Patent Application
20230271109
The present disclosure relates to a method for accurately separating and identifying an oxidized triglyceride in frying oil, and belongs to the technical field of detection.
Due to special flavor and taste, fried food is very popular among people. However, oil will constantly undergo reactions in a frying process, and a large number of polar substances that are harmful to the human body are produced. The polar substances refer to a type of substances such as an oxidized triglyceride (ox-TG), free fatty acids, and triglyceride polymers that have higher polarity than normal triglycerides and are generated by a series of chemical reactions such as oxidation, hydrolysis, and polymerization under the condition of heating or frying of frying oil. Because of the polar substances, the function, sensory evaluation, nutritional value, and flavor of the oil are changed. Studies have shown that the most toxic component of the polar substances is the ox-TG. The ox-TG refers to a triglyceride having an oxidized fatty acid chain and a complicated structure and being difficult to analyze and identify.
At present, the polar substances in the frying oil are mainly separated by a silica gel column chromatography method. According to the method, the purpose of separation is achieved based on the polarity difference between polar components and non-polar components. However, only mixed polar components can be obtained by the method, and ox-TG compounds with highest toxicity cannot be individually separated.
In the present disclosure, based on a molecular imprinting technology, the structure of ox-TG in frying oil is identified by mass spectrometry first. It is found that after frying is conducted for 24 h, the ox-TG mainly includes epoxy ox-TG, hydroxyl ox-TG, and aldehyde ox-TG. Thus, the three types of ox-TG are selected as a template molecule to synthesize a surface molecularly imprinted polymer (SMIPs). Then, a polymer completely matched with the ox-TG template molecule in action site and spatial configuration is synthesized, and the specific ox-TG can be separated by using the SMIPs. According to the present disclosure, OXTG-SMIPs prepared by the molecular imprinting technology has good specificity, stability, and affinity, and accurate separation of the ox-TG in the frying oil can be achieved.
Specifically, the present disclosure has the following technical solution: a method for accurately separating and identifying an oxidized triglyceride in frying oil. The method includes the following steps:
In an embodiment of the present disclosure, in step (a), the substrate material includes any one or more of silica gel, glucan, and titanium dioxide, and is preferably the silica gel.
In an embodiment of the present disclosure, in step (a), the functional monomer is preferably the methacrylic acid or the acrylamide.
In an embodiment of the present disclosure, when the functional monomer is the acrylamide or the 4-vinylpyridine, specific operation of grafting the outer layer of the substrate material with the functional monomer in step (a) includes:
In an embodiment of the present disclosure, when the functional monomer is the methacrylic acid, specific operation of grafting the outer layer of the substrate material with the functional monomer in step (a) includes: adding anhydrous toluene to the substrate material, conducting stirring, and adding triethylamine and 3-(triethoxysilyl)propyl methacrylate dropwise, where a mass ratio of the substrate material to the 3-(triethoxysilyl)propyl methacrylate is 1:1.5 to 1:1.8, the mass of the anhydrous toluene is 20-30 times that of the substrate material, and the mass of the triethylamine is 0.5-2 times that of the substrate material; and after the dropping is completed, carrying out a reaction under the protection of nitrogen for 20-24 h, and then conducting drying to obtain a substrate material grated with the functional monomer.
In an embodiment of the present disclosure, in step (a), structural formulas of the hydroxyl ox-TG, the aldehyde ox-TG, and the epoxy ox-TG are as shown in Formula I to Formula III, respectively:
In an embodiment of the present disclosure, in step (a), a mass ratio of the substrate material grafted with the functional monomer to the template molecule is 5:1 to 10:1, the use amount of the initiator is 2%-4% (m/m) of that of the substrate material, and the use amount of the crosslinking agent is 1.5-2 times (m/m) that of the substrate material.
In an embodiment of the present disclosure, in step (a), different molecularly imprinted polymers (SMIPs) can be prepared by adding different template molecules as required.
In an embodiment of the present disclosure, when any one of the epoxy ox-TG, the hydroxyl ox-TG, and the aldehyde ox-TG is added, a single-template molecularly imprinted polymer (single-template SMIPs), namely hydroxyl OX-TGMIPs (SMIPs1), aldehyde OX-TGMIPs (SMIPs2), or epoxy OX-TGMIPs (SMIPs3), is prepared.
In an embodiment of the present disclosure, when any two of the epoxy ox-TG, the hydroxyl ox-TG, and the aldehyde ox-TG are added, a double-template molecularly imprinted polymer (double-template SMIPs), namely hydroxyl-aldehyde OX-TGMIPs (SMIPs4), hydroxyl-epoxy OX-TGMIPs (SMIPs5), or aldehyde-epoxy OX-TGMIPs (SMIPs6), is prepared.
In an embodiment of the present disclosure, when the epoxy ox-TG, the hydroxyl ox-TG, and the aldehyde ox-TG are added at the same time, a three-template molecularly imprinted polymer (three-template SMIPs), namely hydroxyl-aldehyde-epoxy OX-TGMIPs (SMIPs7), is prepared.
In an embodiment of the present disclosure, the chromatographic column is preferably a glass sand core chromatographic column with a size ϕ of 45 mm*40 cm.
In an embodiment of the present disclosure, when a single-template molecularly imprinted polymer is prepared, the three types of OXTG-SMIPs, namely the SMIPs1, the SMIPs2, and the SMIPs3, are loaded into the chromatographic column at a mass ratio of 1:1:1 to 1:1:2 in step (b).
In an embodiment of the present disclosure, in step (c), the eluting agent is any one of dimethyl sulfoxide and tetrahydrofuran.
In an embodiment of the present disclosure, in step (c), when a mass ratio of the frying oil to the OXTG-SMIPs is in the range of 1:20 to 1:25, a good separation effect is achieved.
In an embodiment of the present disclosure, in step (c), the frying oil is edible oil used for frying and cooking food in families, restaurants, and industrial occasions.
In an embodiment of the present disclosure, the edible oil refers to animal or vegetable oil used in a preparation process of food.
In an embodiment of the present disclosure, the edible oil includes rapeseed oil, peanut oil, hemp oil, corn oil, olive oil, camellia oil, palm oil, sunflower seed oil, soybean oil, sesame oil, flaxseed oil (linseed oil), flower seed oil, fish oil, algae oil, cottonseed oil, rice oil, grape seed oil, walnut oil, peony seed oil, pig fat, cattle fat, goat fat, and combinations thereof. In an embodiment of the present disclosure, in step (d), the eluting agents sequentially include a mixture of acetic acid and methanol at a ratio of 1:8, a mixture of acetic acid and methanol at a ratio of 1:6, and a mixture of acetic acid and methanol at a ratio of 1:4.
In an embodiment of the present disclosure, a method for accurately separating and identifying an oxidized triglyceride in frying oil specifically includes the following steps:
In step (d), the eluting agents sequentially include a mixture of acetic acid and methanol at a ratio of 1:8, a mixture of acetic acid and methanol at a ratio of 1:6, and a mixture of acetic acid and methanol at a ratio of 1:4; and in step (a), structural formulas of the epoxy ox-TG, the hydroxyl ox-TG, and the aldehyde ox-TG are as shown in Formula I to Formula III, respectively:
In an embodiment of the present disclosure, in step (a), the substrate material is titanium dioxide.
In an embodiment of the present disclosure, in step (a), the acrylamide is changed into methacrylic acid. When the methacrylic acid is grafted, only one step is required for completion, and 3-(triethoxysilyl)propyl methacrylate is used as a raw material.
In an embodiment of the present disclosure, in step (c), the eluting agent is tetrahydrofuran.
The present disclosure further provides application of the method in the field of food.
According to the present disclosure, specific OXTG-SMIPs is prepared by a molecular imprinting technology and a Raman spectroscopy technology, the ox-TG in the frying oil can be accurately separated and identified, and the method has the advantages of high affinity, low detection limit and simple operation.
The present disclosure is further described below in conjunction with examples, but the embodiments of the present disclosure are not limited herein.
A determination method and calculation formulas of the RSD, detection limit and recovery rate are as follows: the content of ox-TG obtained after elution is determined by Raman spectroscopy, and the RSD (RSD=standard deviation SD/arithmetic mean X), detection limit (S/N=3) and recovery rate (Recovery=madded/mrecovered) are calculated based on the method.
The substrate materials including silica gel, glucan and titanium dioxide, methacrylic acid, acrylamide, and 4-vinylpyridine mentioned in the following examples and comparative examples were purchased from Bailingwei Chemical Reagent Co., Ltd..
(a) First, 5 g of silica gel as a substrate material was weighed and placed in a 500 mL round-bottomed three-mouth flask, 150 mL of anhydrous toluene was added, 30 ml of aminopropyltriethoxysilane and 10 ml of pyridine were slowly added dropwise under magnetic stirring at room temperature, and magnetic stirring was conducted in a water bath under the protection of nitrogen at 95° C. for 24 h to obtain amino modified SiO2. After drying was completed, 5 g of the amino modified SiO2 was weighed and added to a 250 mL round-bottomed three-mouth flask, 100 mL of anhydrous toluene was added, magnetic stirring was conducted for 15 min, 8 mL of acryloyl chloride and 5 mL of triethylamine were slowly added dropwise, and after the dropping was completed, magnetic stirring was conducted under the protection of nitrogen for 24 h to obtain SiO2@acrylamide.
After drying was completed, 5 g of the SiO2@acrylamide was weighed, 50 mL of DMSO as a solvent, 0.10 g of azodiisobutyronitrile as an initiator, 8.0 g of ethylene glycol dimethacrylate as a crosslinking agent, and 0.80 g of hydroxyl ox-TG as a template molecule were added for a reaction for 24 h, and then an eluting agent (a mixture of acetic acid and methanol at a ratio of 1:4) was added for eluting the template molecule so as to obtain SMIPs1. Another 5 g of the SiO2@acrylamide was taken, and the template molecule was changed into 0.80 g of aldehyde ox-TG and 0.80 g of epoxy ox-TG separately to obtain SMIPs2 and SMIPs3 respectively with other operations same as above.
(b) Characterization of the SMIPs was conducted by Fourier infrared spectroscopy.
(c) The SMIPs1, the SMIPs2, and the SMIPs3 were sequentially loaded into a chromatographic column (as shown in
(d) Sequential elution was conducted: eluting agents including a mixture of acetic acid and methanol at a ratio of 1:8, a mixture of acetic acid and methanol at a ratio of 1:6, and a mixture of acetic acid and methanol at a ratio of 1:4 were added in sequence for elution to obtain hydroxyl triglyceride, aldehyde triglyceride, and epoxy triglyceride.
(e) The content of the ox-TG obtained after elution was determined by a portable Raman spectrometer with potassium thiocyanate (with a characteristic peak at 2,120 cm-1) as an internal standard substance at an excitation light wavelength of 785 nm in a scanning range of 200-400 cm-1, and the test was carried out at room temperature. The epoxy ox-TG has significant characteristic peak values at 810-750 cm-1, 950-840 cm-1, and 1,280-1,240 cm-1. The hydroxyl ox-TG has wide and strong characteristic peaks at 3,700-3,200 cm-1. The aldehyde ox-TG has a significant characteristic peak value at 1,680 cm-1. The steps (c-d) were repeated for five times within one day. The ratio of the peak area of each characteristic peak to the area of an internal standard peak was calculated, the three types of ox-TG were subjected to quantitative treatment, and then the intra-day RSD of the present disclosure was obtained (Table 1).
(f) The steps (c-d) were repeatedly detected at the same time point for five consecutive days. The content of the ox-TG obtained after elution was determined by Raman spectroscopy, and the inter-day RSD based on the method was calculated (Table 1).
(g) The frying oil sample was gradually diluted to obtain various concentrations and then loaded. The steps (c-d) were repeated, and the concentration of the ox-TG in an eluent was determined by Raman spectroscopy. The lowest concentration of the ox-TG that can be detected was recorded, and the detection limit of various types of the ox-TG was obtained (with reference to Table 1).
From Table 1, it can be seen that the maximum RSDintra-day of the present disclosure is 0.6673%, the maximum RSDinter-day is 1.0270%, and the lowest detection limit is 2.0*10-6 g/ ml. The method has the advantages of high accuracy and low detection limit.
The silica gel as a substrate material in step (b) in Example 1 was changed into chitosan or titanium dioxide, and other conditions and parameters were consistent with those in Example 1. Results are as shown in Table 2. It can be seen that the ox-TG in the frying oil can be effectively separated when the substrate material is the silica gel, and the effect is slightly worse when other conditions are used.
The acrylamide as a functional monomer in step (a) in Example 1 was changed into 4-vinylpyridine (dihydroxyvinylpyridine was used as a functional monomer precursor) or methacrylic acid (when the functional monomer was methacrylic acid, the grafting of the functional monomer was required to be completed only in one step, and 3-(triethoxysilyl)propyl methacrylate was used as a raw material), and other conditions and parameters were consistent with those in Example 1. Results are as shown in Table 3. It can be seen that when the functional monomer is the methacrylic acid and the acrylamide, the present disclosure has a better effect.
The eluting agent (dimethyl sulfoxide) in step (c) in Example 1 was changed into tetrahydrofuran or chloroform separately, and other conditions and parameters were consistent with those in Example 2. Results are as shown in Table 4. When the dimethyl sulfoxide and the tetrahydrofuran are used as the eluting agent, polar components in the frying oil can be effectively separated.
The mass ratio of the sample (frying oil sample) to the SMIPs in step (c) in Example 1 was changed from 1:20 into 1:15, 1:25, and 1:30 separately, and other conditions and parameters were consistent with those in Example 2. Results are as shown in Table 5. When the mass ratio of the frying oil sample to the SMIPs is in the range of 1:20 to 1:25, a good separation effect is achieved.
Although the present disclosure has been disclosed as preferred examples as above, the preferred examples are not intended to limit the present disclosure. Various changes and modifications can be made by any person familiar with the technology without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be as defined by the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110395197.3 | Apr 2021 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2021/098379 | Jun 2021 | WO |
| Child | 18301388 | US |
