Food safety and preservation research at the Top Institute Food and Nutrition

Safety and preservation issues with mildly processed foods

Mildly processed, ready-to-use foods are becoming increasingly popular. Due to the mild processing treatments, the sensory and nutritional characteristics of the ingredients are well preserved. However, such treatments do not always result in complete inactivation of the microbes present. For control of microbial growth, these products depend on additional preservation techniques such as refrigeration. In the research program, Food Safety and Preservation, survival strategies that enable food-borne pathogenic and spoilage microbes to survive processing treatments and to grow during product shelf life are being unravelled. The ultimate aim is to identify better ways for controlling these microorganisms in the food production chain.

In this research program, both model strains and industrial isolates of a range of food-borne pathogens such as Listeria monocytogenes, Salmonella, Bacillus cereus, and Clostridium perfringens, and spoilage microbes such as Bacillus subtilis, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, are being investigated. The microbial survival strategies of these microbes are studied at the population level, cell level, and molecular level under conditions that are relevant to the context of food processing. Bioinformatics and advanced genomics approaches are deployed to identify cellular processes and regulatory mechanisms to select targets for controlling and inactivating these microbes.

Microbial survival strategies

In the Food Safety and Preservation research program of the Top Institute Food and Nutrition (TIFN), four microbial survival strategies are being investigated. Insight into these strategies is the first step towards better control of microorganisms in the food production chain.

Formation of spores Several food-borne pathogenic microbes and food spoilage microbes have the capability to form endospores. Such spores are more resistant to preservation treatments than vegetative cells. They easily survive the heat treatments that are generally applied in the processing

Bacillus cereus cells containing spores

of ready-to-prepare, cooked-chilled foods and pasteurized dairy products. They may even survive the processes that are designed to produce ambient-stable foods. Germination and outgrowth of surviving spores during cooling and (chilled) storage can lead to product spoilage and, in the case of pathogenic species, to diseases. In this research program, the pathogenic microbe Bacillus cereus has been studied extensively for the cellular processes and regulatory mechanisms involved in the formation, germination, and outgrowth of spores. Results obtained with this model species are now validated for other spore-forming pathogens and spoilage microbes.

The biofilm mode of life Most microbes, including several food-borne pathogens and spoilage microbes, have the capability to adhere to surfaces and to form biofilms. In such biofilms, cells are embedded in a matrix of excreted polymeric substances that keeps the community attached to the surface, providing a buffer against the environment. As a consequence, biofilms are often difficult to remove completely, and the cells are more resistant to disinfection treatments. Cells can also detach from a biofilm, both in a passive way, as a result of extrinsic factors such as shear, or by mechanisms employed by the biofilm cells themselves. So, biofilms in processing environments may act as a persistent source of spoilage and pathogenic microbes that contaminate the food being produced. Examples of biofilm systems studied by TIFN include Salmonella biofilms that are present on processing equipment used in the slaughtering and processing of pigs, and biofilms of Geobacillus stearothermophilus and Anoxybacillus flavithermus that are formed in the hot sections of the production process of condensed and dried dairy products.

Stress adaptive response Microbes are able to adapt rapidly in response to stresses that they experience in the food production chain. This process of physiological adaptation is called the stress adaptive response, and involves the induction of mechanisms that both repair the damage that was caused and prevent further damage. It confers a transient resistance not only against the stress that induced it, but also against other stresses. So, in the food production chain, exposure to a stress experienced at some point may confer resistance to the next step in the chain, and this increased resistance may lead to a higher number of surviving microbes than expected. TIFN has investigated the microbial responses to several stresses that are relevant in the context of food processing and preservation such as heat, and the preservatives acetic acid, lactic acid, and sorbic acid.

Generation of diversity Food preservation treatments are generally based on the assumption that inactivation of microbes follows first-order kinetics. However, in practice, deviations from first-order kinetics have been observed. A commonly observed pattern is that of a gradually decreasing rate of inactivation over time. This phenomenon is generally referred to as tailing. One of the explanations for tailing is the presence in the population of individuals with increased resistance. In current practice, safety margins included in the process conditions are generally sufficient to take moderate tailing effects into account. With the increasing interest in milder preservation, the presence of resistant subpopulations can become a cause of concern in that it can lead to higher numbers of surviving microbes than were expected.

An example: population diversity of Listeria monocytogenes

TIFN has studied the occurrence of diversity in resistance to preservation treatments in populations of the food-borne pathogen Listeria monocytogenes [Van Boeijen et al, 2008, Journal of Food Protection 71, p 2007]. When such populations were exposed to mild, high- hydrostatic pressure treatments,

Listeria monocytogenes biofilm on stainless steel (Photo: Stijn van der Veen)

tailing inactivation curves were observed, indicating the presence of a sensitive majority population and a resistant subpopulation. This was found for all three strains of Listeria monocytogenes that were studied, suggesting that the presence of resistant subpopulations may be a common property of this pathogen. For two of these strains it was found that the increased resistance was a stable feature of a significant part of the resistant populations. In contrast, no stable, resistant isolates could be identified in the resistant population of the third strain.

We continued our research with a comparative analysis of 24 stable stress-resistant variants that were isolated from one of the three Listeria monocytogenes strains with the aim of assessing their robustness and growth performance in a range of food-relevant conditions [Van Boeijen et al, submitted]. Analysis of stress survival capacity, motility, biofilm formation, and growth under various conditions showed all variants to be not only more resistant to pressure than the wild type, but surprisingly, also more heat resistant. Differences among the variants were observed in acid resistance, growth rate, motility, and biofilm-forming capacity. A cluster analysis of these characteristics revealed that 14 of these 24 variants could be sorted out into three

Example of tailing inactivation of Listeria monocytogenes

groups. The remaining 10 variants did not cluster, implying that each of these variants has a unique phenotype. The largest of the three clusters contained 7 variants that were characterized by a moderate to high resistance to high-hydrostatic pressure, and a high resistance to heat. Genetic analysis revealed that the variants in this cluster had a mutation in the gene that codes for CtsR, the regulator of a specific, adaptive response to heat shocks. Due to this mutation, the regulator function has been lost and the adaptive response is permanently switched on. Analysis of the mechanisms that confer to the other variants their resistance, and the search for the underlying mutations, is ongoing.

Generation of diversity in populations, which for a pathogen is an effective strategy to maximize its chances to survive sudden changes in its environment, is, for the food industry, a major hurdle in the search for milder preservation strategies. By studying such microbial survival strategies, TIFN builds the knowledge base and the tools that enable the food industry to develop a new generation of safe, mildly processed foods.

About the Top Institute Food and Nutrition

Dr. Roy Moezelaar (Photo: Guy Ackermans)

The Food Safety and Preservation research program of the Top Institute Food and Nutrition, led by Dr. Roy Moezelaar (please contact at moezelaar@tifn.nl|), started in 2004 with a total budget for eight years of €12 million (US$18 million).

The Top Institute Food and Nutrition (TIFN) is a public/private partnership carrying out strategic fundamental research of commercial relevance. At TIFN, the research is flexibly organized in themes and projects, which are all aimed at the development of innovative products and technologies that respond to consumer demands for safe, tasty and healthy food. Three interrelated programs: "Nutrition and Health," "Sensory and Structure," and "Bio-ingredients and Functionality" have been established in which the research is jointly decided upon and guided by the industry and research partners. Over 51 patents have been filed, and innovative products and knowledge have been introduced. The results achieved and experiences gained underscore the ambition to increase the size and scope of the research portfolio and attract new partners from the food and nutrition industry. Current industrial partners are CSM, DSM, FrieslandCampina, Unilever and VION. Research partners are Wageningen University and Research Centre, TNO, NIZO food research, Maastricht University and the University of Groningen. 

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