Technical papers
Characterization of Farmed Ovate Pompano (Trachinotus ovatus Linnaeus) Freshness during Ice Storage by Monitoring the Changes of Volatile Profile
2014 Volume 20 Issue 1 Pages 79-84
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2014 Volume 20 Issue 1 Pages 79-84
Changes of the volatile profile in farmed ovate pompano stored in ice were investigated in order to determine fish freshness. The volatile organic compounds were analyzed by head space solid phase microextraction (SPME) and GC/MS analysis. Principal component analysis (PCA) was conducted to characterize the freshness levels with time. PCA could differentiate fish freshness level based on their different volatile profile. Major compounds that contribute to the separation were acetaldehyde, 2-methyl-butane, hexane, 3-hydroxy-2-butanone, benzaldehyde, and hexadecane. The results show that fish volatile profile analysis could be a potential useful and rapid tool to characterize fish freshness during storage in ice.
Fish freshness is an important character of fish quality. Various methods including chemical, microbiological, physical methods and sensory evaluation were developed to evaluate fish freshness. All of these methods have their limitations and disadvantages. Researchers are still developing methods that can precisely and objectively characterize fish freshness. Sensory evaluation method employs several parameters such as the appearance of the skin, eyes, mucus and gills, the firmness of the flesh, and odor. However, sensory analysis method can be subjective and it is also often expensive and time consuming to train a panel to conduct the analysis. Odor is one of the most important parameters in the sensory analysis process for the characterization of fish freshness. Fish develops volatile compounds that are characteristic of fish freshness/spoilage and the aroma significantly influences the consumers' choice of fresh fish. The smell of fish changes rapidly while fish freshness level is decreasing. Consequently, the changing profile of volatile organic compounds in fish could be used to monitor the fish freshness (Triqui and Bouchriti, 2003; Duflos et al., 2005). It is thus interesting to explore the characteristic volatile compounds that could be served as fish freshness markers (Mansur et al., 2003; Duflos et al., 2006; Wierda et al., 2006; Edirisinghe et al., 2007).
Both microbial degradation and the enzymatic or autoxidative lipid oxidation can cause the change of the volatile profile of fish during storage. The fish freshness chemical markers derived from one species of fish can often not be used for another species of fish. For example, the volatile characteristic compounds of the fatty fish could be significantly different from those of lean fish. This makes it difficult to search for species-independent chemical markers (Duflos et al., 2006). Zhang et al. (2009) found that the difference of entire volatile profile characteristics other than individual compounds seemed to provide more precise information for oyster deterioration during storage. The analysis of the entire volatile profile could have to be used in evaluating fish freshness and in searching for species specific chemical markers.
Volatile compounds can be determined by static or dynamic headspace analysis (HS), simultaneous distillation and extraction, vacuum distillation, supercritical fluid extraction and solid phase microextraction (SPME) analysis. SPME is a simple extraction technique which does not require organic solvents and have gained popularity in fish aroma analysis these years (Duflos et al. 2005). The present study also employed SPME to analyze fish volatile compounds.
Ovate pompano (Trachinotus ovatus Linnaeus) fish is cultured in the southern coast of China including Fujian, Guangdong and Hainan province. It makes a great contribution to the local economy development. To our knowledge, the volatile profile of farmed raw ovate pompano fish stored in ice has not been reported by researchers. It is thus of interest to study the volatile compounds of this species of fish and also to monitor the fish freshness by the change of the volatile profile. Ovate pompano fish stored in ice was evaluated by quality index method (QIM). QIM is a seafood freshness grading system and is one of the most commonly used methods for the quality assessment of raw fish (Olafsdottir et al. 1997). The total volatile basic nitrogen (TVB-N) value was also determined.
Materials and Reagents Carboxen (CAR)/polydimethylsiloxane (PDMS) (75 µm) was from Supelco (Bellefonte, PA, USA). CAR/PDMS was conditioned according to the manufacture's recommendation before first use. Chemicals used for TVB-N determination were analytical grade.
Sample Preparation
Live ovate pompano (Trachinotus ovatus Linnaeus) (average weight approximately 450 g) was purchased from local supermarket. Fish were quickly killed at that place and kept on ice and transferred to the lab immediately. All fish were stored in crushed ice at 4°C in self-draining polystyrene boxes for 8 days. The internal temperaute of fish samples was approximately 0°C. Fresh ice was supplemented daily. On day 0, 2, 4, and 8, samples were taken out for the sensory evaluation. After sensory evaluation, the fish were beheaded, eviscerated and cut with a knife into small cubes for the TVB-N and GC-MS analysis.
Sensory Evaluation Fish were evaluated by the quality index method (QIM). A three person panel with previous expereinece in assessing fish freshness conducted the sensory evaluation. Changes in the smell, texture, eye appearance (cornea, form, pupil), skin and gills (color, mucus) were evaluated and scored. The grade of skin, texture, eyes, color of gills ranged from 0 to 2 while mucus on gills and smell ranged from 0 to 3. On different experiment days, fish samples were evaluated by the sensory panel and scored. The points are added up to get the quality index (QI).
Determination of TVB-N TVB-N was determined according to the EU directive 95/149/EC (European Union 1995). Briefly, fish samples (10 g) were homogenized with 90 mL of 0.6 mol/L perchloric acid. Then after filtration, the extract was mixed with sodium hydroxide (20%) and subjected to steam distillation. After distillation, the volatile base compounds were absorbed into boric acid (3%). The distillate containing amounts of volatile base compounds were determined as TVB-N by titration with 0.05 mol/L hydrochloric acid. Results were expressed as mg TVB-N per 100 g fish sample. Five replicates were conducted on each interval of the storage.
SPME Procedure 5 g of chopped fish samples were weighed into 20 mL headspace vials and sealed with polytetrafluorethylene (PTFE)-coated silicone rubber septa. The vial containing the fish sample was maintained at room temperature for 30 min to allow the volatiles in the headspace to reach equilibrium. The vial was then kept at 50°C for 20 min to allow the volatiles to be absorbed into the CAR/PDMS fiber. The fiber was then retrieved and inserted into the GC/MS system.
GC/MS analysis The GC/MS system used in this study was a Shimadzu GC/MS-QP2010 equipped with Rtx-5 MS capillary column (60 m × 0.25 mm, 0.25 µm film; Restek, Bellefonte, PA). Helium was used as the carrier gas. The injector and the detector temperature were set at 250°C and 280°C respectively. The SPME fiber was maintained for 2 min in the injection port following injection. The injections were performed in split mode (ratio 1:10). The column temperature was set at 35°C for 5 min, 35 to 100°C at 10°C/min, 100 to 250°Cat 20°C/min and held there for 5 min. Electron ionization masses were recorded at 70 eV in the mass range between m/z 29 and 500. Volatile compounds were identified by comparing their mass spectra with those contained in the National Institute of Standards and Technology (NIST) 2008 library.
Data Analysis Results from TVB-N and sensory evaluation were analyzed by analysis of variance (ANOVA) and means were separated by the least significant different test at a significant level of 0.05 or less. SYSTAT (Systat software, Inc., Chicago, IL) was used to analyze these data. The peak area of each identified volatile compound integrated from the total ion chromatogram was log 10 transformed. The principal component analysis (PCA) was conducted using the Unscrambler X 10.3 (Camo software, Norway).
TVB-N and Sensory Evaluation Table 1 shows the TVB-N values and sensory evaluation data of the ovate pompano fish during storage in ice for 8 days. TVB-N values andsensory (skin, texture, gills, eyes, smell and QI) scores increased over storage time. The concentration of TVB-N value of freshly caught fish is typically between 5 and 20 mg /100 g flesh (Ababouch et al., 1996). The TVB-N of fresh fish samples on day 0 in this study was 9.2 mg/100 g flesh, which is within this range. Fish are considered unfit for human consumption if the TVB-N values exceed the levels of 30 - 35 mg/100 g. At the end of the storage period, the average TVB-N value of the fish samples (15.6 mg/100 g) was still within acceptable range, but clearly showed signs of spoilage by sensory evaluation. TVB-N values of fish samples between day 0 and day 2, day 2 and day 4, day 4 and day 8 were not statistically significant (P > 0.05). It suggests that TVB-N determination could not completely separate fish samples with different storage time or indicate the different levels of freshness. On the other hand, the QI scores provided more accurate information on the fish freshness levels. QIM clearly differentiated fish samples stored on various days in ice (Table 1).
| Sensory scores | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Storage time (days) | TVB-N*, mg/100 g fish | QI* | Skin | Texture | Gills | Eyes | Smell | |||
| Color | Mucus | Cornea | Form | Pupil | ||||||
| 0 | 9.2 ± 1.0a | 0.4 ± 0.5a | 0.0a | 0.0a | 0.0a | 0.2a | 0.2a | 0.0a | 0.0a | 0.0a |
| 2 | 11.3 ± 3.4ab | 4.4 ± 0.4b | 0.2a | 1.0b | 0.5b | 1.0b | 0.5ab | 0.0a | 0.4a | 0.8b |
| 4 | 13.5 ± 1.4bc | 6.2 ± 1.4c | 0.2a | 1.0b | 0.6b | 1.1b | 0.9bc | 1.0b | 0.4a | 1.0b |
| 8 | 15.6 ± 1.2c | 9.7 ± 0.8d | 0.8b | 1.1b | 0.8b | 1.3b | 1.3c | 1.4b | 1.3b | 1.7c |
*Values are mean ± standard error of five determinations. Means within a column with different letters are significantly different (P < 0.05).
In the SPME/GC/MS analysis, different fibres can influence the extraction efficiency of fish volatile organic compounds (VOCs). The CAR/PDMS fibre was used in this study as this fiber has been widely used in fish VOCs analysis (Mansur et al., 2003). This type of fiber is a bipolar fiber and was found to be very efficient to extract low molecular weight compounds (Fratini et al., 2012). Other parameters that could influence the SPME sampling of VOCs from fish like the sampling time and incubation temperature were chosen based on previous work shown by other researchers (Duflos et al., 2005; Miyasaki et al., 2011).
The 20 volatile compounds in farmed ovate pompano fish were tentatively identified according to the standard mass spectra of the NIST MS library (Table 2). Most compounds identified in this study were also found in other species of seafood by many researchers. The lipid oxidation, microbial action and enzymatic activities can cause the production of these volatile compounds in seafood (Olafsdottir et al., 1997). Ovate pompano fish contains rich polyunsaturated fatty acids, which are prone to oxidation. The lipid oxidation can happen from the time fish was killed. The 1-penten-3-ol, hexanal, heptanal, and nonanal found in this study were derived from the oxidation of unsaturated fatty acids (Yasuhara and Shibamoto, 1995; Cruz-Romero et al., 2008). 1-Penten-3-ol has been identified in various fish species including tuna, gilthead sea bream, oyster, whiting (Duflos et al., 2005; Edirisinghe et al., 2007; Soncin et al., 2009; Zhang et al., 2009). 1-Penten-3-ol was reported to be the product of the action of lipoxygenases on ω-3 PUFA (German et al., 1992; Hu and Pan, 2000). 1-Penten-3-ol could also be caused by Pseudomonas species and can contribute to the stale and putrid off odors in fish (Miller et al., 1973; Olafsdottir et al., 2005). Nakamura et al. (1980) also found that 1-penten-3-ol may be produced by microbial spoilage.
| Storage time (days) | ||||||
|---|---|---|---|---|---|---|
| Peak No. | Retention time (min) | Compounds* | 0 | 2 | 4 | 8 |
| 1 | 2.36 | Acetaldehyde | 5.9 | 5.8 | 6.0 | 6.5 |
| 2 | 2.5 | Ethanol | 6.3 | 5.7 | 6.1 | 6.4 |
| 3 | 2.55 | 2-Methyl-butane | ND | ND | ND | 6.6 |
| 4 | 2.82 | Methylene chloride | 7.1 | 6.9 | 7.0 | 7.1 |
| 5 | 3.08 | Trimethylsilanol | 6.9 | 6.9 | 6.8 | 7.0 |
| 6 | 3.35 | Hexane | 6.3 | 6.3 | 6.1 | 7.2 |
| 7 | 4.84 | 1-Penten-3-ol | 6.3 | 6.2 | 6.0 | 6.4 |
| 8 | 5.14 | 2,3-Pentanedione | 5.6 | 5.9 | 5.5 | 6.1 |
| 9 | 5.58 | 3-Hydroxy-2-Butanone | 5.3 | 5.2 | ND | ND |
| 10 | 7.11 | Toulene | 5.6 | 5.7 | 5.5 | 5.7 |
| 11 | 7.27 | 1-Pentanol | 5.1 | 5.1 | 4.9 | 5.1 |
| 12 | 7.35 | (Z)-2-Penten-1-ol | 5.1 | 5.2 | 4.9 | 5.1 |
| 13 | 8.08 | Hexanal | 5.8 | 6.1 | 5.8 | 6.2 |
| 14 | 9.89 | p-Xylene | 4.8 | 5.0 | 4.8 | 4.9 |
| 15 | 10.54 | 2-Propenoic acid, butyl ester | 5.3 | 5.4 | 5.3 | 5.6 |
| 16 | 10.66 | Heptanal | 4.7 | 5.0 | 4.9 | 5.0 |
| 17 | 11.97 | Benzaldehyde | ND | 5.1 | 5.0 | 5.2 |
| 18 | 12.18 | 3,5,5-trimethyl-2-Hexene | 4.8 | 4.4 | 4.5 | 4.8 |
| 19 | 14.13 | Nonanal | 5.0 | 5.2 | 5.2 | 5.4 |
| 20 | 18.54 | Hexadecane | 4.5 | 4.7 | 4.6 | 5.1 |
*Compounds were identified by comparison with mass spectrum from NIST library. Values were expressed as means of the log 10 transformed peak areas (n=5); ND, not detected.
Aldehydic compounds such as hexanal, heptanal, and nonanal can make a significant contribution to the oxidized rancid odor due to the low threshold of perception. Hexanal is formed from n-6 polyunsaturated fatty acids oxidation and has a low threshold of perception (Josephson, 1991). Its odor is described as oxidized fatty, green, grassy, and penetrating (Turchini et al., 2004). Heptanal was generated by oxidation of n-9 monounsaturated fatty acids and n-6 polyunsaturated fatty acids (Duflos et al., 2006). Heptanal was described to have an earthy and boiled-potato like smell and may cause the fishy odor in cold stored cod (Karahadian and Lindsay, 1989; Olafsdottir et al., 2005; Maqsood and Benjakul, 2011). Heptanal can be used as a reliable indicator of flavor deterioration for fish products, while hexanal is related with the rancidity in meats (Augustin et al., 2006; Ross and Smith, 2006). Nonanal was found to have a green and floral odor and was one of the most important contributors to the raibow trout aroma (Selli et al., 2009). Acetaldehyde can be formed from the oxidized product of eicosapentaenoic acid via α/β double-bonded hydration and retro-aldol condensation (Kawai, 1996; Duflos et al., 2006).
Ethanol and 3-hydroxy-2-Butanone was reported to be associated with the growth of Pseudomonas spp (Olafsdottir et al., 2005). The utilization of carbohydrate sources by spoilage microorganisms caused the production of ethanol. The formation of 2, 3-pentanedione was due to the glycogen metabolism by Carnobacterium maltaromaticum and has been found to have a characteristic butter and cheesy odor (Joffraud et al., 2001; Jaffres et al., 2011). Benzaldehyde was reported to be produced from amino acid degradation (Piveteau et al., 2000). The odour was candy, sweet and almond (Turchini et al., 2004). Toulene may be from the carotenoid oxidation (Piveteau et al., 2000). The origin of p-xylene was unknown. It was also found in oyster and fish.
Trimethylamine was not detected in this study. Miyasaki et al. (2011) reported that there was no trimethylamine from several kinds of sea fish on day 0 during storage in ice. Researchers have suggested that aldehydes and ketones rather than trimethylamine contributed to the fresh fish flavor and trimethylamine is related to the spoilage. The characterized fishy odor caused by volatile trimethylamine was not detected during the storage even on day 8 in this study. It could also be coelulted with other compounds and consequently was not identified (Olafsdottir et al., 2005).
Principal component analysis was conducted to find if fish stored on different days could be separated by their different volatile profiles. Results from PCA were shown in Figure 1. The ratios of contribution for the principal components 1 (PC1) and 2 (PC2) were 69% and 21% respectively. It can be seen from the figure that samples were distinguished by their storage time according to the PC1 value. The PC1 value increased with increasing storage time and suggested the decrease of freshness. The PC2 can only distinguish samples on day 0, 2, and 4, but could not separate samples on day 0 and 8. The PC 2 could be indicator of the freshness of very fresh fish such as fish stored in ice for a very short period.
Principal component analysis for the volatile profile of farmed ovate pompano (Trachinotus ovatus Linnaeus) during storage in ice
The correlation loading figure showed the major compounds that contributed to the separation of the fish samples stored on various days (Figure 2). They were acetaldehyde,2-methylbutane,hexane,3-hydroxy-2-butanone, benzaldehyde, and hexadecane. These 6 compounds could be selected as potential chemical markers for farmed ovate pompano fish freshness. It is noted that the volatile profile of fish samples may not be completely determined by SPME extraction due to the limitation of the polarity of the fiber. Some other volatile compounds that can contribute to the separation significantly could be ignored. A complete extraction of the volatile compounds by combination of different extraction techniques will certainly provide more accurate volatile profile data to be used to characterize fish freshness level.
Correlation loading plot from the principal component analysis
In conclusion, this study investigated the volatile profile of farmed ovate pompano (Trachinotus ovatus Linnaeus) during storage in ice on various days by SPME/GC/MS analysis. The difference in the volatile profile can be used to monitor fish freshness. Some volatile compounds that contributed to the separation significantly were identified. The relative amounts of different volatile compounds or the volatile profile may serve as characteristic finger prints to be used to objectively define the fish freshness level.
Acknowledgements This work has been supported by National Science & Technology Pillar Program in "12th Five Year" Period (2012BAD28B05), Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, P.R. China (NYBJG201206), the Education Department of Fujian Province (JA12192) and SRF for ROCS, SEM.
