The growth and survival of food-borne pathogens in sweet and fermenting brewers' wort
Introduction
Wort is the sweet extract that is fermented by yeast (Saccharomyces cerevisiae) for the production of beer. As the fermentation proceeds, several antimicrobial hurdles arise, as ethanol and carbon dioxide are produced, and the pH and nutrient levels decrease. Along with the added hops, these intrinsic hurdles, and processing hurdles such as wort boiling and often pasteurisation (or sterile filtration), add to the antimicrobial properties of beer. However, it is not until the wort is fermented that some of these hurdles arise, therefore wort may succumb to undesirable microbial growth, especially if pitching is delayed as is common in beer production by continuous fermentation.
Whilst several genera of beer spoilage bacteria have been recognised, previous studies have shown that the survival of pathogens in beer is generally poor (Bendová & Kurzová, 1968, Bunker, 1955, Felsenfeld, 1965, Lentz, 1903, Menz et al., 2009, Sachs-Müke, 1908, Sheth et al., 1988, Zikes, 1903), although Hompesch (1949) showed that Salm. Paratyphi could survive for up to 63 days, albeit from a high inoculation level. Pathogen survival and/or growth is enhanced when antimicrobial hurdles are reduced, for example, L'Anthoën and Ingledew (1996) reported that several pathogens could grow in alcohol-free beer. Further, pathogens have been reported or inferred in some traditional African beers (Pattison et al., 1998, Shayo et al., 2000), and E. coli has been associated with beer dispense (Schindler & Metz, 1990, Taschan, 1996). However, we are aware of only one study in which the survival of pathogens in wort has been investigated, where Bendová and Kurzová (1968) reported on the survival (but not growth) of Shigella flexneri and Salmonella enteritidis in wort.
The four species chosen for this study (Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus) are common causes of food-borne illness, and they have previously been isolated from other beverages. For example, Salmonella Typhimurium and E. coli have been isolated from carbonated soft-drinks (Akond et al., 2009), and E. coli has been transmitted through unpasteurised apple cider (Besser et al., 1993), where it has been shown to survive for extended periods despite a pH below 4.0 (Miller & Kaspar, 1994, Zhao et al., 1993). Such high acid resistance may confer low infective doses, for example E. coli O157:H7 has an infective dose as low as 10–100 cells.
With the advent of such previously unreported acid-resistant strains, coupled with brewing practices trending towards a reduction in antimicrobial hurdles (such as reduced or late hopping, reduced alcohol beers, and unpasteurised beer), it seems prudent to investigate the potential for the growth and survival of pathogenic bacteria in stored and fermenting wort, and to elucidate the effects of the typical antimicrobial hurdles in wort and beer.
Section snippets
Strain information and inoculum preparation
In this work we evaluated the survival of the following bacteria; E. coli O157:H7-VT (N) – NCTC 12900, L. monocytogenes ATCC 7644, Salm. Typhimurium (kindly provided by the University of Melbourne, Melbourne, Australia), and S. aureus (kindly provided by Victoria University, Melbourne, Australia). A brewing strain of S. cerevisiae was used for wort fermentation. Microorganisms were subcultured weekly and maintained at 4 °C on nutrient agar for bacteria, or YPD for the yeast. Overnight cultures
Survival in fermenting wort
The growth and survival of four pathogens in fermenting, hopped (20 IBUs) wort, with an original gravity of 1.040, is shown as Fig. 1. All worts were pitched with S. cerevisiae (time zero), and at time zero and every subsequent 24 h, a set of flasks were inoculated with the pathogen under investigation, to divulge the effect of wort contamination at various points during the fermentation. The production of 5.1% (w/v) ethanol by the brewing yeast is also shown in Fig. 1.
The Gram-negative
Discussion
In this work we have investigated the growth and survival of four food-borne pathogens in wort, given various levels of several important antimicrobial hurdles. We began by assessing survival in fermenting wort by introducing the pathogenic bacteria at various stages during the fermentation, and from this “base wort”, we further elucidated the effects of several key hurdles on the growth and survival of the four pathogens in wort.
The Gram-negative pathogens (E. coli and Salm. Typhimurium) were
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