Color developing card for qualitatively and quantitatively measuring degradation degree of burdock medicine fishy smell as well as preparation method and use method of color developing card

Abstract

The invention discloses an anthocyanidin color developing card for qualitatively and quantitatively detecting the degradation degree of burdock medicine fishy smell and a preparation and use method of the anthocyanidin color developing card. 12 anthocyanidin extracts are distributed on the color developing card, and each anthocyanidin extract at least comprises one of the following four pairs of anthocyanidin, the four pairs of anthocyanin are respectively (a) cyanidin-3-O-beta-D-glucoside and malvidine-3-O-glucoside, (b) cyanidin-3-O-beta-D-glucoside and (c) malvidine-3-O-glucoside; (b) malvidine-3-O-galactoside and paeoniflorin-3-O-arabinoside, and (b) a mixture of malvidine-3- (c) petunidin-3-O-glucoside and peonidin-3-O-glucoside, and (c) a mixture of petunidin-3-O-glucoside and And (d) peonidin-3-O-glucoside and malvidine-3-O-galactoside, which are used as main raw materials. Qualitative and quantitative detection of the degradation degree of the fishy smell of the burdock medicine is realized, and the color developing card for identifying the degradation degree of the fishy smell of the burdock medicine can be stored for a long time and is not limited by preparation and use at present.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, relates to an olfactory visualization technology, and in particular to an anthocyanin color developing card for qualitatively and quantitatively detecting the degradation degree of burdock odor, and a preparation and use method thereof. Background Art

[0002] Burdock is rich in nutrients such as protein, calcium, vitamin A, vitamin B, etc. It has many functions such as strengthening the spleen and stomach, clearing away heat and detoxifying, anti-oxidation, anti-fatigue, regulating immunity, protecting the liver, and promoting the growth of probiotics. It is a natural health product. Since the unique medicinal smell of burdock seriously affects consumers' acceptance of burdock products, the applicant has developed a probiotic fermentation process that can remove the medicinal smell of burdock. In this process, controlling the fermentation degree, that is, the degree of degradation of the medicinal smell of burdock, plays an extremely important role in the improvement of the flavor of burdock. However, the existing methods for judging the degree of degradation of the fishy smell of burdock have shortcomings: 1. Quality control personnel judge based on the changes in color, aroma and taste during the fermentation process of burdock, which has defects such as strong subjectivity, low accuracy and poor reproducibility; 2. Professional testers determine the content of the fishy smell components of burdock by solid phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS). This method has accurate results but requires large-scale experimental equipment, high cost and complex operation, and it is difficult to meet the real-time, fast and convenient requirements of actual production; 3. Olfactory visualization technology identification using porphyrin and pH indicator chemical color developer materials to make a visual sensor array (Chinese patent CN118347997B), but because porphyrin needs to be dissolved in toxic organic solvents such as dichloromethane, it has food safety risks. In addition, they need to be synthesized and purified, which is costly and brings environmental risks.

[0003] Anthocyanin is one of the most widely used natural pigments in the plant kingdom, with colors ranging from orange / red to purple / blue, almost touching the entire visible spectrum. The color development of anthocyanin is affected by the number of hydroxyl groups on the B ring, the position of 0-glycosylation, and the aromatic or aliphatic modification of glycosylation. The oxygen atom on the pyran ring is tetravalent and alkaline, and the phenolic hydroxyl group is acidic, so the structure of anthocyanin changes with the pH value of the medium and presents different colors. The presence of both acidic and alkaline volatile components in the volatile components of burdock will change the pH value, thereby causing the natural pigment to respond. Chinese patent CN202210165053.3 discloses a degradable plastic film with the function of preserving and visually monitoring the freshness of seafood and its preparation method, which realizes the full utilization of all components of sugarcane, that is, using sugarcane peel to prepare sugarcane peel anthocyanin, using sucrose to prepare polylactic acid, and using bagasse to prepare sugarcane fiber, and by effectively combining sugarcane peel anthocyanin, polylactic acid and sugarcane fiber, a degradable plastic film is obtained. Chinese patent CN202311607012.6 A kind of food freshness indicator film with visual polysaccharide base and its preparation method, adding different concentrations of red bean bilberry anthocyanin to agar polysaccharide base to prepare a natural and environmentally friendly food freshness indicator film, which has high sensitivity to the corruption gas NH3, obvious indication effect, strong antioxidant capacity, and better ability to maintain food freshness. Chinese patent CN202311860111.5 discloses a preparation method, equipment and application of a colorimetric microneedle sensor for meat freshness detection, using a deep learning algorithm to train a meat atlas with a sensor attached, forming a meat freshness classification model and constructing a device, uploading real-time images of meat with a sensor attached or storing photos, and detecting the freshness of the meat. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an anthocyanin colorimetric card for qualitatively and quantitatively detecting the degree of degradation of burdock's medicinal smell, and a method for qualitatively and quantitatively detecting the degree of degradation of burdock's medicinal smell using the colorimetric card.

[0005] Invention content: To solve the above technical problems, the present invention adopts the following technical solution: an anthocyanin colorimetric card for qualitatively and quantitatively detecting the degradation degree of burdock odor, wherein the anthocyanin colorimetric card is provided with 12 kinds of anthocyanin extracts, each of which contains at least one of the following four pairs of anthocyanins:

[0006] The 12 anthocyanin extracts are black bean peel extract, acai berry extract, blueberry extract, bilberry extract, bilberry extract, mulberry extract, roselle extract, black rice extract, cranberry extract, blue indigo fruit extract, elderberry extract, and Marselan grape extract;

[0007] The four pairs of anthocyanins are (a) cyanidin-3-O-β-D-glucoside and malvidin-3-O-glucoside; (b) malvidin-3-O-galactoside and peonidin-3-O-arabinoside; (c) petunidin-3-O-glucoside and peonidin-3-O-glucoside; (d) peonidin-3-O-glucoside and malvidin-3-O-galactoside.

[0008] Furthermore, the 12 anthocyanin extracts are obtained by extracting with a mixture of methanol and formic acid and then taking the supernatant after ultrasonic centrifugation.

[0009] Furthermore, the color development card is packed with aluminum foil and sealed with nitrogen.

[0010] Furthermore, the 12 anthocyanin extracts are arranged in a 4×3 lattice.

[0011] The present invention also provides a method for preparing the color developing card, wherein each anthocyanin extract is dissolved in ethanol respectively, and the anthocyanin extracts are sequentially spotted on a polyvinylidene fluoride (PVDF) membrane after ultrasonic vibration.

[0012] The present invention also provides a method for using the color developing card, comprising the following steps:

[0013] (1) Place the burdock sample and the color card in an odor-free environment and react for at least 15 minutes;

[0014] (2) Use image processing software to process the color card images before and after the reaction and generate a feature matrix;

[0015] (3) Input the feature matrix into the random forest RF discriminant model of the degradation degree of the burdock odor, and output the classification result of the degradation degree of the burdock odor;

[0016] (4) The partial least squares regression method (PLSR) was used to establish a regression model between the feature matrix and the contents of the three volatile components of burdock medicinal smell. The goodness of fit of the model was r 2 At 0.700-0.802, the content of the medicinal smell substance components can be determined by the color card; the three volatile components of the medicinal smell of burdock are hexanal, 2-isobutyl-3-methoxypyrazine and 2-sec-butyl-3-methoxypyrazine.

[0017] Furthermore, the burdock odor degradation degree discrimination model in step (3) is an RF model optimized by the frost and ice optimization algorithm RIME.

[0018] Furthermore, the optimal number of principal components of the random forest RF discriminant model in step (3) is set to 7.

[0019] Furthermore, the processing of the color card image before and after the reaction in step (2) includes removing the center point and edge of the anthocyanin extract.

[0020] Furthermore, the process of generating the feature matrix in step (2) is as follows: scanning the color card images before and after the reaction, using image processing software to perform median filtering to remove noise, and obtaining the final color difference image information, thereby generating a feature matrix.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It can accurately, quickly and conveniently qualitatively determine the degree of degradation of burdock medicinal smell, with an accuracy rate of up to 94.60%, and quantitatively determine the contents of three volatile components (hexanal, 2-methoxy-3-sec-butylpyrazine, and 2-methoxy-3-isobutylpyrazine) that form the burdock medicinal smell, thereby improving the detection method of the degree of degradation of burdock medicinal smell; 2. Anthocyanin screening rules suitable for burdock medicinal smell detection are proposed; 3. The color development card for identifying the degree of degradation of burdock medicinal smell can be stored at room temperature for 6 months, and the detection result is equivalent to that of the existing color development card, and is not limited to the current preparation and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photo of the anthocyanin extract color development card for identifying the degradation degree of burdock odor in the present invention; Figure 2 Characteristic images of 19 candidate anthocyanin extracts reacted with burdock samples; Figure 3 The molecular structure diagrams of 4 pairs of anthocyanins contained in the 12 anthocyanin extracts screened by the present invention; Figure 4 It is the characteristic image after the color card reacts with the burdock sample after fermentation for 1h-8h; Figure 5 Characteristic images of the color card and fermented burdock sample at a reaction time of 6min-20min; Figure 6 Schematic diagram of recognition accuracy of PSO-RF model when the number of principal component vectors is 7 and it runs 50 times; Figure 7 Schematic diagram of the recognition accuracy of the RIME-RF model when the number of principal component vectors is 7 and it is run 50 times; Figure 8 It is the color card for quantitative determination of hexanal by PLSR model; Fig. 9 This is the color card for quantitative determination of 2-isobutyl-3-methoxypyrazine by PLSR model; Fig.10 This is a color card for quantitative determination of 2-sec-butyl-3-methoxypyrazine using the PLSR model. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0024] Example 1: A colorimetric card for qualitatively and quantitatively determining the degradation degree of burdock odor

[0025] The color developing card of this embodiment has the following 12 kinds of anthocyanin extracts distributed thereon, which are obtained by screening from 19 kinds of anthocyanin extracts. The digital codes of the anthocyanin extracts are shown in the following Table 1:

[0026] Table 1

[0027] Each anthocyanin extract contains one or two of the following four pairs of anthocyanins: (a) cyanidin-3-O-β-D-glucoside and malvidin-3-O-glucoside (b) malvidin-3-O-galactoside and peonidin-3-O-arabinoside (c) petunidin-3-O-glucoside and peonidin-3-O-glucoside (d) peonidin-3-O-glucoside and malvidin-3-O-galactoside. The color card base is PVDF membrane, and the 12 anthocyanin extracts are arranged in a 4×3 dot matrix.

[0028] The general extraction method of the anthocyanin extract of the present invention is as follows: weigh 5.0 g of the raw material, grind and homogenize it, add a mixed extraction solvent of 50% methanol and 0.5% formic acid, and take the supernatant after ultrasonication and centrifugation to obtain an extract. The extraction step is repeated 3 times, and the volume of the extraction solvent added is 20, 20, and 10 mL respectively. Ultrasonic conditions: 20 ° C, 20 min, 100 W. Centrifugal conditions: 5000 r / min, 10 min, and the 3 extracts are combined.

[0029] Figure 2 To screen 12 anthocyanin extracts from 19 anthocyanins with obvious characteristic images and bright colors that react with fermented burdock samples as color card indicators. Figure 2 As shown, the 19 candidate anthocyanins are: 1, 4, 5, 6, 9, 10, 13, 18, 21, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35. The 12 selected anthocyanins are numbered 6, 10, 18, 21, 26, 28, 29, 30, 31, 32, 34, 35.

[0030] Table 2-1 and Table 2-2 show the anthocyanin composition of 12 anthocyanin extracts determined by HPLC, and the screening rule for anthocyanin extracts that can respond to the degree of degradation of burdock fishy smell is summarized as follows: containing 1 or 2 pairs of the above 4 pairs of anthocyanins.

[0031] Table 2-1 Anthocyanin composition of 12 selected anthocyanin extracts

[0032] Table 2-2 Anthocyanin composition of 12 selected anthocyanin extracts

[0033] ad refers to four pairs of anthocyanidins, (a) cyanidin-3-O-β-D-glucoside and malvidin-3-O-glucoside (b) malvidin-3-O-galactoside and peonidin-3-O-arabinoside (c) petunidin-3-O-glucoside and peonidin-3-O-glucoside (d) peonidin-3-O-glucoside and malvidin-3-O-galactoside.

[0034] Figure 3 These are four pairs of anthocyanin molecular structure diagrams. Figure 4 The characteristic images of the color cards prepared from the 12 selected anthocyanin extracts after reacting with burdock samples fermented for 1-8 hours. The anthocyanin extracts in the color cards can show different amounts and degrees of color change for burdock samples with different degrees of degradation of the medicinal smell. This shows that the 12 anthocyanin extracts are suitable for the preparation of color cards.

[0035] Example 2: Preparation method of a color developing card for qualitatively and quantitatively determining the degradation degree of burdock odor

[0036] Taking the color card described in Example 1 as an example, this embodiment provides a method for preparing a color card for qualitatively and quantitatively determining the degree of degradation of the fishy smell of burdock. Add 0.018 g of each of the 12 anthocyanin extracts mentioned above into a 5 ml centrifuge tube, add 3 ml of 100% ethanol, vortex, and ultrasonicate for 20 minutes to make a 6 mg / ml solution. According to the 4×3 dot matrix arrangement, spot the samples in sequence on a 3.6×4.1 cm PVDF membrane to make a color card. The distance from the edge of the anthocyanin extract in the color card to the edge of the color card is 0.4~0.9 cm to avoid the anthocyanin extract from being accidentally touched when the color card is taken.

[0037] Example 3: A method for using a color developing card for qualitatively and quantitatively determining the degradation degree of burdock odor

[0038] Based on the color development card of Example 1, this example provides a method for identifying the degradation degree of burdock odor, comprising the following steps: (1) Place 20 g of burdock sample fermented for 4 h and the color card in a culture dish (9 cm in diameter, 20 mm in height) in a ventilated and odor-free empty room, and stick the color card on the inside of the top cover of the culture dish. The reaction time is 6, 8, 10, 12, 14, 16, 18, and 20 minutes; (2) Use a scanner (Canon LiDE 300) to scan the color card images before and after the reaction, and use image processing software (MATLAB 2018b) to perform median filtering to remove noise, obtain the final color R, G, B differential image information, and generate the response feature matrix of the color card.

[0039] like Figure 5 ,Compared with other reaction times, the characteristic image of the ,fermented burdock sample for 16 min was obvious and bright in color, ,so 16 min was selected as the reaction time.

[0040] Example 4: A method for using a color developing card for qualitatively and quantitatively determining the degradation degree of burdock odor

[0041] The discrimination model of the degradation degree of burdock odor by the color card of the present invention is established based on a machine learning algorithm, specifically including: the model is trained and optimized by test data of 120×36 dimensions, the data comes from 120 fermented burdock samples (15 samples are fermented per hour, and samples are taken for fermentation for 1, 2, 3, 4, 5, 6, 7, and 8 hours), and each sample can obtain 36 feature image data (each sample has 12 color-sensitive materials, and each color-sensitive material has three feature differences of R, G, and B). The test data is subjected to dimensionality reduction analysis, the number of principal components with a cumulative variance contribution rate of 90% is selected for modeling analysis, and the test data is randomly divided into a training set and a prediction set at a ratio of 3:1. Three classification models (support vector machine SVM, random forest RF convolutional neural network CNN) are introduced for model training, and the accuracy of the prediction set is used as the judgment standard of the model training result. The present invention finally selects the random forest model with the best effect, adopts the RIME algorithm to optimize the RF model, takes the recognition error rate as the objective function, and finally establishes a discrimination model for the degradation degree of the burdock odor.

[0042] Based on the color development card of Example 1, the method for identifying the degradation degree of burdock odor in this example comprises the following steps: (1) Place 20 g of burdock samples fermented for 1, 2, 3, 4, 5, 6, 7, and 8 h in a culture dish (9 cm in diameter, 20 mm in height) in a ventilated, odor-free room with a color card. Stick the color card on the inside of the top cover of the culture dish and react for 16 min. (2) Scan the color card images before and after the reaction with a scanner (Canon LiDE 300), and use image processing software (MATLAB 2018b) to perform median filtering to remove noise, obtain the final color R, G, B differential image information, and generate the response feature matrix of the color card; (3) The characteristic matrix of the samples was input into the RIME-RF model for distinguishing the degradation degree of burdock odor, and the number of principal component vectors was set to 7. The samples fermented for 1, 2, 3, and 4 hours were classified as incomplete degradation, and the samples fermented for 5, 6, 7, and 8 hours were classified as complete degradation.

[0043] The classification accuracy of this model in identifying the degradation degree of burdock odor was 94.60%, see Table 1 and Figure 6 .

[0044] Example 5: Method for using a color developing card for qualitatively and quantitatively determining the degradation degree of burdock odor

[0045] Based on the use method and feature matrix of Example 3, this example provides a method for identifying the degradation degree of burdock odor. The difference between this example and Example 3 is that the discrimination model is an RF model optimized by a particle swarm optimization (PSO) algorithm, and the number of principal component vectors is set to 7; samples fermented for 1, 2, 3, and 4 hours are classified as incomplete degradation, and samples fermented for 5, 6, 7, and 8 hours are classified as complete degradation.

[0046] The classification accuracy of this model in identifying the degradation degree of burdock odor was 94.00%, see Table 3 and Figure 7 .

[0047] Table 3 shows the model qualitative discrimination accuracy

[0048] Example 6: A method for using a color developing card for qualitatively and quantitatively determining the degradation degree of burdock odor

[0049] The color development card of Example 1 was packed in aluminum foil and sealed with nitrogen and stored at room temperature.

[0050] This embodiment provides a method for identifying the degradation degree of burdock odor, comprising the following steps:

[0051] (1) Prepare 28 color development cards, wrap them in aluminum foil, seal them with nitrogen, and store them at room temperature;

[0052] (2) On the 7th, 15th, 30th, 60th, 90th, 120th, and 180th days of storage, four color development cards were taken out and four freshly prepared color development cards were used as controls. They were placed in a sealed container without odor to react with 20 g of burdock samples fermented for 4 hours.

[0053] (3) The feature matrix generation method is the same as in Example 3;

[0054] (4) The determination method is the same as that in Example 3.

[0055] As shown in Table 4, the values ​​in the table are the number of color cards with correct discrimination results. The correct number of discrimination results of the freshly prepared color cards for the degradation degree of the burdock odor is equivalent to the accurate number of the color cards stored for 1-6 months, indicating that the optimal storage time of the color cards for identifying the degradation degree of the burdock odor packed in aluminum foil and sealed with nitrogen is 6 months.

[0056] Table 4 The results of the nitrogen-filled aluminum foil packaging color card on the degradation of the burdock odor during storage

[0057] Example 7 A method for using a color developing card for qualitatively and quantitatively determining the degradation degree of burdock odor

[0058] Based on the method and feature matrix of Example 3, this example provides a method for quantitatively determining the content of burdock medicinal odor substances. The feature matrix of the fermentation 1-8h sample and the content of burdock medicinal odor substances (hexanal, 2-isobutyl-3-methoxypyrazine, 2-sec-butyl-3-methoxypyrazine) were used to establish a regression model through PLSR. The content of burdock medicinal odor substances changed during the fermentation process, as shown in Table 5.

[0059] Table 5 Changes in the content of burdock odor substances during the fermentation process

[0060] like Figure 8-10 As shown in Table 5, the training set r of hexanal 2 =0.8022, prediction set r 2 =0.7952; training set r of 2-isobutyl-3-methoxypyrazine 2 =0.7081, prediction set r2=0.7002; training set r2 of 2-sec-butyl-3-methoxypyrazine 2 =0.7422, prediction set r 2 =0.7133.

Claims

1. An anthocyanin colorimetric card for qualitatively and quantitatively detecting the degradation degree of burdock odor, characterized in that: The anthocyanin color developing card is distributed with 12 kinds of anthocyanin extracts, each of which contains at least one of the following four pairs of anthocyanins: The 12 anthocyanin extracts are black bean peel extract, acai berry extract, blueberry extract, bilberry extract, bilberry extract, mulberry extract, roselle extract, black rice extract, cranberry extract, blue indigo fruit extract, elderberry extract, and Marselan grape extract; The four pairs of anthocyanins are (a) cyanidin-3-O-β-D-glucoside and malvidin-3-O-glucoside; (b) malvidin-3-O-galactoside and peonidin-3-O-arabinoside; (c) petunidin-3-O-glucoside and peonidin-3-O-glucoside; (d) peonidin-3-O-glucoside and malvidin-3-O-galactoside.

2. The color developing card according to claim 1, characterized in that The 12 anthocyanin extracts are obtained by extracting a mixture of methanol and formic acid and then taking the supernatant after ultrasonic centrifugation.

3. The color developing card according to claim 1, characterized in that The color development card is packed with aluminum foil and sealed with nitrogen.

4. The color developing card according to claim 1, characterized in that The 12 anthocyanin extracts are arranged in a 4×3 dot matrix.

5. A method for preparing the color developing card according to claim 1, characterized in that: Each anthocyanin extract was dissolved in ethanol, and spotted on a polyvinylidene fluoride (PVDF) membrane in turn after ultrasonic vibration.

6. A method for using the color developing card according to claim 1, characterized in that: The following steps are involved: (1) Place the burdock sample and the color card in an odor-free environment and react for at least 15 minutes; (2) Use image processing software to process the color card images before and after the reaction and generate a feature matrix; (3) Input the feature matrix into the random forest RF discriminant model of the degradation degree of the burdock odor, and output the classification result of the degradation degree of the burdock odor; (4) The partial least squares regression method (PLSR) was used to establish a regression model between the feature matrix and the contents of the three volatile components of burdock medicinal smell. The goodness of fit of the model was r 2 At 0.700-0.802, the content of the medicinal smell substance components can be determined by the color card; the three volatile components of the medicinal smell of burdock are hexanal, 2-isobutyl-3-methoxypyrazine and 2-sec-butyl-3-methoxypyrazine.

7. The method of use according to claim 6, characterized in that: The burdock odor degradation degree discrimination model described in step (3) is the RF model optimized by the frost and ice optimization algorithm RIME.

8. The method of use according to claim 6, characterized in that: The optimal number of principal components of the random forest RF discriminant model in step (3) is set to 7.

9. The method of use according to claim 6, characterized in that: The processing of the color card image before and after the reaction in step (2) includes removing the center point and edge of the anthocyanin extract.

10. The method of use according to claim 6, characterized in that: The process of generating the feature matrix in step (2) is as follows: scanning the color card images before and after the reaction, using image processing software to perform median filtering to remove noise, and obtaining the final color difference image information, thereby generating the feature matrix.

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