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Recovery, Development and Validation of Appropriate Surrogate Microorganisms in Meat and Poultry Emulsions for In-plant Critical Control Point Validation Studies


The goal of this research is to validate how thermal destruction of a non-pathogenic (surrogate) microorganism (Pediococci sp. NRRL B-2354) relates to that of path owns of concern in meat products (e.g. Listeria monocytogenes and Salmonella). An appropriate surrogate could be used in the factory setting to validate the safety of complex multi-variable thermal processes on a periodic basis. This approach if successful could be used to verify thermal process CCPs in the context of HACCP. Pediococcus sp. NRRL B-2354 (formerly, Micrococcus freudenreichh) was selected as the surrogate because USDA researchers had proposed its use with fluid products due to its high heat resistance and non-pathogenicity and because prior research by the principal investigator revealed similar heat resistance to Salmonella senftenberg 775W when tested in fluid milk. The composition of food matrices can have a profound impact on bacterial heat resistance. Data is lacking on the heat resistance of Pediococcus sp., NRRL B-2354 in relation to Listeria monocytogenes or Salmonella in solid foods including meats.<p>
Trials to determine the heat resistance of selected organisms (e.g. Listeria monocytogenes and Pediococcus) at four temperatures in ground beef is planned. Some challenges of ground beef have been performed with Pediococcus, Salmonella Senftenberg 775W (renown for its high heat resistance), S. Typhimurium DT104, and Listeria monocytogenes.

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Philosophy behind experimental approach<p>
Traditional approaches to this type of work involve experimental designs with one organism per sample. These designs can be very time consuming to implement. However, if all challenge organisms could be added to each sample more rapid generation of data should be possible, assuming no interaction between organisms occurs. In order for this approach to be implemented, the organisms should also be able to be differentiated from one another on recovery medium.<p>
Status: Media studies Direct plating media Studies were performed with eight differential selective media. A medium (PRAB++) able to recover the Pediococcus surrogate was developed that would not recover Salmonella or Listeria strains (manuscript in progress). Media screened for recovery of Listeria to the exclusion of other challenge organisms included Modified Oxford (MOX), and PALCAM. These media inhibited recovery of Salmonella and Pediococci. Salmonella recovery media XLT4, XLD and MLIA inhibited recovery of Pediococcus and Listeria strains while recovering Salmonella strains very well. XLT4 media effectively inhibited background microflora to a greater extent than XLD or MLIA in a preliminary study on Salmonella inoculated ground beef. The S. senftenberg strain was readily distinguishable from the S. Typhimurium DT 104 strain by yellow and black colonies formed on XLT4, respectively.<p>
Recovery of heat inured cells by direct plating
Media for use in thermal studies must also have the ability to recover heat-injured cells. An approach was developed that combined a modified Thin Agar Layer (TAL) approach with spiral plating (manuscript in preparation). Recovery of heat treated Listeria monocytogenes 101M (a meat isolate), S. senftenberg 775W and Pediococcus at 62 C in broth for various lengths of time was determined by plating onto non-selective (TSAYE), selective media [MOX, XLT4 and PRAB (++)] and TSAYE overlaid on each of these selective media. Recovery of challenged organisms was not significantly different between non-selective TSAYE medium and TSAYE overlaid selective media. However, cell recovery on selective agar alone was reduced significantly. This data indicated that the overlay approach to spiral plating of samples would recover heat injured cells.<p>
Universal pre-enrichment broth (UPB) was evaluated for its ability to qualitatively recover Salmonella, Listeria monocytogenes and Pediococcus added at low (e.g. 1 cell per ml) and "high" levels (100 cells per ml) in all possible combinations (e.g. of organism and level). These levels were selected, because they are known to be below the detection limit of qualitative assays used, unless growth of the organisms occurred. Control trials were also assayed with individual strains. The VidasT SLM and LMO assays were used to measure detection of Salmonella, and Listeria monocytogenes, respectively. UPB enrichments were streaked to plates of PRAB++ agar. UPB enrichment effectively recovered all organisms after 24, and 48 hours at 37 C. Consequently, UPB was used as an enrichment media in later trials with ground beef.<p>
Preliminary thermal challenge<p>
Survival in Trypticase Soy Broth (TSB) at 62 C of cultures of S. senftenberg 775W, S. Typhimurium DT104, Pedicococcus sp. NRRL B 2354, and nine strains of Listeria monocytogenes grown in TSB overnight was evaluated. Each overnight culture was added to 5 individual capillary tubes (25 gL) per sampling time/organism combination and immersed in a 62 C water bath for 3, 6, 10 and 15 minutes. Subsequent to thermal treatment capillary tubes were removed, immediately cooled in an ice bath, their surfaces disinfected with ethanol, asceptically broken and their contents added to 5 mls of pre-sterilized enrichment broth. Qualitative assays were performed. Pediococcus sp., and S. senftenberg 775W were recovered in 5 of 5, and 2 of 5 capillary tubes after
15 minutes, respectively. Most other organisms tested did not survive even 3 minutes at 62 C, with the exception of L. monocytogenes strains 1O1M, 802 and LCDC where organisms were recovered from 1 of 5, 1 of 5 and 2 of 5 tubes respectively. None of the Listeria monocytogenes strains tested were recovered at 6 minutes at 62 C. L. monocytogenes strain IOIM was selected for further studies due to its high heat resistance relative to most of other L. monocytogenes strains tested and because it had previously been isolated from a meat product. S. Typhimurium DT104 did not survive in 5 of 5 capillary tubes after 3 minutes at 62 C.<p>
Preliminary thermal trials in Ground Beef
A preliminary study was performed in retail lean ground beef inoculated with 5.37, 6.00, 6.18, and 6.70 logo CFU of Pediococcus, S. senftenberg 775W, L. monocytogenes 101 M, and S. Typhimurium DT 104, respectively. Validation studies were previously done to ensure effective cell distribution during the inoculation and mixing process. Inoculated lean ground beef was then added to three pre-sterilized Whirl-Pakrm bags per sampling time, flattened to 0.051 mm and placed into a thermostatically controlled water bath for trials at 62 C. Samples were removed after 0.67, 5, 15, and 25 minutes, immediately chilled and enriched in UPB. UPB was tested, as described above under "Enrichment". The heat resistance of the isolates was (in order from highest to lowest) Pediococcus (detected in 3 of 3 samples at 25 minutes), S. senftenberg 775W (3/3 at 15 min but no survivors detected at 25 min.), L. monocytogenes IOIM (1 of 3 at 15 min.) and S. Typhimurium DT 104 (0 of 3 at 0.67 min.). This work indicated high resistance of the surrogate compared to L. monocytogenes and S. Typhimurium DT104.
Effect of growth media on the heat resistance of challenge organisms<p>
All four organisms were grown in separate tubes of MRS broth, TSB, and TGY broth to determine the influence of growth media on the heat resistance of the cells. After growth in these media, cells were centrifuge and washed in 1 % beef broth three times and inoculated into pre-sterilized beef broth pre-heated to 62 C to obtain an instantaneous temperature come up time. Analysis of data indicated that growth in MRS resulted in greater Pediococcus thermal tolerance than when cells grew in TGY and TSB. The opposite appeared to be the case for L. monocytogenes and S. senftenberg 775 W. Consequently future experiments were planned where TSB would be the growth medium of choice to diminish thermal resistance of Pediococcus and enhance that of Salmonella and L. monocytogenes.<p>
Second Ground beef trial<p>
A second 62 C trail was performed with inoculated lean (4% fat) ground beef. Inoculum levels Pediococcus sp. NRRL B-2354 were about 5.8 logo cfu per gram; whereas, levels of S. senftenberg 775W, S. Typhimurium , and L. monocytogenes were about 6.7 logo cfu per gram. Samples were taken at 0.75, 2, 3, 4, 5, 6, 7, 8, 10, 15, 25, 35, 45, 55, and 65 minutes. Pediococci remained the most heat resistant of the four strains assays and were quantitatively recoverable at 25 but not 35 minutes, but survivors were detected by enrichment for the entire length of the trial. S. senftenberg 775W were detected by quantitative techniques at 8 but not 10 minute but by enrichment at 55 but not 65 minutes, suggesting the Pediococcus sp. NRRL B-2354 is an acceptable surrogate for S. senftenberg 77W. However, L. monocytogenes 101M was quantitatively detected at 2 but not 4 minutes and detected by enrichment at 3 but
not 4 minutes. S. Typhimurium DT104 was not detected at 0.75 minutes either quantitatively or qualitatively.<p>
Jonesia trial<p>
A preliminary study was done with Jonesia denitrificans (formerly Listeria denitrificans) in broth that suggested this potential Listeria surrogate was not as heat resistant as the L. monocytogenes 101 M strain.<p>
Future plans<p>
A study is planned to evaluate the use of co-inoculated organisms on the heat resistance of individually inoculated strains. An attempt to develop a mathematical model correlating heat resistance of Pediococcus sp. NRRL B 2354 with Listeria monocytogenes IOIM, and S. senftenberg 775W at a variety of temperatures in lean ground beef is planned. The use of S. Typhimurium DT104 will be dropped due to its extreme heat sensitivity compared to L. monocytogenes 101 M. Examination of raw quantitative data from this trial suggests that relatively small reductions (e.g. 0.5 logo cu/g) of Pediococcus sp. NRRL B-2354 in ground beef at 62 C corresponds to much larger reductions of L. monocytogenes 101 M (e.g. 5.5 log o reduction), whereas 1 log o reduction in the Pediococcus surrogate corresponded with a 3.5 logo reduction of S. senftenberg 775W. This information has practical significance in that far smaller numbers of the Pediococcus surrogate need be added to raw meat emulsion blends prior to thermal processing (with appropriate factory-based processes and products) to validate an L. monocytogenes or Salmonella kill in an industrial trial. If this trend (e.g. higher thermal resistance of Pediococcus compared to pathogens) continues at other temperatures it will thus eliminate the need to produce large volumes of the surrogate that would have been required if the surrogate had similar heat resistance to the selected pathogens.<p>


In ground beef containing 12% fat, L. monocytogenes was consistently more heat sensitive than S. Senftenberg at all four temperatures tested. Also, results indicated that thermal treatments that kill E. faecium 2354 in ground beef would also kill L. monocytogenes and Salmonella. In a search for a less heat resistant surrogate than E. faecium 2354, the heat resistance of Pediococcus parvulus HP and Pediococcus acidilactici LP, which are used as commercial meat starter cultures, were compared. These two Pediococcus strains may be alternate surrogates for validation studies when a less heat resistant surrogate is desired; however, studies at additional temperatures are needed with these strains for validation of a temperature range of 58 to 68°C.<p>

Deliverable <p>

Depending on the margin of safety desired, processors could use E. faecium 2354 as a surrogate for validation studies of thermal processes for lean and 12% fat ground beef at 58 and 68°C.

Kornacki, Jeffrey; Doyle, Michael
University of Georgia
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