Masters Theses
Date of Award
8-2003
Degree Type
Thesis
Degree Name
Master of Science
Major
Food Science and Technology
Major Professor
Jochen Weiss
Committee Members
P. Michael Davidson, Barry D. Bruce
Abstract
The objectives of these studies were to determine the influence of chelator combinations on the effectiveness of the food antimicrobial lysozyme against the foodborne pathogenic bacterium, Escherichia coli O157:H7. By itself, lysozyme has little or no activity against Gram-negative bacteria, however, in the presence of chelating agents, the antimicrobial spectrum of lysozyme can be increased to include Gramnegative bacteria such as E. coli O157:H7. Experiments were three-fold. The first series of experiments examined the effects of lysozyme and three chelating agents (ethylenediamine tetraacetic acid (EDTA), disodium pyrophosphate (DSPP), and pentasodium tripolyphosphate (PSTPP)) on growth inhibition of E. coli O157:H7. The second study determined how pH, cation size and strain of E. coli O157:H7 influenced neutralization of lysozyme-chelator combinations. The third experiment used zeta potential measurements to observe the effects of lysozyme and EDTA on surface charge properties of E. coli O157:H7 strain 932. In the first study, the inhibitory effect of 300-1500 μg/mL EDTA and 3,000- 15,000 μg/mL DSPP and PSTPP in combination with 200-600 μg/mL lysozyme was studied at pH 6, 7, and 8 on four strains of Escherichia coli O157:H7 for 48 hours, using a microplate dilution assay. Addition of EDTA enhanced the inhibitory effect of lysozyme against strains of E. coli O157:H7. EDTA concentrations ≥ 300 μg/mL combined with 200 μg/mL lsyzoyme were sufficient to inhibit the strains at pH 6 and 8. At pH 7, 200 μg/mL lysozyme and EDTA concentrations ≥ 1,000 μg /mL were effective in inhibiting three of four strains. DSPP at pH 6, was effective at ≥ 10,000 μg/mL when v combined with 200 μg/mL lysozyme. In contrast, PSTPP increased the inhibitory activity of lysozyme more effectively at pH 8. 200 μg/mL lysozyme was effective against two strains of E. coli O157:H7 when used in conjunction with ≥ 5,000 μg/mL PSTPP. Remaining strains were inhibited by ≥ 10,000 μg/mL PSTPP. Results indicated that inhibition occurred with each lysozyme-chelator combination, however, greater concentrations of polyphosphates were required, compared to EDTA, to increase the antimicrobial spectrum of lysozyme against E. coli O157:H7. The second study analyzed the influence of seven mineral salts on neutralization of the inhibitory properties of lysozyme-chelator combinations. Lysozyme-chelators used were 1500 μg/mL EDTA and 15,000 μg/mL DSPP or PSTPP in the presence of 600 μg/mL lysozyme. Mineral salts were added to lysozyme-chelator combinations at 1.0, 4.0, 7.5, and 10 mM at pH 6.0, 7.0, 8.0 on E. coli O157:H7 strains 932 and H1730. For strain, 932 Ca++ and Mg++ neutralized inhibition by lysozyme and EDTA except for EDTA at pH 6.0. Inhibition of strain H1730 was eliminated by Ca++ and Mg++ except for EDTA at pH 6.0 and Mg++ with DSPP at pH 6.0. Concentrations ≥ 4.0 mM Fe++ neutralized all lysozyme-chelator combinations. Addition of Na+ and K+ to lysozymechelator combinations produced minimal to no reverse inhibition of strain 932. However, Na+ and K+ concentrations were effective in reversing lysozyme-chelator inhibition for strain H1730. The addition of ≥ 1.0 mM Fe+++ or Al+++ was effective in overcoming inhibition of lysozyme and EDTA at pH 6.0, 7.0, and 8.0. It was concluded that calcium and magnesium are important in the reversal of lysozyme-chelator inhibition, whereas the reversal by sodium and potassium were variable and strain dependent. Iron was vi additionally effective in neutralizing lysozyme-chelator inhibition, probably because of its wide range of functions and binding constants to chelators. In the third, zeta potential values were obtained for pure lysozyme and lysozyme- EDTA combinations. EDTA and lysozyme-EDTA combinations were additionally examined to determine the effects of the antimicrobial-chelator combinations on E. coli O157:H7 surface charge. Over a pH range of 5-13, zeta potential measurements for lysozyme ranged from 61.26 to – 42.14 mV, respectively. The presence of EDTA reduced lysozyme zeta potential to 38.13 at pH 5.0 to – 33.45 mV at pH 13.0. Native E. coli O157:H7 possessed a negative charge over a pH range of 5-13. Values ranged from – 11.67 at pH 5.0 to – 36.95 mV at pH 13.0. EDTA and lysozyme-EDTA combinations lowered the zeta potential of E. coli O157:H7. Lysozyme and EDTA together significantly reduced the zeta potential of E. coli O157:H7 strain 932, from – 3.76 at pH 5.0 to – 32.85 mV at pH 13.0. Treatments with EDTA alone further reduced zeta potential values to – 1.28 to – 27.47 mV for pH 5 and 13, respectively. It was concluded that the presence of lysozyme-EDTA combinations and EDTA alone significantly lowered the zeta potential values of E. coli O157:H7 strain 932. The addition of chelators successfully enhanced the antimicrobial spectrum of lysozyme to inhibit E. coli O157:H7. It was found that certain metal ions are responsible for stabilizing the LPS layer and protecting the bacterial cell from the inhibitory activity of lysozyme. Additionally, environmental factors such as pH, cation size/charge, and E. coli O157:H7 strain type influence the chelation of these ions. Furthermore, it was concluded that the synergistic properties of lysozyme and EDTA reduce bacterial surface charge, allowing lysozyme and EDTA to inhibit E. coli O157:H7.
Recommended Citation
Boland, Jeffrey Smith, "Influence of Chelators on the Antimicrobial Activity of Lysozyme Against Escherichia coli O157:H7.. " Master's Thesis, University of Tennessee, 2003.
https://trace.tennessee.edu/utk_gradthes/1905