CN115047057A - Method for detecting lactic acid in wine-making fermentation product - Google Patents
Method for detecting lactic acid in wine-making fermentation product Download PDFInfo
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- CN115047057A CN115047057A CN202210556304.0A CN202210556304A CN115047057A CN 115047057 A CN115047057 A CN 115047057A CN 202210556304 A CN202210556304 A CN 202210556304A CN 115047057 A CN115047057 A CN 115047057A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/622—Ion mobility spectrometry
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- G—PHYSICS
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Abstract
The application relates to the technical field of wine brewing process detection, in particular to a method for detecting lactic acid in wine brewing leavening, which comprises the following steps: (1) weighing a fermentation product sample, and adding an extraction solvent into the fermentation product sample for extraction to obtain an extraction liquid; (2) and filtering the extract liquor and detecting the extract liquor by an ion mobility spectrometry detector. The method and the device have the advantages of accurate detection result, high detection efficiency and low detection cost.
Description
Technical Field
The application relates to the technical field of wine brewing process detection, in particular to a method for detecting lactic acid in wine brewing leavening.
Background
The distiller's yeast mainly plays a role of a saccharification leaven and provides starch substances in the production process of grain wine, and is one of important components in the production of the grain wine, and the quality of the distiller's yeast directly influences the fermentation process of fermented grains, so that the yield and the quality of the wine are influenced. Fermentation of fermented grains requires distiller's yeast to provide microorganisms for saccharification and fermentation, and the fermentation quality of the fermented grains is directly related to the yield and quality of wine. Therefore, the fermentation conditions of the distiller's yeast and the fermented grains are mastered in time, and the method is very important for the production of grain wine.
The lactic acid content is an important monitoring content in the production process of the Maotai-flavor liquor, and can directly reflect the fermentation conditions of the distiller's yeast and the fermented grains. Therefore, the lactic acid content in the yeast and the fermented grains can be mastered in time, and corresponding process improvement can be made according to the lactic acid content by combining the production process, so that the fermentation quality of the yeast and the fermented grains is improved. Therefore, the rapid detection of the lactic acid in the distiller's yeast and the fermented grains has important scientific guiding significance for the production of grain wine and has great practical application value.
A common detection method for lactic acid in white spirit is high performance liquid chromatography, but the detection cost for detecting lactic acid by adopting high performance liquid chromatography is high, the required sample amount is large, the pretreatment is complex, the operation is complex, and the use is limited by instruments, consumables, resources and the like.
Disclosure of Invention
In order to detect lactic acid more quickly and conveniently, the application provides a detection method of lactic acid in a wine-making fermentation product.
The detection method for lactic acid in the wine fermentation product adopts the following technical scheme:
a detection method of lactic acid in wine fermentation comprises the following steps:
(1) weighing a fermentation product sample, and adding an extraction solvent into the fermentation product sample for extraction to obtain an extraction liquid;
(2) and filtering the extract liquor and detecting the extract liquor by an ion mobility spectrometry detector.
Preferably, in the step (1), the fermentation product sample comprises one or a mixture of two of distiller's yeast and fermented grains.
Preferably, in the step (1), the fermentation product sample is distiller's yeast, and the weighed mass of the distiller's yeast sample is 10-1000 mg.
Preferably, in the step (1), the fermented substance sample is fermented grains, and the weighed fermented grain sample has a mass of 10-50 mg.
Preferably, in the step (1), the extraction solvent is isopropanol, and the addition amount of the isopropanol is 0.5-2.0 mL;
preferably, the addition amount of the isopropyl alcohol is 1 mL.
Preferably, in the step (1), the extraction includes: performing vortex oscillation for 1-3 min, centrifuging for 1-3 min, performing solid-liquid separation, collecting liquid, repeatedly extracting the residual solid for 2-3 times, and combining the liquid to obtain an extract;
preferably, the extraction liquid comprises: vortex and oscillate for 2min, then centrifuge for 2min, and collect liquid after solid-liquid separation.
Preferably, in the step (2), the filtering of the extract solution includes: the extract was filtered through a filter membrane with a pore size of 0.22 μm.
Preferably, in the step (2), 2-4 μ L of the filtered extract is diluted by 30-50 times and then enters an ion mobility spectrometry analyzer for detection;
preferably, in the step (2), 3 μ L of the filtered extract is diluted by 40 times and then enters an ion mobility spectrometry detector for detection.
When the dilution multiple is less than 30 times, the concentration of lactic acid in the combined extract is higher, and the instrument is directly polluted; when the dilution factor is more than 50 times, the dilution factor is too high, and the content of lactic acid is small, so that the detection result is influenced.
Preferably, in the step (2), the parameters of the ion mobility spectrometry detector are set as follows: the ionization source is a photoionization source, an anion mode is adopted, the temperature of the migration tube is kept at 90-110 ℃, the sample carrier gas is air, and the flow rate of the sample carrier gas is 180-220 mL/min; the floating gas is air, and the flow rate of the floating gas is 580-620 mL/min; the doping gas is acetone, and the flow rate of the doping gas is 45-55 mL/min;
preferably, the temperature of the migration tube is kept at 100 ℃, the sample carrier gas is air, and the flow rate of the sample carrier gas is 200 mL/min; the floating gas is air, and the flow rate of the floating gas is 600 mL/min; the doping gas is acetone, and the flow rate of the doping gas is 50 mL/min.
Preferably, in the step (2), the detecting by an ion mobility spectrometry detector includes: performing qualitative analysis on the lactic acid according to the migration time, and establishing a linear equation according to the ionic signal intensity and the lactic acid concentration for quantitative analysis; the migration time of the qualitative characteristic peak of the lactic acid is 4.615 ms.
The application has the following beneficial technical effects:
the method extracts the wine brewing leavening, and then detects the extracted extraction liquid through the ion mobility spectrometry analysis detector, so that the lactic acid in the wine brewing leavening is qualitatively and quantitatively analyzed. The application detects the lactic acid in the wine fermentation, the pretreatment is simple, the detection sample amount is small, the detection cost is low, and the lactic acid in the wine fermentation can be detected more quickly.
Drawings
FIG. 1 is an ion mobility spectrum of lactic acid detected by anion mode ion mobility spectrometry of the present application;
FIG. 2 is a standard graph of the determination of lactic acid content;
FIG. 3 is a graph showing ionization and desorption in example 1 using isopropanol as an extraction solvent;
FIG. 4 is a graph showing ionization and desorption using methanol as an extraction solvent in example 1;
FIG. 5 is a graph showing the ionization desorption of ethanol as an extraction solvent in example 1;
FIG. 6 is a comparative graph after a second extraction in example 1 with isopropanol and methanol as extraction solvents;
FIG. 7 is a graph showing ionization and desorption in example 2 using isopropanol at a concentration of 50% as an extraction solvent;
FIG. 8 is a graph showing ionization and desorption in example 2 using 75% isopropyl alcohol as an extraction solvent;
FIG. 9 is an ion mobility spectrum of lactic acid in koji in example 3;
FIG. 10 is an ion mobility spectrum of lactic acid detected from fermented grains in example 4.
Detailed Description
The present application is further described below with reference to the accompanying drawings and examples.
Reagent: methanol (chromatographically pure), ethanol (chromatographically pure), isopropanol (chromatographically pure), lactic acid (concentration 98%).
The instrument comprises the following steps: an ion mobility spectrometry analyzer (model: BBF 005; institute of chemical and physical research, university of Chinese academy of sciences).
The application provides a detection method of lactic acid in a wine brewing fermentation product, which comprises the following steps:
(1) and weighing a fermentation product sample, and adding an extraction solvent into the fermentation product sample for extraction to obtain an extraction liquid.
Weighing a fermentation sample, and when the weighed fermentation sample is distiller's yeast, weighing the distiller's yeast sample by 10-1000 mg; and when the weighed fermented substance sample is fermented grains, weighing the fermented grains with the mass of 10-50 mg. In the detection, two fermentation product samples can be mixed and then subjected to extraction detection. Adding an extraction solvent into a fermentation product sample for extraction, wherein the extraction solvent is isopropanol, the volume of the added isopropanol is 0.5-2.0 mL, and the volume of the added isopropanol is specifically 1 mL. The extraction steps in this application are: adding isopropanol into a fermentation product sample, performing vortex oscillation for 1-3 min, centrifuging for 1-3 min, performing solid-liquid separation, and collecting liquid, wherein the vortex oscillation time is specifically 2min, the centrifugation speed is 12000rpm/min, and the liquid is collected after the solid-liquid separation. And repeatedly extracting the residual solid after the solid-liquid separation for 2-3 times, specifically selecting to repeatedly extract the residual solid for 2 times in the application, and combining the separated liquid to obtain the extraction liquid.
(2) And filtering the extract liquor and detecting the extract liquor by an ion mobility spectrometry detector.
Filtering the extract liquor through a filter membrane with the aperture of 0.22 mu m, taking 2-4 mu L of filtered extract liquor, diluting by 30-50 times, and then, entering an ion mobility spectrometry analysis detector for detection; in the application, 3 mu L of filtered extract is diluted by 40 times and then enters an ion mobility spectrometry analysis detector for detection. The parameters of the ion mobility spectrometry detector are set as follows: the ionization source is a photoionization source, an anion mode is adopted, the temperature of the migration tube is kept at 90-110 ℃, the sample carrier gas is air, and the flow rate of the sample carrier gas is 180-220 mL/min; the floating gas is air, and the flow rate of the floating gas is 580-620 mL/min; the doping gas is acetone, and the flow rate of the doping gas is 45-55 mL/min. The parameters of the ion mobility spectrometry detector in the application are specifically set as follows: the temperature of the migration tube is kept at 100 ℃, the sample carrier gas is air, and the flow rate of the sample carrier gas is 200 mL/min; the floating gas is air, and the flow rate of the floating gas is 600 mL/min; the doping gas is acetone, and the flow rate of the doping gas is 50 mL/min.
Qualitative analysis of lactic acid standard
Pure lactic acid was added to isopropanol as a solvent to prepare a 5. mu.g L standard solution of lactic acid. A micro-sample injector is used for accurately measuring 3 mu L of lactic acid standard solution and injecting the lactic acid standard solution into an ionization region of an ion transfer tube, sample molecules are directly ionized into positive ions and negative ions in the ionization region, hot carrier gas enters a drift region formed by a uniform electric field through an ion gate which is periodically opened, and separation and detection are carried out in the drift region to obtain a detection signal. Fig. 1 is an ion mobility spectrum of lactic acid detected by anion mode ion mobility spectrometry of the present application, and it can be seen from the figure that the target peak of the anion mode lactic acid detection is 4.615 ms.
Quantitative analysis of lactic acid standards
Adding pure lactic acid into isopropanol to prepare 1 mug/mL, 2.5 mug/mL, 5 mug/mL, 7.5 mug/mL and 10 mug/mL of lactic acid standard solutions respectively by taking the isopropanol as a solvent, detecting each lactic acid standard sample by taking 3 muL of injection ion mobility spectrometry analysis detector respectively, and recording the peak-out migration time of the lactic acid and a continuous 0-30S signal tracking change trend curve of the lactic acid peak in a negative ion mode; the software calls a lactic acid peak tracking change trend curve to obtain a signal intensity data summation value, namely peak areas corresponding to different concentrations of lactic acid, so as to obtain a standard curve graph for detecting the content of lactic acid. As shown in FIG. 2, the peak of lactic acid (4.615ms) in the concentration range of 1-10. mu.g/mL is in accordance with the linear equation: y-1470.17 + 2174.84X, X indicating lactate concentration, Y indicating signal intensity, and correlation coefficient 0.9906.
The method extracts the wine brewing leavening, and then detects the extracted extraction liquid through the ion mobility spectrometry analysis detector, so that the lactic acid in the wine brewing leavening is qualitatively and quantitatively analyzed. The application detects the lactic acid in the wine fermentation, the pretreatment is simple, the detection sample amount is small, the detection cost is low, and the lactic acid in the wine fermentation can be detected more quickly.
Example 1 Effect of different extraction solvents on Signal Strength
This application has analyzed the influence of different solvents to the analysis effect, and the solvent of adoption includes: isopropanol, methanol, ethanol. The method comprises the specific steps of respectively taking isopropanol, methanol and ethanol as solvents, and respectively adding pure lactic acid into the three solvents to prepare a 5 mu g/mL lactic acid standard solution. A micro sample injector is used for accurately measuring 3 mu L of lactic acid standard solution, injecting the lactic acid standard solution into an ionization region of an ion transfer tube for detection, the detection results of the three solvents for detecting the lactic acid are shown in figures 3-5, and the detected analytic signal intensity is shown in table 1.
TABLE 1 Signal intensity of different extraction solvents
The experiment inspects the influence of different solvents on the analysis effect of the target object, and selects the solvent with the highest analytic signal intensity as the optimal solvent. As can be seen from fig. 3 to 5 in combination with table 1, when isopropanol was used as the extraction solvent, the photoionization time of lactic acid was short and the lactic acid detection signal was strong; when methanol and ethanol are used as extraction solvents, the photoionization time of the lactic acid is long, and the lactic acid detection signal is weak. This shows that the isopropanol as extraction solvent can promote photoionization of lactic acid to rapidly ionize lactic acid, and that the isopropanol can increase the signal intensity for detecting lactic acid.
In addition, comparing the extraction effects of isopropanol and methanol as extraction solvents, specifically, taking isopropanol as an extraction solvent, accurately measuring 10mg of distiller's yeast sample in a 10mL sample bottle, and adding 1mL of isopropanol solvent for extraction. The extraction steps are as follows: adding 1mL of isopropanol into 10mg of distiller's yeast sample, performing vortex oscillation for 2min, centrifuging for 2min at 12000rpm/min, collecting liquid after solid-liquid separation, performing second extraction on the separated solid, and centrifuging for the second extraction to obtain a graph shown in FIG. 6. The lactic acid extraction of the koji sample with methanol as the extraction solvent and the second extraction centrifugation are shown in FIG. 6. The centrifuge tubes numbered 1-3 in fig. 6 are in a state of being subjected to the second extraction centrifugation using isopropanol as the extraction solvent, and the centrifuge tubes numbered 2-3 in fig. 6 are in a state of being subjected to the second extraction centrifugation using methanol as the extraction solvent. As can be seen from FIG. 6, when isopropanol is used for extracting lactic acid in the koji, the second centrifugation still has deep extraction effect, and obvious solid-liquid separation still occurs. This indicates that isopropanol has a better extraction effect as an extraction solvent. Therefore, isopropanol is used as the extraction solvent in the scheme.
Example 2 comparison of the Effect of Isopropanol concentration on Signal Strength
The application analyzes the influence of different isopropanol concentrations on the analysis effect, and the adopted solvents comprise: 50% isopropanol, 75% isopropanol, 100% isopropanol (chromatographically pure isopropanol), the remaining detection steps being identical to those of example 1. The results of detecting lactic acid with three different concentrations of isopropanol as solvent are shown in fig. 7, fig. 8 and fig. 3, wherein fig. 3 is the result of detecting lactic acid with 100% isopropanol as solvent, and the analytic signal intensity of the detection is shown in table 2.
TABLE 2 Signal intensity at different isopropanol concentrations
| Concentration of isopropyl alcohol | Signal intensity (mv) |
| 50％ | 336 |
| 75％ | 419 |
| 100％ | 2800 |
The experiment inspects the influence of different isopropanol concentrations on the analysis effect of the target object, and selects the isopropanol with the highest analytic signal intensity as the optimal solvent. As can be seen from fig. 7, 8 and 3 in combination with table 2, when isopropanol with a concentration of 100% was used as the extraction solvent, the photoionization time of lactic acid was short and the lactic acid detection signal was much higher than other concentrations; when isopropanol with the concentration of 50% and isopropanol with the concentration of 75% are used as extraction solvents, the photoionization time of lactic acid is long, the detection signal of lactic acid is weak, and the detection result is greatly influenced by the high water content of an ion migration tube of a detector because the ion movement under a detection electric field is detected. This shows that the use of 100% isopropanol as the extraction solvent can promote photoionization of lactic acid to rapidly ionize lactic acid, and that isopropanol can increase the signal intensity for detection of lactic acid.
Example 3
A detection method of lactic acid in a wine-making fermentation product comprises the following steps:
(1) and weighing a fermentation product sample, and adding an extraction solvent into the fermentation product sample for extraction to obtain an extraction liquid.
Specifically, 10mg of a koji sample is accurately weighed in a 10mL sample bottle, and 1mL of an isopropanol solvent is added to the koji sample for extraction. The extraction steps are as follows: adding 1mL of isopropanol solvent into a 10mg distiller's yeast sample, performing vortex oscillation for 2min, centrifuging for 2min at 12000rpm/min, and performing solid-liquid separation to collect liquid. Extracting the residual solid after solid-liquid separation for 2 times, and mixing the liquid to obtain the extract.
(2) And filtering the extract liquor and detecting the extract liquor by an ion mobility spectrometry analysis detector.
Specifically, the extract is filtered through a filter membrane with the aperture of 0.22 μm, and 3 μ L of the filtered extract is diluted by 40 times and then enters an ion mobility spectrometry analyzer for detection. The parameters of the ion mobility spectrometry detector are set as follows: the ionization source is a photoionization source, a negative ion mode is adopted, the temperature of the migration tube is kept at 100 ℃, the sample carrier gas is air, and the flow rate of the sample carrier gas is 200 mL/min; the floating gas is air, and the flow rate of the floating gas is 600 mL/min; the doping gas is acetone, and the flow rate of the doping gas is 50 mL/min.
The ion mobility spectrum of lactic acid in the koji in this example is shown in FIG. 9, from which it can be seen that the ion mobility spectrum coincides with the characteristic peak position of the standard solution for detecting lactic acid. Continuously collecting 4.615ms signal peaks for 0.5min, substituting the recorded accumulated sum signal Y value of the lactic acid peaks into a standard curve equation Y of-1470.17 + 2174.84X, calculating to obtain the concentration of lactic acid, and fitting to calculate the content of the lactic acid in the koji sample to be 0.4g/100 g.
Example 4
A detection method of lactic acid in wine fermentation comprises the following steps:
(1) and weighing a fermentation product sample, and adding an extraction solvent into the fermentation product sample for extraction to obtain an extract.
Specifically, 24mg of fermented grain sample is accurately weighed in a 10mL sample bottle, and 1mL of isopropanol solvent is added into the fermented grain sample for extraction. The extraction steps are as follows: adding 1mL of isopropanol solvent into 10mg of fermented grain sample, performing vortex oscillation for 2min, centrifuging for 2min at 12000rpm/min, and performing solid-liquid separation to collect liquid. Extracting the residual solid after solid-liquid separation for 2 times, and mixing the liquid to obtain the extractive solution.
(2) And filtering the extract liquor and detecting the extract liquor by an ion mobility spectrometry detector.
Specifically, the extract is filtered through a filter membrane with the aperture of 0.22 μm, and 3 μ L of the filtered extract is diluted by 40 times and then enters an ion mobility spectrometry analyzer for detection. The parameters of the ion mobility spectrometry detector are set as follows: the ionization source is a photoionization source, a negative ion mode is adopted, the temperature of the migration tube is kept at 100 ℃, the sample carrier gas is air, and the flow rate of the sample carrier gas is 200 mL/min; the floating gas is air, and the flow rate of the floating gas is 600 mL/min; the doping gas is acetone, and the flow rate of the doping gas is 50 mL/min.
The ion mobility spectrum of lactic acid detected in fermented grains in this example is shown in fig. 10, which shows that the ion mobility spectrum is consistent with the position of the characteristic peak of the standard solution for detecting lactic acid. Continuously collecting 4.615ms signal peaks for 0.5min, substituting the recorded accumulated sum signal Y value of the lactic acid peaks into a standard curve equation Y of-1470.17 + 2174.84X, calculating to obtain the concentration of lactic acid, and fitting to calculate the content of the lactic acid in the fermented grains sample to be 5.4g/100 g.
The method extracts the wine brewing leavening, and then detects the extracted extraction liquid through the ion mobility spectrometry analysis detector, so that the lactic acid in the wine brewing leavening is qualitatively and quantitatively analyzed. The application detects the lactic acid in the wine fermentation, the pretreatment is simple, the detection sample amount is small, the detection cost is low, and the lactic acid in the wine fermentation can be detected more quickly.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A detection method of lactic acid in wine fermentation is characterized by comprising the following steps:
(1) weighing a fermentation product sample, and adding an extraction solvent into the fermentation product sample for extraction to obtain an extraction liquid;
(2) and filtering the extract liquor and detecting the extract liquor by an ion mobility spectrometry detector.
2. The method for detecting lactic acid in fermented product according to claim 1, wherein the fermentation product sample in step (1) comprises one or a mixture of koji and fermented grains.
3. The method for detecting lactic acid in the wine-making fermentation product according to claim 2, wherein in the step (1), the fermentation product sample is distiller's yeast, and the weighed mass of the distiller's yeast sample is 10-1000 mg.
4. The method for detecting lactic acid in fermented products for wine brewing according to claim 2, wherein in the step (1), the fermented product sample is fermented grains, and the mass of the fermented grains sample is 10-50 mg.
5. The method for detecting lactic acid in the wine fermentation product according to claim 1, wherein in the step (1), the extraction solvent is isopropanol, and the addition amount of the isopropanol is 0.5-2.0 mL;
preferably, the addition amount of the isopropyl alcohol is 1 mL.
6. The method for detecting lactic acid in wine-making fermentation product according to claim 1, wherein in the step (1), the extraction comprises: performing vortex oscillation for 1-3 min, centrifuging for 1-3 min, performing solid-liquid separation, collecting liquid, repeatedly extracting the residual solid for 2-3 times, and combining the liquid to obtain an extract;
preferably, the extraction liquid comprises: vortex and oscillate for 2min, then centrifuge for 2min, and collect liquid after solid-liquid separation.
7. The method for detecting lactic acid in wine fermentation according to claim 1, wherein in the step (2), the filtering of the extract comprises: the extract was filtered through a filter membrane with a pore size of 0.22 μm.
8. The method for detecting lactic acid in wine brewing leavening according to claim 7, wherein in the step (2), 2-4 μ L of filtered extract is diluted by 30-50 times and then enters an ion mobility spectrometry analyzer for detection;
preferably, in the step (2), 3 μ L of the filtered extract is diluted by 40 times and then enters an ion mobility spectrometry detector for detection.
9. The method for detecting lactic acid in wine fermentation according to claim 1, wherein in the step (2), the parameters of the ion mobility spectrometry detector are set as follows: the ionization source is a photoionization source, an anion mode is adopted, the temperature of the migration tube is kept at 90-110 ℃, the sample carrier gas is air, and the flow rate of the sample carrier gas is 180-220 mL/min; the floating gas is air, and the flow rate of the floating gas is 580-620 mL/min; the doping gas is acetone, and the flow rate of the doping gas is 45-55 mL/min;
preferably, the temperature of the migration tube is kept at 100 ℃, the sample carrier gas is air, and the flow rate of the sample carrier gas is 200 mL/min; the floating gas is air, and the flow rate of the floating gas is 600 mL/min; the doping gas is acetone, and the flow rate of the doping gas is 50 mL/min.
10. The method for detecting lactic acid in wine fermentation according to claim 1, wherein in the step (2), the detecting by an ion mobility spectrometry detector comprises: performing qualitative analysis on the lactic acid according to the migration time, and establishing a linear equation according to the ionic signal intensity and the lactic acid concentration for quantitative analysis; the migration time of the qualitative characteristic peak of the lactic acid is 4.615 ms.
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