Resistance of micro‐organisms to high pressure is variable and directly related to extrinsic and intrinsic factors. Pressures of 100, 200, 300, 350 and 400 MPa were applied at 20°C for 10 min and at 10°C for 20 min using strains of Gram‐positive and Gram‐negative bacteria, moulds and yeasts, as well as spores of Gram‐positive bacteria. The results showed that at pressures of 100 and 200 MPa, decreases in microbial populations were not significant, whereas the populations of all the micro‐organisms tested decreased considerably at a pressure of 300 MPa. A pressure of 300 MPa at 10°C for 20 min was required to completely reduce the population of Saccharomyces cerevisiae, and a pressure of 350 MPa was needed to reduce most of the Gram‐negative bacteria and moulds. The Gram‐positive bacteria were more resistant, and pressures of 400 MPa were unable to completely reduce their populations. The different pressures employed had little effect on the initial numbers of spores. The initial populations of viable aerobic mesophiles and moulds and yeasts in vegetables (lettuce and tomatoes) decreased 1 log unit at pressures of 300 MPa and above under both sets of experimental treatment conditions. However, treatment at that pressure also resulted in alterations in the organoleptic properties of the samples. In the tomatoes, the skin loosened and peeled away, though the flesh remained firm, and colour and flavour were unchanged. The lettuce remained firm but underwent browning; flavour was unaffected. In vegetables use of moderate pressures in combination with other treatment conditions would appear to be required to reduce the populations of contaminating micro‐organisms while avoiding the undesirable alterations in organoleptic properties that take place at 300 MPa.

Cryo-fracture scanning electron microscopy revealed the changes that cauliflower and spinach leaves undergo after treatment with high hydrostatic pressure. High pressure changed cell permeability and enabled movement of water and metabolites from inside to outside of the cell. The ultrastructure showed that parenchyma organization disappeared in spinach leaf and cavity formation occurred after treatment. However, cauliflower exhibited a firm structure with a soaked appearance and would be more suitable than spinach for high hydrostatic pressure treatment.

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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.

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The effect of high-pressure (HP) treatment on the hydrolysis of dairy whey proteins by trypsin, chymotrypsin and pepsin was analysed. Isostatic pressure (100-300 MPa for 15 min at 37 1C) was applied to the protein substrate prior to its enzymatic hydrolysis. Digestion was also conducted at atmospheric pressure (0.1 MPa) and under high pressure. The extent of hydrolysis was measured by the o-phthaldialdehyde method, the peptide profile was analysed by reverse-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the residual immunochemical reactivity was assessed by an ELISA test using a pool of seven sera from children allergic to bovine milk, an individual serum also positive (positive control) and two sera from non-allergic children (negative controls). The high pressure increased the degree of hydrolysis by the three enzymes used. Chymotrypsin and trypsin showed the highest proteolysis at 100 and 200 MPa followed by pepsin at 300 MPa. The b-lactoglobulin was hydrolysed by trypsin and chymotrypsin at atmospheric and at high pressures, whereas the pepsin only hydrolysed this protein under high pressure. Pepsin and trypsin hydrolysed a-lactalbumin in all cases. In contrast, this protein was not digested by chymotrypsin, irrespective of the pressure applied. An important decrease of immunochemical reactivity was found for pepsin and trypsin hydrolysates obtained under high pressure. The pool of seven sera detected immunoreactivity in the products of chymotrypsin hydrolysis under high pressure, which was not detected when the serum of one patient was used. The results suggest that dairy whey hydrolysates obtained by pepsin and trypsin in combination with HP treatment could be used as a source of peptides in hypo-allergenic infant formulae. r

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