Ph.D.
Department Extension Leader and Professor
Department of Poultry Science
North Carolina State University
Raleigh, NC
U.S.A
Many factors contribute to foodborne disease outbreaks including surface to product contact cross-contamination occurring at the processing plant, in commercial kitchens, and in the home. To address this factor, studies were conducted in my laboratory that demonstrated the efficacy of commercial HabaGUARD® conveyor belts containing a microbial inhibitor to reduce the populations of several foodborne bacterial pathogens.
The objective of this study was to evaluate the effectiveness of HabaGUARD® conveyor belts of varying composition to inhibit Salmonella serotypes, Campylobacter jejuni, Listeria monocytogenes, Escherichia coli O157:H7, and Pseudomonas fluorescens on both treated and control flexible and rigid modular belting materials fabricated from thermoplastic polyurethane, polyethylene, polypropylene, or polyacetal. Two different test protocols were followed; a seed layer test and a quantitative direct inoculation test.
In the seed layer method, test organisms suspended in solidified culture media were placed onto the belt surfaces and then depending on the organism, the belts were incubated at 37 oC or 41oC for 1 to 96 hours. For the quantitative direct inoculation test, liquid suspensions of the bacterial cultures were deposited directly onto the surface of the belt samples, covered with a film to prevent drying, and then incubated for 24 hours at 35°C. The range of inhibition achieved over three replicate trials for both methods was from 2.0 to 7.5 logs. To estimate the shelf life of the HabaGUARD® belts under simulated commercial conditions, the belts were immersed for 8 weeks at room temperature in several commercial cleaners and disinfectants at normal use concentrations and then evaluated for inhibitory activity against L. monocytogenes and E. coli O157:H7 as outlined above for the quantitative direct inoculation protocol. Inhibition ranged from 3.95 to 6.8 and 0.6 to 6.6 logs for Listeria and E. coli, respectively.
Our final objective was focused on estimating the effectiveness of HabaGUARD® belts in preventing bacterial biofilm formation on control and treated conveyor belts. Our preliminary results suggest that the treated HabaGUARD® belts appear to reduce biofilm formation by Listeria monocytogenes. These findings confirm that HabaGUARD® conveyor belts are effective at inhibiting some common foodborne bacterial pathogens associated with human illnesses. Moreover, the belts may also reduce the formation of bacterial biofilms on conveyor belt surfaces and thus reduce the risk of product contamination via contact with contaminated conveyor belts.
Introduction
Food Safety has become one of the most visible issues of recent times. Outbreaks of foodborne illness persist worldwide and in the U.S. food supply even though it is considered one of the safest in the world. Moreover, consumer fears over the safety of animal-derived foods in particular, have led to some erosion of public confidence in the beef, pork, poultry, dairy and seafood industries. Of greatest concern to consumers is contamination of foods with microbial pathogens. The U.S. FoodNet Surveillance system currently monitors sites in seven U.S. regions containing 25.8 million people (7.7% of the U.S. population). In 1999 within the surveillance area, a total of 10,717 confirmed cases of foodborne illnesses were identified in 5 states and of these, 10,248 cases were of bacterial origin. These included 3,884 cases caused by Campylobacter, 4,488 Salmonella cases, 510 E. coli 0157:H7 cases, and 114 Listeria cases (CDC, 1999). There are approximately 2 million cases of foodborne salmonellosis annually in the U.S., resulting in an estimated annual cost of one billion dollars (Roberts, 1988; Budnick, 1990). Outbreaks of foodborne disease are most often attributed to inadequate cooking, temperature abuse, use of contaminated raw ingredients, and cross-contamination (Doyle and Cliver, 1990). Although many of the factors that contribute to foodborne disease outbreaks are directly related to the activities of the consumer or food service worker, the mere presence of these pathogens on raw foods coming from the processing plant contributes significantly to the potential occurrence of foodborne disease outbreaks. Listeria monocytogenes was the number one reason for recalls of contaminated meat and poultry products between 1994-1998 (39). Annually the meat and poultry products as well as other food industries are confronted with recalling their products from the retail and wholesale market due to L. monocytogenes or other bacterial contamination. In 2001, there were 11 food product recalls due to L. monocytogenes contamination. The expense for a recall alone without taking into account liability can run into the millions of dollars. The estimated cost of a single recall may go as high as $76 million.
The development of multiple safety hurdles to control foodborne pathogens and spoilage microorganisms along the food chain (from farm to table) remains a top research priority of several U.S. regulatory agencies (i.e., USDA and FDA) and the Executive Branch of Government (i.e., "Food Safety From Farm to Table" presidential initiative). In particular, their priorities are focused on developing and implementing HACCP programs (Hazard Analysis and Critical Control Point) throughout food processing, distribution, and marketing. The success of these programs is directly related to first identifying the specific hazards and critical control points associated with each product and then introducing effective control(s) for eliminating or reducing the threat from the hazard.
One aspect of the farm to table HACCP program that needs greater attention is the control of bacterial pathogen proliferation and prevention of product cross-contamination as products move through the processing plant. Moreover, the prevention or elimination of biofilms on food contact surfaces remains a high priority research area since little is known on how these micro colonies of microorganisms contribute to product contamination and ultimately the risk associated with food borne illness.
The importance of biofilms to food safety and spoilage warrants a better understanding of their biology, structure, function, and ultimately prevention or elimination. Biofilms consist of bacteria, fungi, and/or protozoa growing alone or in combination that are bound together by an extracellular matrix that is generally attached to a solid or firm surface. They form on surfaces in large part because nutrients are found in higher concentrations than in open liquid. In laboratory studies, surface adherence is best achieved in rich media (Blackman and Frank, 1996). Attachment is facilitated by the microbial excretion of an exopolysaccharide matrix sometimes referred to as a glycocalyx. Microcolonies form within this microenvironment in a manner that leads to microbial communities that allow channels to form between and around the microcolonies. The latter have been likened to a primitive circulatory system where nutrients are brought in and toxic by-products carried out. Microbial cells in liquids that are not in a biofilm are in a planktonic (free floating) state.
From the standpoint of food safety and spoilage, biofilms are important because of their accumulation on foods, food utensils, and food contact surfaces in processing plants, restaurants, or homes; and because they are difficult to remove. While under natural conditions biofilms tend to be composed of mixed cultures, pure biofilm systems are often used in laboratory studies. Some of the solid surfaces employed to study foodborne bacteria biofilms include floor sealant, rubber, stainless, steel, and Teflon. Conveyor belt materials used in food processing plants would be another excellent surface to study since most foods are generally conveyed throughout processing plants on conveyor belts.
From the numerous published studies that have examined biofilm formation in food processing environments, the following statements can be made:
• Although biofilm formation by single cultures in rich media (e.g., tryptic soy broth) may be evident after 24 hours when appropriate growth temperatures are used, three to four days or more are necessary for maximum development. On glass slides that were suspended in a culture medium for three days at 24oC, Listeria monocytogenes grew to about log10 6-7/cm2 (Arizcun, 1998).
- Microorganisms in biofilms are considerably more resistant to remove by commonly used cleaning and sanitizing agents than organisms not associated with biofilms.
- In general, microorganisms in biofilms are more difficult to destroy by lethal agents (Chumkhunthod, 1998; Frank and Koffi, 1990).
- The attachment of a given pathogen to surfaces may be aided by the formation of a mixed-culture biofilm (Bruswell et al., 1998; Leriche and Carpertier, 1995; Sasahara and Zottola, 1993).
- Microorganisms in biofilms may exhibit different physiologic reactions than planktonic forms, and the biofilm may contain cells in the viable but nonculturable state (Carpertier and Cerf, 1993; Chumkhunthod, 1998)
- The use of cleaners and sanitizers in combination rather then singly appears to be more effective in removing biofilm growth (Arizcum et al., 1998; Oh and Marshall, 1966).
- Not all strains of the same species are equally capable of initiating biofilm formation; surface attachment and biofilm development are distinctly two different processes (Michiels et al., 1997; Kim and Frank, 1995).
Based on the scientific literature on biofilms and the significant risk of cross-contamination between contaminated food conveyor belt surfaces and raw and ready-to-eat foods in food processing plants, the ability of the HabaGUARD® conveyor belt system to inhibit food borne pathogens and prevent biofilm formation would represent a significant advancement in food safety hurdle strategies for food processors. Thus, the objectives of our study were to assess and validate the efficacy of HabaGUARD® conveyor belt materials to inhibit bacterial pathogens and prevent biofilm formation.
Specifically, the four objectives were:
- To quantitatively estimate the efficacy of HabaGUARD® conveyor belt materials to inhibit selected foodborne bacterial pathogens and spoilage bacteria,
- To estimate the shelf life of HabaGUARD® conveyor belt materials following extended exposure to commercial cleaners and sanitizers,
- To evaluate the efficacy of HabaGUARD® conveyor belts following 1 to 2 years of commercial use, and
- To evaluate whether the HabaGUARD® conveyor belt system can prevent or reduce biofilm formation involving a food borne pathogen.
Materials and methods
Preparation of bacterial cultures. The bacteria used in this study (and their sources) included S. Typhimurium ATCC BAA-216, S. Enteritidis ATCC 4931, L. monocytogenes ATCC 19114 and 19115, E. coli O157:H7 ATCC 43894 and 8739 (American Type Culture Collection, Rockville, MD), C. jejuni - TER 72927, TER 71940, EPI 36 (obtained from the U.S. Department of Agriculture [USDA]-Agriculture Research Service, Athens, GA), and P. fluorescens (environmental isolate). Stock cultures of Campylobacter and the other test organisms were maintained in phosphate buffered saline (PBS, pH 7.0) and Brain Heart Infusion (BHI) broth (Difco Laboratories, Detroit, Mich.), respectively, each supplemented with 16-20% (vol/vol) glycerol and stored at -80oC. For each Campylobacter strain, individual 10-ml Brucella broth cultures (Difco Laboratories, Detroit, Mich.) were prepared (48 h, 42oC) in a microaerophilic environment (10% CO2, 85% N2, 5% O2) and a 0.1-ml sub-sample of each was transferred to 30 ml of fresh Brucella broth and incubated as described above. For the other bacteria, individual 10-ml BHI broth cultures were prepared (24 h, 37oC) and a 0.1-ml sub-sample of each was transferred to 30 ml of fresh BHI broth and incubated as described above.
Seed layer test protocol. Early to mid stationary phase cells (generally the most resistant to disinfectants) of each test organism (three strain cocktail of Campylobacter jejuni and one strain of E. coli O157:H7) were prepared in the appropriate broth media, harvested by centrifugation (12 min, 5oC, 9,000 X g), washed twice with 0.1% buffered peptone water, and resuspended in 0.1% buffered peptone water to a final population of ~106-9 CFU/ml. One ml of the diluted sample was transferred to 100 ml of an appropriate tempered culture media containing agar (i.e., Brucella agar or BHI agar) to yield a concentration of ~104-7 CFU/ml. Twenty mls of the cell suspension were pipetted through a small hole located in the center of an inverted 100 x 15 mm Petri dish base positioned on top of a similarly sized control and treated HabaGUARD® conveyor belt sample (HabaGUARD® FAB and FNB flexible conveyor belts). After solidification, the Petri dish base was removed and the belt sample incubated under aerobic or microaerophilic (5% O2, 10% CO2, 85% N2, for Campylobacter) conditions for 1, 3, 5, 7 and/or 24 hours (96 hours for Campylobacter) at 35oC for E. coli and 41oC for Campylobacter. After incubation, colonies were enumerated using a Microbiology International Protocol automatic colony counting system. Triplicate samples were tested per organism and the trials run in triplicate or quadruplicate using new or used (washed and UV sterilized for 10 minutes per belt side) belting material for each replicate. Colony counts were converted to log10 values, means calculated, and statistical comparisons made between control and treated samples. In addition to testing the control and treated belts, the initial starting populations for each organism were estimated using the same recovery and plating procedures.
Quantitative direct inoculation test protocol. Five hundred (500) µl of a 10-2 dilution of a twenty-four (24) hour culture of each test organism (S. Typhimurium and S. Enteritidis, L. monocytogenes, E. coli O157:H7, P. fluorescens) in 0.1% sterile buffered peptone water (~106-8/ml) were aseptically transferred to the surface of triplicate belt samples (4 cm2) per treatment (control and treated belts) and then each overlayed with a sterile (4 cm2) sterile stomacher bag film. The inoculated belts were each placed in a sterile Petri dish containing moistened filter paper to prevent desiccation and then incubated at 35oC for 24 hours. After incubation, surviving organisms were recovered from the belt samples and accompanying overlay film using a one minute stomaching procedure in 5 ml of 0.1% buffered water, diluted as needed in 0.1% buffered peptone water, manually or spiral plated on BHI agar and subsequently incubated at 35oC for 48 hours. Triplicate samples were tested per organism and the trials run in triplicate or quadruplicate using new or used (washed and UV sterilized for 10 minutes per belt side) control and treated belting material for each replicate [HabaGUARD® FAB flexible conveyor belt and PE (polyethylene) and POM (polyoxymethylene) rigid modular belting]. Colonies were enumerated and then converted to a log CFU/ml basis. In addition to testing the control and treated belts, the initial starting populations for each organism were estimated using the same recovery and plating procedures.
Shelf life studies. The environmental conditions of a food processing plant (i.e., temperature, pH, presence of organic matter, exposure to cleaners and sanitizers) may affect the migration rate of inhibitor to the conveyor belt surface and thereby influence its immediate and long-term effectiveness and shelf life against targeted microorganisms. To test this assumption, triplicate control and treated HabaGUARD® rigid conveyor belt samples [PE, POM, PP (polypropylene)] were suspended in several common commercial cleaners and sanitizers for 8 weeks at 25oC (assuming a commercial processing plant exposure time of 30 minutes per day, 8 weeks of exposure is equivalent to 7.4 years). The treatments included untreated control belts and belts suspended in water and in cleaning solutions prepared in tap water at the manufacturer's highest recommended use level. Cleaning solutions included a 5% solution of P3-topax® 66 (Henkel Ecolab GmbH & Co., Düsseldorf, an alkaline cleaner containing 1.5% chlorine, pH 11.6-12.0, PE and PP modules), a 2% solution of P3-topax® 91 (Henkel Ecolab, a neutral disinfectant based on quaternary ammonium compounds, pH 7.7- 8.1, PE, PP, and POM modules), a 4% solution of P3-topactive® 200 (Ecolab, an alkaline cleaner, pH 12.8-13.2, PE, PP, and POM modules), a 6% solution of P3-topactive® 500 (Henkel Ecolab, an acidic cleaner, pH 1.7-2.1, PE and PP modules), and a 3% solution of P3-topactive® DES (Henkel Ecolab, a peracetic/acetic acid disinfectant containing hydrogen peroxide, pH 3.2-3.6, PE and PP modules). Following exposure, the belt samples were washed in sterile deionized/distilled water, dried, UV sterilized, and subjected to the quantitative direct inoculation test protocol using the L. monocytogenes and E. coli O157:H7 test strains and procedures as described above. These trials were replicated three times.
Commercial use studies. HabaGUARD® modular PE conveyer belts were placed for 1 to 2 years in two commercial meat-processing operations; a raw pork processing plant and a pilot plant for fully cooked frozen breaded meat and poultry products. The belts were used to convey either raw pork or fully cooked frozen breaded meat and poultry products following the companies' standard processing schedules. Moreover, the belts were subjected to a standard cleaning and sanitizing protocol as employed in each facility. Belt samples were removed at 1 (both plants) and 2 (breaded meat and poultry plant only) year increments, shipped to our laboratory, and tested for their ability to inhibit L. monocytogenes using the quantitative direct inoculation test protocol as described above. The recovered populations were compared to populations recovered from the corresponding PE control belts.
Biofilm studies. BHI and 1% reconstituted skim milk (Hood and Zottola, 1997) were used as the test media. Triplicate HabaGUARD® conveyer belts per belt type were tested to determine their effectiveness in preventing biofilm formation (control and treated PE and POM rigid modular belts). Plastic bottles (Fisher Scientific) containing 500 ml of the sterile test media and the conveyer belts were inoculated with the test culture (L. monocytogenes, initial population ~107 CFU/ml) and incubated at room temperature between 23ºC and 25ºC. The belts were aseptically removed at selected time intervals and rinsed by pumping sterile distilled water across the belt surface at a flow rate of 100 ml/min using a Manostat pump (model 72-640-000, Barnant Company, Barrington, IL). This rinse procedure was used to assure that the belts were washed in a consistent way to remove unattached cells from the belt surface. After rinsing the belts were air dried and stained for twenty minutes in 0.05 mg/ml bisBenzimide (Hoechst 33258, Molecular Probes, Inc.). Following staining, the belts were rinsed with sterile distilled water and air dried. Twenty (20) random 3.058 mm2 fields per belt were viewed using a Nikon Eclipse E600 epifluorescence microscope equipped with a Magnafire CCD camera and the average biofilm area of the 20 samples calculated using the metamorph software. Each image was adjusted such that the stained cells appeared red and the belt surface black. The software was used to convert the intensity of the fluorescence signals emitted from the stained cells to an average biofilm area contained in the 3.058 mm2 image area. The biofilm was visualized using an excitation filter of 340-380 nm, emission filter of 435-485 nm and a dichroic mirror of 400 nm.
Results and discussion
Seed layer test. Figures 1 and 2 summarize the inhibitory activity of new (Figure 1) and used (i.e., following washing and UV sterilization, Figure 2) HabaGUARD® FAB and FNB flexible thermoplastic polyurethane conveyor belts following a 96 hour exposure/incubation period against a three strain cocktail of C. jejuni (seed layer test protocol). To be effective using the seed layer test procedure, the inhibitor must be able to migrate from the interior of the belt to the surface and then onto and throughout the culture medium. Thus, the degree of inhibition is related to the organism's sensitivity to the inhibitor as well as to the migration rate through the belt and culture media. Migration rate would conceivably be dictated by the chemical composition of the belt and media, temperature, charge effects, cross linking, pH, hydrophobicity, viscosity of the culture medium, and other factors.
Compared to the control belts lacking the inhibitor, the treated belts reduced the C. jejuni populations from 4.9 to 5.6 (Figure 1) and 5.6 to 6.2 (Figure 2) log CFU (colony forming units) per new and used belt, respectively. It appears that the washing and UV sterilization procedures did not adversely impact the inhibitory activity of these flexible HabaGUARD® conveyor belts. A 96 hour exposure/incubation time was required for Campylobacter due to its microaerophilic nature, slower growth rate, and longer time required to produce visible colonies. For E. coli O157:H7, the flexible thermoplastic polyurethane HabaGUARD® FNB belts yielded around a 9 log reduction in population following 24 hours of incubation (Figures 3 and 4). No or minimal inhibition was observed for the new FAB and FNB belts following 1, 3, 5, or 7 hours of incubation (Figure 3). However, washed and UV sterilized treated belts produced a 4.5 log reduction after 3 and 5 hours of exposure (FAB belt) and a 5 log reduction after 7 hours (FNB belt). These findings show that the inhibitory agent contained in the flexible HabaGUARD® conveyor belts migrates at a slow rate (between 7 to 24 hours to yield a significant kill). Thus, the inhibitor would not be classified as a disinfectant. Although the data was not completely consistent (i.e., 7 hour FNB belt), our findings suggest that washing effects and/or UV sterilization may actually improve the inhibitory activity, possibly by enhancing the migration of inhibitor to the belt surface. These results clearly show that the HabaGUARD® flexible thermoplastic polyurethane conveyor belts are effective using the seed layer test method.
Quantitative direct inoculation test. Figures 5 (new FAB belts) and 6 (new POM belts) summarize the findings of our quantitative inoculation test against five test microorganisms. The three bars include the initial inoculum population and the bacterial populations following a 24-hour exposure at 37oC to the control and treated belts. The range of inhibition extended from 3.93 to 7.12 logs for the FAB belts and 2.03 to 7.12 logs for the POM belts. The most sensitive organisms included the Salmonella serotypes and Listeria monocytogenes (6.5 to 7.5 log reduction). In contrast, the enteropathogenic E. coli strain exhibited an intermediate level of sensitivity (4 to 6 log reduction) followed by Pseudomonas fluorescens which was the most resistant test organism (2 to 4 log reduction). These findings illustrate that the inhibitor contained within the HabaGUARD® belts exerted a broad spectrum of inhibitory activity against both Gram negative and Gram positive pathogenic and spoilage bacteria. Moreover, there does not appear to be a large difference in the degree of inhibition achieved between the flexible (FAB) and rigid (POM) conveyor belts although the FAB belt yielded more inhibition (approximately 1.7 to 1.9 logs) than the POM belt for the E. coli and P. fluorescens test strains.
Similar levels of inhibition were also observed for the HabaGUARD® rigid PE belt (Figure 7). In this experiment a comparison was made between unused control and treated PE belts in comparison to used treated PE belts. The term "used" in this context refers to treated belts that had been used in at least one or two previous quantitative direct inoculation trials and then were washed with a standard laboratory detergent, rinsed in distilled/deionized water, and UV sterilized (10 minutes per side at a distance of approximately 15 cm). Following this protocol we detected levels of inhibition ranging from 3.63 to 7.52 logs for the unused PE belts and 6.65 to 7.52 logs for the used PE belts.
The degree of sensitivity of the test organisms to the inhibitor was similar to that observed in Figures 5 and 6. Washing and UV sterilization of the treated PE belts produced similar improvements in the level of inhibition seen in Figures 3 and 4 for the flexible FNB and FAB treated belts. Predisposing the belts to the washing and disinfections steps presumably aides in extracting more inhibitor from the belt which results in greater inhibition.
Shelf life studies. Because our previous trials suggested that washing and/or UV disinfection of the belts enhanced the degree of inhibition, our next objective was to evaluate the effect of soaking HabaGUARD® rigid PE, PP, and POM belts in different commercial detergent and disinfectant solutions of varying pH on the degree of inhibition achieved against L. monocytogenes and E. coli O157:H7 using a quantitative direct inoculation protocol. The treated PE, PP, and POM belts were suspended for 8 weeks at 25oC in the recommended use concentrations of these cleaners and sanitizers for food processing plants. This time frame is equivalent to 7.4 years of exposure assuming that under standard operating practices the belts are exposed to the cleaner and sanitizer for no more than 30 minutes per day. Under these conditions, the level of inhibition achieved against L. monocytogenes was 5 to ≥6.2 logs for PE, 5.7 to ≥6.9 logs for PP, and 5.9 to ≥6.9 logs for POM (Figure 8). For E. coli O157:H7, the level of inhibition was 0 to 2.8 logs for PE, 0.2 to 2.3 logs for PP, and 6 to ≥6.6 logs for POM (Figure 8). The POM belts were not subjected to the alkaline/chlorine, acid cleaner, or acid/H2O2 treatments. These findings indicate that there was sufficient inhibitor remaining in the belts following exposure to the cleaners and sanitizers to exert a significant kill against the more sensitive organism, L. monocytogenes. In the case of the more resistant enteropathogenic E. coli test strain, the degree of inhibition achieved with PE and PP was significantly reduced (0-2.8 logs for PE and 0.2 to 2.3 logs for PP) (Figure 9). For the POM belts, the range of inhibition was 6 to ≥6.6 logs.
These findings demonstrate that the excessive soaking conditions used in this shelf life study did significantly reduce the effectiveness of the PE and PP belts against L. monocytogenes, presumably by excessively extracting the inhibitor. No consistent trends were observed as to which cleaner or sanitizer type had the greatest impact on the level of inhibition. However, the results do indicate that the minimum inhibitory concentration of inhibitor necessary to kill E. coli O157:H7 was not available in the PE and PP belts. Presumably the variations in inhibition exhibited between belt types may be related to the relative hydrophilicity or compatibility of the inhibitor to the belt type. PE and PP belts are more hydrophobic than POM and thus the inhibitor which is slightly polar (i.e., more hydrophilic) would be less compatible with the PE and PP belts (more compatible with POM) and would more favourably partition into the surrounding aqueous cleaning/sanitizer medium. However, pH effects, charge, and other factors may also influence the degree of migration of inhibitor from the belt. These findings demonstrate that the useful shelf life of these belts is probably related more to their physical attributes rather than their loss of inhibitory activity. The life of most heavily used conveyor belts in food processing plants (i.e., for conveying poultry meat) is probably far less than 7 years.
Commercial use studies. The shelf life of the HabaGUARD® belts under commercial applications is dictated not only by the physical characteristics and durability of the belts but also the retention of inhibitory activity against the targeted pathogens and spoilage microorganisms. The objective of this study was to determine the level of inhibitory activity remaining in HabaGUARD® modular PE belts after 1 and 2 years of commercial use in a raw pork processing plant and a fully cooked frozen breaded meat and poultry products processing facility. The test organism was L. monocytogenes. As noted in Figure 10, the HabaGUARD® belts maintained significant inhibitory activity against the target pathogen in both the 1 and 2 year samples. Compared to the control populations, the treated belts resulted in population reductions of log 3.87, 3.61, and 3.9 CFU/ml for the 1 year pork processing plant and 1 and 2 year poultry and meat processing plant belt samples, respectively. These findings demonstrate that the shelf life of these belts is apparently not limited by their lack of inhibitory activity.
Biofilm studies. The final objective was focused on determining if the HabaGUARD® belts were capable of preventing biofilm formation by L. monocytogenes on control and treated PE belts. In this study the control and treated belts were suspended in a culture suspension of L. monocytogenes and incubated for varying time periods to facilitate biofilm formation. After incubation, the belts were washed, stained with a fluorescent dye and then examined with an epifluorescent microscope to detect the presence of biofilms. Twenty randomly chosen 3.058 mm2 microscopic fields were observed and analyzed by computer to determine the average biofilm area on control and treated PE belts. A typical photomicrograph of a control and treated PE belt is shown in Figure 11. Those areas appearing white on the picture represent areas of fluorescence caused by the adhering L. monocytogenes biofilm. As noted, the control PE belt had significantly more fluorescence than the treated PE belt indicating that the inhibitor contained in the belt prevented or greatly reduced biofilm formation.
Following a computer analysis of the fluorescence data, the plot shown in Figure 12 depicts the average biofilm area in µm2 contained within the 3.058 mm2 imaging area. After subtracting the control belt's background fluorescence value (894 µm2), our findings show that the inhibitor contained in the PE belts reduced the area of biofilm by 80% or a 5-fold reduction. Although these results are still preliminary, this data indicates that HabaGUARD® conveyor belts may prevent or significantly reduce the development of biofilms by L. monocytogenes. These findings will need to be more fully explored using other belt types as well as individual and mixed bacterial strains.
From Proceedings of the "Midwest Poultry Federation Convention", St. Paul, Minnesota, U.S.A.
