Method for identifying cooked fresh live tai-shan red-scaled fish

Abstract

The application discloses a method for identifying cooked fresh Tai Shan red scale fish, and belongs to the field of food science and technology. Acetic acid, octanal, pentanol, trans-2-hexenal, 2-heptanone, 1-penten-3-ol, trans-2-pentenal, trans-2-butenal, 3-octanone, ethyl hexanoate, butyl butyrate and butyl propionate are used as markers of the cooked fresh Tai Shan red scale fish. When the above-mentioned 12 kinds of markers are contained in a to-be-detected sample at the same time and the peak area of each marker is in a specific range, the sample is determined as the cooked fresh Tai Shan red scale fish. The application has simple pretreatment, and only needs to cut the fish meat into pieces and then directly sample, so that the detection can be completed within 30 minutes, and the cooked fresh Tai Shan red scale fish, cooked frozen Tai Shan red scale fish and cooked fresh Ma-kou fish can be rapidly and accurately identified.

Description

Technical Field

[0001] This invention relates to the field of food science and technology, and in particular to a method for identifying cooked, fresh, live Taishan red-scaled fish. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The Taishan Red-scaled Fish is a rare and precious fish species unique to the Taishan region, prized for its tender flesh and delicious flavor, giving it high commercial value. Currently, it is mainly sold live or cooked live in the market. Due to preservation costs and seasonality, live red-scaled fish commands a much higher price than frozen ones. Many restaurants price their fish high by highlighting its deep-fried live form, but the frying process significantly alters the fish's appearance, making it difficult for consumers and market regulators to determine the source—whether it's live, frozen, or other low-priced fish—based solely on sensory characteristics. This practice of passing off inferior products as superior ones not only harms consumer rights but also disrupts the normal order of the red-scaled fish market.

[0004] Existing methods for identifying fish meat mainly include DNA barcoding, protein electrophoresis, and stable isotope tracing. While DNA barcoding has high species specificity, it is cumbersome, time-consuming, and costly, making it difficult to meet the market's demand for rapid testing. Protein electrophoresis requires high sample freshness and pretreatment; protein denaturation during cooking can render this method ineffective. Stable isotope tracing is primarily used for origin identification and struggles to distinguish between fresh and frozen raw materials. In recent years, gas chromatography-ion mobility spectrometry (GC-IMS) has begun to be applied to food flavor analysis and quality evaluation; however, there is currently no mature technical solution for using this technology to identify the origin of cooked fish meat, particularly for the specific species *Taishan red-scaled fish* and its differentiation from other fish.

[0005] Therefore, there is an urgent need in this field for a method that can quickly and accurately identify cooked fresh Taishan red-scaled fish in order to solve the technical problem of the incompatibility of fish meat appearance after cooking and the difficulty in tracing the source of raw materials. Summary of the Invention

[0006] In view of this, the present invention provides a method for identifying cooked fresh Taishan red-scaled fish. The method utilizes gas chromatography-ion mobility spectrometry to screen the volatile characteristic components in the flesh of cooked fresh red-scaled fish, cooked frozen red-scaled fish, and cooked fresh miscellaneous fish (mouthmouth fish). Based on the characteristic components in the samples, the method can quickly identify cooked fresh, cooked frozen Taishan red-scaled fish, and cooked fresh miscellaneous fish (mouthmouth fish).

[0007] In a first aspect, the present invention provides a method for identifying cooked fresh Taishan red-scaled fish, characterized in that acetic acid, octanal, pentanol, trans-2-hexenal, 2-heptanone, 1-penten-3-ol, trans-2-pentenal, trans-2-butenal, 3-octanone, ethyl hexanoate, butyl butyrate, and butyl propionate are used as markers for cooked fresh Taishan red-scaled fish.

[0008] Preferably, gas chromatography-ion mobility spectrometry is used to identify the marker by the peak volume corresponding to the marker.

[0009] Preferred peak areas of the markers for cooked live Taishan red-scaled fish were as follows: acetic acid 6045.830-8327.408, octanal 1417.380-2175.266, pentanol 2428.995-4180.104, trans-2-hexenal 1881.649-4975.1, 2-heptanone 1488.532-1619.304, 1-penten-3-ol 2621.940-3716.484, and trans-2-pentenal 107. 2.655-4721.987, trans-2-butenal 1634.036-1709.620, 3-octanone 84.191-107.165, ethyl hexanoate 52.475-369.650, butyl butyrate 308.888-847.594, butyl propionate 405.055-610.390; when the flesh of the Taishan red-scaled fish contains the above markers and the peak areas are within the corresponding peak area ranges, it is considered a cooked, fresh Taishan red-scaled fish.

[0010] Preferably, sample pretreatment is performed before gas chromatography-ion mobility spectrometry analysis; The sample pretreatment specifically involves taking cooked Taishan red-scaled fish meat, cutting it into small pieces, and obtaining the sample to be tested.

[0011] Preferably, the cooked Taishan red-scaled fish is directly deep-fried.

[0012] Preferably, the cooked Taishan red-scaled fish includes cooked fresh Taishan red-scaled fish, cooked frozen Taishan red-scaled fish, and cooked fresh chub.

[0013] Preferably, the fish meat includes at least one of back fish meat or belly fish meat.

[0014] Preferably, the gas chromatography-ion mobility spectrometry (GC-IMS) uses headspace injection after incubation. The conditions for headspace sampling are: Incubation temperature: 75-85℃; Incubation time: 10-20min; Injection volume: 500 µL; Splitless injection; Incubation speed: 400-600 r / min; Injection needle temperature: 100-110℃.

[0015] The preferred gas chromatography determination conditions are as follows: Analysis time: 30 min; Column type: MXT-WAX capillary column (15 m × 0.53 mm, 1.0 μm); Column temperature: 55-65℃; Carrier gas: high-purity nitrogen; Injector temperature: 75-90℃.

[0016] Preferably, the measurement conditions for ion mobility spectrometry are as follows: ion migration tube length is 50-55 mm, electric field strength is 480-520 V / cm, temperature is 40-50℃; the drift gas is high-purity nitrogen, and the drift velocity of the high-purity nitrogen is 70-80 mL / min. The chromatographic carrier gas flow rate program was set as follows: 0 to 2 minutes, 2 mL / min; 2 to 10 minutes, increasing from 2 mL / min to 10 mL / min; 10 to 20 minutes, increasing from 10 mL / min to 100 mL / min; 20 to 30 minutes, maintaining at 100 mL / min.

[0017] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) This invention is the first to discover that a combination of 12 volatile substances, namely acetic acid, octanal, pentanol, trans-2-hexenal, 2-heptanone, 1-penten-3-ol, trans-2-pentenal, trans-2-butenal, 3-octanone, ethyl hexanoate, butyl butyrate, and butyl propionate, is stably present in cooked fresh Taishan red-scaled fish with peak areas within a specific range. However, the content levels of these markers differ significantly between cooked frozen red-scaled fish and cooked fresh mixed fish. The above markers cover a variety of flavor compounds, including aldehydes, ketones, alcohols, acids, and esters, and can reflect the freshness and varietal characteristics of raw materials from different metabolic pathways. The combined use avoids the misjudgment that may be caused by a single marker, giving the identification method good specificity and reliability.

[0018] (2) This invention provides a rapid and reliable method for identifying cooked fresh red-scaled fish. Based on the differential analysis method of volatile compounds in fish meat, gas chromatography-ion mobility spectrometry is used to detect volatile compounds in the meat of cooked fresh and frozen red-scaled fish and fresh miscellaneous fish (mouthmouth fish). Potential characteristic markers are found based on the peak volume of the reaction ion peaks. By identifying the composition and content of volatile organic substances in the sample, cooked fresh and frozen red-scaled fish or miscellaneous fish (mouthmouth fish) can be rapidly identified. This method has simple pretreatment and good separation effect, short detection time (completed within 30 minutes), and high feasibility. It can achieve rapid and accurate identification of cooked fresh and cooked frozen Taishan red-scaled fish and other cooked miscellaneous fish (mouthmouth fish) through characteristic markers of volatile substances, and has certain market value. Attached Figure Description

[0019] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0020] Figure 1 The GC-IMS two-dimensional spectra of cooked fresh Taishan red-scaled fish, cooked frozen Taishan red-scaled fish, and cooked fresh Makou fish in Example 1 are shown. Figure 2 The GC-IMS differential spectra of cooked fresh Taishan red-scaled fish, cooked frozen Taishan red-scaled fish, and cooked fresh Makou fish provided in Example 1 are shown. Detailed Implementation

[0021] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this application are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or as per the product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this application. The preferred embodiments and materials described herein are for illustrative purposes only.

[0023] The equipment used in this invention includes: a gas chromatography-ion mobility spectrometry system; and a 0.01 AL204 electronic analytical balance (Mettler-Toledo Instruments (Shanghai)).

[0024] Example 1 (1) Sample pretreatment Group A (Cooked Fresh Group): 30 female or male fish were randomly selected from the collected Taishan red-scaled fish. 15 fish were randomly selected, stunned with ice water, and then slaughtered. The fish were rinsed under running water (the water temperature should not exceed 15℃), and the surface moisture was patted dry with absorbent paper. After the oil temperature in the pan was raised, the fish were deep-fried for 2 minutes. After frying, the fish were removed and cooled at room temperature (25℃). 1g of back and belly meat (both with skin) were taken from each fish, chopped, and placed in headspace vials. Three replicates were prepared for each group.

[0025] Group B (cooked and frozen group): 15 fish were randomly selected from the collected Taishan red-scaled fish, stunned with ice water, slaughtered, rinsed under running water (washing water temperature not exceeding 15℃), and the surface moisture of the fish was patted dry with absorbent paper. After freezing at -18℃ for 3 months, the fish were taken out and thawed for 20 minutes. They were then deep-fried for 2 minutes using the same method. After cooling, 1g of back and belly meat (both with skin) were taken, chopped, and placed in headspace vials. Three replicates were prepared for each group.

[0026] Group C (Cooked Fresh Mandarin Fish Group): 30 female or male mandarin fish were randomly selected from the collected mandarin fish. 15 fish were randomly selected, stunned with ice water, and then slaughtered. The fish were rinsed under running water (temperature not exceeding 15℃) and the surface moisture was patted dry with kitchen absorbent paper. After the oil temperature in the pan was raised, the fish were fried for 2 minutes. After frying, the fish were removed and cooled at room temperature (25℃) to obtain cooked fresh samples. After cooling, 1g of back and belly meat (both with skin) were taken, chopped, and placed in headspace vials. Three replicates were prepared for each group.

[0027] (2) GC-IMS determination The determination was performed using gas chromatography-ion mobility spectrometry (GC-IMS), with headspace injection after incubation.

[0028] The headspace injection conditions were as follows: incubation temperature 80℃; incubation time 15 min; injection volume 500 µL; splitless injection; incubation speed 500 r / min; injection needle temperature 105℃.

[0029] The gas chromatography conditions were as follows: column temperature 60℃; carrier gas was high-purity nitrogen (purity ≥99.999%); carrier gas flow rate program was set as follows: 0 to 2 min, 2 mL / min; 2 to 10 min, linearly increasing from 2 mL / min to 10 mL / min; 10 to 20 min, linearly increasing from 10 mL / min to 100 mL / min; 20 to 30 min, holding at 100 mL / min; injection port temperature 80℃. Gas chromatography conditions are shown in Table 1. Table 1 Gas Chromatography Conditions

[0030] The ion mobility spectrometry conditions were as follows: the ionization source was a tritium source (³H); the migration tube length was 53 mm; the electric field strength was 500 V / cm; the migration tube temperature was 45 °C; the drift gas was high-purity nitrogen (purity ≥99.999%), the flow rate was 75.0 mL / min; the positive ion mode was used; the gate opening time was 100 μs and the gate closing voltage was 90 dgt.

[0031] Place the headspace vial in the sample tray of the autosampler and perform GC-IMS analysis under the conditions described above to collect spectral data of volatile components.

[0032] (3) Results and Analysis The GC-IMS spectra of samples from groups A, B, and C were compared and analyzed. The LAV (Laboratory Analytical Viewer) was used to analyze the differences in volatile organic compounds (VOCs) between cooked fresh and cooked frozen Taishan red-scaled fish. The GC-IMS Library Search software was used to qualitatively identify the unknown volatile components in the samples. Two-dimensional spectra and difference spectra of the volatile components were generated using plugins such as Reporter, Gallery Plot, and Dynamic PCA in the VOCal data processing software for comparison of VOCs between samples. The results are shown below. Figure 1-2 See Table 2. The data in Table 2 are the average values ​​from three parallel experiments.

[0033] Figure 1 Note: (1) The background of the entire figure is blue, and the red vertical line at the horizontal axis 1.0 is the RIP peak (reaction ion peak, normalized).

[0034] (2) The vertical axis represents the retention time (s) of gas chromatography, and the horizontal axis represents the relative migration time (normalized, au).

[0035] (3) Each point on either side of the RIP peak represents a volatile organic compound. The color represents the peak intensity of the substance, ranging from blue to red, with darker colors indicating greater peak intensity.

[0036] As shown in Table 2, the qualitative analysis information of differential volatile substances in cooked fresh, cooked frozen Taishan red-scaled fish and cooked fresh horse mackerel includes: compound name, GAS number, molecular weight, retention index, retention time, migration time, peak volume of different samples, etc. The peak volume of each substance is different, which means that there are differences in the substances among different samples.

[0037] Based on the gas chromatographic retention time and IMS migration time of volatile substances, 99 characteristic volatile components (including dimers and trimers of some substances) of cooked fresh Taishan red-scaled fish, cooked frozen Taishan red-scaled fish, and cooked fresh Makou fish were identified. Among them, the characteristic components of cooked fresh Taishan red-scaled fish, cooked frozen Taishan red-scaled fish, and cooked fresh Makou fish obtained by random screening in this example showed significant differences. Moreover, the peak volumes of characteristic markers in cooked fresh Taishan red-scaled fish, namely acetic acid, octanal, pentanol, trans-2-hexenal, 2-heptanone, 1-penten-3-ol, trans-2-pentenal, trans-2-butenal, 3-octanone, ethyl hexanoate, butyl butyrate, and butyl propionate, were all within the range of characteristic markers, thus proving the effectiveness of the method.

[0038] Table 2 Qualitative volatile compounds of cooked fresh Taishan red-scaled fish, cooked frozen Taishan red-scaled fish, and cooked fresh chub.

[0039]

[0040]

[0041]

[0042] To further visually compare the differences in volatile aroma components of fish meat processed using different techniques and varieties, the spectrum of sample A-1 was selected as a reference. The spectra of other samples were subtracted from the reference spectrum to obtain a comparison chart of the differences between the samples, as shown below. Figure 2 As shown, if the volatile organic compound content in the target sample and the reference is the same, the background after subtraction is white, while red indicates that the concentration of the substance in the target sample is higher than that in the reference, and blue indicates that the concentration of the substance in the target sample is lower than that in the reference.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for identifying cooked, fresh, live Taishan red-scaled fish, characterized in that, Acetic acid, octanal, pentanol, trans-2-hexenal, 2-heptanone, 1-penten-3-ol, trans-2-pentenal, trans-2-butenal, 3-octanone, ethyl hexanoate, butyl butyrate, and butyl propionate were used as markers for cooked fresh Taishan red-scaled fish.

2. The method according to claim 1, characterized in that, The markers were identified by the peak volume corresponding to the markers using gas chromatography-ion mobility spectrometry.

3. The method according to claim 1, characterized in that, The peak areas of the markers for cooked fresh Taishan red-scaled fish were acetic acid 6045.830-8327.408, octanal 1417.380-2175.266, pentanol 2428.995-4180.104, trans-2-hexenal 1881.649-4975.1, 2-heptanone 1488.532-1619.304, 1-penten-3-ol 2621.940-3716.484, and trans-2-pentenal 1072. 655-4721.987, trans-2-butenal 1634.036-1709.620, 3-octanone 84.191-107.165, ethyl hexanoate 52.475-369.650, butyl butyrate 308.888-847.594, butyl propionate 405.055-610.390; when the flesh of the Taishan red-scaled fish contains the above markers and the peak areas are within the corresponding peak area ranges, it is considered a cooked, fresh Taishan red-scaled fish.

4. The method according to claim 1, characterized in that, Sample pretreatment is performed before gas chromatography-ion mobility spectrometry analysis; The sample pretreatment specifically involves taking cooked Taishan red-scaled fish meat, cutting it into small pieces, and obtaining the sample to be tested.

5. The method according to claim 4, characterized in that, The cooked Taishan red-scaled fish is directly deep-fried.

6. The method according to claim 4, characterized in that, The cooked Taishan red-scaled fish includes cooked fresh Taishan red-scaled fish, cooked frozen Taishan red-scaled fish, and cooked fresh chub.

7. The method according to claim 4, characterized in that, The fish meat includes at least one of the back meat or the belly meat.

8. The method according to claim 2, characterized in that, The gas chromatography-ion mobility spectrometry (GC-IMS) method used headspace injection after incubation. The conditions for headspace sampling are: Incubation temperature: 75-85℃; Incubation time: 10-20min; Injection volume: 500 µL; Splitless injection; Incubation speed: 400-600 r / min; Injection needle temperature: 100-110℃.

9. The method according to claim 2, characterized in that, The determination conditions for gas chromatography are as follows: Analysis time: 30 min; Column type: MXT-WAX capillary column (15 m × 0.53 mm, 1.0 μm); Column temperature: 55-65℃; Carrier gas: high-purity nitrogen; Injector temperature: 75-90℃.

10. The method according to claim 1, characterized in that, The measurement conditions for ion mobility spectrometry are as follows: ion migration tube length is 50-55 mm, electric field strength is 480-520 V / cm, temperature is 40-50℃; the drift gas is high-purity nitrogen, and the drift velocity of the high-purity nitrogen is 70-80 mL / min. The chromatographic carrier gas flow rate program was set as follows: 0 to 2 minutes, 2 mL / min; 2 to 10 minutes, increasing from 2 mL / min to 10 mL / min; 10 to 20 minutes, increasing from 10 mL / min to 100 mL / min; 20 to 30 minutes, maintaining at 100 mL / min.

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