Method for detecting active enzyme in solid substance
By detecting the substrate concentration in the environment of active enzymes in solid substances, and using near-infrared spectroscopy and sensors to monitor and control temperature and humidity, the problem of cumbersome active enzyme detection in existing technologies is solved, and a simple and reliable active enzyme activity determination is achieved.
Patent Information
- Application Number
- CN202510894998.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-11-25
AI Technical Summary
In existing technologies, detecting the activity of active enzymes requires removing and purifying the substance containing the active enzyme, which is cumbersome and inefficient, making it difficult to quickly detect the activity of active enzymes on tobacco materials.
By detecting the substrate concentration in the environment of the active enzyme in solid matter, and using a near-infrared spectrometer to detect the substrate concentration at different temperatures, the activity of the active enzyme can be indirectly determined. Combined with gradient heating and humidity control, temperature and humidity sensors are used to monitor and regulate the environment.
A simple and reliable method for detecting active enzymes has been developed, which can quickly determine the activity of active enzymes, reduce the probability of enzyme inactivation, and improve detection efficiency.
Smart Images
Figure CN121006392A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of active enzyme detection, in particular to a method for detecting active enzyme in solid matter. BACKGROUND
[0002] Active enzyme is a kind of biological catalyst with high specificity and high catalytic efficiency to substrate, and the catalysis of enzyme is often used to change the chemical composition of substrate.
[0003] Active enzyme fermentation technology is an effective method for rapidly improving tobacco quality in the process of cigarette processing, and its main process is to uniformly apply different active enzyme preparations to the surface of tobacco material according to the chemical composition of tobacco material, and to ferment under suitable temperature and humidity conditions to make the chemical composition of tobacco tend to be coordinated and the quality to be improved. Active enzyme has adjustability and instability, and has high requirements for the environment. In order to improve the fermentation speed of tobacco material, the catalytic reaction of active enzyme needs to be controlled at a suitable temperature. Different active enzymes have different optimal catalytic reaction temperatures, but at present, when detecting the activity of active enzyme, the substance containing active enzyme is taken out, purified and then detected, which is complicated and low in efficiency. SUMMARY
[0004] Therefore, the purpose of the present application is to provide a method for detecting active enzyme in solid matter, which indirectly detects the activity of active enzyme by detecting the concentration of substrate in the environment of active enzyme, and the detection method is simple and has high reliability.
[0005] The present application solves the above technical problems through the following technical means:
[0006] A method for detecting active enzyme in solid matter, comprising the following steps:
[0007] S1, placing the substrate containing active enzyme on the object table and uniformly distributing it;
[0008] S2, heating the substrate containing active enzyme, and controlling the temperature and humidity of the substrate according to the heating condition;
[0009] S3, detecting the concentration of the substrate at different temperatures by near-infrared spectrometer to determine the activity of active enzyme at this temperature.
[0010] According to the above technical means, by detecting the concentration of the substrate at different temperatures by near-infrared spectrometer under different heating conditions of the substrate containing active enzyme, the activity of active enzyme at this temperature can be indirectly and quickly determined, and the method is simple and has high reliability.
[0011] As a preferred, in the S1 step, the active enzyme is one or a combination of proteinase, amylase, pectinase and cellulase.
[0012] As preferred, in the S1 step, the substrate is one or a combination of tobacco leaves, tobacco stems, tobacco shreds, shreds, and reconstituted tobacco leaves.
[0013] Further preferably, the substrate has a flat thickness of 2-5 cm on the loading platform.
[0014] Further preferably, the substrate has a relative humidity of 60-80%.
[0015] Further preferably, the substrate and the active enzyme are added at a ratio of 1: (0.001-0.05).
[0016] According to the above technical means, by controlling the flat thickness of the substrate and the addition ratio of the active enzyme, the active enzyme can be more evenly distributed in the substrate. At the same time, by controlling the relative humidity of the substrate, the active enzyme can be more effectively fermented.
[0017] As preferred, in the S2 step, when the temperature is raised, gradient heating is used to raise the temperature to 40-75℃ at a rate of 3-5℃ / min.
[0018] According to the above technical means, by gradient heating, different active enzymes can be adapted, thereby avoiding too fast heating and too high temperature, which can cause the inactivation of the active enzyme.
[0019] As preferred, in the S2 step, when the temperature and humidity of the substrate are controlled, a temperature sensor and a humidity sensor are used to monitor the temperature and humidity of the substrate in parallel.
[0020] Further preferably, when the humidity of the substrate is monitored, when the humidity is less than the preset value, the heating is stopped, and the substrate is humidified by a humidifying medium; when the humidity is greater than the preset value, the gradient heating is performed.
[0021] Further preferably, the humidifying medium is pure water.
[0022] According to the above technical means, by monitoring and controlling the temperature and humidity of the substrate, the fermentation of the active enzyme on the tobacco material is more conducive, and the probability of inactivation of the active enzyme is reduced. Moreover, by using pure water as the humidifying medium, the introduction of other impurities that can affect the activity of the active enzyme can be avoided.
[0023] As preferred, in the S3 step, when the near-infrared spectrometer is used to detect the concentration of the substrate at different temperatures, a quantitative model of different active enzymes in the near-infrared spectrometer is called to measure the concentration of the substrate.
[0024] Further preferably, the quantitative model is at least one of a protein quantitative analysis model, a starch quantitative analysis model, a pectin quantitative analysis model, and a cellulose quantitative analysis model.
[0025] Furthermore, in the quantitative model analysis, a first-level model is used for evaluation, and the coefficient of determination (R²) and chi-square function (χ²) are used to evaluate the model's goodness of fit. The first-level model is as follows:
[0026] , where C t Ct is the amount of substrate in the sample at time t (%), C0 is the amount of substrate in the sample at the initial time (%), and k is the first-order reaction rate constant (ht). -1 ).
[0027] Based on the above-mentioned technical means, by using different quantitative analysis models under the fermentation of different active enzymes, the concentration of the substrate is measured, and the degradation activity of the active enzyme is analyzed according to the first-order kinetic model.
[0028] The present application, employing the above-described scheme, has at least the following beneficial effects:
[0029] 1. In this application, the activity of the active enzyme is indirectly detected by using a near-infrared spectrometer to detect the substrate concentration in the environment where the active enzyme is located. The detection method is simple and highly reliable. 2. In this application, by using gradient heating and controlling the temperature and relative humidity of the substrate during the detection process, the impact on the active enzyme can be reduced, thereby making the detection results more reliable. Attached Figure Description
[0030] This application can be further illustrated by the non-limiting embodiments given in the accompanying drawings;
[0031] Figure 1 This is a graph showing the detection results when amylase was used in Example 1 of this application. Detailed Implementation
[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0033] This application discloses a method for detecting active enzymes in solid substances, comprising the following steps:
[0034] S1. Place the substrate containing the active enzyme on the stage and distribute it evenly;
[0035] S2. Heat the substrate containing the active enzyme, and control the temperature and humidity of the substrate according to the heating process;
[0036] S3. Use a near-infrared spectrometer to detect the concentration of the substrate at different temperatures in order to determine the activity of the active enzyme at that temperature.
[0037] By detecting the concentration of substrates containing active enzymes at different temperatures using a near-infrared spectrometer, the activity of the active enzyme at that temperature can be determined indirectly and rapidly. This method is simple and highly reliable.
[0038] The substrates used in this application were obtained from our company's workshop.
[0039] The following examples, using different substrates and active enzymes, will provide a detailed explanation:
[0040] Example 1, Method for Detecting Active Enzymes in Solid Substances
[0041] In this embodiment, tobacco leaves are used as the substrate, and α - Amylase is an active enzyme.
[0042] S1, 100g of product containing α - The tobacco leaves containing amylase were placed on a stage, evenly distributed, and contained α-amylase. - The thickness of the tobacco leaves laid flat with amylase is 2-3 cm, the moisture content of the tobacco leaves is 60-80%, and the tobacco leaves are in contact with α-amylase. - The ratio of amylase added was 1:0.002;
[0043] S2, containing α - The tobacco leaves treated with amylase undergo a gradient heating process in a heating device, with the temperature gradually increased to 55°C at a rate of 5°C / min. The temperature of the tobacco leaves is monitored in real time by a temperature sensor to maintain it at 55±1°C. The relative humidity of the tobacco leaves is monitored in real time by a humidity sensor to ensure that the relative humidity is 70-80%.
[0044] When the temperature of the tobacco leaves is maintained at 55±1℃, a humidity sensor monitors the relative humidity of the tobacco leaves in real time. When the humidity is less than 70%, purified water is sprayed onto the surface of the tobacco leaves through a spray structure in the heating device to restore the relative humidity to 70-80%. When the humidity is greater than 80%, a gradient heating process is implemented, raising the temperature to 60-65℃. During the heating process, the relative humidity of the tobacco leaves is monitored in real time until it returns to 70-80%, at which point the temperature is maintained. Depending on the fermentation duration, the relative humidity of the tobacco leaves is maintained by spraying purified water at the corresponding temperature.
[0045] S3. Using a near-infrared spectroscopy instrument at a tobacco leaf temperature of 55±1℃ and a relative humidity of 70-80%, the content of α-propagation molecules was detected at different fermentation times. -The total starch content in tobacco leaves was used to determine the activity of the amylase at this temperature. Specifically, the starch quantitative analysis model in the near-infrared spectrometer was used to calculate the first-order kinetic constant k under amylase treatment according to the first-order kinetic model, and the coefficient of determination (R²) was used. 2 The model fit is evaluated using the chi-square function (χ²).
[0046] The first-order dynamic model is: In the formula, C t Ct is the amount of substrate in the sample at time t (%), C0 is the amount of substrate in the sample at the initial time (%), and k is the first-order reaction rate constant (ht). -1 ).
[0047] The total starch content at different time points was measured using a near-infrared spectroscopy starch quantification model, as shown in Table 1 below:
[0048] Table 1. Relationship between fermentation time and total starch content
[0049]
[0050] The above formula yields k = 0.0109 / h, with a coefficient of determination R² of 0.9148, which describes the enzymatic degradation process of α-amylase. Furthermore, data from Table 1 show that at a temperature of 55±1℃ and a relative humidity of 70-80%, the total starch content gradually decreases with increasing fermentation time, indicating that α-amylase activity remains good. The change in total starch content decreases between 7 and 10 hours, indicating that α-amylase activity... - The activity of amylase is gradually decreasing.
[0051] Example 2, Method 2 for Detecting Active Enzymes in Solid Substances
[0052] In this embodiment, tobacco leaves are used as the substrate and protease is used as the active enzyme.
[0053] S1. Place 100g of tobacco leaves containing protease on a platform, distribute them evenly, and the thickness of the tobacco leaves containing protease is 2-3cm. The moisture content of the tobacco leaves is 60-70%, and the ratio of tobacco leaves to protease is 1:0.001.
[0054] S2. The tobacco leaves containing protease are subjected to gradient heating in a heating device at a rate of 5℃ / min to 50℃. The temperature of the tobacco leaves is monitored in real time by a temperature sensor to maintain the temperature at 50±1℃. The relative humidity of the tobacco leaves is monitored in real time by a humidity sensor to ensure that the relative humidity of the tobacco leaves is 60-70%.
[0055] When the temperature of the tobacco leaves is maintained at 50±1℃, a humidity sensor monitors the relative humidity of the tobacco leaves in real time. When the humidity is less than 60%, purified water is sprayed onto the surface of the tobacco leaves through a spray structure in the heating device to restore the relative humidity to 60-70%. When the humidity is greater than 70%, a gradient heating process is implemented, raising the temperature to 55-60℃. During the heating process, the relative humidity of the tobacco leaves is monitored in real time until it recovers to 60-70%, at which point the temperature is maintained. Depending on the fermentation time, the relative humidity of the tobacco leaves is maintained by spraying purified water at the corresponding temperature.
[0056] S3. Using a near-infrared spectroscopy instrument at a tobacco leaf temperature of 50±1℃ and a relative humidity of 60-70%, the total protein content in tobacco leaves containing protease was measured at different fermentation times to determine the protease activity at this temperature. Specifically, the protein quantification analysis model in the near-infrared spectroscopy instrument was called, and the first-order kinetic constant k under protease treatment was calculated according to the first-order kinetic model, and the coefficient of determination (R²) was used. 2 The model fit is evaluated using the chi-square function (χ²).
[0057] The first-order dynamic model is: In the formula, C t Ct is the amount of substrate in the sample at time t (%), C0 is the amount of substrate in the sample at the initial time (%), and k is the first-order reaction rate constant (ht). -1 ).
[0058] The total protein content at different time points was measured using a near-infrared spectroscopy protein quantification model, as shown in Table 2 below.
[0059] Table 2. Relationship between fermentation time and total protein content
[0060]
[0061] The above formula yields k = 0.0112 / h, with a coefficient of determination R² of 0.9217, which describes the enzymatic degradation process of the protease. Furthermore, the data in Table 2 show that at a temperature of 50±1℃ and a relative humidity of 60-70%, the total protein content gradually decreases with increasing fermentation time, indicating that the protease activity remains good. The change in total protein content decreases between 8 and 10 hours, indicating that the protease activity gradually declines.
[0062] Example 3, Method 3 for Detecting Active Enzymes in Solid Substances
[0063] In this embodiment, tobacco leaves are used as the substrate and cellulase is used as the active enzyme.
[0064] S1. Place 100g of tobacco leaves containing cellulase on a platform, distribute them evenly, and the thickness of the tobacco leaves containing cellulase is 2-3cm. The moisture content of the tobacco leaves is 75-80%, and the ratio of tobacco leaves to protease is 1:0.005.
[0065] S2. The tobacco leaves containing cellulase are subjected to a gradient heating device at a rate of 5℃ / min to 40℃. The temperature of the tobacco leaves is monitored in real time by a temperature sensor to maintain the temperature at 40±1℃. The relative humidity of the tobacco leaves is monitored in real time by a humidity sensor to ensure that the relative humidity of the tobacco leaves is 75-80%.
[0066] When the temperature of the tobacco leaves is maintained at 40±1℃, a humidity sensor monitors the relative humidity of the tobacco leaves in real time. When the humidity is less than 75%, purified water is sprayed onto the surface of the tobacco leaves through a spray structure in the heating device to restore the relative humidity to 75-80%. When the humidity is greater than 80%, a gradient heating process is implemented, raising the temperature to 45-50℃. During the heating process, the relative humidity of the tobacco leaves is monitored in real time until the relative humidity of the tobacco leaves recovers to 75-80%, and then maintained at this temperature. Depending on the fermentation time, the relative humidity of the tobacco leaves is maintained by spraying purified water at the corresponding temperature.
[0067] S3. Using a near-infrared spectroscopy instrument at a tobacco leaf temperature of 40±1℃ and a relative humidity of 75-80%, the total cellulose content in tobacco leaves containing cellulase was measured at different fermentation times to determine the activity of cellulase at this temperature. Specifically, the cellulose quantitative analysis model in the near-infrared spectroscopy instrument was called, and the first-order kinetic constant k under cellulase treatment was calculated according to the first-order kinetic model, and the coefficient of determination (R²) was used. 2 The model fit is evaluated using the chi-square function (χ²).
[0068] The first-order dynamic model is: In the formula, C t Ct is the amount of substrate in the sample at time t (%), C0 is the amount of substrate in the sample at the initial time (%), and k is the first-order reaction rate constant (ht). -1 ).
[0069] The total cellulose content at different time points was determined using a near-infrared spectroscopy quantitative analysis model, as shown in Table 3 below.
[0070] Table 3. Relationship between fermentation time and total cellulose content
[0071]
[0072] The above formula yields k = 0.0107 / h, with a coefficient of determination R² of 0.9142, which describes the enzymatic degradation process of cellulase. Furthermore, data from Table 3 shows that at a temperature of 40±1℃ and a relative humidity of 75-80%, the total cellulose content gradually decreases with increasing fermentation time, indicating that cellulase activity remains good. The change in total cellulose content decreases between 5 and 10 hours, indicating a gradual decrease in cellulase activity, especially between 7 and 10 hours, where the activity further declines.
[0073] Example 4, Method 4 for Detecting Active Enzymes in Solid Substances
[0074] In this embodiment, tobacco leaves are used as the substrate and pectinase is used as the active enzyme.
[0075] S1. Place 100g of tobacco leaves containing pectinase on a platform, distribute them evenly, and the thickness of the tobacco leaves containing pectinase is 2-3cm. The moisture content of the tobacco leaves is 70-80%, and the ratio of tobacco leaves to protease is 1:0.0012.
[0076] S2. The tobacco leaves containing pectinase are subjected to a gradient heating device at a rate of 5℃ / min to 45℃. The temperature of the tobacco leaves is monitored in real time by a temperature sensor to maintain the temperature at 45±1℃. The relative humidity of the tobacco leaves is monitored in real time by a humidity sensor to ensure that the relative humidity of the tobacco leaves is 70-80%.
[0077] When the temperature of the tobacco leaves is maintained at 45±1℃, a humidity sensor monitors the relative humidity of the tobacco leaves in real time. When the humidity is less than 70%, purified water is sprayed onto the surface of the tobacco leaves through a spray structure in the heating device to restore the relative humidity to 70-80%. When the humidity is greater than 80%, a gradient heating process is implemented, raising the temperature to 45-50℃. During the heating process, the relative humidity of the tobacco leaves is monitored in real time until it returns to 70-80%, at which point the temperature is maintained. Depending on the fermentation duration, the relative humidity of the tobacco leaves is maintained by spraying purified water at the corresponding temperature.
[0078] S3. Using a near-infrared spectroscopy instrument at a tobacco leaf temperature of 45±1℃ and a relative humidity of 70-80%, the total pectin content in tobacco leaves containing pectinase was measured at different fermentation times to determine the activity of pectinase at this temperature. Specifically, the pectin quantitative analysis model in the near-infrared spectroscopy instrument was called, and the first-order kinetic constant k under pectinase treatment was calculated according to the first-order kinetic model, and the coefficient of determination (R²) was used. 2 The model fit is evaluated using the chi-square function (χ²).
[0079] The first-order dynamic model is: In the formula, C t Ct is the amount of substrate in the sample at time t (%), C0 is the amount of substrate in the sample at the initial time (%), and k is the first-order reaction rate constant (ht). -1 ).
[0080] The total pectin content at different time points was measured using a near-infrared spectroscopy pectin quantitative analysis model, as shown in Table 4 below:
[0081] Table 4. Relationship between fermentation time and total pectin content
[0082]
[0083] The above formula yields k = 0.0115 / h, with a coefficient of determination R² of 0.9243, which describes the enzymatic degradation process of pectinase. Furthermore, data from Table 4 shows that at a temperature of 45±1℃ and a relative humidity of 70-80%, the total pectin content gradually decreases with increasing fermentation time, indicating that pectinase activity remains good. The change in total pectin content decreases between 8 and 10 hours, indicating a gradual decrease in pectinase activity, especially between 9 and 10 hours, where pectinase activity further declines.
[0084] In summary, this application indirectly detects the activity of an active enzyme by using a near-infrared spectrometer to detect the substrate concentration in the environment in which the active enzyme is located. The detection method is simple and highly reliable.
[0085] The above provides a detailed description of a method for detecting active enzymes in solid substances according to the present invention. The specific embodiments described are merely for the purpose of helping to understand the method and its core ideas. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.
[0086] It should be noted that: for experimental steps or conditions not specified in the examples, the procedures and conditions described in conventional experimental procedures in the literature of this art can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.
[0087] The above examples are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by anyone under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.
Claims
1. A method for detecting active enzymes in solid substances, characterized in that, Includes the following steps: S1. Place the substrate containing the active enzyme on the stage and distribute it evenly; S2. Heat the substrate containing the active enzyme, and control the temperature and humidity of the substrate according to the heating process; S3. Use a near-infrared spectrometer to detect the concentration of the substrate at different temperatures in order to determine the activity of the active enzyme at that temperature.
2. The detection method according to claim 1, characterized in that, In step S1, the active enzyme is one or more combinations of protease, amylase, pectinase, and cellulase.
3. The detection method according to claim 1, characterized in that, In step S1, the substrate is one or more combinations of tobacco leaves, tobacco stems, shredded tobacco, stem shreds, and reconstituted tobacco leaves.
4. The detection method according to claim 1, characterized in that, The ratio of substrate to active enzyme is 1:(0.001-0.05).
5. The detection method according to any one of claims 1-4, characterized in that, In step S2, a gradient heating method is used during the heating process, with the temperature gradually increased to 40-75℃ at a rate of 3-5℃ / min.
6. The detection method according to any one of claims 1-4, characterized in that, In step S2, when controlling the temperature and humidity of the substrate, a temperature sensor and a humidity sensor are used to monitor the temperature and humidity of the substrate in parallel.
7. The detection method according to claim 6, characterized in that, When monitoring the humidity of the substrate, if the humidity is less than a preset value, the heating is stopped and the substrate is humidified by a humidifying medium; if the humidity is greater than the preset value, the temperature is increased gradually.
8. The detection method according to any one of claims 1-3, characterized in that, In step S3, when the concentration of the substrate at different temperatures is detected using a near-infrared spectrometer, the substrate concentration is measured by calling the quantitative model of different active enzymes in the near-infrared spectrometer.
9. The detection method according to claim 8, characterized in that, The quantitative model is at least one of the following: protein quantitative analysis model, starch quantitative analysis model, pectin quantitative analysis model, and cellulose quantitative analysis model.
10. The detection method according to claim 8, characterized in that, In the quantitative model analysis, a first-order model was used for evaluation, and the coefficient of determination R² and the chi-square function χ² were used to assess the model's goodness of fit. The first-order model is as follows: , where C t C0 is the percentage of substrate in the sample at time t, C0 is the percentage of substrate in the sample at the initial time, and k is the first-order reaction rate constant h. -1 .