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Pathogens in low moisture food

Grzegorz Rachon and Paul Gibbs of Leatherhead Food Research investigate the persistence and survival of pathogens in low moisture food.

Introduction
Although low moisture food is considered as low risk in terms of bacterial contamination, it appears to contribute significantly to the total number of food-borne infections and therefore more attention should be paid to controlling pathogens in these foods.

Outbreaks related to low moisture food
Although Salmonella outbreaks from low-moisture products are relatively rare, they often have an impact on large numbers of people:

  • 1,000 people were affected by paprika powdered potato chips in the 1993 outbreak in Germany;
  • >400 cases were associated with black pepper outbreaks in 1981 and 2009;
  • >200 cases were attributed to toasted oats cereal in the USA between April and June 1998;
  • >400 cases were attributed to peanut butter in the USA between August 2006 and May 2007;
  • >700 cases were attributed to peanut butter and peanut butter-containing products in the USA between 2008 and 2009 [1].
  • 31 individuals were infected with Salmonella in Organic Sprouted Chia Powder between January and June 2014 in 16 US states resulting in several widespread recalls of products containing organic sprouted chia powder and chia seeds in the US.

Due to the large number of unreported cases of salmonellosis for all types of product, the actual number of cases is likely to be much higher.

Persistence of pathogens in low moisture food
Bacteria cannot grow in low moisture foods but can survive extremely well. Cell metabolism is dramatically slowed and proteins conferring heat resistance are produced so that dehydrated cells are more heat resistant with an increased ability to survive.

The main pathogen associated with low moisture foods is Salmonella and the number of outbreaks notified by the Rapid Alert System for Food and Feeds (RASFF) and HorizonScan during the last decade has risen. Public Health England reported 5,937 Salmonella gastro-infection cases in England and Wales up until December in 2014, second only to Campylobacter infections (55,504) [2].

Figure 1 Horizon Scan notifications for Salmonella in low moisture food.HorizonScan recorded 814 notifications worldwide related to food contaminated with Salmonella in 2014, of which 103 (12.7% of total notifications) were related to the persistence of Salmonella in low moisture foods. Powdered spice recorded the highest number of notifications (33) followed by sesame seeds (24), chia seeds powder (12), other powders (13) and nuts (8) (Figure 1). Analyses of these numbers reveals that paprika and chilli powder and chia and sesame seeds are the products associated with the highest number of notifications.

The large number of Salmonella notifications in low moisture foods indicates that current processes for control or elimination of Salmonella are not efficacious or are not correctly implemented. Attention should be focused on monitoring the industrial hygienic and sustainability practices, identifying areas for improvement and education, strengthening the commitment to produce low moisture foods in a safe, responsible manner, verifying current practices and implementing additional treatments if necessary.

Sources and routes into product
Bacteria may enter food by various routes. Cross contamination can occur during agricultural processes starting with the soil itself and including non-potable water (e.g. rivers, streams or ponds/storage reservoirs), organic fertilisers, wild animals and people or machinery involved in harvesting. It is very likely that, at this stage of production, pathogenic bacteria are present in raw materials and therefore constant monitoring is required to minimise their presence. Raw products which in normal circumstances would not have received any treatment require special attention because pathogenic bacteria can easily persist and cross-contaminate other materials or products labelled as ready-to-eat. Any further processes and operations should be performed in maintained areas and strictly controlled. Air, water, personnel, pests and contact materials can be an additional source of contamination. Poor cleaning, inadequate waste water control, inappropriately trained production personnel, a lack of effective pest control or poorly maintained equipment and machinery, are just some of the elements contributing to cross-contamination [3].

Survival mechanism
Effect of storage temperature
Storage conditions in which bacteria cannot grow trigger a survival mechanism. Whilst in conditions supporting growth (i.e. a high moisture environment), increasing the temperature towards the optimum for growth will increase growth kinetics, in conditions not supporting growth (i.e. a low moisture environment), increased temperatures have an opposite effect. At lower storage temperatures (<16°C) the rate of chemical reactions and cell metabolism is significantly slowed and the speed of desiccation is decreased so that physical changes within the bacterial cell and at the cell wall are minimised [4]. At higher temperatures when growth is prohibited by lack of water, desiccation of cells progresses faster than at lower temperatures resulting in bacterial cell wall damage and leakage of cytoplasm. The influence of the temperature will be magnified by additional factors like pH, antimicrobial components, salt and sugar but at lower temperatures there is less bacterial damage.

Desiccation and water activity
The mechanisms of survival of bacteria in a desiccated state depend on the bacterial species, temperature, water activity and food composition. Treatments, such as freeze drying, allow vegetative microbial cells and spores to be stored for many years in a severely desiccated state. Once cells are freeze dried, bacterial cell metabolism completely stops as no water is available; viability can be improved by adding compounds which protect the cell from damage to walls and membranes. Such freeze dried microorganisms are commercially available; for example, yeasts with a shelf life of 2 years, probiotic supplements with 12 months shelf life and microbial cultures with over 10 years shelf life.

Impact of pH
When a cell is placed in an acidic environment, undissociated lipophilic acid molecules, unlike protons and other charged molecules, can pass freely through the membrane from an external environment of low pH, where the equilibrium favours the undissociated molecule, into the cytoplasm, where a higher intracellular pH changes the equilibrium promoting the dissociated state. This results in the release of protons in the intracellular milieu, the acidification of the cytoplasm and an increase in the osmotic pressure. Under these conditions the cell tries to maintain its internal pH by neutralising or exporting the protons released by dissociation of the acid, but this further slows growth as the cell diverts energy to stress resistance mechanisms. If the external pH is sufficiently low and the extracellular concentration of acid high, the burden on the cell becomes too great and the cytoplasmic pH drops to a level where cellular structures, such as proteins, are damaged resulting in cell death. In low moisture foods any interaction between bacteria and the pH of the surrounding environment is slowed down; higher water activity and higher temperatures result in increased interactions promoting bacterial death.

Mutations
Research indicates that microbial populations are not homogeneous [5,6]. Mutations, even within theoretically homogeneous populations, occur randomly and mutants can be more resistant to various environmental conditions. Resistance of mutants to heat, acid, desiccation or antibiotics can be significantly greater than in the corresponding wild-type strains leading to increased survival. Hypermutable regions (more prone to mutation) are over-represented in stress genes and therefore may play an important role in the generation of stress-resistant mutants at high frequency (5). This phenomenon has been demonstrated experimentally in Listeria monocytogenes (6) where a hypermutable region in ctsR, encoding the regulator of class III stress genes, resulted in the generation of stress-resistant mutants within hours of growth in pure clonal cultures. Several studies have highlighted a similar phenomenon in E. coli and Salmonella in which rpoS plays a central role in acid and general stress resistance and also in pathogenicity of Salmonella Typhimurium [7]. RpoS is subject to great genetic variability due to mutations occurring even within clonal populations [8]. Such mutations can either increase resistance or enhance growth, depending on the environmental conditions causing stress during food processing and storage and might enhance virulence or stress tolerance increasing the likelihood of contamination [8].

Food components
The ability of bacteria to survive in different foods is variable. While pH, temperature and water activity play significant roles in survival patterns, food composition is also very important [9]. In low moisture foods, components do not play a nutritional role as growth is inhibited, but can interact with the cell resulting in damage and cell death, or can protect the cell and increase survival in conjunction with additional factors, such as heat.

Effective processes
While it can be difficult to modify some storage conditions, temperature, humidity, packaging material and MAP at various stages of food production can be controlled relatively easily. Use of appropriate processes throughout the food production chain, from harvesting, transportation, and pre-process storage to heat processes and packaging, can result in safer food and can extend shelf life reducing food waste. Although most current processes are adequate, there is significant room for improvement, especially for low moisture foods. Despite the fact that they are often classified as raw but safe, they may contain pathogenic and spoilage bacteria. Cross-contamination caused by dried materials can readily occur as powders spread easily. Similarly, herbs and spices are very often used at the end of processing and nuts are generally eaten without further cooking. The best example of heating processes successfully reducing the presence of Salmonella in low aw (water activity) products is the pasteurising step applied to raw almonds, which was voluntarily adopted by the California almonds industry after two Salmonella outbreaks in early 2000. Almond pasteurisation is now required by law in the U.S, Canada, and Mexico. According to the FDA, pasteurised almonds retain their nutritional profile and since their composition does not fundamentally change, they can still be labelled as raw.

Recent studies
Leatherhead Food Research is undertaking a Forum project, initiated in 2012, with a number of organisations and experts from the food industry, academia and the engineering sector to study survival and heat resistance of pathogens in low moisture foods. The survival of pathogens in foods is being evaluated at various water activities under different storage conditions, e.g. temperature. In addition, a search for surrogate bacteria, which can be used to validate actual in-factory conditions in low moisture foods, is underway and the effects of a pilot-scale pasteuriser on a number of non-pathogenic microorganisms (Biosafety Level 1 - BSL 1) have been evaluated.

Figures 2 and 3The survival of food-associated Salmonella strains in paprika powder and rice flour was found to be greater when stored at low aw and at 15°C compared to 25°C. In some conditions, Salmonella spp. survive better in paprika powder than in rice flour during storage (Figures 2 and 3, aw=0.55, storage temperature = 15°C), but they can be eliminated more readily from paprika powder than from rice flour when heat is applied (Table 1). It appears that natural components of these products or differences in their water sorption characteristics have a significant influence on protection of cells during storage and heat processes. Heat resistance of salmonellae in paprika powder at water activity aw=0.55 was only slightly lower than at aw=0.45, but there are significant differences in heat resistance in rice flour at aw=0.2 and aw=0.55.

Heat resistance (D and z-values)
Strain Paprika powder
  D - value (min)  
 
 
aw 70ºC 75ºC 80ºC 90ºC z-value (ºC)
Salmonella Enteritidis PT 30 ATCC BAA-1045 0.45 12.56±0.29 5.89±0.25 2.82±0.25 - 15.43±0.78
 
 
0.55 9.48±0.95 3.79±0.19 2.29±0.18 - 16.22±0.68
Enterococcus faecium ATCC 8459 0.45 18.61±1.48 9.26±0.62 2.67±0.19 - 11.90±0.87
  0.55 12.62±0.62 4.82±0.18 2.07±0.12 - 12.72±0.20
  Rice flour
Salmonella Enteritidis PT 30 ATCC BAA-1045 0.2 41.14±4.99 24.14±3.59 11.35±0.99   18.28±3.29
  0.55 26.45±1.91 11.60±1.32 3.73±0.55   11.80±1.12
Enterococcus faecium ATCC 8459 0.2 - 38.07±4.79 11.79±0.69 5.07±0.22 11.47±0.57
  0.55 - 9.33±0.09 3.38±0.48 1.54±0.07 12.80±0.31

Survival and heat resistance studies on the Salmonella surrogate, Enterococcus faecium (ATCC 8459), in rice flour and paprika powder have identified limitations in the use of this organism as a surrogate. Although survival patterns of Enterococcus faecium are very similar to Salmonella (Figs 2 and 3), there are differences in heat resistance (Table 1, Figure 3); higher temperatures are required to inactivate salmonellae than to inactivate Enterococcus faecium. Temperatures of 75-90°C are ‘break points’ below which the heat resistance of the surrogate is greater than resistance of salmonellae and above which the heat resistance of the surrogate is lower than that of salmonellae.

Grzegorz Rachon is Senior Research Scientist and Paul Gibbs is Principal Consultant at Leatherhead Food Research
Tel: +44 (0)1372 822243 (219) Email: grachon@leatherheadfood.com Web: www.leatherheadfood.com

The Forum project described is supported by Dr Walter Penaloza, Nestlé Research Centre, Dr. Kimon Andreas Karatzas, University of Reading, Dr Paul Gibbs, Leatherhead Food Research. A pilot scale dry food pasteuriser unit has been designed by Winkworth Machinery Ltd. This work is part of Grzegorz Rachon’s doctorate project with the Food Advanced Training Partnership (Food ATP).

References

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  2. Public Health England, “Health Protection Report,” 14 November 2014. [Online]. Available: https://www.gov.uk/government/uploads/system/uploads/attachment_data/fil.... [Accessed 30 November 2014].
  3. Beuchat, L., Komitopoulou, E., Betts, R., Beckers, H., Bourdichon, F., Joosten, H., Fanning, S., Kuile. B.,, “Persistance and Survival of Pathogens in Dry Foods and Dry Food Processing Environments,” ILSI Europe, Belgium, 2011.
  4. Komitopoulou, E., Penaloza, W.,, “Fate of Salmonella in dry confectionery raw materials,” Journal of Applied Microbiology, vol. 106, pp. 1892-1900, 2009.
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  6. Karatzas, K.A.G., Hocking, P.M., Jorgensen, F., Mattick, K., Leach, S., Humphrey, T.J.,, “Effects of repeated cycles of acid challenge and growth on the phenotype and virulence of Salmonella enterica,” Journal of Applied Microbiology, vol. 105, p. 1640–1648, 2008.
  7. Jørgensen, F., Leach, S., Wilde, S.J., Davies, A., Stewart ,G.S.A.B., Humphrey, T.J.,, “Invasiveness in chickens, stress resistance and RpoS status of wild-type Salmonella enterica subsp. enterica serovar Typhimurium definitive type 104 and serovar Enteritidis phage type 4 strains,” Microbiology, vol. 146, pp. 3227-3235, 2000.
  8. Notley-McRobb L., King T., Ferenci T.,, “rpoS Mutations and Loss of General Stress Resistance in Escherichia coli Populations as a Consequence of Conflict between Competing Stress Responses,” Journal of Bacteriology, vol. 184, pp. 806-811, 2002.
  9. Douglas, L.A.,, “Preservation microbiology and safety: evidence that stress enhances virulence and triggers adaptive mutations,” Trends in Food Science & Technology, vol. 7, pp. 91-95, 1996.
  10. Tajkarimi M.M., Ibrahim, S.A., Cliver, D.O.,, “Antimicrobial herb and spice compounds in food,” Food Control, vol. 21, pp. 1199-1218, 2010.

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