Research ArticleEffects of Freeze-Thaw Cycles on Gel-Forming Ability and ProteinDenaturation in Alaska Pollock Frozen Surimi

Shuji Abe 1 Takuma Asada2 and Kazuhito Kajiwara1

1School of Bioscience and Biotechnology Tokyo University of Technology 1404-1 Katakura Hachioji Tokyo 192-0982 Japan2Graduate School Tokyo University of Technology 1404-1 Katakura Hachioji Tokyo 192-0982 Japan

Correspondence should be addressed to Shuji Abe abeshjstfteuacjp

Received 18 February 2019 Revised 3 May 2019 Accepted 27 May 2019 Published 7 July 2019

Academic Editor Jose A Beltran

Copyright copy 2019 Shuji Abe et al is is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Frozen surimi may be damaged by freeze-thaw cycles (refreezing) under various conditions However few studies have examinedthe deterioration of the quality of refrozen surimi e objective of this study was to determine the deterioration mechanism ofrefrozen surimiWe used Alaska pollock frozen surimi which has been studied extensively for gel formation Refreezing decreasedthe breaking strength and breaking strain of the heated gele length of the line in the diagram between breaking strength and gelstiness (L value which indicates the level of change in the breaking strength and gel stiness with setting time) was alsodecreased In contrast the eect of refreezing on the texture of the gel without the setting process was small e polymerizationrate of myosin heavy chain in refrozen surimi during the setting process was slower than that in nonrefrozen surimi Additionallythe Ca-ATPase activity of surimi was reduced by approximately 30 with each refreezing ese results demonstrate that thereduction in gel-forming ability by refreezing was caused by the decreased polymerization rate of myosin heavy chain because ofmyosin head denaturation We also found that quality deterioration including myobrillar protein denaturation of frozen surimioccurred mostly during rst refreezing rather than during second refreezing Overall refreezing andor repeated refreezing ofsurimi simply decreased the suwari gel-forming ability without changing the characteristics of surimi e primary cause of thedecrease in gel-forming ability induced by refreezing is considered to be the suppression of myosin heavy-chain polymerizationduring setting

1 Introduction

It is well known that refreezing reduces the quality of foodproducts [1 2] Refreezing causes damage such as cell de-struction protein denaturation and surface drying in foodsWhile many studies have examined the eects of refreezingon food products few reports have evaluated food materials

Surimi paste products are important components ofseafood productsemainmaterial in surimi paste products isfrozen surimiemost important property of frozen surimi isits gel-forming ability which depends on the structure ofmyobrillar proteins in surimi Kato et al [3] detected acorrelation between the Ca-ATPase activity of frozen surimiand gel-forming ability Additionally denatured myobrillarprotein in surimi cannot form gel [4] Myobrillar protein insh muscle is susceptible to denaturation by heating andor

freezing because of its sensitivity to changing temperatures [5]In most previous studies the gel-forming ability was evaluatedby conducting a texture test In Japan the punch test has beenused to evaluate the gel-forming ability of frozen surimi Abeet al [6] found a linear relationship between breaking strengthand gel stiness Additionally they revealed that the line po-sition linear slope and plot dispersion depend on the propertyof frozen surimi Figure 1 shows a model of the relationshipbetween gel stiness and breaking strength of two-step heatedgels e relationship diagram of breaking strength versus gelstiness revealed the following (1) When the straight line wasshifted to the right side or when its incline was small the surimiformed a brittle and hard gel (2) When the plots were scat-tered the surimi formed a nonuniformed gel For example theplots of suwari gel with microbial transglutaminase tended tobe scattered (3)When the plots formed clumps the surimi did

HindawiJournal of Food QualityVolume 2019 Article ID 3760368 9 pageshttpsdoiorg10115520193760368

not form a suwari gel ese relationships between breakingstrength and gel stiffness have been used to analyze the gel-forming ability of surimi [6ndash9]

Frozen surimi is manufactured in many countriesHowever frozen surimi factories are at risk of unexpectedtemperature rise of frozen surimi Particularly the warmclimate is one factor causing a rise in the temperature offrozen surimi which can damage the product when thawingand freezing (refreezing) go unnoticed Furthermorerefreezing after a temperature increase may occur duringtransportation us the refreezing risk of frozen surimi isan important problem in the surimi paste product industrySeveral effects of refreezing on the gel-forming ability offrozen surimi have been reported [10ndash12] Another studyshowed that refreezing of frozen surimi reduces its gel-forming ability [13] Furthermore ionic and hydrogenbonds are increased in heat-induced gel prepared fromrefrozen surimi whereas hydrophobic interactions S-Sbonds and more intensive bonding interactions are de-creased [13] However the refreezing problem of frozensurimi in the surimi seafood industry has not yet beensolved To address this problem previous studies examinedthe effects of refreezing times that cannot occur at pro-duction sites Moreover these previous reports did not fullyconsider the reduction of the gel-forming ability byrefreezing ie these reports did not study the change in thesuwari gel-forming ability with setting time erefore thepurpose of this study was to determine the effect ofrefreezing on the relationship between protein denaturationand gel-forming ability of frozen surimi after differentsetting times

2 Materials and Methods

21 Materials Alaska pollock (eragra chalcogramma)frozen surimi (A grade) was obtained from Maruha NichiroCorporation (Tokyo Japan) and then cut into approximately

600 g blocks e cut frozen surimi was stored at minus30degC in afreezer until further use e refrozen surimi was preparedby freezing at minus30degC for at least 24 h after thawing at 4degCovernight (1st F-T cycle surimi) e refreezing process wasrepeated twice (2nd F-T cycle surimi) within 1week Po-tassium chloride calcium chloride 2-amino-2-hydrox-ymethyl-13-propanediol (Tris) maleic acid trichloroaceticacid 2-mercaptoethanol dodecyl sodium sulfate (SDS)urea and bovine serum albumin were purchased fromWakoPure Chemical Industries (Osaka Japan) ATP was pur-chased from Sigma Chemical Co (St Louis MO USA)

22 Preparation of Heat-Induced Gel Frozen surimi blockswere thawed by refrigeration (4degC) overnight and then cutinto small cubes Surimi cubes were ground with water(60mL600 g of surimi) until the surimi cube temperaturewas 1ndash2degC Next 3 NaCl was added and grinding wascontinued until the surimi paste temperature was 7ndash10degCe surimi paste was stuffed into a polyvinylidene chloridetube (diameter 24mm) Next the tubes containing surimipaste were preheated for 0ndash24 h at 20degC in a water bath(suwari gel) At set time points some tubes were removedfrom the water bath and heated for 30min at 90degC Afterheating the gels were chilled immediately in water for over10min ese gels were defined as two-step heated gels egel heated for 30min at 90degC without preheating was definedas the directly heated gel

23 Gel Texture Measurement All gels were cut into piecesof 30 cm in length and 25 cm in diameter Breaking strengthand breaking strain of the gel samples were measured with acreep meter (RHEONER II Yamaden Tokyo Japan) using aspherical plunger that was 5mm in diameter with a de-pression speed of 1mms Gel stiffness was calculated usingthe following equation (1)

gel stiffness(gcm) breaking strength(g)

breaking strain(cm) (1)

24 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electropho-resis (SDS-PAGE) e sample used to analyze the proteinpatterns of heat-induced gel by SDS-PAGE was prepared asdescribed by Numakura et al [14] with slight modificationsGels were added to SDS-urea solution containing 2 SDS2 2-mercaptoethanol 8M urea and 20mM Tris-HCl (pH88) and heated to 100degC for 2mine sample was shaken todissolve the gel in SDS-urea solution on a seesaw shakerovernight e solution was applied to SDS-PAGE by theLaemmli method [15] using 75 separating polyacrylamidegels (e-PAGELreg Atto Tokyo Japan) e gel was stainedusing Coomassie Brilliant Blue

25 Determination of Residual Rate of Myosin Heavy Chain(MHC) and the Concentration of 150 kDa Band e imagedata of SDS-PAGE pattern were obtained using a scannere staining intensity of MHC band and the 150-kDa band

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

Ordinary type

Nonuniformed gel forming type

Brittle and hard gel forming type

Nonformed setting gel type

Figure 1 Model of GS versus BS relationship diagram and indicate various types of surimi

2 Journal of Food Quality

was determined by Image J e rate of staining intensity ofthese two bands was calculated by dividing their stainingintensity by the staining intensity of all bands e MHCresidual rate was calculated assuming that the concentrationof the band with a setting time of 0 hours was 100

26 Preparation of Myofibrillar Protein Suspension fromFrozen Surimi e myofibrillar protein suspension offrozen surimi was prepared as described by Kato et al [16]with slight modificationsawed surimi was added to 01MKCl and 20mM Tris-HCl (pH 70) and then homogenizede homogenate was centrifuged at 7500times g for 20minAfter removing the supernatant 01M KCl and 20mM Tris-HCl (pH 70) were addedis process was repeated until thesupernatant was clear e precipitate was suspended in01M KCl and 20mM Tris-HCl (pH 70) and then filteredthrough a gauze e filtrate was used as the myofibrillarprotein suspension ere was almost no change in therecovered amount of myofibrillar protein from each frozensurimi e prepared myofibrillar protein suspension wasstored on ice until measurement of Ca-ATPase activity

27 Measurement of Ca-ATPase Activity e myofibrillarprotein suspension (25ndash35mgmL) was added to 01MKCl 5mMCaCl2 1mMATP and 25mMTris-maleate (pH70) e reaction was conducted at 25degC e reaction wasstopped by adding 15 perchloric acid at each reactiontime e supernatant was added to sulfate-molybdate acidsolution and Elon reagent e inorganic phosphate con-centration in the supernatant was measured by colori-metric determination at a wavelength of 640 nm Ca-ATPase activity was calculated using the following equa-tion (2)

Ca-ATPase activity ln 1minusPi0( 1113857minus ln 1minusPit( 11138571113864 1113865 times1000

t

times1

Cmmicromolmiddotminminus1 middotmgof proteinminus11113960 1113961

(2)

where t is the time of reaction Pi0 is the inorganic phosphateacid concentration at a reaction time 0min (blank) Pit is theinorganic phosphate acid concentration after the reactionand Cm is the concentration of myofibrillar protein in thereaction reagent

e protein concentration of the myofibrillar proteinsuspension used in the reaction was determined by the biuretmethod using bovine serum albumin as a standard

28 Statistical Analysis Statistical analysis was performedusing the bell curve for Excel (Social Survey Research In-formation Co Ltd Tokyo Japan) e results were com-pared using the least significant difference method atplt 005

3 Results and Discussion

31 Gel Texture Measurement Changes in the breakingstrength and strain of suwari gel prepared from frozensurimi are shown in Figure 2 Compared to that of thecontrol sample the breaking strength of refrozen surimi waslow In contrast the breaking strength of the 1st and 2nd F-Tcycle samples showed nearly the same value e breakingstrain of the control sample was higher than that of the 1stand 2nd F-T cycle samples until a setting time of 3 hHowever all samples showed nearly the same value after 6 hof setting time Figure 3 shows the relationship betweenbreaking strength and gel stiffness of the suwari gel preparedfrom each frozen surimi sample When the plots formedclumps in the relationship diagram of breaking strengthversus gel stiffness the surimi did not form a suwari gel [8]erefore the length of the line in the relationship diagramof breaking strength versus gel stiffness indicates the level ofchange in the breaking strength and gel stiffness with settingtime us we considered that the length would be an in-dicator of suwari gel-forming ability In this study the lengthof the line was defined as the L value e L value in Figure 3is the simplified calculated length of an approximate straightline e length was calculated by substituting the minimumpoint and maximum point which were defined as theshortest and longest lengths from the origin point re-spectively into the equation for the approximate line erelationship between breaking strength and gel stiffness waslinear in the suwari gel prepared from all samples Previousstudies of surimi gel confirmed the linear relationship be-tween breaking strength and gel stiffness [6] We confirmedthe linear relationship with the suwari gel As shown inFigure 1 the plots of the surimi with poor suwari gel-forming ability formed clumps in the breaking strengthversus gel stiffness relationship diagram erefore the Lvalue of the surimi that lost its suwari gel-forming ability waslow As shown in Figure 3 the L value decreased each timethe refreezing was repeated However the slope and locationof the line were largely unchanged by refreezing is in-dicates that refreezing andor repeated refreezing of surimisimply decreased the suwari gel-forming ability withoutchanging the characteristics of surimi

Changes in the breaking strength and strain of the directandor two-step heated gel prepared from frozen surimi areshown in Figure 4 e difference in breaking strength andbreaking strain of the direct heated gel (ie setting time of0 h) prepared for all samples was small In contrast thebreaking strength and breaking strain value of the two-stepheated gel prepared from the control sample was higher thanthat of those prepared from the refrozen sample As ob-served for the suwari gel the breaking strength and breakingstrain of the two-step heated gel prepared from the 1st F-Tcycle sample did not differ from those of the two-step heatedgel prepared from the 2nd F-T cycle sample As shown inFigure 4 the effect of refreezing on frozen surimi wasconfirmed to be low for surimi paste products producedwithout a setting process

Figure 5 shows the relationship between breakingstrength and gel stiffness of the direct andor two-step heated

Journal of Food Quality 3

gel prepared from each frozen surimi sample e definitionof the L value is the same as that in Figure 3e relationshipbetween breaking strength and gel stiffness was linear in theheated gel prepared from all samples e L values of theheated gel prepared from each surimi sample were higherthan those of the suwari gel e value of breaking strengthand gel stiffness increased because of gelation induced byheating e L value of the heated gel decreased each timerefreezing was repeated e reduction in the L value of theheated gel by refreezing was considered to be caused by theloss of suwari gel-forming ability

Previous studies on the indicators of the suwari reactionrate focused only on breaking strength and did not consider

the concept of breaking strain [17 18] In this study the Lvalue which includes the breaking strain (ie gel stiffness iscalculated by dividing breaking strength by breaking strain)in Figures 3 and 5 well reflects the results in Figures 2 and 4as the first refreezing reduced the gel-forming ability morethan the second refreezing erefore the L value may serveas a good indicator of the suwari gel-forming ability

32 SDS-PAGE Patterns and Changes in Staining Intensity ofthe MHC Band and 150 kDa Band e SDS-PAGE patternsof suwari gel prepared from each surimi sample are shown inFigure 6 Furthermore the reduction rate of MHC band with

0

200

400

600

800

0 5 10 15 20 25

Brea

king

stre

ngth

(gf)

Setting time (h)

Control

1st F-T

2nd F-T cycle

(a)

0

5

10

15

0 5 10 15 20 25

Brea

king

stra

in (m

m)

Setting time (h)

Control

1st F-T

2nd F-T cycle

(b)

Figure 2 Changes in breaking strength and breaking strain of suwari gel prepared from each frozen surimi during setting time at 20degCError bar indicates standard deviation

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

Control

Maximum pointMinimum point

L = 875

(a)

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

1st F-T cycle

Maximum pointMinimum point

L = 690

(b)

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

2nd F-T cycle

Maximum pointMinimum point

L = 556

(c)

Figure 3 Relationship between breaking strength and gel stiffness of suwari gel prepared from each frozen surimi sample L value indicatesthe approximate length of the straight line from the minimum point to the maximum point Minimum point and maximum point weredefined as the shortest distance and longest distance from the origin respectively

4 Journal of Food Quality

setting time is show in Figure 7 During setting MHCformed a large polymer that could not be loaded onto theelectrophoresis gel erefore the MHC band of the suwarigel prepared from control surimi decreased for 6 h Incontrast the MHC band of the suwari gel prepared from the1st and 2nd T-F cycle surimi samples decreased more slowlythan that of the control surimi e decreasing rate of theMHC band in the suwari gel prepared from the 1st T-F cyclesurimi was the same as that of the suwari gel prepared fromthe 2nd T-F cycle surimi e SDS-PAGE patterns of thedirect andor two-step gel prepared from each surimi sampleare shown in Figure 8 and the reduction rate of the MHC

band with setting time is shown in Figure 9 e MHC bandof heated gel prepared from control surimi rapidly decreasedover the setting time As observed for the suwari gel by SDS-PAGE the MHC band of the heated gel prepared from the1st and 2nd T-F cycle surimi samples decreased more slowlythan that of control surimi e delay of MHC polymeri-zation by refreezing contributes to the decreases in thebreaking strength and breaking strain and the reduction inthe L value e changes in the staining intensity of the 150-kDa band of suwari gel and two-step heated gel are shown inTable 1 e 150-kDa band appeared by setting e stainingintensity of the 150-kDa band tended to increase when

0 5 10

Control1st F-T2nd F-T cycle

15 20 25Setting time (h)

1500

1000

500

0

Brea

king

stre

ngth

(gf)

(a)

0 5 10 15 20 25Setting time (h)

Brea

king

stra

in (m

m) 15

10

5

0

Control1st F-T2nd F-T cycle

(b)

Figure 4 Changes in breaking strength and breaking strain of direct andor two-step heated gel prepared from each frozen surimi duringthe setting time at 20degC Error bar indicates standard deviation

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

Control

L = 1467

Maximum point

Minimum point

(a)

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

1st F-T cycle

L = 1066

Maximum point

Minimum point

(b)

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

2nd F-T cycle

Maximum point

Minimum point

L = 949

(c)

Figure 5 Relationship between breaking strength and gel stiffness of direct andor two-step heated gel prepared from each frozen surimisample e L value indicates the approximate length of the straight line from the minimum point to the maximum point Minimum pointand maximum point are defined in Figure 3

Journal of Food Quality 5

Control 1st F-T cycle 2nd F-T cycle

kDa

200150120100

85

706050

40

M 0 3 6 12 24 M 0 3 6 12 24 M 0 3 6 12 24Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 6 SDS-PAGE pattern of suwari gel prepared from each frozen surimi M MHC and AC indicate the protein marker myosin heavychain and actin respectively

0

20

40

60

80

100

0 5 10 15 20 25

MH

C re

sidua

l rat

e (

)

Setting time (h)

Control1st F-T cycle2nd F-T cycle

Figure 7 Changes in MHC residual rate of suwari gel prepared from each frozen surimi with setting time

kDa

200150120100

85706050

40

0 3 6 12 24 0 3 6 12 24 0 3 6 12 24

Control 1st F-T cycle 2nd F-T cycle

Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 8 SDS-PAGE pattern of direct andor two-step heated gel prepared from each frozen surimi M MHC and AC indicate the proteinmarker myosin heavy chain and actin respectively

6 Journal of Food Quality

refreezing was repeated Konno and Imamura reported thatthe 150-kDa band appeared to be degraded by proteaseduring setting [19] erefore the MHC of refrozen surimimay be easily degraded by protease Based on these resultsthe primary cause of the decrease in gel-forming abilityinduced by refreezing is considered to be the suppression ofMHC polymerization during setting

33 Changes in Ca-ATPase Activity of Surimi by RefreezingFigure 10 shows the Ca-ATPase activity of each surimisample which decreased by approximately 30 with eachrefreezing is demonstrates that the myosin heads weredenatured by refreezing In a previous study the Ca-ATPaseactivity of surimi decreased linearly with each refreezing[20] However Ca-ATPase activity quickly decreased duringthe 1st refreezing and slowly decreased during the 2ndrefreezing is suggests that myofibrillar protein wasstrongly denatured by the 1st refreezing greatly decreasingthe quality of surimi based on the results of gel texturemeasurement and SDS-PAGE patterns erefore de-naturation of myosin heads by the 1st refreezing greatlyreduces the suwari gel-forming ability However Ca-ATPaseindicates only the denaturation of the myosin head usthe myosin rod needs to be investigated to reveal the effect ofrefreezing on the gel-forming ability of frozen surimi

40

60

80

100

MH

C re

sidua

l rat

e (

)

Control1st F-T cycle2nd F-T cycle

20

00

5 10 15 20Setting time (h)

25

Figure 9 Changes in MHC residual rate of two-step gel prepared from each frozen surimi with setting time

Table 1 e rate of 150 kDa band against all bands in SDS-PAGE of suwari gel and two-step heated gel

Setting time (h)Suwari gel Two-step heated gel

Control 1st F-T cycle 2nd F-T cycle Control 1st F-T cycle 2nd F-T cycle0 06 07 28 37 24 323 61 59 81 56 65 696 109 71 114 74 81 10712 92 92 104 92 107 13224 82 117 123 91 93 130

0

005

01

015

02

Control 1st F-T cycle 2nd F-T cycle

Ca-A

TPas

e act

ivity

(microm

olm

inm

g of

pro

tein

)

a

b

c

Figure 10 Ca-ATPase activity of each frozen surimi Error barindicates standard deviation Different letters indicate a significantdifference (plt 005)

Journal of Food Quality 7

4 Conclusions

In this study the gel-forming ability of refrozen surimi wasconfirmed to be weakened by refreezing In particular thesuwari gel-forming ability was greatly reduced by de-naturation of the myosin head upon refreezing In additionMHC polymerization during setting was suppressed byrefreezing ese findings revealed that decreasing thesuwari gel-forming ability reduced the heated gel textureerefore the influence of refreezing on the texture ofsurimi paste products without the setting process was smalle quality deterioration including myofibrillar proteindenaturation of frozen surimi was greater during the 1strefreezing than during the 2nd refreezing

Data Availability

e data used to support the findings of this study are in-cluded within the supplementary information files

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is work was supported by JSPS KAKENHI (grant no JP15K16194)

Supplementary Materials

S-Figure 1(a) breaking strength raw data (setting gel)S-Figure 1(b) breaking strain raw data (setting gel)S-Figure 2(a) calculation process of gel stiffness (setting gel)S-Figure 2(b) correlation diagram between breakingstrength and gel stiffness (setting gel) S-Figure 3(a)breaking strength raw data (double-heated gel)S-Figure 3(b) breaking strain raw data (double-heated gel)S-Figure 4(a) calculation process of gel stiffness (double-heated gel) S-Figure 4(b) correlation diagram betweenbreaking strength and gel stiffness (double-heated gel)S-Figure 5 amp Table 1 changes in MHC residual rate and150 kDa band of suwari gel prepared from each frozensurimi with setting time S-Figure 6 amp Table 1 changes inMHC residual rate and 150 kDa band of direct andor two-step heated gel prepared from each frozen surimi withsetting time S-Figure 7 Ca-ATPase activity raw data(Supplementary Materials)

References

[1] H Ando M Fukuoka O Miyawaki and T Suzuki ldquoDamageevaluation on freeze-thawing process of food by using NMRrdquoTransactions of the Japan Society of Refrigerating and AirConditioning Engineers vol 23 no 3 pp 305ndash312 2006 inJapanese

[2] S Boonsumrej S Chaiwanichsiri S Tantratian T Suzukiand R Takai ldquoEffects of freezing and thawing on the qualitychanges of tiger shrimp (Penaeus monodon) frozen by air-

blast and cryogenic freezingrdquo Journal of Food Engineeringvol 80 no 1 pp 292ndash299 2007

[3] N Kato H Nozaki K Komatsu and K Arai ldquoA new methodfor evaluation of the quality of frozen surimi from Alaskapollack relationship between myofibrillar ATPase activity andkamaboko forming ability of frozen surimirdquo Bulletin of theJapanese Society of Scientific Fisheries vol 45 no 8pp 1027ndash1032 1979 in Japanese

[4] T Numakura R Mizoguchi I Kimura et al ldquoChanges in gelforming ability and cross-linking ability of myosin heavychain of Alaska pollack surimi denatured by heat treatmentrdquoNippon Suisan Gakkaishi vol 55 no 6 pp 1083ndash1090 1989in Japanese

[5] Y Fukuda E Okazaki and R Wada ldquoEffect of fluctuations oftemperature during frozen storage on denaturation of fishmyofibrillar proteinrdquo Transactions of the Japan Society ofRefrigerating and Air Conditioning Engineers vol 23 no 3pp 335ndash340 2006 in Japanese

[6] Y Abe K Yasunaga S Kitakami Y Murakami T Ota andK-I Arai ldquoQuality of kamaboko gels from walleye pollackfrozen surimis of different grades on applying additive con-taining TGaserdquo Nippon Suisan Gakkaishi vol 62 no 3pp 439ndash445 1996 in Japanese

[7] T Okayama T Ooizumi Y Akahane S-I KitakamiY-I Abe and J Shirai ldquoChanges in physical properties ofheat-induced gel on addition of gluconate associated withsuppression of myosin denaturation in walleye pollack salt-ground surimi during preheatingrdquo Fisheries Science vol 73no 4 pp 931ndash939 2007

[8] S Kitakami Y Murakami K Yasunaga Y Abe N Kato andK-I Arai ldquoHeated gel forming ability of walleye pollackfrozen surimis of various grades as measured by physicalproperties of heated gel and its dependence on proteinconcentrationrdquo Nippon Suisan Gakkaishi vol 75 no 2pp 250ndash257 2009 in Japanese

[9] M Kunimoto T Okumura N Kato and K Arai ldquoChar-acteristic properties of heated gels formed from frozen surimiin terms of protein solubility in various solvents and effect ofaddition of albumen powder on gel-formationrdquo NipponShokuhin Kagaku Kogaku Kaishi vol 61 no 1 pp 19ndash262014 in Japanese

[10] B Y Kim D D Hamann T C Lanier andM CWu ldquoEffectsof freeze-thaw abuse on the viscosity and gel-formingproperties of surimi from two speciesrdquo Journal of Food Sci-ence vol 51 no 4 pp 951ndash956 1986

[11] E J Kang A L Hunt and J W Park ldquoEffects of salinity onphysicochemical properties of Alaska pollock surimi afterrepeated freeze-thaw cyclesrdquo Journal of Food Science vol 73no 5 pp C347ndashC355 2008

[12] B Kong Y Guo X Xia Q Liu Y Li and H ChenldquoCryoprotectants reduce protein oxidation and structuredeterioration induced by freeze-thaw cycles in common carp(Cyprinus carpio) surimirdquo Food Biophysics vol 8 no 2pp 104ndash111 2013

[13] S Abe Y Endo Y Abe et al ldquoe effect of freeze-thaw cycleson the gelation of heat-induced gel from frozen surimirdquoCryobiology and Cryotechnology vol 61 no 1 pp 45ndash532015 in Japanese

[14] T Numakura N Seki I Kimura et al ldquoCross-linking reactionof myosin in the fish paste during setting (Suwari)rdquo NipponSuisan Gakkaishi vol 51 no 9 pp 1559ndash1565 1985 inJapanese

8 Journal of Food Quality

was determined by Image J e rate of staining intensity ofthese two bands was calculated by dividing their stainingintensity by the staining intensity of all bands e MHCresidual rate was calculated assuming that the concentrationof the band with a setting time of 0 hours was 100

26 Preparation of Myofibrillar Protein Suspension fromFrozen Surimi e myofibrillar protein suspension offrozen surimi was prepared as described by Kato et al [16]with slight modificationsawed surimi was added to 01MKCl and 20mM Tris-HCl (pH 70) and then homogenizede homogenate was centrifuged at 7500times g for 20minAfter removing the supernatant 01M KCl and 20mM Tris-HCl (pH 70) were addedis process was repeated until thesupernatant was clear e precipitate was suspended in01M KCl and 20mM Tris-HCl (pH 70) and then filteredthrough a gauze e filtrate was used as the myofibrillarprotein suspension ere was almost no change in therecovered amount of myofibrillar protein from each frozensurimi e prepared myofibrillar protein suspension wasstored on ice until measurement of Ca-ATPase activity

27 Measurement of Ca-ATPase Activity e myofibrillarprotein suspension (25ndash35mgmL) was added to 01MKCl 5mMCaCl2 1mMATP and 25mMTris-maleate (pH70) e reaction was conducted at 25degC e reaction wasstopped by adding 15 perchloric acid at each reactiontime e supernatant was added to sulfate-molybdate acidsolution and Elon reagent e inorganic phosphate con-centration in the supernatant was measured by colori-metric determination at a wavelength of 640 nm Ca-ATPase activity was calculated using the following equa-tion (2)

Ca-ATPase activity ln 1minusPi0( 1113857minus ln 1minusPit( 11138571113864 1113865 times1000

t

times1

Cmmicromolmiddotminminus1 middotmgof proteinminus11113960 1113961

(2)

where t is the time of reaction Pi0 is the inorganic phosphateacid concentration at a reaction time 0min (blank) Pit is theinorganic phosphate acid concentration after the reactionand Cm is the concentration of myofibrillar protein in thereaction reagent

e protein concentration of the myofibrillar proteinsuspension used in the reaction was determined by the biuretmethod using bovine serum albumin as a standard

28 Statistical Analysis Statistical analysis was performedusing the bell curve for Excel (Social Survey Research In-formation Co Ltd Tokyo Japan) e results were com-pared using the least significant difference method atplt 005

3 Results and Discussion

31 Gel Texture Measurement Changes in the breakingstrength and strain of suwari gel prepared from frozensurimi are shown in Figure 2 Compared to that of thecontrol sample the breaking strength of refrozen surimi waslow In contrast the breaking strength of the 1st and 2nd F-Tcycle samples showed nearly the same value e breakingstrain of the control sample was higher than that of the 1stand 2nd F-T cycle samples until a setting time of 3 hHowever all samples showed nearly the same value after 6 hof setting time Figure 3 shows the relationship betweenbreaking strength and gel stiffness of the suwari gel preparedfrom each frozen surimi sample When the plots formedclumps in the relationship diagram of breaking strengthversus gel stiffness the surimi did not form a suwari gel [8]erefore the length of the line in the relationship diagramof breaking strength versus gel stiffness indicates the level ofchange in the breaking strength and gel stiffness with settingtime us we considered that the length would be an in-dicator of suwari gel-forming ability In this study the lengthof the line was defined as the L value e L value in Figure 3is the simplified calculated length of an approximate straightline e length was calculated by substituting the minimumpoint and maximum point which were defined as theshortest and longest lengths from the origin point re-spectively into the equation for the approximate line erelationship between breaking strength and gel stiffness waslinear in the suwari gel prepared from all samples Previousstudies of surimi gel confirmed the linear relationship be-tween breaking strength and gel stiffness [6] We confirmedthe linear relationship with the suwari gel As shown inFigure 1 the plots of the surimi with poor suwari gel-forming ability formed clumps in the breaking strengthversus gel stiffness relationship diagram erefore the Lvalue of the surimi that lost its suwari gel-forming ability waslow As shown in Figure 3 the L value decreased each timethe refreezing was repeated However the slope and locationof the line were largely unchanged by refreezing is in-dicates that refreezing andor repeated refreezing of surimisimply decreased the suwari gel-forming ability withoutchanging the characteristics of surimi

Changes in the breaking strength and strain of the directandor two-step heated gel prepared from frozen surimi areshown in Figure 4 e difference in breaking strength andbreaking strain of the direct heated gel (ie setting time of0 h) prepared for all samples was small In contrast thebreaking strength and breaking strain value of the two-stepheated gel prepared from the control sample was higher thanthat of those prepared from the refrozen sample As ob-served for the suwari gel the breaking strength and breakingstrain of the two-step heated gel prepared from the 1st F-Tcycle sample did not differ from those of the two-step heatedgel prepared from the 2nd F-T cycle sample As shown inFigure 4 the effect of refreezing on frozen surimi wasconfirmed to be low for surimi paste products producedwithout a setting process

Figure 5 shows the relationship between breakingstrength and gel stiffness of the direct andor two-step heated

Journal of Food Quality 3

gel prepared from each frozen surimi sample e definitionof the L value is the same as that in Figure 3e relationshipbetween breaking strength and gel stiffness was linear in theheated gel prepared from all samples e L values of theheated gel prepared from each surimi sample were higherthan those of the suwari gel e value of breaking strengthand gel stiffness increased because of gelation induced byheating e L value of the heated gel decreased each timerefreezing was repeated e reduction in the L value of theheated gel by refreezing was considered to be caused by theloss of suwari gel-forming ability

Previous studies on the indicators of the suwari reactionrate focused only on breaking strength and did not consider

the concept of breaking strain [17 18] In this study the Lvalue which includes the breaking strain (ie gel stiffness iscalculated by dividing breaking strength by breaking strain)in Figures 3 and 5 well reflects the results in Figures 2 and 4as the first refreezing reduced the gel-forming ability morethan the second refreezing erefore the L value may serveas a good indicator of the suwari gel-forming ability

32 SDS-PAGE Patterns and Changes in Staining Intensity ofthe MHC Band and 150 kDa Band e SDS-PAGE patternsof suwari gel prepared from each surimi sample are shown inFigure 6 Furthermore the reduction rate of MHC band with

0

200

400

600

800

0 5 10 15 20 25

Brea

king

stre

ngth

(gf)

Setting time (h)

Control

1st F-T

2nd F-T cycle

(a)

0

5

10

15

0 5 10 15 20 25

Brea

king

stra

in (m

m)

Setting time (h)

Control

1st F-T

2nd F-T cycle

(b)

Figure 2 Changes in breaking strength and breaking strain of suwari gel prepared from each frozen surimi during setting time at 20degCError bar indicates standard deviation

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

Control

Maximum pointMinimum point

L = 875

(a)

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

1st F-T cycle

Maximum pointMinimum point

L = 690

(b)

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

2nd F-T cycle

Maximum pointMinimum point

L = 556

(c)

Figure 3 Relationship between breaking strength and gel stiffness of suwari gel prepared from each frozen surimi sample L value indicatesthe approximate length of the straight line from the minimum point to the maximum point Minimum point and maximum point weredefined as the shortest distance and longest distance from the origin respectively

4 Journal of Food Quality

setting time is show in Figure 7 During setting MHCformed a large polymer that could not be loaded onto theelectrophoresis gel erefore the MHC band of the suwarigel prepared from control surimi decreased for 6 h Incontrast the MHC band of the suwari gel prepared from the1st and 2nd T-F cycle surimi samples decreased more slowlythan that of the control surimi e decreasing rate of theMHC band in the suwari gel prepared from the 1st T-F cyclesurimi was the same as that of the suwari gel prepared fromthe 2nd T-F cycle surimi e SDS-PAGE patterns of thedirect andor two-step gel prepared from each surimi sampleare shown in Figure 8 and the reduction rate of the MHC

band with setting time is shown in Figure 9 e MHC bandof heated gel prepared from control surimi rapidly decreasedover the setting time As observed for the suwari gel by SDS-PAGE the MHC band of the heated gel prepared from the1st and 2nd T-F cycle surimi samples decreased more slowlythan that of control surimi e delay of MHC polymeri-zation by refreezing contributes to the decreases in thebreaking strength and breaking strain and the reduction inthe L value e changes in the staining intensity of the 150-kDa band of suwari gel and two-step heated gel are shown inTable 1 e 150-kDa band appeared by setting e stainingintensity of the 150-kDa band tended to increase when

0 5 10

Control1st F-T2nd F-T cycle

15 20 25Setting time (h)

1500

1000

500

0

Brea

king

stre

ngth

(gf)

(a)

0 5 10 15 20 25Setting time (h)

Brea

king

stra

in (m

m) 15

10

5

0

Control1st F-T2nd F-T cycle

(b)

Figure 4 Changes in breaking strength and breaking strain of direct andor two-step heated gel prepared from each frozen surimi duringthe setting time at 20degC Error bar indicates standard deviation

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

Control

L = 1467

Maximum point

Minimum point

(a)

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

1st F-T cycle

L = 1066

Maximum point

Minimum point

(b)

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

2nd F-T cycle

Maximum point

Minimum point

L = 949

(c)

Figure 5 Relationship between breaking strength and gel stiffness of direct andor two-step heated gel prepared from each frozen surimisample e L value indicates the approximate length of the straight line from the minimum point to the maximum point Minimum pointand maximum point are defined in Figure 3

Journal of Food Quality 5

Control 1st F-T cycle 2nd F-T cycle

kDa

200150120100

85

706050

40

M 0 3 6 12 24 M 0 3 6 12 24 M 0 3 6 12 24Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 6 SDS-PAGE pattern of suwari gel prepared from each frozen surimi M MHC and AC indicate the protein marker myosin heavychain and actin respectively

0

20

40

60

80

100

0 5 10 15 20 25

MH

C re

sidua

l rat

e (

)

Setting time (h)

Control1st F-T cycle2nd F-T cycle

Figure 7 Changes in MHC residual rate of suwari gel prepared from each frozen surimi with setting time

kDa

200150120100

85706050

40

0 3 6 12 24 0 3 6 12 24 0 3 6 12 24

Control 1st F-T cycle 2nd F-T cycle

Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 8 SDS-PAGE pattern of direct andor two-step heated gel prepared from each frozen surimi M MHC and AC indicate the proteinmarker myosin heavy chain and actin respectively

6 Journal of Food Quality

refreezing was repeated Konno and Imamura reported thatthe 150-kDa band appeared to be degraded by proteaseduring setting [19] erefore the MHC of refrozen surimimay be easily degraded by protease Based on these resultsthe primary cause of the decrease in gel-forming abilityinduced by refreezing is considered to be the suppression ofMHC polymerization during setting

33 Changes in Ca-ATPase Activity of Surimi by RefreezingFigure 10 shows the Ca-ATPase activity of each surimisample which decreased by approximately 30 with eachrefreezing is demonstrates that the myosin heads weredenatured by refreezing In a previous study the Ca-ATPaseactivity of surimi decreased linearly with each refreezing[20] However Ca-ATPase activity quickly decreased duringthe 1st refreezing and slowly decreased during the 2ndrefreezing is suggests that myofibrillar protein wasstrongly denatured by the 1st refreezing greatly decreasingthe quality of surimi based on the results of gel texturemeasurement and SDS-PAGE patterns erefore de-naturation of myosin heads by the 1st refreezing greatlyreduces the suwari gel-forming ability However Ca-ATPaseindicates only the denaturation of the myosin head usthe myosin rod needs to be investigated to reveal the effect ofrefreezing on the gel-forming ability of frozen surimi

40

60

80

100

MH

C re

sidua

l rat

e (

)

Control1st F-T cycle2nd F-T cycle

20

00

5 10 15 20Setting time (h)

25

Figure 9 Changes in MHC residual rate of two-step gel prepared from each frozen surimi with setting time

Table 1 e rate of 150 kDa band against all bands in SDS-PAGE of suwari gel and two-step heated gel

Setting time (h)Suwari gel Two-step heated gel

Control 1st F-T cycle 2nd F-T cycle Control 1st F-T cycle 2nd F-T cycle0 06 07 28 37 24 323 61 59 81 56 65 696 109 71 114 74 81 10712 92 92 104 92 107 13224 82 117 123 91 93 130

0

005

01

015

02

Control 1st F-T cycle 2nd F-T cycle

Ca-A

TPas

e act

ivity

(microm

olm

inm

g of

pro

tein

)

a

b

c

Figure 10 Ca-ATPase activity of each frozen surimi Error barindicates standard deviation Different letters indicate a significantdifference (plt 005)

Journal of Food Quality 7

4 Conclusions

In this study the gel-forming ability of refrozen surimi wasconfirmed to be weakened by refreezing In particular thesuwari gel-forming ability was greatly reduced by de-naturation of the myosin head upon refreezing In additionMHC polymerization during setting was suppressed byrefreezing ese findings revealed that decreasing thesuwari gel-forming ability reduced the heated gel textureerefore the influence of refreezing on the texture ofsurimi paste products without the setting process was smalle quality deterioration including myofibrillar proteindenaturation of frozen surimi was greater during the 1strefreezing than during the 2nd refreezing

Data Availability

e data used to support the findings of this study are in-cluded within the supplementary information files

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is work was supported by JSPS KAKENHI (grant no JP15K16194)

Supplementary Materials

S-Figure 1(a) breaking strength raw data (setting gel)S-Figure 1(b) breaking strain raw data (setting gel)S-Figure 2(a) calculation process of gel stiffness (setting gel)S-Figure 2(b) correlation diagram between breakingstrength and gel stiffness (setting gel) S-Figure 3(a)breaking strength raw data (double-heated gel)S-Figure 3(b) breaking strain raw data (double-heated gel)S-Figure 4(a) calculation process of gel stiffness (double-heated gel) S-Figure 4(b) correlation diagram betweenbreaking strength and gel stiffness (double-heated gel)S-Figure 5 amp Table 1 changes in MHC residual rate and150 kDa band of suwari gel prepared from each frozensurimi with setting time S-Figure 6 amp Table 1 changes inMHC residual rate and 150 kDa band of direct andor two-step heated gel prepared from each frozen surimi withsetting time S-Figure 7 Ca-ATPase activity raw data(Supplementary Materials)

References

[1] H Ando M Fukuoka O Miyawaki and T Suzuki ldquoDamageevaluation on freeze-thawing process of food by using NMRrdquoTransactions of the Japan Society of Refrigerating and AirConditioning Engineers vol 23 no 3 pp 305ndash312 2006 inJapanese

[2] S Boonsumrej S Chaiwanichsiri S Tantratian T Suzukiand R Takai ldquoEffects of freezing and thawing on the qualitychanges of tiger shrimp (Penaeus monodon) frozen by air-

blast and cryogenic freezingrdquo Journal of Food Engineeringvol 80 no 1 pp 292ndash299 2007

[3] N Kato H Nozaki K Komatsu and K Arai ldquoA new methodfor evaluation of the quality of frozen surimi from Alaskapollack relationship between myofibrillar ATPase activity andkamaboko forming ability of frozen surimirdquo Bulletin of theJapanese Society of Scientific Fisheries vol 45 no 8pp 1027ndash1032 1979 in Japanese

[4] T Numakura R Mizoguchi I Kimura et al ldquoChanges in gelforming ability and cross-linking ability of myosin heavychain of Alaska pollack surimi denatured by heat treatmentrdquoNippon Suisan Gakkaishi vol 55 no 6 pp 1083ndash1090 1989in Japanese

[5] Y Fukuda E Okazaki and R Wada ldquoEffect of fluctuations oftemperature during frozen storage on denaturation of fishmyofibrillar proteinrdquo Transactions of the Japan Society ofRefrigerating and Air Conditioning Engineers vol 23 no 3pp 335ndash340 2006 in Japanese

[6] Y Abe K Yasunaga S Kitakami Y Murakami T Ota andK-I Arai ldquoQuality of kamaboko gels from walleye pollackfrozen surimis of different grades on applying additive con-taining TGaserdquo Nippon Suisan Gakkaishi vol 62 no 3pp 439ndash445 1996 in Japanese

[7] T Okayama T Ooizumi Y Akahane S-I KitakamiY-I Abe and J Shirai ldquoChanges in physical properties ofheat-induced gel on addition of gluconate associated withsuppression of myosin denaturation in walleye pollack salt-ground surimi during preheatingrdquo Fisheries Science vol 73no 4 pp 931ndash939 2007

[8] S Kitakami Y Murakami K Yasunaga Y Abe N Kato andK-I Arai ldquoHeated gel forming ability of walleye pollackfrozen surimis of various grades as measured by physicalproperties of heated gel and its dependence on proteinconcentrationrdquo Nippon Suisan Gakkaishi vol 75 no 2pp 250ndash257 2009 in Japanese

[9] M Kunimoto T Okumura N Kato and K Arai ldquoChar-acteristic properties of heated gels formed from frozen surimiin terms of protein solubility in various solvents and effect ofaddition of albumen powder on gel-formationrdquo NipponShokuhin Kagaku Kogaku Kaishi vol 61 no 1 pp 19ndash262014 in Japanese

[10] B Y Kim D D Hamann T C Lanier andM CWu ldquoEffectsof freeze-thaw abuse on the viscosity and gel-formingproperties of surimi from two speciesrdquo Journal of Food Sci-ence vol 51 no 4 pp 951ndash956 1986

[11] E J Kang A L Hunt and J W Park ldquoEffects of salinity onphysicochemical properties of Alaska pollock surimi afterrepeated freeze-thaw cyclesrdquo Journal of Food Science vol 73no 5 pp C347ndashC355 2008

[12] B Kong Y Guo X Xia Q Liu Y Li and H ChenldquoCryoprotectants reduce protein oxidation and structuredeterioration induced by freeze-thaw cycles in common carp(Cyprinus carpio) surimirdquo Food Biophysics vol 8 no 2pp 104ndash111 2013

[13] S Abe Y Endo Y Abe et al ldquoe effect of freeze-thaw cycleson the gelation of heat-induced gel from frozen surimirdquoCryobiology and Cryotechnology vol 61 no 1 pp 45ndash532015 in Japanese

[14] T Numakura N Seki I Kimura et al ldquoCross-linking reactionof myosin in the fish paste during setting (Suwari)rdquo NipponSuisan Gakkaishi vol 51 no 9 pp 1559ndash1565 1985 inJapanese

8 Journal of Food Quality

gel prepared from each frozen surimi sample e definitionof the L value is the same as that in Figure 3e relationshipbetween breaking strength and gel stiffness was linear in theheated gel prepared from all samples e L values of theheated gel prepared from each surimi sample were higherthan those of the suwari gel e value of breaking strengthand gel stiffness increased because of gelation induced byheating e L value of the heated gel decreased each timerefreezing was repeated e reduction in the L value of theheated gel by refreezing was considered to be caused by theloss of suwari gel-forming ability

Previous studies on the indicators of the suwari reactionrate focused only on breaking strength and did not consider

the concept of breaking strain [17 18] In this study the Lvalue which includes the breaking strain (ie gel stiffness iscalculated by dividing breaking strength by breaking strain)in Figures 3 and 5 well reflects the results in Figures 2 and 4as the first refreezing reduced the gel-forming ability morethan the second refreezing erefore the L value may serveas a good indicator of the suwari gel-forming ability

32 SDS-PAGE Patterns and Changes in Staining Intensity ofthe MHC Band and 150 kDa Band e SDS-PAGE patternsof suwari gel prepared from each surimi sample are shown inFigure 6 Furthermore the reduction rate of MHC band with

0

200

400

600

800

0 5 10 15 20 25

Brea

king

stre

ngth

(gf)

Setting time (h)

Control

1st F-T

2nd F-T cycle

(a)

0

5

10

15

0 5 10 15 20 25

Brea

king

stra

in (m

m)

Setting time (h)

Control

1st F-T

2nd F-T cycle

(b)

Figure 2 Changes in breaking strength and breaking strain of suwari gel prepared from each frozen surimi during setting time at 20degCError bar indicates standard deviation

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

Control

Maximum pointMinimum point

L = 875

(a)

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

1st F-T cycle

Maximum pointMinimum point

L = 690

(b)

1000

800

600

400

200

0

Brea

king

stre

ngth

(gf)

0 200 400 600Gel stiffness (gfcm)

2nd F-T cycle

Maximum pointMinimum point

L = 556

(c)

Figure 3 Relationship between breaking strength and gel stiffness of suwari gel prepared from each frozen surimi sample L value indicatesthe approximate length of the straight line from the minimum point to the maximum point Minimum point and maximum point weredefined as the shortest distance and longest distance from the origin respectively

4 Journal of Food Quality

setting time is show in Figure 7 During setting MHCformed a large polymer that could not be loaded onto theelectrophoresis gel erefore the MHC band of the suwarigel prepared from control surimi decreased for 6 h Incontrast the MHC band of the suwari gel prepared from the1st and 2nd T-F cycle surimi samples decreased more slowlythan that of the control surimi e decreasing rate of theMHC band in the suwari gel prepared from the 1st T-F cyclesurimi was the same as that of the suwari gel prepared fromthe 2nd T-F cycle surimi e SDS-PAGE patterns of thedirect andor two-step gel prepared from each surimi sampleare shown in Figure 8 and the reduction rate of the MHC

band with setting time is shown in Figure 9 e MHC bandof heated gel prepared from control surimi rapidly decreasedover the setting time As observed for the suwari gel by SDS-PAGE the MHC band of the heated gel prepared from the1st and 2nd T-F cycle surimi samples decreased more slowlythan that of control surimi e delay of MHC polymeri-zation by refreezing contributes to the decreases in thebreaking strength and breaking strain and the reduction inthe L value e changes in the staining intensity of the 150-kDa band of suwari gel and two-step heated gel are shown inTable 1 e 150-kDa band appeared by setting e stainingintensity of the 150-kDa band tended to increase when

0 5 10

Control1st F-T2nd F-T cycle

15 20 25Setting time (h)

1500

1000

500

0

Brea

king

stre

ngth

(gf)

(a)

0 5 10 15 20 25Setting time (h)

Brea

king

stra

in (m

m) 15

10

5

0

Control1st F-T2nd F-T cycle

(b)

Figure 4 Changes in breaking strength and breaking strain of direct andor two-step heated gel prepared from each frozen surimi duringthe setting time at 20degC Error bar indicates standard deviation

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

Control

L = 1467

Maximum point

Minimum point

(a)

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

1st F-T cycle

L = 1066

Maximum point

Minimum point

(b)

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

2nd F-T cycle

Maximum point

Minimum point

L = 949

(c)

Figure 5 Relationship between breaking strength and gel stiffness of direct andor two-step heated gel prepared from each frozen surimisample e L value indicates the approximate length of the straight line from the minimum point to the maximum point Minimum pointand maximum point are defined in Figure 3

Journal of Food Quality 5

Control 1st F-T cycle 2nd F-T cycle

kDa

200150120100

85

706050

40

M 0 3 6 12 24 M 0 3 6 12 24 M 0 3 6 12 24Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 6 SDS-PAGE pattern of suwari gel prepared from each frozen surimi M MHC and AC indicate the protein marker myosin heavychain and actin respectively

0

20

40

60

80

100

0 5 10 15 20 25

MH

C re

sidua

l rat

e (

)

Setting time (h)

Control1st F-T cycle2nd F-T cycle

Figure 7 Changes in MHC residual rate of suwari gel prepared from each frozen surimi with setting time

kDa

200150120100

85706050

40

0 3 6 12 24 0 3 6 12 24 0 3 6 12 24

Control 1st F-T cycle 2nd F-T cycle

Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 8 SDS-PAGE pattern of direct andor two-step heated gel prepared from each frozen surimi M MHC and AC indicate the proteinmarker myosin heavy chain and actin respectively

6 Journal of Food Quality

refreezing was repeated Konno and Imamura reported thatthe 150-kDa band appeared to be degraded by proteaseduring setting [19] erefore the MHC of refrozen surimimay be easily degraded by protease Based on these resultsthe primary cause of the decrease in gel-forming abilityinduced by refreezing is considered to be the suppression ofMHC polymerization during setting

33 Changes in Ca-ATPase Activity of Surimi by RefreezingFigure 10 shows the Ca-ATPase activity of each surimisample which decreased by approximately 30 with eachrefreezing is demonstrates that the myosin heads weredenatured by refreezing In a previous study the Ca-ATPaseactivity of surimi decreased linearly with each refreezing[20] However Ca-ATPase activity quickly decreased duringthe 1st refreezing and slowly decreased during the 2ndrefreezing is suggests that myofibrillar protein wasstrongly denatured by the 1st refreezing greatly decreasingthe quality of surimi based on the results of gel texturemeasurement and SDS-PAGE patterns erefore de-naturation of myosin heads by the 1st refreezing greatlyreduces the suwari gel-forming ability However Ca-ATPaseindicates only the denaturation of the myosin head usthe myosin rod needs to be investigated to reveal the effect ofrefreezing on the gel-forming ability of frozen surimi

40

60

80

100

MH

C re

sidua

l rat

e (

)

Control1st F-T cycle2nd F-T cycle

20

00

5 10 15 20Setting time (h)

25

Figure 9 Changes in MHC residual rate of two-step gel prepared from each frozen surimi with setting time

Table 1 e rate of 150 kDa band against all bands in SDS-PAGE of suwari gel and two-step heated gel

Setting time (h)Suwari gel Two-step heated gel

Control 1st F-T cycle 2nd F-T cycle Control 1st F-T cycle 2nd F-T cycle0 06 07 28 37 24 323 61 59 81 56 65 696 109 71 114 74 81 10712 92 92 104 92 107 13224 82 117 123 91 93 130

0

005

01

015

02

Control 1st F-T cycle 2nd F-T cycle

Ca-A

TPas

e act

ivity

(microm

olm

inm

g of

pro

tein

)

a

b

c

Figure 10 Ca-ATPase activity of each frozen surimi Error barindicates standard deviation Different letters indicate a significantdifference (plt 005)

Journal of Food Quality 7

4 Conclusions

In this study the gel-forming ability of refrozen surimi wasconfirmed to be weakened by refreezing In particular thesuwari gel-forming ability was greatly reduced by de-naturation of the myosin head upon refreezing In additionMHC polymerization during setting was suppressed byrefreezing ese findings revealed that decreasing thesuwari gel-forming ability reduced the heated gel textureerefore the influence of refreezing on the texture ofsurimi paste products without the setting process was smalle quality deterioration including myofibrillar proteindenaturation of frozen surimi was greater during the 1strefreezing than during the 2nd refreezing

Data Availability

e data used to support the findings of this study are in-cluded within the supplementary information files

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is work was supported by JSPS KAKENHI (grant no JP15K16194)

Supplementary Materials

S-Figure 1(a) breaking strength raw data (setting gel)S-Figure 1(b) breaking strain raw data (setting gel)S-Figure 2(a) calculation process of gel stiffness (setting gel)S-Figure 2(b) correlation diagram between breakingstrength and gel stiffness (setting gel) S-Figure 3(a)breaking strength raw data (double-heated gel)S-Figure 3(b) breaking strain raw data (double-heated gel)S-Figure 4(a) calculation process of gel stiffness (double-heated gel) S-Figure 4(b) correlation diagram betweenbreaking strength and gel stiffness (double-heated gel)S-Figure 5 amp Table 1 changes in MHC residual rate and150 kDa band of suwari gel prepared from each frozensurimi with setting time S-Figure 6 amp Table 1 changes inMHC residual rate and 150 kDa band of direct andor two-step heated gel prepared from each frozen surimi withsetting time S-Figure 7 Ca-ATPase activity raw data(Supplementary Materials)

References

[1] H Ando M Fukuoka O Miyawaki and T Suzuki ldquoDamageevaluation on freeze-thawing process of food by using NMRrdquoTransactions of the Japan Society of Refrigerating and AirConditioning Engineers vol 23 no 3 pp 305ndash312 2006 inJapanese

[2] S Boonsumrej S Chaiwanichsiri S Tantratian T Suzukiand R Takai ldquoEffects of freezing and thawing on the qualitychanges of tiger shrimp (Penaeus monodon) frozen by air-

blast and cryogenic freezingrdquo Journal of Food Engineeringvol 80 no 1 pp 292ndash299 2007

[3] N Kato H Nozaki K Komatsu and K Arai ldquoA new methodfor evaluation of the quality of frozen surimi from Alaskapollack relationship between myofibrillar ATPase activity andkamaboko forming ability of frozen surimirdquo Bulletin of theJapanese Society of Scientific Fisheries vol 45 no 8pp 1027ndash1032 1979 in Japanese

[4] T Numakura R Mizoguchi I Kimura et al ldquoChanges in gelforming ability and cross-linking ability of myosin heavychain of Alaska pollack surimi denatured by heat treatmentrdquoNippon Suisan Gakkaishi vol 55 no 6 pp 1083ndash1090 1989in Japanese

[5] Y Fukuda E Okazaki and R Wada ldquoEffect of fluctuations oftemperature during frozen storage on denaturation of fishmyofibrillar proteinrdquo Transactions of the Japan Society ofRefrigerating and Air Conditioning Engineers vol 23 no 3pp 335ndash340 2006 in Japanese

[6] Y Abe K Yasunaga S Kitakami Y Murakami T Ota andK-I Arai ldquoQuality of kamaboko gels from walleye pollackfrozen surimis of different grades on applying additive con-taining TGaserdquo Nippon Suisan Gakkaishi vol 62 no 3pp 439ndash445 1996 in Japanese

[7] T Okayama T Ooizumi Y Akahane S-I KitakamiY-I Abe and J Shirai ldquoChanges in physical properties ofheat-induced gel on addition of gluconate associated withsuppression of myosin denaturation in walleye pollack salt-ground surimi during preheatingrdquo Fisheries Science vol 73no 4 pp 931ndash939 2007

[8] S Kitakami Y Murakami K Yasunaga Y Abe N Kato andK-I Arai ldquoHeated gel forming ability of walleye pollackfrozen surimis of various grades as measured by physicalproperties of heated gel and its dependence on proteinconcentrationrdquo Nippon Suisan Gakkaishi vol 75 no 2pp 250ndash257 2009 in Japanese

[9] M Kunimoto T Okumura N Kato and K Arai ldquoChar-acteristic properties of heated gels formed from frozen surimiin terms of protein solubility in various solvents and effect ofaddition of albumen powder on gel-formationrdquo NipponShokuhin Kagaku Kogaku Kaishi vol 61 no 1 pp 19ndash262014 in Japanese

[10] B Y Kim D D Hamann T C Lanier andM CWu ldquoEffectsof freeze-thaw abuse on the viscosity and gel-formingproperties of surimi from two speciesrdquo Journal of Food Sci-ence vol 51 no 4 pp 951ndash956 1986

[11] E J Kang A L Hunt and J W Park ldquoEffects of salinity onphysicochemical properties of Alaska pollock surimi afterrepeated freeze-thaw cyclesrdquo Journal of Food Science vol 73no 5 pp C347ndashC355 2008

[12] B Kong Y Guo X Xia Q Liu Y Li and H ChenldquoCryoprotectants reduce protein oxidation and structuredeterioration induced by freeze-thaw cycles in common carp(Cyprinus carpio) surimirdquo Food Biophysics vol 8 no 2pp 104ndash111 2013

[13] S Abe Y Endo Y Abe et al ldquoe effect of freeze-thaw cycleson the gelation of heat-induced gel from frozen surimirdquoCryobiology and Cryotechnology vol 61 no 1 pp 45ndash532015 in Japanese

[14] T Numakura N Seki I Kimura et al ldquoCross-linking reactionof myosin in the fish paste during setting (Suwari)rdquo NipponSuisan Gakkaishi vol 51 no 9 pp 1559ndash1565 1985 inJapanese

8 Journal of Food Quality

setting time is show in Figure 7 During setting MHCformed a large polymer that could not be loaded onto theelectrophoresis gel erefore the MHC band of the suwarigel prepared from control surimi decreased for 6 h Incontrast the MHC band of the suwari gel prepared from the1st and 2nd T-F cycle surimi samples decreased more slowlythan that of the control surimi e decreasing rate of theMHC band in the suwari gel prepared from the 1st T-F cyclesurimi was the same as that of the suwari gel prepared fromthe 2nd T-F cycle surimi e SDS-PAGE patterns of thedirect andor two-step gel prepared from each surimi sampleare shown in Figure 8 and the reduction rate of the MHC

band with setting time is shown in Figure 9 e MHC bandof heated gel prepared from control surimi rapidly decreasedover the setting time As observed for the suwari gel by SDS-PAGE the MHC band of the heated gel prepared from the1st and 2nd T-F cycle surimi samples decreased more slowlythan that of control surimi e delay of MHC polymeri-zation by refreezing contributes to the decreases in thebreaking strength and breaking strain and the reduction inthe L value e changes in the staining intensity of the 150-kDa band of suwari gel and two-step heated gel are shown inTable 1 e 150-kDa band appeared by setting e stainingintensity of the 150-kDa band tended to increase when

0 5 10

Control1st F-T2nd F-T cycle

15 20 25Setting time (h)

1500

1000

500

0

Brea

king

stre

ngth

(gf)

(a)

0 5 10 15 20 25Setting time (h)

Brea

king

stra

in (m

m) 15

10

5

0

Control1st F-T2nd F-T cycle

(b)

Figure 4 Changes in breaking strength and breaking strain of direct andor two-step heated gel prepared from each frozen surimi duringthe setting time at 20degC Error bar indicates standard deviation

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

Control

L = 1467

Maximum point

Minimum point

(a)

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

1st F-T cycle

L = 1066

Maximum point

Minimum point

(b)

0

500

1000

1500

2000

0 500 1000

Brea

king

stre

ngth

(gf)

Gel stiffness (gfcm)

2nd F-T cycle

Maximum point

Minimum point

L = 949

(c)

Figure 5 Relationship between breaking strength and gel stiffness of direct andor two-step heated gel prepared from each frozen surimisample e L value indicates the approximate length of the straight line from the minimum point to the maximum point Minimum pointand maximum point are defined in Figure 3

Journal of Food Quality 5

Control 1st F-T cycle 2nd F-T cycle

kDa

200150120100

85

706050

40

M 0 3 6 12 24 M 0 3 6 12 24 M 0 3 6 12 24Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 6 SDS-PAGE pattern of suwari gel prepared from each frozen surimi M MHC and AC indicate the protein marker myosin heavychain and actin respectively

0

20

40

60

80

100

0 5 10 15 20 25

MH

C re

sidua

l rat

e (

)

Setting time (h)

Control1st F-T cycle2nd F-T cycle

Figure 7 Changes in MHC residual rate of suwari gel prepared from each frozen surimi with setting time

kDa

200150120100

85706050

40

0 3 6 12 24 0 3 6 12 24 0 3 6 12 24

Control 1st F-T cycle 2nd F-T cycle

Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 8 SDS-PAGE pattern of direct andor two-step heated gel prepared from each frozen surimi M MHC and AC indicate the proteinmarker myosin heavy chain and actin respectively

6 Journal of Food Quality

refreezing was repeated Konno and Imamura reported thatthe 150-kDa band appeared to be degraded by proteaseduring setting [19] erefore the MHC of refrozen surimimay be easily degraded by protease Based on these resultsthe primary cause of the decrease in gel-forming abilityinduced by refreezing is considered to be the suppression ofMHC polymerization during setting

33 Changes in Ca-ATPase Activity of Surimi by RefreezingFigure 10 shows the Ca-ATPase activity of each surimisample which decreased by approximately 30 with eachrefreezing is demonstrates that the myosin heads weredenatured by refreezing In a previous study the Ca-ATPaseactivity of surimi decreased linearly with each refreezing[20] However Ca-ATPase activity quickly decreased duringthe 1st refreezing and slowly decreased during the 2ndrefreezing is suggests that myofibrillar protein wasstrongly denatured by the 1st refreezing greatly decreasingthe quality of surimi based on the results of gel texturemeasurement and SDS-PAGE patterns erefore de-naturation of myosin heads by the 1st refreezing greatlyreduces the suwari gel-forming ability However Ca-ATPaseindicates only the denaturation of the myosin head usthe myosin rod needs to be investigated to reveal the effect ofrefreezing on the gel-forming ability of frozen surimi

40

60

80

100

MH

C re

sidua

l rat

e (

)

Control1st F-T cycle2nd F-T cycle

20

00

5 10 15 20Setting time (h)

25

Figure 9 Changes in MHC residual rate of two-step gel prepared from each frozen surimi with setting time

Table 1 e rate of 150 kDa band against all bands in SDS-PAGE of suwari gel and two-step heated gel

Setting time (h)Suwari gel Two-step heated gel

Control 1st F-T cycle 2nd F-T cycle Control 1st F-T cycle 2nd F-T cycle0 06 07 28 37 24 323 61 59 81 56 65 696 109 71 114 74 81 10712 92 92 104 92 107 13224 82 117 123 91 93 130

0

005

01

015

02

Control 1st F-T cycle 2nd F-T cycle

Ca-A

TPas

e act

ivity

(microm

olm

inm

g of

pro

tein

)

a

b

c

Figure 10 Ca-ATPase activity of each frozen surimi Error barindicates standard deviation Different letters indicate a significantdifference (plt 005)

Journal of Food Quality 7

4 Conclusions

In this study the gel-forming ability of refrozen surimi wasconfirmed to be weakened by refreezing In particular thesuwari gel-forming ability was greatly reduced by de-naturation of the myosin head upon refreezing In additionMHC polymerization during setting was suppressed byrefreezing ese findings revealed that decreasing thesuwari gel-forming ability reduced the heated gel textureerefore the influence of refreezing on the texture ofsurimi paste products without the setting process was smalle quality deterioration including myofibrillar proteindenaturation of frozen surimi was greater during the 1strefreezing than during the 2nd refreezing

Data Availability

e data used to support the findings of this study are in-cluded within the supplementary information files

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is work was supported by JSPS KAKENHI (grant no JP15K16194)

Supplementary Materials

S-Figure 1(a) breaking strength raw data (setting gel)S-Figure 1(b) breaking strain raw data (setting gel)S-Figure 2(a) calculation process of gel stiffness (setting gel)S-Figure 2(b) correlation diagram between breakingstrength and gel stiffness (setting gel) S-Figure 3(a)breaking strength raw data (double-heated gel)S-Figure 3(b) breaking strain raw data (double-heated gel)S-Figure 4(a) calculation process of gel stiffness (double-heated gel) S-Figure 4(b) correlation diagram betweenbreaking strength and gel stiffness (double-heated gel)S-Figure 5 amp Table 1 changes in MHC residual rate and150 kDa band of suwari gel prepared from each frozensurimi with setting time S-Figure 6 amp Table 1 changes inMHC residual rate and 150 kDa band of direct andor two-step heated gel prepared from each frozen surimi withsetting time S-Figure 7 Ca-ATPase activity raw data(Supplementary Materials)

References

[1] H Ando M Fukuoka O Miyawaki and T Suzuki ldquoDamageevaluation on freeze-thawing process of food by using NMRrdquoTransactions of the Japan Society of Refrigerating and AirConditioning Engineers vol 23 no 3 pp 305ndash312 2006 inJapanese

[2] S Boonsumrej S Chaiwanichsiri S Tantratian T Suzukiand R Takai ldquoEffects of freezing and thawing on the qualitychanges of tiger shrimp (Penaeus monodon) frozen by air-

blast and cryogenic freezingrdquo Journal of Food Engineeringvol 80 no 1 pp 292ndash299 2007

[3] N Kato H Nozaki K Komatsu and K Arai ldquoA new methodfor evaluation of the quality of frozen surimi from Alaskapollack relationship between myofibrillar ATPase activity andkamaboko forming ability of frozen surimirdquo Bulletin of theJapanese Society of Scientific Fisheries vol 45 no 8pp 1027ndash1032 1979 in Japanese

[4] T Numakura R Mizoguchi I Kimura et al ldquoChanges in gelforming ability and cross-linking ability of myosin heavychain of Alaska pollack surimi denatured by heat treatmentrdquoNippon Suisan Gakkaishi vol 55 no 6 pp 1083ndash1090 1989in Japanese

[5] Y Fukuda E Okazaki and R Wada ldquoEffect of fluctuations oftemperature during frozen storage on denaturation of fishmyofibrillar proteinrdquo Transactions of the Japan Society ofRefrigerating and Air Conditioning Engineers vol 23 no 3pp 335ndash340 2006 in Japanese

[6] Y Abe K Yasunaga S Kitakami Y Murakami T Ota andK-I Arai ldquoQuality of kamaboko gels from walleye pollackfrozen surimis of different grades on applying additive con-taining TGaserdquo Nippon Suisan Gakkaishi vol 62 no 3pp 439ndash445 1996 in Japanese

[7] T Okayama T Ooizumi Y Akahane S-I KitakamiY-I Abe and J Shirai ldquoChanges in physical properties ofheat-induced gel on addition of gluconate associated withsuppression of myosin denaturation in walleye pollack salt-ground surimi during preheatingrdquo Fisheries Science vol 73no 4 pp 931ndash939 2007

[8] S Kitakami Y Murakami K Yasunaga Y Abe N Kato andK-I Arai ldquoHeated gel forming ability of walleye pollackfrozen surimis of various grades as measured by physicalproperties of heated gel and its dependence on proteinconcentrationrdquo Nippon Suisan Gakkaishi vol 75 no 2pp 250ndash257 2009 in Japanese

[9] M Kunimoto T Okumura N Kato and K Arai ldquoChar-acteristic properties of heated gels formed from frozen surimiin terms of protein solubility in various solvents and effect ofaddition of albumen powder on gel-formationrdquo NipponShokuhin Kagaku Kogaku Kaishi vol 61 no 1 pp 19ndash262014 in Japanese

[10] B Y Kim D D Hamann T C Lanier andM CWu ldquoEffectsof freeze-thaw abuse on the viscosity and gel-formingproperties of surimi from two speciesrdquo Journal of Food Sci-ence vol 51 no 4 pp 951ndash956 1986

[11] E J Kang A L Hunt and J W Park ldquoEffects of salinity onphysicochemical properties of Alaska pollock surimi afterrepeated freeze-thaw cyclesrdquo Journal of Food Science vol 73no 5 pp C347ndashC355 2008

[12] B Kong Y Guo X Xia Q Liu Y Li and H ChenldquoCryoprotectants reduce protein oxidation and structuredeterioration induced by freeze-thaw cycles in common carp(Cyprinus carpio) surimirdquo Food Biophysics vol 8 no 2pp 104ndash111 2013

[13] S Abe Y Endo Y Abe et al ldquoe effect of freeze-thaw cycleson the gelation of heat-induced gel from frozen surimirdquoCryobiology and Cryotechnology vol 61 no 1 pp 45ndash532015 in Japanese

[14] T Numakura N Seki I Kimura et al ldquoCross-linking reactionof myosin in the fish paste during setting (Suwari)rdquo NipponSuisan Gakkaishi vol 51 no 9 pp 1559ndash1565 1985 inJapanese

8 Journal of Food Quality

Control 1st F-T cycle 2nd F-T cycle

kDa

200150120100

85

706050

40

M 0 3 6 12 24 M 0 3 6 12 24 M 0 3 6 12 24Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 6 SDS-PAGE pattern of suwari gel prepared from each frozen surimi M MHC and AC indicate the protein marker myosin heavychain and actin respectively

0

20

40

60

80

100

0 5 10 15 20 25

MH

C re

sidua

l rat

e (

)

Setting time (h)

Control1st F-T cycle2nd F-T cycle

Figure 7 Changes in MHC residual rate of suwari gel prepared from each frozen surimi with setting time

kDa

200150120100

85706050

40

0 3 6 12 24 0 3 6 12 24 0 3 6 12 24

Control 1st F-T cycle 2nd F-T cycle

Setting time (h) Setting time (h) Setting time (h)

MHC

AC

Figure 8 SDS-PAGE pattern of direct andor two-step heated gel prepared from each frozen surimi M MHC and AC indicate the proteinmarker myosin heavy chain and actin respectively

6 Journal of Food Quality

refreezing was repeated Konno and Imamura reported thatthe 150-kDa band appeared to be degraded by proteaseduring setting [19] erefore the MHC of refrozen surimimay be easily degraded by protease Based on these resultsthe primary cause of the decrease in gel-forming abilityinduced by refreezing is considered to be the suppression ofMHC polymerization during setting

33 Changes in Ca-ATPase Activity of Surimi by RefreezingFigure 10 shows the Ca-ATPase activity of each surimisample which decreased by approximately 30 with eachrefreezing is demonstrates that the myosin heads weredenatured by refreezing In a previous study the Ca-ATPaseactivity of surimi decreased linearly with each refreezing[20] However Ca-ATPase activity quickly decreased duringthe 1st refreezing and slowly decreased during the 2ndrefreezing is suggests that myofibrillar protein wasstrongly denatured by the 1st refreezing greatly decreasingthe quality of surimi based on the results of gel texturemeasurement and SDS-PAGE patterns erefore de-naturation of myosin heads by the 1st refreezing greatlyreduces the suwari gel-forming ability However Ca-ATPaseindicates only the denaturation of the myosin head usthe myosin rod needs to be investigated to reveal the effect ofrefreezing on the gel-forming ability of frozen surimi

40

60

80

100

MH

C re

sidua

l rat

e (

)

Control1st F-T cycle2nd F-T cycle

20

00

5 10 15 20Setting time (h)

25

Figure 9 Changes in MHC residual rate of two-step gel prepared from each frozen surimi with setting time

Table 1 e rate of 150 kDa band against all bands in SDS-PAGE of suwari gel and two-step heated gel

Setting time (h)Suwari gel Two-step heated gel

Control 1st F-T cycle 2nd F-T cycle Control 1st F-T cycle 2nd F-T cycle0 06 07 28 37 24 323 61 59 81 56 65 696 109 71 114 74 81 10712 92 92 104 92 107 13224 82 117 123 91 93 130

0

005

01

015

02

Control 1st F-T cycle 2nd F-T cycle

Ca-A

TPas

e act

ivity

(microm

olm

inm

g of

pro

tein

)

a

b

c

Figure 10 Ca-ATPase activity of each frozen surimi Error barindicates standard deviation Different letters indicate a significantdifference (plt 005)

Journal of Food Quality 7

4 Conclusions

In this study the gel-forming ability of refrozen surimi wasconfirmed to be weakened by refreezing In particular thesuwari gel-forming ability was greatly reduced by de-naturation of the myosin head upon refreezing In additionMHC polymerization during setting was suppressed byrefreezing ese findings revealed that decreasing thesuwari gel-forming ability reduced the heated gel textureerefore the influence of refreezing on the texture ofsurimi paste products without the setting process was smalle quality deterioration including myofibrillar proteindenaturation of frozen surimi was greater during the 1strefreezing than during the 2nd refreezing

Data Availability

e data used to support the findings of this study are in-cluded within the supplementary information files

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is work was supported by JSPS KAKENHI (grant no JP15K16194)

Supplementary Materials

S-Figure 1(a) breaking strength raw data (setting gel)S-Figure 1(b) breaking strain raw data (setting gel)S-Figure 2(a) calculation process of gel stiffness (setting gel)S-Figure 2(b) correlation diagram between breakingstrength and gel stiffness (setting gel) S-Figure 3(a)breaking strength raw data (double-heated gel)S-Figure 3(b) breaking strain raw data (double-heated gel)S-Figure 4(a) calculation process of gel stiffness (double-heated gel) S-Figure 4(b) correlation diagram betweenbreaking strength and gel stiffness (double-heated gel)S-Figure 5 amp Table 1 changes in MHC residual rate and150 kDa band of suwari gel prepared from each frozensurimi with setting time S-Figure 6 amp Table 1 changes inMHC residual rate and 150 kDa band of direct andor two-step heated gel prepared from each frozen surimi withsetting time S-Figure 7 Ca-ATPase activity raw data(Supplementary Materials)

References

[1] H Ando M Fukuoka O Miyawaki and T Suzuki ldquoDamageevaluation on freeze-thawing process of food by using NMRrdquoTransactions of the Japan Society of Refrigerating and AirConditioning Engineers vol 23 no 3 pp 305ndash312 2006 inJapanese

[2] S Boonsumrej S Chaiwanichsiri S Tantratian T Suzukiand R Takai ldquoEffects of freezing and thawing on the qualitychanges of tiger shrimp (Penaeus monodon) frozen by air-

blast and cryogenic freezingrdquo Journal of Food Engineeringvol 80 no 1 pp 292ndash299 2007

[3] N Kato H Nozaki K Komatsu and K Arai ldquoA new methodfor evaluation of the quality of frozen surimi from Alaskapollack relationship between myofibrillar ATPase activity andkamaboko forming ability of frozen surimirdquo Bulletin of theJapanese Society of Scientific Fisheries vol 45 no 8pp 1027ndash1032 1979 in Japanese

[4] T Numakura R Mizoguchi I Kimura et al ldquoChanges in gelforming ability and cross-linking ability of myosin heavychain of Alaska pollack surimi denatured by heat treatmentrdquoNippon Suisan Gakkaishi vol 55 no 6 pp 1083ndash1090 1989in Japanese

[5] Y Fukuda E Okazaki and R Wada ldquoEffect of fluctuations oftemperature during frozen storage on denaturation of fishmyofibrillar proteinrdquo Transactions of the Japan Society ofRefrigerating and Air Conditioning Engineers vol 23 no 3pp 335ndash340 2006 in Japanese

[6] Y Abe K Yasunaga S Kitakami Y Murakami T Ota andK-I Arai ldquoQuality of kamaboko gels from walleye pollackfrozen surimis of different grades on applying additive con-taining TGaserdquo Nippon Suisan Gakkaishi vol 62 no 3pp 439ndash445 1996 in Japanese

[7] T Okayama T Ooizumi Y Akahane S-I KitakamiY-I Abe and J Shirai ldquoChanges in physical properties ofheat-induced gel on addition of gluconate associated withsuppression of myosin denaturation in walleye pollack salt-ground surimi during preheatingrdquo Fisheries Science vol 73no 4 pp 931ndash939 2007

[8] S Kitakami Y Murakami K Yasunaga Y Abe N Kato andK-I Arai ldquoHeated gel forming ability of walleye pollackfrozen surimis of various grades as measured by physicalproperties of heated gel and its dependence on proteinconcentrationrdquo Nippon Suisan Gakkaishi vol 75 no 2pp 250ndash257 2009 in Japanese

[9] M Kunimoto T Okumura N Kato and K Arai ldquoChar-acteristic properties of heated gels formed from frozen surimiin terms of protein solubility in various solvents and effect ofaddition of albumen powder on gel-formationrdquo NipponShokuhin Kagaku Kogaku Kaishi vol 61 no 1 pp 19ndash262014 in Japanese

[10] B Y Kim D D Hamann T C Lanier andM CWu ldquoEffectsof freeze-thaw abuse on the viscosity and gel-formingproperties of surimi from two speciesrdquo Journal of Food Sci-ence vol 51 no 4 pp 951ndash956 1986

[11] E J Kang A L Hunt and J W Park ldquoEffects of salinity onphysicochemical properties of Alaska pollock surimi afterrepeated freeze-thaw cyclesrdquo Journal of Food Science vol 73no 5 pp C347ndashC355 2008

[12] B Kong Y Guo X Xia Q Liu Y Li and H ChenldquoCryoprotectants reduce protein oxidation and structuredeterioration induced by freeze-thaw cycles in common carp(Cyprinus carpio) surimirdquo Food Biophysics vol 8 no 2pp 104ndash111 2013

[13] S Abe Y Endo Y Abe et al ldquoe effect of freeze-thaw cycleson the gelation of heat-induced gel from frozen surimirdquoCryobiology and Cryotechnology vol 61 no 1 pp 45ndash532015 in Japanese

[14] T Numakura N Seki I Kimura et al ldquoCross-linking reactionof myosin in the fish paste during setting (Suwari)rdquo NipponSuisan Gakkaishi vol 51 no 9 pp 1559ndash1565 1985 inJapanese

8 Journal of Food Quality

refreezing was repeated Konno and Imamura reported thatthe 150-kDa band appeared to be degraded by proteaseduring setting [19] erefore the MHC of refrozen surimimay be easily degraded by protease Based on these resultsthe primary cause of the decrease in gel-forming abilityinduced by refreezing is considered to be the suppression ofMHC polymerization during setting

33 Changes in Ca-ATPase Activity of Surimi by RefreezingFigure 10 shows the Ca-ATPase activity of each surimisample which decreased by approximately 30 with eachrefreezing is demonstrates that the myosin heads weredenatured by refreezing In a previous study the Ca-ATPaseactivity of surimi decreased linearly with each refreezing[20] However Ca-ATPase activity quickly decreased duringthe 1st refreezing and slowly decreased during the 2ndrefreezing is suggests that myofibrillar protein wasstrongly denatured by the 1st refreezing greatly decreasingthe quality of surimi based on the results of gel texturemeasurement and SDS-PAGE patterns erefore de-naturation of myosin heads by the 1st refreezing greatlyreduces the suwari gel-forming ability However Ca-ATPaseindicates only the denaturation of the myosin head usthe myosin rod needs to be investigated to reveal the effect ofrefreezing on the gel-forming ability of frozen surimi

40

60

80

100

MH

C re

sidua

l rat

e (

)

Control1st F-T cycle2nd F-T cycle

20

00

5 10 15 20Setting time (h)

25

Figure 9 Changes in MHC residual rate of two-step gel prepared from each frozen surimi with setting time

Table 1 e rate of 150 kDa band against all bands in SDS-PAGE of suwari gel and two-step heated gel

Setting time (h)Suwari gel Two-step heated gel

Control 1st F-T cycle 2nd F-T cycle Control 1st F-T cycle 2nd F-T cycle0 06 07 28 37 24 323 61 59 81 56 65 696 109 71 114 74 81 10712 92 92 104 92 107 13224 82 117 123 91 93 130

0

005

01

015

02

Control 1st F-T cycle 2nd F-T cycle

Ca-A

TPas

e act

ivity

(microm

olm

inm

g of

pro

tein

)

a

b

c

Figure 10 Ca-ATPase activity of each frozen surimi Error barindicates standard deviation Different letters indicate a significantdifference (plt 005)

Journal of Food Quality 7

4 Conclusions

In this study the gel-forming ability of refrozen surimi wasconfirmed to be weakened by refreezing In particular thesuwari gel-forming ability was greatly reduced by de-naturation of the myosin head upon refreezing In additionMHC polymerization during setting was suppressed byrefreezing ese findings revealed that decreasing thesuwari gel-forming ability reduced the heated gel textureerefore the influence of refreezing on the texture ofsurimi paste products without the setting process was smalle quality deterioration including myofibrillar proteindenaturation of frozen surimi was greater during the 1strefreezing than during the 2nd refreezing

Data Availability

e data used to support the findings of this study are in-cluded within the supplementary information files

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is work was supported by JSPS KAKENHI (grant no JP15K16194)

Supplementary Materials

S-Figure 1(a) breaking strength raw data (setting gel)S-Figure 1(b) breaking strain raw data (setting gel)S-Figure 2(a) calculation process of gel stiffness (setting gel)S-Figure 2(b) correlation diagram between breakingstrength and gel stiffness (setting gel) S-Figure 3(a)breaking strength raw data (double-heated gel)S-Figure 3(b) breaking strain raw data (double-heated gel)S-Figure 4(a) calculation process of gel stiffness (double-heated gel) S-Figure 4(b) correlation diagram betweenbreaking strength and gel stiffness (double-heated gel)S-Figure 5 amp Table 1 changes in MHC residual rate and150 kDa band of suwari gel prepared from each frozensurimi with setting time S-Figure 6 amp Table 1 changes inMHC residual rate and 150 kDa band of direct andor two-step heated gel prepared from each frozen surimi withsetting time S-Figure 7 Ca-ATPase activity raw data(Supplementary Materials)

References

[1] H Ando M Fukuoka O Miyawaki and T Suzuki ldquoDamageevaluation on freeze-thawing process of food by using NMRrdquoTransactions of the Japan Society of Refrigerating and AirConditioning Engineers vol 23 no 3 pp 305ndash312 2006 inJapanese

[2] S Boonsumrej S Chaiwanichsiri S Tantratian T Suzukiand R Takai ldquoEffects of freezing and thawing on the qualitychanges of tiger shrimp (Penaeus monodon) frozen by air-

blast and cryogenic freezingrdquo Journal of Food Engineeringvol 80 no 1 pp 292ndash299 2007

[3] N Kato H Nozaki K Komatsu and K Arai ldquoA new methodfor evaluation of the quality of frozen surimi from Alaskapollack relationship between myofibrillar ATPase activity andkamaboko forming ability of frozen surimirdquo Bulletin of theJapanese Society of Scientific Fisheries vol 45 no 8pp 1027ndash1032 1979 in Japanese

[4] T Numakura R Mizoguchi I Kimura et al ldquoChanges in gelforming ability and cross-linking ability of myosin heavychain of Alaska pollack surimi denatured by heat treatmentrdquoNippon Suisan Gakkaishi vol 55 no 6 pp 1083ndash1090 1989in Japanese

[5] Y Fukuda E Okazaki and R Wada ldquoEffect of fluctuations oftemperature during frozen storage on denaturation of fishmyofibrillar proteinrdquo Transactions of the Japan Society ofRefrigerating and Air Conditioning Engineers vol 23 no 3pp 335ndash340 2006 in Japanese

[6] Y Abe K Yasunaga S Kitakami Y Murakami T Ota andK-I Arai ldquoQuality of kamaboko gels from walleye pollackfrozen surimis of different grades on applying additive con-taining TGaserdquo Nippon Suisan Gakkaishi vol 62 no 3pp 439ndash445 1996 in Japanese

[7] T Okayama T Ooizumi Y Akahane S-I KitakamiY-I Abe and J Shirai ldquoChanges in physical properties ofheat-induced gel on addition of gluconate associated withsuppression of myosin denaturation in walleye pollack salt-ground surimi during preheatingrdquo Fisheries Science vol 73no 4 pp 931ndash939 2007

[8] S Kitakami Y Murakami K Yasunaga Y Abe N Kato andK-I Arai ldquoHeated gel forming ability of walleye pollackfrozen surimis of various grades as measured by physicalproperties of heated gel and its dependence on proteinconcentrationrdquo Nippon Suisan Gakkaishi vol 75 no 2pp 250ndash257 2009 in Japanese

[9] M Kunimoto T Okumura N Kato and K Arai ldquoChar-acteristic properties of heated gels formed from frozen surimiin terms of protein solubility in various solvents and effect ofaddition of albumen powder on gel-formationrdquo NipponShokuhin Kagaku Kogaku Kaishi vol 61 no 1 pp 19ndash262014 in Japanese

[10] B Y Kim D D Hamann T C Lanier andM CWu ldquoEffectsof freeze-thaw abuse on the viscosity and gel-formingproperties of surimi from two speciesrdquo Journal of Food Sci-ence vol 51 no 4 pp 951ndash956 1986

[11] E J Kang A L Hunt and J W Park ldquoEffects of salinity onphysicochemical properties of Alaska pollock surimi afterrepeated freeze-thaw cyclesrdquo Journal of Food Science vol 73no 5 pp C347ndashC355 2008

[12] B Kong Y Guo X Xia Q Liu Y Li and H ChenldquoCryoprotectants reduce protein oxidation and structuredeterioration induced by freeze-thaw cycles in common carp(Cyprinus carpio) surimirdquo Food Biophysics vol 8 no 2pp 104ndash111 2013

[13] S Abe Y Endo Y Abe et al ldquoe effect of freeze-thaw cycleson the gelation of heat-induced gel from frozen surimirdquoCryobiology and Cryotechnology vol 61 no 1 pp 45ndash532015 in Japanese

[14] T Numakura N Seki I Kimura et al ldquoCross-linking reactionof myosin in the fish paste during setting (Suwari)rdquo NipponSuisan Gakkaishi vol 51 no 9 pp 1559ndash1565 1985 inJapanese

8 Journal of Food Quality

4 Conclusions

In this study the gel-forming ability of refrozen surimi wasconfirmed to be weakened by refreezing In particular thesuwari gel-forming ability was greatly reduced by de-naturation of the myosin head upon refreezing In additionMHC polymerization during setting was suppressed byrefreezing ese findings revealed that decreasing thesuwari gel-forming ability reduced the heated gel textureerefore the influence of refreezing on the texture ofsurimi paste products without the setting process was smalle quality deterioration including myofibrillar proteindenaturation of frozen surimi was greater during the 1strefreezing than during the 2nd refreezing

Data Availability

e data used to support the findings of this study are in-cluded within the supplementary information files

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is work was supported by JSPS KAKENHI (grant no JP15K16194)

Supplementary Materials

S-Figure 1(a) breaking strength raw data (setting gel)S-Figure 1(b) breaking strain raw data (setting gel)S-Figure 2(a) calculation process of gel stiffness (setting gel)S-Figure 2(b) correlation diagram between breakingstrength and gel stiffness (setting gel) S-Figure 3(a)breaking strength raw data (double-heated gel)S-Figure 3(b) breaking strain raw data (double-heated gel)S-Figure 4(a) calculation process of gel stiffness (double-heated gel) S-Figure 4(b) correlation diagram betweenbreaking strength and gel stiffness (double-heated gel)S-Figure 5 amp Table 1 changes in MHC residual rate and150 kDa band of suwari gel prepared from each frozensurimi with setting time S-Figure 6 amp Table 1 changes inMHC residual rate and 150 kDa band of direct andor two-step heated gel prepared from each frozen surimi withsetting time S-Figure 7 Ca-ATPase activity raw data(Supplementary Materials)

References

[1] H Ando M Fukuoka O Miyawaki and T Suzuki ldquoDamageevaluation on freeze-thawing process of food by using NMRrdquoTransactions of the Japan Society of Refrigerating and AirConditioning Engineers vol 23 no 3 pp 305ndash312 2006 inJapanese

[2] S Boonsumrej S Chaiwanichsiri S Tantratian T Suzukiand R Takai ldquoEffects of freezing and thawing on the qualitychanges of tiger shrimp (Penaeus monodon) frozen by air-

blast and cryogenic freezingrdquo Journal of Food Engineeringvol 80 no 1 pp 292ndash299 2007

[3] N Kato H Nozaki K Komatsu and K Arai ldquoA new methodfor evaluation of the quality of frozen surimi from Alaskapollack relationship between myofibrillar ATPase activity andkamaboko forming ability of frozen surimirdquo Bulletin of theJapanese Society of Scientific Fisheries vol 45 no 8pp 1027ndash1032 1979 in Japanese

[4] T Numakura R Mizoguchi I Kimura et al ldquoChanges in gelforming ability and cross-linking ability of myosin heavychain of Alaska pollack surimi denatured by heat treatmentrdquoNippon Suisan Gakkaishi vol 55 no 6 pp 1083ndash1090 1989in Japanese

[5] Y Fukuda E Okazaki and R Wada ldquoEffect of fluctuations oftemperature during frozen storage on denaturation of fishmyofibrillar proteinrdquo Transactions of the Japan Society ofRefrigerating and Air Conditioning Engineers vol 23 no 3pp 335ndash340 2006 in Japanese

[6] Y Abe K Yasunaga S Kitakami Y Murakami T Ota andK-I Arai ldquoQuality of kamaboko gels from walleye pollackfrozen surimis of different grades on applying additive con-taining TGaserdquo Nippon Suisan Gakkaishi vol 62 no 3pp 439ndash445 1996 in Japanese

[7] T Okayama T Ooizumi Y Akahane S-I KitakamiY-I Abe and J Shirai ldquoChanges in physical properties ofheat-induced gel on addition of gluconate associated withsuppression of myosin denaturation in walleye pollack salt-ground surimi during preheatingrdquo Fisheries Science vol 73no 4 pp 931ndash939 2007

[8] S Kitakami Y Murakami K Yasunaga Y Abe N Kato andK-I Arai ldquoHeated gel forming ability of walleye pollackfrozen surimis of various grades as measured by physicalproperties of heated gel and its dependence on proteinconcentrationrdquo Nippon Suisan Gakkaishi vol 75 no 2pp 250ndash257 2009 in Japanese

[9] M Kunimoto T Okumura N Kato and K Arai ldquoChar-acteristic properties of heated gels formed from frozen surimiin terms of protein solubility in various solvents and effect ofaddition of albumen powder on gel-formationrdquo NipponShokuhin Kagaku Kogaku Kaishi vol 61 no 1 pp 19ndash262014 in Japanese

[10] B Y Kim D D Hamann T C Lanier andM CWu ldquoEffectsof freeze-thaw abuse on the viscosity and gel-formingproperties of surimi from two speciesrdquo Journal of Food Sci-ence vol 51 no 4 pp 951ndash956 1986

[11] E J Kang A L Hunt and J W Park ldquoEffects of salinity onphysicochemical properties of Alaska pollock surimi afterrepeated freeze-thaw cyclesrdquo Journal of Food Science vol 73no 5 pp C347ndashC355 2008

[12] B Kong Y Guo X Xia Q Liu Y Li and H ChenldquoCryoprotectants reduce protein oxidation and structuredeterioration induced by freeze-thaw cycles in common carp(Cyprinus carpio) surimirdquo Food Biophysics vol 8 no 2pp 104ndash111 2013

[13] S Abe Y Endo Y Abe et al ldquoe effect of freeze-thaw cycleson the gelation of heat-induced gel from frozen surimirdquoCryobiology and Cryotechnology vol 61 no 1 pp 45ndash532015 in Japanese

[14] T Numakura N Seki I Kimura et al ldquoCross-linking reactionof myosin in the fish paste during setting (Suwari)rdquo NipponSuisan Gakkaishi vol 51 no 9 pp 1559ndash1565 1985 inJapanese

8 Journal of Food Quality

[15] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227no 5259 pp 680ndash685 1970

[16] N Kato H Uchiyama S Tsukamoto and K Arai ldquoA bio-chemical study of fish myofibrillar ATPaserdquo Bulletin of theJapanese Society of Scientific Fisheries vol 43 no 7pp 857ndash867 1977 in Japanese

[17] N Kato A Hashimoto H Nozaki et al ldquoEffect of temper-ature on the rate for the setting of meat pastes from Alaskapollack white croaker and tilapiardquo Bulletin of the JapaneseSociety of Scientific Fisheries vol 50 no 12 pp 2103ndash21081984 in Japanese

[18] K Yamada T Kajita M Matsumiya and H FukushimaldquoEvaluation of the quality of frozen surimi using suwari re-action speed and activation energyrdquo CyTA-Journal of Foodvol 16 no 1 pp 723ndash729 2018

[19] K Konno and K Imamura ldquoIdentification of the 150 and70 kDa fragments generated during the incubation of saltedsurimi paste of walleye pollackrdquo Nippon Suisan Gakkaishivol 66 no 5 pp 869ndash875 2000 in Japanese

[20] T Wang Z Li N Mi et al ldquoEffects of brown algal phlor-otannins and ascorbic acid on the physiochemical propertiesof minced fish (Pagrosomus major) during freezendashthaw cy-clesrdquo International Journal of Food Science amp Technologyvol 52 no 3 pp 706ndash713 2017

Journal of Food Quality 9

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom