CN114283896B - Modeling method for monitoring component change model in enzymatic reaction process - Google Patents
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Abstract
The invention discloses a modeling method for monitoring a component change model in an enzymatic reaction process, which relates to the technical field of reaction component monitoring and comprises the following steps: preparing a substrate and recording the quality; respectively starting enzymatic reactions of the two reaction kettles at preset time intervals, sampling from the reaction kettles at preset sampling time in the enzymatic reaction process, and collecting near infrared spectrum information of the sample liquid; processing the extracted sample liquid to obtain the concentration of an enzymatic reaction product in the sample liquid at a preset sampling moment; alternately sampling in the two reaction kettles according to preset time until the enzymatic reaction is finished, and recording the quality again; and calculating the substrate concentration at the preset sampling moment, and establishing association between the product concentration and the substrate concentration and near infrared spectrum information according to the preset sampling moment to obtain a quantitative near infrared spectrum model reflecting the change of the product and the substrate concentration in the enzymatic reaction process. The model can monitor the change of the concentration of each component in the reaction liquid on line in real time, further analyze the enzymatic reaction process, and shorten the detection time.
Description
Technical Field
The invention relates to the technical field of reaction component monitoring, in particular to a modeling method for a component change model in an enzymatic reaction monitoring process.
Background
Trehalose (α, α -Trehalose) is a disaccharide, formed by the α -1 linkage of two D-glucose molecules, and is an isomer of maltose. It is a non-reducing sugar, not easy to be hydrolyzed by acid, the glycosidic bond is not easy to be cut by glycosidase, and the molecular formula is C 12 H 22 O 11 And a molecular weight of 342.31. After purification, it usually exists in the form of dihydrate, and trehalose has a remarkable biological protection mechanism and can stabilize the cell membrane of a cell organism and protein molecules to be inactivated without denaturation under some severe conditions, thereby maintaining biological characteristics and life processes, so that the trehalose is also called natural 'life sugar'. The industrial production method is concerned, and the production method of the trehalose mainly comprises a microbial fermentation method, an extraction method, a gene recombination method and an enzymatic synthesis method, wherein the enzymatic synthesis method is the main method applied to the current industrial production. The enzymatic method mainly comprises a phosphorylation pathway and a non-phosphorylation pathway, wherein the former pathway comprises a TPS/TPP pathway and a Tre P pathway. Such reactions require high-energy substances such as UDP and GDP, and the enzymatic reaction is unstable, so that large-scale industrial production is difficult to realize. (ii) a non-phosphorylation pathway,for example, the enzyme system comprises MTSase and MTHase, wherein the MTSase can act on maltooligosaccharide, maltodextrin or amylose with DP more than or equal to 3 to generate alpha, alpha-1,1-glucoside bond with trehalose structure at the tail end of the MTSase; MTHase converts the above-mentioned alpha, alpha-1,1-glucosidic bond specifically by cleavage from the linear end into trehalose, the synthetic pathway is called the TreY/TreZ pathway, which is considered to be the most potential trehalose production method, and therefore the enzymatic process analysis of this pathway is of guiding significance. At present, the trehalose components are mainly detected by High Performance Liquid Chromatography (HPLC) to analyze an enzymatic process, and the method is time-consuming and labor-consuming and is difficult to detect the component change in the enzymatic conversion process on line in real time. Therefore, the invention provides an online monitoring technology capable of timely and continuously feeding back the change of the substrate and the product in the conversion process on line, and has great significance for analyzing the enzymatic reaction process.
Near Infrared Spectroscopy (NIRS) analysis technology is a new "giant" of analysis that has developed the fastest in recent decades, and its emergence can be said to bring a revolution in further analysis technology. The near infrared spectrum analysis technology integrates physics, chemistry, computer science, information science and related technologies, and has been developed into a very active research field. The near infrared technology is a thickness detection and monitoring technology which has no radioactivity, is non-contact and has high measurement precision, high resolution and high reliability; is widely applied in the fields of food, petrochemical engineering, medical engineering and the like.
In the prior art, a detection pool is adopted in the process of monitoring trehalose production by utilizing a near infrared technology, sample liquid is subjected to external circulation treatment instead of in-situ monitoring, so that the reaction temperature of the sample liquid is influenced, and the actual value cannot be timely fed back on line, so that the modeling accuracy is not high. At present, no report of online monitoring of the change of the product and substrate amount in the process of the TreY/TreZ path by using near infrared exists.
Disclosure of Invention
The invention provides a modeling method for monitoring a component change model in an enzymatic reaction process aiming at the problems and technical requirements, wherein a near-infrared spectrum model is established and corrected by utilizing the relation between the concentration of a main product trehalose and the concentration change of a substrate maltodextrin calculated according to the theoretical conversion rate in the process of near-infrared real-time monitoring of TreY/TreZ pathway conversion, and the model is used for monitoring and analyzing the continuous process of the enzymatic reaction.
The technical scheme of the invention is as follows:
a modeling method for monitoring a component change model in an enzymatic reaction process comprises the following steps:
installing a near-infrared monitoring and enzymatic reaction device, wherein the near-infrared monitoring device comprises a computer, a near-infrared spectrometer and a probe thereof, and the near-infrared spectrometer is connected with the computer and used for near-infrared modeling; the enzymatic reaction device comprises at least two reaction kettles;
preparing an initial sample solution in each reaction kettle, and recording the mass of the reaction kettle and the initial sample solution, wherein the initial sample solution comprises maltodextrin serving as a reaction substrate;
respectively starting enzymatic reactions of the two reaction kettles at preset time intervals, immersing a probe into sample liquid of the reaction kettles in the enzymatic reaction process, continuously extracting the sample liquid from the reaction kettles at a preset sampling moment, and acquiring near infrared spectrum information of the sample liquid in the reaction kettles, wherein the near infrared spectrum information corresponds to the preset sampling moment; performing off-line treatment on the extracted sample liquid to obtain the concentration of an enzymatic reaction product in the sample liquid at a preset sampling moment, wherein the product comprises a main product trehalose and byproducts glucose and maltose; when the preset time is reached, transferring the probe to another reaction kettle to repeat the steps of sampling and spectrum collection in the enzymatic reaction process;
after the sampling of the initial reaction stages of the two reaction kettles is finished, the step of immersing the probe into the sample liquid of the reaction kettles is repeatedly executed, the samples are alternately sampled and the spectrums are collected in the two reaction kettles according to the preset time until the enzymatic reaction is finished, and the quality of the reaction kettles and the residual sample liquid is recorded;
and calculating the concentration of maltodextrin at the preset sampling moment according to the two recorded masses and the concentration of each product, and establishing association between the trehalose concentration obtained by offline processing and the calculated maltodextrin concentration according to the preset sampling moment and the collected near infrared spectrum information to obtain a quantitative near infrared spectrum model reflecting the concentration change of the product and the substrate in the process of the TreY/TreZ route.
The further technical scheme is that the method for calculating the maltodextrin concentration at the preset sampling moment according to the two recorded masses and the concentration of each product comprises the following steps:
and calculating the water evaporation volume according to the two recorded masses, wherein the expression of the actual volume of the sample liquid at the preset sampling moment is as follows:
wherein m is Beginning of the design M is the mass of the reaction vessel and the initial sample solution Final (a Chinese character of 'gan') Is the mass of the reaction kettle and the rest sample liquid, rho Water (I) The density of water, V' is the volume of the initial sample liquid, H is the total reaction time, and H is the time corresponding to the corresponding preset sampling time; converting the concentrations of trehalose, glucose and maltose obtained by off-line processing back to the corrected concentrations of the original volume, wherein the expressions are respectively as follows:
c T ＝c t ×V÷V′；
c G ＝c g ×V÷V′；
c M ＝c m ×V÷V′；
wherein, c T 、c G 、c M Corrected concentrations of trehalose, glucose, maltose in sequence at a predetermined sampling time c t 、c g 、c m Sequentially obtaining the concentrations of trehalose, glucose and maltose at the preset sampling moment through off-line treatment;
the expression for calculating the maltodextrin concentration at the predetermined sampling time is:
wherein, c Bottom (C) Is the maltodextrin concentration, c 'at the predetermined sampling time' Bottom The initial sample solution concentration is 1.05 theoretical conversion rate of trehalose and maltose, and 1.11 is glucoseTheoretical conversion of (2).
The further technical scheme is that the method for obtaining the concentration of the enzymatic reaction product in the sample liquid at the preset sampling moment by off-line processing of the extracted sample liquid comprises the following steps:
and (3) stopping enzymatic reaction of the extracted sample liquid by adopting ethanol with a preset concentration, and detecting the concentrations of trehalose, glucose and maltose in the sample liquid at each preset sampling moment by adopting HPLC (high performance liquid chromatography) after diluting by proper times after high-speed centrifugation.
The further technical scheme is that initial sample liquid is prepared in each reaction kettle, and the method comprises the following steps of:
weighing a certain amount of maltodextrin and deionized water, pouring into a reaction kettle, mixing to obtain an initial sample liquid, and recording the volume and the concentration of the initial sample liquid.
The reaction kettle is provided with a temperature control system and a stirring device, wherein the temperature control system comprises a temperature sensor and is used for feeding back the temperature of the sample liquid to the temperature control system in real time, and the stirring device comprises a stirring paddle arranged at the bottom of the reaction kettle;
the two reaction kettles respectively start enzymatic reactions at preset time intervals, and the enzymatic reactions comprise: the method comprises the steps of extending a temperature sensor into reaction kettles to be fixed at a proper position, starting a temperature control system and a stirring device, controlling the temperature of sample liquid at a preset temperature, after maltodextrin is dissolved, respectively putting MTSase and MTHase with different proportions and pullulanase and cyclodextrin enzyme with the same quantity into the two reaction kettles at a preset time interval, and respectively starting enzymatic reaction by staggering reaction time.
The further technical proposal is that the device for monitoring near infrared and enzymatically reacting is arranged, and the device also comprises:
the near-infrared monitoring device also comprises an iron ring with a movable zenith, an optical fiber connected with the near-infrared spectrometer and a probe of the near-infrared spectrometer passes through the iron ring, and the position of the iron ring is adjusted to keep the optical fiber not to be bent;
enzymatic reaction unit still includes iron stand platform, and reation kettle arranges in on the iron stand platform, fixes the probe through the iron stand platform.
The further technical scheme is that the acquisition conditions of the near infrared spectrum information are as follows: near infrared spectrumThe scanning range of the instrument is 3995cm -1 -12000cm -1 Resolution of 16cm -1 The number of scanning times is 5-10, and the scanning is continuous.
The further technical scheme is that the chromatographic conditions for measuring the sample liquid by adopting HPLC are as follows: polyurethane column-Dikama polyamine, 5 μm, 250X 4.6mm; the volume ratio of acetonitrile/water is 75/25; the flow rate is 1.0mL/min, and the detector adopts a parallax refraction detector; the sample injection amount is 20 mu L; the column temperature was 30 ℃ and the parallax temperature was 35 ℃.
The further technical proposal is that the optimum reaction temperature of MTSase is 45-50 ℃, the optimum reaction temperature of MTHase is 48-50 ℃, and the optimum pH values of the MTSase and the MTHase are both 6.0-6.5.
The modeling method further comprises the following steps of static modeling to verify experimental feasibility:
preparing maltodextrin, trehalose, a mixed solution of maltodextrin and trehalose with different concentrations according to the actual conversion rate in the enzyme conversion process, and preparing MTSase and MTHase inactivated enzyme solution added with hydrochloric acid as four sample solutions, wherein the hydrochloric acid is used for controlling the pH value of the inactivated enzyme solution to be 2.0-2.2;
respectively placing four sample liquids with the same volume into a beaker, sequentially immersing a probe into the sample liquids and acquiring near infrared spectrum information; establishing association between the near infrared spectrum information and the known concentration or the added content of the sample to obtain a spectrum model; and collecting near infrared spectrum information of the liquid to be detected and inputting the information into the spectrum model to obtain the concentration of the liquid to be detected, comparing the concentration with the actual concentration of the liquid to be detected, and judging the accuracy of the spectrum model.
The beneficial technical effects of the invention are as follows:
according to the method, in the enzymatic process of the TreY/TreZ approach, near infrared spectrum information of a sample liquid is continuously collected through a probe, and is correlated with the concentration of each product obtained through off-line processing and the concentration of a substrate obtained through calculation according to a preset sampling moment, so that a quantitative near infrared spectrum model capable of reflecting the concentration change of the product and the substrate in the process of the TreY/TreZ approach is obtained, the model can be used for monitoring the concentration change of each component in a reaction liquid in real time in an on-line mode, further the enzymatic reaction process can be analyzed, the detection time is greatly shortened, the model is accurate and reliable, and the monitoring cost of the reaction is reduced.
Drawings
FIG. 1 is a flow chart of a modeling method for monitoring a model of a change in a composition during an enzymatic reaction as provided herein.
FIG. 2 is a schematic view of a near infrared monitoring and enzymatic reaction apparatus provided herein.
Fig. 3 is a near-infrared raw spectral curve of the TreY/TreZ pathway reaction solution provided herein.
Fig. 4 is a near-infrared spectrum curve of the TreY/TreZ pathway reaction solution provided by the present application after being subjected to multivariate scattering correction pretreatment.
FIG. 5 is a plot of predicted values versus truth values for trehalose from the calibration set and the prediction set of the PLS model provided herein.
FIG. 6 is a plot of the prediction values of maltodextrins in the correction set and prediction set of the PLS model provided herein versus true values.
Detailed Description
The following further describes the embodiments of the present invention with reference to the drawings.
A modeling method for monitoring a component change model in an enzymatic reaction process, as shown in FIG. 1, includes the steps of:
step 1: static modeling to verify experimental feasibility.
Preparing maltodextrin, trehalose, a mixed solution of maltodextrin and trehalose with different concentrations according to the actual conversion rate in the enzyme conversion process, and preparing MTSase and MTHase inactivated enzyme solution added with hydrochloric acid as four sample solutions, wherein the hydrochloric acid is used for controlling the pH value of the inactivated enzyme solution to be 2.0-2.2.
10mL portions of sample liquid are respectively put into a 20mL beaker, a near-infrared probe is sequentially immersed into the sample liquid, and near-infrared spectrum information is collected. And (4) establishing association between the near infrared spectrum information and the known concentration or the added content of the sample to obtain a spectrum model. Collecting near infrared spectrum information of the liquid to be detected and inputting the information into the spectrum model to obtain the concentration of the liquid to be detected, comparing the concentration with the actual concentration of the liquid to be detected, and judging the accuracy of the spectrum model. Optionally, the liquid to be measured is one of four sample liquids. Experiments prove that the method has feasibility.
Step 2: and installing a near infrared monitoring and enzymatic reaction device.
As shown in fig. 2, the near infrared monitoring device comprises a computer 1, a near infrared spectrometer 2 and a probe 3 thereof, and an iron ring 4 with a movable zenith. The near-infrared spectrometer 2 is connected with the computer 1 and used for near-infrared modeling, an optical fiber 5 connected with the near-infrared spectrometer 2 and a probe 3 of the near-infrared spectrometer passes through the iron ring 4, and the position of the iron ring is adjusted to keep the optical fiber 5 not to be bent.
The enzymatic reaction device comprises an iron stand 6 and at least two reaction kettles 7 with temperature control systems and stirring devices. The reaction kettle 7 is arranged on the iron support 6, and the probe 3 is fixed through the iron support 6. The temperature control system comprises a temperature sensor 71 for feeding back the temperature of the sample liquid to the temperature control system in real time, and the stirring device comprises a stirring paddle 72 arranged at the bottom of the reaction kettle.
Optionally, three reaction kettles, namely a # 1 reaction kettle, a # 2 reaction kettle and a # 3 reaction kettle, are used in this embodiment.
And step 3: preparing an initial sample liquid in each reaction kettle, recording the mass of the reaction kettle and the initial sample liquid, and recording the mass as m First stage (kg)。
For each reaction kettle, 300g of maltodextrin and 986.2g of deionized water (accurate to 0.01 g) were weighed and poured into the reaction kettle to be mixed to obtain initial sample liquid, and the volume and concentration of the initial sample liquid were recorded as V '=1.2L and c' Bottom ＝250g/L。
And 4, step 4: the enzymatic reactions are respectively started at intervals of preset time between the reaction kettles.
The temperature sensor is stretched into the reaction kettle and fixed at a proper position, namely, the temperature sensor cannot touch the stirring paddle at the bottom of the reaction kettle too deeply and cannot sense the feedback temperature state too shallowly and correctly. And starting the temperature control system and the stirring device, controlling the temperature of the sample liquid to be 50 ℃ at a preset temperature, and optionally, controlling the rotating speed of the stirring paddle to be 80rpm/min. Preheating for 20min after maltodextrin is completely dissolved, respectively putting MTSase/MTHase 1 (5.10 + 7.20mL) into a 1# reaction kettle, putting MTSase/MTHase 1 (9.60 + 2.70mL) into a 2# reaction kettle, and putting MTSase/MTHase 1:6 (1.80 + 10.50mL) into a 3# reaction kettle at preset time intervals; 4.8mL of pullulanase and 1.4mL of cyclodextrin enzyme are added into the three reaction kettles, and enzymatic reactions are started respectively by staggering the reaction time. Optionally, because the reaction is active in the first 1h of the enzymatic reaction (i.e., the initial reaction stage), the predetermined time is 1h, and then the 1# reaction kettle, the 2# reaction kettle and the 3# reaction kettle are sequentially started at an interval of 1 h.
Optionally, the optimal reaction temperature of MTSase is 45-50 ℃, the optimal reaction temperature of MTHase is 48-50 ℃, and the optimal pH values of the two are 6.0-6.5.
And 5: in the enzymatic reaction process, the three reaction kettles are alternately sampled according to the preset sampling time, and the near infrared spectrum information of the sample liquid is collected for modeling.
Step 51: and (3) immersing the probe into the sample liquid of the No. 1 reaction kettle, continuously extracting the sample liquid from the No. 1 reaction kettle at a preset sampling moment, and acquiring the near infrared spectrum information of the sample liquid in the No. 1 reaction kettle, wherein the near infrared spectrum information corresponds to the preset sampling moment.
Optionally, in order to enrich the modeling data, after each group of sampling is finished, the near infrared spectrum information of the sample liquid in the reaction kettle No. 1 is collected again at an interval of about 10 seconds (no sampling is carried out at this time) until the next group of sampling is started.
Step 52: and (3) carrying out off-line treatment on the extracted sample liquid to obtain the concentration of an enzymatic reaction product in the sample liquid at a preset sampling moment, wherein the product comprises a main product trehalose and byproducts glucose and maltose.
And (3) terminating the enzymatic reaction of the extracted sample liquid by using ethanol with the final concentration of 75%, and detecting the concentrations of trehalose, glucose and maltose in the sample liquid at each preset sampling moment by using HPLC (high performance liquid chromatography) after diluting by proper times after high-speed centrifugation.
Optionally, the chromatographic conditions for measuring the sample solution by using HPLC are as follows: polyurethane column-Dikama polyamine, 5 μm, 250X 4.6mm; the volume ratio of acetonitrile/water is 75/25; the flow rate is 1.0mL/min, and the detector adopts a parallax refraction detector; the sample injection amount is 20 mu L; the column temperature was 30 ℃ and the parallax temperature was 35 ℃.
Step 53: and when the set 1h is reached, transferring the probe into a 2# reaction kettle to repeat the steps of sampling and spectrum acquisition in the enzymatic reaction process, namely repeating the steps 51-53 until the sampling in the initial reaction stage of the 3# reaction kettle is finished.
Step 54: alternately sampling and collecting spectra in three reaction kettles by a probe at an interval of 1h according to the sequence of 1# → 2# → 3#, recording the mass of the reaction kettles and the residual sample liquid, and recording the mass as m Final (a Chinese character of 'gan') (kg)。
Optionally, in order to enrich modeling data, after each group of sampling is finished, the near infrared spectrum information of the sample liquid in the reaction kettle is collected again at an interval of about 5min (no sampling at this time) until the next group of sampling is started.
In this embodiment, the near-infrared spectrogram of each sample is acquired according to the above time schedule, the used instrument is an FT-NIR7200 near-infrared spectrometer of brueck, germany, the spectrum acquisition and information processing software is OPUS7.8 software, and the acquisition conditions of the near-infrared spectrum information are as follows: the scanning range of the near infrared spectrometer is 3995cm -1 -12000cm -1 Resolution of 16cm -1 Scanning 10 times, continuously scanning, and wave number 3995cm in FIG. 3 -1 -12000cm -1 NIR spectrum of the sample.
Step 6: the maltodextrin concentration at the predetermined sampling instant was calculated from the two masses recorded and the concentration of each product.
Step 61: and calculating the water evaporation volume according to the two recorded masses, wherein the expression of the actual volume of the sample liquid at the preset sampling moment is as follows:
wherein the unit of V is mL; rho Water (I) Taking 1g/mL as the density of water; h is the total reaction time, and H is the time corresponding to the corresponding preset sampling moment.
Step 62: converting the concentrations of trehalose, glucose and maltose obtained by off-line processing back to the corrected concentrations of the original volume, wherein the expressions are respectively as follows:
wherein, c T 、c G 、c M Corrected concentrations of trehalose, glucose, maltose in sequence at a predetermined sampling time c t 、c g 、c m The concentration obtained by off-line processing of trehalose, glucose and maltose at a preset sampling time is sequentially in g/L.
And step 63: the expression for calculating the maltodextrin concentration is:
wherein, c Bottom (C) The unit is g/L for the maltodextrin concentration at the preset sampling time, 1.05 is the theoretical conversion rate of trehalose and maltose, and 1.11 is the theoretical conversion rate of glucose.
And 7: and establishing the relationship between the trehalose concentration obtained by off-line treatment and the maltodextrin concentration obtained by calculation and the collected near infrared spectrum information through OPUS according to the preset sampling time to obtain a quantitative near infrared spectrum model reflecting the concentration change of the product and the substrate in the process of the TreY/TreZ approach.
In a computer, near infrared spectrum information of a sample is processed by using preprocessing methods such as a first derivative, vector normalization (SNV), multivariate Scattering Correction (MSC), a second derivative and the like so as to eliminate noise influence introduced in spectral measurement, and FIG. 4 is an NIR spectrogram of TreY/TreZ path reaction liquid preprocessed by MSC. And (3) rejecting abnormal samples through main factor regression analysis and Partial Least Squares (PLS) analysis, thereby improving the accuracy and stability of model prediction. The near infrared spectrum information of the reaction liquid is input into the model, the change of the concentration of each component in the reaction liquid can be monitored on line in real time, the enzymatic reaction process can be analyzed, and the detection time is greatly shortened.
The accuracy of the quantitative near-infrared spectrum model established by the method is verified by calculating the evaluation index of the model:
the model evaluation index established in the experiment is R 2 (coefficient of determination), RMSECV (Cross validation allRoot mean square error), RMSEE (root mean square error) and RPD (relative analysis error), calculated as follows:
wherein, y i Is the concentration reference value of the i-th sample, y m Taking the concentration mean value of the mth correction sample, wherein the correction sample is a predetermined number of samples needing to be corrected selected from all samples; differ is the difference between the true value and the predicted value, wherein the true value is the trehalose concentration detected by HPLC and the maltodextrin concentration obtained by calculation, and the predicted value is the component concentration output by the quantitative near infrared spectrum model; SEE is the standard analytical error of the calibration sample, M is the number of calibration samples, R is the dimension; SD is the standard deviation of the corrected sample.
The PLS modeling results of the spectra under the Multivariate Scatter Correction (MSC) preprocessing method are given below:
TABLE 1 PLS modeling of trehalose concentration during TreY/TreZ pathway under MSC pretreatment method
| Pretreatment method | RMSECV | Dimension number | R 2 | RMSEE | RPD |
| Multivariate Scattering Correction (MSC) | 0.111 | 7 | 0.9948 | 0.094 | 13.9 |
As shown in fig. 5, in the figure: the abscissa is the true value, the ordinate is the predicted value, the straight line fitted is the optimum model finally obtained. Table 1 shows the R of the model for predicting trehalose concentration for the modeling effect after MSC treatment 2 =0.995, rmsee =0.094, rpd =13.9. After MSC treatment, the values of RMSECV and RMSEE are the most close to 0, and R of the model 2 The RPD closest to 1,is far more than 2, which shows that the quantitative near infrared spectrum model established by the technical method is accurate and reliable.
TABLE 2 PLS modeling of substrate maltodextrin concentration during TreY/TreZ pathway under MSC pretreatment method
| Pretreatment method | RMSECV | Dimension number | R 2 | RMSEE | RPD |
| Multivariate Scattering Correction (MSC) | 0.123 | 8 | 0.9849 | 0.102 | 8.14 |
As shown in fig. 6, in the figure: the abscissa is the true value, the ordinate is the predicted value, the straight line fitted is the optimal model finally obtained. Table 2 shows the modeling effect of MSC treatment, and the R of the model for predicting the maltodextrin concentration 2 =0.985, rmsee =0.102, rpd =8.14. After MSC treatment, the values of RMSECV and RMSEE are the most close to 0, and R of the model 2 The RPD closest to 1,is far more than 2, which shows that the quantitative near infrared spectrum model established by the technical method is accurate and reliable.
What has been described above is only a preferred embodiment of the present application, and the present invention is not limited to the above examples. It is to be understood that other modifications and variations directly derivable or suggested by those skilled in the art without departing from the spirit and concept of the present invention are to be considered as included within the scope of the present invention.
Claims (10)
1. A modeling method for a model for monitoring a change in a composition during an enzymatic reaction, the modeling method comprising:
installing a near-infrared monitoring and enzymatic reaction device, wherein the near-infrared monitoring device comprises a computer, a near-infrared spectrometer and a probe of the near-infrared spectrometer, and the near-infrared spectrometer is connected with the computer and used for near-infrared modeling; the enzymatic reaction device comprises at least two reaction kettles;
preparing an initial sample solution in each reaction kettle, and recording the mass of the reaction kettle and the initial sample solution, wherein the initial sample solution comprises maltodextrin serving as a reaction substrate;
respectively starting enzymatic reactions of two reaction kettles at preset time intervals, immersing the probe into sample liquid of the reaction kettles in the enzymatic reaction process, continuously extracting the sample liquid from the reaction kettles at a preset sampling moment, and collecting near infrared spectrum information of the sample liquid in the reaction kettles, wherein the near infrared spectrum information corresponds to the preset sampling moment; performing off-line treatment on the extracted sample liquid to obtain the concentration of an enzymatic reaction product in the sample liquid at a preset sampling moment, wherein the product comprises a main product trehalose and byproducts glucose and maltose; when the preset time is reached, transferring the probe to another reaction kettle to repeat sampling and spectrum collection in the enzymatic reaction process;
after the sampling of the initial reaction stages of the two reaction kettles is finished, repeatedly performing the step of immersing the probe into the sample liquid of the reaction kettles, alternately sampling and collecting spectra in the two reaction kettles according to preset time until the enzymatic reaction is finished, and recording the mass of the reaction kettles and the mass of the residual sample liquid;
and calculating the maltodextrin concentration at the preset sampling moment according to the two recorded masses and the concentration of each product, and establishing association between the trehalose concentration obtained by offline processing and the calculated maltodextrin concentration and the collected near infrared spectrum information according to the preset sampling moment to obtain a quantitative near infrared spectrum model reflecting the concentration change of the product and the substrate in the process of the TreY/TreZ approach.
2. A modelling method for monitoring a model of the variation of a composition in an enzymatic reaction process according to claim 1, characterized in that said calculation of the maltodextrin concentration at a predetermined sampling instant from the two masses recorded and the concentration of each product comprises:
and calculating the water evaporation volume according to the two recorded masses, wherein the expression of the actual volume of the sample liquid at the preset sampling moment is as follows:
wherein the content of the first and second substances,
the mass of the reaction kettle and the initial sample liquid,
the mass of the reaction kettle and the residual sample liquid,
is the density of the water and is,
is the volume of the initial sample liquid,Hthe total reaction time is the total reaction time,hthe time corresponding to the corresponding predetermined sampling time; the concentrations of trehalose, glucose and maltose obtained by off-line processing are converted back to the corrected concentrations of the original volume, and the expressions are respectively:
wherein the content of the first and second substances,c T 、c G 、c M the correction concentrations of the trehalose, the glucose and the maltose at the preset sampling time are sequentially obtained,c t 、c g 、c m sequentially obtaining the concentrations of trehalose, glucose and maltose at the preset sampling moment through off-line treatment;
calculating the expression of the maltodextrin concentration at the predetermined sampling time as follows:
wherein the content of the first and second substances,
is the maltodextrin concentration at the predetermined sampling time,
the theoretical conversion of trehalose to maltose is 1.05 and the theoretical conversion of glucose is 1.11 for the initial sample solution.
3. The modeling method for monitoring the component change model in the enzymatic reaction process according to claim 1, wherein the step of performing off-line processing on the extracted sample liquid to obtain the concentration of the enzymatic reaction product in the sample liquid at a predetermined sampling time comprises:
and (3) stopping the enzymatic reaction of the extracted sample solution by adopting ethanol with a preset concentration, and detecting the concentrations of trehalose, glucose and maltose in the sample solution at each preset sampling moment by adopting HPLC (high performance liquid chromatography) after diluting by a proper multiple after high-speed centrifugation.
4. The modeling method for monitoring a model of a change in composition during an enzymatic reaction according to claim 1, wherein said preparing an initial sample solution in each of said reaction vessels comprises, for each of said reaction vessels:
weighing 300g maltodextrin and 986.2g deionized water, pouring the weighed materials into the reaction kettle, mixing to obtain an initial sample solution, and recording the volume and the concentration of the initial sample solution.
5. The modeling method for monitoring the component change model in the enzymatic reaction process according to claim 1, wherein the reaction kettle is provided with a temperature control system and a stirring device, the temperature control system comprises a temperature sensor for feeding back the temperature of the sample liquid to the temperature control system in real time, and the stirring device comprises a stirring paddle arranged at the bottom of the reaction kettle;
the two reaction kettles respectively start enzymatic reactions at preset time intervals, and the enzymatic reactions comprise: and (2) extending the temperature sensor into the liquid in the reaction kettle, fixing the position of a stirring paddle which is not in contact with the bottom of the reaction kettle, starting the temperature control system and the stirring device, controlling the temperature of the sample liquid to be at a preset temperature, after the maltodextrin is dissolved, respectively putting MTSase and MTHase with different proportions and pullulanase and cyclodextrin enzyme with the same amount into the two reaction kettles at preset time intervals, and respectively starting enzymatic reaction by staggering the reaction time.
6. The modeling method for monitoring a composition change model in an enzymatic reaction process according to claim 1, wherein said installing a near infrared monitoring and enzymatic reaction apparatus further comprises:
the near-infrared monitoring device also comprises an iron ring with a movable zenith, an optical fiber connected with the near-infrared spectrometer and a probe of the near-infrared spectrometer penetrates through the iron ring, and the position of the iron ring is adjusted to keep the optical fiber not to be bent;
enzymatic reaction unit still includes iron stand platform, reation kettle arranges in on the iron stand platform, through the iron stand platform is fixed the probe.
7. The modeling method for monitoring a model of a change in a component in an enzymatic reaction process according to claim 1, wherein the collection condition of the near infrared spectrum information is: the scanning range of the near-infrared spectrometer is 3995cm -1 -12000cm -1 Resolution of 16cm -1 The number of scanning times is 5-10, and the scanning is continuous.
8. A modeling method for a model for monitoring changes in composition during an enzymatic reaction according to claim 3, characterized in that the chromatographic conditions for the sample solution measured by HPLC are: polyurethane column-Dikama Polyamino,5 μm,250 × 4.6mm; the volume ratio of acetonitrile/water is 75/25; the flow rate is 1.0mL/min, and the detector adopts a parallax refraction detector; the sample size is 20 muL; the column temperature was 30 ℃ and the parallax temperature was 35 ℃.
9. The modeling method for monitoring a model of change in a component during an enzymatic reaction according to claim 5, wherein the optimum reaction temperature of MTSase is 45-50 ℃, the optimum reaction temperature of MTHase is 48-50 ℃, and both the optimum pH values are 6.0-6.5.
10. A modelling method for monitoring a model for the change in a composition during an enzymatic reaction according to any of claims 1-9, wherein said modelling method further comprises static modelling to verify experimental feasibility:
preparing maltodextrin, trehalose, a mixed solution of maltodextrin and trehalose with different concentrations according to the actual conversion rate in the enzyme conversion process, and preparing MTSase and MTHase inactivated enzyme solution added with hydrochloric acid as four sample solutions, wherein the hydrochloric acid is used for controlling the pH of the inactivated enzyme solution to be 2.0-2.2;
respectively placing four sample liquids with the same volume into a beaker, sequentially immersing the probe into the sample liquids and collecting near infrared spectrum information; establishing association between the near infrared spectrum information and the known concentration or the added content of the sample to obtain a spectrum model; collecting near infrared spectrum information of the liquid to be detected and inputting the information into the spectrum model to obtain the concentration of the liquid to be detected, comparing the concentration with the actual concentration of the liquid to be detected, and judging the accuracy of the spectrum model.
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