CN111751364B - Method for rapidly determining water-soluble protein and total sugar of royal jelly - Google Patents
Method for rapidly determining water-soluble protein and total sugar of royal jelly Download PDFInfo
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- 229940109850 royal jelly Drugs 0.000 title claims abstract description 117
- 235000000346 sugar Nutrition 0.000 title claims abstract description 88
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 46
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 50
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- 238000001514 detection method Methods 0.000 claims abstract description 19
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- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims abstract description 10
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
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Abstract
The utility model relates to the technical field of royal jelly quality detection, and particularly discloses a method for rapidly determining water-soluble protein and total sugar of royal jelly, which comprises the steps of (1) selecting a royal jelly sample; (2) Detecting the water-soluble protein content of the royal jelly sample by adopting a Coomassie brilliant blue staining method; detecting the total sugar content of the royal jelly sample by adopting a potassium ferrocyanide method; (3) scanning the royal jelly sample using a mid-infrared spectrometer; (4) Establishing a first quantitative analysis model and a second quantitative analysis model under full wavelength; (5) Selecting the most suitable pretreatment mode relative to the water-soluble protein content and the most suitable pretreatment mode relative to the total sugar content; (6) screening characteristic wavelengths; (7) Establishing a first partial least square model of the water-soluble protein content and a second partial least square model of the total sugar content; (8) And (5) establishing a prediction model of the water-soluble protein content and the total sugar content of the royal jelly. The utility model has the advantages of simple operation, time and labor saving and higher analysis precision.
Description
Technical Field
The utility model relates to the technical field of quality detection of royal jelly, in particular to a method for rapidly detecting water-soluble protein and total sugar of royal jelly.
Background
The royal jelly is a natural pulp secreted by the lingual gland and the palate gland of a nursing bee, has complex components, contains a large amount of proteins, amino acids, saccharides, lipids, vitamins and minerals, and also contains specific royal jelly acid, wherein the protein content is the largest, especially the protein 1-5 of the royal jelly is an important nutrition and health care factor, has the effects of improving immunity, strengthening physique, promoting intelligence, resisting aging and the like, and has important significance on the royal jelly.
The water-soluble protein and total sugar content in the royal jelly are important indexes for evaluating the quality of the royal jelly, but the quality of the royal jelly is greatly influenced by storage conditions, and the quality degradation of the royal jelly is mainly represented by the degradation of the water-soluble protein, which indicates that the quality of the royal jelly is related to the change of the protein. On the premise of ensuring the freshness of the royal jelly, the protein content measurement is an indispensable work. The Kjeldahl nitrogen determination method in the national royal jelly standard is used for determining the total nitrogen content, wherein the total nitrogen also comprises non-protein nitrogen, and the experimental process is tedious and time-consuming; in the prior art, a tabletting method is used for predicting the protein content by combining infrared spectrum, a sample and a proper amount of potassium bromide are mixed and put into a vacuum drying oven, and then the mixture is pressed into tablets after moisture is dried, and the influence of temperature tends to influence the change of the protein structure, so that a certain error is caused; on the other hand, coomassie brilliant blue staining is relatively rapid, but is still tedious and time-consuming in hundreds of thousands of royal jelly samples.
Therefore, the current detection of the water-soluble protein and the total sugar content of the royal jelly has the problems of complex operation, time and labor consumption and lower analysis precision.
Disclosure of Invention
The utility model aims to solve the technical problems of the existing detection of the water-soluble protein and the total sugar content of the royal jelly, and provides a method for rapidly detecting the water-soluble protein and the total sugar of the royal jelly, which is simple and convenient to operate, saves time and labor and has higher analysis precision.
The technical scheme of the utility model is as follows: the fast royal jelly water-soluble protein and total sugar measuring process includes the following steps,
(1) Selecting a royal jelly sample and preserving under proper conditions;
(2) Detecting the water-soluble protein content of the royal jelly sample by adopting a Coomassie brilliant blue staining method to obtain a water-soluble protein content matrix; detecting the total sugar content of the royal jelly sample by adopting a potassium ferrocyanide method to obtain a total sugar content matrix;
(3) Scanning a royal jelly sample by using a mid-infrared spectrometer to obtain a mid-infrared attenuated total reflection spectrum matrix of the royal jelly sample;
(4) After the mid-infrared attenuated total reflection spectrum matrix obtained in the step (3) is processed in a plurality of pretreatment modes, a first quantitative analysis model under the total wavelength is established with the water-soluble protein content matrix obtained in the step (2) by adopting a partial least square method;
after the mid-infrared attenuated total reflection spectrum matrix obtained in the step (3) is processed in a plurality of pretreatment modes, a second quantitative analysis model under the full wavelength is established with the total sugar content matrix obtained in the step (2) by adopting a partial least square method;
(5) Selecting the most appropriate pretreatment mode relative to the water-soluble protein content according to the correction set correlation coefficient, verification set correlation coefficient and root mean square error in the first quantitative analysis model result at full wavelength;
selecting the most appropriate pretreatment mode relative to the total sugar content according to the correction set correlation coefficient, the verification set correlation coefficient and the root mean square error in the second quantitative analysis model result under the full wavelength;
(6) Screening characteristic wavelengths in the first quantitative analysis model result by using a competitive self-adaptive re-weighting method;
screening characteristic wavelengths in the second quantitative analysis model result by using a competitive self-adaptive re-weighting method;
(7) Taking characteristic wavelength in a first quantitative analysis model result as an independent variable, establishing a first partial least square model of the water-soluble protein content, and selecting a main factor number according to a PRESS value of the first partial least square model;
taking characteristic wavelength in a second quantitative analysis model result as an independent variable, establishing a second partial least square model of the total sugar content, and selecting a main factor number according to a PRESS value of the second partial least square model;
(8) Respectively establishing prediction models of the water-soluble protein content and the total sugar content of the royal jelly according to the determined most suitable pretreatment mode, characteristic wavelength and main factor number,
and a linear fitting equation of the predicted value and the true value of the water-soluble protein content is obtained,
y＝0.9327x+0.003078
wherein x represents a true value of the water-soluble protein content, and y represents a predicted value of the water-soluble protein content;
and a linear fit equation of the predicted and actual values of total sugar content,
y＝1.168x-0.6529
where x represents the true value of the total sugar content and y represents the predicted value of the total sugar content.
The utility model adopts a Coomassie brilliant blue staining method to detect the water-soluble protein content of the royal jelly sample, and obtains a water-soluble protein content matrix; detecting the total sugar content of the royal jelly sample by adopting a potassium ferrocyanide method to obtain a total sugar content matrix; scanning a royal jelly sample by using a mid-infrared spectrometer to obtain a mid-infrared attenuated total reflection spectrum matrix of the royal jelly sample; then, a partial least square method is adopted to establish a quantitative analysis model under full wavelength, and then a corresponding most suitable pretreatment mode suitable for water-soluble proteins and total sugar is selected; the utility model uses a competitive self-adaptive re-weighting method to screen corresponding characteristic wavelengths corresponding to water-soluble proteins and total sugar in quantitative analysis model results; the utility model uses the characteristic wavelength in the quantitative analysis model result as independent variable, establishes a partial least square model of the water-soluble protein content, and selects the main factor number suitable for the water-soluble protein and the total sugar according to the partial least square model PRESS value; according to the determined most suitable pretreatment mode, characteristic wavelength and main factor number, a prediction model suitable for the water-soluble protein content and the total sugar content is established, and finally a linear fitting equation of the predicted value and the true value of the water-soluble protein content and the total sugar content is obtained; the whole detection process utilizes a method combining the attenuated total reflection mid-infrared spectrum technology and the partial least square method, and has the advantages of simple and convenient operation, science, time and labor saving, higher analysis efficiency and higher analysis precision.
Preferably, in the step (1), the royal jelly sample is first preserved at-18 ℃, then the royal jelly sample at-18 ℃ is divided into a plurality of small portions according to the requirement, and preserved at-18 ℃, 4 ℃ and 25 ℃ respectively, and after preserving the 0d, 7d, 14d, 30d, 60d and 84d under different temperature conditions, the content detection and mid-infrared spectrum test of the corresponding royal jelly sample are carried out.
Preferably, in the step (2), the content of the water-soluble protein in each royal jelly sample is detected by adopting a coomassie brilliant blue staining method, so as to obtain a content matrix of the water-soluble protein; detecting the total sugar content of each royal jelly sample by adopting a potassium ferrocyanide method to obtain a total sugar content matrix;
in the step (3), each royal jelly sample is scanned by using a mid-infrared spectrometer to obtain a mid-infrared attenuated total reflection spectrum matrix of the royal jelly sample.
Preferably, in the step (3), the mid-infrared spectrometer is a fourier transform mid-infrared spectrometer, and the mid-infrared spectrometer is provided with an attenuated total reflection accessory; in the step (3), before the mid-infrared spectrometer is used, the drying agent is replaced, and then the mid-infrared spectrometer is preheated for 15min, and then the self-inspection, the performance test and the background spectrum acquisition of the instrument are carried out; the drying agent is a silica gel drying agent which is dried for 12 hours in an oven at the temperature of 120 ℃.
Preferably, the collection background spectrum is a spectrum when air is used as a background; in the step (3), the method for scanning the royal jelly sample by using the mid-infrared spectrometer is that,
and sucking a proper amount of royal jelly sample by using a suction pipe, smearing the royal jelly sample on an attenuated total reflection accessory, and then using a mid-infrared spectrometer to scan the sample, wherein the mid-infrared spectrometer automatically deducts the background spectrum from the spectrum of the sample and then uses the sample spectrum as spectrum data to be analyzed.
Preferably, the application of the royal jelly sample is performed by uniformly covering the circular crystals on the attenuated total reflection accessory. The crystal is ZnSe crystal, and the royal jelly sample is only required to be dripped on the surface of the ZnSe crystal when being smeared, so that the operation is simple and convenient.
Preferably, after each measurement of a royal jelly sample, the circular crystals on the attenuated total reflection accessory are washed with absolute ethyl alcohol and wiped clean.
Preferably, the scanning temperature range of the mid-infrared spectrometer is 15-25 ℃, and the scanning wavelength range is 4000-600 cm -1 Resolution of 4cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The sample scanning times are 16 times, and after each sample is scanned for 3 times, an average value is obtained; the number of the background scanning times is 16, and the average value is obtained after the scanning is completed; the wavelength ranges corresponding to the similar characteristic wavelength independent variables in the first quantitative analysis model result and the second quantitative analysis model result are 1620-1650 cm -1 、1520～1540cm -1 And 600-900 cm -1 。
Preferably, the dosage of each royal jelly sample in the step (2) and the step (3) is 0.03-0.1 g; the scanning time in the step (3) is 25-40 s. More preferably, the dosage of each royal jelly sample in the step (2) and the step (3) is 0.05-0.08 g; the scanning time in the step (3) is 30-35 s. The dosage of the royal jelly sample is less during detection, so that the detection cost is lower; the scanning detection process of the whole instrument needs shorter time, is quick and convenient, and has higher detection efficiency.
Preferably, the change of the wavelength number, the interactive verification mean square error and the wavelength variable regression coefficient in the result of the first quantitative analysis model is reserved in the screening process;
and (3) reserving the wavelength number, the interactive verification mean square error and the change of the regression coefficient of the wavelength variable in the second quantitative analysis model result in the screening process.
Preferably, when the cross-validation mean square error is minimum, all wavelengths of the water-soluble protein and all wavelengths of the total sugar in the corresponding royal jelly sample are selectedThe characteristic wavelength is obtained; the wavelength range corresponding to the characteristic wavelength independent variable in the first quantitative analysis model result and the wavelength range corresponding to the characteristic wavelength independent variable in the second quantitative analysis model result in the step (7) are 2000-500 cm -1 。
Preferably, the preprocessing mode includes one or more of no processing, baseline correction processing, first derivative processing, second derivative processing, smoothing processing, MSC processing, trending correction processing, ATR correction processing, differential derivation processing, SNV processing, and smoothing+snv processing.
The utility model has the following beneficial effects:
the utility model adopts a Coomassie brilliant blue staining method to detect the water-soluble protein content of the royal jelly sample, and obtains a water-soluble protein content matrix; detecting the total sugar content of the royal jelly sample by adopting a potassium ferrocyanide method to obtain a total sugar content matrix; scanning a royal jelly sample by using a mid-infrared spectrometer to obtain a mid-infrared attenuated total reflection spectrum matrix of the royal jelly sample; then, a partial least square method is adopted to establish a quantitative analysis model under full wavelength, and then a corresponding most suitable pretreatment mode suitable for water-soluble proteins and total sugar is selected; the utility model uses a competitive self-adaptive re-weighting method to screen corresponding characteristic wavelengths corresponding to water-soluble proteins and total sugar in quantitative analysis model results; the utility model uses the characteristic wavelength in the quantitative analysis model result as independent variable, establishes a partial least square model of the water-soluble protein content, and selects the main factor number suitable for the water-soluble protein and the total sugar according to the partial least square model PRESS value; according to the determined most suitable pretreatment mode, characteristic wavelength and main factor number, a prediction model suitable for the water-soluble protein content and the total sugar content is established, and finally a linear fitting equation of the predicted value and the true value of the water-soluble protein content and the total sugar content is obtained; the whole detection process utilizes a method combining the attenuated total reflection mid-infrared spectrum technology and the partial least square method, and has the advantages of simple and convenient operation, science, time and labor saving, higher analysis efficiency and higher analysis precision.
Drawings
FIG. 1 is an original mid-infrared spectrogram of royal jelly of the utility model;
FIG. 2 is a diagram of a characteristic wavelength CARS method screening process of a water-soluble protein according to the present utility model;
FIG. 3 is a diagram of the screening process of the characteristic wavelength CARS method of the total sugar of the utility model;
FIG. 4 is a characteristic wavelength distribution diagram of a water-soluble protein of the present utility model;
FIG. 5 is a characteristic wavelength distribution plot of total sugar of the present utility model;
FIG. 6 is a diagram showing the process of selecting the optimal prime factor number for the water-soluble protein according to the present utility model;
FIG. 7 is a graph showing the process of selecting the number of main factors for total sugar optimization in accordance with the present utility model;
FIG. 8 is a graph showing the modeling effect of a characteristic wavelength PLS model of a water-soluble protein according to the present utility model;
FIG. 9 is a graph showing the modeling effect of the characteristic wavelength PLS model of total sugar according to the present utility model.
Detailed Description
The utility model is further illustrated by the following figures and examples, which are not intended to be limiting.
The fast royal jelly water-soluble protein and total sugar measuring process includes the following steps,
(1) Selecting a royal jelly sample and preserving under proper conditions;
wherein the royal jelly sample is selected from different producing areas and different years; specifically, the royal jelly sample is derived from: zhejiang Fuyang, 11 months in 2017; collected in 2019 in 4 months; qinghai, collection in 2019 6; is provided by Hangzhou Kangli food Limited, hangzhou Dexing Royal Limited, nanjing Jiangshan food Limited, respectively.
The method comprises the steps of firstly storing a royal jelly sample at the temperature of minus 18 ℃, then dividing the royal jelly sample at the temperature of minus 18 ℃ into a plurality of small parts according to the requirement, respectively storing the small parts at the temperature of minus 18 ℃, 4 ℃ and 25 ℃, and carrying out content detection and mid-infrared spectrum test of the corresponding royal jelly sample after storing the 0 th d, 7d, 14d, 30d, 60d and 84d of the royal jelly sample at different temperatures.
(2) Detecting the water-soluble protein content of the royal jelly sample by adopting a Coomassie brilliant blue staining method to obtain a water-soluble protein content matrix; detecting the total sugar content of the royal jelly sample by adopting a potassium ferrocyanide method to obtain a total sugar content matrix;
detecting the water-soluble protein content of each royal jelly sample by adopting a coomassie brilliant blue staining method to obtain a water-soluble protein content matrix; detecting the total sugar content of each royal jelly sample by adopting a potassium ferrocyanide method to obtain a total sugar content matrix;
(3) Scanning a royal jelly sample by using a mid-infrared spectrometer to obtain a mid-infrared attenuated total reflection spectrum matrix of the royal jelly sample; wherein the infrared spectrogram is shown in figure 1, the abscissa in figure 1 represents wavenumber, and the ordinate absorptance unit represents absorbance unit;
and scanning each royal jelly sample by using a mid-infrared spectrometer to obtain a mid-infrared attenuated total reflection spectrum matrix of the royal jelly sample.
The mid-infrared spectrometer is a Fourier transform mid-infrared spectrometer, and the mid-infrared spectrometer can be selected from the Fourier transform mid-infrared spectrometer of Bruker company of America, and is provided with an Attenuated Total Reflection (ATR) accessory.
Before the mid-infrared spectrometer is used, the drying agent is replaced, and then the mid-infrared spectrometer is preheated for 15min, and then the self-inspection, the performance test and the background spectrum acquisition of the instrument are carried out.
The drying agent is silica gel drying agent after drying for 12 hours in an oven at 120 ℃.
The collection background spectrum is a spectrum when air is used as a background.
The method for scanning the royal jelly sample by using the mid-infrared spectrometer comprises,
and sucking a proper amount of royal jelly sample by using a suction pipe, smearing the royal jelly sample on an attenuated total reflection accessory, and then using a mid-infrared spectrometer to scan the sample, wherein the mid-infrared spectrometer automatically deducts the background spectrum from the spectrum of the sample and then uses the sample spectrum as spectrum data to be analyzed.
The application of the royal jelly sample is based on uniformly covering the circular crystal on the attenuated total reflection accessory. The crystal is ZnSe crystal, and the royal jelly sample is only required to be dripped on the surface of the ZnSe crystal when being smeared, so that the operation is simple and convenient.
After each measurement of a royal jelly sample, the circular crystals on the attenuated total reflection accessory are cleaned by absolute ethyl alcohol and wiped clean. Thus, cross-contamination between samples can be avoided.
The scanning temperature range of the mid-infrared spectrometer is 15-25 ℃, and the scanning wavelength range is 4000-600 cm -1 Resolution of 4cm -1 。
Sample scanning times are 16 times, and after each sample is scanned 3 times, an average value is obtained; the number of background scans was 16, and the average value was taken after the scanning was completed.
The royal jelly sample at-18 ℃ was divided into 63 small portions, and the analysis results of the water-soluble protein and total sugar content of the royal jelly sample are shown in table 1:
table 1: analysis result of water-soluble protein and total sugar content of royal jelly sample
| Number of samples | Minimum value | Maximum value | Average value of | Standard deviation of | Extremely poor | |
| Water-soluble protein/% | 63 | 3.24 | 4.50 | 3.94 | 0.28 | 1.25 |
| Total sugar/% | 63 | 5.05 | 13.83 | 11.17 | 1.85 | 8.79 |
As can be seen from Table 1, the range of variation of the property values of each index is larger, the range is wider, the range of the conventional index of the royal jelly can be basically covered, and the quantitative model established by the quantitative model has better applicability.
The dosage of each royal jelly sample is 0.03-0.1 g; the scanning time is 25-40 s. More preferably, the dosage of each royal jelly sample is 0.05-0.08 g; the scanning time is 30-35 s. The dosage of the royal jelly sample is less during detection, so that the detection cost is lower; the scanning detection process of the whole instrument needs shorter time, is quick and convenient, and has higher detection efficiency.
(4) After the mid-infrared attenuated total reflection spectrum matrix obtained in the step (3) is processed in a plurality of pretreatment modes, a first quantitative analysis model under the total wavelength is established with the water-soluble protein content matrix obtained in the step (2) by adopting a partial least square method;
after the mid-infrared attenuated total reflection spectrum matrix obtained in the step (3) is processed in a plurality of pretreatment modes, a second quantitative analysis model under the full wavelength is established with the total sugar content matrix obtained in the step (2) by adopting a partial least square method;
as shown in table 2, the preprocessing mode includes one or more of no processing, baseline correction processing, first derivative processing, second derivative processing, smoothing processing, MSC processing, trending correction processing, ATR correction processing, differential derivation processing, SNV processing, and smoothing+snv processing.
Table 2: full-wavelength quantitative model results of water-soluble proteins and total sugars under different pretreatment methods
(5) Selecting the most appropriate pretreatment mode relative to the water-soluble protein content according to the correction set correlation coefficient, verification set correlation coefficient and root mean square error in the first quantitative analysis model result at full wavelength;
selecting the most appropriate pretreatment mode relative to the total sugar content according to the correction set correlation coefficient, the verification set correlation coefficient and the root mean square error in the second quantitative analysis model result under the full wavelength;
as shown in table 3, the most suitable pretreatment mode with respect to the water-soluble protein content is baseline correction, wherein the corresponding correction set correlation coefficient is 0.8702, the validation set correlation coefficient is 0.9611, the correction set predicted root mean square error is 0.1308, and the validation set predicted root mean square error is 0.1165. The modeling effect is good, and the prediction effect is good.
The most suitable preprocessing mode is smoothing processing relative to the total sugar content, wherein the corresponding correction set correlation coefficient is 0.9048, the verification set correlation coefficient is 0.9228, the correction set prediction root mean square error is 0.0080, and the verification set prediction root mean square error is 0.0089. The modeling effect is good, and the prediction effect is good.
Table 3: quantitative model results of water-soluble proteins and total sugars by screening characteristic wavelengths and different pretreatment methods
| Pretreatment mode | RMSECV | R C | RMSEP | R P | Principal factor number |
| Without any means for | 0.1171 | 0.8955 | 0.1259 | 0.9216 | 17 |
| 0.0079 | 0.9030 | 0.0099 | 0.9107 | 17 | |
| First derivative | 0.1125 | 0.9027 | 0.2278 | 0.9055 | 17 |
| 0.0089 | 0.8611 | 0.0046 | 0.9795 | 15 | |
| Second derivative | 0.1142 | 0.9224 | 0.2422 | 0.8270 | 16 |
| 0.0086 | 0.9042 | 0.0044 | 0.9438 | 19 | |
| Baseline correction | 0.1308 | 0.8702 | 0.1165 | 0.9611 | 18 |
| 0.0082 | 0.9040 | 0.0078 | 0.9025 | 18 | |
| Extension ATR | 0.1189 | 0.9030 | 0.1443 | 0.9086 | 18 |
| 0.0099 | 0.8760 | 0.0065 | 0.8903 | 17 | |
| MSC | 0.1355 | 0.8554 | 0.1307 | 0.9308 | 16 |
| 0.0110 | 0.8033 | 0.0069 | 0.9206 | 15 | |
| Detrend correction | 0.1647 | 0.7907 | 0.1742 | 0.8725 | 14 |
| 0.0126 | 0.7849 | 0.0151 | 0.5795 | 12 | |
| Differential derivation | 0.1125 | 0.9027 | 0.2278 | 0.9055 | 17 |
| 0.0103 | 0.8118 | 0.0054 | 0.9698 | 16 | |
| SNV | 0.1315 | 0.8582 | 0.1443 | 0.9057 | 16 |
| 0.0088 | 0.8814 | 0.0076 | 0.9041 | 17 | |
| Smoothing | 0.1641 | 0.7981 | 0.1337 | 0.9180 | 15 |
| 0.0080 | 0.9048 | 0.0089 | 0.9228 | 18 | |
| Smoothing, SNV | 0.1558 | 0.8086 | 0.1293 | 0.9277 | 16 |
| 0.0090 | 0.8777 | 0.0071 | 0.9498 | 18 |
(6) Screening characteristic wavelengths in the first quantitative analysis model result by using a competitive adaptive re-weighting method (CARS);
screening characteristic wavelengths in the second quantitative analysis model result by using a competitive self-adaptive re-weighting method;
the change of the wavelength number, the interactive verification mean square error and the wavelength variable regression coefficient in the first quantitative analysis model result is reserved in the screening process; the screening process is shown in fig. 2, and the abscissa number of sampling runs in fig. 2 represents the number of samples; ordinate number of sampled variable represents the number of sampling variables, RMSECV represents the root mean square error predicted by the water-soluble protein correction set, regression coefficients path represents the regression coefficient path;
the change of the wavelength number, the interactive verification mean square error and the wavelength variable regression coefficient in the second quantitative analysis model result is reserved in the screening process; the screening process is shown in fig. 3, and the abscissa number of sampling runs in fig. 3 represents the number of samples; ordinate number of sampled variable represents the number of sampling variables, RMSECV represents the root mean square error of the total sugar correction set prediction, and regression coefficients path represents the regression coefficient path.
When the cross validation mean square error is minimum, all wavelengths of the water-soluble protein and all wavelengths of the total sugar in the corresponding royal jelly sample are the selected characteristic wavelengths.
The number of the reserved variables shows a decreasing trend along with the increase of the operation times, the interactive root mean square error shows a trend of decreasing first and then increasing last, the independent variables are firstly removed one by one in the sampling process, then the effective variables are removed, and when the interactive verification root mean square error is minimum, the independent variables are all removed. Thus at sample 25, 64 wavelengths of the full wavelength of the water-soluble protein are retained as characteristic wavelengths; as shown in fig. 4, the abscissa Wavenumber in fig. 4 represents the wave number, the ordinate absorptance unit represents the Absorbance unit, and selected variable in fig. 4 represents the selection variable;
for the total sugar of the royal jelly, all irrelevant variables are removed at the 28 th time, and finally 42 characteristic wavelengths are reserved; as shown in fig. 5, the abscissa Wavenumber in fig. 5 represents the wave number, the ordinate absorptance unit represents the Absorbance unit, and selected variable in fig. 5 represents the selection variable.
(7) Taking characteristic wavelength in a first quantitative analysis model result as an independent variable, establishing a first Partial Least Squares (PLS) model of the water-soluble protein content, and selecting a main factor number according to a PRESS value of the first partial least squares model; as shown in fig. 6, the abscissa in fig. 6 is the number of main factors, and the ordinate is the value of the first partial least squares model PRESS; the final selected prime factor number n=18;
taking characteristic wavelength in a second quantitative analysis model result as an independent variable, establishing a second partial least square model of the total sugar content, and selecting a main factor number according to a PRESS value of the second partial least square model; as shown in fig. 7, the abscissa in fig. 7 is the number of main factors, and the ordinate is the value of the second partial least squares model PRESS; the final selected prime factor number n=18;
the wavelength range corresponding to the characteristic wavelength independent variable in the first quantitative analysis model result and the wavelength range corresponding to the characteristic wavelength independent variable in the second quantitative analysis model result are 2000-500 cm -1 The method comprises the steps of carrying out a first treatment on the surface of the Two cases can be seen;
in one case, the wavelength ranges corresponding to the similar characteristic wavelength independent variables in the first quantitative analysis model result and the wavelength ranges corresponding to the similar characteristic wavelength independent variables in the second quantitative analysis model result are 1620-1650 cm -1 、1520～1540cm -1 And 600-900 cm -1 。
In another case, there is a distribution difference at these places, which is consistent with both having a correlation with time of temperature but a difference.
The absorption peak of the water-soluble protein corresponds to a wavelength range of 1700-1520 cm -1 。
The amide band of the water-soluble protein corresponds to a wavelength of 1620cm -1 And 1540cm -1 Mainly c=o stretching vibration; the amide II band of the water-soluble protein has a wavelength range of 1565-1520 cm -1 Mainly C-N stretching vibration and N-H bending vibration.
The absorption peak of the total sugar corresponds to a wavelength range of 900-1250 cm -1 。
The C-O stretching vibration of the reducing sugar in the total sugar corresponds to a wavelength of 1050cm -1 P=O stretching vibration of reducing sugar in total sugar corresponds to wavelength of 1240cm -1 。
(8) Respectively establishing prediction models of the water-soluble protein content and the total sugar content of the royal jelly according to the determined most suitable pretreatment mode, characteristic wavelength and main factor number, as shown in fig. 8 and 9, wherein the abscissa in fig. 8 represents the true value of the water-soluble protein content, and the ordinate represents the predicted value of the water-soluble protein content; the abscissa in fig. 9 represents the actual value of the total sugar content, and the ordinate represents the predicted value of the total sugar content;
and a linear fitting equation of the predicted value and the true value of the water-soluble protein content is obtained,
y＝0.9327x+0.003078
wherein x represents a true value of the water-soluble protein content, and y represents a predicted value of the water-soluble protein content;
and a linear fit equation of the predicted and actual values of total sugar content,
y＝1.168x-0.6529
where x represents the true value of the total sugar content and y represents the predicted value of the total sugar content.
In order to improve the detection efficiency of the royal jelly, a simpler and faster method becomes a powerful choice for the rapid detection of the quality of the royal jelly. At present, no literature for applying the attenuated total reflection mid-infrared spectrum technology to the content of the water-soluble protein and the total sugar content of the royal jelly exists, and the method is the first application in predicting the content of the water-soluble protein and the total sugar content of the royal jelly.
Claims (3)
1. A method for rapidly determining water-soluble protein and total sugar of royal jelly is characterized in that: comprises the steps of,
(1) Selecting a royal jelly sample and preserving under proper conditions;
(2) Detecting the water-soluble protein content of the royal jelly sample by adopting a Coomassie brilliant blue staining method to obtain a water-soluble protein content matrix; detecting the total sugar content of the royal jelly sample by adopting a potassium ferrocyanide method to obtain a total sugar content matrix;
(3) Scanning a royal jelly sample by using a mid-infrared spectrometer to obtain a mid-infrared attenuated total reflection spectrum matrix of the royal jelly sample;
in the step (3), the mid-infrared spectrometer is a Fourier transform mid-infrared spectrometer, and the mid-infrared spectrometer is provided with an attenuated total reflection accessory;
in the step (3), before the mid-infrared spectrometer is used, the drying agent is replaced, and then the mid-infrared spectrometer is preheated for 15min, and then the self-inspection, the performance test and the background spectrum acquisition of the instrument are carried out; the drying agent is a silica gel drying agent which is dried for 12 hours in an oven at the temperature of 120 ℃;
(4) After the mid-infrared attenuated total reflection spectrum matrix obtained in the step (3) is processed in a plurality of pretreatment modes, a first quantitative analysis model under the total wavelength is established with the water-soluble protein content matrix obtained in the step (2) by adopting a partial least square method;
after the mid-infrared attenuated total reflection spectrum matrix obtained in the step (3) is processed in a plurality of pretreatment modes, a second quantitative analysis model under the full wavelength is established with the total sugar content matrix obtained in the step (2) by adopting a partial least square method;
the preprocessing mode comprises one or more of no processing, baseline correction processing, smoothing processing, trending correction processing, ATR correction processing, differential derivation processing, SNV processing and smoothing+SNV processing;
(5) Selecting the most appropriate pretreatment mode relative to the water-soluble protein content according to the correction set correlation coefficient, verification set correlation coefficient and root mean square error in the first quantitative analysis model result at full wavelength;
selecting the most appropriate pretreatment mode relative to the total sugar content according to the correction set correlation coefficient, the verification set correlation coefficient and the root mean square error in the second quantitative analysis model result under the full wavelength;
(6) Screening characteristic wavelengths in the first quantitative analysis model result by using a competitive self-adaptive re-weighting method;
screening characteristic wavelengths in the second quantitative analysis model result by using a competitive self-adaptive re-weighting method;
(7) Taking characteristic wavelength in a first quantitative analysis model result as an independent variable, establishing a first partial least square model of the water-soluble protein content, and selecting a main factor number according to a PRESS value of the first partial least square model;
taking characteristic wavelength in a second quantitative analysis model result as an independent variable, establishing a second partial least square model of the total sugar content, and selecting a main factor number according to a PRESS value of the second partial least square model;
(8) Respectively establishing prediction models of the water-soluble protein content and the total sugar content of the royal jelly according to the determined most suitable pretreatment mode, characteristic wavelength and main factor number,
and a linear fitting equation of the predicted value and the true value of the water-soluble protein content is obtained,
y＝0.9327x+0.003078
wherein x represents a true value of the water-soluble protein content, and y represents a predicted value of the water-soluble protein content;
and a linear fit equation of the predicted and actual values of total sugar content,
y＝1.168x-0.6529
wherein x represents a true value of the total sugar content, and y represents a predicted value of the total sugar content;
in the step (1), a royal jelly sample is firstly preserved at the temperature of minus 18 ℃, then the royal jelly sample at the temperature of minus 18 ℃ is divided into a plurality of small parts according to the requirement, and is preserved at the temperature of minus 18 ℃, 4 ℃ and 25 ℃ respectively, and after preserving the 0d, 7d, 14d, 30d, 60d and 84d at different temperature, the content detection and mid-infrared spectrum test of the corresponding royal jelly sample are carried out;
the collected background spectrum is a spectrum obtained when air is used as a background;
in the step (3), the method for scanning the royal jelly sample by using the mid-infrared spectrometer is that,
sucking a proper amount of royal jelly sample by using a suction pipe, smearing the royal jelly sample on an attenuated total reflection accessory, then using a mid-infrared spectrometer to scan the sample, and automatically deducting the background spectrum from the spectrum of the sample by the mid-infrared spectrometer to obtain spectrum data to be analyzed;
the application of the royal jelly sample is based on uniformly covering the circular crystals on the attenuated total reflection accessory;
after each measurement of a royal jelly sample, absolute ethyl alcohol is used for cleaning and attenuating the circular crystals on the total reflection accessory, and the round crystals are wiped clean;
the scanning temperature range of the mid-infrared spectrometer is 15-25 ℃ and the scanning wavelength is the sameIn the range of 4000-600 cm -1 Resolution of 4cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The sample scanning times are 16 times, and after each sample is scanned for 3 times, an average value is obtained; the number of the background scanning times is 16, and the average value is obtained after the scanning is completed; the wavelength ranges corresponding to the similar characteristic wavelength independent variables in the first quantitative analysis model result and the second quantitative analysis model result are 1620-1650 cm -1 、1520～1540cm -1 And 600-900 cm -1 ；
When the cross validation mean square error is minimum, all wavelengths of the water-soluble protein and all wavelengths of the total sugar in the corresponding royal jelly sample are the screened characteristic wavelengths;
the wavelength range corresponding to the characteristic wavelength independent variable in the first quantitative analysis model result and the wavelength range corresponding to the characteristic wavelength independent variable in the second quantitative analysis model result in the step (7) are 2000-500 cm -1 ；
The crystal is ZnSe crystal;
the change of the wavelength number, the interactive verification mean square error and the wavelength variable regression coefficient in the first quantitative analysis model result is reserved in the screening process;
and (3) reserving the wavelength number, the interactive verification mean square error and the change of the regression coefficient of the wavelength variable in the second quantitative analysis model result in the screening process.
2. The method for rapidly determining the water-soluble protein and total sugar of royal jelly according to claim 1, characterized in that: in the step (2), detecting the water-soluble protein content of each royal jelly sample by adopting a coomassie brilliant blue staining method to obtain a water-soluble protein content matrix; detecting the total sugar content of each royal jelly sample by adopting a potassium ferrocyanide method to obtain a total sugar content matrix;
in the step (3), each royal jelly sample is scanned by using a mid-infrared spectrometer to obtain a mid-infrared attenuated total reflection spectrum matrix of the royal jelly sample.
3. The method for rapidly determining the water-soluble protein and total sugar of royal jelly according to claim 1, characterized in that: the dosage of each royal jelly sample in the step (2) and the step (3) is 0.03-0.1 g; the scanning time in the step (3) is 25-40 s.
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