Introduction

Coilia ectenes related to Osteichthyes, Clupeiformes, and Engraulidae, is an important fish species that migrates from near-ocean waters to freshwater rivers every spring. In China, Coilia ectenes is mainly classified into the Jianghai migratory type and Lake settlement type according to its survival mode. The Jianghai migratory type migrates periodically between river and sea for some reason, which includes physiological requirements, genetic, and environment factors. The Jianghai migratory type includes Yangtze River-Coilia and East China sea-Coilia. The lake settlement type is a kind of Coilia ectenes living in lakes all through its lifetime, including Chao Lake-Coilia and Yellow River-Coilia [1]. It was reported that some Coilia ectenes species do not migrate to freshwater rivers due to genetic or environment factors, so there are significant differences between the river-anchovy and sea-anchovy [2]. Coilia ectenes has a high economic value for its distinctive aroma, intensive umami taste, and high nutritional value. However, its declining production fails to meet the market demands due to overfishing and environmental pollution, thereby contributing to the rise in prices (Yangtze River-Coilia is 8000–12000 yuan/kg, i.e., approximately 1333–2000 US$/kg) and to the development of Coilia ectenes farming.

MonoTrap™ (MT) is a novel absorbent composed of silica gel, activated carbon, and octadecyl silane (ODS). It is widely used in the extraction of polar and non-polar compounds as well as those with high and low boiling points. When the samples pass through the pores of the silica monolithic structure, they are trapped by the ODS groups, which are bound to the silica surface or the activated carbon present inside and outside the structure. This mode of absorption is called monolithic material sorptive extraction (MMSE), which is similar to solid-phase micro extraction (SPME) and stir bar sorptive extraction (SBSE). The headspace MMSE (HS-MMSE) with a larger specific surface area and porous surface [3], has higher adsorption efficiency than the headspace SPME (HS-SPME) and the headspace SBSE (HS-SBSE). HS-MMSE has been applied to plants, coffee, tea, sesame oil, and dairy products [4].

The GC–MS–O technology, which was first proposed by Fuller in 1964, is a useful method for evaluation of odorants from complex materials, including the frequency detection method, the dilution method and the direct intensity method [5]. The frequency detection method was used to detect the odor frequency by assessors and described by Linssen in 1993 [6]. The dilution method is the dilution level at which flavor compounds cannot be smelled by the human nose [7]. The direct intensity method could offer several advantages of reducing sniffing time and errors by recording the changes of odor intensities directly, which is superior to frequency detection and the dilution method [8]. Several researchers have analyzed odor-active compounds in seafoods by GC–MS–O, such as the New Zealand sea urchin Evechinus chloroticus [9], rainbow trout Oncorhynchus mykiss [10], and Yangtze River-Coilia [11]. However, few works have focused on the study of flavor compounds in Coilia ectenes by GC–MS–O associated with odor intensity.

The objective of this study was to identify the important odorants in steamed Yangtze River-Coilia, East China sea-Coilia, Chao Lake-Coilia and Yellow River-Coilia by GC–MS–O (direct intensity method) as associated with the odor intensity method. This study may provide a theoretical basis for further research and development in breeding technologies of Coilia ectenes or other aquatic products.

Materials and methods

Materials

Ten Yangtze River-Coilia (average weight and length 109.5 ± 7.7 g and 26.3 ± 0.6 cm, respectively) and ten East China sea-Coilia (average weight and length 106.1 ± 4.5 g and 25.9 ± 1.1 cm, respectively) were purchased from the Xiaohu Aquatic Products Market (Jiangsu, China) in April 2012; ten Chao Lake-Coilia (average weight and length 25.2 ± 9.4 g and 20.4 ± 2.0 cm, respectively) were purchased from Fuhuang Sungem Food Company (Anhui, China) in October 2012; and ten Yellow River-Coilia (average weight and length 91.5 ± 6.4 g and 29.9 ± 1.7 cm, respectively) were purchased from Dongying Jingming Aquatic Products Company (Shandong, China) in April 2012.

All specimens were transported to the laboratory with dry ice, fishbone and liver were removed, and then stored at −80 °C until required for analysis.

C6–C30 n-alkanes standards were purchased from Sigma–Aldrich Trading Co., Ltd (Shanghai, China). Other reagents used were of analytical grade and purchased from Anpel Laboratory Technologies Co., Ltd (Shanghai, China).

HS-MMSE for volatile compounds extraction

Yangtze River-Coilia, East China sea-Coilia, Chao Lake-Coilia, and Yellow River-Coilia were rinsed under running water, eviscerated, steamed for 20 min to guarantee the Coilia ectenes was fully cooked, and minced. After cooling to room temperature, the Coilia ectenes were homogenized in an ice-bath condition (Model JZ-II, Tianjin Sifang Equipment Ltd, China). Prior to use, MonoTrap™ rods were conditioned in an oven at 250 °C for 30 min to remove any impurities and inserted into the stainless steel MT holders using a set of clean tweezers to avoid contamination, and the stainless steel MT holder was inserted into the MT stand. Nine MT rods were applied in this study for achieving the greatest extraction of volatile compounds in Coilia ectenes. A clean septum was passed through the end of the MT holder using clean tweezers, and a cap was placed on the top of the holder. MT rods were placed at a fixed position in the headspace of a 15 mL-vial (item Nr. VAAP-38015E-1760A-100, Shanghai Anpu Scientific Instrument Co., Ltd.) containing approximately 5.0 g homogenized sample, and a septum was hermetically sealed on the vial, which was kept in a heat-gathering style magnetism mixer for 60 min at 80 °C. Volatile compounds were exposed and adsorbed to both side surfaces of the MT rods. Following adsorption, the MT rods were removed and immediately placed carefully in an adsorption tube, then desorbed via a thermal desorption unit (TDU, Gerstel, Baltimore,Md., USA) and injected via a cold injection system (CIS, Gerstel, Baltimore,Md., USA). The TDU temperature was programmed at 180 °C/min from 50 to 270 °C and maintained for 5 min; the CIS temperature was programmed at 12 °C/s from −40 to 250 °C and maintained for 0.5 min.

Gas chromatography–mass spectrometry–olfactometry (GC–MS–O)

A gas chromatography–mass spectrometer (Model 6980, Agilent Inc., USA) was coupled to an ODP 2 sniffing port (Gerstal). The effluent from the capillary column was split (1:1.5 v/v) between the mass spectrometry detector (MSD) and the ODP-2. Separations in the GC were performed on a DB-5MS capillary column (60 m length × 0.25 mm internal diameter × 0.25 μm film thickness; Agilent Inc., USA), using helium (99.999 % space purity) as the carrier gas (1.2 mL/min). The oven temperature was programmed from 40 to 100 °C at a rate of 5 °C/min, then increased to 180 °C at a rate of 2 °C/min, followed by an increase at 5 °C/min to 240 °C and maintained for 5 min.

MS conditions were as follows: detector interface temperature was 250 °C, ion source temperature was 230 °C, ionization energy was 70 Ev, mass range was 40–450 amu, electron multiplier voltage was 1576 V, and scan rate was 1.8/s. Samples were desorbed at 270 °C with a TDU (Gerstal) directly into the hot injector (250 °C) of the CIS (Gerstal) with simultaneous cryofocusing using liquid nitrogen.

In the GC–MS–O study, ten experienced assessors with previous sniffing experience (five male and five females, 22–30 years of age) were choose as candidates in the GC–MS–O panelist selection. Based on the guidelines established by Pollien and others [12], three assessors (one male and two females) were required to take part in GC–MS–O detection due to their high sensitive olfaction and recorded the aroma characteristics and aroma strength of each sample. Each sample was smelled twice by the assessors. The mean of odor intensity was calculated and the final odor intensities were obtained from the mean values of at least two assessors. During a 50-min sniffing period per sample, assessors ranked the intensity of each odor on a four-point intensity scale: 1 = weak, 2 = moderate, 3 = strong, and 4 = very strong [8].

Statistical analyses

All detections had been replicated three times in this study. Results were expressed as mean ± standard deviation (SD) (n = 3). The SPSS 19.0 software (SPSS Inc., Chicago, III., USA) was applied to check if there were significant differences (p < 0.05) between four Coilia ectenes based on one-way analysis of variance (ANOVA).

Volatile compounds were analyzed by matching the mass spectra (Wiley/NIST 2008) and the linear retention index (LRI) with reference values and odor properties. The LRI was calculated by the following equation,

$$LRI = \left( {\frac{{Rt_{{(x)}} - Rt_{{(n)}} }}{{Rt_{{(n + 1)}} - Rt_{{(n)}} }} + n} \right) \times 100,$$

where Rt(χ) is the retention time of each volatile compound (χ), Rt(n) and Rt(n + 1) are the retention times of n-alkanes eluting directly before and after the compound (χ) under identical chromatographic conditions.

Results

The total ion chromatogram obtained from Coilia ectenes samples is shown in Fig. 1. A total of 63 volatile compounds were identified in steamed meat of Yangtze River-Coilia, East China sea-Coilia, Chao Lake-Coilia, and Yellow River-Coilia on a DB-5ms column (Table 1): including 19 aldehydes, 11 ketones, 11 alcohols, 8 aromatics, 5 hydrocarbons, 6 N-and S- containing compounds, and 3 furans. Among them, 49 were found in Yangtze River-Coilia, 31 in East China sea-Coilia, 30 in Chao Lake-Coilia, and 43 in Yellow River-Coilia. On comparing the compounds found in Coilia ectenes, we found that 20 volatile compounds were common to all four Coilia ectenes. Thirteen volatile compounds (propanal, 2-methyl-2-butenal, (E)-2-pentenal, 2-ethyl-4-pentenal, 2-heptanone, 2,3-octanedione, 5-methyl-2-heptanone, heptanol, (E)-2-octen-1-ol, 2-ethyl-4-methyl-1-pentanol, undecane, N,N-dimethyl-formamide,and N,N-diethyl-formamide) were found exclusively in Yangtze River-Coilia, two volatile compounds (2-butanone, 1-pentadecene) were found in Chao Lake-Coilia, and 9 volatile compounds (2,2-dimethyl-propanal, heptanal, 1H-pyrrole-2,5-dione, 5-ethyl-2(5H)-furanone, limonene, 2-ethyl-1H-pyrrole, 2-propyl-furan, 2-(2-pentenyl)furan, hexadecanoicacid, methyl ester) were found in Yellow River-Coilia.

Fig. 1
figure 1

Total ion chromatograms of volatile compounds of four steamed Coilia ectenes a Yangtze river-Coilia, b East China sea-Coilia, c Chao lake-Coilia, d Yellow river-Coilia

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Table 1 Odor thresholds and peak area of volatile compounds identified in four steamed Coilia ectenes (n = 3)
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A total of 35 odorants (including 33 volatile compounds and 2 unknown compounds) were identified in four Coilia ectenes by the olfactometric analysis (Table 2). Among all the odorants, there were 19 compounds in Yangtze River-Coilia, 21 compounds in East China sea-Coilia, 16 compounds in Chao Lake-Coilia and 20 compounds in Yellow River-Coilia. Based on the direct intensity method, assessors use a scale to measure the perceived intensity of a compound as it elutes, those odorants whose intensities were greater than or equal to 3 could be regarded as important odorants. Results showed that the important odorants including TMA (fishy, ammonia-like), 1-penten-3-ol (mushroom, cabbage), N,N-diethyl-formamide (roasted meaty), hexanal (grassy, earthy), (Z)-4-heptenal (fishy, boiled potato) and benzaldehyde (almond, metallic) in Yangtze River-Coilia; TMA, 1-penten-3-ol, ethylbenzene (nutty, floral), (Z)-4-heptenal, 1-octen-3-ol (mushroom, earthy) and nonanal (oily) in East China sea-Coilia;TMA, 1-penten-3-ol, ethylbenzene, 1-octen-3-ol and nonanal in Chao Lake-Coilia, TMA, 1-penten-3-ol, hexanal, (Z)-4-heptenal,1-octen-3-ol and decanal (green, oily) in Yellow River-Coilia.

Table 2 Odorants (Odor intensity >1) in four steamed Coilia ectenes by GC–O detection (n = 3)
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Discussion

Volatile compounds in four steamed Coilia ectenes

A total of 19 aldehydes were found in four Coilia ectens. The 3-methyl-butanal and 2-methyl-butanal mainly contribute to the nutty almond notes, and may be formed by the Strecker degradation of leucine in the course of the Maillard reaction [13]. This reaction is known to be enhanced at high temperatures [14]. Benzaldehyde is a predominant compound in Yangtze River-Coilia, East China sea-Coilia and Chao Lake-Coilia, which originated from the oxidation of carbon–carbon double bonds in styrene and have been detected in fresh-baked sockeye salmon [15], roasted peanuts [16], and algae [17]. There are high levels of hexanal and nonanal in Chao Lake-Coilia. Hexanal, generally imparting a grassy odor, originates from the degradation of n-6 PUFA oxides and could be used as a degradation indicator of seafood products and meat from terrestrial animals [18]. Nonanal, imparting grassy and oily odors, is the predominant constituent in the autoxidation of linoleic acid [19]. Octanal and decanal may contribute to the more desirable aromas as well as the rancid odors and flavors that appear during the spoilage of fat and fatty foods [20]. Unsaturated straight chain aldehydes mainly contribute to the grassy, fatty, and fishy odors. For example, (Z)-4-heptenal is characterized by a fatty and creamy odor, and this compound may be formed by a retro-aldol condensation of 2,6-nonadienal, which has been shown to be a major odor impact compound in a number of fresh marine creatures [21]. Other saturated aliphatic aldehydes such as octanal and decanal have grassy and oily odors.

Ketones, which are derived from PUFA oxidative degradation [22], have a higher threshold value compared to aldehydes and a relative small contribution to fish aroma [23, 24]. However, the flavor differences among fish were mainly attributed to carbonyl compounds, so ketones have certain influences on flavor development [25]. Yangtze River-Coilia, Chao Lake-Coilia and Yellow River-Coilia have high levels of (E, E)-3, 5-octadien-2-one, which resulted from PUFA auto-oxidation and imparting sweet and roasted odors [4]. The compound 2, 3-pentanedione is an indicator of lipid oxidation in chilled fish muscle [26]. The 2, 3-pentanedione may contribute to roasted meat, caramel, and buttery odors, especially in cooked fillet of European catfish [27]. The compound 3-cyclohepten-1-one presents a high concentration in East China sea-Coilia, and has native and intense aromas that might be associated with the high fat content of East China sea-Coilia.

Alcohols are divided into two main classes: saturated alcohols and unsaturated alcohols. Saturated alcohols have higher flavor threshold values than unsaturated alcohols, therefore the contributions to fish odors is small [28, 29]. Unsaturated alcohols have been identified as the major volatile alcohols in shellfish and cooked alligator meat [30] as well as many other aquatic products. Eleven alcohols were detected in Coilia ectenes; three alcohols (1-penten-3-ol, 1-pentanol, and 1-octen-3-ol) were common to all four species. The compound 1-penten-3-ol is the most abundant alcohol in Yangtze River-Coilia and Yellow River-Coilia, which was generated from PUFA oxidation [31]. It has been reported that 1-penten-3-ol presented in fresh whitefish at high concentration [32]. The compound 1-octen-3-ol presents in Chao Lake-Coilia at high concentration, imparting desirable mushroom-like odors, mainly derived from the hydrogen peroxide degradation of linoleic acid [33].

Eight aromatics were identified in Coilia ectenes and six aromatics (benzene, toluene, ethylbenzene, p-xylene, styrene, and naphthalene) were common in all four species. Among them, toluene and p-xylene are generally believed to originate from environmental pollutants [34], which contributing to unpleasant off-flavors.

Hydrocarbons do not have any odor activities due to their high threshold values [35]. Nevertheless, intermediate alkanes, alkenes, arenes, and a small fraction of heterocyclic compounds may enhance the overall fish flavor [36].

The N- and S-containing aromatic compounds, resulted from Maillard reactions between amino acids and reducing sugars, pyrolysis of amino acids (such as proline) and thiamine, dicarbonyl compounds from Maillard intermediate products, and degradations of thiamine via aldol condensation and aldehyde amine polymerization [31]. The S-containing aromatic compounds originate from the thermal degradation of methionine and cysteine [37], imparting meaty, maotai, and onion garlic flavors [31]. Even though some differences in the types and levels of N- and S-containing aromatic compounds in Coilia ectenes, TMA (53.82 %) was present in all four species. TMA is generally believed to be produced by microbial metabolism in the presence of the precursor TMA oxide (TMAO) [38]. The presence of high levels of TMA in seafoods is undesirable, while TMA may add a pleasant crustacean-like odor at low levels [39].

Furans are a family of substances that play an important role in the flavor formation of Coilia ectenes, three furans have been identified in this study. The 1st odorant is 2-ethyl-furan, which was detected in all four Coilia ectenes, having a sweet corn odor. It was reported that 2-ethyl-furan is the oxidative degradation products of linolenic acid and commonly in crab [40–42]. The other furans, which were only detected in Yellow River-Coilia in this study, have also been reported in crab [43].

Above all, the main compounds associated with fish flavor are C6–C9 olefine aldehydes, enols and olefine ketones. There are more aldehydes, ketones, and alcohols in Yangtze River-Coilia and Yellow River-Coilia than in East China sea-Coilia and Chao Lake-Coilia. It is possible that Yangtze River-Coilia (15.7 ± 1.2 %) and Yellow River-Coilia (18.1 ± 0.5 %) have a higher fat content than East China sea-Coilia (13.6 ± 2.6 %) and Chao Lake-Coilia (8.6 ± 1.0 %), aldehydes, ketones, and alcohols are produced from the oxidation and degradation of lipids. Additionally, environmental factors, e.g., water quality, sediment, algae and microbial species, may have significant effects on the flavor of Coilia ectenes [17]. The aromatic and hydrocarbon levels are similar to the four Coilia ectenes, other flavor compounds (e.g., TMA, dimethyl trisulfide compounds, and alkyl furans) are abundant in Yangtze River-Coilia and Yellow River-Coilia.

Important odorants in four steamed Coilia ectenes

A total of 35 odorants were identified in four Coilia ectenes (Table 2). Most of the odors are present at low levels but have important functions in the flavor development of Coilia ectenes. The characteristic flavors may originate from lipids, mainly short chain aldehydes and ketones [62]. Even though odorants are different among the four species, the main odor characteristics are fishy, grassy, mushroom, fatty, oily and meaty, which are consistent with a previous report [11]. TMA (fishy, ammonia-like), 1-penten-3-ol (mushroom, cabbage), N,N-dimethyl-formamide (roasted meaty), hexanal (grassy, earthy), ehtylbenzene (nutty, floral), (Z)-4-heptenal (fishy, boiled potato), benzaldehyde (almond, metallic), 1-octen-3-ol (mushroom, earthy), nonanal (oily), and decanal (green, oily) were the important odorants in four Coilia ectenes.

TMA levels are inversely proportional to fish quality [39]. TMA was perceived by the assessors to have average odor intensity values of 4 in four Coilia ectenes, showing the fishy and ammonia-like odors.

Unsaturated alcohols have low odor threshold values and with mushroom and metallic-like odors. The 1-penten-3-ol and 1-octen-3-ol are commonly found in freshwater and seawater fish with strong odor activities [31]. The 1-penten-3-ol has an odor intensity value more than 3 in four Coilia ectenes, imparting mushroom and cabbage odors. The 1-octen-3-ol imparted the mushroom and earthy odors, mainly derived from the hydrogen peroxide degradation of linoleic acid [33], and has the lowest value in Yangtze River-Coilia.

Hexanal, contributed to the grassy, earthy scent of the four Coilia ectenes. It has been reported that hexanal mainly derived from the oxidation of linoleic acid and provides the “green” and fatty characters of fish and other seafoods [63]. Ethylbenzene, which has a nutty, floral odor, was previously identified with a relatively high intensity. The (Z)-4-heptenal, is a volatile flavor compound with low odor threshold value, which is characterized by a cooked fish, potato-like odor, might be derived from the water-mediated retro-aldol condensation of (E,Z)-2,6-nonadienal [64]. Chao Lake-Coilia has the highest levels of (E,Z)-2,6-nonadienal. Benzaldehyde, which was assigned an odor intensity of 3 in Yangtze River-Coilia, mainly contributed to the almond and metallic odors. It is derived from the oxidation of benzyl alcohol, which is catalyzed by dehydrogenases [65]. Decanal, which resulted from the oxidation of oleic and linoleic acids [11], has the higher intensity in Yellow River-Coilia than the other three Coilia ectenes, imparting unpleasant green and oily odors.

The compound 2, 3-pentanedione contributes to roasted meaty and caramel odors and buttery and caramel odors in salmon and trout [35]. The (E, E)-3, 5-octadien-2-one, which has a sweet, roast odor, has previously been detected in several seafood products [43].

In addition to known odorants, two unknown odorants were detected by the assessors. The first unknown odorant was detected as having a corn, fruity odor. The second unknown odorant had a garlic, roasted meaty odor. Although these odors could be detected by the assessors, they could not be identified by MS due to their weak signals. Nevertheless, even with weak signals and low estimated concentrations, these compounds may impart significant aromas in steamed Coilia ectenes, and should be evaluated in further studies.