Production

Patcharee
ABSTRACT A [la-oollying agent was prepared from

of Flavouring Agent from Shrimp Heads

and Nongnuch
weight

Teerasuntonwat

Raksakulthai
nucleotides and organic bases and

peptides,

extraction of nitrogenous

compollnds

shrimp heads by with water, sodium

non-nitrogenous compounds comprising organic acids, sugars and inorga~ic constituents (Konosu and Yamaguchi, 1982). Food flavouring agents can be
produced from plants or animals, or can be chemically synthesiscd. However, the flavours from natural

chloride, hydrochloric acid, sodium hydroxide, bromelain, papain and ncutrase. The appropriate conditions of extraction were based on total solids,formaldehydenitrogen, soluble protein CO/ltents and sensory evaluation scores. Shrimp flavour extract was dried using dextrin, dextrose or sodium chloride as binders. Brome/aill was found to be the most promising extracting medium. The appropriate conditions were 0.25% bromclain, pH 6, for 5 hI's at 500( using 50dillm chloride as the binder. The free amino acid compositions of shrimp flavour pmoders prepared with "loater
and bromelaill were similar, but the total amino acid COI1-

substances

are considered

more delicate whereas

chemically synthesised flavours cannot always perfectly duplicate natural flavours. Since Thailand is a major shrimp producing country, there is a large amount of \..Taste which is sold at low
prices for feed production. shrimp \vaste to produce It IS advisable to use

at the present

a flavouring time has to be imported.

agent which

tent in shrimp flavour extracted zuith enzyme was about

2.5-fold higher. The major free ami110
acids

in both fln-

The objectives of this study were to:

(i) find

The major amino acids in the acid hydrolysate of the shrimp flavour powder were glutamic acid, glycine and alanine. Sensory evall/atioll of an aqueous soilition of the preferred flavol/ring agent and the reference krill extract, sho"ll.1Cd no significanl difference IP<O.05! bllt shrimp flapollred crackers containing 2% krill extract had a higher preference score than those prepared with 2% of the shrimp head extract.

vOllring agents were alanine, glycine and arginille.

the appropriate

conditions
in combination

for extraction

of

shrimp fla\'our \vith water, dilute acid, dilute base,

brine and proteases with heat treat. .

ment, and (n) analyse the qualIty and acceptabIlIty vour extracts prepared.
.. .

ot the fla-

INTRODUCTION The average annual increase of shrimp exports from Thailand from 1981 to 1991 was 20.5%. Exports in 1981 were 18,761 ton increasing to 121,239 ton in 1991 (Office o~ Agricultural Economics, 1992). Approximately 7:)% of the shnmp was exported frozen, the rest was processed into dried, boiled or canned products (Office of Agricultural Economics, 1991). Waste from shrimp processing plants varied from 40-80% of the total weight (Afolabi, Oka and Umoh, 1980). Shrin1p waste is used dried or fresh as animal feed but has other value added uses for production of chitin or chitosan (Johnson and Peniston, 1982), carotenoprotein (Simpson and Haard, 1985), and flavouring compounds (Gray, 1966) or directly as an

MATERIALS Raw material Black from -30C were

AND

METHODS

tiger prawn heads (Penealls m01l0dOll Fabricius) a freezing plant in Bangkok were frozen at and kept at -20C until used. Shrimp heads partially thawed and ground in a blender. without enzyme

Extraction

Ground shrimp heads (20 g) were blended with an extracting solution (water, brine, dilute HCI, dilute NaOH) at a ratio of 1:2 (\v Iv), transferred to Erlenmeyer flasks, covered with aluminium foil and incubated at different ten1peratures for different times. After incubation, the mixtures were filtered through

ingredient in oriental prawn crackers (Sophanodora and Buckle, 1988).

The flavour components in fish and shellfish can be divided into two broad groups: nitrogenous compounds including free amino acids, low molecular Ms
TeeraS//Iltommt

Whatman No. -+ paper. The residues \vere rinsed twice with boiled water, each tin1e using an amount of
boiled water equal to that of the extracting solution. The filtrate collected was combined and kept at -20'C for further analysis. The concentrations of Nael were 1, 3 and 10'70 (w Iv), the concentrations of HCI 0.3, 0.5 and 1.0% (\' Iv) and
the concentrations of NaOH All samples were incubated 0.3, 0.5 and 1.0% (w Iv).

was a Graduate~flldelltalld Dr Raksakllltlllli

is all Assistallt Professor at fhe Faculty of Fisheries, Kascfsart University, Bangkok J0900, THAILAND.

at 100C for 60 min.

ASEA.'\'Food Journal Vol. 10,No.4, 1995

131

The incubation temperatures were 37, 50, 100 and 120cC with water, 1% NaCl, 0.3% HCI or 0.3% NaOH. All samples were incubated for 60 min. The incubation times studied were 30, 60, 120 and 150 min. at IOO'C with water, 1% NaCl, 0.3% HCI and 0.3% NaOH. Extraction with enzymes

1987). Soluble protein was determined by the Biuret method (Cooper, 1977). Free and total amino acid contents were analysed using HPLC (Waters Associate) with a Bondapak C 18 column (3.9x300 mm) and peaks recorded with Waters a 484 Detector at 254 mm. Sensory evaluation

Ground shrimp heads were blended with water at a ratio of 1:2 and mixed with enzymes: bromclain activity 1000 (Great Food (Biochem) Co., Ltd., Thailand); papain activity 30000 (extracted with 95% alcohol); or neutrase activity of 0.532 Anson unit/ g (Novo Industrial Co., Ltd.). The mixtures were adjusted to pH 6 and incubated at 50'C for different times. After incubation, the mixtures were boiled for 2-3 min and filtered through Whatman No.4 paper. The residues were rinsed twice with boiled water. The amount of boiled water used each time was the same as the amount of blended water. The combined filtrate was kept at -20ce for further analysis. The concentrations of bromelain and papain were 0.25, 0.5 and 1.0% (w /w) of shrimp heads. The concentrations of neutrasc were 0.5, 1.0 and 2.0% (wi w) of shrimp heads. The incubation conditions studied 7 hrs at 500C and pH 6. Statistical analysis analysed using analysis new multiple range test flavour powder of \vcre 1, 3, 5, 6 and

Difference tests wcre carried out using the Triangle test (Larmond, 1977). Ranking and preference tests with a hedonic scale of 1-9 were also used. Laboratory panels comprised staff members and students of the Department of Fishery Products, Kasetsart University. Krill extract (Reiber & Son, Norway) and Seafood flavour (Nestle Thailand) were used as references. Shrimp flavour extract was heated before presenting it to the judges and shrimp flavour powder was dissolved in hot water at 2 or 3% (w Iv) before testing. Crackers incorporating shrimp flavour powder wcre prepared from tapioca flour, 0.4% (w /w) salt and 2% (w /w) shrimp flavour powder prepared by different methods. Hot water \vas added as needed. The mixture was formed into a cylinder shape of 2.5 em diameter and steamed for 30 min, cooled and kept overnight at -tcC. The refrigerated dough was cut into slices of 2-3 nnn thickness, dried at 50'C for 12 hrs and deep fried. The judges were asked to evaluate odour, taste and overall acceptability of the crackers using a hedonic scale of 1-9 (Larmond, 1977).

RESULTS Composition

AND DISCUSSION

Dati! were statistically variance and Duncan's Preparation of shrimp

Proximate composition, sodium chloride content and freshness of raw material are shown in Table 1. The protein content in shrimp heads was quite high compared to the reported range of 8.9 - 23.2% in whole shrimp (Chotiyanavong, 1981) but fat content was lower than 5% therefore it could be categorised as low fat. TVB-N of the shrimp heads was less than 20 rng%, thus the raw material was considered fresh.

Ground shrimp heads (250 g) were blended with water, brine or dilute HCI (1:2 w/v), incubated at optima] time and temperature. The mixtures were filtered through Whatman No.4 paper and the residues rinsed with hot water until the filtrate collected was lL. Thc filtrate was mixed with 10% (w Iv) dextrin, dextrose or sodium chloride and dried ejt]1er in a hot air oven at 50+5C or in a dehumidifier (Institute of Food Research and Product Development, Kasetsart University) for 24 hrs (dextrin), 48 hrs (dextrose) and 72 hrs (NaCI). The shrimp flavour powder was kept at 4'C until used. Chemical analyses

Formaldehyde nitrogen was determined according to the method described by the Thailand Industrial Standard Institute (]983). Total nitrogen, moisture, ash and sodium chloride content were determined according to AOAC (] 980). Freshness of raw material was detennined using total volatile base (TVB) values (Ng,

132

Extraction

with water

Total solids, formaldehyde nitrogen content and preference scores of shrimp flavour extracted with \vater at different temperatures for 60 min and for different times at 100C sho\ved no significant differences. However, filtration of samples extracted at temperatures lower than 100'e was difficult. Therefore, in the next experinlent, the extraction was at 100C for 30 min. Extraction with NaCl

Ho\vever, at temperatures lower than 100C, filtration of samples was difficult. Incubation time was found to affect total solid content but had no effect on formaldehyde nitrogen and preference scores. Samples incubated for a shorter time contained higher total solids, perhaps due to the effect of heat on solubility of the proteins. \Vhen incubated for longer times, coagulation of proteins occurred and insoluble proteins could not pass through the filter paper, giving lower total solids in the filtrate. For further experiments, the conditions for sodium chloride extraction were 1% at 100C for 30 min. Extraction with HCl

Total solids, formaldehyde nitrogen content and preference scores of shrimp flavour extracted with different concentrations of NaCl solution at 100ce for 60 min are sho\vn in Table 2. Data for extraction with 1% NaCl at 100"e for different times are shown in Table 3. Samples extracted with higher salt concentrations had higher solid contents (1'<0.05) but formaldehyde nitrogen and preference scores \vere not significantly different. Samples extracted at different temperatures were not significantly different either (data not sho\"',1n).

Higher concentrations of HCI resulted in lower preference scores of shrimp flavour extract due to the acid odour but had no effect on total solids and formaldehyde nitrogen content (data not shown). Different extraction times and temperatures also had no effect on total solids, formaldehyde nitrogen content and preference scores but at temperatures lower than 100oe, filtration was difficult. Therefore, 0.3% HCl, at 100C for 30 min was used in the next experiment. Extraction total with NaOH formaldehyde nitrogen content and

Different concentrations of NaOH had no effect on

solids,

preference scores of shrimp flavour extract. Different

extraction times and telnperatures affected formaldehyde nitrogen content but had no effect on solids content and preference scores (data not shown). At SOC, formaldehyde nitrogen was higher than at 37'C. At 100 and 120'e, formaldehyde nitrogen contents were not significantly different and were the lowest. An incubation time of 60 filin yielded the highest formaldehyde nitrogen. All conditions of extraction with NaOH showed that it was not a suitable extracting solution due to the difficulty in filtration since all samples were slimy. Therefore, NaOH was not used in further studies. Extraction with enzymes

Fig. 1 shows that longer incubation times resulted in lower Biuret soluble protein contents with an increase in hydrolysis. Fh'e hours was chosen as the appropriate incubation time since there was little decrease in soluble protein content beyond this tinle.

The

concentration of bromelain affected the formaldehyde nitrogen content in shrimp flavour extract, but had no effect on total solids, soluble protein content and preference scores. At 0.25%, formaldehyde nitrogen content \vas the highest, thus this concentration was used for further studies.
The concentrations of papain and Neutrase had no

133

with Neutrase and papain might be due to the higher free amino groups in the samples since the enzymes hydrolysed peptide bonds which increased free amino groups to react with formalin. Protein content in samples could be separated into two levels i.e., soluble protein in shrimp flavour extracted with bromelain \vas highest and significantly different from those extracted with water, HC!, NaC!, Neutrase or papain. Total solids, ash and sodium chloride contents in shrimp flavour extracted with NaCI were the highest. However, the amount of total solids not only depends upon ash and NaCI contents, but also on soluble protein. Pat content of the shrimp flavour extracts was very low. Preparation of shrimp flavour powder

effect on forn1aldehyde nitrogen, total solids, soluble protein content and preference scores of shrimp flavour extract (P>0.05). Therefore, the lmNest concentrations of enzymes were used in further experiments. Chemical analysis of shrimp flavour extract

Extraction of shrimp flavour was carried out under the following conditions: (i) (ii) (iii) (iv) (v) (vi) With With With With With With water at lOOC for 30 min 1.0% NaC! at lOOOCfor 30 min 0.3% HC! at 100C for 30 min 0.25% bromelain at 50C, pH 6, for 5 hrs 0.25% papain at 50C, pH 6, for 5 hrs 0.50% Neutrase at 50C, pH 6, for 5 hrs

Shrimp flavour powders dried by the two methods \",rere dissolved in hot ,vater at 2% (w Iv) and subjected to the triangle test to evaluate the effect of drying method. Only 5 out of 10 judges could correctly detect the different samples. Since at least 7 out of 10 must be correct to be significantly different (P<0.05), it was concluded that there was no significant difference between the two methods of drying. Dextrose, dextrin and sodium chloride were used as binders during drying of shrimp flavour extract at 10% \v Iv and dried in a hot air oven at 50::!::5C for 48, 24 and 72 hrs, respectively. After drying. the samples were ground and tested. The sample \vith dextrose as a binder was sweet and had a brown colour. It stuck to the drying trays. The odour was like shrimp mixed with caramel. The sample using dextrin as a binder was light brown with very mild shrimp odour but strong potato starch odour. Although, it did not stick to the drying trays and required a shorter drying time, it was not suitable as a flavour binder. Salnples using NaCI as binder required longer drying times and stuck slightly to the drying trays but had the

Formaldehyde nitrogen, Biuret protein, total solids, ash and sodium chloride contents of shrimp flavour extracts are shown in Table 4. The formaldehyde nitrogen contents of shrimp flavour extract prepared with papain and Neutrase ,vere higher than those extracted with bromelain, water, HCl or NaC!. The higher formaldehyde nitrogen in samples prepared

strongest shrimp flavour. In general, shrimp flavour powder is used in soups or snacks and should not be sweet, therefore it was concluded that NaCI was thc most appropriate binder for shrimp flavour powder. Composition The proximate composition and sodium chloridc content of shrimp flavour po\vder extracted by different solutions and enzymes with NaCl as binder are shown in Table 5. Amino acid composition

The total and free amino acid contents of shrimp extract powder prepared with \vater and bromelain and the ratio of individual frce amino acid to total amino acid of shrimp extract powdcr and krill extract are shown in Table 6. The free amino acid contents of shrimp flavour powder extracted \vith water and bromelain do not differ much, but the total amino acid content of a sample extracted with bromelain was much higher. Apparently, bromelain is an endoprotease and produces peptides rather than free amino acids. The peptide amino acid content in shrimp flavour po\vder extracted with bromelain was 3.9 fold that extracted with water. The ratio of free to total amino acids in shrimp flavour

extracted with water was high compared to that extracted with bromelain e.g. Arg was 93.990 in the sample prepared with water whereas in the sample prepared with bromelain, it was only 34.29/:. In krill extract, most of the ratios were high except for Glu which was only 2.9% because most of the glutamic acid \vas in peptide form. Asp in all three samples was also in peptide form. Othcr amino acids with more than 6070 in free form in samples prepared with water wcre His, Ala, Met, lIe, Leu and Phe, and in krill Arg, Ala, Pro, Tyr, Val, Met, Leu and Phe. Mole percentages of total and free amino acid contents of shrimp flavour extractcd with water and bromelain

compared to amino acid content tract are shown in Table I.

of krill flavour ex-

The amino acid compositions of shrimp flavour extracted with brornelain and water did not differ much. The rnajor free amino acids in the two samples were Ala, Gly and Arg but those in krill extract were Gly, Ala, Pro and Leu. The major amino acid residues in shrimp flavour extract and krill extract were Gly, Giu and Ala. The major peptide amino acid was Glu. Raksakulthai and Haard (1992) reported on the correlation between the concentration of peptides and amino acids and the flavour of fish sauce and concluded that both free Glu and pep tides containing Glu were important to the flavour of fish sauce. Konosu and Yamaguchi (1982) reported that the fla\'our of fish and shellfish were from water soluble, low molecular weight components, especially free amino acids. Glycine (Gly), proline (Pro), arginine (Arg), taurine and alanine (Ala) ,vere major amino acids in shrimp flavour. Senso ry evaluation Odo",.. Shrimp flavour powder samples prepared by different solutions and enzymes were dissolved in hot water (3% w Iv) and presented to 13 judges. Judges ranked the samples in order of strongest to mildest odour. The results are shown in Table 8. To ,.erify the differences in the samples, shrimp fla\'our po\vder prepared with bromelain (highest rank total) and a sample extracted with HCI (lo\\'est rank

total) were subjected to the triangle test. Eight out of 12 judges correctly identified the odd samples. It was concluded therefore, that samples prepared with brornelain and HCl ,vere significantly different. Since the sample prepared with bromelain received the highest ranking score, it was compared to reference flavour extracts (commercial samples) using a hedonic preference scale of 1-9. The laboratory sample \vas dissolved in hot water (2 and 3% w Iv) and compared with 2% (w Iv) commercial Krill Extract (Reiber & Sons) and Seafood Flavour (Nestle Co., Ltd.). The results are shown in Table 9. Krill extract is a thick brown liquid with strong shrimp flavour containing 7% NaCl, 65% total solids, 45% crude protein, 0.2% fat and 14% ash. Seafood flavour is a fine pov,,'der of light yellow colour 'with salty taste but the composition is unknown. There was no significant difference between preference scores. Shrimp flavour extracted with bromelain at the concentration of 3% recei\'ed the highest preference score. However due to the very high salt content in ,. .' . t Ile samp Ie llUrIng tIle preparatIOn 0 f sh rImp fl avoure d crackers, only 2% (w Iw) of shrimp flavour powder \vas used. Evaluation of flavour

Shrimp flavour extracted with bromelain in which NaCl was used as binder (at 2% wlw) was added to shrimp flavoured crackers with and \vithout spices (garlic and pepper) to test the effects of spices on the

CONCLUSION Appropriate conditions for extraction of flavour from shrimp heads were as follo,v: (i) (ii) (iii) (iv) (v) (vi) acceptability of the crackers. The crackers were deep fried and 10 judges asked to evaluate the samples in terms of colour, flavour, odour and overall acceptability using a hedonic scale of 1-9. The average sensory scores of crackers wHh and without spices were not significantly different, although the score of the sample with spices was higher than that without spices (data not shown). To eliminate the effects of spices, shrimp flavoured cracker samples were prepared without spices and deep fried for sensory evaluating by 16 panelists. The results are shown in Table 10. Shrimp flavoured crackers containing krill extract had the highest sensory evaluation score. Crackers with shrimp flavour extracted with bromelain were significantly different from control crackers 'Nithout flavour extract, but not significantly different to samples with flavour extract prepared with papain, water, NaCl, neutrase or HC!. GiJdberg (1993) reported that hydrolysates prepared with bromelain had better organoleptic quality than those prepared with other proteinases. with with with with with with water at 100C for 30 min 1.0% NaCl at 1000C for 30 min 0.3 HCl at lOOT for 30 min 0.25% bromeJain at 50C, pH 6, for 5 hrs 0.25% papain at SOGe, pH 6, for 5 hrs 0.5% neutrase at 50GC, pH 6, for 5 hrs

NaOH was not suitable for extraction of flavour due to the difficulty in filtration. Oven-drying or dehumidifying had no effect on acceptability of prepared shrimp flavour and NaCl was the most suitable flavour binder. The major free amino acids in shrimp flavour powder prepared with water and bromelain were alanine, glycine and arginine. The major amino acid residues of the shrimp flavour powder were glutamic acid, glycine and alanine. The major peptide amino acid was glutamic acid.

Chemical analysis and sensory evaluation of shrimp

flavour extracted with different media showed that an enzymic process using bromelain was superior to water, NaCl or HC!. Shrimp flavour extracted with brome]ain was the most acceptable product.

ACKNOWLEDGEMENT The support of the Asian Fisheries Society is gratefully acknowledged. Supply of krill extract from

ASEAN

Food Journal Vol. 10, No.4,

1995

137

Keiber & Son, NG. way and Seafood 1\estle, Thailand is appreciated.

Flavour

from

REFERENCES Afolabi, O.A., Oka, O.L. and Umoh, LB. The use of fish waste as animal feed. Ntr. Rep. Intern. 21:901-906; 1980. AOAC Official methods of analysis. 13th edn. Washington D.C: Association of Official Analytical Chemists; 1980. Chotiyanavong, A. Analysis of fishery products. Bangkok: Kasetsart University; 1981. (In Thai). Cooper, T.G. The tool of biochemistry. John Wiley & Sons Inc.; 1977. Gildberg, A. materials. Enzymic processing Process Biochemistry. New York:

Ng, C.s. Determination of trimethylamine oxide (TMAO-N), trimethylamine (TMA-N), total vilatile basic nitrogen (TVB-1\) by Conway's method. Hasegowa, H. (Ed.) Laboratory manual on analytical methods and procedures for fish and fish products. Singapore: Marine Fisheries Research Dept; 1987. Office of Agricultural Economics. Report on the trend of demand and supply of marine shrimp in Thailand. Bangkok: Division of Agricultural Economic Research, Ministry of Agriculture and Cooperatives, 1991. (In Thai). Office of Agricultural Economics. Report on export of frozen shrimp and squid of Thailand. Bangkok: Division of Agricultural Economic Research, :V!inistry of Agriculture and Cooperatives, 1992. (In Thai). Raksakulthai, N. and Haard, N.F. Correlation between the concentration of peptides and amino acids and the flavour of fish sauce. ASEAN Food Journal. 7: 86-90; 1992. Simpson, B.K. and Haard, N.F. The use of proteolytic enzymes to extract carotenoproteins from shrimp waste. J. Appl. Biochem. 7:212-222; 1985. Sopanodora, P. and Buckle, K.A. Utilization of prawn head in oriental prawn cracker. Maneepun, S., Varangoon, P. and Phithakpol, B. (Eds.) Bangkok: Proceedings of the Food Conference '88, 1988. Thai Industrial Standards Institute. Standard of local fish sauce. Bangkok: Ministry of Industry, 1983. (In Thai).

of marine raw 28:1-15; 1993.

Gray, R.D. Dry flavoring from crustacean. U.s. Patent 3 264 116. August 2, 1966. Gillis, M.T. ed. Seafood Processing. Park Ridge, 1\J: 1\oyes Data Corp. 170-171; 1966. Johnson, E.L. and Peniston, Q.P. Utilization of shellfish waste for chitin and chitosan production. Martin, R.E., Flick, G.J., Hebard, CE. and Ward, D.R. (Eds.) Chemistry and biochemistry of marine food products. Westport, CT: A VI, 415-422; 1982. Konosu, S. and Yamaguchi, K. The flavor components in fish and shellfish. Martin, R.E., Flic, G., Hebard, CE. and Ward, R. (Eds.) Chemistry and biochemistry of marine food products. Westport, CT: A VI. 415-422; 1982. Larmond, E. Laboratory methods for sensory evaluation of food. Department of Agriculture; 1977.

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