Fresh live bighead carp were purchased from the local market (Wuhan, Hubei, China) and transported alive to the laboratory in oxygenated bags with water within 20 min. The fish were treated in ice water to death, then scaled, eviscerated, decapitated, chopped, and frozen to -80℃ for subsequent experiments. The entire experimental procedure followed the research protocols approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University (Wuhan, Hubei, China). Pepsin with an enzyme activity of 10,000 U/g was purchased from Guangzhou Saiguo biotech Co., LTD (Guangzhou, China). Sample buffer and molecular weight markers were bought from Tiangen (Beijing, China). All the chemicals and reagents such as the anilino-8-naphthalenesulfonate (ANS), tris hydrochloride, and maleic acid were of analytical and HPLC grades.
2.2 Preparation of bighead carp myofibrillar proteins (BMP)
Extraction of BMP was performed on ice by previously reported method with some modifications (Li et al. 2021b). Briefly, the samples were stored at -80℃, and thawed in a refrigerator at 4℃ for 18 h prior to extraction. The minced meat from bighead was washed with 0.02 M Tris-Maleate (pH 7.0) at the rate of 1:10 (w/v) and 6,000 r/min in a waring blender for 60 s. After filtration with 300 mesh gauze, the precipitate was rewashed with 0.02 M Tris-Maleate under the same blending and filtration conditions as described above. The precipitate was resuspended in 0.6 M Tris-Maleate (pH 7.0) for 18 h at 4℃ with interval stirring. The mixture was centrifuged at 12,000 g at 4℃ for 20 min. The supernatant was washed with 10 vol (w/v) ultrapure water and centrifuged at 12,000 g for 20 min. The precipitate was washed twice more under that same condition. The precipitate from the last centrifugation was bighead carp myofibrillar protein (BMP), which was stored at 4℃ before use. The Bradford method was used to measure the protein concentration, and the protein concentration of the precipitates was 12.0 mg/mL approximately.
2.3 BMP treatment
2.3.1 Ultra-high hydrostatic pressure (UHP) treatment
The BMP samples were placed in a pressure container filled with water and then treated at 300 MPa for 20 min. Pressure container temperature was maintained at 4℃. One portion of the treated samples were stored at 4℃ and the other portion was lyophilized and stored at -20℃ for subsequent use. Non-UHP treatment BMP was used as control (0.1 MPa). These pressurization parameters were chosen from the results of pre-experiments.
2.3.2 High pressure homogenization (HPH) treatment
The BMP was homogenized twice at 30 MPa in a continuous laboratory-scale high pressure homogenizer (ATS AH-2010, Suzhou, China). Following each homogenization, the BMP dispersion liquid was rapidly cooled to 4 ± 2°C in an ice-water bath. The untreated BMP was used as a control (0.1 MPa). Finally, the samples were stored or lyophilized for further analysis.
2.3.3 UHP followed by HPH (U-H treatment)
Following the same treatment operations in 2.3.1. and 2.3.2, the BMP dispersion liquid post UHP was then subjected to HPH.
2.3.4 HPH followed by UHP (H-U treatment)
H-U treatment conditions were the same with U-H ones with a reverse sequence of HPH and UHP.
2.4 Enzymatic hydrolysis
Following the above-mentioned treatment, pepsin (4000 u/g) was added to the BMP solution (pH 3.0), and reaction was performed at 55℃ for 6 h. The hydrolysis reaction was stopped in a water bath at 95℃ within 10 min. The hydrolysate was centrifuged at 10,000 g for 20 min, and the supernatant was collected. These enzymatic hydrolysis conditions were determined by the results of pre-experiments.
2.5 Total sulfhydryl content (SHT)
The total sulfhydryl content was determined in reference to the method reported in previous study (Liu et al. 2000) with slight modifications. The 1.0 mL protein sample solution was mixed with 9.0 mL Tris-HCl buffer (0.2 mol/L, pH 7.0) containing 8 mol/L urea, 10 mmol/L EDTA, 2% SDS. After 30-min standing, the 4.0 mL mixture solution was further mixed with 0.4 mL DTNB (0.1%, pH 8.0). After 25-min reaction at 40℃, the absorbance of the mixture was measured at 412 nm. The molar extinction coefficient of 13600 L• (mol • cm)-1 was used to calculate the SHT.
2.6 Fourier Transform infrared spectroscopy (FT-IR)
The attenuated total reflection spectra of the lyophilized sample of BMP were obtained within the wavenumber range of 600 to 4000 cm-1 (Nicolet Is50 Fourier transform infrared spectroscopy, Germany) in reference to the previous study with minor modifications (Sharifian et al. 2019). The spectra were fitted within 1700-1600 cm-1 using Peakfit Version 4.04 to obtain gaussian curve (SPSS Inc., Chicago, II, USA). Prior to curve fitting, the data were subtracted from the linear baseline, and Fourier deconvolution was used to differentiate the peak locations. The secondary structures (α-helix, β-sheet, β-turn, and random coil) were calculated according to the curve fitting results.
2.7 Intrinsic Fluorescence Emission Spectroscopy (IFES)
The intrinsic fluorescence is used to evaluate the "denaturation state" of proteins by targeting aromatic amino acids, mainly tryptophan. Fluor photometer (F-7000, Hitachi, Japan) was used to monitor BMP dispersion (0.3 mg/mL) by the previously reported method with minor modifications (Shi et al. 2019). The excitation wavelength was 280 nm and the monitoring range of emission spectrum was from 270 to 450 nm. The scan speed and slit width were 2400 nm/min and 2.5 nm, respectively.
2.8 Surface Hydrophobicity (H0)
ANS titration method was used to measure the surface hydrophobicity (H0) of BMP solution (Haskard and Li-Chan 1998). Different treated BMPs were diluted to the concentrations ranging from 0.1 mg/mL to 0.5 mg/mL with 0.6 M Tris-maleate buffer (pH 7.0). The 2.0 mL of BMP solution was mixed with 10.0 μL of ANS solution (8.0 mmol/L in 0.1 mol/L sodium phosphate buffer, pH 7.0) and reacted for 30 min. A fluorescence spectrophotometer (F-7000, Hitachi, Japan) was used to determine the relative fluorescence intensity at 385 nm excitation wavelength and at 470 nm emission wavelength (slit 3 nm). The initial slope of the fluorescence intensity versus protein concentration curve (calculated by linear regression analysis) was defined as the value of H0.
2.9 Particle size distribution and zeta potential
The particle size and zeta potential of BMP were determined using photon correlation spectroscopy (Zetasizer Nano ZS, Malvern Instruments, UK), as reported by Roy et al. (1999). Briefly, the BMP dispersion liquid (1.0 mg/mL, pH 7.0) was stirred for 1 hour and centrifuged at 2000 g for 1 min to obtain supernatant for analysis.
2.10 BMP stability analysis by multiple light scattering (MLS)
A laser diffraction scanner (Turbiscan LAB, Formulaction, Ramonville St. Agne, France) was used to determine the stability of treated BMP. The scanner was equipped with a pulse near-infrared light source (λ = 880 nm) and two synchronous detectors scanning the height of the sample. Transmission and backscatter data were collected every 40 μm. The scanning frequency was set as every 30 s in the initial 30 min, and the percentage of light backscatter or transmission was measured in the initial 30 min at 25℃. The turbiscan stability index (TSI) calculated by the Turbisoft 2.1 software was used to evaluate the stability of the BMP (Raikos et al. 2017).
2.11 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
The composition of the treated BMP solution was determined through SDS-PAGE in which gels contained 5% and 12% polyacrylamide corresponding to application voltages of +80 V (0.5 h) and +120 V (1.0 h). Specifically, in the presence or absence of β-mercaptoethanol, 100.0 μL (1.5mg/mL) of the treated BMP solution was mixed with 50.0 μL buffer solution. Then, 10.0 μL mixture was added onto the top of the gel. After electrophoresis, the gel was stained with Coomassie brilliant blue (G-250), and was photographed after excess dye removal (Laemmli 1970).
2.12 Morphological characterization
2.12.1 Scanning electron microscopy (SEM)
The microstructure of BMP freeze-dried powder was observed under cold field emission scanning electron microscopy (S-4800, Hitachi, Tokyo, Japan). The different treated BMP powder was subjected to the same crushing treatment. The BMP powder was uniformly adhered to the conductive adhesive and observed after gold spraying.
2.12.2 Transmission electron microscopy (TEM)
The structure of BMP solution was observed at 80 kV acceleration voltage under transmission electron microscopy (Hitachi H-7650) in high contrast imaging mode. BMP was drop cast onto carbon-coated copper grid, negatively stained with 2% sodium ortho-tungstate solution for 30 s, and air dried before imaging.
2.13 Determination of hydrolysis degree
The amino acid peptide nitrogen content in enzymatic hydrolysate and raw material were determined by formaldehyde potentiometric titration (Mahmoud et al. 1992). The Bradford method was used to determine the protein concentration in raw material, and the total nitrogen content in raw material was calculated as protein concentration multiplied by conversion coefficient (6.25). The degree of hydrolysis was calculated according to the following formula.
The degree of hydrolysis = (AN-AN0) /N× 100%
where AN is the content of amino acid peptide nitrogen in enzymatic hydrolysate, AN0 is the initial free amino acid nitrogen (not induced by enzymatic hydrolysis-naturally present in the BMP), and N is the content of total nitrogen in BMP.
2.14 Determination of antioxidant capacity
2.14.1 Scavenging effect of BMP and its enzymatic hydrolysate on DPPH
The scavenging activity of DPPH was determined by previously reported method with some modifications (Xie et al. 2008). The 2.0 mL sample solution was mixed with 2.0 mL DPPH (0.2 mM) and reacted at room temperature in the dark for 30 min. The absorbance of the mixed solution at 517 nm was measured with a spectrophotometer (L5S, Shanghai, China) and expressed as Ai. The DPPH solution was replaced with 95% ethanol, and the above operation was repeated for 3 times. The absorbance of solution (95% ethanol + BMP) was expressed as Aj. The BMP sample was replaced with ultrapure water, and the above operation was repeated for 3 times. The absorbance of solution (95% ethanol + ultrapure water) was recorded as Ac. The radical scavenging capacity was calculated by the following formula:
Inhibition (%) = [1-(Ai-Aj)/Ac] × 100%
2.14.2 Scavenging effect of BMP and its enzymatic hydrolysate on ·OH
The scavenging effect of ·OH was determined by test kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
2.14.3 Chelation of BMP or its enzymatic hydrolysate with Fe2+
The Fe2+ chelation was determined by the previously reported method with some modifications (Decker and Welch 1990). The 1.0 mL the sample solution, 3.7 mL ultra-pure water, 0.1 mL ferrous chloride solution (2.0 mM), and 0.2 mL of phenazine solution (5.0 mM) were mixed and reacted for 10 min. Subsequently, the absorbance of the mixture at 562 nm was measured. The ultrapure water was used as the control. The chelation ability of Fe2+ was calculated according to the following formula.
Chelation (%) = [(Acontrol-Asample)/Acontrol] × 100%
2.14.4 Reducing capacity of BMP and its enzymatic hydrolysate
The reducing capacity of BMP and its enzymatic hydrolysate was measured using the method reported by Yen and Chen (1995) with some modifications. The 2.5 mL potassium ferricyanide solution (1%), 0.5 mL BMP, and 2.5 mL phosphate-buffered saline (0.2 M, pH 6.6) were mixed and incubated at 50℃ for 20 min. Subsequently, 2.5 mL trichloroacetic acid (10%) was added, and the mixture was centrifuged at 3000 g for 10 min. The 2.5 mL supernatant, 0.5 mL ferric chloride solution (0.1%), and 2.5 mL distilled water were mixed and reacted at room temperature for 10 min, and the absorbance of the mixture solution at 700 nm was measured with a spectrophotometer (L5S, Shanghai, China). The higher the absorbance, the stronger the sample reducibility.
2.15 Statistical Analysis
In this study, all experiments were conducted at least in triplicates, and the data were subjected to one-way ANOVA using SPSS software (SPSS 25.0, SPSS Inc., Chicago, IL). The results were expressed as means ± standard deviations (SDs), and p < 0.05 was considered as statistically significant.