Production of Flavouring Agent fromShrimp Heads

Patcharee Teerasuntonwat and Nongnuch Raksakulthai

ABSTRACT weight peptides, nucleotides and organic bases andnon-nitrogenous compounds comprising organic ac-

A [la-oollying agent was prepared from shrimp heads by ids, sugars and inorga~ic constituents (Konosu andextraction of nitrogenous compollnds with water, sodium Yamaguchi, 1982). Food flavouring agents can bechloride, hydrochloric acid, sodium hydroxide, bromelain, produced from plants or animals, or can be chemicallypapain and ncutrase. The appropriate conditions of synthesiscd. However, the flavours from naturalextraction were based on total solids,formaldehydenitrogen, substances are considered more delicate whereassoluble protein CO/ltents and sensory evaluation scores. chemically synthesised flavours cannot always perfectlyShrimp flavour extract was dried using dextrin, dextrose duplicate natural flavours.or sodium chloride as binders. Brome/aill was found to bethe most promising extracting medium. The appropriate Since Thailand is a major shrimp producing country,conditions were 0.25% bromclain, pH 6, for 5 hI's at 500( there is a large amount of \..Taste which is sold at lowusing 50dillm chloride as the binder. The free amino acid prices for feed production. It IS advisable to usecompositions of shrimp flavour pmoders prepared with "loater shrimp \vaste to produce a flavouring agent whichand bromelaill were similar, but the total amino acid COI1- at the present time has to be imported.tent in shrimp flavour extracted zuith enzyme was about2.5-fold higher. The major free ami110 acids in both fln- The objectives of this study were to:vOllring agents were alanine, glycine and arginille. Themajor amino acids in the acid hydrolysate of the shrimp (i) find the appropriate conditions for extraction offlavour powder were glutamic acid, glycine and alanine. shrimp fla\'our \vith water, dilute acid, dilute base,Sensory evall/atioll of an aqueous soilition of the preferred brine and proteases in combination with heat treat-flavol/ring agent and the reference krill extract, sho"ll.1Cd no ment, andsignificanl difference IP

The incubation temperatures were 37, 50, 100 and120cC 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 150min. at IOO'C with water, 1% NaCl, 0.3% HCI and0.3% NaOH.

Extraction with enzymes

Ground shrimp heads were blended with water at aratio of 1:2 and mixed with enzymes: bromclainactivity 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 wereadjusted to pH 6 and incubated at 50'C for differenttimes. After incubation, the mixtures were boiled for2-3 min and filtered through Whatman No.4 paper.The residues were rinsed twice with boiled water. Theamount of boiled water used each time was the sameas the amount of blended water. The combined fil-trate 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. Theconcentrations of neutrasc were 0.5, 1.0 and 2.0% (wiw) of shrimp heads.

The incubation conditions studied \vcre 1, 3, 5, 6 and7 hrs at 500C and pH 6.

Statistical analysis

Dati! were statistically analysed using analysis ofvariance and Duncan's new multiple range test

Preparation of shrimp flavour powder

Ground shrimp heads (250 g) were blended withwater, brine or dilute HCI (1:2 w/v), incubated atoptima] time and temperature. The mixtures werefiltered through Whatman No.4 paper and theresidues rinsed with hot water until the filtratecollected was lL. Thc filtrate was mixed with 10%(w Iv) dextrin, dextrose or sodium chloride and driedejt]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 (dex-trose) and 72 hrs (NaCI). The shrimp flavour pow-der was kept at 4'C until used.

Chemical analyses

Formaldehyde nitrogen was determined according tothe method described by the Thailand Industrial Stand-ard Institute (]983). Total nitrogen, moisture, ash andsodium chloride content were determined accordingto AOAC (] 980). Freshness of raw material was de-tennined using total volatile base (TVB) values (Ng,

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1987). Soluble protein was determined by the Biuretmethod (Cooper, 1977). Free and total amino acidcontents were analysed using HPLC (Waters Associ-ate) with a Bondapak C 18 column (3.9x300 mm) andpeaks recorded with Waters a 484 Detector at 254 mm.

Sensory evaluation

Difference tests wcre carried out using the Triangletest (Larmond, 1977). Ranking and preference testswith a hedonic scale of 1-9 were also used. Laboratorypanels comprised staff members and students of theDepartment of Fishery Products, Kasetsart University.Krill extract (Reiber & Son, Norway) and Seafoodflavour (Nestle Thailand) were used as references.

Shrimp flavour extract was heated before presentingit to the judges and shrimp flavour powder wasdissolved in hot water at 2 or 3% (w Iv) before testing.

Crackers incorporating shrimp flavour powder wcreprepared from tapioca flour, 0.4% (w /w) salt and 2%(w /w) shrimp flavour powder prepared by differentmethods. Hot water \vas added as needed. Themixture was formed into a cylinder shape of 2.5 emdiameter and steamed for 30 min, cooled and keptovernight at -tcC. The refrigerated dough was cut intoslices of 2-3 nnn thickness, dried at 50'C for 12 hrsand deep fried. The judges were asked to evaluateodour, taste and overall acceptability of the crackersusing a hedonic scale of 1-9 (Larmond, 1977).

RESULTS AND DISCUSSION

Composition

Proximate composition, sodium chloride content andfreshness of raw material are shown in Table 1.

The protein content in shrimp heads was quite highcompared to the reported range of 8.9 - 23.2% inwhole shrimp (Chotiyanavong, 1981) but fat contentwas lower than 5% therefore it could be categorisedas low fat. TVB-N of the shrimp heads was lessthan 20 rng%, thus the raw material was consideredfresh.

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Extraction with water

Total solids, formaldehyde nitrogen content and pref-erence scores of shrimp flavour extracted with \vaterat different temperatures for 60 min and for differenttimes at 100C sho\ved no significant differences.However, filtration of samples extracted at tempera-tures lower than 100'e was difficult. Therefore, inthe next experinlent, the extraction was at 100C for30 min.

Extraction with NaCl

Total solids, formaldehyde nitrogen content andpreference scores of shrimp flavour extracted withdifferent concentrations of NaCl solution at 100ce for60 min are sho\vn in Table 2. Data for extractionwith 1% NaCl at 100"e for different times are shownin Table 3.

Samples extracted with higher salt concentrations hadhigher solid contents (1'

effect on forn1aldehyde nitrogen, total solids, solubleprotein content and preference scores of shrimp fla-vour extract (P>0.05). Therefore, the lmNest concen-trations of enzymes were used in further experiments.

Chemical analysis of shrimp flavour extract

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

Formaldehyde nitrogen, Biuret protein, total solids, ashand sodium chloride contents of shrimp flavourextracts are shown in Table 4.

The formaldehyde nitrogen contents of shrimp flavourextract prepared with papain and Neutrase ,verehigher than those extracted with bromelain, water, HClor NaC!.

The higher formaldehyde nitrogen in samples prepared

with Neutrase and papain might be due to the higherfree amino groups in the samples since the enzymeshydrolysed peptide bonds which increased free aminogroups to react with formalin.

Protein content in samples could be separated intotwo levels i.e., soluble protein in shrimp flavour ex-tracted with bromelain \vas highest and significantlydifferent from those extracted with water, HC!, NaC!,Neutrase or papain.

Total solids, ash and sodium chloride contents inshrimp flavour extracted with NaCI were the highest.However, the amount of total solids not only dependsupon ash and NaCI contents, but also on solubleprotein. Pat content of the shrimp flavour extractswas very low.

Preparation of shrimp flavour powder

Shrimp flavour powders dried by the two methods\",reredissolved in hot ,vater at 2% (w Iv) and subjectedto the triangle test to evaluate the effect of dryingmethod. Only 5 out of 10 judges could correctly de-tect the different samples. Since at least 7 out of 10must be correct to be significantly different (P

strongest shrimp flavour. In general, shrimp flavourpowder is used in soups or snacks and should notbe sweet, therefore it was concluded that NaCI wasthc most appropriate binder for shrimp flavour pow-der.

Composition

The proximate composition and sodium chloridccontent of shrimp flavour po\vder extracted bydifferent solutions and enzymes with NaCl as binderare shown in Table 5.

Amino acid composition

The total and free amino acid contents of shrimpextract powder prepared with \vater and bromelainand the ratio of individual frce amino acid to totalamino acid of shrimp extract powdcr and krill extractare shown in Table 6.

The free amino acid contents of shrimp flavourpowder extracted \vith water and bromelain do notdiffer much, but the total amino acid content of asample extracted with bromelain was much higher.Apparently, bromelain is an endoprotease andproduces peptides rather than free amino acids. Thepeptide amino acid content in shrimp flavour po\vderextracted with bromelain was 3.9 fold that extractedwith water.

The ratio of free to total amino acids in shrimp flavour

extracted with water was high compared to thatextracted with bromelain e.g. Arg was 93.990 in thesample prepared with water whereas in the sampleprepared with bromelain, it was only 34.29/:. In krillextract, most of the ratios were high except for Gluwhich was only 2.9% because most of the glutamicacid \vas in peptide form. Asp in all three sampleswas also in peptide form. Othcr amino acids withmore than 6070 in free form in samples prepared withwater wcre His, Ala, Met, lIe, Leu and Phe, and inkrill Arg, Ala, Pro, Tyr, Val, Met, Leu and Phe.

Mole percentages of total and free amino acid contentsof shrimp flavour extractcd with water and bromelain

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compared to amino acid content of krill flavour ex- total) were subjected to the triangle test. Eight outtract are shown in Table I. of 12 judges correctly identified the odd samples. It

was concluded therefore, that samples prepared withThe amino acid compositions of shrimp flavour ex- brornelain and HCl ,vere significantly different.tracted with brornelain and water did not differ much.The rnajor free amino acids in the two samples were Since the sample prepared with bromelain receivedAla, Gly and Arg but those in krill extract were Gly, the highest ranking score, it was compared to referenceAla, Pro and Leu. The major amino acid residues in flavour extracts (commercial samples) using a hedonicshrimp flavour extract and krill extract were Gly, Giu preference scale of 1-9. The laboratory sample \vasand Ala. The major peptide amino acid was Glu. dissolved in hot water (2 and 3% w Iv) and comparedRaksakulthai and Haard (1992) reported on the with 2% (w Iv) commercial Krill Extract (Reiber &correlation between the concentration of peptides and Sons) and Seafood Flavour (Nestle Co., Ltd.). Theamino acids and the flavour of fish sauce and results are shown in Table 9.concluded that both free Glu and pep tides containingGlu were important to the flavour of fish sauce. Krill extract is a thick brown liquid with strong shrimpKonosu and Yamaguchi (1982) reported that the fla- flavour containing 7% NaCl, 65% total solids, 45%\'our of fish and shellfish were from water soluble, crude protein, 0.2% fat and 14% ash. Seafood flavourlow molecular weight components, especially free is a fine pov,,'der of light yellow colour 'with salty tasteamino acids. Glycine (Gly), proline (Pro), arginine but the composition is unknown. There was no(Arg), taurine and alanine (Ala) ,vere major amino significant difference between preference scores.acids in shrimp flavour. Shrimp flavour extracted with bromelain at the

concentration of 3% recei\'ed the highest preferencescore. However due to the very high salt content inSenso ry evaluation I I ,. I . ' f h . fl dt le samp e llUrIng t le preparatIOn 0 s rImp avoure

Odo",.. Shrimp flavour powder samples prepared by crackers, only 2% (w Iw) of shrimp flavour powderdifferent solutions and enzymes were dissolved in hot \vas used.water (3% w Iv) and presented to 13 judges. Judgesranked the samples in order of strongest to mildest Evaluation of flavourodour. The results are shown in Table 8.

Shrimp flavour extracted with bromelain in whichTo ,.erify the differences in the samples, shrimp NaCl was used as binder (at 2% wlw) was added tofla\'our po\vder prepared with bromelain (highest rank shrimp flavoured crackers with and \vithout spicestotal) and a sample extracted with HCI (lo\\'est rank (garlic and pepper) to test the effects of spices on the

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acceptability of the crackers. The crackers were deepfried and 10 judges asked to evaluate the samples interms of colour, flavour, odour and overall acceptabil-ity using a hedonic scale of 1-9.

The average sensory scores of crackers wHh and with-out spices were not significantly different, althoughthe score of the sample with spices was higher thanthat without spices (data not shown). To eliminatethe effects of spices, shrimp flavoured cracker sam-ples were prepared without spices and deep fried forsensory evaluating by 16 panelists. The results areshown in Table 10.

Shrimp flavoured crackers containing krill extract hadthe highest sensory evaluation score. Crackers withshrimp flavour extracted with bromelain weresignificantly different from control crackers 'Nithoutflavour extract, but not significantly different to sam-ples with flavour extract prepared with papain, wa-ter, NaCl, neutrase or HC!. GiJdberg (1993) reportedthat hydrolysates prepared with bromelain had bet-ter organoleptic quality than those prepared with otherproteinases.

ASEAN Food Journal Vol. 10, No.4, 1995

CONCLUSION

Appropriate conditions for extraction of flavour fromshrimp heads were as follo,v:(i) with water at 100C for 30 min(ii) with 1.0% NaCl at 1000C for 30 min(iii) with 0.3 HCl at lOOT for 30 min(iv) with 0.25% bromeJain at 50C, pH 6, for 5 hrs(v) with 0.25% papain at SOGe, pH 6, for 5 hrs(vi) with 0.5% neutrase at 50GC, pH 6, for 5 hrs

NaOH was not suitable for extraction of flavour dueto the difficulty in filtration.

Oven-drying or dehumidifying had no effect onacceptability of prepared shrimp flavour and NaCl wasthe most suitable flavour binder.

The major free amino acids in shrimp flavour powderprepared with water and bromelain were alanine,glycine and arginine. The major amino acid residuesof the shrimp flavour powder were glutamic acid,glycine and alanine. The major peptide amino acidwas glutamic acid.

Chemical analysis and sensory evaluation of shrimpflavour extracted with different media showed thatan enzymic process using bromelain was superior towater, NaCl or HC!. Shrimp flavour extracted withbrome]ain was the most acceptable product.

ACKNOWLEDGEMENT

The support of the Asian Fisheries Society is grate-fully acknowledged. Supply of krill extract from

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Keiber & Son, NG. way and Seafood Flavour from Ng, C.s. Determination of trimethylamine oxide1\estle, Thailand is appreciated. (TMAO-N), trimethylamine (TMA-N), total vilatile

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