Liquor flavoring type daqu esterification force determination and verification method

Abstract

The application discloses a kind of determination and verification method of strong-flavor Daqu esterification, including sample crushing treatment, reaction parameter optimization, esterification, double detection, blank control correction, data difference correction and esterification force calculation;5-factor 4-level orthogonal test system is adopted to obtain the optimal reaction parameter combination of adaptive organic phase reaction system;Organic phase reaction system uses n-heptane as organic phase medium;Double detection is cooperatively implemented using acid reduction method and gas chromatography, respectively obtaining substrate consumption related detection data and product generation related detection data;Blank control correction is obtained by three types of blank test superposition calculation Correction value is used to deduct non-target interference;For the dimensional difference of double detection, hierarchical correction logic is designed for data difference correction, to ensure the consistency and accuracy of detection result;The method of the application is simple in operation, short in detection period, high in precision, suitable for industrialized batch detection, can quickly feedback strong-flavor Daqu quality to guide liquor production.

Description

Technical Field

[0001] This invention relates to the field of baijiu brewing testing technology, specifically to a method for determining and verifying the esterification power of strong-aroma daqu (a type of starter culture). This method is used to rapidly and accurately determine the esterification power of strong-aroma daqu and is applicable to industrial testing scenarios such as daqu quality grading and fermentation parameter control during the production of strong-aroma baijiu. It can efficiently and accurately determine the activity of daqu esterification enzymes. Background Technology

[0002] Daqu (a type of starter culture) is the core saccharification and fermentation agent in the brewing of strong-aroma baijiu. Its esterification power directly determines the efficiency of the formation of main flavor substances such as ethyl hexanoate in the mash, thus affecting the flavor quality and fermentation efficiency of baijiu. It is a key physicochemical indicator for evaluating the quality of daqu. Currently, the industry generally uses the "QB / T 4257-2011 General Analytical Method for Brewing Daqu" to determine the esterification power of daqu. Under specified test conditions, this method utilizes the esterification enzymes in daqu to catalyze the synthesis of esters from free organic acids (hexanoic acid) and ethanol. After 7 days of esterification, the sample solution is distilled, and the total esters (calculated as ethyl hexanoate) generated are determined by saponification, thereby characterizing the esterification power of daqu.

[0003] However, this traditional method still has many obvious shortcomings: 1. The testing cycle is too long: It requires 7 days (168h) of constant temperature static culture. The test results are seriously delayed and cannot guide the production process such as Daqu screening and fermentation parameter control in the cellar in a timely manner. It is difficult to meet the high-efficiency testing needs of modern liquor production. 2. The measurement results are prone to distortion: After the esterification reaction is completed, the esters need to be separated by heating and distillation. During the distillation process, the residual hexanoic acid and ethanol will continue to undergo secondary esterification, leading to an overestimation of the measurement results. In the subsequent boiling water bath saponification, the esters are easily lost due to high-temperature volatilization, further introducing systematic errors. The dual effects cause the measured value to deviate significantly from the true esterification power of Daqu. 3. Poor precision: Traditional methods use an aqueous reaction system. Non-polar substrates such as hexanoic acid have limited solubility in water, and the Daqu (a type of starter culture) particles are prone to sedimentation, resulting in uneven contact between the esterification enzyme and the substrate. Furthermore, the sample needs to be transferred multiple times after esterification, and smearing can easily occur during distillation. Under the influence of multiple factors, the relative standard deviation (RSD) of parallel samples can only be controlled within ≤10%, resulting in poor data repeatability and insufficient reliability. 4. Cumbersome operation process: It includes multiple complex steps such as distillation and saponification reflux, requires special equipment, consumes a large amount of test reagents, consumes a lot of energy, and is labor-intensive, making it unsuitable for large-scale batch testing. 5. Insufficient validation of single methods: Existing methods mostly use single detection means such as saponification, acid reduction or single gas chromatography, which lack cross-validation between different independent methods, making it difficult to eliminate systematic errors caused by single methods, thus limiting the reliability of detection results.

[0004] 6. The blank correction system is imperfect. Using only a single blank correction cannot completely eliminate multiple interferences such as non-enzymatic reactions, reagent impurities, and initial sample conditions, resulting in insufficient detection accuracy. 7. Problem of data discrepancy in dual detection: If the acid reduction method and gas chromatography are used for parallel detection, the two detection dimensions are different (corresponding to substrate consumption and product formation, respectively). The acid reduction method is easily affected by other organic acids in Daqu, and the gas chromatography method is easily affected by impurity peaks, which can easily lead to detection data deviation. The existing technology lacks a scientific correction logic to solve this problem.

[0005] Furthermore, existing related improvement technologies have failed to fully address the aforementioned issues: some schemes only optimize distillation or saponification operation parameters, failing to overcome the inherent limitations of aqueous systems, and the problems of substrate solubility and enzyme-substrate contact efficiency remain unresolved; a few studies, while attempting to use organic phases as reaction media, lack systematic multi-factor parameter optimization, have not designed correction schemes for data differences from dual detection, and have not optimized the definition of esterification power units; other schemes only employ gas chromatography for single detection, still relying on long-term culture or complex pretreatment, failing to balance speed and accuracy. Therefore, developing a method for determining the esterification power of strong-aroma daqu with a short detection cycle, high accuracy, good precision, simple operation, and suitability for industrial applications has become an urgent need in the current field of baijiu brewing testing. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by providing a method for determining and verifying the esterification power of strong-aroma daqu (a type of Chinese liquor). Through scientific parameter optimization, a comprehensive blank correction system, and a reasonable dual-detection correction logic, the method achieves rapid and accurate determination of the esterification power of daqu, meeting the testing needs of industrial production.

[0007] To achieve its objectives, the present invention employs the following technical solution: The method for determining the esterification power of strong-aroma daqu (a type of Chinese liquor) of this invention is characterized by the following steps: sample pulverization, reaction parameter optimization, esterification reaction, dual detection, blank control correction, data difference correction, and esterification power calculation. The reaction parameter optimization employs a 5-factor, 4-level orthogonal experimental system to screen and obtain the optimal combination of reaction parameters suitable for the organic phase reaction system. The organic phase reaction system uses n-heptane as the organic phase medium. The dual detection method combines acid reduction and gas chromatography to obtain substrate consumption-related and product generation-related detection data, respectively. The blank control correction calculates a correction value by superimposing three types of blank experiments to eliminate non-target interference. The data difference correction employs a graded correction logic designed for dimensional differences in dual detection to ensure the consistency and accuracy of the detection results.

[0008] The method for determining the esterification power of strong-aroma daqu (a type of starter culture) of this invention is carried out according to the following steps: Step 1, Sample Grinding Process: Grind the strong-aroma type Daqu into powder, pass it through a 40-60 mesh sieve, take the sieve material, seal and dry it for storage, and obtain Daqu sample with uniform particle size and stable enzyme activity. Step 2: Screening the optimal combination of reaction parameters: A 5-factor, 4-level orthogonal experiment was used to screen the reaction parameters suitable for the n-heptane organic phase system, and to obtain the optimal combination of reaction parameters with good esterification effect and precision; the 5 factors are: hexanoic acid concentration, ethanol concentration, amount of Daqu sample, esterification temperature and esterification time. Step 3, Esterification Reaction: In a 100mL Erlenmeyer flask with a ground glass stopper, add 30mL of n-heptane as the organic phase medium. Then, add 10mL each of hexanoic acid-n-heptane solution and ethanol-n-heptane solution of the corresponding concentrations, along with the corresponding mass of Daqu (a type of starter culture) sample, according to the optimal reaction parameter combination. Mix thoroughly to obtain an optimal reaction system mixture with a total volume of 50mL for the esterification reaction. Seal the Erlenmeyer flask with a stopper and sealing film to prevent the n-heptane from evaporating. Perform the esterification reaction according to the esterification temperature and time specified in the optimal reaction parameter combination to obtain the esterification reaction solution. Step 4: Perform dual detection: Analyze the esterification reaction solution using both the acid reduction method and gas chromatography, obtaining two sets of independent data. The volume V1 of sodium hydroxide standard solution consumed by the acid reduction method and the concentration c of ethyl hexanoate determined by gas chromatography are obtained as follows: 酯 ; Step 4.1: After the esterification reaction is complete, shake the Erlenmeyer flask well and let it stand for 10-15 minutes until the Daqu particles are completely precipitated. Step 4.2, Determination of acid reduction: Take 20 mL of supernatant from the Erlenmeyer flask and transfer it to a 50 mL conical flask. Add 20 mL of distilled water and 2 drops of phenolphthalein indicator. Titrate with 0.05 mol / L sodium hydroxide standard solution until the solution turns slightly red and does not fade within 30 seconds. Record the volume of sodium hydroxide standard solution consumed, V1. Step 4.3, Gas Chromatography Detection: The supernatant was collected in an Erlenmeyer flask, filtered through a 0.22 μm organic phase filter membrane, and the amount of ethyl hexanoate produced was determined by gas chromatography. The concentration c of ethyl hexanoate was calculated using the internal standard method. 酯 ; Step 5, Blank Control Correction: Set up three types of blank tests, namely reagent blank, sample blank and inactivation blank. Calculate the blank correction value by superposition correction method. This value is used to deduct multiple interferences from non-enzymatic reactions, reagent impurities and the initial state of the sample, and obtain the volume correction value V0 of the sodium hydroxide standard solution consumed in the acid reduction method blank and the concentration correction value c0 of the ethyl hexanoate determined by the gas chromatography blank. Step 6, Data Difference Correction: For the differences in detection data from the acid reduction method (substrate consumption dimension) and the gas chromatography method (product formation dimension), a graded correction is performed based on relative deviation determination, as follows, to obtain the initial corrected esterification power values ​​for the two different detection methods: Step 6.1: Calculate the initial value of esterification force: Based on the 1:1 molar ratio of hexanoic acid to ethyl hexanoate, the initial values ​​of esterification power for the acid reduction method are calculated according to equations (1) and (2) respectively. 1. Initial value of esterification force in gas phase method 2 (1); (2); in: c is the concentration of the sodium hydroxide standard solution; V 总 The total volume of the reaction system; 144 is the simplified calculated molar mass of ethyl hexanoate; m is the mass of the Daqu sample; V 取样 The volume of supernatant was taken for titration of the esterification reaction solution using the acid reduction method; 1000 is the unit conversion factor. The definition of esterification power unit (U) is: the number of milligrams of ethyl hexanoate synthesized from hexanoic acid and ethanol per gram of oven-dried Daqu (a type of starter culture) under 35°C conditions for 24 hours. It is expressed as "milligrams per gram • 24 hours (mg / g • 24h)". Step 6.2: Calculate the relative deviation D according to formula (3): (3); The relative deviation D characterizes the degree of deviation in the detection data between two different detection methods; Step 6.3, Set the calibration logic: If D≤3%, it indicates that the data from the two detection methods are in good agreement and there is no significant interference. If 3% < D ≤ 8%: the weighted average method is used for correction. Combining the specificity of the two detection methods, since gas chromatography has stronger specificity for directly detecting characteristic products, the weight of the acid reduction method is set to 0.4 and the weight of gas chromatography is set to 0.6. If D > 8%: After investigating interfering factors, retest. Use high performance liquid chromatography to remove interference from other organic acids using the acid reduction method, and optimize chromatographic conditions using gas chromatography to ensure that the resolution between the ethyl hexanoate peak and the impurity peak is ≥ 1.5. Step 7: Based on the initial esterification power value and the data difference correction results, determine the final esterification power value M per gram of oven-dried Daqu using the following method, as the test result: If D≤3%, then the final value of esterification power is... for: ; If 3% < D ≤ 8%, then the final value of esterification power is... for: = 1×0.4+ 2×0.6; This completes the determination of the esterification power of strong-aroma daqu.

[0009] The method for determining the esterification power of strong-aroma Daqu (a type of Chinese koji) in this invention is also characterized in that: in step 2, the 5 factors and 4 levels refer to: hexanoic acid concentration 0.15-0.30 mol / L, ethanol concentration 0.1-0.4 mol / L, Daqu sample amount 1-15 g, esterification temperature 25-40℃, and esterification time 24-60 h; the orthogonal experiment uses L... 16 (4 5 An orthogonal array was used, with a total of 16 experimental groups. Each group of samples was measured twice in parallel and incubated at a constant temperature. After incubation, the consumption of hexanoic acid in each group was initially determined by the acid reduction method, and the amount of ethyl hexanoate produced was initially determined by gas chromatography. The consumption of hexanoic acid, the amount of ethyl hexanoate produced, and the relative standard deviation (RSD) of parallel samples were used as comprehensive evaluation indicators. The influence of each parameter on the esterification power was intuitively analyzed by the main effect diagram of each factor. The parameter combination with high hexanoic acid consumption, large amount of ethyl hexanoate produced, and small RSD of parallel samples was selected as the optimal reaction parameter combination for the n-heptane organic phase system.

[0010] The method for determining the esterification power of strong-aroma daqu in this invention is characterized by the following: the optimal combination of reaction parameters determined through screening is: hexanoic acid concentration 0.25 mol / L, ethanol concentration 0.3 mol / L, daqu sample amount 1 g, esterification temperature 35℃ and esterification time 24 h. The optimal combination of reaction parameters enables rapid and accurate determination of the esterification power of strong-aroma daqu.

[0011] The method for determining the esterification power of strong-aroma Daqu in this invention is also characterized by the following: in step 1, the sample pulverization process involves pulverizing Daqu and passing it through a 50-mesh sieve; in step 4.1, after the esterification reaction is completed, the settling time after shaking the Erlenmeyer flask is set to 12 minutes to ensure that the supernatant is clear and free of impurities, thus avoiding interference from Daqu particles on the titration results and gas phase detection results.

[0012] The method for determining the esterification power of strong-aroma daqu (a type of Chinese liquor) of the present invention is also characterized by the following: In step 4.3, the chromatographic conditions are set as follows: a DB-WAX capillary column with a specification of 30m×0.25mm×0.25μm; column temperature program: initial temperature 40℃ and hold for 2 min, then increase the temperature to 160℃ at 15℃ / min and hold for 2 min, then increase the temperature to 220℃ at 30℃ / min and hold for 10 min; injection port temperature 230℃, detector temperature 240℃; carrier gas is high-purity nitrogen, flow rate 1.7mL / min; split ratio 45:1; injection volume 1μL.

[0013] The method for determining the esterification power of strong-aroma daqu (a type of Chinese liquor) of this invention is also characterized by the following: the capillary column in the gas chromatography detection can be replaced with HP-INNOWAX, or other types of chromatographic columns that are consistent with DB-WAX capillary columns in terms of separation effect and applicable range. The column temperature program is finely adjusted according to the instrument performance and impurity separation to ensure that the separation degree of ethyl hexanoate peak and other impurity peaks is ≥1.5. The acid reduction method can use high-performance liquid chromatography to assist in the determination of hexanoic acid purity in the system, eliminate the interference of other organic acids, and further improve the accuracy of the detection results of the acid reduction method.

[0014] The method for determining the esterification power of strong-aroma daqu (a type of Chinese liquor) of this invention is also characterized in that: in step 5, the volume correction value V0 of the sodium hydroxide standard solution consumed in the acid reduction method blank and the concentration correction value c0 of ethyl hexanoate determined by the gas chromatography blank are obtained as follows: Reagent blank test: Prepare a reagent mixture without Daqu sample according to the composition of the optimal reaction system mixture, and incubate and detect according to steps 3 and 4. Obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the reagent blank test. 01 And the concentration of ethyl hexanoate c determined by gas chromatography in the reagent blank test. 01 ; Sample blank test: Using the optimal reaction system mixture that has not undergone esterification as the object, dual detection is performed according to step 4 to obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the sample blank test. 02 And the concentration of ethyl hexanoate c determined by gas chromatography in the sample blank test. 02 ; Inactivation blank test: After sterilizing the Daqu sample at 121℃ for 30 min to inactivate enzyme activity, prepare a mixture according to the optimal reaction system mixture composition, and then incubate and detect according to steps 3 and 4. Obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the inactivation blank test. 03 And the concentration of ethyl hexanoate c determined by gas chromatography in the inactivation blank test. 03 Then we have: V0 = V 01 + (V 02 - V 01 ) + (V 03 - V 01 ); c0 = c 01 + (c 02 - c 01 ) + (c 03 -c 01 ).

[0015] The present invention has the following characteristics regarding the verification method for the esterification power of strong-aroma daqu: after the esterification power of strong-aroma daqu is determined, a series of verification tests are conducted, and a qualified determination method is obtained through multi-dimensional verification. The multi-dimensional verification includes verification of the optimal combination of reaction parameters, comparative verification of aqueous and organic phase systems, and 6 parallel precision tests.

[0016] The characteristic of this invention regarding the verification method for the esterification power of strong-aroma daqu (a type of Chinese liquor) that has already been determined also lies in the following series of verification tests: The verification of the optimal reaction parameter combination refers to: conducting verification tests on 3 batches of samples according to the optimal reaction parameter combination, with each sample measured twice in parallel, to determine the esterification power and precision. The comparative verification of the aqueous and organic phase systems refers to: based on the optimal combination of reaction parameters, replacing the reaction medium with water to construct an aqueous phase system, and comparing the esterification power and precision with the n-heptane organic phase system. The aforementioned 6-parallel precision test refers to performing esterification reactions and measurements on the same Daqu sample using the optimal combination of reaction parameters in 6 parallel samples, with an RSD ≤ 5%.

[0017] This invention uses a 5-factor, 4-level orthogonal experimental system to screen and determine the optimal combination of reaction parameters. It combines simultaneous determination using an acid reduction method and gas chromatography, designs a targeted data difference correction logic, and optimizes the definition of the esterification power unit. This enables the determination of the esterification power of Daqu (a type of Chinese liquor) based on a n-heptane organic phase system. Compared with existing technologies, the advantages of this invention are as follows: 1. Scientific and systematic parameter optimization with outstanding innovation: This invention abandons the traditional single-factor optimization method. Through a 5-factor, 4-level orthogonal experiment, it comprehensively screens five key influencing factors and different levels, including hexanoic acid concentration, ethanol concentration, sample amount, temperature, and time. It accurately locates the key factors affecting the esterification reaction. The optimal combination of reaction parameters selected balances esterification effect and detection precision, effectively avoiding the limitations of single-factor optimization and ensuring the scientific nature and stability of the method.

[0018] 2. Dual detection and correction logic closed loop ensures high reliability of results: Cross-validation is performed using the acid reduction method (substrate consumption) and gas chromatography (product formation), and a hierarchical correction logic based on relative deviation is designed. This design effectively solves the data inconsistency problem caused by dimensional differences in dual detection, significantly improving the consistency and accuracy of the final detection results.

[0019] 3. Improved blank correction system significantly enhances detection accuracy: An innovative three-tiered blank correction system—reagent blank, sample blank, and inactivation blank—is constructed, comprehensively eliminating multiple interferences such as non-enzymatic reactions, reagent impurities, and initial sample conditions. This solves the technical problem of incomplete single blank correction in existing technologies, enabling the measurement results to more accurately reflect the actual activity of Daqu esterase.

[0020] 4. The unit definition is intuitive and has strong industrial applicability: The esterification power unit is defined as "mg / g·24h (U)", which can directly reflect the esterification capacity of each gram of daqu (oven-dried) within 24 hours. This is in line with the cognitive habits of enzyme activity indicators in the liquor industry, lowers the threshold for data interpretation, and facilitates rapid comparison and quality control on the production site.

[0021] 5. Improved data authenticity and significantly enhanced precision: This method avoids secondary esterification and ester volatilization losses by omitting distillation and saponification steps; the n-heptane organic phase medium also improves substrate solubility and reduces ester hydrolysis, ensuring data authenticity from the source; based on the 1:1 molar ratio conversion of the esterification reaction, the units of the acid reduction method and gas chromatography are unified, laying a solid foundation for data accuracy; the relative standard deviation (RSD) of parallel samples is ≤5%, which is significantly better than the traditional method (RSD≤10%).

[0022] 6. Significantly shortened testing cycle, meeting the high-efficiency needs of industrial production: Under optimal reaction parameter combinations, the esterification reaction cycle is shortened from 168 hours in the traditional method to 24 hours, improving efficiency by 85.7%. Testing results can be quickly fed back to the production process, providing timely guidance for Daqu grading and fermentation control, significantly improving the overall efficiency of Baijiu production.

[0023] 7. Comprehensive verification system and high method stability: Through multi-dimensional verification experiments (optimal reaction parameter combination verification, system comparison, precision verification, and consistency verification), the stability, applicability, and anti-interference ability of this method have been fully demonstrated. It can meet the stringent requirements of industrial batch testing, and the test results are highly reliable.

[0024] 8. Simple operation and energy saving, with broad application prospects: This method does not require distillation equipment and saponification reflux equipment, which greatly simplifies the operation process; the minimum sample amount is only 1g, which is only 4% of the sample amount of traditional methods, reducing reagent consumption and significantly reducing detection costs, labor intensity and energy consumption; gas phase detection pretreatment only requires simple filtration, the data correction logic is clear and operable, the calculation process can be simplified under optimal conditions, it is suitable for large-scale batch detection, and has good industrial application prospects. Attached Figure Description

[0025] Figure 1 This is a framework diagram for a 5-factor, 4-level orthogonal experimental design.

[0026] Figure 2 This is the main effect diagram of the influence of various factors on esterification force in the orthogonal experiment.

[0027] Figure 3 This is a comparison diagram of the detection process of the method of the present invention and the traditional method.

[0028] Figure 4 This is the standard curve for ethyl hexanoate.

[0029] Figure 5 This is the gas chromatogram of the sample. Detailed Implementation

[0030] The method for determining the esterification power of strong-aroma daqu in this invention includes sample pulverization, reaction parameter optimization, esterification reaction, dual detection, blank control correction, data difference correction, and esterification power calculation. Reaction parameter optimization employs a 5-factor, 4-level orthogonal experimental system to screen and obtain the optimal combination of reaction parameters suitable for the organic phase reaction system. The organic phase reaction system uses n-heptane as the organic phase medium. Dual detection is implemented using a combination of acid reduction and gas chromatography to obtain substrate consumption-related detection data and product generation-related detection data, respectively. Blank control correction is calculated by superimposing three types of blank experiments to obtain correction values, used to eliminate non-target interference. The data difference correction is designed with a hierarchical correction logic to address the dimensional differences in dual detection, ensuring the consistency and accuracy of the detection results.

[0031] 1. Materials and Instruments 1.1 Experimental materials: strong-aroma Daqu sample, hexanoic acid (analytical grade), ethanol (analytical grade), deionized water, phenolphthalein indicator, sodium hydroxide standard solution (0.05 mol / L), ethyl hexanoate (chromatographic grade), n-amyl acetate (chromatographic grade), etc.

[0032] 1.2 Test instruments: Gas chromatograph (equipped with flame ionization detector and DB-WAX capillary column), electronic balance (accuracy 0.001 g), constant temperature water bath (accuracy ±0.5℃), pulverizer, standard sieve, desiccator, burette, Erlenmeyer flask, etc.

[0033] 2. Test methods 2.1 Screening for the optimal combination of reaction parameters To optimize the reaction parameters in the determination of esterification power of strong-aroma daqu and improve the precision, stability and accuracy of the determination method, this invention selects five key reaction parameters that have a significant impact on the esterification reaction and the determination of esterification power as factors for investigation. Each factor is set with four levels, and orthogonal experimental design is used to optimize and screen multiple factors and levels to determine the optimal combination of reaction parameters.

[0034] 2.1.1 Orthogonal Experimental Design Based on the characteristics of the esterification reaction of strong-aroma daqu (a type of starter culture), hexanoic acid concentration, ethanol concentration, sample amount, esterification temperature, and esterification time were selected as factors to be investigated, denoted by symbols A, B, C, D, and E, respectively. Each factor was set with four levels, and L was selected as the optimal level. 16 (4 5 The orthogonal experimental setup is arranged as shown in the attached table. Figure 1 As shown.

[0035] 2.1.2 Results and Analysis of Orthogonal Experiments Sixteen experimental groups were tested twice in parallel. The reaction mixture (30 mL n-heptane + corresponding substrate + Daqu sample, total volume 50 mL) was prepared according to the orthogonal array parameters. The Erlenmeyer flasks were sealed with stoppers and incubated at the corresponding temperature and time. After incubation, 20 mL of the supernatant from each group was taken for preliminary determination of hexanoic acid consumption using the acid reduction method. Another supernatant was filtered and used for preliminary determination of ethyl hexanoate formation by gas chromatography. The effects of each factor on esterification power were analyzed using main effect plots to determine the optimal parameter combination. See the appendix for detailed main effect plots of each factor. Figure 2 .

[0036] Appendix Figure 2 middle Figure 2 A represents the main effect diagram of the effect of hexanoic acid concentration. As can be seen from the figure, the esterification power first increases and then decreases with increasing hexanoic acid concentration, reaching its maximum value at level A3, indicating that A3 is the optimal level of hexanoic acid concentration. Appendix Figure 2 middle Figure 2 B is the main effect diagram of the effect of ethanol concentration. As can be seen from the figure, the esterification power first increases and then decreases with the increase of ethanol concentration, and reaches the maximum value at the B3 level, indicating that B3 is the optimal level of ethanol concentration. Appendix Figure 2 middle Figure 2 C represents the main effect diagram of the effect of ethanol concentration. As can be seen from the figure, the esterification power gradually decreases with the increase of sample amount, and reaches the maximum value at the C1 level, indicating that C1 is the optimal level for sample amount. Appendix Figure 2 middle Figure 2 D is the main effect diagram of the effect of ethanol concentration. As can be seen from the figure, the esterification power first increases and then decreases with increasing temperature, reaching its maximum value at the D3 level, indicating that D3 is the optimal temperature level. Appendix Figure 2 middle Figure 2 E is the main effect diagram of the effect of ethanol concentration. As can be seen from the figure, the esterification power generally decreases with the extension of reaction time, and reaches its maximum value at the E1 level, indicating that E1 is the optimal time level.

[0037] The optimal combination of reaction parameters for determining the esterification power of strong-aroma Daqu (a type of Chinese koji) was determined to be A3B3C1D3E1, namely, hexanoic acid concentration of 0.25 mol / L, ethanol concentration of 0.3 mol / L, sample amount of 1 g, esterification temperature of 35℃, and esterification time of 24 h. Under this parameter combination, the esterification reaction can proceed fully, and the measured esterification power values ​​are stable and accurate, which can best reflect the esterification ability of strong-aroma Daqu.

[0038] 2.2 Screening based on the settling time of the reaction solution After the esterification reaction is complete, shake the Erlenmeyer flask well and let it stand for 10-15 minutes, preferably 12 minutes, until the Daqu particles are completely precipitated; so that the supernatant is clear and free of impurities, and the Daqu particles do not interfere with the titration results and gas phase detection results.

[0039] 10 minutes is sufficient for basic sedimentation, enabling rapid initial sedimentation of Daqu particles, suitable for scenarios requiring a certain level of experimental efficiency; 12 minutes is the optimal duration, allowing Daqu particles to settle completely, with a clear supernatant free of impurities. This avoids interference from particle residue due to insufficient sedimentation time, and also prevents prolonged experimental cycles due to excessive time, balancing sedimentation effect and detection efficiency; 15 minutes provides more thorough sedimentation, suitable for Daqu samples with fine particles and easy suspension, further avoiding interference from incomplete solid-liquid separation, ensuring the accuracy and stability of titration and gas chromatography results.

[0040] 2.3 Verification Experiment In a specific implementation of this invention, the verification method for the measured esterification power of strong-aroma daqu is as follows: after completing the measurement of the esterification power of strong-aroma daqu, a series of verification tests are carried out. The qualified measurement method is obtained through multi-dimensional verification. The multi-dimensional verification includes verification of the optimal reaction parameter combination, verification of the aqueous phase and organic phase system comparison, and 6 parallel precision tests.

[0041] Optimal reaction parameter combination verification refers to: conducting verification tests on 3 batches of samples according to the optimal reaction parameter combination, with each sample measured twice in parallel, to determine the esterification power and precision; The comparative verification of aqueous and organic phase systems refers to: based on the optimal combination of reaction parameters, replacing the reaction medium with water to construct an aqueous phase system, and comparing the esterification power and precision with the n-heptane organic phase system. 6. Parallel precision test refers to: performing esterification reaction and determination of the same Daqu sample in 6 parallel samples according to the optimal reaction parameter combination, requiring RSD≤5%.

[0042] Specific verification data are shown in Table 1.

[0043] The verification test results show that the method for determining the esterification power of strong-aroma daqu under the optimal parameter combination has high precision, good stability, and good reproducibility. The test results are accurate and reliable, and it can be used as a standard method for determining the esterification power of strong-aroma daqu.

[0044] 3. Procedure for determining esterification power Based on the optimal combination of reaction parameters selected above, the method for determining the esterification power of strong-aroma daqu in the specific implementation of this invention is carried out according to the following steps: Step S1, Sample pulverization: Pulverize the strong-aroma Daqu (a type of starter culture) and pass it through a 40-60 mesh sieve, preferably a 50 mesh sieve; take the sieve-passing material, seal and dry it for storage, and obtain a Daqu sample with uniform particle size and stable enzyme activity; Step S2, esterification reaction: Accurately pipette 10.0 mL each of hexanoic acid-n-heptane solution (0.25 mol / L) and ethanol-n-heptane solution (0.3 mol / L) into a 100 mL ground glass stoppered conical flask. Weigh 1.0 g of Daqu powder (accurate to 0.01 g) into the conical flask, add 30 mL of n-heptane, shake well, stopper, and seal the conical flask with sealing film. Incubate at 35℃ for esterification for 24 h. Step S3: Perform dual detection: Analyze the esterification reaction solution using both the acid reduction method and gas chromatography, obtaining two sets of independent detection data. The volume V1 of sodium hydroxide standard solution consumed by the acid reduction method and the concentration c of ethyl hexanoate determined by gas chromatography are obtained as follows: 酯 : Step S3.1: After the esterification reaction is completed, shake the Erlenmeyer flask well and let it stand for 12 minutes until the Daqu particles are completely precipitated; make the supernatant clear and free of impurities to avoid interference from the Daqu particles on the titration results and gas phase detection results. Step S3.2, Determination of acid reduction: Take 20 mL of supernatant from the Erlenmeyer flask and place it in a 50 mL Erlenmeyer flask. Add 20 mL of distilled water and 2 drops of phenolphthalein indicator. Titrate with 0.05 mol / L sodium hydroxide standard solution until the solution turns slightly red and does not fade within 30 seconds. Record the volume V1 of sodium hydroxide standard solution consumed. Step S3.3, Gas Chromatography Detection: The supernatant was drawn into an Erlenmeyer flask, filtered through a 0.22 μm organic phase filter membrane, and the amount of ethyl hexanoate produced was determined by gas chromatography. The concentration c of ethyl hexanoate was calculated using the internal standard method. 酯 ; The specific chromatographic conditions were set as follows: a DB-WAX capillary column with dimensions of 30m × 0.25mm × 0.25μm; column temperature program: initial temperature 40℃ for 2 min, then increased to 160℃ at 15℃ / min and held for 2 min, then increased to 220℃ at 30℃ / min and held for 10 min; injection port temperature 230℃, detector temperature 240℃; carrier gas was high-purity nitrogen at a flow rate of 1.7mL / min; split ratio 45:1; injection volume 1μL; and the concentration of ethyl hexanoate (c) was calculated using the internal standard method. 酯 Standard curve preparation: Prepare a series of ethyl hexanoate standard solutions ranging from 5 to 5000 mg / L, and determine the concentrations under the chromatographic conditions described above. Plot a standard curve with peak area on the ordinate and concentration on the abscissa, with a correlation coefficient R² ≥ 0.995. The standard curve and chromatogram are attached. Figure 4 and attached Figure 5 As shown.

[0045] The capillary column used in gas chromatography can be replaced with HP-INNOWAX, or other types of columns that are consistent with DB-WAX capillary columns in terms of separation effect and applicability. The column temperature program can be fine-tuned according to the instrument performance and impurity separation to ensure that the resolution of the ethyl hexanoate peak and other impurity peaks is ≥1.5. The acid reduction method can be assisted by high performance liquid chromatography to determine the purity of hexanoic acid in the system, eliminate the interference of other organic acids, and further improve the accuracy of the acid reduction method detection results. Step S4, Blank Control Correction: Three types of blank tests are set up: reagent blank, sample blank, and inactivation blank. The blank correction value is calculated by superposition correction method to eliminate multiple interferences from non-enzymatic reactions, reagent impurities, and the initial state of the sample. The volume correction value V0 of sodium hydroxide standard solution consumed in the acid reduction method blank and the concentration correction value c0 of ethyl hexanoate determined by gas chromatography blank are obtained as follows: Reagent blank test: Prepare a reagent mixture without Daqu sample according to the composition of the optimal reaction system mixture, and incubate and detect according to steps S2 and S3. Obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the reagent blank test. 01 And the concentration of ethyl hexanoate c determined by gas chromatography in the reagent blank test. 01 ; Sample blank test: Using the optimal reaction system mixture that has not undergone esterification as the object, dual detection is performed according to step S3 to obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the sample blank test. 02 And the concentration of ethyl hexanoate c determined by gas chromatography in the sample blank test. 02 ; Inactivation blank test: After sterilizing the Daqu sample at 121℃ for 30 min to inactivate enzyme activity, prepare a mixture according to the optimal reaction system mixture composition, and then incubate and detect according to steps S2 and S3. Obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the inactivation blank test. 03 And the concentration of ethyl hexanoate c determined by gas chromatography in the inactivation blank test. 03 Then we have: V0 = V 01 + (V 02 - V 01 ) + (V 03 - V 01 ); c0 = c 01 + (c 02 - c 01 ) + (c 03 -c 01 ).

[0046] Step S5, Data Difference Correction: For the differences in detection data from the acid reduction method (substrate consumption dimension) and the gas chromatography method (product formation dimension), a graded correction is performed based on relative deviation determination, as follows, to obtain the initial esterification power values ​​for the two different detection methods after correction: Step S5.1: Calculate the initial value of esterification force: Based on the 1:1 molar ratio of hexanoic acid to ethyl hexanoate, the initial values ​​of esterification power for the acid reduction method are calculated according to equations (1) and (2) respectively. 1. Initial value of esterification force in gas phase method 2 (1); (2); in: c is the concentration of the sodium hydroxide standard solution, in mol / L; V 总 The total volume of the reaction system is expressed in mL. 144 is the simplified calculated molar mass of ethyl hexanoate, in g / mol; m is the mass of the Daqu sample, in g. V 取样The volume of the supernatant taken for titration of the esterification reaction solution by acid reduction method is in mL; 1000 is the unit conversion factor. The definition of esterification power unit (U) is: the number of milligrams of ethyl hexanoate synthesized from hexanoic acid and ethanol per gram of oven-dried Daqu (a type of starter culture) under 35°C conditions for 24 hours. It is expressed as "milligrams per gram • 24 hours (mg / g • 24h)". Step S5.2: Calculate the relative deviation D according to formula (3): (3); The relative deviation D characterizes the degree of deviation in the detection data between two different detection methods; Step S5.3: Set the calibration logic: If D≤3%, it indicates that the data from the two detection methods are in good agreement and there is no significant interference. If 3% < D ≤ 8%: the weighted average method is used for correction. Combining the specificity of the two detection methods, since gas chromatography has stronger specificity for directly detecting characteristic products, the weight of the acid reduction method is set to 0.4 and the weight of gas chromatography is set to 0.6. If D > 8%: After investigating interfering factors, retest. Use high performance liquid chromatography to remove interference from other organic acids using the acid reduction method, and optimize chromatographic conditions using gas chromatography to ensure that the resolution between the ethyl hexanoate peak and the impurity peak is ≥ 1.5. Step S6: Based on the initial esterification power value and the data difference correction results, determine the final esterification power value per gram of oven-dried Daqu using the following method. As a result of the test: If D≤3%, then the final value of esterification power is... For: M If: 3%<D≤8%: then the final value of esterification power for: = 1×0.4+ 2×0.6; The determination of the esterification power of strong-aroma daqu was completed using these methods.

[0047] Compared with traditional methods, the method of this invention has significant technical advantages, as detailed in Table 2. A comparison of the detection process is attached. Figure 3 As shown.

[0048] Table 1: Results of a series of verification tests

[0049] Table 2: Comparison of the technical advantages of this invention and traditional methods

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the esterification power of strong-aroma daqu (a type of Chinese starter culture), characterized by: The process includes sample pulverization, reaction parameter optimization, esterification reaction, dual detection, blank control correction, data difference correction, and esterification force calculation. The reaction parameter optimization employs a 5-factor, 4-level orthogonal experimental system to screen and obtain the optimal combination of reaction parameters suitable for the organic phase reaction system. The organic phase reaction system uses n-heptane as the organic phase medium. The dual detection method combines acid reduction and gas chromatography to obtain substrate consumption and product formation data, respectively. The blank control correction is calculated by superimposing three types of blank tests to obtain a correction value, which is used to subtract non-target interference; the data difference correction is designed with hierarchical correction logic for the dimensional differences of dual detection to ensure the consistency and accuracy of the detection results.

2. The method for determining the esterification power of strong-aroma daqu according to claim 1, characterized in that: Follow these steps: Step 1, Sample Grinding Process: Grind the strong-aroma type Daqu into powder, pass it through a 40-60 mesh sieve, take the sieve material, seal and dry it for storage, and obtain Daqu sample with uniform particle size and stable enzyme activity. Step 2: Screening the optimal combination of reaction parameters: A 5-factor, 4-level orthogonal experiment was used to screen the reaction parameters suitable for the n-heptane organic phase system, and to obtain the optimal combination of reaction parameters with good esterification effect and precision; the 5 factors are: hexanoic acid concentration, ethanol concentration, amount of Daqu sample, esterification temperature and esterification time. Step 3, Esterification Reaction: In a 100mL Erlenmeyer flask with a ground glass stopper, add 30mL of n-heptane as the organic phase medium. Then, add 10mL each of hexanoic acid-n-heptane solution and ethanol-n-heptane solution of the corresponding concentrations, along with the corresponding mass of Daqu (a type of starter culture) sample, according to the optimal reaction parameter combination. Mix thoroughly to obtain an optimal reaction system mixture with a total volume of 50mL for the esterification reaction. Seal the Erlenmeyer flask with a stopper and sealing film to prevent the n-heptane from evaporating. Perform the esterification reaction according to the esterification temperature and time specified in the optimal reaction parameter combination to obtain the esterification reaction solution. Step 4: Perform dual detection: Analyze the esterification reaction solution using both the acid reduction method and gas chromatography, obtaining two sets of independent data. The volume V1 of sodium hydroxide standard solution consumed by the acid reduction method and the concentration c of ethyl hexanoate determined by gas chromatography are obtained as follows: 酯 ; Step 4.1: After the esterification reaction is complete, shake the Erlenmeyer flask well and let it stand for 10-15 minutes until the Daqu particles are completely precipitated. Step 4.2, Determination of acid reduction: Take 20 mL of supernatant from the Erlenmeyer flask and transfer it to a 50 mL conical flask. Add 20 mL of distilled water and 2 drops of phenolphthalein indicator. Titrate with 0.05 mol / L sodium hydroxide standard solution until the solution turns slightly red and does not fade within 30 seconds. Record the volume of sodium hydroxide standard solution consumed, V1. Step 4.3, Gas Chromatography Detection: The supernatant was collected in an Erlenmeyer flask, filtered through a 0.22 μm organic phase filter membrane, and the amount of ethyl hexanoate produced was determined by gas chromatography. The concentration c of ethyl hexanoate was calculated using the internal standard method. 酯 ; Step 5, Blank Control Correction: Set up three types of blank tests, namely reagent blank, sample blank and inactivation blank. Calculate the blank correction value by superposition correction method. This value is used to deduct multiple interferences from non-enzymatic reactions, reagent impurities and the initial state of the sample, and obtain the volume correction value V0 of the sodium hydroxide standard solution consumed in the acid reduction method blank and the concentration correction value c0 of the ethyl hexanoate determined by the gas chromatography blank. Step 6, Data Difference Correction: For the differences in detection data from the acid reduction method (substrate consumption dimension) and the gas chromatography method (product formation dimension), a graded correction is performed based on relative deviation determination, as follows, to obtain the initial corrected esterification power values ​​for the two different detection methods: Step 6.1: Calculate the initial value of esterification force: Based on the 1:1 molar ratio of hexanoic acid to ethyl hexanoate, the initial values ​​of esterification power for the acid reduction method are calculated according to equations (1) and (2) respectively.

1. Initial value of esterification force in gas phase method 2 （1）； （2）； in: c is the concentration of the sodium hydroxide standard solution; V 总 The total volume of the reaction system; 144 is the simplified calculated molar mass of ethyl hexanoate; m is the mass of the Daqu sample; V 取样 The volume of supernatant was taken for titration of the esterification reaction solution using the acid reduction method; 1000 is the unit conversion factor. The definition of esterification power unit (U) is: the number of milligrams of ethyl hexanoate synthesized from hexanoic acid and ethanol per gram of oven-dried Daqu (a type of Chinese liquor) under 35°C conditions for 24 hours. It is expressed as mg / g·24h. Step 6.2: Calculate the relative deviation D according to formula (3): （3）； The relative deviation D characterizes the degree of deviation in the detection data between two different detection methods; Step 6.3, Set the calibration logic: If D≤3%, it indicates that the data from the two detection methods are in good agreement and there is no significant interference. If 3% < D ≤ 8%: the weighted average method is used for correction. Combining the specificity of the two detection methods, since gas chromatography has stronger specificity for directly detecting characteristic products, the weight of the acid reduction method is set to 0.4 and the weight of gas chromatography is set to 0.

6. If D > 8%: After investigating interfering factors, retest. Use high performance liquid chromatography to remove interference from other organic acids using the acid reduction method, and optimize chromatographic conditions using gas chromatography to ensure that the resolution between the ethyl hexanoate peak and the impurity peak is ≥ 1.

5. Step 7: Based on the initial esterification power value and the data difference correction results, determine the final esterification power value M per gram of oven-dried Daqu using the following method, as the test result: If D≤3%, then the final value of esterification power is... for: ; If 3% < D ≤ 8%, then the final value of esterification power is... for: = 1×0.4+ 2×0.6; This completes the determination of the esterification power of strong-aroma daqu.

3. The method for determining the esterification power of strong-aroma daqu according to claim 1, Its characteristic is that in step 2, the 5 factors and 4 levels refer to: hexanoic acid concentration 0.15-0.30 mol / L, ethanol concentration 0.1-0.4 mol / L, Daqu sample amount 1-15 g, esterification temperature 25-40℃, and esterification time 24-60 h; the orthogonal experiment uses L 16 (4 5 An orthogonal array was used, with a total of 16 experimental groups. Each group of samples was measured twice in parallel and incubated at a constant temperature. After the culture was completed, the consumption of hexanoic acid in each group of reaction solutions was initially determined by the acid reduction method, and the amount of ethyl hexanoate produced was initially determined by gas chromatography. The consumption of hexanoic acid, the amount of ethyl hexanoate produced, and the relative standard deviation (RSD) of parallel samples were used as comprehensive evaluation indicators. The influence of each parameter on the esterification power was intuitively analyzed by the main effect diagram of each factor. The parameter combination with high hexanoic acid consumption, large amount of ethyl hexanoate produced, and small RSD of parallel samples was selected as the optimal reaction parameter combination for the n-heptane organic phase system.

4. The method for determining the esterification power of strong-aroma daqu according to claim 3, characterized in that: The optimal combination of reaction parameters determined through screening is as follows: hexanoic acid concentration 0.25 mol / L, ethanol concentration 0.3 mol / L, Daqu sample amount 1 g, esterification temperature 35℃ and esterification time 24 h. The optimal combination of reaction parameters enables rapid and accurate determination of the esterification power of strong-aroma Daqu.

5. The method for determining the esterification power of strong-aroma Daqu according to claim 2, characterized in that in step 1, the sample pulverization treatment is to pulverize Daqu and pass it through a 50-mesh sieve; in step 4.1, after the esterification reaction is completed, the settling time after shaking the Erlenmeyer flask is set to 12 minutes to make the supernatant clear and free of impurities, so as to avoid Daqu particles from interfering with the titration results and gas phase detection results.

6. The method for determining the esterification power of strong-aroma daqu according to claim 2, characterized in that... In step 4.3, the chromatographic conditions are set as follows: a DB-WAX capillary column with dimensions of 30m × 0.25mm × 0.25μm; column temperature program: initial temperature 40℃ and hold for 2 min, then increase the temperature to 160℃ at 15℃ / min and hold for 2 min, then increase the temperature to 220℃ at 30℃ / min and hold for 10 min; injection port temperature 230℃, detector temperature 240℃; carrier gas is high-purity nitrogen, flow rate 1.7mL / min; split ratio 45:1; injection volume 1μL.

7. The method for determining the esterification power of strong-aroma daqu according to claim 6, characterized in that: The capillary column used in the gas chromatography detection can be replaced with HP-INNOWAX, or other types of columns that are consistent with the DB-WAX capillary column in terms of separation effect and applicable range. The column temperature program can be fine-tuned according to the instrument performance and impurity separation to ensure that the resolution of the ethyl hexanoate peak and other impurity peaks is ≥1.

5. The acid reduction method can use high performance liquid chromatography to assist in the determination of the purity of hexanoic acid in the system, eliminate the interference of other organic acids, and further improve the accuracy of the detection results of the acid reduction method.

8. The method for determining the esterification power of strong-aroma daqu according to claim 2, characterized in that, in step 5, the volume correction value V0 of the sodium hydroxide standard solution consumed in the acid reduction method blank and the concentration correction value c0 of ethyl hexanoate determined by the gas chromatography blank are obtained as follows: Reagent blank test: Prepare a reagent mixture without Daqu sample according to the composition of the optimal reaction system mixture, and incubate and detect according to steps 3 and 4. Obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the reagent blank test. 01 And the concentration of ethyl hexanoate c determined by gas chromatography in the reagent blank test. 01 ; Sample blank test: Using the optimal reaction system mixture that has not undergone esterification as the object, dual detection is performed according to step 4 to obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the sample blank test. 02 And the concentration of ethyl hexanoate c determined by gas chromatography in the sample blank test. 02 ; Inactivation blank test: After sterilizing the Daqu sample at 121℃ for 30 min to inactivate enzyme activity, prepare a mixture according to the optimal reaction system mixture composition, and then incubate and detect according to steps 3 and 4. Obtain the volume V of sodium hydroxide standard solution consumed by the acid reduction method in the inactivation blank test. 03 And the concentration of ethyl hexanoate c determined by gas chromatography in the inactivation blank test. 03 Then we have: V0 = V 01 + (V 02 - V 01 ) + (V 03 - V 01 ); c0 = c 01 + (c 02 - c 01 ) + (c 03 -c 01 )。 9. A method for verifying the esterification power of a strong-aroma type of daqu (a type of starter culture), characterized in that... After completing the determination of the esterification power of strong-aroma daqu, a series of verification tests were carried out. The qualified determination method was obtained through multi-dimensional verification, which included verification of the optimal reaction parameter combination, comparative verification of aqueous and organic phase systems, and 6 parallel precision tests.

10. The verification method for the determined esterification power of strong-aroma daqu as described in claim 9, characterized in that... In the series of verification tests: The verification of the optimal reaction parameter combination refers to: conducting verification tests on 3 batches of samples according to the optimal reaction parameter combination, with each sample measured twice in parallel, to determine the esterification power and precision. The comparative verification of the aqueous and organic phase systems refers to: based on the optimal combination of reaction parameters, replacing the reaction medium with water to construct an aqueous phase system, and comparing the esterification power and precision with the n-heptane organic phase system. The aforementioned 6-parallel precision test refers to performing esterification reactions and measurements on the same Daqu sample using the optimal combination of reaction parameters in 6 parallel samples, with an RSD ≤ 5%.

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