Materials
Chicken egg white lysozyme (Phygene Scientific Reagent Co. Ltd., Fuzhou, China), Micrococcus lysodeikticus [M. lysodeikticus] (BeNa Culture Collection, Xinyang City, Henan, China), 8-Anilinonaphthalene-1-sulfonic acid [ANS] (Shanghai Macklin Biochemical Co. Ltd., Shanghai, China) 5,5-dithio-bis-(2-nitrobenzoic acid) [DTNB] (Shanghai Ryon Biological Technology Co. Ltd., Shanghai, China).
[ Figure 1 about here]
High voltage cold atmospheric plasma machine
A high voltage cold atmospheric plasma (HV-CAP) machine (Nanjing Suman Plasma Technology Co., Ltd. Nanjing, China) with dielectric barrier discharge (DBD) configuration was employed. A detailed description of the machine was given in our earlier article (Nasiru et al. 2021). The device was operated at three different high voltage power configurations of 120, 140, and 160 kV at a frequency of 120 Hz and a treatment time of 3 min.
Ultrasound device
Ultrasound device (KH5200DE, Kunshan Hechuang Ultrasonic Instrument Co., Ltd., Qingshan Lake Technological City, Hangzhou, China) was used and operated at an intensity of 95 % for 10 min at 4 to pre-treat the lysozyme.
Experimental plan
The effects of ultrasound-assisted HV-CAP on lysozyme activity and its mechanism were evaluated in two groups and a control sample. The first group comprised HV-CAP treated samples, while the second group was pre-treated with ultrasound before HV-CAP treatment. The treated samples were divided as follows:
CK – Control (No HV-CAP or ultrasound pre-treatment)
T1 – 120 kV/120 Hz/3 min
T2 – 140 kV/120 Hz/3 min
T3 – 160 kV/120 Hz/3 min
T4 – 120 kV/120 Hz/3 min + ultrasound pre-treatment
T5 – 140 kV/120 Hz/3 min + ultrasound pre-treatment
T6 – 160 kV/120 Hz/3 min + ultrasound pre-treatment
Ultrasound pre-treatment: 95 %/10 min/4
Lysozyme preparation
Lyophilized chicken egg lysozyme powder was dissolved in sodium phosphate buffer (20 mM, pH 7.6). The protein concentration of lysozyme was 2.12 mg/mL using the Biuret reagent method with Bovine Serum Albumin (BSA) as standard.
Lysozyme activity assay
The turbidimetric assay method was used based on Zhao and Yang (2008). As a substrate, a lyophilized M. lysodeikticus powder (0.36 mg/mL) was dissolved in sodium phosphate buffer (20 mM, pH 7.6). 2.3 mL of the substrate was put in a cuvette for each sample and stored at 25 °C with continual magnetic stirring. Initially, 0.3 mL of lysozyme sample was transferred into a 2.6 mL reaction volume in the cuvette. A decrease in absorbance of mixed M. lysodeikticus suspension was measured at an interval of 0.5 s at 450 nm using a UV-vis spectrophotometer (Shimadzu 2600 UV-vis spectrophotometer, Shimadzu Corporation, Kyoto, Japan). The decrease in absorbance against time was plotted from the absorbance data that was recorded, and the activity of each sample (450/min) was evaluated. An enzyme unit equals to a decrease in turbidity of 0.0001/min at 450 nm; thus, the activity per mL of HV-CAP and ultrasound-assisted HV-CAP treated lysozyme samples were evaluated as below:
Relative residual activity
Lysozyme's relative residual activity (RRA) was calculated as a percentage of HV-CAP, and ultrasound-assisted HV-CAP treated lysozyme solutions activity compared to control. After the activity assay, all samples were stored in ice-bath water at 0 .
[ Figure 2 about here]
Relative protein concentration
The relative protein concentration (RPC) was expressed as a percentage of the soluble protein concentration of the HV-CAP, and ultrasound-assisted HV-CAP treated lysozyme solution compared to that of the control. A 0.45 μm filter was used to filter the samples, and the absorbance was measured at room temperature with a UV-vis spectrophotometer (Shimadzu 2600 UV-vis spectrophotometer, Shimadzu Corporation, Kyoto, Japan). The RPC was expressed as:
Where; A280 is the absorbance at 280 nm of the treated lysozyme solution while AC280 is the absorbance of the control sample.
Turbidity
Turbidity of the control and HV-CAP and ultrasound-assisted HV-CAP treated lysozyme were evaluated according to Zhao and Yang (2008) using a spectrophotometer. 3 mg/mL of treated lysozyme solution was recorded at 650 nm using a UV-vis spectrophotometer (Shimadzu 2600 UV-vis spectrophotometer, Shimadzu Corporation, Kyoto, Japan). The turbidity was calculated as the transmittance percentage (T %).
Tertiary structure of lysozyme
Ultraviolet absorption spectra
A UV-vis spectrophotometer was used to analyse the UV spectra of the treated and control samples at 280 nm in a phosphate buffer (20 mM, pH 7.6) (Shimadzu 2600 UV-vis spectrophotometer, Shimadzu Corporation, Kyoto, Japan). The absorption spectra were measured from 200 to 320 nm.
Exposed sulfhydryl groups
The sulfhydryl content (SHF) of the treated and control lysozyme samples were evaluated based on Zhao et al. (2007). The treated lysozyme was diluted 10 times in Tris-glycine buffer (0.1 M Tris–(hydroxymethyl)–aminomethane (Tris), 0.1 M glycine, and 4 mM ethylenediamine–tetraacetic acid (EDTA) disodium salt, pH 7.0). 4 mg/mL 5',5-dithiobis (2-nitrobenzoic acid) [DTNB] in Tris-glycine–buffer was used to make Ellman's reagent, and 10 μL was added to the protein solution. After being maintained at ambient temperature for 10 min, the mixture was centrifuged at 19,000 g at 4 for 15 min (Allegra 64R Centrifuge, Beckman Coulter, IN, USA). In a blank sample, DTNB was substituted with Tris-glycine buffer. The supernatant was read at an absorbance of 412 nm using a spectrophotometer (Varioskan Flash, Thermo Scientific, USA). The sulfhydryl content was expressed as below:
Where; A412 is the absorbance at 412 nm, C is the lysozyme concentration (mg/mL), and D is the dilution factor.
Surface hydrophobicity
The surface hydrophobicity of treated and control lysozyme samples was evaluated by Zhao et al. (2007). The relative fluorescence intensity was used to assess the surface hydrophobicity of lysozyme. After dissolving the treated lysozyme (2 mg/mL) in phosphate buffer (20 mM, pH 7.6), 10 mL of 8 mM 8-Anilino-1-naphthalene sulfonic acid (ANS) in phosphate buffer (20 mM, pH 7.6) was added to 2 mL of the lysozyme dispersion. Using a spectrofluorometer (Varioskan Flash, Thermo Scientific, USA), the relative fluorescence intensity of lysozyme and ANS was measured at 390 nm at room temperature (excitation wavelength, slit 2.5 nm), 410 – 650 nm (emission wavelength, slit 2.5 nm), and a speed of 10 nm/s.
[ Figure 3 about here]
Intrinsic fluorescence spectroscopy
The intrinsic fluorescence spectra of HV-CAP and ultrasound-assisted HV-CAP treated and control lysozyme samples were measured in a phosphate buffer (20 mM, pH 7.6) at room temperature using a spectrofluorometer (Varioskan Flash, Thermo Scientific, USA) at 295 nm (excitation wavelength, slit: 2.5 nm), 320 – 400 nm (emission wavelength, slit: 2.5 nm) and a scanning speed of 10 nm/s.
Carbonyl content
According to Wu et al. (2014), the HV-CAP and ultrasound-assisted HV-CAP treated lysozyme carbonyl contents were evaluated. The lysozyme reaction utilised the 2,4-dinitrophenylhydrazine (DNPH) and the carbonyl groups. 0.35 mL (5 mg/mL) of lysozyme solutions were blended with 1 mL 10 mM DNPH in 2 N HCl and incubated for 2 h at 20 . A blank was made by mixing 1 mL of 2 N HCl with an equivalent volume of protein solution without DNPH. Then 0.45 mL 40 percent trichloroacetic acid was added and vortexed (Crystal VM-01U, Suzhou Jiemei Electronics Co., Ltd., Jiangsu, China) for 20 min before centrifuging for 10 min at 10,000 g (Allegra 64R Centrifuge, Beckman Coulter, IN, USA). The pellet was washed 3 times in a 1.5 mL ethyl acetate (1:1, v/v) solution. The protein was suspended in 1.0 mL 6 M guanidine hydrochloride solution and vortexed every 5 min in a water bath (37 ) for 20 min. At 367 nm, the absorbance was measured (Varioskan Flash, Thermo Scientific, USA). The results were expressed as μmoles carbonyl groups per mg of soluble protein with a molar extinction coefficient of 36,000 M-1 cm-1.
Lipid oxidation
Lipid oxidation of the HV-CAP and ultrasound-assisted HV-CAP treated lysozyme were determined according to Wang et al. (2021). Using a homogeniser (IKA T25 digital ULTRA, Germany) at a speed of 10,000 g for 30 s, 3 mg of lysozyme powder was dissolved in 15 mL deionised water and 0.05 mL butylated hydroxyl toluene (BHT) (4.2 percent in ethanol). The homogenate was centrifuged for 5 min at 2,000 g (Allegra 64R Centrifuge, Beckman Coulter, IN, USA). Then 2 mL of thiobarbituric acid (TBA) at a concentration of 20 mM was added. The mixture was then boiled in a water bath for 5 min at 90 . The mixture's temperature was then lowered, and it was centrifuged for 15 min at 4000 g. The supernatant was read at 532 nm (Varioskan Flash, Thermo Scientific, USA), and the results were expressed as mg malondialdehyde (MDA) per kg lysozyme.
Particle size distribution
The particle size and zeta potential of HV-CAP and ultrasound-assisted HV-CAP treated lysozyme (0.3 mg/mL) were measured according to the method of Qian et al. (2021) using Zetasizer Nano ZS analyser (Malvern Instruments Ltd., Worcestershire, UK) at 25 .
Secondary structure of lysozyme
Fourier transform infrared-attenuated total reflectance spectroscopy
The Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy investigated the HV-CAP, and ultrasound-assisted HV-CAP treated lysozymes' secondary structure. The treated lysozyme powder (3 mg) was placed on the diamond crystal of Nicolet iS10 FTIR-ATR Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The spectra of the lysozyme samples were scanned at a resolution of 32 cm−1 with 100 scans from 4000 to 400 cm−1.
Circular dichroism spectroscopy
The Circular dichroism (CD) spectroscopy was utilised further to study the HV-CAP and ultrasound-assisted HV-CAP effects on the treated lysozyme. The CD measurement was conducted using a Jasco spectrometer (J-1500, Jasco Corporation, Japan). The treated lysozyme was scanned from 240 to 200 nm with a 5 nm bandwidth, 1 nm pitch, and 100 nm/min speed using a 1 mm path length cuvette. Each lysozyme sample was dissolved in phosphate buffer (20 mM, pH 7.6) to a 0.3 mg/mL final concentration.
Relative Molar Ellipticity
The relative molar ellipticity at 222 and 208 nm of lysozyme were described as a percentage of the molar ellipticity of the HV-CAP-treated and ultrasound-assisted HV-CAP-treated lysozyme samples relative compared to the control.
Statistical analysis
Origin Graphing and Statistical Analysis Software was used to perform statistical analyses and data plots (Version 2021b, OriginLab Corporation, Massachusetts, USA). Tukey's test evaluated statistical differences using a one-way analysis of variance (ANOVA). 0.05 was used as the statistical significance level. All the trials were repeated three times.
[ Figure 4 about here]