CN114965838A - Construction method and identification method of identification model of malt processed product - Google Patents
Construction method and identification method of identification model of malt processed product Download PDFInfo
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Abstract
The invention discloses a construction method and an identification method of an identification model of a malt processed product, wherein the identification method of the malt processed product adopts a high performance liquid chromatography to obtain a liquid chromatogram of an object to be detected; the liquid phase map comprises at least 2 significant difference components and 1 internal standard component, the relative peak area of the significant difference components compared with the internal standard component is obtained, the relative peak area of each significant difference component obtained by detecting the object to be detected is compared with an identification model, and the judgment of the type of the processed malt product is realized. The method can quickly and accurately distinguish different processed malt products and the types of the aqueous extracts thereof, and is very simple and convenient to operate.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine identification, in particular to a construction method and an identification method of an identification model of a malt processed product.
Background
The fructus Hordei Germinatus is processed product of mature fruit of Hordeum vulgare L. of Gramineae plant by germinating and drying. Soaking wheat grains in water, keeping at proper temperature and humidity, and drying in the sun or at low temperature until young buds grow to about 5 mm. Is a common clinical digestant, has sweet and mild nature and taste, and has the effects of invigorating qi, promoting digestion, strengthening spleen, stimulating appetite, promoting lactation and relieving swelling. According to different processing methods, the raw malt, the roasted malt and the scorched malt are divided. The processed products have different efficacies, and the raw malt has the effects of strengthening the spleen and stomach, soothing the liver and promoting the circulation of qi, and is used for treating spleen deficiency, anorexia and milk stasis; stir-baked wheat germ is indicated for indigestion and milk loss of women, while scorched wheat germ is more apt to digest and resolve stagnation.
In the literatures of ' malt alkaloid chemical substance and pharmacodynamic research based on UPLC-Q-TOF-MS/MS and network pharmacology, Hubei university of traditional Chinese medicine 2021(09) ' and ceramic break ', the UPLC-MS/MS means is used for identifying chemical components in different processed products of malt, 33 components are identified in total, and the phenomena that the produced malt and the roasted malt are mainly alkaloid and the scorched malt is more reduced and is mainly organic acid are found; and a method for measuring the contents of N-methyltyramine, barley malt alkali and arundoin in different processed products of the malt is established, and the content changes of the N-methyltyramine, the barley malt alkali and the arundoin are clarified. Meanwhile, other documents disclosed in the prior art also disclose a malt HPLC fingerprint spectrum, 19 common peaks are determined, and component changes in different processed products are introduced.
However, the change of the components disclosed in the prior art is judged by the content of the corresponding characteristic components, and the content detection is required in the judging process, so that the operation is complex; in addition, the content of each original component in the raw materials is different, and the determination of the type of the processed product by only the content of the characteristic component is not accurate, so the prior art still has the problem that different malt processed products cannot be effectively and quickly distinguished.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the problem that the prior art does not disclose that different malt processed products can be effectively and rapidly distinguished; therefore, the identification model and the construction method thereof which can effectively and quickly distinguish different types of the malt processed products and the method for identifying the types of the malt processed products by using the constructed identification model are provided.
The model construction method for identifying the malt processed product comprises the following steps:
performing high performance liquid chromatography detection on the malt processed products of known types to obtain chromatograms of the malt processed products;
screening at least 10 common peaks with larger separation degree and peak area in the chromatogram of all the malt processed products as characteristic peaks;
normalizing the peak areas of the characteristic peaks, comparing the aggregation distribution conditions of the characteristic peaks of various malt processed products by adopting a principal component analysis method, and respectively performing pairwise analysis on all the malt processed product types by utilizing orthogonal partial least squares discriminant analysis to obtain at least 2 significant difference components and 1 internal standard component;
and obtaining the relative peak area of the significant difference component in the chromatogram compared with the internal standard component, and dividing the ratio range of the significant difference component in various processed malt products according to the ratio of the relative peak areas of the significant difference component, wherein the ratio range is the identification model of the processed malt product types.
The principal component analysis method and the orthogonal partial least square discriminant analysis can be realized by adopting SIMCA statistical analysis software or SPSS statistical analysis software;
when the significant difference component and the internal standard component are partitioned by adopting SIMCA statistical analysis software, the significant difference component is a component with VIP more than or equal to 1.5, and the internal standard component is a component with VIP less than 1.5.
The processed malt products comprise raw malt, roasted malt and scorched malt.
The number of the characteristic peaks is more than or equal to 15.
The identification model of the malt processed product is constructed by adopting the model construction method for identifying the malt processed product.
An identification model of a malt processed product comprises 2 significant difference components and 1 internal standard component, wherein the 2 significant difference components are respectively 5-hydroxymethylfurfural and ferulic acid, and the internal standard component is tricin; the relative peak area ranges obtained for significant difference components compared to the internal standard component are:
charred malt: the relative peak area of the peak of the 5-hydroxymethylfurfural is more than or equal to 1.20;
raw malt: the relative peak area of the peak of the 5-hydroxymethylfurfural is less than 1.20, and the relative peak area of the peak of the ferulic acid is more than 0.93;
and (3) malt frying: the relative peak area of the peak of the 5-hydroxymethylfurfural is less than 1.20, and the relative peak area of the peak of the ferulic acid is less than or equal to 0.93.
The significant difference component comprises at least another 8 components; the characteristic peak of the medicagine is peak 8, the characteristic peak of the 5-hydroxymethylfurfural is peak 2, and the characteristic peak of the ferulic acid is peak 11; the characteristic peaks corresponding to the other 8 components are named as peak 3, peak 4, peak 6, peak 7, peak 9, peak 10, peak 13 and peak 14 in sequence according to the peak emergence time; the relative retention time of each characteristic peak is ± 10% of a predetermined value, and the predetermined value includes: peak 2 of 0.32, peak 3 of 0.40, peak 4 of 0.66, peak 6 of 0.75, peak 7 of 0.80, peak 8 of 1.00, peak 9 of 1.08, peak 10 of 1.09, peak 11 of 1.12, peak 13 of 1.42, peak 14 of 1.46;
the relative peak area ranges obtained for the significant difference components compared to the internal standard components are:
raw malt: peak 2 is less than 1.20, peak 3 is less than 0.70, peak 4 is greater than 1.45, peak 6 is less than or equal to 0.74, peak 7 is less than or equal to 1.95, peak 9 is greater than 0.37, peak 10 is greater than 0.90, peak 11 is greater than 0.93, peak 13 is greater than 0.30, and peak 14 is less than or equal to 0.28;
and (3) malt frying: peak 2 is less than 1.20, peak 3 is more than or equal to 0.70 and less than or equal to 1.50, peak 6 is more than 0.74, peak 7 is more than 1.95, peak 9 is more than or equal to 0.21 and less than or equal to 0.37, peak 10 is more than 0.90, peak 11 is more than or equal to 0.93, peak 13 is more than or equal to 0.30, and peak 14 is more than 0.28;
charred malt: peak 2 is greater than or equal to 1.20, peak 3 is greater than 1.50, peak 4 is less than 1.45, peak 6 is greater than 0.74, peak 7 is greater than 1.95, peak 9 is less than 0.21, peak 10 is less than or equal to 0.90, peak 13 is less than or equal to 0.30, and peak 14 is less than or equal to 0.28.
A method for identifying processed malt product comprises obtaining liquid chromatogram of the substance to be detected by high performance liquid chromatography; and obtaining the relative peak area of the significant difference component compared with the internal standard component, and comparing the relative peak area of each significant difference component obtained by detecting the object to be detected with the identification model constructed by the model construction method for identifying the malt processed product or the identification model of the malt processed product to judge the type of the malt processed product.
And when the number of the significant difference components is more than or equal to 10, judging the number of the relative peak areas of the characteristic peaks of the significant difference components in the object to be detected falling into the corresponding characteristic peak value range in the identification model, and if the number of the characteristic peaks falling into the value range is more than or equal to 80% of the total number of the significant difference components, judging that the object to be detected belongs to the corresponding malt processed product type in the identification model.
The object to be detected is a processed malt product or an aqueous extract of the processed malt product; the water extract of the processed malt product is water extract, concentrated solution, drying solution or formula granules.
The chromatographic conditions of the high performance liquid chromatography are as follows:
a chromatographic column: chromatographic column with HSS T3 bonded silica gel as filler; the detection wavelength is 305-315 nm; taking acetonitrile as a mobile phase A, taking 0.08-0.12% formic acid water solution as a mobile phase B, and eluting according to the following gradient elution program, wherein:
the column temperature of a chromatographic column in the high performance liquid chromatography is 28-32 ℃; the chromatographic column has specification of 2.1 × 150mm, 1.8 μm; the flow rate is 0.285-0.315 ml/min, and the number of theoretical plates is not less than 5000 calculated according to the tricin peak.
The preparation process of the test solution used in the high performance liquid chromatography comprises the following steps: and (3) adding a solvent into the substance to be detected, and filtering after treatment to obtain a filtrate, wherein the filtrate is the test solution.
In the preparation process of the test solution, the treatment is ultrasonic treatment; the solvent is methanol water solution with the volume fraction of 40-60%.
The ultrasonic treatment time is 20-60 minutes.
The technical scheme of the invention has the following advantages:
1. according to the model construction method for identifying the malt processed product and the formula particles thereof, the significant difference components of different processed products can be effectively determined, the identification model for judging the types of the malt processed products can be obtained by combining the design of the ratio range of the relative peak areas of the significant difference components, and the types of different processed products can be effectively and quickly distinguished by comparing the ratio range of the characteristic peak of the significant difference component in the object to be detected, which is compared with the relative peak area of the S peak, with the identification model, so that the operation is simple and convenient.
2. In the method for identifying the processed malt products, the significant difference components in the model for identifying the types of the processed malt products at least comprise 5-hydroxymethylfurfural and ferulic acid, the internal standard component is tricin, and the types of different processed malt products can be effectively and quickly distinguished only by detecting the ratio result of the relative peak area of the characteristic peak of the significant difference component of the object to be detected compared with the characteristic peak of the tricin and comparing the ratio result with the range of the relative peak area of the characteristic peak corresponding to each significant difference component in the identification model.
3. In the detection process, the relative peak areas of characteristic peaks of individual characteristic peaks are small or the process and other reasons possibly cause that the relative peak areas of the characteristic peaks of the significant difference components in the processed product have discrete values, so that the result is easy to be inaccurate, therefore, the identification method in the invention provides a range value of the relative peak areas of the characteristic peaks of at least 10 significant difference components in the identification model, the type of the processed malt product to which the object to be detected belongs can be determined as long as more than 80% of the characteristic peaks conform to a ratio, and the judgment result is more accurate and reliable.
4. The identification method can also be used for judging the end point of the processing technology, so that the quality of processed products is more stable; meanwhile, the quantitative characterization and calculation of the object to be detected are not needed; the data does not need to be imported into software for secondary analysis, and time and labor are saved.
5. The invention further optimizes the high performance liquid chromatography condition in the identification method, can obtain at least 10 characteristic peaks with obviously changed component contents in the processing process, and effectively provides effective premise for accurately identifying the types of the processed malt products.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a liquid phase map of three malt preparations of example 1 of the present invention.
Detailed Description
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The instrument comprises the following steps: waters ultra-high performance liquid chromatograph (PDA ultraviolet detector), electronic balance (ML204T type, mettler toledo instruments ltd), electronic balance (JJ500 type, double jie test instruments factory, orthodox), ultrasonic cleaner (KQ-100DE type, ultrasonic instruments ltd, kunshan), water bath constant temperature oscillator (THZ-82 type, jintan jingda instruments ltd), chromatographic column: waters ACQUITY
HSS T3(2.1*150mm，1.8μm)。
Reagent testing: acetonitrile (Merck) is chromatographically pure; phosphoric acid (Thermo) is chromatographically pure; the water is distilled water (drochen); other reagents were analytically pure.
Example 1
The model construction method for identifying the malt processed product comprises the following specific processes:
1. taking 14 batches of samples (raw malt particles, roasted malt particles and scorched malt particles) of formula particles of different processed products, grinding, preparing a test sample solution according to the following method, detecting, and obtaining a liquid phase spectrum.
Wherein, the batch numbers of the raw malt particles are respectively as follows: 20003572, 20003582, 20003592, 20003602;
the batch numbers of the roasted malt particles are respectively: 19019753, 19017301, 20042891, 20006521, 20006511;
the batch numbers of the scorched malt particles are respectively: 20022481, 20022491, 20005381, 20005391, 20005401.
(1) Preparation of control solutions: weighing tricin, 5-hydroxymethyl furfural and ferulic acid reference substance, adding 50% methanol to dissolve, and making into solutions containing 5 μ g, 10 μ g and 1 μ g per 1mL respectively;
(2) preparation of a test solution: taking a proper amount of the product, grinding, precisely weighing about 1.0g, placing in a conical flask, precisely adding 25ml of 50% methanol, sealing, weighing, carrying out ultrasonic treatment (power 500W and frequency 40kHz) for 20 minutes, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, taking 15ml of subsequent filtrate, evaporating to dryness, dissolving in a 5ml volumetric flask with 50% methanol, fixing the volume, shaking up, and filtering to obtain the product as a test sample.
(3) Chromatographic conditions
A chromatographic column: HSS T3 bonded silica gel is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the granularity is 1.8 mu m;
column temperature: 30 ℃;
mobile phase: acetonitrile is taken as a mobile phase A, and 0.1% formic acid aqueous solution is taken as a mobile phase B;
flow rate of mobile phase: 0.3 mL/min;
sample introduction amount: 3 mu L of the solution;
detection wavelength: 310 nm;
the number of theoretical plates is not less than 5000 according to the calculation of the medicaginine peak;
the mobile phase was subjected to gradient elution according to table 1 below, specifically:
TABLE 1
The liquid phase profiles of the 14 batches were obtained following the elution procedure described in table 1 above.
2. Processing of liquid phase map data
(1) Obtaining a common peak:
using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012.1 version), taking the liquid phase spectra of the first batch of S1 as reference spectra, analyzing and comparing the liquid phase spectra of the three processed product particles, and finally selecting 15 peaks with larger peak area and better separation effect from the common peaks as characteristic peaks, wherein the 15 characteristic peaks are sequentially named as peak 1-peak 15 in the embodiment, and chromatograms of different processed products are shown in fig. 1.
(2) And acquiring significant difference components and internal standard components in the characteristic peaks.
Software capable of distinguishing the internal standard component from the component with the significant difference in the present invention includes, but is not limited to, SIMCA statistical analysis software or SPSS statistical analysis software, and in this embodiment, the significant difference component and the internal standard component of the 14 batches of samples are obtained by using the SIMCA statistical analysis software as an example.
Specifically, 15 characteristic peak areas of 14 batches of samples (raw malt particles, roasted malt particles and scorched malt particles) are subjected to normalization processing, SIMCA-P14.1 software is adopted for pattern recognition, a principal component analysis method of a PCA module is utilized for comparing the aggregation distribution conditions of the raw malt, the roasted malt and the scorched malt, two-two analysis is respectively carried out by utilizing orthogonal partial least squares discriminant analysis (OPLS-DA), difference Variables (VIP) are screened by the OPLS-DA, significant difference components corresponding to the characteristic peaks are screened by taking VIP >1.5 as a standard, and the screened significant difference components can be selected as quality markers for processing product identification.
The analysis results of every two processed products show that the continuous change of different component contents from raw malt to scorched malt in the processing process can be seen through the ratio, some peaks are sensitive to heat and change obviously in the stage of frying, so that the method can be used for distinguishing raw products from processed products, and some peaks are insensitive, can be decomposed and converted after being heated for a long time, and can be used for distinguishing raw malt, scorched malt and scorched malt granules. From the screening results, it was found that the peaks 3, 6, 7, 9, 11, 13 and 14 can distinguish between malt and roasted malt granules, the peaks 2, 3, 4, 6, 7, 9, 10 and 13 can distinguish between malt and roasted malt granules, and the peaks 2, 3, 9, 10 and 14 can distinguish between roasted malt and roasted malt granules. Peak 3 and Peak 9 can distinguish raw product from processed product (parched malt and scorched malt).
Meanwhile, differential Variables (VIP) are screened through OPLS-DA, one component of which VIP IS less than 1.5 IS selected as an Internal Standard (IS), the internal standard has small content change in the processing process, the separation effect IS good, and the peak area IS moderate. The Internal Standard (IS) selected in this example was the medicacin of peak 8.
(3) The range of relative peak areas of the significant difference component compared to the internal standard component in each type of malt preparation was determined.
Taking the peak areas of the significant difference components selected in the step (2) and the internal standard as a ratio to obtain relative peak areas, and counting the ratio ranges of the relative peak areas of the different significant difference components of different processed products, wherein the ratio ranges of the peak areas of the significant difference components of the 14 batches of different processed products are shown in the following table 2:
TABLE 2
Through the ratio range of the peak areas of the significant difference components, the critical value for identifying different processed products can be determined, and the result of determining the critical value in this embodiment is shown in table 3 below:
TABLE 3
Note: indication of extent not applicable
In the present invention, the relative peak area ranges of at least two significant difference components capable of distinguishing three kinds of processed malt products can be directly screened out from table 3, and the relative peak area ranges can be used as a model for distinguishing the kinds of processed malt products, for example, 5-hydroxymethylfurfural at peak 2 and ferulic acid at peak 11 are screened out, and the kinds of processed malt products can be distinguished through the relative peak area ranges of the two significant difference components. The identification model consisting of these two significant difference components is:
scorched malt: the relative peak area of the peak of the 5-hydroxymethylfurfural is more than or equal to 1.2;
raw malt: the relative peak area of the peak of the 5-hydroxymethylfurfural is less than 1.2, and the relative peak area of the peak of the ferulic acid is more than 0.93;
and (3) malt frying: the relative peak area of the peak of the 5-hydroxymethylfurfural is less than 1.2, and the relative peak area of the peak of the ferulic acid is less than or equal to 0.93.
Furthermore, because the characteristic peaks in the processed products have discrete values due to smaller peak areas of individual characteristic peaks or other reasons such as the process, in order to obtain higher identification accuracy, the ratio ranges in table 3 are all included in the identification model of the types of the processed products of malt, and the ratio conforming range of more than 80% (i.e. more than 8) characteristic peaks specified in the embodiment can be used for determining the types of the samples, so that the determination result is more accurate.
Example 2
The discrimination method of the malt processed product adopts the discrimination model obtained in the embodiment 1 for judgment, and the specific process is as follows:
3 batches of each of the formulation granules of a known kind of malt processed product were obtained, and the batch number of the formulation granules of each malt processed product is shown in table 4 below.
TABLE 4
The same high performance liquid chromatography detection method as that of example 1 was used to perform detection to obtain a liquid chromatogram, and the peak area of 10 characteristic peaks of peaks 2, 3, 4, 6, 7, 9, 10, 11, 13, and 14 was calculated to obtain the ratio to the internal standard, and the results are shown in table 5 below.
TABLE 5 relative peak area of each significant difference component for three malt preparation formulations
From the results obtained in table 5 above, it can be seen that the relative peak area ranges of the significant difference components therein all match the identification model shown in table 3. Therefore, the identification model can be effectively used for identifying the raw malt, roasted malt and scorched malt formula particles, and the identification result has high accuracy.
Example 3
This example differs from example 1 in the preparation conditions of the test solution, specifically:
1. solvent volume investigation of test solution
Respectively taking raw malt formula granules (batch number: 20003572), weighing 1g in parallel, precisely weighing, placing in a conical flask with a plug, precisely adding 15ml, 25ml and 50ml of 50% methanol, weighing, performing ultrasonic extraction (power 500W and frequency 40kHz), taking out, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking up, filtering, precisely weighing a certain volume of subsequent filtrate (60% of the added volume), evaporating to dryness, dissolving in a 5ml volumetric flask with 50% methanol, fixing the volume, shaking up, and filtering to obtain the product. And (4) calculating the relative retention time and the relative peak area of the No. 1-15 characteristic peak by taking the No. 8 peak as a reference peak S, and calculating the RSD.
The relative retention times and relative peak areas of the characteristic peaks of the different volume extraction samples are shown in tables 6 and 7.
TABLE 6 examination of extraction solvent volume relative retention time table
TABLE 7 table of relative peak areas for volume examination of extraction solvents
The results show that when the volumes of the extraction solvent are 15ml, 25ml and 50ml, the relative peak areas of the characteristic peaks are greatly influenced, but the RSD is less than 10%, and the judgment requirement is met. The volume of the extraction solvent was determined to be 25ml, taking into account the relative peak area.
2. Ultrasonic time inspection of test solution
Respectively taking raw malt formula particles (batch number: 20003582), weighing 1g in parallel, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 50% methanol, weighing, carrying out ultrasonic extraction (power 500W, frequency 40kHz) for 20 min, 40 min and 60 min, taking out, cooling, weighing again, complementing the loss weight with 50% methanol, shaking up, filtering, precisely weighing 15ml of subsequent filtrate, evaporating to dryness, dissolving in a 5ml volumetric flask with 50% methanol, fixing the volume, shaking up, and filtering to obtain the product to be tested. And (4) calculating the relative retention time and the relative peak area of the No. 1-15 characteristic peak by taking the No. 8 peak as a reference peak S, and calculating the RSD. The results of the relative retention time and the relative peak area are shown in tables 8 and 9.
TABLE 8 relative Retention time Table for different ultrasound time surveys
TABLE 9 relative peak area table for different ultrasonic time investigation
The results show that the RSD of the relative peak areas of the characteristic peaks is less than 10% and the relative retention time is less than 0.5% when the ultrasonic time is 20 minutes, 40 minutes and 60 minutes, so that the extraction time can be 20-60 minutes.
Example 4
This example was used to methodological validate the high performance liquid chromatography conditions of example 1, including:
1. repeatability verification
Taking 6 parts of raw malt granules (batch number: 20003592), determining according to the method of example 1 to obtain a characteristic map thereof, and calculating the relative retention time and the relative peak area of No. 1-15 characteristic peaks by taking the No. 8 peak as a reference peak S; and calculates the RSD. The results are shown in tables 10 and 11.
TABLE 10 relative retention time Table for feature map repeatability tests
TABLE 11 relative peak area table for feature map repeatability survey
According to the repeatability investigation result, the relative retention time RSD of each characteristic peak is in a range of less than 0.2%, and the relative peak areas RSD of other characteristic peaks are in a range of 0.00% -6.13%, which shows that the characteristic peak of the characteristic spectrum has good repeatability.
2. Precision verification
Taking raw malt particles (batch number: 20003592), preparing 6 samples at different times by different analysts, carrying out intermediate precision test, determining according to a text method to obtain a characteristic spectrum, and calculating the relative retention time and the relative peak area of each characteristic peak by taking the No. 8 peak as a reference peak S; results RSD values were calculated from the results of the repeatability tests as shown in table 12.
TABLE 12 relative Retention Peak area Table for intermediate precision examination
From the above results, it can be seen that: the relative peak area RSD of each characteristic peak of samples prepared by different personnel and different time is in the range of 0.00-6.03%, which shows that the intermediate precision of the analysis method is good.
3. Stability verification
Taking raw malt particles (batch number: 20003592), preparing a test solution according to a text method, respectively measuring for 0, 3, 6, 9, 12 and 24 hours according to the text method to obtain a characteristic spectrum of the test solution, and calculating the relative retention time and the relative peak area of each characteristic peak by taking a peak No. 8 as a reference peak S; and calculates the RSD. The results are shown in tables 13 and 14.
TABLE 13 stability relative Retention time Table
TABLE 14 table of relative peak areas for stability
From the above stability test results, it was found that 15 characteristic peaks in the sample solution both had an internal relative retention time and a relative peak area of less than 3%, and the sample was considered to be stable within 24 hours at room temperature.
Example 5
A method for identifying a malt processed product adopts the identification model obtained in the example 1 for judgment, and comprises the following specific steps:
the known types of malt processed products and their formulation granules were obtained in 3 batches, and the batch numbers of the malt processed products and their formulation granules were the same as those shown in table 4 in example 2.
Chromatographic conditions of chromatographic column: HSS T3 bonded silica gel is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the granularity is 1.8 mu m; column temperature: 28 ℃; mobile phase: acetonitrile is taken as a mobile phase A, and 0.12% formic acid aqueous solution is taken as a mobile phase B; flow rate of mobile phase: 0.315 mL/min; sample introduction amount: 3 mu L of the solution; detection wavelength: 305 nm;
a liquid chromatogram was obtained by the same mobile phase gradient detection as in example 1, and the peak area of 10 characteristic peaks of peaks 2, 3, 4, 6, 7, 9, 10, 11, 13, and 14 was calculated as a ratio to the internal standard, and the results are shown in table 15 below.
Table 15: relative peak area of each significant difference component of three malt processed product formula particles
Note: indication of range not applicable, no relative peak area calculation
The results obtained from the above table effectively indicate that: the identification method of the invention has higher accuracy and can be used for identifying the raw malt, the roasted malt and the scorched malt formula granules.
Example 6
A method for identifying a malt processed product adopts the identification model obtained in the example 1 for judgment, and comprises the following specific steps:
the known types of malt processed products and their formulation granules were obtained in 3 batches, and the batch numbers of the malt processed products and their formulation granules were the same as those shown in table 4 in example 2.
Chromatographic conditions of chromatographic column: HSS T3 bonded silica gel is used as a filler, the column length is 150mm, the inner diameter is 2.1mm, and the granularity is 1.8 mu m; column temperature: at 32 ℃; mobile phase: acetonitrile is taken as a mobile phase A, and 0.08% formic acid aqueous solution is taken as a mobile phase B; flow rate of mobile phase: 0.285 mL/min; sample injection amount: 3 mu L of the solution; detection wavelength: 315 nm;
a liquid chromatogram was obtained by the same mobile phase gradient detection as in example 1, and the peak area of 10 characteristic peaks of peaks 2, 3, 4, 6, 7, 9, 10, 11, 13, and 14 was calculated as a ratio to the internal standard, and the results are shown in table 16 below.
TABLE 16 relative peak area of each significant difference component for three malt processed product formula granules
The results obtained from the above table effectively indicate that: the identification method of the invention has higher accuracy and can be used for identifying the raw malt, the roasted malt and the scorched malt formula granules.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. The model construction method for identifying the malt processed product is characterized by comprising the following steps:
performing high performance liquid chromatography detection on the malt processed products of known types to obtain chromatograms of the malt processed products;
screening at least 10 common peaks with larger separation degree and peak area in the chromatogram of all the malt processed products as characteristic peaks;
normalizing the peak areas of the characteristic peaks, comparing the aggregation distribution conditions of the characteristic peaks of various malt processed products by adopting a principal component analysis method, and respectively carrying out pairwise analysis on all the malt processed product types by utilizing orthogonal partial least squares discriminant analysis to obtain at least 2 significant difference components and an internal standard component;
and obtaining the relative peak area of the significant difference component in the chromatogram compared with the internal standard component, and dividing the ratio range of the significant difference component in various processed malt products according to the ratio of the relative peak areas of the significant difference component, wherein the ratio range is the identification model of the processed malt product types.
2. The model building method of claim 1, wherein the principal component analysis method and the orthometric partial least squares discriminant analysis are implemented by using SIMCA or SPSS statistical analysis software;
when the significant difference component and the internal standard component are partitioned by adopting SIMCA statistical analysis software, the significant difference component is a component with VIP more than or equal to 1.5, and the internal standard component is a component with VIP less than 1.5.
3. The model construction method according to claim 1 or 2, wherein the kind of malt processed product includes raw malt, roasted malt, scorched malt;
the number of the characteristic peaks is more than or equal to 15.
4. An identification model for a malt processed product, which is constructed by the method for constructing a model for identifying a malt processed product according to any one of claims 1 to 3.
5. An identification model of a malt processed product is characterized by comprising 2 significant difference components and 1 internal standard component, wherein the 2 significant difference components are respectively 5-hydroxymethylfurfural and ferulic acid, and the internal standard component is tricin; the relative peak area ranges obtained for significant difference components compared to the internal standard component are:
scorched malt: the relative peak area of the peak of the 5-hydroxymethylfurfural is more than or equal to 1.20;
raw malt: the relative peak area of the peak of the 5-hydroxymethylfurfural is less than 1.20, and the relative peak area of the peak of the ferulic acid is more than 0.93;
and (3) malt frying: the relative peak area of the peak of the 5-hydroxymethylfurfural is less than 1.20, and the relative peak area of the peak of the ferulic acid is less than or equal to 0.93.
6. The discriminatory model of claim 5 wherein the significant difference component comprises at least an additional 8 components; the characteristic peak of the medicagine is peak 8, the characteristic peak of the 5-hydroxymethylfurfural is peak 2, and the characteristic peak of the ferulic acid is peak 11; the characteristic peaks corresponding to the other 8 components are named as peak 3, peak 4, peak 6, peak 7, peak 9, peak 10, peak 13 and peak 14 in sequence according to the peak emergence time; the relative retention time of each characteristic peak is ± 10% of a predetermined value, and the predetermined value includes: peak 2 of 0.32, peak 3 of 0.40, peak 4 of 0.66, peak 6 of 0.75, peak 7 of 0.80, peak 8 of 1.00, peak 9 of 1.08, peak 10 of 1.09, peak 11 of 1.12, peak 13 of 1.42, peak 14 of 1.46;
the relative peak area ranges obtained for significant difference components compared to the internal standard component are:
raw malt: peak 2 is less than 1.20, peak 3 is less than 0.70, peak 4 is greater than 1.45, peak 6 is less than or equal to 0.74, peak 7 is less than or equal to 1.95, peak 9 is greater than 0.37, peak 10 is greater than 0.90, peak 11 is greater than 0.93, peak 13 is greater than 0.30, and peak 14 is less than or equal to 0.28;
and (3) malt frying: peak 2 is less than 1.20, peak 3 is more than or equal to 0.70 and less than or equal to 1.50, peak 6 is more than 0.74, peak 7 is more than 1.95, peak 9 is more than or equal to 0.21 and less than or equal to 0.37, peak 10 is more than 0.90, peak 11 is more than or equal to 0.93, peak 13 is more than or equal to 0.30, and peak 14 is more than 0.28;
charred malt: peak 2 is greater than or equal to 1.20, peak 3 is greater than 1.50, peak 4 is less than 1.45, peak 6 is greater than 0.74, peak 7 is greater than 1.95, peak 9 is less than 0.21, peak 10 is less than or equal to 0.90, peak 13 is less than or equal to 0.30, and peak 14 is less than or equal to 0.28.
7. A method for identifying a malt processed product is characterized in that a high performance liquid chromatography is adopted to obtain a liquid chromatogram of a substance to be detected; and obtaining the relative peak area of the significant difference component compared with the internal standard component, and comparing the relative peak area of each significant difference component obtained by detecting the object to be detected with the identification model constructed by the model construction method for identifying the processed malt product according to any one of claims 1 to 3 or the identification model for identifying the processed malt product according to any one of claims 4 to 6, so as to realize the judgment of the type of the processed malt product.
8. The identification method according to claim 7, wherein when the number of the significant difference components is greater than or equal to 10, the number that the relative peak area of the characteristic peak of each significant difference component in the object to be detected falls within the numerical range of the corresponding characteristic peak in the identification model is determined, and if the number of the characteristic peaks falling within the numerical range is greater than or equal to 80% of the total number of the significant difference components, the object to be detected is determined to belong to the corresponding malt processed product type in the identification model.
9. The identification method according to claim 7 or 8, wherein the test substance is a malt processed product or an aqueous extract of a malt processed product; the water extract of the processed malt product is water extract, concentrated solution, drying solution or formula granules.
10. The identification method according to any one of claims 7 to 9, wherein the chromatographic conditions of the high performance liquid chromatography are:
a chromatographic column: chromatographic column with HSS T3 bonded silica gel as filler; the detection wavelength is 305-315 nm; taking acetonitrile as a mobile phase A, taking 0.08-0.12% formic acid water solution as a mobile phase B, and eluting according to the following gradient elution program, wherein:
preferably, the column temperature of a chromatographic column in the high performance liquid chromatography is 28-32 ℃; the specification of the column was 2.1 x 150mm, 1.8 μm; the flow rate is 0.285-0.315 ml/min, and the number of theoretical plates is not less than 5000 calculated according to the alfalfa peak;
preferably, the preparation process of the sample solution used in the high performance liquid chromatography comprises the following steps: adding a solvent into a substance to be detected, processing and filtering to obtain a filtrate, wherein the filtrate is a test solution; more preferably, in the preparation process of the test solution, the treatment is ultrasonic treatment; the solvent is methanol water solution with the volume fraction of 40-60%;
preferably, the sonication time is 20 to 60 minutes.
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