Method for detecting lactic acid in a wine fermentation

Abstract

The application relates to the technical field of liquor-making process detection, in particular to a detection method of lactic acid in liquor-making fermentation, which comprises the following steps: (1) weighing a fermentation sample, adding an extraction solvent to the fermentation sample for extraction to obtain an extraction liquid; (2) filtering the extraction liquid and detecting the same through an ion mobility spectrum analysis detector. The application has the advantages of accurate detection result, high detection efficiency and low detection cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of liquor-making process detection, in particular to a detection method of lactic acid in liquor-making fermentation. BACKGROUND

[0002] The wine starter in the production process of grain liquor mainly plays the role of saccharifying fermentation agent and provides starch material, and is one of the important components of grain liquor production. The quality of the wine starter directly affects the fermentation process of the fermented grains, thereby affecting the liquor yield and the quality of the liquor. The fermentation of the fermented grains requires the wine starter to provide saccharifying microorganisms, and the fermentation quality is directly related to the yield and quality of the liquor. Therefore, timely grasping the fermentation status of the wine starter and the fermented grains is crucial for the production of grain liquor.

[0003] The lactic acid content is an important monitoring content in the production process of Maotai-flavor liquor, and the lactic acid content can directly reflect the fermentation status of the wine starter and the fermented grains. Therefore, timely grasping the lactic acid content in the wine starter and the fermented grains, combined with the production process, can make corresponding process improvements according to the lactic acid content to improve the fermentation quality of the wine starter and the fermented grains. Therefore, the rapid detection of lactic acid in the wine starter and the fermented grains has important scientific guiding significance for the production of grain liquor and has great practical application value.

[0004] The commonly used detection method of lactic acid in liquor is high-performance liquid chromatography, but the detection cost of lactic acid detection by high-performance liquid chromatography is high, the sample amount required is large, the pretreatment is complex, and the operation is cumbersome, which is limited by instruments, consumables, resources, etc. SUMMARY

[0005] In order to be able to detect lactic acid more quickly and conveniently, the present application provides a detection method of lactic acid in liquor-making fermentation.

[0006] The detection method of lactic acid in liquor-making fermentation provided by the present application adopts the following technical scheme:

[0007] A detection method of lactic acid in liquor-making fermentation, comprising the following steps:

[0008] (1) A fermentation sample is weighed, an extraction solvent is added to the fermentation sample for extraction, and an extraction liquid is obtained;

[0009] (2) The extraction liquid is filtered and detected by an ion mobility spectrum analyzer.

[0010] Preferably, in step (1), the fermentation sample includes one or a mixture of both of the wine starter and the fermented grains.

[0011] Preferably, in step (1), the fermentation sample is the wine starter, and the mass of the wine starter sample is 10-1000 mg.

[0012] Preferably, in the step (1), the fermentation sample is fermented grains, and the mass of the fermented grains sample is 10-50 mg.

[0013] Preferably, in the step (1), the extraction solvent is isopropyl alcohol, and the addition amount of the isopropyl alcohol is 0.5-2.0 mL.

[0014] Preferably, the addition amount of the isopropyl alcohol is 1 mL.

[0015] Preferably, in the step (1), the extraction includes: first vortex oscillation for 1-3 min, then centrifugation for 1-3 min, collection of the liquid after solid-liquid separation, repeated extraction of the remaining solid for 2-3 times, and collection of the liquid to obtain the extraction liquid.

[0016] Preferably, the extraction includes: first vortex oscillation for 2 min, then centrifugation for 2 min, and collection of the liquid after solid-liquid separation.

[0017] Preferably, in the step (2), the filtration of the extraction liquid includes: filtration of the extraction liquid through a filter membrane with a pore size of 0.22 μm.

[0018] Preferably, in the step (2), 2-4 μL of the filtered extraction liquid is taken, diluted by 30-50 times, and then subjected to detection by the ion mobility spectrometry analyzer.

[0019] Preferably, in the step (2), 3 μL of the filtered extraction liquid is taken, diluted by 40 times, and then subjected to detection by the ion mobility spectrometry analyzer.

[0020] When the dilution multiple is less than 30 times, the lactic acid concentration in the combined extraction liquid is relatively high, which directly pollutes the instrument; when the dilution multiple is greater than 50 times, the dilution multiple is too high, and the lactic acid content is relatively small, which affects the detection result.

[0021] Preferably, in the step (2), the parameter settings of the ion mobility spectrometry analyzer are as follows: the ionization source is a photoionization source, a negative ion mode is adopted, the temperature of the drift tube is kept at 90-110℃, the sample carrier gas is air, the flow rate of the sample carrier gas is 180-220 mL / min; the drift gas is air, the flow rate of the drift gas is 580-620 mL / min; the doping gas is acetone, and the flow rate of the doping gas is 45-55 mL / min.

[0022] Preferably, the temperature of the drift tube is kept at 100℃, the sample carrier gas is air, the flow rate of the sample carrier gas is 200 mL / min; the drift gas is air, the flow rate of the drift gas is 600 mL / min; the doping gas is acetone, and the flow rate of the doping gas is 50 mL / min.

[0023] Preferably, in the step (2), the detection by the ion mobility spectrometry detector comprises: qualitative analysis of lactic acid according to the migration time, and quantitative analysis according to a linear equation established according to the ion signal intensity and the lactic acid concentration; the qualitative characteristic peak of lactic acid has a migration time of 4.615 ms.

[0024] The present application has the following beneficial technical effects:

[0025] The present application extracts the wine fermentation material, and then detects the extracted extract by an ion mobility spectrometry detector, so as to qualitatively and quantitatively analyze the lactic acid in the wine fermentation material. The present application can detect the lactic acid in the wine fermentation material more quickly with simple pretreatment, less sample amount, and low detection cost. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is an ion mobility spectrum of the present application for detecting lactic acid in a negative ion mode;

[0027] Figure 2 is a standard curve for detecting the content of lactic acid;

[0028] Figure 3 is an ionization desorption diagram of Example 1 using isopropyl alcohol as an extraction solvent;

[0029] Figure 4 is an ionization desorption diagram of Example 1 using methanol as an extraction solvent;

[0030] Figure 5 is an ionization desorption diagram of Example 1 using ethanol as an extraction solvent;

[0031] Figure 6 is a comparison diagram of Example 1 after a second extraction using isopropyl alcohol and methanol as extraction solvents;

[0032] Figure 7 is an ionization desorption diagram of Example 2 using isopropyl alcohol with a concentration of 50% as an extraction solvent;

[0033] Figure 8 is an ionization desorption diagram of Example 2 using isopropyl alcohol with a concentration of 75% as an extraction solvent;

[0034] Figure 9 is an ion mobility spectrum of Example 3 for detecting lactic acid in wine starter;

[0035] Figure 10 is an ion mobility spectrum of Example 4 for detecting lactic acid in fermented grains. DETAILED DESCRIPTION

[0036] The present application is further described below in combination with the drawings and examples.

[0037] Reagents: methanol (chromatographic pure), ethanol (chromatographic pure), isopropanol (chromatographic pure), lactic acid (concentration of 98%).

[0038] Instrument: ion mobility spectrometry detector (model: BBF005; Dalian Institute of Chemical Physics, Chinese Academy of Sciences).

[0039] The application provides a detection method of lactic acid in a brewing fermentation product, comprising the following steps:

[0040] (1) A fermentation product sample is weighed, and an extraction solvent is added to the fermentation product sample for extraction to obtain an extraction solution.

[0041] The fermentation product sample is weighed, the mass of the koji sample is 10-1000 mg when the weighed fermentation product sample is koji, and the mass of the fermented grains sample is 10-50 mg when the weighed fermentation product sample is fermented grains. The two kinds of fermentation product samples can also be mixed and then extracted for detection. The extraction solvent is isopropanol, and 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 step in the application is: the isopropanol is added to the fermentation product sample, vortexed for 1-3 min, and then centrifuged for 1-3 min, and the liquid is collected after solid-liquid separation, the vortexing time is specifically 2 min, the centrifugation time is specifically 2 min, the centrifugation speed is 12000 rpm / min, and the liquid is collected after solid-liquid separation. The remaining solid after solid-liquid separation is extracted for 2-3 times, and the remaining solid is extracted for 2 times in the application, and the separated liquids are combined to obtain the extraction solution.

[0042] (2) The extraction solution is filtered and detected by an ion mobility spectrometry detector.

[0043] The extraction solution is filtered through a filter membrane with a pore size of 0.22 μm, 2-4 μL of the filtered extraction solution is diluted 30-50 times, and then introduced into an ion mobility spectrometry analyzer for detection. In this application, 3 μL of the filtered extraction solution is diluted 40 times, and then introduced into an ion mobility spectrometry analyzer for detection. The parameter settings of the ion mobility spectrometry analyzer are as follows: the ionization source is a photoionization source, the negative ion mode is adopted, the temperature of the drift tube is kept at 90-110°C, the sample carrier gas is air, the sample carrier gas flow rate is 180-220 mL / min; the drift gas is air, the drift gas flow rate is 580-620 mL / min; the doping gas is acetone, and the doping gas flow rate is 45-55 mL / min. In this application, the parameter settings of the ion mobility spectrometry analyzer are as follows: the temperature of the drift tube is kept at 100°C, the sample carrier gas is air, the sample carrier gas flow rate is 200 mL / min; the drift gas is air, the drift gas flow rate is 600 mL / min; the doping gas is acetone, and the doping gas flow rate is 50 mL / min.

[0044] Qualitative analysis of lactic acid standard

[0045] Pure lactic acid is added to isopropanol to prepare a 5 μg / L lactic acid standard solution with isopropanol as the solvent. 3 μL of the lactic acid standard solution is accurately measured by using a microsyringe and injected into the ionization zone of the ion mobility tube. The sample molecules are directly ionized into positive and negative ions in the ionization zone. The hot carrier gas enters the drift zone formed by the uniform electric field through the periodically opened ion gate, and is separated and detected in the drift zone to obtain the detection signal. Figure 1 The ion mobility spectrum of lactic acid in the negative ion mode of this application is shown in the figure. As can be seen from the figure, the target peak of lactic acid in the negative ion mode is 4.615 ms.

[0046] Quantitative analysis of lactic acid standard

[0047] Pure lactic acid is added to isopropanol to prepare lactic acid standard solutions with concentrations of 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL and 10 μg / mL, respectively. 3 μL of each lactic acid standard sample is injected into the ion mobility spectrometry analyzer for detection. The peak migration time of lactic acid in the negative ion mode and the signal tracking trend curve within 0-30 S of the lactic acid peak are recorded. The signal intensity data and the number of values corresponding to the peak area of lactic acid at different concentrations are obtained by calling the lactic acid peak tracking trend curve, and the standard curve graph of the detected lactic acid content is obtained. The results are shown in Figure 2 The lactic acid peak (4.615 ms) in the concentration range of 1-10 μg / mL conforms to the linear equation relationship, and the linear equation is Y=-1470.17+2174.84*X, where X represents the lactic acid concentration and Y represents the signal intensity. The correlation coefficient is 0.9906.

[0048] The application extracts the wine fermentation product, and then detects the extracted solution by an ion mobility spectrum analyzer, so as to qualitatively and quantitatively analyze the lactic acid in the wine fermentation product. The application detects the lactic acid in the wine fermentation product, and has the advantages of simple pretreatment, small sample amount, low detection cost, and faster detection of the lactic acid in the wine fermentation product.

[0049] Example 1: Influence of different extraction solvents on signal intensity

[0050] The application analyzes the influence of different solvents on the analysis effect, and the solvents used include isopropyl alcohol, methanol and ethanol. The specific steps are as follows: isopropyl alcohol, methanol and ethanol are used as solvents, and pure lactic acid is added to the three solvents to prepare a lactic acid standard solution of 5 μg / mL. A microsyringe is used to accurately take 3 μL of the lactic acid standard solution and inject it into the ion mobility tube ionization zone for detection. The detection results of the three solvents for lactic acid are as shown in Figures 3 to 5 , and the analysis signal intensity of the detection is as shown in Table 1.

[0051] Table 1: Signal intensity of different extraction solvents

[0052]

[0053]

[0054] The experiment investigates the influence of different solvents on the analysis effect of the target, and selects a solvent with the highest analysis signal intensity as the optimal solvent. Figures 3 to 5 It can be seen from Table 1 that when isopropyl alcohol is used as the extraction solvent, the photoionization time of lactic acid is short, and the lactic acid detection signal is strong; when methanol and ethanol are used as the extraction solvents, the photoionization time of lactic acid is long, and the lactic acid detection signal is weak. This shows that using isopropyl alcohol as the extraction solvent can promote the photoionization of lactic acid to make lactic acid ionize quickly, and isopropyl alcohol can improve the signal intensity of lactic acid detection.

[0055] In addition, the extraction effects of isopropyl alcohol and methanol as extraction solvents are compared. Specifically, 10 mg of koji sample is accurately taken into a 10 mL sample bottle, 1 mL of isopropyl alcohol solvent is added for extraction. The extraction steps are as follows: 1 mL of isopropyl alcohol is added to 10 mg of koji sample, vortexed for 2 min first, and then centrifuged for 2 min at a speed of 12000 rpm / min. After solid-liquid separation, the liquid is collected, and the separated solid is subjected to a second extraction. The second extraction centrifugation diagram is as shown in Figure 6 . Using methanol as the extraction solvent, the koji sample is subjected to lactic acid extraction according to the above method, and the second extraction centrifugation diagram is as shown in Figure 6 . Figure 6The centrifuge tube with the middle number 1-3 is a state diagram after the second centrifugation after the second extraction with isopropanol as the extraction solvent, Figure 6 The centrifuge tube with the middle number 2-3 is a state diagram after the second centrifugation after the second extraction with methanol as the extraction solvent. Figure 6 It can be seen that when isopropanol is used to extract lactic acid in koji, the second centrifugation still has a deep extraction effect, and obvious solid-liquid separation phenomenon still occurs. This shows that isopropanol as an extraction solvent has better extraction effect. Therefore, isopropanol is used as the extraction solvent in the present scheme.

[0056] Example 2 Influence of isopropanol concentration on signal intensity

[0057] The present application analyzes the influence of different isopropanol concentrations on the analysis effect, and the solvents used include: 50% isopropanol, 75% isopropanol, 100% isopropanol (chromatographically pure isopropanol), and the remaining detection steps are the same as in Example 1. The detection results of lactic acid detected by three different concentrations of isopropanol as solvent are shown in Figure 7 , Figure 8 and Figure 3 , wherein Figure 3 is the detection result of lactic acid detected by 100% isopropanol as solvent, and the resolved signal intensity is shown in Table 2.

[0058] Table 2 Signal intensity of different isopropanol concentrations

[0059] Isopropanol concentration Signal strength (mv) 50％ 336 75％ 419 100％ 2800

[0060] This experiment investigates the influence of different isopropanol concentrations on the analysis effect of the target, and selects the isopropanol with the highest resolved signal intensity as the optimal solvent. It can be seen from Figure 7 , Figure 8 and Figure 3 in combination with Table 2 that when the concentration of isopropanol used as the extraction solvent is 100%, the photoionization time of lactic acid is shorter, and the detection signal of lactic acid is much higher than that of other concentrations; when the concentration of isopropanol used as the extraction solvent is 50% and 75%, the photoionization time of lactic acid is longer, and the detection signal of lactic acid is weaker. The ion mobility tube of the detector is the movement of ions under the detection electric field, and a higher water content will have a greater impact on the detection result. This shows that using isopropanol with a concentration of 100% as the extraction solvent can promote the photoionization of lactic acid to ionize lactic acid quickly, and isopropanol can improve the signal intensity of lactic acid detection.

[0061] Example 3

[0062] A method for detecting lactic acid in a wine fermentation product, comprising the following steps:

[0063] (1) Weigh the fermentation product sample, and add an extraction solvent to the fermentation product sample for extraction to obtain an extraction liquid.

[0064] Specifically, 10 mg of koji sample was accurately weighed in a 10 mL sample bottle, and 1 mL of isopropyl alcohol solvent was added to the koji sample for extraction. The extraction steps were as follows: 1 mL of isopropyl alcohol solvent was added to 10 mg of koji sample, vortexed for 2 min first, then centrifuged for 2 min at a speed of 12000 rpm / min, and the liquid was collected after solid-liquid separation. The remaining solid after solid-liquid separation was extracted twice, and the liquid was combined to obtain the extract.

[0065] (2) The extract was filtered and detected by an ion mobility spectrum analysis detector.

[0066] Specifically, the extract was filtered through a filter membrane with a pore size of 0.22 μm, and 3 μL of the filtered extract was diluted 40 times and then introduced into the ion mobility spectrum analysis detector for detection. The parameter settings of the ion mobility spectrum analysis detector were as follows: the ionization source was a photoionization source, the negative ion mode was used, the temperature of the migration tube was kept at 100°C, the sample carrier gas was air with a flow rate of 200 mL / min; the drift gas was air with a flow rate of 600 mL / min; and the doping gas was acetone with a flow rate of 50 mL / min.

[0067] The ion mobility spectrum of lactic acid in koji detected in this example is shown in Figure 9 The results showed that the characteristic peak position was consistent with that of the standard lactic acid solution. The signal peak was continuously collected for 0.5 min, and the recorded lactic acid peak cumulative addition signal Y value was introduced into the standard curve equation Y = -1470.17 + 2174.84*X to calculate the lactic acid concentration. The lactic acid content in the koji sample was calculated to be 0.4 g / 100 g.

[0068] Example 4

[0069] A method for detecting lactic acid in a wine fermentation product, comprising the following steps:

[0070] (1) Weigh the fermentation product sample, add the extraction solvent to the fermentation product sample for extraction, and obtain the extract.

[0071] Specifically, 24 mg of fermented grains sample was accurately weighed in a 10 mL sample bottle, and 1 mL of isopropyl alcohol solvent was added to the fermented grains sample for extraction. The extraction steps were as follows: 1 mL of isopropyl alcohol solvent was added to 10 mg of fermented grains sample, vortexed for 2 min first, then centrifuged for 2 min at a speed of 12000 rpm / min, and the liquid was collected after solid-liquid separation. The remaining solid after solid-liquid separation was extracted twice, and the liquid was combined to obtain the extract.

[0072] (2) The extract was filtered and detected by an ion mobility spectrum analysis detector.

[0073] Specifically, the extraction solution is filtered through a filter membrane with a pore size of 0.22 μm, 3 μL of the filtered extraction solution is diluted 40 times, and then introduced into the ion mobility spectrum analyzer for detection. The parameter settings of the ion mobility spectrum analyzer are as follows: the ionization source is a photoionization source, the negative ion mode is adopted, the temperature of the migration tube is kept at 100°C, the sample carrier gas is air, the sample carrier gas flow rate is 200 mL / min; the drift gas is air, the drift gas flow rate is 600 mL / min; the doping gas is acetone, and the doping gas flow rate is 50 mL / min.

[0074] The ion mobility spectrum of lactic acid in the fermented grains detected in the embodiment is shown in FIG. 1. Figure 10 As can be seen from the figure, the characteristic peak position is consistent with that of the standard lactic acid solution. The signal peak is continuously collected for 0.5 min, the recorded lactic acid peak cumulative addition signal Y value is brought into the standard curve equation Y = -1470.17 + 2174.84 * X, the lactic acid concentration is calculated, and the lactic acid content in the fermented grains sample is calculated to be 5.4 g / 100 g.

[0075] The present application extracts the brewing fermentation material, and then detects the extracted extraction solution by using the ion mobility spectrum analyzer, so as to qualitatively and quantitatively analyze the lactic acid in the brewing fermentation material. The present application detects the lactic acid in the brewing fermentation material, and has the advantages of simple pretreatment, small sample amount, low detection cost, and faster detection of the lactic acid in the brewing fermentation material.

[0076] The present embodiment is only an explanation of the present application, and is not a limitation of the present application. Those skilled in the art can make modifications to the present embodiment without creative contribution after reading the present specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for detecting lactic acid in a brewing ferment, characterized by, The method comprises the following steps: (1) weighing the fermentation sample, adding an extraction solvent to the fermentation sample for extraction to obtain an extraction liquid; (2) filtering the extraction liquid and detecting it by an ion mobility spectrum analysis detector; In step (1), the fermentation sample comprises one or a mixture of both of starter and fermented grains. In step (1), the mass of the starter sample is 10-1000 mg, the mass of the fermented grain sample is 10-50 mg, the extraction solvent is isopropyl alcohol, and the addition amount of the isopropyl alcohol is 0.5-2.0 mL. In step (1), the extraction comprises vortex oscillation for 1-3 min, centrifugation for 1-3 min, collection of the liquid after solid-liquid separation, and repeated extraction of the remaining solid for 2-3 times, and then the liquids are combined to obtain the extraction liquid. In step (2), 2-4 μL of the filtered extraction liquid is diluted by 30-50 times and then introduced into the ion mobility spectrum analysis detector for detection. In step (2), the ion mobility spectrum analysis detector is set as follows: the ionization source is a photoionization source, the negative ion mode is adopted, the temperature of the migration tube is kept at 90-110℃, the sample carrier gas is air with a flow rate of 180-220 mL / min, the drift gas is air with a flow rate of 580-620 mL / min, and the doping gas is acetone with a flow rate of 45-55 mL / min.

2. The method for detecting lactic acid in a brewing fermentation product according to claim 1, characterized in that: The addition amount of the isopropyl alcohol is 1 mL.

3. The method for detecting lactic acid in a brewing ferment according to claim 1, characterized by, The extraction liquid is vortex oscillated for 2 min and then centrifuged for 2 min, and the liquid is collected after solid-liquid separation.

4. The method for detecting lactic acid in a brewing ferment according to claim 1, characterized by, In step (2), the filtration of the extraction liquid comprises filtering the extraction liquid through a filter membrane with a pore size of 0.22 μm.

5. The method for detecting lactic acid in a brewing ferment according to claim 1, characterized by, In step (2), 3 μL of the filtered extraction liquid is diluted by 40 times and then introduced into the ion mobility spectrum analysis detector for detection.

6. The method for detecting lactic acid in a brewing ferment according to claim 1, characterized by, The temperature of the migration tube is kept at 100℃, the sample carrier gas is air with a flow rate of 200 mL / min, the drift gas is air with a flow rate of 600 mL / min, and the doping gas is acetone with a flow rate of 50 mL / min.

7. The method according to claim 1, wherein the method is characterized by, In step (2), the detection by the ion mobility spectrum analysis detector comprises qualitative analysis of lactic acid according to the migration time and quantitative analysis according to a linear equation established based on the ion signal intensity and the lactic acid concentration; the characteristic peak migration time of lactic acid is 4.615 ms.

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