Method and system for evaluating acetic acid production capacity of Luzhou-flavor Daqu

Abstract

The invention provides a method and a system for evaluating acetic acid production capacity of strong-flavor Daqu, and relates to the technical field of microbial function analys.The method comprises the steps of obtaining kinetic parameters of the acetic acid production capacity of a main stress response stage representing the upper limit of the Daqu acid production capacity through pulse injection of gaseous ethanol, and then conducting pulse injection of atomized lactic acid serving as a metabolism perturbator to obtain the acetic acid production capacity of the Daqu. Obtaining kinetic parameters representing the acetic acid production capacity of the microbial system under external pressure in a metabolic disturbance recovery stage, and evaluating the acetic acid production capacity of the Luzhou-flavor Daqu based on the kinetic parameters of the acetic acid production capacity in the main stress response stage and the kinetic parameters of the acetic acid production capacity in the metabolic disturbance recovery stage. The method solves the problem of low efficiency of evaluating the acetic acid production capacity of the existing strong-flavor Daqu, and is suitable for evaluating the acetic acid production capacity of the Daqu.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial function analysis, in particular to a method and system for evaluating the acetic acid production capacity of Luzhou Daqu. BACKGROUND

[0002] As the core saccharifying and fermenting agent for the production of Chinese Luzhou-flavor liquor, the microbial community enriched in Luzhou Daqu is the key to driving the entire brewing process and ultimately determining the flavor and quality of the liquor. The quality of Daqu directly affects the stability of the pit micro-ecosystem and the synthesis of main flavor substances such as ethyl caproate. In these complex biochemical reactions, acetic acid, as one of the precursors for the synthesis of ethyl acetate, the main flavor substance, is an important indicator for evaluating the function of Daqu. Therefore, accurate and rapid evaluation of the acetic acid production capacity of Luzhou Daqu is of great significance to stabilize and improve the quality of liquor products.

[0003] The main technical means for evaluating the acetic acid production capacity of Luzhou Daqu is usually to conduct laboratory-scale culture experiments under simulated actual fermentation conditions. This method generally involves inoculating Daqu samples into liquid or solid culture medium and conducting static culture for several days or even weeks under constant temperature and humidity conditions. After the culture is completed, the final cumulative concentration of acetic acid in the culture is determined by chromatographic analysis or other means, which is used as the basis for evaluating the acetic acid production capacity. For example, CN113702535 A discloses a method for evaluating the quality of Daqu, in which the acetic acid content in the fermentation broth is used as an actual acetic acid production indicator for the sample Daqu.

[0004] Using the acetic acid content in the fermentation broth as an acetic acid production indicator requires a long culture period, resulting in extremely low evaluation efficiency, which cannot meet the needs of rapid screening and immediate quality control of large batches of raw materials in modern production processes. More importantly, this method can only provide a static and single-dimensional evaluation result. The final acetic acid production measured by this method only reflects the maximum acetic acid production potential of the microbial community under ideal conditions, but it cannot reveal the dynamic functional characteristics of the microbial community in the actual brewing complex environment. For example, it cannot distinguish between two samples with the same final acetic acid production but different acetic acid production rates, and such rate differences are crucial for the regulation of the fermentation process. SUMMARY

[0005] The technical problem solved by the present application is to provide a method and system for evaluating the acetic acid production capacity of Luzhou Daqu, which solves the problem of low evaluation efficiency of the acetic acid production capacity of existing Luzhou Daqu.

[0006] The technical solution adopted by the present application to solve the above technical problem is a method for evaluating the acetic acid production capacity of Luzhou Daqu, comprising the following steps: S1, suspending a granular Luzhou Daqu sample in a buffer solution without a carbon source in a closed reaction chamber to form a sample suspension; S2. A first preset dose of gaseous ethanol is pulsed into the reaction chamber, and the rate of change of gas concentration in the reaction chamber is monitored in real time. Based on the rate of change of gas concentration, the kinetic parameters of acetic acid production capacity in the main stress response stage are obtained until the acetic acid production rate reaches a steady state. The kinetic parameters of acetic acid production capacity in the main stress response stage include one or more of the following: maximum acetic acid production rate, metabolic initiation time, maximum oxygen consumption rate, and respiratory entropy. S3. Once the acetic acid production rate reaches a steady state, a second preset dose of metabolic disturbance agent, which is atomized lactic acid, is pulsed into the reaction chamber. The rate of change in gas concentration in the reaction chamber is continuously monitored in real time. Based on the rate of change in gas concentration, the kinetic parameters of acetic acid production capacity during the metabolic disturbance recovery phase are obtained until the acetic acid production rate reaches a new steady state. The kinetic parameters during the metabolic disturbance recovery phase include one or more of the metabolic resistance index and the metabolic recovery rate constant. S4. The acetic acid production capacity of strong-aroma daqu was evaluated based on the kinetic parameters of acetic acid production capacity during the main stress response stage and the kinetic parameters of acetic acid production capacity during the metabolic disturbance recovery stage.

[0007] Furthermore, the formula for calculating the acetic acid formation rate is as follows: ,in, Indicates the rate of acetic acid formation. This indicates the volume of gas in the reaction chamber. This indicates the quality of the strong-aroma daqu sample. This indicates the rate of change in acetic acid concentration.

[0008] Furthermore, the formula for calculating respiratory entropy is: ,in, Represents respiratory entropy. Indicates the rate of carbon dioxide formation. This indicates the rate of oxygen consumption.

[0009] Furthermore, the acetic acid formation rate reaches a steady state when the relative standard deviation of the acetic acid formation rate is less than 2%.

[0010] Furthermore, the formula for calculating the metabolic resistance index is as follows: ,in, Indicates the metabolic resistance index. This indicates the minimum rate of acetic acid production after lactic acid injection. This indicates the instantaneous acetic acid generation rate during the plateau period before lactic acid injection.

[0011] Furthermore, the formula for calculating the metabolic recovery rate constant is as follows: ,in, This represents the rate of acetic acid formation at time t. This represents the rate of acetic acid formation in the new steady state. This indicates the minimum rate of acetic acid production after lactic acid injection. This represents the metabolic recovery rate constant. This indicates the time corresponding to the lowest acetic acid production rate after lactic acid injection.

[0012] Furthermore, the acetic acid production capacity of strong-aroma daqu is evaluated based on the kinetic parameters of acetic acid production capacity during the main stress response phase and the metabolic disturbance recovery phase. This includes normalizing the kinetic parameters of acetic acid production capacity during the main stress response phase and the metabolic disturbance recovery phase, and then weighting and fusing them to obtain a comprehensive acetic acid production capacity index of strong-aroma daqu. The acetic acid production capacity of strong-aroma daqu is evaluated through the index.

[0013] Furthermore, the sample reaction module also includes a temperature control unit, which is used to maintain the temperature of the reaction chamber at a preset temperature value.

[0014] This invention also provides a system for evaluating the acetic acid production capacity of strong-aroma daqu (a type of starter culture), realizing the acetic acid production capacity evaluation method of strong-aroma daqu as described above. The system includes a sample reaction module, a pulse injection module, a gas concentration monitoring module, and a kinetic parameter calculation module. The sample reaction module includes a reaction chamber made of chemically inert material for containing a suspension of strong-aroma daqu sample. The pulse injection module is used to pulse-inject gaseous ethanol and atomized lactic acid into the reaction chamber. The gas concentration monitoring module is used to monitor the rate of change of gas concentration in the reaction chamber. The kinetic parameter calculation module is used to obtain kinetic parameters of acetic acid production capacity based on the rate of change of gas concentration, and to evaluate the acetic acid production capacity of strong-aroma daqu based on the kinetic parameters of acetic acid production capacity.

[0015] Furthermore, the sample reaction module also includes a magnetic stirrer, which is used to maintain the suspension of the Daqu sample particles in the buffer solution.

[0016] The beneficial effects of this invention are as follows: This invention provides a method and system for evaluating the acetic acid production capacity of strong-aroma daqu (a type of starter culture). By pulse-injecting gaseous ethanol, kinetic parameters characterizing the upper limit of daqu's acetic acid production capacity during the main stress response phase are obtained. Then, by pulse-injecting atomized lactic acid as a metabolic disturbance agent, kinetic parameters characterizing the acetic acid production capacity of the microbial system during the metabolic disturbance recovery phase under external pressure are obtained. Based on these kinetic parameters, the acetic acid production capacity of strong-aroma daqu is evaluated, solving the problem of low evaluation efficiency in existing methods. This invention provides a method for evaluating the acetic acid production capacity of strong-aroma daqu, expanding acetic acid production capacity from a potential dimension to a composite dimension including metabolic resilience and recovery capacity. This provides a more comprehensive description of the acetic acid production capacity of strong-aroma daqu, and can quantitatively distinguish strong-aroma daqu samples with the same maximum acetic acid production rate but different metabolic stability. It can provide direct and quantitative decision-making basis for optimizing process parameters such as raw material selection, temperature control, and turning timing during the daqu production process. Attached Figure Description

[0017] Figure 1 This is a flowchart illustrating a method for evaluating the acetic acid production capacity of strong-aroma daqu provided by the present invention. Detailed Implementation

[0018] This invention addresses the problem of low efficiency caused by the long cultivation period required for evaluating the acetic acid production capacity of strong-aroma koji (a type of starter culture) based on the acetic acid content in the fermentation broth. It provides a method and system for evaluating the acetic acid production capacity of strong-aroma koji. This method evaluates the acetic acid production capacity of strong-aroma koji by using kinetic parameters of acetic acid production capacity during the main stress response phase and the metabolic disturbance recovery phase. This eliminates the need for a long cultivation period and provides a more comprehensive description of the acetic acid production capacity of strong-aroma koji. It can quantitatively distinguish strong-aroma koji samples with the same maximum acid production rate but different metabolic stability, providing direct and quantitative decision-making basis for optimizing process parameters such as raw material selection, temperature control, and turning timing during the koji-making process.

[0019] like Figure 1 As shown, the present invention provides a method for evaluating the acetic acid production capacity of strong-aroma daqu (a type of starter culture), comprising the following steps:

[0020] S1. In a sealed reaction chamber, the granular strong-aroma koji sample is suspended in a buffer solution without a carbon source to form a sample suspension.

[0021] Specifically, the process of obtaining granular strong-aroma koji samples is as follows: the strong-aroma koji samples are pulverized using a planetary ball mill or equivalent pulverizing equipment, and the pulverized samples are passed through a standard sieve. The particles retained on the 60-mesh standard sieve are collected to obtain sample powder with uniform particle size distribution, thereby reducing experimental errors introduced by the difference in sample specific surface area.

[0022] The buffer solution can be a phosphate buffer solution, prepared by dissolving sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate in deionized water, and adjusting its final pH to 4.5 using phosphoric acid or sodium hydroxide. This buffer solution does not contain glucose, sucrose, or other carbohydrates that can be directly utilized by microorganisms. The purpose is to ensure that the acetic acid production observed in subsequent stages is entirely derived from the exogenously added ethanol substrate, thereby isolating and accurately evaluating the function of the target metabolic pathway.

[0023] The sample mass in the sample suspension can be 2.0 g, and the buffer volume can be 40 ml, thus forming a sample suspension with a solid-liquid mass-to-volume ratio of 1:20.

[0024] To maintain the suspension of sample particles in the buffer solution, a magnetic stirrer can be used to stir at a speed of 300 rpm. To ensure a constant temperature during fermentation, the temperature of the reaction chamber can be set and maintained at a preset value, such as 35°C, using a temperature control unit. To ensure the stability of the gas content in the reaction chamber under initial conditions, a constant flow of zero-grade air or high-purity nitrogen is introduced into the top space of the reaction chamber to purge the gas. When the fluctuation of the concentration of background gases such as acetic acid, carbon dioxide, and oxygen is less than a preset threshold (e.g., the relative standard deviation of the concentration is less than 1%) within 5 consecutive minutes, the stability of the gas content in the reaction chamber is considered, and this is used as a reference for gas concentration.

[0025] S2. A first preset dose of gaseous ethanol is pulsed into the reaction chamber, and the rate of change of gas concentration in the reaction chamber is monitored in real time. Based on the rate of change of gas concentration, the kinetic parameters of acetic acid production capacity during the main stress response phase are obtained until the acetic acid production rate reaches a steady state. The kinetic parameters of acetic acid production capacity during the main stress response phase include one or more of the following: maximum acetic acid production rate, metabolic initiation time, maximum oxygen consumption rate, and respiratory entropy.

[0026] Specifically, the duration of the pulse injection of a first preset dose of gaseous ethanol into the reaction chamber is between 100 and 500 milliseconds, and the total amount of ethanol injected is set to instantaneously form a target concentration of 5 to 20 ppmv (parts per million volume concentration) in the gas phase of the reaction chamber.

[0027] After ethanol injection, the instantaneous acetic acid production rate rises rapidly, reaching a peak, then may experience a slight decline, eventually entering a relatively stable rate range. This process is the main stress response phase. The entry of the instantaneous acetic acid production rate into a relatively stable rate range indicates that the acetic acid production rate has reached steady state. A criterion for this is that the relative standard deviation of the acetic acid production rate is less than 2%.

[0028] Metabolic initiation time refers to the time elapsed from the injection of the first preset dose of ethanol to the point when the instantaneous rate of acetic acid production reaches its maximum.

[0029] The formula for calculating the acetic acid formation rate is: ,in, Indicates the rate of acetic acid formation. This indicates the volume of gas in the reaction chamber. This indicates the quality of the strong-aroma daqu sample. The rate of change in acetic acid concentration is used to find the maximum acetic acid formation rate. Similarly, replacing the rate of change in acetic acid concentration with the rate of change in oxygen concentration yields the oxygen consumption rate, thus finding the maximum oxygen consumption rate. Replacing the rate of change in acetic acid concentration with the rate of change in carbon dioxide concentration yields the carbon dioxide formation rate. The rates of change in acetic acid, oxygen, and carbon dioxide concentrations can be obtained by taking the first derivative of the corresponding concentration versus time curves. For concentration data, the Savitzky-Golay smoothing filter algorithm can be used to process the data, smoothing the data points through polynomial least squares fitting, effectively filtering out high-frequency noise.

[0030] The formula for calculating respiratory entropy is: ,in, Represents respiratory entropy. Indicates the rate of carbon dioxide formation. This indicates the rate of oxygen consumption.

[0031] S3. Once the acetic acid production rate reaches a steady state, a second preset dose of metabolic disturbance agent, which is atomized lactic acid, is pulsed into the reaction chamber. The rate of change of gas concentration in the reaction chamber is continuously monitored in real time. Based on the rate of change of gas concentration, the kinetic parameters of the acetic acid production capacity during the metabolic disturbance recovery phase are obtained until the acetic acid production rate reaches a new steady state. The kinetic parameters of the metabolic disturbance recovery phase include one or more of the metabolic resistance index and the metabolic recovery rate constant.

[0032] Specifically, the atomized lactic acid is formed by an ultrasonic atomization unit and injected by a carrier gas. The duration of the pulse injection is also precisely controlled between 100 and 500 milliseconds. The total amount of injected lactic acid is set to instantaneously form a target concentration of 2 to 10 ppmv in the gas phase of the reaction chamber.

[0033] After lactic acid injection, the acetic acid production rate will experience a rapid decline due to metabolic inhibition, reaching a minimum point. The microbial system then begins to adapt and degrade lactic acid, gradually restoring the acetic acid production capacity. The acetic acid production rate then rebounds and reaches a new steady state. This process is the metabolic disturbance recovery phase.

[0034] The formula for calculating the metabolic resistance index is: ,in, Indicates the metabolic resistance index. This indicates the minimum rate of acetic acid production after lactic acid injection. This indicates the instantaneous acetic acid generation rate during the plateau period before lactic acid injection.

[0035] The formula for calculating the metabolic recovery rate constant is: ,in, This represents the rate of acetic acid formation at time t. This represents the rate of acetic acid formation in the new steady state. This indicates the minimum rate of acetic acid production after lactic acid injection. This represents the metabolic recovery rate constant. This indicates the time corresponding to the lowest acetic acid production rate after lactic acid injection.

[0036] S4. The acetic acid production capacity of strong-aroma daqu was evaluated based on the kinetic parameters of acetic acid production capacity during the main stress response stage and the kinetic parameters of acetic acid production capacity during the metabolic disturbance recovery stage.

[0037] Specifically, the kinetic parameters of acetic acid production capacity during the main stress response phase and the metabolic disturbance recovery phase can be directly used as indicators to evaluate the acetic acid production capacity of strong-aroma koji. Alternatively, the kinetic parameters of acetic acid production capacity during the main stress response phase and the metabolic disturbance recovery phase can be normalized and then weighted and fused to obtain a comprehensive acetic acid production capacity index for strong-aroma koji. The acetic acid production capacity of strong-aroma koji can be evaluated through this index. The calculation formula for the index is as follows: ,in, This indicates the acetic acid production capacity index of strong-aroma daqu (a type of starter culture). This indicates the total number of dynamic parameters selected. Indicates the first The weights of each dynamic parameter, and , Indicates the first The normalized dynamic parameter values.

[0038] This invention also provides a system for evaluating the acetic acid production capacity of strong-aroma daqu (a type of starter culture), realizing the acetic acid production capacity evaluation method of strong-aroma daqu as described above. The system includes a sample reaction module, a pulse injection module, a gas concentration monitoring module, and a kinetic parameter calculation module. The sample reaction module includes a reaction chamber made of chemically inert material for containing a suspension of strong-aroma daqu sample. The pulse injection module is used to pulse-inject gaseous ethanol and atomized lactic acid into the reaction chamber. The gas concentration monitoring module is used to monitor the rate of change of gas concentration in the reaction chamber. The kinetic parameter calculation module is used to obtain kinetic parameters of acetic acid production capacity based on the rate of change of gas concentration, and to evaluate the acetic acid production capacity of strong-aroma daqu based on the kinetic parameters of acetic acid production capacity.

[0039] The kinetic parameters include the kinetic parameters of acetic acid production capacity during the main stress response phase and the kinetic parameters of acetic acid production capacity during the metabolic disturbance recovery phase.

[0040] The sample reaction module also includes a magnetic stirrer, which is used to maintain the suspension of the Daqu sample particles in the buffer solution.

[0041] The sample reaction module also includes a temperature control unit, which is used to maintain the temperature of the reaction chamber at a preset temperature value.

[0042] In this invention, kinetic parameters can be used to quantitatively distinguish strong-aroma koji samples with the same maximum acid production rate but different metabolic stability, providing a direct and quantitative basis for optimizing process parameters such as raw material selection, temperature control, and turning timing during koji making.

Claims

1. A method for evaluating the acetic acid production capacity of strong-aroma daqu (a type of starter culture), characterized in that, Includes the following steps: S1. In a closed reaction chamber, granular strong-aroma koji samples are suspended in a buffer solution without a carbon source to form a sample suspension. S2. A first preset dose of gaseous ethanol is pulsed into the reaction chamber, and the rate of change of gas concentration in the reaction chamber is monitored in real time. Based on the rate of change of gas concentration, the kinetic parameters of acetic acid production capacity in the main stress response stage are obtained until the acetic acid production rate reaches a steady state. The kinetic parameters of acetic acid production capacity in the main stress response stage include one or more of the following: maximum acetic acid production rate, metabolic initiation time, maximum oxygen consumption rate, and respiratory entropy. S3. Once the acetic acid production rate reaches a steady state, a second preset dose of metabolic disturbance agent, which is atomized lactic acid, is pulsed into the reaction chamber. The rate of change in gas concentration in the reaction chamber is continuously monitored in real time. Based on the rate of change in gas concentration, the kinetic parameters of acetic acid production capacity during the metabolic disturbance recovery phase are obtained until the acetic acid production rate reaches a new steady state. The kinetic parameters during the metabolic disturbance recovery phase include one or more of the metabolic resistance index and the metabolic recovery rate constant. S4. The acetic acid production capacity of strong-aroma daqu was evaluated based on the kinetic parameters of acetic acid production capacity during the main stress response phase and the kinetic parameters of acetic acid production capacity during the metabolic disturbance recovery phase.

2. The method for evaluating the acetic acid production capacity of strong-aroma daqu according to claim 1, characterized in that, The formula for calculating the acetic acid formation rate is: ,in, Indicates the rate of acetic acid formation. This indicates the volume of gas in the reaction chamber. This indicates the quality of the strong-aroma daqu sample. This indicates the rate of change in acetic acid concentration.

3. The method for evaluating the acetic acid production capacity of strong-aroma daqu according to claim 1, characterized in that, The formula for calculating respiratory entropy is: ,in, Represents respiratory entropy. Indicates the rate of carbon dioxide formation. This indicates the rate of oxygen consumption.

4. The method for evaluating the acetic acid production capacity of strong-aroma daqu according to claim 1, characterized in that, The acetic acid formation rate reaches a steady state when the relative standard deviation of the acetic acid formation rate is less than 2%.

5. The method for evaluating the acetic acid production capacity of strong-aroma daqu according to claim 1, characterized in that, The formula for calculating the metabolic resistance index is: ,in, Indicates the metabolic resistance index. This indicates the minimum rate of acetic acid production after lactic acid injection. This indicates the instantaneous acetic acid generation rate during the plateau period before lactic acid injection.

6. The method for evaluating the acetic acid production capacity of strong-aroma daqu according to claim 1, characterized in that, The formula for calculating the metabolic recovery rate constant is: ,in, This represents the rate of acetic acid formation at time t. This represents the rate of acetic acid formation in the new steady state. This indicates the minimum rate of acetic acid production after lactic acid injection. This represents the metabolic recovery rate constant. This indicates the time corresponding to the lowest acetic acid production rate after lactic acid injection.

7. The method for evaluating the acetic acid production capacity of strong-aroma daqu according to claim 1, characterized in that, The acetic acid production capacity of strong-aroma daqu is evaluated based on the kinetic parameters of acetic acid production capacity during the main stress response phase and the metabolic disturbance recovery phase. This includes normalizing the kinetic parameters of acetic acid production capacity during the main stress response phase and the metabolic disturbance recovery phase, and then weighting and fusing them to obtain a comprehensive acetic acid production capacity index of strong-aroma daqu. The acetic acid production capacity of strong-aroma daqu is evaluated through the index.

8. A system for evaluating the acetic acid production capacity of strong-aroma daqu (a type of starter culture), characterized in that, The method for evaluating the acetic acid production capacity of strong-aroma daqu as described in claim 1 includes a sample reaction module, a pulse injection module, a gas concentration monitoring module, and a kinetic parameter calculation module. The sample reaction module includes a reaction chamber made of chemically inert material, used to contain a suspension of strong-aroma daqu samples. The pulse injection module is used to pulse-inject gaseous ethanol and atomized lactic acid into the reaction chamber. The gas concentration monitoring module is used to monitor the rate of change of gas concentration within the reaction chamber. The kinetic parameter calculation module is used to obtain kinetic parameters of acetic acid production capacity based on the rate of change of gas concentration, and to evaluate the acetic acid production capacity of strong-aroma daqu based on these kinetic parameters.

9. The acetic acid production capacity evaluation system of strong-aroma daqu according to claim 8, characterized in that, The sample reaction module also includes a magnetic stirrer, which is used to maintain the suspension of the Daqu sample particles in the buffer solution.

10. The acetic acid production capacity evaluation system of strong-aroma daqu according to claim 8, characterized in that, The sample reaction module also includes a temperature control unit, which is used to maintain the temperature of the reaction chamber at a preset temperature value.

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