Present address: Ethan B. Solomon, USDA-ARS-ERRC, 600 E, Mermaid Lane, Wyndmoor, PA 19038-8598, USA.
Dallas G. Hoover, Department of Animal and Food Sciences, University of Delaware, Newark, DE 19716-2150, USA (e-mail: firstname.lastname@example.org).
Aims: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems.
Methods and Results: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25°C suspended in Bolton broth, phosphate buffer (0·2 m, pH 7·3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken pureé. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300–325 MPa for 10 min at 25°C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50–75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature.
Significance and Impact of the Study: These data demonstrated that HPP at levels of ≤400 MPa, can inactivate C. jejuni in both model and food systems.
Campylobacter jejuni has emerged over the past 10 years to become a leading cause of food-borne gastroenteritis in the US and perhaps the world (Solomon and Hoover 1999). Campylobacter comprised ca one-third of all laboratory diagnosed cases reported to FoodNet in 2002 (Anon. 2003). Campylobacter jejuni is zoonotic with many animals serving as reservoirs for human disease (Saeed et al. 1993). The most important vehicles of transmission of C. jejuni to human beings are undercooked or mishandled poultry products (Deming et al. 1987), raw milk and contaminated water (Blaser et al. 1979). The Centers for Disease Control estimates the overall infection rate of Campylobacter at 1000 cases per 100 000 population, accounting for over 2 million cases per year in the US (Nachamkin 2001).
The past decade has also seen a great increase in consumer demand for more fresh and minimally processed food products. The food industry has responded with development of alternative processing methods to produce higher quality foods more economically. High hydrostatic pressure processing (HPP) is one of the alternative methods that has emerged commercially. It offers benefit over traditional thermally processed foods in that micro-organisms and detrimental enzymes can be inactivated by HPP without significant changes in the taste, texture, colour or nutritional value of the food (Shearer et al. 2000). Most of the common bacterial food-borne pathogens, such as Salmonella, Vibrio spp., Escherichia coli O157:H7, Yersinia enterocolitica and Listeria monocytogenes, have been investigated in a variety of food systems and buffers, at various temperatures and for various pressure durations (Metrick et al. 1989; Styles et al. 1991; Patterson et al. 1995; Stewart et al. 1997; Ellenberg and Hoover 1999). Generally, the vegetative Gram-positive organisms are more recalcitrant to pressure inactivation than Gram-negative bacteria; however, HPP is effective at reducing or eliminating most vegetative forms of bacteria at pressures in the range of 300–800 MPa.
Poultry products and raw milk are the two most common vehicles of transmission of C. jejuni to human beings. Both of these products possess a relatively short shelf life and a high consumer demand for freshness, such that application of HPP may be beneficial. To date, there has been only one study reporting the effects of HPP on C. jejuni (Shigehisa et al. 1991); however, the pressure resistance of C. jejuni in poultry or milk products was not determined. The objective of this study was to investigate the response of C. jejuni to HPP when present in several model foods.
Materials and methods
Strains and culture preparation
Campylobacter jejuni ATCC 35919 and 35921 were obtained from R. Thunberg (US Food and Drug Administration, Washington, DC, USA) and stored in 75% Bolton broth (product no. CM 0983; Oxoid, Basingstoke, UK) and 25% glycerol at −80°C. Working cultures were prepared by streaking a loopful of frozen stock onto plates of Karmali agar (product no. CM 0935; Oxoid) supplemented with ferrous sulphate, sodium metabisulphite and sodium pyruvate, all at 0·25 g l−1 (FBP) (Sigma, St Louis, MO, USA), and incubating 48 h at 42°C under microaerophilic conditions (5% O2, 10% CO2, 85% N2) generated using a CampyPak envelope (BBL, Sparks, MD, USA) within an anaerobic jar. After three successive transfers, cultures were used. Working cultures were maintained throughout the study by restreaking fresh plates of Karmali agar every 2–3 days. Broth cultures were prepared 2 days prior to use by inoculating 50 ml of Bolton broth supplemented with FBP as well as Vitox supplement (Oxoid) and 0·5 ml OxyraseTM (Oxyrase Inc., Mansfield, OH, USA). The tubes were then incubated at 42°C for 48 h.
Pressure experiments in microbiological media
Five tubes per experiment were centrifuged at 400 g and resuspended in 10 ml of fresh Bolton broth supplemented as above. The tubes were then combined to give a 50-ml volume with a plate count of ca 108 CFU ml−1. Ten millilitres of this inoculum was placed into sterile polyester pouches (3M, Minneapolis, MN, USA) and sealed. The pouches were then placed into the pressure unit (Autoclave Engineers Isostatic Press; Autoclave Engineers, Erie, PA, USA) and pressurized at 25°C for 10 min at pressures ranging from 50 to 400 MPa. This pressure unit used water as its pressurization medium. The adiabatic heat of water is ca 3°C for every 100-MPa increase in pressure; therefore, a 400-MPa treatment would raise the temperature inside the pressure chamber ca 12°C above ambient. One inoculated and sealed pouch was not pressurized and served as the control. Pouches were removed from the pressure vessel, dipped in 70% ethanol, and aseptically opened using sterile scissors. Survivors were enumerated by dilution and spread plating onto Karmali agar. The pressure resistance of C. jejuni in phosphate buffer (0·2 m, pH 7·3) was determined in an analogous manner. Experiments were conducted in duplicate and repeated twice.
Pressure experiments in food systems
Cultures of C. jejuni 35919 and 35921 were prepared as above, and then resuspended in 50 ml of ultra-high temperature (UHT) whole milk (Parmalat, Wallington, NJ, USA), UHT skim milk, soya milk or chicken pureé (Gerber, Fremont, MI, USA). Ten-millilitre aliquots of each food product were sealed in polyester pouches and pressurized between 100 and 400 MPa for 10 min at 25°C. Pouches were removed, aseptically opened and cells were enumerated as described above. Food system experiments were conducted in duplicate and repeated twice.
Inoculated pack studies with UHT whole milk
Cultures were prepared as above and resuspended in 50 ml of UHT whole milk. Ten-millilitre aliquots were placed into pouches and pressurized at either 325 or 350 MPa for 10 min at 25°C, then held at 4, 25 or 42°C for 7 days. Samples were enumerated prior to pressurization, immediately after pressurization, and on days 1, 3, 5 and 7 (as described earlier). Three replicates were performed for each experiment.
The inactivation of C. jejuni 35919 and 35921 following 10-min treatments in Bolton broth and phosphate buffer is shown in Fig. 1. As can be seen, both strains were reduced from levels of ca 108 CFU ml−1 to <10 CFU ml−1 by a 10-min process at 300–325 MPa. Survival remained quite stable up through 200 MPa, then dropped precipitously afterward, with every increase of 25 MPa resulting in the inactivation of an additional 2–2·5 log cycle of C. jejuni. Figure 2 shows the response of C. jejuni 35921 to HPP while inoculated into UHT whole milk, UHT skim milk, soya milk and chicken pureé. The responses of C. jejuni 35919 to these conditions were very similar to the responses of C. jejuni 35921 (data not shown). It was evident that the food systems offered a much greater pressure-protective effect for the cells of C. jejuni than did Bolton broth or phosphate buffer. Populations of 108 CFU ml−1 were reduced to <100 CFU ml−1 by 10-min treatments of 375 MPa or more, as opposed to the 300–325 MPa required when the cells were suspended in Bolton broth or phosphate buffer. There appeared to be no effect until pressures above 250 MPa were reached, and even 350 MPa offered only a 4-log reduction. Inoculated pack studies (Fig. 3) were performed to determine the ability of cells of C. jejuni 35919 to withstand an initial pressurization and recover in UHT whole milk at refrigeration temperature (4°C), room temperature (25°C) and incubation temperature (42°C). In addition to poultry products, raw milk has been found to be the other primary vehicle for the transmission of C. jejuni to human beings (Blaser et al. 1979). Doyle and Roman (1982) showed that C. jejuni can survive well for up to 2 weeks in sterile milk. Pressures of 325 and 350 MPa were used for these experiments. As can be seen in Fig. 3, 325 MPa was able to affect only a 2–3 log initial decrease, whereas 350 MPa was sufficient to produce a 5-log reduction. It was clear that postprocess survival was a function of the temperature at which the sample was held. Following a 350-MPa treatment, storage at 4°C allowed the organism to be detected up through day 3. After an initial treatment of 325 MPa, storage at 4°C allowed the cells to survive in high numbers through day 7. Postprocess storage at ambient temperature allowed the cells to survive only through day 3, but not beyond. Following pressurization at both 325 and 350 MPa, neither strain could be detected even after just 1 day of storage at 42°C.
High pressure processing has been evaluated over the past decade as a method for the inactivation of pathogenic micro-organisms in foods. It has been found to be effective, even at ambient temperatures, against Gram-negative pathogens such as Salmonella, Vibrio spp. and E. coli. Pressures ranging from 300 to 800 MPa have been commonly reported to reduce the levels of these organisms below detectable limits in a variety of menstrua. It appears that C. jejuni is no exception and fits well among the other Gram-negative pathogens in that it is inactivated by 10-min treatments of 300–400 MPa at ambient temperature. In this, the pressure resistance is similar to that of pathogenic Vibrio species (Berlin et al. 1999). This seems intuitive because Campylobacter was originally classified as a member of the family Vibrionaceae. Both C. jejuni 35919 and 35921 exhibited the same basic trend of increased inactivation with increasing pressure, regardless of the pressurization medium; however, 35919 seemed somewhat more resistant in food systems. Significantly more pressure was required to inactivate C. jejuni in food systems than in Bolton broth or phosphate buffer. These results are similar to findings with other food pathogens. Escherichia coli and Salmonella were more sensitive to pressure treatment in phosphate buffer than in richer media such as chicken pureé and UHT milk (Metrick et al. 1989; Styles et al. 1991). Foods may offer protection by reducing the osmotic pressure across the cytoplasmic membrane to aid in maintaining its integrity.
Inoculated pack studies were performed to determine the ability of cells of C. jejuni to withstand initial pressurization and recover in UHT whole milk at 4, 25 and 42°C. Accordingly, 350 MPa was sufficient to cause at least a 5-log reduction in counts in UHT whole milk, while 325 MPa was sufficient to inactivate only 2–3 log. These studies were performed to determine the correlation between the rigorousness of the initial treatment and the ability of cells to survive under different incubation temperatures. Campylobacter jejuni exposed to 325 MPa survived until day 7 only when held at 4°C. This could be anticipated as low temperatures have been shown to enhance the survivability of C. jejuni (Lazaro et al. 1999; Chan et al. 2001). Grigoriadis et al. (1997) recovered cells of C. jejuni from inoculated hamburger patties held at −18°C for 90 days. Medema et al. (1992) found that nonculturable but viable cells of C. jejuni survived for more than 30 days when held in sterile water at 15°C, but only 3 days when held under the same conditions at 25°C. Pressure-stressed cells of C. jejuni probably behave in a similar fashion, surviving better in colder environments that are conducive to slower metabolic reactions and protein synthesis than elevated temperatures.
Presently, the pressure magnitudes used to process commercial foods lies within the ca range of 425–580 MPa (Farkas and Hoover 2000); treatment times are ca 5–7 min, sometimes less. Results presented in this paper suggest that currently used commercial HPP parameters will effectively compromise and probably eliminate C. jejuni from pressure-processed foods given the pressure sensitivity of C. jejuni.