Microbial reclamation of fish processing wastes for the production of fish sauce

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<ul><li><p>Enzyme and Microbial Technology 33 (2003) 154162</p><p>Microbial reclamation of fish processing wastes forthe production of fish sauce</p><p>Ing-Lung Shih a, Lien-Guei Chen b, Ton-Shi Yu b, Wen-Teish Chang b, San-Lang Wang b,a Department of Environmental Engineering, Da-Yeh University, Chang-Hwa 51505, Taiwan</p><p>b Department of Bioindustry Technology, Da-Yeh University, Chang-Hwa 51505, TaiwanReceived 2 January 2003; accepted 27 March 2003</p><p>Abstract</p><p>In order to enhance the economical values of bonito and reduce the environmental problems caused by the wastes from bonito processing,the possibility of utilizing fish wastes for fish sauce production was investigated. Fish sauces were prepared experimentally from bonitowastes with or without the addition of various enzymes including viscera itself, soybean koji, and ang-khak. Preparations with the wholefish were also carried out for comparison. Fish sauce products with similar quality were obtained when the wastes and the whole fish wereused as the raw materials. Fish sauce with better sensory flavor was obtained from the ang-khak treatment. Volatile compounds of fish saucesamples were separated, identified by Gas Chromatography-Mass Spectroscopy. A total of 23 volatile compounds were identified, whichmay contribute to the aroma of the fish sauces. These aroma compounds of the fish sauces were mainly from lipids, amino compounds,and sugars of the raw materials, in which lipid was the major contributor. 2003 Elsevier Science Inc. All rights reserved.</p><p>Keywords: Fish sauce; Fish waste; Volatile compounds; Sensory evaluation</p><p>1. Introduction</p><p>Fish is one of the major commodities in Taiwan fromwhich a large domestic and export revenue is earned. How-ever, a large amount of waste materials, which generallyamount to 50% of the weight of the fish materials, weregenerated accompanying fishery processing. These fisherywastes can cause serious environmental problems if dis-posed improperly. On the contrary, these fishery wastes canbe valuable bio-resources because they contain an abundantsupply of valuable bio-materials, such as proteins, lipids,enzymes, and chitins [1]. Presently, fishery wastes are re-processed into fish or shrimp powders and fish or shrimpsolubles. These recovered products are low in market val-ues, making the recovery process uneconomical. Therefore,there is necessity of converting these wastes into highervalue products [2].</p><p>Fishery products of bonito include dry bonito and cannedbonito. In a regularly manufacturing processing, the headsand viscera are wastes. These wastes take away a largeportion of the fish. In addition, treatment of these wastes is</p><p> Corresponding author. Tel.: +886-2-2809-6078;fax: +886-2-2809-1892.</p><p>E-mail address: sabulo@mail.dyu.edu.tw (S.-L. Wang).</p><p>tedious and expensive. Studies showed that bonito is abun-dant in unsaturated fatty acids such as DHA and EPA whichare of good nutritional value [3]. In addition to the inherentnutritional value, bonito emits mellow aroma when madeinto sauce that is a rich food flavor additive. The purpose ofthis study is to investigate the suitable method for conver-sion of bonito wastes into valuable fish sauce, and evaluatethe quality of the sauce thus produced. Fish sauce is widelyused in Southeast Asia and to some extent in some parts ofthe world, which is commonly used as a condiment to addflavor to the flatness in taste in rice, a staple food of thesoutheast people. It also provides a supplement to some forthe protein requirements of the people. The utilization ofbonito wastes for fish sauce production are both economicaland environmental advantageous.</p><p>2. Materials and methods</p><p>2.1. Materials</p><p>Bonitos were purchased from a local fishery plant. Thefish were frozen catches from the Pacific Ocean. Soybeankoji (Aspergilus oryzae) and ang-khak (Monacus purpureus)were purchased from a local supermarket.</p><p>0141-0229/$ see front matter 2003 Elsevier Science Inc. All rights reserved.doi:10.1016/S0141-0229(03)00083-8</p></li><li><p>I.-L. Shih et al. / Enzyme and Microbial Technology 33 (2003) 154162 155</p><p>2.2. Experimental preparations</p><p>Frozen bonitos were defrosted at room temperature for 1 hbefore cutting into flesh body, head, fins, and viscera. Ex-periments were divided into eight treatments for fermenta-tion. Each treatment contains different combinations of fishbody parts with or without the addition of koji or ang-khak.These eight treatments were as the following: (1) visceraand wastes: Bonito wastes were blended and sterilized at121 C for 15 min. Blended bonito wastes were mixed withviscera (1:2, w/w) and a 20% salt solution. The mixture wasput into a porcelain vase for fermentation. (2) Viscera andwhole fish: Whole bonitos were blended and sterilized at121 C for 15 min, which was then mixed with viscera (l:2,w/w) and a 20% salt solution. The mixture was put intoa porcelain vase for fermentation. (3) Bonito wastes only:Blended bonito wastes and a 20% salt solution were inocu-lated into a porcelain vase for fermentation. (4) Whole fishonly: Blended whole bonitos and a 20% salt solution wereinoculated into a porcelain vase for fermentation. (5) Soy-bean koji and bonito wastes: Blended and sterilized bonitowastes were mixed with a 38% soybean koji. The mixturewas stored at room temperature for 2 days before a 20%salt solution was added, and then put into a porcelain vasefor fermentation. (6) Soybean koji and whole fish: Blendedand sterilized whole bonitos were mixed with a 38% soy-bean koji. The mixture was stored at room temperature for2 days before a 20% salt solution was added, and then putinto a porcelain vase for fermentation. (7) Ang-khak andbonito wastes: Blended and sterilized bonito wastes weremixed with a 38% Ang-khak. The mixture was stored atroom temperature for 2 days before a 20% salt solution wasadded, and then put into a porcelain vase for fermentation.(8) Ang-khak and whole fish: Blended and sterilized wholebonitos were mixed with a 38% ang-khak. The mixture wasstored at room temperature for 2 days before a 20% saltsolution was added, and then put into a porcelain vase forfermentation.</p><p>The pH, protein contents, and free amino acids were an-alyzed during the fermentation at room temperature up to 5months. Enzymatic activities and aromatic flavor were alsoanalyzed regularly.</p><p>2.3. Chemical analyses</p><p>The pH and protein contents were determined as de-scribed in standard methods [4]. The contents of reducingsugar and total sugar were measured by Millers method[5] and phenol-sulfuric method [6], respectively. Trimethy-lamine (TMA) and volatile basic nitrogen (VBN) weremeasured following Castell et al.s method [7], and Cobbet al.s method [8], respectively. The compositions of freeamino acids were determined by directly submitting super-natant samples to a Beckman system 6300E amino acidanalyzer. Color was measured using a M80 color Measuring</p><p>System (Nippon Denshoku Inc., Co., Japan). In this colornotation system, L indicates lightness darkness, Aindicates red-green, and B indicates yellow-blue. Thisinstrument was calibrated using a white standard plate.</p><p>2.4. Assay of enzyme activity</p><p>For enzyme activity analysis, the samples of fish sourcewere dialyzed against a 50 mM phosphate buffer (pH 7.0)at 40 C for 24 h and served as enzyme solutions. Proteaseactivity was assayed at 37 C with Hammarsten casein(Merck, Germany) as a substrate. Three-fourth of 1 ml of1.33% casein in a 50 mM phosphate buffer (pH 7.0,) wasincubated for 5 mm at 37 C followed by adding 0.25 ml ofthe enzyme solution. After 20 min, the reaction was termi-nated by adding 1 ml of a 0.44 M trichloroacetic acid (TCA)solution. The reaction was kept at 37 C for 20 min and fil-tered through Whattman No. 5 filter paper. 0.5 ml of the fil-trate was added with 2.5 ml of 0.44 M Na2CO3 solution and0.5 ml of phenol reagent After mixing completely, the reac-tion mixture was kept at 37 C for 20 min. The absorbanceof the solution was measured at 660 nm. One unit (U) wasdefined as the amount of enzyme that released 1g equiva-lent of tyrosine per minute. Amylase activity was measuredwith soluble starch (Wako Pure Chemical Ind., Japan) as asubstrate. Enzyme solution (0.1 ml) was mixed with 0.4 mlof 1.5% soluble starch in a 50 mM phosphate buffer (pH7.0). After incubation at 37 C for 20 min, the mixture wascentrifuged and the increased reducing sugar was measuredas glucose by the Millers method [5]. One unit (U) ofamylase activity was defined as the amount of enzyme thatliberates 1mol equivalent of reducing sugar per minute.</p><p>2.5. Volatile compound analyses</p><p>2.5.1. Sample preparationDuring 5-month fermentation period, liquid portion of the</p><p>fish sauce was occasionally drawn from the broth and usedfor analysis. This liquid portion was centrifuged for 20 minat 10,000 g and 4 C to remove the residual particles.</p><p>2.5.2. Isolation of the volatile compoundsFive hundred grams of the resultant supernatant prepared</p><p>above was steam distilled and extracted with 60 ml of redis-tilled methylene chloride in a modified LikensNickersonapparatus for 3 h. After the steam distillation was done,the extract was dehydrated by anhydrous sodium sulfate.After filtration, the compound mixtures isolated were con-centrated in a fractionation column filled with glass beads.The flavor concentrate was further concentrated by nitrogenflow to a minimum volume. The weight of the flavor isolatewas recorded.</p><p>2.5.3. Gas Chromatography (GC) analysisA Hitachi G-3000 gas chromatograph equipped with a</p><p>fused silica capillary column (50 m 0.32 mm i.d., 1m</p></li><li><p>156 I.-L. Shih et al. / Enzyme and Microbial Technology 33 (2003) 154162</p><p>thickness; DB-WAX, J&amp;W Inc.) and a flame ionization de-tector (FID) was used to analyze the volatile compounds.The operating conditions were as following: injector temper-ature, 250 C; detector temperature, 270 C; nitrogen carrierflow rate, l.2 ml/min; temperature program, 40 C (5 min),2 C/min, 240 C (60 min). A split ratio of 50 to 1 was used.</p><p>2.5.4. Quantification of the volatile compoundsQuantification of a volatile compound in the fish sauce</p><p>was based on its peak area on the GC chromatogram againstthe weight of the flavor concentrate isolated from the fishsauce as described above. The response factor for each com-pound on FID was assumed to be one. The concentration ofa volatile compound in the fish sauce was estimated usingthe following formula:Concentration (ppb)</p><p>=(area of a compound/total area of compounds)weight of the isolate (g)</p><p>500 (g) 106</p><p>2.5.5. Gas Chromatography-Mass Spectrometry(GC-MS) analysis</p><p>The concentrated isolate was analyzed using a GC-MS,a Hewlett-Packard 5890 gas chromatograph coupled to aHewlett-Packard MSD equipped with a direct split interface,and a fused silica capillary column (50 m 0.32 mm i.d.,1m thickness; DB-WAX, J&amp;W Inc.). The operating con-ditions were the same as those used in the GC analysisdescribed above. Mass spectra were obtained by electronionization at 70 eV and an ion source temperature of 250 C.</p><p>2.5.6. Identification of the volatile compoundsThe volatile compounds in the isolate were identified</p><p>based on the data collected on the GC-MS. The assignmentof volatile compounds was accomplished by comparing thespectral data with those of authentic compounds availablefrom the Browse-Wiley computer library, NBS computerlibrary, or previously published literature data.</p><p>Table 1Some chemical properties of the fish sources</p><p>Property Treatment</p><p>1 2 3 4 5 6 7 8</p><p>pH 7.0 6.5 7.1 6.6 5.9 5.3 5.5 4.7Total sugar (mg/ml) 4.6 3.6 4.6 5.9 10.8 17.4 16.1 12.1Reducing sugar (mg/ml) 0.9 1.0 1.4 1.1 1.7 2.0 3.4 1.7Protein (mg/ml) 5.0 7.0 5.0 10.0 8.0 9.0 6.0 8.0Amylase activity (U/ml) 95 100 20 5.0Protease activity (U/ml) 36 35 80 44 86 60 50 NaCl (g/100 g) 20.3 24.2 21.4 24.2 23.2 25.1 21.0 23.5TMA (mg/100 g) 1.3 26.7 4.0 37.3 33.3 26.7 41.3 25.3VBN (mg/100 g) 448 490 308 420 448 280 364 224L 40 38 38 36 35 36 36 39A 9 7 10 7 5 7 8 12B 10 6 8 5 4 5 5 10</p><p>2.6. Sensory evaluation</p><p>Quantitative descriptive analysis (QDA) was performedto determine the sensory characteristics of fish sauces.Analyses were carried out with an internal panel of eightmembers. Each treatment was coded and presented to panelmembers in isolation in a sensory laboratory. Memberswere asked to compare sensory attribute of each treatment(bitter, sweet, savory umami, alcohol, cheesy, sour, am-monia, meaty, caramel) on a 10-point scale, 0 accountsfor a not perceivable intensity while 9 accounts for anextreme intensity of an attribute.</p><p>2.7. Statistical analysis</p><p>The data were processed with SAS statistical package forthe analysis of variances and deviation of standard deviationsamong observations per data set.</p><p>3. Results and discussions</p><p>To monitor the change during the course of fermentation,several chemical properties such as pH, protein contents,and free amino acids were analyzed during the fermentation.Enzymatic activities and aromatic flavor were also analyzedregularly. The properties of the fish sauces of all the treat-ments are compared in Table 1. The change of pH during thecourse of fermentation is shown in Fig. 1. During the firstmonth of fermentation, the medium of each set of experi-ment was found to be acidic; however, pH of the mediumincreased thereafter. The rise of pH is probably due to theincrease of alkaline VBN during the fermentation period;the final VBN values of each set of treatments are shown inTable 1. TMA and VBN are indices for fishiness odor, withthe thresholds of 6 mg/100 g and 50 mg/100 g, respectively.Unfortunately, Table 2 shows that the TMA and VBN con-tents of most of the products produced in this study exceeded</p></li><li><p>I.-L. Shih et al. / Enzyme and Microbial Technology 33 (2003) 154162 157</p><p>Fig. 1. Changes in pH value of the fish sauce during ripening at room temperature. The reaction was done under the conditions described in Section 2.The symbols , , , , , , and represent treatments 18, respectively.</p><p>Table 2Free amino acid composition (mg/100 ml) of the fish sourceAmino acid Treatment</p><p>1 2 3 4 5 6 7 8</p><p>EssentialArg 0.6 0.7 0.7 1.0 1.0 1.9 4.2His 19.8 124.4 75.2 73.2 47.6 62.0 38.8 68.6Ile 137.7 372.9 129.0 245.8 227.8 263.0 181.3 253.0Leu 59.0 129.5 51.8 90.4 84.7 97.0 69.4 95.2Lys Met 22.0 66.8 21.2 37.5 28.3 33.6 23.4 34.4Phe 72.8 193.4 11.0 116.8 92.8 124.8 82.7 144.2Thr 21.2 21.0 19.6 33.6 30.7 39.1 26.3 42.9Tyr 40.6 32.8 35.1 25.9 15.1 32.5 29.8 46.9Val 39.2 87.6 36.2 51.8 47.8 52.4 41.5 51.5</p><p>Total (a) 412.3 1029 379.8 675.7 575.8 705.4 495.1 740.9Nonessential</p><p>Ala 42.8 80.0 27.1 47.3 43.9 50.8 41.3 57.8Asp 39.3 19.4 58.4 14.3 13.8 20.6 24.8 12.9Glu 259.2 Asn Gln 0.5 1.3 0.8 2.7 0.8 0.9 Gly 36.9 78.8 29.9 40.1 41.4 45.5 35.7 45.3Pro 20.5 41.6 9.4 13.1 12.6 19.1 12.1 20.2Ser 0.5 0.7 0.8 20.8 37.2 21.4 32.7Cys 1.7 2.9 1.1 1.3 1.6 1.4 1.4 1.2</p><p>Total (b) 142.2 483.2 127.4 119.6 134.9 175.5 136.7 170.1Grand total (a + b) 554.5 1512.2 507.2 795.3 710.7 880.9 631.8 911.0</p></li><li><p>158 I.-L. Shih et al. / Enzyme and Microbial Technology 33 (2003) 154162</p><p>the limits. The exceptions are TMA concent...</p></li></ul>

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