9
Enzyme and Microbial Technology 33 (2003) 154–162 Microbial reclamation of fish processing wastes for the production of fish sauce 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 b Department of Bioindustry Technology, Da-Yeh University, Chang-Hwa 51505, Taiwan Received 2 January 2003; accepted 27 March 2003 Abstract 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 bonito wastes with or without the addition of various enzymes including viscera itself, soybean koji, and ang-khak. Preparations with the whole fish were also carried out for comparison. Fish sauce products with similar quality were obtained when the wastes and the whole fish were used as the raw materials. Fish sauce with better sensory flavor was obtained from the ang-khak treatment. Volatile compounds of fish sauce samples were separated, identified by Gas Chromatography-Mass Spectroscopy. A total of 23 volatile compounds were identified, which may 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. Keywords: Fish sauce; Fish waste; Volatile compounds; Sensory evaluation 1. Introduction Fish is one of the major commodities in Taiwan from which a large domestic and export revenue is earned. How- ever, a large amount of waste materials, which generally amount to 50% of the weight of the fish materials, were generated accompanying fishery processing. These fishery wastes can cause serious environmental problems if dis- posed improperly. On the contrary, these fishery wastes can be valuable bio-resources because they contain an abundant supply 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 shrimp solubles. These recovered products are low in market val- ues, making the recovery process uneconomical. Therefore, there is necessity of converting these wastes into higher value products [2]. Fishery products of bonito include dry bonito and canned bonito. In a regularly manufacturing processing, the heads and viscera are wastes. These wastes take away a large portion of the fish. In addition, treatment of these wastes is Corresponding author. Tel.: +886-2-2809-6078; fax: +886-2-2809-1892. E-mail address: [email protected] (S.-L. Wang). tedious and expensive. Studies showed that bonito is abun- dant in unsaturated fatty acids such as DHA and EPA which are of good nutritional value [3]. In addition to the inherent nutritional value, bonito emits mellow aroma when made into sauce that is a rich food flavor additive. The purpose of this study is to investigate the suitable method for conver- sion of bonito wastes into valuable fish sauce, and evaluate the quality of the sauce thus produced. Fish sauce is widely used in Southeast Asia and to some extent in some parts of the world, which is commonly used as a condiment to add flavor to the flatness in taste in rice, a staple food of the southeast people. It also provides a supplement to some for the protein requirements of the people. The utilization of bonito wastes for fish sauce production are both economical and environmental advantageous. 2. Materials and methods 2.1. Materials Bonitos were purchased from a local fishery plant. The fish were frozen catches from the Pacific Ocean. Soybean koji (Aspergilus oryzae) and ang-khak (Monacus purpureus) were purchased from a local supermarket. 0141-0229/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0141-0229(03)00083-8

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

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Page 1: Microbial reclamation of fish processing wastes for the production of fish sauce

Enzyme and Microbial Technology 33 (2003) 154–162

Microbial reclamation of fish processing wastes forthe production of fish sauce

Ing-Lung Shiha, Lien-Guei Chenb, Ton-Shi Yub, Wen-Teish Changb, San-Lang Wangb,∗a Department of Environmental Engineering, Da-Yeh University, Chang-Hwa 51505, Taiwan

b Department of Bioindustry Technology, Da-Yeh University, Chang-Hwa 51505, Taiwan

Received 2 January 2003; accepted 27 March 2003

Abstract

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.

Keywords:Fish sauce; Fish waste; Volatile compounds; Sensory evaluation

1. Introduction

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].

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

∗ Corresponding author. Tel.:+886-2-2809-6078;fax: +886-2-2809-1892.

E-mail address:[email protected] (S.-L. Wang).

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.

2. Materials and methods

2.1. Materials

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.

0141-0229/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved.doi:10.1016/S0141-0229(03)00083-8

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2.2. Experimental preparations

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.

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.

2.3. Chemical analyses

The pH and protein contents were determined as de-scribed in standard methods[4]. The contents of reducingsugar and total sugar were measured by Miller’s 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

System (Nippon Denshoku Inc., Co., Japan). In this colornotation system, “L” indicates lightness darkness, “A”indicates red-green, and “B” indicates yellow-blue. Thisinstrument was calibrated using a white standard plate.

2.4. Assay of enzyme activity

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 1�g 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 Miller’s method[5]. One unit (U) ofamylase activity was defined as the amount of enzyme thatliberates 1�mol equivalent of reducing sugar per minute.

2.5. Volatile compound analyses

2.5.1. Sample preparationDuring 5-month fermentation period, liquid portion of the

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.

2.5.2. Isolation of the volatile compoundsFive hundred grams of the resultant supernatant prepared

above was steam distilled and extracted with 60 ml of redis-tilled methylene chloride in a modified Likens–Nickersonapparatus 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.

2.5.3. Gas Chromatography (GC) analysisA Hitachi G-3000 gas chromatograph equipped with a

fused silica capillary column (50 m× 0.32 mm i.d., 1�m

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thickness; DB-WAX, J&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.

2.5.4. Quantification of the volatile compoundsQuantification of a volatile compound in the fish sauce

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)

=(area of a compound/total area of compounds)

×weight of the isolate(g)

500(g) × 10−6

2.5.5. Gas Chromatography-Mass Spectrometry(GC-MS) analysis

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.,1�m thickness; DB-WAX, J&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.

2.5.6. Identification of the volatile compoundsThe volatile compounds in the isolate were identified

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.

Table 1Some chemical properties of the fish sources

Property Treatment

1 2 3 4 5 6 7 8

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) 448 490 308 420 448 280 364 224

L 40 38 38 36 35 36 36 39A 9 7 10 7 5 7 8 12B 10 6 8 5 4 5 5 10

2.6. Sensory evaluation

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.

2.7. Statistical analysis

The data were processed with SAS statistical package forthe analysis of variances and deviation of standard deviationsamong observations per data set.

3. Results and discussions

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 inTable 1. The change of pH during thecourse of fermentation is shown inFig. 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 2shows that the TMA and VBN con-tents of most of the products produced in this study exceeded

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Fig. 1. Changes in pH value of the fish sauce during ripening at room temperature. The reaction was done under the conditions described inSection 2.The symbols�, �, �, �, �, �, and � represent treatments 1–8, respectively.

Table 2Free amino acid composition (mg/100 ml) of the fish source

Amino acid Treatment

1 2 3 4 5 6 7 8

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

Total (a) 412.3 1029 379.8 675.7 575.8 705.4 495.1 740.9

NonessentialAla 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

Total (b) 142.2 483.2 127.4 119.6 134.9 175.5 136.7 170.1

Grand total (a+ b) 554.5 1512.2 507.2 795.3 710.7 880.9 631.8 911.0

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the limits. The exceptions are TMA concentration of treat-ments 1 and 3, which are 1.3 mg/100 g and 4.0 mg/100 g,respectively. As shown inTable 1, treatments 5–8 still hada final pH below 6; however, these acidic pH were suitablefor the sour taste in the final fish source. Although the rea-son is still not clear, it is noteworthy that the pH trend ofbonito fermentation is contrary to that of bean sauce fer-mentation[9]. The sugar contents in the supernatant liquorof all treatments were measured after 5-month fermentation,and are shown inTable 1. The supernatant liquor producedby using soybean koji (treatments 5 and 6) and ang-khak(treatments 7 and 8) as inocula contained high sugar con-centrations. The total sugar contents of treatments 5–8 are10.8, 17.4, 16.1, and 12.1 mg/l, respectively. In contrast, thetotal sugar contents of treatments 1–4 are 4.6, 3.6, 4.6, and5.9 mg/l, respectively. The reducing sugar contents for treat-ments 5–8 are 1.7, 2.0, 3.4, and 1.7 mg/l in contrast to 0.9,1.0, 1.4, and 1.1 mg/l for treatments 1–4. The sugar con-tents of supernatant liquor produced by soybean koji andang-khak inoculation are much higher than those producedwithout the same inoculation. The difference is probablydue to the fact that these two inocula themselves have highsugar content[10]. The reason that koji and ang-khak wereused as inocula is that these two are nonpathogenic and fre-quently used in food processing to add to the aroma, nutri-tion, and color of the fermentation produced. For example,ang-khak is frequently used in Chinese red-wine fermenta-tion, and soybean koji for bean sauce fermentation[11–13].As shown inTable 1, amylase activities were not detectablein the supernatant liquor of treatments 1–4. In contrast, amy-lase activities were 95, 100, 20, and 5 U/ml in that of treat-ments 5–8, respectively. This result is consistent with thefact that higher sugar contents were detected in treatments5–8. However, the relationships between amylase activitiesand sugar contents were aberrant.

In fish sauce fermentation, supernatant liquor is produced,which increase in its soluble nitrogen contents over a periodof months. This soluble nitrogen which includes proteins,peptides, and amino acids is mainly derived from breakdownof the fish protein by the enzymes of the fish. Therefore,it is essential to analyze the protease, peptide, and aminoacid constituents in the fish sauce. As shown inTable 1,the protein contents in the experiments containing bonitowaste were lower than those in the experiments containingthe whole fish (e.g. treatment 1 versus treatment 2, treatment3 versus treatment 4, treatment 5 versus treatment 6, treat-ment 7 versus treatment 8). However, the difference is notvery significant. The free amino acid contents of the ripenliquor of each set of experiments are shown inTable 2. Itis evident that the amino acid contents, whether essential ornonessential, of the experiments containing bonito wasteswere lower than those containing the whole fish. This trendis similar to that found in contents. Since the quality ofany protein or hydrolysate originates mainly from its essen-tial amino acids content, it is particularly noteworthy thatthe essential amino acids content are much higher than the

nonessential ones in all eight treatments. The relationshipbetween the individual amino acid and the flavor has beenreported. For example, glutamic acids are responsible forpalatable “umami” taste; glycine and alanine for sweetness;proline, arginine, methionine, and leucine for bitterness. Inthis study, asparagine, glutamine, cystein, lysine, and argineconcentrations were low, despite the facts that lysine andarginine are essential amino acids. In all experimental treat-ments, the essential amino acid components obtained werecomposed mainly of isoleucine, phenyalamine, leucine, andmoderately of tereonine, valine, tyrosine, methionine, andhistidine. The nonessential group was moderate in asparticacids, glycine, alanine, and proline. The total amino acidsconcentration of all the products prepared in this study arelower in comparison to those of a commercial product. Thismay be due to the facts that a high amount of salts wereadded and the lack of heat thickening. It must be remem-bered that hydrolysis is total place under high salt conditionswhich could be detrimental to the proteolytic activity of theenzymes present in fermentation broth. Proteolytic enzymesare essential in the fish sauce production. Delicate relation-ships of protease activities and aroma of fish has been re-ported [14]. For example, the effect of adding bromelain,papain, or ficin, on the rate of hydrolysis and extent of theconversion of insoluble fish protein to soluble nitrogen wasfully examined[14]. The protease activity during the courseof fermentation is shown inFig. 2, and the final protease ac-tivity of the ripen liquor of each set of experiment are shownin Table 1. As shown inFig. 2, treatments 1 and 2, both con-tained viscera, exhibited higher protease activities during thefirst month of ripening as compared to the other treatments.However, these activities were quickly reduced in the restof the fermentation period. Unfortunately, there is no clearconclusion that can be drawn on the relationship betweenprotease activity and soluble nitrogenous components.

To the consumer, aroma is a prime indicator of the qualityof a fish. Part of the aroma, such as nitrogenous compoundsformed in the supernatant liquor could have an effect onconsumer acceptability; others such as volatile compoundsare equally important in terms of the flavor and nutritionalvalues. Volatile compounds identified in the fish sauce prod-ucts of this study are shown inTable 3. A total of 23 volatilecompounds were identified, and they are separated into fivegroups according to their probable original sources, i.e. (1)sugar origin: limonene, furfuryl alcohol; (2) lipids origin:2,4-hexadienal, heptanoic acid, (E,E)-3,5-octadien-2-one,octanoic acid, decanal, methyl hexadecanoic acid, ethyltetradecanoic acid, ethyl hexadecanoic acid, dodecanol(isomer 1), ethyl pamitoleate, dodecanol (isomer 2), ethyloctadecanoic acid, methyl octadecanoic acid, tetradecanoic,and hexadecanoic acid; (3) amino compounds origin (i.e.proteins and peptides): isoamyl alcohol and dimethyl trisul-fide; (4) both sugar and amino compound origin: ethanoland 1-propanol; (5) both amino compound and lipid ori-gin: butanoic acid and 3-methyl butanoic acid. Most ofthe volatile compounds found in this study are originated

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Fig. 2. Changes in protease activity of the fish sauce during ripening at room temperature. The reaction was done under the conditions described inSection 2. The symbols�, �, �, �, �, �, and � represent treatments 1–8, respectively.

Table 3Volatile compounds identified in the fish sauce products

Source Compound Mw Formula

Sugars Limonene 136 C10H18

Furfuryl alcohol 98 C5H6O2

Lipids 2,4-Hexadienal 96 C6H8OHeptanoic acid 130 C7H14O2

(E,E)-3,5-Octadien-2-one 124 C8H16O2

Octanoic acid 144 C8H16O2

Decanal 156 C10H20OMethyl hexadecanoic acid 270 C17H34O2

Ethyl tetradecanoic acid 256 C16H32O2

Dodecanol (isomer 1) 184 C12H24OEthyl palmitoleate 282 C18H34O2

Dodecanol (isomer 2) 184 C12H24OEthyl octadecanoic acid 312 C20H40O2

Methyl octadecanoic acid 296 C19H36O2

Tetradecanoid acid 228 C14H28O2

Hexadecanoic acid 256 C16H32O2

Amino compounds Isoamyl alcohol 88 C5H12ODimethyl trisulfide 126 C2H6S2

Sugars and amino compounds Ethanol 46 C2H6O1-Propanol 60 C3H8O

Lipids and amino compounds Butanoic acid 88 C4H8O2

3-Methyl butanoic acid 88 C5H10O2

Volatile compounds are separated into five groups according to their probable original sources.

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Table 4The concentration of the individual and total volatile compound in each set of treatments

Compound Formula Concentration (ppm)

No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8

Ethanol C2H6O –a – – – – – – 0.252,4-Hexadienal C6H8O 0.21 0.04 – 0.03 0.16 – 0.07 0.041-Propanol C3H8O 0.03 – – 0.06 0.04 0.01 0.03 0.38Isoamyl alcohol C5H12O 0.28 0.14 0.17 1.67 0.26 0.42 4.60 0.70Limonene C10H18 0.63 0.53 0.16 – – 0.30 0.15 0.34Dimethyl trisulfide C2H6S2 0.14 0.06 0.05 0.10 0.38 0.05 0.04 0.01Heptanoic acid C7H14O2 0.02 0.02 – 0.05 0.01 0.04 0.05 0.06(E,E)-3,5-Octadien-2-one C8H12O 0.32 0.31 0.31 – 0.82 0.05 – 0.06Butanoic acid C4H8O2 0.02 0.03 3.52 0.10 0.02 – 0.01 1.53Octanoic acid C8H16O2 – 0.38 – 1.62 – 1.42 0.85 –3-Methyl butanoic acid C5H10O2 – – – – 0.08 – – 1.25Furfuryl alcohol C5H6O2 0.73 – 0.35 – – – – 1.69Decanal C10H20O 0.02 0.04 – 0.05 0.04 0.76 0.03 –Methyl hexadecanoic acid C17H34O2 0.11 0.10 0.37 1.24 0.99 0.26 0.57 0.14Ethyl tetradecanoic acid C16H32O2 0.04 0.02 0.20 0.43 0.07 0.23 0.08 5.91Ethyl hexadecanoic acid C18H36O2 0.02 0.07 0.30 0.17 0.08 0.12 0.08 3.79Dodecanol (isomer 1) C12H24O2 0.20 0.50 0.50 0.19 0.15 0.52 0.16 7.12Ethyl palmitoleate C18H34O2 0.03 0.04 0.12 0.33 – 0.02 0.02 7.12Docecanol (isomer 2) C12H24O 0.59 0.38 3.22 0.35 0.43 0.02 0.10 0.27Ethyl octadecanoic acid C20H40O2 – – – 0.04 – 0.24 – 2.49Methyl octadecanoic acid C19H36O2 0.23 0.30 0.28 – 0.10 1.78 0.47 7.21Tetradecanoic acid C14H28O2 2.44 3.30 – 5.20 6.02 – 1.59 5.26Hexadecanoic acid C16H32O2 4.23 1.86 – 9.60 7.47 2.56 2.17 4.82

Total 10.3 8.19 9.5 21.2 17.1 8.8 11.1 50.4

a Not detected.

from lipid, a result consistent with the fact that fish tissuescontained high amount of unsaturated lipids. The notionthat these compounds were probably derived from lipidoxidation implies a fact that besides those of amylase andproteases, the actions of lipid oxidation initiating enzymeswere important during the fermentation process.

The concentrations of the individual and total volatilecompounds in each set of treatments are shown inTable 4.Evidently, the total volatile compounds in the experimentscontaining bonito waste were lower than those in exper-iments containing the whole fish (e.g. treatment 1 versustreatment 2, treatment 3 versus treatment 4, treatment 5 ver-sus treatment 6, treatment 7 versus treatment 8). Among allthe treatments that contained a whole fish as the substrate,the treatment 8 that uses ang-khak as inoculum producedan exceptionally large amount of volatile compounds. Thevolatile compounds are mainly tetradecanoic acid and hex-adecanoic acid in most of the treatments, with the exceptionof the treatment 8 in which high amounts of ethyl-estersof fatty acids were also produced. These fatty esters werepresumably generated from the esterification of fatty acidsliberated from fish oil by lipase or other lipid oxidationinitiating enzymes and ethyl alcohol that existed as one ofthe fermentation products.

Studies on the volatile compounds of fish sauces arescant, although a comparative study on the aromas of threekind of fish sauces—Shottsuru, Nampla, and Nourcmam—by using GC and GC-MS has been reported[15]. These

three fish sauces revealed aromas different from each other.A total of 50, 44, and 49 volatile compounds were identifiedin Shottsuru, Nampla, and Nourcmam, respectively. Acids,alcohols, nitrogen-containing compounds, sulfur-containingcompounds, lactones, esters, phenols, carbonyls, and hydro-carbons were among the main groups of volatile compoundsidentified. The differences in the aromas of the sampleswere thought to be due to the differences in the level ofconcentrations of the major acids. It was also concludedin Scanceda’s study that there were differences in the totalaroma of the commonly used and representative samples offish sauces from different countries and these differenceswere attributed to the aroma characteristics of the major aswell as the minor volatile compounds. It is hard to comparethe results of Scanceda’s study to that of our present study,since limited information as to the production conditions aswell as the species of fishes used in preparing Shottsuru,Nampla, and Nourcmam were known, so were their influ-ence on the total aroma. Besides, the methods used in bothstudies varied from the collection of volatile compounds tothe columns and analytic conditions of chromatography.

The use of trash fish to produce an acceptable fish sauceby traditional methods has been investigated but did notmeet with any success[14]. In this study, we demonstratedthat both bonito waste and whole fish can be used to pro-duced fish sauces with reasonable qualities in terms ofaroma, nutrition, and color. In addition, the commercialavailable soybean koji and ang-khak were suitable inocula

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Fig. 3. The sensory profiles of all treatments based on the QDA test—eight attributes by eight panelists.

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for the preparations. Although the products produced withthe whole fish as substrate and ang-khak as inoculum ap-peared to have slightly better qualities, the fish saucesproduced in all treatments were tasted and no particularlystrong or unpleasant flavor was found with any of them,indicating that no apparent spoilage had occurred. The sen-sory profiles of all treatments based on the QDA test—eightattributes by eight panelists—are shown inFig. 3.

This study is preliminary to whether bonito waste can befermented into suitable fish sauce. Therefore, elaboration offermentation conditions, such as precise control of pH tem-perature, agitation speed, time, etc., has not been attempted.These factors are currently being investigated in the hope toachieve a product of commercial quality. The use of bonitowaste has undoubtedly produced a fish source without anyloss of nutritional quality and with an increased saving incapital costs.

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