CN107677669A - 20 to 30 degree wine alcoholic strength simplicity rapid assay methods - Google Patents
20 to 30 degree wine alcoholic strength simplicity rapid assay methods Download PDFInfo
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- CN107677669A CN107677669A CN201710924890.9A CN201710924890A CN107677669A CN 107677669 A CN107677669 A CN 107677669A CN 201710924890 A CN201710924890 A CN 201710924890A CN 107677669 A CN107677669 A CN 107677669A
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- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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Abstract
Alcoholic strength is that alcohol product must survey index, lack at present have concurrently accurately and reliably, easy quick and economical and practical alcoholic strength assay method.The present invention can be unwind using DNA fragmentation in containing ethanol system causes its absorbance under 260nm wavelength to change, so as to establish a kind of new alcoholic strength assay method.The present invention screens suitable short double chain DNA fragment mixed system such as SEQ ID NO for 20 to 30 degree wine：Shown in 12, the calibration curve equation for establishing its absorbance x and wine sample alcoholic strength y is y=9.2499x+18.422（20≤y≤30）.The alcoholic strength assay method of the present invention is applied to the measure of 20 to 30 degree wine, has good accuracy and reliability, safer without distilling, it is easy to operate rapid, required wine sample amount is seldom and cheap, is suitable for brewery 20 to 30 and spends the measure practice of wine alcoholic strength.
Description
Technical field
The present invention relates to ethanol detection method, more particularly to a kind of ethanol content of 20 to 30 degree wine is the simplicity of alcoholic strength
Rapid assay methods.
Background technology
Ethanol is the main component of wine, and its content is one of important indicator of wine quality, is referred to as alcoholic strength in national standard, is led to
Often it refers at 20 DEG C, the ethanol milliliter number contained in every 100 ml wine liquids.Alcoholic strength is that must surveying for alcohol product refers to
Mark, assay method has much at present, and common includes alcoholic strength meter method, density bottle method, gas chromatography and near infrared spectroscopy
Deng.Both are the methods based on modern analytical technique for gas chromatography and near infrared spectroscopy, have that detection sensitivity is high, knot
Fruit is accurate, can be with high throughput assay the advantages that, but there is also the deficiencies of equipment is expensive, complex operation and testing cost are high, because
It is actual that this is not suitable for brewery's detection.Alcoholic strength meter method and density bottle method are most commonly used that in brewery, but both approaches detect
Before be both needed to the advance distillation of alcoholic drink, after removing impurity, then alcohol water blend is measured.Time-consuming for whole detection process,
Step is more, stability is poor, it is impossible to meets the needs of quick detection development.
In summary, need badly at present establish accurately and reliably, easy quick and economical and practical alcoholic strength assay method, for
The invention provides based on specific ultrashort DNA fragmentation for 20 to 30 degree wine（That is oligodeoxynucleotide chain）In different alcoholic strength wine samples
Middle denaturation degrees difference causes under 260nm wavelength absorbance difference, and establishes a kind of new alcoholic strength assay method i.e.
Meet this demand.DNA double chain be lean on base between hydrogen bond and accumulation force maintain, but some external factors such as high temperature,
In the presence of extreme pH, organic reagent such as urea, formamide, methanol and ethanol, these maintenance energies can be destroyed, so that
DNA double chain, which is unwind, becomes single-stranded, causes its absorbance under 260nm to be significantly increased（Also known as hyperchromicity）.It is special using this
Property, suitable DNA systems are built, wine sample is added thereto processing, using the DNA systems absorbance between alcoholic strength in wine sample
Association, so as to realize the measure of alcoholic strength.
The content of the invention
It is an object of the invention to provide the easy rapid assay methods of 20 to 30 degree wine alcoholic strengthes.
The technical solution adopted for the present invention to solve the technical problems is the easy quick measure side of 20 to 30 degree wine alcoholic strengthes
The key step of method is as follows：
（1）400ul room temperatures 20 to 30 degree wine wine sample to be measured is pipetted into the ELISA Plate of sky, adds the agent of 50ul denaturations and 50ul
Short double chain DNA fragment mixed system simultaneously fully stands 5min after mixing, while sets control wells to add 50ul for 400ul wine samples to be measured
Denaturation agent adds 50ul distilled water, and wine sample to be measured and control make even row survey three times, be placed in ELISA Plate after sample process is good
In ELIASA.
（2）Set absorbance of the ELIASA program under 260nm wavelength in determination sample hole and control wells, then sample
The mean light absorbency that mean light absorbency subtracts control is the actual absorbance of short double chain DNA fragment mixed system in sample.Will
The actual absorbance x of DNA systems substitutes into relational expression y=9.2499x+18.422 in sample（20≤y≤30）In converse wine sample
Alcoholic strength y.
Wherein, step（1）Described in 20 to 30 degree wine refer to alcoholic strength be more than or equal to 1 and less than or equal to 20 liquor
Product；The wine sample temperature is normal room temperature, it is preferable that control is between 30.0 ± 5.0 DEG C；Described denaturation agent
It is the urea of 5% mass volume fraction and the formamide mixed aqueous solution of 8% volume volume fraction, i.e., per 100ml mixed aqueous solutions
In contain 5g urea and 8ml formamides；Described short double chain DNA fragment mixed system includes the 2 short He of double chain DNA fragment 1
2, its sequence is respectively such as SEQ ID NO：Shown in 1-2, each short double chain DNA fragment concentration of bar is 10 μm of ol/l.
The present invention screens suitable short double chain DNA fragment mixed system, and discloses its extinction in 20 to 30 degree wine
Relation between degree and alcoholic strength, the easy rapid assay methods of 20 to 30 degree wine alcoholic strengthes are constructed on this basis.This hair
Bright alcoholic strength assay method is applied to the measure of 20 to 30 degree wine, has good accuracy and reliability, without distilling ratio
Safer, easy to operate rapid, required wine sample amount is seldom and cheap, is suitable for brewery 20 to 30 and spends wine alcoholic strength measure
Practice.
Brief description of the drawings
Fig. 1 is absorbance-alcoholic strength standard curve that the present invention 20 to 30 spends wine alcoholic strength measure new method.Abscissa is
Add the absorbance of short double chain DNA fragment mixed system and the artificial wine sample of denaturation agent processing under 260nm, ordinate is it
Alcoholic strength.Y=9.2499x+18.422 in figure be the softwares of SPSS 23.0 fitting absorbance x and alcoholic strength y relational expression, R2
=0.9996 represent be matched curve the coefficient of determination.
Embodiment
Below in conjunction with specific embodiment, the present invention is expanded on further.
The present invention is additionally added urea and the formamide mixing of optimized certain concentration in wine sample to be measured and DNA systems
Liquid, it is easy to primarily to making DNA fragmentation be in one in the environment of denaturation, so that alcohol determining method is with higher
Sensitivity.Short double-stranded DNA system provided by the invention contains 2 DNA fragmentations, by SEQ ID NO：Order incremental 1-2 its
Melting temperature is to rise, therefore increasing with alcoholic strength, and the type and quantity of denaturation double-strand are consequently increased in system, because
It is positively related between this absorbance and alcoholic strength.In addition, DNA denaturation is realized in a narrow scope, therefore need
Screened and condition optimizing so that border is that have to the DNA fragmentation of different melting temperatures under the specified conditions in DNA systems
It is superimposed what is continued.The narrow mobility scale of this gentle denaturant in ethanol（Within 10ml）With the ultrashort fragments of DNA（Deficiency
15bp）, it has been investigated that the melting extent of reaction of Denaturant for ethanol concentration and specific DNA systems can be realized within the specific limits
Linear fit.Because second alcohol and water accounts for main body in wine, solid content is very low therefore it influences on absorbance of the system in 260nm
Very little.In addition, there is provided only add the wine sample of denaturation agent to eliminate as much as influence of the background to absorbance as control.
In system of the present invention with the conditions of, less than 20 degree of alcoholic strength is still insufficient to allow any short double chain DNA fragment that partial denaturization occurs,
And more than 30 degree of alcoholic strength has been enough to make all short double chain DNA fragments be denatured completely, therefore alcoholic strength provided by the invention
Standard curve is only applicable to the alcoholic strength measure of 20 to 30 degree wine.In the detection of drinks product quality, AAS is conventional
Means, can determine including colourity, methanol and specific amino acids etc., therefore spectrophotometer is conventional equipment.
Embodiment 1
Embodiment 1 is the system that the present invention 20 to 30 spends alcoholic strength bioassay standard curve in wine alcoholic strength simplicity rapid assay methods
Make.
（1）Artificial 20 to 30 degree wine sample is prepared
With corresponding pipette take respectively 20.0,22.0,24.0,26.0,28.0 and 30.0ml absolute ethyl alcohols in 100ml volumetric flasks
In, it is continuously added distilled water and slightly shakes and stand repeatedly, final constant volume to 100ml is stored at room temperature 10min, that is, obtained not
With artificial 20 to the 30 degree wine sample of alcoholic strength.
（2）Artificial 20 to 30 degree wine sample pre-treatment
The artificial wine sample of 400ul 20 to 30 degree to be measured is pipetted with liquid-transfering gun（30.0℃）Into the common ELISA Plate of sky, 5% is first added
The urea of quality volume fraction and the formamide mixed aqueous solution 50ul of 8% volume volume fraction add as denaturation agent
Such as SEQ ID NO：Short double chain DNA fragment mixed system shown in 1-2（Concentration per bar segment is 10 μm of ol/l）50ul, then
Total system is 500ul, and 5min is stood after fully mixing.It should be noted that the urea of 5% mass volume fraction and 8% volume volume
The formamide of fraction refers to the formamide of the urea and 8ml in 100ml distilled water dissolved with 5g, and short double chain DNA fragment is by upper
The complementary single strand mixed in equal amounts of Hai Shenggong bioengineering Co., Ltd synthesis forms.
Control wells are set to add 50ul denaturation agent to add 50ul distilled water for artificial 20 to the 30 degree wine samples of 400ul simultaneously, i.e.,
Compare to substitute short double chain DNA fragment mixed system with distilled water, each artificial 20 to 30 degree wine sample and corresponding control are made even
ELISA Plate three times, ELIASA is placed in after sample preparation is good by row（The Multiskan GO of Thermo companies of the U.S.）In.
（3）It is fitted alcoholic strength assay method standard curve
Absorbance of the ELIASA program under 260nm wavelength in determination sample hole and control wells is set, then the average suction of sample
Luminosity（Three parallel to be averaged）Subtract the mean light absorbency of control（Three parallel to be averaged）The reality of DNA systems as in sample
Border absorbance, as shown in table 1.
The scatterplot of absorbance and corresponding alcoholic strength is observed, finds 20.0,22.0,24.0,26.0,28.0 and
Linear trend is presented in point corresponding to 30.0 this 6 alcoholic strengthes, is fitted to obtain the relational expression between alcoholic strength y and absorbance x
For y=9.2499x+18.422（20≤y≤30）, and R2=0.9996 very close 1 shows that linear relationship is notable, then this is alcohol
Spend assay method standard curve.
The alcoholic strength standard curve data of table 1
| Mean light absorbency x | Alcoholic strength y |
| 0.165 | 20.0 |
| 0.393 | 22.0 |
| 0.612 | 24.0 |
| 0.812 | 26.0 |
| 1.028 | 28.0 |
| 1.257 | 30.0 |
Embodiment 2
Embodiment 2 is the practical measurement to certain 20 to 30 degree wine alcoholic strength using new method of the present invention.
（1）Wine sample pre-treatment to be measured
Select Long Yan Chen Gang Jiu（Long Yan Chen Gang Jiu industry Co., Ltd produces）This commercially available yellow rice wine, takes two bottles（Catalyzed wine 1 and catalyzed wine
2）Carry out alcoholic strength measure.The 400ul wine samples are pipetted with liquid-transfering gun（30.0℃）Into the common ELISA Plate of sky, 5% matter is first added
The urea of volume fraction and the formamide mixed aqueous solution 50ul of 8% volume volume fraction are measured as denaturation agent, add as
SEQ ID NO：Short double chain DNA fragment mixed system shown in 1-2（Concentration per bar segment is 10 μm of ol/l）50ul, then always
System is 500ul, and 5min is stood after fully mixing.
Control wells are set to add 50ul denaturation agent to add 50ul to distill aqueous systems for 400ul wine samples to be measured simultaneously（Cumulative volume
Also it is 500ul）, each wine sample to be measured and corresponding control make even row three times, ELISA Plate are placed in into ELIASA after sample preparation is good
（The Multiskan GO of Thermo companies of the U.S.）In.
（2）Wine sample alcoholic strength measure to be measured
Absorbance of the ELIASA program under 260nm wavelength in determination sample hole and control wells is set, then the average suction of sample
Luminosity（Three parallel to be averaged）Subtract the mean light absorbency of control（Three parallel to be averaged）The reality of DNA systems as in sample
Border absorbance, as shown in table 2.The relational expression substituted between alcoholic strength y and absorbance x is y=9.2499x+18.422（20≤y≤
30）, calculate alcoholic strength result（Table 2）.In addition, the alcoholic strength of the wine sample is determined using density bottle method simultaneously, as a result such as the institute of table 2
Show.
The alcoholic strength numerical value of the inventive method of table 2 and density bottle method measure
| Yellow rice wine | Absorbance values | The alcoholic strength that the inventive method is surveyed | The alcoholic strength that density bottle method is surveyed | Deviation % |
| Catalyzed wine 1 | 0.182 | 20.11 | 20.05 | 0.30 |
| Catalyzed wine 2 | 0.181 | 20.10 | 20.08 | 0.10 |
In table 2, on the basis of deviation refers to the alcoholic strength that is measured by density bottle method, alcoholic strength and its difference that the inventive method measures
Account for the percentage of alcoholic strength meter method result.As shown in Table 2, using the two bottles of catalyzed wine alcoholic strengthes and density of the inventive method measure
Bottle method measurement result deviation be respectively 0.30% and 0.10%, meet within 1% national regulations alcoholic strength measure it is accurate
Degree.
Embodiment 3
Embodiment 3 is to pick up people to commercially available Jiang little Bai callings using new method of the present invention to drink white wine（Chongqing Jiang Ji chateaus Co., Ltd
Production）The measure of the alcoholic strength of this 20 to 30 degree wine, takes altogether two bottles（Jiang little Bai 1 and river little Bai 2）.Operated with reference to embodiment 2,
It the results are shown in Table 3.In addition, this wine alcoholic strength measure is carried out using density bottle method simultaneously.
The alcoholic strength numerical value of the inventive method of table 3 and density bottle method measure
| White wine | Absorbance values | The alcoholic strength that the inventive method is surveyed | The alcoholic strength that density bottle method is surveyed | Deviation % |
| Jiang little Bai 1 | 0.707 | 24.96 | 25.18 | 0.87 |
| Jiang little Bai 2 | 0.709 | 24.98 | 25.19 | 0.83 |
As shown in Table 3, send yellow rice wine alcoholic strength and density bottle method measurement result inclined in the two bottles of red Fujian of green grass or young crops determined using the inventive method
Difference is respectively 0.87% and 0.83%, and the accuracy of the alcoholic strength measure of national regulations is met within 1%.
Embodiment 4
Embodiment 4 is to commercially available blueberry ice wine using new method of the present invention（Daxing'an Mountainrange arctic ice blueberry chateau Co., Ltd produces）
The measure of the alcoholic strength of this 20 to 30 degree wine, takes altogether two bottles（Blueberry ice wine 1 and blueberry ice wine 2）.Grasped with reference to embodiment 2
Make, the results are shown in Table 4.In addition, this wine alcoholic strength measure is carried out using density bottle method simultaneously.y=9.2499x+18.422
The alcoholic strength numerical value of the inventive method of table 4 and density bottle method measure
| Yellow rice wine | Absorbance values | The alcoholic strength that the inventive method is surveyed | The alcoholic strength that density bottle method is surveyed | Deviation % |
| Blueberry ice wine 1 | 1.104 | 28.63 | 28.51 | 0.42 |
| Blueberry ice wine 2 | 1.101 | 28.61 | 28.47 | 0.49 |
As shown in Table 4, using the two bottles of Lanling Meijiu alcoholic strengthes and density bottle method measurement result deviation point of the inventive method measure
Not Wei 0.42% and 0.49%, meet within 1% national regulations alcoholic strength measure accuracy.
In summary, alcoholic strength assay method of the invention is applied to the measure of 20 to 30 degree wine, has well accurate
Property and reliability；It is safer without distilling compared with alcoholic strength meter method or density bottle method, operate 10min and simple, convenient and rapid；Separately
Wine sample amount is seldom needed for outer, and testing cost is very cheap less than 1 yuan/sample, is suitable for the such wine alcoholic strength measure practice of brewery.
Sequence table
<110>Huanghai Sea rosy clouds
<120>20 to 30 degree wine alcoholic strength simplicity rapid assay methods
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atatatatat actg 14
<210> 2
<211> 15
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atatatatat actga 15
Claims (3)
1.20 to 30 degree wine alcoholic strength simplicity rapid assay methods, it is characterised in that alcoholic strength simplicity rapid assay methods it is main
Step is：
（1）400ul room temperatures 20 to 30 degree wine wine sample to be measured is pipetted into the ELISA Plate of sky, adds the agent of 50ul denaturations and 50ul
Short double chain DNA fragment mixed system simultaneously fully stands 5min after mixing, while sets control wells to add 50ul for 400ul wine samples to be measured
Denaturation agent adds 50ul distilled water, and wine sample to be measured and control make even row survey three times, be placed in ELISA Plate after sample process is good
In ELIASA；
（2）Set absorbance of the ELIASA program under 260nm wavelength in determination sample hole and control wells, then sample is averaged
The mean light absorbency that absorbance subtracts control is the actual absorbance of short double chain DNA fragment mixed system in sample；By this reality
Border absorbance x substitutes into relational expression y=9.2499x+18.422 the alcoholic strength y for conversing wine sample to be measured, in the range of 20≤y≤30
Effectively.
2. 20 to 30 degree wine alcoholic strength simplicity rapid assay methods according to claim 1, it is characterised in that step（1）In
Described short double chain DNA fragment mixed system includes 2 specific short double chain DNA fragments, and its sequence is respectively such as SEQ ID NO：1
With SEQ ID NO：Shown in 2, each short double chain DNA fragment concentration of bar is 10 μm of ol/l.
3. 20 to 30 degree wine alcoholic strength simplicity rapid assay methods according to claim 1, it is characterised in that step（1）In
Described denaturation agent is the urea of 5% mass volume fraction and the formamide mixed aqueous solution of 8% volume volume fraction.
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|---|---|---|---|
| CN201710924890.9A CN107677669A (en) | 2017-10-02 | 2017-10-02 | 20 to 30 degree wine alcoholic strength simplicity rapid assay methods |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419172A (en) * | 2008-09-25 | 2009-04-29 | 上海锦隆生物科技有限公司 | A kind of ethanol content detecting reagent in saliva |
| CN101825576A (en) * | 2009-11-30 | 2010-09-08 | 无锡灵特生物技术有限责任公司 | Method and kit for rapid detection of ethanol content in microbial fermentation solution |
| CN104977296A (en) * | 2015-06-30 | 2015-10-14 | 江苏大学 | Novel alcohol degree detection method and apparatus |
| CN105223346A (en) * | 2014-06-16 | 2016-01-06 | 北京雅康博生物科技有限公司 | Detect method and the kit of DNA methylation |
-
2017
- 2017-10-02 CN CN201710924890.9A patent/CN107677669A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101419172A (en) * | 2008-09-25 | 2009-04-29 | 上海锦隆生物科技有限公司 | A kind of ethanol content detecting reagent in saliva |
| CN101825576A (en) * | 2009-11-30 | 2010-09-08 | 无锡灵特生物技术有限责任公司 | Method and kit for rapid detection of ethanol content in microbial fermentation solution |
| CN105223346A (en) * | 2014-06-16 | 2016-01-06 | 北京雅康博生物科技有限公司 | Detect method and the kit of DNA methylation |
| CN104977296A (en) * | 2015-06-30 | 2015-10-14 | 江苏大学 | Novel alcohol degree detection method and apparatus |
Non-Patent Citations (3)
| Title |
|---|
| 张枫等: "《医学化学基础 第2版》", 30 June 2010 * |
| 王泉云等: "偶联酶法测定微量乙醇的研究", 《华西医学》 * |
| 黄国霞等: "四种黄酮类化合物与DNA相互作用关系研究", 《食品科学》 * |
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