Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: Prevention and decontamination of foodborne pathogen contamination on fresh fruits and vegetables was investigated. Irrigation water can serve as an important vector for the transmission of foodborne pathogens onto ready to eat fresh fruits and vegetables. As a means to prevent the contamination of fruits and vegetables at the production level, irrigation water was decontaminated using ultraviolet light. An ultraviolet light exposure of 14.2 mJ/cm2 was used to treat irrigation water from different sources and varying qualities. Due to the delicate nature of leafy greens, decontamination methods are limited. The most commonly used disinfection methods are chemical disinfectants such as chlorinated compounds and peroxyacetic acid. However, chemical disinfection has been shown to achieve only a 2-log reduction for pathogens on leafy greens. In an attempt to develop more effective decontamination methods for leafy greens, ultraviolet light, mild heat, and ethyl pyruvate were investigated as leafy green disinfection methods. Ultraviolet light was shown to achieve 1.8 log reduction with E. coli O157:H7 with 62.5 mJ/cm2 exposure. Combination treatments of UV with acidified sodium hypochlorite and 50C resulted in a greater than 5-log reduction. Ethyl pyruvate vapor was investigated as a gaseous disinfection method for leafy greens. It is currently used as a flavoring ingredient in foods and has GRAS status. Samples were treated with 0, 40, 100, and 400 ul/L) concentration of gaseous ethyl pyruvate in a 2.6 L enclosed container. The efficacy of ethyl pyruvate on E. coli O157:H7(GFP) was monitored over the course of 7 days at 4C and 10C.The lowest concentration of ethyl pyruvate (40 ul/L) resulted in a 1.4 log CFU/g reduction E. coli O157:H7(GFP) on green onions at 4C in 7 days, and 2.5 log CFU/g at 10C in 5 days (P < 0.05). In baby spinach, the same concentration and incubation time resulted in 0.9 log CFU/g and 1.4 log CFU/g (P < 0.05) of E. coli O157:H7(GFP) at 4C in 7 days and 10C in 5 days, respectively. On green onions, the highest concentration of ethyl pyruvate (400 ul/L) significantly reduced the population of E. coli O157:H7(GFP) by ~5.0 log CFU/g in 7 days at 4C, and 5 days at 10C (P < 0.05). It was also determined that the same concentration was significantly effective in reducing E. coli O157:H7(GFP) populations by 4.3 log at 4C in 7 days and approximately 7 log at 10C in 3 days with baby spinach (P <0.05). PARTICIPANTS: Lisa Jones, PhD student; Zeki Durak, PhD student; Ms.Laura Strawn, Ph.D. candidate; Martin Wiedmann, Professor; Christ Smart, Associate Professor of Plant Pathology; John Churey, Research Support Specialist; Randy Worobo, PI; Elizabeth Bihn, Senior Extension Associate; New York State Fruit and Vegetable Growers TARGET AUDIENCES: Consumers, fruit and vegetable growers, fruit and vegetable processors PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Ultraviolet light exposure of 14.2 mJ/cm2 was shown to be an effective decontamination method for irrigation water. A greater than 5-log reduction was achieved with all strains E. coli O157:H7 and Salmonella that were tested in varying turbidity irrigation waters. This laboratory scale ultraviolet treatment systems has a maximum output of 420 gallons/hour but the inactivation kinetics for varying turbidities and dissolved solids has been established by this research. The scaled up versions required for irrigation water demands could be developed by an industrial or commercial partner. The treatment of irrigation water would greatly enhance the safety of fresh fruits and vegetables that will be consumed raw. The results of the ethyl pyruvate decontamination method indicate that ethyl pyruvate is an effective antimicrobial that could be used to enhance the safety of fresh produce. The delivery system could be incorporated into existing packaging technology to allow for release of ethyl pyruvate during transportation or during holding at wholesale distribution.
Publications
- Manns DC, Churey JJ, Worobo RW. 2012. Functional assignment of YvgO, a novel set of purified and chemically characterized proteinaceous antifungal variants produced by Bacillus thuringiensis SF361. Appl Environ Microbiol. Feb 3. [Epub ahead of print]
- Assatarakul, K., Churey, J.J., Manns, D.C., and Worobo, R.W. 2012. Patulin Reduction in Apple Juice from Concentrate by UV Radiation and Comparison of Kinetic Degradation Models between Apple Juice and Apple Cider. J. Food Prot. 75(4): 717-24.
- Chiang IY, Worobo RW, Churey JJ, Henick-Kling T. 2012. Growth Inhibition of Foodborne Pathogens by Oenococcus oeni. J Food Sci. Jan;77(1):M15-9.
- PW.Landscape and Meteorological Factors Affecting Prevalence of Three Foodborne Pathogens in Fruit and Vegetable Farms. Appl Environ Microbiol. 2012 Nov 9. [Epub ahead of print]
- Durak, M. Z., Churey, J.J., Gates, M., Sacks, G.L., and Worobo, R.W. 2012. Decontamination of green onions and baby spinach by vaporized ethyl pyruvate. J. Food Prot. 75(6): 1012-22.
- Durak, M.Z., Churey, J.J., and Worobo, R.W. 2012. Efficacy of UV, acidified sodium hypochlorite, and mild heat for decontamination of surface and infiltrated Escherichia coli O157:H7 on green onions and baby spinach. J. Food Prot. 75(7): 1198-206.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Irrigation water decontamination was investigated using a commercially available UV treatment unit (CiderSure 3500). Water samples from various sources and times were collected and inoculated with E. coli O157:H7 and Salmonella spp. Using the algorithm developed for turbid unfiltered cider, the UV treatment achieved a greater than 5-log reduction for E. coli O157:H7 and Salmonella spp. The modeling of inactivation kinetics for both pathogens in response to varying turbidity levels was performed and an algorithm achieving a consistent 5-log reduction was determined. Current studies are focused on determining scaling up to meet the irrigation demands for varying produce farm sizes. Post harvest decontamination methods (UV, mild heat, chlorine) were evaluated for their individual and combined effectiveness with inoculated fresh produce. UV light (500 mJ/cm2) was shown to reduce E. coli O157:H7 populations by 1 log CFU/g with dip-inoculated samples, the population was reduced by 1.7 log at 90 mJ/cm2 and 2.7 log at 1000 mJ/cm2 level for spot-inoculated green onions. Chlorine treatments alone were capable of reducing E. coli O157:H7 populations by 0.6 log CFU/g for dip inoculated samples, whereas for spot-inoculated samples treated with chlorine resulted in a 3.5 log CFU/g log reduction. Even though mild heat treatments alone showed no significant differences, a further 0.5 log reduction was observed when used in combination with chlorine (200 ppm at 50C). The combination of selected UV exposure (90 mJ/cm2) and chlorine (200 ppm at 50C) treatments showed a total of 4.7 log reduction with a five-strain cocktail of E. coli O157:H7 spot-inoculated green onions. Additional UV exposure (500 mJ/cm2), yielded a 2.1 log CFU/g for dip-inoculated samples. PARTICIPANTS: Lisa Jones, PhD student; Zeki Durak, PhD student; Ms.Laura Strawn, Ph.D. candidate; Martin Wiedmann, Professor; Christ Smart, Associate Professor of Plant Pathology; John Churey, Research Support Specialist; Randy Worobo, PI; Elizabeth Bihn, Senior Extension Associate; New York State Fruit and Vegetable Growers TARGET AUDIENCES: Consumers, fruit and vegetable growers, fruit and vegetable processors PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Pre- and post-harvest intervention methods are needed to enhance the safety of fresh and fresh cut produce. Focus at the pre-harvest level minimizes the pathogen entry by identifying entry points into the produce production chain at the farm level. Irrigation water represents a high risk for pathogen entry and decontamination methods using UV light may be a possible water treatment method that does not involve chemical disinfection which is affected by organic load of water, pH, and can persist for long periods of time in the soil and have toxicity to plants. Post-harvest decontamination methods currently employed by produce processors include peroxyacetic acid and chlorine based disinfection chemicals. The effectiveness of these disinfection methods are limited. The alternative decontamination methods being studied in this project may provide an improved method for reducing pathogen levels on finished processed produce.
Publications
- M. Zeki Durak, John J. Churey, and Randy W. Worobo. 2011. Decontamination of Green Onions and Spinach Using Gaseous Ethyl Pyruvate. International Association for Food Protection Annual Meeting, Milwaukee, WI
- M. Zeki Durak, John J. Churey, and Randy W. Worobo. 2011. Postharvest Intervention Methods and Combined Treatments to Decontaminate Spinach. International Association for Food Protection Annual Meeting, Milwaukee, WI
- E. A. BIHN, John J. Churey, K. J. Mangione, C. A. Ford, W. F. Lyons and R. W. Worobo. 2011. Assessing the Quality of Surface Water Utilized for Fresh Produce Production in New York. International Association for Food Protection Annual Meeting, Milwaukee, WI
- L.K. Strawn, E. D. Fortes, E. A. Bihn, K. K. Nightingale, R. W. Worobo and M. Wiedmann. 2011. Pathogens in Produce Production Environments: A Longitudinal Study of Fruit and Vegetable Farms in New York State. International Association for Food Protection Annual Meeting, Milwaukee, WI
- Smart, C.D., Dunn, A.R., Jones, L.A. and Lange, H.W. (2011) Management Strategies for Phytophthora Blight. Proceedings of the Mid-Atlantic Fruit and Vegetable Convention (p. 81-82).
- Jones, L.A., Worobo, R.W. and Smart, C.D. (2011) Preliminary Results of Irrigation Water Sampling. Proceedings of the 2011 Fruit and Vegetable Expo, Syracuse, NY. On-line at http://www.hort.cornell.edu/expo/proceedings.php
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Irrigation water decontamination was investigated using a commercially available UV treatment unit (CiderSure 3500). Water samples from various sources and times were collected and inoculated with E. coli O157:H7 and Salmonella spp. Using the algorithm developed for turbid unfiltered cider, the UV treatment achieved a greater than 5-log reduction for E. coli O157:H7 and Salmonella spp. The modeling of inactivation kinetics for both pathogens in response to varying turbidity levels was performed and an algorithm achieving a consistent 5-log reduction was determined. Decontamination methods (UV, mild heat, chlorine) were evaluated for their individual and combined effectiveness with inoculated green onions and spinach. UV light (500 +/- 20 mJ/cm2) was shown to reduce E. coli O157:H7 populations by 1 +/- 0.2 log CFU/g with dip-inoculated samples, the population was reduced by 1.7 +/- 0.2 log at 90 +/- 7 mJ/cm2 and 2.7 +/- .2 log at 1000 +/- 40 mJ/cm2 level for spot-inoculated green onions. Chlorine treatments alone were capable of reducing E. coli O157:H7 populations by 0.6 +/- 0.3 log CFU/g for dip inoculated samples, whereas for spot-inoculated samples treated with chlorine resulted in a 3.5 +/- 0.3 log CFU/g log reduction. Even though mild heat treatments alone showed no significant differences, a further 0.5 log reduction was observed when used in combination with chlorine (200 ppm at 50C). The combination of selected UV exposure (90 +/- 7 mJ/cm2) and chlorine (200 ppm at 50C) treatments showed a total of 4.7 +/- 0.3 log reduction with a five-strain cocktail of E. coli O157:H7 spot-inoculated green onions. Additional UV exposure (500 mJ/cm2), yielded a 2.1 +/- 0.3 log CFU/g for dip-inoculated samples. PARTICIPANTS: Dr. Randy Worobo, PI, Cornell University; Mr. John Churey, Research Support Specialist, Cornell University; Mr. Guoping Feng, Ph.D. student, Cornell University; Mr. David Manns, Ph.D. student, Cornell University; Mr. Zeki Durak, Ph.D. student, Cornell University; Ms. Giselle Guron, Master student, Cornell University, Elizabeth Buerman, undergraduate student, Cornell University TARGET AUDIENCES: Produce growers and processors, regulatory agencies, academia PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Irrigation water represents a significant entry point for pathogens. Currently the only water treatment option that exists for produce growers is chlorination which potentially accumulates in the soil. The feasibility of UV light as a treatment for irrigation water was confirmed by determining the UV death kinetics for pathogenic E. coli and Salmonella strains in irrigation water with varying water qualities (turbidity, solids). UV light was also shown to be effective in reducing E. coli O157:H7 levels in spinach and green onions but alone was not capable of achieving a 5-log reduction. In combination with other decontamination treatments (chemical disinfectants that include peracetic acid, or chlorinated compounds) the combination treatment will significantly reducing potentially contaminating pathogens on finished produce.
Publications
- Durak MZ, Churey JJ, Danyluk MD, Worobo RW. 2010. Identification and haplotype distribution of Alicyclobacillus spp. from different juices and beverages. Int J Food Microbiol. Sep 1;142(3):286-91. Epub 2010 Jul 14.
- Dong Q, Manns DC, Feng G, Yue T, Churey JJ, Worobo RW. 2010. Reduction of patulin in apple cider by UV radiation. J Food Prot. 2010 Jan;73(1):69-74.
- Feng G, Churey JJ, Worobo RW. 2010. Thermoaciduric Clostridium pasteurianum spoilage of shelf-stable apple juice. J Food Prot. Oct;73(10):1886-90.
- Feng, G., J.J. Churey, and R. W. Worobo. Naturally occurring bacteriocinogenic lactic acid bacteria as bioprotective cultures to enhance the safety of sprouts. International Association of Food Protection Annual Meeting, Anaheim, CA, August 2010.
- Durak, M.Z., J.J. Churey, and R. W. Worobo. Inactivation of E. coli O157:H7 on green onions. 2010. IFT Annual Conference. Chicago, IL.
- Strawn L.K., E. D. Fortes, E. A. Bihn, R. W. Worobo and M. Wiedmann. Isolation and Detection of Listeria monocytogenes, E. coli O157:H7, non-O157 shiga toxin producing E. coli and Salmonella from produce farms. International Association of Food Protection Annual Meeting, Anaheim, CA, August 2010.
- Strawn L.K., E. D. Fortes, E. A. Bihn, R. W. Worobo and M. Wiedmann. Isolation and Detection of Listeria monocytogenes, E. coli O157:H7, non-O157 shiga toxin producing E. coli and Salmonella from produce farms. Cornell University Infection and Pathobiology Annual Retreat, Owego, NY, October 2010. Best Poster Award
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Genetic and chemical characterization of antimicrobial peptides and proteins produced by bacterial isolates from food has continued. Two strains, SF361 (Bacillus thuringiensis) and CUGF08 (Enterococcus mundtii) were found to produce antibacterial peptides and the antibacterial peptides were purified and chemical characterization found thurincin H (produced by B. thuringiensis) to be 3139.51 Da and mundticin L (produced by E. mundtii) to be 4301.8 Da. Both are unique antibacterial peptides. Thurincin H was found to be composed of a unique 14 amino acid N-terminal extension and the structural gene was tandem repeated three times and transcribed as a single transcript. It is believed that this may tandem repeated structural gene allows for higher levels of antibacterial production by the producer bacterium compared to most commonly found, single transcript bacteriocins. The unusual amino acid sequence suggests a unique tertiary structure. Additional structural characterization is currently underway. Mundticin L antibacterial peptide was found to exhibit a high activity against Listeria monocytogenes. The genetic characterization of the production, immunity, and regulation genes were identified and found to be highly homologous to mundticin KS and enterocin CRL35, but had unique substitutions in the YGNGVX N-terminal of the bacteriocin. This conserved N-terminal sequence was thought to be a key feature in Listeria-active bacteriocins. The comparison of the activities for the three homologous bacteriocins revealed similar activities against Listeria spp. and the conservative substitution of the 5th residue in the conserved N-terminal sequence is not essential for the anti-Listeria activity. The CUGF08 isolate was added to Listeria monocytogenes inoculated alfalfa sprouts as a potential bioprotective culture to control the growth of foodborne pathogens. The levels of both the producer and Listeria monocytogenes were monitored over a 72 hour sprouting period and it was found to be capable of inhibiting the growth of Listeria monocytogenes. PARTICIPANTS: Dr. Randy Worobo, PI, Cornell University; Mr. John Churey, Research Support Specialist, Cornell University; Mr. Guoping Feng, Ph.D. student, Cornell University; Dr. Hyungjae Lee, Postdoctoral Associate, Cornell University; Mr. David Manns, Ph.D. student, Cornell University; Mr. Zeki Durak, Ph.D. student, Cornell University; Ms. Giselle Guron, undergraduate student, Cornell University TARGET AUDIENCES: produce growers and processors, regulatory agencies, academia PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Fresh produce, particularly seed sprouts continue to be responsible for foodborne outbreaks. Effective decontamination methods have not been identified for fresh produce and sprouts without altering the organoleptic or sensory properties. The identification of potential bioprotective cultures that inhibit the growth of foodborne pathogens associated with produce may serve as an additional barrier to enhance the safety while not altering the organoleptic or nutitional quality.
Publications
- Lee H, Churey JJ, Worobo RW. 2009. Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced by Bacillus thuringiensis SF361. FEMS Microbiol Lett. 299(2):205-13. Epub 2009 Sept 1.
- Feng G, Guron GK, Churey JJ, Worobo RW. 2009. Characterization of mundticin L, a class IIa anti-Listeria bacteriocin from Enterococcus mundtii CUGF08. Appl Environ Microbiol. Sep;75(17):5708-13. Epub 2009 Jul 6.
- Lee H, Churey JJ, Worobo RW. 2009. Isolation and characterization of a protective bacterial culture isolated from honey active against American Foulbrood Disease. FEMS Microbiol Lett. 2009 Jul;296(1):39-44. Epub 2009 Apr 15.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Research emphasis on the chemical and genetic characterization of novel antifungal and antibacterial compounds produced by bacteria (bacteriocins) exhibiting activity against fruit and vegetable pathogens is continuing. These findings have been published in peer reviewed journals to expand the existing knowledge of these antimicrobial compounds. PARTICIPANTS: Dr. Hyungjae Lee is a postdoctoral researcher that is responsible for the genetic characterization of the antifungal protein and all the thuricin H research. Mr. David Manns is a PhD student who is involved in the mode of action studies of the antifungal protein. Mr. Manns is planning on completing his PhD studies in 2010. Mr. Guoping Feng is a PhD student who is responsible for the genetic characterization of an antibacterial peptide. He will be completing his PhD studies in 2009. Ms. Giselle Guron is a Cornell undergraduate student that is working on the identification of the immunity protein and genes involved in immunity for an antibacterial peptide (bacteriocin). TARGET AUDIENCES: The research described in this project is involved in training graduate and undergraduate students. The target audience is other researchers in the fruit and vegetable safety or antimicrobial protein research areas. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Thuricin H is an antibacterial protein that is produced by Bacillus thuringiensis that was isolated from domestic honey. Genetic characterization of the genes responsible for the production of the antibacterial peptide was found to be encoded by a tandem repeating locus of the structural gene which has not been previously reported for antibacterial compounds. RNA transcriptional analysis showed the tandem repeats are transcribed as a single transcript from a promotor upstream of the first structural gene. It is postulated that the tandem array results in higher expression levels of the antibacterial compound compared to the typical single structural gene. This finding suggests that engineering of other structural genes for antimicrobial peptides may increase expression levels and improve the inhibition against sensitive pathogens. Research on the novel antifungal compound produced by a Gram positive bacterium has revealed an additional protein that is similar in sequence and may contribute to a portion of the antifungal activity exhibited by the strain. Two dimensional gel electrophoresis and subsequent ESI MALDI TOF revealed the size and sequence, respectively. It appears that different promotor initiation sites results in two different mature proteins that exhibit different levels of activity. Complementation studies are planned to determine if there is a synergistic activity and the overall percentage of activity associated with each protein. The mode of action of the antifungal proteins against different indicator microorganisms. The chemical and genetic characterization of these novel antimicrobial compounds will provide a deeper understanding of how these peptides and proteins exhibit their activity against sensitive pathogens.
Publications
- Lee H, Churey JJ, Worobo RW. 2008. Antimicrobial activity of bacterial isolates from different floral sources of honey. Int J Food Microbiol. 126(1-2):240-244.
- Lee H, Churey JJ, Worobo RW. 2008. Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25. J Appl Microbiol. 105(3):663-673.
- Bonsi, I.A., Thimothe, J., Koo, M., Worobo, R.W., Padilla-Zakour, O.I. 2008. Pinot noir grape polyphenolic extracts: Chemical composition and antimicrobial effects against Streptococcus mutans and Alicyclobacillus acidoterrestris. IFT Program and Abstract Book Annual Meeting, New Orleans, LA (056-01).
- Feng, G., Churey, J.J., Worobo, R.W. 2008. Isolation of two bacteriocin-producing lactic acid bacteria from alfalfa seeds. IFT Program and Abstract Book Annual Meeting, New Orleans, LA (010-29).
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The novel antifungal compound produced by a bacterial strain originally isolated from honey has been purified and chemically characterized as described in previous reports. The genetic elements coding for the antifungal protein have been characterized at the DNA level. The entire coding region for the preprotein has been sequenced and analyzed. From the analysis, the protein does not show any homology to other antifungal proteins and peptides from the iturin or surfactant groups. High DNA homology was observed to genes identified in bacterial genome sequences but the function had not been assigned to the putative proteins encoded by these genes. DNA sequencing and analysis of the upstream and downstream regions of the gene coding for the antifungal protein did not show any additional open reading frames. It is not known whether an immunity protein is required or present which is usually immediately downstream of the antimicrobial peptide or protein. In addition, transport
and regulatory elements for the antifungal protein were not observed. It is likely that these essential proteins are located distal to the gene coding for the antifungal protein. Based on the DNA sequence, the preprotein has a leader or signal peptide that is cleaved in the active mature antifungal protein. The antifungal proteins exhibits a broad spectrum of activity that includes but is not limited to Penicillium, Byssochlamys and Aspergillus. The antifungal protein did not show activity against a limited range of yeast tested that included Saccharomyces and Candida.
PARTICIPANTS: Randy W. Worobo (PI) John J. Churey (Research Support Specialist) Hyungjae Lee (postdoctoral researcher)
Impacts
The antifungal protein represents a novel antifungal protein that is active against both human clinical and foodborne pathogens. Existing antifungal proteins and peptides are limited due to cellular toxicity by pore formation on the cell membranes of eukaryotic cells. Due to the size of this antifungal protein and preliminary tests, cell toxicity due to cell membrane pore formation does not occur. There is a potential for this protein to be used as an orally administered antifungal agent for human therapy or as an antifungal compound for foods to prevent spoilage or protection against mycotoxin producing molds.
Publications
- Feng G., Churey J.J., Worobo R.W. 2007. Thermal inactivation of Salmonella and Escherichia coli O157:H7 on alfalfa seeds. J. Food Prot. 70(7):1698-703.
- Matak K.E., Sumner S.S., Duncan S.E., Hovingh E., Worobo R.W., Hackney C.R., Pierson M.D. 2007. Effects of ultraviolet irradiation on chemical and sensory properties of goat milk. J. Dairy Sci. 90(7):3178-86.
- Feng G., Churey J.J., Worobo R.W. 2007. Effects of Heat Treatment on Survival of Salmonella spp. and Escherichia coli O157:H7 on Alfalfa Seeds. IAFP Program and Abstract Book Annual Meeting, Lake Bueno Vista, FL (P1-43)
- Lee H., Churey J.J., Worobo R.W. 2007. Isolation and characterization of a honey and honeybee sac protective bacterial culture against American Foulbrood Disease. IFT Program and Abstract Book Annual Meeting, Orlando, FL (053-10)
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Progress 01/01/06 to 12/31/06
Outputs
The genetic and chemical characterization of the antifungal and antibacterial proteins produced by the bacterial strain isolated from a domestic honey sample continues to progress. The gene encoding for the antifungal protein has been located on a 3.4 kb chromosomal fragment of the producer strain. A 600 base pair probe is being used as a probe to assist in cloning the fragment encoding the protein. The purification of the antifungal protein was accomplished by initial ammonium sulfate precipitation followed by column chromotography and then subjecting the active fraction to 2 dimensional gel electrophoresis. Two identical protein spots exhibiting antifungal activity against Byssochlamys fulva were observed. Both spots were transferred to a solid membrane by Western blotting and the active proteins were excised and analyzed by ESI-MALDI-TOF. N-terminal sequencing revealed both proteins had the identical amino acid sequence suggesting the difference in size and
isoelectric point of the proteins may be due to post-translational modifications of the proteins. The purified antifungal protein was found to have a molecular weight of 14,400 Daltons. The antibacterial protein produced by the same producer bacterium was found to show high homology to a recently identifed bacteriocin produced by Bacillus cereus with a similar spectrum of activity. It is suspected that the antibacterial peptides are the same.
Impacts
The antifungal compound produced by the bacterial strain isolated from honey is a novel antifungal compound. This novel compound may be the first antifungal compound identified to date that does not have renal toxicity and thus may have oral therapeutic applications for humans and animals. This antifungal compound may have application outside of food safety area and may be suitable for treatment and prevention of human fungal infections and diseases.
Publications
- Lee, H. 2006. Antibacterial and antifungal proteins produced by Bacillus subtilis H215 and Bacillus thuringiensis SF361 isolated from honey. Ph.D. Thesis, Cornell University.
- Mundo, M., Churey, J.J. and R. W. Worobo. 2006. Partial Purification and Characterization of the Non-peroxide Antibacterial Agent from Manuka Honey . International Association for Food Protection Annual Conference, Calgary, Alberta, Canada. Poster P5-72.
- Mundo, M. 2006. Isolation and purification of antibacterial compounds from honey and bee honeystomachs for potential use as novel food preservatives. Ph.D. Thesis, Cornell University.
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Progress 01/01/05 to 12/31/05
Outputs
The antibacterial and antifungal peptides produced by a bacterium isolated from domestic sunflower honey have been purified by ammonium sulfate precipitation, ion exchange chromatography and finally RP-HPLC. The antifungal peptide was found to be unstable under acidic conditions while extremely stable under alkaline conditions. The purified antimicrobial compounds were subjected to MALDI-TOF, ESI-MS and Edman degradation to elucidate the molecular mass and amino acid sequence of both antimicrobial peptides. The molecular weight and amino acid sequences of both peptides do not show any homology to any known antimicrobial peptides when searched on NCBI databases. Based on the amino acid sequences derived from Edman degradation, degenerate oligonucleotide probes were designed and used as probes and reverse PCR primers to isolate the chromosomal fragment encoding for the antimicrobial peptides. The chromosomal fragment encoding for the antifungal peptide has been
identified and cloning is presently underway. The antibacterial peptide was found to contain extensive post-translational modifications and the limited N-terminal sequence identified modified and unusual amino acids possibly suggesting a lantibiotic compound. Using the limited amino acid sequence data, the identification and cloning of the chromosomal fragment encoding the antibacterial peptide is currently being performed using reverse PCR. Additional bacterial isolates from various honey samples were selected for further characterization of the antimicrobial compound. Based on 16s rDNA sequencing, the majority of the selected isolates were Paenibacillus polymyxa and partial purification indicated the production of a variety of different polymyxin antibiotics.
Impacts
The antibacterial and antifungal compounds produced by this single bacterial strain may have potential to inhibit bacterial foodborne pathogens as well as inhibit mycotoxin producing mold species that are associated with fruits and vegetables. The safety of fruits and vegetables may be enhanced either using the bacterial strain as a protective bacterial culture or the purified antimicrobial compounds themselves.
Publications
- M. Moake, Padilla-Zakour, O. I., and R. W. Worobo. 2005. Comprehensive review of patulin control methods in foods. Comprehensive Rev. in Food Sci. and Food Safety. 1: 8-21.
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Progress 01/01/04 to 12/31/04
Outputs
A total of more than 5000 bacterial isolates from 10 different domestic honey samples were isolated and tested for antibacterial and antifungal activity against Gram negative (E. coli O157:H7, Salmonella enterica, Vibrio parahaemolyticus) Gram positive (Bacillus subtilis, Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens) and a heat resistant mold (Byssochlamys fulva). Percentages of antimicrobial inhibition production for the strains isolated from each of the honey samples were calculated and ranged from 11 to 77 percent. A single bacterial strain isolated from Sunflower honey showed a broad range of antibacterial and antifungal activity. The strain was selected for further chemical and genetic characterization of the antimicrobial compounds. Two different proteins were found to be responsible for the inhibitory activities. Purification of these two proteins is presently being performed using ion exchange and hydrophobic
interaction chromotography followed by solvent precipitation and finally HPLC. The antibacterial peptide was completely purified and subjected to N-terminal sequencing to elucidate the N-terminal amino acid sequence and MALDI-TOF to determine the exact mass of the mature peptide. The amino acid sequence of the antibacterial peptide was compared to the database of identified proteins using the NCBI-BLAST database. No homology to any existing proteins or peptides was identified. The identification of the genes responsible for the production and maturation of the antibacterial peptide are currently being elucidated. The purification and chemical characterization of the antifungal peptide produced by the same bacterial isolate is currently being performed and will be subjected to N-terminal sequencing and mass spectrometry.
Impacts
Selected bacterial strains isolated from various fruits and vegetables that produce inhibitory compounds may act as protective cultures to prevent the outgrowth of pathogenic bacteria associated with fruits and vegetables.
Publications
- Mundo, Melissa A., Olga I. Padilla-Zakour, and Randy W. Worobo. 2004. Growth Inhibition of Foodborne Pathogens and Food Spoilage Organisms by Select Raw Honeys. International Journal of Food Microbiology.97: 1-8.
- Hu, H., Churey, J.J. and R. W. Worobo. 2004. Heat treatments to enhance the safety of contaminated mung bean seeds. Journal of Food Prot. 67, 6: 1257-1260.
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Progress 01/01/03 to 12/31/03
Outputs
The sequence of the 50 kb chromosomal fragment encoding polymyxin M has been completed. Sequence analysis has revealed the presence of ten modules involved in the activation and condensation of the ten amino acids that constitute polymyxin M. All the modules show the same organization pattern and high homology to existing nonribosomal peptide synthetases. Unique to the polymyxin M biosynthetic operon is the presence of two tandem ATP binding cassette transporter genes. The role of the second ATP binding cassette transporter is unknown and has not been identified before. The final sequence assembly is being completed and additional regulatory elements for the production of polymyxin M are being investigated upstream of the first activation/condensation module. A second broad spectrum bacteriocinogenic strain isolated from a domestic honey source is being investigated. Using 16s rRNA sequencing, the strain was identified as Bacillus thuringiensis. Using deferred
inhibition assays, the strain was shown to produce a proteinaceous antimicrobial compound(s) that were active against Gram positive (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, Bacillus subtilis), Gram negative (Escherichia coli O157:H7, Salmonella enterica, Vibrio parahaemolyticus.) and fungi (Byssochlamys fulva). The antimicrobial peptide(s) were purified by ammonium sulfate precipitation followed by DEAE chromotography and finally by RP-HPLC on a C-18 column with acetonitrile as the solvent. The various fractions were tested for activity and it appears that there are at least three different peptides that are responsible for the range of activity observed with the native bacterial culture. Sufficient amounts of each of the active peptides have been RP-HPLC purified and the mass spectral analysis and N-terminal sequence of the different peptides is currently being performed. Further chemical and structural characterization will be
performed.
Impacts
Selected bacterial strains isolated from various fruits and vegetables that produce inhibitory compounds may act as protective cultures to prevent the outgrowth of pathogenic bacteria associated with fruits and vegetables.
Publications
- Martin, N., Hu, H., Moake, M., Churey, J. J., Whittal, R., Worobo, R. W. and J. C. Vederas. 2003. Isolation, structural characterization, and properties of mattacin (polymyxin M), a cyclic peptide antibiotic produced by Paenibacillus kobensis M. Journal of Biological Chemistry. 278, 15:13124-12132.
- Hu, H., 2003. Biochemical and genetic characterization of polymyxin M produced by Paenibacillus kobensis. Ph.D. dissertation. Cornell University, Ithaca, NY.
- Lee, H., Mundo, M. A., Churey, J. J., Padilla-Zakour, O. I., and Worobo, R. W. 2003. Antimicrobial activity exhibited by bacteria isolated from selected US honey samples. Poster 29G-2. IFT Annual Meeting. Chicago, IL
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Progress 01/01/02 to 12/31/02
Outputs
One of the laboratory bacterial isolates that exhibited a broad spectrum of activity against Gram positive and Gram negative foodborne pathogens and spoilage bacteria has been identified by 16s rRNA sequencing as Paenibacillus kobensis. The antimicrobial compound produced by this strain has been purified using various cation exchange and HPLC chromotography. The mass spectrometry yielded a mass of 1157 Da. 1D HNMR was performed and the results identified the antimicrobial peptide to be polymyxin M. The strain was electroporated with pLTV3, a transpositional mutagenesis vector and a mutant library was created. Sixteen mutants were isolated that were negative or showed partial production of polymyxin M. The transposon interrupted genes were recovered from the chromosome of the mutant strains and cloned to allow for sequencing of the interrupted region. Sequencing and subsequent DNA homology comparisons identified homologies to non-ribosomal peptide synthetases and ATP
binding binding cassette transporters. A 50-kb chromosomal fragment showing homology to the interrupted genes was cloned in a cosmid vector and subsequently partially digested with Sau3AI. Fragments ranging in size from 1-1.5 kb were cloned and a library of 100 clones is presently being sequenced to identify the biosynthetic genes responsible for the production of polymyxin M. In addition, a bacteriocin from Streptococcus bovis HC5 was purified and characterized in collaboration with Dr. Jim Russell (Cornell University, USDA). The bacteriocin was purified using ammonium sulfate precipitation followed by separation on SP sepharose. The crude bacteriocin was applied to a C18 column on HPLC with acetonitrile as the carrier solvent. The active fractions were pooled, concentrated and applied to a C16 column with ethanol as the carrier solvent. The active fraction corresponded to a single peak which was lyophilized for further characterization studies. The purified sample was subject to
MALDI-TOF and the mass of the peptide was determined to be 2440 Da. N-terminal sequencing of the purified peptide was performed and the amino acid sequence was compared to identified proteins and peptides on the NCBI database. The only peptide showing homology to the bovicin HC5 bacteriocin was the lantibiotic precursor of Streptococcus pyogenes SF370. The amino acid sequence and the homology to a known lantibiotic suggests that bovicin HC5 is a novel lantibiotic.
Impacts
The identification of novel peptides that are active against spoilage bacteria and foodborne pathogens will enhance the safety and quality of foods.
Publications
- Mantovani, H.C., Hu, H., Worobo, R.W. and J.B. Russell. 2002. Purification and characterization of a bacteriocin (bovicin HC5) from Streptococcus bovis HC5. Microbiology. 148:3347-52.
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Progress 01/01/01 to 12/31/01
Outputs
The bacteriocin genes of three bacteriocinogenic lactic acid bacteria strains are currently being cloned. Two of the three strains have been transformed with a plasmid that contains a transposon and a temperature sensitive origin of replication. A library of transpositional mutants has been created by elevating the growth temperature to 41 celcius. The mutant libraries have been screened for loss of bacteriocin production. The loss of bacteriocin production is due to the insertional inactivation of the bacteriocin gene or additional genes involved in bacteriocin production. The various bacteriocin minus mutants are presently being characterized by cloning the inactivated region of the chromosome. The region will be further characterized by DNA sequencing and compared to existing characterized bacteriocin genes to determine if the bacteriocin structural genes are unique. The third bacteriocin operon is being characterized by reverse genetics which entails using the
partial amino acid sequence to create a degenerate oligonucleotide probe that is based on the partial N-terminal amino acid sequence. From the bacteriocin structural gene, it will be possible to identify additional proximal genes and determine if they are involved in bacteriocin production for that particular strain.
Impacts
The identification of unique peptides may have application in foods to enhance the safety of the food as well as reduce spoilage.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
We have focused our research on 5 different bacteriocinogenic strains that were isolated by our laboratory. We have completed the purification of all 5 strains and obtained partial sequence of the N-terminus using Edman degradation. One of the 5 bacteriocins contains the conserved YGNGV region in the N-terminus but the activity spectrum is atypical for Class II bacteriocins. The remaining 4 all appear to be unique bacteriocins with little homology to previously identified bacteriocins. The CU216 bacteriocin displays high activity and specificity towards the Genus Alicyclobacillus which are important spoilage organisms in fruit and vegetable beverage industry. Using purified CU216, we were able to eliminate Alicyclobacillus from apple juice. Presently, research is focused on the isolation and identification of the bacteriocin genes of these 5 antimicrobial peptides to completely elucidate the N-terminal extension and complete amino acid sequence of the bacteriocin
peptide.
Impacts
These bacteriocins may have potential in dairy starter cultures to inhibit Listeria monocytogenes and other common spoilage organisms to enhance the safety of the product as well as extend the shelf life of these products.
Publications
- Franz C. M. A. P., van Belkum M. J., Vederas J. C. R. W. Worobo and Stiles M. E. 2000. Genes Responsible for Production and Immunity of Carnobacteriocin A: the Immunity Protein Confers Cross-protection to Enterocin B. Microbiology Mar;146 (Pt 3):621-31.
- Oh S., S. H. Kim and R.W. Worobo. 2000. Characterization and purification of a bacteriocin produced by a potential probiotic culture, Lactobacillus acidophilus 30SC. J Dairy Sci. Dec;83(12):2747-52.
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Progress 01/01/99 to 12/31/99
Outputs
Three bacteriocinogenic lactic acid bacteria strains have been purified using hydrophobic interaction chromatography, FPLC and HPLC. The N-terminus of two of the bacteriocins have been elucidated by Edman degradation. One of the bacteriocins shows homology to Class II while the other bacteriocin shows no homology to identified bacteriocins to date. Both bacteriocins are active against Listeria monocytogenes as well as other Gram-positive bacteria (lactic acid bacteria, Bacillus sp.). Degenerate oligonucleotide probes have been designed and synthesized from the N-terminal amino acid data. Presently, research to locate and clone the structural gene(s) responsible for the bacteriocin activity is underway.
Impacts
These bacteriocins may have potential in dairy starter cultures to inhibit Listeria monocytogenes and other common spoilage organisms to enhance the safety of the product as well as extend the shelf life of these products.
Publications
- Franz, C. M. A. P, Worobo, R. W., Quadri, L. E. N., Schillinger, U., Holzapfel, W. H., Vederas, J. C. and Stiles, M. E. 1999. Atypical Genetic Locus for Enterocin B Production in Enterococcus faecium BFE 900. 65:2170-2178.
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Progress 01/01/98 to 12/31/98
Outputs
During the last year, three bacteriocinogenic strains were isolated various food and environmental samples. One strain is identified as a Lactococcus lactis while the other two other organisms remain to be identified to the Genus and species level. All three strains are capable of inhibiting select foodborne pathogens that include Listeria spp. and Bacillus cereus. Listeria monocytogenes is a key target organism because it is ubiquitous in the environment and is capable of causing foodborne illness in a wide range of food from meats and dairy to fruits and vegetables. Inhibition of this key pathogen is a main objective of this work. Present and future research involves the purification of the antimicrobial peptides/proteins (bacteriocins) of these bacteriocinogenic strains. The purified bacteriocins will be used to study the mode of action on susceptible bacteria as well as to determine the N-terminal amino acid sequence of the peptide to commence the location and
sequence of the gene(s) responsible for the antimicrobial activity.
Impacts
(N/A)
Publications
- Biet, F., Berjeaud, J. M., Worobo, R. W., Cenatiempo, Y. and Fremaux, C. 1998. Heterologous expression of mesentericin Y105 using the general secretion pathway (GSP) or the dedicated transport system in Gram-positive and Gram-negative bacteria. Microbiology (Reading) 144:2845-54.
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