Food Chemisuy 406 (2028) 135007 Contents lists available nt ScienceDirect Food Chemistry ELSEVIER journal homepage: www elsevier.com/locate/foodchem ® Quality improvement of tilapia fillets by light salting during repeated sae freezing-thawing: Contribution of structural rearrangement and molecular interactions Qingqing Jiang", Shiyu Huang", Yunfan Du", Jianbo Xiao”, Mingfu Wang‘, Xichang Wang", Wenzheng Shi’, Yueliang Zhao" * cote of Fd Scns and Tcl, Shag Engiering seach Caer of Aqua Pra Proce & Prout, Shag 1201306, Cha Una, Sangha =prmint of Anal Chena and Fd Sc Fao Fo Se and Tosh, Ley of Vig - Ouese Camps £2008 Owens, Spin ta fr Avs Say, Shes Unters, She, Ce ARTICLE INFO ABSTRACT ower ‘he present sty evaluated the fies and undeying mechanisns of Ugh salting on quality properties of tap filet ing repented Srecring thawing. Light salting Was found 0 improve wate-‘olding capacity and feceetted texture softening in lpia files during repeated feezng thawing. Instead of tse istrtion and heterogeneous aggregates ia enol gioups, light alg promoted nyotbll lsassenbly and formation ofa Lee cided rtelntetwk wt he shbllaed nyo roti. The myoAs preset an aera anor ‘Tope ce phon penance wth th lof Mines, reroring the reans ory wel ant slat om ‘binds in salted groups during repeated feezing thawing. Te trctral earrangement caused by light salting faeltated the elaigement of water-holding space, uarsfrnation of ussue wate, and tsue feoveraliy Inpeoving water-holding capacity and textue properties of tapi Sts during fering tating. The Sdn Provided novel insight into che inprovement of quality propos of tain lets by ight salting when subjeced to drastic temperature Muctuatons. 1. Introduetion ‘lain has become one ofthe most essential cured freshwater fish species witha total world produetion of 4514.6 thousand tons in 2020 (AO, 2022), beeause ofits rapid growl and easy cultivation, and it is mainly traded as frozen whole fish or files (Cao ets, 2022; Subs ‘il, 2015). NaCLhas been used as a preservative for aquatic produets since ancient times, which ean reduce water activity and inbiit mi crobial growth and reproduction (Inguglia, Zliang, Tivar, Kerty, & Burgess, 2017). tis also found that NaCl can cause structural changes of myofibrillar proteins and affeet water holding capacity, texture prop: erties, and color characterises of muscle foods (Pete al, 2019), ‘where the varying degrees are dependent on salting conditions, espe lally sale concentration (Jiang, Jia et al, 2019; Jiang, Nakazawa, Hu, ‘osako, & Okazaki, 20198). Because exeessive sodium intake causes hypertension, cardiovascular disease, and other health problems (He, “Tan, Mi, 4 MacGregor, 2020), fish produets with a relatively low salt * Conesponding authors nt (<8), refered (0 a lightly salted Ash products become increasingly popular with che development of iahome economy furenty, which can be consunied or cooked directly withont desal nation and rehydration procedures (Shi, Cul, Lo, & Zhow, 2014; Jiang, Du, Nakazawa eta, 2029; Jiang, Du, Huang et al, 2022). share highly perishable ue co rich nutrients, high water content, and fragile tissue structure (Subbalal eal, 2015). The eontent and existing state of water in musele are altered, which affects the tissue Integrity and water-holding capacity during feezing-thaving. Phase transitions (liquid ~ solid and solid — liquid) and the resultant physt- cochemical changes due co concentration, extrusion, damages, ete. are tainly responsible forthe quality deterioration In feozen fish and fish produets (Jang, Jiaet al, 2019; Jiang, Du, Nakazawa et al, 2022). The ‘degrees of protein denaturation/agsregation and tissue destruction are closely related tothe morphological properties of lee crystals, including the size and distribution (Jiang, Nakazawa, Hu, Osako, & Okazaki, 2019; Jiang, Du, Nokizawa et sl, 2022), Large extracellular fee Email addres: wshigsbos.eiton QW. SAD, yueliag2110@162.com, lebanon. edt.en (. Zh). psd ory/10.1016 fodehen, 2022.135007 Received & September 2022; Recelved la 1eised form 21 November 2022: Accepted 25 November 2022 ‘Availabe online 28 Noverer 2022 ‘O3088146/0 2022 Eee Lic Al rights reserve,Okage crystals can destabilize tissue matrix, resulting in decreased recover: ability after thawing, which will be exacerbated by temperature Buc Tuntions due to recrystlliation during storage and transportation (Nakazawa & Okazaki, 2020; Zhan, Son, Zhi, & Wang, 2018). The transverse relaxation time (7) i frequently applied to asses the dis tribution, relative content, and mobility of water in foods using low field.nuclesr magnetic resonance (LENMR) (Cha, Roth, Jessen, Jakobsen, & Lerfell, 2022). Bound water (Tap, 0-1-10 ms), usually bound to maeromolecular substances, stich as protein, is highly stable in food systems, inmobilized water (Tz1, 10-150 ms) exists typically in the myofibrillar structure, and free water (Tz, > 150 ms) is usualy present ‘ouside che myofibrils ori the spaces betweet! myofibers. The mobility ‘nd relative contents of the water in diferent states can be compared sing the peak summit times Ty, Tap, and Ta, andthe area ratios ofthe corresponding peaks Pp, Pa, and Pz, respectively (Shi t al, 2020) To reduce the drip lass and texture sofeening of fozen fish and fish products is critical subject for both scientists and entrepreneurs. thas been verified that light salting under proper conditions ean enhance the water holding expacity,rexture properties, and stability of tuna flesh during frozen storage in our previons work inn etal. 201%; ong, Dia, Nakzavea ef a, 2022), NaCl is also found (0 enhance the water holding capacity of chicken breast meat after freezing (Petracci, Rimini, Mulder, & Cavani, 2013). There is, however, lite Information ‘available on the preservative effects of presalting on the quality atc ures of tilapia fillets subjected to drastic temperature fluctuations Different from the extracted myofibrillar proteins ora specifi protein, ‘musee tissue is characterized by varying levels of ordered structure, Inchuding thick and thin filments, sarcomeres, myobrils, myofibers, ‘ete, and the integrity of tise oF erie importance for muscle foods quality (Nakazawa & Okezaki, 2020; Zhan et al, 2018). It was by. pothesized that the quality changes in fish fillets after brining in NaCl solutions were related tothe structural rearrangement and molecular Interactions in tissue during repeated freezing-thawing. Therefore, the present study compared the changes inthe water holding capacity and texture properties of lightly salted tilapia fillets with those of tir wn salted counterparts subjected to various freeze-thaw cycles. Moreover, the underlying mechanisms would be revealed from the perspectives of siruetural roarangement aid molecular interactions sing multiple histological and physicochemical techniques, 2. Materials and methods 2.1, Sample preparation ‘Thirty live Ulapla sh were purchased froma local aquatle matket ‘and transported (othe laboratory with an oxygen supply system. The fish were 589.3 + 23.1 gin weight and 30.6 0.5 em in length, which ‘were measured using an electronic balance (MTS600, Shenzhen Melfi Electronic Co.,ltd, China) and a measuring tape (DLS795, Deli Group Co, It, Chins). The fish were killed by a perenssive blow tothe herd prior o eviscerating and washing with running water (14.0 = 0.5 °C) ‘within 30 min. The fish wsed in the prosent study were handed bie manely according co the aguatie animal health code (orld Organize ton for Animal Health, 2012), Tapia fillets were obtained in the sizeof 10.0 em « 2.0 em. % 1.0 em from the dors part snd divided into ewo ‘groups randomly. Lightly salted tilapia filets (Group 8) were prepared by soaking 100 g of files in 200 g of 9 % NaC! solution for one ho (4 °C) to achieve a target salt content of 1.5 9 in tissue based on our previous results Jiang, Du, Huang etal, 2022) Fresh samples with a salt content of 0.26 86 without salting were used as the control group (Group ©). The water content was 78.3 % and 77.6 96 for Group C and ‘Group S, respectively, andthe values were not significantly different (p > 0.05), The salted and unsalted files were divided ino five pats with ‘ix pieces in each part. The fillets were frozen and stored at — 18 °C for thee days before thawing at 4 °C tothe central temperature of 0 °C, which was measured using ® temperature recorder (MIK-R200T, Foe Chom 406 (2029) 135097 Hangzhou Asmik Technology Group Co, It, China). The above steps were repeated for 0, 1, 3, 5; and 7 tines, and the samples with the comresponding numberof freeze-thaw cycles were obtained. 2.2. Detennination of water-holding capacity ‘The water-holding capacity of filets was assayed following Siang, Du, Huang tel. (2022). The thawing los, centrifuging loss, and water binding index were caleaated using formula (1)-(3) Thang toss (%) = (Ws); x 100 @ Cemvitging loss (%) = (Ws — W,)/Ws x 100 @ Water — binding inde (%) = (an % W3)/( % Ws) > 100, @ where Wy and W were the weight before and after freezing thawing, respectively; Ws and Ws were the weight before and after centrifuge tion, respectively; ay and oy were the warer content before after centrifugation, respectively. 23, Measurement of transverse relaraton tne (T2) Following Lan et al. (2020) the transverse relaxation time was determined with slight modifications using # MesoMR23.060H.1 pulse NMR nnalvzer (Shanghai Nivmiai Electronic Technology Co,, Chia) with @ nuclear magnetic rube (70 mm in diameter). The proton reso: nance frequency Was 23.148 MH2, and the magnetic streagth was 0.54 7. The transverse relaxation tine (Tz) was determined using the Carr Purcell Melboont ill sequence at $2 °C, and the dats were normal aed afer inversion. 24. Measurement of texture properties ‘The texture properties of tilapia fillets were measured inthe size of 2.0 em x 2.0 em» 1.0 em sing a texture analyzer (TA.XT Plus, Stable Mieto System, UR). Te eylinrieal probe was Psy the rigger value of 5 1589s applied perpendicular ro the myofibers, andthe test rate was 1.0 ‘m/s with the highest compression ratio of 40S 25, Determination of protein solubiley ‘The protein solubility was measured following Fy mare, Baron, ane socobsen (2008) with slight modifications. The sample (1.00 ¢) was rized with 40 mL of 20 mmol/L Tris-HCI buffer (0.6 mol/L Nacl, pH 7.5) and then homogenized. After one hour in an ice bath and eentt- fugation (9100 x g, 4 °C, 15 min), the protein concentration (nig/tL) was measured by the biuret metho. The protein solubility was ealew laced using formula (4): Protein slubity (5 JG, x 100 w where C; was the protein concentration before centrifugation Ca was the protein concentration after centrifugation (ng/mL). vga); 26, Determination of contents of roral and active sulfyary groups Following Jiang, Du, Huang et al. (2022) the contents of total and active sulfiydeyl groups of myofibrillar proteins were determined, The sample (1.00 g) was mixed with 20 a. of 20 mmol/L phosphate buller A (0.1 mol/L NaCl, 1 mmol/L EDTA, pH 7.0). After homogenization and centrifugation (9100%g, 4 °C, 15 min), the sediment was washed with 15 mi of buffer A rvce, 25 ml. of 20 mmol/L phosphate bur (0.6 ‘mol/L Natl, pl 7.0) was added aod homogenized. After one hou a ice bath, the homogenate was filtered using two layers of gauze, The syofibrillar protein suspension (1 miL) was added to 9 ml of 0.2 mol/L ‘Tris-HCI (8 mOV/, urea, 19 SDS, 3 nmol/L EDTA, pH 8.0), which vasOkage ‘referred as bufer C. The above mixture (4 mi) was added to 0.5 mL of (0.2 mol/L Tris HCI (10 nmol/L 5,9" thio bis (2 niwobenzoie acid), pH 8.0), The absorbance (a) was measured at 412 nm after reaction for 25 min (40°C), using buffer Bs the blank contol. The detection method of| the reaetive sulfhydryl groups was the same ns that of the rotl sith” dry! groups, except thet urea was not added in buffer C. The value was ‘calculated using formula (9) Contem of slfydryt groups (nmol/mg MP) = (ax m)/(e x C) «10 (5) ‘where n was the dilution factor; ¢ was 18,600 L/(molem concentration of myofibrillar proteins (mg/m). was the 27. Sodlum dodecyl sulfte-polyacrylamide ge electrophoresis (SDS. PAGE) SDS-PAGE was condicred to evaluate the protein changes in dlapia samples during repeated freezingthawing following Jiang et (20190), The minced sample (1,00 g) was mixed with 40 mb of 20 mol/L Tris HCI (0.1 mol/L NaCl, pH7.5) and homogenized co analyze total proteins (TP). After centrifugation (0100 x g, 4°C, 10 min), the ‘supernatant was used to analyze water-soluble proteins (WE). The pellet ‘was homogenized with the above buffer, and the filtrate was obteined to alyze myoribillar protelns (MP) after ilration vsing two layers of ‘gauze, The concentrations ofthe above proteins were determined using the biurec method, and bovine serum albumin was used as the standard. ‘The protein solutions/suspensions were mixed with 20 mmol/L Tris HCL (pH18.8), containing mol/L utes, 2% SDS, and 2% p-mercaptoethane), heated at 98°C for 10min, aud cooled to the room temperate. Precast polyacrylamide ges witha gradient concentration of 5 %~20.% (Sangon Biotech Co,, Id, China) were ased, and 6 ig of each sample was uplosded, The gels were stained with Coomassie brilliant blne, and Photos were caken after destaining until the protein bands were clear. 2.8. Scanning electron microscopy (SEM) ‘The thawed samples were fined in 2.5 % glutaraldehyde overnight, (4°C. After weshing with 0.1 mol/L phosphate butfer (pH 7.3) three times, dhe specimens (0.5 em 0.5 em 0.3 em) were dehydrated using ‘ethanol solitons, freeze dried, coated with gold, and observed using 8 'SU3000 scanning electron microscope (Hitachi, High Technologies (Shanghai) Co, Id, Chine, 2.9, Light microscopy (LM) Following Jang, Ji otal. 2019), the thawed samples were xed in ‘carnoy's fixative overnight (4 °C). The fixed samples were washed using ‘deionized water three times, The specimens in the size of 0.5 em x 0.5 ‘em > 0.5 em were dehydrated using ethanol solutions with inereasing concentrations (50 %-100 8). The specimens were embedded in paraffin, sectioned (4 yn), dried, dewaxed, stained with eosin solution, ‘and observed using a MSSOOW light microscope (Shanghei Meizs Pre cision Instrument Co, Id, China) 2.10. ‘Transmision electron microscopy (TEM) The samples were cut into small pieces in the size of 1.0 mm > 1.0, rim > 1.0 mim and fixed in the fixative for eleetron mieroseopy. The specimens were washed wih 0.1 mol/1 phosphate buffer (pH 7.4) three times. After post-fixing with 196 osmium tetroxide in 0.1 mol/L phos phate buffer (pH 7.4) fr two hous, the specimens were rinsed with 0.1 ‘mol/L phosphate buffer (pH 7.4 three times, The ultrastructural images were observed using a HT7800 transmission electron microscope Hitwchi Co, ld, Japan. Foe Chom 406 (2029) 135097 2.11. Statistical analyses The data were analyzed by one-way analysis of variance using '53823.0, and multiple comparisons of means were conducted at p < 5 wsing Duncan's method. The daca were presented as means standard deviations, 3. Results and discussion, 1, Effect of lig salting on the water holding capacty of tlapia fillets tring repeated freezing thawing Waterholding capacity, the holding expacity of the internal or external moistre in musele during processing and storage, is sally Indicated by the dip loss during thawing, centrifugation, ete. (Chan fecal, 2022) Asshown in Fi. 1, the hawing loss decreased significantly after salting, which was likely tributed tothe enlarged water holding spaces inside the salted tise due ro swelling (in tal, 2019). tn the presence of NaC, the electrostatic repulsion between myofibrillar po teins increased andthe selling of myofibrillar lattice and solubilization of myofibrillar proteins oceured (Patt etal, 2019). As the number of freeze-thaw eycles inreased, the thawing lss of Group $ was low and did not change obviously (p > 0.05), while the thawing loss of Group © increased. drastically (p < 0.05). The centrifuging loss of Group C increased mntkedly after the first freeze-thaw eyele (9 < 0.05), and it became constant ater 3 or more freeze-thaw eycles, which might be due to the excessive drip loss during thawing. After freezing thawing, the centrifuging less was much lower inthe lightly salted fillets than the in their control counterparts (@ < 0.05). The water-binding index decreased in both Group C and Group S with the increasing freeze-thaw cycles, which was atrbuted to the destruction of musee structure and aggregation and/or denaturation of myofibrillar proteins (Diao, Cheng, Wang, & Xie, 2021) After 7 freeze-thaw eyeles, the water-holding cx Dacity of Group S was higher chan that of Group C, and the water binding index was 17.28 % higher than that in Group € (p < 0.05). ‘Te results indicated that ight salting improved the storage stability of tilapia filets and high water holding capacity maintained even after repeated frezing thawing 32. Effect of ight sling on the water distribution and mbit in tapia files during repeated freezing thawing ‘The distibution and mobility of water in lightly salted tapi Ales were assessed using LF NMR as affected by freeze-thaw cycles, The {ypleal To relaxation tue curve (se Pig. $1) had three peaks, whe reflected the differences in existing states of water in fish les. As shown In Table 1, the Tay nd Tn control iapia fillets inereased significantly after he fist feeze-haw eyele and salting (p < 0.05), suggesting that the mobility of bound water and immobilized water increased due to mechanical damages or the presence of fons. The valle of Tas was almost the same in Group $ (21) and Group © (23) when the number of freeze-thaw eyeles increased, indicating the bound water was more likely 10 be niodified in dhe inital postmortem period. Repeated freezing-thawing had no significant effect on Tz: in Group 8 (p> 0.05), ight be because light salting countetacted the mechanical damages of ise, and the water formed by ice erystals was absorbed by the salted tissue mote realy during thawing (eng, Js el, 2019). A8 the freeze-dhawr cycles increased, Tap declined drastically in Group C, tnd the declined mobility of free water was ascribed tothe loss of free water as demonstrated by the inereased thawing drip. Te was worth noting that Tze could not be detected in Grom S after freezing thawing, which was consistent with the finding of Li etal. (2022). Qin et al (2017) also found that the swelling of the myofibrillar lattice conti ted tothe inumobilization offre water and reduced its mobility, The disappearance ofthe peak and the low thawing drip in de lightly salted filets indicated that presalting combined. with freezing-thawingdanger (A) Foe Chom 406 (2029) 135097 (B) “0 cc es ce os aS ; z eu { a as i a a ee $ =” z if : © i 1 Ele) le | | che Ew e | le er || Wee Sata meses aa Freeze-thaw cycles Freeze-thaw eyeles (©) 100 ce coco ms g ab Fat be abe og 2 gn ol ct = oes oa Eye ES | Freeze-thaw eycles Fig. 1, Changes in water-holding capacity of tightly ste lap ills during index: conto simples; igh salted tap iets repeated feezing thawing. (A) Thawing loss (8) Centsifgng loss, (C) Water-binding ‘Table 1 ‘Changes in peak summit ines and the area ratios ofthe coesponding peaks (transverse relaxation) in ight salted api lets during repeated feezing thawing Tein) i350 aysae iasaa®aos00 assoi aasom caso aasox oasay a7aun Talm) 527420 Sega SSO BBs LIs SAT 4OO! Setar Saye Seek Sara AOL oe aoe Tim) 13195 m2 work ag sok os x 8 x x Py) 21404" = B04 seas? zoe" Baas 23405" ea 2940 ryt) 977402 omeLoM wrs4ns> e774 IS? MALI 974s 02 ora 7 cost Pot 0240s" sot tears to2eis este aasos x s Data aresiown as means + standad deviations (a6) Dtain the same iow with difevent lowercase ets ave igniicanly diferent (p< 0.05): conned samples Sigil sate Files N: undetected accelerated the transformation of tissue water, whicl was partially responsible forthe enbanoed water-holding eapaciy. The relative contents of different water ean be evaluated by the aren ‘atios of the corresponding peaks from the transverse relaxation time ‘curve. There was na considerable difference in Pa after light salting (P > 0105), and freeze-thaw eyeles had no matked effect on Pay in Group C ‘and Group S(p > 0.05) twas ikly attributable tothe unirozen bound water under the present experimental conditions, because the bound water would not be affected until the temperature was ~ 40 °C or lower (G5, 2, Sun, 2018; Miyewaki, 2018 Nakazawa & Okazaki, 2020). As the freeze-thaw eyeles increased, Poo increased with & simultaneous decrease in Pat in Group C, which indicated the transformation of Immobilized water to free water. Ic was probably because the growth and reerystallization of ice crystals destroyed the structure of muscle tissue and myofibrils In fish flesh seriously, allowing the water to translocate tothe external space of myoRDbrils, extracellular spaces oF exude continuously when subjected to severe temperature fluctuations Moreover, the water could hardly be reabsorbed by the damaged myo. fibrils during thawing (Zing i, Diao, Kong, & Xia, 2017), Kewas worth noting that, Pa of Group S was not significantly affected by freeze-thaw eyeles (@ < 0.05), which was mainly related to the water-holding ex pacity. Moreover, the disappearance of the peak for free water and the lovr thowing drip im che lightly salted filles indicated that preslting combined with feezing-thawing accelerated the transformation of ts ste water to immobilized water, which was responsible forthe eulanced water holding capacity. I might be due 10 the solubilization ofdanger myofibrillar proteins and the formation of an ordered protein network, shifting free water to immobilized or bound water (Wag, Feng, Wang, ‘ssig, & Xia, 2021). The water holding eapacity was related to the protein properties and the mirostrenire of tissue by affecting the protein-water interactions and the water-holding space (Shino et 2016), which could be reflected by the water mobility and trans. formation (Li, Wang, Kong, Shi, & Xia, 2019), 3.3. pect of Hg sating onthe texture properties of rilapa fers during rapected freeing thawing Texture properties, one of the essential quality tats for muscle foods, are closely related tothe physical properties and organizational structure of related products. Texture softening was observed in Group , whiel was presented by the continue decrease in hardness until 3 freeze-thaw cycles (see Fig. 2A). Thereafter, i became constant, ind ‘ating the hardness changed drantatcaly in the eaty stages. Thowgh the hardness decreased ater salting tne value was higher in Group S than thar in group C with the same freeze-thaw eyeles (p< 0.05). Te was suggested tac presalting could bean alternative meas to alleviate the problem of texture softening in frozen fillets. As shown in Fig. 2B, the springiness of lapa fillets increased after salting, and the spinginess of Group decreased markedly alter freezing thanving (p < 0.05). The springiness of Group $ was, however, higher than that of Group C with) 5 freeze-thaw eyeles (? < 0.05), whieh might be related to the disas sembly of myofibrils and formation of intermolecular interactions dur ing salting and freezing thawing (Jiang, Jia et aly 2019; Jiang et al ‘2019b), No difference in cohesiveness was observed after sling, but the cohesiveness increased significantly in Group § after freezing thawing (© < 005), which was likely associated with the inerease of (A) sev (B) Foe Chom 406 (2029) 135097 protein-protein interactions (Jiang etal, 20196). There was no differ fence in the cohesiveness berween the two groups after 7 freeze-thaw yee (p > 0.05), 4. Bier of light salting on the protein properties in iapa files during repeated freezing thawing Properties of muscle proteins, especially the myofibrillar proteins are closely related to the quality of fish and fish products. As shown in Fig. 3A, compared with the fresh samples, light salting ad no significant effet on the protein solubility of ilapia filles (p > 0.05). After 7 freeze-thaw eyeles, the protein solubility decreased by 439 in Group C, which might be eaused by protein aggregation and denaturation (io! ssorebrov,Bikevik Bantle, 2016). Presalting decelerated the decrease n protein solubility <0.05), bu the values of protein solubility could not reflect the existing stare of myofibrillar protein in tissues actually, so more analyses were needed. ‘The proteins from Group C did uot change significantly from the SDS. PAGE profile during the process (see Fis. 5B), but light salting affected the giant myofibrillar structural protein, ctin, which was marked by arrows. As the freeze-thaw eyles incrensed, the myosin heavy chain in the MP fraction and ttn 2 in the MP fraction from Group $ decreased gradually, which might precipitate with other myofibrillar proteins during repeated freezing-thawing. The proteins with Tow molecular weight in the WP fraction fom the salted Ales (marked by parenthesis) tended to disappear with the increasing freeze-thaw eyeles. These pro: teins were considered tobe tropomyosin, tropouin, myosin light ehains, and their degradative products (Lie, 2022), Moreover, the formation of large aggregates by non-disulfde covalent bonds was observed in the Lightly salted fillets after 3 or mate freeze-thaw cyeles, which could not 4 cc ms fall oc es 000 bee BS » ors} ofl] bed od dep Dek 3 1 ti of 3m ae 4 E a. ol 050| 200 1 haa ba el] ¢ re HI ale as : [| Cee ay ogo EE Freeze-thaw eycles © os coc ms a fe hp eles ae = | d gu é oa oo Freeze-thaw cycles sae Sigh sled aia les 2. Changes in texture properties of lightly salted tapi lets dung repeated feenlng thawing, (A) Hardness (8) Springiness;(C) Cohesvenes.C: cottodanger Foe Chom 406 (2029) 135097 (A) Protein solubility (%) s pare ale si Freezethaw cycles (B) < 7 c s c s Moass 701387 Movas7 01387 Movs? 01389 eee eee wee eee ey za LLU SoG D pa: — 7 Gah EAH Titi HHHHHRE ett i 200 oo oo MIC 200 >) yoyo on uac y, \Saae nae BeE ; SSeLSceeeee SB oom Bon aoe q qg BEsESSHeees Hho se ak oo ae oi SOSSEEE > DESSEEDEBEE SaaGRn NE? Seeee BSesscccace Feennncccce 7 we we Ce a) Oye aoe MET ce Eouthd oe meer re . be ede ae ze | 2 Ew Be 33 Hy Som ab, g 3 reteset frkere-han es Fig. 3. Changes in proein prope in lightly sate apa lets dang repeated freezing thawing (A) Protein solubility (8) SDS-PAGE profiles (©) conten of total sully gros: (D) Content of ative slfiyy! gioups. C= contol samples: igh sated lap fillets; Me protein naukers| TP oil proteins, MP tuyofbilae protein faction; WP: water solible protein acon MHC: myosin heavy cin: Atzow cate tn; Paerheis indeats alte proteins be cleaved in the solution containing mercaptoethanol and accumu- declined significantly as well a the aetve sulfhydryl groups after § oF lated above the stacking ge. Iwas suggested thac presalingredkiced the more freeze-thaw eyeles in both gronps, bt the Vales were higher i stability of muscle proteins, and they tended to interact during repeated Group S with the same freeze-thavecyeles(p < 0.05). It was suggested freezing-thawing thar repented freezing-thawing accelerated the oxidation of sulthyary] Sulihydyl groups in proteins are easy to be oxidized into disulfide groups (Nikoo, Benjakl, Gavlighi, Xu, & Regenstein, 2019), Du et al. bonds with a decrease In the contents of total or active sulfiydryl (2021) found that the contents of total and active sulfhydryl groups of groups. The declined content of sulfhydryl groups Is an indicator of MP from mirror carp deereased with the Ineeasing freeze-thaw eyels. procein oxidation, With the exposure of dhe buried sulfydry groups co The generation of large ice crystals could disrupt the cellar integrity the surface, the content of active sulthydtyl groups would increase during repeated freezing-hawing, leading tothe release of heme, ion, Inially (Zhang, Fang, Hao, & Zhang, 2018). AS shown In Fig, 3 the and other prooxidaat substances, which could exacerbate the oxidation content of ative sulfhydryl groups was higher in Group S than that in. of proteins (\ikoo & Benak, 2015). The loss of pro-oxidant substances Gronp © afer salting snd the first freeze-thaw eyele (p < 0.05), while during salting combined with the reduced mechanesl damages to ts the content of tora sulfhydryl groups vas similar in these wo groups (> sues by ice crystals might contribute to the lower degree of protein > 0.05), indicating thatthe rate of structural changes exceeded the rate oxidation in light salted tilapia fillets when subjected to severe ten ‘of oxidation in te inital stage. The content of total sulfhydryl groups perature Auctuaions (Nakazawa & Okazaki, 2020),danger 4.5. Effect of light salting othe histological properties of lpia fillets daring repeated freezing cain Different histological technologies were applied to stay the struc toe of tilapia risses at varios levels using SEM, LM, and TEM, and the results are presented in is. 4. Alarge numberof voids were observed in Group C after the fist freeze-thaw eyele from the SEM micrographs, which were the mechanical damages to tissues during frezing thawing Ginng er, 2019). The voids in Group C became larger and larger with the inerensing freeze-thaw eyeles. The tissues were distorted gradually (Gee Fig, 4), which decreased the water-holding eapacity of fsh Resh (Gee Pig, 1). The transfer of water from the intra myofibilar spaces co the extra-myofibrillar spaces resulted in the formation of large lee crystals during repeated freezing thawing and vice versa, The severe mechanical damages and the appearance of Inge voids in tissues after thawing were related to the decrease in the content of immobilized water during repeated freezing-thawing (liens etal, 2019). Compared with Group €, Gronp $ could stil maintain che brous structure with & (A) (B) © Foe Chom 406 (2029) 135097 close connection between myofibers even after 7 freeze-thavr cycles from the transverse SEM images, indicating thatthe lightly salted tissues hed beter recoverability during thavsing. Moreover, no intracellular voids were observed in Group $ from the LM images, which was likely responsible for the maintenance of high water-holding capacity and springiness in lightly salted samples even after 7 freeze-thaw cycles (Kang et al, 2016) ‘As shoving, 4C, the structure of myofibrils in Group C was lear, Intaet, and orderly before freezing, while the myofibrils in Group S were rot neady arranged, with faint Zlines and blurred bands. 1¢ was indicated that the excraction/solubilization of myofibrils oeeurred in Lightly salted sample, though the value of proven solubility was not significantly different from that in Group before freezing See Fig. 3A) Alor 7 freeze-thaw eyeles the linear boundaries of myofibrils cannot be distinguished, and the sarcomere strctute could hardly be recognized ln Group S. Compated with the samples before freezing, the Zines in lightly salted filets changed a litle, which could be still discerned vaguely after? freeze-thaw eycles. Notably, the cleat M-lines in lightly ‘Changes in histological proprtis in igh salted pla filles during repeated feezing thawing, Mlcographs fom (A) scanning electron miesoscop; (8) light eroscope;(C)taasmision election mroscope. C: conta sanples: light slted lpia lets AoW ineates mechanical damages alter thawing, ZZ line; eM-ine A: Ahad ba enor,danger solted samples disappeared after 7 freeze-thaw cycles, which might contribute (0 the disassembly of myofibrils and the solubilization of proteins from the A bands. It was consistent with the previous finding "ha the structural breakdown of sarcomere was initiated atthe A-band region (Wi, Farouk, Clerens, & Rosenvolé, 2014). The solubilized ‘myofibrillar proteins would interact more readily without the con saints of M-line, in whieh the immobilized water could be hel, resulting in the transformation of free water to inumobilized water (see “Tnble 1). The rarrangemtent of myofibrillar proteins andthe formation ‘of protein nerwork were considered to contribute tothe high recover ‘ability and the improved water-holding capacity and springiness in lighdy salted tapis Alles during repeated trezing thawing. It was supposed that the fragments of myofibrils caused by lee crystals ‘control samples could not form an ordered protein network becatse of steric hindrance, reslting in the deterioration in quality properties during repeated freezing-thaing, Moreover the better preservation of ‘mitochondria was observed in lighily salted fillets after 7 freeze-thaw ‘eyeles, ut further study s needed to reves! its specific relationship with the quality changes 6. Contribution of structural rearrangement and molecular interactions ‘om quai’ nprovement by Hight satin lap filers during repeated Freeing thawing ‘The underlying mechanisms of he effect of ight salt holding capacity and texture properties of tilapia filets freezing thawing are illustrated in Vig. 5. The orderly structure of fish tissue is broken aftr salting, demonstrated by the malposition of myo- fibrils andthe unclear Z-lines and L-band. After light salting, swelling oF ‘myofilament lattice in tilapia fillets occurs, wile the components ofthe sareomierestrucare ae largely preserved, Solubilization of myofibrillar proveins and/or unfolding of proteins might occur, but a limited fextent, die to the regulation of structural proteins. After repeated freezing thawing, the ordered structure of myofibrils in the unsalted Uisues disappears completely, and large gaps form between the myof- brils. Moreover, che differen width of myofibrils in the thawed fillets Indicates distortion and fragmentacon of myofibrils when subjected co severe temperature luetuations. Suffering from great pressures fom the oe crystals due to the augmented volume, the feagments of tissues might form heterogeneous aggregate, resulting in poor water retaining ea pacity and low recoverability after thawing. Therefore, high drip loss ‘and worsening texture properties are observed in the unsalted fish files after repeated freezing-thawing. Notably, the myofibrils in the lightly as Meanie dam mgs Foe Chom 406 (2029) 135097 salted filets appear to be fused, withthe faint traces of Z-lines after repeated freezing thawing. ‘The sarcomere structure can hardly be recognized, with the severe loss of Milnes, Repeated freezing thawing promotes the redistrbution of NCI nd water, an the solubilization of myofibrillar proteins in sues, The myofbril disassembly and the for nation ofan ordered protein network with the solubilized myofibrillar proteins contribute to the enlarged water holding space, transformation of tissue water, and tissue recoverability during salting and repeated freezing thawing Problents, inchiding high thawing drip loss and rexture softening plage the industries of frozen fish and fish products fr along period, Wile cause quality deterioration, resouree waster, and economic loses. ‘The lightly salted flletsmsintain the enhaneed water holding capacity and decelerate texture softening even after mmitiple freeze-thaw eyees “The present findings provide altemative menns to reduce the dip loss ‘nd alleviate the problem of texture softening in frozen fish and fish product 4. Conclusions nthe preseat study, it 89s found that light salting contebued tothe tintenance of high water-holding capacity and the deceleration of texture softening in dlapa fillets during repeated freezing thawing. The "hinwing loss of tilapin lets was decreased rom 16.9% 0 1.49 y ight salting after 7 freeze-thave cycles (p < 0.05). The relative content of immobilized water was higher chan 97.1 8, and the peak for free water disappeared in lightly salted fillets after freezing thawing. On the contrast, che immobilized water was decreased from 97.7 96 10 83.8% In tunsated fille afer 7 freeze-thaw eyeles, with a conewrrentinerease free water from 0.2 eto 144% (p < 0.05) The transformation from free water t0 immobilized or bound water and the reduced mobility were ascribed tothe solubilization of myofibrillar proteins and the formation of an ordered protein network by light salting during repeated freezing thawing Light salting interfered with the degradation of strstural proteins (ttn), contributing to the preserved hardness of fish filles Light salting promoted the myofiril disassembly and the formation of dered protein aetwork with the solubilized axyoRbrilar proteins, Which facilitated the enlargement of water-holding space, trans formation of tisue water, and high issue recoverabliy during repeated freezing-thawing, The finding provided novel insight into the improve. tment of quality properties f tilapia filles by light salting when sub Jected 10 drastic temperature fluctuations. Beiter preservation of mitochondria was observed in he lighdy salted files ater freezing sexy interactions UP Rerprzaion ==: Se rime casa > Texture Sa softening Fig. 5. Schematic ilistation of eranced ~ Improving water ty propestes in lozen aia files by Hig sling eng repented fleeing thawing.danger ‘thawing, but further study is needed 10 reveal its specific relationship ‘wid the quality changes of fish flesh, especialy the oxidative stability ‘and the uiderlying mechanisms. Qingging Jiang: Coneeptualization, Investigation, Formal analysis Writing ~ original draft, Writing — review & editing. Shiyw Huang: Investigation, Formial atalysis, Waiting — original draft. Yunfan Da: Conceptualization, Investigation, Formal analysis. Jianbo Xiao: Writing “review & editing: Mingfu Wang: Writing - review & editing. Xichang ‘Wang: Concepnialization, Writing ~ review & editing. Wenzheng Shi ‘Writing ~ review & editing, Supervision, Yueliang Zhao: Writing re view & editing, Supervision. Declaration of Competing Interest ‘The authors declare that they have no known competing financial {interests or personal relationships that could have appeared to influence the work reported in this pape: Dat wil be made available on request Acknowledgements This work was supported by National Key RAD Program of china (No. 2019¥FD0002003), Shanghai “Chenguang” Progeam (No. 20068), and National Natural Sclence Foundation of china (1901807) Appendix A. Supplementary data Supplementary datato this article can be found onlineat itps://doi. ‘og/10.1016/5f00dehem.2022.135097, References: oo. Feng A Be Wang Ja i Nin Gi (022). Te econ ‘mechani t fone yng bon the sey of lpi let poacts Food Frei 2), 816-87 ‘Ohan, :5 oth, Bese Fe Jahon, A Ry ell J (202). Water hog ‘ropes ot Ane sainon. Congebonsne Revie Road Sic nd Fd Se, Pu. 77 9. cog 55 Wang XH Li: Yang, HM, Wang. Hl, Wang HLT Tam, MQ. (019) intence a mui eset es on guy characteris of Bot ‘enimenbranons msl With ephan on te ss ad btn y MR oR a See 147, 98-52 Dio, YD han, XY Wa 8 8, WS (2021) Eee of mersion fee ‘tho on mater oli caps fe aaa ater ization nas cp ‘sing arn sap, nse ued of etn, £2, 381-0 Du Ky LL Jy Doo CH en, Yl Pa KOU, eX. F200 et tee sructarng tela be miosuuctne sn moto prot sauce of tor eanp (Cpr eo L} laced Y eae poceses. nt Pod Sele nd Technolgy, 199, Ae 110570 hao. Loita jmez0 10570 ‘yma Sy baron © Py dace, (2008). Onda of pd and poten in hate ‘mule (rach wvtrn) ince snd wae mince dig rong Sore ed Chemistry, 140), 57-65. 0 202) The sate of Wola Reis ad aquscle 2022: Towards De ‘restoration ome oe nd Are Onarzaon Ed, Tan My Ma 8s Mackneger, 6-200) Sl oduct o pest [jpeenin scene tet JACC stata sew ural f he ‘Arrears Clg of Car, 751), 652-547. Ingo, Zang My Tor BK, Rey: Aegon © M2017) Sle ‘econ westeginin proceed men pcs Arevew, Teen Fond See nd Teco 39,7078 tang. 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