Kinetics and mechanism of bacterial inactivation by ultrasound waves and sonoprotective effect of milk components

Open Access
- Author:
- Motwani, Neetu
- Graduate Program:
- Food Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- February 26, 2008
- Committee Members:
- Stephanie Doores, Thesis Advisor/Co-Advisor
- Keywords:
- L. monocytogenes
Ultrasound waves
Milk
E. coli
Sonoprotection - Abstract:
- The need for alternative food processing technologies that can be used to pasteurize foods while maintaining their fresh-like qualities is increasing due to increasing consumer demand for minimally processed foods. Ultrasound technology is one of the emerging alternate food processing technologies and uses sound waves with frequencies higher than 20 kHz to inactivate microorganisms. Microbial inactivation by ultrasound waves (USW) is attributed to cavitation which involves formation, growth and collapse of bubbles in a liquid medium. Although bacterial inactivation by USW has been studied extensively in buffers, the potential of USW to inactivate bacteria in real foods like milk still needs to be investigated. The first objective of this study was to determine the impact of sonication medium and growth phase on inactivation kinetics of E. coli and L. monocytogenes. Preliminary studies to select sonication frequency were conducted by comparing the inactivation of mid-log phase cells of E. coli upon treatment with high frequency (650, 765 and 850 kHz) and low frequency (24 kHz) USW in phosphate buffer. Ultrasound waves with high frequency were ineffective in inactivating E. coli, while low frequency ultrasound waves decreased the number of survivors by ~ 3 logs in 2.5 minutes. Therefore, 24 kHz ultrasound waves were used for this study. Mid-log or mid-stationary phase cells of E. coli and L. monocytogenes were treated with 24 kHz ultrasound waves in phosphate buffer, whole milk or skim milk. Log phase cells of E. coli and L. monocytogenes were more sensitive to ultrasound treatment than stationary phase cells in all three sonication media. Escherichia coli exhibited non-log linear inactivation kinetics with tailing while L. monocytogenes exhibited log linear inactivation kinetics throughout. Ultrasound treatment exhibited minimal injury to bacteria suggesting that inactivation of bacteria by USW was following all or nothing phenomenon. The D values for inactivation of mid stationary-phase cells of E. coli were 2.19, 2.43 and 2.41 min in phosphate buffer, whole milk and skim milk, whereas those for L. monocytogenes were 7.63, 9.31 and 8.61 min, respectively. Significantly higher (p < 0.05) D values for inactivation of E. coli and L. monocytogenes in whole and skim milk as compared to phosphate buffer suggested that milk exerts a sonoprotective effect on these bacteria. However, the D values for both the organisms were not significantly different between whole and skim milk indicating that fat in milk does not exert a sonoprotective effect on these bacteria. In the second part of the study the sonoprotective effect of milk components, lactose, casein and β lactoglobulin, on E. coli and L. monocytogenes was investigated. Lactose, casein, or β lactoglobulin was added to simulated milk ultrafiltrate (SMUF) at a concentration of 5, 3 and 0.3g/100 ml, respectively. Casein was added to SMUF as a non-micellar, sodium caseinate or a micellar, phosphocasein, to investigate the differences in protection conferred due to its physical form. In another experiment, all three components were added to SMUF to determine the combined protective effect of these components on bacteria. Presence of casein in micellar and non-micellar forms did not result in a significant change in the D values compared to SMUF for inactivation of both organisms. Addition of whey protein β lactoglobulin to SMUF also did not result in a significant change in D value for E. coli while that for L. monocytogenes increased significantly (p < 0.05). Presence of lactose in SMUF, however, resulted in significant increase (p < 0.05) in D values for inactivation of both the organisms. The D values for E. coli in SMUF and SMUF + lactose were 2.84 and 3.42 min; while those for L. monocytogenes were 7.3 and 8.52 min, respectively, suggesting that lactose was conferring a protective effect on these bacteria. Moreover, D values obtained in SMUF + lactose were not significantly different from those in skim milk for either organism, suggesting that amongst the components tested lactose alone was conferring a significant protective effect to bacteria. There was no significant increase in D values when E. coli and L. monocytogenes were sonicated in SMUF containing all the three components suggesting that milk components do not exert any additive or synergistic protective effect against ultrasound treatment. Scanning electron microscopy of ultrasound-treated cells of E. coli and L. monocytogenes was conducted to see the morphological changes in cell structure upon sonication. The SEM images demonstrated that ultrasound treatment resulted in physical damage to cell wall and cell membrane of E. coli and L. monocytogenes cells, leading to their death.