Antibacterial activity of lysozyme analysis

Lysozyme antibacterial activity on the Micrococcus Lysodeikticus (ML) bacterial strain was studied using a microplate absorbance reader apparatus (MultiSkan FC from Thermo Fisher Scientific) and a 96 well microfiber sterile plate from Thermo Fisher.

Micrococcus Lysodeikticus (ML) lyophilized cells were purchased from SIGMA-ALDRICH. These tests were carried out according to Toro et al.²² and Lee et al.²³. The assay started adding 20 µL of lysozyme solution in a well with 200 µL of bacteria culture (0.3 mg mL⁻¹). The procedure was repeated twice with the same bacteria culture and the complete assay was performed two more times with different cultures to assess the reproducibility of the measurement. The solutions were stirred for 30 s before measurements and were incubated at 30 °C throughout the measurement.

Turbidity modification (ΔA_450nm) was measured at 450 nm for 10 min with intervals of 15 s and the collected data was plotted as a function of time.

A pre-study was conducted for optimizing the method (investigation and results analysis) to study the protein antibacterial properties. Following the article of Prasad et al.²⁴, the absorbance, the logarithm and the reciprocal of lysozyme absorbance against the bacteria substrate were plotted against time. The linearity for activity-time function was observed only in the plot of 1/Absorbance (1/A) as a function of time (data not shown). This indicated that the reaction between the lysozyme and the Micrococcus Lysodeikticus is a second order reaction.

The reaction rate was estimated from the slope of the 1/A_450nm versus time graph and the activity of the lysozyme samples was calculated using Eq. (²).

where A_u is the activity of the lysozyme, Slope is the slope from 1/A_450nm vs. time graph, [LSZ] is the concentration of lysozyme in the well in mg mL⁻¹, [ML] is the concentration of Micrococcus Lysodeikticus in the well in mg mL⁻¹.

Normalization was done using the reference lysozyme sample (untreated lysozyme) value to give an index of activity Eq. (³).

where I_Au is the index of activity, A_u, A_{u native} are the calculated activities of the treated and the reference lysozyme, respectively.

In the current study, statistical test was performed with two pair t-test using OriginPro 2019 software. A confidence level of 95% was selected to estimate the significance and a difference of statistical significance was defined for P < 0.05.