Food Chemistry 158 (2014) 162–170

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Food Chemistry

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Seafood-like flavour obtained from the enzymatic hydrolysisof the protein by-products of seaweed (Gracilaria sp.)

http://dx.doi.org/10.1016/j.foodchem.2014.02.1010308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +66 2 470 7752; fax: +66 2 470 7781.E-mail addresses: nutta.lao@kmutt.ac.th, nattalao@hotmail.com (N. Laohakunjit).

Natta Laohakunjit ⇑, Orrapun Selamassakul, Orapin KerdchoechuenDivision of Biochemical Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 49 Tientalay 25 Rd.,Takham, Bangkhuntien, Bangkok 10150, Thailand

a r t i c l e i n f o a b s t r a c t

Article history:Received 31 October 2013Received in revised form 9 February 2014Accepted 21 February 2014Available online 1 March 2014

Keywords:BromelainEnzymatic hydrolysisThermal processed flavourSeafood-like flavourSeaweed

An enzymatic bromelain seaweed protein hydrolysate (eb-SWPH) was characterised as the precursor forthermally processed seafood flavour. Seaweed (Gracilaria fisheri) protein after agar extraction was hydro-lysed using bromelain (enzyme activity = 119,325 U/g) at 0–20% (w/w) for 0.5–24 h. Optimal hydrolysisconditions were determined using response surface methodology. The proposed model took into accountthe interaction effect of the enzyme concentration and hydrolysis time on the physicochemical propertiesand volatile components of eb-SWPH. The optimal hydrolysis conditions for the production of eb-SWPHwere 10% bromelain for 3 h, which resulted in a 38.15% yield and a 62.91% degree of hydrolysis value.Three free amino acids, arginine, lysine, and leucine, were abundant in the best hydrolysate. Ten volatileflavours of the best eb-SWPH were identified using gas chromatography/mass spectrometry. The pre-dominant odourants were hexanal, hexanoic acid, nonanoic acid, and dihydroactinidiolide. The thermallyprocessed seafood flavour produced from eb-SWPH exhibited a roasted seafood-like flavouring.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Thermally processed flavourings have become widely used assavoury flavouring agents in various products, such as soups,sauces, snacks, and ready meals. The meaty or beefy flavour is gen-erated by heating a combination of protein hydrolysate, aminoacids, and reducing sugars under controlled conditions throughthe Maillard reaction (Manley & Ahmedi, 1995). Thermal process-ing of various plant protein sources such as soy (Wu & Cadwallad-er, 2002), wheat (Lee, Chung, & Kim, 2012), and Brassica (Guo, Tian,& Small, 2009) has produced meaty or beefy flavours to replace fla-vouring agents from animal protein sources. Protein hydrolysatesfrom marine animal sources, such as fish, shrimp, clam, crab, andseafood by-products, have generally been used to produce seafoodflavourings. A high flavour quality is difficult to ensure for seafoodflavouring that is produced from marine animal sources because oftheir high susceptibility to lipid oxidation and the high cost ofremoving excess fat (Imm & Lee, 1999). For centuries, seaweedhas been used in the preparation of soups and foods due to itspleasing flavour. Seaweed by-products after agar extraction aregood sources of plant protein and contain taste-active amino acids,

such as aspartic acid, glutamic acid, arginine, and lysine, in addi-tion to a low fat content.

Gracilaria fisheri is one of the widespread red seaweeds that iscultivated in Asian countries. In the Southern part of Thailand, asmall amount of G. fisheri has been used for agar production, inhuman diets, and for abalone or shrimp feeds. In human diets,the consumption of seaweed does not provide sufficient nutritionalvalue, due to the abundance of carbohydrates and fibre (Dawczyn-ski, Schubert, & Jaheris, 2007). After removing the agar of G. fisheri,the by-product is rich in proteins, which contain high amounts ofessential amino acids. However, the potential peptides and aminoacids in various plant sources, including G. fisheri, are only partiallydigested using gastrointestinal enzymes, and they are lost duringthe cooking process (Sarmadi & Ismail, 2010). The production ofpotential peptides of G. fisheri by human gastrointestinal digestionis still limited, due to the complex protein of seaweed with lowdigestibility and poor solubility at acidic pH (Fleurence, 1999),and the uncontrolled hydrolysis of gastrointestinal enzymes(Sarmadi & Ismail, 2010). An effective process to improve thephysicochemical characteristics, volatile compounds, and organo-leptic quality of plant proteins is selective enzymatic hydrolysisunder controlled conditions, which generates available peptidesand amino acids with less salt and carcinogenic compounds, suchas mono- and dichloropropanols or 3-monochloropropane-1,2-diol(3-MCPD), than acid hydrolysis (Weir, 1992).

N. Laohakunjit et al. / Food Chemistry 158 (2014) 162–170 163

The application of hydrolytic enzymes to hydrolyse plant pro-tein can enhance the flavour of the protein hydrolysate becauseit increases the amount of amino acids and low molecular weightpeptides that possess unique taste properties, including sweet,salty, sour, bitter, and umami tastes (Su et al., 2012). The typesof protease and protein substrate affect the functional propertiesand flavour profile of the protein hydrolysate (McCarthy, O’Calla-ghan, & O’Brien, 2012). Various commercial proteases are em-ployed in enzymatic hydrolysis, one of which is bromelain. Stembromelain (EC 3.4.22.32), which is a cysteine endoproteinase, is ex-tracted from the core of the stem of pineapple fruit (Ananas como-sus), which is a common and abundant plant grown in Thailand.Bromelain is a potential enzyme for the production of enzymaticprotein hydrolysates and is widely used in various food productsbecause of its broad specificity in the cleavage of peptide bondsand its stable activity over a wide pH range (pH 4.0–8.0). Althoughmany studies have reported the functional properties (antioxidantand bioactive capacities) and flavour of plant protein hydrolysatesproduced using bromelain hydrolysis (Liu & Chiang, 2008; Sonklin,Laohakunjit, & Kerdchoechuen, 2011), a hydrolysate from G. fisheriusing bromelain to generate seafood flavour has not been charac-terised. Nevertheless, the flavour of plant protein hydrolysatesdoes not necessarily impart a meaty flavouring. Therefore, thermalprocessing may improve the taste and odour of the protein hydrol-ysates (Manley & Ahmedi, 1995). The production of seafood flavourfrom thermally processed enzymatic hydrolysates of seaweed pro-tein requires investigation. Therefore, the objectives of this studywere to produce a protein hydrolysate from G. fisheri using brome-lain and to characterise the physicochemical properties and vola-tile components of this protein hydrolysate followed by asensory evaluation. The enzymatic bromelain seaweed proteinhydrolysate (eb-SWPH) was used as the precursor of a thermallyprocessed seafood flavouring agent. Moreover, response surfacemethodology (RSM) was applied to evaluate the optimal hydrolysisconditions.

2. Materials and methods

2.1. Materials and chemicals

G. fisheri was harvested from a commercial seaweed pond in theChaiya District of the Surat Thani Province in the Southern part ofThailand during November 2009. Stem bromelain (E.C. 3.4.22.32;119,325 U/g) was kindly provided by Hong Mao Biochemicals Co.,Ltd. located in Rayong, Thailand. All reagents were of analyticalgrade (Mallinckrodt Chemicals, St Louis, MO).

2.2. Preparation of seaweed protein

Seaweed (G. fisheri) by-products after agar extraction werecollected from local food grade agar processing factories in SuratThani province, Thailand. The method of agar extraction wasconfidential (according to the manufacturer). The by-productswere ground and sieved through an 80-mesh screen. The powdercontained 27.84% protein, 0.60% fat, 9.07% ash, 20.65% fibre, and41.83% carbohydrate (dry weight). The powder was packed in plas-tic bags and stored at �20 �C prior to proteolytic hydrolysis.

2.3. Preparation of the liquid seaweed protein hydrolysates

For the production of the liquid protein hydrolysates, 10 g ofseaweed by-product were dispersed in 500 ml of sterile water,and the pH of the dispersions was adjusted to pH 6.0 with 1 NHCl. The dispersions were then pre-incubated at 50 �C for 10 minto ensure optimal bromelain activity. The dispersions were then

incubated at 50 �C for 0.5, 3, 6, 12, 18, and 24 h with bromelainat enzyme/substrate ratios (E/S) of 0%, 5%, 10%, 15%, and 20%(weight of enzyme/weight of seaweed by-products), which corre-spond to enzymatic activities of 0; 27,415; 54,831; 82,247; and109,663.2 U/g, respectively. One unit was defined as the amountof enzyme that liberated 1 mg of tyrosine in 10 min at a pH of6.0 and a temperature of 50 �C. The reaction was terminated atthe end of the hydrolysis time by heating at 95 �C for 15 min.The dispersions were centrifuged at 5000g for 15 min at 25 �C,and the supernatant was collected and filtered through filter paper(Whatman No. 1). The eb-SWPH solutions were stored in brownglass bottles at �20 �C prior to physicochemical analysis. All testswere performed in triplicate.

2.4. Determination of the physicochemical properties of the liquid eb-SWPH

The physicochemical properties (colour, total salt, and degree ofhydrolysis) of the liquid eb-SWPHs that were produced under allconditions were characterised.

The colour of the liquid eb-SWPHs was evaluated using the CIEL�a�b� system (Sathivel et al., 2003). The L�, a�, and b� values of3-ml samples were measured using a colorimeter (MiniScan� EZ,HunterLab, Reston, VA). These values were used to quantifyperceived colour differences between hydrolysates with and with-out bromelain. Colour difference (DE) was calculated as follows:

DE ¼ ½ðL�2 � L�1Þ2 þ ða�2 � a�1Þ

2 þ ðb�2 � b�1Þ2�

1=2

where L�1, a�1, and b�1 represent the L�, a�, and b� values, respectively,of the liquid seaweed by-products from identical conditionswithout bromelain hydrolysis. L�2, a�2, and b�2 represent the L�, a�,and b� values, respectively, of the hydrolysed liquid eb-SWPHsusing different bromelain concentrations and hydrolysis times.

Total salt values of the liquid eb-SWPHs were reported as thepercentage of chloride and were analysed using the direct titrationof liquid eb-SWPHs with silver nitrate solution according to themethod of AOAC (2005).

The degree of hydrolysis (DH) was followed by the method ofSathivel et al. (2003). The% DH defined as the percentage of solubleprotein in trichloroacetic acid (TCA). A sample aliquot (10 ml) wasmixed with 10 ml of 20% TCA and then centrifuged at 5000g for15 min at 25 �C. The soluble nitrogen in the supernatant and thetotal nitrogen were determined using the Kjeldahl method. Thedegree of hydrolysis was calculated using the following equation:

%degree of hydrolysisðDHÞ ¼ 20% TCA Soluble NTotal N

� 100

where total N represents the amount of nitrogen in the hydrolysedprotein solution, and 20% TCA soluble N represents the amount ofsoluble protein in TCA.

The % yield was estimated according to the method of Sonklinet al. (2011). To calculate the percent yield (% yield), the liquideb-SWPHs were concentrated using a rotary evaporator (R-200;Büchi, Flawil, Switzerland) at 40 �C and 5 kPa for 2 h, resulting ina paste-like material. Percent yields were calculated as follows:

%Yield ¼ weight of paste� like hydrolysateweight of liquid seaweed protein

� 100

2.5. Experimental design for optimization using response surfacemethodology (RSM)

Optimal eb-SWPH hydrolysis conditions were established usingresponse surface methodology (RSM). Colour, % total salt, % yield,and % DH were used as the criteria. A 5 � 6 factorial in a completely

164 N. Laohakunjit et al. / Food Chemistry 158 (2014) 162–170

randomised design (CRD) was used to obtain the combination ofvalues that optimised the reaction. Two of the independent vari-ables were the five enzyme concentrations (x1 = 0, 5, 10, 15, and20% w/w) and the six hydrolysis times (x2 = 0.5, 3, 6, 12, 18, and24 h). To predict the optimal point, the response surface (y) wasdescribed with the aid of a second-order polynomial model accord-ing to Eq. (1) as follows:

y ¼ b0 þ b1x1 þ b2x2 þ b12x1x2 þ b11x21 þ b22x2

2 ð1Þ

where y represents the predicted response (colour, % total salt, %yield, and % DH); x1 and x2 represent independent variables; b0 rep-resents an offset term; b1 and b2 represent linear effects; and b12

represents an interaction effect. The model was used to evaluatethe effect of each independent variable.

2.6. Special analyses of the paste-like protein hydrolysate

The liquid eb-SWPH that was produced from the optimal condi-tions was concentrated using a rotary evaporator (Büchi R-200) at40 �C and 5 kPa for 2 h, and the resulting paste-like eb-SWPH wasanalysed for amino acid content and volatile compounds followedby a sensory evaluation.

2.6.1. Determination of the amino acid composition of the paste-likeeb-SWPH

The total amino acid composition of the paste-like eb-SWPHwas compared to the total amino acid composition of the seaweed(G. fisheri) by-products using high-performance liquid chromatog-raphy (HPLC), following the AOAC method (2005). Prior to thedetermination of the total amino acid composition, the seaweedby-product powder was treated with 6N HCl at 110 �C for 24 hand derivatised using the AccQ-Fluor reagent followed by HPLCanalysis (Alliance 2695; Waters Corporation, Milford, MA). The freeamino acid content of the paste-like eb-SWPH was determined byanalysis without acid hydrolysis. The acid-hydrolysed seaweed by-products and the concentrated paste-like eb-SWPH samples werethen analysed using HPLC coupled to a fluorescence detector(JASCO FP2020, Japan), with the excitation and emission wave-lengths set at 250 and 395 nm, respectively. A Hypersil GOLDcolumn (4.6 mm � 100 mm, 5 lm, Thermo Scientific, USA) wasused and was eluted at a flow rate of 1 ml/min in gradient modeusing a mixture of eluent A (sodium acetate buffer; pH 4.90) andeluent B (60% acetonitrile). The amino acid composition was iden-tified and quantified by comparison with a standard mixture ofamino acids (Sigma–Aldrich, St. Louis, MO) and was reported asgrams of amino acid per 100 g of protein in the sample solution).

2.6.2. Determination of the volatile compound composition of thepaste-like eb-SWPH

The composition of volatile compounds in the paste-likeeb-SWPH was analysed on a headspace solid-phase microextrac-tion-gas chromatography-mass spectrometry (HS–SPME-GC–MS)system (789DA MS, Agilent Technologies, Santa Clara, CA) (Sonklinet al., 2011). Each sample (3 ml) was placed into a 20-ml vial andheated at 90 �C for 10 min in a GC–MS heating block for headspaceanalysis. Volatile compounds were absorbed onto an SPME fibre(50/30 lm DVB/Carboxen™/PDMS StableFlex™; Supelco, Belle-fonte, PA) for 20 min. After equilibrium, the SPME fibre was des-orbed into the injector port at 240 �C for 20 min, and the injectorwas operated in splitless mode. Helium was used as the carriergas at a constant velocity of 1.0 ml/min. Volatile compounds wereseparated using a DB-Wax capillary column (30 m � 0.25 mm,0.25 lm film thickness; J&W Scientific Inc., Folsom, CA). The oventemperature program was as follows: initial temperature of55 �C; increased to 180 �C at 5 �C/min; increased to 200 �C at

8 �C/min; and held at 200 �C for 10 min. Volatile compounds weredetected using MSD (scan range of m/z 35–350) at 230 �C. Theidentification of compounds was based on the comparison of theirretention time and mass spectrum with data in the Wiley 275 andNIST libraries at a quality match greater than 85%. A series of n-al-kanes (C8–C20) was analysed by direct injection on the GC–MS toobtain retention index (RI) values. The RI data were compared withpreviously published literature values.

2.6.3. Sensory evaluation of the paste-like eb-SWPHSensory profiling was performed on the paste-like eb-SWPH.

Quantitative descriptive analysis was applied for sensory evalua-tion. The intensity of each attribute was scored on a scale rangingfrom 0 to 9 by 15 semi-trained panellists consisting of universitystudents (4 males and 11 females between the ages of 22 and30 years old) and 5 professional panellists. All semi-trained panel-lists (university students) were previously trained in the descrip-tive sensory analysis of various food samples and in thedefinition of reference solutions in at least two training sessions.The attributes included four odours for seaweed, crab, shrimp,and caramel and four tastes of umami, sweet, salty, and bitter.Dried seaweed, burnt sugar, crab flavour powder, shrimp flavourpowder (Firmenich SA, Meyrin, Switzerland), and sucrose were dis-solved in hot distilled water (5% w/v) and used as reference solu-tions for the seaweed, caramel, crab, shrimp odour, and sweettastes, respectively. Reference solutions for the umami, saltiness,and bitter tastes were 4 mM of monosodium glutamate (pH 5.6)(Ajinomoto Co., Ltd., Samutprakan, Thailand), 12 mM NaCl, and1.5 mM caffeine, respectively. The paste-like eb-SWPH samples(5 ml) were dissolved in 10 ml of hot distilled water and servedin opaque disposable plastic cups.

2.7. Preparation of seafood processed flavour

Concentrated paste-like eb-SWPH that was produced from theoptimal treatment conditions was further processed to prepareseafood processed flavour (SPF). The thermal treatment of paste-like eb-SWPH was performed according to a general proceduremodified by Manley and Ahmedi (1995). A mixture of glucose, ri-bose, taurine, arginine, alanine, glycine, and paste-like eb-SWPHat 2:1:2:2:2:8:15 g was dissolved in 100 ml of distilled water.The pH was adjusted to 5.5 with 1 N HCl, and the solution was thenheated at 95 �C for 120 min. After the reaction, the thermal reac-tion products were cooled to room temperature. The SPF was con-centrated using a rotary evaporator (Büchi R-200) at 40 �C and5 kPa. The concentrated SPF paste was analysed for colour, volatilecompounds, and sensory profile. Colour measurements of the SPFpaste were determined by analysing 3 ml of the concentrated SPFpaste using a colorimeter (MiniScan� EZ; HunterLab, Reston, VA).The L*, a*, and b* values and the hue angle were measured to quan-tify the colour intensity or colour saturation. Determination of thevolatile compounds in the concentrated SPF paste was performedusing DHS–SPME–GC–MS, with conditions similar to those previ-ously described for the evaluation of volatile compounds in theconcentrated paste-like eb-SWPH. Descriptive sensory analysis ofthe concentrated SPF paste was performed using the fifteensemi-trained and five professional panellists and the identicalattributes that were used to evaluate the concentrated paste-likeeb-SWPH. The concentrated paste-like eb-SWPH that was pro-duced from the optimal conditions was used as the control solu-tion. For evaluation, the panellists were asked to evaluate thesample using a 9-point scale (�4 to 4; weaker than the controlto stronger than the control). Concentrated SPF paste was dissolvedin hot distilled water at a ratio of 1:2 (v/v) and served in opaque,disposable plastic cups.

Fig. 1. Response surfaces for the effect of enzyme concentration and hydrolysistime on (A) yield and (B) % degree of hydrolysis of liquid eb-SWPHs.

N. Laohakunjit et al. / Food Chemistry 158 (2014) 162–170 165

2.8. Statistical analysis

All assays were conducted in triplicate and analysed by analysisof variance (ANOVA) and Duncan’s multiple-range test (DMRT)using Version 9 of the SAS program (SAS Institute Inc., Cary, NC).The 3D surface plots of the experimental model were generatedusing the Statistica Program 5.0 (StatSoft Inc., Tulsa, OK). Sensoryanalysis was performed based on a randomised complete block de-sign (RCBD), and the means were separated using DMRT and signif-icant differences between each sample were obtained by ANOVAusing the SAS Program.

3. Results and discussion

3.1. Physicochemical properties of the eb-SWPH

The by-products of agar-extracted seaweed (G. fisheri) are asuitable protein source to produce protein hydrolysates becausethey contain a high amount of protein (28%) and a low amountof fat (0.60%). Thus, the by-products of agar-extracted G. fisheriwere used as the protein source for enzymatic hydrolysis usingbromelain. The high protein content of the seaweed by-productsoffers the potential to convert the by-products of agar productioninto value-added products.

The DE values of liquid eb-SWPHs were calculated from the L�,a�, and b� values. Statistical analysis indicated that the enzymeconcentration and the hydrolysis time did not influence the DEof the liquid eb-SWPHs. The DE values of the liquid eb-SWPHsranged from 1.92 to 4.03. The results indicated that the colour ofthe liquid eb-SWPHs was similar to the colour of the controlhydrolysate (non-enzymatic hydrolysis). The colour appearanceof the liquid eb-SWPHs was light yellow-green because of theirseaweed pigments. The perception of food may be influenced bycolour; therefore, the colour of the eb-SWPH may be a goodindicator of its use as a flavouring agent.

The total salt content of the liquid eb-SWPHs was not affectedby the enzyme concentration or the hydrolysis time (p > 0.05).The values for total salt content ranged from 0.43% to 0.48%. Thetotal salt content of the liquid eb-SWPH was lower than that ofthe seaweed by-products hydrolysed by acid, which resulted inan extremely high content of salt (17.10%) as reported by Weir(1992). Although salt could improve the taste of food, high levelsof salt lead to increased blood pressure (Law, 2000). Therefore,eb-SWPHs can be alternatives for healthy food products.

Fig. 1A and B illustrate the influence of the hydrolysis time andenzyme concentration on the proteolytic activity of the enzyme.The % yield and % DH indicated the efficiency of the enzyme tocleave peptide bonds at various concentrations and hydrolysistimes. Therefore, the % yield and % DH were further optimisedusing a statistical model established by RSM, which was effectivein replacing traditional kinetic experiments to quantify the effectsof each parameter and the interaction between factors (Guo et al.,2009). Regression coefficients for the two response variables (%yield and % DH) under different treatment conditions are presentedin Table 1. Regression coefficient (r2) values for all of the responsevariables were higher than 0.80, indicating that the generatedmodels adequately explained the data variation and significantlyrepresented the actual relationships between reaction parameters.RSM was applied for % yield and % DH using the following modelEqs. (2) and (3), respectively:

%Yield ¼ 32:440þ 0:500x1 � 0:012x21 þ 0:331x2 � 0:009x2

2 ð2Þ

%DH ¼ 44:382þ 1:736x1 þ 0:702x2 � 0:020x1x2 � 0:044x22 ð3Þ

where x1 represents the enzyme concentration, and x2 representsthe hydrolysis time.

The interaction between the enzyme concentration (x1) and thehydrolysis time (x2) on the yield was significant (p 6 0.01). Theyield demonstrated a positive linear correlation between the en-zyme concentration (x1) and hydrolysis time (x2) but a negativecorrelation to an interaction of x2

1 and x22. Considering the enzyme

concentration, the high value for the estimated regression coeffi-cient (b1 = 0.500) exerted the maximum influence on the yield,which explained the observed curve. The % yields for all of the en-zyme concentrations increased rapidly in the initial phase ofhydrolysis (0–3 h) and then increased slightly after 6 h. The liquideb-SWPH that was produced from an enzyme concentration of 20%bromelain and a hydrolysis time of 3 h resulted in the highest %yield (38.15%). However, the % yield of this liquid eb-SWPH didnot increase with increasing hydrolysis times of 6, 12, 18, and24 h (Fig. 1A). Seaweed by-products protein was mostly composedof alkali-soluble proteins; the hydrolysis of bromelain at pH 6.0(optimal bromelain activity) seems to be more effective than the

Table 2Predicted and experimental values of liquid eb-SWPH hydrolysed by 10% bromelainfor 3 h.

Trial Conditions %Yield %DH

Observed a Predicted Observed a Predicted

1 x1 = 5, x2 = 3 35.98 ± 0.21ns 35.89ns 54.66 ± 1.39ns 54.42ns

2 x1 = 10,x2 = 3

38.15 ± 0.19ns 38.29ns 62.91 ± 1.38ns 62.65ns

3 x1 = 10,x2 = 6

38.44 ± 0.33ns 39.25ns 62.61 ± 1.43ns 62.97ns

x1 Represents enzyme concentration and x2 represents hydrolysis time.ns means of %yield and %DH compared by t-test are not significantly different

(p > 0.05),a Observed %yield and %DH values are expressed as means ± SD (n = 3).

Table 1Regression coefficients of a quadratic model estimated by multiple linear regressionsfor the% yield and% DH of the liquid eb-SWPHs.

Factors Regression coefficient

%Yield %DH

Constant 32.440** 44.382**

Linearx1 0.500** 1.736**

x2 0.331** 0.702**

Quadraticx2

1�0.012** �0.002

x22

�0.009** �0.044*

Interactionx1x2 �0.002 �0.020*

Statistical analysis for the modelr2 0.853 0.891Adjusted r2 0.812 0.860

x1 = enzyme concentration and x2 = hydrolysis time.** Significant at p 6 0.01;* Significant at p 6 0.05.

Table 3Amino acid composition and taste components of G. fisheri by- products treated with6 N HCl to complete hydrolysis, and paste-like eb-SWPH hydrolysed by 10%bromelain for 3 h.

Amino acidsa Amino acid content (mg/100 g of protein)*

G. fisheri by-products + HCl Paste-like eb-SWPH

Essential amino acidHistidine 752 ± 7.07 10.0 ± 0.71Threonine 3371 ± 49.5 90.0 ± 2.12Cystine 915 ± 28.3 0 ± 0.00Tyrosine 1576 ± 51.6 177 ± 4.24Valine 2600 ± 56.6 93.3 ± 2.12Methionine 744 ± 9.90 110 ± 3.54Lysine 3371 ± 99.0 267 ± 9.19Isoleucine 2000 ± 21.2 53.3 ± 1.41Leucine 4482 ± 53.7 213 ± 2.83Phenylalanine 3256 ± 35.4 93.3 ± 0.28

Non-essential amino acidAspartic acid 8108 ± 31.1 40 ± 0.42Serine 3932 ± 14.1 100 ± 1.65Glutamic acid 7973 ± 120 46.7 ± 2.83Glycine 4011 ± 141 73.3 ± 1.56Arginine 5091 ± 212 310 ± 4.88Alanine 5109 ± 70.7 157 ± 4.67Proline 3002 ± 106 46.7 ± 0.42Total 60,291 ± 360 1880 ± 33.91

Taste componentBitterb 20845.45 693.33Umamic 16080.3 86.67Sweetd 17503.03 656.67Saltye 915.15 0.00Tastelessf 4946.97 443.33Total 60290.9 1880

a Amino acids in the seaweed by-products were in free amino acid form andamino acids in peptide chains.

b Bitter was calculated from the sum of histidine + valine + methionine +isoleucine + leucine + phenylalanine + glycine + proline.

c Umami was calculated from the sum of aspartic acid + glutamic acid.d Sweet was calculated from the sum of threonine + serine + arginine + alanine.e Salty was calculated from cystine.f Tasteless was calculated from the sum of tyrosine + lysine.

* Values are means and standard deviations of triplicate measurements.

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human gastrointestinal digestion. Because of the remaining poly-saccharides which limited the digestibility of enzyme and lowactivity of the crude enzyme (174,285 U/g), the % yield was nottoo high.

Regression coefficients indicated that the enzyme concentrationand the hydrolysis time demonstrated a significant linear effect onthe% DH (Table 1). The high value for the estimated regressioncoefficient resulted from the enzyme concentration (b1 = 1.736),indicating that the enzyme concentration was the most importantlinear variable influencing the % DH. A positive value indicated thatthe % DH values increased with increasing enzyme concentration,as reported in previous studies (Guo et al., 2009; Sonklin et al.,2011). Similar to the % yield, the hydrolysis time (0–3 h) initiallyaffected the% DH of the liquid eb-SWPH. After 3 h of hydrolysis,the % DH reached more than half of the % DH at 6 h of hydrolysis,and the % DH value reached a plateau after 6 h (Fig. 1B). The % DHdramatically increased when the enzyme concentration was in-creased from 5% to 10%. However, the % DH values of the liquideb-SWPH using 15% and 20% bromelain were not significantly dif-ferent due to saturated enzyme/substrate or inhibitory effects ofthe end-products (McCarthy, O’Callaghan, & O’Brien, 2012).

To aid in the visualisation, response surface plots and a contourplot of % yield and % DH were used to determine the optimalhydrolysis conditions. Fig. 1A and B show the three-dimensionalresponse surface plots and contour plot for the independent vari-ables (concentration and hydrolysis time) on % yield and % DH,respectively. The overlay plot shows the zone of optimisation, indi-cating that the optimal enzyme concentrations and hydrolysistimes were 10–20% and 3–12 h, respectively. Therefore, the hydro-lysis of seaweed by-products using 10% bromelain for 3 h was se-lected as the optimal conditions for producing liquid eb-SWPHbecause these conditions resulted in the highest % DH and % yieldin the shortest hydrolysis time.

The model equation to predict the optimal response values wasverified by determining the best conditions using the statistical mod-el. Observed and predicted values are listed in Table 2. The experi-mental results were similar to the predicted values for % yield and% DH using RSM. These results confirmed the suitability of the modelto predict the optimal conditions for liquid eb-SWPH hydrolysis.

3.2. Amino acid composition of the seaweed (G. fisheri) by-productsand the paste-like eb-SWPH

The amino acid compositions of the acid-hydrolysed seaweedby-products and the enzyme-hydrolysed paste-like eb-SWPH that

was produced using 10% bromelain for 3 h were determined usingHPLC and are shown in Table 3. Seventeen amino acids were foundin the seaweed by-products that were treated with 6N HCl at110 �C for 24 h, which resulted in complete hydrolysis to freeamino acids. The results indicated that the protein in the seaweedby-products contained a large amount of essential amino acids.Aspartic acid was the most abundant, accounting for 13.25% ofthe total amino acids. Other abundant amino acids found in theseaweed by-products include glutamic acid, alanine, arginine,and lysine. These amino acids are the most abundant amino acidsin various seaweed species (Dawczynski et al., 2007). Aspartic acid

N. Laohakunjit et al. / Food Chemistry 158 (2014) 162–170 167

exhibits interesting properties in flavour development, andglutamic acid is the major component in the taste sensation ofumami (which means ‘‘delicious’’ in Japanese), which is describedas savoury with a meat or broth-like taste (Kato, Rhue Mee, &Nishimura, 1989).

The selectivity and specificity of bromelain to hydrolyseproteins into small peptides resulted in a larger amount of smallpeptides than free amino acids in the paste-like eb-SWPH. The to-tal amount of free amino acids in the paste-like eb-SWPH was5.64 mg/100 g, which was lower than that found in the seaweedby-products (162 mg/100 g) subjected to complete hydrolysisusing 6N HCl (Table 3). From the result of amino acid compositions,it could be explained that bromelain might be partially hydrolysedto free amino acids (Liu & Chiang, 2008). However, the result wasconfirmed by molecular weight distribution estimated by sodiumdodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE).Electrophoresis profile of eb-SWPH was composed of four bandswith apparent molecular weights distributed at 21, 17, 14 and lessthan 2 kDa (data not shown). Among these bands, the bandsappeared at molecular weight less than 2 kDa were predominant.This could signify that bromelain could hydrolyse native proteinsof seaweed by-products into oligo-peptides and di or tri peptidewhich contributed to the taste of eb-SWPH, especially umami tasteas reported in previous evidence (Su et al., 2012). The proportion offree amino acids to total amino acids indicated that peptides werepresent in the paste-like eb-SWPH (Sonklin et al., 2011). In ourstudy, sixteen free amino acids derived from enzymatic hydrolysiswere identified. One sulphur-containing (methionine), sevenhydrophobic (alanine, valine, leucine, isoleucine, proline, methio-nine, and phenylalanine), four hydrophilic (glycine, serine, threo-

Table 4Volatile compounds of paste-like eb-SWPH from the hydrolysis of seaweed protein by 10

Volatile compounda RIa,b Peak area (108)

eb-SWPH

Methylpyrazine 1282 –Hexanal 1339 5.692,5-Dimethylpyrazine 1345 –2,3-Dimethylpyrazine 1363 –2-Ethyl-5-methylpyrazine 1408 –Trimethylpyrazine 1422 –3-Ethyl-2,5-dimethylpyrazine 1478 –Tetramethylpyrazine 1492 –2-Ethyl-1-hexanol 1505 –Diethylmethylpyrazine 1509 –Benzaldehyde 1537 0.682,3-Butanediol 1558 –Estragole 1681 –2-Acetyl-3,5-dimethylpyrazine 1701 –5-Methylfurfuryl alcohol 1738 –Hexanoic acid 1739 5.59Heptanoic acid 1855 1.76Octanoic acid 1950 1.642-Methylquinoxaline 1982 –Eugenol 2141 –Nonanoic acid 2202 2.892,4-di-tert-butylphenol 2301 –Dihydroactindiolide 2315 4.43Decanoic acid 2361 2.10Dodecanoic acid 2517 1.471-hexadecanol 2393 –Dibutyl phthalate 2678 –Hexadecanoic acid 2875 1.60

a All compounds were identified by comparison with mass spectra and retention indeb RI (retention index) calculated with a DB-Wax stationary phase using a series of alkc Odour descriptions were cited from www.flavornet.org and recent reports.A Yu & Chen, 2010.B Morita et al., 2001

nine, and tyrosine), two acidic (aspartic and glutamic acid), andthree basic (lysine, arginine, and histidine) amino acids were foundin the paste-like eb-SWPH that was hydrolysed using 10% brome-lain for 3 h. Among the identified amino acids, arginine was themost abundant amino acid and accounted for 0.93 mg/100 g. Thesefindings may reflect the preference of stem bromelain to catalysethe hydrolysis of N2-benzoyl-L-arginine ethyl ester and N2-ben-zoyl-L-arginine amide via an acyl-enzyme mechanism that resultsin the release of arginine during bromelain hydrolysis (Wharton,1974). Other abundant amino acids in the paste-like eb-SWPH in-cluded lysine (0.80 mg/100 g), leucine (0.64 mg/100 g), and tyro-sine (0.53 mg/100 g), of which leucine gives a bitter taste, andlysine and tyrosine are tasteless. The amino acids found in thepaste-like eb-SWPH were less abundant than those in the hydroly-sed seaweed by-products. For example, cysteine was not found inthe products of enzymatic hydrolysis, but cystine (two covalentlylinked cysteines) was found, indicating that free cysteines formeda disulphide bond during enzymatic hydrolysis, which results inthe absence of cysteine in the paste-like eb-SWPH (Sonklin et al.,2011).

As shown in Table 3, free amino acids were divided into severalclasses based on their taste characteristics. The ratio of bitter/uma-mi and sweet/umami for the seaweed by-products was 1.30 and1.09, respectively, whereas the corresponding ratios for thepaste-like eb-SWPH were 8.00 and 7.58, respectively. These resultsindicated that the bitter and sweet characteristic tastes of thepaste-like eb-SWPH were greater than those of the seaweedby-products due to bromelain hydrolysis. The amount of hydro-philic amino acids in the paste-like eb-SWPH was lower than theamount of hydrophobic amino acids because endoproteinases

% bromelain for 3 h and seafood processed flavour (SPF).

Odour descriptionc Positive relation to

SPF

6.32 Fishy-ammoniacal notes– Resin, flower, green40.04 Nutty, roasted CrabA, shrimpB

3.01 Roasted potato, coffee crabA, shrimpB

9.64 Roasted, nutty54.12 Powdery cocoa, potato Crab, shrimpB

19.0431.89 Nutty, brown cocoa0.71 Oily, sweet14.32 Nutty, meaty1.22 Sweet, oily, nutty, woody CrabA, shrimpB

2.58 Creamy, caramel CrabA

0.18 Sweet, spice1.36 Roasted, nutty1.65 –– Sour, fatty, sweat, cheese– Cheesy, waxy, sweaty– Fatty, rancid, vegetable2.44 Toasted coffee, nutty2.10 Clove like, savoury notes– Green, fat0.82 fermented sausage1.55 Sweet– Fatty, rancid– Coconut, fatty, waxy0.24 Waxy, floral1.39 – CrabA

– Heavy waxy, creamy

x database.anes between C8 and C20 as reference standards.

168 N. Laohakunjit et al. / Food Chemistry 158 (2014) 162–170

possess a broad specificity for the hydrolysis of proteins at hydro-phobic amino acid residues and non-polar amino acid residues atthe C-terminus of the peptide remain, as has been reviewed in pre-vious studies (Wharton, 1974).

3.3. Volatile compounds in the paste-like eb-SWPH

Ten volatile compounds in the paste-like eb-SWPH that washydrolysed using 10% bromelain for 3 h were identified and arelisted in Table 4. Major volatile compounds included hexanal, hex-anoic acid, nonanoic acid, and dihydroactinidiolide with% peakareas at 5.69, 5.59, 2.89, and 4.43 � 108, respectively. Odourdescription of the volatile compounds revealed that the paste-likeeb-SWPH had sweet, slightly green, and fatty odour characteristics.Fatty acids and hexanal were reported as the major and minorflavour compounds in the original seaweed by-products. Thesecompounds are derived from the degradation of polyunsaturatedfatty acids either by auto-oxidation or by the action of enzymes,including lipoxygenase and fatty acid hydroperoxide lyase (LePape, Grua-Priol, Prost, & Demaimay, 2004). The intensity of thefatty acids (heptanoic acid, octanoic acid and nonanoic acid) andsweet volatiles (benzaldehyde) in the paste-like eb-SWPH in-creased during hydrolysis (Fig. 2A). This finding was in agreement

Fig. 2. Gas chromatography chromatogram of (A) eb-SWPH from hydrolysed seaweedfrom eb-SWPH.

with previous observations by Imm and Lee (1999). Importantly,hexanal, benzaldehyde, hexanoic acid, and octanoic acid that werefound in the eb-SWPH have also been detected in other marinespecies, such as shrimp, crab, and fish (Josephson & Lindsay,1986; Yu & Chen, 2010). Among the volatile components observedin the paste-like eb-SWPH, the major fatty acid components wereheptanoic acid, octanoic acid, nonanoic acid, decanoic acid,dodecanoic acid, and hexadecanoic acid. These fatty acids havebeen reported as the precursors to seafood flavours (Josephson &Lindsay, 1986).

3.4. Sensory evaluation of the paste-like eb-SWPH

The sensory profile of the paste-like eb-SWPH was seaweedodour, crab odour, shrimp odour, caramel odour, umami, sweet,salty, and bitter taste, which received scores of 7.6, 1.8, 1.6, 1.0,4.2, 3.5, 2.0, and 1.0, respectively. The panellists described the fla-vour characteristic of the paste-like eb-SWPH as tasteless, but itpresented an umami taste and a seaweed odour. The odour attri-butes of the paste-like eb-SWPH that were determined by the sen-sory evaluation were consistent with the volatile profile identifiedusing GC–MS (Table 4). The presence of fatty acids in the paste-likeeb-SWPH may explain the seaweed aroma of the hydrolysate.

protein by-products by 10% bromelain for 3 h and (B) seafood processed flavour

Fig. 3. Sensory profiles of seafood processed flavour from the hydrolysis ofeb-SWPH by 10% bromelain for 3 h. 0 = control line (eb-SWPH), �, �� = significantlystronger than control, �p 6 0.05, ��p 6 0.01 versus control (Student’s t test).

N. Laohakunjit et al. / Food Chemistry 158 (2014) 162–170 169

Moreover, the distribution of the free amino acids and small pep-tide may influence the sensory perception of the hydrolysate. Forexample, leucine may impart a bitter taste, but alanine and argi-nine may impart a sweet taste (Kato et al., 1989). The umami tastewas characterised by the presence of free glutamic acid and aspar-tic acid, which were present in high amounts in the paste-like eb-SWPH. Although free glutamic and aspartic acid were present atlow levels in the paste-like eb-SWPH, the paste-like eb-SWPH gavea moderate umami taste, which may arise from the remainingsmall peptide content rather than the presence of free amino acids.It should be noted that small peptide (molecular weight less than2 kDa) might contribute to the taste of eb-SWPH, especially umamitaste as reported in previous evidence (Su et al., 2012). From thisresult, the modification of the bromelain hydrolysis is an effectiveway to improve flavour of seaweed by-products, as compared toacid hydrolysis which fully hydrolyses all proteins to free aminoacids.

Although the paste-like eb-SWPH contained a large amount ofbitter amino acids (histidine, valine, methionine, and proline),the panellists could not detect the bitter taste because the concen-trations of these amino acids were lower than the bitter tastethreshold level of the panellists (Kato et al., 1989). Moreover, thebitterness of the peptides may be correlated with their averagehydrophobicity and chain length. Accordingly, the bitter tasteintensity of the protein hydrolysates depended on the% DH andprotease specificity. Therefore, small peptides may be generatedwith an increase in the% DH, which may account for the low bitter-ness of the paste-like eb-SWPH.

3.5. Seafood processed flavour (SPF)

Although the paste-like eb-SWPH contained amino acids foundin marine meats, the flavour of the paste-like eb-SWPH was notsufficiently strong to represent a specific seafood flavour. For creat-ing a seafood flavour, not only reducing sugars (glucose and/or ri-bose) but also amino acids (glycine, alanine, and arginine) wereused as reactants. Especially, taurine (a sulphur-containing aminoacid) found in crustaceans (Konosu & Yamaguchi, 1987) was addedto enhance seafood profile flavour. It has been reported that theseamino acids could react with reducing sugars to give compounds asbenzaldehyde, heterocyclic compounds and pyrazines with apleasant sweet odour profile as found in crab and shrimp (Wong,Abdul, & Mohamed, 2008). Therefore, the paste-like eb-SWPHhydrolysed by 10% bromelain for 3 h was used as a precursor towhich the reactants were added to enhance the flavour character-istics and the perception qualities of the paste-like eb-SWPHthrough thermal processing.

The colour of the concentrated SPF paste was dark brown, withL*, a*, b*, and hue values of 17.2, 22.6, 28.9, and 37.87, respectively.The colour of the concentrated SPF paste was dark due to the pres-ence of brown pigments that originated from brown nitrogenouspolymers and melanoidins, which were produced from the thermalreactions. Colour development was promoted by heating sugar (viacaramelisation) and through condensation between reducing sug-ars and amino acids in the Maillard reaction (Manley & Ahmedi,1995).

Solid-phase microextraction of the headspace of the concen-trated SPF paste followed by GC–MS analysis enabled the detectionof 20 volatile compounds (Table 4). Aldehydes and pyrazines,which naturally occur in foods and processed flavour products,were the predominant compounds found in the concentrated SPFpaste (Fig. 2B). The Maillard reaction between sugar and the addedamino acids in addition to the amino acids present in the paste-likeeb-SWPH, including glycine and lysine, generated large amounts ofalkylpyrazines, pyrazines, and furan derivatives (Oh, Shu, & Ho,1991). Roasted and savoury flavours may be related to heterocyclic

compounds, such as pyrazines. 2,5-Dimethylpyrazine, 2,3-dimeth-ylpyrazine, trimethylpyrazine and 2,3-butanediol were the pre-dominant aroma-active compounds in cooked shrimp and crab(Morita, Kubota, & Aishima, 2001; Yu & Chen, 2010). As reportedby Yu and Chen (2010), 2,3-butanediol, which has a creamy andcaramel aroma, was the most important contributor to the aromaof steamed crab meat and was considered to be important incooked crustaceans due to its potent, creamy aroma. Fatty acidssuch as hexanoic acid, heptanoic acid, and nonanoic acid, whichare considered important components of seaweed flavour, werenot found in the concentrated SPF paste due to the thermal degra-dation of lipids.

As shown in Fig. 3, the seafood odour (crab and shrimp odour),caramel odour, umami taste, and sweet taste of the concentratedSPF paste were significantly increased. In contrast, the seaweedodour of the concentrated SPF paste was significantly decreasedduring the thermal process compared to the paste-like eb-SWPH(used as a control). The concentrated SPF paste exhibited seaweed,crab, shrimp, and caramel odour and umami, sweet, salty, and bit-ter tastes, with values of �3.71, 3.04, 3.22, 2.60, 1.71, 2.31, 0.09,and 0.98, respectively. The panellists described the concentratedSPF paste as a roasted seafood-like flavour. This description wasconsistent with the GC–MS analysis.

The increase in the sweet taste of the concentrated SPF pastecompared with that of the paste-like eb-SWPH may be due tothe added sweet amino acids (arginine and alanine), whereas theincrease in the bitter taste may arise from the Maillard reactionproducts, including melanoidin (Manley & Ahmedi, 1995). Theumami taste was related to monosodium glutamate and to the so-dium salt of glutamic acid, which may be produced during the pHadjustment in the production of the SFP, resulting in a greaterumami score than the paste-like eb-SWPH (Kato et al., 1989). Freeamino acids and peptide amino acids in the paste-like eb-SWPHplay an important role in the flavour of the concentrated SPF paste(Su et al., 2012). Because the crab and shrimp characteristics of theconcentrated SPF paste were not significantly different, the flavourof the concentrated SPF paste was described as a crustacean-likeflavour. However, the concentrated SPF paste may potentially beused as a seafood flavour enhancer, which might be useful inaccentuating or extending the basic seafood flavour of eachproduct.

170 N. Laohakunjit et al. / Food Chemistry 158 (2014) 162–170

4. Conclusions

A seaweed protein hydrolysate was produced under optimalconditions using 10% bromelain for 3 h. After bromelain hydrolysis,arginine, lysine, and leucine were the most abundant free aminoacids. As indicated by the panellists, the paste-like eb-SWPH wastasteless, but it exhibited an umami taste and a seaweed odour.Predominant odorants of the paste-like eb-SWPH included sweet,green, fatty, and seaweed odour characteristics. The paste-likeeb-SWPH demonstrates great potential as a precursor to thermallyprocessed seafood-like flavour that can be used as a flavour supple-ment and as a savoury flavour source for various seafood products.

Acknowledgements

This work was supported by the Thailand Institute of Scientificand Technological Research and the National Research UniversityProject (NRU) of Thailand, Higher Education Commission.

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