Analysis of volatile components in a Chinese fish sauce, Fuzhou Yulu, by gas chromatography-mass spectrometry

Analysis of volatile components in a Chinese fish sauce, Fuzhou Yulu, by gas chromatography-mass spectrometry 1 2 first-author †E-mail:yuanfan@jmu.edu.cnpsu@zju.edu.cn †E-mail:yuanfan@jmu.edu.cnpsu@zju.edu.cn yuanfan@jmu.edu.cn 2 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yuanfan@jmu.edu.cnpsu@zju.edu.cn †E-mail:yuanfan@jmu.edu.cnpsu@zju.edu.cn yuanfan@jmu.edu.cn School of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310029, China School of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310029, China School of Bioengineering, Jimei University, Xiamen 361021, China School of Bioengineering, Jimei University, Xiamen 361021, China Volatile components of Fuzhou Yulu, a Chinese fish sauce, were analyzed by gas chromatography-mass spectrometry (GC-MS), and two pretreatment methods, i.e., purge and trap (P&T) GC-MS and ethyl acetate extraction followed by GC-MS, were compared. P&T-GC-MS method determined 12 components, including sulfur-containing constituents (such as dimethyl disulfide), nitrogen-containing constituents (such as pyrazine derivatives), aldehydes and ketones. Ethyl acetate extraction followed by GC-MS method detected 10 components, which were mainly volatile organic acids (such as benzenepropanoic acid) and esters. Neither of the two methods detected alcohols or trimethylamine. This study offers an important reference to determine volatile flavor components of traditional fish sauce through modern analysis methods. Fish sauce Fuzhou Yulu Volatile component Purge and trap Gas chromatography-mass spectrometry (GC-MS) INTRODUCTION 1 Yulu, also known as a fish sauce, is a clear brown liquid, produced by fermenting small fish and shrimp or byproducts from aquatic products processing, which have been preconditioned with salt (Sanceda et al., 2003a 2003a ; 2003b 2003b ). Fermentation results from the action of endogenous enzyme or additional microbial enzymes (Fukami et al., 2004a 2004a ; 2004b 2004b ; Siringan et al., 2006 2006 ; Dissaraphong et al., 2006 2006 ). The regions of fish sauce producing and consumption are comparatively scattered mainly in the southeast of Asia, east coast of China, Japan, and the north of Philippines. As one of the most famous brands of fish sauces in China, Fuzhou Yulu has a maximal yield and has been exported to 16 nations or regions in the world. Because of its unique, traditional flavor, the fish sauce has been widely consumed as one of the condiments for cooking, although the use of amount is considerably tiny. While there have been many reports focusing on the nutrient compositions of fish sauces (Ijong and Ohta, 1995 1995 ; Brillantes and Samosorn, 2001 2001 ; Park et al., 2001 2001 ; 2002a 2002a ; 2002b 2002b ; Sanceda et al., 2003a 2003a ; 2003b 2003b ), limited reports about their volatile flavor components are available. In the present study, we analyzed the volatile components in Fuzhou Yulu, a typical and representative Chinese fish sauce by using gas chromatography-mass spectrometry (GC-MS), and compared two pretreatment methods, purge and trap (P&T) GC-MS and ethyl acetate extraction followed by GC-MS. The component fingerprint we described here can be used as a control for the comparison between different brands of fish sauce. In addition, the flavor component analysis method we used here can also be applied to other traditional food products. MATERIALS AND METHODS 1 Materials 2 During the production of Fuzhou Yulu, all raw materials were fermented with fish enzymes and salt-resistant microorganisms at natural temperature for 10~12 months, and high concentration of salt was used to prevent spoilage. Generally, the fermented mash was extracted and passed through the filters three times to produce the first, second, and third extracts, respectively. In this study, we only chose the first extract for analysis, as the first extraction sauce represents the most unique flavors of different brands of fish sauces. All samples were collected from 8 factories in Fuzhou, Fujian Province, China. All these producers use traditional brewing techniques without adding any food additives. All chemicals used were of analytical grade and obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Preparation of samples 2 1. Ethyl acetate extraction 3 The 50 ml sample was mixed with 20 ml ethyl acetate and loaded into a 250-ml separatory funnel; after shaking vigorously, the funnel was laid still for 20 min; the upper organic phase was collected. Then repeated this process three times. The final extract was stored at 4 °C before GC-MS analysis. 2. Purge and trap (P&T) 3 The 5 ml sample was introduced into a glass vessel at 60 °C and purged with 60 ml/min of N 2 for 11 min. Stainless steel cartridges packed with 100 mg Tenax TA (previously deactivated under N 2 at 250 °C for 2 h) were used as traps. After that, the cartridges were capped at 210 °C for 4 min, with a split ratio of 2:1. 3. Determination by GC-MS 3 The GC-MS system used for analysis was Agilent Technologies 6890N with Agilent 5975 inert XL Mass selective Detector (Agilent Technologies Co., Ltd., Palo Alto, USA) operating at 70 eV in EI (electron ionization) mode. Data acquisition and analysis were performed by using Enhanced Data Analysis software, which is supplied by the manufacturer. Substances were separated on a fused silica capillary column, HP-5MS flexible glass capillary gas chromatography column (30 m×0.25 mm×0.25 μm, Agilent Technologies Co., Ltd., Palo Alto, USA). The column was kept at 35 °C for 4 min, heated from 35 to l00 °C at a programmed rate of 30 °C/min and held at l00 °C for 2 min, and then heated from 100 to 300 °C at a rate of 15 °C/min and held at 300 °C for 10 min. The temperatures for the injection port, ion source, quadrupole, and interface were set at 250, 230, 150, 280 °C, respectively. Helium with a flow rate of 0.5 ml/min was used as carrier gas, and split ratio was 2:1. The parameter was monitored in full scan mode with a scan range of m / z 40~400. RESULTS 1 Analytical results of purge and trap 2 The GC-MS with P & T chromatographic profile of the sample is shown in Fig. 1 1 , and the list of the principal volatile compounds in Fuzhou Yulu is given in Table 1 1 . A total of 12 peaks were identified, and 10 matched to the known compounds: dimethyl disulfide, 2-heptanone, 2,6-dimethyl pyrazine, benzaldehyde, dimethyl trisulfide, 2-ethyl-6-methyl pyrazine, 2-methyl-5-(1-methylethyl) pyrazine, acetophenone, 3-ethyl-2,5-dimethyl pyrazine, dimethyl tetrasulfide, respectively (Table 1 1 ). The match degrees of these 10 peaks were all above 78%. The other two peaks failed to match to any known compounds due to their low match degrees (Table 1 1 ). RT : retention time Peak No. RT (min) Component name Match (%) 1 2.694 Acetone 9 2 3.401 Formic acid ethenyl ester 9 3 5.616 Dimethyl disulfide 96 4 7.548 2-Heptanone 91 5 7.872 2,6-Dimethyl pyrazine 72 6 8.660 Benzaldehyde 95 7 8.822 Dimethyl trisulfide 95 8 9.178 2-Ethyl-6-methyl pyrazine 90 9 9.954 2-Methyl-5-(1-methylethyl) pyrazine 87 10 10.199 Acetophenone 87 11 10.314 3-Ethyl-2,5-dimethyl pyrazine 91 12 12.212 Dimethyl tetrasulfide 78 Analytical results of extracts by ethyl acetate 2 The GC-MS chromatographic profiles of the solvent (ethyl acetate) and the ethyl acetate extracted sample are shown in Figs. 2a and 2b 2a and 2b , respectively. The comparison of principal volatile compounds between solvent and the extracted sample is given in Table 2 2 . As shown in Fig. 2a 2a , all peaks of ethyl acetate have flown out within 7 min. Therefore, the peaks after 7 min in Fig. 2b 2b belong to the extracted sample. Except for ethyl acetate, propanoic acid ethyl ester, toluene, acetic acid and butyl ester which exist in extractant, 2-methyl propanoic acid, butanoic acid, 3-methyl butanoic acid, 3-methyl pentanoic acid, 2-methyl butanoic acid, 2-methyl hexanoic acid, benzeneacetic acid, benzenepropanoic acid, phthalic acid isobutyl octyl ester, and 1,2-benzenedicarboxylic acid butyl 2-methylpropyl ester were also determined in ethyl acetate extracted samples. In addition, diethyldithiophosphinic acid and 2,3,4-trihydroxybenzaldehyde could not be confirmed due to their low match degrees. RT : retention time Peak No. RT (min) Component name Match (%) 4 Ethyl acetate 1~14 3.490~4.386 Ethyl acetate 90~72 15 5.240 Propanoic acid ethyl ester 72 16 5.976 Toluene 95 17 6.589 Acetic acid, butyl ester 90 4 4 Fuzhou Yulu 1~27 3.401~4.386 Ethyl acetate 90~72 28 5.240 Propanoic acid ethyl ester 64 29 5.976 Toluene 91 30 6.323 2-Methyl propanoic acid 64 31 6.593 Acetic acid, butyl ester 90 32 6.876 Butanoic acid 91 33 7.350 3-Methyl butanoic acid 81 34 7.583 2-Methyl hexanoic acid 78 35 12.429 Benzeneacetic acid 90 36 13.376 Benzenepropanoic acid 96 37 17.520 Diethyldithiophosphinic acid 43 38 17.791 Phthalic acid isobutyl octyl ester 90 39 18.192 2,3,4-Trihydroxybenzaldehyde 47 40 18.298 2,3,4-Trihydroxybenzaldehyde 43 41 18.429 1,2-Benzenedicarboxylic acid butyl 2-methylpropyl ester 93 DISCUSSION 1 Effect of different method on analytical results 2 In previous studies (Shimoda et al., 1996 1996 ; Michihata et al., 2002 2002 ), a group of volatile compounds in fish sauce were identified by using headspace gas analysis, which includes alcohols, nitrogen-containing constituents, sulfur-containing constituents and aldehydes. However, none of fatty acid or long-chain alcohols was detected by this method. Considering the powerful analytical ability of GC-MS in determining volatile flavor components, we successfully applied GC-MS analysis on fish sauce. The results reveal comprehensive constitution of volatile components in fish sauce, with some components detected for the first time, such as acetophenone. We also compared the two different pretreatment methods in preparing the samples, i.e., P&T and ethyl acetate extractions. We found considerable differences between these two methods. When the fish sauce sample was pretreated with P&T method, GC-MS could detect sulfur-containing constituents and nitrogen-containing constituents, i.e., aldehydes and ketones. When the fish sauce sample was treated by ethyl acetate extraction, GC-MS could detect organic acids and esters. These data indicate that different sampling injection and different pretreated method were able to detect different components. Therefore, in order to determine a more comprehensive fingerprint of the volatile components of fish sauce, we need to collect different samples widely and to combine different analytical methods as well. Characteristics of volatile components of Fuzhou Yulu 2 A total of 22 volatile components of fish sauce were detected in this study, including sulfur-containing constituents, nitrogen-containing constituents, aldehydes, ketones, organic acids, and esters. While the results show a great similarity between Fuzhou Yulu and other brands of fish sauces, the characteristics of Fuzhou Yulu were also detected. Fuzhou Yulu and other brands of fish sauces were all rich in dimethyl sulfides, pyrazine derivatives and volatile organic acids, but the constitution of organic acids in Fuzhou Yulu was slightly different to that in others. In this study, only phthalic acid isobutyl octyl ester and 1,2-benzenedicarboxylic acid butyl 2-methylpropyl ester were detected in Fuzhou Yulu, but others reported that fish sauce has many different types of esters (Shimoda et al., 1996 1996 ). There were a few kinds of aldehydes and ketones in Fuzhou Yulu, especially simple aldehydes and ketones. In addition, lower alcohols and trimethylamine were not detected in Fuzhou Yulu. CONCLUSION 1 In summary, we analyzed the volatile components in Fuzhou Yulu by GC-MS, and compared two pretreatment methods, P&T-GC-MS and ethyl acetate extraction followed by GC-MS. We found that the main volatile components of Fuzhou Yulu are similar to those of other brands of fish sauces, but it also has its unique characteristics, which is probably due to producers’ proprietary usage of raw materials and the influence of local climate. 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