Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy

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Abstract
Application of high hydrostatic pressure (200, 300, 350 and 400 MPa) at 5 °C for 30 min to different micro‐organisms, including Gram‐positive and Gram‐negative bacteria, moulds and yeasts, proved to be more effective in inactivating these organisms than treatments at 20 °C for 10 min and at 10 °C for 20 min. Moulds, yeasts, Gram‐negative bacteria and Listeria monocytogenes were most sensitive, and their populations were completely inactivated at pressures between 300 and 350 MPa. The same conditions of pressure, temperature, and time were applied to different vegetables (lettuce, tomato, asparagus, spinach, cauliflower and onion), achieving reductions of from 2–4 log units in both viable mesophiles and moulds and yeasts at pressures of between 300 and 400 MPa. Sensory characteristics were unaltered, especially in asparagus, onion, tomato and cauliflower, though slight browning was observed in cauliflower at 350 MPa. Flow cytometry was applied to certain of the microbial populations used in the above experiment before and after the pressurization treatment. The results were indicative of differing percentage survival rates depending on micro‐organism type, with higher survival rates for Gram‐positive bacteria, except L. monocytogenes, than in the other test micro‐organisms. Growth of survivors was undetectable using the plate count method, suggesting that micro‐organisms suffering from pressure stress were metabolically inactive though alive. The pressurization treatments did not inactivate the peroxidase responsible for browning in vegetables. Confocal microscopic examination of epidermal tissue from onion showed that the enzyme had been displaced to the cell interior. Use of low temperatures and moderately long pressurization times yielded improved inactivation of micro‐organisms and better sensorial characteristics of the vegetables, and should lower industrial costs.