Rapid identification method for head and back muscles and tail muscles of salmon salmon

Abstract

The invention provides a rapid identification method for head and back muscles and tail muscles of salmon salmon, and belongs to the technical field of biological variety identification. The method comprises the following steps: respectively preparing samples of head and back muscles and tail muscles of the salmon salmon; respectively carrying out pretreatment and GC-IMS detection on the prepared sample to obtain each volatile component and peak intensity thereof; on the basis of the peak intensities of the volatile components, principal component analysis and orthogonal partial least square discriminant analysis are carried out respectively, the peak intensities with significant differences and the corresponding volatile components are screened out, and rapid identification of the head and back muscles and the tail muscles of the salmon salmon is achieved. According to the method, complex pretreatment is not needed, the samples can be directly detected and classified, the influence of pretreatment reagents on the samples is reduced, false positive results are reduced, the detection time is only 30 minutes, and the detection time is greatly shortened compared with traditional PCR and other technologies.

Description

Technical Field

[0001] The invention belongs to the technical field of biological species identification, and in particular relates to a method for quickly identifying head and back muscles and tail muscles of a seven-color salmon. Background Art

[0002] Colorful salmon (Salvelinus Fontinalis), scientifically known as American char, belongs to the Salmoniformes, Salmonidae, and genus Salmon. It is one of the five famous salmon along with lake salmon, Arctic char, dory salmon, and bull salmon. It is native to the Labrador region in eastern Canada. Because of its high crude fat and crude protein content in its muscles, especially the rich essential amino acids, which are higher than the FAO / WHO standards, it is a nutritionally balanced freshwater fish variety. Therefore, it can be used as a very ideal protein supplement source in the human daily diet and has a high edible value.

[0003] The nutritional content and muscle quality of fish are affected by many factors, such as the muscle part, nutrition, and environment of the fish. The nutrition and flavor of fish from different parts vary greatly. Colorful salmon are mainly sold as sashimi (raw fish) and smoked fish slices, and both methods generally require the fish skin to be peeled off and sliced ​​and sold directly or smoked. Colorful salmon meat is milky white, and the color of the fish meat on the back of the head and the color of the fish meat on the tail are difficult to distinguish through sensory inspection. Some unscrupulous vendors even use white spot trout meat to impersonate American trout meat, which harms the interests of consumers.

[0004] Studies have shown that the protein content of the head and back muscles of the seven-color salmon is 84.13-88.95% (dry basis content), and the protein content of the tail muscles is 69.35-75.39% (dry basis content); the fat content of the head and back muscles is 5.81-9.42% (dry basis content), and the fat content of the tail muscles is 2.46-5.02% (dry basis content). The head and abdominal muscles are rich in monounsaturated fatty acids, with oleic acid accounting for the highest 93.18% of monounsaturated fatty acids, while oleic acid accounts for 79.26% of monounsaturated fatty acids in the tail muscles. Oleic acid can regulate blood lipid levels, lower cholesterol, effectively reduce the occurrence of hypercholesterolemia and cardiovascular diseases, and reduce the risk of coronary heart disease. It can be seen that the nutritional value of the head and back muscles is higher than that of the tail muscles. Therefore, different parts of the seven-color salmon have their own characteristics. If a zoning pricing strategy is implemented according to the parts, on the one hand, it can improve the income level of fishermen, and on the other hand, it can better protect the interests of consumers and encourage consumers to make accurate purchases.

[0005] At present, there are many technical methods for the authenticity identification of fish products, such as near-infrared spectroscopy, denaturing high-performance liquid chromatography, enzyme-linked immunosorbent assay, two-dimensional electrophoresis, AFLP markers (amplified fragment length polymorphism), PCR-RFLP (restriction fragment length polymorphism) and DNA barcode technology. However, they all have certain defects, such as near-infrared needs to rely on a large number of basic experiments to establish a database; high-performance liquid chromatography and enzyme-linked immunosorbent assay require complex pre-treatment and may produce false positives; electrophoresis, PCR, etc. require a long experimental time and analysis time cycle. However, the technology for distinguishing different parts of fish meat has not been reported. Therefore, it is of great application significance to establish a method for rapid identification of the head and back muscles and tail muscles of seven-color salmon. Summary of the invention

[0006] The present invention provides a rapid identification method for the head and back muscles and tail muscles of the seven-color salmon. The method adopts a volatile fingerprint spectrum and can quickly and reliably distinguish the head and back muscles and tail muscles of the seven-color salmon. At the same time, the method does not require complex pre-treatment, can directly detect and classify the samples, reduces the influence of the pre-treatment reagents on the samples themselves, reduces false positive results, and the detection time is only 30 minutes, which greatly shortens the detection time compared with traditional PCR and other technologies.

[0007] In order to achieve the above object, the present invention provides a method for quickly identifying the head and back muscles and tail muscles of a seven-color salmon, comprising the following steps:

[0008] Prepare samples of head and back muscles and tail muscles of discus salmon respectively;

[0009] The prepared samples were pre-treated and tested by GC-IMS to obtain the volatile components and their peak intensities;

[0010] Based on the peak intensities of the above-mentioned volatile components, principal component analysis and orthogonal partial least squares discriminant analysis were performed respectively to screen out peaks with significant differences in peak intensity and their corresponding volatile components, thereby achieving rapid identification of the head and back muscles and tail muscles of the rainbow salmon.

[0011] Preferably, the prepared samples of the head and back muscles and tail muscles of the rainbow salmon are peeled and cut into 1-2 mm pieces.

[0012] Preferably, the prepared samples are pre-processed respectively, specifically:

[0013] Weigh 3 g of sample into a 20 mL headspace vial and incubate at 40-45 °C and 250 rpm for 20 min;

[0014] Use a 45°C headspace injection needle to inject 800 μL of gas headspace into the MXT-WAX capillary column.

[0015] Preferably, the prepared gas samples are subjected to GC-IMS detection respectively, and the detection conditions are:

[0016] Use nitrogen as carrier gas and set the flow rate to 2 mL / min for 2 min, then increase to 15 mL / min within 8 min, 50 mL / min within 5 min, 100 mL / min within 5 min, and 150 mL / min within 5 min, and maintain for 5 min until stopping;

[0017] The pretreated gas sample was driven from the ionization source to the ionization chamber in the positive ion mode, using a 9.8 cm drift tube operated at 45 °C, 150 mL / min nitrogen flow rate, and the capillary column temperature was 60 °C.

[0018] Preferably, for distinguishing the head and back muscles of rainbow salmon, there are 15 peaks with significantly different peak intensities, and the corresponding 15 volatile components are α-phellandrene, γ-pinene, 2-heptanone, N-nitroso-N-ethylaniline, methyl cinnamate, ethyl hexanoate (monomer), ethyl hexanoate (dimer), 2-methylpropanol, styraxyl alcohol, butyl isovalerate, 4-isopropylbenzyl alcohol, ethyl propionate, 4-heptanone, n-butanol and camphor terpene.

[0019] Preferably, for distinguishing the tail muscles of rainbow salmon, there are 9 peaks with significantly different peak intensities, and the corresponding 9 volatile components are n-propanol, cis-jasmone, 3-nonanone, hydroxyacetone, 3-hydroxy-2-butanone, 2-phenyl-2-butenal, phenylpropionaldehyde, diethyl malonate and 4,5-dimethylthiazole.

[0020] Preferably, the peaks with significantly different peak intensities and their corresponding volatile components are specifically:

[0021]

[0022]

[0023] The present invention also provides a method for quickly identifying whether commercially available fish meat belongs to the head and back muscles or the tail muscles of the seven-colored salmon, comprising the following steps:

[0024] Take commercially available skinless colorful salmon meat and cut it into 1-2 mm pieces as samples;

[0025] The sample to be tested is subjected to the pretreatment as described in the above technical solution and the GC-IMS detection as described in the above technical solution to obtain each volatile component and its peak intensity;

[0026] Based on the peak intensities of the above-mentioned volatile components, principal component analysis and orthogonal partial least squares discriminant analysis are performed respectively to screen out peaks with significant differences in peak intensity and their corresponding volatile components, and the screened volatile components and their peak intensities are compared with the corresponding volatile components and their peak intensities with significant differences in peak intensity in the above-mentioned technical scheme, so as to quickly determine whether the sample to be tested belongs to the head and back muscles or the tail muscles of the rainbow salmon.

[0027] Preferably, the detection time of the method is 30 minutes and the accuracy rate reaches 100%.

[0028] Compared with the prior art, the advantages and positive effects of the present invention are:

[0029] 1. The present invention provides a method for rapid identification of the head and back muscles and tail muscles of rainbow salmon. The method adopts a volatile fingerprint spectrum, which can mark the gas phase mass spectrometry spectrum of the common peaks of each component in the sample, and can quickly and reliably distinguish the head and back muscles and tail muscles of rainbow salmon, greatly reducing false positive results.

[0030] 2. This method does not require complicated pre-treatment and can directly detect and classify samples, reducing the impact of pre-treatment reagents on the samples themselves. When this method is used for daily testing, the detection time is only 30 minutes, which greatly shortens the detection time compared with traditional PCR and other technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the numbering of the parts of the seven-colored salmon sample provided in Example 1 of the present invention;

[0032] Figure 2 A 3D fingerprint of volatile components of different parts of seven-color salmon provided in Example 1 of the present invention;

[0033] Figure 3 A gallery of volatile components of different parts of the seven-color salmon provided in Example 1 of the present invention, wherein: each Indicates a volatile substance. The redder and larger the dot is, the higher the content of the substance is. The color of the dot changes from yellow to light blue, indicating that the content of the substance in the corresponding sample gradually decreases. The order of dot identification from high to low content can be expressed as

[0034] Figure 4 A schematic diagram of using PCA analysis of volatile components to classify the head, back and tail muscles of seven-color salmon into two categories provided in Example 1 of the present invention;

[0035] Figure 5 A 3D fingerprint of volatile components of different parts of seven-color salmon provided in Example 2 of the present invention;

[0036] Figure 6 A gallery of volatile components of different parts of the seven-color salmon provided in Example 2 of the present invention, wherein: each Indicates a volatile substance. The redder and larger the dot is, the higher the content of the substance is. The color of the dot changes from yellow to light blue, indicating that the content of the substance in the corresponding sample gradually decreases. The order of dot identification from high to low content can be expressed as

[0037] Figure 7 A schematic diagram of using volatile component PCA analysis to divide the head, back and tail muscles of rainbow salmon into two categories provided in Example 2 of the present invention.

[0038] Figure 8 The principal component analysis and discrimination results of volatile components in muscles of different parts of seven-color salmon provided in the rapid detection application of the present invention. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1

[0041] Sample preparation:

[0042] Take the skinned muscles of different parts of the rainbow salmon (No. 1) (No. AH, head and back: AD and tail: EH) and cut them into 1-2 mm pieces. Figure 1 shown.

[0043] Sample preparation:

[0044] Weigh 3 g into a 20 mL headspace bottle and incubate at 40°C, 250 rpm for 20 min. Use a 45°C headspace injection needle to inject 800 μL of gas headspace into a MXT-WAX (15 m × 0.53 mm, 1.0 μm df) capillary column.

[0045] Sample GC-IMS detection:

[0046] Use nitrogen (purity 99.99%) as carrier gas, set the flow rate at 2 mL / min, continue for 2 min, then increase to 15 mL / min within 10 min, increase to 50 mL / min within 15 min, increase to 100 mL / min within 20 min, increase to 150 mL / min within 25 min, and maintain for 50 min until stopping;

[0047] The pretreated gas sample was driven from the ionization source to the ionization chamber in the positive ion mode, using a 9.8 cm drift tube operated at 45 °C, 150 mL / min nitrogen flow rate, and the capillary column temperature was 60 °C.

[0048] GC-IMS test results:

[0049] Qualitative analysis of volatile substances: The volatile organic compound spectra collected from the head and back muscles and tail muscles of the seven-color salmon were analyzed using Vocal0.4.03 software. Figure 2 Using the GC-IMS Library Search software, the unknown volatile components in the sample were qualitatively analyzed in two dimensions using the 2020 NIST gas phase retention index database and IMS database built into the software. The results are shown in Figure 3 and Table 1-2. Figure 3 The volatile components of different parts of the seven-color salmon (numbered AH) detected by GC-IMS are shown to be 38 in total, including 14 common volatile components and 24 distinguishing volatile components. Table 1 shows the 24 volatile components detected for different parts to distinguish the head and back muscles and tail muscles of the seven-color salmon and their corresponding peak intensities. Table 2 shows the qualitative data of the 24 volatile components and the peak intensity judgment range obtained based on different experimental examples.

[0050] Quick identification:

[0051] Based on the peak intensities of the above volatile components, principal component analysis and orthogonal partial least squares discriminant analysis were performed to screen out the peaks with significantly different peak intensities and their corresponding volatile components, so as to achieve rapid identification of the head and back muscles and tail muscles of the seven-color salmon. Figure 3 shown.

[0052] Among them, there are 15 peaks with significantly different peak intensities for distinguishing the head and back muscles of discus salmon, and the corresponding 15 volatile components are α-phellandrene, γ-pinene, 2-heptanone, N-nitroso-N-ethylaniline, methyl cinnamate, ethyl hexanoate (monomer), ethyl hexanoate (dimer), 2-methylpropanol, styraxyl alcohol, butyl isovalerate, 4-isopropylbenzyl alcohol, ethyl propionate, 4-heptanone, n-butanol and camphor terpene.

[0053] For distinguishing the tail muscles of the seven-color salmon, there are 9 peaks with significantly different peak intensities, and the corresponding 9 volatile components are n-propanol, cis-jasmone, 3-nonanone, hydroxyacetone, 3-hydroxy-2-butanone, 2-phenyl-2-butenal, phenylpropanal, diethyl malonate and 4,5-dimethylthiazole. Table 2 shows the qualitative parameters of the volatile components with significant differences obtained through screening and the corresponding peak intensity ranges summarized for the head and back muscles and tail muscles.

[0054]

[0055]

[0056]

[0057] Example 2

[0058] Sample preparation:

[0059] The skinned muscles of different parts (numbers AH, head and back: AD and tail: EH) of the rainbow salmon (number 2) were cut into pieces of 1-2 mm.

[0060] Sample preparation:

[0061] Weigh 3 g into a 20 mL headspace bottle and incubate at 45°C, 250 rpm for 20 min. Use a 45°C headspace injection needle to inject 800 μL of gas sample headspace into a MXT-WAX (15 m × 0.53 mm, 1.0 μm df) capillary column.

[0062] Sample GC-IMS detection:

[0063] Use nitrogen (purity 99.99%) as carrier gas, set the flow rate at 2 mL / min, continue for 2 min, then increase to 15 mL / min within 8 min, increase to 50 mL / min within 5 min, increase to 100 mL / min within 5 min, increase to 150 mL / min within 5 min, and maintain for 5 min until stopping;

[0064] The pretreated gas sample was driven from the ionization source to the ionization chamber in the positive ion mode, using a 9.8 cm drift tube operated at 45 °C, 150 mL / min nitrogen flow rate, and the capillary column temperature was 60 °C.

[0065] GC-IMS test results:

[0066] Qualitative analysis of volatile substances: Vocal0.4.03 was used to perform differential analysis on the volatile organic compound profiles of the head and back muscles and tail muscles of the seven-color salmon. Figure 5Using the GC-IMS Library Search software, the unknown volatile components in the sample were qualitatively analyzed in two dimensions using the 2020 NIST gas phase retention index database and IMS database built into the software. The results are shown in Figure 6 and Table 3. Figure 6 The volatile components of different parts of the red salmon (numbered AH) detected by GC-IMS are 38 in total, including 14 common volatile components and 24 distinguishing volatile components. Table 3 shows the 24 volatile components detected for different parts to distinguish the head and back muscles and tail muscles of the red salmon and their corresponding peak intensities.

[0067] Quick identification:

[0068] Based on the peak intensities of the above volatile components, principal component analysis and orthogonal partial least squares discriminant analysis were performed to screen out the peak intensities with significant differences and their corresponding volatile components, so as to achieve rapid identification of the head and back muscles and tail muscles of the seven-color salmon. Figure 7 shown.

[0069] Among them, there are 15 peaks with significant differences in distinguishing the head and back muscles of discus salmon, and the corresponding 15 volatile components are α-phellandrene, γ-terpinene, 2-heptanone, N-nitroso-N-ethylaniline, methyl cinnamate, ethyl hexanoate (monomer), ethyl hexanoate (dimer), 2-methylpropanol, styraxyl alcohol, butyl isovalerate, 4-isopropylbenzyl alcohol, ethyl propionate, 4-heptanone, n-butanol and camphor terpene.

[0070] For distinguishing the tail muscles of the seven-color salmon, there are 9 peaks with significant differences, and the corresponding 9 volatile components are n-propanol, cis-jasmone, 3-nonanone, hydroxyacetone, 3-hydroxy-2-butanone, 2-phenyl-2-butenal, phenylpropanal, diethyl malonate and 4,5-dimethylthiazole. The above results are the same as those in Example 1. The qualitative parameters of the volatile components with significant differences obtained by screening and the corresponding peak intensity ranges summarized for the head and back muscles and tail muscles are shown in Table 2.

[0071]

[0072]

[0073] Rapid detection application

[0074] Unknown parts of seven-color salmon were taken, numbered corresponding to unknown 1-4, and the fish sample preparation, pretreatment and GC-IMS detection conditions were the same as those in the above-mentioned Example 2. After 30 minutes of detection, the detection results are shown in Table 4.

[0075] Table 4

[0076]

[0077] Based on the peak intensity data of each volatile component in Table 3, principal component analysis and orthogonal partial least squares discriminant analysis were performed, and compared with the peak intensities with significant differences and their corresponding volatile components screened out in the present invention, as shown in FIG. Figure 8 As shown, unknown fish 1 and 2 can be quickly identified as the back muscles of the head of the rainbow salmon, and unknown fish 3 and 4 are the tail muscles of the rainbow salmon, with a discrimination rate of 100%.

[0078] Comparative Example 1

[0079] In the document "Construction and validation of a multiplex PCR detection system for identifying six common edible meats" (Jiang Zhiwei, Xia Ruocheng, Tao Ruiyang, et al. Construction and validation of a multiplex PCR detection system for identifying six common edible meats [J]. Journal of Forensic Medicine, 2024, 40(3): 254-260.), a rapid, accurate and sensitive multiplex PCR detection method was established to simultaneously identify six common edible meats (cattle, sheep, chicken, pig, goose, duck) and evaluate its application value in the identification of adulteration of meat food. This method requires a DNA extraction kit and a fluorescence quantifier, and the sequencing work needs to be assisted by a third-party testing company (Shanghai Bioengineering). The experimental process involves primer design, sample DNA extraction, PCR product amplification, electrophoresis detection, sequencing, etc. The procedure is complicated, the PCR procedure time is about 75 minutes, and DNA may be degraded, resulting in PCR amplification failure.

[0080] Comparative Example 2

[0081] The document "Study on the Identification of Hippocampus abdominalis Leeson Based on Trait Identification and Specific PCR Technology" uses trait identification and polymerase chain reaction (PCR) technology to study the main seahorse cultured product in my country, Hippocampus abdominalis Leeson, to provide data support for the development of identification technology for this species. Based on the cytochrome C oxidase subunit I (COI) gene sequence of Hippocampus abdominalis Leeson and other seahorses, the specific single nucleotide polymorphism (SNP) sites of Hippocampus abdominalis Leeson were screened, and primers PFHM-Primer-F and PFHM-Primer-R were designed exclusively for Hippocampus abdominalis Leeson, which can quickly identify Hippocampus abdominalis Leeson and provide a reference for the identification and subsequent development of new seahorse resources in my country. The experiment requires the use of complex equipment such as gradient PCR instrument, PowerPac universal electrophoresis instrument, gel imaging system, etc. The CO I universal primer PCR amplification time of sample DNA is 26 minutes, and the seahorse-specific PCR amplification time is 55 minutes, but when the template amount is too high, the PCR amplification efficiency will be inhibited, resulting in false negative results.

[0082] In summary, the two comparative examples respectively utilize multiplex PCR and classic COI gene sequence screening SNP technology to identify edible meat and seahorse species. Although they have similar patent purposes to the present application, compared with them, the method of the present invention has simple pre-treatment and convenient operation, which can greatly shorten the detection time and has a higher judgment rate.

Claims

1. A method for quickly identifying the head and back muscles and tail muscles of a seven-color salmon, characterized in that: The following steps are involved: Prepare samples of head and back muscles and tail muscles of discus salmon respectively; The prepared samples were pre-treated and tested by GC-IMS to obtain the volatile components and their peak intensities; Based on the peak intensities of the above-mentioned volatile components, principal component analysis and orthogonal partial least squares discriminant analysis were performed respectively to screen out peaks with significant differences in peak intensity and their corresponding volatile components, thereby achieving rapid identification of the head and back muscles and tail muscles of the rainbow salmon.

2. The rapid identification method according to claim 1, characterized in that: The prepared samples of the head and back muscles and tail muscles of the rainbow salmon were skinned and cut into 1-2 mm pieces.

3. The rapid identification method according to claim 1, characterized in that: The prepared samples were pre-processed separately, specifically: Weigh 3 g of sample into a 20 mL headspace vial and incubate at 40-45 °C and 250 rpm for 20 min; Use a 45°C headspace injection needle to inject 800 μL of gas headspace into the MXT-WAX capillary column.

4. The rapid identification method according to claim 3, characterized in that: The prepared samples were subjected to GC-IMS detection under the following conditions: Use nitrogen as carrier gas and set the flow rate to 2 mL / min for 2 min, then increase to 15 mL / min within 8 min, 50 mL / min within 5 min, 100 mL / min within 5 min, and 150 mL / min within 5 min, and maintain for 5 min until stopping; The pretreated samples were driven from the ionization source to the ionization chamber in the positive ion mode, using a 9.8 cm drift tube operated at 45 °C, 150 mL / min nitrogen flow rate, and the capillary column temperature was 60 °C.

5. The rapid identification method according to claim 1, characterized in that: For distinguishing the head and back muscles of rainbow salmon, there are 15 peaks with significant differences in peak intensity, and the corresponding 15 volatile components are α-phellandrene, γ-terpinene, 2-heptanone, N-nitroso-N-ethylaniline, methyl cinnamate, ethyl hexanoate (monomer), ethyl hexanoate (dimer), 2-methylpropanol, styraxyl alcohol, butyl isovalerate, 4-isopropylbenzyl alcohol, ethyl propionate, 4-heptanone, n-butanol and camphor terpene.

6. The rapid identification method according to claim 1, characterized in that: For distinguishing the tail muscles of discus salmon, there are 9 peaks with significant differences in peak intensity, and the corresponding 9 volatile components are n-propanol, cis-jasmone, 3-nonanone, hydroxyacetone, 3-hydroxy-2-butanone, 2-phenyl-2-butenal, phenylpropanaldehyde, diethyl malonate and 4,5-dimethylthiazole.

7. The rapid identification method according to claim 5 or 6, characterized in that: The peaks with significant differences in peak intensity and their corresponding volatile components are as follows: 。 8. A method for quickly identifying whether commercially available fish meat belongs to the head and back muscles or the tail muscles of a red salmon, characterized in that: The following steps are involved: Take commercially available skinless colorful salmon meat and cut it into 1-2 mm pieces as samples; The sample to be tested is subjected to the pretreatment as claimed in claim 3 and the GC-IMS detection as claimed in claim 4 to obtain each volatile component and its peak intensity; Based on the peak intensities of the above-mentioned volatile components, principal component analysis and orthogonal partial least squares discriminant analysis are performed respectively to screen out peaks with significant differences in peak intensity and their corresponding volatile components, and the screened volatile components and their peak intensities are compared with the corresponding volatile components and their peak intensities with significant differences in peak intensity in claim 7, so as to quickly determine whether the sample to be tested belongs to the head and back muscles or the tail muscles of the rainbow salmon.

9. The method according to claim 8, characterized in that The detection time of the method is 30 minutes, and the accuracy rate reaches 100%.

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