Porphyra haitanensis producing area determination method based on mineral element fingerprint technology

Abstract

The invention discloses a porphyra haitanensis producing area determination method based on a mineral element fingerprint technology. The method comprises the following steps: measuring the mineral element content of a known porphyra haitanensis sample by using an inductively coupled plasma mass spectrometer, fitting a variable importance index VIP value by using an orthogonal partial least square discriminant method, taking the mineral element with the VIP value greater than 1.0 as a characteristic mineral element, and constructing a porphyra haitanensis producing area discriminant functional expression in combination with linear discriminant analysis. The origin of the porphyra haitanensis is traced, and the origin of an unknown porphyra haitanensis sample can be distinguished. The overall average discrimination accuracy of the porphyra haitanensis producing area by using the discrimination model is 94.2%, the discrimination accuracy is high, the stability is good, the repeatability is high, the uncertainty based on subjective identification and inherent labels of porphyra haitanensis is avoided, and the method has wide popularization and application values.

Description

Technical Field

[0001] The present invention belongs to the technical field of determining the true attributes of the origin of aquatic products, and more specifically, relates to a method for determining the origin of Porphyra haitanensis based on mineral elements. Background Art

[0002] Pyropia haitanensis is an important economic red algae widely distributed in coastal areas of China. It is widely regarded as a food with high nutritional value, rich in protein, vitamins and minerals, and can be used as a source of healthy substances such as high concentrations of porphyrins, vitamin B12 and taurine. In addition, the algae is rich in a variety of bioactive ingredients and has anti-cancer and anti-inflammatory properties.

[0003] In recent years, with the expansion of market demand for Porphyra haitanensis, the phenomenon of mixed origins has become increasingly prominent. The properties of Porphyra haitanensis from different origins are very similar after processing, which leads to illegal vendors often mixing inferior products with those from other origins, seriously affecting consumer rights and market stability. Therefore, it is particularly important to carry out research on the origin traceability of Porphyra haitanensis.

[0004] Since the production of agricultural products is closely related to its growth environment, the change rules of element composition vary greatly due to factors such as natural climate, topography, soil and biological metabolism type, which leads to certain regional differences in the content of various mineral elements in the same plant from different regions. This difference provides a unique identification for the origin of agricultural products and becomes an important reference indicator for the traceability of agricultural product origin. Based on this theory, the origin of agricultural products can be traced by detecting the content and type of mineral elements in agricultural products. The mineral element fingerprint technology, with its advantages of high sensitivity and rapid analysis, has become a powerful tool in the study of agricultural product origin traceability. In view of the different sea environments in which Porphyra haitanensis is cultivated, the content of various mineral elements in its algae is closely related to its growth environment, which will directly affect the content of various mineral elements in its algae. Therefore, it is highly feasible to effectively distinguish and trace the differences in the content of various mineral elements in Porphyra haitanensis from different main producing areas, and it can be used to establish the origin identification method and traceability system of Porphyra haitanensis.

[0005] Inductively coupled plasma mass spectrometry (ICP-MS) has become a widely used technical means in the field of mineral element determination due to its high sensitivity, wide linear range and ability to simultaneously determine multiple elements. However, in the research and application of origin traceability of Porphyra haitanensis, methods and related patents for traceability based on mineral element content are still relatively scarce. Existing identification technologies for Porphyra haitanensis mainly rely on its apparent characteristics, such as size, shape, color, gloss, moisture content and taste. Although these characteristics have certain identification value, they are difficult to achieve quantitative evaluation and have limited popularity. Therefore, developing an origin traceability technology for Porphyra haitanensis that is fast, accurate, quantifiable, easy to promote and suitable for statutory testing is of great significance for promoting the healthy development of the Porphyra haitanensis industry and ensuring food safety. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the technical gap in the origin tracing of Porphyra haitanensis, and provide a method for identifying the origin of Porphyra haitanensis based on mineral element fingerprint technology. The method uses an inductively coupled plasma mass spectrometer to determine the content of mineral elements in Porphyra haitanensis, uses the orthogonal partial least squares discriminant method to screen out mineral elements with VIP values ​​greater than 1.0 as characteristic mineral elements, and combines linear discriminant analysis to construct a discriminant function formula for the origin of Porphyra haitanensis to achieve the origin tracing of Porphyra haitanensis. When it is necessary to determine the origin of Porphyra haitanensis of unknown origin, it is only necessary to perform ICP-MS determination on its sample, and then input the determination result into the constructed discriminant function formula for the origin of Porphyra haitanensis, calculate the absolute value of each origin discriminant function result, and the origin corresponding to the maximum value of the absolute value of the result is the origin of the Porphyra haitanensis sample of unknown origin, thereby achieving accurate traceability and identification of the origin of Porphyra haitanensis.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] The present invention provides a method for tracing the origin of Porphyra haitanensis based on mineral element fingerprint technology, comprising the following steps:

[0009] S1: Collect samples of Porphyra haitanensis cultivated in five sea areas in Shandong, Jiangsu, Zhejiang, Fujian and Guangdong, and remove impurities, dry, crush and microwave digest;

[0010] S2: using an inductively coupled plasma mass spectrometer (ICP-MS) to determine the contents of mineral elements Mg, Fe, Cu, Zn, Ca, K, Na, Mn, S, P, B, Li, Al, Ni, Be, Cr, Ag, Co, Mo, Sn, As, Se, Cd, Sb, Ba, Ti, Pb, Si, Sr, and Zr in the Porphyra haitanensis sample;

[0011] S3: Use the orthogonal partial least squares discriminant method to fit the variable importance index VIP value, screen the mineral elements Mg, Cu, K, Na, P, Ag, Sn, Se, Cd, and Zr with VIP values ​​greater than 1.0 as characteristic mineral elements, and use the linear discriminant method to use the characteristic mineral elements to establish a discriminant function formula for the origin of Porphyra haitanensis as Shandong, Jiangsu, Zhejiang, Fujian, and Guangdong;

[0012] The discriminant function F1 of Shandong Porphyra is:

[0013] F1=0.024×Mg+9.62×Cu+0.00006394×K+0×Na+0.009×P-50.335×Ag+82.818×Sn+36.324×Se+18.739×Cd-48.072×Zr-148.307;

[0014] The discriminant function F2 of Jiangsu Porphyra is:

[0015] F2=0.013×Mg-3.239×Cu-0.002×K+0×Na+0.051×P-28.135×Ag+84.391×Sn+60.586×Se-13.718×Cd-4.507×Zr-168.524;

[0016] The discriminant function F3 of Zhejiang Porphyra is:

[0017] F3=0.016×Mg+17.983×Cu+0.001×K+0×Na+0.013×P-247.096×Ag+65.081×Sn+10.053×Se+18.965×Cd-36.337×Zr-212.975;

[0018] The discriminant function F4 of Porphyra haitanensis is:

[0019] F4=0.021×Mg-3.998×Cu-0.001×K+0.000778×Na+0.051×P-60.054×Ag+120.254×Sn+45.764×Se-28.302×Cd+25.854×Zr-148.307;

[0020] The discriminant function F5 of Porphyra haitanensis is:

[0021] F5=0.008×Mg-5.238×Cu-0.002×K+0.00225×Na+0.05×P+104.669×Ag+99.167×Sn+93.539×Se-10.007×Cd-14.242×Zr-160.519;

[0022] S4: Use the ICP-MS method in the step S2 to determine the content of the characteristic mineral elements in the sample of Porphyra haitanensis of unknown origin, substitute the content of the characteristic mineral elements into the calculation results of the discriminant function formulas F1, F2, F3, F4, and F5 in the step S3, in which the origins of Porphyra haitanensis are Shandong, Jiangsu, Zhejiang, Fujian, and Guangdong, and compare the absolute values ​​of the results of F1, F2, F3, F4, and F5. When the absolute value of F1 is the largest, the origin of the Porphyra haitanensis to be tested is Shandong; when the absolute value of F2 is the largest, the origin of the Porphyra haitanensis to be tested is Jiangsu; when the absolute value of F3 is the largest, the origin of the Porphyra haitanensis to be tested is Zhejiang; when the absolute value of F4 is the largest, the origin of the Porphyra haitanensis to be tested is Fujian; when the absolute value of F5 is the largest, the origin of the Porphyra haitanensis to be tested is Guangdong.

[0023] Preferably, the drying temperature of the Porphyra haitanensis sample is 60° C., and the sample is dried to a constant weight.

[0024] Preferably, the content of mineral elements in the Porphyra haitanensis sample is the dry weight concentration value of Porphyra haitanensis.

[0025] Preferably, in step S1, a nitric acid solution with a concentration of 65% is used to digest the Porphyra haitanensis sample.

[0026] Preferably, the working conditions of the microwave digestion in step S1 are: heating from room temperature to 180°C, power 1800W, 15 minutes; maintaining a constant temperature of 180°C, power 1800W, 25 minutes; cooling from 180°C to 55°C, power 0W, 20 minutes.

[0027] Preferably, the working conditions of the inductively coupled plasma mass spectrometer in step S2 are: nebulizing gas flow rate 0.7 L / min, auxiliary gas flow rate 0.8 L / min, radio frequency power 1200 W, and cooler flow rate 14 L / min.

[0028] Preferably, the mineral element content of the Porphyra haitanensis sample solution determined by ICP-MS in steps S2 and S4 is quantitatively analyzed by an external standard method, and the stability of the instrument is monitored with reference to the National Center for Standard Material Research standard GBW10021 (GSB-12).

[0029] Preferably, the modeling parameters of the orthogonal partial least squares discriminant method in step S3 are: 7 cross validation times and 200 maximum iterations.

[0030] In addition, the application of the method in distinguishing Porphyra haitanensis from different origins should also be within the protection scope of the present invention.

[0031] The present invention has the following beneficial effects:

[0032] The present invention proposes a method for tracing the origin of Porphyra haitanensis based on mineral element fingerprint technology. For the first time, the method of combining multiple mineral elements with chemometrics is applied to the determination of the origin of Porphyra haitanensis. By constructing an efficient classification and discrimination model, the fingerprint information of mineral elements is further analyzed, which can accurately reflect the unique geographical characteristics of Porphyra haitanensis from different origins. The overall average discrimination accuracy can reach 94.2% through verification of the discrimination model. The method has high detection accuracy, strong sensitivity and high reliability, and effectively avoids the uncertainty and limitations of traditional methods based on subjective identification and inherent labels.

[0033] The chemometrics technology used in the present invention can also identify important characteristic mineral elements that affect origin identification, providing a new perspective and strong technical support for optimizing the key indicators for tracing the origin of Porphyra haitanensis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings are incorporated in and constitute a part of this specification to provide a further understanding of the embodiments. The accompanying drawings illustrate embodiments and together with the description serve to explain the principles of the present invention.

[0035] Figure 1 A flowchart of an implementation of a specific embodiment is shown;

[0036] Figure 2 A score diagram of an OPLS-DA model for distinguishing Porphyra haitanensis from different origins according to a specific embodiment is shown;

[0037] Figure 3 A VIP score result of distinguishing Porphyra haitanensis from different origins is shown in a specific embodiment;

[0038] Figure 4 A scatter plot of FDA discrimination of Porphyra haitanensis from different origins in a training set of a specific embodiment is shown;

[0039] Figure 5 A confusion matrix diagram of FDA discrimination of Porphyra haitanensis from different origins in a validation set according to a specific embodiment is shown. DETAILED DESCRIPTION

[0040] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.

[0041] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] Example 1 Establishment and verification of a method for determining the origin of Porphyra haitanensis

[0043] Samples of Porphyra haitanensis from the sea areas of Shandong, Jiangsu, Zhejiang, Fujian and Guangdong were collected for impurity removal, drying, crushing and microwave digestion. The mineral element content was detected using an inductively coupled plasma mass spectrometer (ICP-MS). The variable importance index VIP value was fitted to the measurement results using orthogonal partial least squares discriminant analysis (OPLS-DA), and the mineral elements with VIP values ​​greater than 1.0 were screened as characteristic mineral elements. 70% of the collected samples were randomly selected as training set samples, and 30% of the samples were selected as validation set samples. The characteristic mineral elements of the training set samples were used in combination with linear discriminant analysis (FDA) to construct discriminant function formulas for five Porphyra haitanensis production areas. Finally, the characteristic mineral elements of the validation set samples were used to verify the discriminant function formula of the production area. The example process of this embodiment is as follows: Figure 1 As shown, the specific implementation steps are as follows:

[0044] 1. Collection of Porphyra haitanensis Samples

[0045] From September to December 2023, samples of Porphyra haitanensis were collected from the sea areas of Shandong, Jiangsu, Zhejiang, Fujian and Guangdong, including 9 strains each from Shandong and Jiangsu, 36 strains each from Zhejiang and Guangdong, and 63 strains from Fujian, for a total of 153 strains.

[0046] 2. Sample Pretreatment

[0047] First, the Porphyra haitanensis sample was removed from impurities, dried at 60°C to constant weight, and then pulverized; then 0.2 g (accurate to 0.0001 g) of the pretreated Porphyra haitanensis sample was weighed into a digestion inner tank; then 5 ml of 65% nitric acid solution was added for immersion, and digestion was performed according to the working conditions of the microwave digester shown in Table 1; finally, the clarified solution obtained after digestion was transferred into a 25 ml volumetric flask, the washing solution was combined, the volume was fixed with 1% nitric acid, and the mixture was mixed for use, and a reagent blank was made for a blank test.

[0048] Table 1: Microwave digestion instrument working conditions

[0049] stage Temperature / ℃ Power / W Time / min 1 (heating) Room temperature~180 1800 15 2 (keep) 180 1800 25 3 (cooling) 180～55 0 20

[0050] 3. Determination of mineral element content

[0051] ICP-MS was used to determine the content of mineral elements in Porphyra haitanensis. The instrument working conditions were: atomizing gas flow rate: 0.7L / min; auxiliary gas flow rate: 0.8L / min; radio frequency power: 1200W; cooling gas flow rate: 14L / min. The contents of 30 mineral elements Mg, Fe, Cu, Zn, Ca, K, Na, Mn, S, P, B, Li, Al, Ni, Be, Cr, Ag, Co, Mo, Sn, As, Se, Cd, Sb, Ba, Ti, Pb, Si, Sr, and Zr in the sample were determined. Quantitative analysis was performed using the external standard method. Each sample was tested 3 times, and the stability of the instrument was monitored by referring to the National Standard Material Research Center GBW10021 (GSB-12). 1% HNO 3 The mixed standard solutions with different concentration gradients were prepared by the medium and the standard curve was drawn. The results showed that the linear relationship of each mineral element to be tested was good and met the analysis requirements.

[0052] 4. Screening characteristic mineral elements and constructing a functional discrimination model for Porphyra haitanensis from different origins

[0053] The OPLS-DA model was used to analyze the 30 mineral element data of the above-mentioned Porphyra haitanensis samples from different origins. During the modeling process, the number of cross-validations was 7 and the maximum number of iterations was set to 200. The scoring results of the fitted OPLS-DA model are shown in Figure 2. Figure 2 As shown, the cumulative contribution of latent variables R 2 X=0.869,R 2 Y=0.801,Q 2 (cum) = 0.765, indicating that the model is stable and efficient, and the model is not overfitted. The established model can be applied to the identification of the origin of Porphyra haitanensis. The score chart shows that the samples of Porphyra haitanensis from Shandong, Jiangsu, Zhejiang, Fujian, and Guangdong can be classified into clusters, and the origin classification has obvious regional characteristics, proving that mineral elements can leave special fingerprints of Porphyra haitanensis in specific regions, which is an effective indicator for origin tracing.

[0054] In order to screen the multi-type characteristic mineral elements that affect the differentiation of Porphyra haitanensis production areas, the OPLS-DA model was fitted with the importance of variables (VIP) scores of 30 mineral elements. Figure 3 After screening, it was found that there were 10 weight factors with VIP values ​​greater than 1.0, namely P, Cu, Cd, Ag, Mg, K, Se, Na, Sn, and Zr, accounting for 30% of the total elements used for modeling, so P, Cu, Cd, Ag, Mg, K, Se, Na, Sn, and Zr were selected as characteristic mineral elements.

[0055] The above 10 characteristic mineral elements were substituted into SPSS, and the discriminant function formula of the five origins of Porphyra haitanensis was constructed using FDA analysis. The scatter distribution diagram is shown in Figure 4As shown in the figure, the discriminant function of the origin of Porphyra haitanensis is as follows:

[0056] The discriminant function F1 of Shandong Porphyra is:

[0057] F1=0.024×Mg+9.62×Cu+0.00006394×K+0×Na+0.009×P-50.335×Ag+82.818×Sn+36.324×Se+18.739×Cd-48.072×Zr-148.307;

[0058] The discriminant function F2 of Jiangsu Porphyra is:

[0059] F2=0.013×Mg-3.239×Cu-0.002×K+0×Na+0.051×P-28.135×Ag+84.391×Sn+60.586×Se-13.718×Cd-4.507×Zr-168.524;

[0060] The discriminant function F3 of Zhejiang Porphyra is:

[0061] F3=0.016×Mg+17.983×Cu+0.001×K+0×Na+0.013×P-247.096×Ag+65.081×Sn+10.053×Se+18.965×Cd-36.337×Zr-212.975;

[0062] The discriminant function F4 of Porphyra haitanensis is:

[0063] F4=0.021×Mg-3.998×Cu-0.001×K+0.000778×Na+0.051×P-60.054×Ag+120.254×Sn+45.764×Se-28.302×Cd+25.854×Zr-148.307;

[0064] The discriminant function F5 of Porphyra haitanensis is:

[0065] F5=0.008×Mg-5.238×Cu-0.002×K+0.00225×Na+0.05×P+104.669×Ag+99.167×Sn+93.539×Se-10.007×Cd-14.242×Zr-160.519.

[0066] Wherein, P, Cu, Cd, Ag, Mg, K, Se, Na, Sn, and Zr represent the concentration of the element.

[0067] Furthermore, after measuring the concentrations of the above 10 characteristic elements in the Porphyra haitanensis samples of unknown origin, the discriminant function is entered into the discriminant function, the results of the discriminant function are calculated, and the absolute values ​​of the results are compared. The origin corresponding to the largest absolute value of the result is the origin of the Porphyra haitanensis samples of unknown origin.

[0068] 5. Discriminant model verification

[0069] The collected Porphyra haitanensis samples were randomly divided into two parts: 30% of the samples were used as validation set samples, and 70% of the samples were used as training set samples. The model was constructed using the characteristic mineral element data of the training set samples, and the characteristic mineral element data of the validation set samples were introduced into the model to obtain the discrimination accuracy of the validation set samples. The accuracy of the production area classification was measured by the discrimination accuracy. The overall average discrimination accuracy of the validation set samples of the present invention is shown in Figure 5 It is shown as 94.2%. It can be shown that the origin discrimination model of Porphyra haitanensis constructed by the present invention can realize the accurate traceability and identification of Porphyra haitanensis from different origins, and has certain application value.

[0070] The above describes the specific implementation of the present application, but the implementation of the present invention is not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention shall be equivalent replacement methods and are included in the protection scope of the present invention. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A method for determining the origin of Porphyra haitanensis based on mineral element fingerprint technology, characterized in that: The following steps are involved: S1: Based on the linear discriminant method, the characteristic mineral elements of Porphyra haitanensis are used to establish a discriminant function formula for the origin of Porphyra haitanensis. The discriminant function formula is as follows: The discriminant function F1 of Shandong Porphyra is: F1=0.024×Mg+9.62×Cu+0.00006394×K+0×Na+0.009×P-50.335×Ag+82.818×Sn+36.324×Se+18.739×Cd-48.072×Zr-148.307; The discriminant function F2 of Jiangsu Porphyra is: F2=0.013×Mg-3.239×Cu-0.002×K+0×Na+0.051×P-28.135×Ag+84.391×Sn+60.586×Se-13.718×Cd-4.507×Zr-168.524; The discriminant function F3 of Zhejiang Porphyra is: F3=0.016×Mg+17.983×Cu+0.001×K+0×Na+0.013×P-247.096×Ag+65.081×Sn+10.053×Se+18.965×Cd-36.337×Zr-212.975; The discriminant function F4 of Porphyra haitanensis is: F4=0.021×Mg-3.998×Cu-0.001×K+0.000778×Na+0.051×P-60.054×Ag+120.254×Sn+45.764×Se-28.302×Cd+25.854×Zr-148.307; The discriminant function F5 of Porphyra haitanensis is: F5=0.008×Mg-5.238×Cu-0.002×K+0.00225×Na+0.05×P+104.669×Ag+99.167×Sn+93.539×Se-10.007×Cd-14.242×Zr-160.519; S2: Determine the content of the characteristic mineral elements in the Porphyra haitanensis sample of unknown origin, substitute the content of the characteristic mineral elements into the discriminant function formula of the Porphyra haitanensis origin and calculate the result, compare the absolute values ​​of the discriminant function formula results, and determine the origin corresponding to the maximum absolute value of the discriminant function formula results as the origin of the Porphyra haitanensis sample of unknown origin.

2. The method according to claim 1, characterized in that The variable importance index VIP value is fitted based on the orthogonal partial least squares discriminant method, and the elements with VIP values ​​greater than 1.0 in the mineral elements of Porphyra haitanensis are screened as the characteristic mineral elements.

3. The method according to claim 1 or 2, characterized in that: The characteristic mineral elements include Mg, Cu, K, Na, P, Ag, Sn, Se, Cd, and Zr.

4. The method according to claim 2, characterized in that The mineral elements of the porphyra haitanensis include Mg, Fe, Cu, Zn, Ca, K, Na, Mn, S, P, B, Li, Al, Ni, Be, Cr, Ag, Co, Mo, Sn, As, Se, Cd, Sb, Ba, Ti, Pb, Si, Sr, and Zr.

5. The method according to claim 2, characterized in that: The modeling parameters of the orthogonal partial least squares discriminant method are: 7 cross-validation times and 200 maximum iterations.

6. The method according to claim 2, characterized in that The determination of the content of mineral elements in Porphyra haitanensis comprises the following steps: A1: Samples of Porphyra haitanensis cultivated in the sea areas of Shandong, Jiangsu, Zhejiang, Fujian and Guangdong were collected for impurity removal, drying, crushing and microwave digestion treatment; A2: Using an inductively coupled plasma mass spectrometer to determine the content of mineral elements in the Porphyra haitanensis.

7. The method according to claim 6, characterized in that The drying temperature of the A1 Porphyra haitanensis sample is 60° C., and the sample is dried to a constant weight.

8. The method according to claim 6, characterized in that The working conditions of the A1 Porphyra zhonghaitanensis sample in the microwave digestion instrument are: heating from room temperature to 180°C, power 1800W, 15 minutes; maintaining a constant temperature of 180°C, power 1800W, 25 minutes; cooling from 180°C to 55°C, power 0W, 20 minutes.

9. The method according to claim 6, characterized in that The working conditions of the inductively coupled plasma mass spectrometer in A2 are: nebulizing gas flow rate 0.7 L / min, auxiliary gas flow rate 0.8 L / min, radio frequency power 1200 W, and cooling gas flow rate 14 L / min.

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