CN114107434A - Method for evaluating lactic acid production capacity of Daqu in high throughput - Google Patents
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Abstract
The invention relates to the quality evaluation of Daqu in the technical field of microbial fermentation engineering, in particular to a method for evaluating the lactic acid production capacity of Daqu. The method comprises the following steps: (1) and (3) processing of a sample: adding Daqu leaching liquor into MRS culture medium, culturing, and collecting supernatant; (2) preparation of reaction solution: adding lactate oxidase and horseradish peroxidase into the buffer solution; (3) preparing a standard curve of a lactic acid standard substance; (4) diluting the supernatant in the step (1), adding the diluted supernatant into the reaction solution in the step (2), oscillating the diluted supernatant for reaction, and measuring the absorbance; calculating the content of the lactic acid according to the standard curve in the step (3); the sample is a Daqu leaching liquor. The determination method provided by the invention is particularly suitable for determining the content of lactic acid produced by a Daqu sample, and has high accuracy and high correlation with the content of lactic acid determined by an HPLC method. Compared with the conventional liquid phase analysis method, the method for analyzing the lactic acid production capacity of the Daqu has the characteristics of simple and accurate pretreatment, high speed and the like when being used for determining the lactic acid content in the Daqu fermentation liquor, and provides a new method for quickly detecting and analyzing the lactic acid content of a large number of samples.
Description
Technical Field
The invention relates to the quality evaluation of Daqu in the technical field of microbial fermentation engineering, in particular to a method for evaluating the lactic acid production capacity of Daqu.
Background
The Daqu is a leaven which is prepared by taking wheat as a raw material, properly crushing the wheat, adding water and mother yeast, stirring, pressing into blocks and fermenting, and has the functions of saccharification, fermentation, aroma enhancement and the like in the process of fermenting the white spirit. A large number of microorganisms are enriched and screened in the fermentation process of the yeast for making the hard liquor, a special microbial community system containing bacteria, molds and yeasts is formed, and the microorganisms and various enzymes produced by metabolism of the microorganisms are important sources of the microorganisms and the enzymes in the later white liquor brewing process. The yeast is the bone of the wine, the quality of the Daqu can directly influence the quality of the white spirit, and the important point is that the quality of the Daqu can directly influence the accumulation of lactic acid in the fermentation process of fermented grains. Lactic acid bacteria are a general term for a class of microorganisms capable of fermenting sugars to produce lactic acid, and are commonly present in the fermentation process of white spirit, and have important influence on the quality of the white spirit. In the wine making process, the Daqu is one of important sources of lactic acid bacteria, and the lactic acid bacteria can regulate the pH value of the fermented grains through metabolic synthesis of lactic acid, so that the community structure of microorganisms of the fermented grains is influenced, and the fermentation process can be influenced by interaction with other microorganisms. Therefore, the lactic acid production capacity of the Daqu fermentation can directly influence the lactic acid yield of fermented grains in the production process of white spirit, thereby influencing the quality of the produced white spirit.
Most of the existing methods for evaluating the yeast are sensory analysis, physical and chemical index detection, microorganism composition analysis and the like, and the evaluation indexes are the evaluation on the yeast making quality but cannot directly reflect the influence of the yeast on the subsequent wine making fermentation process. Meanwhile, the analysis method of the lactic acid content of the fermentation sample is mainly a High Performance Liquid Chromatography (HPLC) method, which has the advantages of accurate analysis, few pretreatment steps and the like, but the HPLC method can only analyze a single sample at each time, and the analysis takes long time and has low flux, so that the requirement of primary screening of a large number of samples in the actual production process cannot be met.
Disclosure of Invention
The yeast is a starter, a saccharifying agent and a raw material, the fermentation acid-producing capacities of different yeasts are different, and the fermentation acidity of fermented grains in the later-stage wine production can be influenced by the yeast with different acid-producing capacities.
In some embodiments, the present invention provides a method for determining the content of Daqu lactic acid, the method comprising the steps of: (1) treatment of the samples: adding Daqu leaching liquor into MRS culture medium, culturing, and collecting supernatant; (2) preparation of reaction solution: adding lactate oxidase and horseradish peroxidase into the buffer solution; (3) preparing a standard curve of a lactic acid standard substance; (3) diluting the supernatant in the step (1), adding the diluted supernatant into the reaction solution in the step (2), oscillating the diluted supernatant for reaction, and measuring the absorbance; calculating the content of the lactic acid according to the standard curve in the step (3); the sample is a Daqu leaching liquor.
When the indicator color development method is used for measuring the content of the lactic acid in the Daqu leach liquor fermentation liquor, the components of the Daqu leach liquor fermentation liquor are complex, namely the content of the lactic acid in the fermentation liquor is measured by using the experimental conditions of the indicator color development method with good linear effect of a standard curve, the detection error is large, and the correlation with the content of the lactic acid measured by an HPLC method is low. The inventor finds that the determination method provided by the invention is particularly suitable for determining the content of lactic acid produced by the Daqu sample, has high accuracy and high correlation with the content of lactic acid determined by an HPLC method. Compared with the conventional liquid phase analysis method, the method for analyzing the lactic acid production capacity of the Daqu has the characteristics of simple and accurate pretreatment, high speed and the like when being used for determining the lactic acid content in the Daqu fermentation liquor, and provides a new method for quickly detecting and analyzing the lactic acid content of a large number of samples.
In some embodiments, the step of preparing the Daqu leaching solution comprises: weighing Daqu in a sterile shake tube, adding sterile water, and vortex oscillating for more than 1.5min to obtain leaching solution.
In some embodiments, the ratio of the koji to water is from 0.5 to 4 g: 6-9.5 mL.
In some embodiments, the ratio of the koji to water is 1-2 g: 8-9 mL.
In some embodiments, the ratio of the koji to water is 1.0 g: 9 mL.
In some embodiments, the Daqu is selected from Daqu obtained by fermenting wheat and used in the production process of liquor, and high-temperature Daqu used in the production process of Maotai-flavor liquor.
In some embodiments, the step of sample processing comprises: adding MRS liquid culture medium and Daqu leaching liquor in a volume ratio of 12-19: 1-8, the culture temperature is 30-45 ℃, and the culture time is 3-7 days.
In some embodiments, the volume ratio of the MRS liquid medium to the Daqu leaching solution is 15-18: 2-5.
In some embodiments, the ratio of the MRS liquid medium to the Daqu extract is 9: 1.
in some embodiments, the incubation temperature is 35-40 ℃ and the incubation time is 5-7 days.
In some embodiments, the components of the buffer solution include: anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, N-ethyl-N- (2 hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and 4-aminoantipyrine.
In some embodiments, the buffer solution contains 3-7g/L of anhydrous potassium dihydrogen phosphate, 6-10g/L, N g/L of anhydrous disodium hydrogen phosphate, 0.5-3g/L of ethyl-N- (2 hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, and 0.05-0.3g/L of 4-aminoantipyrine.
In some embodiments, the buffer solution comprises 4.0-6.0g/L of anhydrous potassium dihydrogen phosphate; further preferably 5.6 g/L.
In some embodiments, the buffer solution contains 7.0 to 9.0g/L, more preferably 8.7g/L, of anhydrous disodium hydrogen phosphate.
In some embodiments, the buffer solution comprises 1-2g/L of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt.
In some embodiments, the buffer solution contains 1.85g/L of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt.
In some embodiments, the buffer solution contains 0.05 to 0.2g/L of 4-aminoantipyrine, and more preferably 0.08 g/L.
In some embodiments, the reaction solution comprises 400U/L of lactate oxidase 100-S and 700U/L of horseradish peroxidase 200-S.
In some embodiments, the reaction solution comprises 350U/L of lactate oxidase 200-; further preferably 250U/L.
In some embodiments, the reaction solution contains 500U/L horseradish peroxidase 300-; further preferably 400U/L.
In some embodiments, the concentration of the lactic acid standard ranges from 0.01 to 1 g/L.
In some embodiments, the concentration of the lactic acid standard ranges from 0.01 to 0.5 g/L.
In some embodiments, the concentration of the lactic acid standard ranges from 0.01 to 0.2 g/L.
In some embodiments, the concentration of the lactic acid standard ranges from 0.01 to 0.15 g/L.
In some embodiments, in step (4), the dilution factor of the supernatant is 10-60 fold.
In some embodiments, in step (4), the volume ratio of the supernatant diluent to the reaction solution is 1-4: 20-50 ℃, the oscillation reaction temperature is 35-40 ℃, and the oscillation time is 5-25 min.
In some embodiments, in step (4), the volume ratio of the supernatant diluent to the reaction solution is 2: 20-40, and the oscillation time is 10-15 min; preferably, the oscillation time is 10 min.
In some embodiments, the invention provides the use of the method for assessing the lactic acid production capacity of a yeast. The invention firstly provides the evaluation index of the quality of the yeast for making hard liquor by taking the lactic acid production capacity of the yeast for making hard liquor, and provides a new idea for further perfecting the quality evaluation system of the yeast for making hard liquor.
Drawings
FIG. 1 is a standard curve for lactic acid content determination.
FIG. 2 is a comparison of the results of measuring the lactic acid production content of Daqu by the method of example 2 and HPLC.
FIG. 3 shows the variation of lactic acid content in fermentation broth of Daqu in example 3 for different days of cultivation.
FIG. 4 is the color change of the fermentation broth of Daqu in example 3 for different days of cultivation.
FIG. 5 is a correlation analysis of the determination of the lactic acid content of Daqu by the indicator color development method and the HPLC method in comparative example 1.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention. Herein, the sources of the yeast are: high-temperature Daqu used in the brewing process of Maotai-flavor liquor.
EXAMPLE 1 preparation of a Standard Curve for lactic acid content
(1) Preparing a buffer solution: 5.60g of anhydrous potassium dihydrogen phosphate, 8.7g of anhydrous disodium hydrogen phosphate, 1.85g of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (EHSPT, or TOOS), and 0.08g of 4-aminoantipyrine (4-APP), 980mL of ultrapure water was added, and the mixture was stored at 4 ℃ for later use.
(2) Preparing a reaction solution: adding Lactate Oxidase (LOD) and horseradish Peroxidase (POD) into the buffer solution in the step (1) to enable the concentrations of the lactate oxidase and the horseradish peroxidase to be 250U/L and 400U/L respectively, and preparing reaction liquid;
(3) preparing a gradient diluted lactic acid standard solution (lactic acid standard): diluting the lactic acid standard substance with ultrapure water to obtain final concentrations of 0, 0.04, 0.05, 0.06, 0.07 and 0.08 g/L;
(4) taking 10 mu L of the lactic acid standard solution with each concentration in the step (3), adding 200 mu L of the reaction solution prepared in the step (2), after oscillation reaction, measuring the absorbance at 555nm by using an enzyme-labeling instrument, wherein the oscillation reaction temperature is 37 ℃, and the reaction time is 10 min;
(5) and drawing a standard curve according to the concentration and the absorbance of the lactic acid standard solution.
The standard curve is shown in figure 1.
EXAMPLE 2 determination of lactic acid-producing ability of Daqu
(1) Preparation of Daqu leaching liquor
Weighing 1.0g of Daqu in a sterile shake tube, adding 9.0mL of sterile water, and vortex shaking for 2min to obtain a leaching solution.
(2) And (3) processing of a sample: adding 900 μ L of MRS liquid culture medium into the well plate, leaching 100 μ L of Daqu, culturing at 37 deg.C for 5 days, and collecting supernatant.
(3) Preparation of buffer solution: 5.60g of anhydrous potassium dihydrogen phosphate, 8.7g of anhydrous disodium hydrogen phosphate, 1.85g of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (EHSPT, or TOOS) and 0.08g of 4-aminoantipyrine (4-APP) were added to ultrapure water to obtain anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and 4-aminoantipyrine in the buffer solution, wherein the concentrations of the anhydrous potassium dihydrogen phosphate, the anhydrous disodium hydrogen phosphate, the N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and the 4-aminoantipyrine were 5.71g/L, 8.88g/L, 1.89g/L and 0.82g/L, respectively.
(4) Preparation of reaction solution: and (3) adding Lactate Oxidase (LOD) and horseradish Peroxidase (POD) into the buffer solution in the step (3) to prepare a reaction solution, wherein the concentrations of the lactate oxidase and the horseradish peroxidase are respectively 250U/L and 400U/L.
(5) Diluting the supernatant of the step (2) by 50 times with ultrapure water, adding 10 μ L of the diluted supernatant into 200 μ L of the reaction solution prepared in the step (4), carrying out oscillation reaction at 37 ℃ for 10min, and then measuring the absorbance at 555nm by using an enzyme-labeling instrument.
From the measured absorbance values of the 16 groups of samples, the content of lactic acid was calculated by using the standard curve of example 1, as shown in table 1.
TABLE 1
Test example 1
The supernatant from step (2) of Experimental example 2 was taken and subjected to HPLC analysis:
1mL of the fermentation solution was centrifuged at 1,2000rpm at 4 ℃ for 10min, and the supernatant was diluted to an appropriate volume with ultrapure water, and then filtered by membrane filtration (0.22 μm) to obtain a filtrate, which was then analyzed for lactic acid.
A chromatographic column: a 4.6X 50mm, 1.8 μm Agilent ZORBX RRHT SB-C18 chromatography column;
and (3) computer program: the column temperature was 30 ℃ and contained 0.12% (vol/vol) phosphoric acid aqueous solution (A) and 100% methanol (B) as mobile phases at a flow rate of 0.2mL/min, a detection wavelength of 214nm and a sample introduction volume of 10. mu.L. A standard solution of lactic acid was prepared with ultrapure water in the concentration ranges of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0g/L, and the lactic acid was measured by the above analytical method, and a standard curve was drawn with the lactic acid peak area as ordinate and the concentration as abscissa.
The content of lactic acid in the fermentation broth was calculated according to the standard curve, and the measurement results of each group are shown in table 2.
The concentration values of the same samples measured in example 2 were fitted to the concentration values of the same samples measured in test example 1, and the fitted curve is shown in FIG. 2.
TABLE 2
Example 3 variation of lactic acid production content of Daqu at different incubation times
(1) Preparation of Daqu leaching liquor
Weighing 1.0g of Daqu in a sterile shake tube, adding 9.0mL of sterile water, and vortex shaking for 2min to obtain a leaching solution.
(2) And (3) processing of a sample: adding 900 μ L of MRS liquid culture medium into the well plate, leaching 100 μ L of Daqu, culturing at 37 deg.C for 1d, 2d, 3d, 4d, 5d and 6d, and collecting supernatant.
(3) Preparation of buffer solution: to 980mL of ultrapure water were added 5.60g of anhydrous potassium dihydrogen phosphate, 8.7g of anhydrous disodium hydrogen phosphate, 1.85g of N-ethyl-N- (2 hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (EHSPT, or TOOS), and 0.08g of 4-aminoantipyrine (4-APP), to give a buffer solution containing 5.71g/L, 8.88g/L, 1.89g/L, and 0.82g/L of anhydrous potassium dihydrogen phosphate, disodium hydrogen phosphate anhydrous, N-ethyl-N- (2 hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, and 4-aminoantipyrine, respectively.
(4) Preparation of reaction solution: and (3) adding Lactate Oxidase (LOD) and horseradish Peroxidase (POD) into the buffer solution in the step (3) to prepare a reaction solution, wherein the concentrations of the lactate oxidase and the horseradish peroxidase are respectively 250U/L and 400U/L.
(5) Diluting the supernatant of the step (2) by 50 times, adding 10 mu L of the diluted supernatant into 200 mu L of the reaction solution prepared in the step (4), carrying out oscillation reaction at 37 ℃ for 10min, and then measuring the absorbance at 555nm by using an enzyme-labeling instrument.
The lactic acid content was calculated from the measured absorbance values of each group and the standard curve of example 1.
The results of the measurements of the respective groups are shown in table 3, and fig. 3 and 4.
TABLE 3
| Cultivation time (sky) | Absorbance value |
| 1 | 0.688±0.052 |
| 2 | 1.319±0.54 |
| 3 | 2.165±0.155 |
| 4 | 2.837±0.133 |
| 5 | 3.203±0.321 |
| 6 | 3.233±0.246 |
Comparative example 1
In order to verify the accuracy of the indicator color development method for detecting the lactic acid in the sample, 50 samples are selected for liquid fermentation, and the lactic acid in the fermentation liquid is simultaneously measured by the color development method and HPLC.
The preparation method of the Daqu leaching solution and the sample treatment method are the same as in example 2.
(1) The specific experimental procedures of the indicator color development include (including preparation of a standard curve and determination of sample values using the standard curve):
fermentation liquor: preparing by using phosphate buffer solution with pH 7.6 and 10mM concentration;
lactic acid standard solution: preparing according to seven concentrations of 0, 0.1, 0.2, 0.5, 1,2 and 4 g/L;
preparing an indicator: preparing 0.05% bromothymol blue aqueous solution;
according to the test scheme, bromothymol blue aqueous solutions with different lactic acid concentration gradients are respectively prepared, indicator solutions are added into standard liquid and fermentation liquid according to the proportion of 4%, then absorbance measurement is carried out at the wavelength of 615nm, and the lactic acid content in the fermentation liquid is calculated according to the standard curve.
(2) The HPLC experimental method was the same as in test example 2.
The results of the measurement experiment are shown in FIG. 5. As can be seen from fig. 5, when the indicator color method is used for measuring the content of lactic acid in the fermentation broth of the Daqu leaching solution, the detection error is large because the components of the fermentation broth of the Daqu leaching solution are complex, i.e., the content of lactic acid in the fermentation broth is measured by using the experimental conditions of the indicator color method with good linear effect of the standard curve. The correlation with the content of lactic acid measured by HPLC method is low.
Claims (10)
1. A method for measuring the content of Daqu lactic acid is characterized by comprising the following steps:
(1) and (3) processing of a sample: adding Daqu leaching liquor into MRS culture medium, culturing, and collecting supernatant;
(2) preparation of reaction solution: adding lactate oxidase and horseradish peroxidase into the buffer solution;
(3) preparing a standard curve of a lactic acid standard substance;
(4) diluting the supernatant in the step (1), adding the diluted supernatant into the reaction solution in the step (2), oscillating the diluted supernatant for reaction, and measuring the absorbance; calculating the content of the lactic acid according to the standard curve in the step (3);
the sample is a Daqu leaching liquor.
2. The method of claim 1, wherein the step of preparing the Daqu leach solution comprises:
weighing Daqu in a sterile shake tube, adding sterile water, and performing vortex oscillation for more than 1.5min to obtain a leaching solution;
preferably, the ratio of the Daqu to water is 0.5-4 g: 6-9.5 mL;
preferably, the ratio of the Daqu to water is 1-2 g: 8-9 mL;
preferably, the ratio of the Daqu to water is 1.0 g: 9 mL;
preferably, the Daqu is selected from Daqu fermented from wheat used in the production process of liquor, and comprises high-temperature Daqu, medium-temperature Daqu and low-temperature Daqu;
preferably, the Daqu is selected from high-temperature Daqu used in the production process of Maotai-flavor liquor.
3. The method of claim 1, wherein the step of sample processing comprises: adding MRS liquid culture medium and Daqu leaching liquor in a volume ratio of 12-19: 1-8, the culture temperature is 30-45 ℃, and the culture time is 3-7 d;
preferably, the volume ratio of the MRS liquid medium to the Daqu leaching liquor is 15-18: 2-5;
preferably, the volume ratio of the MRS liquid medium to the Daqu leaching liquor is 9: 1;
or preferably, the culture temperature is 35-40 ℃, and the culture time is 5-7 d.
4. The method of claim 1, wherein the composition of the buffer solution comprises: anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, N-ethyl-N- (2 hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, 4-aminoantipyrine;
preferably, the buffer solution contains 3-7g/L of anhydrous potassium dihydrogen phosphate, 6-10g/L, N g/L of anhydrous disodium hydrogen phosphate, 0.5-3g/L of sodium salt of ethyl-N- (2 hydroxy-3-sulfopropyl) -3-methylaniline and 0.05-0.3g/L of 4-aminoantipyrine;
preferably, the buffer solution contains 4-6g/L of anhydrous potassium dihydrogen phosphate; further preferably 5.6 g/L;
preferably, the buffer solution contains 7-9g/L of anhydrous disodium hydrogen phosphate, and more preferably 8.7 g/L;
preferably, the buffer solution contains 1-2g/L of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt;
preferably, the buffer solution contains 1.85g/L of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt;
preferably, the buffer solution contains 0.05-0.2g/L of 4-aminoantipyrine, and further preferably 0.08 g/L.
5. The method as claimed in claim 1, wherein the reaction solution comprises lactate oxidase 100-400U/L, horseradish peroxidase 200-700U/L;
preferably, the reaction solution contains 350U/L of lactate oxidase 200-; further preferably 250U/L;
preferably, the reaction solution contains 500U/L of horseradish peroxidase 300-; further preferably 400U/L.
6. The method of claim 1, wherein the concentration of the lactic acid standard ranges from 0.01 to 1 g/L;
preferably, the concentration range of the lactic acid standard substance is 0.01-0.5 g/L;
preferably, the concentration range of the lactic acid standard substance is 0.01-0.2 g/L;
preferably, the concentration range of the lactic acid standard substance is 0.01-0.15 g/L.
7. The method of claim 1, wherein in step (4), the dilution factor of the supernatant is 10-60.
8. The method of claim 1, wherein in step (4), the volume ratio of the supernatant diluent to the reaction solution is 1-4: 20-50 ℃, the oscillation reaction temperature is 35-40 ℃, and the oscillation time is 5-25 min.
9. The method of claim 1, wherein in step (4), the volume ratio of the supernatant diluent to the reaction solution is 2: 20-40, and the oscillation time is 10-15 min; preferably, the oscillation time is 10 min.
10. Use of the method of claims 1-9 for evaluating the lactic acid production capacity of a koji.
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