Method for rapidly detecting odor active substances in fish meat

Abstract

The invention provides a method for rapidly detecting odor active substances in fish meat, and belongs to the technical field of detection of the odor active substances in the fish meat. The method for rapidly detecting the odor active substances in the fish meat comprises the following steps: mixing the fish meat, a saturated sodium chloride solution and 2, 4, 6-trimethylpyridine, heating at 85 DEG C for 40 minutes, and extracting by a headspace solid-phase microextraction method; and performing gas chromatography-mass spectrometry, identifying the compounds, calculating to obtain the content of each volatile compound component relative to the internal standard, calculating to obtain OAV, and screening to obtain the odor active substances in the fish meat according to the condition that OAV is greater than or equal to 1. According to the method, the odor active substances in the sample are screened by combining the quantitative value of each volatile component by the internal standard with the odor threshold value of each substance, and the compound which really contributes to the overall odor of the fish meat can be determined. In addition, by designing different extraction temperatures and extraction time, the odor active substances in the fish meat can be simply, conveniently and quickly detected and obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of detecting odor active substances in fish meat, and in particular relates to a method for rapidly detecting odor active substances in fish meat. Background Art

[0002] The quality of fish, especially its smell, is a key factor affecting consumer consumption behavior. Therefore, it is very important to accurately determine the composition of volatile odor compounds in fish. At present, the area normalization method is often used to quantify the detected compounds in the determination of volatile components in food, such as patent publication number CN116577447A (a method for determining flavor volatile substances in tea) and patent publication number CN109001361A (a method for detecting flavor substances in rapeseed oil). The quantitative results of volatile compounds obtained using this method are often expressed as the percentage of the component in the total volatile compounds in the sample. This quantitative method is not suitable for horizontal comparison of multiple samples, and it is also impossible to identify the odor active substances in the sample (i.e., the substances that actually contribute to the overall odor of the sample) through the odor threshold of each substance (the lower limit of the perceptible content of the substance). The volatile components detected range from dozens to hundreds, making the subsequent analysis of the sample as a whole very difficult. Moreover, there is a problem in the current art that directly determines the conditions for volatile compound detection based on the type and content of the measured substance: that is, it is impossible to determine whether the detected substance can actually be smelled by mammals / consumers by just looking at the content. For example, the concentration of substance A is as high as 1000ng / g, and the content of substance B is only 5ng / g. However, if the threshold of substance A is 1200ng / g, then even if the concentration of substance A is very high, it has no effect on the actual smell, and if the threshold of substance B is 4ng / g, then even if the concentration of substance B looks very low, it has an effect on the overall smell. Therefore, how to accurately and quickly detect the odor-active substances in fish meat that actually affect the smell of fish meat has always been a technical problem that needs to be solved in the art. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a method for quickly detecting odor-active substances in fish meat, which can accurately and quickly detect and obtain the compounds that truly affect the overall odor of fish meat.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a method for rapidly detecting odor active substances in fish meat, comprising the following steps: placing fish meat and a saturated sodium chloride solution in a headspace bottle, adding an internal standard 2,4,6-trimethylpyridine, heating at 85°C for 40 minutes, inserting a headspace microextraction injection needle into the headspace bottle and continuing heating; performing gas chromatography-mass spectrometry analysis after the extraction, identifying compounds, and calculating the content of each volatile compound component relative to the internal standard according to compound relative content=unknown substance ion peak area / internal standard peak area×internal standard substance content; calculating the odor activity value OAV according to odor activity value OAV=compound relative content / odor threshold value of the compound, and screening the odor active substances in the fish meat according to OAV≥1.

[0006] Preferably, the final concentration of the internal standard 2,4,6-trimethylpyridine is 5 mg / L.

[0007] Preferably, the fish meat includes grass carp meat.

[0008] Preferably, the chromatographic conditions in the gas chromatography-mass spectrometry analysis are: chromatographic column: HP-5MS (30m×0.25mm×0.25μm); column temperature: initial temperature 50°C maintained for 2min, increased to 180°C at 5°C / min and maintained for 5min, then increased to 250°C at 10°C / min and maintained for 5min; inlet temperature: 250°C; transmission line temperature: 280°C; carrier gas flow rate: 1.0mL / min; split ratio: no split.

[0009] Preferably, the mass spectrometry conditions in the gas chromatography-mass spectrometry analysis are: ion source temperature: 230° C.; quadrupole temperature: 150° C.; mass spectrometry: EI source; mode: full scan 40-600 m / z.

[0010] Preferably, the method for identifying the compound is: comparing the mass spectrum of the compound with the NIST20 database, preliminarily identifying the compound by comparing the mass spectrum of the standard, calculating the retention index RI according to the improved Kováts method, comparing the RI with the RI of each existing substance, and further identifying the compound.

[0011] Preferably, the odor active substances in the fish meat obtained by screening include n-octanal, octanol, nonanal, trans-2-decenal, trans-2,4-decadienal, trans-2-octenal, 1-octen-3-ol and 1-nonanol.

[0012] Preferably, the mass volume ratio of fish meat to saturated sodium chloride solution is 2-4 g:5 mL.

[0013] Preferably, the temperature for continued heating is 85° C., and the time for continued heating is 30 minutes.

[0014] Beneficial effects of the present invention:

[0015] The present invention achieves the purpose of quantifying volatile components in fish meat by introducing a stable internal standard. The present invention screens out odor active substances in the sample by combining the quantitative value of each volatile component with the odor threshold of each substance through the internal standard, and can determine the compounds that truly contribute to the overall odor of fish meat. The present invention determines the most suitable conditions for detecting volatile compounds in fish meat by designing different extraction temperatures and extraction times, optimizes the detection method, and can simply and quickly detect and obtain odor active substances in fish meat. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The total ion current chromatogram. DETAILED DESCRIPTION

[0017] The invention provides a method for rapidly detecting odor active substances in fish meat, comprising the following steps: placing fish meat and a saturated sodium chloride solution in a headspace bottle, adding an internal standard 2,4,6-trimethylpyridine, heating at 85°C for 40 minutes, inserting a headspace microextraction injection needle into the headspace bottle and continuing heating; performing gas chromatography-mass spectrometry analysis after the extraction, identifying compounds, and calculating the content of each volatile compound component relative to the internal standard according to compound relative content=unknown substance ion peak area / internal standard peak area×internal standard substance content; calculating the odor activity value (OAV) according to odor activity value (OAV)=compound relative content / odor threshold value of the compound, and screening the odor active substances in the fish meat according to OAV≥1.

[0018] In the present invention, the fish meat preferably includes grass carp meat; the mass volume ratio of the fish meat and the saturated sodium chloride solution is preferably 2-4 g:5 mL, more preferably 3 g:5 mL. In the present invention, the final concentration of the internal standard 2,4,6-trimethylpyridine is preferably 5 mg / L; the type of the headspace microextraction injection needle is preferably divinylbenzene / carboxymethyl / polymethylsiloxane (50 / 30 μm, 1 cm, DVB / CAR / PDMS, Supelco, PA, USA). In the present invention, the temperature for continued heating is preferably 85°C, and the time for continued heating is preferably 30 min.

[0019] In the present invention, the chromatographic conditions in the gas chromatography-mass spectrometry analysis are preferably: chromatographic column: HP-5MS (30m×0.25mm×0.25μm); column temperature: initial temperature 50°C maintained for 2min, increased to 180°C at 5°C / min and maintained for 5min, then increased to 250°C at 10°C / min and maintained for 5min; injection port temperature: 250°C; transmission line temperature: 280°C; carrier gas flow rate: 1.0mL / min; split ratio: no split. The mass spectrometry conditions in the gas chromatography-mass spectrometry analysis are preferably: ion source temperature: 230°C; quadrupole temperature: 150°C; mass spectrometry: EI source; mode: full scan 40-600m / z.

[0020] In the present invention, the method for identifying the compound is preferably: comparing the mass spectrum of the compound with the NIST20 database, preliminarily identifying the compound by comparing the mass spectrum of the standard, calculating the retention index RI according to the improved Kováts method, comparing the RI with the RI of each existing substance, and further identifying the compound. In the present invention, comparing the mass spectrum of the standard means comparing the ion fragment peaks in the NIST20 database with the fragment peaks of the unknown substance detected in the fish meat, and obtaining a matching degree, screening substances with a matching degree of more than 70%, and preliminarily identifying the obtained compound. In the present invention, when calculating the retention index RI by the improved Kováts method, the conventional improved Kováts method in the art can be used for calculation, such as the improved Kováts method in Wu, N., Gu, S., Tao, N., Wang X., Ji, S., 2014. Characterization of important odorants in steamed 341 male Chinese mitten crab (Eriocheir sinensis) using gas chromatography-mass spectrometry olfactometry. Journal of Food Science 79: C1250-1259. doi: 10.1111 / 1750-3841.12511. The present invention uses two identification methods, standard mass spectrometry and RI, to identify the compound, which has higher accuracy.

[0021] In the present invention, the odor threshold of the compound can be obtained from the prior art, such as the odor threshold of n-octanal disclosed in the prior art is 0.00001ppm. In the present invention, the odor active substances in fish meat obtained by screening include n-octanal, octanol, nonanal, trans-2-decenal, trans-2,4-decadienal, trans-2-octenal, 1-octen-3-ol and 1-nonanol. In the present invention, the odor active substances refer to substances that actually contribute to the overall odor of fish meat.

[0022] Since extraction conditions such as extraction temperature and time will directly affect the types and contents of flavor substances obtained subsequently, no relevant exploration work has been conducted on the appropriate extraction temperature and time for fish and its products, and the extraction temperature and time are often determined based on the personal experience of researchers. The present invention adopts headspace solid phase microextraction combined with gas chromatography-mass spectrometry analysis to determine the optimal conditions for extracting volatile substances in fish meat based on comprehensive judgment of odor active substances and types. The detection method of the present invention can accurately and quickly detect the types of odor active substances in fish meat.

[0023] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] The gas chromatograph-mass spectrometer used in the following examples is model 7890B-7000D, manufactured by Agilent.

[0027] Example 1

[0028] A method for rapidly detecting odor active substances in fish meat comprises the following steps:

[0029] (1) 3 g of minced grass carp meat was placed in a 20 mL headspace bottle, 5 mL of saturated sodium chloride solution was added, and internal standard 2,4,6-trimethylpyridine was added to make the final concentration of 2,4,6-trimethylpyridine 5 mg / L. The extraction conditions were: heating at 85 °C for 40 min, then the headspace microextraction needle was inserted into the headspace bottle, and heating was continued at 85 °C for 30 min.

[0030] (2) Desorption at 250℃ injection port for 5 minutes, and the following gas chromatography-mass spectrometry analysis was performed: the chromatographic column was HP-5MS (30m×0.25mm×0.25μm), and the chromatographic separation conditions were: column temperature: initial temperature 50℃ maintained for 2 minutes, increased to 180℃ at 5℃ / min and maintained for 5 minutes, then increased to 250℃ at 10℃ / min and maintained for 5 minutes; injection port temperature: 250℃; transfer line temperature: 280℃; carrier gas flow rate: 1.0mL / min; split ratio: no split; mass spectrometry conditions were: ion source temperature: 230℃, quadrupole temperature: 150℃, mass spectrometry: EI source, mode: full scan 40-600m / z. The total ion current chromatogram obtained is shown in the figure below: Figure 1 shown.

[0031] (3) Matching the ion fragment peaks of the unknown compound with the NIST20 database, that is, comparing the ion fragment peaks in the NIST20 database with the fragment peaks of the unknown substance detected in the fish meat to obtain the matching degree, screening substances with a matching degree of more than 70%, and preliminarily identifying the obtained compounds.

[0032] The retention index (RI) of the compounds obtained by preliminary identification was calculated according to the improved Kováts method, and the RI was compared with the RI of each existing substance to further identify the compound. The substances with a matching degree of ≥70% were screened. The RI of each substance is obtained by querying existing literature, for example, the following two documents can be queried: 1. Zhou, XX, Chong, YQ, Ding, YT, Gu, SQ, Liu, L., 2016. Determination of the effects of different washing processes on aroma characteristics in silver carp mince by MMSE-GC-MS, e nose and sensory evaluation. Food Chemistry 207: 205-213. doi: 10.1016 / j.foodchem.2016.03.026. 2. Frank, D., Poole, S., Kirchhoff, S., Forde, C., 2009. Investigation of sensory and volatile characteristics of farmed and wild barramundi (Lates calcarifer) using gas chromatography-olfactometry massspectrometry and descriptive sensory analysis. Journal of Agricultural and Food Chemistry 57:10302-10312.doi:10.1021 / jf902030y.

[0033] (4) The content of each volatile component relative to the internal standard was calculated according to the following calculation formula: relative content of compound (ng / g) = unknown substance ion peak area / internal standard peak area × internal standard substance content × 1000; then the odor activity value (OAV) was calculated according to the following formula: OAV = relative content of compound (ng / g) / odor threshold of the compound (ng / g), where the odor threshold of the compound was obtained by consulting existing literature, such as the following literature: Giri, A., Osako, Ka., Ohshima, T., 2010. Identification and characterisation of headspace volatiles of fish miso, a Japanese fish meat based fermented paste, with special emphasis on effect of fish species and meat washing. Food Chemistry 120: 621-631. doi: 10.1016 / j.foodchem.2009.10.036.

[0034] According to OAV≥1, odor active substances, i.e. compounds that actually contribute to the flavor of grass carp meat, were screened out, and the results are shown in Table 1. A total of 8 volatile substances were identified as odor active substances in grass carp meat: n-octanal, octanol, nonanal, trans-2-decenal, trans-2,4-decadienal, trans-2-octenal, 1-octen-3-ol, and 1-nonanol.

[0035] Table 1 Identification results of odor active substances in grass carp meat

[0036]

[0037] Example 2

[0038] Comparison of the effects of different extraction temperatures and times on the final test results

[0039] The difference from Example 1 is that the extraction conditions in step (1) are adjusted as shown in Table 2, and the rest are the same as Example 1.

[0040] Table 2 Settings of different extraction conditions

[0041]

[0042] When further identifying the compounds in step (3), the substances with a matching degree ≥ 70% were screened to obtain the number of volatile compounds under each extraction condition. The relative content of the compounds was calculated according to step (4), and the results are shown in Table 3. According to OAV ≥ 1, the volatile compounds under each extraction condition were screened, as shown in Table 4.

[0043] Table 3 Number of volatile compounds and relative internal standard content under different extraction conditions

[0044] Serial number Extraction temperature (℃) Extraction time (min) Number of volatile compounds Volatile compound content (ng / g) 1 30 20 11 11528.35 2 50 20 19 6585.99 3 70 20 24 4109.45 4 85 20 24 2338.45 5 30 30 19 17488.20 6 50 30 23 6520.21 7 70 30 26 2572.68 8 85 30 31 2021.60 9 30 40 15 22706.27 10 50 40 20 3931.15 11 70 40 35 2811.11 Example 1 85 40 46 5413.05

[0045] Table 4 Volatile compounds under different extraction conditions finally screened out according to OAV≥1

[0046]

[0047]

[0048] It can be seen from Tables 3 and 4 that different extraction temperatures and times will directly affect the difficulty of detecting odor active substances in fish meat.

[0049] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for rapidly detecting odor-active substances in fish meat, characterized in that: The method comprises the following steps: placing fish meat and a saturated sodium chloride solution in a headspace bottle, adding an internal standard 2,4,6-trimethylpyridine, heating at 85°C for 40 minutes, inserting a headspace microextraction injection needle into the headspace bottle and continuing heating; after completion, performing gas chromatography-mass spectrometry analysis to identify the compounds, and calculating the content of each volatile compound component relative to the internal standard according to compound relative content=unknown substance ion peak area / internal standard peak area×internal standard substance content; calculating the odor activity value OAV according to odor activity value OAV=compound relative content / odor threshold of the compound, and screening the odor active substances in the fish meat according to OAV≥1.

2. The method according to claim 1, characterized in that The final concentration of the internal standard 2,4,6-trimethylpyridine was 5 mg / L.

3. The method according to claim 1, characterized in that The fish meat includes grass carp meat.

4. The method according to claim 1, characterized in that The chromatographic conditions in the gas chromatography-mass spectrometry analysis were as follows: chromatographic column: HP-5MS; column temperature: initial temperature 50°C, maintained for 2 min, increased to 180°C at 5°C / min, maintained for 5 min, then increased to 250°C at 10°C / min, maintained for 5 min; injection port temperature: 250°C; transfer line temperature: 280°C; carrier gas flow rate: 1.0 mL / min; split ratio: no split.

5. The method according to claim 1, characterized in that The mass spectrometry conditions in the gas chromatography-mass spectrometry analysis were: ion source temperature: 230°C; quadrupole temperature: 150°C; Mass spectrometry: EI source; mode: full scan 40-600 m / z.

6. The method according to claim 1, characterized in that The method for identifying the compound is: comparing the mass spectrum of the compound with the NIST20 database, preliminarily identifying the compound by comparing the mass spectrum of the standard, calculating the retention index RI according to the improved Kováts method, comparing the RI with the RI of each existing substance, and further identifying the compound.

7. The method according to claim 1, characterized in that The odor-active substances screened in fish meat include octanal, octanol, nonanal, trans-2-decenal, trans-2,4-decadienal, trans-2-octenal, 1-octen-3-ol and 1-nonanol.

8. The method according to claim 1, characterized in that The mass volume ratio of fish meat and saturated sodium chloride solution is 2-4g:5mL.

9. The method according to claim 1, characterized in that: The temperature of the continued heating is 85° C., and the time of the continued heating is 30 minutes.

Images