Weiqing Ln,Xioyu Hu,Xiohong Sun,*,Xi Zhng,Jing Xie,**

a College of Food Science and Technology,Shanghai Ocean University,Shanghai,201306,China

b Shanghai Aquatic Products Processing and Storage Engineering Technology Research Center,Shanghai,201306,China

c Shanghai Professional Technology Service Platform on Cold Chain Equipment Performance and Energy Saving Evaluation,Shanghai,201306,China

d National Experimental Teaching Demonstration Center for Food Science and Engineering(Shanghai Ocean University),Shanghai,201306,China

A R T I C L E I N F O

Keywords:

Litopenaeus vannamei

Repetitive freeze-thaw

Muscle microstructure

Water migration

Proteins

A B S T R A C T

The objective of this study was to evaluate the effects of the number of freeze-thaw(F-T)cycles on moisture migration,protein degradation,microstructure and quality in Litopenaeus vannamei.The quality of samples with different F-T cycles were determined by low-field nuclear magnetic resonance(LF-NMR),magnetic resonance imaging(MRI)and scanning electron microscopy(SEM),combined with sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE),texture properties analysis(TPA),color difference,polyphenol oxidase(PPO)activity,total volatile basic nitrogen(TVB-N),total viable count(TVC),and sensory evaluation.The results showed that F-T cycles caused a significantly increase in transverse relaxation time in T22 and T23 and a decreased brightness of pseudo-color diagram after 4 F-T cycles,indicating that water mobility increased as immobilized water was shifted to free water.The texture of samples declined as well,especially after 4 F-T cycles.The rapid growth of PPO activity caused a decrease in brightness(L＊)and an increase in redness(a*)after 3 F-T cycles.The increase of TVB-N,TVC,and sensory score explained the changes in shrimp quality which became obvious after 3 F-T cycles and unacceptable after 6 cycles.Meanwhile,fewer than 4 F-T cycles accelerated protein aggregation,while denaturation occurred after 4 cycles.Therefore,repeated F-T cycles could accelerate the changes of protein,microstructure,water distribution,and quality deterioration especially after 3 F-T cycles,with a threshold was 6 F-T cycles.

1.Introduction

Pacific white shrimp(Litopenaeus vannamei),indigenous to the Pacific coast of South America,has been introduced and widely cultured in China successfully(Zhang,Wang,Dong,& Lu,2016).It has rapidly become one of the popular and most profitable shrimp species,accounting for 85% of the farmed shrimp production in China,owing to its prime quality,high nutritional value,and delicacy(Li&Xiang,2013;Thanasak,Soottawat,& Kitiya,2018).However,shrimp is easily spoiled by protein degradation and bacterial and high cathepsin activity(Dong,Xu,Ahmed,Li,&Lin,2018).In addition,the shelf-life of shrimp is also limited by melanosis or red stain due to the appearance of black or red spots during storage(Thanasak & Soottawat,2019).Frozen storage is a common method for the preservation of aquatic products(Kittiphattanabawon,Benjakul,Visessanguan,& Shahidi,2012).Thawing is necessary for frozen food in order to facilitate the subsequent food processing and,it is inevitable that temperature changes or repeated F-T cycles will occur in retail stores,restaurants or families(Mehran Yasemi,2017).Unsold shrimps are usually frozen by retailers and then sold as iced prawns,which can also make repeated F-T cycles.The integral structure and physicochemical quality of shrimp are largely influenced by it.Protein oxidation and denaturation are probably accelerated by lipid oxidation caused by F-T cycles and may decrease the edible quality,such as poor taste and discoloration(Haghshenas et al.,2015;Shi et al.,2019).

F-T cycles can destroy the texture of meat and transfer or redistribute its moisture,which decreases the water capacity and the sensory quality of meat(Passakorn & Soottawat,2016).As water is the main component of meat products,especially in aquatic products,the quality and stability of aquatic product will be influenced by the physical state of water to a large extent(Passakorn & Soottawat,2016).LF-NMR can be used to describe the change of moisture distribution and transfer in meat by measuring the proton relaxation.Carla et al.(2013)explored the water redistribution in Pacific white shrimp treated with sodium tripolyphosphate during frozen storage by LF-NMR and observed that LF-NMR can be used to investigate the water migration and quality changes in thawed shrimp during the freeze-thaw process under different treatments.Proteins also contribute to the desirable sensory quality and the physicochemical properties of muscle foods are so dependent on it(Zhang,Hao,Cao,Tang,Zhang,& Deng,2018).It has been found that F-T cycles can lead to water loss,protein degradation,and muscle fibers damage in instant sea cucumber(Tan,Lin,Zu,Zhu,&Cheng,2018).Zhang et al.have confirmed that the repeated F-T cycles could destroy the muscle structure and disturb the stability of physicochemical properties in shrimp(Zhang,Fang,Hao,& Zhang,2018).

The physicochemical changes of shrimp via multiple F-T cycles can be evaluated using the techniques from Zhang et al.(2018).Nevertheless,few studies evaluate the effect of the number of F-T cycles on shrimp and the threshold of F-T cycles by the comparison between the changes of moisture migration,muscle microstructure,protein degradation and physicochemical indexes.The aim of our research was to investigate the maximum number of F-T cycles during storage by physicochemical indexes and found the correlation between the changes of water migration,muscle microstructure,protein degradation and quality in shrimp influenced by the number of F-T cycles.

2.Materials and methods

2.1.Sample preparation

Samples of fresh Pacific white shrimp weighing a total of 1.6 kg(average weight of 12.0±1.0 g)were transported from a local market(Pudong,Shanghai,China)to the laboratory within 30 min and euthanized with ice water.Thereafter,in order to simulate the temperature fluctuations or repeated freeze-thaw cycles during commercial sale process and circulation,they were stored at－20°C for 12 h and then thawed with crushed ice at 4°C for 12 h.This sequence represented one F-T cycle.Then the thawed samples were treated by F-T cycles until total 8 F-T cycles were completed.

2.2.Low-field nuclear magnetic resonance(LF-NMR)& magnetic resonance imaging(MRI)

LF-NMR measurements were carried out by using a Niumag Benchtop Pulsed NMR Analyzer(Niumag Electric Corporation,Shanghai,China)after each F-T cycle in accordance with Wang et al.(2018).Samples were briefly placed in cylindrical tubes before transverse relaxation times were measured at 32°C with a resonance frequency of 21.0 MHz using the Carr-Purcell-Meiboom-Gill(CPMG)sequence.Data were acquired from 5000 echoes of 8 scan repetitions and analyzed by MultiExp Inv.Analysis software from the Niumag Electric Corporation.At least three replicates were performed per test.

The MRI was measured in accordance with Wang et al.(2018)after determining the relaxation time and the difference of moisture content was measure via a 2D proton density image for the transverse section of sample.The proton density imaging of samples was obtained via MSE imaging sequences with determining conditions of repeat waiting times(TR)=500 ms and echo time(TE)=18.2 ms.The signal-to-noise ratio and image sharpness were adjusted and the resulting image map was produced by a summation of 8 repeated scans.A proton density map was produced by unified mapping and false color from Shanghai Niumag Electronic Technology Co.,LTD.

2.3.Scanning electron microscopy(SEM)

SEM analysis of samples was carried out every 2 F-T cycles as described by Zhang,Fang,et al.(2018)and Zhang,Hao,et al.(2018).The samples were cut in 5 cm×5 cm×2 cm pieces along the direction of muscle fibers.Then samples were fixed with 2.5% glutaraldehyde solution for 24 h and rinsed 3 times by 0.1 mol/L phosphate buffer(pH 7.0).After 2 h at room temperature,samples were rinsed with distilled water,followed by a gradient dehydration with ethanol solutions of different concentrations and repeated tert-butanol elution 3 times.The processed samples were freeze-dried overnight and gold-coated(Eiko IB3,JEOL Ltd.,Tokyo,Japan)for 5 min before they were observed with a scanning electron microscope(JSM-6390LV,JEOL Ltd.,Tokyo,Japan).

2.4.Sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDSPAGE)

Myofibrillar proteins were extracted in accordance with Ma,Zhang,Deng,and Sun(2015)and Zhang,Fang,et al.(2018)and Zhang,Hao,et al.(2018).SDS-PAGE analysis was performed every 2 F-T cycles on 10% resolving gel and 4% stacking gel as described by Kulraphat,Sorada,Dudsadee,and Vilai(2016)and Lu,Zhang,Li,and Luo(2017).The voltage of stacking and resolving gels were 80 and 120 V,respectively.The gel was stained with Coomassie brilliant blue for 2 h and then decolorized in ethanol/glacial acetic acid decolorization solution until the protein band and the gel background were clear.Compared with the protein standard(ThermoFisher Scientific Co.,Shanghai,China),the polypeptide bands of shrimp protein could be recognized through relative molecular weight.

2.5.Physicochemical analysis

After each F-T cycle,samples were taken for the determination of physicochemical indexes.

2.5.1.Texture properties analysis(TPA)

TPA was measured by using a texture analyzer(Model TA-XT2,Texture Analyzer,Texture Technologies Corp.,Scarsdale,NY,USA)and parameter settings referred to(Zhang et al,2015a,b,c).The second ventral muscle of shelled shrimp sample was used for TPA analysis and in order to simulate the chewing process,a P/5 flat probe(5 mm diameter)was used.

2.5.2.Color difference

The color of shrimp samples was measured by using an automatic color difference meter(DC-P3,Shanghai Go On Chemical Co.,Ltd.,Shanghai,China)according to the method described by(Zhang et al,2015a,b,c).Color difference was determined in two zones(head and body)of the shelled shrimp.

2.5.3.Polyphenol oxidase(PPO)activity

The PPO extraction was carried out according to Qian et al.(2014)and its activity was determined according to the description of Sara,Seyed,Mahmoud,and Sohrab(2015).L-DOPA was used as the substrate and the determination wavelength was 530 nm.The PPO activity was expressed by the increase of absorbance value(A)in unit time(min).

2.5.4.Total volatile base nitrogen(TVB-N)

Five grams of shrimp muscle were taken for TVB-N measurement by using a kjeltec™2300 auto sampler system(FOSS Companies,Shanghai,China)following the methods of Yuan,Lv,Tang,Zhang,and Sun(2016).TVB-N values were expressed as mg N/100 g sample.

2.6.Microbiological analysis

Microbiological analyses were performed after every F-T cycle and were determined following the methods of Liao et al.(2018).According to them,plate count agar(PCA)was used to determine the total viable count(TVC)and the culture temperature was 37°C for 48 h.Finally,it was expressed as the log of colony forming units(CFU)per gram of sample.

2.7.Sensory evaluation

Sensory evaluation of shrimp samples was carried out from five aspects(appearance,color,odour,texture,and overall likeness)by six trained panelists who were not allergic to shrimp and had no preference for shrimp according to Xu,Liu,Wang,Hong,and Luo(2017).For each aspect,the sensory scores were given by using a 1-6 descriptive hedonic scale,in which 6 represented the best characteristics.The final sensory score was obtained by adding up the scores of different aspects and was unacceptable when below 15.Shrimps from each treatment group were taken after every F-T cycle up to 8 F-T cycles for sensory evaluation.

2.8.Statistical analysis

The SPSS package(SPSS 17 for Windows,SPSS Inc,Chicago,IL,USA)was used for statistical analyses and Duncan's test was used for significance analysis of intra-group differences,in which the means differed significantly atP＜0.05.The data were shown in means±standard deviation(SD)of triplicate measurements.

3.Results

3.1.LF-NMR

The result of LF-NMR showed the water redistribution and the damage of myofibrillar structure to some extent.Table 1 shows the percentage of change in moisture content(T21,T22,T23)in various water states(bound water,immobilized water,and free water)under different F-T cycles.Relaxation time ofT22increased significantly with F-T cycles and there was a sharp rise inT22value after 3 F-T cycles(Table 1),signaling that the conversion of immobilized water to free water was dominant after 3 F-T cycles for shrimp.TheA23values of samples showed an opposite trend toA22during different F-T cycles.Kulraphat et al.(2016)have found that for incompact muscle tissue,free water was more difficult to retain.The water migration and loss started to become serious after 3 F-T cycles.

The proton density weighted imaging of shrimp that experienced repeated F-T cycles were translated into pseudo color of1H-MRI(Fig.1).Generally,brighter in the image(the more red present in the pseudo-color diagram),the stronger the signal of water protons and,the higher the water content(Tan et al.,2018).The brightness of pseudocolor diagram decreased from high to low with the increase of F-T cycles(Fig.1).The changes were most evident after 4 F-T cycles,indicating that the water loss and quality deterioration became more serious.

Table 1Changes in the percentage of T2i in shrimp with different freeze-thaw cycles.

3.2.Changes in the microstructure of muscle tissue

The microstructures of samples that were subjected to 2,4,6 and 8 F-T cycles were compared with that of fresh shrimp(Fig.2)Transverse sections,of fresh samples showed a well-organized structure and,muscle bundles had no evident changes at the 2nd F-T cycle,but were more separated after 4 F-T cycles(The white arrow in the figure was used to highlight the changes.).The shrinkage of fibers gave rise to crevices and the destruction of muscle fiber integrity at the 6th and 8th cycle.

3.3.SDS-PAGE patterns of protein

The patterns of shrimp muscle protein during different F-T cycles were shown in Fig.3.An understanding of protein oxidative degradation in shrimp during different F-T cycles was important for studying antioxidants which protect protein.According to methods in Ma et al.(2015),we can find four protein bands:Paramyosin(approximately 100 kDa),actin(approximately 45 kDa),troponin T(approximately 34 kDa)and myosin light chain(MLC;approximately 20 kDa)(Ma et al.,2015).The results showed that the intensity of actin,troponin T and MLC after 4 F-T cycles decreased markedly as compared with the intensity fresh samples.Bands began to disappear gradually with F-T cycles.

3.4.Physicochemical analysis

3.4.1.TPA

The texture of fish will contribute to its tastiness to some extent(Farajzadeh,Motamedzadegan,Shahidi,& Hamzeh,2016).The changes of hardness,springiness and chewiness of samples were represented in Table 2.The results showed that these properties were significantly reduced at the 1st and 2nd F-T cycle(P＜0.05),after which there was a gentle downward trend throughout the later period,with the lowest values belonging to the 8th F-T cycle.

3.4.2.Changes in color difference

Color parameters in shrimp over various F-T cycles were shown in Fig.4.For fresh shrimp,lightness(L＊)and redness(a＊)values of shrimp heads were 45.64±1.15 and－0.84±0.03 and that of shrimp abdomens were 47.25±0.62 and－1.11±0.17.Then,compared to the fresh samples,no significant difference was found for both the head and abdomen of shrimp in the 1st to 2nd F-T cycle,but was significantly lower after 3rd cycle(P＜0.05).The 6th and 7th F-T cycles resulted in lowerL＊values of sample compared to the 4th and 5th cycles(P＜0.05).Furthermore,thea＊values of shrimp head were increased from－0.84±0.03 to 5.38±0.14 and those of body were also increased from－1.11±0.17 to 1.94±0.05 after the 8th cycles(P＜0.05).There was no significant difference in samples between the 1st and 2nd F-T cycles.Thea＊values were gradually increased from 4th to 8th F-T cycles both in the head and the abdomen of shrimp(P＜0.05).

3.4.3.Changes in PPO activity

The changes of PPO activity in Pacific white shrimp under different F-T cycles were shown in Fig.5.The PPO activity of samples in 0-2 F-T cycles decreased dramatically(P＜0.05)(Fig.5).This might be because when the storage temperature was less than 35°C,PPO activity was positively correlated with temperature and the temperature of shrimp from the outside environment to the low temperature environment was reduced resulting in the decrease of PPO activity.An increase in PPO activity was observed with F-T cycles(P＜0.05).From the 2th F-T cycle,a growth trend of PPO activity was observed and the growth rate was significantly high.

Fig.1.Pseudo color of 1H-MRI in Pacific white shrimp with different freeze-thaw cycles.(For interpretation of the references to color in this figure legend,the reader is referred to the Web version of this article.)

3.4.4.Changes in TVB-N value

The effects of repeated F-T cycles on TVB-N value were shown in Table 3.The TVB-N value of fresh samples was 5.39±0.25 mg N/100 g and increased continuously during F-T cycles.Compared with the 1st or 2nd F-T cycles,TVB-N value of sample after 3th F-T cycles significantly increased(P＜0.05).These results reflected the lipid oxidation and the growth of microorganisms during repeated F-T cycles,especially after 4 F-T cycles.

3.5.Microbiological analysis

The quality loss in shrimps is closely related to the growth of spoilage microbes.The changes in total viable count(TVC)of samples during different F-T cycles(Table 3).The TVC of fresh shrimp samples was 3.88±0.01 lg CFU/g before increasing significantly to 5.14±0.09 lg CFU/g after 4 F-T cycles and to 6.54±0.05 lg CFU/g after 7 F-T cycles.

3.6.Changes of sensory evaluation

The sensory scores of shrimp samples with repeated F-T cycles were shown in Table 3.Consistent with the changes of texture parameters and color difference,sensory scores of shrimp samples decreased significantly with the F-T cycles,especially at the 3rd F-T cycle.The shrimp samples became unacceptable at the 6th F-T cycle,according to Wang et al.(2018).Combined with the changes of TVB-N value and TVC,these suggested that F-T cycles could induce the deterioration of shrimp quality,especially after 3 cycles,with an acceptable threshold of F-T cycles at 6.

4.Discussion

4.1.LF-NMR

The decrease inT22relaxation time indicated that as F-T cycles progressed,immobilized water shifted to free water and the content of water in muscle tissue decreased.During repeated F-T cycles,the different size and location of ice crystals formed during each freeze-thaw cycle damaged the protein network and the reduction of water content was closely related to the damage of cell membranes and myofibrillar protein caused by the repeated formation of ice crystals,especially after 3 cycles(Christian,Graham,& Stephen,2015;Passakorn & Soottawat,2016).The damaged myofibrillar network made immobilized water difficult to retain.Repeated F-T cycles could also cause the water to be continuously transferred from intracellular to extracellular regions and perimysium leakage or the change some conformations of protein and increase the content of free water and drip loss(Leygonie,Britz,&Hoffman,2012;Anese et al.,2012).In addition,the reduction of available macromolecules in protein side chains bound to water molecules also caused the release of these bonds or immobilized water and the remarkable increase in free water,especially after 3 F-T cycles(Table 1)(McDonnell et al.,2013).Regardless,the recrystallization of water causing mechanical damage of muscle tissue and protein oxidative denaturation were the primary cause of water migration and loss.The results indicated that F-T cycles induce water migration and loss,especially after 3F-T cycles,as compared to the fresh shrimp.

4.2.Changes in the microstructure of muscle tissue

Fig.2.Scanning electron microscopy micrographs(magnification:5000×)of the shrimp samples with different freeze-thaw cycles.The sample under 0 F-T cycle represented fresh shrimp.

Fig.3.SDS-PAGE patterns of muscle proteins in shrimp treated with different freeze-thaw cycles.0,2,4,6,8 at the top of picture represent the number of F-T cycle.The sample under 0 F-T cycle represented fresh shrimp.

A less compact structure likely resulted from protein denaturation and endomysium disruption during repeated F-T cycles.The reduction in the structural integrity of muscle protein might be due to the repeated formation of ice crystals causing physical damage(Zhang et al,2015a,b,c).The looser structure of muscle tissue from the disruption of myofiber contributed to the lower texture properties values of shrimp samples.The myofibrillar protein denaturation accompanied by the disruption of protein structure was induced by F-T cycles,which was aligned with the result of LF-NMR in instances,such as the lowered water-holding capacity of muscle.This indicated that repeated F-T cycles more seriously damaged to the microstructure,texture properties and muscle protein especially after 4 F-T cycles.

Table 2Texture properties of Pacific white shrimp over different freeze-thaw cycles.

4.3.SDS-PAGE patterns of protein

Frozen storage leads to protein oxidative denaturation(Nikoo &Benjakul,2015).For SDS-PAGE,the decrease may be caused by the proteolysis,denaturation or oxidation of protein during F-T cycles(Ma et al.,2015).At the same time,the release of some enzymes from mitochondria into the sarcoplasm may facilitate the protein fragmentation,especially at 4th F-T cycle,is caused by the disruption of muscle cells during repeated F-T cycles(Hultmann,Phu,Tobiassen,Aas-Hansen,& Rustad,2012).The results of SDS-PAGE indicated that protein fragmentation was accompanied by the aggregation of protein during repeated F-T cycles and the protein oxidative degradation was accelerated by increasing F-T cycles,especially after 4 F-T cycles.

Fig.4.Color of Pacific white shrimp with different freeze-thaw cycles.

4.4.TPA

Consistent with previous research,the hardness of rainbow trout fillets orProcambarus clarkiidecreased significantly during frozen storage were reported(Jouki,Mortazavi,Yazdi,Koocheki,& Khazaei,2014;Shi et al.,2018).The study showed that repeated F-T cycles could cause a clear linear decrease in the hardness of shrimp samples and the softening of shrimp muscle during storage,which produced detrimental side effects in the textural properties of shrimp.This decrease in texture properties caused by the repeated formation of ice crystals was associated with the loss in the integrity of muscle fibers and the weakening structure of muscle,which implied a lower resistance to shear force(Passakorn & Soottawat,2016).The result suggested that compared to fresh shrimp samples,repeated F-T cycles were more likely to accelerate the destruction in muscle fiber integrity,the increase of drip loss and the deterioration of texture properties,especially at the 1st and 2nd cycles.

4.5.Changes in color difference

The reduction of theL＊values agreed with the change of melanosis in shrimps(Arancibia,López-Caballero,Gómez-Guillén,& Montero,2015).Farajzadeh et al.(2016)have reported that theL＊value of shrimps coated with chitosan-gelatin during the refrigerated storage decreased and the color measurement results were consistent with the changes of melanosis scores in this study.In combination with the research of Bindu et al.(2013),repeated F-T cycles might promote the release of astaxanthins in nature by inducing the dissociation of carotenoprotein(Bindu,Ginson,Kamalakanth,Asha,& Srinivasa Gopal,2013).The increase ofa＊values might be induced by protein denaturation or associated with the pronounced release of free pigments caused by the destruction of actin-myosin causing red color(Bindu et al.,2013).Temperature fluctuations caused by repeated F-T cycles could promote protein denaturation and discoloration in the surface of shrimp,especially after 3 F-T cycles.

4.6.Changes in PPO activity

The melanosis of shrimp is related to the role of PPO.PPO could cause the oxidation of tyrosine to produce melanin resulting in browning(Barjinder & P Srinivasa,2017).When shrimp is stressed,colorless monohyericphenol and oxygen generates colorless bisphenol,which is oxidized into quinones substances and then combined with amino acid so that proteins began to produce black spots(Arancibia et al.,2015).It has been found that PPO was released and activated due to the damage of cell membrane or organelles,even without repeated melting and reformation of ice crystals during F-T cycles(Zhang et al,2015a,b,c).It is clear that the increase in the number of F-T cycles aggravated the release of PPO and the increase in PPO activity,especially after 2th F-T cycles.

4.7.Changes in TVB-N value,microbiological analysis and sensory evaluation

Generally,the TVB-N value of 20 mg N/100 g protein in shrimp represented the threshold of freshness and below 30 mg N/100 g protein was considered acceptable(Liao et al.,2018;Yang,Xie,& Qian,2017).TVB-N values were unacceptable following the 6th F-T cycle.These results revealed that F-T cycles could cause the oxidative degradation of protein,especially after 4 cycles,for seafood products,the threshold of freshness about TVC value is 5 lg CFU/g and a TVC value above 6 lg CFU/g represents an unacceptable quality(Liao et al.,2018).With the blackening,reddening,and putrification of samples during repeated F-T cycles,sensory characteristics such as color and odor deteriorated,which was reflected in the decrease in sensory scores.After 3F-T cycles,the sensory scores of samples decreased significantly and were lower than 15(acceptable threshold)after the 6th F-T cycle,which was consistent with the change in color difference and texture.Based on these indicators,the quality of shrimps treated with different F-T cycles started to deteriorate visibly at the 3rd F-T cycle and became unacceptable at the 6th F-T cycle.

5.Conclusions

In summary,multiple F-T cycles cause mechanical damage of muscle tissue and protein oxidative denaturation,especially after 3 F-T cycles,which prolonged relaxation time(T22andT23)significantly.Immobilized water(A22)and brightness of the pseudo-color diagram decreased.Protein aggregation was measured from SEM and the result of SDS-PAGE demonstrated the protein degradation that,was accelerated by multiple F-T cycles,especially after 4 F-T cycles,caused a sharp decrease in texture properties.In addition,the reformation of ice crystals caused by repeated F-T cycles resulted in the deterioration of texture properties and color,which was closely related to the damage of muscle microstructure and PPO activity.Consistent with TVC,TVB-N value and sensory score results,the deterioration of color and texture properties became noticeable after 3 F-T cycles and unacceptable after 6F-T cycles.The changes of sensory quality,TPA,TVB-N,TVC,protein degradation and nutritive value were not obvious in the first 3F-T cycles,were noticeable after 3F-T cycles,and were unacceptable after 6FT cycles.

Conflicts of interest

We declare that we have no conflict of interest.

Histogram is on behalf ofL＊and line graph is on behalf ofa＊.Each data is the mean values per treatment and bars represent the standard deviation from triplicate determinations.

Each data is the mean values per treatment and bars represent the standard deviation from triplicate determinations.Different letters on the top of data bars indicate significant differences(p＜0.05)between mean values.

Acknowledgement

The study was financially supported by China Agricultural Research System(CARS-47-G26),Shanghai promote agriculture by applying scientific & technological advances projects(2016No.1-1),Ability promotion project of Shanghai Municipal Science and Technology Commission Engineering Center(16DZ2280300);Key Laboratory of Refrigeration and Conditioning Aquatic Products Processing,Ministry of Agriculture and Rural Affairs.The project was supported by Key Laboratory of Refrigeration and Conditioning Aquatic Products Processing,Ministry of Agriculture and Rural Affairs(Grant No.KLRCAPP2018-11).