effect of some additives.pdf

Original article

Effect of some additives on the inhibition of lizardfish

trimethylamine-N-oxide demethylase and frozen storage stability

of minced flesh

Kittima Leelapongwattana,1 Soottawat Benjakul,1* Wonnop Visessanguan2 & Nazlin K. Howell3

1 Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90112, Thailand

2 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phaholyothin Rd.,

Klong 1, Klong Luang, Pathumthani 12120, Thailand

3 School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK

(Received 03 April 2006; Accepted in revised form 26 September 2006)

Summary Effects of some additives on the inhibition of trimethylamine-N-oxide demethylase (TMAOase) from

lizardfish (Saurida tumbil) muscle were investigated. Sodium citrate and pyrophosphate could inhibit

TMAOase activity in a concentration-dependent manner, most likely because of their chelating property.

Sodium alginate was the hydrocolloid possessing the inhibitory activity towards TMAOase (P < 0.05).

During the storage of lizardfish mince at )20 �C for 24 weeks, the addition of 0.5% sodium alginate and

0.3% pyrophosphate in combination with 4% sucrose and 4% sorbitol as the cryoprotectants resulted in the

retarded increase in TMAOase activities with the coincidental lowered formation of dimethylamine and

formaldehyde (FA), compared with the control (without additives) (P < 0.05). The loss in solubility of

muscle proteins was also impeded with the addition of those compounds, suggesting their role in the

inhibition of TMAOase as well as the retardation of protein denaturation induced by FA.

Keywords Additive, dimethylamine, formaldehyde, frozen storage, lizardfish, trimethylamine-N-oxide, trimethylamine-N-oxide demethylase.

Introduction

Frozen storage is considered very important for thepreservation of fish. Depending on intrinsic factorsincluding species and season as well as technologicalfactors such as handling practices prior to freezing,freezing rate, storage temperature, and the presence ofprotective barriers against oxidation, the practical stor-age life of frozen fish may vary substantially (Carecheet al., 1999). During frozen storage of some fish species,formaldehyde (FA) and dimethylamine (DMA) areformed stoichiometrically from trimethylamine-N-oxide(TMAO) by demethylation, catalysed mainly by tri-methylamine-N-oxide demethylase (TMAOase) (Amanoet al., 1963; Yamada & Amano, 1965). The producedFA can react with a number of chemical groupsincluding a number of amino acid residues, terminalamino groups, and various low molecular weightcompounds, leading to the denaturation and cross-linking of proteins (Nielsen & Jorgensen, 2004). This is

accompanied by the losses in textural property andfunctional properties (Careche et al., 1999). To retardthe freeze-toughening of fish, numerous chemical addi-tives have been used (DaPonte et al., 1986). Bothsodium citrate and H2O2 were found to slow the rateof DMA formation in frozen gadoid mince (Parkin &Hultin, 1982). Sodium pyruvate and H2O2 were shownto decrease the extent of toughening in fillets of cod andminced red hake during frozen storage (Racicot et al.,1984). Additionally, the protective effects of sugars,phosphates and several carbohydrates on the denatur-ation of fish muscle during frozen storage were alsoexamined (Noguchi, 1984).Lizardfish (Saurida spp.) have been considered as a

potential raw material for high-grade surimi in Japan,because of their high yield, white colour, good flavour,and high gel-forming ability (Morrissey & Tan, 2000).Lizardfish are generally considered as the low-market-value fish in Southeast Asia because of their appearanceand susceptibility to spoilage. As a consequence, thelarge quantities are landed as trawl by catch. InThailand, lizardfish have been used to produce thelow-grade surimi, which is mainly served for low-priced

*Correspondent: Fax: +66-7421-2889;

e-mail: [email protected]

International Journal of Food Science and Technology 2008, 43, 448–455448

doi:10.1111/j.1365-2621.2006.01466.x

� 2007 Institute of Food Science and Technology Trust Fund

fishcake products (Benjakul et al., 2003b). The gel-forming ability of this fish decreased rapidly duringpostharvested handling (Yasui & Lim, 1987). This wasassociated with proteolysis as well as the formation ofFA (Benjakul et al., 2003b). FA production is consid-ered as one of the major causes of the liability oflizardfish in the frozen state (Benjakul et al., 2005).Therefore, the addition of some compounds capable ofinhibiting TMAOase as well as stabilising the proteinstructure during the extended storage should be thepromising approach to retard the quality deteriorationof this species. This study aimed to examine theinfluence of various additives on TMAOase activityand the physicochemical and biochemical changes oflizardfish mince during frozen storage at )20 �C.

Materials and methods

Chemicals

All chemicals for TMAOase activity assay were pur-chased from Wako Pure Chemical Industries, Ltd(Tokyo, Japan). TMAO was obtained from AldrichChemical Company, Inc (Milwaukee, WI, USA). Acetyl-acetone, Triton X-100, maltodextrin DE 20, sodiumpyruvate, sodium tripolyphosphate and sodium pyro-phosphate were purchased from Fluka (Bucks, Switzer-land). Tris (hydroxymethyl) aminomethane, hydrogenperoxide, and chlorine were obtained from Merck(Darmstadt, Germany). l-ascorbic acid was procuredfrom Sigma Chemical Co. (St. Louis, MO, USA).

Fish sample

Lizardfish (Saurida tumbil) of the average weight of 200–250 g per fish, caught off the Songkhla Coast along theGulf of Thailand and off-loaded approximately 36–48 hafter catching, were purchased from the dock inSongkhla. Fish kept in ice with fish/ice ratio of 1:2(w/w) were transported to the Department of FoodTechnology, Prince of Songkla University, Hat Yaiwithin 3 h. Upon arrival, fish were headed, gutted andwashed with water. The flesh was separated from skinand ground in a meat grinder (MX-T2G National,Tokyo, Japan).

Preparation of TMAOase crude extract from lizardfishmuscle

The TMAOase extract was prepared according tomethod of Benjakul et al. (2004) with some modifica-tions. To prepare the crude extract of TMAOase, finelychopped muscle of lizardfish was added with 2 vol. ofchilled 20 mm Tris–acetate buffer, pH 7.0 containing0.1 m NaCl and 0.1% Triton X-100. The mixture washomogenised for 3 min using a homogeniser (IKA

Labortechnik, Selangor, Malaysia) at a speed of12000 r.p.m. The homogenate was centrifuged at19 400 ·g for 1 h at 4 �C using a Sorvall Model RC-BPlus centrifuge (Sorvall, Newtown, CT, USA). Thesupernatant obtained was referred to as ‘TMAOasecrude extract’.

Trimethylamine-N-oxide demethylase activity assay

The TMAOase activity was assayed using TMAO as asubstrate in the presence of selected cofactors (Benjakulet al., 2004). To 2.5 mL of assay mixture (24 mm Tris–acetate containing 24 mm TMAO, 2.4 mm cysteine,2.4 mm ascorbate and 0.24 mm FeCl2, pH 7.0), 0.5 mLof enzyme solution was added to initiate the reaction. Thereaction was conducted at 25 �C for 80 min and 1 mL of10% trichloroacetic acid was added to terminate thereaction. The reaction mixture was then centrifuged at8000 ·g for 15 min and the supernatant was subjected toDMA determination. One unit of TMAOase is defined asthe activity, which releases 1 nmol DMA per minute.

Effects of some additives on TMAOase activity

To study the effect of various additives on TMAOaseactivity, TMAOase crude extract was mixed with anequal volume of stock solutions of additives to obtainthe designated final concentrations. The mixture wasallowed to stand at room temperature (26–28 �C) for10 min. The residual activity was then determined. Theadditives exhibiting the highest inhibitory activitytowards TMAOase were chosen for further study.

Effects of selected additives on physicochemicaland biochemical changes of lizardfish mince duringfrozen storage

Fish sample preparationThe lizardfish mince (0.5 kg) containing cryoprotectants[4% (w/w) sucrose and 4% (w/w) sorbitol] was mixedwith the selected additives showing the highest inhibi-tory effects on TMAOase using a mixer (KitchenAid,St. Joseph, MI, USA). Additionally, the mince with nocryoprotectants and additive was used as the control.All samples were packaged in a polyethylene bag andsealed tightly. All samples were kept at )20 �C for6 months. At definite time intervals (0, 2, 4, 6, 8, 12, 16,20 and 24 weeks), samples were removed, thawed withrunning water (26–28 �C) to obtain the core temperatureof 0–2 �C and subjected to TMAOase activity deter-mination and chemical analyses.

Trimethylamine-N-oxide determinationThe sample (2.5 g) was added with 10 mL of 5%trichloroacetic acid and homogenised with a homoge-niser) at a speed of 12 000 r.p.m. for 2 min. The

Effect of additives on inhibition of lizardfish TMAOase K. Leelapongwattana et al. 449

� 2007 Institute of Food Science and Technology Trust Fund International Journal of Food Science and Technology 2008, 43, 448–455

homogenate was centrifuged at 3000 ·g for 15 min. Thesupernatant containing TMAO was used for analysis.TMAO was determined after reduction to TMA by themethod of Benjakul et al. (2004). The supernatant(2 mL) was added to 2 mL of 1% TiCl3 and incubatedat 80 �C for 90 s, followed by cooling with a runningwater. TMA was determined according to the method ofConway & Byrne (1936). TMAO was then calculatedafter subtracting the indigenous TMA content in thesamples and expressed as lmol g)1.

Dimethylamine and free formaldehyde determinationThe sample (2.5 g) was added with 10 mL of 5%trichloroacetic acid and homogenised with a homoge-niser at a speed of 12 000 r.p.m. for 2 min. Thehomogenate was centrifuged at 3000 ·g for 15 minand the supernatant was removed. To the pellet, 5 mLof 5% trichloroacetic acid was added and homogenisedas described previously. The supernatants were com-bined and neutralised to pH 6.0–6.5 and the finalvolume was made up to 25 mL using distilled water. Thesupernatant was then used for DMA and FA determi-nations as described by Benjakul et al. (2004).

Protein solubilityProtein solubility was determined as described byBenjakul & Bauer (2000) with a slight modification.To 2.5 g sample, 22.5 mL of 0.6 m KCl was added andthe mixture was homogenised for 30 s. The homogenatewas stirred at room temperature (25–27 �C) for 4 h,followed by centrifuging at 12 000 ·g for 30 minat 4 �C. To 10 mL of supernatant, 2.5 mL of cold50% (w/v) trichloroacetic acid was added to obtain thefinal concentration of 10%. The precipitate was washedwith 10% trichloroacetic acid and then solubilised in0.5 m NaOH. Protein content was determined by theBiuret method (Robinson & Hodgen, 1940) usingbovine serum albumin as a standard. The solubilitywas expressed as the percentage of the soluble protein,compared with total protein in the sample.

Statistical analysis

Data were subjected to analysis of variance and meancomparisons were carried out using Duncan’s multiplerange test (Steel & Torrie, 1980). Statistical analysis wasperformed using the Statistical Package for Social Science(spss 10.0 for windows; SPSS Inc., Chicago, IL, USA).

Results and discussion

Effect of some additives on lizardfish muscle TMAOaseactivity

The effects of various additives on TMAOase activityfrom lizardfish muscle are shown in Table 1. TMAOase

activity was strongly inhibited by sodium pyrophos-phate (P < 0.05). In the presence of 0.2% and 0.4%sodium pyrophosphate, the activities of 68.3% and76.4% were inhibited, respectively. In addition, sodiumcitrate at a concentration of 0.4% also showed the highinhibitory effect on TMAOase activity (61.3% inhibi-tion). Pyrophosphate and citrate might function as thechelating agents, which can sequester the ions located inthe active site or those required for TMAOase activity.Ferrous or ferric ions are found in TMAOase active siteand determine the activity of this enzyme (Parkin &Hultin, 1986). Krueger & Fennema (1989) reportedthat sodium citrate significantly decreased DMAproduction of Alaska pollack fillets throughout thestorage at )10 �C. However, ethylenediaminetetraaceticacid (EDTA) showed no inhibitory activity against

Table 1 Effect of various additives on the inhibition of TMAOase

activity from lizardfish muscle

Additives Final concentration % Inhibition*

Chelator

EDTA 1 mM 0 a

2 mM 0 a

Sodium citrate 0.2% 31.58 ± 0.12 l

0.4% 61.32 ± 0.24 m

Anionic compounds

Sodium tripolyphosphate 0.2% 0 a

0.4% 5.57 ± 0.19 c

Sodium pyrophosphate 0.2% 68.30 ± 1.60 n

0.4% 76.42 ± 1.46 o

Oxidising agent

H2O2 10 ppm 16.73 ± 1.38 i

30 ppm 24.17 ± 1.79 j

Chlorine 10 ppm 0 a

30 ppm 0 a

Hydrocolloids

Carageenan 0.25% 5.35 ± 0.14 bc

0.5% 11.18 ± 0.75 fgh

Carboxymethylcellulose 0.25% 3.38 ± 0.21 b

0.5% 12.48 ± 0.10 gh

Locust bean gum 0.25% 5.15 ± 0.09 bc

0.5% 6.78 ± 0.07 cd

Sodium alginate 0.25% 9.32 ± 0.28 ef

0.5% 30.28 ± 0.51 kl

Xanthan gum 0.25% 10.08 ± 0.05 ef

0.5% ND

Others

Sodium pyruvate 0.2% 8.49 ± 0.74 de

0.4% 12.84 ± 1.58 h

Maltodextrin DE 20 4% 10.77 ± 0.22 fg

8% 28.58 ± 0.01 j

ND, not determined. Xanthan gum at 0.5% was not completely soluble

and was not used for inhibition study.

*Values are given as mean ± SD from triplicate determinations.

Different letters in the same column indicate significant differences

(P < 0.05).

Effect of additives on inhibition of lizardfish TMAOase K. Leelapongwattana et al.450

International Journal of Food Science and Technology 2008, 43, 448–455 � 2007 Institute of Food Science and Technology Trust Fund

TMAOase from lizardfish muscle. Some metal chelatorsincluding potassium cyanide and EDTA were shownto inhibit mackerel lipoxygenase, a non-heme-iron-containing enzyme (Banerjee, 2006).For oxidising agents, only H2O2 showed the inhibi-

tory activity towards TMAOase and the greater inhibi-tion was noticeable as the concentrations increased(P < 0.05). Krueger & Fennema (1989) found theinhibition effect of 0.85% (v/v) H2O2 on DMA forma-tion of Alaska pollack (Theragra chalcogramma) filletsduring frozen storage at )10 �C. Racicot et al. (1984)also reported that the presence of H2O2 in red hakemince at concentration as low as 0.05% (w/w) reducedDMA production during 7 weeks of frozen storage at)12 �C. H2O2 is thought to create oxidising conditionsin the fish muscle and affect the reducing conditionsnecessary for TMAOase activity (Parkin & Hultin, 1982;Racicot et al., 1984). Nevertheless, the negligible inhi-bition towards TMAOase from lizardfish muscle wasfound when chlorine was used (Table 1). Generalinhibitors of redox reactions, such as H2O2, were strongoxidants and normally promote the inhibition of enzy-matic TMAO breakdown (Phillippy & Hultin, 1993).Additionally, TMAO demethylation can be stimulatedor inhibited by compounds involved in redox reactionswith an electron transfer mechanism (Sikorski &Kostuch, 1982).For hydrocolloids, sodium alginate at a level of

0.5% showed the highest inhibition (30.3%), whereasnon-ionic (locust bean gum) and ionic (xanthan gum,carboxymethylcellulose) gums had a slight inhibitoryeffect on TMAOase activity. Sodium alginate is apolysaccharide consisting of guluronic acid and man-nuronic acid residues and complexes with metal ionsvia its carboxyl groups on polyguluronate units(Gupta et al., 2002). Carrageenan was found to inhibitthe TMAOase activity in a concentration-dependentmanner. Carrageenan is a polysaccharide chain com-prising galactose units and 3,6-anhydro galactose,both sulphated and non-sulphated, joined by alternat-ing a-1,3, b-1,4 glycosidic linkages (Wong, 1989).Sulphate groups might form the salt bridge with ionsneeded for TMAOase activity or chelate with ferrousor ferric ions in the active site of enzyme. From theresult, carboxymethylcellulose, locust bean gum andxanthan gum also showed the inhibitory effect onTMAOase activity, depending on the concentrationused. Carboxymethylcellulose contains a hydrophiliccarboxyl group (Wong, 1989), which can interact withmono and divalent cations (Whistler & Daniel, 1990).Therefore, the inhibitory effect of anionic and ionicgums might be a result of the interaction of ionicgroups of gum with the ions required for TMAOaseactivity.The addition of sodium pyruvate and maltodextrin

also resulted in the decreases in TMAOase activity. High

inhibition was noticeable with increasing concentrations(P < 0.05). However, Krueger & Fennema (1989)reported that treatment of Alaska pollack fillets with0.85 m sodium pyruvate had no significant effect onDMA formation. Maltodextrins were strong inhibitorsof FA production at )10 �C, showing the inhibitionlevels greater than 30% over the entire storage period(Herrera et al., 1999). Herrera et al. (2000) found thatadding maltodextrins to minced blue whiting muscleinhibited FA production during storage at )10 and)20 �C.As a result of the highest inhibitory effect of sodium

pyrophosphate (0.3%) and sodium alginate (0.5%)among all additives tested on TMAOase activity, theywere chosen for further use in combination withcryoprotectants (4% sucrose and 4% sorbitol) to lowerthe protein alterations taking place in frozen storedlizardfish mince induced by TMAOase activity.

Changes in TMAOase activity in lizardfish muscleduring frozen storage

TMAOase activity in lizardfish muscle was monitoredduring frozen storage at )20 �C. A marked increase inactivity was found in all samples within 4 weeks ofstorage (P < 0.05) (Fig. 1). Thereafter, TMAOaseactivity continuously decreased up to 24 weeks. Anincrease in TMAOase activity within the first 6 weeksmight result from the disruption of cell membraneduring frozen storage. The formation and accretion of

TM

AO

ase

activ

ity (

units

g–1

sam

ple)

0

2

4

6

8

10

12

14

16

18

20

Storage time (weeks)

Control Cryo Cryo + Al Cryo + 0.1P

Cryo + 0.3P Cryo + 0.1P/Al Cryo + 0.3P/Al

Figure 1 Changes in TMAOase acitivity in lizardfish mince without

and with various additives during frozen storage at )20 �C for

24 weeks. Cryo, 4% sucrose and 4% sorbitol; Cryo + Al, cryopro-

tectants with 0.5% sodium alginate; Cryo + 0.1P, cryoprotectants

with 0.1% pyrophosphate; Cryo + 0.3P, cryoprotectants with 0.3%

pyrophosphate; Cryo + 0.1P/Al, cryoprotectants with 0.1% pyro-

phosphate and 0.5% sodium alginate; Cryo + 0.3P/Al, cryoprotec-

tants with 0.3% pyrophosphate and 0.5% sodium alginate; control

without cryoprotectants and additives. Bars represent standard

deviation from triplicate determinations.



ice crystals, dehydration, and increase in unfrozenphase led to the changes in muscle tissues as well as celldamage (Shenouda, 1980). Therefore, freezing candisrupt muscle cells, resulting in the release of mitoch-ondrial and lysosomal enzymes into sarcoplasm, espe-cially with increasing frozen storage time (Hamm,1979). The subsequent decrease in activities was poss-ibly caused by the denaturation of TMAOase and thelower extraction efficacy of TMAOase from the muscle,owing to the protein aggregation, particularly withincreasing storage time. Generally, the control (withoutcryoprotectants and additives) had higher TMAOaseactivity, compared with samples containing cryopro-tectants and additives throughout the storage(P < 0.05). When the mince was mixed with cryopro-tectants prior to frozen storage, those compounds wereable to retard the denaturation of protein or disruptionof cells as evidenced by the lower TMAOase released.From the result, the differences in TMAOase activityamong all samples were noticeable. In the presence ofcryoprotectants, the samples with a higher level ofpyrophosphate comprised the lower TMAOase activity.The lowest TMAOase activity was found in the frozenstored lizardfish mince containing sodium pyropho-sphate (0.3%) and sodium alginate (0.5%) throughoutthe storage. The lowered activity of TMAOase inmince added with pyrophosphate and alginate waspostulated to be due to the inhibition of TMAOase bythose compounds. Both pyrophosphate and alginatewith anionic groups, phosphate and carboxyl groups,most likely chelated the ions localised in the active siteof enzyme or required for its activity.

Changes in trimethylamine-N-oxide, dimethylamine andformaldehyde contents of lizardfish mince during frozenstorage

Formation of DMA and FA in lizardfish mince isdepicted in Fig. 2a and b. In general, DMA and FAcontents increased continuously as the storage timeincreased up to 24 weeks of storage (P < 0.05). Thegreatest changes were observed in the control sample(without cryoprotectants and additives), where DMAand FA contents were 0.92 and 0.57 lmol g)1 sample,respectively, after 24 weeks of storage. Tejada et al.(2002) also found the formation of DMA and FAduring frozen storage of hake mince. It has beensuggested that the action of the TMAOase system inseveral species of the family Gadidae is responsible forrapid textural deterioration (Amano et al., 1963).Equimolar quantities of DMA and FA are formedfrom TMAO (Dingle et al., 1977). On the contrary,the sample containing 0.3% sodium pryrophosphateand 0.5% sodium alginate showed the lowest changesin DMA and FA formation throughout the storagetime. However, no marked differences in DMA and

FA contents were observed among other samples.Ayyad & Aboel-Niel (1991) reported that frozenmince of mackerel added with 0.5% alginate showedthe lower DMA and FA contents, compared with thecontrol sample. From the results, the lower amount ofFA measured was noticeable, compared with that ofDMA produced. FA is reactive with free amino acidsand proteins (Dingle & Hines, 1975; Castell & Smith,1973). This most likely caused the loss in FAextractability from the muscle. As a consequence, thelower content of free FA was found, compared withtotal FA content, which is equivalent to DMA

DM

A (

µmol

e g–1

sam

ple)

TM

AO

(µm

ole

g–1 s

ampl

e)FA

(µm

ole

g–1 s

ampl

e)

(a)

(c)

(b)

0

0.2

0.4

0.6

0.8

1


0

0.2

0.4

0.6

0.8

1


0

0.5

1

1.5

2

2.5

3

3.5


Control Cryo Cry + Al Cryo + 0.1P


Figure 2 Changes in DMA (a), FA (b) and TMAO (c) contents in

lizardfish mince with and without various additives during frozen

storage at )20 �C for 24 weeks. Bars represent standard deviation

from triplicate determinations. Key: see Fig. 1 caption.



amount. The deterioration in quality of frozen storedfish because of the breakdown of TMAO is basicallyattributed to the formation of FA rather than DMA(Gill et al., 1979).TMAO content gradually decreased throughout the

storage time (Fig. 2c) with concomitant increases inDMA and FA (Fig. 2a and b). Increases in DMA andFA contents indicated the breakdown of TMAO inlizardfish mince. The highest decreasing rate of TMAOwas also found in the control sample, compared withother samples. Parkin & Hultin (1982) suggested thatthe relative low approximate Km for TMAO of thesystem, indicating that TMAOase could function at arelatively high activity until most of the TMAO isconverted. Although lizardfish mince was kept underfrozen storage, the TMAOase activity still maintainedand played an important role in textural changes offrozen lizardfish mince because of the low activationenergy needed of this enzyme. Benjakul et al. (2003a)reported that the activation energy of TMAOase fromlizardfish kidney was 30.5 kJ mol)1 K)1. From theresults, the lowest changes in DMA, FA and TMAOcontents were found in sample containing both sodiumalginate and pyrophosphate. Lowered TMAOase activ-ity caused by those two compounds might be associ-ated with the retarded formation of FA and DMA inthe lizardfish mince.

Changes in protein solubility of lizardfish mince duringfrozen storage

Solubility of muscle protein in lizardfish mince in 0.6 m

KCl decreased continuously during the prolongedstorage at )20 �C (P < 0.05) (Fig. 3). The sharpdecrease was noticeable within the first 2 weeks.Among all samples, the control without cryoprotec-tants and additives had the lowest protein solubility.With the addition of cryoprotectants, higher solubilitywas observed, regardless of the additive used. How-ever, sample added with cryoprotectants and 0.5%alginate had no differences in solubility, compared withthe sample mixed with only cryoprotectants. From theresult, the addition of sodium pyrophosphate andalginate in combination with cryoprotectants (4%sucrose and 4% sorbitol) lowered the decrease inprotein solubility. Decreases in protein solubility dur-ing frozen storage were noticeable in minced trout(Moral et al., 1986), minced cod (Tejada et al., 1996)and hake actomyosin (Del Mazo et al., 1999). Tejadaet al. (2003) also found the decrease in proteinsolubility of hake muscle during storage at )20 �Cfor 365 days. Generally, less protein could be extractedwith salt solutions where the greater amount of FAwas produced (Sotelo et al., 1995).From the result, the inhibitory activity of 0.5%

sodium alginate and 0.3% pyrophosphate towards

TMAOase (Table 1) might be associated with thedecreased FA production in lizardfish mince (Fig. 2b).As a result, the loss of protein solubility of the samplecontaining both compounds was arrested. Herreraet al. (1999) postulated that FA molecules wouldinitially bind easily accessible reactive groups, and itwould cause some chemical modifications in the nativestructure. Apart from the inactivation of TMAOase bypyrophosphate and alginate, the restriction of molecu-lar diffusion caused by those compounds and cryopro-tectants added could lessen the movement of reactantsfor the reactions, both enzymatic reactions and proteindenaturation. Additionally, cryostabilisers presumablyplay an important part in the inhibition of thereactivity of FA by reducing the exposure and avail-ability of reactive groups, as well as by restricting thediffusion of the FA molecules towards these groups(Herrera et al., 1999).

Conclusions

Sodium pyrophosphate and sodium citrate showed thehigh inhibitory effect on TMAOase activity fromlizardfish muscle. Sodium alginate also inhibitedTMAOase to some extent. The addition of 0.3%pyrophosphate together with 0.5% alginate in combi-nation with cryoprotectants could lower TMAOaseactivity as well as retard protein denaturation inducedby FA in lizardfish mince during frozen storage.

Acknowledgment

This work was supported by the Thailand ResearchFund under the Royal Golden Jubilee Ph.D Program toKittima Leelapongwattana (PHD/0145/2545).

)%( ytilibuloS

0

20

40

60

80

100


Control Cryo Cryo + Al Cryo + 0.1P


Figure 3 Changes in protein solubility of lizardfish mince with and

without various additives during storage at )20 �C for 24 weeks.

Bars represent standard deviation from triplicate determinations. The

solubility was expressed as the percentage of soluble protein relative

to total protein content in the sample. Key: see Fig. 1 caption.



References

Amano, K., Yamada, K. & Bito, M. (1963). Content of formaldehydeand volatile in different tissues of gadoid fish. Bulletin of theJapanese Society of Scientific Fisheries, 29, 860–864.

Ayyad, K. & Aboel-Niel, E. (1991). Effect of addition of somehydrocolloids on the stability of frozen minced fillet of mackerel.Carbohydrate Polymer, 15, 143–149.

Banerjee, S. (2006). Inhibition of mackerel (Scomber scombrus) musclelipoxygenase by green tea polyphenols. Food Research International,39, 486–491.

Benjakul, S. & Bauer, F. (2000). Physicochemical and enzymaticchanges of cod muscle proteins subjected to different freeze-thawcycles. Journal of the Science of Food and Agriculture, 80, 1143–1150.

Benjakul, S., Visessanguan, W. & Tanaka, M. (2003a). Partialpurification and characterization of trimethylamine-N-oxidedemethylase from lizardfish kidney. Comparative Biochemistry andPhysiology Part B, 135, 359–371.

Benjakul, S., Visessanguan, W. & Tueksuban, J. (2003b). Changes inphysico-chemical properties and gel-forming ability of lizardfish(Saurida tumbil) during post-mortem storage in ice. Food Chemistry,80, 535–544.

Benjakul, S., Visessanguan, W. & Tanaka, M. (2004). Inducedformation of dimethylamine and formaldehyde by lizardfish kidneytrimethylamine-N-oxide demethylase. Food Chemistry, 84, 297–305.

Benjakul, S., Visessanguan, W., Thongkaew, C. & Tanaka, M. (2005).Effect of frozen storage on chemical and gel-forming propertiesof fish commonly used for surimi production in Thailand.Food Hydrocolloids, 19, 197–207.

Careche, M., Herrero, A.M., Rodriguez-Casado, A., Del Mazo, M.L.& Carmona, P. (1999). Structural changes of hake (Merlucciusmerluccius L.) fillets: effects of freezing and frozen storage. Journal ofAgricultural and Food Chemistry, 47, 952–959.

Castell, C.H. & Smith, B. (1973). Measurement of formaldehyde in fishmuscle using TCA extraction and the Nash reagent. Journal of theFisheries Research Board of Canada, 30, 91–98.

Conway, E.J. & Byrne, A. (1936). An absorption apparatus for themicro determination of certain volatile substances. I. The micro-determination of ammonia. Biochemical Journal, 27, 419–429.

DaPonte, D.J.B., Roozen, J.P. & Pilnik, W. (1986). Effect of additions,irradiation, and heating on the activity of trimethylamine oxidase infrozen stored minced fillets of whiting. Journal of Food Technology,21, 33–43.

Del Mazo, M.L., Torrejon, P., Careche, M. & Tejada, M. (1999).Characteristics of the salt-soluble fraction of hake (Merlucciusmerluccius) fillets stored at )20 and )30 �C. Journal of Agriculturaland Food Chemistry, 47, 1372–1377.

Dingle, J.R. & Hines, J.A. (1975). Protein instability in frozen fleshfrom fillets and frames of several commercial Atlantic fishes duringstorage at )5 �C. Journal of the Fisheries Research Board of Canada,32, 775–783.

Dingle, J.R., Keith, R.A. & Lall, B. (1977). Protein instability in frozenstorage induced in minced muscle of flat fishes by mixture of muscleof red hake. Canadian Institute of Food Science and TechnologyJournal, 10, 143–146.

Gill, T.A., Keith, R.A. & Smith Lall, B. (1979). Textural deteriorationof red hake and haddock muscle in frozen storage as related tochemical parameters and changes in the myofibrillar proteins.Journal of Food Science, 44, 661–667.

Gupta, M.N., Jain, S. & Roy, I. (2002). Immobilized metal affinitychromatography without chelating ligands: purification of soybeantrypsin inhibitor on zinc alginate beads. Biotechnology Progress, 18,78–81.

Hamm, R. (1979). Delocalization of mitochondrial enzymes duringfreezing and thawing of skeletal muscle. In: Protein at LowTemperatures, Advances in Chemistry Series (edited by O.R.Fennema). p. 191.Washington, DC: American Chemical Society.

Herrera, J.J., Pastoriza, L., Sampedro, G. & Cabo, M. (1999). Effectof various cryostabilizers on the production and reactivity offormaldehyde in frozen-stored minced blue whiting muscle. Journalof Agricultural and Food Chemistry, 47, 2386–2397.

Herrera, J.J., Pastoriza, L. & Sampedro, G. (2000). Inhibition offormaldehyde production in frozen-stored minced blue whiting(Micromesistius poutassou) muscle by cryostabilizers: an approachfrom the glassy state theory. Journal of Agricultural and FoodChemistry, 48, 5256–5262.

Krueger, D.J. & Fennema, O.R. (1989). Effect of chemical additives ontoughening of fillets of frozen Alaska Pollack (Theragra chalco-gramma). Journal of Food Science, 54, 1101–1106.

Morrissey, M.T. & Tan, S.M. (2000). World resources for surimi. In:Surimi and Surimi Seafood (edited by J.W. Park). Pp. 1–21. NewYork, NY: Marcel Dekker.

Moral, A., Tejada, M. & Borderias, A.J. (1986). Stability of frozentrout I. Treated and untreated minces stored at )12, )18, and)24 �C. Journal of Food Biochemistry, 10, 37–46.

Nielsen, M.K. & Jorgensen, B.M. (2004). Quantitative relationshipbetween trimethylamine oxide aldolase activity and formaldehydeaccumulation in white muscle from gadiform fish duringfrozen storage. Journal of Agricultural and Food Chemistry, 52,3814–3822.

Noguchi, B. (1984). Characteristic of fish meat protein in processing.In: Science of Food Protein (edited by F. Yamauhi). Pp. 195–208.Tokyo: Syokuhin Shizai Kenkykai.

Parkin, K.L. & Hultin, H.O. (1982). Some facts influencing theproduction of dimethylamine and formaldehyde in minced andintact red hake muscle. Journal of Food Processing and Preservation,6, 73–97.

Parkin, K.L. & Hultin, H.O. (1986). Characterization of trimethyla-mine-N-oxide (TMAO) demethylase activity from fish musclemicrosomes. Journal of Biochemistry, 100, 77–86.

Phillippy, B.Q. & Hultin, H.O. (1993). Distribution and somecharacteristics of trimethylamine-N-oxide (TMAO) demethylaseactivity of red hake muscle. Journal of Food Biochemistry, 17,235–250.

Racicot, L.D., Lundstrom, R.C., Wilhelm, K.A., Ravesi, E.M. &Licciardello, J.J. (1984). Effect of oxidizing and reducing agents ontrimethylamine oxide demethylase activity in red hake muscle.Journal of Agricultural and Food Chemistry, 32, 459–464.

Robinson, H.W. & Hodgen, C.G. (1940). The biuret reaction in thedetermination of serum protein. I. A study of the conditionnecessary for the production of the stable color which bears aquantitative relationship to the protein concentration. Journal ofBiological Chemistry, 135, 707–725.

Shenouda, S.Y.K. (1980). Theories of protein denaturation duringfrozen storage of fish flesh. In: Advance in Food Research (edited byC.O. Chichester). p. 275. New York, NY: Academic Press.

Sikorski, Z.E. & Kostuch, S. (1982). Trimethylamine-N-oxidedemethylase: its occurrence, properties, and role in technologicalchanges in frozen fish. Food Chemistry, 9, 213–222.

Sotelo, G.C., Pineiro, C. & Perez-Martin, R.I. (1995). Denaturation offish proteins during frozen storage: Role of formaldehyde.Zeitschrift fur Lebensmittel-Untersuchung und-Forschung, 200,14–23.

Steel, R.G.D. & Torrie, J.H. (1980). Principles and Procedures ofStatistics: A Biometrical Approach. Pp. 862. New York, NY:McGraw-Hill.

Tejada, M., Careche, M., Torrejon, P. et al. (1996). Protein extractsand aggregates forming in minced cod (Gados morhua) duringfrozen storage. Journal of Agricultural and Food Chemistry, 44,3308–3314.

Tejada, M., Mohamed, G.F. & Huidobro, A. (2002). Addition ofsardine to hake minces and subsequent effect on dimethylamine andformaldehyde formation. Journal of the Science of Food andAgriculture, 82, 351–359.



Tejada, M., Huidobro, A. & Mohamed, G.F. (2003). Comparison ofgilthead sea bream (Sparus aurata) and hake (Merluccius merluccius)muscle proteins during iced and frozen storage. Journal of theScience of Food and Agriculture, 83, 113–122.

Whistler, R. & Daniel, J.R. (1990). Functional of polysaccharides infoods. In: Food Additives (edited by A.R. Branen, P.M. Davidson &S. Salminen). Pp. 395–423. New York, NY: Marcel Dekker.

Wong, D.W.D. (1989). Mechanism and Theory in Food Chemistry. Pp.129–143. New York, NY: Van Nostrand Reinhold.

Yamada, K. & Amano, K. (1965). Studies on the biological formationof formaldehyde and dimethylamine in fish and shellfish. VII. Effectof methylene blue on the enzymatic formation of formaldehyde anddimethylamine from trimethylamine oxide. Bulletin of the JapaneseSociety of Scientific Fisheries, 31, 1030–1037.

Yasui, A. & Lim, P.Y. (1987). Changes in chemical and physicalproperties of lizardfish meat during iced and frozen storage. NipponShokuhin Kogyo Gakkaishi, 34, 54–60.



Documents

effect of some additives.pdf