Correlation analysis of microstructure, protein pattern, and thermal properties of Procambarus clarkia subjected to different cryogenic treatments

Abstract The objective of this work was to investigate the freezing and storage temperature (−80 and −18℃) on the microstructure, protein pattern, and thermal properties of red swamp crayfish after one‐week storage, and a Pearson correlation analysis was performed among these attributes. After cryogenic treatments for short‐term storage, Tp (pretein denaturation temperature) was significantly raised (p < .05) except for samples frozen at −80℃ prior to store at −18℃ (−80/‐18). Samples frozen and stored at −80℃ (−80/‐80) had lower number and sum area of white regions in histology, higher intensity of most protein bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) image, and relatively higher Tp and ΔH (p < .05), while −80/‐18 samples had lower intensity of most protein bands and TP 2, and higher number and sum area of white regions and ΔH 2 (p < .05). Pearson's analysis results showed the intensive TN T and MLC 1 band could be potentially considered as the markers of tissue integrity and protein degradation. Therefore, the three attributes could be applied to comprehensively assess the quality of frozen aquatic products, and −80/‐80 treatment was appropriate for crayfish preservation.

product, as its convenience and tasty, is becoming more and more popular for young families and considered as rinsing star product in E-commerce.
Freezing is an effective technique to preserve food, especially in food industries. Low temperature could effectively inhibit the reproduction of microbial and retard the quality deterioration of food products (Leygonie et al., 2012). Several works have been done to investigate the effects of freezing on the meat quality during frozen storage (Choi et al., 2017a(Choi et al., , 2017bDalvi-Isfahan et al., 2016;Shi, Tao, et al., 2018). Beside texture, microstructure, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and differential scanning calorimetry (DSC) are widely applied to observe the arrangement of muscle structure , semiquantitative analysis in the protein patterns (Yin & Park, 2015), and monitor the protein denaturation during thermal processing (Shao et al., 2018). Our previous research has reported the freezing temperature (liquid nitrogen, −80, −30, and −18℃) and storage duration (1, 4, 12, and 24 weeks) on the properties of red swamp crayfish . Generally, the biochemical and physical quality of frozen crayfish showed a trend of gradual deterioration with the increase of storage duration. However, it also noticed that no significant changes in the hardness for all cryogenic treatments at week 1, which was not consistent with the trend of Ca 2+ -ATPase activity and sulfhydryl content of crayfish protein. The correlations among these evaluation attributes have not been reported yet. Therefore, the objective of this research was to investigate the effects of cryogenic condition on the microstructure, protein pattern, and thermal properties of red swamp crayfish frozen for oneweek storage and explore the internal relationships among these attributes by Pearson's correlation analysis.

| Experimental design and materials
Fresh alive red swamp crayfish (Procambarus clarkia) was purchased on May from Qiyimen fresh market, Wuhan. Once arrival to the laboratory, crayfish with the uniform size (9.8 ± 0.5cm) was picked out and cleaned with ice water and then vacuum packed individually. Each ten crayfish (random picked) were set as one batch and subjected to different cryogenic processing for one week. For further analysis, frozen samples were first thawing at 4℃ for 12h and then equilibrating to room temperature. Fresh samples were set as the control.

| Histology
Sample preparation of micromorphology observation was performed according to the method of Jiang et al., (2019). Crayfish tail muscle was dehydration, paraffin embedding, sectioning (cross cut) and dyeing treatment, and then observed by the optical microscope (Eclipse Ci, Nikon, Tokyo, Japan). The magnification of scanned images was adjusted to 1,500 times, and images were collected using Pannoramic Viewer (1.15.3, 3DHISTECH Ltd, Budapest, Hungary).
The increase of white regions in the images refers to the damage of cell structure. The number and sum area of white regions, namely WN and WA, respectively, were recorded by Image-Pro Plus 6.0 (Media Cybernetics, Inc, Rockville, MD, USA) according to the method describe by (Shi, Tao, et al., 2018).

| SDS-PAGE
The protein pattern of crayfish was revealed using SDS-PAGE according to Jiang et al., (2018) with minor modifications. Ground crayfish tail muscle was homogenized (HBM-400B, IKA, Germany) with 10-fold SDS (5%) at 8,000 rpm for 1min. Then, homogenate was water bathed at 90℃ for 1 hr, followed by centrifugation (GL-21 M, Xiangyi Inc., Changsha, China) at 4,000 rpm for 10 min to obtain the supernatant protein solution. After adjusted the concentration to 1.5 mg/ml, the protein sample was mixed with a loading buffer (1MTris-HCl (pH 6.8), 50% glycerol, 10% SDS, 14.4 mM βmercaptoethanol, 1% bromophenol blue and ddH 2 O) in the ratio of 1:2, following by heating at 90℃ for 5 min. Stacking and separating gels were made using 5% and 10% acrylamide solutions, respectively. Aliquots of samples (7 μl) were loaded onto the gel and subjected to electrophoresis at 120 V. After running, gel was stained with 0.125% Coomassie brilliant blue R-250 and destained in a solution containing 50% methanol and 10% acetic acid. Gel image was taken using a Gel Doc XR scanner (Bio-Rad Laboratories, Milan, Italy). The molecular weight of protein bands was determined by comparing with the protein standard (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China). The intensity of protein bands was analyzed by Image J 1.52a (National Institute of Health, Wayne Rasband, USA) according to a method described by Zhu et al., (2017).

| Thermal properties
Thermal properties of crayfish tail muscle were measured by differential scanning calorimetry (DSC) using a calorimeter (200F3, Netzsch, Germany). The DSC analyzer was calibrated using indium, and an empty aluminum pan was used as a reference. The samples (about 19~23mg) were prepared in aluminum pans and sealed hermetically, and heated from 10℃ to 90℃ at a rate of 5℃/min. Thermal transitions of Tp (peak of denaturation temperature) and enthalpy of denaturation (ΔH) were calculated and recorded by Proteus software (Netzsch, Germany).

| Statistical analysis
GraphPad Prism (GraphPad Software Inc, La Jolla, CA, USA) was performed for graph drawing. ANOVA and Duncan's multiple range tests were used to estimate the statistical significance with a level of p <.05 using SPSS (IBM Cop., Armonk, New York, United States). DPS (Zhejiang University, Hangzhou, China) was applied for Pearson's correlation coefficients calculation and correlation analysis.  Plus was applied to analyze the histologic images for digital data.

| Microstructure
The white regions in the image were seen as the gaps among the tissue cells. Generally, the more and larger the white regions, the higher level of cell damage. According to Table 1 The quality of frozen food material was influenced by several processing factors, such as freezing rate , storage temperature, and duration (Lee & Park, 2016). In this research, the crayfish was frozen stored for one week, in the purpose of maximum maintaining the freshness of samples. However, the histomorphology of crayfish muscle was still negative impacted by the cryogenic  processing, which indicated the quality deterioration of frozen crayfish.  believed fast freezing rate was better for food perseveration, since the small and uniform ice crystals were formed during the fast freezing processing without harm the muscle tissue. In this research, WA and WN of −80/-80 group (freezing rate of 0.325℃ /min) was lower than those of −18/-18 group (freezing rate of 0.038℃ /min), which proved the Kaale's opinion. Besides, the stability of storage temperature greatly affected the sample quality (Romotowska et al., 2017), which was also confirmed by the high increase rate of WA and WN of the −80/-18 group (Table 1). The −80/-18 treatment exhibited a disordered muscle structure (Figure 2c), which might be contributed by the warming effect during frozen storage (Shi, Tao, et al., 2018) and the magnifying effect due to the vulnerable short tissue fiber of crayfish. Comparatively speaking, −80/-80 treatment was appropriate for crayfish preservation while −80/-18 was not.

| SDS-PAGE
The protein pattern and band intensities of crayfish subjected to different cryogenic treatments was shown in Figure 2 and  Figure 3 and Table 2 displayed the thermal properties red swamp crayfish subjected to different cryogenic treatments. Figure 3 exhibited three thermal denaturation peaks between 40 and 90℃, referring to head of myosin (Peak 1), rod tail of myosin (Peak 2), and actin (Peak 3), respectively (Korzeniowska et al., 2013). It was noticed that the position and attitude of three peaks for different treatment were slight moved. As shown in Table 2, Tp (peak of denaturation temperature) of crayfish muscle was significantly raised (p <.05) after frozen stored for one week, except for the Tp2 of −80/-18 treatment. However, no obvious change of Tp was observed among the cryogenic treatments (except Tp1 of −80/-18 treatment), which suggested it was a moderate denaturation of myofibrillar protein contributed by short-term frozen storage.

| DSC
The value of ΔH (enthalpy of denaturation) revealed the energy required for protein denaturation. It is clearly observed that the ΔH of −18/-18 treatment was significantly lower than samples freezing at −80℃ (p <.05), suggesting that the change of protein conformation was easily happened when freezing under a slow rate. Big and inhomogeneous ice crystals were formed accompanied by slow rate freezing , which could lead to cell damage and cytosol loss and resulted in quality deterioration of frozen crayfish product. While on the condition of fast freezing rate, the ΔH of three peaks of −80/-80 treatment were slightly higher than −80/-18 treatment (p >.05), indicating the storage at lower temperature was benefit for maintaining the stability of muscle protein product.

| Pearson's correlation analysis
The Pearson correlation coefficients among the protein band inten-  cryogenic treatments for short-term storage, cell structure was partially damaged, and protein aggregation and denaturation were presented. Furthermore, WN was negatively correlated (p <.01) with the band intensity of TN T, and MLC 1 band intensity was positively correlated (p <.01) with the intensity of small proteins (including TN I, MLC 2, and 3). Therefore, this study suggested (1) −80/-80 treatment was appropriate for crayfish preservation while −80/-18 was not;

| CON CLUS IONS
(2) the intensive TN T band might suggest the tissue integrity of muscle; and (3) the intensive MLC 1 band could be considered as the valid label to protein degradation.

ACK N OWLED G M ENT
The authors would like to thank the financial support from

CO N FLI C T O F I NTE R E S T
The authors declared that there are no conflicts of interest regarding the publication of this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.