Inactivation of Problematic Microorganisms 22221 Spores and Vegetative Bacteria

Most fungal conidiospores and ascospores can usually be inactivated at pressures between 300 and 450 MPa at ambient temperature, but exceptions exist. For example, in a study on dormant Talaromyces macrosporus ascospores, mild treatment (200 to 500 MPa, 20°C) activated dormant ascospores but caused little or no inactivation. Higher pressures (500 to 700 MPa, 20° C) were required to inactivate the ascospores; however, a treatment of 700 MPa after 60 minutes only reduced the spore population by less than 2log10 units, indicating the resistance of the ascospores to high pressure [7].

Hayashi [8] found that pressures of 200 MPa effectively killed yeasts and molds in freshly squeezed orange juice at ambient temperature. Ogawa et al. [9] stated that after HPP (400 MPa and 23°C) both freshly squeezed orange juice and orange juice that had been inoculated with yeasts and molds showed no increase in total counts after 17 months of storage at 4°C. Aleman et al. [10] reported treatment at 340 MPa for 15 minutes extended the shelf life of fresh-cut pineapple. Populations of Gram-negative bacteria, yeasts, and molds could be reduced by at least 1 log10 at pressures of 300 and 350 MPa for inoculated lettuce and tomatoes, even though the tomato skins loosened and peeled away and lettuce browned in this range of pressures [11]. Raso et al. [12] used pressure to inactivate ascospores and vegetative cells of Zygosac-charomyces bailii suspended in apple, orange, pineapple, cranberry, and grape juices. HPP at 300 MPa for 5 minutes reduced the population of vegetative cells and ascospores by almost 5log10 units and 0.5 to 1 log10 units, respectively.

Parish [13] applied pressures between 500 and 350 MPa to nonpasteurized Hamlin orange juice (pH 3.7) inoculated with Saccharomyces cerevisiae and found that the D values for ascospores and vegetative cells inoculated into the pasteurized orange juice were from 4 to 76 sec and from 1 to 38 sec, respectively. The native microbiota in the orange juice had D values from 3 to 74 sec in the pressure range of 500 and 350 MPa. The corresponding z values were 123, 106 and 103 MPa for ascospores, vegetative cells, and native microbiota, respectively.

Zook et al. [14] used fruit juices and a model juice buffer (pH 3.5 to 5.0) as suspension media to determine pressure inactivation kinetics of S. cerevisiae ascospores. Approximately 0.5 x 106 to 1.0 x 106ascospores/ml were pressurized at 300 to 500 MPa in juice or buffer. D values were 8 sec to 10.8 min at 500 and 300 MPa, respectively; the corresponding z values were 115 and 121 MPa. No differences (P > 0.05) in D values (at constant pressure) or z values among buffers or juices at any pH were determined, suggesting little influence of pH in this range.

Bacterial endospores are the most difficult life forms to eliminate with hydrostatic pressure; spores of bacillus have been exposed to >1,724 MPa (250,000 psi) and remained viable [15]. Applications of pressure alone will not inactivate bacterial endospores. Hurdle technology that utilizes pressure in combination with other process technologies (including pressure pulsing) are proposed to improve spore inactivation rates. Mild elevated heat (e.g., 40 to 55°C) with pressure treatment is required for substantial reduction of spore loads [16,17]. Sterilization requires higher temperatures resulting in a definite cooked appearance of the food.

Bacterial spores have been shown to demonstrate variable pressure resistances with respect to sporulation conditions. The anhydrous structure and dimensions of the spore are believed to contribute to the pressure resistance of bacterial spores, causing a major challenge to produce shelf-stable low-acid food products [18]. Spores can germinate at different combinations of temperature and pressure [19]. The initiation of spore germination results in loss of resistance. Two-exposure treatments (i.e., twin pressure pulse) have been proposed to enhance the inactivation of spores by HPP [20]. The concept is that the first exposure at low pressure results in spore germination, and the second exposure at a higher pressure inactivates the germinated spores and vegetative cells. Unfortunately, it appears that not all spores are germinated by pressure and not all germinated spores appear to be inactivated by pressure [21].

Oh and Moon [22] investigated the effect of pH on the initiation of spore germination and inactivation of Bacillus cereus KCTC 1012 spores using pressures to 600 MPa. The pH of the sporulation medium affected inactivation of B. cereus spores under pressure more than the suspension medium pH. B. cereus spores obtained through sporulation at pH 6.0 showed greater resistance to pressure than those sporulated at pH 7.0 and 8.0 at 20, 40, and 60°C.

To date, successful commercial preservation of foods utilizing HPP depends upon the use of post-treatment refrigeration or a product pH below 4.5 to block the germination of spores of C. botulinum and other sporeforming bacteria. Production of commercially sterile low-acid foods such as meat, milk, and vegetables must overcome the extreme pressure resistance of spores.

Similar as found in fungi, vegetative forms of bacteria are normally more easily inactivated by pressure than spores. Linton et al. [23] investigated the inactivation of a pressure-resistant strain of Escherichia coli O157:H7 (NCTC 12079) in orange juice over the pH range 3.4 to 5.0. The sterile orange juices were adjusted to various pH levels (3.4, 3.6, 3.9, 4.5, and 5.0) and inoculated with E. coli O157:H7 at 108 CFU/ml. A 6 log10 inactivation was obtained after 5 minutes at 550 MPa and 20°C at every pH evaluated except pH 5.0 (~5.5log10); this pressure combined with mild heat (30°C) resulted in a 6 log10 inactivation at pH 5.0.

There were considerable variations in bacterial pressure resistance in different types of fruit juices. Teo et al. [24] reported HPP treatment at low temperature (15°C) had the potential to inactivate E. coli O157:H7 strains. A three-strain cocktail of E. coli O157:H7 (SEA13B88, ATCC 43895, and 932) was found to be most sensitive to pressure in grapefruit juice (8.3 log10 reduction) and least sensitive in apple juice (0.4log10 reduction) when pressurized at 615 MPa for 2 minutes at 15° C. The resistance difference might come from the various pH values and the presence of natural antimicrobials in different fruit juices.

Wuytack et al. [25] applied pressures of 250, 300, 350, and 400 MPa to reduce the microbial loads of garden cress, sesame, radish, and mustard seeds that were immersed in water and treated at 20°C for 15 minutes. The percentages of seeds germinating on water agar were recorded to 11 days after pressure treatment. Radish and garden cress seeds were the most pressure-sensitive and pressure-resistant types, respectively. For example, after a 250 MPa treatment, radish seeds displayed 100% germination nine days later than untreated controls, while garden cress seeds attained 100% germination one day after the controls. Garden cress seeds were inoculated with suspensions of seven different kinds of bacteria (starting inocula 107CFU/g). Treatment at 300 MPa for 15 minutes and 20°C resulted in 6log10 reductions of Salmonella Typhimurium, E. coli MG1655, and Listeria innocua, >4log10 reductions of

Shigella flexneri and the pressure-resistant stain E. coli LMM1010, and a 2log10 reduction of Staphylococcus aureus; however, Enterococcus faecalis was not inactivated.

Ramaswamy et al. [26] applied 150 to 400 MPa to apple juices inoculated with E. coli 29055 at 25°C for 0 to 80 minutes. The surviving cells with and without injury were differentiated through the use of brain-heart infusion agar (BHIA) and violet-red bile agar (VRBA). It was found that D values of E. coli decreased with an increase in pressure, and pressure D values from BHIA (survivors including injured cells) were higher than from VRBA (survivors excluding injured cells), indicating that a greater number of cells were initially injured than killed with HPP treatment. The associated z values (pressure range to result in a decimal change in D values) were 126 and 140 MPa on BHIA and VRBA, respectively.

22.2.2.2 Viruses

The first examination of the pressure sensitivity of viruses was by Giddings et al. [27] who found that a 920 MPa exposure was required to inactivate tobacco mosaic virus (TMV). Since that early work, it now appears that most human viruses are substantially more pressure-sensitive than TMV. Human immunodeficiency viruses (HIV) can be reduced by 104 to 105 viable particles after exposure to 400 to 600 MPa for 10 minutes [28], but some viruses can be inactivated at even lower levels of pressures. For example, Brauch et al. [29] reported that pressures of 300 to 400 MPa significantly killed bacteriophage (0x, X and T4), and Shigehisa et al. [30] found that an 8 log10 plaque-forming unit (PFU) population of herpes simplex virus type 1 could be eliminated by a 10-minute exposure to 400 MPa, and a 5 log10 PFU population of human cytomegalovirus was inactivated by a 10-minute exposure to 300 MPa. Shigehisa et al. [31] later reported that a 5.5log10 tissue culture infectious dose of HIV type 1 was eliminated after a 10-minute exposure to 400 MPa at 25°C.

According to Kingsley et al. [32], a 7log10 PFU/ml hepatitis A virus (HAV) stock in tissue culture medium was reduced to nondetectable levels after exposure to >450 MPa for 5 minutes. Titers of HAV were reduced in a time- and pressure-dependent manner between 300 and 450 MPa, but poliovirus titer was unaffected by a 5-minute treatment at 600 MPa. Salts had a protective effect on viruses because dilution with seawater increased the pressure resistance of HAV. Experiments involving RNase protection indicated that viral capsids might remain intact during pressure treatment, suggesting that inactivation was due to subtle alterations of viral capsid proteins. A 7log10 tissue culture infectious dose of feline calicivirus, a Norwalk virus surrogate, was completely inactivated by exposure to 275 MPa or above after 5 minutes, indicating that HAV and feline calicivirus could be inactivated by pressure.

Currently, there are few publications available addressing pressure inactivation of viruses in fruit and vegetables products. It can be anticipated that investigations will more closely evaluate inactivation of viruses by HPP

given the recent food safety issues concerning fecally contaminated fresh produce.

22.2.2.3 Parasites

Human feces are not just a source of human viruses, but also a source of human parasites. Raw fruits and vegetables can become fecally contaminated with parasites that include the protozoans Giardia intestinalis, Cryptosporidium parvum, Cyclospora cayetanensis, and the helminth parasites Fasciola hepatica, Ascaris lumbricoides, and Ascaris suum [33]; however, few articles are available regarding pressure inactivation of parasites in or on fresh fruits and vegetables. Slifko et al. [34] applied 550 MPa to apple and orange juices in which Cryptosporidium parvum oocysts were suspended. After a 30-second exposure, C. parvum oocysts were inactivated by at least 3.4log10, and an exposure to 550 MPa for more than 60 seconds efficiently rendered the oocysts nonviable and noninfectious.

Recently, HPP was used to inactivate parasites from muscle tissues and fish. A pressure of 200 MPa for 10 minutes inactivated all anisakis larvae isolated from fish tissues either in distilled water or in a physiological isotonic solution between 0 and 15° C; when exposed to 140 MPa for 1 hour, all larvae were killed [35]. Dong et al. [36] pressure-inactivated Anisakis simplex larvae inoculated in king salmon and arrowtooth flounder. Complete kill of the larvae (ranging from 13 to 118) contained in fish fillets was obtained by treatments of 414 MPa for 0.5 to 1 minute, 276 MPa for 1.5 to 3 minutes, and 207 MPa for 3 minutes; however, it was stated that the application of HPP to raw fish was limited because of the significant whitening of the flesh of HPP-treated fish fillets (P < 0.05).

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