steps: taking 0.3g of Antarctic krill oil, diluting it to 10mL with chloroform / methanol (1 / 3, v / v), taking 100μL in a graduated test tube (the amount of krill oil used is 3mg), adding 50L of 20mM BHT (dissolved in chloroform / methanol (1 / 3, v / v)), 25L of 3mM DPPP (1,3-bis(diphenylphosphine)propane, dissolved in chloroform / methanol (1 / 3, v / v)), and then diluting it to 10mL with chloroform / methanol (1 / 3, v / v), incubating in a 70°C water bath for 150min, vortexing and oscillating for 10s every 30min, and terminating the reaction by ice bathing for 10min after the incubation time is up, and leaving it at room temperature for 5min. The fluorescence intensity is measured under 351nm excitation light, 380nm emission light, and 2.5nm slit width.

[0028] 1. Optimization of various parameters in the detection method:

[0029] (1) Ratio of chloroform / methanol mixed solvent used

[0030] Different ratios of chloroform / methanol mixed solvents were applied in the detection method, including chloroform / methanol ratios of 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1, and the fluorescence intensity of the prepared samples was used as the evaluation index.

[0031] (2) Incubation temperature

[0032] The water bath incubation temperatures in the detection method were set to 50° C., 60° C., 70° C., 80° C., and 90° C. The fluorescence intensity of the prepared samples was investigated.

[0033] (3) Incubation time

[0034] The water bath incubation time in the detection method was set to 60 min, 90 min, 120 min, 150 min, 180 min, and 210 min, respectively. The fluorescence intensity of the prepared samples was investigated.

[0035] (4) DPPP addition amount

[0036] The amount of DPPP added in the detection method (concentration of 3 mM) was set to 10 L, 15 L, 25 L, 35 L, and 50 L. The fluorescence intensity of the prepared samples was examined.

[0037] (5) Oil sample quality

[0038] The mass of the oil samples used in the detection method was set to 30 mg, 6 mg, 3 mg, 1.5 mg, 1.2 mg, 1 mg, 0.8 mg, and 0.6 mg, respectively. The hydroperoxide content of the oil samples was determined.

[0039] 2. Methodological validation:

[0040] (1) Blank experiment

[0041] Add 50L 20mM BHT (dissolved in chloroform / methanol (1 / 3, v / v)) and 25L 3mM DPPP (dissolved in chloroform / methanol (1 / 3, v / v)) to a brown test tube, then make up to 10mL with chloroform / methanol (1 / 3, v / v), incubate in a 70°C water bath for 150min, put in an ice bath for 10min to terminate the reaction, leave at room temperature for 5min, and measure the fluorescence intensity.

[0042] (2) Linear range, detection limit, and quantification limit

[0043] Use cumene hydroperoxide to prepare standard solutions of different concentrations, ranging from 0.00005mM to 0.3mM, draw a standard curve of fluorescence intensity and standard solution concentration, and then evaluate the linear range. Through the blank experiment and the establishment of the standard curve, obtain the response value deviation and the slope of the standard curve, and calculate the detection limit and quantification limit. Calculation formula:

[0044] Detection limit = 3.3 response value deviation / slope of standard curve;

[0045] Limit of quantitation = 10 response deviation / slope of standard curve.

[0046] (3) Spiked recovery rate

[0047] Take 0.3g of Antarctic krill oil, dilute to 10mL with chloroform / methanol (1 / 3, v / v), take 100μL in a graduated test tube (the amount of krill oil used is 3mg), add 50L 20mM BHT (chloroform / methanol (1 / 3, v / v) to dissolve), 25L 3mM DPPP (chloroform / methanol (1 / 3, v / v) to dissolve), then add different volumes of cumene hydroperoxide standard solution, and then dilute to 10mL with chloroform / methanol (1 / 3, v / v), and the subsequent operation is the same as in Example 1. The concentrations of the added cumene hydroperoxide after dilution are 0.001mM (high spike concentration) and 0.0005mM (low spike concentration), respectively.

[0048] (4) Precision

[0049] The samples with two spiked concentrations prepared in (3) were selected and their hydroperoxide contents were measured 6 times according to the method of Example 1, and the precision was calculated.

[0050] 3. Comparison between this experimental method and the titration method in the national standard:

[0051] Five fish oil samples with different oxidation degrees were selected, and their peroxide values ​​were determined using this experimental method and the titration method in the national standard (GB5009.227-2016 Determination of Peroxide Value in Food) to compare whether there were any differences in the determination results of the two determination methods.

[0052] 4. Results and Discussion

[0053] The present invention first optimizes the five experimental condition parameters selected in the detection method, then performs methodological verification on the established method, and finally compares the established method with the titration method. The results are as follows Figure 1-Figure 6 shown.

[0054] Depend on Figure 1 It can be seen that when the ratio of chloroform to methanol is 1:3, the fluorescence intensity of the sample is the highest. Therefore, the ratio of the mixed solvent is selected to be 1:3, and the mixed solvent of this ratio is used to dissolve the solid reagent and as a reaction medium. Figure 2 It can be seen that the fluorescence intensity of the prepared sample increases with the increase of the system reaction temperature. When the temperature reaches 70℃, the fluorescence intensity is the highest. If the temperature continues to rise, the fluorescence intensity decreases significantly. Therefore, the system reaction temperature is selected to be 70℃. Figure 3 It can be seen that as the reaction time increases, the fluorescence intensity of the prepared sample gradually increases. After the system reaction time exceeds 150 minutes, the fluorescence intensity has no obvious increase. Therefore, the system reaction time is selected as 150 minutes. Figure 4This is the effect of the amount of DPPP added on the fluorescence intensity. When the amount of DPPP added is small, the fluorescence intensity of the prepared sample increases with the increase of the amount of DPPP added. When the amount of DPPP added exceeds 25 μL, the fluorescence intensity does not increase significantly. This may be because the hydroperoxide in the oil sample has been fully reacted. Therefore, the amount of DPPP added is selected to be 25 μL. Figure 5 This is the effect of the amount of Antarctic krill oil used on the amount of hydroperoxide (ROOH) measured in the sample. As can be seen from the figure, the amount of oil sample used has an important influence on the determination of hydroperoxide content. When the oil sample is used too much or too little, it will lead to a decrease in the measured content. When the amount is kept within a certain range, the measured content of ROOH is higher. Antarctic krill oil is rich in phospholipids and has a high viscosity. When the amount of oil sample used is large, the oil sample may be difficult to fully disperse in the solvent, resulting in insufficient reaction between ROOH and DPPP in the oil, resulting in a weakening of the fluorescence intensity. When the amount of oil sample used is small, ROOH in the oil sample can fully react with DPPP, but the matrix effect of DPPP is more obvious, which seriously affects the measured concentration of ROOH. Therefore, the optimal amount of oil sample is selected as 3 mg.

[0055] The optimized detection method was validated by methodology. The linear range of the inspection method was 0.3mmol / kg-6.7mmol / kg, the detection limit was 0.17mmol / kg, and the quantitative limit was 0.3mmol / kg. The calibration curve was y=309886x+368.59 (R2=0.9945). In addition, the spike recovery rate was 95.81% for high concentration (spiked concentration 0.001mM) and 120.28% for low concentration (0.0005mM). Precision (n=6): ROOH content was 0.0005mM (0.5%), and content was 0.001mM (1.88%). As can be seen from the above, this method has high sensitivity, good accuracy and stability, and meets the requirements for the determination of ROOH content in oil samples.

[0056] The established detection method (fluorescence method) and titration method were used to determine the peroxide value of five fish oil samples. The five fish oil samples had different oxidation degrees. The results are as follows: Figure 6 As shown in the figure, there is no significant difference between the peroxide values ​​of the five samples determined by the fluorescence method and the titration method. Therefore, this result further verifies the reliability of the fluorescence method for determining the peroxide value of oils and fats.

[0057] At present, the determination method of the peroxide value of oils and fats mainly adopts the iodine titration and potentiometric titration in the national standard ("GB5009.227-2016 Determination of Peroxide Value in Foods"), and these two methods have certain limitations when determining dark oils and fats with high phospholipid content. When the traditional iodine titration method determines the peroxide value, the titration end point is determined according to the color development of starch and iodine. The dark color of the oil seriously interferes with the judgment of the titration end point. When the phospholipid content in the oil is high, emulsification is prone to occur during the titration process using sodium thiosulfate solution, which is not conducive to the full reaction of elemental iodine and sodium thiosulfate, affecting the accuracy of the determination result. After searching, the inventor only found two foreign literatures that used fluorescence to determine the peroxide value content, but the samples in the literature were emulsions and fish oils, and fish oils were light-colored, low-phospholipid content samples, and did not involve color interference. In addition, the amount of fish oil used in the detection method in the literature is not applicable when applied to Antarctic krill oil, and the present invention proves that the amount of shrimp oil used is a key parameter in the determination method ( Figure 5 This is also the difference between the present invention and the method in the literature.

[0058] In addition, examples of samples applicable to the present invention include: Antarctic krill oil, sea cucumber oil, scallop oil, hot pot oil and other dark-colored oils or oils with a high phospholipid content. The method of the present invention can be used to detect the peroxide value, which can overcome the influence of the color of the oil and has a wide range of applications.

[0059] The various aspects, embodiments, and features of the present invention should be considered to be illustrative in all aspects and not limiting of the present invention, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0060] In the preparation method of the present invention, the order of each step is not limited to the order listed. For those skilled in the art, without creative work, the order of each step is also within the protection scope of the present invention. In addition, two or more steps or actions can be performed simultaneously.

[0061] Finally, it should be noted that the specific embodiments described herein are merely examples of the present invention, and are not intended to limit the implementation methods of the present invention. A person skilled in the art of the present invention may make various modifications or supplements to the specific embodiments described, or replace them in a similar manner. It is not necessary and impossible to provide all examples of all implementation methods here. However, these obvious changes or modifications derived from the essential spirit of the present invention still fall within the scope of protection of the present invention, and interpreting them as any additional limitation is contrary to the spirit of the present invention.

Claims

1. A method for detecting the peroxide value of Antarctic krill oil, characterized in that: Excess DPPP was used to react with the hydroperoxide in Antarctic krill oil, and the fluorescence intensity after the reaction was measured. The hydroperoxide content in the krill oil was calculated based on the corresponding standard curve of fluorescence intensity and hydroperoxide concentration, thereby determining the peroxide value of the Antarctic krill oil.

2. The detection method according to claim 1, characterized in that The method comprises the following steps: taking 100 μL of chloroform / methanol solution containing 2-5 mg into a graduated test tube, adding 40-60 μL of 20 mM BHT (2,6-di-tert-butyl-p-cresol) solution and 20-35 μL of 3 mM DPPP solution, and then making the volume to 10 mL with chloroform / methanol, incubating in a water bath at 60-70°C for 145-220 minutes, and terminating the reaction in an ice bath after the incubation time is up; and measuring the fluorescence intensity under 351 nm excitation light, 380 nm emission light and 2.5 nm slit width.

3. The detection method according to claim 2, characterized in that: Take 0.2-0.5 g of Antarctic krill oil and dilute it to 10 mL with chloroform / methanol to prepare a chloroform / methanol solution containing Antarctic krill oil.

4. The detection method according to claim 2, characterized in that: The solvents of BHT and DPPP solutions were chloroform / methanol.

5. The detection method according to any one of claims 2 to 4, characterized in that: The volume ratio of chloroform to methanol in chloroform / methanol is 4:1 to 1:

4.

6. The detection method according to claim 2, characterized in that: During the incubation period, vortexing was performed for 10 s every 6 min for the first hour and vortexing was performed for 10 s every 30 min for the next 1.5 h.

7. The detection method according to claim 2, characterized in that: After the incubation time is up, place in an ice bath for 10 to 15 minutes to terminate the reaction.

8. The detection method according to claim 2, characterized in that: After the incubation, the reaction was placed at room temperature for 3 to 5 minutes and vortexed for 10 seconds before measurement.

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