WO2020118526A1

WO2020118526A1 - Method for detecting capsaicin in oil

Info

Publication number: WO2020118526A1
Application number: PCT/CN2018/120331
Authority: WO
Inventors: 李金峰; 柯跃斌; 黎诚耀; 夏俊杰; 彭朝琼; 肖云军; 吕子全; 何洁
Original assignee: 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所）
Priority date: 2018-12-10
Filing date: 2018-12-11
Publication date: 2020-06-18
Also published as: CN109991412B; CN109991412A

Abstract

A method for detecting capsaicin in oil The method comprises the following steps: using immunoaffinity to separate and extract capsaicin from oil in advance; and using immunochromatography to perform detection on the separated and extracted capsaicin molecules. Using immunoaffinity to separate and extract the capsaicin molecules from oil comprises diluting an oil sample with a solution containing methanol and nano titanium dioxide and adding immunomagnetic beads which can specifically bind to capsaicin molecules to capture capsaicin molecules therein. In the method, combination of immunochromatography and immunoaffinity greatly reduces workload associated with detection of capsaicin in oil, and increases detection sensitivity.

Description

一种检测油脂中辣椒碱成分的方法技术领域 Method for detecting capsaicin in oil and fat

[0001] 本发明属于生物技术领域，具体涉及一种检测油脂中辣椒碱成分的方法。 [0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for detecting capsaicin components in oils and fats.

背景技术 Background technique

[0002] “地沟油”也称“泔水油”“潲水油”，泛指在生活中存在的各类劣质油，主要由废弃油脂经沉淀去杂，脱臭脱色的一种质量极差、极不卫生的非食用油。但一些不法商贩受利益驱动而不顾人民群众生命安全，私自生产加工“地沟油”并作为食用油低价销售给小餐馆，给消费者的身体健康和生命安全带来了严重的危害。然而，检测地沟油非常困难。地沟油是一个极不标准的研究对象，没有固定的组成，也没有典型的样品，给检测带来很大的不确定性。复杂多变的成分导致无法为地沟油找到特征指标，并且正常食用油的特征指标也无法在地沟油的检测中适用，为地沟油的判定带来了很大的困难。 [0002] "Ditch oil" is also called "swill oil" "潲水油", which refers to all kinds of inferior oils in life, mainly from waste oil through precipitation to remove impurities, deodorization and decolorization of a very poor quality, extremely Hygienic non-edible oil. However, some unscrupulous traders are driven by interests and disregard the safety of the people's lives. They produce and process "ditch oil" and sell it to small restaurants as low-cost cooking oil, causing serious harm to consumers' health and life safety. However, it is very difficult to detect drain oil. Ditch oil is an extremely non-standard research object with no fixed composition and no typical samples, which brings great uncertainty to the detection. The complex and variable composition makes it impossible to find the characteristic index for the drainage oil, and the characteristic index of the normal edible oil cannot be applied in the detection of the drainage oil, which brings great difficulty to the determination of the drainage oil.

[0003] 辣椒作为中国人常用的调味料广泛出现在各种菜肴中，特别是川菜中出现更多，而辣椒中的辣椒碱是一种易溶于油脂的物质，经过简单的提取难以有效去除干净。因此，检测油脂中的辣椒碱成为鉴别油脂质量的选项之一。目前，检测辣椒碱的方法主要有分光光度法、色谱法、传感器法、免疫学方法等，但是这些方法仍旧普遍存在前处理复杂，耗时长的问题，而且灵敏性也往往满足不了精炼油脂检测的要求。 [0003] Chili peppers are widely used in Chinese cuisine as a seasoning in various dishes, especially in Sichuan cuisine, and the capsaicin in chili peppers is a substance that is easily soluble in oils and is difficult to effectively remove after simple extraction. clean. Therefore, the detection of capsaicin in oil has become one of the options for identifying the quality of oil. At present, the methods for detecting capsaicin mainly include spectrophotometry, chromatography, sensor method, immunology method, etc., but these methods still generally have the problems of complicated pre-processing and time-consuming, and the sensitivity is often not enough for refined oil detection. Claim.

发明概述 Summary of the invention

技术问题 technical problem

问题的解决方案 Solution to the problem

技术解决方案 Technical solution

[0004] 本发明就油脂中辣椒碱检测费时费力、效能差这一问题提供一种简便易行的检测方法。 [0004] The present invention provides a simple and easy detection method for the problem that capsaicin detection in oils is time-consuming, labor-intensive, and has poor efficacy.

[0005] 本发明所要解决的技术问题通过以下技术方案予以实现： [0005] The technical problem to be solved by the present invention is achieved by the following technical solutions:

[0006] 一种检测油脂中辣椒碱成分的方法，该方法包括以下步骤：预先采用免疫亲和法对油脂中的辣椒碱分子进行分离提取，然后采用免疫层析法对分离提取到的辣椒碱分子进行检测，所述采用免疫亲和法对油脂中的辣椒碱分子进行分离提取是指采用含有甲醇、纳米二氧化钛的溶液稀释油脂样品，然后添加与辣椒碱分子特异性结合的免疫磁珠捕获其中的辣椒碱分子。 [0006] A method for detecting the capsaicin component in oils and fats, the method includes the following steps: using immunoaffinity in advance Method to separate and extract the capsaicin molecules in the oil and fat, and then to detect the separated and extracted capsaicin molecules by the immunochromatography method. The separation and extraction of the capsaicin molecules in the oil and fat by the immunoaffinity method refers to The solution of methanol and nano titanium dioxide is used to dilute the oil sample, and then the immunomagnetic beads that specifically bind to the capsaicin molecule are added to capture the capsaicin molecule.

[0007] 如上所述检测有之中辣椒碱成分的方法，所述二氧化钛的粒径为 2nm-25nm [0007] As mentioned above, there is a method for detecting the capsaicin component, the particle size of the titanium dioxide is 2nm-25nm

[0008] 如上所述的一种检测油脂中辣椒碱成分的方法，免疫层析法是指采用免疫层析试纸条进行检测，所述免疫层析试纸条包括依次搭接粘贴到 PVC材质的底板上的样品垫、层析膜、吸水纸，层析膜上有检测区和质控区，检测区包被有辣椒素 - 载体蛋白偶联物。 [0008] As described above, a method for detecting the capsaicin component in oils and fats, immunochromatography refers to the use of immunochromatographic test strips for detection, the immunochromatographic test strips include lap and paste to PVC material The sample pad, chromatography membrane, and absorbent paper on the bottom plate of the chromatography membrane have a detection area and a quality control area, and the detection area is coated with a capsaicin-carrier protein conjugate.

[0009] 如上所述的一种检测油脂中辣椒碱成分的方法，所述免疫磁珠在制备时采用 5- 氨基戊酸进行封闭。 [0009] As described above, a method for detecting the capsaicin component in oils and fats, the immunomagnetic beads are blocked with 5-aminovaleric acid during preparation.

[0010] 如上所述的一种检测油脂中辣椒碱成分的方法，所述免疫磁珠在制备过程中，采用含有水解乳蛋白的复溶液重悬颗粒。 [0010] As described above, a method for detecting the capsaicin component in oils and fats, in the preparation process of the immunomagnetic beads, a double solution containing hydrolyzed milk protein is used to resuspend the particles.

[0011] 如上所述的一种检测油脂中辣椒碱成分的方法，免疫磁珠捕获辣椒碱分子之后 [0011] As described above, a method for detecting the capsaicin component in oils and fats, after the immunomagnetic beads capture the capsaicin molecule

，用含有 S9表面活性剂（Tetronic 1307）的重悬液重悬免疫磁珠。 , Resuspend the immunomagnetic beads with a resuspended solution containing S9 surfactant (Tetronic 1307).

发明的有益效果 Beneficial effects of invention

有益效果 Beneficial effect

[0012] 油脂具有粘稠的特点，在免疫层析试纸条上的层析性能差，因此，若要检测油脂中的成分，要么将目标物质提取出来再进行检测（提取导致检测方法的繁琐），要么对油脂进行大幅度稀释再进行检测（稀释导致检测灵敏性的降低）。 [0012] Oils and fats are viscous and have poor chromatographic performance on immunochromatographic test strips. Therefore, to detect components in oils or fats, either the target substance is extracted and then detected (extraction leads to cumbersome detection methods ), either dilute the oil and fat before testing (dilution reduces the sensitivity of the test).

5见有免疫层析法，需要从大体积的油脂中萃取，然后要对将大体积的有机溶剂进行吹干操作，步骤繁琐、耗时（需要几个小时，甚至更久）、对仪器依赖高，不便于高通量检测。本发明提供的一种检测油脂中辣椒碱成分的方法，该方法首先用免疫磁珠对油脂中的辣椒碱分子进行富集，解决了繁杂的前处理问题（甲醇和纳米二氧化钛的联合应用，既解决了油脂的粘稠问题，还保证了免疫磁珠上抗体的活性）。其次，采用免疫层析法快速检测，降低了检测成本，缩短了检测时间。解决了现有油脂中辣椒碱检测费时费力、效能差的问题（整个检测流程下来，只需要 70min，甚至更短的时间；只需要处理小体积样品即可满足检测需求，为高通量检测提供了方便) 。 5 See there is immunochromatography, which requires extraction from a large volume of oil and fat, and then the operation of drying the large volume of organic solvent is cumbersome and time-consuming (it takes a few hours or even longer) and depends on the instrument High, not convenient for high-throughput detection. The present invention provides a method for detecting capsaicin in oils and fats. This method first enriches capsaicin molecules in oils and fats with immune magnetic beads, which solves the complicated pretreatment problem (the combined application of methanol and nano-titanium dioxide, both Solve the viscosity problem of oil and fat, and also ensure the activity of antibodies on immunomagnetic beads). Secondly, the rapid detection by immunochromatography reduces the detection cost and shortens the detection time. It solves the problem of time-consuming, laborious and poor performance in the detection of capsaicin in existing oils and fats (the entire detection process takes only 70 minutes or even shorter time; only a small volume of samples can be processed to full Meet the needs of testing, which provides convenience for high-throughput testing).

发明实施例 Invention Example

本发明的实施方式 Embodiments of the invention

[0013] 下面用实施例对本发明进行详细的说明，实施例仅是本发明的优选实施方式，不是对本发明的限定。 [0013] The following examples will be used to describe the present invention in detail, the examples are only preferred embodiments of the present invention, rather than limiting the present invention.

[0014] 实施例 1样品前处理方案 Example 1 Sample preparation scheme

[0015] 1. 免疫磁珠的制备： [0015] 1. Preparation of immunomagnetic beads:

[0016] (1) 取 lml磁珠 ( lOmg/ml, 货号 MS300/Carboxyl，日本 JSR公司) ，将磁珠置于磁场中，收集磁珠，弃掉上清； [0016] (1) Take 1ml magnetic beads (10mg/ml, article number MS300/Carboxyl, Japan JSR company), put the magnetic beads in the magnetic field, collect the magnetic beads, discard the supernatant;

[0017] (2) 用 lml PBS (磷酸盐缓冲溶液， pH7.4) 重悬磁珠，加入 5mg EDC(l-(3 -二甲氨基丙基 )-3 -乙基碳二亚胺盐酸盐)，室温震荡 15min; [0017] (2) Resuspend the magnetic beads with 1 ml PBS (phosphate buffer solution, pH 7.4), add 5 mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt), shake at room temperature for 15min;

[0018] (3) 将磁珠置于磁场中，收集磁珠，去除上清；用 lml PBS (pH7.4) 重悬颗粒；重复本步骤 1次； [0018] (3) Place the magnetic beads in a magnetic field, collect the magnetic beads, and remove the supernatant; resuspend the particles with 1 ml of PBS (pH7.4); repeat this step once;

[0019] (4) 加入抗辣椒碱抗体 20mg，室温混匀 2h; [0019] (4) Add anti-capsaicin antibody 20mg, mix at room temperature for 2h;

[0020] (5) 加入封闭剂，继续反应 2h; [0020] (5) Add a blocking agent and continue the reaction for 2h;

[0021] (6) 将磁珠置于磁场中，收集磁珠，去除上清；用 lml PBS (pH7.4) 重悬颗粒；重复本步骤 2次； [0021] (6) Place the magnetic beads in a magnetic field, collect the magnetic beads, and remove the supernatant; resuspend the particles with 1 ml of PBS (pH7.4); repeat this step 2 times;

[0022] (7) 用 10ml复溶液重悬颗粒，置于 4°C保存。 [0022] (7) Resuspend the particles with 10ml of double solution and store at 4°C.

[0023] 2. 取 2g油脂，加入 8ml [0023] 2. Take 2g of fat and add 8ml

样品稀释液，加入 40pL免疫磁珠，旋转混匀 lh (20°C-40°C，推荐 37°C) 。 Add 40pL immunomagnetic beads to the sample diluent and spin to mix for 1h (20°C-40°C, 37°C recommended).

[0024] 3. 将步骤 2的混匀溶液置于磁场中，收集免疫磁珠。 [0024] 3. Place the mixed solution of step 2 in a magnetic field and collect the immunomagnetic beads.

[0025] 4. 用 lml PBST (含吐温 -20的磷酸盐缓冲液， pH7.4, 吐温 20含量 0.1%(W/V，质量浓度 )) 洗涤磁珠 1次，并将颗粒悬浮于 20(VL重悬液中，备用。 4. Wash the magnetic beads once with 1 ml PBST (phosphate buffer containing Tween-20, pH7.4, Tween 20 content 0.1% (W/V, mass concentration)), and suspend the particles in 20 (in VL resuspension, set aside.

[0026] 5. 试纸条的制备： [0026] 5. Preparation of test strips:

[0027] ( 1) 按照按照 lpL/cm喷涂浓度为 1.5mg/mL的辣椒素 -BSA偶联物，作为检测区，按照 lpL/cm喷涂浓度为 0.2mg/ml的羊抗鼠 IgG作为质控区，置于室温晾干，备用。 [0027] (1) The capsaicin-BSA conjugate sprayed at a concentration of 1.5 mg/mL according to lpL/cm was used as the detection zone, and the goat anti-mouse IgG sprayed at a concentration of 0.2 mg/ml according to lpL/cm was used as a quality control Area, let dry at room temperature and set aside.

[0028] (2) 将样品垫、层析膜、吸水纸依次搭接粘贴到 PVC (聚氯乙烯) 材质的底板上，层析膜的检测区靠近样品垫、质控区靠近吸水纸检测线裁切成 4mm宽的免疫层析试纸条。 [0028] (2) The sample pad, the chromatographic membrane, and the absorbent paper are attached to the bottom of the PVC (polyvinyl chloride) material in this order On the board, the detection area of the chromatographic membrane is close to the sample pad, and the quality control area is close to the absorbent paper detection line and cut into 4mm wide immunochromatographic test strips.

[0029] 6. 将免疫层析试纸条样品垫端***步骤 4中悬浮的磁珠中，室温静置 lOmin。 [0029] 6. Insert the pad end of the immunochromatographic test strip sample into the magnetic beads suspended in step 4, and let stand at room temperature for 10 min.

[0030] 7. 判读结果： [0030] 7. Interpretation results:

[0031] A、可以用肉眼直接判读： ⑴ 检测区信号强于质控区信号，说明不含有辣椒碱，或辣椒碱的含量低于本方法的检出限。（2）检测区信号等于或弱于质控区信号，说明样品中含有辣椒碱成分。 [0031] A. It can be directly interpreted with the naked eye: ⑴ The signal in the detection zone is stronger than the signal in the quality control zone, indicating that it does not contain capsaicin, or the content of capsaicin is lower than the detection limit of this method. (2) The signal in the detection zone is equal to or weaker than the signal in the quality control zone, indicating that the sample contains capsaicin.

[0032] B、还可以用磁性信号检测仪对试纸条进行扫描检测。同样，（1）检测区信号强于质控区信号，说明不含有辣椒碱，或辣椒碱的含量低于本方法的检出限。 [0032] B. The magnetic signal detector can also be used to scan and test the test paper strip. Similarly, (1) The signal in the detection zone is stronger than the signal in the quality control zone, indicating that it does not contain capsaicin, or the capsaicin content is lower than the detection limit of this method.

（2）检测区信号等于或弱于质控区信号，说明样品中含有辣椒碱成分。 (2) The signal in the detection zone is equal to or weaker than the signal in the quality control zone, indicating that the sample contains capsaicin.

[0033] 实施例 2 Example 2

[0034] 免疫磁珠封闭剂对于检测结果的影响：以实施例 1为基础，采用不同的封闭剂（见表 1）封闭磁珠。不同的封闭剂对检测灵敏性有明显的影响， 5 -氨基戊酸检测下限最佳，深水鱼明胶则最差。用 Tris进行封闭的颗粒则在油水混合物中容易发生聚集，特别是在磁场中，聚集之后难以打散，导致接下来的检测操作无法进行。 [0034] Effect of immunomagnetic bead blocking agent on the detection result: Based on Example 1, different magnetic blocking agents (see Table 1) were used to block the magnetic beads. Different blocking agents have a significant effect on the sensitivity of detection. The lower limit of detection of 5-aminovaleric acid is the best, and that of deep-water fish gelatin is the worst. The particles sealed with Tris are easy to aggregate in the oil-water mixture, especially in the magnetic field, which is difficult to disintegrate after aggregation, resulting in the inability to carry out the following detection operations.

[0035] 表 1封闭剂对于结果的影响 Table 1 Effect of blocking agent on results

[] []

[表 1] [Table 1]

[0036] 实施例 3 Example 3

[0037] 免疫磁珠复溶液对于检测结果的影响：以实施例 1为基础，比较了不同复溶液（表 2）对于检测结果的影响，从表中可以看出，添加了水解乳蛋白的复溶液，对于检测灵敏性的提升有明显改善。 [0037] The effect of the immune magnetic bead complex solution on the detection result: Based on Example 1, the effect of different complex solutions (Table 2) on the detection result was compared, and it can be seen from the table that the complex with hydrolyzed milk protein added The solution has significantly improved the detection sensitivity.

[0038] 表 2复溶液对于检测结果的影响 [0038] Table 2 The effect of complex solution on the test results

[] 幽 [] You

[0039] 实施例 4 [0040] 样品处理方法对于检测结果的影响：以实施例 1为基础，比较了不同样品稀释液（表 3 -表 6）对于检测结果的影响，从表中可以看出，甲醇和纳米二氧化钛都能够有效改善检测的灵敏性，而两者联合应用效果更佳。甲醇、纳米二氧化钛的浓度，纳米二氧化钛的粒径这些因素都会影响检测下限。 Example 4 [0040] The effect of the sample processing method on the test results: Based on Example 1, the impact of different sample dilutions (Table 3-Table 6) on the test results was compared, as can be seen from the table, both methanol and nano titanium dioxide It can effectively improve the sensitivity of detection, and the combined application of the two is better. Factors such as the concentration of methanol, nano-titania, and the particle size of nano-titania all affect the lower detection limit.

[0041] 表 3样品稀释液对于检测结果的影响 [0041] Table 3 The effect of sample dilutions on the detection results

[] [表 3] [] [table 3]

[0042] 表 4样品稀释液中甲醇含量对于检测结果的影响 Table 4 The effect of methanol content in the sample dilution on the detection result

[] []

[表 4] [Table 4]

[0043] 表 5样品稀释液中纳米二氧化钛颗粒浓度对于检测结果的影响 [0043] Table 5 The effect of the concentration of nano-titania particles in the sample dilution on the detection results

[] []

[表 5] [table 5]

[0044] 表 6样品稀释液中纳米二氧化钛颗粒粒径对于检测结果的影响 [0044] Table 6 The influence of the particle size of the nano-titanium dioxide particles on the detection results in the sample dilution

[] []

[表 6] [Table 6]

[0045] 实施例 5 Example 5

[0046] 重悬液对于检测结果的影响：以实施例 1为基础，比较了不同重悬液 (表 7) 对于检测结果的影响，从表中可以看出，不同重悬液对于检测的灵敏性影响并不大，但对于阴性样本的检测信号有着明显的影响，添加了 0.5% S9的重悬液对于检测信号的提升有明显改善。 [0046] The impact of the resuspended solution on the detection results: Based on Example 1, the effect of different resuspended solutions (Table 7) on the test results was compared, as can be seen from the table, the sensitivity of different resuspended solutions on the detection The effect is not large, but it has a significant impact on the detection signal of negative samples. The resuspended solution with 0.5% S9 has a significant improvement in the detection signal.

[0047] 表 7重悬液对于检测结果的影响 Table 7 The effect of resuspension on the test results

[] [表 7] [] [Table 7]

[0048] 实施例 6 Example 6

[0049] 1. 免疫磁珠的制备： 1. Preparation of immunomagnetic beads:

[0050] (1) 取 lml磁珠 ( lOmg/ml, 货号 MS300/Carboxyl，日本 JSR公司) ，将磁珠置于磁场中，收集磁珠，弃掉上清； [0050] (1) Take 1ml magnetic beads (10mg/ml, article number MS300/Carboxyl, Japan JSR company), put the magnetic beads in the magnetic field, collect the magnetic beads, discard the supernatant;

[0051] (2) 用 lml PBS (磷酸盐缓冲溶液， pH7.4) 重悬磁珠，加入 5mg EDC(l-(3 -二甲氨基丙基 )-3 -乙基碳二亚胺盐酸盐)，室温震荡 15min; [0051] (2) Resuspend the magnetic beads with 1 ml of PBS (phosphate buffer solution, pH 7.4), add 5 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt), shake at room temperature for 15min;

[0052] (3) 将磁珠置于磁场中，收集磁珠，去除上清；用 lml PBS (pH7.4) 重悬颗粒；重复本步骤 1次； [0052] (3) Place the magnetic beads in a magnetic field, collect the magnetic beads, and remove the supernatant; resuspend the particles with 1 ml of PBS (pH7.4); repeat this step once;

[0053] (4) 加入抗辣椒碱抗体 20mg，室温混匀 2h; [0053] (4) Add anti-capsaicin antibody 20mg, mix at room temperature for 2h;

[0054] (5) 加入封闭剂 (5 -氨基戊酸，工作浓度 0.5% (W/V) ) , 继续反应 2h; [0054] (5) Add blocking agent (5-aminovaleric acid, working concentration 0.5% (W/V)), continue the reaction for 2h;

[0055] (6) 将磁珠置于磁场中，收集磁珠，去除上清；用 lml PBS (pH7.4) 重悬颗粒；重复本步骤 2次； [0055] (6) Place the magnetic beads in a magnetic field, collect the magnetic beads, and remove the supernatant; resuspend the particles with 1 ml of PBS (pH7.4); repeat this step 2 times;

[0056] (7) 用 10ml复溶液 (PBS， 1%水解乳蛋白， pH7.4) 重悬颗粒，置于 4°C保存 [0056] (7) Resuspend the pellet with 10ml of double solution (PBS, 1% hydrolyzed milk protein, pH7.4) and store at 4°C

[0057] 2. 取 2g油脂，加入 8ml样品稀释液 ( I0mg/ml 纳米二氧化钛悬液（悬浮在含有 20%（V/V）甲醇的 PBS中， pH7.4）加入 40^L免疫磁珠，旋转混匀 lh （推荐 3⁷°C）。 [0057] 2. Take 2g of fat and oil, add 8ml of sample diluent (10mg/ml Nano TiO2 suspension (suspended in PBS containing 20% (V/V) methanol, pH7.4), add 40^L immunomagnetic beads, and rotate to mix for 1 h (3 ⁷ °C recommended).

[0058] 3. 将步骤 2的混匀溶液置于磁场中，收集免疫磁珠。 [0058] 3. Place the mixed solution of step 2 in a magnetic field and collect the immunomagnetic beads.

[0059] 4. 用 lml PBST （含吐温 -20的磷酸盐缓冲液， pH7.4, 吐温 20含量 0.1%（W/V，质量浓度））洗涤磁珠 1次，并将颗粒悬浮于 20（VL重悬液（PBS， 0.5% 4. Wash the magnetic beads once with 1 ml of PBST (phosphate buffer containing Tween-20, pH7.4, Tween 20 content 0.1% (W/V, mass concentration)), and suspend the particles in 20 (VL resuspended solution (PBS, 0.5%

（W/V） S9 , pH7.4）中，备用。 (W/V) S9, pH7.4), spare.

[0060] 5. 试纸条的制备： [0060] 5. Preparation of test strips:

[0061] （1）按照按照 lpL/cm喷涂浓度为 1.5mg/mL的辣椒素 -BSA偶联物，作为检测区，按照 lpL/cm喷涂浓度为 0.2mg/ml的羊抗鼠 IgG作为质控区，置于室温晾干，备用。 [0061] (1) The capsaicin-BSA conjugate sprayed at a concentration of 1.5 mg/mL according to lpL/cm was used as the detection zone, and the goat anti-mouse IgG sprayed at a concentration of 0.2 mg/ml according to lpL/cm was used as the quality control Area, let dry at room temperature and set aside.

[0062] （2）将样品垫、层析膜、吸水纸依次搭接粘贴到 PVC （聚氯乙烯）材质的底板上，层析膜的检测区靠近样品垫、质控区靠近吸水纸检测线裁切成 4mm宽的免疫层析试纸条。 [0062] (2) Paste the sample pad, chromatography membrane, and absorbent paper on the bottom plate of PVC (polyvinyl chloride) material in sequence, the detection area of the chromatography membrane is close to the sample pad, and the quality control area is close to the absorbent paper detection line Cut into 4mm wide immunochromatographic test strips.

[0063] 6. 将免疫层析试纸条***步骤 4中悬浮的磁珠中，室温静置 lOmin。 [0063] 6. Insert the immunochromatographic test strip into the magnetic beads suspended in step 4, and let stand at room temperature for 10 min.

[0064] 7. 判读结果： [0064] 7. Interpretation result:

[0065] A、可以用肉眼直接判读： ⑴ 检测区信号强于质控区信号，说明不含有辣椒碱，或辣椒碱的含量低于本方法的检出限。（2）检测区信号等于或弱于质控区信号，说明样品中含有辣椒碱成分。 [0065] A. It can be directly interpreted with the naked eye: ⑴ The signal in the detection zone is stronger than the signal in the quality control zone, indicating that it does not contain capsaicin, or the content of capsaicin is lower than the detection limit of this method. (2) The signal in the detection zone is equal to or weaker than the signal in the quality control zone, indicating that the sample contains capsaicin.

[0066] B、还可以用磁性信号检测仪对试纸条进行扫描检测。同样，（1）检测区信号强于质控区信号，说明不含有辣椒碱，或辣椒碱的含量低于本方法的检出限。 [0066] B. It is also possible to scan and test the test paper strip with a magnetic signal detector. Similarly, (1) The signal in the detection zone is stronger than the signal in the quality control zone, indicating that it does not contain capsaicin, or the capsaicin content is lower than the detection limit of this method.

[0067] 对比例 1 Comparative Example 1

[0068] 5见有技术方案（参照“食用植物油外源污染物辣椒素及黄曲霉毒素免疫检测技术研究 .杨青青 .2016”） [0068] 5 See there are technical solutions (refer to "Research on Immunodetection Technology of Capsaicin and Aflatoxin Exogenous Pollutants in Edible Vegetable Oil. Yang Qingqing. 2016")

[0069] 称取调和食用植物油 100 g 加入 800 mL甲醇后，震荡混匀 1分钟，再在 50°C条件下超声提取 10分钟，冷却至室温后， 4°C、 4000 rpm离心 5分钟，取 400 m L 上层甲醇相，氮气吹干，加 10 mL 10%甲醇 -PBS复溶后得到 5倍浓缩提取液。用胶体金免疫层析方法对提取液进行检测。 [0070] 对照方法（参照国家标准（GB/T 30388-2013辣椒及其油树脂总辣椒碱含量的测定高效液相色谱法）进行）。 [0069] Weigh 100 g of edible vegetable oil, add 800 mL of methanol, mix by shaking for 1 minute, then extract ultrasonically at 50°C for 10 minutes, cool to room temperature, centrifuge at 4°C, 4000 rpm for 5 minutes, take 400 mL of the upper methanol phase was blown dry with nitrogen, and reconstituted with 10 mL of 10% methanol-PBS to obtain a 5-fold concentrated extract. The extract was detected by colloidal gold immunochromatography. [0070] Control method (refer to national standard (GB/T 30388-2013 pepper and its oleoresin determination of total capsaicin content by high performance liquid chromatography)).

[0071] 分别用实施例 6、对比例 1和对照方法三个方案检测 23分油脂样品，检测结果如表 8所示，本发明技术方案实施例 6与对照方案 C检测结果完全一致，而现有技术方案对比例 1则出现 3例假阳性， 2例漏检（假阴性）。 [0071] Each of the three schemes of Example 6, Comparative Example 1 and the control method were used to test 23 points of oil and fat samples. The test results are shown in Table 8. The test results of Example 6 of the technical scheme of the present invention are completely consistent with the control scheme C. There are technical solutions for Comparative Example 1 with 3 false positives and 2 missed tests (false negatives).

[0072] 表 8检测油脂样品 [0072] Table 8 Detection of oil samples

[] [表 8] [] [Table 8]

[0073] 以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制，但凡采用等同替换或等效变换的形式所获得的技术方案，均应落在本发明的保护范围之内。 [0073] The above-mentioned examples only express the implementation of the present invention, and the description is more specific and detailed, but it should not be construed as a limitation of the patent scope of the present invention, which is obtained in the form of equivalent replacement or equivalent transformation All technical solutions should fall within the protection scope of the present invention.

Claims

权利要求书 Claims

[权利要求 1] 一种检测油脂中辣椒碱成分的方法，其特征在于，该方法包括以下步骤：预先采用免疫亲和法对油脂中的辣椒碱分子进行分离提取，然后采用免疫层析法对分离提取到的辣椒碱分子进行检测，所述采用免疫亲和法对油脂中的辣椒碱分子进行分离提取是指采用含有甲醇、纳米二氧化钛的溶液稀释油脂样品，然后添加与辣椒碱分子特异性结合的免疫磁珠捕获其中的辣椒碱分子。 [Claim 1] A method for detecting the capsaicin component in oils and fats, characterized in that the method comprises the following steps: the capsaicin molecules in the oils are separated and extracted by immunoaffinity in advance, and then the immunochromatographic method is used for Separation and extraction of capsaicin molecules for detection. The separation and extraction of capsaicin molecules in oils and fats by immunoaffinity means that the oil and fat sample is diluted with a solution containing methanol and nano titanium dioxide, and then added to specifically bind to capsaicin molecules. The immunomagnetic beads capture the capsaicin molecule.

[权利要求 2] 如权利要求 1所述的一种检测油脂中辣椒碱成分的方法，其特征在于，所述二氧化钦的粒径为 2nm-25nm [Claim 2] The method for detecting capsaicin in oils and fats as claimed in claim 1, characterized in that the particle size of the dioxin is 2nm-25nm

[权利要求 3] 如权利要求 1所述的一种检测油脂中辣椒碱成分的方法，其特征在于，免疫层析法是指采用免疫层析试纸条进行检测，所述免疫层析试纸条包括依次搭接粘贴到 PVC材质的底板上的样品垫、层析膜、吸水纸，层析膜上有检测区和质控区，检测区包被有辣椒素-载体蛋白偶联物。 [Claim 3] The method for detecting the capsaicin component in oils and fats according to claim 1, characterized in that the immunochromatography method uses immunochromatographic test strips for detection, and the immunochromatographic test strips The strip includes a sample pad, a chromatographic membrane, and a water-absorbent paper that are sequentially attached to the bottom plate made of PVC. The chromatographic membrane has a detection area and a quality control area. The detection area is coated with a capsaicin-carrier protein conjugate.

[权利要求 4] 如权利要求 1所述的一种检测油脂中辣椒碱成分的方法，其特征在于，所述免疫磁珠在制备时采用 5 -氨基戊酸进行封闭。 [Claim 4] A method for detecting capsaicin in oils and fats as claimed in claim 1, characterized in that the immunomagnetic beads are blocked with 5-aminovaleric acid during preparation.

[权利要求 5] 如权利要求 1所述的一种检测油脂中辣椒碱成分的方法，其特征在于，所述免疫磁珠在制备过程中，采用含有水解乳蛋白的复溶液重悬颗粒。 [Claim 5] A method for detecting capsaicin in oils and fats according to claim 1, characterized in that, in the preparation process of the immunomagnetic beads, the particles are resuspended using a double solution containing hydrolyzed milk protein.

[权利要求 6] 如权利要求 1所述的一种检测油脂中辣椒碱成分的方法，其特征在于，免疫磁珠捕获辣椒碱分子之后，用含有 S9表面活性剂的重悬液重悬免疫磁珠。 [Claim 6] The method for detecting the capsaicin component in fats and oils according to claim 1, characterized in that after the capsaicin molecules are captured by the immunomagnetic beads, the immunomagnetic is resuspended with a resuspending solution containing S9 surfactant Beads.