CN106568848A - Method for detecting ethanol in living lactobacillus beverage - Google Patents
Method for detecting ethanol in living lactobacillus beverage Download PDFInfo
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- CN106568848A CN106568848A CN201510651237.0A CN201510651237A CN106568848A CN 106568848 A CN106568848 A CN 106568848A CN 201510651237 A CN201510651237 A CN 201510651237A CN 106568848 A CN106568848 A CN 106568848A
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- ethanol
- detection
- lactobacillus beverage
- type lactobacillus
- viable type
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 267
- 235000013361 beverage Nutrition 0.000 title claims abstract description 98
- 241000186660 Lactobacillus Species 0.000 title claims abstract description 94
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 94
- 238000000034 method Methods 0.000 title claims abstract description 74
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 108
- 238000001514 detection method Methods 0.000 claims abstract description 98
- 239000007788 liquid Substances 0.000 claims description 36
- 239000007789 gas Substances 0.000 claims description 29
- 239000006228 supernatant Substances 0.000 claims description 21
- 238000012360 testing method Methods 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 239000012159 carrier gas Substances 0.000 claims description 16
- 238000004587 chromatography analysis Methods 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 238000005070 sampling Methods 0.000 claims description 8
- 238000004817 gas chromatography Methods 0.000 claims description 7
- 239000012086 standard solution Substances 0.000 claims description 5
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims 1
- 239000008267 milk Substances 0.000 claims 1
- 210000004080 milk Anatomy 0.000 claims 1
- 235000013336 milk Nutrition 0.000 claims 1
- 230000008901 benefit Effects 0.000 abstract description 9
- 239000000203 mixture Substances 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 19
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 12
- 239000012535 impurity Substances 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 239000012528 membrane Substances 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- IMBKASBLAKCLEM-UHFFFAOYSA-L ferrous ammonium sulfate (anhydrous) Chemical compound [NH4+].[NH4+].[Fe+2].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O IMBKASBLAKCLEM-UHFFFAOYSA-L 0.000 description 4
- 230000005484 gravity Effects 0.000 description 4
- 229910052700 potassium Inorganic materials 0.000 description 4
- 239000011591 potassium Substances 0.000 description 4
- 206010019133 Hangover Diseases 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 235000013618 yogurt Nutrition 0.000 description 3
- 206010017533 Fungal infection Diseases 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002309 gasification Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 238000010926 purge Methods 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000006200 vaporizer Substances 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Sampling And Sample Adjustment (AREA)
- Dairy Products (AREA)
Abstract
The invention provides a method for detecting ethanol in a living lactobacillus beverage. The method comprises the following steps: (1) mixing a living lactobacillus beverage with acetonitrile, subjecting the mixture to a centrifugal treatment, and collecting the supernate; and (2) subjecting the supernate to chromatographic detection, and determining the ethanol content of the living lactobacillus beverage based on the detection result. The provided method at least has one advantage of convenient operation, rapidness, high precision, and high accuracy.
Description
Technical field
The present invention relates to field of food.In particular it relates to detect ethanol in viable type lactobacillus beverage
Method.
Background technology
Easily by yeast-infection in viable type lactobacillus beverage production process, cause product swollen bag occur, affect
Product quality.The metabolic characteristic of ethanol is produced using yeast fermentation, by the content for detecting ethanol, can be sentenced
Whether pregnancy ceased product are by yeast-infection.
However, at present the method for ethanol still has much room for improvement in detection viable type lactobacillus beverage.
The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.For this purpose, one of the present invention
Purpose is the method for providing ethanol in a kind of detection viable type lactobacillus beverage, the method is easy to operate, quick,
Precision is high or accuracy is strong.
It should be noted that the present invention is completed based on the following discovery of inventor：
At present, detecting the method for ethanol in beverage mainly includes hydrometer method and potassium dichromate-Ferrous ammonium sulfate oxygen
Change reductometry.When gravimeter is floated in a liquid, the gravity phase of the liquid that the gravity of itself is arranged with it
Deng proportion gauge method mainly uses the principle and detected.However, hydrometer method typically cannot be measured accurately
Low content ethanol.Potassium dichromate-Ferrous ammonium sulfate oxidimetry is that potassium dichromate is used in sulfuric acid medium
Ethanol in oxidised samples, in the presence of phenanthroline ferrum indicator solution is as indicator, is titrated with Ferrous ammonium sulfate
Excessive potassium dichromate, according to the second in the addition of potassium dichromate and the consumption calculating sample of Ferrous ammonium sulfate
Alcohol content.However, the method is excessively loaded down with trivial details, and accuracy is relatively low.
The present inventor has found through many experiments, carries out after viable type lactobacillus beverage is mixed with acetonitrile
Centrifugal treating, and supernatant is collected, chromatography detection is carried out to supernatant, and based on resulting testing result
Determine the content of ethanol in viable type lactobacillus beverage.Thus, the side of ethanol in viable type lactobacillus beverage is detected
Method has at least one of following advantages：Easy to operate, quick, precision is high and accuracy is strong.
In a first aspect of the present invention, the present invention proposes a kind of side of ethanol in detection viable type lactobacillus beverage
Method.Embodiments in accordance with the present invention, the method includes：(1) the viable type lactobacillus beverage is mixed with acetonitrile
Centrifugal treating is carried out after conjunction, and collects supernatant；And (2) carry out chromatography detection to the supernatant,
And the content of ethanol in the viable type lactobacillus beverage is determined based on resulting testing result.Using viable type
Ethanol can be dissolved in acetonitrile phase and the impurity such as protein the characteristics of do not dissolve in acetonitrile phase in lactobacillus beverage, by acetonitrile
Mix with viable type lactobacillus beverage to be measured, extract ethanol, reduce other impurities and subsequent detection is done
Disturb.Thus, detect that the method for ethanol in viable type lactobacillus beverage has at least one of following advantages：Operation
Easy, quick, precision is high and accuracy is strong.
Embodiments in accordance with the present invention, the method for ethanol can also have in above-mentioned detection viable type lactobacillus beverage
Following additional technical feature：
Embodiments in accordance with the present invention, based on 1 milliliter of viable type lactobacillus beverage, the consumption of acetonitrile is
1～10 milliliter.Thus, the method for ethanol can in detection viable type lactobacillus beverage according to embodiments of the present invention
Further to have easier, quick operation, higher precision or stronger accuracy.
Embodiments in accordance with the present invention, the centrifugation is to carry out 3～10 according to the rotating speed of 3000rpm～8000rpm
Minute.Thus, the method for ethanol can enter one in detection viable type lactobacillus beverage according to embodiments of the present invention
Step has easier, quick operation, higher precision or stronger accuracy.
Embodiments in accordance with the present invention, before step (2) is carried out, filter in advance to the supernatant,
And resulting filtrate is carried out into the chromatography detection.Thus, detection viable type according to embodiments of the present invention
In lactobacillus beverage the method for ethanol can further have easier, quick operation, higher precision or
The stronger accuracy of person.
Embodiments in accordance with the present invention, the filtration adopts 0.22 micron of filter membrane.Thus, according to the present invention
The method of ethanol can further have easier, quick behaviour in the detection viable type lactobacillus beverage of embodiment
Work, higher precision or stronger accuracy.
Embodiments in accordance with the present invention, the viable type lactobacillus beverage is Yoghourt.Thus, according to of the invention real
Applying the method for ethanol in the detection viable type lactobacillus beverage of example can further have easier, quick behaviour
Work, higher precision or stronger accuracy.
Embodiments in accordance with the present invention, before the chromatography detection is carried out, in advance by second in the supernatant
The concentration of alcohol is adjusted to 1mg/100mL～32mg/100mL.Thus, detection according to embodiments of the present invention is lived
The method of ethanol can further have easier, quick operation, higher precision in bacterial type lactobacillus beverage
Degree or stronger accuracy.
Embodiments in accordance with the present invention, the chromatography is detected as gas chromatography detection, the gas chromatography
Detection is carried out according to following condition：Chromatographic column：CD-2560 posts；Injector temperature：180℃；Detection
Device temperature：200℃；Sampling volume：1 microlitre；Flow rate of carrier gas：2.0 ml/min；Carrier gas is high pure nitrogen,
Purity 99.999%；Split ratio is 20：1；Heating schedule：80 DEG C of initial temperature, is kept for 1 minute；With 10 DEG C
/ minute rises to 150 DEG C, is kept for 2 minutes；10 minutes total times.Thus, inspection according to embodiments of the present invention
Surveying the method for ethanol in viable type lactobacillus beverage can further have easier, quick operation, higher
Precision or stronger accuracy.
In a second aspect of the present invention, the present invention proposes a kind of side of ethanol in detection viable type lactobacillus beverage
Method.Embodiments in accordance with the present invention, the method includes：The first step, draws 5mL viable type lactobacillus beverages
Sample to be tested is settled to scale in 25mL volumetric flasks with acetonitrile, mixes, and is centrifuged under the rotating speed of 5000rpm
5 minutes, supernatant is collected, cross 0.22 μm of organic filter membrane, obtain prepare liquid.Second step, to liquid to be detected
Carry out gas chromatographic detection：(1) gas chromatogram detection limit is determined：Can be with by 3 times of signal to noise ratios (S/N=3)
Ethanol is carried out qualitative, 10 times of signal to noise ratios (S/N=10) can be carried out quantitatively to ethanol.It is in content
During 2mg/100mL, peak height signal is 2146.8, and noise is 70, and signal to noise ratio now is 30.7, based on 10
Times signal to noise ratio, the quantitative detection for determining instrument is limited to 0.65mg/100mL, because prepare liquid is entered according to sample
5 times of row dilutes what is obtained, and then calculates the detection of sample and be limited to 3.25mg/100mL.Wherein, signal to noise ratio=
Peak height signal/noise；Quantitative detection limit=(10 × concentration of alcohol × noise)/peak height signal.(2) standard curve
Drafting：Ethanol in standard solution is detected with gas chromatograph, with peak area Y to its concentration X
(mg/100mL) the standard curve Y=50.19385*X of ethanol, r are obtained2=0.9999, its correlation coefficient is more than
0.99, illustrate that linear relationship is good.(3) concentration of alcohol in prepare liquid：With gas chromatograph to second in prepare liquid
Alcohol is detected that peak area corresponding concentration on standard curve is the content of ethanol in prepare liquid.(4) treat
Concentration of alcohol in test sample sheet：Extension rate is multiplied by according to the content of ethanol in prepare liquid and is calculated viable type to be measured
The content of ethanol in lactobacillus beverage.Gas chromatographic detection condition：Detector is flame ionization ditector
(FID), chromatographic column be CD-2560 posts (100 meters × 0.25 millimeter, 0.2 micron of Φ), injector temperature：
180℃；Detector temperature：200℃；Sampling volume：1 microlitre；Flow rate of carrier gas：2.0 ml/min；Carry
Gas is high pure nitrogen, purity 99.999%；Split ratio is 20：1；Heating schedule：80 DEG C of initial temperature, protects
Hold 1 minute；150 DEG C are risen to 10 DEG C/min, is kept for 2 minutes；10 minutes total times.Thus, according to
The method of ethanol can further have easier, fast in the detection viable type lactobacillus beverage of the embodiment of the present invention
The operation of speed, higher precision or stronger accuracy.
Additionally, embodiments in accordance with the present invention, the method tool of ethanol in present invention detection viable type lactobacillus beverage
There are at least one of following advantages：
1st, embodiments in accordance with the present invention, can be dissolved in acetonitrile phase using ethanol in viable type lactobacillus beverage and
The characteristics of impurity such as protein do not dissolve in acetonitrile phase, acetonitrile is mixed with viable type lactobacillus beverage to be measured, is carried
Ethanol is taken out, interference of the other impurities to subsequent detection is reduced.
2nd, embodiments in accordance with the present invention, are carried out using gas chromatography to ethanol in viable type lactobacillus beverage
Detection, can relatively accurately determine ethanol content, and detection time is short, the response rate is high.
The additional aspect and advantage of the present invention will be set forth in part in the description, partly will be from explained below
In become obvious, or by the present invention practice recognize.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will from the description with reference to accompanying drawings below to embodiment
Become obvious and easy to understand, wherein：
The method that Fig. 1 shows ethanol in detection viable type lactobacillus beverage according to an embodiment of the invention
Schematic flow sheet；
Fig. 2 shows the side of ethanol in detection viable type lactobacillus beverage in accordance with another embodiment of the present invention
The schematic flow sheet of method；And
Fig. 3 shows the gas chromatogram of ethanol in viable type lactobacillus beverage according to an embodiment of the invention
Figure.
Specific embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining
The present invention, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for describing purpose, and it is not intended that indicating
Or hint relative importance or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ",
One or more this feature can be expressed or be implicitly included to the feature of " second ".Further, exist
In description of the invention, unless otherwise stated, " multiple " are meant that two or more.
It should be noted that the present invention is completed based on the following discovery of inventor：
At present, detecting the method for ethanol in beverage mainly includes hydrometer method and potassium dichromate-Ferrous ammonium sulfate oxygen
Change reductometry.When gravimeter is floated in a liquid, the gravity phase of the liquid that the gravity of itself is arranged with it
Deng proportion gauge method mainly uses the principle and detected.However, hydrometer method typically cannot be measured accurately
Low content ethanol.Potassium dichromate-Ferrous ammonium sulfate oxidimetry is that potassium dichromate is used in sulfuric acid medium
Ethanol in oxidised samples, in the presence of phenanthroline ferrum indicator solution is as indicator, is titrated with Ferrous ammonium sulfate
Excessive potassium dichromate, according to the second in the addition of potassium dichromate and the consumption calculating sample of Ferrous ammonium sulfate
Alcohol content.However, the method is excessively loaded down with trivial details, and accuracy is relatively low.
The present inventor has found through many experiments, carries out after viable type lactobacillus beverage is mixed with acetonitrile
Centrifugal treating, and supernatant is collected, chromatography detection is carried out to supernatant, and based on resulting testing result
Determine the content of ethanol in viable type lactobacillus beverage.Thus, the side of ethanol in viable type lactobacillus beverage is detected
Method has at least one of following advantages：Easy to operate, quick, precision is high and accuracy is strong.
Thus, the method that the present invention proposes ethanol in a kind of detection viable type lactobacillus beverage, will be made below
Describe in detail.
In a first aspect of the present invention, the present invention proposes a kind of side of ethanol in detection viable type lactobacillus beverage
Method.With reference to Fig. 1, embodiments in accordance with the present invention, the method for ethanol can in the detection viable type lactobacillus beverage
To comprise the following steps, thus, the method for ethanol has following excellent in final detection viable type lactobacillus beverage
At least one of point：Easy to operate, quick, precision is high and accuracy is strong.
S100 mixes, centrifugation, collects supernatant
Centrifugal treating is carried out after viable type lactobacillus beverage is mixed with acetonitrile, and collects supernatant.Due to viable bacteria
Ethanol content is relatively low in type lactobacillus beverage, and containing many impurity.If directly carrying out chromatograph detection after centrifugation,
Testing result has error, and also chromatographic column can be damaged.Inventor obtains acetonitrile and makees through many experiments screening
For extractant.Containing materials such as substantial amounts of protein in viable type lactobacillus beverage, some extractants can be by ethanol
Together extract with the impurity such as protein, and the impurity such as protein can make follow-up gas chromatographic detection produce mistake
Difference, additionally, the impurity such as protein can also cause to damage to gas chromatogram instrument.Because protein does not dissolve in acetonitrile,
To in non-acetonitrile phase, ethanol is completely dissolved in acetonitrile to make protein precipitation in extraction process.Thus, detection is lived
The method of ethanol has at least one of following advantages in bacterial type lactobacillus beverage：Easy to operate, quick, precision
Degree is high and accuracy is strong.
Embodiments in accordance with the present invention, based on 1 milliliter of viable type lactobacillus beverage, the consumption of acetonitrile is 1～10
Milliliter.Inventor obtains optimum acetonitrile content through many experiments optimization.Acetonitrile content is very few, it is impossible to extract completely
Get ethanol.Additionally, the cumulative volume based on both acetonitrile and viable type lactobacillus beverage is constant, if acetonitrile content
Very few, then the used in amounts of viable type lactobacillus beverage will increase accordingly, but contain in viable type lactobacillus beverage
There is substantial amounts of water, because water has larger evaporation swelling volume, sample during gas chromatographic detection will be caused
Gasification swelling up bushing pipe, the sample of gasification is returned into carrier gas and purging gas circuit, due to the temperature of the purging gas circuit of carrier gas
Degree is much lower compared with vaporizer, and sample can be condensed in this, be blown into analysis system by gas in analysis later and formed
Ghost peak, has undesirable effect to testing result.Acetonitrile content excessively by the concentration of alcohol,diluted, makes testing result
It is inaccurate.Thus, the method for ethanol can be entered in detection viable type lactobacillus beverage according to embodiments of the present invention
One step has easier, quick operation, higher precision or stronger accuracy.
Embodiments in accordance with the present invention, centrifugation is to carry out according to the rotating speed of 3000rpm～8000rpm at room temperature
3～10 minutes.The rotating speed of centrifugation is too low or the time it is too short can prevent ethanol from being dissolved completely in acetonitrile, and
Have excessive impurity to be dissolved in acetonitrile phase.Thus, detection viable type lactic acid bacteria according to embodiments of the present invention
The method of ethanol can further have easier, quick operation, higher precision or stronger in beverage
Accuracy.
Embodiments in accordance with the present invention, viable type lactobacillus beverage is Yoghourt.Thus, according to embodiments of the present invention
Detection viable type lactobacillus beverage in ethanol method can further have easier, quick operation, compared with
High precision or stronger accuracy.
S200 chromatographs are detected
Chromatography detection is carried out to supernatant, and viable type lactobacillus beverage is determined based on resulting testing result
The content of middle ethanol.
Referring to Fig. 2, embodiments in accordance with the present invention, before step S200 is carried out, supernatant is entered in advance
Row filters S110, and resulting filtrate is carried out into chromatography detection S200.
Embodiments in accordance with the present invention, filter using 0.22 micron of filter membrane.Thus, according to present invention enforcement
Example detection viable type lactobacillus beverage in ethanol method can further have easier, quick operation,
Higher precision or stronger accuracy.
Term " filter membrane " used in the present invention is known to those skilled in the art.For the material of filter membrane,
The present invention does not make considered critical, every to play a role in filtering, and does not affect the filter membrane that ethanol content changes all may be used
For in the present invention.Specific example of the invention, present invention filter membrane used is organic filter membrane, model
13mm × 0.22 μm, aperture is 0.22 μm.
Embodiments in accordance with the present invention, viable type lactobacillus beverage is Yoghourt.Thus, according to embodiments of the present invention
Detection viable type lactobacillus beverage in ethanol method can further have easier, quick operation, compared with
High precision or stronger accuracy.
Embodiments in accordance with the present invention, before chromatography detection S200 is carried out, in advance by ethanol in supernatant
Concentration adjust to 1mg/100mL～32mg/100mL.Gas chromatogram has different detections to different material
Limit, concentration of alcohol is too high, causes peak area excessive, as a result has deviation, and concentration of alcohol is too low, peak area mistake
It is little, can also make result produce deviation.Thus, in detection viable type lactobacillus beverage according to embodiments of the present invention
The method of ethanol can further have easier, quick operation, higher precision or stronger accurate
Property.
Embodiments in accordance with the present invention, chromatography is detected as gas chromatography detection, the gas chromatography detection
Carry out according to following condition：Chromatographic column：CD-2560 posts；Injector temperature：180℃；Detector temperature
Degree：200℃；Sampling volume：1 microlitre；Flow rate of carrier gas：2.0 ml/min；Carrier gas is high pure nitrogen, pure
Degree 99.999%；Split ratio is 20：1；Heating schedule：80 DEG C of initial temperature, is kept for 1 minute；With 10 DEG C
/ minute rises to 150 DEG C, is kept for 2 minutes；10 minutes total times.Specific example of the invention, chromatograph
The size of post CD-2530 posts is 100 meters × 0.25 millimeter × 0.2 micron.Thus, it is according to embodiments of the present invention
The method of ethanol can further have easier, quick operation, higher in detection viable type lactobacillus beverage
Precision or stronger accuracy.
In a second aspect of the present invention, the present invention proposes a kind of side of ethanol in detection viable type lactobacillus beverage
Method.Embodiments in accordance with the present invention, the method includes：The first step, draws 5mL viable type lactobacillus beverages
Sample to be tested is settled to scale in 25mL volumetric flasks with acetonitrile, mixes, and is centrifuged under the rotating speed of 5000rpm
5 minutes, supernatant is collected, cross 0.22 μm of organic filter membrane, obtain prepare liquid.Second step, to liquid to be detected
Carry out gas chromatographic detection：(1) gas chromatogram detection limit is determined：Can be with by 3 times of signal to noise ratios (S/N=3)
Ethanol is carried out qualitative, 10 times of signal to noise ratios (S/N=10) can be carried out quantitatively to ethanol.It is in content
During 2mg/100mL, peak height signal is 2146.8, and noise is 70, and signal to noise ratio now is 30.7, based on 10
Times signal to noise ratio, the quantitative detection for determining instrument is limited to 0.65mg/100mL, because prepare liquid is entered according to sample
5 times of row dilutes what is obtained, and then calculates the detection of sample and be limited to 3.25mg/100mL.Wherein, signal to noise ratio=
Peak height signal/noise；Quantitative detection limit=(10 × concentration of alcohol × noise)/peak height signal.(2) standard curve
Drafting：Ethanol in standard solution is detected with gas chromatograph, with peak area Y to its concentration X
(mg/100mL) the standard curve Y=50.19385*X of ethanol, r are obtained2=0.9999, its correlation coefficient is more than
0.99, illustrate that linear relationship is good.(3) concentration of alcohol in prepare liquid：With gas chromatograph to second in prepare liquid
Alcohol is detected that peak area corresponding concentration on standard curve is the content of ethanol in prepare liquid.(4) treat
Concentration of alcohol in test sample sheet：Extension rate is multiplied by according to the content of ethanol in prepare liquid and is calculated viable type to be measured
The content of ethanol in lactobacillus beverage.Gas chromatographic detection condition：Detector is flame ionization ditector
(FID), chromatographic column be CD-2560 posts (100 meters × 0.25 millimeter, 0.2 micron of Φ), injector temperature：
180℃；Detector temperature：200℃；Sampling volume：1 microlitre；Flow rate of carrier gas：2.0 ml/min；Carry
Gas is high pure nitrogen, purity 99.999%；Split ratio is 20：1；Heating schedule：80 DEG C of initial temperature, protects
Hold 1 minute；150 DEG C are risen to 10 DEG C/min, is kept for 2 minutes；10 minutes total times.Thus, according to
The method of ethanol can further have easier, fast in the detection viable type lactobacillus beverage of the embodiment of the present invention
The operation of speed, higher precision or stronger accuracy.
To sum up, embodiments in accordance with the present invention, the method tool of ethanol in present invention detection viable type lactobacillus beverage
There are at least one of following advantages：
1st, embodiments in accordance with the present invention, can be dissolved in acetonitrile phase using ethanol in viable type lactobacillus beverage and
The characteristics of impurity such as protein do not dissolve in acetonitrile phase, acetonitrile is mixed with viable type lactobacillus beverage to be measured, is carried
Ethanol is taken out, interference of the other impurities to subsequent detection is reduced.
2nd, embodiments in accordance with the present invention, are carried out using gas chromatography to ethanol in viable type lactobacillus beverage
Detection, can relatively accurately determine ethanol content, and detection time is short, the response rate is high.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that under
The embodiment in face is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted in embodiment
Particular technique or condition, according to the technology or condition described by document in the art or according to the description of product
Book is carried out.Agents useful for same or the unreceipted production firm person of instrument, be can pass through city available from conventional products.
In the following embodiments, if do not clearly stated, the test apparatuses that adopt and reagent for：
Without methanol/ethanol：Chromatographically pure, density 0.789g/mL；Acetonitrile：Chromatographically pure, purity is 99.9%.
The preparation of standard solution：Accurately draw without the μ L of methanol/ethanol 316.9 in 25mL volumetric flasks, use acetonitrile
Scale is settled to, ethanol content is made into for 10mg/mL intermediate standard liquid.Draw 10mg/mL when using respectively
Middle interstitial fluid 0.02mL, 0.04mL, 0.08mL, 0.16mL, 0.32mL distinguish in 10mL volumetric flasks
Be settled to scale with acetonitrile, shake up, obtain ethanol content for 2mg/100mL, 4mg/100mL, 8mg/100mL,
The standard working solution of five Concentraton gradient of 16mg/100mL, 32mg/100mL.
Embodiment 1
In this embodiment, the ethanol in viable type lactobacillus beverage detected through the following steps：
The first step, draws 5mL viable type lactobacillus beverage samples to be tested in 25mL volumetric flasks, uses acetonitrile
Scale is settled to, is mixed, be centrifuged 5 minutes under the rotating speed of 5000rpm, collect supernatant, crossing 0.22 μm has
Machine filter film, obtains prepare liquid.
Second step, to liquid to be detected gas chromatographic detection is carried out：
(1) gas chromatogram detection limit is determined
Qualitative, 10 times of signal to noise ratio (S/N=10) energy can be carried out to ethanol by 3 times of signal to noise ratios (S/N=3)
It is enough that ethanol is carried out quantitatively.When content is 2mg/100mL, peak height signal is 2146.8, and noise is 70,
Signal to noise ratio now is 30.7, and based on 10 times of signal to noise ratios, the quantitative detection for determining instrument is limited to 0.65mg/
100mL, obtains, and then calculates the detection of sample being limited to because prepare liquid carries out 5 times of dilutions according to sample
3.25mg/100mL.Wherein, signal to noise ratio=peak height signal/noise；Quantitative detection limit=(10 × concentration of alcohol × make an uproar
Sound)/peak height signal.
(2) drafting of standard curve
Ethanol in standard solution is detected with gas chromatograph, with peak area Y to its concentration X
(mg/100mL) the standard curve Y=50.19385*X of ethanol, r are obtained2=0.9999, its correlation coefficient is more than
0.99, illustrate that linear relationship is good.
(3) concentration of alcohol in prepare liquid
Ethanol in prepare liquid is detected with gas chromatograph, peak area corresponding concentration on standard curve is
For the content of ethanol in prepare liquid.
(4) concentration of alcohol in sample to be tested
Extension rate is multiplied by according to the content of ethanol in prepare liquid to be calculated in viable type lactobacillus beverage to be measured
The content of ethanol.
Gas chromatographic detection condition：
Detector be flame ionization ditector (FID), chromatographic column be CD-2560 posts (100 meters of Φ ×
0.25 millimeter, 0.2 micron), injector temperature：180℃；Detector temperature：200℃；Sampling volume：1
Microlitre；Flow rate of carrier gas：2.0 ml/min；Carrier gas is high pure nitrogen, purity 99.999%；Split ratio is 20：
1；Heating schedule：80 DEG C of initial temperature, is kept for 1 minute；150 DEG C are risen to 10 DEG C/min, 2 are kept
Minute；10 minutes total times.
Embodiment 2
According to ethanol in the method detection viable type lactobacillus beverage of embodiment 1, difference is, in the first step,
Absorption 5mL viable type lactobacillus beverage samples to be tested are in 10mL volumetric flasks；
Centrifugation is to carry out 10 minutes under rotating speed according to 3000r/min.
Embodiment 3
According to ethanol in the method detection viable type lactobacillus beverage of embodiment 1, difference is, in the first step,
Absorption 5mL viable type lactobacillus beverage samples to be tested are in 50mL volumetric flasks；
Centrifugation is to carry out 3 minutes under rotating speed according to 8000r/min.
Comparative example 1
According to ethanol in the method detection viable type lactobacillus beverage of embodiment 1, difference is, in the first step,
Absorption 3mL viable type lactobacillus beverage samples to be tested are in 50mL volumetric flasks.
Comparative example 2
According to ethanol in the method detection viable type lactobacillus beverage of embodiment 1, difference is, in the first step,
Absorption 7mL viable type lactobacillus beverage samples to be tested are in 10mL volumetric flasks.
Comparative example 3
According to ethanol in the method detection viable type lactobacillus beverage of embodiment 1, difference is, in second step,
Chromatographic column is CP Sil 19CB (30 meters × 0.32 millimeter, 0.25 micron of Φ).
Comparative example 4
According to ethanol in the method detection viable type lactobacillus beverage of embodiment 1, difference is, in second step,
GC conditions：
Detector be flame ionization ditector (FID), chromatographic column be CD-2560 posts (100 meters of Φ ×
0.25 millimeter, 0.2 micron)；
Injector temperature：160℃；
Detector temperature：200℃；
Sampling volume：1 microlitre；
Flow rate of carrier gas：1.0 ml/mins, carrier gas is high pure nitrogen, purity 99.999%；
Split ratio is 20：1；
Heating schedule：120 DEG C of constant temperature is kept for 18 minutes.
The Analysis of test results of ethanol in viable type lactobacillus beverage
1st, the measure of concentration of alcohol
It is as shown in table 1 according to the concentration of alcohol that embodiment 1～3 and the detection of comparative example 1～4 are obtained, as a result show
Embodiment 1～3, comparative example 1 and comparative example 4 accurate can detect ethanol content in sample, but as schemed
Shown in 3, comparative example 3 is detected using chromatographic column CP Sil 19CB, and resulting collection of illustrative plates hangover is serious,
Quantitative experiment cannot be carried out；Because extension rate is less in comparative example 2, it is impossible to protein is precipitated completely, sample
Liquid is muddy, it is impossible to carry out quantitative experiment.
The testing result of the concentration of alcohol of table 1
2nd, response rate checking
Add ethanol in viable type lactobacillus beverage sample and obtain mark-on sample, the addition concentration of ethanol is
4mg/100mL、8mg/100mL、40mg/100mL.According to embodiment 1 method detection mark-on sample and
The concentration of ethanol not plus in target viable type lactobacillus beverage, and the response rate is calculated according to testing result, wherein returning
In yield (%)=100 × (concentration of alcohol in mark-on sample-plus concentration of alcohol in target sample)/mark-on sample
Ethanol theoretical concentration, as a result as shown in table 2.As a result show, the ethanol ult rec that embodiment 1～3 is obtained
Between 89.5%～108%, the method response rate can meet the needs of detection, and for comparative example 1～4, it is right
The response rate of ratio 1 is between 99.6%～100.2%, but because extension rate is larger, and response signal is less, low
Integration manually is needed during concentration point, workload is increased；Because extension rate is less in comparative example 2, it is impossible to make
Protein is precipitated completely, and sample liquid is muddy, it is impossible to carry out quantitative experiment；Chromatographic column hangover used in comparative example 3
Seriously, it is impossible to carry out quantitative experiment；The response rate of comparative example 4 is between 86.8%～103.9%, and the response rate is slightly lower
In embodiment 1～3 and analysis time is longer.
The response rate of table 2 is verified
3rd, precision checking
Embodiment 1～3 and the duplicate detection 6 of comparative example 1～4 are used respectively to the mark-on sample of different addition concentration
It is secondary, the relative standard deviation (RSD%) of 6 testing results is calculated, as a result as shown in table 3.As a result show,
The relative standard deviation of embodiment 1～3 is less than 15%, and method precision can meet the needs of detection,
And in comparative example 1～4, the precision of comparative example 1 between 4.8%～5.4%, due to extension rate in comparative example 2
Less, it is impossible to protein is precipitated completely, sample liquid is muddy, it is impossible to carry out precision test；Institute in comparative example 3
It is serious using chromatographic column hangover, it is impossible to carry out precision test；The response rate of comparative example 4 is between 2.3%～4.2%.
The precision of table 3 is verified
In the description of this specification, reference term " one embodiment ", " another embodiment ", " some realities
Apply example ", " example ", the description of " specific example " or " some examples " etc. mean with reference to the embodiment or show
Specific features, structure, material or the feature that example is described is contained at least one embodiment or example of the present invention
In.In this manual, identical embodiment or example are not necessarily referring to the schematic representation of above-mentioned term.
And, the specific features of description, structure, material or feature can in any one or more embodiments or
Combine in an appropriate manner in example.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：
Without departing from the present invention principle and objective in the case of these embodiments can be carried out various changes, modification,
Replace and modification, the scope of the present invention is limited by claim and its equivalent.
Claims (9)
1. it is a kind of detection viable type lactobacillus beverage in ethanol method, it is characterised in that include：
The first step, draws 5mL viable type lactobacillus beverage samples to be tested in 25mL volumetric flasks, uses acetonitrile
Scale is settled to, is mixed, be centrifuged 5 minutes under the rotating speed of 5000rpm, collect supernatant, crossing 0.22 μm has
Machine filter film, obtains prepare liquid.
Second step, to liquid to be detected gas chromatographic detection is carried out：
(1) gas chromatogram detection limit is determined
Qualitative, 10 times of signal to noise ratio (S/N=10) energy can be carried out to ethanol by 3 times of signal to noise ratios (S/N=3)
It is enough that ethanol is carried out quantitatively.When content is 2mg/100mL, peak height signal is 2146.8, and noise is 70,
Signal to noise ratio now is 30.7, and based on 10 times of signal to noise ratios, the quantitative detection for determining instrument is limited to
0.65mg/100mL, obtains because prepare liquid carries out 5 times of dilutions according to sample, and then calculates sample
Detection is limited to 3.25mg/100mL.Wherein, signal to noise ratio=peak height signal/noise；Quantitative detection limit=(10 × second
Determining alcohol × noise)/peak height signal.
(2) drafting of standard curve
Ethanol in standard solution is detected with gas chromatograph, with peak area Y to its concentration X
(mg/100mL) the standard curve Y=50.19385*X of ethanol, r are obtained2=0.9999, its correlation coefficient is more than
0.99, illustrate that linear relationship is good.
(3) concentration of alcohol in prepare liquid
Ethanol in prepare liquid is detected with gas chromatograph, peak area corresponding concentration on standard curve is
For the content of ethanol in prepare liquid.
(4) concentration of alcohol in sample to be tested
Extension rate is multiplied by according to the content of ethanol in prepare liquid to be calculated in viable type lactobacillus beverage to be measured
The content of ethanol.
Gas chromatographic detection condition：
Detector be flame ionization ditector (FID), chromatographic column be CD-2560 posts (100 meters of Φ ×
0.25 millimeter, 0.2 micron), injector temperature：180℃；Detector temperature：200℃；Sampling volume：1
Microlitre；Flow rate of carrier gas：2.0 ml/min；Carrier gas is high pure nitrogen, purity 99.999%；Split ratio is 20：
1；Heating schedule：80 DEG C of initial temperature, is kept for 1 minute；150 DEG C are risen to 10 DEG C/min, 2 are kept
Minute；10 minutes total times.
2. it is a kind of detection viable type lactobacillus beverage in ethanol method, it is characterised in that include：
(1) centrifugal treating is carried out after the viable type lactobacillus beverage is mixed with acetonitrile, and collects supernatant；
And
(2) chromatography detection is carried out to the supernatant, and the work is determined based on resulting testing result
The content of ethanol in bacterial type lactobacillus beverage.
3. method according to claim 2, it is characterised in that based on 1 milliliter of viable type lactic acid
Bacteria beverage, the consumption of acetonitrile is 1～10 milliliter.
4. method according to claim 2, it is characterised in that the centrifugation be according to 3000rpm～
The rotating speed of 8000rpm carries out 3～10 minutes.
5. method according to claim 2, it is characterised in that before step (2) is carried out, in advance
The supernatant is filtered, and resulting filtrate is carried out into the chromatography detection.
6. method according to claim 5, it is characterised in that the filtration is using 0.22 micron of filter
Film.
7. method according to claim 2, it is characterised in that the viable type lactobacillus beverage is acid
Milk.
8. method according to claim 2, it is characterised in that before the chromatography detection is carried out,
The concentration of ethanol in the supernatant is adjusted to 1mg/100mL～32mg/100mL in advance.
9. method according to claim 2, it is characterised in that the chromatography is detected as gas chromatogram
Method detects that the gas chromatography detection is carried out according to following condition：
Chromatographic column：CD-2560 posts；
Injector temperature：180℃；
Detector temperature：200℃；
Sampling volume：1 microlitre；
Flow rate of carrier gas：2.0 ml/min；
Carrier gas is high pure nitrogen, purity 99.999%；
Split ratio is 20：1；
Heating schedule：80 DEG C of initial temperature, is kept for 1 minute；150 DEG C are risen to 10 DEG C/min, 2 are kept
Minute；10 minutes total times.
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Citations (3)
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|---|---|---|---|---|
| US3896659A (en) * | 1973-08-27 | 1975-07-29 | Bacardi And Company Ltd | Method for determining the ethanol content of alcoholic beverages |
| CN1715907A (en) * | 2004-07-02 | 2006-01-04 | 中国科学院兰州化学物理研究所 | The gas chromatography analysis method of micro ethanol in the blood of human body |
| CN104330485A (en) * | 2014-10-24 | 2015-02-04 | 西安米亿生物科技有限公司 | Detection method for content of alcohol in concentrated juice |
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2015
- 2015-10-08 CN CN201510651237.0A patent/CN106568848B/en active Active
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|---|---|---|---|---|
| US3896659A (en) * | 1973-08-27 | 1975-07-29 | Bacardi And Company Ltd | Method for determining the ethanol content of alcoholic beverages |
| CN1715907A (en) * | 2004-07-02 | 2006-01-04 | 中国科学院兰州化学物理研究所 | The gas chromatography analysis method of micro ethanol in the blood of human body |
| CN104330485A (en) * | 2014-10-24 | 2015-02-04 | 西安米亿生物科技有限公司 | Detection method for content of alcohol in concentrated juice |
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| WANG ML 等: "A rapid method for determination of ethanol in alcoholic beverages using capillary gas chromatography", 《JOURNAL OF FOOD AND DRUG ANALYSIS》 * |
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