CN106770019A - A kind of assay method of Itanlian rye soluble sugar content - Google Patents
A kind of assay method of Itanlian rye soluble sugar content Download PDFInfo
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- CN106770019A CN106770019A CN201710115673.5A CN201710115673A CN106770019A CN 106770019 A CN106770019 A CN 106770019A CN 201710115673 A CN201710115673 A CN 201710115673A CN 106770019 A CN106770019 A CN 106770019A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
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Abstract
The invention discloses a kind of assay method of the Itanlian rye soluble sugar content that can quickly determine Itanlian rye soluble sugar content.The assay method provides a kind of forecast model of soluble sugar real content in Itanlian rye based on near-infrared spectrum technique, on the basis of known a large amount of sample real contents, gather the near-infrared original spectrum of sample, set up the Quantitative Analysis Predictive Model of the Itanlian rye soluble sugar content based on near-infrared spectrum technique and chemometrics method, then, it is only necessary to by Itanlian rye sample to be measured by obtaining Itanlian rye sample powder to be measured after pretreatment；And gather its near-infrared primary light spectrogram, spectra collection process time is short, and detection is can be carried out after near infrared spectrum is gathered, with it is simple to operate, detection is rapid, detection efficiency is high, accuracy of detection is high the features such as.It is adapted to evaluate field popularization and application in Forage Nutrition Quality.
Description
Technical field
Field is evaluated the present invention relates to Forage Nutrition Quality, and in particular to a kind of survey of Itanlian rye soluble sugar content
Determine method.
Background technology
Itanlian rye (Lolium multiflorum Lam.) is that grass family Lolium is annual or more year life is herded
Grass, is suitable to be grown in one of warmer climate humid region, good forage of Shi Ju worlds cultivation meaning, saves the south China more
(city) has the cultivation of larger area.In recent years, with the adjustment of the National agricultural industrial structure, herbivorous stock raising is fast-developing,
Itanlian rye is because the advantages of its yield is high, the vigorous, strong adaptability of winter-spring season growth, nutritive value enrich, solving China south
The not enough problem of square winter-spring season domestic animal forage grass, has played extremely important effect, especially in the cereal-forage rotation of south China area
It is that the excellent Varieties of Lolium that Herbage harvest is high, nutritional quality is good is particularly important to Animal husbandry production.Educated in Itanlian rye
Plant in work, yield kind high is not only cultivated, while also to take into account the quality of herbage.
Glucide is one of important composition composition of composition plant, is also the primary raw material and stock of metabolism
Matter.Soluble sugar (Water soluble carbohydrates, WSC) includes reduction monose and energy water in plant sample
Solution into the reduction sucrose of monose, maltose and can partial hydrolysis be the starch of glucose.The content of WSC is influence herbage in herbage
Nutritional quality and the key factor of ensiling, WSC contents are high, and herbage quality is good, modulate ensilage success rate and quality also compared with
Height, conversely, WSC contents are low, ensiling is then difficult successfully.Therefore research WSC contents in Itanlian rye dynamic change for
Forage grass production, breeding have great importance.It is domestic at present to determine main in conventional wet chemical method for herbage WSC ---
Based on anthrone Sulphuric acid colorimetry, but exist larger complex operation, error, high cost, time-consuming, poisonous and harmful chemicals pollution
The outstanding problems such as environment, it is difficult to the appropriate kind of WSC contents is filtered out from substantial amounts of Varieties of Lolium (being) or sample
(being) or sample, therefore, a kind of method that can quickly determine WSC contents in Itanlian rye is found out, Itanlian rye is educated
Plant and herbage quality control is significant.
The content of the invention
The technical problems to be solved by the invention are to provide one kind and can quickly determine Itanlian rye soluble sugar content
Itanlian rye soluble sugar content assay method.
The technical solution adopted for the present invention to solve the technical problems is：The measure of the Itanlian rye soluble sugar content
Method, comprises the following steps：
A, set up Itanlian rye soluble sugar content near-infrared forecast model；The Itanlian rye soluble sugar content
Near-infrared forecast model is adopted and set up with the following method, specifically includes following steps：
A, collection Itanlian rye sample, the Itanlian rye sample include different cultivars, different lines, different bearing
The fresh grass sample of phase, different planting and different parts, is respectively pre-processed the Itanlian rye sample of above-mentioned collection,
The method of the pretreatment is as described below：The Itanlian rye sample of collection is first finished 20min in 105 DEG C of environment, so
After being dried in 65 DEG C of environment afterwards, powder is ground into micropulverizer and 40 mesh sieves excessively obtain Itanlian rye sample powder
End；
B, the Itanlian rye sample powder for obtaining step a carry out respectively near infrared spectra collection obtain each spend more it is black
The near-infrared primary light spectrogram of wheat straw sample powder；
C, the near-infrared original spectrum of all Itanlian rye sample powders is analyzed using PCA algorithms, rejects tool
There is the Itanlian rye sample of similar spectral, remaining Itanlian rye sample is representative Itanlian rye sample；
D, the representative Itanlian rye sample obtained to step c using anthrone Sulphuric acid colorimetry carry out soluble sugar one by one
Content is measured and obtains each representative Itanlian rye sample solubility sugared content value；
Representative Itanlian rye sample is divided into school by the representative Itanlian rye sample solubility sugared content value of e, foundation
Positive collection and checking collection two parts, specific method are as follows：The representative Itanlian rye soluble sugar content value that will be obtained first from
It is small to being ranked up greatly, then taken every 31 as checking collect, remaining as calibration set, and adjust representativeness spend more it is black
The minimum value and maximum of soluble sugar content in wheat straw, it is calibration set to be allowed to incorporate into；
F, by the atlas of near infrared spectra of calibration set sample and the importing OPUS/ of the soluble sugar content value of calibration set sample
In QUENT5.5 commercialization quantitative spectrochemical analysis softwares, first, the near infrared spectrum to calibration set sample in full spectral limit is carried out
Pretreatment, then sets up prediction calibration model to calibration set sample using partial least-squares regression method combination validation-cross, according to
The parameter of near-infrared quantitative calibration models is evaluated prediction calibration model, the near-infrared evaluated prediction calibration model
Quantitative calibration models parameter includes coefficient of determination R2, validation-cross coefficient of determination R2Cv, validation-cross root-mean-square error RMSECV,
Its coefficient of determination R2It is 90.20%, validation-cross coefficient of determination R2Cv is 87.77%, and validation-cross root-mean-square error RMSECV is
3.11, optimal spectrum pretreatment is determined for min-max is normalized, best modeled spectral regions are 6101.9~5446.2cm-1With
4601.5~4246.7cm-1, optimum factor number is 7, residual according to mahalanobis distance, THE PRINCIPAL FACTOR ANALYSIS figure, spectrum in evaluation procedure
The Itanlian rye calibration set sample of difference figure and chemical analysis value residual plot result rejecting abnormalities, so as to obtain optimal spending more
The optimal near-infrared prediction calibration model of rye-grass soluble sugared content；Then, near infrared spectrum and the checking of collection sample will be verified
Checking is analyzed in the prediction calibration model of the soluble sugar content importing foundation for collecting sample obtain prediction checking model；Connect
, the prediction checking model of gained is evaluated using the parameter of near-infrared quantitative verification model, prediction checking model is entered
The parameter of the near-infrared quantitative verification model that row is evaluated includes external certificate coefficient of determination R2Ev and checking root-mean-square error
RMSEP, its external certificate coefficient of determination R2Ev is 91.28%, and checking root-mean-square error RMSEP is 2.56, in evaluation procedure
It is black according to spending more for mahalanobis distance, THE PRINCIPAL FACTOR ANALYSIS figure, spectrum residual plot and chemical analysis value residual plot result rejecting abnormalities
Wheat straw checking collection sample, so as to obtain optimal Itanlian rye soluble sugar content near-infrared forecast model；
B, by Itanlian rye sample to be measured according to described in step a preprocess method process after obtain it is to be measured spend more it is black
Wheat straw sample powder；
C, the Itanlian rye sample powder to be measured that step B is obtained is carried out near infrared spectra collection obtain it is to be measured spend more it is black
The near-infrared primary light spectrogram of wheat straw sample powder；
D, the Itanlian rye that will be obtained in the near-infrared primary light spectrogram and step f of Itanlian rye sample powder to be measured
Soluble sugar content near-infrared forecast model is imported into the commercial quantitative spectrochemical analysis softwares of OPUS/QUENT 5.5, by model
Operational analysis, you can obtain the content of soluble sugar in Itanlian rye sample to be measured.
It is further that breeding time of the Itanlian rye includes tillering stage, jointing stage, boot stage, heading stage, blooms
Phase, productive phase, maturity period.
Be further, the planting type of the Itanlian rye include broadcasting sowing planting type, culturing and transplanting seedlings planting type,
Applied nitrogen planting type.
It is further that the collection position of the Itanlian rye includes stem, leaf, complete stool.
It is further in stepb, Itanlian rye sample powder to be carried out closely respectively using method as described below
Infrared spectrum is gathered, specifically, taking appropriate Itanlian rye sample powder, is put into Bruker MPA type Fourier transformation NIRS instrument
In specimen cup, sample is shakeout naturally, set Instrument working parameter, it is closely red to gather sample under the conditions of being 25 ± 0.5 DEG C in temperature
External spectrum, obtains the first time near infrared light spectrum of the sample, then, specimen cup is placed into after the sample in specimen cup is taken out
In, sample is shakeout naturally, sample near infrared spectrum is gathered again, second near infrared light spectrum of the sample is obtained, then
First time near infrared light spectrum and second near infrared light spectrum are carried out averagely, obtaining the near-infrared of the Itanlian rye sample
Primary light spectrogram.
It is further that the Bruker MPA types Fourier transformation NIRS Instrument working parameters are set as：Spectral range
4000~12500cm-1, resolution ratio 8cm-1, scanning times 64 times.
It is further, using the commercial quantitative spectrochemical analysis softwares of OPUS/QUENT 5.5 to first time near infrared light spectrum
With the near-infrared primary light spectrogram that second near infrared light spectrum averagely obtain Itanlian rye sample.
It is further in step d, to be carried out one by one using the representative Itanlian rye sample of anthrone Sulphuric acid colorimetry
Soluble sugar content is measured and obtains each representative Itanlian rye sample solubility sugared content value；
It is further that in step f, the near infrared spectrum to calibration set sample in full spectral limit is led using single order
Number, second dervative, subtract straight line, first derivative+MSC (multiplicative scatter correction), first derivative+subtract straight line,
The normalization of order derivative+vector, without Pretreated spectra, min-max normalization, vector normalization, eliminate constant offset and
11 kinds of preprocessing procedures of multiplicative scatter correction.
The beneficial effects of the present invention are：The assay method of Itanlian rye soluble sugar content of the present invention is provided
The forecast model of soluble sugar real content in a kind of Itanlian rye based on near-infrared spectrum technique, in known a large amount of samples
On the basis of product real content, the near-infrared original spectrum of sample is gathered, set up and be based on near-infrared spectrum technique and stoichiometry
The Quantitative Analysis Predictive Model of the Itanlian rye soluble sugar content of method, then, it is only necessary to which to be measured is spent more into rye
Careless sample is by obtaining Itanlian rye sample powder to be measured after pretreatment；And its near-infrared primary light spectrogram is gathered, spectrum is adopted
Collection process time is short, and detection, in addition to early stage sets up forecast model, whole near-infrared are can be carried out after near infrared spectrum is gathered
Detection process only needs short a few minutes, with it is simple to operate, detection is rapid, detection efficiency is high the characteristics of, and the method is
On the basis of known a large amount of sample real contents, the near-infrared original spectrum of sample is gathered, set up and be based near infrared spectrum
The Quantitative Analysis Predictive Model of the Itanlian rye soluble sugar content of technology and chemometrics method, its accuracy of detection is very
Height, additionally, detection method of the invention need not add any organic reagent, will not damage the health of testing staff, more will not
The problems such as because causing environmental pollution using chemical reagent, more safety and environmental protection, have for Itanlian rye production and breeding work
There is particularly important meaning.
Brief description of the drawings
Fig. 1 is 404 parts of atlas of near infrared spectra of Itanlian rye sample described in embodiment；
Fig. 2 is 123 parts of atlas of near infrared spectra of representative Itanlian rye sample described in embodiment；
Fig. 3 be calibration set sample in embodiment near infrared spectrum optimal pretreatment mode min-max normalize into
Best modeled composes the atlas of near infrared spectra in area after row pretreatment；
Fig. 4 is cross validation root-mean-square error RMSECV, validation-cross coefficient of determination R in embodiment2 cvAs main composition is tieed up
Several variation diagrams；
Fig. 5 is to verify related between collection sample solubility sugared content chemical score and near infrared spectrum predicted value in embodiment
Property figure.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
The assay method of the Itanlian rye soluble sugar content, comprises the following steps：
A, set up Itanlian rye soluble sugar content near-infrared forecast model；The Itanlian rye soluble sugar content
Near-infrared forecast model is adopted and set up with the following method, specifically includes following steps：
A, collection Itanlian rye sample, the Itanlian rye sample include different cultivars, different lines, different bearing
The fresh grass sample of phase, different planting and different parts, is respectively pre-processed the Itanlian rye sample of above-mentioned collection,
The method of the pretreatment is as described below：The Itanlian rye sample of collection is first finished 20min in 105 DEG C of environment, so
After being dried in 65 DEG C of environment afterwards, powder is ground into micropulverizer and 40 mesh sieves excessively obtain Itanlian rye sample powder
End；
B, the Itanlian rye sample powder for obtaining step a carry out respectively near infrared spectra collection obtain each spend more it is black
The near-infrared primary light spectrogram of wheat straw sample powder；
C, the near-infrared original spectrum of all Itanlian rye sample powders is analyzed using PCA algorithms, rejects tool
There is the Itanlian rye sample of similar spectral, remaining Itanlian rye sample is representative Itanlian rye sample；
D, the representative Itanlian rye sample obtained to step c using anthrone Sulphuric acid colorimetry carry out soluble sugar one by one
Content is measured and obtains each representative Itanlian rye sample solubility sugared content value；
Representative Itanlian rye sample is divided into school by the representative Itanlian rye sample solubility sugared content value of e, foundation
Positive collection and checking collection two parts, specific method are as follows：The representative Itanlian rye soluble sugar content value that will be obtained first from
It is small to being ranked up greatly, then taken every 31 as checking collect, remaining as calibration set, and adjust representativeness spend more it is black
The minimum value and maximum of soluble sugar content in wheat straw, it is calibration set to be allowed to incorporate into；
F, by the atlas of near infrared spectra of calibration set sample and the importing OPUS/ of the soluble sugar content value of calibration set sample
In QUENT5.5 commercialization quantitative spectrochemical analysis softwares, first, the near infrared spectrum to calibration set sample in full spectral limit is carried out
Pretreatment, then sets up prediction calibration model to calibration set sample using partial least-squares regression method combination validation-cross, according to
The parameter of near-infrared quantitative calibration models is evaluated prediction calibration model, the near-infrared evaluated prediction calibration model
Quantitative calibration models parameter includes coefficient of determination R2, validation-cross coefficient of determination R2Cv, validation-cross root-mean-square error RMSECV,
Its coefficient of determination R2It is 90.20%, validation-cross coefficient of determination R2Cv is 87.77%, and validation-cross root-mean-square error RMSECV is
3.11, optimal spectrum pretreatment is determined for min-max is normalized, best modeled spectral regions are 6101.9~5446.2cm-1With
4601.5~4246.7cm-1, optimum factor number is 7, residual according to mahalanobis distance, THE PRINCIPAL FACTOR ANALYSIS figure, spectrum in evaluation procedure
The Itanlian rye calibration set sample of difference figure and chemical analysis value residual plot result rejecting abnormalities, so as to obtain optimal spending more
The optimal near-infrared prediction calibration model of rye-grass soluble sugared content；Then, near infrared spectrum and the checking of collection sample will be verified
Checking is analyzed in the prediction calibration model of the soluble sugar content importing foundation for collecting sample obtain prediction checking model；Connect
, the prediction checking model of gained is evaluated using the parameter of near-infrared quantitative verification model, prediction checking model is entered
The parameter of the near-infrared quantitative verification model that row is evaluated includes external certificate coefficient of determination R2Ev and checking root-mean-square error
RMSEP, its external certificate coefficient of determination R2Ev is 91.28%, and checking root-mean-square error RMSEP is 2.56, in evaluation procedure
It is black according to spending more for mahalanobis distance, THE PRINCIPAL FACTOR ANALYSIS figure, spectrum residual plot and chemical analysis value residual plot result rejecting abnormalities
Wheat straw checking collection sample, so as to obtain optimal Itanlian rye soluble sugar content near-infrared forecast model；
B, by Itanlian rye sample to be measured according to described in step a preprocess method process after obtain it is to be measured spend more it is black
Wheat straw sample powder；
C, the Itanlian rye sample powder to be measured that step B is obtained is carried out near infrared spectra collection obtain it is to be measured spend more it is black
The near-infrared primary light spectrogram of wheat straw sample powder；
D, the Itanlian rye that will be obtained in the near-infrared primary light spectrogram and step f of Itanlian rye sample powder to be measured
Soluble sugar content near-infrared forecast model is imported into the commercial quantitative spectrochemical analysis softwares of OPUS/QUENT 5.5, by model
Operational analysis, you can obtain the content of soluble sugar in Itanlian rye sample to be measured.
The assay method of Itanlian rye soluble sugar content of the present invention provides a kind of based near infrared spectrum
The forecast model of soluble sugar real content in the Itanlian rye of technology, on the basis of known a large amount of sample real contents,
The near-infrared original spectrum of sample is gathered, setting up the Itanlian rye based on near-infrared spectrum technique and chemometrics method can
The Quantitative Analysis Predictive Model of dissolubility sugared content, then, it is only necessary to by Itanlian rye sample to be measured by being obtained after pretreatment
To Itanlian rye sample powder to be measured；And its near-infrared primary light spectrogram is gathered, spectra collection process time is short, near in collection
Detection is can be carried out after infrared spectrum, in addition to early stage sets up forecast model, whole near infrared detection process only needs short several
Minute, with it is simple to operate, detection is rapid, detection efficiency is high the characteristics of, and the method is true in known a large amount of samples
On the basis of content, the near-infrared original spectrum of sample is gathered, set up and be based on near-infrared spectrum technique and chemometrics method
Itanlian rye soluble sugar content Quantitative Analysis Predictive Model, its accuracy of detection is very high, additionally, detection of the invention
Method need not add any organic reagent, will not damage the health of testing staff, more will not be because causing ring using chemical reagent
The problems such as border is polluted, more safety and environmental protection, have particularly important meaning for Itanlian rye production and breeding work.
The accuracy of detection of forecast model has direct relation with the species of collection sample, in order to further increase forecast model
Accuracy of detection, need to gather the Itanlian rye sample of different growing, breeding time of the Itanlian rye include tillering stage,
Jointing stage, boot stage, heading stage, florescence, productive phase, maturity period.
It is further that accuracy of detection and the species of collection sample of forecast model have direct relation, in order to further
Increase the accuracy of detection of forecast model, the Itanlian rye sample of different planting, the cultivation of the Itanlian rye need to be gathered
Training mode includes broadcasting sowing planting type, culturing and transplanting seedlings planting type, applied nitrogen planting type.
It is further that accuracy of detection and the species of collection sample of forecast model have direct relation, in order to further
Increase the accuracy of detection of forecast model, the Itanlian rye sample of different parts, the collection portion of the Itanlian rye need to be gathered
Position includes stem, leaf, complete stool.
In order that the near-infrared primary light spectrogram of the Itanlian rye sample for obtaining is more accurate true, in stepb, adopt
Near infrared spectra collection is carried out respectively to Itanlian rye sample powder with method as described below, specifically, take spending more in right amount
Rye grass sample powder, is put into MPA type Fourier transformation NIRS instrument specimen cups, sample is shakeout naturally, setting instrument work
Parameter, gathers sample near infrared spectrum, the first time near infrared light spectrum of the sample is obtained, then, by the sample in specimen cup
Placed into after taking-up in specimen cup, sample is shakeout naturally, sample near infrared spectrum is gathered again, obtain second of the sample
Near infrared light spectrum, then to first time near infrared light spectrum and second near infrared light spectrum averagely, obtain this and spend more
The near-infrared primary light spectrogram of rye grass sample.
It is further that the Bruker MPA types Fourier transformation NIRS Instrument working parameters are set as：Spectral range
4000~12000cm-1, resolution ratio 8cm-1, scanning times 64 times.
Operate for convenience, using the commercial quantitative spectrochemical analysis softwares of OPUS/QUENT 5.5 to first time near infrared spectrum
Value and second near infrared light spectrum averagely obtain the near-infrared primary light spectrogram of Itanlian rye sample.
It is further in step d, to be carried out one by one using the representative Itanlian rye sample of anthrone Sulphuric acid colorimetry
Soluble sugar content is measured and obtains each representative Itanlian rye sample solubility sugared content value；
It is further that in step f, the near infrared spectrum to calibration set sample in full spectral limit is led using single order
Number, second dervative, subtract straight line, first derivative+MSC (multiplicative scatter correction), first derivative+subtract straight line,
The normalization of order derivative+vector, without Pretreated spectra, min-max normalization, vector normalization, eliminate constant offset and
11 kinds of preprocessing procedures of multiplicative scatter correction.
Embodiment
A, set up Itanlian rye soluble sugar content near-infrared forecast model；The Itanlian rye soluble sugar content
Near-infrared forecast model is adopted and set up with the following method, specifically includes following steps：
A, collection Itanlian rye sample, the Itanlian rye sample is the Itanlian rye of 2015-2016 collections
Sample, including 16 states examine kinds, 22 new lines, and collection breeding time includes tillering stage, jointing stage, boot stage, heading stage, opens
Florescence, productive phase, seven breeding times of maturity period, collection position include stem, leaf, complete stool, gather the planting type of Itanlian rye
Including broadcasting sowing planting type, culturing and transplanting seedlings planting type, applied nitrogen planting type, Itanlian rye sample has 403 parts altogether,
105 DEG C of de-enzyme 20min, after 65 DEG C of drying, powder are ground into micropulverizer and 40 mesh sieves are crossed, and obtain Itanlian rye sample
Product powder；
B, the Itanlian rye sample powder for obtaining step a carry out respectively near infrared spectra collection obtain each spend more it is black
The near-infrared primary light spectrogram of wheat straw sample powder；Specifically, taking appropriate Itanlian rye sample powder, Bruker companies are put into
In the MPA type Fourier transformation NIRS instrument specimen cups of production, sample is shakeout naturally, when loading sample, sample is kept as far as possible
Useful load, real close degree are consistent with surfacing, and sample loading volume is half of specimen cup capacity or so, setting instrument work ginseng
Number is 4000~12500cm of Spectral range-1, resolution ratio 8cm-1, scanning times 64 times are adopted using the built-in reference of instrument as correction
Collection sample spectra；Spectra collection is carried out under the conditions of 25 ± 0.5 DEG C of room temperature, the first time near infrared light spectrum of the sample is obtained,
Then, placed into after the sample in specimen cup is taken out in specimen cup, sample is shakeout naturally, sample near infrared light is gathered again
Spectrum, obtains second near infrared light spectrum of the sample, and collection is twice to down-sample the spectral drift for causing, then adopting
With the commercial quantitative spectrochemical analysis softwares of OPUS/QUENT 5.5 to first time near infrared light spectrum and second near infrared light spectrum
Carry out averagely, obtaining the near-infrared primary light spectrogram of Itanlian rye sample, sample primary light spectrogram is as shown in Figure 1；
C, the primary light spectrogram that will be obtained in step b import the commercial spectrum of OPUS/QUENT 5.5 of Bruker companies of Germany
In quantitative analysis software, the near-infrared original spectrum of all Itanlian rye sample powders is analyzed using PCA algorithms, picked
Except the Itanlian rye sample with similar spectral, remaining 123 parts of Itanlian rye samples are representative Itanlian rye sample,
Its sample primary light spectrogram is as shown in Figure 2；
D, the representative Itanlian rye sample obtained to step c using anthrone Sulphuric acid colorimetry carry out soluble sugar one by one
Content is measured and obtains each representative Itanlian rye sample solubility sugared content value；
Described anthrone Sulphuric acid colorimetry is：Engraved using Suzhou section《Plant soluble sugar kit --- micromethod》Carry out
Determine, instrument is the Multiskan of Thermo scientific companies productionTMGo ELIASAs, its step is as follows：
1st, in sample soluble sugar extraction：0.035~0.050g sample powders are weighed in 2.0mL centrifuge tubes, is added
1mL distilled water grinds to form homogenate in high-throughput tissue grinder, and 95 DEG C of water-bath 10min (in the process, keep centrifugation lid
Cover tightly, to prevent moisture loss), after cooling, under the conditions of 25 DEG C, 8000g centrifugation 10min take supernatant in 10mL centrifuge tubes
In, 10mL is settled to distilled water, shake up standby；
2nd, soluble sugar content is determined：ELIASA preheats 30min, adjusting wavelength to 620nm before the assay；By in kit
Reagent one add 2.5mL reagents two to be configured to working solution, fully used after dissolving, it is more difficult to which dissolving can heating stirring；By sample
Add 1.5mL centrifuge tubes to be reacted with reagent, 40 μ L samples, 40 μ L distilled water, 20 μ L working solutions and 200 are added in measure pipe
The μ L concentrated sulfuric acids, sample is replaced in blank tube with isometric distilled water；Above-mentioned mixed liquor is mixed, 95 DEG C of water-bath 10min are placed in
(lid being covered tightly, to prevent moisture loss), after being cooled to room temperature, takes 200 μ L and is transferred in 96 hole elisa Plates, at 620nm, point
Du Qu not blank tube and the absorbance for determining pipe；If note △ A more than 1 (△ A=A determine pipe-A blank tubes), it is necessary to by sample
Product distilled water diluting, meanwhile, corresponding extension rate is multiplied by computing formula；
3rd, the calculating of soluble sugar content：According to formula --- soluble sugar (%)=[(△ A+0.07)+4.275 × V1]
1000 × 100=2.34 of ÷ (W × V1-V2) ÷ × (△ A+0.07) ÷ W ÷ 10.Wherein, V1 is addition sample volume, 40 μ L；
V2 is addition extracting liquid volume, 10mL；W is sample dry weight g；
Representative Itanlian rye sample is divided into school by the representative Itanlian rye sample solubility sugared content value of e, foundation
Positive collection and checking collection two parts, specific method are as follows：The representative Itanlian rye soluble sugar content value that will be obtained first from
Small 1 then to be taken every 3 and is collected as checking to being ranked up greatly, remaining is used as calibration set；I.e. representative 123 parts
In Itanlian rye sample, there are 93 parts as calibration set, 30 parts collect as checking, and adjust soluble sugar in Itanlian rye
The minimum value and maximum of content, it is calibration set to be allowed to incorporate into.
F, by the atlas of near infrared spectra of calibration set sample and the importing OPUS/ of the soluble sugar content value of calibration set sample
In QUENT5.5 commercialization quantitative spectrochemical analysis softwares, first, the near infrared spectrum to calibration set sample in full spectral limit is carried out
Pretreatment, in full spectral limit to the near infrared spectrum of calibration set sample using first derivative, second dervative, subtract one it is straight
Line, first derivative+MSC (multiplicative scatter correction), first derivative+subtract straight line, first derivative+vector normalization, do not have
Pretreated spectra, min-max normalization, vector normalization, elimination constant offset and 11 kinds of spectrum of multiplicative scatter correction are pre-
Processing method, then sets up prediction calibration model, root to calibration set sample using partial least-squares regression method combination validation-cross
Prediction calibration model is evaluated according to the parameter of near-infrared quantitative calibration models, determines optimal spectrum preprocess method, it is main into
Part factor number and best modeled spectral regions, finally predicting the minimum value of the validation-cross root-mean-square error RMSECV of calibration model
Correspondence optimal spectrum preprocess method, best modeled spectral regions and optimal main Composition Factor number, Fig. 3 is the near red of calibration set sample
External spectrum best modeled after the normalization of optimal pretreatment mode min-max is pre-processed composes the atlas of near infrared spectra in area；
Table 1 is best modeled spectrum area and model parameter under different preprocessing procedures, as described in table 1, the final optimal spectrum for determining
Pre-process as min-max is normalized, best modeled spectral regions are 6101.9~5446.2cm-1With 4601.5~4246.7cm-1, optimum factor number is 7；According to mahalanobis distance, THE PRINCIPAL FACTOR ANALYSIS figure, spectrum residual plot and chemical analysis in evaluation procedure
The Itanlian rye calibration set sample of the result rejecting abnormalities such as value residual plot, so as to obtain optimal Itanlian rye soluble sugar
The optimal near-infrared prediction calibration model of content, includes to the near-infrared quantitative calibration models parameter that prediction calibration model is evaluated
Coefficient of determination R2, validation-cross coefficient of determination R2Cv, validation-cross root-mean-square error RMSECV；Then, using checking collection sample pair
Prediction calibration model carries out external certificate, and the specific method of described external certificate is to utilize to predict calibration model to checking light harvesting
Spectrum is predicted, i.e., select to set up fixed in the commercial quantitative spectrochemical analysis softwares of OPUS/QUENT 5.5 of German Bruker companies
2 methods are measured, will verify that the near infrared spectrum of collection sample and the soluble sugar content of checking collection sample import the prediction correction set up
Checking is analyzed in model and obtains prediction checking model；Then, using the parameter of near-infrared quantitative verification model to gained
Prediction checking model is evaluated, according to mahalanobis distance, THE PRINCIPAL FACTOR ANALYSIS figure, spectrum residual plot and chemistry in evaluation procedure
The Itanlian rye checking collection sample of the result rejecting abnormalities such as assay value residual plot, it is soluble so as to obtain optimal Itanlian rye
Sugared content near-infrared forecast model；The near-infrared quantitative verification model parameter that prediction checking model is evaluated is tested including outside
Card coefficient of determination R2Ev and checking root-mean-square error RMSEP.That is prediction calibration model has coefficient of determination R higher2, validation-cross
Coefficient of determination R2Cv and relatively low RMSECV, prediction checking model has external certificate coefficient of determination R higher2Ev and relatively low
During RMSEP values, the near-infrared forecast model is applied to the measure of Itanlian rye soluble sugar content；Fig. 4 is cross validation
Root-mean-square error RMSECV, validation-cross coefficient of determination R2With the variation diagram of main composition dimension, Fig. 5 is that checking collection sample can to cv
Dependency graph between dissolubility sugared content chemical score and near infrared spectrum predicted value；It is described optimal many by optimizing evaluation
The forecast model of flower rye-grass soluble sugared content, its coefficient of determination R2It is 90.20%, validation-cross coefficient of determination R2Cv is
87.77%th, external certificate coefficient of determination R2Ev is that 91.28%, RMSECV is that 3.11 and RMSEP is 2.56；The prediction correction
Model eliminates 4 abnormal samples, i.e. calibration set sample for 89；Described prediction checking model does not have abnormal sample, that is, test
Card collection sample is 30；
Table 1
B, by Itanlian rye sample to be measured according to described in step a preprocess method process after obtain it is to be measured spend more it is black
Wheat straw sample powder；
C, the Itanlian rye sample powder to be measured that step B is obtained is carried out near infrared spectra collection obtain it is to be measured spend more it is black
The near-infrared primary light spectrogram of wheat straw sample powder；
D, the Itanlian rye that will be obtained in the near-infrared primary light spectrogram and step f of Itanlian rye sample powder to be measured
Soluble sugar content near-infrared forecast model is imported into the commercial quantitative spectrochemical analysis softwares of OPUS/QUENT 5.5, by model
Operational analysis, you can obtain the content of soluble sugar in Itanlian rye sample to be measured.
Following table is the content value of the soluble sugar that 38 parts of Itanlian rye samples to be measured are measured using the above method；
As seen from the above table, using spending more that the assay method of Itanlian rye soluble sugar content of the present invention is determined
With its actual value closely, its accuracy of detection is very high for the measured value of rye-grass soluble sugared content.
Embodiment described above is only that the preferred embodiment of the present invention is described, not to model of the invention
Enclose and be defined.Any those of ordinary skill in the art, are not departing from the situation of Spirit Essence of the invention and technical scheme
Under, may be by above-mentioned methods and techniques content and many possible variations and modification are made to technical solution of the present invention.Cause
This, every content without departing from technical solution of the present invention, according to technical spirit of the invention to variation made for any of the above embodiments
And modification, belong in the range of technical solution of the present invention protection.
Claims (9)
1. a kind of assay method of Itanlian rye soluble sugar content, it is characterised in that comprise the following steps：
A, set up Itanlian rye soluble sugar content near-infrared forecast model；The Itanlian rye soluble sugar content is closely red
Outer forecast model is adopted and set up with the following method, specifically includes following steps：
A, collection Itanlian rye sample, the Itanlian rye sample include different cultivars, different lines, different growing,
The fresh grass sample of different planting and different parts, is respectively pre-processed the Itanlian rye sample of above-mentioned collection, institute
The method for stating pretreatment is as described below：The Itanlian rye sample of collection is first finished 20min in 105 DEG C of environment, then
After being dried in 65 DEG C of environment, powder is ground into micropulverizer and 40 mesh sieves excessively obtain Itanlian rye sample powder
End；
B, the Itanlian rye sample powder for obtaining step a carry out near infrared spectra collection and obtain each Itanlian rye respectively
The near-infrared primary light spectrogram of sample powder；
C, the near-infrared original spectrum of all Itanlian rye sample powders is analyzed using PCA algorithms, rejecting has phase
Like the Itanlian rye sample of spectrum, remaining Itanlian rye sample is representative Itanlian rye sample；
D, the representative Itanlian rye sample obtained to step c using anthrone Sulphuric acid colorimetry carry out soluble sugar content one by one
It is measured and obtains each representative Itanlian rye sample solubility sugared content value；
Representative Itanlian rye sample is divided into calibration set by the representative Itanlian rye sample solubility sugared content value of e, foundation
Collect two parts with checking, specific method is as follows：The representative Itanlian rye soluble sugar content value that will be obtained first from it is small to
It is ranked up greatly, 1 is then taken every 3 and is collected as checking, remaining adjusts representative Itanlian rye as calibration set
The minimum value and maximum of middle soluble sugar content, it is calibration set to be allowed to incorporate into；
F, by the atlas of near infrared spectra of calibration set sample and the importing Bruker of the soluble sugar content value of calibration set sample
In the commercial quantitative spectrochemical analysis softwares of OPUS/QUENT 5.5, first, to the near infrared light of calibration set sample in full spectral limit
Spectrum is pre-processed, and prediction straightening die is then set up to calibration set sample using partial least-squares regression method combination validation-cross
Type, the parameter according to near-infrared quantitative calibration models is evaluated prediction calibration model, and prediction calibration model is evaluated
Near-infrared quantitative calibration models parameter include coefficient of determination R2, validation-cross coefficient of determination R2Cv, validation-cross root-mean-square error
RMSECV, its coefficient of determination R2It is 90.20%, validation-cross coefficient of determination R2Cv is 87.77%, validation-cross root-mean-square error
RMSECV is 3.11, determine optimal spectrum pretreatment be min-max normalization, best modeled spectral regions be 6101.9~
5446.2cm-1With 4601.5~4246.7cm-1, optimum factor number is 7, according to mahalanobis distance, main factor point in evaluation procedure
The Itanlian rye calibration set sample of analysis figure, spectrum residual plot and chemical analysis value residual plot result rejecting abnormalities, so that
To the optimal optimal near-infrared prediction calibration model of Itanlian rye soluble sugar content；Then, the near red of collection sample will be verified
Checking is analyzed in the prediction calibration model that the soluble sugar content importing of external spectrum and checking collection sample is set up to be predicted
Checking model；Then, the prediction checking model of gained is evaluated using the parameter of near-infrared quantitative verification model, to prediction
The parameter of the near-infrared quantitative verification model that checking model is evaluated includes external certificate coefficient of determination R2Ev and checking are square
Root error RMSEP, its external certificate coefficient of determination R2Ev is 91.28%, and checking root-mean-square error RMSEP is 2.56, is being evaluated
During according to mahalanobis distance, THE PRINCIPAL FACTOR ANALYSIS figure, spectrum residual plot and chemical analysis value residual plot result rejecting abnormalities
Itanlian rye checking collection sample, so as to obtain optimal Itanlian rye soluble sugar content near-infrared forecast model；
B, by Itanlian rye sample to be measured according to described in step a preprocess method process after obtain Itanlian rye to be measured
Sample powder；
C, the Itanlian rye sample powder to be measured that step B is obtained is carried out near infrared spectra collection obtain Itanlian rye to be measured
The near-infrared primary light spectrogram of sample powder；
D, the Itanlian rye that will be obtained in the near-infrared primary light spectrogram and step f of Itanlian rye sample powder to be measured are solvable
Property sugared content near-infrared forecast model imported into the commercial quantitative spectrochemical analysis software of OPUS/QUENT 5.5, by model calculation
Analysis, you can obtain the content of soluble sugar in Itanlian rye sample to be measured.
2. the assay method of Itanlian rye soluble sugar content according to claim 1, it is characterised in that：It is described to spend more
The breeding time of rye grass includes tillering stage, jointing stage, boot stage, heading stage, florescence, productive phase, maturity period.
3. the assay method of Itanlian rye soluble sugar content according to claim 1, it is characterised in that：It is described to spend more
The planting type of rye grass includes broadcasting sowing planting type, culturing and transplanting seedlings planting type, applied nitrogen planting type.
4. the assay method of Itanlian rye soluble sugar content according to claim 1, it is characterised in that：It is described to spend more
The collection position of rye grass includes stem, leaf, complete stool.
5. the assay method of Itanlian rye soluble sugar content according to claim 1, it is characterised in that：In step b
In, near infrared spectra collection is carried out using method as described below respectively to Itanlian rye sample powder, specifically, taking appropriate
Itanlian rye sample powder, is put into Bruker MPA type Fourier transformation NIRS instrument specimen cups, sample is shakeout naturally, if
Determine Instrument working parameter, sample near infrared spectrum is gathered under the conditions of being 25 ± 0.5 DEG C in temperature, the first time for obtaining the sample is near
Infrared light spectrum, then, places into specimen cup after the sample in specimen cup is taken out, and sample is shakeout naturally, gathers again
Sample near infrared spectrum, obtains second near infrared light spectrum of the sample, then to first time near infrared light spectrum and second
Secondary near infrared light spectrum averagely obtain the near-infrared primary light spectrogram of the Itanlian rye sample.
6. the assay method of Itanlian rye soluble sugar content according to claim 5, it is characterised in that：It is described
BrukerMPA type Fourier transformation NIRS Instrument working parameters are set as：4000~12500cm of Spectral range-1, resolution ratio 8cm-1, scanning times 64 times.
7. the assay method of Itanlian rye soluble sugar content according to claim 6, it is characterised in that：Using
OPUS/QUENT 5.5 is commercial, and quantitative spectrochemical analysis software enters to first time near infrared light spectrum and second near infrared light spectrum
Row is average, obtains the near-infrared primary light spectrogram of Itanlian rye sample.
8. the assay method of Itanlian rye soluble sugar content according to claim 1, it is characterised in that：In step d
In, carry out soluble sugar content one by one using the representative Itanlian rye sample of anthrone Sulphuric acid colorimetry and be measured to obtain every
Individual representative Itanlian rye sample solubility sugared content value.
9. the assay method of Itanlian rye soluble sugar content according to claim 1, it is characterised in that：In step f
In, in full spectral limit to the near infrared spectrum of calibration set sample using first derivative, second dervative, subtract straight line, one
Order derivative+MSC (multiplicative scatter correction), first derivative+subtract straight line, first derivative+vector normalization, there is no spectrum pre-
Treatment, min-max normalization, vector normalization, 11 kinds of Pretreated spectra sides of elimination constant offset and multiplicative scatter correction
Method.
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| CN114018865A (en) * | 2021-11-18 | 2022-02-08 | 河北农业大学 | Method for constructing near-infrared model of peanut sucrose content with different seed coat colors |
| CN114018866A (en) * | 2021-11-18 | 2022-02-08 | 河北农业大学 | Method for constructing near-infrared model of total sugar content of peanuts with different seed coat colors |
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| CN114428061A (en) * | 2021-12-16 | 2022-05-03 | 皖西学院 | Method for predicting total polysaccharide content in fiddlehead based on ultraviolet-visible-near infrared spectrum |
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| CN115468930A (en) * | 2022-10-19 | 2022-12-13 | 华中农业大学 | Method for rapidly determining sugar content produced by barley stalk enzymolysis |
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| CN107314986A (en) * | 2017-06-12 | 2017-11-03 | 华中农业大学 | A kind of method that utilization near infrared spectrum detects rape stem soluble sugar content |
| CN108548792A (en) * | 2018-03-12 | 2018-09-18 | 河南省农业科学院 | A kind of fast non-destructive detection method of peanut kernel soluble sugar content |
| CN114018865A (en) * | 2021-11-18 | 2022-02-08 | 河北农业大学 | Method for constructing near-infrared model of peanut sucrose content with different seed coat colors |
| CN114018866A (en) * | 2021-11-18 | 2022-02-08 | 河北农业大学 | Method for constructing near-infrared model of total sugar content of peanuts with different seed coat colors |
| CN114384041A (en) * | 2021-11-18 | 2022-04-22 | 河北农业大学 | Method for constructing near-infrared model of soluble sugar content of peanuts with different seed coat colors |
| CN114428061A (en) * | 2021-12-16 | 2022-05-03 | 皖西学院 | Method for predicting total polysaccharide content in fiddlehead based on ultraviolet-visible-near infrared spectrum |
| CN114813627A (en) * | 2022-04-24 | 2022-07-29 | 广东省农业科学院环境园艺研究所 | Dendrobium nobile mannose content detection method based on near infrared spectrum |
| CN115468930A (en) * | 2022-10-19 | 2022-12-13 | 华中农业大学 | Method for rapidly determining sugar content produced by barley stalk enzymolysis |
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