Fig 1.
A) enzymatic formation of allicin from alliin; B) the coupled reaction of pyruvic acid, a co-product of reaction alliin with alliinase, with NADH and LDH used for UV-VIS spectroscopic assay.
Table 1.
Conditions for testing of enzyme stability at 25°C and 37°C.
Fig 2.
A) 10% SDS-PAGE of isolated garlic alliinase; B) kinetic analysis of alliinase catalyzed reaction.
The reaction mixture (0.1 mL) contained alliinase (5 μg/mL) and various concentrations of alliin (0 to 50 mM), the reaction temperature was 25°C, Tricine-KOH (pH 8) buffer was used for the assays.
Fig 3.
Effect of pH (A) and type of used buffer (B) on the alliinase activity at 25°C.
Fig 4.
A) Time-dependent enzyme stability and activity of alliinase in Na-PB, PBS and Tricine-KOH buffer. All results are normalized to alliinase activity measured at 0 h in Tricine-KOH buffer; B) effect of temperature on alliinase activity in Tricine-KOH buffer.
Table 2.
Effect of time and storage temperature on alliinase activity in Tricine-KOH buffer.
Fig 5.
Effect of salts on the initial activity of alliinase (A) and 3 h (B) incubation at 25°C.
All results are normalized to alliinase activity measured at 0 h (25°C, Tricine-KOH buffer, no additives). Means followed by the same letter (a–f) were not significantly different (P>0.01, ANOVA, Tukey’s test).
Fig 6.
A) The effect of additives on enzyme stability at 25°C; B) comparison of the individual and combined effect of 4 mM ascorbic acid (AA) and 50 mM NaCl on enzyme stability.
All results are normalized to alliinase activity measured at 0 h (25°C, Tricine-KOH buffer) without additives; * results did not differ statistically from the reference at given time (P>0.01, ANOVA, Dunnett’s test).
Fig 7.
A) Effect of storage conditions on the activity of lyophilized alliinase; B) calculated half-life of alliinase vs relative humidity and storage temperature.
The initial activity of the lyophilized powder stored at -20°C was taken as a reference value.
Fig 8.
A) Example of disk diffusion susceptibility testing of E. coli, P. putida and S. epidermidis (numbers correspond to alliinase concentration in mg/mL), ATB—kanamycin, E—enzyme, S—substrate; B) influence of alliinase concentration (0.001 to 10 mg/mL) in combination with 100 mM alliin on the diameter of inhibition zones.
Error bars represent standard deviation based on three independent experiments.
Fig 9.
Effect of alliinase (A) and alliin (B) concentration on the inhibition of E. coli.
The inhibition zone for kanamycin (50 mg/mL, 20 μL) served as a reference value in both cases; x-axis in logarithmic scale; error bars represent standard deviation based on three independent experiments.
Fig 10.
Cell viability assay of bacterial suspension (300 μL) incubated for 0 to 30 min with a mixture of alliin (100 μL; 100 mM) and alliinase (100 μL; 1 mg/mL).
Ten fields or more were analyzed in triplicates; the scale bar corresponds to 50 μm; the total number of bacteria at a given time was taken as 100%.
Fig 11.
Bactericidal effect of the allicin evaluated by the plate count method.
Left—agar plates with bacterial colonies after plating of the sample; Right—quantification of plate count method. The total count of emerging colonies from the untreated sample was taken as 100%.
Fig 12.
Cell morphology of E. coli, P. putida and S. epidermidis before (no allicin—The left column), right after (time 0 min—Middle column) and after 30 min upon allicin addition (the right column); Arrows show the first appearance of morphological changes.