Ultrasound treatment of frozen crayfish with chitosan Nano-composite water-retaining agent: Influence on cryopreservation and storage qualities

Journal Pre-proof Ultrasound treatment of frozen crayfish with chitosan Nanocomposite water-retaining agent: Influence on cryopreservation and storage qualities

Yanan Sun, Min Zhang, Bhesh Bhandari, Chao-hui Yang PII:

S0963-9969(19)30556-3

DOI:

https://doi.org/10.1016/j.foodres.2019.108670

Reference:

FRIN 108670

To appear in:

Food Research International

Received date:

23 July 2019

Revised date:

2 September 2019

Accepted date:

9 September 2019

Please cite this article as: Y. Sun, M. Zhang, B. Bhandari, et al., Ultrasound treatment of frozen crayfish with chitosan Nano-composite water-retaining agent: Influence on cryopreservation and storage qualities, Food Research International (2018), https://doi.org/10.1016/j.foodres.2019.108670

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© 2018 Published by Elsevier.

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Ultrasound treatment of frozen crayfish with chitosan Nano-composite water-retaining agent：Influence on cryopreservation and storage qualities Yanan Suna,c, Min Zhanga,b,* [email protected], Bhesh Bhandarid, Chao-hui Yangc,e a

State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, Jiangsu, China

b

Jiangsu Province Key Laboratory of Advanced Food Manufacturing Equipment and Technology,

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International Joint Laboratory on Food Safety, Jiangnan University, Jiangnan University, China

Jiangnan University, China d

Yangzhou Yechun Food Production & Distribution Co., Yangzhou 225200, Jiangsu, China

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e

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School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD, Australia

*

Corresponding author at: School of Food Science and Technology, Jiangnan University,

Abstract

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214122 Wuxi, Jiangsu Province, China.

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The synergistic impact of ultrasound and Nano-water retaining agent on the cryoprotective effect on crayfish during frozen storage was investigated. The samples

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soaked in water-retaining agent (WRA), ultrasonic 60W treatment combined with water-retaining agent (WRA-US60W) and ultrasonic 80W treatment combined with water-retaining agent (WRA-US80W) were frozen storage at -18 ℃. The indices of frozen storage in 0, 7, 14, 21, 28, 35 and 42 days were measured. The results showed that there was a significant difference in the soaking weight gain, thawing loss, water content and water activity between control group and WRA groups (P < 0.05). The ultrasound combined with WRA treatment showed better water retention effect. The water holding capacity of heat induced gel decreased continuously during the frozen storage period, and the WRA-US60W group exhibited significantly higher values than that of other treatment groups (P < 0.05). The texture characteristics of hardness, elasticity and chewiness had a significant change (P < 0.05). The development of total volatile base nitrogen content, myofibrillar protein content and Ca2+-ATPase activity

Journal Pre-proof of muscle protein were significantly delayed by WRA-US60W treatment, maintaining the integrity of tissue structure. Therefore, WRA-US60W treatment was found effectively improving the quality of crayfish during frozen storage. Keyword

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Ultrasound, nanocomposites, water holding capacity, xylitol, frozen storage

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1. Introduction The crayfish have high water content, which makes them prone to spoilage and deterioration under the action of oxidation reaction (Jenkelunas et al., 2018), microorganisms and endogenous enzymes, resulting in the decline of quality and economic value (Wang et al., 2019). At present, there are two main sales modes of crayfish: fresh sale and frozen sale. Although fresh sale can maximize the quality of crayfish, it is limited by specific areas and seasons. Consequently, frozen sale is

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mostly used for crayfish production in off-season or export products abroad, to maintain the nutritional value of crayfish to maximum (Li et al., 2017; Gao et al.,

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2019). However, the ice crystals formed during frozen storage can easily damage

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muscle cells, increase the protein denaturation and juice loss during thawing, leading to the decrease of flavor and nutritional value of crayfish (Zhang et al., 2017). During

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storage small ice crystals gradually disappear, while large ice crystals grow larger and

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larger, which can aggravate the mechanical damage inside the frozen products (Jenkelunas et al., 2018; Gao et al., 2019). At the same time, the pH and ionic strength

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of unfrozen water change due to the increased concentration of solution caused by the freezing of water into ice, leading to changes of intracellular glia and the

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conformation of protein (Avtar et al., 2018; Xia et al., 2009). With the extension of freezing time, ice crystallization results in the destruction of the cellular micro-structure, loss of the original cell reconciliation function, the drip loss increases during thawing. Consequently, the muscles become hard and fragile, the color dark, and the flavor and nutritional value decreased (Hong et al., 2013; Giddings et al., 2010). At present, carbohydrates are widely used as water-retaining agents in frozen aquatic products. The mechanism of action is that sugars can change the state of bound water embedded in protein molecules, replace the bound water on the surface of protein molecules and bind with it consequently inhibit the protein denaturation (Zhang et al., 2018a; Ma et al., 2015; Huff-Lonergan et al., 2005). Xylitol is a kind of polyhydroxy glycol, which contains many hydroxyl groups that can bind to water

Journal Pre-proof molecules by hydrogen bonds (Campodeaño et al., 2011). Chitosan has good prospects in food processing as a natural, non-toxic and efficient food preservative and film-forming agent (Pan et al., 2014). Relevant experiments (Wang et al., 2014) have found that chitosan can form a film layer on the surface of chilled meat, water loss can be effectively prevented. Sodium alginate has good water solubility and excellent chelating ability to Ca2+, Mg2+ in water (Ma et al., 2015). Therefore, the mechanism of water retention is that Ca2+ and Mg2+ in muscle may be chelated by sodium alginate to prevent the loss of water.

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Ultrasound can be transmitted in solid, liquid and gas media, and its mechanism

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can be divided into cavitation effect, heating effect and mechanical effect (Duan et al., 2008; Mothibe et al., 2011). Cavitation effect is mainly related to the propagation of

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ultrasound in liquid media, during which the positive pressure and negative pressure

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are generated alternately in the process of ultrasonic action (Islam et al., 2014; Zheng

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et al., 2006). The heating effect can be divided into two forms: instantaneous thermal effect and continuous thermal effect (Kang et al., 2016; Liao et al., 2015). Mechanical

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effect is caused by the continuous high-speed vibration of particles in the medium, resulting in changes of mechanical quantities such as velocity, sound intensity and

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pressure (Xin et al., 2013). Ultrasound-assisted soaking can accelerate the diffusion of water-retaining agent into crayfish and make the water-retaining agent fully interact with materiel. In the process of acoustic cavitation, the liquid medium is subjected to physical forces such as shear and oscillation. When the shear rate continues to increase until the Brownian motion and overcome the molecular collision, the cavity bubbles become more orderly in the flow field, showing less resistance to flow, resulting in a decrease in liquid viscosity (Dey et al., 2013; Pakhale et al., 2016). The pulses generated by ultrasound directly affect the structural space of myofibrillar protein molecules, which can improve the water holding capacity, hardness and chewiness meat (Zhang et al., 2018c; Kang et al., 2017; Fan et al., 2019; Cai et al., 2018a). There are few reports on the improvement of water-holding capacity of aquatic products by ultrasound. Polyphosphate is a conventional water retaining agent used for aquatic product,

Journal Pre-proof which can effectively reduce the loss of water and nutrients in the process of processing, transportation and storage (Chen et al., 2016). However, excessive use of phosphate will not only make food develop unpleasant metallic astringency in practical applications, but also lead to deterioration of product flavor, rough structure, and unpleasant aftertaste. Moreover, the long-term excessive phosphate content in the diet will combine with the intestinal calcium to form water-insoluble orthophosphate, thus reducing the human body's absorption of calcium. In this study, a low phosphorus water retaining agent was mixed with chitosan, xylitol, sodium alginate, sodium

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chloride and sodium tripolyphosphate in water. The wet composite mixtures were

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prepared into nanoparticles by colloidal milling and ultrasonic treatment. At the same time, ultrasonic treatment was applied during the soaking process with water retaining

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agent. Therefore, this work was undertaken to evaluate the influence of ultrasonic

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treatment combined with nano-sized composites on the water retention of frozen

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crayfish.

2. Materials and methods

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2.1. Preparation of nano-water retaining agent The xylitol (1.6 g), chitosan (0.6 g), sodium tripolyphosphate (0.5 g), sodium

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chloride (0.2 g), and sodium alginate (0.1 g) powders were mixed in 100 mL distilled water. Then, the mixture was treated by colloidal mill until homogeneity for 20 min and the subsequent solution was treated with 600W ultrasound for 30 min. Finally, the treated mixture solution was centrifuged for 10 min at 5000 r/min to obtain Nano-composite water retaining agent. 2.2. Raw materials and ultrasound treatment Fresh crayfishes (body length 10-15 cm, weight about 30 g) was obtained from Yechun Food Co., Ltd in Yangzhou, China. The fresh crayfishes were transported back to the laboratory in insulated box filled with crushed ice. Individual crayfish were selected, wiped with gauze to remove excess water on the surface. The second dorsal sarcomere of crayfish was perforated for the penetration of water-retaining agent. Samples of different treatments are shown in the table 1. The shelled crayfish

Journal Pre-proof were frozen quickly in ultra-low temperature freezer, so that the central temperature of the product could reach -10 ℃ within half an hour. Then, the crayfishes were frozen at -18 ℃ refrigerator and sampled once every 7 days to determine the relevant water retention index. 2.3. Evaluation of water retention 2.3.1. Soaking weight gain and thawing loss Soaking weight gain and thawing loss was measured as described by Yuan et al. (2018). The crayfish were soaked in a water-retaining agent with different treatment

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groups. After 1 hour, the surface moisture was removed and weighed, the soaking

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weight gain was calculated

m1  m 100 m

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Soaking weight gain (%) =

After frozen storage, crayfishes were taken out, thawed naturally at room

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and the thawing loss was calculated.

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temperature (around 25 ℃) for 2 hours, then drained on gauze for 10 min, weighed

m1  m2 100 m1

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Thawing loss (%) =

m is the weight of crayfish before soaking, g; m1 is the weight of crayfish after

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soaking, g; m2 is the quantity of thawed crayfish, g. 2.3.2. Water holding capacity of heat-induced gel The water holding capacity of heat-induced gel was measured as described by Yuan et al. (2018). The following formula was used to calculate the WHC in the sample: Gel water retention (WHC, %) =

w1  w 100 w2  w

w1 is the quantity of gel after centrifugation + centrifugation tube, w2 is the quantity of the gel before centrifugation + centrifugation tube, w is the quantity of centrifugal tube. 2.3.3. Total volatile base nitrogen (TVB-N) The total volatile base nitrogen was measured as described by Li, et al. (2019).

Journal Pre-proof The following formula was used to calculate the content of TVB-N in the sample: X=

(v1  v 2 )  c14 100 m  v / v0

X=the content of TVB-N in the sample (mg/100g), V1=The test solution consumes the volume of standard titration solution of hydrochloric acid (mL), V2=Reagent blank consumes the volume of standard titration solution of hydrochloric acid (mL), C=Concentration of standard titration solution of hydrochloric acid (mol/L), M-Sample mass (g), V=accurately absorbed filtrate volume (mL), V0=Total

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volume of sample solution in milliliter (mL), 100=the conversion coefficients.

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2.3.4. Myofibrillar protein content

Myofibrillar protein content was measured as reported by Lee et al. (2019).

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Samples of crayfish were weighed (5.0 g), minced and added with 10 times Tris-

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maleic acid buffer (20 mmol/L, pH 7.0, containing 0.05mol/L KCl), homogenized for

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1 min, and centrifuged at low temperature (4 ℃) for 15 min at 20000 r/min. Ten times the amount of Tris-maleic acid buffer (20 mmol/L, pH 7.0, containing 0.6 mol/L KCl) was added to the precipitation. The homogenate was extracted at 4 ℃ for 1 hour after

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homogeneity for 1 min, then centrifuged at 9 000 r/min for 10 min at 4 ℃. The

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supernatant obtained was the myofibrillar protein solution. 2.3.5. Ca2+-ATPase activity of myofibrillar protein The Ca2+-ATPase activity of myofibrils from the treated samples was preformed according to the methods of Zhang, et al. (2017). The extracted myofibrillar protein solution was diluted to 5 mg/mL with KCl of 0.6 mol/L and pH 7.0, and then determined by ATPase test kit. 150 μL myofibrillar protein (5 mg/mL) was reacted with kit reagent at 37°C for 10 min, then the reaction was terminated by kit reagent. The supernatant was mixed with kit reagent at 45°C after centrifugation at 3500 g for 5 min. The mixture was taken out for 20 min, cooled to room temperature and the absorbance was measured at 660 nm. 2.3.6. Texture Texture characteristics of crayfish meat was measured using the texture analyzer (TAXT2i, Stable Micro Systems Ltd., UK) as reported by Berizi et al. (2018). The P/5

Journal Pre-proof (5 mm diameter) flat-bottomed cylindrical probe was used to simulate human teeth chewing food. The samples were compressed twice and tested under TPA mode. Each group of experiments was repeated for 8 times, and the average value was taken after removing the outlier value. 2.3.7. LF-NMR and MRI analysis The status and distribution of water in muscle tissue during storage was determined by low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) according to the method from Fan et al. (2019). The

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samples were placed in the center of the RF coil of the permanent magnetic field for

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T2 acquisition. The relaxation time T2 and proton density M2 were obtained by inversion of the spectra. Relaxation time T2 indicates the fluidity of water, while

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2.3.8. Hematoxylin-eosin staining (HE)

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proton density M2 indicates the content of water at a corresponding relaxation time.

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The quick-frozen sample was cut into 5 mm × 5 mm × 5 mm pieces with the knife. The fixative was dripped on a small disc, placed in the sample, then embedded with fixative, and pre-cooled by the freezing slicer for 30 min. Fixed samples were

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sliced by slicer with a thickness of 10 μm. The sliced samples were stained with

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hematoxylin dye for 5 min, washed gently with distilled water, differentiated for 30 s, then immersed in distilled water for 15 min, placed in eosin dye for 1 min, and rinsed gently with distilled water for 2 min. After dehydration, transparency and sealing, the samples were rinsed with 95% ethanol for 2-3 s twice, then rinsed with 100% ethanol for 2-3 s, put in 100% ethanol for 1 min, dipped in xylene carboxylic acid for 1 min, and then rinsed twice with xylene for 1 min. Finally, the sample was sealed with neutral gum and observed under light microscope. 2.4. Statistical analysis SPSS 21 (SPSS Inc., Chicago, IL, USA) was applied to perform analysis of variance (ANOVA). Significant difference was determined among the treatments using Duncan’s test with 95% the level of confidence (P < 0.05).

3. Results and discussion

Journal Pre-proof 3.1. Particle size distribution of water retaining agent The xylitol and chitosan in the composite mixture of water-retaining agent are kind of polymers having a large polymer "skeleton" and carry a large number of polar hydrophilic groups such as -COOH, -OH, -NH2, and can adsorb hundreds of times or even higher water of their own quantity. For the water-retaining agent of the same quantity, the smaller the particle size, the larger the specific surface area, the higher the water absorption rate, the shorter the time required for water absorption saturation. In addition, the smaller the particle size, the higher the rate of release of water to the

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sample when the water is under stress. It can be seen from Fig 1 that the particle size

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of the ultrasound-treated water-retaining agent solution is mainly distributed between 164.2 d. nm-615.1 d. nm, and 24.5% of the particles with an average particle size of

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295.3 d. nm.

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3.2. Effect of different treatments on water content and water activity

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Water content has a direct impact on the sensory characteristics of crayfish meat, but it will gradually decrease with the increase of freezing time, which can result in

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dehydration of crayfish meat tissue and deterioration in sensory characteristics (Mcdonnell et al., 2013). Water activity (Aw) indicates the degree to which water in

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food is utilized by microorganisms, and also reflects the characteristics of food quality, such as fluidity, hardness, flavor, processing adaptability and storage resistance. The effects of different treatments on water content and water activity of frozen crayfish are shown in Fig. 2. During the frozen storage, the water content of crayfish in each group decreased significantly (P < 0.05). The water content and water activity of crayfish muscle tissue in control group and treatment group did not change significantly (P > 0.05) during the frozen storage for 0-14 days. After 42 days of frozen storage, the water content of crayfish meat in WRA-US 60W group (65.32%) was significantly higher than that in other treatment groups (P < 0.05). This may be due to the proper power of ultrasound treatment increased the phosphate ion of the water retaining agent into the muscle, as a consequence the pH of the muscle increased, and the hydrophilicity of actin enhanced, which inhibited the water loss from the muscles of crayfish meat. The water content in muscle tissue of crayfish

Journal Pre-proof treated with WRA-US80W (64.57%), WRA (63.54%) and control group (61.08%) from high to low, showed that ultrasound combined with water retaining agent soaking treatment could effectively reduce the water loss of crayfish. In addition, xylitol can change the state of bound water in protein molecules potentially by replacing the bound water on the surface of protein molecules and binding with protein molecules (Zhang et al., 2018d). The water activity of crayfish with distilled water treatment group (0.9513) was significantly higher than that in other treatment groups (P < 0.05), followed by WRA group (0.9431), WRA-US80W group (0.940)

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and WRA-US60W group (0.9378) after frozen storage. The water activity of the

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WRA-US60W group was significantly lower than that of other treatments groups (P < 0.05), which may be the association of water molecules in muscle tissue which was

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affected by molecular weight, spatial structure of xylitol and the distribution of free

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hydroxyl groups.

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3.3. Effect of different treatment on soaking weight gain and thawing loss Water loss, fat detachment and water-soluble nitrogen compounds outflow are

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the main changes of muscle during frozen storage, among which the greatest impact was the water loss. The change of thawing juice loss during storage is the main

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manifestation of water retention, which can be used to estimate the quality of frozen crayfish. During the freezing storage, the muscle tissue is compressed by the formation of ice crystals damaging the cell membrane structure which results in the increased loss of cell juice during thawing. The effects of different treatment groups on the water retention of frozen crayfish are shown in Fig 3. The results showed that, the soaking weight gain of treatment groups were significantly higher than the control group. During 0-14 days of frozen storage, the thawing loss of crayfish in different treatments did not change significantly (P > 0.05). After that, the thawing loss in the treatment groups and the control group were significantly different (P < 0.05). When the frozen storage was 42d, the thawing loss in the control group was the highest (11.74%), followed by the WRA group (10.22%), WRA-US80W group (10.11%) and WRA-US60W group (9.58%). The water holding capacity of crayfish decreases continuously, mainly due to the formation of a large number of ice crystals in the

Journal Pre-proof tissue cells, which will squeeze the tissue structure, aggravating the physical damage of the tissue structure, and then causing the loss of juice in the tissue cells during the thawing process. The hydrophobicity of frozen crayfish was improved by xylitol molecules, which may promote the transformation of free water into bound water through the free hydroxyl group of sugar in the tissues, so that the temperature of "eutectic point" could be decreased and the amount of ice crystal formation could be reduced (Zhang et al., 2018b and d). 3.4. Effect of different treatment on water holding capacity of heat-induced gel

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The water holding capacity (WHC) of heat-induced gel mainly refers to the water

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retention of myofibrillar protein (Yuan et al., 2018). According to Fig. 4, the WHC of crayfish heat induced gel decreased continuously during the frozen storage period.

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The WHC of heat-induced gel decreased from 56.33% to 34.56% at 42 d in the

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control group, which was 38.65% lower than that in the original treatment. However,

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the WHC of the heat-induced gel from the samples treated with WRA was lower than that of the control group, which decreased from the initial 56.35%, 56.36% and

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56.36% to 39.11%, 42.66% and 40.04% at 42d, compared with the original, which decreased by 30.59%, 24.31% and 28.96%, respectively. The extraction rate of

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myofibrillar protein is affected by ion species and pH. The addition of sodium chloride (NaCl) and phosphate can improve the ionic strength of meat, so that myosin and actin (the important components of myofibrillar protein) could be dissolved effectively. The cavitation effect of ultrasound increases the space between myofibrillar proteins, which can combine more moisture and increase the water retention of the gel (Tzanakis et al., 2017). The natural long-chain molecular structure of sodium alginate can also penetrate into muscle by capillary force and interact with protein, increasing the space of muscle fibers, and making water enter into the structure of myofibril. At the same time, the membrane formed by chitosan and sodium alginate covering the surface of muscle can make the infused water difficult to squeeze out. The synergistic effect of NaCl, chitosan, sodium alginate and phosphate applied as water retaining agent improved the WHC of heat-induced gelation. 3.5. Effect of different treatment on total volatile base nitrogen (TVB-N)

Journal Pre-proof The protein in crayfish meat and the protein nitrogen produced by decomposition are further decomposed into ammonia salt or ammonia, and finally converted into volatile base nitrogen. Total volatile base nitrogen (TVB-N) is the objective index to evaluate the changes in the quality of aquatic products, whose content is inversely proportional to the freshness. The edible range of TVB-N value for frozen crayfish meat is less than 15 mg/100 g according to the national standard. Fig. 5 showed that TVB-N value of crayfish increased with the increase of frozen storage time, but all of experimental groups had lower than 15 mg/100 g value, which accord with the

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requirements of national sanitary standards for freshly frozen animal aquatic products.

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The TVB-N value of control group increased the fastest, while the samples treated with WRA and ultrasound combined with WRA had obvious effect on inhibiting the

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increase of TVB-N value (P < 0.05). The internal structure of the crayfish will be

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destroyed by freezing storage process, leading to the decomposition of proteins and

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nitrogen-containing compounds. In addition, the TVB-N gradually accumulate with the extension of freezing storage time. After the 42 days of freezing storage, the

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TVB-N value of control group was 4.48 mg /100 g, the TVB-N value of WRA group was 4.02 mg /100 g, and the TVB-N value of WRA-US60E, WRA-US80W group was

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3.64 mg /100 g and 3.79 mg /100 g, respectively. The TVB-N values of control group and treatment group were significantly different (P < 0.05). It can be seen that the deterioration of freshness of frozen crayfish can be effectively alleviated by ultrasound combined with water retaining agent. 3.6. Effect of different treatment on myofibrillar protein content and Ca2+-ATPase activity Myofibril protein is a salt soluble protein. The soluble protein content is a main characterization indicator to reflect the frozen denaturation of crayfish myofibril protein. The functional characteristics of protein mainly depends on the myofibril protein. The formation of hydrogen bond, hydrophobic bond and disulfide bond, as well as the interaction between ions lead to the decrease of salt-soluble protein content. The salt-soluble protein content can also reflect the denaturation of protein to a certain extent. During freezing storage, actomyosin molecules form non-covalent

Journal Pre-proof bonds with each other due to the precipitation of ice crystals formed by partially bound water of proteins, which further leads to the formation of insoluble coagulation and decrease the actomyosin dissolution. Furthermore, actomyosin denaturation also produce alkali-soluble protein that is insoluble in high ionic strength but soluble in lye. This also leads to decreased solubility of actomyosin during frozen storage (Cai et al., 2018b; Yan et al., 2018). During the period of frozen storage, the total amount of myofibril protein showed an overall decreasing trend, and the dissolution rate of treated with WRA was

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significantly higher than that in the control group (P < 0.05). After 14 days of freezing

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storage, myofibril protein dissolution in the control group had decreased to 67.36% as compared with that of sample immediately after freezing. The myofibril protein

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dissolution decreasing rates of crayfish soaked with WRA, WRA-US60W and

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WRA-US80W were 16.61%, 13.28% and 13.35%, respectively. The dissolution of

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myofibrillar protein decreased slowly from 35 to 42 days after frozen. By 42 days, the dissolution in the control group had decreased to 45.67% at the beginning of freezing

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storage, the other three groups treated with water retaining agent and ultrasound had decreased by 41.73%, 33.56% and 35.88%, respectively. It can be seen that the

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myofibrillar protein of crayfish in the control group underwent serious denaturation during freezing storage. Thus, the water retaining agent had anti-freezing protective effect on the protein reducing the loss of myofibrillar protein during freezing storage. The dissolution of myofibrillar protein in WRA-US60W group was significantly higher than that in other treatment groups (P < 0.05), which indicated that appropriate power ultrasound treatment was helpful to maintain myofibrillar protein of crayfish. The content of myofibrillar protein in 80W ultrasound treatment group was lower than that in 60W ultrasound treatment group, possibly because the high intensity of ultrasound may destroy the spatial structure of myofibrillar protein, resulting in reduced dissolution. Ca2+-ATPase activity as an important indicator of protein properties was used to determine the integrity of crayfish myofibrillar protein. Ca2+-ATPase activity of the four groups of samples decreased significantly with the increase of freezing storage

Journal Pre-proof time (P < 0.05), which was due to the tertiary structure of protein change by ice crystals and an increase in ionic strength in the system. In addition, the oxidation of sulfhydryl group also has an effect on its activity, resulting in a decrease in the activity value (Yan et al., 2018). The higher the activity value of Ca2+-ATPase, the more stable the protein property and the less the freezing denaturation. The activity of Ca2+-ATPase in control group decreased the most (79.46%), decreased the least (67.70%) in WRA-US60W group, while decreased to 75.56% and 73.01% in WRA group and WRA-US80W group, respectively. After 42 days of frozen storage, the

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Ca2+-ATPase activities of control group, WRA group, WRA-US60W group and

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WRA-US80W group were 0.46, 0.55, 0.73 and 0.61 U/mg, respectively, and there were significant differences in Ca2+-ATPase activities among the four groups (P <

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0.05). The 60W ultrasound-assisted soaking can increase the contact area between the

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water retaining agent and the sample. At the same time, ultrasound has the cavitation

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effect on muscle fiber, reducing the formation of ice crystals. However, the 80W ultrasonic power is too large, possibly damaging the structure of muscle fibril and

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leading to the inactivation of enzyme. During storage, enzymatic activity decreased possibly due to the extrusion and puncture of cell membranes by ice crystals. The

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ionic concentration increased due to the water molecules turning into ice and causing unfrozen water more concentrated.

In consequence, the biochemical reaction will be

accelerated, causing Ca2+-ATPase activity decreased. It can be seen that the appropriate power of ultrasound combined with water retaining agent had a good effect on inhibiting the freezing denaturation of shrimp myofibrillar protein. 3.7. Effect of different treatments on texture of crayfish meat The effects of different treatments on the texture characteristics such as hardness, elasticity and chewiness of frozen crayfish are shown in Table 2. With the prolongation of frozen storage time, the hardness of muscle in different treatment groups decreased significantly (P < 0.05), but WRA-US groups were significantly lower than that in control group (P < 0.05). This may be due to the formation of ice crystals during frozen storage damaging the muscle cells and causing the denaturation of muscle proteins leading to the decrease in hardness of muscle. Moreover, some

Journal Pre-proof researchers believe that calpain and cathepsin enzyme can act on myofibrillar protein and promote its degradation (Wang et al., 2018). Elasticity reflects the deformation degree of food under external forces and the recovery degree after withdrawal. From Table 1, the muscle elasticity of fresh crayfish (frozen for 0 d) was 6.69 mm, which showed a significant downward trend in the process of frozen storage (P < 0.05). The muscle elasticity of crayfish changed significantly (P < 0.05) after freezing for 0-28 days. After 42 days of frozen storage, the elasticity values (5.74, 5.71 and 5.68 mm) of sample treated with WRA-US60W, WRA-US80W and WRA were significantly

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higher than those treated with distilled water (control) (P < 0.05). This may be due to

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changes in muscle protein during freezing storage, resulting in changes of elasticity. The chewiness (fresh shrimps 27.11) of frozen crayfish decreased with the

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prolongation of frozen storage time. The frozen samples for 0~21 days, the value of

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chewability in each treatment group decreased significantly (P < 0.05), which was

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presumably caused by the destruction of muscle tissue by ice crystals. During 28-42 days of frozen storage, the change of chewability value of muscle was not significant

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(P > 0.05), which might be related to the change of water activity. Water is the main basis of the texture characteristics of crayfish meat. The texture change is directly

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related to the change of water content and water activity during storage. After 42 days of frozen storage, the chewiness of sample treated with WRA-US60W was significantly higher than that treated with distilled water, WRA-US80W and WRA (P < 0.05). It can be seen from the above that WRA-US treatment can retard the texture deterioration of crayfish.

3.8. Effect of different treatments on water status Fig 7 shows that there were three forms of water in crayfish muscle: combined water T21 (0.01-10 ms), immobilised water T22 (10-100 ms) and free water T23 (>100 ms) (Sánchez-Alonso et al., 2014; Sánchez-Alonso et al., 2012). The percentage of the integral area around the transverse coordinates of the peaks of T21, T22 and T23 is the moisture content of each state, which is recorded as A21, A22 and A23, respectively. Compared with fresh samples, A22 of each group showed evidently decreasing trend after frozen storage, as compared to control group which is decreased by 39.45%. But

Journal Pre-proof there was no significant change of A21 and A23. It may be that some of the crayfish tissues were destroyed during freezing storage, and the binding force of immobilised water was decreased. The A22 of the control group decreased significantly compared with sample treatment with WRA and WRA-US groups (P < 0.05). This may be because the sodium alginate solution promoted the "bare" process caused by the relaxation of myofibrillar structure, thus keeping water in the gel immobilised. The decrease of A22 was the smallest in the WRA-US60W group after freezing storage, which may be due to the influence of muscle tissue structure on water holding

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capacity and water molecular status. The water holding capacity of muscles mainly

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depends on the attraction between the sarcoplasmic protein molecules and the polarized groups of water molecules (Sánchez-Alonso et al., 2014). Among them,

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most of the water is absorbed into the sarcoplasmic within the cell membrane, and a

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small part is trapped outside the cell membrane of muscle fibers through capillary

(Sánchez-Alonso et al., 2012).

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force, which is tightly surrounded by the perimysium, reducing the fluidity

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Fig 8 shows the effects of fresh samples and different ultrasound-assisted water-retaining agents on magnetic resonance imaging (MRI) after frozen storage.

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Under four different treatments, the water content and state of samples changed significantly, which was consistent with the results of Fig. 7. In food, T1 and T2 weighted imaging represent the distribution of short relaxation (bound water) and long relaxation (free water), respectively. The more obvious the red, the greater the intensity of NMR signal, and the more obvious the blue, the weaker the intensity of NMR signal. From Fig 8, the crawfish meat is composed of relatively short collagen fibers, with the increase of frozen storage time, a large amount of water in the crawfish body was lost. The collagen fibers were possibly degenerated, cross-linked and contracted, which reduced the crawfish volume. After 20 days of freezing storage, the water signal of crawfish treated with different method was weaker than that of fresh samples, and the immobilised water signal of crawfish treated with WRA was significantly higher than that of control group. The immobilised water of crawfish treated with WRA-US60W transformed into bound water more obviously. Weak

Journal Pre-proof signals were detected in samples treated by each group from freezing storage for 30 d, and too long freezing storage time will damage the structure of collagen fiber network, thus reducing the water holding capacity of crawfish. 3.9. Effect of different treatments on microstructure The volume increase of ice crystal has a great influence on the quality of frozen aquatic products. This makes muscle fibers deformed by compression, and even local tissue may rupture. After thawing, the loss of tissue juice is increased, and the sensory quality and nutritional value are reduced. HE staining results of frozen crawfish

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muscle tissue are shown in Fig. 9. Fresh shrimp (Fig. 9 A) muscle structure was

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relatively complete, muscle fibers were arranged tightly and regularly, and there was only a few small gaps between the tissue structure. After 42 days of freezing storage,

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the structure of crawfish meat in control group (Fig. 9 B) was destroyed and the order

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of connective tissue was disrupted most seriously. There were a large number of gaps

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between tissues and tissues, and most of the tissues were broken which may be that the formation of ice crystals during freezing storage caused mechanical damage to

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muscle cells n during freezing storage, causing the increases of spacing between the muscle bundles myoblasts and internal hole. The muscle fibers of WRA group (Fig. 9

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C) and WRA-US60W group (Fig. 9 D) were arranged densely, and their relative integrity was still good, and there was no obvious extrusion or distortion. The WRA-US80W group (Fig. 9 E) showed slightly more tissue damage than the other groups, and the number of muscle fiber gap was relatively large, but it was still significantly better than control group. The results showed that the muscle stability of crawfish during frozen storage was better, and the formation and growth of ice crystals could be inhibited by ultrasound combined with water retaining agent. Zhang et al. (2018) also found that the sugar solution could penetrate into the tissue gap of shrimps during soaking, which could inhibit the growth of ice crystals and reduce the mechanical damage of ice crystals to the structure of shrimps, thus stabilizing the integrity of shrimp tissue structure.

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4. Conclusions The thawing loss, water activity and TVB-N content of the crayfish can be significantly reduced by treating with chitosan Nano-composite water-retaining agent combined with ultrasonic 60W during the frozen storage. Meanwhile, the water content, soaking weight gain, heat induced gel water retention, texture characteristics, myofibrillar protein content and Ca2+-ATPase activity of protein in muscle can be effectively maintained. Microstructure observation showed that after 40 days of

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freezing storage, the tissue structure of crayfish meat treated with WRA-US60W was relatively complete compared with control group, similar to that of fresh crawfish

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meat. The effect of ultrasound on the functional properties of protein is mainly based

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on its changes in protein structure, which makes the crawfish tissue loose by the cavitation and mechanical vibration of ultrasound. Therefore, an optimum ultrasonic

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treatment can improve the functional properties of protein. It can be seen that a

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composite mixture of NaCl, xylitol, chitosan and sodium alginate as antifreeze and US treatment can have a synergistic effect and can be an alternative of conventional

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phosphate water-retaining agents in aquatic products.

Acknowledgments

This work was financially supported by National Key R&D Program of China (Contract No. 2018YFD0400801), National First-class Discipline Program of Food Science and Technology (No.JUFSTR20180205) and Jiangsu Province Key Laboratory Project of Advanced Food Manufacturing Equipment and Technology (No. FMZ201803).

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References Avtar, S., & Benjakul, S. (2018). Proteolysis and its control using protease inhibitors in fish and fish products: A review. Comprehensive Reviews in Food Science and Food Safety, 17, 496-509. Berizi, E., Hosseinzadeh, S., Shekarforoush, S. S., & Barbieri, G. (2018). Microbial, chemical, textural and sensory properties of coated rainbow trout by chitosan combined with pomegranate peel extract during frozen storage. International

of

Journal of Biological Macromolecules, 106, 1004-1013. Cai, L., Cao, M., Cao, A., Regenstein, J., Li, J., & Guan, R. (2018a). Ultrasound or

ro

microwave vacuum thawing of red seabream (Pagrus major) fillets. Ultrasonics

-p

Sonochemistry, 47, 122-132.

Cai, L., Feng, J., Cao, A., Tian, H., Wang, J., Liu, Y., Gong, L., & Li, J. (2018b).

re

Effect of partial substitutes of NaCl on the Cold-set gelation of grass carp

lP

myofibrillar protein mediated by microbial transglutaminase. Food and Bioprocess Technology, 11, 1876-1886.

na

Campodeaño, L., Tovar, C. A., Borderías, J., & Fernándezmartín, F. (2011). Gelation process in two different squid (dosidicus gigas) surimis throughout frozen

Jo ur

storage as affected by several cryoprotectants: thermal, mechanical and dynamic rheological properties. Journal of Food Engineering, 107, 107-116. Chen, Q. M., Fu, C. L., Wang, S. Y., & Rao, P. F. (2016). The effect of polyphosphates on improving water holding capacity of fish fillet and its action mechanism. Journal of Chinese Institute of Food Science and Technology. 16, 65-71. China. (2016). GB5009.228-2016 Determination of total volatile base nitrogen in food. Beijing: National Health and Family Planning Commission of the People’s Republic of China. Dey, S., & Rathod, V. K. (2013). Ultrasound assisted extraction of β-carotene from Spirulina platensis. Ultrasonics Sonochemistry, 20, 271-276. Duan, X., Zhang, M., Li, X., & Mujumdar, A. S. (2008). Ultrasonically enhanced osmotic pretreatment of sea cucumber prior to microwave freeze drying. Drying

Journal Pre-proof Technology, 26(4), 420-426. Fan, K., Zhang, M., & Jiang, F. J. (2019). Ultrasound treatment to modified atmospheric packaged fresh-cut cucumber: Influence on microbial inhibition and storage quality. Ultrasonics Sonochemistry. Gao, W. H., Rui H., & Xin-An Z. (2019). Synergistic effects of ultrasound and soluble soybean polysaccharide on frozen surimi from grass carp. Journal of Food Engineering, 240, 1-8. Giddings, G. G., & Hill, L. H. (2010). Relationship of freezing preservation

of

parameters to texture-related structural damage to thermally processed

ro

crustacean muscle. Journal of Food Processing and Preservation, 2, 249-264. Hong, H., Luo, Y., Zhou, Z., Bao, Y., Lu, H., & Shen, H. (2013). Effects of different

-p

freezing treatments on the biogenic amine and quality changes of bighead carp

re

(Aristichthys nobilis) heads during ice storage. Food Chemistry, 138, 1476-1482.

lP

Huff-Lonergan, E., & Lonergan, S. M. (2005). Mechanisms of water-holding capacity of meat: the role of postmortem biochemical and structural changes. Meat

na

Science, 71, 194-204.

Islam, M. N., Zhang, M., & Adhikari, B. (2014). The inactivation of enzymes by

1-21.

Jo ur

ultrasound-a review of potential mechanisms. Food Reviews International, 30,

Jenkelunas, P. J., & Ecy, L. C. (2018). Production and assessment of pacific hake (merluccius productus) hydrolysates as cryoprotectants for frozen fish mince. Food Chemistry, 239, 535-543. Kang, D. C., Gao, X. Q., Ge, Q. F., Zhou, G. H., & Zhang, W. G. (2017). Effects of ultrasound on the beef structure and water distribution during curing through protein degradation and modification. Ultrasonics Sonochemistry, 38, 17-325. Kang, D. C., Zou, Y. H., Cheng, Y. P., Xing, L. J., Zhou, G. H., & Zhang, W. G. (2016). Effects of power ultrasound on oxidation and structure of beef proteins during curing processing. Ultrasonics Sonochemistry, 33 (4), 7-53. Lee, C. H., & Chin, K. B. (2019). Evaluation of various salt contents on quality characteristics with or without curdlan of pork myofibrillar protein gels and the

Journal Pre-proof development of low-salt pork sausages. International Journal of Food Science and Technology, 54, 550-557. Li, X. X., Liu, S., Su, W., Cai, L., & Li, J. (2017). Physical quality changes of precooked Chinese shrimp, Fenneropenaeus chinensis, and correlation to water distribution and mobility by low-field NMR during frozen storage. Journal of Food Processing and Preservation, 41, e13220. Li, Y. L., Tang, X. Y., Shen, Z. X., & Dong, J. (2019). Prediction of total volatile basic nitrogen (TVB-N) content of chilled beef for freshness evaluation by using

of

viscoelasticity based on airflow and laser technique. Food Chemistry, 287,

ro

126-132.

Liao, N., Zhong, J., Ye, X., Lu, S., Wang, W., Zhang, R., Xu, J., Chen, S., & Liu, D.

-p

(2015). Ultrasonic assisted enzymatic extraction of polysaccharide from

re

Corbicula fluminea: characterization and antioxidant activity. LWT--Food

lP

Science and Technology, 60, 1113-1121.

Ma, L. K., Zhang, B., Deng, S. G., & Xie, C. (2015). Comparison of the

na

cryoprotective effects of trehalose, alginate, and its oligosaccharides on peeled shrimp (Litopenaeus vannamei) during frozen storage. Journal of Food Science,

Jo ur

80, 540-546.

Mothibe, K. J., Zhang, M., Nsor-Atindana, J., & Wang, Y. C. (2011). Use of ultrasound pretreatment in drying of fruits: drying rates, quality attributes, and shelf life extension. Drying Technology, 29(14), 1611-1621. Pakhale, S. V., & Bhagwat, S. S. (2016). Purification of serratiopeptidase from Serratia marcescens NRRL B 23112 using ultrasound assisted three phase partitioning. Ultrasonics Sonochemistry, 31, 532-538. Pan, S., & Wu, S. (2014). Effect of chitooligosaccharides on the denaturation of weever myofibrillar protein during frozen storage. International Journal of Biological Macromolecules, 65, 549-552. Sánchez-Alonso, I., Martinez, I., & Sánchez-Valencia, J. (2012). Careche M., Estimation of freezing storage time and quality changes in hake (merluccius merluccius, L) by low field nmr. Food Chemistry, 135, 1626-1634.

Journal Pre-proof Sánchez-Alonso, I., Moreno, P., & Careche, M. (2014). Low field nuclear magnetic resonance (lf-nmr) relaxometry in hake (merluccius merluccius, l.) muscle after different freezing and storage conditions. Food Chemistry, 153, 250-257. Tzanakis, I., Lebon, G. S., Eskin, D. G., & Pericleous, K. A. (2017). Characterizing the cavitation development and acoustic spectrum in various liquids. Ultrasonics Sonochemistry, 34, 651-662. Wang, H. B., Pan, S. K., & Wu, S. J. (2014). Chitooligosaccharides suppress the freeze-denaturation of actomyosin in aristichthys nobilis surimi protein.

of

International Journal of Biological Macromolecules, 63, 104-106. Kinetics

ro

Wang, J., Tang, J., Rasco, B., Sablani, S. S., Ovissipour, M., Qu, Z. (2018).

of quality changes of shrimp (litopenaeus setiferus) during pasteurization. Food

-p

and Bioprocess Technology, 11, 1027-1038.

re

Wang, X. Y., & Xie, J. (2019). Evaluation of water dynamics and protein changes in

lP

bigeye tuna (Thunnus obesus) during cold storage. LWT--Food Science and Technology, 108, 289-296.

na

Xia, X., Kong, B., Liu, Q., & Liu, J. (2009). Physicochemical change and protein oxidation in porcine longissimus dorsi as influenced by different freeze-thaw

Jo ur

cycles. Meat Science, 83, 239-245. Xin, Y., Zhang, M., & Adhikari, B. (2013). Effect of trehalose and ultrasound-assisted osmotic dehydration on the state of water and glass transition temperature of broccoli (brassica oleracea l. var. botrytis l.). Journal of Food Engineering, 119(3), 640-647.

Yuan, L., Dang, Q., Mu, J., Feng, X., & Gao, R. (2018). Mobility and redistribution of waters within bighead carp (aristichthys nobilis) heat-induced myosin gels. International Journal of Food Properties, 21, 834-848. Zhang, B., Fang, C. D., Hao, G. J., & Zhang, Y. Y. (2018a). Effect of kappa-carrageenan oligosaccharides on myofibrillar protein oxidation in peeled shrimp (Litopenaeus vannamei) during long-term frozen storage. Food Chemistry, 245, 254-261. Zhang, B., Hao, G. J., Cao, H. J., Tang, H., Zhang, Y. Y., & Deng, S. G. (2018b). The

Journal Pre-proof cryoprotectant effect of xylooligosaccharides on denaturation of peeled shrimp (litopenaeus vannamei) protein during frozen storage. Food Hydrocolloids, 77, 228-237. Zhang, B., Yang, H. C., Tang, H., Hao, G. J., Zhang, Y. Y., & Deng, S. G. (2017). Insights into Cryoprotective Roles of Carrageenan Oligosaccharides in Peeled Whiteleg Shrimp (Litopenaeus vannamei) during Frozen Storage. Journal of Agricultural and Food Chemistry, 65, 1792-1801. Zhang, M., Niu, H., Chen, Q., Xia, X., & Kong, B. (2018c). Influence of

of

ultrasound-assisted immersion freezing on the freezing rate and quality of

ro

porcine longissimus muscles. Meat Science, 136, 1-8.

Zhang, Y. Y., Zhang, B., Hao, G. J., Deng, X. Y., & Tang, H. (2018d). Effect of Sugar

-p

Alcohols on Water Retention in Frozen Shrimp (Litopenaeus vannamei). Food

re

Science, 39, 170-175.

lP

Zheng, L., & Sun, D.W. (2006). Innovative applications of power ultrasound during

Jo ur

16-23.

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food freezing processes: a review. Trends in Food Science & Technology, 17 (1),

Journal Pre-proof Figure and table captions Fig. 1. Fig. 1. Particle size distribution of compound water retaining agent. Fig. 2. Effect of ultrasound synergistic Nano-WRA treatment on soaking weight gain (A) and thawing loss (B) of crayfish during frozen storage. Fig. 3. Effect of ultrasound synergistic Nano-WRA treatment on moisture content (Lines curves) and water activity (Bars chart) of crayfish during frozen storage. Fig. 4. Effect of ultrasound synergistic Nano-WRA treatment on water holding capacity of heat-induced gels of crayfish during frozen storage.

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Fig. 5. Effect of ultrasound synergistic Nano-WRA treatment on total volatile base

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nitrogen of crayfish during frozen storage.

Fig. 6. Effect of ultrasound synergistic Nano-WRA treatment on myofibrillar protein

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content (Line curves) and Ca2+-ATPase activity (Bar chart) of crayfish during frozen

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storage.

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Fig. 7. Effect of ultrasound synergistic Nano-WRA treatment on distribution of transverse relaxation times (T2) of crayfish after frozen storage.

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Fig. 8. Water-selective transverse images of crayfish with ultrasound synergistic Nano-WRA treatment after frozen storage.

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Fig. 9. Results of HE staining of crayfish with ultrasound synergistic Nano-WRA treatment after frozen storage.

Table 1 The different treatment methods of raw materials. Table 2 Effect of ultrasound synergistic Nano-WRA treatment on texture of crayfish after frozen storage.

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Fig. 1. Particle size distribution of compound water retaining agent.

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(B) Fig. 2. Effect of ultrasound synergistic Nano-WRA treatment on soaking weight gain (A) and thawing loss (B) of crayfish during frozen storage.

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Fig. 3. Effect of ultrasound synergistic Nano-WRA treatment on moisture content (Lines curves) and water activity (Bars chart) of crayfish during frozen storage.

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Fig. 4. Effect of ultrasound synergistic Nano-WRA treatment on water holding capacity of heat-induced gels of crayfish during frozen storage.

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Fig. 5. Effect of ultrasound synergistic Nano-WRA treatment on total volatile base nitrogen of crayfish during frozen storage.

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Fig. 6. Effect of ultrasound synergistic Nano-WRA treatment on myofibrillar protein content (Line curves) and Ca2+-ATPase activity (Bar chart) of crayfish during frozen storage.

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Fig. 7. Effect of ultrasound synergistic Nano-WRA treatment on distribution of transverse relaxation times (T2) of crayfish after frozen storage.

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Fig. 8. Water-selective transverse images of crayfish with ultrasound synergistic Nano-WRA treatment after frozen storage.

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A

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Fig. 9. Results of HE staining of crayfish with ultrasound synergistic Nano-WRA treatment after frozen storage A: Fresh sample; B: Samples of distilled water; C: Samples of Nano-WRA; D: Samples of ultrasound (60W) synergistic Nano-WRA; E: Samples of ultrasound (80W) synergistic Nano-WRA

Journal Pre-proof Table 1. The different treatment methods of raw materials. 10 crayfishes were immersed in 1 L distilled water

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Group 2

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10 crayfishes were immersed in 1 L Nano-water retaining agent (WRA)

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Group 3

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10 crayfishes were immersed in 1 L WRA (immersion for 5 min, 60W ultrasound for 1 min)

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Group 4

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Group 1

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10 crayfishes were immersed in 1 L WRA (immersion for 5 min, 80W ultrasound for 1 min)

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Table 2. Effect of ultrasound synergistic Nano-WRA treatment on texture of crayfish after frozen storage. 28

35

42

control

635.31 ±8.47

608.66 ±6.33

563.98 ±8.98

511.65 ±9.34

460.02 ±8.66

427.84 ±7.89

401.20 ±8.94

WRA

635.31 ±8.47

614.33 ±9.76

580.11 ±8.74

530.31 ±7.89

496.22 ±9.99

451.53 ±6.43

430.16 ±8.11

WRA-US 60W

635.31 ±8.47

616.32 ±9.13

607.26 ±8.33

570.76 ±8.40

534.42 ±7.47

497.36 ±7.88

460.21 ±7.63

WRA-US 80W

635.31 ±8.47

615.32 ± 10.22

600.44 ±9.77

560.33 ±6.41

520.11 ±8.55

487.66 ±8.78

450.38 ±9.29

control

6.69± 0.22

6.44± 0.15

6.23± 0.13

6.01± 0.11

5.71± 0.11

5.62± 0.10

5.55± 0.06

WRA

6.69± 0.22

6.53± 0.13

6.37± 0.12

6.19± 0.09

5.83± 0.11

5.76± 0.14

5.68± 0.10

WRA-US 60W

6.69± 0.22

6.60± 0.12

6.52± 0.11

6.33± 0.09

6.01± 0.07

5.89± 0.09

5.74± 0.11

WRA-US 80W

6.69± 0.22

6.57± 0.13

5.93± 0.15

5.80± 0.09

5.71± 0.09

control

27.11 ±0.45

WRA

27.11 ±0.45

WRA-US 60W WRA-US 80W

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6.30± 0.12

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Chewiness

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25.17 ±0.36

22.63 ±0.31

18.66 ±0.33

15.19 ±0.29

13.76 ±0.35

12.45 ±0.38

25.78 ±0.32

23.43 ±0.37

19.69 ±0.31

17.23 ±0.25

15.34 ±0.36

13.38 ±0.33

27.11 ±0.45

26.76 ±0.30

25.54 ±0.32

21.45 ±0.37

19.44 ±0.27

18.26 ±0.29

15.23 ±0.30

27.11 ±0.45

26.21 ±0.32

25.09 ±0.37

20.54 ±0.35

18.23 ±0.29

17.77 ±0.37

14.98 ±0.31

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Elasticity

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Hardness

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6.46± 0.11

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Conflicts of interest

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There are no conflicts to declare.

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Ultrasound treatment of frozen crayfish with chitosan Nano-composite water-retaining agent：Influence on cryopreservation and storage qualities

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Graphical abstract:

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Highlights 

The chitosan Nano-WRA was prepared by colloid mill and ultrasound set-up.



The synergistic influence of ultrasound and WRA on the cryoprotective of crayfish was investigated.



The WHP, myofibrillar protein and Ca2+-ATPase activity of frozen crayfish was

The ultrasonic combined with WRA can delay the storage quality deterioration of

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the crayfish.

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improved.