Determination of Catalase Activity with an Electrochemical Biosensor for Monitoring Microbial Contamination and for Estimating Sanitation Levels in Meat and Seafood Products
Principal Investigator |
Dr. Dong-Hyun Kang |
Completion Date |
January 31, 2006 |
Mission |
Develop a rapid and sensitive testing method for estimation of total microbial loads in milk, fish and meat. |
Problem Addressed
For food safety, the detection of key pathogens and spoilage micro-orangisms is critical. However, current technology for testing is time-consuming adding that much more time between the farmer and consumer. One common method called conventional plating takes up to 48 hours. Some rapid methods have been proposed but often require 8 to 12 hours of growth time. In milk, psychrotrophs like Pseudomonas species are the main spoilage bacteria. Pseudomonas’ high catalase activity has been correlated to microbial counts in fish and meat. Conventional methods for measuring catalase activity require at least 1 hour and are expensive. An alternative method is needed that could produce results in a much shorter time-frame at an affordable price.
Goal
To develop a rapid and sensitive method for estimation of total microbial loads in milk, fish and meat based on the determination of catalase activity.
Implications
With some refinement, the proposed method has the potential of assisting milk processors in rapidly identifying contaminated milk before unloading tankers and therefore identifying farms that need to improve their sanitation procedures. With better control and estimates of the quality of milk delivered to milk processors, it is possible to better estimate milk shelf life. The method could also be used for fish and meat.
Procedures
Catalase catalyzes the oxidation of peroxides. In the presence of catalase, hydrogen peroxide is rapidly oxidized to water and oxygen. During 2002-2003 researchers approached the measurement of microbial catalase activity by electrochemically measuring the consumption of H2O2, fabricating platinum disk miniature electrodes, platinizing the electrode and covering it with a dialysis membrane, and studying the effect of H2O2 concentration in the rate of reaction.
In 2003-2004 researchers found that: 1) Unless O2 was removed from the solutions, an increase in O2 concentration produced the evolution of O2 gas. 2) Measurement of headspace O2 was carried out using a fiber optic oxygen probe. The sensitivity of the probe was not as high as expected and had the disadvantage of ambient light interference. 3) Manometric measurement of the production of O2 was the best approach. 4) Increasing temperature increases catalase activity and increases the sensitivity of the assay. 5) No increase in enzyme activity was observed when researchers attempted to increase the rate of reaction by chemically disrupting the cell membrane of Pseudomonas Aeruginosa suspensions with two different lysis buffers. 6) The proposed manometric method allowed significantly differentiating 104 from 105 CFU using cell suspensions making the method suitable for milk grading. However, the limit of detection remains too high for any practical application for meat and fish.
Techniques and Technologies Developed
From this research we gained a better understanding of microbial catalase activity. Researchers developed a manometric system that has the potential to be used to develop other enzyme assays for enzymes that produce gas. Also, this method has the potential to be used to measure microbial metabolism. For example, we recently correlated the production of CO2 with coliforms in milk. The cost of CO2 probes is 20 times larger than the cost of the pressure transducers we used for this research.
Publications/Journal Articles From Project
Publications in refereed journal articles, as well as paper presentations at professional meetings of economists will be pursued.