Changes in the volatile components of squid (illex argentinus) for different cooking methods via headspace–gas chromatography–ion mobility spectrometry

Abstract Squid products are becoming more and more popular with consumers because of their high yields and nutrition, including novel textures with desirable sensory properties. However, it has not been determined whether the cooking method has effects on the flavor of the squid. In this study, the aroma and volatile substances of squid samples from different cooking methods (boiled, steamed, sous vide) were determined and analyzed by headspace–gas chromatography–ion mobility spectrometry and differentiated by using, as well, an electronic nose and sensory evaluation. A total of 43 characteristic flavor compounds were identified. Based on the signal intensity of the identified violate compounds, we established a fingerprint of heat‐treated squid from different cooking methods. Due to the long‐term low‐temperature heating conditions under vacuum, the flavor of sous vide squid is different from steamed and boiled squid, and it has unique special flavor compounds. Different cooking methods can affect the aroma of squid, providing support for the industrial production of squid.


| INTRODUC TI ON
Illex Argentines, the Argentinian squid, is the most important cephalopod species and major component of commercial squid in the southwest Atlantic (Waluda, Rodhouse, Podestá, Trathan, & Pierce, 2001). The volume of cephalopods, including squid, cuttlefishes, and octopuses, harvested and consumed in the world during 2017 was 3,772,567 t (data from FISHSTAT, FAO). The edible portion of squid is as high as 80%, which is about 20% higher than that of fish in general. As a kind of invertebrate, the muscle protein of squid is slightly different from that of ordinary fish. The muscle protein of squid contains some paramyosin that has no ATPase activity to influence the protein stability, gel strength, and other properties of squid products (Mirzaei & Regnier, 2006), limiting the processing of squid. At present, research on the processing technology of squid can be divided into two categories. One is the optimization and improvement of traditional squid processing technology, such as squid rings (Tomac, Cova, Narvaiz, & Yeannes, 2017), dried squid (Dong, Zhu, Li, & Li, 2013), and where vacuum packaging prevents food oxidation, reduces flavor and moisture loss during cooking, prevents the growth of aerobic bacteria, and improves shelf life (Baldwin, 2012). Additionally, precise temperature controlling can maintain the color of foods.
Christensen et al. (Christensen et al., 2013) found that low-temperature and long-time (LTLT) heating can reduce the strength of connective tissue and increase the solubility of collagen, which makes beef as tender as veal. Roldán, Antequera, Martin, Mayoral, and Ruiz (2013) found the lamb loin tenderness positively correlated with cooking time (6, 12 hr, and 24 hr). However, as the cooking temperature increased, the weight loss of lamb loin increased, and the moisture content decreased. SV can improve lamb loin brightness and redness, as well as ensure biosafety at 60°C. Moreover, beef cooked by SV (59°C, 5 hr) did not exhibit an increase in thiobarbituric acid reactive substances (TBARS) and warmed-over flavor (WOF) after 30 d of storage (Hansen, Knøchel, Juncher, & Bertelsen, 1995). Many scholars have also evaluated the safety of SV. After cooking at 50-62°C, the number of mesophiles and psychrotrophic bacteria in beef decreased significantly (Botinestean, Keenan, Kerry, & Hamill, 2016;Hansen et al., 1995;Vaudagna et al., 2002). Moreover, the number of Escherichia coli and mesophiles in pork cooked at 53°C for several hours also decreased significantly (Becker, Boulaaba, Pingen, Röhner, & Klein, 2015;Salaseviciene, Vaiciulyte-Funk, & Koscelkovskienė, 2014). To reduce the nutrient loss while optimizing overall quality and shelf life of meat during cooking, lots of food processors use SV technique to replace traditional cooking methods, such as frying, microwaving, and grilling (del Pulgar, Gazquez, & Ruiz-Carrascal, 2012;Roldan, Antequera, Perez-Palacios, & Ruiz, 2014). Thus far, scholars have only focused on SV cooking technology for the protection of food nutrients, quality improvement, and food safety. In addition, they think that the lower heating temperature of SV produces less flavor (Calkins & Hodgen, 2007;Cross, Stanfield, & Koch, 1976). Therefore, there are fewer studies on the flavor of SV. The flavor characteristics of squid by different cooking methods have yet to be reported.
Ion mobility spectrometry (IMS) is a technology that analyzes trace chemical substances based on differences in the migration speed of different ions in the gas field in an electric field. IMS is a chemical substance analysis technology developed in the late 1960s and early 1970s (Eiceman, Karpas, & Hill, 2013). The most prominent advantages of IMS are that it can ionize analytes at atmospheric pressure, and the detection limit is as low as ng/L (Armenta, Alcala, & Blanco, 2011). IMS has been applied to the detection of drugs (Verkouteren & Staymates, 2011), chemical warfare agents (Rearden & Harrington, 2005), and explosives (Asbury, Klasmeier, & Hill, 2000). With the development and improvement of IMS technology, it has gradually been applied in food inspection, such as food adulteration (Garrido-Delgado, Muñoz-Pérez, & Arce, 2018), determination of food quality (Ivanov, Bilgucu, Ivanova, & Dimitrova, 2020;Snyder, Harden, Davis, Shoff, & Maswadeh, 1995), food process control (Karpas, Guamán, Calvo, Pardo, & Marco, 2012), and chemical food safety (Gloess, Yeretzian, Knochenmuss, & Groessl, 2018). Therefore, the main objective of the present study was to analyze by GS-IMS the volatile components of squid and differentiate the samples based on their volatile compound profile, using, as well, an electronic nose and sensory evaluation. Through principal component analysis, the establishment of fingerprints, and heat map analysis, the differences and correlations of volatile flavor substances in squid from different cooking methods were explored. The results provide a foundation for the replacement of traditional cooking methods by SV technology.

| Sample preparation
The Argentinian squid (approximate net weight 400 g) was purchased from an aquatic products market in Qingdao city, Shandong Province of China. The squid specimens were kept refrigerated with flake ice inside polystyrene boxes provided with a lid and holes for drainage. Samples were transported to the laboratory at −18°C.
Prior to cooking, squid specimens were separated into the head, foot (wrist), and ketone body with scissors after thawing and washing. The average weight of the ketone body of squid was 20 ± 4 g (n = 16). Each squid specimen length, width, and thickness of the ketone body were 4 ± 1 cm, 4 ± 1 cm, and 1 ± 0.4 mm, respectively.
All squid were randomly divided into four groups to prepare for processing and kept at 4°C until further analysis. From each specimen, one specimen remained raw (RAW) as a control, and the others were subjected to cooking methods.

| Cooking methods
Different cooking methods were selected as the preparation procedures for squid samples in this study: boiling (BO), steaming (ST), and sous vide (SV).

| BO method
The squid samples were placed in a stainless steel pot of boiling water (2 L) for 5 min. After cooking, the samples were removed and drained on absorbent papers.

| ST method
The squid samples were placed in a steam oven (SCC WE 61, Germany Rational) at 100°C for 5 min. After cooking, the samples were removed and drained on absorbent papers.

| SV method
The samples were put into a plastic vacuum bag (nylon/polyethylene pouches, operating temperature of −20°C/121°C) and sealed using a vacuum sealer (DZ-260, Dajiang Holding Group Electric Co. LTD, China). The samples then were cooked in temperature-controlled water bath (60°C) for 30 min (Cui, Dubova, & Mo, 2019).
All the samples were soaked in ice water after cooking for 30 min and stored at −20°C until analysis, including the control group.

| Sensory evaluation
The sensory evaluation team consisted of 20 people, between 18 and 25 years old, all of whom had undergone sensory evaluation training. A hedonic scale method from 1 to 9 points was adopted, where 1 means extremely disliked and 9 means extremely liked.

| Electronic nose analysis
The electronic nose (PEN 3, Germany AirSense) was used to preliminarily evaluate the aroma profile of the squid samples. It was conducted by the following procedure. Taking the center of the squid sample to be tested, each sample (1 g) was prepared in a 20 ml gas chromatographic analysis bottle at room temperature for 30 min.
The injection rate was 600 ml/min; the carrier gas flow rate was 600 ml/min; and the measurement time was 60 s. Thereafter, the sensors were purged by clean dry air for 180 s. The parameters were optimized, and each analysis was repeated 3 times.

| Isolation of volatile organic compounds in squid
Volatile compounds of squid from different cooking methods were identified by a GC-IMS flavor analyzer (FlavourSpec®, Dortmund, Germany). Each squid sample (2 g) was placed in a 20 ml headspace vial and sealed. Samples were subsequently incubated at 60°C for 15 min. Finally, 500 μL headspace was injected automatically via a heated syringe (65°C) into the heated injector of the GC-IMS equipment under conditions reported below. Three parallel samples were analyzed from the same processing method.

| Chromatographic conditions
The gas chromatographic separation was performed at 60°C on a FS-SE-54-CB-1 capillary chromatographic column (15 m × 0.53 mm, 1 μm). High-purity nitrogen (99.99%) was as a carrier gas with a flow rate of 150 ml/min and a programmed flow as follows: 2 ml/min for 2 min; linear increase to 100 ml/min over 18 min; and total run time of 20 min. To avoid cross-contamination, the syringe automatically flushed for 30 s with nitrogen gas before each analysis and 5 min after each analysis.

| Data analysis
The sensory data analysis was conducted using SPSS 13.0 software (IBM). Principal component analysis (PCA) of electronic nose was carried out with Win Muster software. Volatile organic components (VOCs) were analyzed by laboratory analytical viewer (LAV) and GC × IMS Library Search (FlavourSpec ® ).

| Sensory evaluation
Sensory analysis of squid samples from different cooking methods was performed. Different cooking methods have different sensory evaluations of squid ( Figure 1). The squid cooked by ST and BO contracted and curled to varying degrees, while the squid cooked by SV contracted less and did not curl. There was no difference in the color of the squid processed by the three cooking methods.
In terms of texture, the SV sample was excellent and the softest.
In terms of aroma and flavor, ST and BO were very outstanding, with a strong aroma of cooked squid. The SV samples had the best score in appearance, texture, and preference, while the ST samples performed best in terms of aroma and flavor. The comprehensive sensory score of SV samples was the best because of their tender texture and smoother appearance. However, the aroma and flavor of the SV samples were obviously insufficient and different from ST and BO. Further research is needed.

| Analysis of flavor substances of squid via electronic nose
Compared with traditional sensory analysis, electronic nose is simple, fast, objective, and intuitive. According to the different response values of the electronic nose sensor to the aroma components of squid in different cooking methods, an intuitive radar chart was established, as shown in Figure 2a. The radar chart analysis method is mainly employed to study the sensors. Using this method, the contribution rate of each sensor to the squid sample can be distinguished, so as to investigate which type of volatile components play predominant roles in distinguishing the sample. From Figure 2a, the difference in volatile flavor compounds from different squid samples was mainly in the sensors W2W, W5S, W1W, and W1C, which are aromatic compounds, sulfur organic compounds, and nitrogen oxide compounds. It can be seen from the radar chart that the flavor of squid is greatly influenced by aromatic compounds. Conversely, the BO and ST squid samples had more obvious responses at W1W and W2W, which can be contributed to sulfide. Specific volatile components need further verification.
In order to highlight the aroma differences in squid from different cooking methods, a PCA scatter diagram (Figure 2b) was generated according to electronic nose data of the overall flavor substance composition. PCA is a multivariate statistical analysis technique that examines the correlation between multiple variables and reveals the internal structure between multiple variables through a few principal components (Jo et al., 2013). Generally, the PCA model is selected as the separation model when the cumulative contribution rate reaches 60% (Wu et al., 2015). PCA of aromatic substances from different heat-treated squids (5 detection signals from 48s to 52s) was performed, and the result shows that the first principal component contribution rate was 95.86%; the second principal component contribution rate was 3.30%; and the cumulative contribution rate was 99.16%. This indicates that the two principal components can adequately represent predominant characteristics of the sample.
Each sample in the group was relatively concentrated in a specific range and has a precise distance from the clustered areas of other groups. RAW samples and SV samples were clustered separately (the farthest distance in the PCA chart), while BO and ST were clustered together. This suggests that these methods have a high degree of similarity. Moreover, the results indicate that different cooking methods lead to differences in squid aroma.

| Differences in VOCs in squid samples
A series of physical and chemical changes occur after meat is heated, which significantly affects the quality of the processing (Shahidi, Rubin, D'Souza, Teranishi, & Buttery, 1986). HS-GC-IMS analyzed the differences between volatile compounds in heat-treated squid samples. Figure 3a shows the 3D topographic plot spectrum of volatile flavor compounds. From Figure 3a, the VOCs of squid from different cooking methods are very similar, but the signal intensities are slightly different. After heat treatment, the content of most flavor compounds changes to varying degrees.
Although the differences in volatile flavor compounds can be visualized in the 3D spectrum, a 2D plot spectrum is more evident.   Figure 3 shows the differences in VOCs in squid prepared from different cooking methods intuitively, but it is difficult to accurately F I G U R E 1 Sensory scores of squid in different cooking methods. a, Sensory scores of appearance, aroma, flavor, texture, and preference of squid prepared from different cooking methods. b, Comprehensive sensory score of squid from different cooking methods. BO: boiled squid; ST: steamed squid; SV: sous vide cooking squid determine the specific substance in the 2D and 3D spectrum maps.

| Identification of VOCs in squid from different cooking methods
Through GC-IMS separation, the differences in volatile substances in the squid samples are elucidated. According to the retention time and ion migration time of volatile substances in gas chromatography, 99 volatile substances were detected, and 43 specific volatile substances were determined (Table 1). Due to different concentrations of VOCs, certain compounds might produce multiple signals or spots (dimers or trimers).

| Fingerprints of squid samples
In order to compare the differences in VOCs more comprehensively, the gallery plot plug-in of LAV software was used to generate the peak signal of the topographic plots of squid samples (Figure 4). In Figure 4, each row represents all selected signal peaks from a squid sample, and each column represents the signal peak of the same volatile organic compound. Brightness indicates the content of a substance. The higher the brightness, the higher the content of the substance.
The complete VOC information for each sample and the differences between the samples are depicted in Figure 4

| D ISCUSS I ON
Sensory evaluation is one of the critical indicators of food quality.
In this study, the sensory evaluation could distinguish the squid Consumers' perceptions can be used for the assessment in the product development, which can significantly save time and labor costs (Vieira et al., 2020). It also suggested that evaluation based on preferred attribute elicitation methodology had the advantage of providing data on the importance of attributes for product acceptance (da Costa et al., 2020). Besides, in the sensory evaluation, F I G U R E 3 Gas chromatography-ion mobility spectrometry of squid from different cooking methods. a, Three-dimensional topographic plots and chromatograms of squid samples. The y-axis represents the retention time of the gas chromatograph; the x-axis represents the ion migration time for identification, and the z-axis represents the peak height for quantification. b, Two-dimensional topographic plots and chromatograms of squid samples. RAW: raw squid; BO: boiled squid; ST: steamed squid; SV: sous vide cooking squid. The ordinate represents the retention time, and the abscissa represents the migration time. The red vertical line represents the reaction ion peak (RIP), and the migration time after normalization was 8.0 ms. Each point on the right of RIP represents a volatile substance. Color represents the signal intensity of the substance. White indicates low intensity, and red indicates high intensity. The darker the color, the greater the intensity   products, and lipid oxidation products (Shahidi, 1998). Different cooking methods affect the flavor of meat products (Grosch, 1982), especially the volatile components (Macleod, Seyyedain-Ardebili, & Chang, 1981 (Jeong, O, Shin, & Kim, 2018).
Volatile compounds of cooked meat include products of lipid and fatty acid oxidation, for example, aliphatic hydrocarbons, aldehydes, ketones, alcohols, carboxylic acids, and esters (Mottram, 1998).
Aldehydes are the main products of the oxidative degradation of fatty acids and the characteristic aroma components of meat (Mottram, 1998). In this study, 43 VOCs were identified, including 14 aldehydes. The threshold value of aldehydes is relatively low, which contributes significantly to the flavor in cooked squid. In the experiment, 3-methylthiopropanal was present in all four sample types. 3-Methylthioprapanol was first identified as a volatile component in cooked squid by Kubota, Matsukage, Sekiwa, and Kobayashi (1996) It is a "baked potato" descriptor and considered an essential contributor to the scent of squid (Carrascon, Escudero, Ferreira, & Lopez, 2014) as well as cooked lobster (Lee, Suriyaphan, & Cadwallader, 2001). However, the difference in the contents of aldehydes in squid from different heating methods has an important impact on the sample odor. Ketones are also products of fatty acid oxidation, generally the products of automatic oxidation of unsaturated fatty acids (Thomas, 1971). There are ten ketones in the 43 VOCs detected in ST, BO, and SV samples, such as 3-hydroxybutan-2-one, 3-pentanone, acetone, 2-butanone, and 2-heptanone.
In particular, furaneol (4-hydroxy-2,5-dimethyl-3(2H)furanone) in RAW samples is described as a "caramel-like" flavor, and it is a flavor component of strawberry (Buechi, Demole, & Thomas, 1973) and pineapple (Rodin, Himel, Silverstein, Leeper, & Gortner, 1965). Kubota et al. (1996) considered furaneol a substance that contributes to the sweetness of squid after cooking. Ester compounds are produced by the esterification of alcohols and acids (Domínguez, Gómez, Fonseca, & Lorenzo, 2014) and esters are found in many flavors of crustacean fish products that are cooked and heated (Tanchotikul & Hsieh, 1989). SV squid has the highest content of n-propyl acetate and acetic acid ethyl ester of all samples tested. The volatile substance n-propyl acetate is a short-chain aliphatic ester that produces a pleasant fruity aroma. It is a component of natural flavors (Mahapatra, Kumari, Garlapati, Banerjee, & Nag, 2009). This indicates the SV con ditions are conducive to the esterification reaction and can yield more desirable flavor in squid.  . In this study, n-propyl acetate and acetic acid ethyl ester (highlighted in group H in Figure 4) were produced after SV heating; however, they were not detected in BO and ST.
Moreover, by adding reducing sugar or other flavor precursors, SV food flavor can also be enhanced (Roldan, Loebner, et al., 2015).
In addition to this, SV technology produces high-quality sensory characteristics of meat products. Studies have shown that a slow heating rate is key to generating tender meat (Cover, 1943) and maintaining the meat core temperature closed to 60°C for a long time (Laakkonen, Sherbon, & Wellington, 1970). These observations have been further confirmed in the SV technique (Becker et al., 2015;Christensen et al., 2013;Roldan et al., 2013). SV means not only higher sensorial quality but LTLT heating can also minimize the nutrient loss (Rondanelli et al., 2017), improve bioaccessibility (da Silva et al., 2017), ensure the safety of food (El Kadri, Alaizoki, Celen, Smith, & Onyeaka, 2020;Nissen, Rosnes, Brendehaug, & Kleiberg, 2002), and extend the shelf life (Diaz, Nieto, Banon, & Garrido, 2009;Kim, Hong, Lim, Park, & Lee, 2015;Nissen et al., 2002). Given the many advantages of SV and the growing demand for nutritious and convenient foods, the application of SV technology in the industrial production of squid is feasible.

| CON CLUS IONS
Through sensory evaluation and electronic analysis, it was found that different cooking methods have a great influence on the flavor of the squid. SV squid flavor is different from that of ST and BO. Additionally, a method was developed to evaluate the characteristic volatile compounds of squid samples from different cooking methods by establishing the fingerprint with HS-GC-IMS.
A total of 43 volatile substances, including some dimers, were detected by GC-IMS analysis from samples of squid from different cooking methods. The predominant compounds were mainly aldehydes, ketones, alcohols, and esters. Given the many advantages of SV technology, it could be used for the industrial production of squid.

| INFORMED CON S ENT
Written informed consent was obtained from all study participants.

ACK N OWLED G M ENTS
This work was supported by Henan Province Technology System for Conventional Freshwater Fish Industries (S2014-10-G02). We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

CO N FLI C T S O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.