Effects of the clumping of bacterial cells on their heat resistance theoretical research

1. Effects of the clumping of bacterial cells on their heat resistance theoretical research Petra Klodnerová Supervisor: Dr. Ing. Miroslav Čeřovský

2. Problems of cells clumping • bacteria and their spores present insufficient numbers may clump before or during heating • heat resistance of bacterial clump increases • thermal characteristics (D- and z-value) of single cells are not valid in presence of their clumps → heating by sterilization or pasteurization is not sufficient

3. Death curves by clumping of cells • deviation of the curve from a straight line = tailing death curves

4. most of known bacterial cell surfaces are negatively charged under the physiological conditions physico-chemical point of view Lifshitz–Van der Waals interactions acid–base interactions electrostatic interactions hydrophobic interactions disulfide bonds (S-S) sulfur groups bridged by divalent ions (S-Ca-S, S-Mg-S) biochemical point of view interactions between protease-sensitive molecules on the cell surfaces, called lectins, that bind to carbohydrate receptors on a partner cell Forming the clumps

5. Cause of higher heat resistance of microorganisms clumps (1) • Time to reach equilibrium temperature at the center of the clump? • the clump will, in theory, never reach 100 % of the temperature of the heating fluid Tab. I: Longest time to reach equilibrium temperature in clumps of bacteria heated in dry air at 130 °C. n …number of cells in the clump R … radius of clump t0.99 … time elapsed for the center bacterium to attain 99 % of the temperature of the heating fluid

6. Cause of higher heat resistance of microorganisms clumps (2) • Size and number of cells inside the clumps? • the number nof single spores constituting the clump, d (microns) in diameter n= 0.74 x d3 • e.g. clumps of spores Bacillus: • space clump < 5 μm contains 90 spores • it was indicated that the spore clumps containing 102 spores were five times more heat resistance than a single spore (viz. table)

7. Cause of higher heat resistance of microorganisms clumps (3) • It´s not possible to explain the reason of higher thermal resistance of bacterial clumps with theory of biofilm: • physiology of cells changes by the adhesion on the solid surface • other mechanisms of heat resistance of cells in biofilm

8. Cause of higher heat resistance of microorganisms clumps (4) • Stress alarmones and heat-shock proteins of the cells: • the killed cells may still contain agents, forming as result of heating, which confer stress tolerance on living organisms in the middle of the clump • killed cells can induce other stress responses in living organisms • cellular content leaking from the killed cells may serve as source of nutrient for revitalization of heat injured cells

9. Conclusions • Cells within the clumps are bound with a very large number of strong bonds. • The heat-up time of clumps in vapor or liquid is not significant with usual sterilization time. • Killed cells on the surface of clumps are isolating layer and protect cells in the middle of clumps for some time from the sterilization temperature. • Killed cells contain stress agents which confer stress tolerance on living organisms. • Cellular content leaking from the killed cells may serve as source of nutrient for revitalization of heat injured cells.