Effect of prior growth temperature, type of enrichment medium, and temperature and time of storage on recovery of Listeria monocytogenes following high pressure processing of milk

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Abstract

A five-isolate cocktail of Listeria monocytogenes (103 cfu/ml in skim or whole raw milk) was subjected to 450 MPa for 900 s or 600 MPa for 90 s. The effects of prior growth temperature, type of milk (skim vs. whole), type of recovery-enrichment media (optimized Penn State University [oPSU] broth, Listeria Enrichment Broth [LEB], Buffered LEB [BLEB], Modified BLEB [MBLEB], and milk), storage temperature and storage time on the recovery of L. monocytogenes were examined. Optimized PSU broth significantly increased the recovery of L. monocytogenes following high pressure processing (HPP), and was 63 times more likely to recover L. monocytogenes following HPP, compared to LEB, BLEB and MBLEB broths (p<0.05; Odds Ratio=63.09, C.I. 23.70–167.96). There was a significant main effect for prior growth temperature (p<0.05). However, this relationship could not be interpreted given the significant interaction effects between temperature and both pressure and milk type. HPP-injured L. monocytogenes could be recovered using both LEB and oPSU broths after storage of milk at 4, 15 and 30 °C, with recovery being maximal after 24 to 72 h of storage; however, recovery yield dropped to 0% after prolonged storage of milk at 4 and 30 °C. In contrast, storage of milk at 15 °C yielded the most rapid rate of recovery and the highest recovery yield (100%), which remained high throughout the 14 days of storage at 15 °C. The above factors need to be taken into consideration when designing challenge studies to insure complete inactivation of L. monocytogenes and possibly other foodborne pathogens during high pressure processing of foods.

Introduction

High hydrostatic pressure can inactivate microorganisms in various foods, including milk used to make cheese (Drake et al., 1997), with only minimal effects on texture, nutrition and flavor (Cheftel, 1995). Therefore, this technology is of increasing commercial interest as consumers continue to seek more “fresh” minimally processed foods that are safe, nutritious and of high quality. Various foods treated by high pressure processing (HPP) that meet this demand are now on the international food market. Fluid foods including milk are ideally suited for HPP, because high pressure is rapidly and effectively transmitted throughout liquid foods with little harm to food quality. As a result, a large number of studies have focused on HPP inactivation of microorganisms in milk (Patterson et al., 1995, Simpson and Gilmour, 1997, Alpas and Bozoglu, 2000, Hayman, 2001, McClements et al., 2001, Dogan and Erkmen, 2004).

Listeria monocytogenes is a ubiquitous foodborne pathogen with a very high hospitalisation and case-fatality rate (Mead et al., 1999). It has caused significant foodborne outbreaks due to consumption of ready-to-eat (RTE) dairy products (Fleming et al., 1985, James et al., 1985, Sutherland et al., 2003) and RTE meat products (CDC, 1998, CDC, 2002). In addition to thermal resistance, L. monocytogenes is also typically more resistant to HPP treatments than Gram-negative foodborne pathogens (Alpas and Bozoglu, 2000, Tholozan et al., 2000). Various factors have been shown to affect the resistance of L. monocytogenes to HPP treatments including: (1) growth temperature and growth phase prior to HPP (McClements et al., 2001, Mackey et al., 1995); (2) pressure and time of processing; (3) type of food (Patterson et al., 1995, Simpson and Gilmour, 1997, Alpas and Bozoglu, 2000, Dogan and Erkmen, 2004); (4) pH and type of acid (Stewart et al., 1997, Alpas et al., 2000, Ritz et al., 2000); and (5) presence of bacteriocins (Alpas and Bozoglu, 2000).

Considerable research has been conducted on the recovery of heat-injured cells of L. monocytogenes in both enrichment media (Knabel et al., 1990, Busch and Donnelly, 1992) and foods (McCarthy et al., 1990). Detection of foodborne pathogens typically has required a two-step approach, primary (nonselective) enrichment, followed by secondary (selective) enrichment (Hitchins, 1998). However, this approach is not ideal as injured cells are often not detected, due to overgrowth by background flora during nonselective enrichment and/or inhibition of recovery of injured cells during selective enrichment. Research by Mendonca and Knabel (1994) and Teo and Knabel (2000) led to the development of a one-step, recovery-enrichment system, optimized PSU (oPSU) broth (Teo et al., 2001) that overcame the above dilemma to yield consistent detection of heat-injured L. monocytogenes from pasteurized foods by both conventional and molecular methods (Teo and Knabel, 2000, Knabel, 2002, Parameswaran and Knabel, 2003).

Little research, however, has focused on the recovery and growth of L. monocytogenes in either enrichment broths or foods following HPP treatments. Hayman (2001) found that cells of L. monocytogenes grown at 15 °C did not recover in Listeria Enrichment Broth (LEB) immediately after HPP treatment of milk at 450 MPa for 600, 750 and 900 s or 600 MPa for 90 s. Some LEB enrichments were positive for L. monocytogenes after the HPP-treated milk was stored at 4 °C for 3 days; however, the number of enrichments positive for L. monocytogenes decreased to 0 as storage time increased to 15 days (Hayman, 2001).

Given the above characteristics of L. monocytogenes, it is imperative that this pathogen be completely destroyed by pasteurization treatments, including HPP. Previous research by Knabel et al. (1990) revealed that growth of L. monocytogenes at the relatively high temperature of 43 °C significantly increased its heat resistance. This temperature was therefore used for growth of L. monocytogenes in the present study, because cows shedding L. monocytogenes in their milk can have fevers this high (Doyle et al., 1987) and cells grown at this temperature reportedly maintain thermotolerance in milk for 24 h during refrigerated storage (Farber et al., 1992). Therefore, the purpose of the present study was to determine the effect of: (1) prior growth temperature (15 and 43 °C); (2) type of recovery-enrichment broth (LEB, Buffered LEB, Modified BLEB, aerobic and anaerobic oPSU broth, and milk); and (3) temperature and time of storage, on recovery of L. monocytogenes following high pressure processing of whole and skim milk.

Section snippets

Isolates

Lyophilised cultures of the following isolates of L. monocytogenes were acquired from the Food Research Ryde (FRR) Bacterial Culture Collection of Food Science Australia (North Ryde, NSW, Australia): FRR B2472 (Strain Scott A, a clinical specimen); FRR B2473 (from ice cream); FRR B2538 (from a dairy factory floor); FRR B2543 (from soft cheese); and FRR B2545 (a clinical isolate from blood). These five L. monocytogenes isolates were selected due to their relevance to foodborne disease and dairy

Results

Preliminary data (not shown) indicated that both prior growth at 43 °C and recovery in oPSU broth dramatically increased the recovery of L. monocytogenes following HPP treatment of skim milk at 450 MPa for 900 s, compared to prior growth at 15 °C and recovery in LEB. However, after HPP-treated milk was stored for 24 h at 4 °C, L. monocytogenes could be detected using both oPSU and LEB. In addition, direct plating of HPP-treated milk on Oxford agar revealed rapid growth of L. monocytogenes in

Discussion

The results of the present study confirm previous reports that L. monocytogenes is sublethally injured during HPP (Tholozan et al., 2000, McClements et al., 2001, Ritz et al., 2002). Gervilla et al. (1997) observed log-linear destruction of L. innocua grown at 37 °C during HPP of ewe's milk. However, they plated immediately after treatment onto Listeria selective agar, which probably would not have allowed detection of HPP-injured cells. Many of the results in the present study might be

Acknowledgements

We would like to thank Jacinda Dariotis from the Statistical Consulting Center at The Pennsylvania State University for statistical analysis of the data.

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