Bacterial sporeformers, such as Clostridium botulinum and Bacillus cereus and their toxins, do not get industry’s attention like Listeria monocytogenes and E. coli O157:H7, but that doesn’t mean these microbial contaminants aren’t on the radar screen for food manufacturers. This may be related to the fact that since the implementation of the low acid canned food regulations, there has been a precipitous decline in the number of incidents of reported botulism outbreaks in the U.S. But, as food companies strive both to develop products with extended shelf life and meet regulatory mandates for improved food safety and defense, these spoilage and potential public health contaminants must be addressed anew.

The National Center for Food Safety and Technology (NCFST), a unique food safety and engineering research consortium composed of scientists from academia, industry and the U.S. Food and Drug Administration (FDA) located at the Illinois Institute of Technology’s Moffet Campus, has been investigating microbial behavior of sporeforming organisms in order to foster scientific and technical exchange to better understand the science and research needs. NCFST experts note that while little spore research has been conducted since the 1970s, due to the advent of new food preservation (sterilization) technologies, there is a need to look at more closely the testing methods for determining resistance, the mechanisms of spore inactivation, and methods of production control. By studying resistance of spore populations to various food preservation technologies, particularly novel technologies, the food industry will have a better opportunity to create ambient shelf-stable, low-acid food products that consumers want, as well to implement new technologies to attain production efficiencies and food safety goals.

This Food Safety Magazine (FSM) panel, comprised of NCFST experts and members, discusses the importance of supporting new research in this field and why it is vital in helping government and industry achieve food safety objectives (FSOs).

FSM: Why at this juncture has NCFST undertaken such a big initiative relative to spore-forming bacteria?

Martin Cole: Twenty years ago, I was most fortunate to have Grahame Gould as my first boss in industry. Grahame and his co-worker Jerry Dring were famous for their pioneering work on spores. In those days, there was still a buzz around spore research and there were many labs around the word carrying out excellent research in the area. In hindsight, this was probably the beginning of the end for large-scale spore research. Cutbacks in basic research in companies and food safety attention turning to Listeria, Salmonella and pathogenic E. coli, not to mention other worthy candidates, led to a steady decline in spore research.

Today, with a few exceptions (notably, Peter Setlow in the U.S. and Mike Peck in the U.K.), there is very little spore research being carried out in the food area. Perhaps one might think that we know all we need to know about spores; certainly the excellent safety record of the canning industry might suggest this. So, why would we think of embarking on a research program on spores now? Well, to paraphrase Hoffer, in times of change the learned find themselves beautifully equipped to deal with a world that no longer exists. The truth is that during the past 20 years the demands on the food business have indeed changed—and changed in ways that now put the need to control spores at the forefront of things again. I can see three major trends driving a renewed interest in spores: consumer desire for products that are both fresh-like and convenient, an increase in international trade in food, and new developments in risk management.

The desire for freshness and convenience has led to the development of minimal processes where spores are the limiting factor. The increase in international food trade is being led by processed foods, which are growing twice as fast as commodities and set to double by 2050. International trade tends to need longer product shelf life and again raises the issue on controlling spores. Finally, although the rules of canning are based largely on data that was derived 90 years ago, new developments in risk management offer the promise of improving our understanding on the basis for currently used sterilization processes. These traditional processes have served us extremely well but may be in danger of slowing innovation in this area. Any innovation in an area involving hazards such as Clostridium botulinum will need to be carried out in a prudent and safe manner by those who have a mechanistic understanding of spore inactivation.

Dan Brown: I agree. Consumers are looking for fresher, natural and refrigerated products with extended shelf life, and today, technologies exist to produce these types of products. We need to ensure the application of these technologies will produce safe products. The Center is unique, enabling both industry and government to work together to arrive at a successful application of technology that meets the objectives of both groups.

Cindy Stewart: Since its inception 19 years ago, the NCFST has actively conducted research and provided expertise in the field of bacterial sporeformers, with particular emphasis on C. botulinum and its toxin. Traditionally, this effort was focused on supporting LACF regulations via the NCFST/FDA scientists and engineers. More recently, the Center has expanded its spore expertise to include pathogenic and spoilage Bacillus spp., to support both its initiatives in the novel preservation technologies, aseptic processing and food defense programs.

As Martin and Dan stated, the food industry’s focus on meeting consumer’s demands for convenient, “minimally processed,” more natural, high quality foods has driven the need for innovation in food processing and preservation systems. The need to be competitive and profitable in the global marketplace has driven industry to explore ways to extend the shelf life of food products, while retaining quality, so that the products can be delivered to markets at greater and greater distances from manufacturing sites.

Additionally, the recent needs to address food defense issues, has had industry and government assessing the vulnerability of the entire food chain, and consideration of handling foods intentionally contaminated with nontraditional agents, including bacterial spores and toxins, has been included in the risk assessments and research to address gaps in our current body of knowledge.

John Larkin: Much of our current operational standards/controls for extended shelf-life and shelf-stable foods that the food industry operates under, with regards to an agreed target level of safety, has come from experimental validation of the adequacy of a given process. Most of the time these experimental evaluations cannot be translated to new and/or modified processes. As an example, peroxide is used to sterilize package material, and there are agreed set treatment levels for its use that are linked to the target level of safety. If a processor were to incorporate high pressure with peroxide as an improvement to package sterilization, it would be potentially unreasonable to assume that the same treatment dose should apply, because both the mechanism(s) and kinetics of destruction would not be the same as for peroxide alone. This results in a different type of link between the target level of safety and the treatment validated by way of a surrogate to the organism of concern. The anticipation is that as we understand the mechanisms by which spores are destroyed, our ability to appropriately develop links between the required treatment dose and the one that is measured by way of validation will be made less costly and less experimentally intensive.

Larry Keener: From my perspective, NCFST, owing to its unique relationships with industry, academia and government, is in a unique position to provide global leadership in validating novel preservation technologies, given that much of this validation effort will rely on bacterial spores. NCFST’s member base is composed of scientist from the food industry’s leading companies. It is precisely these companies that are likely to see the benefits in taking new products derived from new preservation methods to market. Clearly, it is of great value to industry to have NCFST at the forefront of this activity.

FSM: We’ve using canning as a primary food preservation method for 90 years. What are the advances in preservation technology that look promising for producing high quality, safe food?

Larkin: Yes, we have been canning for well over 90 years, but the canning industry has been very progressive in the implementation of new and novel technologies. As long as the mechanism of destruction did not change, the experimental validation of the adequacy of the process was primarily relegated to the collection of process delivery parameters. For example, if a new processing system was developed to treat a new packaging application (i.e., foil-laminated pouch) the experimental validation conducted on the new process focused on the ability of the process to deliver and control the heating rate of the package. It was assumed that the mechanism of destruction of the microbial hazards within the package was similar to other thermally processed food and did not require new agreed target levels of safety (i.e., target sterilization value), but only a measurement of the differing death kinetics of the process were needed.

As I look at “soon to be implemented” technologies that might change our current ability to describe the destruction rate of both the surrogate and the pathogen of concern for canned foods, the three major areas that I see are high pressure, formulated chemical sterilants, and combination treatments involving multiple destruction mechanisms. The apparent targets these new technologies are directed at are either improvement in fresh like quality or improved package/convenience for the consumer.

Keener: That’s right. Nicolas François Appert invented a preservation process that is frequently referred to as Appertization nearly a century ago. Since that time we have learned that this method can be an effective means of food preservation. Along the way, however, many people have succumbed to illness or mortality after being exposed to foods that have been preserved using this method. During the Spanish-American War, for example, it is alleged that more American soldiers were killed by consuming canned food than by enemy combatants.

The organism responsible for those outbreaks was the sporeforming anaerobe, C. botulinum. Botulism, the disease that results from exposure to botulinal toxin is (until the advent of antitoxin) frequently fatal. There are a number of well-documented cases of deaths associated with both commercial and home canned low acid food products. An infamous case, involving canned potato soup, gave rise to new food safety regulations in the U.S., circa 1972. The Low Acid Canned Foods (LACF) regulations of the FDA (21 CFR 113), which were later adopted globally, have had the impact of dramatically reducing the incidence of human botulism around the world. So, Appert and his countryman Peter Durand did a good job of stuffing food into jars and cans and exposing them to heat. However, they did this in the absence of proper regard for the spores present in those food products.

We now know that in order to produce safe low acid or acidified food that we must understand, fundamentally, the spores likely to be present in the food. Our understanding must include the mechanics of their inactivation. In large measure, the LACF regulations, coupled with advancements in process control capability, have resulted in the near elimination of botulism in commercial canning operations. Notice, I did not say the absolute elimination since we continue to have the odd occasional outbreak. Most recently there has been an incident of botulism reported in a commercially processed carrot juice.

My point is that there are a number of emerging/novel processing methods that may well lend themselves useful in preserving low acid and or acidified foods. We now understand the impact of heat on C. botulinum spores. The literature is replete with reported D and Z values for this organism and its surrogate (PA 3679) in a wide range of foods. By contrast, there is relatively little reliable data available for the novel methods. So, in going forward with advancing novel preservation method such as pulsed electric fields (PEF), high power ultrasound, microwaves, high pressure processing (HPP) and/or pressure assisted thermal sterilization (PATS), pulsed light, cold plasma or other of the many methods currently under investigation, we must understand how spores held in the food matrix respond to the lethal effects of these novel processes. Likewise, it is critical going forward that we take advantage of the learning offered by Appert’s experiences of the last century.

Brown: As Larry mentions in his list, the three novel technologies that stand out in my mind are PEF for fluid products, and microwave and HPP for both fluid and solid products. There are a number of commercially produced products in the market today using these technologies; however, currently only pasteurized products and shelf-stable jams and jellies are being produced using these technologies. PATS appears to have the potential to produce products of high nutritional and sensorial quality that are commercially sterile.

Stewart: A recent advance in the commercial sterilization of low-acid food products is the complete inactivation of vegetative microbial cells and spores by pressure assisted thermal sterilization. PATS is a combined process where both pressure and temperature contribute to sterilization by the inactivation of spores and enzymes. PATS starts the high pressure treatment at elevated temperatures (e.g. 60–90°C) and uses thermodynamically induced adiabatic heating to raise the temperature of products rapidly and homogeneously. The temperature increase from adiabatic compression is reversible. When pressure is released the product experiences instantaneous cooling.

A clear advantage of PATS compared to traditional heat processing is this theoretical rapid and homogeneous temperature change (heating and cooling) of the product without dependence on product size. In real practice, potential heat transfer effects may alter this homogeneous temperature change due to property differences between the food product and the processing environment. For these reasons, the commercialization of PATS will be dependent on the development of multidisciplinary coordinated programs to assess the efficacy and facilitate the successful development and commercial implementation of this technology.

Recent advances in PATS mean that the Holy Grail of a new sterilization process that leaves food products shelf-stable but with a fresh-like quality (such as fruits and vegetables) or of restaurant quality (such as multi-component meals) could be attainable within the next five years. High-pressure vessels capable of combining both heat and pressure to packaged food products have the possibility of causing a paradigm change in food preservation of a magnitude not seen since the advent of freezing or canning.

Cole: Yes, and in relation to packaging, it is important to note that many of the advances industry has made involve going from heavy rigid cans to using semi-rigid containers and pouches through aseptic processing and filling. A number of non-thermal technologies look promising but perhaps the most promising for shelf-stable products is PATS.

FSM: What has been the impact of a paucity of spore research at advancing preservation technologies to ensure either improved product quality or address food safety?

Keener: The fact is that the nonthermal preservation techniques that are being investigated around the world hold the promise of high quality, nutritious, good tasting, and safe foods. However, it isn’t a given that bacterial spores will respond in the same way to the lethal effects of these novel methods as they do to conventional thermal methods. In the interest of food safety and public health it is imperative that research is conducted that will clearly establish the lethality of the process that is being investigated.

In conducting this research it is very attractive to use models and methods that we’ve grown accustomed to as a result of our familiarity with heat processing. This approach may or may not be a good idea. Perhaps the leading reason for this not being a good approach is due to a lack of understanding of the specifics of inactivation kinetics when using an unproven preservation technique. There are many questions that need to be answered before we can safely assume an equivalent lethal effect between methods. These questions can only be answered through research and as we know, since the emergence of E. coli 0157:H7, and L. monocytogenes in the 80s and 90s there hasn’t been a much work with bacterial spores.

Prior to the advent of the Jalisco cheese outbreak, for example, I was heavily involved with spore research. The focus of much of that work was aimed at providing a means for validating the decontamination efficacy of then-novel aseptic packaging machines. Bacterial spores were used to measure and calculate the decontaminating capacity within the sterile packaging zone and for the product contact surfaces of packaging materials. My research projects involved developing methods for demonstrating resistance and characterizing media that could be used for spore production. This work involved characterizing the heat and H202 resistance of both Bacillus subtilis A and Bacillus globigii, the organisms widely used to judge the lethality of these decontamination processes.

At that time there were also very active spore research programs at UC-Davis, Penn State University, Guelph University, and also within the private sector. Unilever, for example, had an extraordinarily active program. They also had several leading spore researchers, including Professor Gould, on their staff. With the emergence of Listeria and E. coli O:157H:7 as significant foodborne pathogen and the great success of the LACF regulations in reducing the global incidence of botulism in canned foods, there wasn’t a compelling case for industry or government to fund spore research. Clearly, the focus then was on managing the disease burden attributed to these newly characterized pathogens. Consequently, spore research around the world slowed rather dramatically. As progress was being made with novel nonthermal methods, however, there was a corresponding need to understand the impact of these methods on spores and therefore a resurgence of need for research.

Brown: Most of the recently published spore data has been dealing with the destruction of Bacillus anthracis spores and spore survival during space travel since that apparently is where the research dollars can be found. The government’s select agent program has made research work using C. botulinum difficult and expensive to do. We need to have good sampling methods that ensure a representative sample can be taken and analytical methods that can be used to quantitatively determine the level of spores in a product just prior to processing. The majority of the information in the literature on the incidence of C. botulinum in products is qualitative, but quantitative data is needed or conservative estimates have to be made on the initial number of spores present in the product to be processed. With evolving technologies, such as PATS, there is a lack of reliable kinetic spore destruction information in the literature. We need kinetic data on spore forming pathogens such as C. botulinum, C. perfringens and Bacillus cereus.

Cole: Yes, and another issue is that because the science of canning has not advanced over the years, it has made the determining of equivalency of alternative processes difficult to determine, as we discussed earlier.

Stewart: I would add that bacterial spores pose unique research challenges, as well. Bacterial spores are difficult to work with in the laboratory. Production of “good” spore crops (i.e., high numbers, appropriate level of resistance) is difficult. Also, there are few opportunities for students to conduct their master’s or doctoral research in the bacterial spore field, and thus, we have fewer trained scientists entering the workforce who can rapidly begin a spore research program.

Add to this the fact that since 9/11, C. botulinum is regulated as a select agent, which limits the number of laboratories in the U.S. that can conduct research with this microorganism.

Larkin: As I alluded to earlier, the lack of information concerning the spore destruction mechanisms for a given process forces process authorities, when validating a new technology system, to physically measure the effect of the process on both the pathogen of concern and any identifiable organism(s) that can be used as a surrogate to the pathogen of concern. This translates into two additional major research activities besides the existing need to identify the critical control points to the process that need to be set, monitored, and recorded to make sure that the correct lethal treatment has been delivered to all of the processed food. The first is to understand how the processing parameters associated with the system affect the destruction rate of the pathogen of concern, assuming that the pathogen of concern is known. The second would be to determine if a suitable non-pathogenic organism can be found that would mimic the kinetics of the pathogen of concern, and still allow for the necessary measurement of the target level of safety. Since there is limited information about how preservation technologies affect spores, the process authority may need to also conduct some experiments to determine the appropriate organism of concern, and which strain(s) of the organism of concern would be the appropriate target of safety.

FSM: In the context of existing regulatory framework, how will NCFST’s efforts in providing research on bacteria sporeformers advance the FSO model for risk assessment?

Stewart: Risk assessment has traditionally been focused on making chemical or microbial risks as low as reasonable. This approach leads to difficulties, particularly since trade is becoming increasingly global, not only because the technological capabilities of different countries, and even different companies within the same country, remain varied, but also, the idea of what is considered “reasonable” differs from country to country; and acceptable risk may be culturally defined.

The International Commission on Microbiological Specifications for Foods (ICMSF) has recently proposed a scheme for the management of microbial hazards for foods, which includes the concept of Food Safety Objectives (FSOs) to address these issues. An FSO provides a basis for measuring the effectiveness/adequacy of control systems adopted by industry, government and/or regulatory agencies. This scheme offers flexibility for the food industry by allowing the use of alternative, but equivalent, means for achieving the same FSO. Using this framework, the food industry will have a scientifically sound basis for development, validation and commercialization of new or novel food processing technologies as well as traditional processes, which while ensuring safety, will allow for the production and marketing of higher quality (sensory and nutritional quality) food products. The NCFST, in a collaboration project with its industry members and FDA is developing a framework that will allow for the development and evaluation of different, yet equivalent, processes, including those used for the production of low-acid shelf-stable products, and for those processes which are used for manufacturing extended shelf life refrigerated products.

Keener: As mentioned previously, the current regulatory framework for canned foods around the world is defined by 21 CFR 113 and its related parts. The legal basis for these regulations is found at 402(a) 4 of the U.S. Federal Food Drug and Cosmetic Act. This provision of the law states that a food is adulterated if has been prepared, packed or held under insanitary conditions whereby it may have been rendered injurious to the health. In terms of canned foods, these regulations require that they are processed to achieve commercial sterility. Commercial sterility is an interesting concept that is frequently confused with terminal sterilization. Commercial sterility is achieved when the treated food is free of all viable microorganisms capable of reproducing in the food under normal non-refrigerated conditions of storage and distribution. It is clear from this definition that bacteria that will not grow out in the food are allowed. Terminal sterility, on the other hand, requires the absence of all bacteria. So, succinctly, the regulatory intent of the commercial sterility clause is preventing the introducing canned foods into the marketplace that may contain harmful bacteria or their toxins.

It is interesting that many food safety practitioners report that commercial sterility means a heat treatment with the equivalent of a 12D process. However, there is no mention of this as a requirement in the regulations. The genesis of the 12D process is not exactly clear. It is clear that such a potent process would be more than capable of inactivating the spores of C. botulinum. Moreover, one would expect that such a rigorous process would have a deleterious impact on the nutritional and quality attributes of foods so treated. It is hard, for me, to conceive of a food that is intended for human consumption that would have such a high initial load in the first instance. Certainly, one would not expect to find botulinal spores in human foods at anywhere near those levels.

In fact, this point concerning the initial load has everything to do with the FSOs but not necessarily with current food safety guidelines. It makes sense that if one is going to establish a process that will be effective in reducing or eliminating a bacterial population from a food system, that it is important fundamentally to have an understanding of that organism’s initial count at the onset of that process. The FSO concept has been developed around this very basic premise. Essentially, the FSO requires information on initial loading of the target organism and the killing/inactivation capacity of the reduction (processing) step(s) that are to be applied. The true strength of the FSO concept is in its flexibility. It is neither prescriptive, nor proscriptive, in its approach to food safety. For these reasons, the FSO approach is very compatible with innovation and bringing new preservation technologies to the market.

Cole: Yes, the use of the FSO concept is being used as framework to help determine and define commercial sterility using a consideration of the initial microbial load, the process and likely outgrowth. Having a mechanistic understanding of spore inactivation will support the development of this risk management framework and improve our confidence in new combinations of hurdles for achieving commercial sterility.

Larkin: I am going to limit my comments to low-acid canned foods. LACF regulations appear to be the major area of interest to food processors. I believe this is because of the large market for improvement in quality. The LACF regulations may appear rigid, but in fact, with regards to identifying the appropriate treatment dose that must be applied to the process to render the food product commercially sterile, the regulation is flexible. There are a number of different procedures a process authority might consider when establishing the appropriate processing requirements.
I can’t go into all the details, but, suffice it to say, I believe the FSO framework can function easily within the current LACF regulations. For the FSO process to work for LACF products, one area of study that is going to be important is an understanding of how to quantify spore germination prevention. You may ask what do I mean by this? Currently, we understand that specific public health organisms of concern for shelf-stable foods can typically be prevented from germination if the pH is less than 4.6 or if the water activity is less than 0.85. However, we do not have a universally accepted understanding as to how to quantify this preventive control. As we learn about the mechanisms of growth and destruction, the expectation is that we will be able to quantify their control. This should then allow us to determine if a food product with a pH of 4.8, salt content of 2.0%, and a water activity of 0.94 will prevent the growth or germination of the pathogen of concern. The only way to determine this now is to conduct inoculation/incubation studies.

Brown: The primary goal of the FSO model is to translate a risk level to a measure that the food processor can use to ensure the production of safe wholesome food. For commercially sterile products the primary microorganism of concern is C. botulinum and research is needed to understand the kinetics of bacterial spores destruction at various temperature and pressure. Knowledge of the kinetics of spore destruction is critical so that the pr