Inactivation of pathogens and spoilage bacteria

It is widely understood that the increase in the incidence of foodborne illness associated with consumption of fresh fruit and vegetable commodities is positively correlated with a shift in agricultural practices. This shift is due to the relatively18 recent evolution of societies from subsistence farming to large-scale operations, as well as automation of harvesting, changes in processing and in consumer demands19 (Seo and Frank, 1999). This statement also applies to foodborne illness associated with meat products, and other foods.

The predominant Gram-positive bacterial pathogens of concern are Clostridium botulinum (and perfringens), Listeria monocytogenes and Staphylococcus aureus and Bacillus cereus. Clostridium botulinum, the causative agent in botulism, is an anaerobic, spore-forming, Gram-positive bacilli, associated with low-acid, canned and modified atmosphere packaged (MAP) meats and vegetables. In the spore form, Clostridium botulinum is highly resistant to antimicrobial processes. Clostridium botulinum produces several of the most potent and deadly neurotoxins (BoNT) known. Purified type A toxin was reported to have a 50% lethal dose (in humans) of approximately 1 ng/kg of body weight (Jay, 2000). Botulism was at one time a frequent and life-threatening foodborne illness. Although less common today due to the establishment of the strict 12 D process for the inactivation of Clostridia in canned and MAP food products, occasional incidences of botulism are reported annually, due to mishandling during storage, transport or at the consumer end (Jay, 2000).

Another foodborne illness agent, frequently associated with dairy products, 'ready-to-eat' frankfurters and leaf vegetables is Listeria monocytogenes (L. monocytogenes). L. monocytogenes is a Gram-positive, non-spore-forming bacilli, described as a facultative intracellular parasite ubiquitous in poultry, beef, pork (including processed deli meats) and dairy processing, in addition to fresh fruits, vegetation, nuts, seafood, water and soil. The growth range temperature is highly variable for this organism and L. monocytogenes has been cultured at 10C and up to 45 0C, however the standard laboratory incubation (in laboratory) temperature for optimal growth lies between 30 0C and 37 0C. L.

18. 'Recent' describes the period of time (within the last 2000-5000 years) with respect to the age of the earth.

19. Trends, such as the increased demand for 'organic' and nutri-ceutical foods.

monocytogenes is able to survive pasteurization temperatures and may be considered a hardy organism, for this reason (Marth, 1994).

Listeria monocytogenes is one of the key foodborne pathogens of concern for modern food processors and a 'zero tolerance' policy is mandated for food processing plants in the United States and elsewhere. L. monocytogenes relies on a potent lippopolysaccharide toxin (LPS) to sensitize erythrocytes, leading to what is generally characterized as a granulomatosis in the host, also known as listeriosis. Listeriosis has a variable incubation period with onset usually within a few days and up to 28 days following ingestion of infected foods. The disease in humans is of particular concern to pregnant women, immuno-compromised individuals (such as persons undergoing chemotherapy or those suffering from diabetes, cirrhosis of the liver or AIDS).

Listeria spp. have two distinct advantages over other pathogens with regards to their ability to survive and proliferate in and on contaminated food products. First, Listeria spp. are psychrotrophic organisms that can proliferate at refrigeration temperatures (1 to 70C) and secondly, documented cases of Listeria spp. retaining viability and proliferating in temperatures as high as 45 oC (Jay, 2000) have appeared in literature. Some Listeria spp. strains are known to be catalase positive, thus resistant to attack by H2O2 and some are suspected of surviving pasteurization in dairy products (Jay, 2000). Because of the potential for surviving thermal pasteurization, resistance to high-intensity light is indicated.

Staphylococcus aureus, a non-spore-forming, Gram-positive cocci, produces the potent enterotoxin staphylotoxin. The toxin is produced as a result of reheating and holding buffet foods at temperatures below 60 oC. Deli meats, cheese fermentation brines and pre-packaged frozen meals are also a frequent vector for Staphylococcus aureus intoxication (Jay, 2000). Staphylococcus aureus intoxication is considered to be one of the most prevalent causes of food associated gastroenteritis (Jay, 2000). Bacillus cereus, a Gram-positive, spore-forming bacilli, is also a producer of both heat-stable and heat-labile enterotoxins. B. cereus toxins are responsible for gastro enteric illness in humans and viable cells are frequently isolated in low numbers from grains (rice), meat, raw milk and spices (Jay, 2000).

High-intensity light treatment is an appropriate intervention for pathogen control on foods such the surfaces of deli meat and cheese slices for control of Listeria monocytogenes and Staphylococcus aureus, particularly because deli meats are 'ready-to-eat' food items that are extensively handled during slicing and packaging, yet, no further cooking step is applied in most cases (Stermer et al., 1987; Norje et al., 1990). Likewise, high-intensity light may be less appropriate for control of Clostridium botulinum and perfringens in opaque and dense emulsion products such as canned vegetables and stews, but might be useful for inactivation of Clostridia from modified atmosphere packaged low-acid products such as corned beef slabs and sausage products in clear plastic packs.

Typically, 1 to 35 pulses of light in the range of 1 to 2 J/cm2 are selected to inactivate bacteria. Researchers observed a 2 log reduction of the Salmonella population on chicken wings inoculated with either 1.0 x 105CFU/cm2 or 1.0 x 102CFU/cm2 after treatment with high-intensity light flashes (Dunn et al., 1995; Food and Drug Administration, 2000). Similarly, a 2 log reduction of Listeria innocua20 on hot dogs treated with high-intensity pulsed light was reported. Reductions of Salmonella enterica (serovar Enteritidis) on egg shells, up to 8 logs were observed following exposure to 8 flashes at 0.5 J/cm2 radiation value (Dunn, 1996; Kuo et al., 1997; Food and Drug Administration, 2000). High-intensity light is well suited to decontaminate processing equipment, a frequent vector for dissemination of Gram-negative bacteria in meat processing. Packaging materials treated with high-intensity light will also benefit by reducing cross-contamination.

Other microorganisms of concern are located in the protozoan group and include flagellate protozoans, such as Giardia lamblia (giardiasis causing agent), Entamoeba histolytica (amebiasis or amoebic dysentery causing agent) and Toxoplasma gondii (toxoplasmosis causing agent), the latter of which can produce a terminal secondary infection in immuno-compromised individuals. The protozoan group are typically associated with contaminated drinking and processing water and infected under-cooked meats (Jay, 2000). High-intensity light applied to transparent liquids is an effective decontamination treatment. PurePulse Technologies, Inc. is currently testing a model of high-intensity light treatment equipment that can effectively decontaminate water at a rate of four gallons per minute (PurePulse Technologies Inc., 1999). However, if water sources are turbid and contain abundant opaque organic materials or nonuniform particulates, high-intensity light treatment will not be appropriate.

Common fungal pathogens associated with food are producers of potent, concentrated mycotoxins. Mycotoxins are associated with foods such as cheese products, nut meals, beer, raisins, soybeans, coffee bean grains, country cured hams, fruits and vegetables. Mycotoxins frequently associated with foods are Aflatoxins, from the organism Aspergillus flavus, Ochratoxins, from organisms A. ochraceus, A. alliceus and other closely related species, Citrinins, from the organisms Penicillium citrinum and P. viridicatum, Patulins, from the organisms Penicillium expansum, P. patulum, some Aspergillus spp. and some Bsysochlamys spp.

High-intensity light is an effective method for reducing the mold spores, provided that they are on the food surface or in a clear medium. Mold spores of Aspergillus niger are readily reduced by greater than 7 logs following treatment with 'a minimal number of flashes' at 1 J/cm2 power density (Dunn et al., 1995; Food and Drug Administration, 2000).

There are several notable human pathogens in the phylum Nematoda, including but not limited to Trichinella spiralis, causative agent of trichinosis in humans, resulting from eating undercooked meat products21 derived from

20. Listeria incocua is a non-pathogenic (to humans) species often used in testing as a 'surrogate' species; this species is closely related to pathogenic Listeria monocytogenes.

21. Specifically pork and 'exotic meats', such as bear meat.

infected animals. Several agencies world wide, including the World Health Organization, as well as the Food and Drug Administration of the United States, reviewed and improved treatments for the control of Trichinella spiralis in swine and pork meat.22 Due to the fact that Trichinae burrow deep into muscle tissue of infected animals and are not limited to the meat surface, high-intensity light is not currently used to inactivate microorganisms in intra-muscular meat tissue.

In a study at the University of Strathclyde, Royal College, Glasgow it was determined that viable counts of pathogenic bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Salmonella Enteritidis and Escherichia coli23 were reduced up to 2 log following exposure to 200 of low UV light pulses (each pulse duration approximately 100 ns) and up to 6 logs with high UV content light. The generator used was charged to a voltage of 30kV, with a source capacitance of 6.4nF and a source impedance of 3.25 The duration of each pulse was 85 ns. There was no detected increase in the temperature of the food samples due to the fact that each pulse/sec consumed no more than 3 w of electricity, therefore energy absorbed at the food surface level was low.

To determine the comparative resistance of frequent food associated pathogens, representative Gram-positive and Gram-negative pathogens were grown on agar medium exposed to 100-1000 pulses/sec of a PPET (pulse power energizing technique) producing light at wavelengths of 254 nm and 365 nm, respectively. It was determined that Listeria monocytogenes is more resistant to UV light than Staphylococcus aureus, Salmonella Enteritidis,24 Escherichia coli and Bacillus cereus in order of greatest to least resistant (Doyle, 1999). Further, a single pulse of 512 sec duration using PPET, was able to reduce the Listeria monocytogenes population on agar plates by 6 logs in 512 ps (Doyle, 1999). Gram-positive bacteria, and particularly Gram-positive bacterial spores, are more resistant to high-intensity and UV light treatments than vegetative cells, such as Gram-negative bacterial cells, due to the protective endospore coat of spores. Spores are highly heat resistant and tolerate desiccation far better than vegetative cells. Resistance of bacterial spores is somewhat a function of the medium or food equipment under analysis (Doyle, 1999).

22. The World Health Organization approved the use of 1-2 Kilograys of irradiation (typically from accelerated electron beam or Cobalt isotope source).

23. Listeria spp., Bacillus spp. and Staphylococcus spp. are Gram-positive bacteria, likewise, serovars of Salmonella enterica and Escherichia spp. are Gram-negative bacteria.

24. The genus Salmonella has undergone several taxonomical changes in recent years. Currently, there are only two species of Salmonella (enterica) and Bongori, S. enterica has 5 subspecies (II. salamae, Ilia. arizonae, Illb. diarizonae, IV. houtenae and VI. indica) with the former group V. subspecies transferred to the new species (Bongori). There are over 2,000 serovars (not italicized) such as 'Typhimurium', 'Mbandaka' and 'Havana'. Older literature may refer to Salmonella enteritidis (italicized) or Salmonella Typhimurium (not italicized) both designations now refer to S. enterica serovars.

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