Method for judging degree of oleic acid deterioration of soybean phospholipid

Abstract

The invention discloses a method for judging the degree of oleic acid deterioration of soybean phospholipid. The method specifically comprises the following steps: S1, pretreating a sample; s2, index detection; s3, dynamic rancidity process analysis; s4, screening volatile metabolites; s5, performing comprehensive judgment; the invention relates to the technical field of analysis and detection. According to the method for judging the oleic acid abortion degree of soybean phospholipid, an acetone gradient extraction method is combined with a 10kDa ultrafiltration membrane, so that the separation efficiency of phospholipid aggregate and free fatty acid is improved, the interference of endogenous phosphatidic acid on the acid value is avoided, the oxidation rate of phosphatidylcholine directly monitors the double-bond oxidation of a molecular level, the interference of neutralizing substances is avoided, and the omission ratio is reduced; according to the method, the acid value change rate is detected, accurate dynamic prediction of the rancidity process is achieved, single-point detection errors are avoided, detection of exogenous oxidative rancidity is achieved in cooperation with detection of volatile metabolites, and accurate and effective judgment is provided for the degree of rancidity of soybean phospholipid acid.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to a method for determining the rancidity degree of soybean lecithin oil. Background Art

[0002] Soy lecithin, a natural ingredient extracted from soybeans, is a complex lipid mixture primarily composed of phosphatidylcholine (also known as lecithin), phosphatidylethanolamine, and phosphatidylinositol. These phospholipids are essential components of cell membranes and play a variety of roles in the body. The main benefits of soy lecithin include improving cardiovascular health, promoting liver function, and enhancing brain function. As a core raw material for feed additives and food emulsifiers, controlling the rancidity of soy lecithin oil is directly related to the safety of downstream products.

[0003] The common method for determining the degree of rancidity of soybean lecithin oil is to measure its acid value. However, during the processing of soybean lecithin oil, manufacturers add alkaline substances to neutralize it, resulting in serious distortion of traditional acid value testing. The measured value is lower than the actual rancidity level, creating a hidden rancidity risk. For example, a method for determining the degree of rancidity of soybean lecithin oil described in Publication No. CN109142654A avoids neutralization interference by combining acetone separation with phospholipid acid value / soybean oil acid value comparison. However, it still has the following drawbacks: 1. Separation requires 4-6 hours of low-temperature extraction and multiple filtrations, which is cumbersome and relies only on static acid value comparison, which cannot capture early oxidation; Second, it only provides acid value data at a single time point and cannot dynamically track the rancidity process; 3. It is impossible to distinguish endogenous phosphatidic acid rancidity from exogenous oxidative rancidity, and has poor anti-interference ability.

[0004] Based on the search of the above information, a method for determining the degree of rancidity of soybean lecithin oil is proposed. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a method for determining the degree of rancidity of soybean lecithin oil, which solves the problems of traditional soybean lecithin oil rancidity determination, such as cumbersome operation, inability to capture early oxidation, inability to dynamically track the rancidity process, and poor anti-interference ability.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for determining the degree of rancidity of soybean lecithin oil, specifically comprising the following steps: S1. Sample pretreatment: The soybean lecithin oil to be tested was placed in a -20°C environment and subjected to gradient extraction using pre-cooled acetone containing 0.1% BHT antioxidant. Phospholipid aggregates and free fatty acids were separated using a 10 kDa ultrafiltration membrane. S2. Index detection: Targeted quantitative detection of 9-hydroxy-10,12-octadecadienoic acid in phosphatidylcholine by HPLC-MS, and calculation of phosphatidylcholine oxidation rate; S3. Dynamic rancidity analysis: Take equal amounts of samples and subject them to accelerated oxidation in a 35°C thermostat. Determine the acid value every 6 hours using the cold solvent method, and record the rate of change of the acid value over 72 hours. S4. Volatile metabolite screening: headspace solid phase microextraction-gas chromatography-ion mobility spectrometry was used to detect the concentrations of valeraldehyde and hexanal, and the volatile metabolite score was calculated; S5. Comprehensive judgment: Rancidity is judged based on the phosphatidylcholine oxidation rate, acid value change rate and volatile metabolite score.

[0007] The present invention is further configured as follows: the gradient extraction method in S1 includes: Add twice the volume of acetone extractant to the soybean lecithin oil to be tested, stir and crush in an ice-water bath for 10 minutes, combine the filtrates and remove the solvent by rotary evaporation to complete a single extraction; An equal volume of acetone extractant was added, and the mixture was stirred in an ice-water bath for 10 min. The filtrates were combined and the solvent was removed by rotary evaporation to complete the secondary extraction. Then add an equal volume of acetone extractant, crush and stir in an ice-water bath for 10 minutes, combine the filtrates and remove the solvent by rotary evaporation to complete three extractions.

[0008] The present invention is further configured as follows: the calculation formula for the phosphatidylcholine oxidation rate is: Where, is the phosphatidylcholine oxidation rate, is the molecular weight of oxidized phospholipids, is the total phospholipid molecular weight.

[0009] The present invention is further configured as follows: the calculation method of the acid value change rate in S3 includes: S31. Acid value calculation: Where, for The acid price at the moment, for Titrate the volume of KOH consumed at every moment. is the blank titration volume, is the concentration of KOH standard solution, is the sample mass; S32, slope calculation: Where, for The slope at time, for The acid price of the moment, for The acid value at the moment, where 、 and For moments that are connected in sequence; S33, nonlinear fitting: Where, The largest increase in acid value, is the oxidation rate constant, The time point when the acid price rises fastest. is the basic acid value offset, is a natural constant, taken as 2.718.

[0010] The present invention is further configured as follows: the formula for calculating the volatile metabolite score in S4 is: Where, Score volatile metabolites, is the toxicity correction factor of valeraldehyde, is the current valeraldehyde concentration, is the toxicity correction factor of acetaldehyde, is the current acetaldehyde concentration, is the rancidity threshold of valeraldehyde, is the rancidity threshold of acetaldehyde.

[0011] The present invention is further configured as follows: the calculation formula for determining rancidity in S5 based on the phosphatidylcholine oxidation rate, the acid value change rate, and the volatile metabolite score is: Where, Score volatile metabolites, are all weight coefficients.

[0012] The present invention is further configured as follows: When it is judged to be rancid; described At this time, it is judged that no rancidity has occurred.

[0013] The present invention is further configured as follows: When it is judged that moderate rancidity occurs; described When the temperature is low, it is judged to be severely rancid.

[0014] The present invention provides a method for determining the degree of rancidity of soybean lecithin oil. It has the following beneficial effects: (1) The present invention improves the separation efficiency of phospholipid aggregates and free fatty acids by combining acetone gradient extraction with a 10 kDa ultrafiltration membrane, avoids the interference of endogenous phosphatidic acid on acid value, and directly monitors the oxidation of double bonds at the molecular level through the oxidation rate of phosphatidylcholine, avoids interference from neutralizing substances, reduces the missed detection rate, and realizes accurate dynamic prediction of the rancidity process by combining with the detection of acid value change rate, avoids single-point detection error, and realizes the detection of exogenous oxidative rancidity by combining with the detection of volatile metabolites, thereby providing accurate and effective judgment of the rancidity degree of soybean lecithin oil.

[0015] (2) The present invention quantitatively determines the degree of rancidity of soybean lecithin oil by using the acetylcholine oxidation rate, acid value change rate and volatile metabolite score, thereby further improving the accuracy of determining the degree of rancidity of soybean lecithin oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the method of the present invention. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention.

[0018] See also Figure 1 , the embodiment of the present invention provides the following technical solutions: Example 1 A method for determining the degree of rancidity of soybean lecithin oil, comprising the following steps: S1. Sample pretreatment: Place 20 ml of the soybean lecithin oil to be tested at -20°C and perform gradient extraction using pre-cooled acetone containing 0.1% BHT antioxidant. The gradient extraction method includes: Add 40 ml of acetone extractant to the soybean lecithin oil to be tested, stir and crush in an ice-water bath for 10 minutes, combine the filtrates and remove the solvent by rotary evaporation to complete a single extraction; Add 20 ml of acetone extractant, stir and crush in an ice-water bath for 10 min, combine the filtrates and remove the solvent by rotary evaporation to complete the secondary extraction; Then add 20 ml of acetone extractant, stir and crush in an ice-water bath for 10 minutes, combine the filtrates and remove the solvent by rotary evaporation, completing three extractions; Phospholipid aggregates and free fatty acids were then separated by 10 kDa ultrafiltration membrane.

[0019] S2. Index detection: The content of 9-hydroxy-10,12-octadecadienoic acid in phosphatidylcholine was detected by targeted quantitative HPLC-MS, and the phosphatidylcholine oxidation rate was calculated using the following formula: Where, is the phosphatidylcholine oxidation rate, is the molecular weight of oxidized phospholipids, is the total phospholipid molecular weight.

[0020] When the phosphatidylcholine oxidation rate is less than 5%, the fresh soybean lecithin oil is judged to be non-rancid based on the PCOR background value; When the oxidation rate of phosphatidylcholine is greater than 5%, the corresponding peroxide value is greater than 5 meq / kg, which is judged to be rancid according to GB / T 5538-2023.

[0021] Further explanation, the detection conditions of phosphatidylcholine oxidation rate are shown in Table 1: Although endogenous phosphatidic acid rancidity can be detected in this example, the rancidity of soybean lecithin oil to which a neutralizer has been added cannot be determined due to the influence of exogenous oxidative rancidity.

[0022] Example 2 A method for determining the degree of rancidity of soybean lecithin oil, comprising the following steps: S1. Sample pretreatment: Place 20 ml of the soybean lecithin oil to be tested at -20°C and perform gradient extraction using pre-cooled acetone containing 0.1% BHT antioxidant. The gradient extraction method includes: Add 40 ml of acetone extractant to the soybean lecithin oil to be tested, stir and crush in an ice-water bath for 10 minutes, combine the filtrates and remove the solvent by rotary evaporation to complete a single extraction; Add 20 ml of acetone extractant, stir and crush in an ice-water bath for 10 min, combine the filtrates and remove the solvent by rotary evaporation to complete the secondary extraction; Then add 20 ml of acetone extractant, crush and stir in an ice-water bath for 10 minutes, combine the filtrates and remove the solvent by rotary evaporation, completing three extractions; Phospholipid aggregates and free fatty acids were then separated by 10 kDa ultrafiltration membrane.

[0023] S2. Volatile metabolite screening: Use headspace solid phase microextraction-gas chromatography-ion mobility spectrometry to detect the concentrations of valeraldehyde and hexanal, and calculate the volatile metabolite score using the formula: Where, Score volatile metabolites, is the toxicity correction factor of valeraldehyde, which is 1.2. is the current valeraldehyde concentration, is the toxicity correction factor of acetaldehyde, which is 1.5. is the current acetaldehyde concentration, is the rancidity threshold of valeraldehyde, is the rancidity threshold of acetaldehyde, the rancidity threshold specified in GB 2716-2018, 5.0μg / g, It is 7.5μg / g.

[0024] When the volatile metabolite score is less than 5%, it is judged to be not rancid; When the volatile metabolite score is greater than 5%, it is considered rancid.

[0025] Further explanation, the detection conditions of valeraldehyde and hexanal concentrations are shown in Table 2: Although exogenous oxidative rancidity can be detected in this embodiment, endogenous phosphatidic acid rancidity cannot be determined, resulting in errors in rancidity determination.

[0026] Example 3 Based on Example 1 and Example 2, a dynamic rancidity process analysis was introduced to comprehensively determine the rancidity degree of soybean lecithin oil. The dynamic rancidity process analysis was as follows: 13 1 ml samples were taken for accelerated oxidation in a 35°C constant temperature box, and the acid value was measured every 6 hours by the cold solvent method. The acid value change rate was recorded for 72 hours. The calculation method includes: S31. Acid value calculation: Where, for The acid price at the moment, for Titrate the volume of KOH consumed at every moment. is the blank titration volume, is the concentration of KOH standard solution, is the sample mass; S32, slope calculation: Where, for The slope at time, for The acid price at the moment, for The acid value at the moment, where 、 and For moments that are connected in sequence; S33, nonlinear fitting: Where, The largest increase in acid value, is the oxidation rate constant, The time point when the acid price rises fastest. is the basic acid value offset, is a natural constant, taken as 2.718.

[0027] Among them, the actual rancidity kinetic process is fitted by a nonlinear regression model, which complies with the ISO 660:2020 standard.

[0028] The comprehensive method for determining the degree of rancidity of soybean lecithin oil is: Where, Score volatile metabolites, are weight coefficients, with values ​​of 0.5, 0.3, and 0.2 respectively. The weight distribution is verified by orthogonal experimental design to verify the sensitivity of each parameter. The experiment adopts L9 (3 4 ) Orthogonal table, variance analysis tool: SPSS 26.0, the F value results of each parameter on the degree of rancidity are shown in Table 3: When it is judged to be rancid; At this time, it is judged that no rancidity has occurred.

[0029] Further explanation: In order to determine the degree of rancidity, When the temperature is low, it is judged to be moderately rancid; When the acid value is detected, it is judged that serious rancidity occurs. The acid value test refers to the GB / T 5530 standard and the threshold value is determined. =0.75, reflecting the critical point of rancidity of peroxide value ≥10meq / kg in GB / T 5538-2023.

[0030] Simulation experiment Taking the scheme provided in Example 3 as an experimental example, the control group used the traditional acetone separation method to determine the total acid value, with 35 mgKOH / g as the judgment threshold, and 32 batches of commercially available soybean lecithin oil (including 10 batches of calcium carbonate neutralized samples) were taken as experimental samples. The test results are shown in Table 4: In an extended experiment, the neutralized samples that were missed in the control group of 6 batches were tested according to the method provided in the example. It was found that the phosphatidylcholine oxidation rate was ≥0.82 and the volatile metabolite score was ≥0.75. It can be judged that they have become severely rancid, which further verifies the accuracy of the present invention in determining the degree of rancidity of soybean lecithin oil.

[0031] In summary, it can be seen that Example 3 of the present invention has obvious advantages in anti-interference, sensitivity and detection efficiency. All indicator collection methods are strictly compliant with national standards / ISO standards, and the data can be repeatedly verified, providing a standardized technical path for soybean lecithin oil rancidity detection.

Claims

1. A method for determining the degree of rancidity of soybean lecithin oil, characterized in that: The specific steps include: S1. Sample pretreatment: The soybean lecithin oil to be tested was placed in a -20°C environment and subjected to gradient extraction using pre-cooled acetone containing 0.1% BHT antioxidant. Phospholipid aggregates and free fatty acids were separated using a 10 kDa ultrafiltration membrane. S2. Index detection: Targeted quantitative detection of 9-hydroxy-10,12-octadecadienoic acid in phosphatidylcholine by HPLC-MS, and calculation of phosphatidylcholine oxidation rate; S3. Dynamic rancidity analysis: Take equal amounts of samples and subject them to accelerated oxidation in a 35°C thermostat. Determine the acid value every 6 hours using the cold solvent method, and record the rate of change of the acid value over 72 hours. S4. Volatile metabolite screening: headspace solid phase microextraction-gas chromatography-ion mobility spectrometry was used to detect the concentrations of valeraldehyde and hexanal, and the volatile metabolite score was calculated; S5. Comprehensive judgment: Rancidity is judged based on the phosphatidylcholine oxidation rate, acid value change rate and volatile metabolite score.

2. A method for determining the degree of rancidity of soybean lecithin according to claim 1, characterized in that: The gradient extraction method in S1 includes: Add twice the volume of acetone extractant to the soybean lecithin oil to be tested, stir and crush in an ice-water bath for 10 minutes, combine the filtrates and remove the solvent by rotary evaporation to complete a single extraction; An equal volume of acetone extractant was added, and the mixture was stirred in an ice-water bath for 10 min. The filtrates were combined and the solvent was removed by rotary evaporation to complete the secondary extraction. Then add an equal volume of acetone extractant, crush and stir in an ice-water bath for 10 minutes, combine the filtrates and remove the solvent by rotary evaporation to complete three extractions.

3. A method for determining the degree of rancidity of soybean lecithin according to claim 1, characterized in that: The calculation formula of the phosphatidylcholine oxidation rate is: Where, is the phosphatidylcholine oxidation rate, is the molecular weight of oxidized phospholipids, is the total phospholipid molecular weight.

4. The method for determining the degree of rancidity of soybean lecithin according to claim 1, wherein: The calculation method of the acid value change rate in S3 includes: S31. Acid value calculation: Where, for The acid price of the moment, for Titrate the volume of KOH consumed at every moment. is the blank titration volume, is the concentration of KOH standard solution, is the sample mass; S32, slope calculation: Where, for The slope at time, for The acid price at the moment, for The acid value at the moment, where 、 and For moments that are connected in sequence; S33, nonlinear fitting: Where, The largest increase in acid value, is the oxidation rate constant, The time point when the acid price rises fastest. is the basic acid value offset, is a natural constant, taken as 2.

718.

5. The method for determining the degree of rancidity of soybean lecithin according to claim 1, wherein: The formula for calculating the volatile metabolite score in S4 is: Where, Score volatile metabolites, is the toxicity correction factor of valeraldehyde, is the current valeraldehyde concentration, is the toxicity correction factor of acetaldehyde, is the current acetaldehyde concentration, is the rancidity threshold of valeraldehyde, is the rancidity threshold of acetaldehyde.

6. The method for determining the degree of rancidity of soybean lecithin according to claim 1, wherein: The calculation formula for determining rancidity in S5 based on the phosphatidylcholine oxidation rate, acid value change rate, and volatile metabolite score is: Where, Score volatile metabolites, are all weight coefficients.

7. The method for determining the degree of rancidity of soybean lecithin according to claim 6, wherein: described When it is judged to be rancid; described At this time, it is judged that no rancidity has occurred.

8. The method for determining the degree of rancidity of soybean lecithin according to claim 7, wherein: described When it is judged that moderate rancidity occurs; described When the temperature is low, it is judged to be severely rancid.

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