Method for removing heterocyclic amine substances in edible oil and application thereof

Abstract

The invention relates to the field of food, and particularly discloses a method for removing heterocyclic amine substances in edible oil and application of the method. The removing method comprises the following steps: decoloring, degreasing and desugaring natural pollen particles to obtain a sporopollen adsorbing material; and fully mixing the agaricus bisporus adsorbing material with edible oil, and carrying out solid-liquid separation to obtain the edible oil without the heterocyclic amine substances. Experimental results show that at least 12 heterocyclic amines in edible oil can be effectively removed through the agaricus bisporus adsorbing material, and the removal rate of part of substances reaches up to 95% or above. Meanwhile, no negative influence is generated on the quality and flavor of the edible oil. The efficient heterocyclic amine removal technology not only improves the safety of the edible oil, but also retains the original nutritional ingredients and unique flavor of the edible oil.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food, more particularly, it relates to a method for removing heterocyclic amine substances in edible oil and application thereof. BACKGROUND

[0002] Heterocyclic amines (HAAs) are a class of polycyclic aromatic compounds with carcinogenic and mutagenic effects produced by pyrolysis of proteins, amino acids and creatine during high-temperature heating of proteinaceous foods. More than 30 heterocyclic amines have been identified, which are classified into aminoimidazole-azaarenes and amino-carbolines. Heterocyclic amines in food pose a serious threat to human health. Studies have shown that the mutagenicity of heterocyclic amines is 100 times that of aflatoxin B1 and 2,000 times that of benzopyrene. Even if the content of heterocyclic amines in food is very small, the food safety risk it brings cannot be ignored. The European Union once issued a safety warning, requiring that the daily intake of heterocyclic amines in food should not exceed 1 mg.

[0003] Edible oil also has the risk of generating heterocyclic amines during preparation, which is mainly because roasting plant raw materials is a key step in edible oil production. For example, during the high-temperature roasting (above 200°C) of peanuts for a short time (20-40 minutes), a series of complex food chemical reactions such as Maillard reaction and lipid oxidation occur, forming unique peanut oil flavor substances; and during high-temperature roasting and other thermal processing, harmful heterocyclic amine substances are also generated. These substances not only affect the safety of edible oil, but also may cause cell exhaustion and induce various diseases, and even have the risk of teratogenicity and carcinogenicity.

[0004] Studies have shown that the heterocyclic amine pollution level in edible oil is in the order of sesame oil > peanut oil > sunflower oil > soybean oil. Therefore, how to effectively remove heterocyclic amine substances in edible oil is crucial. Currently, some researchers use quaternary ammonium salt (choline chloride) and its water-based eutectic solvent as extractant to remove β-carboline heterocyclic amines in sesame oil. This method can effectively remove heterocyclic amines in sesame oil while retaining the original flavor of sesame oil, and the total removal rate of heterocyclic amines can reach more than 90%. However, in this study, the amount of choline chloride used is as high as 0.5 g / g. The introduction of such a large amount of choline chloride will cause a huge load on the adsorption and separation process, and may introduce new harmful substances. At the same time, the removal process of water-based eutectic solvent involves the addition of water, which will affect the oil production process of pressed sesame oil. Therefore, it is urgent to develop an efficient, green and safe method for removing heterocyclic amines in edible oil. SUMMARY

[0005] To solve the above problems, the application provides a method for removing heterocyclic amine substances in edible oil and application thereof.

[0006] The inventive concept of the application is as follows: Pollen, as a naturally occurring substance, is a microsporidium of seed plants, which can produce male gametes for pollination. The outer wall of the pollen grain is composed of a complex and stable biological polymer, which is called sporopollenin, and has strong resistance to chemical corrosion and high temperature. Fourier transform infrared spectrum analysis of the isolated sporopollenin shows that there is a carboxylic acid group connected with an unsaturated ether bond, indicating that the pollen grain has the potential to be a hydrophilic polar adsorbent.

[0007] Therefore, by modifying the pollen grain, a solid adsorbent material can be obtained by decolorization, degreasing and desugaring process. The material is naturally green, non-immunogenic and chemically stable, and most importantly, it can effectively adsorb heterocyclic amine harmful substances through hydrogen bonding, π-π and hydrophobic interaction, thereby reducing the content of heterocyclic amines in peanut oil products as much as possible without affecting the quality and flavor of peanut oil, and further reducing the safety risk of edible oil products.

[0008] The application adopts the following technical solutions: In a first aspect, the application provides a method for removing heterocyclic amine substances in edible oil, which comprises: After the natural pollen particles are subjected to decolorization, degreasing and desugaring treatment, a sporopollenin adsorbent material is obtained; The sporopollenin adsorbent material is mixed with edible oil, subjected to ultrasonic treatment, centrifuged, and the precipitate is removed to obtain edible oil from which heterocyclic amine substances are removed; Preferably, the removed heterocyclic amine substances include: 1,4-dimethyl-9H-pyrido[4,3-b]indol-3-amine acetate, 6-methyl-pyrido[3',2':4,5]imidazo[1,2-a]pyridine-2-amine hydrochloride hydrate, pyrido[3',2':4,5]imidazo[1,2-a]pyridine-2-amine dihydrochloride, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 3-methylimidazo[4,5-f]quinoxaline-2-amine, 2-amino-3,4,7,8-tetramethyl-3H-imidazo[4,5-f]quinoxaline, 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoline, 1-methyl-9H-pyrido[3,4-b]indole, 9H-pyrido[3,4-b]indole.

[0009] Further, the mass ratio of the edible oil to the spore powder adsorption material is 1:0.01-0.2.

[0010] Further, the ultrasonic treatment time is 5-30 min, and the ultrasonic power is 200-800 W.

[0011] Further, the preparation method of the spore powder adsorption material comprises: The precursor is obtained by removing the fat and pigment in the natural pollen particles through Soxhlet extraction under the condition of continuous reflux at a constant temperature of 55-85 ℃ for 18-48 hours with methanol as the extraction solvent; The precursor is washed multiple times with a mixed solvent system of acetonitrile, water and formic acid to remove the sugar substances, and the product is dried to obtain the spore powder adsorption material.

[0012] Further, in the mixed solvent system, the volume ratio of acetonitrile, water and formic acid is 68-72:26-30:2.

[0013] Further, in the process of removing impurities from the natural pollen particles through Soxhlet extraction, the siphon frequency is controlled to be 3-12 times / h.

[0014] In the second aspect, the application provides a method for determining heterocyclic amine substances in edible oil, which comprises: The edible oil is treated by the removal method: after the spore powder adsorption material is mixed with the edible oil and treated by ultrasonic treatment, centrifugation is performed to obtain supernatant and precipitate, and the supernatant is the edible oil from which the heterocyclic amine substances are removed; An organic solvent containing ammonia is added to the precipitate, vortex mixing is performed, and then centrifugation is performed, which is repeated 1-5 times to obtain an eluate, and the eluate is detected by LC-MS / MS to calculate the removal rate of various heterocyclic amine substances in the edible oil.

[0015] In summary, the application has the following beneficial effects: 1. The spore powder adsorption material used in the application is a natural material obtained by decolorizing, degreasing and desugaring natural pollen particles, which exhibits significant safety and non-toxicity characteristics. Experimental results show that at least 12 kinds of heterocyclic amines (Trp-P-1, Glu-P-2, MeIQ, MeIQx, IQ, PhIP, 4,8-DiMeIQx, 7,8-DiMeIQx, 4,7,8-DiMeIQx, Glu-P-2, Harman, Norharman) in edible oil can be effectively removed by the spore powder adsorption material, and the removal rate of some substances is as high as 95% or more. At the same time, the quality and flavor of the edible oil are not negatively affected. This high removal rate not only ensures the purity of the final product, but also retains the original nutritional ingredients and unique flavor of the edible oil.

[0016] 2. The application only needs to mix the spore powder adsorption material with edible oil after ultrasonic treatment and centrifugal separation to obtain healthy and safe edible oil in the process of removing heterocyclic amine substances. The whole treatment process is simple, and the introduction of exogenous chemicals is effectively avoided, which is safer and more efficient. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a scanning electron microscope image of natural pine pollen and spore powder adsorption material provided in Experimental Example 1 of the application; Figure 2 is the influence of different state pollens on the removal rate of heterocyclic amines provided in Experimental Example 2 of the application; Figure 3 is the influence of different adsorbent material dosages on the removal rate of heterocyclic amines. DETAILED DESCRIPTION

[0018] The embodiments of the application will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only for illustration of the application and should not be regarded as limiting the scope of the application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and the reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0019] The specific embodiments of the application will be described in detail below. It should be understood that the specific embodiments described herein are only for illustration and explanation of the application, and are not intended to limit the application. EXAMPLES Example 1

[0020] The present embodiment provides a method for removing heterocyclic amine substances from edible oil, which comprises: (1) 20 g of natural pine pollen was placed in a Soxhlet extractor, and methanol was used as the extraction solvent. The sample was continuously refluxed at 85°C for 24 hours, and the siphon frequency was controlled at 5 times / hour to remove fat and pigments. Subsequently, the sample was washed with acetonitrile / water / formic acid (70:28:2, v / v / v) for 5-6 times to remove sugars. Finally, the sample was placed in an oven and dried at 60°C for 12 hours to obtain the spore powder adsorption material.

[0021] (2) 1 g of peanut oil sample and 175 mg of spore powder adsorption material were accurately weighed into a 10 mL centrifuge tube, and ultrasonic treatment was performed for 15 min. Then the centrifuge tube was centrifuged at 10000 r / min for 5 min at 4°C, and the supernatant was separated, which was the edible oil with heterocyclic amine substances removed. Example 2

[0022] The embodiment provides a method for removing heterocyclic amine substances in edible oil, which is different from the method in the embodiment 1 in the preparation process of the spore powder adsorption material. (1) 20 g of natural pine pollen is placed in a Soxhlet extractor, methanol is used as an extraction solvent, continuous reflux is carriedied out at 73 DEG C for 30 hours, the siphon frequency is controlled to be 5 times / h, and then the fat and pigment are removed. Subsequently, the sample is washed 5-6 times with acetonitrile / water / formic acid (72:26:2 v / v / v) to remove the saccharide. Finally, the sample is placed in an oven and dried at 60 DEG C for 12 hours, and the spore powder adsorption material is obtained. Embodiment 3

[0023] The embodiment provides a method for removing heterocyclic amine substances in edible oil, which is different from the method in the embodiment 1 in the preparation process of the spore powder adsorption material. (1) 20 g of natural pine pollen is placed in a Soxhlet extractor, methanol is used as an extraction solvent, continuous reflux is carriedied out at 73 DEG C for 30 hours, the siphon frequency is controlled to be 5 times / h, and then the fat and pigment are removed. Subsequently, the sample is washed 5-6 times with acetonitrile / water / formic acid (72:26:2 v / v / v) to remove the saccharide. Finally, the sample is placed in an oven and dried at 60 DEG C for 12 hours, and the spore powder adsorption material is obtained. Embodiment 4

[0024] The embodiment provides a method for removing heterocyclic amine substances in edible oil, which is different from the method in the embodiment 1 in the removal step of the heterocyclic amine. (2) 1 g of the sesame oil sample and 100 mg of the spore powder adsorption material are accurately weighed in a 10 mL centrifuge tube, and ultrasonic treatment is carriedied out for 30 min; then the centrifuge tube is centrifuged at 4 DEG C and 10000 r / min for 5 min, and the supernatant is separated, and the edible oil from which the heterocyclic amine substances are removed is obtained. Embodiment 5

[0025] The embodiment provides a method for removing heterocyclic amine substances in edible oil, which is different from the method in the embodiment 1 in the removal step of the heterocyclic amine. (3) 1 g of the sesame oil sample and 100 mg of the spore powder adsorption material are accurately weighed in a 10 mL centrifuge tube, and ultrasonic treatment is carriedied out for 30 min; then the centrifuge tube is centrifuged at 4 DEG C and 10000 r / min for 5 min, and the supernatant is separated, and the edible oil from which the heterocyclic amine substances are removed is obtained. Embodiment 6

[0026] The embodiment provides a method for removing heterocyclic amine substances in edible oil, which comprises the following steps:

[0027] (1) 20 g of natural rape pollen was placed in a Soxhlet extractor, methanol was used as the extraction solvent, and continuous reflux was carried out at 85°C for 24 hours, with the siphon frequency controlled at 7 times / hour, to remove fat and pigments. Subsequently, the sample was washed 5-6 times with acetonitrile / water / formic acid (70:28:2 v / v / v) to remove sugars. Finally, the sample was placed in an oven and dried at 60°C for 12 hours, to obtain the spore powder adsorbent material.

[0028] (2) 1 g of peanut oil sample and 175 mg of spore powder adsorbent material were accurately weighed into a 10 mL centrifuge tube, and ultrasonic treatment was performed for 15 min. Subsequently, the centrifuge tube was centrifuged at 10000 r / min for 5 min at 4°C, and the supernatant was separated, to obtain the edible oil from which heterocyclic amines had been removed. Comparative Example Comparative Example 1

[0029] The method for removing heterocyclic amines from edible oil in the present comparative example differs from that of Example 1 in that an equal amount of untreated natural pine pollen is used to replace the spore powder adsorbent material, i.e. 175 mg of natural pine pollen is directly mixed with 1 g of peanut oil in step (2) and ultrasonic treatment is performed for 15 min. Comparative Example 2

[0030] The method for removing heterocyclic amines from edible oil in the present comparative example differs from that of Example 1 in that an equal amount of choline chloride is used to replace the spore powder adsorbent material, i.e. 175 mg of choline chloride is mixed with 1 g of peanut oil in step (2) and ultrasonic treatment is performed for 15 min. Application Example

[0031] The present application example provides a method for determining heterocyclic amines in edible oil: The method described in the above examples is used to remove heterocyclic amines from edible oil, and the types and contents of the removed heterocyclic amines are qualitatively and quantitatively detected, and the removal rate is calculated.

[0032] The specific method is as follows: (1) Sample preparation: 1 g of edible oil sample and 175 mg of spore powder adsorbent material (same as in Example 1) were accurately weighed into a 10 mL centrifuge tube, and ultrasonic treatment was performed for 15 min. Subsequently, the centrifuge tube was centrifuged at 10000 r / min for 5 min at 4°C, and the supernatant was separated, to obtain the practical oil from which heterocyclic amines had been removed. 4 mL of 1% ammonia-methanol solution was then added to the centrifuge tube, and vortexed for 3 min. Subsequently, the centrifuge tube was centrifuged at 10000 r / min for 5 min at 4°C, and the supernatant was collected. The above elution step was repeated once, and the supernatants from the two times were combined. Finally, the supernatant was filtered through a 0.22 mm PTFE organic filter membrane, and then subjected to instrument detection.

[0033] (2) Instrument detection: Chromatographic conditions: An Agilent EclipsePlus C18 RRHD column (100 mm × 2.1 mm, 1.8 μm) was used; mobile phase A was an aqueous solution containing 1 mM ammonium acetate and 0.1% formic acid, and mobile phase B was acetonitrile. The gradient elution program is shown in Table 1. The flow rate was 0.3 mL / min, the injection volume was 2 mL, and the column temperature was 40°C.

[0034] Table 1. Mobile phase gradient elution program

[0035] Mass spectrometry conditions: Twelve heterocyclic amines were detected using an electrospray ionization (ESI) source coupled with multiple reaction monitoring (MRM) in positive ionization mode. The curtain gas was 35 psi, the impingement gas mode was medium, the ionization voltage was +5500 V, the ionization temperature was set to 550°C, the nebulizer gas was 55 psi, and the auxiliary heater was 55 psi. All experimental data were processed using MultiQuant™ 3.0 software (ABSCIEX).

[0036] (3) Test results UPLC-MS / MS coupled with multiple reaction monitoring (MRM) mode was used for qualitative and quantitative analysis of heterocyclic amines. In full scan mode, a mixed standard solution of heterocyclic amines was sequentially injected to generate the corresponding parent ions. Then, collisional fragmentation was performed to obtain the two product ions with the highest response for quantitative and qualitative analysis, respectively.

[0037] By the above method, 12 heterocyclic amines were detected in the precipitate after edible oil separation. Their names and classifications are shown in Table 2: Table 2 Names and classifications of twelve heterocyclic amines

[0038] Table 3 lists the optimal MRM parameters for the above 12 heterocyclic amines.

[0039] Table 3 Corresponding MRM parameters for analysis of 12 heterocyclic amines using UPLC-MS / MS

[0040] (4) Removal rate: The above detection method was used to detect 12 heterocyclic amines (as shown in the table below) in the edible oils before and after removal in Examples 1 (peanut oil), 4 (sesame oil), 5 (sunflower oil) and Comparative Examples 1-2 (peanut oil). The removal rates of the 12 heterocyclic amines were calculated, and the results are shown in Table 4.

[0041] Table 4. Removal rate of heterocyclic amines in sesame oil in Examples and Comparative Examples

[0042] As shown in Table 3, compared to natural pine pollen (Comparative Example 1) and choline chloride (Comparative Example 2), the sporopollen adsorption material used in this application has a high removal rate for 12 heterocyclic amines in edible oil, with a removal rate of over 95% for most heterocyclic amines. Furthermore, the sporopollen adsorption material used in this application is a plentiful, green, and non-toxic material, and has no negative impact on the quality and flavor of edible oil. Experimental Example 1

[0043] Characterization of pollen adsorption materials Scanning electron microscopy (SEM) was used to characterize the surface morphology of natural pine pollen and spore pollen adsorption materials.

[0044] The results are as follows Figure 1 As shown: Figure 1 (a, b, c) are scanning electron micrographs of natural pine pollen. As can be seen, the natural pine pollen grains are intact. Air sac-like structures are symmetrically distributed on both sides of the pine pollen grains, their outer walls enveloped by pollen sheaths. Germination pores are clearly visible, forming a complete pine pollen morphology.

[0045] Figure 1 (d, e, f) are scanning electron micrographs of the sporopollen adsorption material prepared in Example 1. As can be seen from the figures, after treatment, the air sac structure of the pine pollen was significantly ruptured, accompanied by the collapse of the surface morphology. Experimental Example 2

[0046] This experiment investigated the effects of pollen of different morphologies on the adsorption of heterocyclic amines. Using the methods of Application Example 1, we systematically compared the removal of heterocyclic amines from edible oils using four different pine pollen forms: natural pine pollen, cracked pine pollen, a spore pollen adsorption material (same as in Example 1), and a cracked spore pollen adsorption material. The cracked pine pollen was obtained by cracking the natural pine pollen using a defatting and decolorization method; the cracked spore pollen adsorption material was prepared using the methods of Example 1 using cracked pine pollen as the raw material.

[0047] The experimental results are as follows Figure 2As shown, P-Pine Pollen, PB-Pine Pollen, B-Pine Pollen, Pine Pollen represent spore powder adsorbent material, broken wall spore powder adsorbent material, broken wall pine pollen, pine pollen respectively. As can be seen from the figure, under the condition of 100 mg / kg standard concentration, there is a significant difference in the removal efficiency of 12 kinds of heterocyclic amines by different forms of pine pollen samples p <0.05), taking the removal rate of heterocyclic amines as the evaluation standard, the removal efficiency of four kinds of pine pollen samples on 12 kinds of heterocyclic amines is in turn: spore powder adsorbent material (≥90%), broken wall spore powder adsorbent material (≥80%), broken wall pine pollen (≥70%), natural pine pollen (≥60%). Further analysis of spore powder adsorbent material and broken wall spore powder adsorbent material, there is a significant difference between the two in removing heterocyclic amines p <0.05), the removal rate of spore powder adsorbent material on 12 kinds of heterocyclic amines is more than 90%. Experimental Example 3

[0048] This experimental example investigates the effect of the amount of spore powder adsorbent material on the adsorption of heterocyclic amines Using the method of Application Example 1, the effect of different amounts of spore powder adsorbent material (250 mg, 175 mg, 100 mg, 75 mg, 50 mg) on the removal of heterocyclic amines in peanut oil was systematically investigated at a higher standard level (100 g / kg).

[0049] The results are shown in Figure 3 As the amount of spore powder adsorbent material increases, the removal effect of heterocyclic amines shows a gradually increasing trend. When the amount of pretreated pine pollen increases to 175 mg and 250 mg, the removal effect of heterocyclic amines tends to be stable, and the removal effect is more than 95%. At the same time, there is no significant difference between the two in removing heterocyclic amines (P>0.05).

[0050] This specific embodiment is only an explanation of the present application, and is not a limitation of the present application. Those skilled in the art can make modifications to this embodiment without creative contribution after reading the present specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for removing heterocyclic amines from edible oil, characterized in that: It includes: After decolorization, degreasing and desugaring of natural pollen particles, spore-pollen adsorption materials are obtained; The sporopollen adsorption material is fully mixed with edible oil, and after solid-liquid separation, the edible oil from which heterocyclic amine substances are removed is obtained.

2. The method for removing heterocyclic amines from edible oil according to claim 1, wherein The mass ratio of the edible oil to the sporopollen adsorption material is 1:0.01-0.

2.

3. The method for removing heterocyclic amines from edible oil according to claim 1, wherein The removal methods include but are not limited to ultrasound, vortex, and stirring.

4. The method for removing heterocyclic amines from edible oil according to claim 1, wherein The natural pollen particles include one or more of insect-pollinated pollen, wind-pollinated pollen or natural spores.

5. The method for removing heterocyclic amines from edible oil according to any one of claims 1 to 4, characterized in that: The preparation method of the pollen adsorption material comprises: Using methanol as the extraction solvent, continuously reflux at a constant temperature of 55-85°C for 5-48 hours, and removing fat and pigment contained in natural pollen grains by Soxhlet extraction to obtain a precursor; The precursor is washed multiple times with a mixed solvent system of acetonitrile, water and formic acid to remove sugar substances, and the product is dried to obtain the sporopollen adsorption material.

6. The method for removing heterocyclic amines from edible oil according to claim 5, wherein: In the mixed solvent system, the volume ratio of acetonitrile, water and formic acid is 68-72:26-30:

2.

7. The method for removing heterocyclic amines from edible oil according to claim 5, wherein: During the Soxhlet extraction of natural pollen particles, the siphoning frequency was controlled at 3-12 times / h.

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