Rapid identification method for muscles of rainbow trout, golden trout and coloured trout

Abstract

The invention provides a rapid identification method for muscles of rainbow trout, golden trout and coloured trout, and belongs to the technical field of biological variety identification. The method comprises the following steps: respectively preparing muscle samples of rainbow trout, golden trout and coloured trout; 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 intensity of each volatile component, principal component analysis and orthogonal partial least square discriminant analysis are respectively carried out, the peak intensity with significant difference and the corresponding volatile component are screened out, and rapid identification of muscles of rainbow trout, golden trout and coloured trout is realized. According to the method, complex pretreatment is not needed, samples can be directly detected and classified, the influence of a pretreatment reagent on the samples is reduced, rainbow trout meat, golden trout meat and coloured trout meat can be rapidly and reliably distinguished, false positive results are reduced, the detection time is only 30 minutes, and compared with PCR, molecular marker and other technologies, the detection time is greatly shortened.

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 the muscles of rainbow trout, golden trout and rainbow salmon. Background Art

[0002] Among the salmon and trout farmed varieties, rainbow trout (Oncorhynchus mykiss), golden trout (Golden Oncorhynchusmykiss) and colorful salmon (Salvelinus fontinalis) are relatively common. Among them, rainbow trout is the largest farmed in my country and belongs to the genus Oncorhynchus; golden trout is a mutant of rainbow trout, which is a golden strain obtained through breeding; colorful salmon, scientifically known as American char, belongs to the genus Salvelinus, is a kind of euryhaline freshwater salmon, native to eastern Canada. The nutritional content and muscle quality of fish are affected by many factors such as fish species, nutrition, environment, etc. Different species of fish have different nutrition and flavor.

[0003] The phenomenon of mislabeling fish products and selling inferior products as good ones still exists, which seriously damages the rights and interests of consumers. At present, it is difficult for ordinary consumers to distinguish the rainbow trout, golden trout and rainbow salmon circulating in the market. There are many technical methods for identifying the authenticity 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 barcoding technology. However, there are 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 have false positives; electrophoresis, PCR, etc. require a long experimental time and analysis time cycle. Therefore, it is of great application significance to establish a fast and efficient method for identifying different varieties of salmon and trout. Summary of the invention

[0004] The present invention provides a method for rapid identification of rainbow trout, golden trout and red salmon meat. The method adopts volatile fingerprints and can provide objective quantitative standards for the distinction and grading of different varieties of salmon and trout. At the same time, the method does not require complex pre-treatment, can directly detect and classify samples, reduces the influence of pre-treatment reagents on the samples themselves, can quickly and reliably distinguish rainbow trout, golden trout and red salmon meat, 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.

[0005] In order to achieve the above object, the present invention provides a method for rapid identification of rainbow trout, golden trout and red salmon muscle, comprising the following steps:

[0006] Prepare muscle samples of rainbow trout, golden trout, and discus salmon respectively;

[0007] The prepared samples were pre-treated and tested by GC-IMS to obtain the volatile components and their peak intensities;

[0008] 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 rainbow trout, golden trout and rainbow salmon muscles.

[0009] Preferably, the muscle samples of rainbow trout, golden trout and rainbow salmon are prepared by peeling the upper back muscles and cutting them into 1-2 mm pieces.

[0010] Preferably, the prepared samples are pre-processed respectively, specifically:

[0011] Weigh 3 g of sample into a 20 mL headspace vial and incubate at 40 °C and 250 rpm for 20 min;

[0012] Use a 45°C headspace injection needle to inject 800 μL of gas headspace into the MXT-WAX capillary column.

[0013] Preferably, the prepared samples are subjected to GC-IMS detection respectively, and the detection conditions are:

[0014] 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;

[0015] The pretreated samples were driven from the ionization source to the ionization chamber in positive ion mode, using a 9.8 cm drift tube operated at 45 °C, 150 mL / min nitrogen flow rate, and a capillary column temperature of 60 °C.

[0016] Preferably, for distinguishing rainbow trout, there are 9 peaks with significant differences in peak intensity, and the corresponding 9 volatile components are pentanol, 3-pentanone, phenylacetaldehyde, o-cresol, trans-2-pentenoic acid, n-hexanal, 3-nonanone, citronellal and 2-ethyl-6-methylpyrazine.

[0017] Preferably, for distinguishing golden trout, there are two peaks with significant differences in peak intensity, and the corresponding two volatile components are 2,6-dimethylaniline and α-pinene, respectively.

[0018] Preferably, for distinguishing the rainbow salmon, there are three peaks with significant differences in peak intensity, and the corresponding three volatile components are p-isopropylbenzyl alcohol, isopentanol and phenylacetic acid.

[0019] Preferably, the peak intensities corresponding to the volatile components having significant differences in peak intensities are specifically:

[0020] Volatile components Golden Trout Colorful salmon rainbow trout Amyl alcohol 780.69-980.26 714.08-939.67 2272.95-2703.59 3-Pentanone 440.98-491.90 503.28-701.85 1389.99-1408.19 Phenylacetaldehyde 1238.98-1729.71 876.42-1125.46 2685.85-3209.49 o-Cresol 1252.91-1696.78 1293.40-1633.56 3773.51-3965.02 n-Hexanal 747.41-775.18 411.05-579.35 858.71-1317.96 trans-2-pentenal 504.68-530.99 454.05-568.95 751.83-845.21 3-Nonanone 164.72-224.04 178.67-222.39 890.61-1148.33 Citronellal 44.81-57.15 28.19-48.69 269.90-312.30 2-Ethyl-6-methylpyrazine 111.54-166.92 136.18-153.27 551.76-712.07 2,6-Dimethylaniline 584.89-1205.58 188.77-330.13 337.63-375.78 α-Pinene 717.88-801.59 302.65-321.08 421.95-466.86 p-Isopropylbenzyl alcohol 1819.63-2922.86 5921.26-7096.29 2973.02-5298.82 Isoamyl alcohol 3827.09-4009.74 4906.98-5002.7 1862.83-2436.66 Phenylacetic acid 5972.41-7374.88 7482.18-8299.78 697.90-750.93

[0021] The present invention also provides a method for quickly identifying whether commercially available fish meat is rainbow trout, golden trout or rainbow salmon, comprising the following steps:

[0022] The upper back muscles of commercially available fish were skinned and cut into 1-2 mm pieces as samples;

[0023] 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;

[0024] 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 rainbow trout, golden trout or discus salmon.

[0025] Preferably, the detection time of the method is 30 minutes, and the discrimination rate reaches above 94%.

[0026] Compared with the prior art, the advantages and positive effects of the present invention are:

[0027] 1. The present invention provides a method for rapid identification of rainbow trout, golden trout and rainbow salmon muscles. The method adopts volatile fingerprints and can indicate the gas phase mass spectrometry of the common peaks of each component in the sample, which can provide an objective and quantitative standard for distinguishing and grading different varieties of salmon and trout.

[0028] 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. It can quickly and reliably distinguish the muscles of rainbow trout, golden trout and discus salmon, greatly reducing false positive results.

[0029] 3. When this method is used for daily testing, the detection time only takes 30 minutes, which greatly shortens the detection time compared with traditional PCR and other technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The three-dimensional response spectrum of volatile components of muscle samples of different species of salmon and trout provided in Example 1 of the present invention;

[0031] Figure 2 The fingerprints of volatile components of different salmon and trout muscles provided in Example 1 of the present invention;

[0032] Figure 3 The principal component analysis and discrimination results of the volatile components of different salmon and trout muscles provided in Example 2 of the present invention;

[0033] Figure 4 The three-dimensional response spectrum of volatile components of muscle samples of different species of salmon and trout provided in Example 2 of the present invention;

[0034] Figure 5 The fingerprints of volatile components of different salmon and trout muscles provided in Example 2 of the present invention;

[0035] Figure 6 The principal component analysis and discrimination results of the volatile components of different salmon and trout muscles provided in Example 2 of the present invention;

[0036] Figure 7 The principal component analysis and discrimination results of volatile components of different salmon and trout muscles provided in the rapid detection application of the present invention. DETAILED DESCRIPTION

[0037] 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.

[0038] Example 1

[0039] Sample preparation:

[0040] The upper back muscles of three kinds of fish, namely rainbow trout (No. 1), golden trout (No. 1) and rainbow salmon (No. 1), were skinned and cut into 1-2 mm pieces.

[0041] Sample preparation:

[0042] 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.

[0043] Sample GC-IMS detection:

[0044] Use nitrogen (purity 99.999%) 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;

[0045] The pretreated samples were driven from the ionization source to the ionization chamber in positive ion mode, using a 9.8 cm drift tube operated at 45 °C, 150 mL / min nitrogen flow rate, and a capillary column temperature of 60 °C.

[0046] GC-IMS test results:

[0047] Qualitative analysis of volatile substances: The volatile organic compounds of rainbow trout, golden trout and colorful salmon meat were analyzed by Vocal 0.4.03. Figure 1 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 2 and Table 1.

[0048] like Figure 2 As shown, 33 volatile components were detected from the three fish by GC-IMS (see relevant information in columns 1-7 of Table 1), of which rainbow trout, golden trout and rainbow salmon meat had a total of 8 volatile components, and 25 volatile substances could be used to distinguish different fish.

[0049] Quick identification:

[0050] Based on the peak intensity of the above volatile components, principal component analysis and orthogonal partial least squares discriminant analysis were performed, such as Figure 3 As shown, peaks with significant differences in peak intensity and their corresponding volatile components were screened out to achieve rapid identification of rainbow trout, golden trout, and red salmon muscles, with a discrimination rate of 94.44%.

[0051] Among them, there are 9 peaks with significant differences in peak intensity for distinguishing rainbow trout, and the corresponding 9 volatile components are pentanol, 3-pentanone, phenylacetaldehyde, o-cresol, trans-2-pentenoic acid, n-hexanal, 3-nonanone, citronellal and 2-ethyl-6-methylpyrazine.

[0052] For distinguishing golden trout, there are two peaks with significant differences in peak intensity, and the corresponding two volatile components are 2,6-dimethylaniline and α-pinene.

[0053] For distinguishing the rainbow salmon, there are three peaks with significant differences in peak intensity, and the corresponding three volatile components are p-isopropylbenzyl alcohol, isopentanol and phenylacetic acid.

[0054]

[0055]

[0056] Example 2

[0057] Sample preparation:

[0058] The upper back muscles of three kinds of fish, rainbow trout (No. 2-4), golden trout (No. 2-4) and rainbow salmon (No. 2-4), were skinned and cut into 1-2 mm pieces.

[0059] Sample preparation:

[0060] 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 sample headspace into a MXT-WAX (15 m × 0.53 mm, 1.0 μm df) capillary column.

[0061] Sample GC-IMS detection:

[0062] 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;

[0063] The pretreated samples were driven from the ionization source to the ionization chamber in positive ion mode, using a 9.8 cm drift tube operated at 45 °C, 150 mL / min nitrogen flow rate, and a capillary column temperature of 60 °C.

[0064] GC-IMS test results:

[0065] Qualitative analysis of volatile substances: Vocal0.4.03 was used to perform differential analysis of volatile organic compounds between rainbow trout, golden trout and colorful salmon meat. Figure 4 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 5 and Table 2.

[0066] like Figure 5As shown, 33 volatile components were detected in the three fish by GC-IMS (see relevant information in columns 1-7 of Table 2), of which rainbow trout, golden trout and rainbow salmon had a total of 8 volatile components, and 25 volatile substances could be used to distinguish different fish.

[0067] Quick identification:

[0068] Based on the peak intensity of the above volatile components, principal component analysis and orthogonal partial least squares discriminant analysis were performed, such as Figure 6 As shown, peaks with significant differences in peak intensity and their corresponding volatile components were screened out to achieve rapid identification of rainbow trout, golden trout, and rainbow salmon muscles with a discrimination rate of 100%.

[0069] Among them, there are 9 peaks with significant differences in peak intensity for distinguishing rainbow trout, and the corresponding 9 volatile components are pentanol, 3-pentanone, phenylacetaldehyde, o-cresol, trans-2-pentenoic acid, n-hexanal, 3-nonanone, citronellal and 2-ethyl-6-methylpyrazine.

[0070] For distinguishing golden trout, there are two peaks with significant differences in peak intensity, and the corresponding two volatile components are 2,6-dimethylaniline and α-pinene.

[0071] For distinguishing the rainbow salmon, there are three peaks with significant differences in peak intensity, and the corresponding three volatile components are p-isopropylbenzyl alcohol, isopentanol and phenylacetic acid.

[0072] The above results are the same as in Example 1.

[0073]

[0074]

[0075]

[0076] Rapid detection application

[0077] Unknown fish (corresponding to one of golden trout, rainbow trout and colorful salmon) were selected and numbered AF respectively, and the fish sample preparation, pretreatment and GC-IMS detection conditions were the same as those in the above-mentioned Example 1. After 30 minutes of detection, the detection results are shown in Table 3.

[0078] Table 3

[0079]

[0080]

[0081] 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 7 As shown, unknown fish A and F can be quickly identified as golden trout, unknown fish D and E as rainbow trout, and unknown fish B and C as rainbow salmon, with a discrimination rate of 100%.

[0082] Comparative Example 1

[0083] 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.

[0084] Comparative Example 2

[0085] 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.

[0086] 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 rapid identification of rainbow trout, golden trout and red salmon muscle, characterized in that: The following steps are involved: Prepare muscle samples of rainbow trout, golden trout, and 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 rainbow trout, golden trout and rainbow salmon muscles.

2. The rapid identification method according to claim 1, characterized in that: The muscle samples of rainbow trout, golden trout and rainbow salmon were prepared by removing the skin of the upper back muscles and cutting them into pieces of 1-2 mm.

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 °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 positive ion mode, using a 9.8 cm drift tube operated at 45 °C, 150 mL / min nitrogen flow rate, and a capillary column temperature of 60 °C.

5. The rapid identification method according to claim 1, characterized in that: For distinguishing rainbow trout, there are 9 peaks with significant differences in peak intensity, and the corresponding 9 volatile components are pentanol, 3-pentanone, phenylacetaldehyde, o-cresol, trans-2-pentenoic acid, n-hexanal, 3-nonanone, citronellal and 2-ethyl-6-methylpyrazine.

6. The rapid identification method according to claim 1, characterized in that: For distinguishing golden trout, there are two peaks with significant differences in peak intensity, and the corresponding two volatile components are 2,6-dimethylaniline and α-pinene.

7. The rapid identification method according to claim 1, characterized in that: For distinguishing the rainbow salmon, there are three peaks with significant differences in peak intensity, and the corresponding three volatile components are p-isopropylbenzyl alcohol, isopentanol and phenylacetic acid.

8. The rapid identification method according to any one of claims 5 to 7, characterized in that: The peak intensities corresponding to the volatile components with significant differences in peak intensity are as follows: 。 9. A method for quickly identifying whether commercially available fish meat is rainbow trout, golden trout or rainbow salmon, characterized in that: The following steps are involved: The upper back muscles of commercially available fish were skinned and cut 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 8, so as to quickly determine whether the sample to be tested belongs to rainbow trout, golden trout or discus salmon.

10. The method according to claim 9, characterized in that The detection time of the method is 30 minutes, and the discrimination rate reaches more than 94%.

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