In recent years, consumption of fresh vegetables has greatly increased and consumer's demand for fresh or minimally-processed fresh food (MPF) is on the rise. The risk of contamination by spoilage and pathogenic microorganisms, and growth of these contaminants are of concern because most of these products are consumed without thermal processing. In addition, the recent outbreaks of foodborne diseases caused by Escherichia coli O157:H7, Hepatitis A, and Camphylobacter jejuni raised serious questions about the safety of minimally processed food.
Chlorine has been used to decrease the microbial load during vegetable processing; however, it can react with organic compounds, resulting in the formation of carcinogenic trihalomethane compounds. Therefore, many studies were done to find suitable alternatives to chlorine. For decades, researchers recognized the germicidal action and oxidizing potential of ozone. Ozone applications in the food industry are mostly related to decontamination of product surfaces and water treatment. Ozone has been used with mixed success to inactivate contaminant microflora on meat, poultry, eggs, fish, fruits, vegetables and dry foods. Additional research is needed to elucidate the kinetics and mechanisms of microbial inactivation by ozone and to optimize its use in food applications.
The objectives of this research were: 1) to study the patterns of inactivation of selected spoilage ( Pseudomonas fluorescens , Leuconostoc mesenteroides ) and pathogenic ( Listeria monocytogenes , Escherichia coli O157:H7) bacteria by ozone; 2) to explore mechanisms of microbial inactivation by ozone; and 3) to optimize the efficiency of ozonation procedures in lettuce processing.
To study microbial inactivation patterns, a batch and three continuous reaction systems were tested. In the batch reactor, all tested microorganisms showed a similar pattern of inactivation. Microbial inactivation occurred immediately after addition of ozone with little change in counts thereafter. The population decreased 2.7 to 7.5 log cfu/mL in 30 seconds, when 1 mg/L ozone was used for the treatment. E. coli O157:H7 was the most resistant and L. monocytogenes was the least resistant to ozone inactivation. A dose-response model having two segments appeared adequate in describing inactivation of tested microorganisms.
A continuous reaction system, using a membrane filter, was found suitable to study patterns of microbial inactivation as it minimizes the auto-decomposition of ozone during the reaction and thus a uniform concentration of ozone can be applied throughout the treatment. Inactivation kinetics for all the tested microorganisms revealed concave downward curves. E. coli O157:H7 was the most resistant and L. monocytogenes was the least resistant to ozone inactivation when tested in this continuous reactor. Application of 2.5 mg/L ozone decreased the count by 5 to 6 log 10 cfu/mL in 40 seconds. A log-log dose-response model described inactivation data appropriately.
Interaction of ozone with microorganisms was elucidated by detecting cell injury and examining treated cells with a scanning electron microscope. When ozone was used at 0.55 to 1.85 mg/L, 51 to 78% of survivors were injured. The injured population was greater at intermediate rather than high C*t (mg/L x sec) values. Among the tested microorganisms, P. fluorescens was the most while L. monocytogenes was the least injured by ozone treatments.
Electron microscopic analysis showed that ozone caused damage to the cellular structure; this damage was more pronounced in gram-negative than gram-positive bacteria. When treated with ozone, gram-positive bacteria seemed to lose some mucoid material outside the cell wall, whereas gram-negative cells tended to collapse and lose cellular components. Therefore, apparent structural integrity of the cell can not be correlated with degree of inactivation of microorganisms by ozone.
The feasibility of using ozone to decontaminate a minimally processed product (i.e., shredded lettuce) was explored. Different ozonation media and delivery methods were tested. Ozonation media include ozonated water, bubbled ozone gas into water, and gaseous ozone. Delivery methods were tested to ensure intimate contact between ozone and the treated product. Delivery of ozone was facilitated by stirring, sonication and stomaching. Average decontamination by water washing, ozonated water, bubbling ozone was 0.9, 1.1, and 1.4 log 10 cfu/g, respectively. Bubbling ozone was significantly better than water treatment and also more efficient way of lettuce disinfection than ozonated water treatment. Among the delivery methods, high-speed stir was better for the decontamination of lettuce than other methods. Maximum inactivation of natural microflora (1.9 log cfu/g) was achieved by bubbling ozone with high-speed stir. Therefore, it was concluded that bubbling ozone treatment with high-speed stir will be the most efficient and applicable way for lettuce disinfection.
In conclusion, inactivation studies on bacterial cell suspensions clearly indicate that ozone is a potent antimicrobial agent. Bactericidal action of ozone varies with the microorganism. Ozone at low concentrations damages the outer membrane of gram-negative bacteria and thus cause dramatic changes in the cell structure. Similar concentrations of ozone cause less damage to the cell wall of gram-positive bacteria, but the agent causes intercellular damage and effectively inactivates the cell. Ozone is a less potent antimicrobial agent against microorganisms in food than in pure cell suspensions. Successful application of ozone in food processing depends on developing methods to ensure good contact between this agent and target microorganisms on the treated food.