Method for analyzing characteristic flavor of Hubei flavor fermented mandarin fish by adopting GC-IMS visualization technology

Abstract

The invention discloses a method for rapidly analyzing the characteristics of Hubei flavor fermented mandarin fish based on a GC-IMS visualization technology, and belongs to the technical field of aquatic product flavor analysis and detection. Headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) is adopted for detecting different Hubei flavor fermented mandarin fish; gC-IMS volatile flavor substance data and a GC-IMS fingerprint spectrum are obtained, different Hubei flavor fermentation difference flavor substances are obtained through principal component analysis and PLS-DA analysis, and finally the key flavor substances of Hubei flavor fermented mandarin fish are obtained through analysis by adopting a relative flavor activity value. The GC-IMS visualization technology is applied, and the method has the advantages that sample pretreatment is not needed, operation is easy, sensitivity is high, and subjectivity of technicians is eliminated. According to the method, rapid identification and detection of different Hubei flavored fermented mandarin fish can be realized, and a new technical means is provided for flavor evaluation of Hubei flavored fermented mandarin fish.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic product flavor detection, and more specifically relates to a method for analyzing the characteristic flavor of Hubei-style fermented mandarin fish by adopting GC-IMS technology. Background Art

[0002] Mandarin fish is a precious freshwater fish unique to my country. In 2022, the total output of mandarin fish farming in China was 401,000 tons, with an economic value of more than 24 billion yuan. The mandarin fish farming industry in Hubei Province has developed rapidly in recent years. The total output of farming has jumped from the fourth place in the country in 2013 to the second place in the country, accounting for about one-fifth of the total output of mandarin fish farming in the country. Fermented mandarin fish is one of my country's traditional flavor fermented fish products with a history of more than 200 years. It is deeply favored by consumers because of its tender meat, few bones and more meat, petal-shaped meat, delicious taste and unique flavor. After the mandarin fish meat is fermented by microorganisms, the protein and fat in it are broken down into amino acids and fatty acids, which not only greatly improves digestibility, but also increases the essential amino acids for the human body. At the same time, small molecules such as amino acids and fatty acids are further degraded to form the main flavor components of fermented mandarin fish.

[0003] Fermented mandarin fish products are mainly distributed in Anhui, Hunan and Hubei provinces. Due to different fermentation processes and regional characteristics, fermented mandarin fish products with regional characteristics such as "Hui style", "Xiang style" and "E style" are formed. At present, the research on fermented mandarin fish mainly focuses on the analysis of volatile flavor substances in traditional fermented mandarin fish in Anhui. Li Chunping et al. used solid phase microextraction combined with gas chromatography-mass spectrometry (GC-MS) to analyze the volatile organic compounds (VOCs) of Huizhou fermented mandarin fish. Through odor activity value (OAV) analysis, a total of 13 flavor active substances were identified, among which butyric acid, trimethylamine and linalool made important contributions to the overall flavor quality. Yang et al. used GC-MS to analyze the VOCs of 14 brands of fermented mandarin fish. The study found that 5 VOCs, including 2-ethyl-1-hexanol, eucalyptol, 1-octen-3-ol, nonanal and indole, were important flavor active substances. Although the samples in this study involved Anhui, Hubei and Hunan, Anhui samples accounted for 78.6%, while Hubei and Hunan only accounted for 21.4%. According to the currently disclosed technical information, there is little technical information on the systematic study of the flavor characteristics of Hubei-flavor fermented mandarin fish. Lei Yuelei et al. (patent name is a production method for Hubei-flavor fermented mandarin fish, Chinese patent publication number is CN106261964 B) disclosed a production method for Hubei-flavor fermented mandarin fish. In addition, Lei Yuelei et al. published a technical article on the process optimization of Hubei-flavor fermented mandarin fish. The reports on the technical documents of Hubei-flavor fermented mandarin fish did not clarify the characteristic flavor of Hubei-flavor fermented mandarin fish, which seriously restricted the origin traceability and flavor regulation of Hubei-flavor fermented mandarin fish.

[0004] Gas chromatography-ion mobility spectrometry (GC-IMS) is an analytical technique that identifies ions based on the difference in the migration rate of different gas-phase ions in the gas phase in an electric field. It is a fast, convenient and low-cost analytical method. GC-IMS technology has well solved the problem of low resolution of IMS and greatly broadened the application scope of traditional IMS. GC-IMS visualization technology has been widely used in the analysis of aquatic product flavors. Therefore, the present invention will propose a method for analyzing the characteristic flavor of Hubei-flavor fermented mandarin fish using GC-IMS visualization technology, in order to provide a reference for the origin identification and flavor regulation of Hubei-flavor fermented mandarin fish. Summary of the invention

[0005] The invention proposes a method for analyzing the characteristic flavor of Hubei-style fermented mandarin fish by utilizing GC-IMS visualization technology, and the method is also applicable to the flavor characteristic analysis of other foods.

[0006] The present invention is specifically achieved through the following technical solutions:

[0007] A method for analyzing the characteristic flavor of Hubei-style fermented mandarin fish using GC-IMS technology, characterized in that the specific steps are:

[0008] S1: Fermented mandarin fish sample preparation: Hubei-style fermented mandarin fish was cut into two pieces along the middle of the spine, and then the fish skin was removed, the back muscles were collected, and the muscles were mixed evenly using a processor;

[0009] S2: Fermented mandarin fish sample test: 5 g sample was weighed and placed in a 20 mL headspace bottle. The sample was analyzed using the FlavourSpec flavor analyzer and the raw data was obtained after the sample was removed from the machine.

[0010] S3: Fermented mandarin fish flavor data analysis: The raw data was analyzed using the analysis software of the FlavourSpec flavor analyzer to obtain qualitative and peak volume data, flavor fingerprints, etc. Then principal component analysis, partial least squares discriminant analysis, and relative odor activity value calculation were used to screen differential flavor substances and key flavor substances.

[0011] Preferably: in step S2, in the gas phase ion mobility spectrometry unit, the analysis time is 20 min. The chromatographic column is a MXT-5 capillary chromatographic column 15 m×0.53 mm×1 μm, the column temperature is 45°C, the IMS temperature is 45°C, and the carrier gas is 99.999% high-purity N2; automatic headspace injection unit: the injection volume is 500 μL, the incubation time is 15 min, the incubation temperature is 60°C, the injection temperature is 85°C, the incubation speed is 500 rpm, and the carrier gas flows according to the program: 2 mL / min for 2 min, 100 mL / min for 20 min, and the drift gas flow rate is 150 mL / min.

[0012] Preferably, in step S3, a flavor fingerprint is constructed using the Gallery Plot plug-in of the FlavourSpec flavor instrument to compare the common and differential flavor substances of the Hubei-style fermented mandarin fish.

[0013] Preferably, in step S3, the volatile components of the sample are qualitatively analyzed and the peak volumes are calculated by using the GC×IMS Library SearchNIST database and IMS database built into the FlavourSpec flavor analyzer.

[0014] Preferably: in step S3, principal component analysis and partial least squares discriminant analysis are performed using the MetaboAnalyst online platform based on the qualitative and peak volume data.

[0015] Preferably: in step S3, when using the MetaboAnalyst online platform for analysis, the data is first standardized with a logarithm of 10, and data scaling is selected, and the output results are used to check whether the data and samples satisfy the normal distribution. If they satisfy the normal distribution, subsequent analysis is performed.

[0016] Preferably: when partial least squares discriminant analysis is used, flavor components with VIP≥1 are regarded as differential flavor substances.

[0017] Preferably, in step S3, the peak volume is normalized and converted into relative percentage content using the GC-IMS qualitative and peak volume data, and the ROAV is calculated according to the odor relative activity value formula, and linalool is selected as the ROAV in the calculation. 芳樟醇 =100, ROAV of other components to be tested i Then calculate according to the following formula:

[0018] ROAV i =C i ×0.22×100 / (T i ×C 芳樟醇 )

[0019] Among them, C i is the relative content of volatile compounds in the tested component, %; C 芳樟醇 is the relative content of linalool, %; T i is the odor threshold of the component i in water, μg / kg; 0.22 is the odor threshold of linalool in water, μg / kg; components with ROAV≥1 calculated by the above formula are considered to be key flavor substances.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The analytical method for analyzing the characteristic flavor of Hubei-style fermented mandarin fish using GC-IMS visualization technology proposed in the present invention is simple, convenient and easy to operate, and the entire analytical operation process is also suitable for flavor analysis of other foods.

[0022] 2) The method proposed in the present invention screened out 9 different flavor substances from different Hubei-flavor fermented mandarin fish, which were 3-hydroxy-2-butanone, butyric acid, 1-octen-3-ol, ethyl propionate, 2-methyl-1-pentanol, 3-methylbutanal, 3-methyl-1-butanol, (E)-ocimene and camphenol, and screened out 7 key flavor substances with important contributions, which were 3-methylbutanal, linalool, ethyl acetate, ethyl isobutyrate, ethyl 2-methylbutyrate, p-cymene and ethyl butyrate. These flavor substances provide a reference for the origin tracing and flavor regulation of Hubei-flavor fermented mandarin fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The fingerprints of volatile flavor compounds of fermented mandarin fish muscle with different Hubei flavors;

[0024] Figure 2 The GC-IMS flavor substance data (percentage content) obtained for different Hubei-flavor fermented mandarin fish samples conformed to normal distribution after Log10 transformation;

[0025] Figure 3 The principal component analysis (PCA) of volatile flavor compounds of fermented mandarin fish with different Hubei flavors was conducted;

[0026] Figure 4 The PLS-DA score diagram of volatile flavor compounds of fermented mandarin fish with different Hubei flavors;

[0027] Figure 5 The VIP score diagram of volatile flavor substances in fermented mandarin fish with different Hubei flavors obtained by PLS-DA analysis. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0029] Example 1

[0030] Figure 1 It is the fingerprint of the identified VOCs in different Hubei-style fermented mandarin fish. Figure 1Each column represents a fermented mandarin fish sample, and each row represents the same VOCs in the muscles of fermented mandarin fish with different Hubei flavors. The darker the red signal, the higher the content of the substance, and the lighter the red signal, the lower the content of the substance. Compounds with the same name in the fingerprint represent monomers (M) and dimers (D). In order to better understand the characteristic flavors of fermented mandarin fish with different Hubei flavors, the red dotted frame is used to circle the A area according to the brightness of the signal ( Figure 1 ),from Figure 1 It can be seen that the volatile components of different Hubei-flavor fermented mandarin fish show great differences, but the samples contain a higher concentration of A (red dotted box), which is mainly composed of 2-methyl-1-propanol, α-terpinene, β-ocimene, β-pinene, limonene, p-cymene-D, p-cymene-M, (E)-ocimene, β-pyroene, etc. These substances are mainly produced by spices such as pepper and chili.

[0031] Example 2

[0032] In order to further screen the characteristic substances of fermented mandarin fish from different sources, the VOCs with higher relative contents and lower thresholds were selected as key VOCs. For different Hubei-flavor fermented mandarin fish (D1, D2, D3 and D4), linalool (ROAV stan =100) as an important key flavor substance. Because linalool not only has a relatively high content in the selected Hubei-flavor fermented mandarin fish samples, but also has a low threshold (0.22μg / kg), scholars have previously screened linalool as a key aroma active compound of fermented mandarin fish (OAV=662.94). As shown in Table 1, there are 14 flavor substances with ROAV greater than 1 in different Hubei-flavor fermented mandarin fish samples, among which the key flavor substances with important contributions are 3-methylbutanal, linalool, ethyl acetate, ethyl isobutyrate, ethyl 2-methylbutyrate, para-cymene, and ethyl butyrate. Linalool has a distinct floral scent, and linalool mainly comes from spices such as pepper and red pepper peel commonly used in the preparation of traditional fermented mandarin fish. 3-Methylbutanal has an apple aroma and oily taste, and has a low flavor threshold, which may come from the effect of microorganisms on leucine. Ethyl butyrate is one of the four major esters in white wine, and its aroma is similar to kiwi and pineapple, and can produce a variety of fruit and other aromas. In summary, the analysis of ROAV of volatile flavor substances in fermented mandarin fish with different Hubei flavors can effectively find the VOCs that play a key role in fermented mandarin fish products, thereby inferring the possible process and ingredients of fermented mandarin fish products, which is of great significance for origin identification and flavor regulation.

[0033] Table 1 Relative odor activity values ​​(ROAV) of volatile flavor compounds in fermented mandarin fish with different Hubei flavors

[0034] Volatile flavor substances Odor threshold (μg / kg) D1 D2 D3 D4 3-Methylbutyraldehyde 1.1 15.25 6.28 9.18 4.12 Octanal 0.587 0.90 0.80 1.01 0.58 Nonanal 1.1 1.02 0.99 1.14 0.92 3-Methyl-1-butanol 4 1.58 2.18 1.92 1.92 1-Octen-3-ol 1.5 0.59 0.87 1.34 1.01 Linalool 0.22 100.00 100.00 100.00 100.00 3-Hydroxy-2-butanone 14 2.37 0.95 0.32 0.14 Ethyl acetate 5 10.74 5.76 4.73 5.42 Ethyl butyrate(D) 0.9 3.65 7.71 3.53 2.75 Ethyl butyrate(M) 0.9 6.39 5.93 4.62 3.55 Ethyl isobutyrate 0.089 20.24 30.72 11.26 14.96 Ethyl 2-methylbutyrate 0.013 45.32 49.51 34.82 33.83 Ethyl hexanoate(M) 2.2 1.13 0.93 1.12 0.79 p-Cymene(M) 5.01 14.23 12.92 11.30 9.86 p-Cymene(D) 5.01 10.80 10.72 9.09 9.98 2-Acetyl-1-pyrroline(M) 0.1 12.65 8.58 9.14 8.63 2-Acetyl-1-pyrroline(D) 0.1 2.24 2.09 1.90 1.55

[0035] Example 3

[0036] The qualitative and peak volume-converted relative content data obtained from D1, D2, D3, and D4 Hubei-style fermented mandarin fish samples were input into the MetaboAnalyst online platform (https: / / www.metaboanalyst.ca / ) and then the data were standardized using Log10, as shown in Figure 2 . Figure 2 As shown in the figure, after Log10 standardization, the sample concentration conforms to the normal distribution, which will be beneficial to the subsequent principal component analysis and partial least squares discriminant analysis.

[0037] Example 4

[0038] Based on the identified VOCs obtained by GC-IMS, the relative content was calculated by signal intensity normalization, and the data were processed by Log10 standardization to make the data conform to the normal distribution. The principal component score diagram of VOCs in fermented mandarin fish from different sources was drawn using the MetaboAnalyst6.0 online tool ( Figure 3 ).from Figure 3 It can be seen that the variance contribution rates of principal component 1 (PC1) and principal component 2 (PC2) are 57.3% and 18.4% respectively, and the cumulative variance contribution rate reaches 75.7%, indicating that the model can better represent the characteristics of most samples. Figure 3 It can be seen that D1, D2, D3 and D4 can be effectively distinguished, indicating that there are differences in their flavor characteristics. Therefore, partial least squares discriminant analysis will be used to screen the different flavor substances in the future. Based on the relative content data of 52 volatile organic compounds (VOCs) (including monomers and dimers) identified by GC-IMS technology, after standardization, a PLS-DA model was established to distinguish the different flavor substances of fermented mandarin fish with different Hubei flavors. Figure 4 The score graph of PLS-DA analysis of VOCs in different Hubei-flavor fermented mandarin fish based on the PLS-DA model is shown. Figure 4 It can be seen that the cumulative explained variance is 69.8%, and the different Hubei-flavor fermented mandarin fish (D1, D2, D3 and D4) are located in different areas and can be effectively distinguished. Generally speaking, compounds with VIP>1 are considered to be significantly different flavor compounds, such as Figure 5As shown, the VIP values ​​of 8 volatile flavor substances (including monomers and dimers) are greater than 1, namely 3-hydroxy-2-butanone (VIP=4.3315), butyric acid (VIP=3.037), 1-octen-3-ol (VIP=1.8648), ethyl propionate (VIP=1.8464), 2-methyl-1-pentanol (VIP=1.5485), 3-methylbutanal (VIP=1.1436), 3-methyl-1-butanol (VIP=1.0889), (E)-ocimene (VIP=1.0481) and camphene (VIP=1.0187). Ke Zehua et al. used GC-MS to study the volatile flavor substances of 6 different brands of fermented mandarin fish in Anhui Province and found that linalool was common to the 6 fermented mandarin fish. In addition, Wang Wei et al. also found that linalool was the key volatile flavor substance to distinguish fresh mandarin fish from fermented mandarin fish in Anhui Province. Yang et al. used GC-MS to find that 2-ethyl-1-hexanol was the differential volatile flavor substance to distinguish different brands of fermented mandarin fish. 2-ethyl-1-hexanol provided fresh and floral fragrance to fermented mandarin fish.

Claims

1. A method for analyzing the characteristic flavor of Hubei-style fermented mandarin fish using GC-IMS technology, characterized in that: The specific steps are: S1: Fermented mandarin fish sample preparation: Hubei-style fermented mandarin fish was cut into two pieces along the middle of the spine, and then the fish skin was removed, the back muscles were collected, and the muscles were mixed evenly using a processor. S2: Fermented mandarin fish sample test: Weigh 5 g sample, place in a 20 mL headspace bottle, analyze using FlavourSpec flavor analyzer, and get raw data off the machine. S3: Fermented mandarin fish flavor data analysis: The raw data was analyzed using the analysis software of the FlavourSpec flavor analyzer to obtain qualitative and peak volume data, flavor fingerprints, etc. Then principal component analysis, partial least squares discriminant analysis, and relative odor activity value calculation were used to screen differential flavor substances and key flavor substances.

2. The method according to claim 1, characterized in that: In step S2, in the gas phase ion mobility spectrometry unit, the analysis time is 20min. The chromatographic column is a MXT-5 capillary chromatographic column 15m×0.53mm×1μm, the column temperature is 45°C, the IMS temperature is 45°C, and the carrier gas is 99.999% high-purity N2; automatic headspace injection unit: the injection volume is 500μL, the incubation time is 15min, the incubation temperature is 60°C, the injection temperature is 85°C, the incubation speed is 500rpm, and the carrier gas flows according to the program: 2mL / min for 2min, 100mL / min for 20min, and the drift gas flow rate is 150mL / min.

3. The method according to claim 1, characterized in that: In step S3, the Gallery Plot plug-in of the FlavourSpec flavor analyzer is used to construct a flavor fingerprint map to compare the common and differential flavor substances of Hubei-style fermented mandarin fish.

4. The method according to claim 1, characterized in that: In step S3, the volatile components of the sample are qualitatively analyzed and the peak volumes are calculated using the GC×IMS Library Search NIST database and IMS database built into the FlavourSpec flavor analyzer.

5. The method according to claim 1, characterized in that: In step S3, principal component analysis and partial least squares discriminant analysis were performed using the MetaboAnalyst online platform based on the qualitative and peak volume data.

6. The method according to claim 1, characterized in that: In step S3, when using the MetaboAnalyst online platform for analysis, the data is first standardized with a logarithm of 10, and data scaling is selected. The output results are used to check whether the data and samples satisfy the normal distribution. If they satisfy the normal distribution, subsequent analysis is performed.

7. The method according to claim 1, characterized in that: In step S3, when partial least squares discriminant analysis is used, flavor components with VIP≥1 are regarded as differential flavor substances.

8. The method according to claim 1, characterized in that: In step S3, the peak volume is normalized and converted into relative percentage content using the GC-IMS qualitative and peak volume data, and the ROAV is calculated according to the odor relative activity value formula, and linalool is selected as the ROAV in the calculation. 芳樟醇 =100, ROAV of other components to be tested i Then calculate according to the following formula: ROAV i =C i ×0.22×100 / (T i ×C 芳樟醇 ) Among them, C i is the relative content of volatile compounds in the tested component, %; C 芳樟醇 is the relative content of linalool, %; T i is the odor threshold of the component i in water, μg / kg; 0.22 is the odor threshold of linalool in water, μg / kg; components with ROAV≥1 calculated by the above formula are considered to be key flavor substances.

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