CN107643262A - 8 to 10 degree yellow rice wine non-sugar solidity simplicity rapid assay methods - Google Patents
8 to 10 degree yellow rice wine non-sugar solidity simplicity rapid assay methods Download PDFInfo
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Abstract
Present invention absorbance difference in the case where different water cause 260nm wavelength with denaturation degrees difference in the ethanolic aqueous system of ethanol mass volume ratio using specific double chain DNA fragment, so as to set up a kind of 8 to 10 brand-new degree Non-sugar Solidity in Chinese Rice Wine assay methods.Specific double chain DNA fragment such as SEQ ID NO:Shown in 1, the relational expression between its absorbance x and ethanolic aqueous system reclaimed water and the mass volume ratio y of ethanol is y=62.317x+21.658.The non-sugar solidity assay method of the present invention is applied to the measure of 8 10 degree of yellow rice wine, has good accuracy and reliability, safer without long time drying, and easy to operate rapid, required wine sample amount is seldom, and testing cost is cheap.
Description
Technical field
The present invention relates to non-sugar solidity detection method, it is easy to be fast to spend yellow rice wine non-sugar solidity for more particularly to one kind 8 to 10
Fast assay method.
Background technology
Yellow rice wine is the characteristic wine kind in China, its bright color, give off a strong fragrance, sweetness is delicious, flavor is mellow, and containing abundant
Amino acid, small molecule carbohydrate, organic acid, vitamin and trace element etc..Yellow rice wine is because its is nutritious, alcoholic strength is low and has
There are unique flavor taste and effect and liked deeply by broad masses of the people.Non-sugar solidity is that non-in addition to sugar is waved in wine sample
The general designation of hair property component, its accounting very little in yellow rice wine is generally less than 3%.Although content is low, non-sugar solidity acts on very
Greatly, it has been largely fixed color and luster, flavour, nutrition and effect of yellow rice wine, is that one of yellow rice wine quality grade evaluation is important
Index.According to national standard GB/T 13662-2008《Yellow rice wine》Middle parameter, same type of Non-sugar Solidity in Chinese Rice Wine content is higher,
The quality of yellow rice wine is better, taste is better.
In existing yellow rice wine national standard the non-sugar solidity content in wine sample, this method are determined using 4h oven drying methods
Time-consuming, and quality is not up to constant weight after wine sample drying 4h being present, is unfavorable for quick measure, accurate measurement and batch
Detection.In addition, also have the wine sample non-sugar solidity rapid assay methods established based near infrared spectrum, but this method needs
By means of valuable equipment, and need to expend higher testing cost.Therefore need badly at present foundation accurately and reliably, it is easy quick
And economical and practical yellow rice wine non-sugar solidity assay method.
Ethanol content is alcoholic strength and total sugar content is the index that must survey of yellow rice wine, and the wine sample quality of unit volume can be held very much
Easily weighed, if can now measure the gross mass of ethanolic aqueous system in wine sample, wine sample quality subtracts ethanolic aqueous system quality and subtracted again
Total reducing sugar is gone to can be obtained by non-sugar solidity quality.The present invention is using specific double chain DNA fragment in different water and ethanol mass body
Denaturation degrees difference causes under 260nm wavelength absorbance difference in the ethanolic aqueous system of product ratio, a kind of complete so as to set up
8 to 10 new degree Non-sugar Solidity in Chinese Rice Wine assay methods, have accurately and reliably, it is easy to be quick and economical and practical the characteristics of, fit
Actual demand is detected together in brewery.
The content of the invention
It is an object of the invention to provide 8 to 10 degree yellow rice wine non-sugar solidity simplicity rapid assay methods.
The technical solution adopted for the present invention to solve the technical problems is that 8 to 10 degree yellow rice wine non-sugar solidities are easy to be quick
The key step of assay method is as follows:
(1)In yellow rice wine Quality Detection, first determining its alcoholic strength v according to existing national standard, this must survey index, while weighed 1L wine
Sample mass M o, determine the temperature of wine sample and inquire about ethanol density p accordingly.
(2)320ul room temperatures yellow rice wine wine sample to be measured is pipetted into the ELISA Plate of sky, the agent of 40ul denaturations is added and 40ul is special
Determine double chain DNA fragment and stand 4min after fully mixing, while set control wells to add 40ul denaturations for 320ul wine samples to be measured
Agent adds 40ul distilled water, and wine sample to be measured and control make even row survey three times, ELISA Plate are placed in ELIASA after sample process is good.
(3)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 x of specific double chain DNA fragment in sample.X is substituted into
The mass volume ratio y of wine sample reclaimed water and ethanol can be then calculated in relational expression y=- 62.317x+21.658, then total solid in wine sample
Content M=Mo-10 × ρ × v-10 × y × v namely M=Mo-10 ×(ρ+y)× v, and then non-sugar solidity content m=M-Mt, Mt are
Total sugar content.
Wherein, step(1)Described in yellow rice wine refer to that National Standard Method measure alcoholic strength v is more than or equal to 8.0 and is less than or equal to
10.0 yellow rice wine;The alcoholic strength v is that yellow rice wine Quality Detection must survey index, and just to have originally directly to use;The 1L wine sample
Mass M o assay method is:When determining wine sample alcoholic strength by National Standard Method, the appearance equipped with wine sample is weighed after measuring 100ml wine samples
Measuring bottle weight, then it is 100ml wine sample weight again that the weight, which deducts spatial capacity bottle, and 1L wine sample mass M o are can obtain through conversion, its
Unit is g/L;The wine sample temperature is normal room temperature, it is preferable that control is between 30.0 ± 5.0 DEG C.
Wherein, step(2)Described in denaturation agent be 2% mass volume fraction urea and 2% volume volume fraction
Formamide mixed aqueous solution, i.e., per 100ml denaturation agent in the urea containing 2g and 2ml formamide;Described is specific
Double chain DNA fragment, its sequence such as SEQ ID NO:Shown in 1, concentration 15umol.
Wherein, step(3)Described in mean light absorbency refer to the arithmetic mean of instantaneous values of three parallel absorbances;It is described total
Solid content M=Mo-10 × ρ × v-10 × y × v, wherein 10 × ρ × v, which is ethanol density, is multiplied by alcoholic strength i.e. 100ml
The alcohol volume content of wine sample is multiplied by with 10, it is possible to obtains the ethanol quality in 1L wine samples;And 10 × y × v is ethanol volume
The mass volume ratio of water and ethanol is multiplied by multiplied by with 10, it is possible to obtain the water quality in 1L wine samples, then 10 ×(ρ+y)× v is real
It is exactly the quality sum of ethanolic aqueous system in 1L wine samples on border;Therefore total solids content M=Mo-10 ×(ρ+y)× v, namely 1L
It is then the total solids content in 1L wine samples that wine sample gross mass, which subtracts ethanolic aqueous system quality therein, and its unit is g/L;And then
Non-sugar solidity content subtracts total sugar content namely m=M-Mt equal to total solids content, and total sugar content Mt is the inspection of yellow rice wine quality
That surveys must survey index, and just having originally directly to use, and its unit is g/L.
The present invention screens its sequence of specific double chain DNA fragment such as SEQ ID NO:Shown in 1, and it is disclosed in 8-10
Spend ultraviolet absorptivity in yellow rice wine and, with the relation between the water and the mass volume ratio of ethanol in system, construct 8- on this basis
The easy rapid assay methods of 10 degree of yellow rice wine non-sugar solidities.It is yellow that the non-sugar solidity assay method of the present invention is applied to 8-10 degree
The measure of wine, there is good accuracy and reliability, easy to operate rapid, required wine safer without long time drying
Sample amount is seldom, and testing cost is cheap, is suitable for such yellow rice wine non-sugar solidity measure practice in brewery.
Brief description of the drawings
Fig. 1 is the standard curve that the present invention 8 to 10 spends yellow rice wine non-sugar solidity measure new method, is related to specific double-stranded DNA
Absorbance x and ethanolic aqueous system reclaimed water and ethanol of the fragment under 260nm mass volume ratio y relation.Y in figure=-
62.317x+21.658 be the softwares of SPSS 23.0 fitting x and y relational expression, R2=0.9971 expression is determining for matched curve
Determine coefficient.
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 the ethanolic aqueous system quality based on this
Assay method has higher sensitivity.The specific double chain DNA fragment such as SEQ ID NO that present invention screening and optimization obtain:1 institute
Show, it has been investigated that its water and ethanol in the absorbance and system under 260nm in 8 to 10 alcoholic strengthes this narrow scopes
Mass ratio can realize linear fit.In system of the present invention with the conditions of, less than 8 degree of alcoholic strength is still insufficient to allow specific double-strand
DNA fragmentation is denatured, and more than 10 degree of alcoholic strength has been enough to make specific double chain DNA fragment be denatured completely, therefore the present invention provides
Standard curve be only applicable to the non-sugar solidity measure of yellow rice wine between 8-10 degree.Because second alcohol and water accounts for main body in wine, Gu
Shape thing content is very low therefore it influences very little to absorbance of the system in 260nm, and uv-absorption maximum wavelength is 260nm's
Specific double chain DNA fragment at concentrations up to 15umol, on the other hand on the one hand can improve detection sensitivity can exclude background
Interference.In addition, it can disappear as far as possible as control there is provided the wine sample for only adding denaturation agent and being not added with specific double chain DNA fragment
Except influence of the background to absorbance.Drinks product quality detection in, AAS is conventional means, can determine including
Colourity, methanol and specific amino acids etc., therefore spectrophotometer is conventional equipment.
Embodiment 1
Embodiment 1 is that the present invention 8 to 10 spends yellow rice wine non-sugar solidity simplicity rapid assay methods reclaimed water and ethanol mass volume ratio
The making of bioassay standard curve.
(1)Artificial ethanolic aqueous system is prepared
8.00ml, 8.40ml, 8.80ml, 9.20ml, 9.60ml and 10.00ml absolute ethyl alcohol are measured respectively with pipettor in 6
In 100ml volumetric flasks, volumetric flask be placed on assay balance be correspondingly separately added into 92.00g, 91.60g, 91.20g, 90.80g,
90.40g and 90.00g distilled water, cover tightly capacity bottle cap and overturn fully mixing repeatedly, be stored at room temperature 10min, finally given not
It is quality/ethanol volume of water with water and ethanol mass volume ratio(It is corresponding be respectively 11.500,10.905,10.364,9.870,
9.417 with 9.000)Artificial ethanolic aqueous system.
(2)Artificial ethanolic aqueous system pre-treatment
320ul room temperatures artificial ethanolic aqueous system to be measured is pipetted respectively with liquid-transfering gun into the common ELISA Plate of sky, first adds 2% matter
The urea of volume fraction and the formamide mixed aqueous solution 40ul of 2% volume volume fraction are measured as denaturation agent, add as
SEQ ID NO:Specific double chain DNA fragment shown in 1(15umol)40ul, then total system is 400ul, is stood after fully mixing
4min.It should be noted that the urea of 2% mass volume fraction and the formamide of 2% volume volume fraction refer to 100ml distilled water
In dissolved with 2g urea and 2ml formamide, specific double chain DNA fragment is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd
Complementary single strand mixed in equal amounts form.
Control wells are set to add the above-mentioned denaturation agent of 40ul to add 40ul distilled water for the artificial ethanolic aqueous systems of 320ul simultaneously,
I.e. control is to substitute specific double chain DNA fragment with distilled water, and each artificial ethanolic aqueous system and corresponding control make even row three times,
ELISA Plate is placed in ELIASA after sample preparation is good(The Multiskan GO of Thermo companies of the U.S.)In.
(3)Artificial ethanolic aqueous system reclaimed water and ethanol mass volume ratio 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)Specific double-stranded DNA as in sample
The actual absorbance x of fragment, as shown in table 1.
Absorbance x and corresponding ethanolic aqueous system reclaimed water and ethanol mass volume ratio y scatterplot are observed, discovery is in
Existing linear trend(Fig. 1), it is y=- 62.317x+21.658 to be fitted to obtain relational expression, and R2=0.9971 shows linear relationship
Well, then this is ethanolic aqueous system reclaimed water and ethanol mass volume ratio assay method standard curve.
The ethanolic aqueous system reclaimed water of table 1 and ethanol mass volume ratio standard curve data
Mean light absorbency x | Water and ethanol mass volume ratio y |
0.163 | 11.500 |
0.173 | 10.905 |
0.180 | 10.364 |
0.190 | 9.870 |
0.196 | 9.417 |
0.203 | 9.000 |
Embodiment 2
Embodiment 2 is the reality that the non-sugar solidity for the yellow rice wine wine sample for being 8 to mark alcoholic strength using new method of the present invention is carried out
Measure.
(1)Select commercially available dragon's fountain yellow rice wine(Dalian Changxing Island Wine Co., Ltd produces), body mark alcoholic strength is 8 degree.It is accurate
Need to determine alcoholic strength again for the sake of really, therefore determine the alcoholic strength of this yellow rice wine using alcoholic strength meter method based on existing yellow rice wine national standard
For 8.2.Through weighing and converting, the quality of this yellow rice wine of 1L is 994.225g.Wine sample temperature is 30 DEG C, through looking into ethanol at this temperature
Density be 0.781g/ml.
(2)Yellow rice wine wine sample pre-treatment to be measured
320ul yellow rice wine wine samples to be measured are pipetted into the common ELISA Plate of sky with liquid-transfering gun, first add the urine of 2% mass volume fraction
Element and the formamide mixed aqueous solution 40ul of 2% volume volume fraction are added such as SEQ ID NO as denaturation agent:1 institute
The specific double chain DNA fragment shown(15umol)40ul, then total system is 400ul, and 4min is stood after fully mixing.
Control wells are set to add 40ul denaturation agent to add 40ul to distill aqueous systems for 320ul yellow rice wine wine samples to be measured simultaneously(Always
Volume is also 400ul), each yellow rice wine wine sample to be measured and corresponding control make even row three times, put ELISA Plate after sample preparation is good
In ELIASA(The Multiskan GO of Thermo companies of the U.S.)In.
(3)Yellow rice wine wine sample non-sugar solidity 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)Specific double-stranded DNA as in sample
Actual absorbance x=0.169 ± 0.001 of fragment, the relation substituted between water and ethanol mass volume ratio y and absorbance x
Formula is y=- 62.317x+21.658, calculates water and ethanol mass volume ratio y=11.126, as shown in table 2.Then total solid in wine sample
Thing content M=Mo-10 × ρ × v+10 × y × v is M=994.225-10 × 0.781 × 8.2-10 × 11.126 × 8.2, is computed
For 17.851g/L.
The empirical value of 2 embodiment of table 2
Yellow rice wine | Wine Essence Degree | Ethanol is close Degree/g × ml-1 | 1L wine sample matter Amount/g | Water and ethanol Mass volume ratio | The inventive method measure Total solid/g × L-1 | National Standard Method determines total Solid content/g × L-1 | Partially Poor % |
Dragon's fountain Yellow rice wine | 8.2 | 0.781 | 994.225 | 11.126 | 17.851 | 17.746 | 0.59 |
In addition, it is 17.746g/L to determine total solids content according to the 4h oven drying methods of national standard.If the total solid measured with National Standard Method
On the basis of thing content, then the inventive method measures numerical value and its deviation are 0.59%(Total solid that the inventive method measures with
The difference of national standard measured value accounts for the percentage of national standard measured value), meet the measurement accuracy of total solid.Finally again by total solid
Content subtracts the content that total sugar content obtains non-sugar solidity.Due to being to take commercially available finished wine to survey, its total sugar content is only marked
Scope is noted, its total reducing sugar is 15.1-40g/L if half-dry type yellow rice wine.In factory in practice, there is the specific measure numerical value of total reducing sugar, can
Directly bring use.
Embodiment 3
Embodiment 3 is the reality that the non-sugar solidity for the yellow rice wine wine sample for being 9 to mark alcoholic strength using new method of the present invention is carried out
Measure.
Select commercially available Jimo yellow rice wine(Co., Ltd of Shandong Jimo Rice Winery produces), body mark alcoholic strength is 9 degree.It is accurate to rise
Seeing needs to determine alcoholic strength again, thus based on existing yellow rice wine national standard use alcoholic strength meter method determine the alcoholic strength of this yellow rice wine for
8.9.Through weighing and converting, the quality of this yellow rice wine of 1L is 1069.672g.Wine sample temperature is 30 DEG C, through looking into ethanol at this temperature
Density be 0.781g/ml.
It is measured with reference to embodiment 2, actual absorbance x=0.182 ± 0.002, correlation values refer to table 3.If with national standard
On the basis of the total solids content that method measures, then the inventive method measures numerical value and its deviation are 0.47%, meet total solid
Measurement accuracy.Total solids content is finally subtracted into the content that total sugar content obtains non-sugar solidity again.Due to being to take city
Finished wine is sold to survey, its total sugar content only marks scope, and total reducing sugar is 15.1-40g/L if half-dried.In factory in practice, have total
The specific measure numerical value of sugar, can directly bring use.
The empirical value of 3 embodiment of table 3
Yellow rice wine | Wine Essence Degree | Ethanol is close Degree/g × ml-1 | 1L wine sample matter Amount/g | Water and ethanol Mass volume ratio | The inventive method measure Total solid/g × L-1 | National Standard Method determines total Solid content/g × L-1 | Deviation % |
Jimo Yellow rice wine | 8.9 | 0.781 | 1069.672 | 10.316 | 82.039 | 81.654 | 0.47 |
In summary, non-sugar solidity assay method of the invention is applied to the measure of 8-10 degree yellow rice wine, has well accurate
Property and reliability;It is safer without long time drying compared with national standard 4h oven drying methods, operate 10min and simple, convenient and rapid;In addition
Required wine sample amount is seldom, and testing cost is very cheap less than 1 yuan/sample, is suitable for such yellow rice wine non-sugar solidity in brewery and surveys
Fixed practice.
Sequence table
<110>Chen Rukai
<120>8 to 10 degree yellow rice wine non-sugar solidity simplicity rapid assay methods
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ctagcaatac 10
Claims (3)
1.8 to 10 degree yellow rice wine non-sugar solidity simplicity rapid assay methods, it is characterised in that the yellow rice wine non-sugar solidity is easy
Rapid assay methods mainly comprise the following steps:
(1)In yellow rice wine Quality Detection, first determining its alcoholic strength v according to existing national standard, this must survey index, and v needs to meet
8.0≤v≤10.0, while weighed 1L wine sample mass M o, determine the temperature of wine sample and inquire about ethanol density p accordingly;
(2)320ul room temperatures yellow rice wine wine sample to be measured is pipetted into the ELISA Plate of sky, the agent of 40ul denaturations is added and 40ul is specific double
Chain DNA fragment simultaneously fully stands 4min after mixing, while sets control wells to add 40ul denaturation agent to add for 320ul wine samples to be measured
40ul distilled water, wine sample to be measured and control make even row survey three times, and ELISA Plate is placed in ELIASA after sample process is good;
(3)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 x of specific double chain DNA fragment in sample, and x is substituted into relation
The mass volume ratio y of wine sample reclaimed water and ethanol can be then calculated in formula y=- 62.317x+21.658, then total solids content in wine sample
M=Mo-10×(ρ+y)× v, and then non-sugar solidity content m=M-Mt, Mt are total sugar content.
2. 8 to 10 degree yellow rice wine non-sugar solidity simplicity rapid assay methods according to claim 1, it is characterised in that step
(2)Described in specific double chain DNA fragment, its sequence such as SEQ ID NO:Shown in 1, concentration 15umol.
3. 8 to 10 degree yellow rice wine non-sugar solidity simplicity rapid assay methods according to claim 1, it is characterised in that step
(2)Described in denaturation agent be 2% mass volume fraction urea and 2% volume volume fraction formamide mixing it is water-soluble
Liquid.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101216419A (en) * | 2007-12-28 | 2008-07-09 | 陈郁 | Method for quickly detecting yellow wine quality index |
CN102759607A (en) * | 2012-07-23 | 2012-10-31 | 福州大学 | Method for rapidly determining non-sugar solid in yellow rice wine |
-
2017
- 2017-11-06 CN CN201711074974.4A patent/CN107643262A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101216419A (en) * | 2007-12-28 | 2008-07-09 | 陈郁 | Method for quickly detecting yellow wine quality index |
CN102759607A (en) * | 2012-07-23 | 2012-10-31 | 福州大学 | Method for rapidly determining non-sugar solid in yellow rice wine |
Non-Patent Citations (3)
Title |
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张枫等: "《医学化学基础 第2版》", 31 December 2010 * |
王泉云等: "偶联酶法测定微量乙醇的研究", 《华西医学》 * |
黄亚东: "《白酒生产技术 第2版》", 31 December 2014, 中国轻工业出版社 * |
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