阅读说明：本技术 一种快速检测花椒果皮中总酰胺含量的电化学方法 (The electrochemical method of total ceramide content in a kind of quick detection Chinese prickly ash pericarp ) 是由 邹小波 赵镭 张迪 史波林 孙晓霞 于 2019-07-23 设计创作，主要内容包括：本发明属于食品检测领域,涉及一种快速检测花椒果皮中总酰胺含量的电化学方法；步骤为:将干燥花椒果皮粉碎过筛,干粉经无水乙醇提取、洗渣并液、稀释定容,得到花椒酰胺提取液；将提取液同电解质溶液混合配置成花椒酰胺检测工作液,其中解质溶液为含有乙醇的磷酸缓冲液；在多次加入标准物羟基-α-山椒素的过程中测定检测工作液的差分脉冲伏安曲线,绘制多次标准加入法工作曲线图,计算得出检测溶液中的总酰胺含量,换算得到花椒果皮样品中的总酰胺含量,即羟基-α-山椒素当量值；本发明解决了现有花椒总酰胺定量检测方法无法实现快速、低成本、准确性兼得的问题,本检测方法能够对花椒果皮中总酰胺含量进行快速、便携、准确检测。(The invention belongs to field of food detection, are related to a kind of electrochemical method for quickly detecting total ceramide content in Chinese prickly ash pericarp；Step are as follows: by the dry broken sieving of Chinese prickly ash fruit peel powder, dry powder obtains fagaramide extracting solution through dehydrated alcohol extraction, washery slag and liquid, dilution constant volume；Extracting solution is mixedly configured into fagaramide detection working solution with electrolyte solution, wherein electrolyte solution is the phosphate buffer containing ethyl alcohol；The differential pulse voltammetry curve of measurement detection working solution during reference substance hydroxyl-alpha-sanshool is repeatedly added, draw Multiple Standard Addition Method working curve diagram, the total ceramide content in detection solution is calculated, conversion obtains the total ceramide content in Chinese prickly ash peel sample, i.e. hydroxyl-alpha-sanshool equivalent value；The present invention solves the problems, such as that existing Chinese prickly ash total ceramide quantitative detecting method cannot achieve quick, inexpensive, accuracy and get both, this detection method can carry out quick, portable, accurate detection to total ceramide content in Chinese prickly ash pericarp.)

1. the electrochemical method of total ceramide content in a kind of quickly detection Chinese prickly ash pericarp, which comprises the following steps:

(1) the drying Chinese prickly ash pericarp for removing wilting or brown stain, crushes, screens through sieve, obtain zanthoxylum powder；

(2) step (1) resulting zanthoxylum powder is weighed, dehydrated alcohol A is added in quality W, and concussion shakes up, carries out under normal temperature condition Ultrasound extraction, obtains crude extract；Crude extract is centrifuged, supernatant is collected；It collects filter residue and dehydrated alcohol B is separately added, shake It is centrifuged again afterwards, collects supernatant；It so repeats, mixes the supernatant repeatedly collected, as detection mother liquor, record total volume V, Refrigeration is stand-by；Entire step operates under the conditions of being protected from light；

(3) configuration electrolysis detects solution first, and the group of electrolysis detection solution becomes dehydrated alcohol and the mixing of PBS buffer solution is molten Liquid；The detection mother liquor that aspiration step (2) is prepared, volume are denoted as V₁, with electrolysis detection solution dilution, it is molten to obtain sample detection Liquid A, volume are denoted as V₂, utilize the differential pulse voltammetry curve of fagaramide substance in three-electrode system detection mixed solution；

Second of absorption detection mother liquor, volume V1, being diluted to volume with electrolytic solution is V2, prepares solution to be detected again, Then α-SOH the titer that volume is V3 is added, obtains mixed solution, utilizes Chinese prickly ash acyl in three-electrode system detection mixed solution The differential pulse voltammetry curve of amine substance；

Third time draws detection mother liquor, and volume V1, being diluted to volume with electrolytic solution is V2, prepares solution to be detected again, α-SOH the titer of 2 times of V3 volumes is added, utilizes the differential pulse of fagaramide substance in three-electrode system detection mixed solution Volt-ampere curve；

4th absorption detects mother liquor, and volume V1, being diluted to volume with electrolytic solution is V2, prepares solution to be detected again, α-SOH the titer of 3 times of V3 volumes is added, utilizes the differential pulse of fagaramide substance in three-electrode system detection mixed solution Volt-ampere curve；The rest may be inferred, repetitive operation；

Finally using the mass concentration of α-SOH titer as abscissa, corresponding peak current is that ordinate does linear regression, corresponding Differential pulse voltammetry peak current be that ordinate does linear regression curves, the absolute value of linear regression curves and abscissa intersection point, Total ceramide content as in sample detection solution, is denoted as m；

(4) the total ceramide content in sample detection solution obtained according to step (3), is further examined by following formula scales Survey the total ceramide content in mother liquor, i.e. α-SOH equivalent content:

In the formula, M: total ceramide content in Chinese prickly ash sample, μ g/g；M: the total ceramide content in sample detection solution, μ g/mL； V1: the detection mother liquor volume used when preparing sample detection solution；V2: the volume of sample detection solution；V: detection mother liquor is overall Product, mL；W: zanthoxylum powder quality, g；A: systematic error compensation coefficient.

2. the electrochemical method of total ceramide content, feature in a kind of quickly detection Chinese prickly ash pericarp according to claim 1 It is, in step (1), the mesh number of the sieve is 20~40 mesh.

3. the electrochemical method of total ceramide content, feature in a kind of quickly detection Chinese prickly ash pericarp according to claim 1 It is, in step (2), the amount ratio of the zanthoxylum powder and dehydrated alcohol A are 5-25g:25-100mL；The ultrasound extraction time For 20~40min.

4. the electrochemical method of total ceramide content, feature in a kind of quickly detection Chinese prickly ash pericarp according to claim 1 It is, in step (2), the amount ratio of the filter residue being collected after centrifugation and dehydrated alcohol B are 1g:1-6mL；The centrifugal condition Are as follows: 2000~3000r/min of revolving speed, time are 3~8min.

5. the electrochemical method of total ceramide content, feature in a kind of quickly detection Chinese prickly ash pericarp according to claim 1 It is, in step (3), the volume ratio of dehydrated alcohol and PBS buffer solution is 1~3:7~9 in the electrolysis detection solution.

6. the electrochemical method of total ceramide content, feature in a kind of quickly detection Chinese prickly ash pericarp according to claim 1 It is, in step (3), the volume V of the detection mother liquor₁With the volume V of sample detection solution₂The ratio between be 0.03-0.5:10.

7. the electrochemical method of total ceramide content, feature in a kind of quickly detection Chinese prickly ash pericarp according to claim 1 It is, in step (3), the volume V of the detection mother liquor₁With the volume V of the α-SOH titer of addition₃The ratio between be 3-12:1； α-SOH the concentration of standard solution is 1.0~10.0g/L.

8. the electrochemical method of total ceramide content, feature in a kind of quickly detection Chinese prickly ash pericarp according to claim 1 It is, in step (3), the three-electrode system group becomes glass-carbon electrode, Ag/AgCl electrode and platinum electrode；Differential pulse volt The testing conditions parameter of peace method are as follows: forward scan ,+0.6V-+1.2V；Potential step 4mV；Amplitude 0.05V；Pulsewidth 0.05s；It adopts Sample width 0.0167s；Pulse period 0.5s.

9. the electrochemical method of total ceramide content, feature in a kind of quickly detection Chinese prickly ash pericarp according to claim 1 It is, in step (3), the number of the repetitive operation is 4~6 times.

Technical field

The invention belongs to field of food detection, are related to a kind of electrochemical method for quickly detecting total ceramide content in Chinese prickly ash.

Background technique

Chinese prickly ash is one of Chinese tradition " eight big flavouring ", is liked deeply by its people because it is with unique numb taste.Cause The main component of Chinese prickly ash fiber crops taste is chain polyunsaturated fatty acid amide, is referred to as fagaramide, wherein hydroxyl-alpha-sanshool (Hydroxyl- α-sanshool, α-SOH), hydroxy-beta-sanshool (Hydroxyl- α-sanshool, α-SOH), hydroxyl-γ- Sanshool (Hydroxyl- α-sanshool, α-SOH), hydroxyl-ε-sanshool (Hydroxyl- α-sanshool, α-SOH) are Most representative amide substance, accounts for 98% or more of total ceramide content.

The height of fagaramide content be measure Chinese prickly ash quality an important indicator and Chinese prickly ash quality grading it is important Foundation.The instrument detection method that fagaramide content can accurately be measured at present is high performance liquid chromatography (HPLC) or efficient liquid Phase chromatographic tandem mass spectrography (HPLC-MS/MS)；A kind of utilization hplc device inspection is disclosed in patent document 201711217038.4 The method for surveying the fagaramide content in Chinese prickly ash sample, but high performance liquid chromatograph device is expensive, operation sequence is more complex, inspection Survey environmental requirement it is high and can not portable use, these limitations hinder the large-scale application of this method in the industry.

It includes: mentioning for total ceramide medicinal extract that existing literature, which discloses a kind of method for rapidly detecting content of numb-taste components, It takes；Amide is changed into ammonium nitrogen；Ammonium nitrogen assay.This method belongs to a kind of titration method of indirect determination fagaramide, Wherein total ceramide extracts and amide was changed into the pretreatment process total time-consuming such as ammonium nitrogen up to 2-6 hours；And titration operation journey Sequence is many and diverse, and titration end-point is easily judged by accident.Currently, there has been no published method can be achieved at the same time fagaramide content it is quick, Portable, accurate, inexpensive detection.

Summary of the invention

The technical problem to be solved by the present invention is to complicated for operation for fagaramide method in detection Chinese prickly ash sample at present, at The problems such as this is higher, provide it is a kind of it is simple, convenient quickly, testing cost it is low, can be realized to Chinese prickly ash total in Chinese prickly ash raw material Amide content carries out the electrochemical method of quick, accurate, portable detection.

A kind of technical solution of present invention solution above-mentioned technical problem are as follows: electrification for quickly detecting total ceramide content in Chinese prickly ash Method, the specific steps are as follows:

(1) Chinese prickly ash pericarp preparation of samples；

It removes and wilts or the drying Chinese prickly ash pericarp of brown stain, crush, screened through sieve, obtain zanthoxylum powder, it is cold under the conditions of -20 DEG C It hides to be detected；

(2) preparation of mother liquor is detected；

Step (1) resulting zanthoxylum powder is weighed, dehydrated alcohol A is added in quality W, and concussion shakes up, carries out under normal temperature condition Ultrasound extraction, obtains crude extract；Crude extract is centrifuged, supernatant is collected；It collects filter residue and dehydrated alcohol B is separately added, shake It is centrifuged again afterwards, collects supernatant；It so repeats, mixes the supernatant repeatedly collected, as detection mother liquor, record total volume V, Refrigeration is stand-by；Entire step operates under the conditions of being protected from light；

(3) differential pulse voltammetry is analyzed；

Hydroxyl-alpha-sanshool standard of certain volume is added dropwise to sample detection solution according to the principle of Multiple Standard Addition Method Solution (α-SOH), followed by the differential pulse voltammetry curve of fagaramide substance in three-electrode system detection mixed solution；

The group of configuration electrolysis detection solution, electrolysis detection solution becomes the mixed solution of dehydrated alcohol and PBS buffer solution；It inhales The detection mother liquor for taking step (2) to be prepared, volume are denoted as V₁, with electrolysis detection solution dilution, sample detection solution A is obtained, Volume is denoted as V₂, utilize the differential pulse voltammetry curve of fagaramide substance in three-electrode system detection mixed solution；

Second of absorption detection mother liquor, volume V1, being diluted to volume with electrolytic solution is V2, is prepared again to be detected molten Then α-SOH titer is added in liquid, volume is added and is denoted as V3, obtains mixed solution, detects mixed solution using three-electrode system The differential pulse voltammetry curve of middle fagaramide substance；

Third time draws detection mother liquor, and volume V1, being diluted to volume with electrolytic solution is V2, is prepared again to be detected molten α-SOH the titer of 2 times of V3 volumes is added in liquid, utilizes the difference of fagaramide substance in three-electrode system detection mixed solution Pulse Voltammetry curve；

4th absorption detects mother liquor, and volume V1, being diluted to volume with electrolytic solution is V2, is prepared again to be detected molten α-SOH the titer of 3 times of V3 volumes is added in liquid, utilizes the difference of fagaramide substance in three-electrode system detection mixed solution Pulse Voltammetry curve；The rest may be inferred, repetitive operation；

Finally using the mass concentration of α-SOH titer as abscissa, corresponding peak current is that ordinate does linear regression, Corresponding differential pulse voltammetry peak current is that ordinate does linear regression curves, and linear regression curves are absolute with abscissa intersection point Value, as the total ceramide content in sample detection solution, are denoted as m；

(4) total ceramide content calculates；

The total ceramide content in sample detection solution obtained according to step (3), is further obtained by following formula scales Detect the total ceramide content in mother liquor, i.e. α-SOH equivalent content:

In the formula, M: total ceramide content in Chinese prickly ash sample, μ g/g；M: in sample detection solution total ceramide content (according to Calculated according to Multiple Standard Addition Method working curve), μ g α-SOH equivalent/mL；V1: the detection used when preparing sample detection solution Mother liquor volume；V2: the volume of sample detection solution；V: detection mother liquor total volume, mL；W: zanthoxylum powder quality, g；A: systematic error Compensation coefficient.

Preferably, in step (1), the mesh number of the sieve is 20~40 mesh.

Preferably, in step (2), the amount ratio of the zanthoxylum powder and dehydrated alcohol A are 5-25g:25-100mL；It is described super Sound extraction time is 20~40min.

Preferably, in step (2), the centrifugal condition is equal are as follows: 2000~3000r/min of revolving speed, time are 3~8min.

Preferably, in step (2), the amount ratio of the filter residue and dehydrated alcohol B are 1g:(1-6) mL.

Preferably, in step (3), electrolysis detection solution in the volume ratio of dehydrated alcohol and PBS buffer solution be 1~3:7~ 9；The concentration of PBS buffer solution is 0.1M.

Preferably, in step (3), the volume V of the detection mother liquor₁With the volume V of sample detection solution₂The ratio between be (0.03-0.5): 10.

Preferably, in step (3), the volume V of the detection mother liquor₁With the volume V of the α-SOH titer of addition₃The ratio between For (3-12): 1；α-SOH the concentration of standard solution is 1.0~10.0g/L.

Preferably, in step (3), three-electrode system group becomes working electrode (glass-carbon electrode), reference electrode (Ag/AgCl Electrode), auxiliary electrode (platinum electrode)；The testing conditions parameter of differential pulse voltammetry are as follows: forward scan ,+0.6V-+1.2V； Potential step 4mV；Amplitude 0.05V；Pulsewidth 0.05s；Sampling width 0.0167s；Pulse period 0.5s.

Preferably, in step (3), the number of the repetitive operation is 4~6 times.

The utility model has the advantages that

(1) present invention establishes the electrochemical detection method of total fagaramide content in Chinese prickly ash pericarp for the first time, with current state Inside and outside existing fagaramide detection method is compared, and instrument used in this method has spy portable, inexpensive, easy to operate Point.

(2) bare glassy carbon electrode used in electrochemical detection method of the present invention, without carrying out complexity to electrode Moditied processing, it is easy to operate.

(3) electrochemical detection method detection accuracy of the present invention is high, the error rate with HPLC-MS method measurement result Lower than 6%, detection speed is fast (differential pulse voltammetry scanning is completed in 2 minutes), can be used for carrying out Chinese prickly ash total ceramide fast Fast accurate quantitative analysis.

(4) electrochemical detection method of the present invention is expected to the alternative that development is HPLC-MS, to Chinese prickly ash and its system The detection and analysis of Chinese prickly ash total ceramide are of great significance in product.

Detailed description of the invention

Fig. 1 is the differential pulse voltammetry figure that standard addition method detects fagaramide content.

Fig. 2 is that standard addition method working curve calculates schematic diagram.

Fig. 3 is high effective liquid chromatography for measuring value and the matched curve of electrochemical process measured value.

Specific embodiment

In order to illustrate more clearly of the contents of the present invention, the present invention is made further below with reference to specific embodiment It is bright, however, it should be noted that these embodiments are only to illustrate to be used, and be not necessarily to be construed as the limitation that the present invention is implemented.

1, laboratory sample, main agents and instrument

Sample: using the drying Chinese prickly ash of 7 kinds of places of production clear (being local harvesting) as test object, they are respectively as follows: 1 Number sample, southern green pepper level-one (Hanyuan County, sichuan Province)；No. 2 samples, Jiangjin pericarpium zanthoxyli schinifolii (Chongqing Jiangjin)；No. 3 samples, Jinyang pericarpium zanthoxyli schinifolii (Sichuan gold Sun)；No. 4 samples, Shao lead to pericarpium zanthoxyli schinifolii (Yunnan Shao is logical)；No. 5 samples lie prostrate green pepper level-one (Gansu Linxia)；No. 6 samples, level-one pericarpium zanthoxyli bungeani (Shaanxi Fuping)；No. 7 samples, level-one pericarpium zanthoxyli bungeani (Laiwu in Shandong province).

Reagent: hydroxyl-alpha-sanshool (α-SOH), hydroxy-beta-sanshool (β-SOH), hydroxyl-γ-sanshool (γ-SOH) Standard items are purchased in Chengdu Mai Desheng Science and Technology Ltd., and purity is greater than 98%.Water used in experimentation is deionization Water, experiment reagent are that analysis is pure.

Instrument: the three-electrode system of electrochemical workstation (CHI660E, Shanghai Chen Hua Instrument Ltd.) is: work electricity Extremely 3mm diameter glass carbon disk electrode (model C HI104), reference electrode are Ag/AgCl electrode (model C HI111), auxiliary electrode For platinum electrode (model C HI115).