1 The Relatinship Between Acid and Neutral Prtease Activities and the ncidence f Sft Cked Texture in the Muscle Tissue f Pacific Hake Merluccius Prductus nfected with Kuda Panifrmis and/r K. Thyrsitis, and Held fr Varying. Times Under Different Pre-Freeze Chilled Strage Cnditins. H. Tsuyuki and S.N. Williscrft Department f Fisheries and Oceans Technlgy Services Branch 6640 N.W. Marine Drive Vancuver, British Clumbia, Canada V6T 1 X2 and Z. Kabata and D.J. Whitaker Department f Fisheries and Oceans Pacific Bilgical Statin Fisheries Research Branch Nanaim, British Clumbia, Canada V9R 5K6 August 1982 Fisheries and Aquatic Sciences Canadian Technical Reprt N.1130
2 Canadian Technical Reprt f Fisheries and Aquatic Sciences These reprts cntain scientific and technical infrmatin that represents an imprtant cntributin t existing knwledge but which fr sme reasn may nt be apprpriate fr primary scientific (i.e. Jurnal) publicatin. Technical Reprts are directed primarily twards a wrldwide audience and have an internatinal distributin. N restrictin is placed n subject matter and the series reflects the brad interests and plicies f the Department f Fisheries and Oceans, namely, fisheries management, technlgy and develpment, cean sciences, and aquatic envirnments relevant t Canada. Technical Reprts may be cited as full publicatins. The crrect citatin appears abve the abstract f each reprt. Each reprt will be abstracted in AquatiC Sciences and Fisheries Abstracts and will be indexed annually in the Department's index t scientific and technical publicatins. Numbers in this series were issued as Technical Reprts f the Fisheries Research Bard f Canada. Numbers were issued as Department f the Envirnment, Fisheries and Marine Service, Research and Develpment Directrate Technical Reprts. Numbers were issued as Department f Fisheries and the Envirnment, Fisheries and Marine Service Technical Reprts. The current series name was changed with reprt number 925. Details n the availability f Technical Reprts in hard cpy may be btained frm the issuing establishment indicated n the frnt cver. Rapprt technique canadien des sciences halieutiques et aquatiques Ces rapprts cntiennent des renseignements scientifiques et techniques qui cnstituent une cntributin imprtante aux cnnaissances actuelles mais qui, pur une raisn u pur une autre, ne semblent pas apprpries pur la publicatin dans un jurnal scientifique. n'y a aucune restrictin quant au sujet, de fait, la serie reflete la vaste gamme des interets et des plitiques du Ministere des Peches et des Oceans, ntamment gestin des peches, techniques et develppement, sciences ceaniques et envirnnements aquatiques, au Canada. Les Rapprts techniques peuvent etre cnsideres cmme des publicatins cmpletes. Le titre exact paraltra au haut du resume de chaque rapprt, qui sera publie dans la revue Aquatic Sciences and Fisheries Abslracts et qui figurera dans 'index annuel des publicatins scientifiques et techniques du Ministere. Les numers de cette serie nt ete publies it titre de Rapprts techniques de 'Office des recherches sur les pecheries du Canada. Les numers , it titre de Rapprts techniques de la Directin generaje de la recherche et du develppement, Service des peches et de la mer, ministere de l'envirnnement. Les numers nt ete publies it titre de Rapprts techniques du Service des peches et de la mer, Ministere des Peches et de 'Envirnnement. Le nm de la serie a ete mdifie it partir du numer 925. ; La page cuverture prte e nm de 'etablissement auteur u 'n peut se prcurer les rapprts sus cuverture cartnnee. t
3 - i - Canadian Technical Reprt f Fisheries and Aquatic Sciences N August 1982 The Relatinship between Acid and Neutral Prtease Activities and the ncidence f Sft Cked Texture in the Muscle Tissue f Pacific Hake (Merluccius prductus) infected with Kuda panifrmis and/r ~ thyrsitis, and Held fr Varying Times under Different Pre-freeze Chilled Strage Cnditins. by H. Tsuyuki and S.N. Williscrft Department f Fisheries and Oceans Technlgy Services Branch 6640 N.W. Marine Drive Vancuver, B.C. V6T 1X2 and z. Kabata and D.J. Whitaker Department f Fisheries and Oceans Fisheries Research Branch Pacific Bilgical Statin Nanaim, B.C. V9R 5K6
4 - ii - Minister f Supply and Services Canada 1982 Cat. N. Fs 97-6/1130 rssn
5 iii TABLE OF CONTENTS Page N. List f Tables iv list f Figures v Abstract v; Re s ume vii i ntrductin 1 Materials and Methds 3 Parasite Evaluatin 4 Prtease Activity 5 Cking Methd 5 Texture... 6 Results and Discussin 6. Parasite nfectin Prtease Activity temperat ure and ph effects 8. Strait f Gergia Ha ke A) Prtease Activity ll V. C) West B) Cked Flesh Texture A) B) Relatinship f nfectin, Cast Hake Prtease Activity Cked Flesh Texture Prtease Activity and Texture C) Relatinship f nfectin, Prtease Activity and Texture.14 V. Effect f Pst-Mrtem Chilled Strage Time 15 A c k n wl e d 9 erne n t s. 16 References 17
6 - i v - LST OF TABLES Page N. Tab 1 e Pacific Hake Landings (tnnes) Table Table Prevalence f Kuda infectins in hake ff the west cast f Vancuver sland 21 Prevalence f Kuda infectins in hake ff the west cast f Vancuver sland 22 Table V Table V Prevalence f Kuda thyrsitis in Gergia Strait hake 23 Prevalence f Kuda thyrsitis in Gergia Strait hake. 24 Table V Prtease(s) activities and textures relating t infectin 25
7 - v - LST OF FGURES Page N. Figure 1- Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Sampling 10catins 26 ph ph C (nn-infected} C (nn-infected} C and 60 C (infected} C and 60 C (infected} 32 Firmness/pst mrtem chill time n ice 33 Acid prtease/pst mrtem chill time n ice 34 Neutral prtease/pst mrtem chill time n ice 35 Firmness/pst mrtem chill time in RSW 36 Acid prtease/pst mrtem chill time in RSW 37 Neutral prtease/pst mrtem chill time in RSW 38 Prtease(s}/pst mrtem chill time n ice and in RSW 39
8 - vi - ABSTRACT Tsuyuki, H., S.N. Williscrft, Z. Kabata, and D.J. Whitaker The relatinship between acid and neutral prtease activities and the incidence f sft cked texture in the muscle tissue f Pacific hake (Merluccius prductus) infected with Kuda ~anifrmis and/r ~ thyrsitis, and held fr varying times un er different pre-freeze chilled strage cnditins. Fish. Mar. Serv Tech. Rep. N One f the mre intractable quality prblems assciated with Pacific hake is the relatively high incidence f develpment f sft texture during the cking prcess. The assciatin f this texture prblem with prtelytic activity r with the presence f parasite has nt been clearly understd despite lng years f study. n this investigatin strng prtelytic activities have been demnstrated in the acid and neutral ph ranges. The prtelytic activity in the acid range has been fund t increase greatly in muscle tissues f Pacific hake infected with Kuda thyrsitis frm bth the Strait f Gergia and the west cast f Vancuver sland. This enzyme(s) is heat labile and des nt appear t degrade thse structural prteins respnsible fr maintaining firm texture after cking. Pacific hake which are uninfected and thse infected with ~ thyrsitis d nt result in unacceptably sft cked flesh texture. Muscle tissues exhibiting strng prtelytic activity in the neutral ph range develped unacceptably sft r mushy texture during the slw cking prcess. The strng neutral prtease activity was assciated with muscle tissues infected nly with the white pseudcysts r the yung frms f ~ panifrmis either alne r as a mixed infectin with ~ thyrsitis. The enzyme(s) was fund t be relatively heat stable with an activity ptimum at C, a transitinal temperature in the cking prcess. The parasite ~ panifrmisis fund nly in hake frm the west cast, indicating insignificant mixing f the tw ppulatins. The develpment f black pseudcysts in lder infectins by either Kuda species is cnsidered a defence mechanism f the muscle tissues. This prcess depsits melanin granules which eventually lead t the destructin f the parasite and the assciated prtease activity. While acid as well as neutral prtease activities in the muscle tissues
9 - vi i - increase directly with the intensity f the yunger frm f infectin with ~ panifrmis. increased activity in the acid ph range can be caused by infectin with K. thyrsitis alne. Prtelytic activity measured in the acid range therefre is nt necessarily assciated with the develpment f sft texture. Failure t distinguish nt nly the difference between the tw parasites but als their age will lead t incnsistencies in relating the develpment f sft texture t the presence f parasite. Hake stred with gd icing and RSW practices maintain quality fr up t 5 days. Subsequent develpment f sft texture after slw cking is related primarily t the presence f the parasite ~ panifrmis.
10 - viii - - ~S~E Tsuyuki, H., S.N. Williscrft, Z. Kabata, and D.J. Whitaker La relatin entre l'activite prtelytique en milieu acide et neutre et l'incidence d'une texture mlle a l'etat cuit, dans le tissu musculaire de la merluche du Pacifique (Merluccius prductus) infectee de Kuda panifrmis et/u K. thyrsitis, et gardee pendant des perides variables sus differentes ~nditins d'entrepsage a frid avant la cngelatin. Fish. Mar. Ser.Tech. Rep. N L'un des prblemes les plus difficiles a resudre quant a la qualite de la merluche du Pacifique est celui de l'incidence relativement elevee de chair a texture mlle au curs de la cuissn. Malgre de lngues annees d'etudes l'assciatin de ce prbleme de texture a l'activite prtelytique u a la presence de parasites n'a pas ete cmprise clairement. Dans cette etude n a demntre des activites prtelytiques prnncees au ph acide u neutre. On a truve que l'activite prtelytique dans le milieu acide augmentait frtement dans les tissus musculaires de la merluche du Pacifique infectee par le Kuda thryrsitis, autant dans celie pechee dans le detrit de Gergia que celie prise sur la cte uest de l'lle de Vancuver. Cet enzyme(s) est labile a la chaleur et ne semble pas degrader les prteines structurelles respnsables de la preservatin de la texture ferme apres CUlssn. La merluche du Pacifique, qu'elle s it infectee u nn de ~ thyrsitis, ne prduit pas de chair qui sit mlle au pint d'etre inacceptable apres cuissn. Les tissus musculaires demntrant une frte activite prtelytique en milieu a ph neutre develppent une texture mlle u en bullie au curs du prcessus a cuissn lente. La frte activite prtelytique en milieu neutre etait assciee uniquement aux tissus musculaires infectes par les pseudcystes blancs u aux frmes jeunes de ~ panifrmis, sit seule, sit en infectin cmbinee avec K. thyrsitis. L'enzyme(s) s'est mntre relativement stable a la chaleur avec une activite ptimale a C, crrespndant a la temperature transitire durant le prcessus de CUlssn. Le parasite ~ panifrmis se truve seulement chez la merluche de la cte uest, indicant un melange insignifiant des deux ppulatins. develppement de pseudcystes nirs dans les infectins plus anciennes par chacune des especes Kuda est cnsidere cmme un mecanisme de defence du tissu musculaire. Ce prcessus cnduit a la depsitin de granules de melanine qu Le
11 - i x - causent eventuellement la destructin du parasite et l'activite prtelytique assciee. Tandis que l'activite prtelytique, tant acide que neutre, dans le tissu musculaire augmente directement avec l'intensite le l'infectin par les frmes jeunes de ~ panifrmis, une activite accrue dans le milieu a ph acide peut etre causee par une infectin de ~ thyrsitis uniquement. Pur cela l'activite prtelytique mesuree en milieu a ph acide n'est pas frcement assciee au develppement de texture mlle. Le defaut d'etablir une distinctin, nn seulement entre les deux parasites, mais egalement entre leur age, cnduira a des incnsistances reliant le develppement de texture mlle a la presence de parasites. La merluche entrepsee de maniere apprpriee dans la glace u dans l'eau de mer refrigeree maintient sa qualite jusqu'a 5 jurs. Le develppement subsequent de texture mlle apres cuissn lente est principalement relie a la presence du parasite K. panifrmis.,
12 - 1 - NTRODUCTON Early interest in Pacific hake (Merluccius prductus) centered arund the ptentiality f develping an industry based n prductin f fish meal and pet fd. A hake meal plant came int peratin in 1966 but clsed shrtly thereafter in 1967 (Dassw and Beardsley, 1973). n a 1977 survey by bttm trawl and hydracustic mid-water trawl in the NPFC statistical areas, the resurce has been estimated t be in excess f ne millin tnnes (Dark et al. 1980). The stcks in the Strait f Gergia have been reprted t be abut 100,000 tnnes (Thmpsn and McFarlane, 1982). The landings ver the past decade in the NPFC statistical areas and ff the cast f British Clumbia are shwn in Table. The flesh f this species, hwever, is generally regarded t be f lw quality with pr keeping characteristics. Nevertheless the magnitude f the resurce stimulated interest since the middle 1960's in increasing utilizatin, particularly as human fd. Sme assessment studies were carried ut n the quality f flesh under chilled strage cnditins (Dassw et ale 1970) and n the stability and desirability characteristics f fillets and minced flesh blcks (Crawfrd et ale 1972a,b, 1979). Cmpsitinal analyses f hake frm Puget Sund and the west cast f the United States were carried ut (Patashnik et ale 1970). The develpment f xidative rancidity in the subcutaneus fatty layer is ne f the quality prblems encuntered during frzen strage. Quality lss f this nature can be minimized by the applicatin f gd glazing practices and lw frzen strage temperature f -29 C (Dassw et ale 1970) and by the use f antixidants (Crawfrd et ale 1972a). Hwever, the mre intractable prblem impeding wider utilizatin lies in the significantly high incidence f develpment f mushy texture in slw cked fillets (Dassw et ale 1970; Patashnik et ale 1982). Varius reprts have indicated that the prtelytic activity respnsible fr the autlysis can be destryed by rapid cking (Dassw et ale 1970) such as by micrwave (Patashnik et ale 1982) r by deep frying in fat (Crawfrd et ale 1972a,b, 1979; Patashnik et ale 1982). Cmmn xidizing agents such as hydrgen perxide and ptassium brmate have been reprted t inhibit prtelysis (Spinelli and Steinberg, 1978) but the methd has nt been used cmmercially. The relatinship between pre-freeze chilled strage time and qual ity has nt been established with certainty. There appears, hwever, t be general
13 - 2 - agreement that thrugh icing r strage f hake in refrigerated seawater immediately after capture is essential, as well as rapid prcessing t allw freezing within several hurs (Dassw and Beardsley, 1973, Dassw et ale 1970; Miyauchi, 1977; Swansn and Ridenhur, 1978). The develpment f mushy texture in ven baked fillets frm hake caught in the ffshre waters f Washingtn and Oregn has been reprted t he assciated in sme way with the presence f myxsprean parasite in the flesh (Dassw et ale 1970). Hwever even very recently it has been reprted that the severity f parasitizatin in fish as determined by visual presence f parasitic cysts may nt crrelate with the degree f sftening f cked flesh (Patashnik et ale 1982). The same reprt als shws that the mst severely parasitized fish fillets culd have bth high and lw prtelytic activity as measured with hemglbin substrate under acidic cnditins. Cnvers~y, high prtelytic activity was shwn in muscle tissues frm fillets shwing nly trace infectin. Clearly, cnclusive evidence has nt yet been established t relate the develpment f unacceptably sft texture after cking with either the degree f parasite infestatin r with the extent f prtelytic activity under the cnditins measured. This study was undertaken t examine bth the characteristics f the prteases invlved in prtelytic activity and the cntributin f parasites present in the flesh tward the develpment f mushy texture in cked flesh f Pacific hake. The identificatin and prevalence f parasites in hake frm the Strait f Gergia and the west cast f Vancuver sland was carried ut at the Pacific Bilgical Statin. The parasitlgical investigatin and the prtein and texture studies were cnducted n the same fish t prvide direct relatinship between mushy texture develpment and the presence f a specific parasite. Frm this study it has already been revealed that the musculature f Pacific hake is infected with tw species f Myxsprea, Kuda thyrsitis and a previusly unknwn species which the authrs named Kuda panifrmis (Kabata and Whitaker, 1981). The new species was specific nly t specimens btained frm the west cast f Vancuver sland.
14 - 3 - MATERALS AND METHODS Fish used in the study were frm tw areas f suthern British Clumbia, the west cast f Vancuver sland and the Strait f Gergia (Fig. 1). The west cast r "ffshre" sample was btained in midwater trawl by M/T Callistratus in 60 fathms in September, 1980, rughly fifty kilmeters suth f Cape Beale (48 30'O"N. x 'O"W.). Within ne hur f capture 35 fish were filleted and the fillets plate frzen immediately and stred at -28 C t serve as "zer time" samples. A ttal f 160 fish was immediately chilled n ice; an additinal 160 fish were placed in refrigerated seawater (R.S.W.) at a temperature f -1.5 t O C. At intervals f frm 4 t 120 hurs pst mrtem, 25 fish were remved frm each f the tw chilled strage systems and fi 11 eted. The fi 11 ets were immedi ately pl ate frzen and stred at -28 C. One f the f"illets frm each fish was examined by the Pacific Bilgical Statin in Nanaim fr the presence f prtzan parasites. secnd fillet was subsequently assayed fr autlytic prtease activity and the cked texture was determined. The inter-relatinships which existed between the degree f infectin by Kuda, the autlytic activity, and the cked texture f the fillets were then examined. Tw separate samples f hake were btained frm the Strait f Gergia. The first, in March 1980, was btained by midwater trawl in 198 fathms by M/V Ocean King in lwer Gergia Strait (49 13'l"N. x 'O"W.). A ttal f fifty fish was immediately iced and at varius pst mrtem times f up t 3 days, lts f ten fish were remved, plate frzen and stred at -28 C. N parasitlgical r prtease evaluatins were cnducted n these samples but the textures f the cked flesh were determined. A secnd sample f 100 hake frm the Strait f Gergia was btained in January, 1981, by mi dwater trawl in 60 fathms by M/V Sharl ene K. apprximately 27 km suth f Savary sland (49 47'12"N. x 'O"W.). All fish were iced immediately and after 2 days apprximately half f the ttal was filleted, the fillet frm ne half examined fr parasites by the Pacific Bilgical Statin and the ther half frzen and held at -28 C fr the determinatin f prtelytic activity. The remaining iced fish were handled and prepared in an identical manner after 3 days icing. Additinally, the cked textures f all fish were determined. The
15 - 4 - Parasite Evaluatin Fr the purpse f ascertaining the prevalence and intensity f Kuda infectins, each fillet was divided hrizntally alng the lateral line and vertically int three apprximately equal parts, s that six areas were identified: area anterir and abve the lateral line, area belw it, area in the middle and abve the lateral line, area V belw it, area V psterir and abve the lateral line, area V belw it. Fillets were examined under dissectin micrscpe, directly after defrsting. Each area was macerated, several fibres at a time, and all abnrmal fibres were checked under cmpund micrscpe, at x900 magnificatin, with the aid f phase cntrast illuminatin. Numbers f infected fibres in each area were recrded separately. Presence f 20 r fewer such fibres was arbitrarily classified as light infectin, between 21 and 100 medium infectin and ver 100 heavy infectin. Spres were studied with the aid f light micrscpe and with scanning electrn micrscpe. Methds used in that study are described elsewhere (Kabata and Whitaker, 1981). Fr the purpse f ascertaining an verall average infectin level fr each fillet, a mean infectin density (MD) was calculated frm the data reflecting the intensity f parasite infectin in the six sub-sampled areas. An example f its calculatin fllws: Each f the six areas (see PARASTE EVALUATON) f each fillet was designated t be infected t a light, medium r heavy degree, if at all. L light infectin 20 r fewer infected fibres M = medi um " = between 20 and 100 infected fi bres H = heavy " mre than 100 infected fibres A typical fillet might be described: Area V V V, nfectin H M H M M L Fr the purpse f calculatin, L was arbitrarily assigned a value f 20, M a value f 60, and H a value f 100. The "MD" fr the abve fillet wuld be: 400/6 = 66.7.
16 - 5 - Prtease Activity The preparatin f hmgenates fr the determinatin f ttal prtelytic activity f muscle against endgenus substrates fllwed the methd f Wjtwiez and Odense (1972). Each fillet was nt systematically sub-sampled in the manner described earlier fr the parasite evaluatin. nstead, fur r five sub-sectins ttalling 5-10 gm were remved frm representative areas f each fillet and pled. The verall prtelytic activity was subsequently determined in dupl icate fr each fillet. The determinatin f autlysis was made accrding t Lwry et ale (1951) as summarized by McDnald and Chen (1965). The prtease activity values represent the extent f prtelysis at a standardized white muscle cncentratin f 3.5 mg/ml. Preliminary wrk n representative numbers f hake established the cnditins t be used in the systemic assay f prtelytic activities in the raw fillets. These parameters included incubatin time, temperature(s) and ph(s) f reactin and will be discussed in a later sectin. Tw buffers were used thrughut. Reactin medium in the acidic range was maintained by a citric acid-sdium citrate buffer (Bichemists Handbk, Ed. Cyril Lng, 1961) and in the neutral and alkaline range with Klthff's brax-phsphate buffer (C.R.C. Handbk f Bichemistry. Selected Data fr Mlecular Bilgy, ed. Sber, 1968). Cking Methd Frzen fillets were thawed by immersin in duble plyethylene bags fr 1.25 hurs in 20 C water. The thawed white muscle was then cut int pieces 5 x 5 x 1.0 cm thick, wrapped in a single layer f aluminum fil and either steamed fr 10 min (fast ck) r baked in a pre-heated 177 C fr 25 min (slw ck). The hake btained in the Strait f Gergia during March, 1980, were cked by steaming while all ther hake were cked by baking. The cked muscle was cled t rm temperature prir t evaluatin f texture.
17 - 6 - Texture The determinatin f firmness f the cked flesh was made with the Ottawa Texture Measuring System (O.T.M.S. Mdel ). The peratinal parameters have been detailed previusly (Bilinski et ale 1977). The 10% and 20% range selectins were used. RESULTS AND DSCUSSON. Parasite nfectin Examinatin f fillets reserved fr parasitlgical study revealed that the West Cast r ffshre hake were infected with tw species f the genus Kuda. One f these species was identified as Kuda thyrsitis (Gilchrist, 1924). Althugh the spres fund in these fish differed in sme details frm thse riginally described as ~ thyrsitis, the verall resemblance was clse enugh t suggest that they belng t that species. ~ thyrsitis has been previusly recrded frm Merluccius capensis, the Suth African hake (Priebe, 1967). The ther species has nt been previusly knwn and it is cnsidered a new species. ts descriptin, as well as that f ~ thyrsitis, has been published elsewhere (Kabata and Whitaker, 1981). The name given t the new species is Kuda panifrmis. The Gergia Strait hake harbured nly ne species, ~ thyrsitis. Althugh an ccasinal straying f the secnd species, ~ panifrmis, might still be discvered, it is nw certain that it des nt reside in the Strait f Gergia. All infrmatin n which this accunt has been based, cmes frm a single ffshre sample. t is pssible, therefre, that mre extensive sampling will result in a mdificatin f cnclusins presented in it. The signs f infectin prduced by Kuda thyrsitis are very similar t thse prduced by Kuda panifrmis, s that the identity f the pathgen can be determined nly by examinatin f the spres under cmpund micrscpe. The parasite penetrates the cre f individual muscle fibres and spreads alng them withut damaging sarclemma, until each fibre is filled with spres. These fibres, knwn as pseudcysts, islate the parasite, s that the cnnective tissue f the hst is nt prvked int reactin. They are
18 - 7 - slightly distended and are externally recgnizable by their pale yellwish clur. t appears that the nly marked defensive reactin f the fish is the depsitin f melanin granules arund the area invaded by the parasite. The timing f the nset f this reactin and its rate cannt be determined n the basis f the present infrmatin. t is clear, hwever, that the reactin is prvked by the presence f the parasite and that it cntinues fr sme time, causing a gradual accumulatin f melanin and a crrespnding change in the clur f the fibre. Turning initially light brwn, it becmes dark brwn and eventually black. This melanin depsitin has an bviusly detrimental effect n the spres, which are prgressively cmpressed, crumpled and, in the end, destryed. As mentined earlier, the ffshre ppulatin f hake was infected with tw species f Kuda, which ccurred separately in different fish r frmed mixed infectins. Of the 322 fish examined, nly 34 (ie. 10.5%) were free f Kuda. The number infected with Kuda thyrsitis alne was 104 (32.3%) and with Kuda panifrmis 125 (38.8%). Mixed infectins were fund in 59 fish (18.3%). Althugh nearly 90% f hake were infected, the intensity f infectin was nt unifrm, many fish carrying nly a few pseudcysts. The infectin was distributed thrugh the thickness f the flesh, infected fibres being lcated with apparently equal frequency clse t the inner bundary f the bdy wall, deep within the muscle and in subcutaneus layers. The distributin f the parasite in different areas f the bdy was less unifrm, as can be seen in Tables and. The infectin was generally heaviest in the anterir part f the fillet and tended t diminish in a psterir directin. Pseudcysts f ~ thyrsitis were predminantly ld, judging frm their dark cluratin, nly relatively few pale pseudcysts being present. This species des nt appear t prduce heavy infectins. Only 3% f areas carried heavy ld infectins; the prprtin f medium infectin was als lw, the highest number being 10% fr area T. Light infectins were much mre cmmn (74% in area ), but here als ld pseudcysts predminated. This prepnderance f black infected fibres suggests that Kuda thyrsitis infects hake at a lw rate, tending t accumulate black fibres with age but seldm prducing very many pseudcysts at the same time. Perhaps this might be taken as an indicatin f an ld and well-established relatinship between Merluccius prductus and Kuda thyrsitis.
19 - 8 - The situatin with Kuda panifrmis is quite different. Figures in Table shw that it prduces many mre heavy infectins and that mst f thse infectins are f recent rigin. n the light categry, ld infectins are mre cmmn than the recent nes; in the medium categry the psitin is intermediate. These figures create the impressin that, althugh bth species f Kuda might begin t infect hake at abut the same time, subsequent curse f infectin is quite different. Whereas the infectin rate with ~ thyrsitis diminishes with the age f the fish, that with K. panifrmis cntinues and perhaps even increases. Hence the latter species is capable f prducing massive numbers f yung, pale pseudcysts. When cmpared with the infectin pattern f ~ thyrsitis, that f ~ panifrmis seems t suggest that it is a mre recent parasite f hake, with which it still remains in a rather unstable equilibrium. t is, therefre, mre likely t be severely damaging t its hst than is ~ thyrsitis. The picture presented by mixed infectins is, nt unexpectedly, intermediate between that resulting frm infectins with single species. Bth the juvenile and adult ppulatins f hake in the Strait f Gergia were infected with ~ thyrsitis nly. As might be expected, the pattern f infectin in the adult fish resembles that f the ffshre infectins with this parasite (Table V and V). There were relatively mre heavy infectins, but in all three categries (heavy, medium and light) the ld, black pseudcysts predminated ver the new nes, the rati f ld t new being rughly 2: 1.. Prtease Activity - temperature and ph effects Fish muscle prteinases active in the acid ph range have been demnstrated in a number f species f fish (cited in Makindan and keda, 1969c). Recently a carp muscle cathepsin D was descrihed which hydrlysed myfibrils prepared frm the same species at ph 3-4 but nt abve ph 6.0 althugh sme hydrlysis ccurred at higher ph with casein as substrate (l"1akindan et al. 1982). The participatin f this enzyme in autlysis was cnsidered dubtful as the pst-mrtem ph f carp muscle ranged between (Makindan et al. 1980). Fish muscle prteinases active in the slightly alkaline r neutral ph
20 - 9 - range with casein as substrate have been described in a number f investigatins (Makindan and keda, 1969a,b, 1971, 1976; Makindan et ale 1979; wata et ale 1974a,b). These prteases in bth the crude and partially purified frm pssessed ptimal activities in the slightly alkaline ph range and at high temperatures f arund 60 C with casein substrate and were demnstrated in a number f fish species (Makindan and keda, 1969a,b; wata et ale 1974a,h,c). The alkaline prtease frm the muscle f a number f fish shws little r n activity against casein at lwer incubatin temperatures f arund C (Makindan and keda, 1969b; wata et ale 1974a,b). A partially purified prtease with ptimum activities at a ph f 8 and at elevated temperatures was demnstrated in mullet muscle using urea denatured hemglbin as substrate (Deng, 1981). While the ph ptimum f this enzyme was nt tested against muscle prteins f mullet, it was nted that significant residual activity existed at ph 6.0, the same ph f mullet muscle. The still significant level f activity at this ph was implicated in the reductin f shear frce value f mullet flesh at r near the temperature ptimum f the enzyme. A partially purified neutral prteinase frm carp muscle has been described (Makindan et ale 1979). Althugh its activity tward carp muscle myfibrils has nt yet been demnstrated, it has been stated that endpeptidase activity twards bth intact and partially hydrlysed hemglbin suggests that the neutral prteinase may playa significant rle in autlysis f fish meat. The rle f the ther endpeptidases in carp muscle, cathepsin 0 and alkaline prteinase, is thught t be difficult t relate t autlysis f fish muscle (Makindan et ale 1979). Cathepsin 0 is inactive in the neutral ph range and alkaline prteinase is inactive at temperatures belw 55 C. Hwever, the rle f alkaline prteinase during the increase in temperature in the slw cking prcess is anther matter. Bth the ph and temperature fr ptimal activity f prteases are influenced cnsiderably by the substrate used fr measuring the activity f the enzyme. Muscle prtease activity measured by splitting f denatured hemglbin frm surces ther than fish is invariably greater than that measured by using muscle prteins frm the same species as substrate and may nt reflect the true activity f the tissue (Wjtwicz and Odense, 1972). Furthermre, fish hemglbins are nt expected t be present in significant levels in the muscle tissues f fish with white flesh. As the develpment f
21 mushy texture reflects the hydrlysis f structural prteins f the musculature, it is mre meaningful t measure the prtease activity against myfibrillar prteins frm the same species. Frm the present study it is evident that the prtelytic activity f Pacific hake muscle at ph 3.8 is ptimal at temperatures between C (Fig. 2). This was fund in representative numbers f bth Gergia Strait hake infected with nly ~ thyrsitis and west cast hake infected with bth. ~ thyrsitis and the new parasite ~ panifrmis. The activity at this ph decreased at higher temperatures. Differences in the magnitude f prtelysis existed between samples but the shape f the temperature curves was quite similar. As the mean ph f unbuffered hake muscle samples was fund t be 6.88 with a range f 6.74 t 7.02 prtease activity at ph near neutrality was cnsidered relevant t the understanding f texture prblems f the musculature at temperatures preceding the inactivatin pint f the enzyme. At ph 6.7 nne f the Gergia Strait hake, whether infected with ~ thyrsitis r nt, exhibited significant prtelytic activity at any temperature. The prtease frm west cast hake, n the ther hand, displayed a temperature ptimum between C (Fig. 3) at ph 6.7. Althugh variatins in activity were bserved between fish, the shape f the temperature curves was abut the same. Where prtelysis is measured by autlysis the activity ptima are generally brader than thse reprted in the literature using single prtein substrates. At 35 C the muscle hmgenates f nn-infected hake frm bth the Strait f Gergia and the west cast display ptimal prtelytic activity at arund ph 4 (Fig. 4). At 60 C the prtelysis curve fr the abve samples differed nt nly by gegraphic area but als by a shift in the acid prtease ph ptima frm 4 t 4.5 (Fig~ 5). This bserved shift in ptima may reflect factrs such as the pssible existence in muscle tissues f several acid prteases with different temperature ptima r the differential heat stability f nn-enzyme and enzyme prteins influencing the cmpsite activity pattern. n the neutral ph range prtease activity f uninfected Gergia Strait hake is minimal while that f uninfected west cast hake indicate a secnd peak f activity (Fig. 5). t is nt knwn at this time whether sme prtease activity in the neutral ph range is an inherent gegraphic difference r whether it is a
22 result f infectin with ~ panifrmis at a level undetectable even by thrugh examinatin. Because at least sme ffshre hake pssess significant prtelytic activity bth in the acid and neutral ph ranges and because literature reprts that neutral r sl ightly alkaline prteases may be implicated in the autlytic mechanism, all samples were rutinely measured fr prtelysis at ph 3.8 and 35 C fr acid prtease and at ph 6.7 and 60 C fr neutral prtease.. Strait f Gergia Hake A. Prtease Activity Strait f Gergia hake heavily infected with ~ thyrsitis exhibit maximum prtelysis nly in the acid ph range whether at 35 C r 60 C (Fig. 6). At the higher temperature, hwever, maximum prtelysis ccurs at a slightly higher ph as was als bserved in Fig. 5. When prtelysis is measured at ph 3.8 and 6.7, average activity at the lwer ph is cnsiderably higher (Table V). When the samples frm Gergia Strait infected with parasite were divided int thse in which the ~ thyrsitis existed mainly in the yung white pseudcyst frm (W+B±) and in the lder black pseudcyst frm (W-B+), significant differences in prtelytic activity can be seen. Muscle tissues infected with yung parasite shw an average prtease activity almst 7 fld higher at the acid ph cmpared t that at neutral ph. Fr tissue in which the infectin is ld, the difference is 3.5 fld. B. Cked Flesh Texture Muscle tissue in which the infectin is new shw a higher average acid prtease value and a lwer OTMS shear press frce (Table V) cmpared t tissue with lder infectin. Hwever, nne f the Strait f Gergia samples were cnsidered t sft r mushy in texture after slw cking. While acid prtease activity may be cntributing t sme prtelysis at flesh ph f 6.88
23 ~ 12 - the extent is nt sufficient t result in texture that is unacceptably sft. The texture f fast cked samples frm an additinal 50 hake sampled in March, 1980 was als similar. C. Relatinship f nfectin, Prtease Activity and Texture While the yung frm (W+B±) f infectin by ~ thyrsitis was crrelated with the acid prtease activity [crr. ceff (p<.ol}j and neutral prtease activity [crr. ceff (p<.ol}j, there was n crrelatin f either prtease activity r the presence f the parasite in either frm with the firmness f cked flesh f hake frm Gergia Strait. The prtease assciated with hake frm Gergia Strait has very little activity in the neutral ph range (Fig. 6). Frm the texture standpint the hake frm Gergia Strait is smewhat similar t thse described fr the same species frm Puget Sund. nfectin f the Gergia Strait hake with ~ thyrsitis des nt result in the develpment f mushy texture during slw cking. V. West Cast Hake A. Prtease Activity At 35 C west cast hake infected with bth Kuda species r with ~ panifrmis alne pssess prtease activity in the acid ph range with an ptimum at abut 3.8 and very little activity in the neutral ph range (Fig. 7). At 60 C, hwever, the prtease activity is evident ver a brad ph spectrum including bth the acid and neutral range. The activity f bth prteases frm infected west cast hake is cnsiderably higher than that bserved frm uninfected hake frm the same area (cmpare Fig. 5 and 7). The mst striking difference in the prtease activity prfile between infected hake frm the tw gegraphic sampling areas is that at 60 C, a transitinal temperature in the cking prcess, Gergia Strait hake infected with ~ thyrsitis has very little activity in the neutral ph range (Fig. 6) while the west cast hake infected with ~ panifrmis exhibit strng activity in
24 the sar;le ph range (Fig. 7, Table V). The prtease activity prfile f muscle tissues frm west cast hake bth uninfected and infected with ~ thyrsitis alne is very similar t that f the Strait f Gergia infected with the same species f parasite. Muscle tissues with yung and ld infectin with ~ thyrsitis shw sme difference in acid prtease activity but fr these samplings at least, nt as large as that bserved fr Gergia Strait hake. t is imprtant t emphasize here that west cast hake in which infectin with ~ panifrmis is ld display enzyme activity values fr bth the acid and neutral prteases smewhat similar t thse f uninfected fish and thse infected nly with ~ thyrsitis frm bth gegraphic areas. Muscle tissues with mixed infectin shw acid and neutral prtease values intermediate t thse infected with ~ panifrmis alne r with ~ thyrsitis alne. B. Cked Flesh Texture Uninfected west cast hake as well as thse infected with ~ thyrsitis in either the yung r ld frm are firm in texture after slw cking (Table V). Gergia Strait hake infected with ~ thyrsitis, while nt cnsidered t sft r mushy, gave cnsistently lwer OTMS readings. nherent gegraphic differences in the tw islated ppulatins (Kabata and Whitaker, 1981) and the larger size f west cast hake in this study culd accunt fr the difference. West cast hake infected with K. panifrmis in the yung (W+B±) frm gave lw OTMS readings after slw cking and all samples were cnsidered excessively sft and mushy. Thse samples infected with ~ panifrmis in the ld (W-B+) frm did nt develp a mushy texture. As stated earlier in the discussin under Parasite nfectin, the depsitin f melanin granules appears t be a defensive mechanism f the muscle tissues fr the prcess culminates in the destructin f the spres. The lw neutral prtease activity tgether with the firmness f the cked fillets frm muscle tissues f west cast hake which has a prevalence f ld black pseudcysts als appears t reflect the destructin f the ability f the parasite t prduce prtelytic enzymes.
25 Samples infected with bth Kuda species resulted in varying degrees f sftness and mushiness and all were rejected n this basis. C. Relatinship f nfectin, Prtease Activity and Texture West cast hake infected with K. panifrmis in the W+R± frm shw a strng psitive crrelatin with bth neutral prtease activity [crr. ceff (p<.ol)] and acid prtease activity [crr. ceff (p<.ol)] and a strng negative crrelatin with firmness [crr. ceff (p<.ol)]. n this investigatin the develpment f sft cked texture was assciated nly with west cast hake infected with ~ panifrmis alne r tgether with ~ thyrsitis. Bth Gergia Strait hake, which are infected nly with ~ thyrsitis, and west cast hake infected with nly the same species d nt result in the develpment f mushy texture in cked flesh. The relatinship f prtease activity and autlysis f the musculature is cmplex. The literature reprts that the muscle tissues f many species f fish have prtease activity in the acid and slightly alkaline ph ranges. Hwever, detailed examinatin fr the presence f parasites has nt been an integral part f thse investigatins. Frm the present study it is evident that infectin f the musculature f Pacific hake with ~ thyrsitis results in a large increase in prtease activity in the acid ph range. Hwever, infectin with ~ panifrmis results in a large increase in prtease activity in bth the acid and neutral range. Whether the prtease activities assciated with the parasite at these ph ranges are the same as thse reprted in uninfected fish is nt knwn. This investigatin has revealed that all Pacific hake shwing strng prtease activity in the presence f parasite at 60 C result in develpment f mushy texture in cked flesh. Acid prtease activity in muscle tissues cntributed by ~ thyrsitis des nt cause mushy texture at flesh ph althugh sme degree f sftening may be ccurring. The cntributin t mushy texture by acid prtease activity in thse specimens als shwing strng neutral prtease activity, as in hake infected with ~ panifrmis alne, can nt yet be ruled ut. Hwever, prtease frm K. panifrmis shw activity at 60 C ver a brad ph spectrum with an ptimum near neutrality. t is reprted in
26 the literature that fish muscle acid prteases with ph ptima f abut are nt cnsistent with the pst-mrtem ph f flesh which is near neutrality. Makindan et ale (1982) reprted that cathepsin D frm, carp muscle hydrlyses carp myfibrils at ph 3-4 but nt at 6.0. Neutral r slightly alkaline prtease activity is mre favrably viewed as being invlved in the autlytic prtelysis f fish muculature. Acid prtease activity in parasite infected Pacific hake muscle is heat sensitive, becming relatively inactive at the elevated temperatures expected during the cking prcess. Hydrlysis which can be demnstrated by prtease activity in the infected hake muscle tissue in the acid ph range des nt appear t be spl itting t any great extent at muscle ph, thse structural prteins which maintain firm texture. v. The effect f pst-mrtem chilled strage time n the acid and neutral prtease activities and its relatinship t the develpment f sft texture. The effect f pst-mrtem chilled strage time n ice and in RSW, fr up t 120 hurs, n the develpment f sft texture in Pacific hake during slw cking is shwn in Fig. 8 and 11 respectively. Samples were segregated ~ gegraphic area and by species f Kuda with which the fish were infected. The west cast samples were frzen immediately n-bard and after varius lengths f chilled strage time fr subsequent analyses fr cked flesh texture and fr prtelytic activities. mmediate freezing was nt pssible with samples frm the Strait f Gergia as the trawlers were nt equipped with n-bard freezing facilities. During strage n ice the shear frce values f all west cast hake after cking drpped sharply during the initial chilled strage perids f 4-8 hurs and subsequently remained either stable r declined very slwly within the limits f the experiment (Fig. 8). When the muscle tissues were segregated by the species f parasite present, all west cast hake infected with ~ panifrmis shwed the lwest shear values and the textures after cking were cnsidered clearly unacceptable beynd 4 hurs f chilled strage. These samples, regardless f chilled strage time, up t and including zer time, cnsistently prduced
27 shear values nly apprximately half thse f fish uninfected r infected with ~ thyrsitis alne. Nne f the latter was judged t sft fr up t 120 hurs f ice strage. Chilled strage fr up t 120 hurs results in cked flesh f acceptable firmness prvided the hake is nt infected with ~ panifrmis alne r in a mixture with ~ thyrsitis. Althugh infectin \~ith the frmer was nt separated by yung and ld frms, it is presumed that thse fish with the lder frm wuld have a firm texture as indicated in Table V. Fig. 9 and 10 shw that bth the acid and neutral prteases are high in fish infected with ~ panifrmis and the level f activity decreases slightly with increased chilled strage time. Cnversely, west cast hake infected nly with ~ thyrsitis display lwer activities fr bth prteases, decreasing little r nne up t 120 hurs. n additin prtelysis appears t be at a virtual standstill thrughut the chilled strage perid f 120 hurs as shwn by lw blank values (Fig. 14). The presence r absence f ~ panifrmis, rather than ice strage time t 5 days, appears t be the main determining factr in the acceptability f the cked flesh frm the standpint f texture. The results btained fr strage in RSW are essentially the same as fr ice strage and nly the results are shwn (Fig. 11, 12, 13). ACKNOWLEDGMENT We wish t thank Mr. J. Cheng f the Technlgy Services Branch fr cllecting and treating the west cast hake samples and Mrs. O.M.A. Nrdin, frmerly f the Branch, fr assistance in the early phase f the prtease assay.
28 REFERENCES BLNSK, E., LAU, Y.C. and JONAS, R.E.E Objective measurement and cntrl f the firmness f canned herring. Fish. and Mar. Serv Technical Reprt N. 727, 22 pp. CRAWFORD, D.L., LAW, O.K. and MCGLL, L.S. 1972a. Shelf life stability and acceptance f frzen Pacific hake (Merluccius prductus) fillet prtins. J. Fd Sci. 37:801. CRAWFORD, D.L., LAW, O.K., BABBTT, J.K. and MCGLL, L.S. 1972b. Yield and acceptabi 1 ity f machi ne separated mi nced fl esh frm srne mari ne fd fish. J. Fd Sci. 37:551. CRAWFORD, D.L., LAW, O.K., BABBTT, J.K. and MCGLL, L.S Cmparative stability and desirability f frzen Pacific hake fillet and minced flesh blcks. J. Fd Sci. 44: DARK, T.A., NELSON, M.D., TRAYNOR, J.J. and NUNNALLEE, E.P The distributin, abundance, and bilgical characteristics f Pacific whiting, Merluccius prductus, in the Califrnia-British Clumbia regin during July-September Marine Fisheries Review, i (3-4): DASSOW, J.A. and BEARDSLEY, A.J The United States experience with Pacific hake, Merluccius prductus. Fd and Agriculture Organizatin f the United Natins, Technical Cnference n Fishery Prducts, Tky, Japan, 14 pp. DASSOW, J.A., PATASHNK, M. and KOURY, B.J Characteristics f Pacific hake, Merluccius rrductus, that affect its suitability fr fd. U.S. Fish and Wild ife Service, Circular 332, Seattle, Bureau f Cmrnercial Fisheries, DENG, J.C Effect f temperature n fish alkaline prtease, prtein interactin and texture quality. J. Fd Sci. 46: FORRESTER, C.R., BAKKALA, R.G., OKADA, K. and SMTH, J.E. n Press. Grundfish, shrimp and herring fisheries in the Bering Sea and Nrtheast Pacific - histrical catch statistics, NPFC Bulletin N. GLCHRST, J.D.F A prtzal parasite Chlrmyxum thyrsites sp.n. f the Cape sea-fish, the Snek (Thyrsites atun, Euphr.). Trans. Ry. Sc. S. Afr. 11: 263. WATA, K., KOBASH, K. and HASE, J. 1974a. Studies n muscle alkaline prtease -. Sme enzymatic prperties f carp muscular alkaline prtease. Bull. Japan. Sc. Sci. Fi she 40(2): WATA, K., KOBASH, K. and HASE, J. 1974b. Studies n muscle alkaline prtease -. Distributin f alkaline prtease in muscle f freshwater fish, marine fish and in internal rgans f carp. Bull. Japan. Sc. Sci. Fish. 40(2):
29 WATA, K., KOBASH, K. and HASE J. 1974c. Studies n muscle alkaline prtease - V. Cmparisn f physicchemical and enzymatic prperties f alkaline prtease frm white craker and barracuda muscle with thse f the enzyme frm carp muscle. Bull. Japan. Sc. Sci. Fish. 40: KABATA, Z. and WHTAKER, D Tw species f Kuda (Myxsprea: Multivalvulida) parasitic in the flesh f Merluccius prductus (Ayres, 1855) (Pisces:Telestei) in the Canadian Pacific. Can. J. Zl. 59: LONG, C Bichemists' Handbk, E. and F.N. SPON Ltd., 22 Henrietta Street, Lndn, W.C pp. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J Prtein measurement with the Flin Phenl Reagent. J. Bil. Chem. 193: MCDONALD, C.E. and CHEN, L.L The Lwry Mdificatin f the Flin Reagent fr determinatin f prteinase activity. Anal. Bichem. 10: MAKNODAN, Y., AKASAKA, T., TOYOHARA, H. and KEDA, S Purificatin and prperties f carp muscle cathepsin D. J. Fd Sci. 47: MAKNODAN, Y., HROTSUKA, M. and KEDA, S Neutral prteinase f carp muscle. J. Fd Sci. 44: MAKNODAN, Y., HROTSUKA, M. and KEDA, S Autlysis f carp muscle. Bull. Japan. Sc. Sci. Fish. ~: MAKNODAN, Y. and KEDA, S. 1969a. Studies n fish muscle prtease -. On the existence f tw kinds f prteinases active in acid and in slightly alkaline ph range. Bull. Japan. Sc. Sci. Fish. ~(7): b.. Purificatin and prperties f a prteinase active in slightly alkaline ph range. Bull. Japan. Sc. Sci. Fish. 35(8): c.. Purificatin and prperties f a prteinase active in acid ph range. Bull. Japan. Sc. Sci. Fish. ~(8): Sc V. On the existence f cathepsins A, Band C. Bull. Japan. Sci. Fish. lz.(10): V. On the existence f prtease active in neutral ph range. Bull. Japan. Sc. Sci. Fish. 42(6): MYAUCH, D. in hake United States fd fishing industry takes a new interest Fishing News nternatinal ~(11): PATASHNK, M., BARNETT, H.J. and NELSON, R.W Prximate cmpsitin f Pacific hake. U.S. Fish and Wildlife Service, Circular 332,
30 PATASHNK, M., GRONNGER, H.S., BARNETT, M., KUDO, G. and KOURY, B Pacific whiting, Merluccius prductus:. Abnrmal muscle texture caused by Myxspridian-induced prtelysis. Marine Fisheries Review, 44(5): PREBE, K Uber das Vrkmmen vn Myspridian-Pseudcysten in der Musculatur vn Sudatlantischen Seehecht (Merluccius capensis). Arch. Lebensmittelhyg. Hannver ~: SPNELL, J. and STENBERG, M.A nhibitin f prtelysis in Pacific hake fillets. Marine Fisheries Review, 40(10):52. SWANSON, R. and RDENHOUR, R.L Ptential fr develpment f a Nrthern Califrnia fishery fr Pacific hake. Humbldt State University Sea Grant Reprt N. HSU-SG-13, Arcata, Cal ifrnia, prepared fr Natinal Oceanic and Atmspheric Administratin, Rckville, Maryland, 28 pp. THOMPSON, J.M. and MCFARLANE, G.A Distributin and abundance f Pacific hake and walleye pllck in the Strait f Gergia, Mar.24-May 2, Can. Tech. Rep. Fish. Aquat. Sci (in press). WOJTOWCZ, M.B. and ODENSE, P.H Cmparative study f the muscle catheptic activity f sme marine species. J. Fish. Res. Bd. Canada 29:85-90.
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