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- Title
- ENRICHMENT OF COXIELLA BURNETII FROM BOVINE AND NON-BOVINE MILKS USING AN AXENIC LIQUID CULTURE MEDIUM
- Creator
- Shi, Manman
- Date
- 2016, 2016-07
- Description
-
Non-bovine milks are becoming more popular as beverages and for making cheeses, however little are understood regarding how pathogens are...
Show moreNon-bovine milks are becoming more popular as beverages and for making cheeses, however little are understood regarding how pathogens are inactivated in these milks during pasteurization. Recently, a tissue culture-PCR method has been developed to quantify inactivation of Coxiella burnetii, the obligate intracellular bacterium which is the reference pathogen for milk pasteurization. Unfortunately, this method is time-consuming and quite laborious. A potential improvement over the ICC-PCR method may be the use of a new specialized liquid medium which has been shown to allow growth of pure cultures of C. burnetii outside of host cells. In this study, ACCM-2 was evaluated for its’ ability to enrich C. burnetii from bovine and non-bovine milks as a potential alternative to tissue culture enrichment for Most Probably Number (MPN) quantification of inactivation of C. burnetii in milks. C. burnetii in bovine whole milk and cream (as a surrogate for a high fat product), camel, water buffalo, and goat milk were grown in ACCM-2 media (1:10) in 10 mL volumes in T-25 flasks at 37 °C under 5% CO2 and 2.5% O2 with sampling over 14 days. C. burnetii levels were determined by quantitative PCR (qPCR) against a standard curve for the Coxiella-specific IS1111a gene. C. burnetii in all milks grew at least 3 logs over 10 days with the exception of the water buffalo milk which did not allow growth. Bovine milk allowed the least growth and additional studies determined that the detection limit for growth of C. burnetii from this milk is about 6 ge/mL. Additional studies showed that adjustment of the pH from ~5.0 to 4.75 results in much improved growth of C. burnetii from bovine milk. Adjustment of pH did not allow growth from water buffalo milk, however additional dilution of the milk did allow improved C. burnetii growth but the added dilution also decreases the detection limit of the proposed new MPN-PCR assay. Further studies were completed to determine how thermal injury affects recover and growth of Coxiella burnetii in ACCM-2 media after thermal processing at 64 °C for up to 8 minutes. Although injury did increase the lag phase period and reduce the log phase growth rate from these samples, recovery and growth of at least 0.5 log was possible from all samples indicating that this media should be useful as an alternative enrichment method for use in an MPN format for quantification of thermal inactivation of C. burnetii in both bovine and non-bovine milks.
M.S. in Food Safety and Technology, July 2016
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- Title
- Efficacy of Organic Acid Treatments for the Reduction of Listeria Monocytogenes on Hard Boiled Eggs
- Creator
- Khouja, Bashayer
- Date
- 2022
- Description
-
Ready-to-eat hard-boiled eggs (HBEs) are a popular and convenient choice for consumers and food servicers. Recentrecalls of hard-boiled eggs...
Show moreReady-to-eat hard-boiled eggs (HBEs) are a popular and convenient choice for consumers and food servicers. Recentrecalls of hard-boiled eggs have highlighted the susceptibility of contamination with Listeria monocytogenes. HBEs are generally treated with antibacterials to ensure the safety and quality of the product. While citric acid is often used, research has determined it is not effective in some situations; therefore, the assessment of additional organic acids is necessary. This study examined the efficacy of acetic, lactic, and malic acid on the reduction of L. monocytogenes on HBEs after a 24- hour treatment trials and 28 days storage trials. Fresh eggs were cooked in boiling water, peeled, and stored at 4°C for 24h before use. For treatment trials, HBEs were dip- inoculated with a 4-strain cocktail of rifampicin resistant L. monocytogenes, resulting in 8 log CFU/egg. Following air drying, hard-boiled eggs were treated at 5 or 25°C with 2% acetic, lactic, or malic acid. L monocytogenes populations were enumerated in intervals up to 24h by homogenization of HBEs with BLEB and cultivation on BHIrif. For pre- treatment storage trials, HBEs were first dip- inoculated with a rifampicin- resistant 4- strain L. monocytogenes cocktail for 20 min, resulting in 1 log CFU/egg, air dried for 10 min, followed by treatment with 2% acetic, lactic, or malic acid for 24 h at either 5 or 25°C. For post- treatment inoculation trials, HBEs were first soaked in 2% acetic, lactic, or malic acid for 24 h at either 5 or 25°C, air dried for 10 min, spot-inoculated at 1 log CFU/egg, and then dried for 20 min. All HBEs were individually stored in bags at 5°C for up to 28 days. The presence of L. monocytogenes was determined at intervals during storage by enrichment with BLEB on BHIrif and Brilliance Listeria Agar. Triplicate eggs were assessed for each timepoint, and three independent trials were conducted. Data were analyzed by Student’s T-test, ANOVA, and Fisher’s exact test, p≤0.05. The initial inoculation level of L. monocytogenes on HBEs was 8.27±0.37 log CFU/egg. After 24 h treatment, all L. monocytogenes populations were significantly reduced on HBEs. At 5°C, populations were reduced by 3.15±0.70, 3.46±0.02, and 4.78±0.23 log CFU/egg. Compared to 5°C, a significantly higher population reduction occurred with acetic and lactic acid when treatment occurred at 25°C. The inactivation of L. monocytogenes on HBEs for the storage trials was associated with the order of the contamination: pre-or post-the acid treatment. Prior storage, L. monocytogenes was detected on 100% of the HBEs. Malic acid pre-treatment was significantly effective in eliminating L. monocytogenes on HBEs at 5 and 25°C, while acetic acid was effective only at 5°C. All acids did not eliminate L. monocytogenes in the case of post-treatment contamination at any tested temperature. The results of this study aid in understanding the efficacy of organic acid treatments against L. monocytogenes on HBEs. Results are useful in the development of preventive controls and guidelines to ensure the safety of HBEs.
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