B01001: Physiological and microstructural factors controlling the survival and lag time of foodborne pathogens

Study Duration: April 1997 to August 2000

Contractor: Institute of Food Research

The Ministry of Agriculture, Fisheries and Food (MAFF) Micromodel has proved valuable to the industry by allowing companies to reduce the number of studies necessary to ensure the safety of products. In some products it has been possible to allow longer shelf lives, based on model predictions, resulting in substantial financial benefit.

However, there are situations, mainly related to survival rather than growth rates, where the predictions are less reliable. For example, there are circumstances where a small fraction of bacterial pathogens such as salmonella survive an industrial process, such as drying. These can cause food poisoning when the dried food (such as an instant soup powder) is rehydrated by consumers at low temperatures.

Other examples exist where manufacturers feel that safety margins based on the model�s predictions of survival rates may be over cautious. This is because they are based on the responses obtained with physiologically active cells that may not apply to all food situations.

Micromodel predictions are based on data obtained from cells that were not exposed to stress and so there is the possibility that lag times could be dangerously overestimated in some circumstances.

These three situations are an example of our current inability to reliably predict the factors involved in microbial survival (such as survival rates, lag times, resuscitation or death rates) in foods. This study aims to improve our ability to predict these survival factors by taking an account of the statistical distribution of the responses within a cell population.

The project has six objectives:

1. To identify which factors enable a small fraction of a cell population to survive a stress treatment (such as osmotic stress).

2. To measure the range of lag times that can be reasonably expected within unadapted cell populations.

3. To determine the extent to which survival rates can be increased and lag times reduced by deliberate preadaption and selection of cells within the population.

4. To develop a complex probability distribution for predicting survival rates within a cell population and the distribution of lag times.

5. To apply the principles to a spray-drying operation.

6. To transfer these principles to industry.

The results showed that the structure of food, as seen under a microscope, is a major factor in determining the survival of a small percentage of bacterial foodborne pathogens in food. The 'microstructural effect' was examined for E. coli, Salmonella typhimurium and Pseudomonas putida and this was most pronounced when the growth conditions (temperature, pH and water activity) promoted survival instead of growth.

This effect is caused by the presence of microscopic 'water-rich' niches in a diverse food matrix. These niches permit a small number of cells to survive. Experiments with E. coli mutants have shown that oxygen toxicity does not contribute significantly even when there are air-filled pores in the food matrix.

Bacterial strains that can synthesise capsules of carbohydrates outside the cells can survive much longer as they create their own local water-rich environment immediately surrounding the cells. This may also be time-dependent where there is slow microscopic phase separation. These results suggest that it may eventually be possible to microengineer the structure of food to reduce bacterial survival.

Variations in cell survival, resuscitation and growth can arise because of differences between populations of stressed and unstressed cells. In particular it has been shown that E. coli cells can exist in different physiological states. These have different tolerances to thermal and osmotic stress that can affect the lag time (the period of time during which there is no apparent cell replication). The more stress a cell is placed under, the longer the lag time.

Mild stress kills vulnerable cells but severe stress forces the surviving cells to adapt, resulting in a longer lag time. Therefore, increasing the osmotic stress of the growth medium causes a shift towards longer mean lag times.

Survival rates can be increased and lag time reduced by selecting cells that have adapted to these conditions. This depends on whether the cells can move independently, known as motility. Cells that are unable to move (non-motile) will instead form a microcolony. If a cell with a very short lag time is removed from culture and placed in new media, the resulting cells will still display a range of lag times compared to non-selected colonies of bacteria.

This is probably due to the differing conditions within a growing microcolony. Cells in the middle are more stressed than those on the outside – due to a lower pH, lack of nutrients and a higher concentration of metabolic end products. Cells in the centre will therefore have longer lag times than cells on the outside.

This pre-adaptation also depends on the type of stress. Osmotic stress (induced by xylitol) has no effect on the lag time of E. coli; but preadaptation to reduced temperature and reduced nutrients such as amino acids does reduce lag time. These results indicate that the history of a cell culture and whether it is motile or not is critical in determining the lag time distribution.

A complex probability distribution has been developed which takes into account all these factors. However, it is not yet fully predictive, as the number and type of subpopulations need to come from experimental data. Spray drying was tested as a method to alter food microstructure, but it was not appropriate.

Final report is available from the Agency's Information centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: infocentre@foodstandards.gsi.gov.uk).

Contact: For any enquiries concerning this research project, please contact the relevant programme contact or email: science@foodstandards.gsi.gov.uk