CN114184632A - Method for identifying selenium-rich black garlic of green marine products - Google Patents
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Abstract
The invention provides a method for identifying selenium-rich black garlic of green marine products, and relates to the technical field of food detection. The invention uses methanol water solution to extract the black garlic; by using1Acquiring fingerprint spectrums of the selenium-rich black garlic produced in Qinghai and the black garlic produced in other known producing areas by an H-NMR technology, and performing supervised clustering analysis by using an orthogonal partial least square method to obtain an OPLS-DA model; and predicting the black garlic sample to be determined in the producing area by using the model. The method takes the methanol water solution as the extraction reagent, can extract target compounds such as amino acid and the like in the black garlic to the maximum extent, obtains more accurate black garlic fingerprint, and is simple and convenient to operate; the invention adopts1Qualitative and quantitative data of various active ingredients of the black garlic can be obtained by one-time detection of an H-NMR technology, the constructed OPLS-DA model is stable and reliable, the green marine selenium-rich black garlic can be accurately, simply and quickly identified, and a scientific basis is provided for quality control and anti-counterfeiting of the green marine selenium-rich black garlic.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a method for identifying selenium-rich black garlic produced in Qinghai.
Background
The black garlic product is prepared by fermenting fresh raw garlic with skin in a high-temperature and high-humidity environment. After the black garlic is fermented, the taste becomes soft, glutinous, fragrant and sweet, the black garlic is suitable for eating, the nutritive value is increased, and the biological activity is enhanced. Researches show that the black garlic has the functions of eliminating fatigue, enhancing the inoxidizability, protecting the liver, promoting sleep, reducing blood pressure and blood fat, reducing cholesterol, resisting diabetes, resisting tumors and the like.
There are many factors affecting the quality of black garlic, and the most important factor is the production area of black garlic. The content of reducing sugar, amino nitrogen, total phenol, moisture and 5-HMF (hydroxymethyl furfural) in black garlic in different producing areas can be different (Li is grown, Chinese fruit dish, 2018,38(09): 5-9). The special geographical environment of the green hydantoin creates the unique quality of the black garlic, the green hydantoin is rich in selenium which is a necessary trace element and is vital to health, and the lack of selenium in a human body can cause the reduction of immunity, the increase of the coronary heart disease rate and the liver disease rate. With the improvement of health consciousness of people, the selenium-rich black garlic produced in Qinghai is popular with more and more people. However, the variety of black garlic products in the market is various, and the merchants of the black garlic which can be used as green marine selenium-rich black garlic are not lacked, but at present, no method for effectively identifying the green marine selenium-rich black garlic from other black garlic products exists.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for identifying selenium-rich black garlic from green marine products. The method provided by the invention can accurately, simply and quickly identify the selenium-rich black garlic from the green sea, and provides a scientific basis for quality control and anti-counterfeiting of the selenium-rich black garlic from the green sea.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for identifying selenium-rich black garlic of green marine products, which comprises the following steps:
(1) respectively extracting black garlic samples in known producing areas to obtain black garlic extracts; the black garlic samples of the known producing areas comprise selenium-rich black garlic produced in Qinghai and black garlic samples of other known producing areas; the reagent for extraction is methanol water solution;
(2) respectively obtaining nuclear magnetic resonance hydrogen spectrums of the black garlic extract in the step (1), and carrying out normalization processing on the nuclear magnetic resonance hydrogen spectrums to obtain nuclear magnetic full spectrum data;
(3) importing the nuclear magnetic full spectrum data in the step (2) into SMICA software, taking the nuclear magnetic full spectrum data as an X value and a production place as a Y value, and performing supervised clustering analysis by adopting an orthogonal partial least square method to obtain an OPLS-DA model;
(4) acquiring nuclear magnetic full spectrum data of the black garlic sample of the origin to be determined by adopting the methods in the steps (1) to (2); introducing the nuclear magnetic full spectrum data of the black garlic sample of the origin to be determined into the OPLS-DA model in the step (3) for prediction to obtain a predicted value; if the predicted score value is larger than 0.5, judging that the black garlic sample of the producing area to be determined is selenium-rich black garlic produced in Qinghai; and if the predicted score value is less than 0.5, judging that the black garlic sample of the origin to be determined is the black garlic of other origins.
Preferably, the black garlic samples of other known producing areas in the step (1) comprise one or more of Guangdong river source, Hebei Heshui, Jiangsu Xuzhou, Shandong Jinxiang and Yunnan Dali black garlic samples.
Preferably, the method for extracting in step (1) comprises the following steps:
respectively peeling the black garlic samples of the known producing areas and then rolling to obtain mud-shaped samples;
and mixing the pasty sample with a methanol water solution and a phosphate buffer solution, sequentially carrying out vortex oscillation, ultrasonic treatment and centrifugation on the obtained mixed solution, and concentrating the supernatant to dryness to obtain the black garlic extract.
Preferably, the volume fraction of the methanol aqueous solution is 20-90%; the dosage ratio of the muddy sample to the methanol water solution is 0.1g: 1-5 mL.
Preferably, the pH value of the phosphate buffer solution is 2-8, and the concentration is 0.05-3 mol/L; the dosage ratio of the mud sample to the phosphate buffer solution is 0.1g: 20-100 μ L.
Preferably, the vortex oscillation time is 3-10 min; the ultrasonic time is 30-60 min; the centrifugation speed is 10000-12000 rpm, and the time is 10-20 min; the method for concentrating to dryness is rotary evaporation, the temperature of the rotary evaporation is 45-50 ℃, and the time is 3-5 hours.
Preferably, the method for obtaining the nuclear magnetic resonance hydrogen spectrum of the black garlic extract in the step (2) comprises the following steps:
mixing the black garlic extract with a deuterated reagent, and sequentially performing vortex oscillation, ultrasonic treatment and centrifugation on the obtained mixed solution to obtain a supernatant; tetramethylsilane is added into the deuterated reagent to be used as an internal standard substance;
and taking the supernatant to perform nuclear magnetic resonance detection to obtain the nuclear magnetic resonance hydrogen spectrum.
Preferably, the deuterated reagent is deuterated methanol or deuterium oxide; the dosage ratio of the black garlic extract to the deuterated reagent is 0.1g to 0.6-1.0 mL.
Preferably, the vortex oscillation time is 3-10 min; the ultrasonic time is 10-20 min; the centrifugation speed is 10000-12000 rpm, and the time is 10-20 min.
Preferably, the nuclear magnetic resonance detection conditions are as follows: 5mm multi-core broadband observation probe, wherein the observation frequency is 600.13MHz, and the standard ZG30 pulse sequence is adopted for acquisition; the parameters are set as follows: the spectrum width SW is 19.8ppm, the center point O1P of the spectrum is 6.175ppm, the spectrum resolution FIDRES is 0.363Hz, and the scanning times NS is 16.
The invention provides a method for identifying selenium-rich black garlic of green marine products, which extracts black garlic products by using methanol aqueous solution; then adopt1H-NMR technology for obtaining selenium-rich black garlic and other products from QinghaiCarrying out supervised clustering analysis on the selenium-rich black garlic produced in Qinghai and the black garlic produced in other known producing areas by utilizing an orthogonal partial least square method to obtain an OPLS-DA model; and predicting the black garlic sample to be determined in the producing area by using the model so as to identify whether the black garlic sample is selenium-rich black garlic produced in Qinghai. The methanol-water solution is used as the extraction reagent, and the target compounds such as amino acid and the like in the black garlic product can be extracted to the maximum extent, so that a more accurate black garlic fingerprint is obtained, and the operation is simple and convenient; the invention adopts1Qualitative and quantitative data of various effective components of the black garlic can be obtained by one-time detection of an H-NMR technology, the difference characteristic components of the green marine selenium-enriched black garlic and other black garlic in a producing area are determined, the constructed OPLS-DA model is stable and reliable, the green marine selenium-enriched black garlic can be accurately, simply and rapidly identified, and a scientific basis is provided for quality control and anti-counterfeiting of the green marine selenium-enriched black garlic.
Drawings
FIG. 1 shows the extract of selenium-rich black garlic from Qinghai and black garlic from other producing areas at 600MHz in example 11H-NMR spectrum;
FIG. 2 shows the selenium-enriched black garlic produced in Qinghai and the black garlic produced in other places in example 11An OPLS-DA model diagram of an H-NMR spectrum;
FIG. 3 is a VIP chart showing the difference characteristic peaks of selenium-enriched black garlic of Qinghai Hainanlu and black garlic of other producing areas in example 1;
FIG. 4 is a model diagram of OPLS-DA of selenium-enriched black garlic of green marine Enlu and other black garlic of origin integrated with example 1 and example 2;
FIG. 5 is a cross-validation graph of the OPLS-DA model of selenium-enriched black garlic of green marine Enlu and other black garlic of origin integrated with example 1 and example 2;
FIG. 6 is a graph of the predicted score of the OPLS-DA model of black garlic in different producing areas;
FIG. 7 is a ROC graph showing the prediction accuracy of black garlic from Qinghai and other producing areas for integrating the embodiment 1 and the embodiment 2;
FIG. 8 shows the selenium-enriched black garlic extracts of Qinghai producing areas in example 1, comparative example 1 and comparative example 21H-NMR spectrum, in which a is of the black garlic extract of Qinghai origin of example 11H-NMR spectrum b of black garlic extract from Qinghai producing area of comparative example 11H-NMR spectrum, c is that of the black garlic extract of Qinghai producing area of comparative example 21H-NMR spectrum.
Detailed Description
The invention provides a method for identifying selenium-rich black garlic of green marine products, which comprises the following steps:
(1) respectively extracting black garlic samples in known producing areas to obtain black garlic extracts; the black garlic samples of the known producing areas comprise selenium-rich black garlic produced in Qinghai and black garlic samples of other known producing areas; the reagent for extraction is methanol water solution;
(2) respectively obtaining nuclear magnetic resonance hydrogen spectrums of the black garlic extract in the step (1), and carrying out normalization processing on the nuclear magnetic resonance hydrogen spectrums to obtain nuclear magnetic full spectrum data;
(3) importing the nuclear magnetic full spectrum data in the step (2) into SMICA software, taking the nuclear magnetic full spectrum data as an X value and a production place as a Y value, and performing supervised clustering analysis by adopting an orthogonal partial least square method to obtain an OPLS-DA model;
(4) acquiring nuclear magnetic full spectrum data of the black garlic sample of the origin to be determined by adopting the methods in the steps (1) to (2); introducing the nuclear magnetic full spectrum data of the black garlic sample of the origin to be determined into the OPLS-DA model in the step (3) for prediction to obtain a predicted value; if the predicted score value is larger than 0.5, judging that the black garlic sample of the producing area to be determined is selenium-rich black garlic produced in Qinghai; and if the predicted score value is less than 0.5, judging that the black garlic sample of the origin to be determined is the black garlic of other origins.
The method comprises the steps of respectively extracting black garlic samples in known producing areas to obtain black garlic extracts; the black garlic samples of the known producing areas comprise selenium-rich black garlic produced in Qinghai and black garlic samples of other known producing areas. In the present invention, the other known black garlic samples preferably include one or more of Guangdong river source, Hebei Heshui, Jiangsu Xuzhou, Shandong Jinxiang and Yunnan Dali black garlic samples. In the present invention, the extraction reagent is an aqueous methanol solution.
In the present invention, the method of extraction preferably comprises the steps of:
respectively peeling the black garlic samples of the known producing areas and then rolling to obtain mud-shaped samples;
and mixing the pasty sample with a methanol water solution and a phosphate buffer solution, sequentially carrying out vortex oscillation, ultrasonic treatment and centrifugation on the obtained mixed solution, and concentrating the supernatant to dryness to obtain the black garlic extract.
The invention has no special requirement on the specific operation method of rolling, and the black garlic sample of the known production area can be rolled into a paste. After obtaining the muddy sample, the invention also preferably dries the muddy sample; the drying is preferably vacuum drying or freeze drying.
In the invention, the volume fraction of the methanol aqueous solution is preferably 20-90%, and more preferably 50-80%; according to the invention, the volume fraction of the methanol aqueous solution is controlled to be 20-90%, so that target compounds such as amino acid and the like in the black garlic product can be extracted to the maximum degree, if the volume fraction of the methanol aqueous solution is less than 20%, part of fat-soluble amino acid and lipid in the black garlic product cannot be extracted, and if the volume fraction of the methanol aqueous solution is more than 90%, part of water-soluble amino acid in the black garlic product is not extracted completely. In the invention, the dosage ratio of the muddy sample to the methanol aqueous solution is preferably 0.1g: 1-5 mL, and more preferably 0.1g: 3-5 mL. In the invention, the pH value of the phosphate buffer solution is preferably 2-8, and more preferably 5-8; the concentration of the phosphate buffer solution is preferably 0.05-3 mol/L, and more preferably 1-2 mol/L; the dosage ratio of the muddy sample to the phosphate buffer is preferably 0.1g: 20 to 100. mu.L, more preferably 0.1g: 30-60 mu L. According to the invention, the methanol water solution and the phosphate buffer solution are preferably added into the muddy sample in sequence for mixing. The source of the aqueous methanol solution and the phosphate buffer solution is not particularly required in the present invention, and the buffer solution may be prepared by a commercially available method known to those skilled in the art or by a self-preparation method known to those skilled in the art. In the invention, the vortex oscillation time is preferably 3-10 min, and more preferably 5-8 min; the ultrasonic time is preferably 30-60 min, and more preferably 30-50 min; the centrifugation speed is preferably 10000-12000 rpm, more preferably 11000-12000 rpm, and the centrifugation time is preferably 10-20 min, more preferably 10-15 min. According to the invention, through the vortex oscillation, the effective components in the black garlic are fully mixed; through the ultrasonic treatment, the target and effective components in the black garlic are fully dissolved out; through the centrifugation, substances which cannot be dissolved in the black garlic are completely precipitated, so that the nonuniformity of the solution is reduced, and a sharp nuclear magnetic spectrum peak is obtained. In the present invention, the method of concentrating to dryness is preferably rotary evaporation; the rotary evaporation temperature is preferably 45-50 ℃, and the time is preferably 3-5 h; the rotary evaporation is preferably carried out in a vacuum concentrator.
According to the invention, the methanol aqueous solution is used as an extraction reagent, so that target compounds such as amino acid and the like in the black garlic product can be extracted to the greatest extent, and a more accurate black garlic fingerprint is obtained; and the extraction method is simple and convenient to operate.
After the black garlic extract is obtained, the invention respectively obtains the nuclear magnetic resonance hydrogen spectra of the black garlic extract (1H-NMR) is carried out, normalization processing is carried out on the nuclear magnetic resonance hydrogen spectrum, and nuclear magnetic full spectrum data are obtained.
In the present invention, the method for obtaining the nmr hydrogen spectrum of the black garlic extract preferably comprises the following steps:
mixing the black garlic extract with a deuterated reagent, and sequentially performing vortex oscillation, ultrasonic treatment and centrifugation on the obtained mixed solution to obtain a supernatant; tetramethylsilane is added into the deuterated reagent to be used as an internal standard substance;
and taking the supernatant to perform nuclear magnetic resonance detection to obtain the nuclear magnetic resonance hydrogen spectrum.
In the present invention, the deuterated reagent is preferably deuterated methanol or deuterium oxide; the dosage ratio of the black garlic extract to the deuterated reagent is preferably 0.1 g/0.6-1.0 mL. In the present invention, the amount of Tetramethylsilane (TMS) added is preferably 0.03% by mass of the deuterated reagent. In the invention, the vortex oscillation time is preferably 3-10 min, and more preferably 3-5 min; the ultrasonic time is preferably 10-20 min, and more preferably 10-15 min; the centrifugation speed is preferably 10000-12000 rpm, more preferably 11000-12000 rpm, and the centrifugation time is preferably 10-20 min, more preferably 10-15 min.
After the supernatant is obtained, the invention preferably takes the supernatant to be placed in a 5mm nuclear magnetic tube for nuclear magnetic resonance detection. In the invention, the nuclear magnetic detection instrument is preferably a Brookfield AV-600MHz nuclear magnetic resonance spectrometer; the conditions for the nuclear magnetic resonance detection are preferably as follows: 5mm multi-core broadband observation probe, wherein the observation frequency is 600.13MHz, and the standard ZG30 pulse sequence is adopted for acquisition; the parameter settings are preferably: the spectrum width SW is 19.8ppm, the center point O1P of the spectrum is 6.175ppm, the spectrum resolution FIDRES is 0.363Hz, and the scanning times NS is 16.
After obtaining the nuclear magnetic resonance hydrogen spectrum, the invention carries out normalization processing on the nuclear magnetic resonance hydrogen spectrum to obtain nuclear magnetic full spectrum data. In the present invention, the method for normalizing the nuclear magnetic resonance hydrogen spectrum preferably includes the steps of: for each of the NMR spectra1Fourier transform is carried out on the H-NMR spectrum, after phase adjustment and baseline correction are carried out, chemical shift calibration is carried out by using the TSP signal peak center position of 0.00; performing phase correction and baseline correction on the nuclear magnetic resonance hydrogen spectrum by using TOPSPIN software, eliminating the influence of a water peak and a methanol peak, and deleting the nuclear magnetic peak; and finally, normalizing each nuclear magnetic peak value.
After nuclear magnetic full spectrum data are obtained, the nuclear magnetic full spectrum data are imported into SMICA software, the nuclear magnetic full spectrum data are used as X values, producing areas are used as Y values, supervised clustering analysis is carried out by adopting an orthogonal partial least square method, and an OPLS-DA model is obtained. The present invention does not require the SMICA software specifically, and SMICA software known to those skilled in the art may be used. In the embodiment of the invention, the selenium-rich black garlic produced in Qinghai and the black garlic samples produced in other producing areas are obviously separated in the OPLS-DA model, the selenium-rich black garlic produced in Qinghai is gathered on the left side of the OPLS-DA model, and the black garlic samples produced in other producing areas are gathered on the right side of the OPLS-DA model.
After obtaining the OPLS-DA model, the invention adopts the method of the technical scheme to obtain the nuclear magnetic full spectrum data of the black garlic sample of the origin to be determined; introducing the full nuclear magnetic spectrum data of the black garlic sample of the origin to be determined into the OPLS-DA model of the technical scheme for prediction, and if the predicted score value is greater than 0.5, judging that the black garlic sample of the origin to be determined is selenium-enriched black garlic produced in Qinghai; and if the predicted score value is less than 0.5, judging that the black garlic sample of the origin to be determined is the black garlic of other origins. In the invention, in the black garlic samples of the known producing areas, the score value of the black garlic of the Qinghai product is about 1.0, and the score values of the black garlic samples of other known producing areas are about 0.0, so that when the predicted score value of the black garlic sample of the producing area to be determined is greater than 0.5, the score value is closer to the score value of the black garlic of the Qinghai product, and the black garlic sample of the producing area to be determined is judged to be the black garlic of the Qinghai product, otherwise the black garlic of the other producing areas.
The invention adopts1Qualitative and quantitative data of various active ingredients of the black garlic can be obtained by one-time detection of an H-NMR technology, the constructed OPLS-DA model is stable and reliable, the green marine selenium-rich black garlic can be accurately, simply and rapidly identified, and a scientific basis is provided for quality control and anti-counterfeiting of the green marine selenium-rich black garlic.
The method for identifying selenium-enriched black garlic from green marine products provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Constructing an OPLS-DA model:
preparing a black garlic sample: preparing green marine selenium-rich black garlic (produced by Qinghai Enlu Biotechnology Co., Ltd.) and other five black garlic of different producing areas (Guangdong river source, Hebei Heshui, Xuzhou Jiangsu, Shandong Jinxiang and Yunnan Dali) 7 each, peeling, uniformly grinding into black garlic paste samples, and freeze-drying for later use. Each black garlic sample was treated as follows:
(1) extracting black garlic: accurately weighing 0.1g of mashed black garlic samples from different producing areas respectively, adding 3mL of 80 vol% methanol aqueous solution and 50 μ L of 2mol/L phosphate buffer solution (pH 7.4), performing vortex oscillation for 5min, ultrasonic treatment for 30min and centrifugation for 10min (centrifugation speed 12000rpm) in sequence, and concentrating and evaporating 1.5mL of supernatant in a vacuum rotary evaporator at 50 ℃ for 3h to obtain the black garlic extract.
(2) And (3) nuclear magnetic resonance detection: adding 600 mu L of deuterated methanol (containing 0.03 wt% of TMS internal standard) into the black garlic extract obtained in the step (1) for redissolving, and after vortex oscillation for 3min, ultrasonic treatment for 10min and centrifugation for 10min (centrifugation speed 12000rpm) in sequence, taking 550 mu L of supernatant into a 5mm nuclear magnetic tube for determination; the measuring instrument uses a Brookx AV-600MHz nuclear magnetic resonance spectrometer and a 5mm multi-core broadband observation (BBO) probe, the observation frequency is 600.13MHz, the standard ZG30 pulse sequence is adopted for collection, and the detailed experimental parameters are as follows: the nuclear magnetic resonance hydrogen spectrum was obtained with experimental parameters SW of 19.8ppm, O1P of 6.175ppm, FIDRES of 0.363Hz, and NS of 16.
According to the chemical shift of metabolites, literature reports and partial standards, the main metabolites of the black garlic extract are phytosterol, leucine, isoleucine, valine, lactic acid, alanine, leucine, glutamic acid, gamma-aminobutyric acid, malic acid, citric acid, methionine, aspartic acid, alpha-ketoglutaric acid, taurine, choline, methanol, 1, 5-lemon dimethyl ester, glucose, beta-fructose, mannose, beta-glucose, threonine, arginine, sucrose and formic acid. Selenium-rich black garlic produced in Qinghai and black garlic samples produced in other five different producing areas1The H-NMR spectrum is shown in figure 1, and the metabolites of the black garlic extract represented by each number in figure 1 are as follows: 1. plant sterol, 2. leucine, 3. isoleucine, 4. valine, 5. lactic acid, 6. alanine, 7. leucine, 8. glutamic acid, 9. gamma-aminobutyric acid, 10. malic acid, 11. citric acid, 12. methionine, 13. aspartic acid, 14. alpha-ketoglutaric acid, 15. taurine, 16. choline, 17. methanol, 18.1, 5-lemon-dimethylester, 19. glucose, 20. beta-fructose, 21. mannose, 22. beta-glucose, 23. threonine, 24. arginine, 25. sucrose, 26. formic acid.
(3) Nuclear magnetic data processing: first for each of the NMR spectra1Fourier transform is carried out on the H-NMR spectrum, after phase adjustment and baseline correction are carried out, chemical shift calibration is carried out by using the TSP signal peak center position of 0.00; performing phase correction and baseline correction on the nuclear magnetic resonance hydrogen spectrum by using TOPSPIN software; and finally, carrying out normalization processing on each nuclear magnetic peak value to obtain nuclear magnetic full spectrum data.
(4) Constructing an OPLS-DA model: and (4) importing the nuclear magnetic full spectrum data of each black garlic sample obtained in the step (3) into SMICA software, and performing supervised clustering analysis on the black garlic produced in Qinghai and the black garlic produced in other known producing areas by adopting an orthogonal partial least square method to obtain an OPLS-DA model, as shown in figure 2. As can be seen from FIG. 2, the selenium-rich black garlic group produced in Qinghai is obviously separated from the black garlic groups produced in other places, the selenium-rich black garlic groups produced in Qinghai are all gathered on the left side of the OPLS-DA model, and the black garlic groups produced in other places are all gathered on the right side of the OPLS-DA model.
And (3) drawing a composition Variable Importance (VIP) graph of the black garlic extract produced in Qinghai and black garlic extract produced in other five producing areas (the VIP graph is obtained by an OPLS-DA model, the abscissa and ordinate are original graphs, and the color is VIP value), as shown in figure 3. The redder the color of the nuclear magnetic peak contributes more to the discrimination between groups, and the bluer the color contributes less to the discrimination between groups, and in general, the greater the VIP value, the greater the difference in significance. As can be seen from FIG. 3, the difference characteristic peaks of the selenium-rich black garlic produced in Qinghai and the black garlic produced in other five places are as follows: the relative quantitative statistics of the different characteristic peaks of phytosterol, leucine, valine, lactic acid, glutamic acid, malic acid, aspartic acid, mannose, beta-glucose, sucrose, formic acid, selenium-rich black garlic of Qinghai Enlu and black garlic of other producing areas are shown in Table 1. As shown in Table 1, the selenium-rich black garlic produced in Qinghai has relatively higher contents of phytosterol, leucine, valine, lactic acid, glutamic acid, malic acid, mannose and formic acid and relatively lower contents of aspartic acid, beta-glucose and sucrose compared with the black garlic produced in other five places. Wherein leucine and valine are branched chain amino acids, and the leucine has effects in repairing muscle, controlling blood sugar, increasing growth hormone production, and burning visceral fat; when the valine is insufficient, the central nervous system function of the brain is disordered, and limb tremor occurs due to ataxia, and diseases such as liver failure and the like are usually treated clinically by using injection of branched chain amino acids such as valine and the like. The branched chain amino acid is one of eight essential amino acids required by human body, and the human body can not synthesize the branched chain amino acid by himself.
TABLE 1 relative quantitative statistics of the difference characteristic peaks of selenium-rich black garlic from Qinghai Enlu and black garlic from other producing areas
Example 2
The method for predicting the producing areas of the black garlic in different producing areas by using the OPLS-DA model established in the embodiment 1 to identify the selenium-rich black garlic produced in Qinghai comprises the following steps:
preparation, extraction and application of black garlic sample1H-NMR measurement: the black garlic samples of 6 producing areas described in example 1 were extracted by selecting 8 black garlic samples from each producing area (i.e., 48 samples in total) according to the procedure of step (1) in example 1, and then subjected to nuclear magnetic resonance examination according to the procedure of step (2) in example 1 to obtain samples of each black garlic sample1An H-NMR spectrum;
the nuclear magnetic data processing process comprises the following steps: for each of the NMR spectra1Fourier transform is carried out on the H-NMR spectrum, after phase adjustment and baseline correction are carried out, chemical shift calibration is carried out by using the TSP signal peak center position of 0.00; performing phase correction and baseline correction on the nuclear magnetic resonance hydrogen spectrum by using TOPSPIN software; finally, normalization processing is carried out on each nuclear magnetic peak value to obtain the nuclear magnetic full spectrum data of the sample in the embodiment 2; reintegrating the samples of example 1 and example 21H-NMR full spectrum data;
and (3) data analysis: 8 sample data of each producing area in example 2, wherein 4 sample data are integrated with the sample in example 1 to be used as a working group (Workset) to perform OPLS-DA modeling (the larger the sample size of the OPLS-DA model is, the better the stability of the model is, so that a part of the samples in example 2 and the samples in example 1 are subjected to modeling again), a reconstructed OPLS-DA model graph (namely a scores score graph, as shown in FIG. 4, the number in FIG. 4 is the sample number), the working group samples gathered on the left side in FIG. 4 are the working group of selenium-enriched black garlic produced in Qinghai, and the working group gathered on the right side are the working groups of black garlic produced in other producing areas; the remaining 4 samples from each source were used for identification of the source as a prediction group (Predictionset) for the source to be determined.
The reconstructed OPLS-DA model is subjected to 200 times of permutation test (validation test), the arrangement sequence of the classification variables Y is changed randomly for many times to obtain different random Q values, and the model validity is further tested (completed by the SMICA software), with the result shown in fig. 5. In fig. 5, R2 represents the interpretability of the model for the classification variable Y, Q2 represents the predictability of the model, and fig. 5 shows that Q2 is negative, and the intercept of the regression line representing Q2 and the vertical axis is less than zero, which indicates that the original model has good robustness and no overfitting phenomenon.
The nuclear magnetic hydrogen spectrum data of the prediction group is imported into an OPLS-DA model shown in figure 4, and the pre-measured score is calculated. The prediction scores of the prediction group samples numbered 10-13 in the figure 4 in the model are all larger than 0.5 and are closer to the score of the working group (the score is about 1.0) of selenium-rich black garlic produced in Qinghai (figure 6); and predicting that the black garlic sample numbered 10-13 is the selenium-rich black garlic produced in Qinghai, and verifying that the black garlic sample numbered 10-13 is the selenium-rich black garlic produced in Qinghai indeed, wherein the prediction is accurate, namely the constructed OPLS-DA model can be used for accurately identifying the selenium-rich black garlic produced in Qinghai.
The results of curve analysis of the Receiver Operating Characteristics (ROC) of the OPLS-DA model predictive sample are shown in FIG. 7. In fig. 7, the abscissa is FPR (false positive classification rate), the ordinate is TPR (true positive classification rate), and fig. 7 shows that the green marine selenium-enriched black garlic has a good distinguishing effect with other black garlic producing places (AUC 1.0, AUC >0.9 shows that the distinguishing effect is good, 1.0 is the best, and 100% of the distinguishing effect is correct), so the method provided by the present invention can be used for rapidly distinguishing the green marine selenium-enriched black garlic.
Comparative example 1
Extracting green marine selenium-rich black garlic: the procedure of example 1, step (1), was changed to "3 mL of the aqueous methanol solution containing 80% by volume in 3 mL" and 3mL of the solution was added80% chloroform aqueous solution ", the rest is the same as example 1, to obtain black garlic extract, and performing nuclear magnetic resonance detection to the black garlic extract to obtain1H-NMR spectrum.
In FIG. 8, a is the extract (80% methanol in water) of black garlic from Qinghai province of example 11H-NMR spectrum; in FIG. 8, b is the extract (80% chloroform aqueous solution) of black garlic of Qinghai origin in comparative example 11H-NMR spectrum. It can be seen that b in FIG. 8 shows that the extraction of target compounds such as amino acids with 80% chloroform aqueous solution is greatly reduced.
Comparative example 2
Extracting green marine selenium-rich black garlic: changing the 'adding 3mL of 80% methanol aqueous solution by volume' into 'adding 3mL of 80% isopropanol aqueous solution by volume' in the step (1) of the example 1, and the rest is the same as the example 1 to obtain the black garlic extract, and performing nuclear magnetic resonance detection on the black garlic extract to obtain the black garlic extract1The H-NMR spectrum was as shown in c in FIG. 8.
C in FIG. 8 shows that the 80% aqueous isopropanol solution extracted the same compound as the 80% aqueous methanol solution, but the residual isopropanol had double and multiple peaks interfering at 1.2 and 4.0ppm, etc.
The embodiment shows that the methanol aqueous solution is used as the extraction reagent, the target compounds such as amino acid and the like in the black garlic can be extracted to the maximum extent, the methanol peak is a single peak of 3.3ppm, the interference is less, the more accurate black garlic fingerprint is obtained, and the operation is simple and convenient; the invention adopts1Qualitative and quantitative data of various active ingredients of the black garlic can be obtained by one-time detection of an H-NMR technology, the constructed OPLS-DA model is stable and reliable, the green marine selenium-rich black garlic can be accurately, simply and rapidly identified, and a scientific basis is provided for quality control and anti-counterfeiting of the green marine selenium-rich black garlic.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The method for identifying the selenium-rich black garlic of the green marine products is characterized by comprising the following steps of:
(1) respectively extracting black garlic samples in known producing areas to obtain black garlic extracts; the black garlic samples of the known producing areas comprise selenium-rich black garlic produced in Qinghai and black garlic samples of other known producing areas; the reagent for extraction is methanol water solution;
(2) respectively obtaining nuclear magnetic resonance hydrogen spectrums of the black garlic extract in the step (1), and carrying out normalization processing on the nuclear magnetic resonance hydrogen spectrums to obtain nuclear magnetic full spectrum data;
(3) importing the nuclear magnetic full spectrum data in the step (2) into SMICA software, taking the nuclear magnetic full spectrum data as an X value and a production place as a Y value, and performing supervised clustering analysis by adopting an orthogonal partial least square method to obtain an OPLS-DA model;
(4) acquiring nuclear magnetic full spectrum data of the black garlic sample of the origin to be determined by adopting the methods in the steps (1) to (2); introducing the nuclear magnetic full spectrum data of the black garlic sample of the origin to be determined into the OPLS-DA model in the step (3) for prediction to obtain a predicted value; if the predicted score value is larger than 0.5, judging that the black garlic sample of the producing area to be determined is selenium-rich black garlic produced in Qinghai; and if the predicted score value is less than 0.5, judging that the black garlic sample of the origin to be determined is the black garlic of other origins.
2. The method according to claim 1, wherein the other known origin black garlic samples in step (1) comprise one or more of Guangdong Heyuan, Hebei Hengshui, Xuzhou Jiangsu, Shandong Jinxiang and Yunnan Dali black garlic samples.
3. The method according to claim 1, wherein the method of extracting in step (1) comprises the steps of:
respectively peeling the black garlic samples of the known producing areas and then rolling to obtain mud-shaped samples;
and mixing the pasty sample with a methanol water solution and a phosphate buffer solution, sequentially carrying out vortex oscillation, ultrasonic treatment and centrifugation on the obtained mixed solution, and concentrating the supernatant to dryness to obtain the black garlic extract.
4. The method according to claim 3, wherein the volume fraction of the methanol aqueous solution is 20-90%; the dosage ratio of the muddy sample to the methanol water solution is 0.1g: 1-5 mL.
5. The method according to claim 3, wherein the phosphate buffer has a pH of 2 to 8 and a concentration of 0.05 to 3 mol/L; the dosage ratio of the mud sample to the phosphate buffer solution is 0.1g: 20-100 μ L.
6. The method according to claim 3, wherein the time of the vortex oscillation is 3-10 min; the ultrasonic time is 30-60 min; the centrifugation speed is 10000-12000 rpm, and the time is 10-20 min; the method for concentrating to dryness is rotary evaporation, the temperature of the rotary evaporation is 45-50 ℃, and the time is 3-5 hours.
7. The method according to claim 1, wherein the step (2) of obtaining the NMR spectrum of the black garlic extract comprises the following steps:
mixing the black garlic extract with a deuterated reagent, and sequentially performing vortex oscillation, ultrasonic treatment and centrifugation on the obtained mixed solution to obtain a supernatant; tetramethylsilane is added into the deuterated reagent to be used as an internal standard substance;
and taking the supernatant to perform nuclear magnetic resonance detection to obtain the nuclear magnetic resonance hydrogen spectrum.
8. The method of claim 7, wherein the deuterated reagent is deuterated methanol or deuterium oxide; the dosage ratio of the black garlic extract to the deuterated reagent is 0.1g to 0.6-1.0 mL.
9. The method according to claim 7, wherein the time of the vortex oscillation is 3-10 min; the ultrasonic time is 10-20 min; the centrifugation speed is 10000-12000 rpm, and the time is 10-20 min.
10. The method according to claim 7, wherein the conditions for nuclear magnetic resonance detection are: 5mm multi-core broadband observation probe, wherein the observation frequency is 600.13MHz, and the standard ZG30 pulse sequence is adopted for acquisition; the parameters are set as follows: the spectrum width SW is 19.8ppm, the center point O1P of the spectrum is 6.175ppm, the spectrum resolution FIDRES is 0.363Hz, and the scanning times NS is 16.
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| CN115631796A (en) * | 2022-10-13 | 2023-01-20 | 济宁市农业科学研究院 | Garlic biological fingerprint spectrum construction and identification method, terminal equipment and storage medium |
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| CN115631796A (en) * | 2022-10-13 | 2023-01-20 | 济宁市农业科学研究院 | Garlic biological fingerprint spectrum construction and identification method, terminal equipment and storage medium |
| CN115631796B (en) * | 2022-10-13 | 2024-04-09 | 济宁市农业科学研究院 | Garlic biological fingerprint construction and identification method, terminal equipment and storage medium |
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