8Aggregation of minced hake during frozen storage

7/28/2019 8Aggregation of minced hake during frozen storage

1/6

Eur Food Res Technol (1999) 209 :209214 Q Springer-Verlag 1999

ORIGINAL PAPER

Purificacin Torrejn 7 Mara Luisa del MazoMargarita Tejada 7 Mercedes Careche

Aggregation of minced hake during frozen storage

Received: 17 November 1998

M. Careche (Y) 7 M.L. del Mazo 7 M. Tejada 7 P. TorrejnInstituto del Fro (CSIC), Ciudad Universitaria s/n,E-28040 Madrid, Spain

Abstract Aggregation in minced hake muscle (Merluc-cius merluccius) during storage at 20 7C was studied inconditions where there is progressive deterioration offunctionality and texture as measured by apparent vis-cosity and shear resistance. Natural actomyosin was ex-tracted with 0.6 M NaCl over a period of 49 weeks. In-soluble residue was extracted successively with 2% so-dium dodecyl sulphate (SDS) and 2% SDS plus 5% b-mercaptoethanol (ME) solutions. SDSpolyacrylamidegel electrophoresis was performed on the extractedfractions. The results showed a 75% decrease in 0.6 MNaCl extractability by the end of the storage period. In-itially the remaining precipitate was all extracted in 2%

SDS and although the amount extracted in this solutionincreased as storage time progressed, its proportion de-creased, accounting for as little as 4050% of the pro-tein aggregate by the end of storage. The proteins mostinvolved in formation of the aggregate not extracted in0.6 M NaCl were myosin and actin. Neither of theseproteins was fully recovered in the fractions extractedwith 0.6 M NaCl, 2% SDS, or 2% SDS plus 5% ME,and therefore it was inferred that they were formingpart of the aggregates, bound by covalent bonds.

Key words Hake 7 Mince 7 Frozen storage 7

Aggregation 7 Actomyosin

Introduction

Hake is a valuable species for the Spanish market, ac-counting for 27% of all fish consumed. Frozen products

based on minced fish are becoming very popular. How-

ever, minced hake suffers functional and textural dete-rioration during prolonged frozen storage [1, 2]; shearresistance values can increase up to 2.5-fold over ayears storage at 18 7C [2], which limits practical stor-age life. These changes are attributed largely to denatu-ration and or aggregation of myofibrillar proteins. Inorder to overcome these problems it is desirable to de-termine the protein deterioration occurring in the fleshof this fish species.

The importance of secondary interactions and coval-ent bonds in the aggregation of these proteins has beendescribed for several different species and conditions

[313]. Myosin and actin are the proteins that are mostlikely to be unextractable, not only in salt but also insolutions that rupture secondary interactions and disul-phide bonds [9, 1114]. However, the importance ofeach bond type and protein involved probably dependson differences in species and storage conditions.

In minced hake, formaldehyde (FA) is thought toplay a role in the aggregation of myofibrillar proteins,since with conditions that inhibit formation of FA andincrease lipid oxidation a slower rate of deteriorationhas been found [2], whereas in non-FA forming speciesthe presence of oxidised lipids leads to a decrease in

functionality [15]. The effect of FA has been studiedwith isolated natural actomyosin (NAM) and it hasbeen shown that the degree of insolubilisation, the typeof aggregation (as measured by the extractability inagents that break secondary interactions and disulphidebonds), and the involvement of myosin in the forma-tion of covalent bonds can all be species dependent [16,17]. In hake NAM, the effect of FA is very pronounced,rendering a non-extractable residue in sodium dodecylsulphate (SDS) plus b-mercaptoethanol (ME) [16, 18]or in urea plus ME [19] after several weeks of frozenstorage.

This work examines aggregation in minced hakemuscle, a FA-forming species, during frozen storage at20 7C, a temperature where there is progressive deteri-oration of functionality and texture.


2/6

210

Materials and methods

Fish source. Hake (Merluccius merluccius L.) in early post-rigorcondition was purchased from a local supplier in March 1994. Themean length and weight of the fish after gutting were81.7B1.7 cm and 2.0B0.5 kg, respectively. A total of six individu-al fish was used. The fish were headed, gutted and washed with

iced water to remove blood, etc. The fish were filleted and thefillets minced in a Baader model 694 apparatus using a drum with3 mm diameter holes. The final temperature of the mince was4 7C. The mince was packaged in trays, each containing approxi-mately 600 g, and immediately frozen in a horizontal plate freezeruntil the thermal centre reached 20 7C. Fish blocks were cut, va-cuum packed in Cryovac BB-1 bags (10.66 KPa) and stored at20 7C for up to 49 weeks. Analyses were performed at 0, 5, 8, 14,22 and 49 weeks. The mincing yield was 65.5% of input. For eachcontrol, minced samples were taken out, thawed (if this was re-quired for the technique), and triplicate analyses were performed,unless stated otherwise.

Proximate analyses and pH. Crude protein content was analysedby the Kjeldahl method [20] using a conversion factor of 6.25 [21].

Crude fat was measured [22, 23]. Moisture and ash were mea-sured by AOAC recommended methods [20]. The results wereexpressed as percentage of muscle. The pH was determined [24]at room temperature.

Apparent viscosity. This was determined in a homogenate ofmuscle in 5% NaCl, pH 7 (1: 4) (w:v) [25] using a Rotary Viscom-eter Brookfield Mod LVTD (Brookfield Engineering Labs,Stoughton, Mass., USA). Results were expressed in centipoise(cP).

Shear resistance. The samples were thawed for 15 h at 4 7C,packed into cylindrical stainless steel containers (ht 30 mm!dia30 mm) and then heated at 100 7C for 5 min in a water bath. Thecontainers were hermetically sealed with screw-fitting tops andbottoms, and each contained approximately 25 g of muscle. Thesamples in the containers were cooled for 30 min in ice water thenkept refrigerated until the measurements were made. These sam-ples were cut into slices of 15 mm thickness. Each determinationwas performed on two slices, and six determinations were per-formed per sampling time. Determinations were performed on anInstron Universal Testing Machine, model 4501 (Instron Engi-neering, Canton, Mass., USA) fitted with a Kramer shear cell[26], at a maximum force of 5 kN and a speed of 100 mm/min. Theresults were analysed using the Instron Series IX software (Auto-mated Materials Testing System V.5). The maximum load wasmeasured and expressed as newtons per gram.

Dimethylamine. The amine extracts were prepared from 20 g of

muscle extracted with 60 ml of 6% trichloroacetic acid (TCA),homogenised for 1 min in an Omnimixer (Omni International,Waterbury, Conn., USA) at setting 4. One millilitre of benzeneand 1 mL KOH 65% (w/v) were added to 1 ml TCA extract. Thetubes were heated at 60 7C for 10 min and shaken for 2 min. Di-methylamine (DMA) was quantified by gas chromatography [27,28], with some modifications. The gas chromatograph (PerkinElmer 8500, Perkin Elmer, Beaconsfield, UK) was equipped witha glass column (1.75 m!2 mm i.d.) and a flame ionisation detec-tor (FID). The column was packed with 25 cm untreated80100 mesh Chromosorb 103 and 150 cm 4% Carbowax20 Mc0.8% KOH on Carbopack B. After conditioning (10 mlN2!min

1, 210 7C), deionised water (10 ml) was injected severaltimes, as recommended by Supelco Inc. [29]. The operating condi-tions were: initial temperature 115 7C; final temperature 200 7C;

rate of temperature rise 307C!

min

1

; initial hold 5 min; time atfinal temperature 12 min; injection port and detector temperature250 7C; N2 flow rate 25 ml!min

1; FID sensitivity 1010; samplesize 2 mL. Peaks were identified on the basis of sample coinci-dence with the relative retention times of standards (DMA). Peak

areas were corrected by calculating the response factors of thestandards in relation to the internal standard (N-propylamine).Results were expressed as micromoles DMA per gram.

Free Formaldehyde. This was determined by the method of Cas-tell and Smith [30]. The colorimetric reaction was measured bythe method of Nash [31]. Results were expressed as micromolesof formaldehyde per gram.

NAM extraction in 0.6 M NaCl. Samples were thawed and NAMwas extracted with 0.6 M NaCl from 100 g of the mixture [32] asfollows. The mince was chopped, and then washed with 5 volumesof phosphate buffer pH 7.5 (3.38 mM potassium dihydrogen phos-phate/15.5 mM disodium hydrogen phosphate). The mixture wascentrifuged at 5000g for 15 min (05 7C) (RC 5B refrigerated cen-trifuge, Sorvall Instruments, DuPont, Wilmington, Del., USA)and the precipitate was washed twice following the same processas before. The resulting precipitate was homogenised in an Om-nimixer with 3 volumes of 0.8 M NaCl pH 7.5 (3.38 mM potassiumdihydrogen phosphate/15.5 mM disodium hydrogen phosphate)for 3 min at setting 6 in an ice-water bath. The homogenate wastransferred to a beaker and the homogeniser vase was rinsed with2 volumes of the above 0.8 M NaCl solution and added to the

previous protein extract. After standing for 2 h in an ice-waterbath, the protein extract was centrifuged for 20 min at 5000g(05 7C). The supernatant and the precipitate were separated.The precipitate was labelled P1 and kept for further extractions inSDS and ME as described in the next section. The supernatantwas diluted with 10 volumes of cold water (02 7C) and left tostand in ice water for about 20 min until the protein precipitated.The top layer was siphoned off and the rest, containing the pro-tein suspended in water, was centrifuged for 15 min at 5000g(05 7C). Then 3M NaCl (50 mM Tris maleate pH 7.0) was addedto the precipitate to bring the salt concentration up to 0.6 MNaCl. The mixture was filtered through nylon gauze to removeany adhering traces of connective tissue, then dialysed against0.6 M NaCl (50 mM Tris maleate pH 7.0) overnight in a refrigera-tion chamber. This dialysed fraction was labelled S1. One extrac-

tion was performed per sampling time. Protein concentration inthe supernatants was determined by the method of Lowry [33, 34]and verified with the method of Kjeldahl [20]. Results were ex-pressed as grams extracted of actomyosin per gram of total pro-tein content in the muscle.

Extractability of aggregates. The protein not extractable with0.6 M NaCl (P1), which formed during frozen storage, was treatedwith 4 volumes of 2.5% SDS (Merck, Darmstadt, Germany) withmagnetic stirring for 10 min at room temperature. After centri-fuging for 15 min at 5000g a supernatant was collected and thepellet was washed again with 1 volume of 2% SDS and centri-fuged as above. Both supernatants were mixed to give fraction S2.Any remaining precipitate (P2) was treated with 2% SDS plus5% ME as before, to obtain an extracted fraction S3 and in somecases an insoluble precipitate (P3). The purpose of these twosteps was to break down non-covalent bonds and disulphidebonds, respectively. The amount of soluble protein in fractions S2and S3 and in P3 was determined by the method of Kjeldahl [20].Results were expressed as a percentage of P1.

Polyacrylamide gel electrophoresis. All extracted fractions (S1,S2 and S3) were analysed by SDS-PAGE in a Phastsystem hori-zontal apparatus (Pharmacia LKB Biotechnology, Uppsala,Sweden) using 12.5% polyacrylamide gels. Samples were treatedwith 2% SDS, 5% ME and 0.002% bromophenol blue, heated for5 min in a boiling water bath and centrifuged (Sorvall Microspin24S, Sorvall Instruments) at 10000g for 1 min [35]. Aliquots(1 ml) containing 1 mg ml1 protein were applied. Electrophoresis

conditions were 4 mA/gel, 250 V and 3 W. Protein bands werestained with Coomassie brilliant blue (Pharmacia LKB Biotech-nology) [36]. Gels were analysed on an Image Analyzer (BioImage and Visage, Millipore Corporation, Ann Arbor, Mich.,USA). The molecular masses of the main component proteins in


3/6

211

Fig. 1 Apparent viscosity (dotted bars) and shear resistance(striped bars) of minced hake stored at 20 7C. Different letters (a,b, c, d, e) in the same bar type indicate significant differences

the samples were estimated by comparing their mobility with thatof a standard high-molecular-mass protein mix (ferritin, 220 kDasubunit; albumin, 67 kDa; catalase, 60 kDa subunit; lactate dehy-drogenase, 36 kDa subunit; ferritin, 18.5 kDa subunit) (Pharma-cia LKB Biotechnology). Quantitative estimation of myosin hea-vy chain (MHC) and actin (Ac) bands in each of the extractedfractions (S1, S2 and S3) was performed by measurement of theintegrated optical density (IOD), previously checked for linearity.

Results were expressed as IOD per microgram of protein. Elec-trophoretic profiles gave an estimation of qualitative changes andwere used to study the rest of the protein bands appearing in theelectrophoresis gels.

Statistical analysis. One-way analysis of variance was used to de-termine the effect of time of storage. The level of significance wasset at P~0.05. These analyses were performed using the programBMDP7D (BMDP Statistical Software, Los Angeles, Calif.,USA).

Results

Proximate analyses and pH

The values of 17.4B0.2% crude protein, 1.3B0.1%crude fat, 82.6B0.2% moisture, 0.9B0.1% ash andpH 6.67 were within the expected value range for thisspecies [2]. Apparent viscosity decreased significantlyduring frozen storage (Fig. 1). Initial values were com-parable to those reported previously in minced muscleof hake [37], although the observed decrease was lesspronounced in the present case. There was an increasein shear resistance (Fig. 1) with storage time, similar tothat found by Careche and Tejada [38] for minced mus-

cle of hake stored at 18 7C. The initial DMA values(Fig. 2) were similar to those reported previously [37]in minced hake muscle. These values increased duringstorage, although at a lower rate than had previouslybeen found for this species [37]. Free FA content(Fig. 2) also increased during storage. In weeks 22 and49, bound formaldehyde, measured as DMA minus freeFA, was appreciable. Bound FA has been reported toindicate reaction of this compound with fish compo-nents such as proteins or low-molecular-weight sub-stances present in the muscle [39, 40].

Fig. 2 Dimethylamine (striped bars) and free formaldehyde (dot-ted bars) of minced hake stored at 20 7C. Different letters (a, b, c,d, e, f) in the same bar type indicate significant differences

Fig. 3 Extractability of minced hake proteins stored at 207C: anatural actomyosin (NAM) extracted in 0.6 M NaCl (S1); b pro-

tein extracted in 2% SDS (S2), 2% SDS plus 5% b-mercaptoetha-nol (S3) from the aggregate not extracted in 0.6 M NaCl, and anon-extractable fraction (P3)

Extractability in 0.6 M NaCl, 2% SDS and 2% SDSplus 5% ME

Extractability in 0.6 M NaCl decreased gradually dur-ing storage (Fig. 3a) (fraction S1), reaching 25% of the

initial value after 49 weeks. Analyses of the proteinsnot extractable in 0.6 M NaCl were carried out fromtime 0 since 10% of the protein was initially unextract-able. Treatment with 2% SDS (fraction S2) initially ex-tracted all the protein from the fraction not extracted inNaCl (P1), and the amount of protein extracted into S2increased with storage time. However, the proportionof this with respect to the total protein in P1 decreased,so that by the end of the storage period it was 50% ofthe aggregate formed (Fig. 3b). The amount and thepercentage extracted by 2% SDS plus 5% ME from theresidue insoluble in NaCl and 2% SDS (fraction S3)

had increased considerably by the end of storage. Instudies of the solubility of frozen-stored muscle of redhake fillets, Owusu-Ansah and Hultin [11] also foundan increase in the proportion of protein solubilised bySDS plus ME. There is also evidence for this from oth-er gadoid species such as minced and filleted cod mus-cle [7, 12, 13]. The amount and proportion of proteinnot extracted by successive treatments (P3) had in-creased by the end of storage. Increasing non-extracta-ble protein has also been reported in cod mince andfillets at our laboratory [12, 13]; there, the percentageof unextractable protein with respect to aggregate not

extractable in saline solutions (P1) was found to belower than in the present study [12].


4/6

212

Table 1 One-way analyses of variance of the variables listed be-low as a function of time. Different letters in the same row indi-cate significant differences

Weeks

0 5 8 14 22 49

MHC (S1) a b c c c cAc (S1) ab a ab ab b c

MHC (S2) a b ab b a abAc (S2) a b a a a aMHC (S3) P a a b ab abAc (S3) P ab a b c d

Fig. 4 Myosin heavy chain (MHC) () and actin (Ac) ( )obtained from the SDS-PAGE (12.5%) in fractions S1 (}), S2(G) and S3 (L) from minced hake stored at 20 7C

Fig. 5 SDS-PAGE (12.5%) of fractions S1, S2, and S3 fromminced hake extracted at 0, 14 and 49 (S1), 0 and 49 (S2), and 5and 49 (S3) weeks of frozen storage at 20 7C: 1 application zone;2 peak in the stacking/resolving interphase; 3 MHC ; 4 proteins ofmolecular weight between 200 and 45 kDa; 5 actin; 6 tropomyo-sin, troponins and myosin light chains

Protein composition of fractions S1, S2 and S3

Fraction S1

The proportion of IOD of the MHC in the fraction ex-tracted with 0.6 M NaCl (Fig. 4, Table 1) decreased sig-nificantly as storage progressed. Values fell below theinitial levels from week 14 with no subsequent changesthereafter. This decline in the proportion of MHC ex-tracted in saline solution was lower than that of mincedcod stored in the same conditions [12]. The proportionof Ac also decreased in the saline extract (Fig. 4, Ta-ble 1) over the storage period. Ac has been reported tobe more stable than MHC during frozen storage in oth-er species [1114, 41, 42]. In the present experiment,the percentage loss sustained by these two proteins atthe end of the storage period was similar, but they dis-appeared at different rates. The decrease of the totalamount of both MHC and Ac over time was more evi-

dent than the decrease of the proportion of these pro-teins, since extractability of this fraction declined(Fig. 3). The electrophoretic profiles of the NAM ex-tracted in 0.6 M NaCl (Fig. 5) show that during storagethere was a slight increase in the proportion of proteinbands of lower molecular weight than Ac extracted infraction S1. Peaks 1 and 2 correspond to protein aggre-

gates that did not penetrate the gel. Proteins in theseaggregates are believed to be linked by non-disulphidecovalent bonds and have been reported in other species[7, 1113, 43].

Fraction S2

In the fraction extracted with 2% SDS the proportionof MHC (Fig. 4, Table 1) remained virtually unchangedover time. Values were higher than in fraction S1(0.6 M NaCl) for the same storage time. The propor-tion of Ac (Fig. 4) also remained unchanged. Despite alack of increase in the proportion of MHC and Ac, thetotal amount of both protein bands increased in thisfraction during storage. The electrophoretic profile

(Fig. 5) shows that the proportion of peak 1 increasedwith time of storage, indicating the increasing presenceof protein aggregates with non-disulphide covalentbonding in the fractions extracted with SDS. This wasreported for this fraction in cod minces stored at 7 and20 7C [7, 12], and in Greenland halibut [9]. At 49weeks, fewer bands other than MHC and Ac were ap-parent in the resolving gel than at 0 week.

Fraction S3

The proportion of MHC did not differ as a function oftime (Fig. 4, Table 1) and was similar to fraction S2.There was a loss in the proportion of Ac and no otherchanges were observed in the electrophoretic profile


5/6

213

(Fig. 5) as storage progressed. As with fraction S2,there was an increase in the total amount of these twoprotein bands in fraction S3. Constant proportions ofMHC and Ac in fractions S2 and S3 have also beenfound in minced cod stored at 20 7C [42].

Discussion

As these results show, with the passage of time there isa decrease in muscle functionality, an increase in shearresistance, and an increase in DMA and FA, indicatingprogressive deterioration of the minced hake muscleover frozen storage. There is also an increase in theprotein not extracted by 0.6 M NaCl, here named P1. Inthese conditions of functional and textural deteriora-tion during storage, there appears to be an increase inthe quantity of bonds or interactions and/or in the inci-dence of stronger bonds or interactions. This was evi-

denced by the fact that protein in P1 was fully extractedby 2% SDS at the outset of storage but only 50% wasextracted by the end; also the proportion and the totalamount of protein extracted by SDS plus ME in-creased, as did the amount of protein not extracted byeither solution.

The time-dependent decrease in the proportion offraction S2 does not necessarily imply a decrease in theinvolvement of secondary interactions in aggregation;this involvement could have been masked by the for-mation of other types of bonds that were not brokenwith these extraction conditions. The decrease in ex-

tractability in SDS and the increase in SDS plus MEand unextractable protein further suggests that thenumber of disulphide and non-disulphide covalentbonds increases with time, since they were presenteither in sufficient quantity or at given locations to pre-vent extraction of the aggregate other than by usingagents that break such bonds. Previous experiments incod showed that extractability in NaCl, SDS and SDSplus ME is dependent on the storage temperature orthe integrity of the muscle [12, 13]. Comparing the ex-tractability of minced cod [12] and hake, it can be seenthat aggregation, measured as extractability in a series

of agents that cleave secondary interactions and disul-phide bonds, is different in these two cases. In hake,loss of extractability in 0.6 M NaCl occurred earlier butdeveloped more slowly over storage time, so that by theend there was less protein aggregate (P1) than in cod.In both cases, about 50% of protein was not extractedby SDS, but in hake there was more involvement ofnon-disulphide covalent bonding, as evidenced by ahigher percentage of P3 than in cod.

The decrease in the proportion of MHC in fractionS1 implies enrichment of the P1 fraction in MHC.Moreover, while the proportion of MHC in fraction S1

tended to decrease, it remained constant in fractions S2and S3, so that the differences between S1 and S2 or S3increased with storage. This may have been becausepart of the MHC was not quantified owing to its in-

volvement in non-disulphide covalent bonds that wereextracted from the different fractions or because it re-mained in aggregate P3 as an unextractable residue.The decrease in the proportion of actin in S1 duringstorage also implies enrichment of the P1 fraction inthis protein. Again, the excess Ac in P1 was not fullyextracted in S2 or S3 by the end of storage, and there-fore it was not quantified because it formed non-disul-phide covalent bonds, which were extracted in the var-ious fractions, or else it could not be extracted and re-mained to enrich P3. Thus, in minced muscle of hake,both MHC and Ac are involved in both covalent andnon-covalent bonding. Comparison of the changes ob-served in extractability and in the proportion of MHCand Ac in the hake extracts with the findings withminced cod [12] suggests that while in cod myosin is thepredominant protein in P1 and in the formation of non-disulphide covalent bonds [12], in hake both myosinand Ac are involved.

FA causes aggregation of proteins in model systemsof myosin or actomyosin [18, 4446] and in muscle ithas been highly correlated to decreases in functionalproperties and loss of textural attributes [2, 6, 38, 47].In the present experiment we found an increase inDMA and FA formation and a decrease in extractabili-ty as a function of time of frozen storage, corroboratingthe findings of other authors. The effect of FA does notonly depend on the amount formed during frozen stor-age since the action of FA on muscle proteins, as men-tioned earlier, is dependent on species. We have ob-served [16, 18] that hake NAM stored in the presence

of FA at subzero temperatures has the potential of be-ing denatured and heavily aggregated by this agent sothat NAM loses extractability in salt and the aggre-gated protein could only be partially extracted withSDS and SDS plus ME. It has been found that the per-centage of MHC and Ac in the salt-extracted solutionin the absence of FA does not change significantly dur-ing storage [16, 19], whereas when FA is added there isa sharp loss of the MHC band. In this FA-treatedNAM, MHC has been proposed to be involved in theformation of covalent bonds [16, 18, 19]. However, inthe present study the extent to which the loss of MHC

is due to the presence of FA remains to be deter-mined.

This paper provides further information on the ag-gregation of fish myofibrillar proteins and shows thatunder conditions where there is progressive loss offunctionality and texture, the aggregation pattern ofminced hake muscle proteins as measured by the ex-tractability in NaCl, SDS and SDS plus ME solutionschanges with time of storage. Both myosin and actinparticipate in the aggregation to a similar extent by theend of the storage, but at different rates. Althoughthese results differ from previous findings for minced

cod studied under similar conditions [12], we do not yetknow to what extent the quantitative and qualitativedifferences in the aggregation pattern and in the pro-teins involved are due to species differences or to other


6/6

214

factors, including those relating to the physiologicalchanges induced by the biological state of the fish.These factors will need to be studied systematically toestablish a broader picture of cold-induced aggrega-tion.

Acknowledgements Thanks are due to Dr. Almudena Huidobro

for assistance with the dimethylamine assays and the statisticalanalysis. This work was financed by the Spanish ALI 94-0954-C02-01 and EU project FAIR-CT95-1111.

References

1. Jimnez Colmenero F, Tejada M, Borderas AJ (1988) J FoodBiochem 12:159170

2. Careche M, Tejada M (1990) Food Chem 36 :1131283. Connell JJ (1965) J Sci Food Agric 16:7697834. Connell JJ (1975) J Sci Food Agric 26: 192519295. Dingle JR, Keith RA, Lall B (1977) Can Inst Food Sci Tech-

nol J 10:143146

6. Gill TA, Keith RA, Smith Lall B (1979) J Food Sci44:661667

7. Matthews AD, Park GR, Anderson EM (1980) Evidence forthe formation of covalent cross linked myosin in frozen-stored cod minces. In: Connell JJ, Staff of Torry ResearchStation (eds) Advances in fish science and technology. Fish-ing News Books Ltd, Surrey, UK, pp 438444

8. Laird WM, Mackie IM, Hattula T (1980) Studies of thechanges in the proteins of cod-frame minces during frozenstorage atP15 7C. In: Connel JJ, Staff of Torry Research Sta-tion (eds) Advances in fish science and technology. FishingNews Books Ltd, Surrey, UK, pp 428434

9. Lim H, Haard NF (1984) J Food Biochem 8: 16318710. Rehbein H, Karl H (1985) Z Lebensm Unters Forsch

180:373378

11. Owusu-Ansah YJ, Hultin HO (1986) J Food Sci51:14021406

12. Tejada M, Careche M, Torrejn P, Del Mazo ML, Solas MT,Garca ML, Barba C (1996) J Agric Food Chem44:33083314

13. Careche M, Del Mazo ML, Torrejn P, Tejada M (1998) JAgric Food Chem 46:15391546

14. LeBlanc EL, LeBlanc RJ (1989) J Food Sci 54 :82783415. Careche M, Tejada M (1990) Food Chem 37 :27528716. Tejada M, Torrejn P, Del Mazo ML, Careche M (1997) Sea-

food from producer to consumer, integrated approach toquality. In: Luten JB, Borresen T, Oehlenschlger J (eds)Proceedings of the International Seafood Conference on theoccasion of the 25th anniversary of the WEFTA, Noordwij-kerhout, The Netherlands, 1995. (Developments in FoodScience, vol 38) Elsevier Science, Amsterdam, pp 265280

17. Careche M, Cofrades S, Carballo J, Jimnez Colmenero F(1998b) J Agric Food Chem 46:813819

18. Del Mazo ML, Huidobro A, Torrejn P, Tejada M, CarecheM (1994) Z Lebensm Unters Forsch 198:459464

19. Cofrades S (1994) Funcionalidad y caractersticas fisico-qum-icas de actomiosina procedente de distintas especies. Influen-cia del estado congelado. PhD thesis, Facultad de Farmacia,Universidad Complutense de Madrid

20. AOAC (1984) Official Methods of Analysis, 14th edn. Asso-ciation of Official Analytical Chemists, Washington DC

21. Lillevik HA (1970) The determination of total organic nitro-gen. In: Joslyn MA (ed) Methods in food analysis. Physical

chemical and instrumental methods of analysis. AcademicPress, New York22. Blight EG, Dyer WJ (1959) Can J Biochem Phys

37:91191723. Knudsen I, Reimers K, Berner L, Jensen NC (1985) A modi-

fication of Bligh and Dyers oil extraction method reducingchloroform vapour outlet. O.E.A./II Western European FishTechnologists Association (WEFTA), Hamburg, Germany

24. Vyncke W (1981) pH of fish muscle: comparison of methods.Western European Fish Technologist Association (WEF-TA), Copenhagen, Denmark

25. Borderas AJ, Jimnez Colmenero F, Tejada M (1985) Ma-rine Fish Rev 47:4345

26. Kramer A, Burkardt GJ, Rogers HP (1951) Canner 112: 3427. Lundstrom RC, Racicot LD (1983) J Assoc Off Anal Chem

66:1158116328. Prez-Martn RI, Franco JM, Molist P, Gallardo (1987) Int J

Food Sci Technol 22:50951429. Supelco (1971) Amine analysis. Bulletin 737D, Superco Inc,

Bellaforte. PA, USA30. Castell CH, Smith B (1973) J Fish Bd Can 30: 919831. Nash T (1953) Biochem J 55: 41642132. Kawashima T, Arai K, Saito T (1973) Bull Jpn Soc Sci Fish

39:20721433. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) J

Biol Chem 193:26527534. Peterson GL (1979) Anal Biochem 100:20122035. Hames BD (1985) One-dimensional polyacrylamide gel elec-

trophoresis. In: Hames BD, Rickwood D (eds) Gel electro-phoresis of proteins. A practical approach. IRL Press, Ox-

ford, pp 19136. Phast-System Users Manual (1990) Pharmacia LKB Biotech-

nology AB, Uppsala, Sweden37. Tejada M, Careche M (1988) Rev Agroqum Tecnol Aliment

28:10911438. Careche M, Tejada M (1991) Z Lebensm Unters Forsch

193:53353739. Banda MCM, Hultin HO (1983) J Food Proc Preserv

7:22123640. Owusu-Ansah YJ, Hultin HO (1987) J Food Biochem

11:173941. Connell JJ (1960) Biochem J 75: 53042. Connell JJ (1969) J Sci Food Agric 11: 51552243. Ragnarsson K, Regenstein JM (1989) J Food Sci 54: 81982344. Torrejn P (1996) Agregacin de protenas de msculo de

pescado congelado. Accin del formaldehdo. PhD thesis,Universidad Complutense de Madrid, Spain

45. Ang JF, Hultin HO (1989) J Food Sci 54:81481846. Careche M, Li-Chan ECY (1997) J Food Sci 62 :71772347. Tokunaga T (1980) Bull Tokai Reg Res Lab Jpn 101: 1129

8Aggregation of minced hake during frozen storage

Documents

Transcript of 8Aggregation of minced hake during frozen storage