Plasma bubbling of milk
The optimized plasma bubbling parameter setup was configured at 200 volts (V), 10 liters per hour (L/h), time interval of 15 minutes (min), and 100 mL sample volume. The atmospheric gas was supplied as feed gas to carry out the plasma bubbling and it bubbled through the milk sample. This parameter set up successfully decontaminated E. coli, coliform, yeast, and mould. The treated and control (raw cow milk) samples were tested for E.coli, coliform, yeast, and mould counts using MacConkey agar (Parseelan et al., 2018), violet red bile agar (Ray & Speck 1978) and chloramphenicol yeast glucose agar (Torkar and Teger 2006) respectively. The current study was observed both control and the optimized set-up parameters for plasma bubbling to better understand the protein quality with respect to control sample.
Extraction Of Casein Protein
Casein protein was extracted from control, optimized plasma bubble treated sample and then compared with standard casein protein purchased commercially (Sigma-Aldrich, USA). For extraction of casein, the milk was purchased from a local vendor in Thanjavur. An extensive comparison was made for the commercial, control and treated casein protein sample. The extraction procedure of casein protein was followed according to Pavia et al., (1998). Briefly, 100 mL volume of milk was centrifugated at 4000 rpm for 20 minutes at ambient temperature for removing the fats or lipids from the mixture. After centrifugation, the lipids containing supernatant were removed. Further, the milk was transferred to beakers and pH (Laqua pH1100, Horiba Scientific, Singapore) was adjusted using 0.2N HCL until it reaches the isoelectric point at pH 4.6 which resulted in precipitation of casein. The curdy precipitate was separated from the supernatant using filter paper (Whatman no 1) of size 25*50 mm and allowed for sedimentation of casein protein. Then, the sedimented precipitate was washed using distilled water to remove the salts, followed by washing with diethyl ether and ethanol in the ratio of 1:1 i.e., 50% of diethyl ether: 50% ethanol to remove excess fat. The precipitated casein protein was lyophilized (Lyophilizer LYO 10 T, Borg Scientific, Chennai) with vacuum at temperature of -55°C to -60°C for 18 hours following Deshwal et al., (2020).
Ftir- Characterization
The FTIR analysis was performed for standard, control and treated casein protein sample to observe the secondary structures of the casein upon the plasma bubbling treatment. The spectrum using 50 Nicolet FTIR spectrophotometer (Thermoscientific, USA) was analyzed with a scan range from 400-4000cm-1 and a resolution of 4 cm-1 (Rutten et al., 2011).
Sample Preparation For Maldi-tof
The sample preparation for MALDI-TOF was followed by Sodium Dodecyl Sulphate–Poly Acrylamide Gel Electrophoresis (SDS-PAGE) using 10% (w/v) gels according to Laemmli, (1970). Known quantities (2–3 µg) of casein protein along with standard molecular weight protein markers were loaded in appropriate wells for running SDS-PAGE. The silver staining was used to stain the gel (Morrissey, 1981). The gel slice was diced to small pieces, de-stained using destaining solution (ammonium bicarbonate: acetonitrile = 1:1) for 10-minute intervals (3–4 times) until the gel pieces become translucent white. The gels were dehydrated completely using acetonitrile in SpeedVac instrument, rehydrated with Dithiothreitol (DTT) by incubation for 1hour. After incubation, the DTT solution was removed and the gel pieces were treated with Iodoacetamide (IDA) for 45min. The supernatant was removed and the gel was incubated with ammonium bicarbonate solution for 10 mins and then dehydrated with acetonitrile for 10 mins in SpeedVac till complete dryness. A concentration of 15µL trypsin solution was added and incubated overnight at 37°C. The digested solution was transferred to fresh Eppendorf tubes. The gel pieces were extracted thrice with extraction buffer Acetonitrile and Trifluoracetic acid (ACN and 0.1% TFA = 1:1 ratio) and the supernatant was collected each time into the Eppendorf and then SpeedVac was used for complete dryness.
MALDI-TOF
The purified casein (control and treated) proteins bands obtained in SDS-PAGE were excised and further subjected to trypsin digestion and were analyzed using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS Bruker Daltonics Ultraflex III). Briefly, dried pepmix was suspended in TA buffer (ACN and 0.1% TFA = 1:2 ratio). The peptides obtained were mixed with α-Cyano-4-Hydroxycinnamic Acid (HCCA) matrix in 1:1 ratio and the resulting 2µl was spotted onto the MALDI plate. After air drying the sample was analyzed on the MALDI TOF/TOF ULTRAFLEX III instrument and further analysis was done with flex analysis software for obtaining the peptide mass fingerprint. The obtained masses were submitted for Mascot search in “concerned” database for protein identification. These peptide mass values were searched against the databases such as National Center for Biotechnology Information Non-Redundant Proteins (NCBI nr), SWISS-PROT to obtain information about the identity of protein.
Peptide Toxicity Prediction
Generally, peptide is being studied as a potential target for therapeutic candidate. However, some peptides showed toxicity towards the eukaryotic cell. Therefore, it is important to estimate the toxicity level before use for in vitro toxicity studies. We have aimed to check the toxicity level of identified peptide molecule using online servers. We have examined toxicity of the peptide using three servers which has shown non-toxic and non-allergen to the human. The predicted peptide (RYLGY) was analyzed for the toxicity using ToxinPred (http://crdd.osdd.net/raghava/toxinpred/) (Gupta et al., 2013), VaxiJen (http://www.ddg-pharmfac.net/vaxijen/vaxijen/vaxijen.html) (Chukwudozie et al., 2021) and AllerTOP v.2.0 (https://www.ddg-pharmfac.net/allertop/method.html) (Dimitrov et al., 2014) servers.
Preparation And Docking Studies
The crystal structure of the ACE2-RBD complex (PDB ID: 6M0J) was obtained from protein data bank (PDB) database (http://www.rcsb.org/pdb). The structure was prepared using protein preparation wizard implemented in the Schrodinger software suite 2021 (http://www.schrodinger.com/) (Mariadasse et al., 2015). The OPLS-2015 (Optimized Potential for Liquid Simulations) (Choubey et al., 2017) all-atom force field incorporated in the Schrodinger software was used for the energy minimization to obtain the improved quality of the ACE2-RBD complex. H-bonds, ionization, missing side chains, and tautomerization of the residues were incorporated besides removal of steric clashes. According to previous report by Kong et al., (2019), the ACE2-RBD interface region was considered as peptide-binding region confirmed through the CoDockPP server (http://codockpp.schanglab.org.cn/) and sitemap module. The grid was generated in the ACE2-RBD interface region of the complex. Further, the peptide (RYLGY) molecule was built using Avogadro software and minimized through the protein preparation wizard of Schrodinger software suite 2021. The minimized structure of ACE2-RBD and peptide molecule was considered for the docking studies using Schrodinger software.
Molecular Dynamics Simulation Studies
The energy minimized ACE2-RBD and ACE2-RBD-peptide complex were used for the molecular dynamics simulation studies by GROMACS (GROningen Machine for Chemical Simulations) version molecular dynamics package 5.1 (Choubey et al., 2017). The GROMACS 56a force field was employed for ACE2-RBD and ACE2-RBD-peptide complex to obtain the energy minimized conformation. Both, the complex systems were fixed with cubic boxes with the dimension of 1 nm and solvated with SPC216 water models. Periodic boundary conditions (PBC) were applied in all directions for the approximation of infinite systems. Further, the net charges of both the systems were neutralized by adding appropriate ions (Na+) counterions. Steepest Descent Algorithm (SDA) was used for energy minimization of the ACE2-RBD and ACE2-RBD-peptide complex through the 50000 steps with a maximum tolerance of 1000 KJ mol–1 nm–1. The complexes were equilibrated with two steps; the first step includes the NVT ensemble (constant number of particles, volume, and temperature) for 100 ps to stabilize the system at 310 K, and the second step, the NPT (constant number of particles, pressure, and temperature) equilibrium using coupling reference pressure of 1 bar (atm) for 100 ps. All bond lengths were constrained with the LINCS (Linear Constraint Solver) algorithm. The PME (Particles Mesh Ewald) electrostatic and periodic boundary conditions were applied in all directions. A cut-off value of 9 Å for coulomb interactions and 10 Ǻ for Van der Waals interactions were assigned. This pre-equilibrated system of the complexes were subjected to 100 ns molecular dynamic simulations (Richard, et al.,2019).
Mmpbsa Analysis
The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method was used to calculate the binding free energies of ACE2-RBD and ACE2-RBD-peptide complex (Eq-1). Here, the last 2 ns frames of the trajectories were obtained to calculate the binding free energy analysis as a method described previously (Richard, et al., 2019).
∆Gbind =Gcomplex– (Gprotein + Gligand)
Where, the ∆Gcomplex is the total free energy of the complex, whereas, ∆Gprotein and ∆Gligand are the total free energies of ACE2-RDB and ACE2-RDB-peptide complex.