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THE TRANSCRIPTIONAL RESPONSE OF ARTIFICIALLY-INOCULATED LISTERIA MONOCYTOGENES DURING THE MANUFACTURE OF UNPASTEURIZED GOUDA CHEESE
Title
THE TRANSCRIPTIONAL RESPONSE OF ARTIFICIALLY-INOCULATED LISTERIA MONOCYTOGENES DURING THE MANUFACTURE OF UNPASTEURIZED GOUDA CHEESE
Creator
Carstens, Christina K.
Date
2017, 2017-05
Description
Listeriosis outbreaks indicate that L. monocytogenes contamination is an issue for various food types, such as unpasteurized (raw) cheese....
Show moreListeriosis outbreaks indicate that L. monocytogenes contamination is an issue for various food types, such as unpasteurized (raw) cheese. Current regulation prevents the interstate sale and distribution of raw cheese and mandates a ≥60 day aging period at ≥2˚C to ensure product safety; yet studies demonstrate that pathogens can persist during aging. Environmental stress can alter the transcriptomic profiles of pathogens; however, these surveys are rarely conducted in food matrices. This study aimed to assess the transcriptomic profiles of L. monocytogenes strain F2365 during environmental stressors inherent throughout cheesemaking. First, quantitative polymerase chain reaction (qPCR) was used to monitor transcription levels of nine L. monocytogenes genes involved in virulence, stress response, energy transport, and metabolism during osmotic stress (11% NaCl) and varying temperature/time (5˚C 24 h, 25˚C 24 h, 38˚C 30 min) conditions in Brain Heart Infusion (BHI) broth, raw milk, and pasteurized milk. Generally, the genes prfA, lmo1381, lmo0963, and lmo1875 were down-regulated, whereas the genes lmo1864, lmo0914, lmo0348, lmo1428, and lmo1264 were up-regulated. Virulence gene, prfA, was most down-regulated when L. monocytogenes was grown in raw milk with salt at 25˚C (4774.72±838.14 fold; relative to 24 h growth at 37˚C). The stress response gene lmo0914, encoding σB, was most up-regulated when L. monocytogenes was grown in BHI at 25˚C with salt (14.61±7.72 fold). Additionally, transcription levels of the nine genes were assessed at points during the laboratory-scale manufacture of Gouda cheese made with raw milk artificially-inoculated with L. monocytogenes via qPCR. Similar differential regulation for both prfA and lmo0914 in L. monocytogenes was observed during cheesemaking. The gene lmo1864, encoding a putative pore-forming hemolysin, was up-regulated throughout the cheesemaking process, but was most up-regulated after stirring the curd (449.81±432.53 fold). Ultimately, these results indicate that the lmo1864 gene may play a role in L. monocytogenes survival during cheesemaking. Methods developed in this study can be used to assess the risk of L. monocytogenes, not only during cheesemaking, but during the ≥60-day aging process. Overall, these results contribute to the understanding of L. monocytogenes survival mechanisms during the cheesemaking process.
M.S. in Food Safety and Technology, May 2017
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SURVIVAL OF LISTERIA MONOCYTOGENES AND ESCHERICHIA COLI O157:H7 DURING AGING OF GOUDA CHEESE MADE USING UNPASTEURIZED MILK
Title
SURVIVAL OF LISTERIA MONOCYTOGENES AND ESCHERICHIA COLI O157:H7 DURING AGING OF GOUDA CHEESE MADE USING UNPASTEURIZED MILK
Creator
Natarajan, Vidya
Date
2018, 2018-05
Description
The FDA code of federal regulations states that cheeses made using unpasteurized milk must be aged for a period of at least 60 days to...
Show moreThe FDA code of federal regulations states that cheeses made using unpasteurized milk must be aged for a period of at least 60 days to minimize the inherent risks associated with unpasteurized milk. However, there have been several foodborne outbreaks associated with 60-day aged semi-soft cheeses made using unpasteurized milk, specifically Gouda cheese. In this study, Gouda cheese was manufactured using unpasteurized milk artificially-inoculated with Listeria monocytogenes (1 or 3 log CFU/mL) and Escherichia coli O157:H7 (1 log CFU/mL). The Gouda cheese was pressed, brined, waxed, and aged at 10°C for 90 (for the 1 log CFU/mL) or 150 (for the 3 log CFU/mL) days. Samples were assessed during cheese manufacture and aging for survival of the pathogen as well as for the population dynamics of the native microflora including Enterobacteriaceae, yeast and mold, lactic acid bacteria, and mesophilic bacteria. In addition, cheese samples during aging were also analyzed for property characteristics including salt and moisture content, fat in solid content, pH, and water activity. Results determined that the population levels of both pathogens significantly increased during manufacture. During aging of the Gouda cheese, E. coli O157:H7 was capable of survival only until 49 days and was henceforth not detected via enrichment. For L. monocytogenes, pathogen populations were 2.07±0.12 log and 1.26±0.00 log CFU/g at 60 and 90 days of aging, respectively, for the 1 log CFU/mL initial inoculation level. Compared to day 60 (2.31±0.92 log CFU/g) of aging, the population of L. monocytogenes for the Gouda cheese made with the 3 log CFU/mL initial inoculation level was significantly higher (p<0.05) on both 90 and 150 d of aging (4.62±0.25 and 6.00±0.72 log CFU/g, respectively). During aging, the populations of lactic acid and mesophilic bacterial were significantly higher than other microflora categories. The population of yeast and mold displayed an increasing trend in population, whereas Enterobacteriaceae populations were highly unsteady. Increases in lactic acid bacterial populations were accompanied by decreases in pH and pathogen populations. These results indicate that the characteristics of Gouda cheese and the native microflora population may play a pivotal role in survival and growth of pathogens. Overall, this study suggests that the current 60-day aging regulation, while sufficient to control E. coli O157:H7, may not be suitable to control the risk of L. monocytogenes in Gouda cheese.The population of yeast and mold displayed an increasing trend in population, whereas Enterobacteriaceae populations were highly unsteady. Increases in lactic acid bacterial populations were accompanied by decreases in pH and pathogen populations. These results indicate that the characteristics of Gouda cheese and the native microflora population may play a pivotal role in survival and growth of pathogens. Overall, this study suggests that the current 60-day aging regulation, while sufficient to control E. coli O157:H7, may not be suitable to control the risk of L. monocytogenes in Gouda cheese.
M.S.in Food Safety and Technology, May 2018
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