The comparison of ultrasound-assisted immersion freezing, air freezing and immersion freezing on the muscle quality and physicochemical properties of common carp (Cyprinus carpio) during freezing storage

Accepted Manuscript The comparison of ultrasound-assisted immersion freezing, air freezing and immersion freezing on the muscle quality and physicochemical properties of common carp (Cyprinus carpio) during freezing storage Qinxiu Sun, Fangda Sun, Xiufang Xia, Honghua Xu, Baohua Kong PII: DOI: Reference:

S1350-4177(18)31418-4 https://doi.org/10.1016/j.ultsonch.2018.10.006 ULTSON 4339

To appear in:

Ultrasonics Sonochemistry

Received Date: Revised Date: Accepted Date:

16 September 2018 29 September 2018 5 October 2018

Please cite this article as: Q. Sun, F. Sun, X. Xia, H. Xu, B. Kong, The comparison of ultrasound-assisted immersion freezing, air freezing and immersion freezing on the muscle quality and physicochemical properties of common carp (Cyprinus carpio) during freezing storage, Ultrasonics Sonochemistry (2018), doi: https://doi.org/10.1016/ j.ultsonch.2018.10.006

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The comparison of ultrasound-assisted immersion freezing, air freezing and immersion freezing on the muscle quality and physicochemical properties of common carp (Cyprinus carpio) during freezing storage

Qinxiu Sun, Fangda Sun, Xiufang Xia, Honghua Xu, Baohua Kong *

College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China

Corresponding author Tel.: +86-451-55191794; fax: +86-451-55190577 E-mail address: [email protected] (B. Kong)

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Abstract This study investigated the impact of ultrasound-assisted immersion freezing (UIF), air freezing (AF), and immersion freezing (IF) on the ice crystal size, protein thermal stability, and physicochemical properties of common carp (Cyprinus carpio) muscle during frozen storage. UIF samples had smaller ice crystals throughout the storage period than AF and IF samples did, which led to less damage to the muscle tissue. Low-field nuclear magnetic resonance analysis revealed that UIF reduced the mobility and loss of immobilized and free water. The thawing and cooking losses in the UIF samples were significantly lower than those in the IF and AF samples (P < 0.05). The AF samples had a higher shear force (P < 0.05) than UIF and IF samples did at the beginning of storage, and then the shear force reduced rapidly. During the 90-180 days, the shear force of the UIF samples was higher than that of the AF and IF samples (P < 0.05). Decreases in the Tmax and enthalpies were observed for all of the treatments during storage, and the UIF samples had a higher protein thermal stability than AF and IF samples did. The UIF samples showed lower thiobarbituric acid reactive substance and total volatile basic nitrogen values during storage than the AF and IF samples did (P < 0.05). Principal component analysis showed that there were significant correlations between the freezing methods and the ice crystal size, protein thermal stability and physicochemical characteristics of frozen muscles. Overall, UIF was an effective way to inhibit the deterioration of frozen fish during frozen storage.

Keywords: Common carp (Cyprinus carpio); Ultrasound-assisted immersion freezing; Frozen storage; Ice crystal; Protein thermal stability; Physicochemical properties 2

1. Introduction Common carp (Cyprinus carpio) is an important fish species in the world. China is the largest producer of common carp, accounting for approximately 70% of the country’s freshwater fish production. Due to its high water activity and abundant nutrients, fish is easily perishable [1]. To reduce spoilage, different preservative methods, such as drying [2], cooling [3], and freezing [4], have been employed. Frozen storage is a common preservation method for fish and fish products [5]. Despite microbial growth being effectively inhibited, quality deterioration and lipid oxidation still occur during frozen storage. The quality of frozen food is greatly affected by the size of the ice crystals and their location inside the meat [6]. Freezing rate has a great influence on the size and uniformity of ice crystals. Fast freezing produces fine and uniform extra- and intra-cellular ice crystals and causes little damage to food structures; slow freezing produces large and irregular extra-cellular ice crystals, which leads to the destruction of muscle structure and reduces the sensory acceptability of food [7]. In addition, the size of ice crystals can grow with extended storage time [8]. The increase in freezing rate could effectively reduce the size of ice crystal, thus reducing the destructive effect caused by ice crystals. In recent years, many efforts have been made by researchers to look for new fast freezing methods. Power ultrasound with low-frequency and high intensity (20-100 kHz with 10-1000 W cm−2) has been proven to be extremely useful in shortening freezing time [9] [10]. Ultrasound can produce cavitation and microstreaming, which can induce the nucleation of ice crystals, promote the recrystallization of ice crystals, and accelerate mass transfer and heat transfer, thus shortening freezing time [11]. Compared to other freezing methods, 3

ultrasound-assisted immersion freezing (UIF) has several advantages: the technique is chemically non-invasive and does not require direct contact with the product, and the use of UIF does not present legislative difficulties [12]. Although the effect of UIF on the quality of foodstuff has been studied by several researchers, the reports mainly focus on vegetables [13], fruits [12] and ice cream [14]. Moreover, most studies were carried out on the freezing process and freezing rate, while studies on the effect of UIF on quality of aquatic products during storage are limited. In our previous study, the influence of UIF on the freezing rate and quality of porcine longissimus muscles under different ultrasound powers was evaluated [10]. The results showed that samples treated with UIF at 180 W ultrasound powers had significantly increased freezing rates and reduced the phase transition times compared with control samples without ultrasound assistance. We also evaluated the effect of UIF on quality, water distribution and microstructure of common carp (Cyprinus carpio) muscle (unpublished paper). The results revealed that samples treated with 175 W ultrasound power had the shortest freezing time and reduced ice crystal size. In the present work, the effects of UIF on the change of ice crystal size, water distribution, protein thermal stability and physicochemical properties of common carp (Cyprinus carpio) muscle during frozen storage were investigated.

2. Materials and Methods 2.1. Chemicals Haematoxylin, eosin, xylene, ammonium molybdate, and sulphuric acid were purchased 4

from the Solabio Corporation (Beijing, China). All chemicals and reagents were of analytical grade.

2.2. Preparation of fish chops A total of 45 common carp with an average live weight of 1100 ± 60 g were obtained from a local market (Harbin, China) and transferred immediately to the laboratory. The fish were immediately anaesthetized, beheaded, eviscerated, and cut into approximately 5-cm-long chops perpendicular to the length of the fish, as shown in Fig. 1. For each fish, 2 chops were obtained, so 90 chops were obtained in total. The first half of the fish was termed chop A, and the second half of the fish, chop B. The weight of each chop was approximately 210 ± 15 g. Each chop was washed with flowing tap water and packed in a polyethylene zipper bag. All samples were stored in a 4 °C refrigerator for 12 h before freezing. There were three treatments; for each treatment, 30 chops were used for analysis, and at each storage time for each treatment, 6 chops were used (3 chop A and 3 chop B). The whole chop A was used to determine thawing loss and cooking loss, and the back of chop A was used to determine shear force. The back of chop B was used to determine differential scanning calorimetry (DSC) and low field-nuclear magnetic resonance (LF-NMR), and the belly of chop B was used to determine total volatile base nitrogen (TVB-N), thiobarbituric acid reactive substance (TBARS), and microstructure. The fish samples were frozen using three different freezing processes: air freezing (AF), immersion freezing (IF), and UIF. The AF samples were frozen in a common refrigerator (-25.0 ± 0.5 °C) until the centre temperature of the fish chops reached approximately -18 °C. The ultrasound-assisted immersion freezer was custom-made by Nanjing Xianou Co., Ltd. 5

(Nanjing, China). The structure of the freezer was same as described by Zhang et al. [10], with 95% ethanol plus 5% fluoride as a coolant. The size of the freezing tank was 30 cm × 22 cm × 26 cm. At the bottom of the freezing tank, there were 10 ultrasound transducers. For IF and UIF, fish chops were placed 10 ± 0.5 cm above the bottom of the tank, and the freezing temperature was set at -25 ± 0.5 °C. The output power of the equipment was set at 0 (IF) and 175 W (UIF) with a frequency of 30 kHz. Ultrasound with a 30 sec on/30 sec off cycle was applied for 9 min after the centre temperature of the fish chops had decreased to 0 °C. The freezing process was considered to be completed when the geometric centre temperature reached approximately -18 °C. After being frozen, all the samples were stored at -18 ± 1 °C for 180 days. Samples were collected on days 0, 30, 60, 90, and 180 for analysis.

2.3. Light microscopy observation The microstructure of the fish samples was observed as described by Zhang et al. [10]. The frozen samples (0.5 × 0.5 × 1 cm3) were cut into 4-μm-thick slices perpendicular or parallel to the orientation of the muscle fibres in a Leica CM1850 cryostat (CM1850, Leica, Germany). The sample slices were placed on glass slides and stained in haematoxylin staining solutions for 15 min and then transferred in 1% hydrochloric acid alcohol solution to make the colour uniform. The slices were washed by running tap water for 15 min. Then, the samples were stained in eosin for 10 min. The slice samples were then dehydrated with absolute ethanol (70%, 80%, 90%, and 100%, each for 2 min). Finally, they were cleared in xylene and then mounted in a resinous medium. All the samples were observed with a light microscope (Olympus BX41, Olympus Optical Co. Ltd., Tokyo, Japan) (magnification 10 × 10) fitted with a digital camera (Nikon DS-5M, Nikon, Japan). The diameter of ice crystals of samples was analysed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, 6

USA). 2.4. Low-field nuclear magnetic resonance LF-NMR relaxation measurements were done according to the methods of Zhang et al. [10] with a minispec mq20 LF-NMR analyser (Bruker Optik GmbH Ettlingen, Germany). Thawed samples (4 °C) were cut into rectangular blocks (1 × 1 × 3 cm3) and placed in nuclear magnetic resonance tubes. The transverse relaxation time (T2) was measured using the Carr-Purcell-Meiboom-Gill pulse sequence and the CONTIN algorithm after normalizing the raw data. 2.5. Determination of thawing loss, cooking loss and shear force Thawing loss was determined according to the method of Xia, Kong, Liu, and Liu [15]. Briefly, the samples were weighed immediately before thawing (W0) and then the frozen samples were thawed to a geometric centre temperature of 4 °C and blotted dry with paper towels and weighed (W1) immediately. The thawing loss was calculated with following equation:

Thawing loss (%) 

W0  W1  100% W0

The cooking loss was determined by the procedure described by Faridnia, et al. [16]. The thawed sample was weighed (W1) and then cooked in a water bath at 85 °C until the centre temperature of the sample reached 75 °C. The cooked sample was immediately blotted dry and weighed (W2). The cooking loss was calculated as:

Cooking loss (%) 

W1 - W2  100% W1 7

After determining cooking loss, the fish samples were used to determine shear force. From each chop, four cylindrical cores (1.27 cm in diameter) were obtained parallel to the muscle fibres, and 5 chops were used for shear force analysis. The shear force (N) was determined perpendicular to the fibre direction using a texture analyser (Stable Micro System; TA: XT2i, England) with a knife blade (HDP/BSW). A 25 kg load cell was used, and the cross-head speed of the knife blade was 5 mm/sec. 2.6. Determination of thiobarbituric acid reactive substance and total volatile base nitrogen The thiobarbituric acid reactive substance (TBARS) value was measured as described by Sun, Chen, Li, Zheng, and Kong [17]. An accurately weighed finely chopped meat sample (2 g) was placed in a 25 mL screw cap test tube, 3 mL of thiobarbituric acid (TBA) solution and 17 mL of trichloroacetic acid-hydrochloric acid solution were subsequently added. The mixture was heated in boiling water (100 °C) for 30 min. After being cooled to room temperature, a 4 mL aliquot of the suspension was mixed by vortexing with 4 mL of chloroform for 1 min, followed by centrifugation with a centrifuge (AllegraTM, 64R, Beckman, Germany) at 1000×g for 10 min. The absorbance of the supernatant was read at 532 nm. The TBARS value, expressed as mg of malonaldehyde (MDA)/kg of muscle sample, was calculated by using the following equation:

TBARS(mg MDA/ kg) 

A532  9.48 Ws

Where A532 is the absorbance (532 nm) of the assay solution, Ws is the meat sample weight (g), and “9.48” is a constant derived from the dilution factor and the molar extinction coefficient (152 000 M-1 cm-1 ) of the red TBA reaction product.

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The TVB-N value was detected as in the procedure described by Sun, Zhao, Chen, Zhang, and Kong [18]. Briefly, 10 g of minced fish samples were mixed with 100 mL distilled water and shocked for 30 min at 4 °C, and the sample solution was then filtered. Then, 5 mL of filtrate and 5 mL of magnesium oxide solution (10 g/L) were mixed and then distilled with Kjeldahl for 5 min; the receiving solution was 10 mL of boric acid solution (20 g/L) mixed with 2-3 drops of indicator (2 g/L methyl red-ethanol solution and 1 g/L methylene blue; 1:1, v/v). The absorption solution was titrated with hydrochloric acid standard titration solution (0.01 mol/L), and the blue-violet colour was used as the end point. The TVB-N value was calculated as follows:

TVB - N (mg/100g) 

(V1  V2 )  c 14 100 m  5 / 100

V1 is the volume of sample solution consumed hydrochloric acid standard solution (mL); V2 is the volume of hydrochloric acid standard solution consumed in the control group (mL); c is the concentration of hydrochloric acid standard solution (mol/L); and m is the mass of the sample (g). 2.7. Differential scanning calorimetry The endothermal transitions of muscle samples were registered using a differential scanning calorimeter (TA multi cell DSC, USA). Temperature calibration was performed using an Indium thermogram. Common carp samples of 8-12 mg were accurately weighed into standard aluminium pans, sealed and then heated from 25 to 100 °C at a scanning rate of 5 °C/min under a nitrogen atmosphere. An empty pan was set as the reference. Enthalpy values (△H) and peak transition temperature (Tmax) were estimated from the thermogram by 9

Pyris-12 software (Perkin-Elmer Instruments, USA). 2.8. Statistical analysis Data were expressed as the mean values ± standard deviations and analysed by the general linear model procedure of the Statistix 8.1 software package (Analytical Software, St Paul, MN, USA). The significance of the main effects was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison, and the confidence interval was set at 95% (P < 0.05). Principal component analysis (PCA) was performed using SPSS 22.0 (Analytical Software, USA). Three batches of fish (replicates) were produced. For each batch of samples, all measurements were run in triplicate.

3. Results and discussion 3.1. Microstructure of the ice crystal of muscle tissues Ice crystal size and uniformity are crucial to the final quality of frozen foods. The expansion pressure of ice crystals can cause irreversible damage to the structure of muscle tissue. Fig. 2 represents the microstructure of common carp muscle (A: cross section; B: longitudinal section; C: average diameter of the ice crystal). The pink is the muscle fibre, and the white is the hole left by the ice crystal. As shown in Fig. 2A and 2B, at day 0, for the AF samples, ice crystals were clearly evident, large and irregular. As is known, large, irregular ice crystals are responsible for muscle tissue damage, which in turn affects the quality of fish during extended frozen storage. A better muscle structure was observed in the UIF samples than that of the AF samples. This may be explained by the cavitation produced by ultrasound, 10

which could be used as a nucleus to induce the formation of ice crystals, and the collapse of cavitation bubbles broke the pre-existing ice crystals into smaller sizes that again acted as primary nuclei. In addition, microstreaming produced by the motion of cavitation bubbles enhanced heat and mass transfer. All of this likely accelerated the freezing rate and promoted the formation of small and uniform ice crystals. To better understand the change of ice crystal size during storage, Image-Pro Plus software was used to analyse the average diameter of ice crystals (Fig. 2C). The average ice crystal diameters of the AF, IF, and UIF samples were 37.73, 26.55, and 21.47 μm at 0 days and rapidly increased to 86.12, 57.93, and 49.02 μm at 180 days, respectively (P < 0.05). The AF samples had significantly larger average ice crystal diameters than did the IF and UIF samples, and the UIF samples expressed the smallest average ice crystal diameters (P < 0.05) during storage. At 180 days, the average diameter of ice crystals in the UIF samples was 43.22% lower than that of the AF samples. Inside the frozen food, there were three phases, namely, the solid phase (ice crystals of different sizes), liquid phase (residual unfrozen aqueous solution), and gas phase (water vapor). The water vapor pressures of both the solution in the liquid phase and small ice crystals were larger than that of large ice crystals. Therefore, the water and water vapor moved from the high vapor pressure to the lower vapor pressure and constantly adhered and condensed onto the large ice crystals. As a result, the large ice crystals increased in number, and the small ice crystals gradually decreased in number and disappeared. The formation of large ice crystals increased the degree of mechanical damage to the cells, leading to denaturation of the protein and increasing thawing and nutrient losses. After freezing, the UIF samples had more uniform ice crystals than the AF and IF samples, so the degree of ice crystal growth during storage in the UIF samples was less than that of others. This indicated 11

that UIF could effectively reduce the increased rate of ice crystal formation during frozen storage. Tu et al. [13] also reported that ultrasound offered a useful approach for minimizing the damage of ice crystals to cell structures. 3.2. Low-field nuclear magnetic resonance The water distribution in frozen food is one of the key indexes to explain the water-holding capacity and quality changes of food during storage. LF-NMR can be applied to determine the distribution of water. As depicted in Fig. 3A, 3B and 3C, there were three peaks, referred to as T2b (0-10 ms), T21 (10-100 ms), and T22 (100-1000 ms). T2b represents bound water, which is tightly bound to protein side-chains and some hydrophilic groups on macromolecules; T21 represents immobilized water that exists in the myofibrillar protein network; and T22 represents free water that exists within the space among fibre bundles depending on capillary force [19]. As shown in Fig. 3D, there was no significant difference for T2b among the three freezing conditions at 0 days (P > 0.05), which indicated that the freezing methods had no noticeable effect on T2b relaxation time during the freezing process. This may be because the bound water is tightly bound to proteins, and it is very resistant to freezing or heating [20]. During frozen storage, the T2b relaxation time was prolonged with increased storage time; the T2b of the AF samples was longer than that of the IF and UIF samples, and the UIF samples had the shortest T2b relaxation time. The change in the T2b relaxation time was related to the degeneration of myofibrillar proteins. When the protein structure is altered, the amount of water bound by protein molecules through non-covalent interaction is reduced [21]. Large and irregular ice crystals in the AF samples destroyed cell structures, resulting in the release of cell 12

contents, thus promoting protein denaturation. Thus, the longer T2b relaxation time of the AF samples revealed that the bound water content in AF samples was lower than that of the IF and UIF samples during the frozen storage. From Fig. 3E, the T21 relaxation time increased significantly (P < 0.05) with increased storage time. The UIF samples had the lowest T21 among all the samples, which was 19.82% lower than that of the AF samples at 180 days (P < 0.05). The increased T21 revealed that extended frozen storage time resulted in a certain level of the immobile water changing to free water, and UIF reduced this change. These results may be because frozen storage leads to water redistribution and the formation of extracellular ice crystals, which produce great mechanical damage to cell membranes, and further cause denaturation of sarcolemma, and myofibrillar protein [10]. In addition, the oxidation of muscle proteins during freezing storage could influence the structure of muscle and induce the aggregation of proteins by increasing carbonyl content, turning SH groups into S-S groups and transforming α-helix into β-sheet, thus reducing the water holding capacity of protein [22]. The application of UIF shortens the freezing time and promotes the formation of small and uniform ice crystals, thus reducing the damage to fish muscle tissues, and thus, the ability of protein to bind water was better than others. The increasing in the T21 relaxation time during storage could be explained by the increased damage produced by the growth of ice crystals. The change in T22 (free water) was similar with T21 (immobilized water) (Fig. 3F). This result revealed that free water in the UIF samples had lower mobility and was more closely trapped in muscle tissue than in the other freezing treatments, thereby reducing the thawing and cooking losses. Therefore, we can conclude that relaxation times have some relationship 13

with water-holding capacity, and shortened T2 relaxation times could be used as an indirect reflection of improved water-holding capacity. 3.3. Thawing loss, cooking loss and shear force Thawing loss has been reported to be an important indicator for frozen fish quality [23]. The increase in thawing loss not only affects the quality of products but also reduces the weight of products, ultimately causing economic loss. As shown in Fig. 4A, the thawing losses of the AF, IF, and UIF samples were 4.61%, 3.83%, and 3.11% at 0 days, and they increased to 6.90%, 5.47%, and 4.95% at 180 days, respectively (P < 0.05). The AF samples had significantly higher thawing losses than the IF and UIF samples, and the UIF samples had the lowest thawing losses (P < 0.05) during 180 days of frozen storage. The increase in thawing loss during storage may be attributed to the growth of ice crystals, which squeeze adjacent muscle fibres and damage tightly arranged muscle tissue [19]. During the thawing process, due to the destruction of muscle fibres, the water held by capillary force decreases, which is unlikely to be reabsorbed into the interior of the cell. Moreover, the damage to the cell membrane also causes cell fluid to leak out. In addition, fat oxidation may lead to protein degeneration and structure changes, thus reducing the water-binding ability of protein. During the UIF process, the advancing speed of ice crystals inside and outside the cells was faster than the rate of water transfer, so small and uniform ice crystals were formed inside and outside the cells. Therefore, the ice crystals of UIF samples were remarkably smaller than those of the AF samples, as shown in Fig. 2, even though they grew during the storage period. Similar results were obtained by Islam et al. [11], who found that the application of UIF significantly decreased the thawing loss of frozen mushrooms. 14

Cooking loss includes a large amount liquid and little soluble matter lost from meat during heating. Cooking loss is mainly caused by the damage of muscle structures due to heat-induced myofibrillar protein degeneration [24] Cooking loss had a similar change trend to thawing loss. At 0 days, the cooking loss of the UIF sample was 4.95%, which was significantly lower than that of the AF samples (6.91%) and IF samples (5.47%) (P < 0.05). At the end of storage (180 days), the cooking loss of the UIF samples was still the lowest among all the samples. The difference in the cooking loss of fish samples treated by different freezing conditions can be explained by the size of ice crystals, destruction of muscle tissue and denaturation of protein [25]. The water-holding capacity of meat is greatly influenced by change of the structure of the myofibrillar protein, particularly myosin [26]. The shear force reflects the change in muscle tenderness [27]. At the beginning of storage, the AF samples showed higher shear force than the IF and UIF samples (P < 0.05), and there were no significant differences between the IF and UIF samples (P > 0.05). The higher shear force in the AF samples at the beginning of storage may be due to the higher thawing and cooking loss, which leads to a hardness increase and tenderness decrease. The shear force in all samples showed a decreased trend during frozen storage, and the decreasing speed of the AF samples was obviously higher than that of the IF and UIF samples. At 180 days, the shear force of the IF and UIF samples were significantly higher than that of AF samples, and the UIF samples had the highest shear force (P < 0.05). The decreased shear force during storage may be due to the destruction of muscle fibres and changes of protein structure, which may be attributed to the growth of ice crystals [28]. For the AF samples, large ice crystals formed by a slow freezing rate (Fig. 2) may cause more serious muscle tissue damage and protein structure changes, which lead to more loss of shear force. In 15

addition, some enzymes, including the endogenous protein enzymes, could also decompose the proteins of connective tissue, such as desmin, connectin, and troponin, thus breaking down the tight structure of the muscle tissue [27]. 3.4. Thiobarbituric acid reactive substance and total volatile base nitrogen The TBARS value is one of the main indicators of lipid oxidation in meat products [29]. TBARS provides information on the amount of secondary oxidation products, which are primarily derived from the degradation of polyunsaturated fatty acids and contribute to the unpleasant flavours of food [30]. In addition, lipid oxidation may cause the proteins oxidation, which will alter the protein structure, cause the peptide chain scission, decrease the protein solubility, and change the protein functional properties, such as water-holding capacity and texture-forming ability [31, 32]. Although biochemical reactions are retarded to a great extent under freezing condition, some non-negligible oxidative degradation still occurs. As shown in Fig. 5A, the TBARS values of all samples increased during storage, which indicated that the lipid oxidation increased with the extension of frozen storage time (P < 0.05). The TBARS value was approximately 0.20 mg MDA/kg at 0 days, and it rapidly increased to 0.48, 0.43, and 0.37 mg MDA/kg at 180 days for the AF, IF, and UIF samples, respectively (P < 0.05), Generally, when TBARS value is less than 0.664 mg MDA/kg, foods are considered still fresh [33, 34]. The AF samples showed a significantly higher TBARS than the IF and UIF samples, and the UIF samples had the lowest TBARS (P < 0.05) during 60 to 180 days of frozen storage, which indicates that the fat oxidation rate of the UIF samples was lower than that of the AF and IF samples. Some studies showed that freezing and thawing of muscle tissues can accelerate lipid oxidation due to ice crystals that can damage cells and cause the 16

release of oxidants, especially non-heme iron [15, 35]. During frozen storage, the growth of ice crystals disrupts the integrity of muscle cells; thus, the oxidative precursors inside cell are released and accelerate the oxidation reaction [36]. The UIF produced small and uniform ice crystals in muscle tissue by relative fast freezing rates, which may be a major reason for a lower extent of lipid oxidation in the UIF samples over a long time of frozen storage. In addition, UIF could inhibit the lipids oxidation by decreasing lipase, phospholipase and lipoxygenase activities [37]. TVB-N is a major indicator to assess muscle freshness [38, 39]. It quantifies a large number of basic volatile compounds, such as ammonia, trimethylamine, methylamine, dimethylamine, etc., which are produced through the degradation of protein and non-protein nitrogen compounds. The initial TVB-N values of AF, IF, and UIF samples were 5.48, 5.47 and 5.18 mg/100 g, respectively (Fig. 5B), which showed no significant difference (P > 0.05). With extended storage, the increase rate of TVB-N in the AF samples was faster than that in the

IF and UIF samples. At 180 days, the TVB-N value of the UIF samples was 13.30 mg/100 g, which was 10.38% and 27.20% lower than that of the IF and AF samples, respectively (P < 0.05), indicating that fast freezing rate can reduce TVB-N formation in fish samples during frozen storage. The increase in the TVB-N value during storage may be attributed to the combination of microbiological and autolytic activities [5]. High TVB-N values are not desirable and are related to unpleasant flavour in meat. In the present study, after 180 days of storage, the TVB-N values of all samples ranged from 13.43 to 18.27 mg/100 g, which still remained below the limit (35 mg/100 g) above which foods are always considered to be inedible for human beings [40].

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3.5. Differential scanning calorimetry Differential scanning calorimetry (DSC) is a quick and simple technique to determine the protein denaturation temperature, and it is also a direct indicator of protein spatial structure (i.e., protein tertiary and quaternary structure). The peak temperatures (Tmax) represent the temperature of protein denaturation and reflect the stability of protein structure, while enthalpy (△H) shows the quantity of necessary energy to induce protein denaturation [41]. The DSC results of the frozen fish samples are shown in Fig. 6 and Table 1. There were two characteristic peaks at temperatures 47.79-55.76 °C (Tmax1) and 68.23-76.63 °C (Tmax2), corresponding to the denaturation temperature of myosin and actin, respectively. Similar to us, Campo-Deaño, Tovar, Borderías, and Fernández-Martín [42] found two major endothermic peaks in squid (Dosidicus gigas) surimis. The maxima transition temperature (Tmax) of the fish muscle decreased to lower temperatures with extended frozen storage time. The Tmax1 of the fish muscles in AF, IF, and UIF samples were 52.47, 54.17, and 55.76 °C at 0 days and decreased to 45.25, 47.79, and 50.22 °C at 180 days, respectively (P < 0.05). The Tmax2 in AF, IF, and UIF samples were 73.70, 75.21, and 76.64 °C at 0 days and decreased to 68.24, 70.78, 72.64 at 180 days, respectively (P < 0.05). The Tmax1 and Tmax2 of the UIF samples were significantly higher than those of the AF and IF samples, and the AF samples had the lowest Tmax1 and Tmax2 over 180 days of frozen storage (P < 0.05). From the Tmax, the UIF samples had a higher thermal stability than the IF and AF samples throughout the frozen storage period. Protein denaturation includes the dissociation of intramolecular bonds (major, non-covalent bonds and a few covalent bonds), so it is an endothermic process, producing endothermic peaks. A significant decrease in the transition temperature showed that the 18

thermal stability of the muscle protein decreased during frozen storage, which might be due to the dissociation of the subunits in myosin and actin denaturation [43]. The UIF samples had smaller and more uniform ice crystals than the other two groups; thus, the degree of myosin denaturation was less serious [44]. Similarly, the △H1 and △H2 of myosin and actin denaturation were substantially decreased during frozen storage (P < 0.05). The △H1 and △H2 of the UIF samples were significantly higher than those of the AF and IF samples, and the AF samples had the lowest △H over 180 days of frozen storage (P < 0.05). At the end of storage, the △H1 and △H2 of the UIF samples were 0.842 and 0.198 J/g, which were 26.6% and 33.8% higher than those of AF samples (P < 0.05), respectively. Generally, the loss of △H could be related to the increased protein aggregation and weakening of hydrogen bonds induced by protein denaturation. The formation and size increase of ice crystals during freezing and frozen storage would cause cell disruption and protein denaturation. UIF resulted in small ice crystals during frozen storage, which might lead to less extensive protein denaturation and protein structure destruction. 3.6. Principal component analysis It is well-known that the freezing condition plays an important role in muscle physical properties, but there was not an objective method to measure this influence. Now, it is possible to establish a correlation between freezing condition and ice crystal size, protein thermal stability and physicochemical characteristics of frozen common carp. Therefore, PCA was performed to obtain linear combinations of muscle microstructure, water distribution, DSC and the physical characteristics throughout the storage period for the three groups of frozen samples. 19

As shown in Fig. 7A, the PCA results revealed that the 96.08% of the total variance in the data is explained by the first two principal components (PCs) (91.90% PC1 and 4.18% PC2), which indicates there is strong interrelation between freezing conditions and quality of frozen fish during frozen storage. Fig. 7B shows the plane of the first two PCs; the properties of the fish are located in quadrants 1, 2, 3, and 4. The freezing conditions in two-dimensional space, with PC1 and PC2 as loading factors, are shown in Fig. 7C. The treatments, inferred to be located in quadrants 1, 2, 3, and 4, were correlated with the variables in the corresponding quadrants. From a physicochemical point of view, the effect of freezing condition is substantial in frozen fish, since samples frozen by AF, IF, and UIF stored for the same time have remarkably different physicochemical properties, suggesting that these changes can be essentially attributed to the freezing condition. The projection of the samples onto the space of the two first PCs (Fig. 7C) showed a clearly difference among fish frozen by different conditions and stored for different times. During storage of the AF, IF, and UIF samples, the first group was characterized by AF-60, -90, and -180; IF-90 and-180; and UIF-180 samples (Fig. 7C); these were the samples with the highest values of the average diameter of ice crystals, thawing loss, cooking loss, TBARS, TVB-N, T2b, T21, and T22. The second group was includes AF 0 and 30 days, IF 0, 30, 60 days, and UIF 0, 30, 60, and 90 days samples, with the highest shear force value, Tmax1, Tmax2, △H1, and △H2. Based on these results, changes in the AF, IF, and UIF samples that occurred during frozen storage affected fish quality at 30-180 days, 60-180 days, and 90-180 days, respectively; thus, a shelf life of 30, 60, and 90 days for AF, IF, and UIF, respectively, are considered to be good for common carp. Furthermore, thawing loss, cooking loss, TBARS, TVB-N, T2b, T21, and T22 were quite closely 20

linked to the average diameter of ice crystals. It is hypothesized that the increase in ice crystals is primarily responsible for the quality deterioration of frozen fish.

4. Conclusions The results of this study showed that UIF inhibited the growth of ice crystals, decreased the mobility and loss of immobilized and free water, reduced thawing and cooking loss, and retarded the increase in TBARS and TVB-N during storage when compared to AF and IF. The UIF was more efficient in keeping the original muscle tissue state, and the UIF sample had higher thermal stability than AF and IF samples. Overall, UIF is an effective way to reduce the deterioration of frozen fish during frozen storage.

Acknowledgements This study was funded by the National Natural Science Foundation of China (grant no. 31771990) and the National Key Research and Development Plan during the 13th five-year plan period (grant no. 2016YFD0401504-03).

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27

Figure captions Fig. 1. Schematic diagram showing the experimental design Fig. 2. Changes in the microstructure cross section (A) and longitudinal section (B), and the average diameter of the ice crystals (C) of common carp muscles frozen by air freezing (AF), immersion freezing (IF), and ultrasound-assisted immersion freezing (UIF) during frozen storage. The means for the same freezing method with different uppercase letters (A-E) differ significantly (P < 0.05); the means between freezing methods on the same days with different lowercase letters (a to d) differ significantly (P < 0.05). Fig. 3. Changes in the water distribution and T2 relaxation time of common carp muscles frozen by air freezing (AF), immersion freezing (IF), and ultrasound-assisted immersion freezing (UIF) during frozen storage. A: AF sample; B: IF sample; C: UIF sample. The means for the same freezing method with different uppercase letters (A-E) differ significantly (P < 0.05); the means between freezing methods on the same days with different lowercase letters (a to d) differ significantly (P < 0.05). Fig. 4. Changes in the thawing loss (A), cooking loss (B), and shear force (C) of common carp muscles frozen by air freezing (AF), immersion freezing (IF), and ultrasound-assisted immersion freezing (UIF) during frozen storage. The means for the same freezing method with different uppercase letters (A-E) differ significantly (P < 0.05); the means between freezing methods on the same days with different lowercase letters (a to d) differ significantly (P < 0.05). Fig. 5. Changes in TBARS (A) and TVB-N (B) of common carp muscles frozen by air 28

freezing (AF), immersion freezing (IF), and ultrasound-assisted immersion freezing (UIF) during frozen storage. TVB-N, total volatile basic nitrogen; TBARS, thiobarbituric acid reactive substances. The means for the same freezing method with different uppercase letters (A-E) differ significantly (P < 0.05); the means between freezing methods on the same days with different lowercase letters (a to d) differ significantly (P < 0.05). Fig. 6. Differential scanning calorimetry (DSC) of common carp muscles frozen by air freezing (AF), immersion freezing (IF), and ultrasound-assisted immersion freezing (UIF) during frozen storage. Fig. 7. Principal component analysis (PCA) of the properties of common carp muscles frozen by air freezing (AF), immersion freezing (IF), and ultrasound-assisted immersion freezing (UIF) during frozen storage. A: Correlation of variables to factors in PCA based on factor loadings; B: loadings for the first two principal components; C: weighed PCA bi-plot of scores. TVB-N, total volatile basic nitrogen; TBARS, thiobarbituric acid reactive substances.

29

5 cm 210 g

5 cm 210 g

Remove

Remove

A

B

UIF

IF

AF

-18 oC

0, 30, 60, 90, 180 days DSC

Thawing loss

A

B

Cooking loss

LF-NMR Microstructure

Cutting force

TBARS/TVB-N DSC TBARS /TVB-N

LF-NMR

Thawing /Cooking loss

Shear force

Microstr ucture

Fig. 1.

30

A

0days

30days

60days

90days

180days

0days

30days

60days

90days

180days

AF

IF

UIF

B AF

IF

C

Average diameter of the ice crystal (um)

UIF

100

80

Aa

AF IF UIF

Aa

60

Ab

Ba Bb Ca

40

Ca

Cb

Ac Bc

Cb Cc

Dc

Db Ec

20

0 0

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60

90

180

Storage time (days) 31

Fig. 2.

32

7

A

D

Aa

Nor m a lize

Ab

AF IF UIF

6

Ba

d a m p litu

T2b (ms)

Ca

T 21

Ac Bb

5

Bc

Cb Da Cc

Db

4

d e (% )

T 2b

Ea

Db EaEa

3

T 22

2 0

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60

90

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Storage time (days)

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70

E

d Nor m a lize

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AF IF UIF

50

a m p litu d e

T21 (ms)

Aa

40

DaDab Db

Ea

Ab

Ca CabCb

Bb

60

90

Ac Bc

Eb Ec

30 20

(% )

10 0 0

30

180

Storage time (days)

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250

AF IF UIF

Ab Ba

Ac

Ca Bb

a m p litu d e

T22 (ms)

d Nor m a lize

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Aa

F

150 Da CDab

Da

100

Bc

Cb BCc

CDb Db

Db

(% )

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Fig. 3.

33

Thawing loss (%)

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Eb

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Fig. 4.

34

0.50

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A AF IF UIF

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Ab Ba

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TVB-N (mg/100g)

18

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Fig. 5.

35

A Endothermic heat flow (j/g)

AF-0 AF-30 AF-60 AF-90 AF-180

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Fig. 6.

36

0.5

PC1 (91.90% )

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0.948

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0.939

0.328

Var iables

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0.947 -0.87

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Fig. 7.

37

Table 1. Changes in the maximum transition temperature (Tmax) and denaturation enthalpy (△H) of myosin (peak I) and actin (peak II) of common carp muscle frozen by air freezing (AF), immersion freezing (IF), and ultrasound-assisted immersion freezing (UIF) during frozen storage. Storage time (days)

Tmax1 (°C)

Tmax2 (°C)

△H1 (J/g)

△H2 (J/g)

A-D

0

30

60

90

180

AF

52.47±0.25Ac

51.39±0.17Ac

49.54±0.97Bc

47.42±0.85Cc

45.25±0.35Dc

IF

54.17±0.29Ab

52.87±0.91ABb

51.93±0.23BCb

50.39±0.74Cb

47.79±0.69Db

UIF

55.76±0.42Aa

54.55±0.62ABa

53.67±0.39BCa

52.51±0.63Ca

50.22±0.59Da

AF

73.70±0.20Ac

72.06±0.84Ac

71.52±0.45Bc

69.84±0.38Cc

68.24±0.78Dc

IF

75.21±0.27Ab

74.23±0.31ABb

73.13±0.40BCb

71.93±0.42CDb

70.78±0.41Db

UIF

76.64±0.59Aa

76.07±0.21Ba

74.99±0.70Ba

73.55±0.49Ca

72.64±0.74Da

AF

1.217±0.025Ac

1.113±0.014Bc

0.874±0.013Cb

0.824±0.011Dc

0.665±0.006Ec

IF

1.286±0.019Ab

1.174±0.013Bb

0.926±0.019Ca

0.851±0.012Db

0.811±0.010Eb

UIF

1.379±0.009Aa

1.266±0.011Ba

0.953±0.012Ca

0.878±0.005Da

0.842±0.010Ea

AF

0.243±0.005Ab

0.217±0.006Bb

0.204±0.004Bb

0.180±0.010Cb

0.148±0.006Db

IF

0.255±0.006Ab

0.233±0.005Bab

0.216±0.005Cb

0.203±0.006Da

0.184±0.006Da

UIF

0.296±0.011Aa

0.250±0.009Ba

0.230±0.006BCa

0.212±0.004CDa 0.198±0.007Da

Means in the same row with different letters differ significantly (P < 0.05); a-c for the same

index, means in the same column with different letters differ significantly (P < 0.05).

38

Highlights 1. Ultrasound immersion freezing (UIF) reduced the ice crystal size in common carp muscle. 2. UIF inhibited the protein denaturation and fat oxidation of common carp muscle. 3. UIF reduced the mobility and loss of immobilized and free water. 4. UIF decreased the thawing and cooking loss of common carp muscle. 5. UIF is an efficient method to maintain the quality of frozen common carp muscle.

39