A combination reagent and its application in the detection of peroxide value in oils and fats.

Abstract

This invention specifically relates to a combined reagent and its application in the detection of peroxide value in oily foods. The invention first optimizes the extraction process of oil components in food. The food to be tested is fully dispersed in anhydrous ethanol, a masking agent is added to complex metal ions, and then a glacial acetic acid-n-hexane mixture is added, followed by thorough shaking and centrifugation. The upper organic phase is retained, and the solvent is removed to obtain the oil components. Those skilled in the art can use a colorimetric method to detect the peroxide value of oil components, effectively shortening the time required for sample pretreatment, simplifying the operation, and making it more suitable for on-site testing.

Description

Technical Field

[0001] This invention relates to the field of peroxide value detection technology for oils and fats, and in particular to a combination reagent and its application in the field of peroxide value detection for oils and fats. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Peroxide value (POV) reflects the degree of oxidation of oils and fats. This indicator indirectly characterizes the oxidative rancidity process of oils and fats during processing and storage by measuring the content of hydroperoxides. Consuming oils and fats with excessive peroxide values ​​can directly threaten health, including damaging the gastrointestinal digestive system and liver function. It may also accelerate the progression of chronic cardiovascular diseases such as atherosclerosis, increase the risk of cardiovascular diseases such as myocardial infarction, and the free radicals produced by the decomposition of peroxides can attack cell membranes and DNA, accelerate aging, and induce cancer.

[0004] With the continuous improvement of national health awareness, the consumption scale of nutritional and health food in my country has exceeded 400 billion yuan, with an average annual growth rate of 12%. Its share in urban residents' dietary expenditure has increased to 8.7%. This is mainly due to the surge in demand for functional nutritional supplements among modern people, especially health foods containing fat-soluble functional ingredients such as Omega-3 and Vitamin E, which account for over 35% of the market share. These products generally use high-end plant oils such as camellia oil and flaxseed oil as carriers, with an oil content typically ranging from 30% to 60%, significantly higher than the lipid ratio of ordinary foods.

[0005] However, high oil content makes health foods more susceptible to oxidative rancidity. Studies show that health foods containing functional oils such as DHA algal oil and evening primrose oil have a 28% risk of exceeding the peroxide value standard after 6 months of storage at room temperature. Oil oxidation not only destroys active ingredients such as vitamin E and coenzyme Q10, but also produces harmful substances such as malondialdehyde, directly weakening the product's health benefits and posing food safety risks. The national standard GB 5009.227-2023 specifies the method for determining the peroxide value in food, applicable to the determination of peroxide value in food, edible animal and vegetable oils, and margarine. According to the relevant provisions in "5.1.2.4 Powdered Oil Products", for powdered oil products containing complex components such as protein and starch, enzymes must first be added to hydrolyze the protein or starch components, and polar reagents such as acetone and petroleum ether are added to the hydrolysis products to extract the fat components in the food to be tested.

[0006] However, for special medical foods, milk powder, infant formula, or compound nutritional supplements, these types of foods contain a series of fatty components with similar polarities, such as medium-chain triglycerides (MCTs) like caprylic acid (C8) and capric acid (C10), long-chain triglycerides (LCTs) like palmitic acid and stearic acid, monounsaturated fatty acids, and polyunsaturated fatty acids, as well as a significant amount of starchy energy components and protein components. Sample extraction using national standard methods has a high probability of emulsification, which is difficult to eliminate. Furthermore, the enzymatic hydrolysis and petroleum ether vacuum distillation processes require low-temperature heating, which accelerates the formation of free radicals from unsaturated fatty acids in the oils, leading to higher peroxide values. The saturated potassium iodide reagent and starch indicator used in the titration stage must be prepared fresh each time. Therefore, the above methods are basically only suitable for laboratory environments and cannot be applied to on-site testing.

[0007] Colorimetric detection offers the advantage of ease of operation and is more suitable for on-site inspections. Commonly used reagents include ferrous ions and colorimetric agents; for example, potassium thiocyanate is used to detect the degree to which ferrous ions are oxidized to ferric ions, thereby determining the peroxide value. However, when testing certain powdered oily foods, such as medical foods, milk powder, infant formula, or compound nutritional supplements, the presence of metal ions, such as calcium, magnesium, and ferrous ions, in these foods can cause significant background interference in colorimetric detection. Addressing these problems in existing technologies, the inventors believe that powdered oily foods occupy an important place in people's daily lives. In particular, the quality control of medical foods and infant formula is crucial to the health of patients and infants. Therefore, strengthening the quality and safety control of compound oily foods such as medical foods and infant formula is of great significance. Summary of the Invention

[0008] To address the shortcomings of existing national standard methods and provide a standardized method for peroxide value detection based on colorimetric principles, this invention optimizes the extraction process of oil components in oily foods. By adding a masking agent to complex the solution system with excess metal ions, it not only eliminates the influence on subsequent colorimetric reagents but also effectively reduces emulsification during extraction, improving the stability of the detection results. This invention achieves rapid (total time <2 hours) and highly sensitive detection of peroxide values ​​through optimized composite solvent systems, the introduction of novel masking agents, and spectrophotometric detection, making it particularly suitable for food quality inspection scenarios requiring batch screening.

[0009] Based on the above-mentioned technical effects, the present invention provides the following technical effects: In a first aspect, a combination reagent is provided, comprising anhydrous ethanol, a masking agent, sodium chloride, glacial acetic acid, n-hexane, ferrous salt, and thiocyanate reagent.

[0010] Furthermore, the masking agent is a premixed solution of EDTA and sodium fluoride, wherein the mass fraction of EDTA is 0.04~0.06% and the mass fraction of sodium fluoride is 0.08~0.12%. In other embodiments, the masking agent is a mixture of EDTA and sodium fluoride in a specified amount, which is then compressed into tablets or granules. Since EDTA and sodium fluoride are solids at room temperature, the masking agent can also be formulated into a quantitative solid material, which is more conducive to rapid on-site detection.

[0011] Furthermore, the volume ratio of glacial acetic acid to n-hexane is 1~3:7~9.

[0012] Furthermore, the ferrous salt is selected from one or a combination of several of ferrous chloride (FeCl2), ferrous sulfate (FeSO4), potassium ferrous sulfate (K2Fe(SO4)2), and potassium ferrous oxalate (K2Fe(C2O4)2).

[0013] Furthermore, the thiocyanate reagent is preferably an alkali metal thiocyanate, such as potassium thiocyanate (KSCN), sodium thiocyanate (NaSCN), or ammonium thiocyanate (NH4SCN).

[0014] Secondly, the application of the combined reagents described in the first aspect in the field of peroxide value detection in oily foods is provided.

[0015] The application described in the second aspect above is a detection method based on the combined reagents, and also includes a detection product based on the combined reagents, such as a detection kit.

[0016] Thirdly, a method for detecting the peroxide value of oily foods is provided, including the following steps: (1) Extraction of oil components: Add the sample to be tested into anhydrous ethanol and disperse it for 20 min to 1 h. Add a masking agent and shake to mix for 5 min to 10 min. Then add glacial acetic acid-n-hexane mixed reagent, shake well and centrifuge. Keep the upper organic phase and remove the solvent to obtain the oil components. (2) Quantitative detection of peroxide value of oil components: Add ferrous ions to the oil components obtained in step (1), react at 35~45℃ in the dark for 8~12 min, add thiocyanate reagent, shake well and let stand for 3~7 min, detect absorbance at a wavelength of 480~500 nm, and obtain the peroxide value of the sample to be tested by quantitative calculation.

[0017] In the above detection method, anhydrous ethanol is used as the initial dispersion medium. Its moderate polarity disrupts the lipoprotein binding state in the matrix of the food to be tested, causing the protein components to denature, thereby fully releasing the fats, metal ions, etc. that were originally bound to the protein. The mixed reagent of glacial acetic acid (strongly polar) and n-hexane (non-polar) uses the difference in polarity gradient to allow the fat components to remain in the organic phase, while the ethanol and masking agent remain in the aqueous phase, thereby achieving the extraction and separation of the fat components. The polarity coverage of the glacial acetic acid-n-hexane mixed solvent is wider, and it can extract both polar and non-polar lipids at the same time, which is especially suitable for complex foods containing phospholipids. It has been verified that the above pretreatment method can improve the oil recovery rate by up to 70% compared with the single petroleum ether system in the national standard method. In step (2), ferrous ions react with the peroxide (ROOH) in the oil under acidic conditions to undergo a redox reaction: the generated Fe³ + It forms a red complex (Fe(SCN)3) with potassium thiocyanate, and its absorbance is linearly related to the peroxide value.

[0018] To prevent the food being tested from undergoing continuous lipidation during the testing process, this invention relates to adding a masking agent to complex the metal ions (such as Fe) in the solution system. 3+ Fe 2+ Cu 2+ Mg 2+ Ca 2+ The masking agent is a combination of EDTA and sodium fluoride, which can block the ability of lipid oxidation to be catalyzed. It can also remove iron and ferrous ions in the solution system in advance to avoid interfering with subsequent colorimetric detection.

[0019] Furthermore, the method described in the third aspect is particularly suitable for detecting foods with complex ingredients and a high content of oil, such as special-purpose medical foods, formula milk powder, soy milk powder, non-dairy creamer, or compound nutritional supplements.

[0020] The above step (1) also has the following preferred implementation: In some embodiments of the present invention with better effects, an inorganic salt is added to the solution system before adding the glacial acetic acid-n-hexane mixed reagent. This can effectively reduce the probability of emulsification in the above solution system. The inorganic salt is preferably a component with high solubility in the aqueous phase, such as sodium chloride, potassium chloride, or ammonium sulfate. The amount of inorganic salt added is 1% to 3% by mass. The salting-out effect reduces the solubility of oils in the ethanol-water system, accelerates the separation of the organic phase, and the centrifugation step further shortens the phase separation time to within 10 minutes.

[0021] Furthermore, the centrifugation speed is 2500~3500 rpm, and the centrifugation time is 4~8 min.

[0022] Furthermore, the dosage ratio of the test sample, anhydrous ethanol, masking agent, and glacial acetic acid-n-hexane mixed reagent is 4~6g:8~12mL:18~22mL:8~12mL:38~42mL.

[0023] In step (2) above, there is also the following preferred implementation method: The dosage ratio of the oil component, ferrous ions and thiocyanate reagent is 0.1g:0.0008~0.0012g:0.0008~0.0012g.

[0024] The quantitative calculation method is as follows: Prepare hydrogen peroxide solutions of various concentrations as standards, replace the oil components in step (2) with hydrogen peroxide solutions, and obtain the corresponding absorbance. Establish a standard curve of hydrogen peroxide concentration versus absorbance, and calculate the amount of peroxide in the above oil components through the standard curve.

[0025] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention first optimizes the extraction process of oil components from powdered oily foods. According to the national standard GB 5009.227-2023 for the testing of powdered oily products, the sample size must be at least 100g, and the amount of petroleum ether or acetone consumed during pretreatment must be at least three times the volume to achieve the 5.0g extraction standard. The optimized oil component extraction process of this invention (salting out + centrifugation, <1 hour) significantly shortens the sample processing time compared to the method in the national standard (rotary evaporation / long-term soaking, 24-28 hours), requiring less sample volume; the processing reagents are simple, avoiding the use of chloroform (carcinogenic) and potassium iodide (easily photodegraded), thus enhancing operational safety.

[0026] 2. In the quantitative detection stage, this invention uses a colorimetric method to detect the amount of Fe(SCN)3 complex at 480 nm, requiring approximately 8-12 minutes. This is significantly simpler than the iodine titration method in the national standard GB 5009.227-2023 (which requires the preparation of sodium thiosulfate titration and starch indicator, and the detection time is over 30 minutes). This method directly measures absorbance at a wavelength of 480 nm, avoiding iodine volatilization loss and endpoint determination errors. Furthermore, the molar absorptivity of the Fe(SCN)3 complex at 480 nm reaches 1.1 × 10⁻⁶. 4 L·mol - ¹·cm -¹ The detection limit is as low as 0.02 meq / kg, which is superior to the iodometric method (0.05 meq / kg), and it is expected to serve as a standard method for peroxide value detection based on colorimetric methods. In addition, the present invention also provides a new masking agent material, namely a quantitative solid material, which is particularly suitable for on-site detection, as it can be used immediately without the need for weighing.

[0027] 3. Reports in this field regarding peroxide value detection methods for powdered and compound nutritional supplements such as infant formula primarily employ iodometric titration for quantitative detection, with a lack of reports on colorimetric methods. The main challenge lies in the inability to eliminate interference from metal ions in the sample. To address this deficiency, this invention proposes adding a masking agent to complexate the metal ions in the original solution system. This prevents further oxidation of the sample during detection and effectively removes interference from metal ions on the colorimetric reagent. Verification has shown that the method of this invention exhibits good accuracy and stability in detecting infant formula and special medical foods, is easy to operate, and is more suitable for rapid on-site testing applications. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 The absorbance charts are of a series of reference standards obtained by the colorimetric method described in Example 1. Figure 2 The results of extraction and layering in Example 1 (A) and Comparative Example 2 (B) are shown. Detailed Implementation

[0030] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1 1. Materials and Methods 1.1 Sample Material Sample to be tested: Soy milk powder (brand: VIVI), the fat source is whole milk powder and peanut oil; Special medical purpose complete nutritional formula powder (brand: Fekahua Rui), the fat source is mainly low erucic acid rapeseed oil and sunflower seed oil, hereinafter referred to as "special medical food"; Infant formula (brand: Friso), fat source is blended vegetable oil; Non-dairy creamer (model: 90A, rich flavor) primarily contains hydrogenated vegetable oil as its fat source.

[0034] 1.2 Oil Extraction Methods The sample to be tested was passed through a 60-mesh sieve. 5g of the sieved solid powder (accurate to 0.01g) was accurately weighed and added to 10mL of water at 37℃ for dispersion. The powder was then transferred to a 200mL iodine flask, and 20mL of anhydrous ethanol was added to disperse it fully. The iodine flask was placed on a shaker and shaken at 60rpm / min~100rpm / min for 30min at room temperature.

[0035] After shaking, add 10 mL of 0.05% EDTA-0.1% sodium fluoride (mass fraction) mixture to the above solution system, and continue shaking at 100 rpm / min for 5 to 10 min. Add sodium chloride to the above mixture until the mass fraction of sodium chloride is 1% to 3%.

[0036] Add 40 mL of glacial acetic acid-n-hexane (volume ratio 2:8) mixture, vortex for 5 min, centrifuge at 3000 rpm for 5 min, take the upper organic phase, blow the solvent dry with nitrogen, weigh and record the mass of the extracted oil.

[0037] The oil extraction rate is calculated as follows: 2. Peroxide value determination 2.1 Colorimetric detection Experimental group: Weigh 0.1g of extracted oil, add 5 mL of ferrous sulfate solution (0.2g / L), and react in a 40℃ water bath in the dark for 10 min. Add 2.5 mL of potassium thiocyanate solution (0.5g / L), shake well, and let stand for 5 min.

[0038] Using deionized water as a blank control and a sample without ferrous sulfate as a control group, the absorbance (A) of the samples was measured at a wavelength of 480 nm.

[0039] Blank control: Deionized water was used instead of oil extract. Color development and absorbance were measured according to the "Experimental Group" procedure, and the result was A.空白 .

[0040] Reference solution: Accurately prepare a 1.0 M H₂O₂ solution as the standard solution, dilute with n-hexane to obtain a 0.1 M hydrogen peroxide standard solution, and perform a series of dilutions (1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 0.1 M). Take 0.1 mL of each solution, perform the colorimetric operation according to the "Experimental Group", and measure the absorbance A. 对照 Establish (A) 对照 -A 空白 The standard curve of H2O2 solution concentration versus H2O2 concentration, such as Figure 1 As shown.

[0041] The peroxide content in the experimental group's oils was calculated based on the above standard curve. The peroxide value was calculated as follows: C ‎: Peroxide concentration (μmol / L) corresponding to the standard curve; V ‎: Total volume of the colorimetric reaction system (mL); m ‎: Mass of extracted oil (g).

[0042] 2.2 Titration method for detection Referring to "5.2 Determination of Samples" in Method 1 of the National Food Safety Standard GB2009.227-2023, the peroxide value of the oils in the above-mentioned test samples was determined by iodometric titration.

[0043] Experimental group: Weigh 2-3g (accurate value 0.001g) of the oil sample and place it in a 250mL iodine flask. Add 30mL of chloroform-glacial acetic acid solution and shake on a shaker at 50-80rpm to completely dissolve the sample. Add 1.00mL of saturated potassium iodide solution, seal the flask tightly, shake gently, and then place it in the dark to equilibrate for 3min.

[0044] After removing the solution, add 100 mL of water, shake well, and immediately titrate with sodium thiosulfate standard solution (concentration of 0.002 mol / L). When the solution in the iodine flask turns pale yellow, add 1 mL of starch indicator. The solution turns blue. Continue titrating and shake vigorously until the blue color in the solution just disappears.

[0045] Blank control group: No sample was added, and the remaining operations were the same as the above determination method.

[0046] The experimental group and the control group were set up in parallel. The results were expressed as the arithmetic mean of two independent measurements and were retained to two significant figures.

[0047] The peroxide value of the above samples was calculated according to the following formula (1): (1) In the above formula (1): X 1. Peroxide value of the food to be tested, g / 100g; V The volume of sodium thiosulfate standard solution consumed in the sample titration, in mL; V 0 ， The volume of sodium thiosulfate standard solution consumed in the titration of the blank control group, in mL; c The concentration of the sodium thiosulfate standard solution, in mol / L; m Mass of the sample, g; The above testing procedures should be carried out under conditions that avoid direct sunlight. Parallel tests should be set up for each group, and the average result should be taken.

[0048] To demonstrate the feasibility of applying the above method to the detection of oils in compound food ingredients, this invention uses the national standard method, titration detection, and the method in Example 1 to measure different types of oils. The results are shown in Table 1 below: Table 1. Results of peroxide value (g / 100g) tests for different types of compound food ingredients In Table 1 above, the units for peroxide value and mean value are g / 100g.

[0049] The "national standard" refers to extracting oil samples according to the relevant provisions in "5.1.2.4 Powdered Oil Products" of the National Food Safety Standard for Determination of Peroxide Value in Food (GB2009.227-2023), and detecting the peroxide value of the extracted oil samples according to the steps in "2.2 Titration Method" above. During the detection process using this method, emulsification occurred with a certain probability in all parallel experimental groups of the four food products, particularly in special medical foods and infant formula. Approximately three to four of the five parallel groups showed varying degrees of emulsification, which could be eliminated by gentle shaking or high-speed centrifugation (5000 r / min, 15℃, 3 min).

[0050] The term "titration method" refers to obtaining an oil sample from the food to be tested using the "1.2 oil extraction method" and then detecting the peroxide value of the extracted oil sample using the steps in "2.2 titration method detection".

[0051] The “colorimetric method” refers to obtaining the oil sample from the food to be tested using the “1.2 Oil Extraction Method”, and then detecting the peroxide value of the extracted oil sample using the steps in “2.1 Colorimetric Detection”.

[0052] As can be seen from the results in Table 1 above, the peroxide values ​​obtained using the oil extraction method provided by this invention, regardless of whether titration or colorimetric methods are used, are basically consistent with the results obtained using the national standard method. This proves that the oil sample extraction method provided by this invention can accurately preserve the degree of oil peroxidation in the food being tested. In terms of reproducibility, the colorimetric method shows better reproducibility, a more stable trend, and faster detection speed.

[0053] Comparative Example 1 In this embodiment, another method for detecting the peroxide value of oil components in food is provided, which differs from Example 1 in that the oil extraction method is as follows: The sample to be tested was passed through a 60-mesh sieve. 5g of the sieved solid powder (accurate to 0.01g) was accurately weighed and added to a 100mL iodine flask. Twice the volume of anhydrous ethanol was added to disperse the powder thoroughly. The mixture was then placed on a shaker at 60-100 rpm for 30 minutes at room temperature. 4mL of water was added to the solution, and the mixture was gently shaken. 50mL of petroleum ether was then added, and the mixture was shaken at 100 rpm for 5-10 minutes. The mixture was allowed to stand overnight at 4°C. The petroleum ether layer was to be retained. The solvent was removed by vacuum distillation. 50mL of a 2:3 mixture of isooctane and glacial acetic acid was added, along with 1mL of saturated potassium iodide. The mixture was then allowed to react in the dark for 5 minutes. A starch indicator was added, and the solution was titrated with a 0.002mol / L sodium thiosulfate standard solution.

[0054] When the food to be tested is infant formula or a special medical food, the above method is used to extract the oil components. After standing overnight, the separation is not obvious, and some emulsification is still present after centrifugation and shaking. (e.g.) Figure 2 As shown in Figure B), it cannot meet the detection purpose of this invention.

[0055] Comparative Example 2 In this embodiment, another method for detecting the peroxide value of oil components in food is provided, referring to patent CN106908567B. The specific determination method is as follows: The sample to be tested was passed through a 60-mesh sieve. 5g of the sieved solid powder (accurate to 0.01g) was accurately weighed and dissolved in 10mL of water at 37℃. The solution was then transferred to a 200mL iodine flask. 100mL of an extraction solvent of hexane-isopropanol (volume ratio 3:1) was added. The mixture was stirred magnetically at 600 rpm for 15 min at room temperature, sonicated at 30kHz for 60s, and centrifuged at 7500g for 10 min. The upper organic phase was collected to obtain organic phase A. The remaining portion was again added to an extraction solvent of hexane-isopropanol (3:1). The mixture was stirred magnetically at 600 rpm for 15 min at room temperature, sonicated at 30kHz for 60s, and centrifuged at 7500g for 10 min. The upper organic phase was collected to obtain organic phase B. Organic phases A and B were mixed and filtered through anhydrous sodium sulfate. The solvent in the filtrate was evaporated to obtain the oil component, which was dissolved in a chloroform-glacial acetic acid solution. Quantitative detection was achieved by titration with a sodium thiosulfate standard solution.

[0056] Comparative Example 3 In this embodiment, another method for detecting the peroxide value of oil components in food is provided, referring to patent CN107315065A. The specific determination method is as follows: The sample to be tested was passed through a 60-mesh sieve. 40g of the sieved solid powder (accurate to 0.01g) was accurately weighed and dissolved in 40g of water at 37℃. The solution was then transferred to a 500mL iodine flask, and 40mL of anhydrous ethanol was added to disperse it fully. The mixture was then placed on a shaker at room temperature and shaken at a speed of 60rpm / min~100rpm / min for 30min.

[0057] Add 50 mL of diethyl ether to the above system, extract for 1 min, and allow to stand for separation to obtain the diethyl ether fraction. Add 50 mL of petroleum ether to the remaining solution system, extract for 0.5 min, and allow to stand for 20 min to obtain the petroleum ether fraction. Pour both the diethyl ether and petroleum ether fractions into a stoppered flat-bottomed flask, passing the solution through anhydrous sodium sulfate to remove water. Remove the solvent by distillation to obtain the oily component, which is dissolved in a chloroform-glacial acetic acid solution. Quantitative detection is achieved by titration with a sodium thiosulfate standard solution.

[0058] The following verification of the oil extraction effect and peroxide value detection results in Example 1 (colorimetric method), Comparative Example 2 and Comparative Example 3 is shown in Table 2 below: Table 2. Results of oil extraction rate (%) and peroxide value (g / 100g) in Example 1 and Comparative Examples 2-3 In Table 2 above, the "national standard" refers to extracting oil samples according to the relevant provisions in "5.1.2.4 Powdered oil products" of the National Food Safety Standard GB2009.227-2023 for Determination of Peroxide Value in Food, and detecting the peroxide value of the extracted oil samples according to the steps in "2.2 Titration Method".

[0059] The method described in "Example 1" involves obtaining an oil sample from the food to be tested using the "1.2 Oil Extraction Method" and then detecting the peroxide value of the extracted oil sample using the steps in "2.1 Colorimetric Detection".

[0060] In the detection of soy milk powder, the method in Example 1 yielded the highest oil extraction rate of 89.73%. The extraction effects of Comparative Examples 2 and 3 were similar to those in Example 1. This may be because the fat source in soy milk powder is mainly peanut oil, which is relatively easy to separate. However, when the national standard method was applied to the detection of soy milk powder, the oil extraction rate was only 66.61%, and the fluctuations in repeated experiments were significant.

[0061] In the testing of special medical foods, according to the national standard method, papain and amylase need to be added for enzymatic hydrolysis. The average oil extraction rate was 51.39%, and the repeatability was poor, which may be because the enzymatic hydrolysis process in each experimental group could not be guaranteed to be completely consistent. During the testing using Comparative Examples 2 and 3, obvious turbidity appeared in the organic phase layer in both cases.

[0062] Similarly, in the detection of infant formula and non-dairy creamer, the method of Example 1 showed the highest oil extraction rate, with virtually no emulsification occurring during the detection process, and good stability in repeated testing. It is expected to serve as a standardized method for detecting the peroxide value of oily foods based on colorimetric methods.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a combination reagent in the detection of peroxide value in oils and fats, characterized in that, The combined reagents include anhydrous ethanol, masking agent, sodium chloride, glacial acetic acid, n-hexane, ferrous salt, and thiocyanate reagent; The masking agent is a premixed solution of EDTA and sodium fluoride, wherein the mass fraction of EDTA is 0.04~0.06% and the mass fraction of sodium fluoride is 0.08~0.12%. The volume ratio of glacial acetic acid to n-hexane is 1~3:7~9.

2. The application of the combined reagent as described in claim 1 in the field of peroxide value detection in oils and fats, characterized in that, The ferrous salt is selected from one or a combination of several of ferrous chloride, ferrous sulfate, potassium ferrous sulfate, and potassium ferrous oxalate; Alternatively, the thiocyanate reagent may be selected from potassium thiocyanate, sodium thiocyanate, or ammonium thiocyanate.

3. A method for detecting the peroxide value of oily foods, characterized in that, The oily food is a special-purpose medical food, formula milk powder, soy milk powder, non-dairy creamer, or compound nutritional supplement. The detection method includes the following steps: (1) Extraction of oil components: The sample to be tested is added to anhydrous ethanol and dispersed thoroughly for 20 min to 1 h. A masking agent is added and shaken to mix for 5 min to 10 min. An inorganic salt is added to the solution system. The inorganic salt is selected from sodium chloride, potassium chloride, and ammonium sulfate. The mass fraction of the inorganic salt added to the solution system is 1% to 3%. Then, a glacial acetic acid-n-hexane mixed reagent is added. After shaking thoroughly, the mixture is centrifuged, and the upper organic phase is retained. The solvent is removed to obtain the oil components. The masking agent is a premixed solution of EDTA and sodium fluoride. The mass fraction of EDTA is 0.04% to 0.06%, and the mass fraction of sodium fluoride is 0.08% to 0.12%. (2) Quantitative detection of peroxide value of oil components: Add ferrous ions to the oil components obtained in step (1), react at 35~45℃ in the dark for 8~12 min, add thiocyanate reagent, shake well and let stand for 3~7 min, detect absorbance at a wavelength of 480~500 nm, and obtain the peroxide value of the sample to be tested by quantitative calculation.

4. The method for detecting the peroxide value of oily foods as described in claim 3, characterized in that, In step (1), the centrifugation speed is 2500~3500 rpm and the centrifugation time is 4~8 min; Alternatively, the dosage ratio of the test sample, anhydrous ethanol, masking agent, and glacial acetic acid-n-hexane mixed reagent is 4~6g:8~12mL:18~22mL:8~12mL:38~42mL.

5. The method for detecting the peroxide value of oily foods as described in claim 3, characterized in that, In step (2), the dosage ratio of the oil component, ferrous ions and thiocyanate reagent is 0.1g:0.0008~0.0012g:0.0008~0.0012g.

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