WO2023097933A1 - 一种利用荧光探针定量检测酒类样品中乙醛的方法 - Google Patents

一种利用荧光探针定量检测酒类样品中乙醛的方法 Download PDF

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WO2023097933A1
WO2023097933A1 PCT/CN2022/081626 CN2022081626W WO2023097933A1 WO 2023097933 A1 WO2023097933 A1 WO 2023097933A1 CN 2022081626 W CN2022081626 W CN 2022081626W WO 2023097933 A1 WO2023097933 A1 WO 2023097933A1
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acetaldehyde
wine
fluorescent probe
samples
concentration
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French (fr)
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刘春凤
刘宜嵩
陈珊珊
李崎
王金晶
郑飞云
钮成拓
许鑫
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江南大学
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Priority to US18/155,227 priority Critical patent/US20230194495A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/14—Beverages
    • G01N33/146—Beverages containing alcohol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N21/88—Investigating the presence of flaws or contamination
    • G01N21/94—Investigating contamination, e.g. dust
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to a method for detecting acetaldehyde in wine samples by using a fluorescent probe, belonging to the field of wine quality control.
  • Aldehydes are the main carbonyl compounds in wine, and acetaldehyde is the volatile aldehyde with the highest content in wine. And the content in wine accounts for about 60%-90% of the total aldehydes.
  • Acetaldehyde is recognized as a Class 2B carcinogen by the International Agency for Research on Cancer (IARC), that is, it may be carcinogenic to humans.
  • IARC International Agency for Research on Cancer
  • acetaldehyde was listed as a class I carcinogen, that is, there is sufficient evidence of carcinogenicity to humans.
  • the content of acetaldehyde directly affects the flavor and aging of wine. When the concentration of acetaldehyde is low, there will be a fruity aroma. When the concentration is high, it will produce a pungent pungent smell and bring a bad grassy taste to the wine, shortening the shelf life of the wine flavor. , Even drinking can cause adverse reactions in the human body.
  • GC and HPLC gas chromatography
  • GC and HPLC methods are usually combined with solid-phase micro Extraction or derivatization treatment technology.
  • GC and HPLC methods involve the use of expensive instruments, complex operating procedures, and long detection time.
  • equipment debugging and maintenance are usually required, which leads to high detection costs. Therefore, an efficient method is established. It is imperative to analyze and detect acetaldehyde in wine samples at low cost, which will help to effectively control the content of acetaldehyde in wine, thereby improving the flavor of wine and improving the competitiveness of my country's wine industry.
  • the technical problem to be solved by the present invention is to provide a method for detecting acetaldehyde in wine samples using a fluorescent probe. Aldehyde concentration, so as to realize the quantification of acetaldehyde concentration, making the evaluation of acetaldehyde content in wine products more efficient, scientific and effective
  • the fluorescent probe involved in the present invention has the following structural formula:
  • a kind of method that utilizes fluorescent probe to detect the content of acetaldehyde in wine sample comprises the following steps:
  • the fluorescent probe of structure shown in formula (I) is dispersed in organic solvent, obtains fluorescent probe solution; Then fluorescent probe solution, hydrochloric acid solution and a series of acetaldehyde standard solutions of known concentration are mixed respectively, The reaction is carried out at 0-10°C, and after the reaction is completed, a mixed system is obtained;
  • step (1) the fluorescent probe solution, hydrochloric acid solution and wine sample are mixed and reacted, then the fluorescence intensity of the mixed system is measured, and the wine is calculated by the quantitative detection model in step (2).
  • the concentration of acetaldehyde in the sample is calculated.
  • the volume ratio of the fluorescent probe solution, hydrochloric acid solution, and acetaldehyde standard solution is 2:1:1.
  • the organic solvent in step (1) is acetonitrile and DMSO, and the volume ratio of acetonitrile to DMSO is 10:1.
  • the concentration of the fluorescent probe solution is 600 mg/L.
  • the hydrochloric acid solution is prepared by using acetonitrile as a solvent to prepare the hydrochloric acid solution.
  • the pH of the hydrochloric acid solution is 2; the mass fraction is 2.96%.
  • a series of acetaldehyde standard solutions with known concentrations range in concentration from 0 to 200 mg/L. Specifically, it can be 0mg/L, 10mg/L, 20mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L.
  • the reaction time is 40-60 minutes; specifically, 50 minutes is optional.
  • the temperature of the reaction may specifically be 5°C.
  • the fluorescence intensity is the fluorescence intensity at an emission wavelength of 553 nm.
  • 485nm is the excitation wavelength
  • 553nm is the emission wavelength
  • the wine sample also needs to be pretreated as follows:
  • the distillation process is completed within 1 min, and the distillate is received under an ice bath using a 10 mL stoppered colorimetric tube.
  • the wine samples include liquor, rice wine and wine samples.
  • the specific process of the detection method is as follows:
  • the fluorescent probe can be purchased or obtained by self-made.
  • the self-made synthesis method is as follows: weigh 500mg NBD-Cl in a flask, and wrap the outer wall with tin foil to avoid light. Add 50ml of chloroform to fully dissolve NBD-Cl. Add 50ml 5% hydrazine hydrate (3.1ml 80% hydrazine hydrate + 46.9ml methanol), purging with nitrogen, seal and let it stand for 1h, a yellow-brown precipitate precipitates and suction filter, wash the filter cake with dichloromethane to obtain a solid probe product; the corresponding synthetic route is:
  • the principle of the detection of acetaldehyde using the fluorescent probe in the present invention is: the strong electron-donating group hydrazine provides electrons to the electron-withdrawing group nitro, and the fluorescent group is affected by the photoelectron-induced transfer effect, and the radiation transition of the electron is blocked, and the fluorescence suppressed.
  • the fluorescent probe interacts with acetaldehyde, the hydrazine group no longer provides electrons to the nitro group, cutting off the photoelectron-induced transfer effect, and the fluorescence is restored, which belongs to the enhanced fluorescent probe.
  • the invention uses fluorescent probes to detect acetaldehyde in wine samples.
  • Benzoxadiazoles belong to a class of common fluorescent groups, and changing the substituents at the 4-position and 7-position will produce different emission characteristics.
  • the 4-position substituent is designed as a nitro group as a strong electron-withdrawing group
  • the 7-position substituent is designed as a hydrazine group as a reactive group to form a photoinduced electron transfer effect.
  • the reactive group is combined with acetaldehyde
  • the acetaldehyde competes for the electrons transferred from the hydrazine group to the nitro group, and the photoinduced electron transfer process is destroyed, resulting in enhanced fluorescence emission.
  • the detection and analysis of acetaldehyde content in wine samples can be realized.
  • the method provided can solve the problem of background interference, and the removal of background interference by distillation is the first time in the application of fluorescent probes to detect actual samples.
  • the method provided is highly effective: the detection limit of acetaldehyde (LOD) is 3.6 ⁇ 10 -8 mol/L; the fluorescent probe has a good linear relationship with the concentration of acetaldehyde in the range of 0-200mg/L, and the linear range is wide;
  • the results of the degree experiment show that the RSD in the simulated solution system is 5.30%, and at the same time, the RSD in the real wine sample system is 3.72%; in the simulated solution system, the recovery rate is 93.87-99.75%. In the real wine sample system, the recovery rate was 94.02-108.12%.
  • the evaluation method provided by the invention has been used in the detection and analysis of finished wine samples and beer fermentation process samples.
  • Figure 1 is the fluorescence selectivity diagram of the fluorescent probe, the excitation wavelength is 485nm, and the emission wavelength is 553nm.
  • Fig. 2 is the anti-interference diagram of the fluorescent probe, the excitation wavelength is 485nm, and the emission wavelength is 553nm.
  • Fig. 3 is a standard curve and a linear range diagram of the fluorescent probe recognizing acetaldehyde, the excitation wavelength is 485nm, and the emission wavelength is 553nm.
  • Figure 4 is the relationship between the concentration factor and the recovery rate in the distillation process, the excitation wavelength is 485nm, and the emission wavelength is 553nm.
  • Fig. 5 is a graph showing the applicable range of detection conditions for the recognition of acetaldehyde by the fluorescent probe, the excitation wavelength is 485nm, and the emission wavelength is 553nm.
  • Figure 6 shows the reproducibility of the method for identifying acetaldehyde with fluorescent probes, the excitation wavelength is 485nm, and the emission wavelength is 553nm.
  • Fig. 7 is a graph showing the tracking and detection of acetaldehyde by fluorescent probes in the beer fermentation process, the excitation wavelength is 485nm, and the emission wavelength is 553nm.
  • acetaldehyde The content of acetaldehyde in the sample is determined by fluorescent probe, the specific operation process and experimental conditions are as follows:
  • the specific operation is as follows, take 50mL wine sample (among them, if you choose beer and other gas-containing samples, you need to add a drop of defoamer; if you choose liquor, rice wine and wine samples, you need to dilute 25 times with distilled water), use diacetyl distillation
  • the device distills wine samples, stop receiving when the distillate is close to 10mL, and make up to 10mL with distilled water to obtain distillate A, which is ready for testing.
  • Detection conditions for 96-well ELISA plate 50 ⁇ L acid solution, 50 ⁇ L distillate A, 100 ⁇ L fluorescent probe solution, placed in an incubator at 5°C for 50 minutes at low temperature. The fluorescence intensity was measured on a fluorescence spectrometer, with 485nm as the excitation wavelength and 553nm as the emission wavelength.
  • the same beer sample was used in this part of the experiment to optimize the detection conditions. Optimization factors include temperature, reaction time, acid concentration, and probe concentration.
  • This experiment is designed to react at 0, 5, 15, 25, and 35°C, and then measure the response value of acetaldehyde in the sample, as shown in Figure 5.
  • the study found that as the system temperature gradually increased, the response value of acetaldehyde gradually decreased, and the response value of acetaldehyde at 0°C and 5°C did not change much. From the perspective of energy consumption, the equilibrium temperature is selected at 5°C to achieve the best detection effect and the highest sensitivity.
  • the probe solution is light yellow, and the background interference of the system itself will affect the final fluorescence intensity.
  • the recovery rate of the method is investigated in the simulated solution system and the real wine sample system respectively.
  • acetaldehyde simulated system add 50 ⁇ L/100 ⁇ L/200 ⁇ L of acetaldehyde stock solution ( 1g/L), prepare 3 parallel samples for each spiked amount; in the real wine system, add 50 ⁇ L/100 ⁇ L/200 ⁇ L acetaldehyde stock solution (1g/L) to 50mL beer, add 500 ⁇ L/ 1000 ⁇ L/2000 ⁇ L acetaldehyde stock solution (1g/L), add 250 ⁇ L/500 ⁇ L/1000 ⁇ L acetaldehyde stock solution (1g/L) to 50mL rice wine and wine, prepare 3 parallel samples for each spiked amount. Then, the above-mentioned fluorescent probe detection method was used to detect the content of acetaldehyde, and the results are shown in Table 1.
  • Samples 1-3 are acetaldehyde standard solutions
  • samples 4-6 are beer samples
  • samples 7-9 are liquor samples
  • samples 10-12 are rice wine samples
  • samples 13-15 are wine samples.
  • the probe has the highest response value to acetaldehyde, and has a higher response value to some microgram-level carbonyl compounds in beer, such as n-propionaldehyde, n-butyraldehyde, and isobutyraldehyde, but the response The value is lower than that of acetaldehyde, and the response value of milligram-level carbonyl compounds in beer is low, only 3.5%-7.6% of the response value of acetaldehyde.
  • the probe has almost no response to alcohol esters. The response values of the main flavor substances in wine except acetaldehyde were negligible.
  • Samples 1-17 are beer samples
  • samples 18-19 are liquor samples
  • samples 20-21 are rice wine samples
  • samples 22-23 are wine samples.
  • acetaldehyde can be detected in 23 kinds of wine samples, and the average content of acetaldehyde in beer, white wine, rice wine and wine samples are 17.80mg/L, 151.59mg/L, 65.09mg/L and 26.79mg/L, with an average RSD of 3.72%.
  • Sig. 0.756>0.05
  • Acetaldehyde is mainly produced by biological and chemical pathways in the wine brewing process. Real-time monitoring of acetaldehyde content is of great significance to the quality control of wine.
  • the detection of acetaldehyde content in wine products by fluorescent probe can ensure the accuracy of the measurement results. Degree, and avoid the use of expensive large-scale detection equipment, simple and fast, cost-saving.
  • the inoculum size is 1.5 ⁇ 10 7 CFU/mL, during the fermentation process, follow the steps (1), (2) and (3) of the above-mentioned Example 4
  • the sample detection method the acetaldehyde content in the fermented liquid is sampled and analyzed every day, and the results are as shown in Figure 6.
  • Example 7 Anti-interference performance of the detection method on carbonyl compounds and main flavor substances in wine samples
  • the interfering substances should include carbonyl compounds and alcohol ester compounds that are abundant in wine samples.
  • the concentration selection is based on the highest reported content, and the acetaldehyde content uses the average concentration of each wine.
  • the concentration of each analyte is: acetaldehyde 10mg/L; propionaldehyde 0.3mg/L; n-butyraldehyde 0.3mg/L; isobutyraldehyde 0.3mg/L; isovaleraldehyde 0.1mg /L; Heptanal 0.2mg/L; Octanal 0.2mg/L; Furfural 2mg/L; 5-Hydroxymethylfurfural 8mg/L; 2.3-Butanedione 1mg/L; 2.3-Pentanedione 0.5mg/L ; Acetoin 5mg/L; Methylglyoxal 0.1mg/L; N-propanol 25mg/L; Isoamyl alcohol: 100mg/L; Ethyl acetate 50mg/L; Add 50 ⁇ L of acid solution H and 100

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Abstract

本发明公开了一种利用荧光探针检测酒类样品中乙醛的方法，属于酒类质量控制领域。本发明检测方法是基于荧光探针测定酒类中的乙醛，在pH＝2.0的酸性条件下，利用光电子诱导转移原理实现荧光探针与乙醛的特异性结合。在0-200mg/L范围内与乙醛浓度呈良好的线性关系，检测限LOD为3.6×10 -8mol/L，样品的回收率在94.02-108.12％之间。检测方法具有廉价、线性范围广、灵敏度高、以及快速准确等优势。此荧光探针成功用于酒类样品和啤酒发酵过程样品的检测分析。

Description

[根据细则91更正 27.05.2022]　一种利用荧光探针定量检测酒类样品中乙醛的方法 技术领域
本发明涉及一种利用荧光探针检测酒类样品中乙醛的方法，属于酒类质量控制领域。
背景技术
酒类的风味物质对其口感和香气起着极其重要的作用，醛类是酒类中存在的主要羰基化合物，其中乙醛是酒类中含量最高的挥发性醛类，在啤酒、白酒、黄酒以及葡萄酒中的含量约占总醛的60％-90％。
乙醛被国际癌症研究署(International Agency for Research on Cancer，IARC)确认为2B类致癌物，即可能对人类致癌。2009年乙醛被列为I类致癌物，即对人类致癌证据充分。乙醛的含量直接影响着酒类的风味和老化，乙醛浓度低时有水果香，浓度高会产生辛辣的刺激性气味并给酒类带来恶劣的青草味，缩短酒类风味的保鲜期，甚至饮用后会引起人体的不良反应。
至今，有许多酒类样品中乙醛含量的检测方法，最为常见的是气相色谱法(GC)和高效液相色谱法(HPLC)，为了提高准确性，GC和HPLC法通常会结合固相微萃取或衍生化处理技术。但是GC和HPLC法涉及昂贵仪器的使用、复杂的操作程序以及较长的检测时间，同时为保证检测准确性通常需要对设备进行调试和维护，进而导致较高的检测成本，因此建立一种高效分析和低成本检测酒类样品中乙醛的方法势在必行，这有助于有效控制酒类中乙醛的含量，进而改善酒类的风味，提高我国酒类行业的竞争力。
发明内容
本发明要解决的技术问题是提供一种利用荧光探针检测酒类样品中乙醛的方法，荧光探针本身具有较弱的荧光，与乙醛作用后荧光发生增强，而且荧光强度正比于乙醛浓度，从而实现对乙醛浓度的定量，使得对酒类产品中乙醛含量的评价更加高效、科学、
全面。
本发明涉及的的荧光探针，其结构式如下：
Figure PCTCN2022081626-appb-000001
本发明提供的一种利用荧光探针检测酒类样品中乙醛含量的方法，包括如下步骤：
(1)将式(I)所示结构的荧光探针分散在有机溶剂中，获得荧光探针溶液；然后将荧光探针溶液、盐酸溶液与一系列已知浓度的乙醛标准溶液分别混合，0-10℃下进行反应，反应结束后，获得混合体系；
(2)利用荧光光谱仪上测定混合体系的荧光强度，并利用荧光强度及其相应乙醛标准溶液的浓度进行线性关联，获得定量检测模型；
(3)按照步骤(1)的过程，将荧光探针溶液、盐酸溶液与酒类样品混合反应，然后测定混合体系的荧光强度，并通过步骤(2)中的定量检测模型，计算得到酒类样品中乙醛的浓度。
在本发明的一种实施方式中，荧光探针溶液、盐酸溶液、乙醛标准溶液的体积比为2：1：1。
在本发明的一种实施方式中，步骤(1)中所述有机溶剂为乙腈、DMSO，乙腈与DMSO的体积比为10：1。
在本发明的一种实施方式中，荧光探针溶液的浓度为600mg/L。
在本发明的一种实施方式中，盐酸溶液是利用乙腈为溶剂配制盐酸溶液。
在本发明的一种实施方式中，盐酸溶液的pH为2；质量分数为2.96％。
在本发明的一种实施方式中，一系列已知浓度的乙醛标准溶液的浓度范围为0-200mg/L。具体可为0mg/L、10mg/L、20mg/L、50mg/L、100mg/L、150mg/L、200mg/L。
在本发明的一种实施方式中，反应的时间为40-60min；具体可选50min。
在本发明的一种实施方式中，反应的温度具体可选5℃。
在本发明的一种实施方式中，荧光强度为发射波长553nm处的荧光强度。
在本发明的一种实施方式中，定量检测模型为F 553nm＝346.14C+45.17，R 2＝0.9954；C的单位为mg/L。
在本发明的一种实施方式中，485nm为激发波长，553nm为发射波长。
在本发明的一种实施方式中，酒样样品还需经过如下预处理：
啤酒等含气样品需滴加一滴消泡剂，白酒、黄酒以及葡萄酒样品需要用蒸馏水稀释25倍； 取50mL酒样，使用双乙酰蒸馏装置蒸馏酒样，当馏出液接近10mL时停止接收，并用蒸馏水补足到10mL，得到馏出液A，即为酒样样品。
在本发明的一种实施方式中，蒸馏过程在1min内完成，馏出液在冰浴下使用10mL具塞比色管接收。
在本发明的一种实施方式中，酒样包括白酒、黄酒以及葡萄酒样品。
在本发明的一种实施方式中，所述检测方法具体过程如下：
(1)以乙腈为溶剂配制质量分数为2.96％的盐酸溶液；以乙腈为溶剂、DMSO为助溶剂(10：1，v/v)配制600mg/L的荧光探针溶液；
(2)用蒸馏水配制0mg/L、10mg/L、20mg/L、50mg/L、100mg/L、150mg/L、200mg/L浓度的乙醛溶液；在96孔酶标板中加入50μL盐酸溶液，50μL标准乙醛溶液，100μL荧光探针溶液，置于5℃培养箱中低温反应50min；在荧光光谱仪上测定荧光强度F 553nm，以乙醛浓度C为横坐标，荧光强度为纵坐标，得到标准工作曲线，线性回归方程为：
F 553nm＝346.14C+45.17，R 2＝0.9954，C的单位为mg/L；
(3)取酒样50mL(啤酒等含气样品需滴加一滴消泡剂，白酒、黄酒以及葡萄酒样品需要用蒸馏水稀释25倍)，使用双乙酰蒸馏装置蒸馏酒样，当馏出液接近10mL时停止接收，并用蒸馏水补足到10mL，得到馏出液A；
(4)在96孔酶标板中加入50μL盐酸溶液，50μL馏出液A，100μL荧光探针溶液，置于5℃培养箱中低温反应50min后在荧光光谱仪上检测，将荧光强度带入步骤(2)的线性回归方程中，得到浓度C，样品的浓度为：
C 样品＝C/N(N为样品的浓缩倍数，样品为啤酒时N＝5，样品为白酒、黄酒或葡萄酒时N＝0.2)。
在本发明的一种实施方式中，所述荧光探针可购买或者自制获得。自制的合成方法为：称取500mg NBD-Cl于烧瓶中，外壁包裹锡纸避光。加入50ml氯仿使NBD-Cl充分溶解。加入50ml 5％水合联氨(3.1ml 80％水合联氨+46.9ml甲醇)，氮气吹扫后封口静置1h，析出黄褐色沉淀并抽滤，用二氯甲烷清洗滤饼，得到固体探针产品；相应的合成路线为：
Figure PCTCN2022081626-appb-000002
本发明使用荧光探针的检测乙醛的原理是：强供电子基团肼基向吸电子基团硝基提供电子，荧光基团受光电子诱导转移效应影响，电子的辐射跃迁被阻断，荧光受到抑制。当荧光探针与乙醛作用后，肼基不再向硝基提供电子，切断了光电子诱导转移效应，荧光得以恢复，属于增强型荧光探针。本发明利用荧光探针对酒类样品中的乙醛进行检测。苯并噁二唑属于一类较为常见的荧光基团，改变4位和7位的取代基会产生不同的发射特点。本发明将4位取代基设计为硝基作为强吸电子基团，7位取代基设计为肼基作为反应基团，形成光诱导电子转移效应。反应基团与乙醛结合后，乙醛争夺了肼基向硝基转移的电子，光诱导电子转移过程被破坏，引起荧光发射增强。利用这一原理可以实现对酒类样品中乙醛含量的检测分析。
本发明的有益效果是：
1.提供的荧光探针合成路线简单，提供的方法廉价、简便且高效。
2.提供的方法可以解决背景干扰问题，通过蒸馏法去除背景干扰在荧光探针检测实际样品的应用中属于首次。
3.提供的方法有效性较高：乙醛检测限LOD为3.6×10 -8mol/L；荧光探针在0-200mg/L范围内与乙醛浓度呈现良好线性关系，线性范围广；精密度实验结果显示，在模拟溶液体系中的RSD为5.30％，同时，在真实酒样体系中的RSD为3.72％；在模拟溶液体系中，回收率为93.87-99.75％，相比之下，在真实酒样体系中，回收率为94.02-108.12％。本发明提供的评价方法已用于成品酒样品和啤酒发酵过程样品的检测分析。
附图说明
图1为荧光探针的荧光选择性图，激发波长485nm，发射波长553nm。
图2为荧光探针的抗干扰性图，激发波长485nm，发射波长553nm。
图3为荧光探针识别乙醛的标准曲线和线性范围图，激发波长485nm，发射波长553nm。
图4为蒸馏过程浓缩倍数与回收率的关系，激发波长485nm，发射波长553nm。
图5为荧光探针识别乙醛检测条件适用范围图，激发波长485nm，发射波长553nm。
图6为荧光探针识别乙醛的方法重现性，激发波长485nm，发射波长553nm。
图7为荧光探针对啤酒发酵过程乙醛跟踪检测图，激发波长485nm，发射波长553nm。
具体实施方式
实施例1定量检测模型的构建
(1)以乙腈为溶剂配制质量分数为2.96％的盐酸溶液(pH＝2)；以乙腈为溶剂、DMSO 为助溶剂(10：1，v/v)配制600mg/L的荧光探针溶液；
(2)用蒸馏水配制一系列浓度(0mg/L、10mg/L、20mg/L、50mg/L、100mg/L、150mg/L、200mg/L)的乙醛标准溶液；在96孔酶标板中加入50μL盐酸溶液(盐酸浓度为2.96wt％)，50μL乙醛标准溶液，100μL荧光探针溶液，置于5℃培养箱中低温反应50min，获得混合体系；
(3)在荧光光谱仪上测定混合体系在553nm处的荧光强度F 553nm，以乙醛浓度C为横坐标，荧光强度为纵坐标，得到标准工作曲线(如图3所示)，线性回归方程为：F 553nm＝346.14C+45.17，R 2＝0.9954，C的单位为mg/L。检测乙醛的线性范围在0-200mg/L，线性范围广，检测限LOD为3.6×10 -8mol/L。
实施例2模拟酒样样品中乙醛的检测
利用荧光探针对样品中的乙醛含量进行测定，具体操作过程及实验条件如下：
1、样品处理：
向50ml蒸馏水中加入500μL乙醛溶液(1000mg/L)，配制为10mg/L的标准溶液，使用双乙酰蒸馏装置对标准样品进行蒸馏，浓缩倍数与回收率结果如图4，结果发现在浓缩倍数为不大于5倍时回收率效果较好。考虑到蒸馏效率，应选择浓缩倍数为5倍。
具体操作如下，取酒样50mL(其中，若酒样选择啤酒等含气样品，需滴加一滴消泡剂；若选择白酒、黄酒以及葡萄酒样品，需要用蒸馏水稀释25倍)，使用双乙酰蒸馏装置蒸馏酒样，当馏出液接近10mL时停止接收，并用蒸馏水补足到10mL，得到馏出液A，待测备用。
2、96孔酶标板检测条件：50μL酸溶液，50μL馏出液A，100μL荧光探针溶液，置于5℃培养箱中低温反应50min。在荧光光谱仪上测定荧光强度，485nm为激发波长，553nm为发射波长。
3、乙醛含量的计算：
将荧光强度带入线性回归方程F 553nm＝346.14C+45.17中得到浓度C，C的单位为mg/L，样品的浓度为：
C 样品＝C/N(N为蒸馏的浓缩倍数，样品为啤酒时N＝5，样品为白酒、黄酒或葡萄酒时N＝0.2)。
4、检测条件的优化：
为避免不同样品之间的差别带来的干扰，本部分实验采取同一个啤酒样品进行检测条件的优化。优化因素包括温度、反应时间、酸浓度和探针浓度。
(1)温度
本实验设计在0，5，15，25，35℃下反应，然后测定样品中乙醛响应值，如图5所示。研究发现随着体系温度逐渐的升高，乙醛的响应值逐渐降低，0℃和5℃温度下的乙醛响应值变化不大。从能耗角度考虑，将平衡温度选择在5℃，检测效果最好，灵敏度最高。
(2)反应时间
考察不同平衡时间0，5，10，20，25，40，50，60，70，80，90，100min时，样品中乙醛的响应值变化，如图5所示。结果发现：平衡时间在0-50min内，乙醛的响应值均随着时间的增加而增大；当平衡时间大于50min时，乙醛的响应值变化不大，探针与乙醛反应完全，因此选择反应时间为50min为佳。
(3)pH
该探针具有一定的pH敏感性能，探针对乙醛(10mg/L)的响应在不同pH下进行评估，如图5所示。结果发现，在pH＝2.0时，乙醛的响应值达到最大。因此本研究选择在pH＝2.0的条件下进行检测，盐酸溶液的质量分数则为2.96％。
(4)探针浓度
探针溶液呈淡黄色，体系本身的背景干扰会影响到最终的荧光强度，本实验研究了探针浓度在100、200、300、400、500、600、700、800mg/L情况下，和乙醛(100mg/L)在pH＝2.0的乙腈溶液中的响应值变化，如图5所示。结果发现在探针浓度为300mg/L时乙醛的响应值最大，600mg/L浓度时响应值次之。进一步探究，发现：300mg/L的探针浓度仅在0-80mg/L内呈现良好的线性关系，F 553nm＝305.03C+80.15，R 2＝0.9923；而600mg/L的探针浓度可以在0-200mg/L内呈现良好的线性关系，F 553nm＝346.14C+45.17，R 2＝0.9954。从线性范围角度考虑探针浓度为600mg/L更符合实际要求，尤其是针对白酒等乙醛含量较高的酒类样品。
实施例3检测方法的加标回收验证
分别在模拟溶液体系和真实酒类样品体系中考察方法的回收率，在乙醛模拟体系中：分别向50mL乙醛标准溶液样品(10mg/L)中加入50μL/100μL/200μL乙醛储备溶液(1g/L)，每个加标量制备3个平行样品；在真实酒类体系中，分别向50mL啤酒中加入50μL/100μL/200μL乙醛储备溶液(1g/L)，向50mL白酒中加入500μL/1000μL/2000μL乙醛储备溶液(1g/L)，向50mL黄酒和葡萄酒中加入250μL/500μL/1000μL乙醛储备溶液(1g/L)，每个加标量制备3个平行样品。然后采用上述的荧光探针检测方法进行乙醛含量的检测，结果如表1所示。
表1加标回收率实验结果(n＝3)
Figure PCTCN2022081626-appb-000003
Figure PCTCN2022081626-appb-000004
注：样品1-3为乙醛标准溶液、样品4-6为啤酒样品、样品7-9为白酒样品、样品10-12为黄酒样品、样品13-15为葡萄酒样品。
由表1可以看出，乙醛在模拟溶液体系和真实酒样体系中的回收率范围分别为93.87-99.75％和94.02-108.12％。该方法的回收率较高，方法的有效性较好。
实施例4检测方法对不同干扰分析物的选择性
向96孔酶标板中加入50μL盐酸溶液H，100μL荧光探针溶液，并加入50μL以下相同质量浓度(15mg/L)的分析物：乙醛、5-羟甲基糠醛、糠醛、乙偶姻、2.3-戊二酮、2.3-丁二酮、丙酮、羟基丙酮、丙酮醛、正丙醛、正丁醛、异丁醛、异戊醛、己醛、壬醛、苯乙醛、乙二醛、丙醇、正丁醇、异丁醇、异戊醇、β-苯乙醇、乙酸、乳酸、乙酸乙酯、乙酸异戊酯、己酸乙酯、乳酸乙酯，置于5℃培养箱中低温反应50min在荧光光谱仪上测定荧光强度。由图1可以发现，在相同质量浓度下，探针对乙醛的响应值最高，对啤酒中部分微克级别羰基化合物响应值较高，如正丙醛、正丁醛、异丁醛，但响应值不及乙醛，而对啤酒中毫克级别羰基化合物响应值较低，仅为乙醛响应值的3.5％-7.6％，同时可以发现探针对醇酯类物质几乎没有响应，对白酒、黄酒以及葡萄酒中除乙醛外的主要风味物质响应值微乎其微。
实施例5真实酒类样品中乙醛的检测
收集23种酒类样品，按照上述实施例4步骤(1)、(2)、(3)记载的样品检测方法，检测其中的乙醛含量结果如表2所示，各类样品的平均值和标准偏差结果如表3所示。
表2不同酒类样品中乙醛含量(n＝3)
Figure PCTCN2022081626-appb-000005
Figure PCTCN2022081626-appb-000006
注：样品1-17为啤酒样品、样品18-19为白酒样品、样品20-21为黄酒样品、样品22-23为葡萄酒样品。
表3不同酒类样品乙醛含量平均值和标准偏差
Figure PCTCN2022081626-appb-000007
由表2可以看出，23种酒样中均可检测出乙醛，啤酒、白酒、黄酒、葡萄酒样品中乙醛的平均含量分别为17.80mg/L、151.59mg/L、65.09mg/L以及26.79mg/L，平均RSD为3.72％。经SPSS显著性差异分析，Sig.＝0.756>0.05，同时Sig.(双尾)＝0.666>0.05，说明两种方法的检测结果无显著性差异。乙醛在酒类酿造过程中主要由生物途径和化学途径产生，实时监测乙醛含量对酒类质量管控具有重要意义，荧光探针法检测酒类产品中乙醛含量既可以保证测定结果的准确度，又避免了昂贵的大型检测仪器的使用，简便快捷，节约成本。
选取其中三份样品进行检测方法重现性的测试，将三份样品冷冻保存，在6天内进行6次平行检测，结果如图6所示，结果表明该检测方法的重现性较好。
实施例6啤酒发酵过程中乙醛的检测
将三种不同啤酒酵母接种到12°P麦汁中进行发酵实验，接种量1.5×10 7CFU/mL，发酵过程中，按照上述实施例4步骤(1)、(2)、(3)记载的样品检测方法，每天取样分析发酵液中乙醛含量，结果如图6所示。
由图7可以看出，发酵过程乙醛先增加至最大值，而后缓慢下降。发酵4天时，三种发酵液样品乙醛浓度都达到峰值。
实施例7检测方法对酒类样品中羰基化合物及主要风味物质的抗干扰性能
模拟真实酒类样品，证实探针的抗干扰性。干扰物质应包含羰基化合物以及酒类样品中含量丰富的醇酯类化合物，浓度的选择以报道过的最高含量作为依据，乙醛含量则使用各酒类的平均浓度。以啤酒抗干扰性分析样品为例，各分析物浓度为：乙醛10mg/L；丙醛0.3mg/L；正丁醛0.3mg/L；异丁醛0.3mg/L；异戊醛0.1mg/L；庚醛0.2mg/L；辛醛0.2mg/L；糠醛2mg/L；5-羟甲基糠醛8mg/L；2.3-丁二酮1mg/L；2.3-戊二酮0.5mg/L；乙偶姻5mg/L；丙酮醛0.1mg/L；正丙醇25mg/L；异戊醇：100mg/L；乙酸乙酯50mg/L；乙酸异戊酯10mg/L。向96孔酶标板中加入50μL酸溶液H，100μL荧光探针溶液，并分别加入上述抗干扰分析物，置于5℃培养箱中低温反应50min在荧光光谱仪上测定荧光光谱图。由图2可以发现，在553nm处只有乙醛表现出较强的荧光信号，因此荧光探针适用于啤酒等酒类样品的复杂体系。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种利用荧光探针检测酒类样品中乙醛含量的方法，其特征在于，包括如下步骤：
    (1)将式(I)所示结构的荧光探针分散在有机溶剂中，获得荧光探针溶液；然后将荧光探针溶液、盐酸溶液与一系列已知浓度的乙醛标准溶液分别混合，0-10℃下进行反应，反应结束后，获得混合体系；
    Figure PCTCN2022081626-appb-100001
    (2)利用荧光光谱仪上测定混合体系的荧光强度，并利用荧光强度及其相应乙醛标准溶液的浓度进行线性关联，获得定量检测模型；
    (3)按照步骤(1)的过程，将荧光探针溶液、盐酸溶液与酒类样品混合反应，然后测定混合体系的荧光强度，并通过步骤(2)中的定量检测模型，计算得到酒类样品中乙醛的浓度。
  2. 根据权利要求1所述的方法，其特征在于，荧光探针溶液、盐酸溶液、乙醛标准溶液的体积比为2：1：1。
  3. 根据权利要求1所述的方法，其特征在于，步骤(1)中所述有机溶剂为乙腈、DMSO；乙腈与DMSO的体积比为10：1。
  4. 根据权利要求1所述的方法，其特征在于，荧光探针溶液的浓度为600mg/L。
  5. 根据权利要求1所述的方法，其特征在于，盐酸溶液是利用乙腈为溶剂配制盐酸溶液。
  6. 根据权利要求1所述的方法，其特征在于，盐酸溶液的pH为2。
  7. 根据权利要求1所述的方法，其特征在于，一系列已知浓度的乙醛标准溶液的浓度范围为0-200mg/L。
  8. 根据权利要求1所述的方法，其特征在于，荧光强度为发射波长553nm处的荧光强度。
  9. 根据权利要求1所述的方法，定量检测模型为F 553nm＝346.14C+45.17，R 2＝0.9954；C的单位为mg/L。
  10. 根据权利要求1所述的方法，其特征在于，酒样样品还需经过如下预处理：
    啤酒类含气样品需滴加一滴消泡剂，白酒、黄酒以及葡萄酒样品需要用蒸馏水稀释25倍；然后取50mL酒样，使用双乙酰蒸馏装置蒸馏酒样，当馏出液接近10mL时停止接收，并用蒸馏水补足到10mL，得到馏出液A，即为酒样样品。
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