Progress 07/01/17 to 06/30/20
Outputs
Target Audience:The target audiences reached during this reporting periodincludes all stakeholders in the food industry including producers, farmers, regulators,retailers, industry groups and educators. Our primary audience is value added dairy producers. Producers were reachedthrough publications, workshops, presentations, as well as extension, outreach, and consulting activities.Graduate and undergraduate students were also reached through mentorship and laboratory instruction. Changes/Problems: Initial trials demonstrated that none of the original eight protective cultures were capable of controlling EHEC in raw milk incubated according to a cheesemaking temperature profile. This did not provide us with a promising candidate for cheesemaking trials under objective 3. Through additional outside funding we were able to screen additional protective cultures against the pathogens targeted in this proposal as well as additional non-O157 shiga toxin-producing E. coli (STEC). Through that work, we identified a culture that was capable of inhibiting both O157 and non-O157 shiga toxin- producing E. coli (STEC). Therefore, we used this protective culture in cheesemaking trials under objective 3 and included additional STEC beyond just O157 to the cocktail. What opportunities for training and professional development has the project provided? The project provided one on one training opportunities for two graduate students as well as nine undergraduate students increasing their proficiency in agriculture related laboratory research. The graduate students working on this project attended both the 2018 and 2019 International Association for Food Protection Annual Meetings to present their results. This provided a unique opportunity for professional development to increase their knowledge and expertise in the field of food safety and microbiology. How have the results been disseminated to communities of interest? Results and other insight gained from this research have been shared with stakeholders through the PIs extension work withsmall-scale value-added dairy processors. We were able to reach a number of stakeholders thorough educational presentations at the 2018 and 2019 International Association for Food Protection Annual Meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Knowledge was gained on the compatibility of commercial protective cultures with commercial cheese making microbes as well as their efficacy against foodborne pathogens under various conditions including the manufacture of cheese. Two graduate students and nine undergraduate students also learned new microbiological techniques and methods. 1: Identify compatible combinations of commercial protective and cheesemaking cultures. With the assistance of a graduate student and several undergraduate research students, we completed both direct and deferred antagonism assays to determine compatibility of 10 protective cultures with each of 8 starter and 4 ripening bacteria cultures, as well as 3 yeast and 9 mold cultures used in the manufacture of cheese. The impact of protective cultures on the acidification profiles of starter cultures was also determined. We collected data on the inhibition, or lack thereof, of starter and ripening cultures used in cheese production. These data identified protective cultures that are compatible and those that are potentially incompatible with cultures necessary for the production of cheese. For example, one culture was found to inhibit all starter and ripening cultures in the direct agar assay but did not affect acidification during co-culture. This protective culture was not inhibitory against yeast and molds. Two of the three yeast strains and all blue molds were not inhibited by the presence and growth of protective cultures, while varying degrees of inhibition were observed for white molds. These data represent new knowledge and will be shared with stakeholders so that they can make informed decisions about which cultures they can use and those that they may want to avoid based on the starter and ripening cultures currently used in the manufacture of specific cheeses. It is expected that this change in knowledge will lead to a change in action once shared. 2: Determine the antimicrobial activity of commercial protective cultures against L. monocytogenes and EHEC. With the assistance of two graduate students and several undergraduate research students, we conducted experiments to determine the antimicrobial activity of 8 commercial protective cultures against Listeria monocytogenes and Escherichia coli O157:H7. Using cell free supernatants (CFS) of protective cultures we identified synergistic and antagonistic combinations of protective cultures against these pathogens. In those experiments, concentrations of CFS that inhibited pathogen growth to half that of the control (IC50) were identified and compared. Diagonal measurement of n-way drug interactions methodology was used to identify and analyze interactions using fractional inhibitory concentration scoring calculated from observed and expected IC50 values. We found that supernatants from all protective cultures were able to inhibit the growth of both pathogens at varying levels. Although no synergistic combinations were identified against EHEC, combinations of cultures were synergistic against Listeria monocytogenes. These experiments generated new knowledge regarding the efficacy of eight commercial PCs for the control of foodborne pathogens. The experiments described also represent a novel sampling and scoring method to identify synergistic and antagonistic combinations of protective cultures. Using the data collected, we completed co-culture assays in raw milk containing both the target pathogen and protective cultures individually and in combinations through a simulated cheesemaking temperature profile of 4 h at 35°C followed by 18 h at 20°C. None of the 8 protective cultures, or their combinations, tested in this project were inhibitory to the growth of EHEC in raw milk compared to control. However, through a synergistic project we identified a culture of Hafnia alvei that inhibited the growth of STEC O157 andnon-O157 STECin coculture to ~2 log, and ~3.5 logCFU/mL lower than pathogen controls, respectively.Protective cultures were more effective against L. monocytogenes. We identified two protective cultures that were capable on inhibiting L. monocytogenes growth though the entire incubation period. We also identified a single culture that reduced L. monocytogenes counts to <1 log CFU/mL by 24 h and inhibited further growth though the duration of incubation. In contrast, the control reached ~6 log CFU/mL. We also identified one protective culture combination with an additive effect and one synergistic combination. The synergistic combination reduced L. monocytogenes counts to undetectable levels by hour 96 with no subsequent regrowth. This work demonstrates the effectiveness of the protective cultures for the control of pathogens in an intended dairy matrix (raw milk) and under simulated use parameters (temperature and time). Differences in effectiveness against different pathogens inform future applications in the production of cheese and other dairy products. 3: Determine the effect of commercial protective cultures onL. monocytogenesand EHEC during the manufacture and storage of soft-ripened cheese manufactured from raw milk. With the assistance of a graduate student and several undergraduate research students we evaluated two commercially available protective cultures for the control ofListeria monocytogenesand shiga-toxin producingEscherichia coli(STEC)in the manufacture and ripening of a surface-mold- ripened soft cheese. Cheese was manufactured in the lab from raw milk intentionally contaminated with cocktails of either pathogen at a concentration of ~2 log CFU/mL prior to manufacture. Commercially available PCs previously identified as effective against each pathogen were added to contaminated milk according to manufacturer's instructions. After de-hooping and dry salting, cheeses were ripened in a controlled environmental chamber for 14 days prior to storage at 4°C through day 63 and 7°C for another 8 weeks. Pathogens were enumerated throughout the cheesemaking process and bi-weekly throughout ripening and storage. Counts of STEC, but not L. monocytogenes, increased during the milk ripening stage while both pathogens were concentrated in the cheese curd over the course of manufacture and pressing. MeanL. monocytogenescounts in control and treatment cheeses decreased slightly by day 35 and increased gradually thereafter. In contrast, STEC counts decreased on both the control and treatments throughout ripening and storage.Overall, aging alone was not sufficient to eliminateL. monocytogenesor STEC in a surface-mold-ripened soft cheese made with raw milk with both pathogens detectable in cheeses 120 days after manufacture. The addition of protective bacterial cultures with demonstrated efficacy in raw milk did not provide additional control of these pathogens under the conditions tested.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Gensler, C.A., Brown, S.R.B., Aljasir, S.F, and D. D'Amico. 2020. Compatibility of commercially produced protective cultures with common cheesemaking cultures and their antagonistic effect on foodborne pathogens. J Food Prot.. doi: 10.4315/JFP-19-614.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Aljasir, S.F., Gensler, C., Sun, L. and D'Amico, D.J., 2020. The efficacy of individual and combined commercial protective cultures against Listeria monocytogenes, Salmonella, O157 and non-O157 shiga toxin-producing Escherichia coli in growth medium and raw milk. Food Control, 109: p.106924. https://doi.org/10.1016/j.foodcont.2019.106924
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Gensler, Catherine, "Evaluation of Commercial Protective Cultures for the Control of Listeria monocytogenes and Shiga Toxin-Producing Escherichia coli in Raw Milk Cheese" (2019). Master's Theses. 1412.
https://opencommons.uconn.edu/gs_theses/1412
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Aljasir, S., Gensler, C., and D. D'Amico. 2019. Determining the Effect of Individual or Combined Protective Cultures on the Growth of Listeria monocytogenes and Shiga Toxin-Producing Escherichia coli in Raw Milk. Journal of Food Protection. 82 (suppl.):243.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Gensler, C, and D. D'Amico. 2019. Evaluation of Commercially Available Protective Cultures to Control Listeria monocytogenes and Shiga-Toxin Producing Escherichia coli in Soft, Surface-Mold Ripened Raw Milk Cheese. Journal of Food Protection. 82 (suppl.):59.
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Progress 07/01/18 to 06/30/19
Outputs
Target Audience:The target audience reached during this reporting period includes all stakeholders in the dairy food product industry including producers, regulators, retailers, industry groups, regulators, retailers, industry groups and educators. Producers were reached through extension, outreach, and consulting activities. Regulators, industry groups, scientists and educators were reached through presentations at an international food safety conference. Efforts on this project, including laboratory instruction, also reached undergraduate and graduate students involved in, and mentored during, this work. Changes/Problems:Initial trials demonstrated that none of the original eight protective cultures were capable of controlling EHEC in raw milk incubated according to a cheesemaking temperature profile.This did not provide us with a promising candidate for cheesemaking trials under objective 3. Through additional outside funding we were able to screen additional protective cultures against the pathogens targeted in this proposal as well as additional non-O157 shiga toxin-producing E. coli (STEC).Through that work, we identified a culture that was capable of inhibiting both O157 and non-O157 shiga toxin-producing E. coli (STEC). Therefore, we decided to use this culture in cheesemaking trials under objective 3 and to include additional STEC beyond just O157 to the cocktail. What opportunities for training and professional development has the project provided?The project provided one on one training opportunities for two graduate students as well as nine undergraduate students increasing their proficiency in agriculture related laboratory research. The graduate students working on this project are traveling to the 2019 International Association for Food Protection Annual Meeting to present their results. This will also provide a unique opportunity for professional development to increase their knowledge and expertise in the field of food safety and microbiology. How have the results been disseminated to communities of interest?Results and other insight gained from this research have been shared with stakeholders through the PIs extension work with small-scale value-added dairy processors. We were able to reach a number of stakeholders thorough educational presentations at the 2019 International Association for Food Protection Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will complete the cheesemaking trials under objective 3 to determine the effect of commercial protective cultures on L. monocytogenes and STEC during the manufacture and storage of cheese manufactured from raw milk.
Impacts
What was accomplished under these goals?
Knowledge was gained on the compatibility of commercial protective cultures with commercial cheese making microbes as well as their efficacy against foodborne pathogens under various conditions including the manufacture of cheese. Two graduate students and nine undergraduate students also learned new microbiological techniques and methods. 1: Identify compatible combinations of commercial protective and cheesemaking cultures. With the assistance of a graduate student and several undergraduate research students, we completed both direct and deferred antagonism assays to determine compatibility of 10 protective cultures with each of 8 starter and 4 ripening bacteria cultures, as well as 3 yeast and 9 mold cultures used in the manufacture of cheese. The impact of protective cultures on the acidification profiles of starter cultures was also determined. We collected data on the inhibition, or lack thereof, of starter and ripening cultures used in cheese production. These data identified protective cultures that are compatible and those that are potentially incompatible with cultures necessary for the production of cheese. For example, one culture was found to inhibit all starter and ripening cultures in the direct agar assay but did not affect acidification during co-culture. This protective culture was not inhibitory against yeast and molds. Two of the three yeast strains and all blue molds were not inhibited by the presence and growth of protective cultures, while varying degrees of inhibition were observed for white molds. These data represent new knowledge and will be shared with stakeholders so that they can make informed decisions about which cultures they can use and those that they may want to avoid based on the starter and ripening cultures currently used in the manufacture of specific cheeses. It is expected that this change in knowledge will lead to a change in action once shared. 2: Determine the antimicrobial activity of commercial protective cultures against L. monocytogenes and EHEC. With the assistance of two graduate students and several undergraduate research students, we conducted experiments to determine the antimicrobial activity of 8 commercial protective cultures against Listeria monocytogenes and Escherichia coli O157:H7. Using cell free supernatants (CFS) of protective cultures we identified synergistic and antagonistic combinations of protective cultures against these pathogens. In those experiments, concentrations of CFS that inhibited pathogen growth to half that of the control (IC50) were identified and compared. Diagonal measurement of n-way drug interactions methodology was used to identify and analyze interactions using fractional inhibitory concentration scoring calculated from observed and expected IC50 values. We found that supernatants from all protective cultures were able to inhibit the growth of both pathogens at varying levels. Although no synergistic combinations were identified against EHEC, combinations of cultures were synergistic against Listeria monocytogenes. These experiments generated new knowledge regarding the efficacy of eight commercial PCs for the control of foodborne pathogens. The experiments described also represent a novel sampling and scoring method to identify synergistic and antagonistic combinations of protective cultures. Using the data collected, we completed co-culture assays in raw milk containing both the target pathogen and protective cultures individually and in combinations through a simulated cheesemaking temperature profile of 4 h at 35°C followed by 18 h at 20°C. None of the 8 protective cultures, or their combinations, tested in this project were inhibitory to the growth of EHEC in raw milk compared to control. Protective cultures were more effective against L. monocytogenes.We identified two protective cultures that were capable on inhibiting L. monocytogenes growth though the entire incubation period. We also identified a single culture that reduced L. monocytogenes counts to <1 log CFU/mL by 24 h and inhibited further growth though the duration of incubation. In contrast, the control reached ~6 log CFU/mL. We also identified one protective culture combination with an additive effect and one synergistic combination. The synergistic combination reduced L. monocytogenes counts to undetectable levels by hour 96 with no subsequent regrowth. This work demonstrates the effectiveness of the protective cultures for the control of pathogens in an intended dairy matrix (raw milk) and under simulated use parameters (temperature and time). Differences in effectiveness against different pathogens inform future applications in the production of cheese and other dairy products. 3: Determine the effect of commercial protective cultures on L. monocytogenes and EHEC during the manufacture and storage of soft-ripened cheese manufactured from raw milk. Using the data generated in objectives 1 and 2, we selected a single protective culture for the challenge studies in cheese. Through supplemental funding from another sponsor, we identified a protective culture that was capable of inhibiting both O157 and non-O157 shiga toxin-producing E. coli (STEC). Therefore, we selected this culture for the cheese challenge studies. Cheese was manufactured in the lab from raw milk intentionally contaminated with 6 and 7 strain cocktails of either LM or STEC, respectively, at a concentration of ~2 log CFU/mL prior to manufacture. Commercially available PCs previously identified as effective against each pathogen were added to contaminated milk at a concentration of ~6 log CFU/mL according to manufacturer instructions. After de-hooping and dry salting, cheeses were ripened in a controlled environmental chamber (12°C, 93% RH) for 14 days prior to storage at 4°C up to day 63 and 7°C for another 8 weeks. Pathogens were enumerated throughout the cheesemaking process and bi-weekly throughout ripening and storage. Two independent batches of each control and treatment were used for analysis. STEC, but not LM, concentration increased over milk ripening. Both pathogens were concentrated in the cheese curd over the course of manufacture and pressing. Mean LM concentration in control and treatment cheeses decreased slightly by day 35, relative to starting inoculation, and increased gradually out to day 120. LM counts were associated with the extent of cheese ripening, which varied between trials. STEC was detectable in cheese out to day 120. No differences in pathogen counts were observed between control and treatment cheeses.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Gensler, C., and D. D'Amico. 2018. Determining the Efficacy of Protective Cultures for the Control of Listeria monocytogenes and Non-O157 Shiga Toxin-producing Escherichia coli in Raw Milk for Cheesemaking. Journal of Food Protection. 81 (suppl.):63
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Sun, L., and D. D'Amico. 2018. Antimicrobial Activity of Commercial Protective Cultures against Listeria monocytogenes and Escherichia coli O157:H7. Journal of Food Protection. 81 (suppl.):37
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Gensler, C, and D. D'Amico. 2019. Evaluation of Commercially Available Protective Cultures to Control Listeria monocytogenes and Shiga-Toxin Producing Escherichia coli in Soft, Surface-Mold Ripened Raw Milk Cheese. IAFP 2019
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Aljasir, S., Gensler, C., and D. D'Amico. 2019. Determining the Effect of Individual or Combined Protective Cultures on the Growth of Listeria monocytogenes and Shiga Toxin-Producing Escherichia coli in Raw Milk. IAFP 2019
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Progress 07/01/17 to 06/30/18
Outputs
Target Audience:The target audience reached during this reporting through period includes all stakeholders in the dairy food product industry including scientists, producers, food safety professionals, regulators, retailers, industry groups and educators. Our primary audience is value-added dairy producers. Producers were reached through extension, outreach, and consulting activities. Efforts on this project, including laboratory instruction, also reached undergraduate and graduate students involved and mentored during this work. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided one on one training opportunities for two graduate students as well as nine undergraduate students increasing their proficiency in agriculture related laboratory research. The graduate students working on this project are traveling to the 2018 International Association for Food Protection Annual Meeting to present their results. This will also provide a unique opportunity for professional development to increase their knowledge and expertise in the field of food safety and microbiology. How have the results been disseminated to communities of interest?Results and other insight gained from this research have been shared with stakeholders through the PIs extension work with small-scale value-added dairy processors. We were able to reach a number of stakeholders thorough educational presentations at the 2018 International Association for Food Protection Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will complete the coculture trials and select protective culture treatments to be used in cheese manufacturing experiments. We will then determine the effect of commercial protective cultures on L. monocytogenes and EHEC during the manufacture and storage of soft-ripened cheese manufactured from raw milk.
Impacts
What was accomplished under these goals?
1: Identify compatible combinations of commercial protective and cheesemaking cultures. With the assistance of a graduate student and several undergraduate research students, we completed both direct and deferred antagonism assays to determine compatibility of 10 protective cultures with each of 8 starter and 4 ripening bacteria cultures, as well as 3 yeast and 9 mold cultures used in the manufacture of cheese. The impact of protective cultures on the acidification profiles of starter cultures was also determined. We collected data on the inhibition, or lack thereof, of starter and ripening cultures used in cheese production. These data identified protective cultures that are compatible and those that are potentially incompatible with cultures necessary for the production of cheese. For example, one culture was found to inhibit all starter and ripening cultures in the direct agar assay but did not affect acidification during co-culture. This protective culture was not inhibitory against yeast and molds. Two of the three yeast strains and all blue molds were not inhibited by the presence and growth of protective cultures, while varying degrees of inhibition were observed for white molds. These data represent new knowledge and will be shared with stakeholders so that they can make informed decisions about which cultures they can use and those that they may want to avoid based on the starter and ripening cultures currently used in the manufacture of specific cheeses. It is expected that this change in knowledge will lead to a change in action once shared. 2: Determine the antimicrobial activity of commercial protective cultures against L. monocytogenes and EHEC. With the assistance of two graduate students and several undergraduate research students, we conducted experiments to determine the antimicrobial activity of 8 commercial protective cultures against Listeria monocytogenes and Escherichia coli O157:H7. Using cell free supernatants (CFS) of protective cultures we identified synergistic and antagonistic combinations of protective cultures against these pathogens. In those experiments, concentrations of CFS that inhibited pathogen growth to half that of the control (IC50) were identified and compared. Diagonal measurement of n-way drug interactions methodology was used to identify and analyze interactions using fractional inhibitory concentration scoring calculated from observed and expected IC50 values. We found that supernatants from all protective cultures were able to inhibit the growth of both pathogens at varying levels. Although no synergistic combinations were identified against EHEC, combinations of cultures were synergistic against Listeria monocytogenes. These experiments generated new knowledge regarding the efficacy of eight commercial PCs for the control of foodborne pathogens. The experiments described also represent a novel sampling and scoring method to identify synergistic and antagonistic combinations of protective cultures. Using the data collected, we conducted co-culture assays in raw milk containing both the target pathogen and protective culture at 35°C for 24 h or a simulated cheesemaking temperature profile of 4 h at 35°C followed by 18 h at 20°C and 7 days at 12°C. Thus far we have not identified a protective culture that substantially inhibits the growth of EHEC under either temperature condition. Protective cultures are more effective against L. monocytogenes.Thus far we have identified two cultures that inhibited Listeria growth to ≤ 0.5 log CFU/mL after incubation under both temperature conditions. This work demonstrates the effectiveness of the protective cultures for the control of pathogens in an intended dairy matrix (raw milk) and under simulated use parameters (temperature and time). Differences in effectiveness against different pathogens inform future applications in the production of cheese and other dairy products. 3: Determine the effect of commercial protective cultures on L. monocytogenes and EHEC during the manufacture and storage of soft-ripened cheese manufactured from raw milk. Nothing to report during this reporting period for this goal.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Gensler, C., and D.J. D'Amico. Determining the Efficacy of Protective Cultures for the Control of Listeria monocytogenes and non-O157 STEC Escherichia coli in Raw Milk for Cheesemaking. To be presented at the 2018 IAFP Annual Meeting.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Sun, L. and D.J. D'Amico.Antimicrobial Activity of Commercial Protective Cultures Against Listeria monocytogenes and Escherichia coli O157:H7. To be presented at the 2018 IAFP Annual Meeting.
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