Rapid Methods and Applications of Natural Ingredients for Food Safety and Security

NON-TECHNICAL SUMMARY: Food safety and pathogen control have become a central concern in modern food science due to outbreaks of foodborne infections/intoxications and potential of bioterrorism attacks. Foodborne illnesses account for billions of dollars of economic losses annually. Since safety of the public food supply is a major concern, this research will focus on destruction, detection and control of foodborne pathogens in foods to monitor food safety and security from farm to fork. <P>

APPROACH: The first section of this project is to focus on developing rapid sample preparation and enrichment methods followed by rapid detection methods to achieve truly rapid, sensitive, and reliable detection of foodborne pathogens. The resistance and injury of foodborne pathogens after traditional and novel processing will also be studied using innovative recovery methods. These rapid methods and information will be applied to the food industry and also be introduced nationally and internationally to promote food safety. This section includes: 1.Real-time detection of microbial contamination in meat products using a non-instrumental microrespirometer technique (NMR), which measures the CO2 evolution rate (CER) as an indicator of general biological activities to predict spoilage. We will test the hypothesis that this NMR method can be applied as a reliable rapid method (less than 2 h) for real time detection of total spoilage/contaminating bacteria in food samples with and without previous freezing. We will also develop NMR methods for the specific detection of coliforms, Escherichia coli, and Enterococcus in naturally contaminated and spiked food samples using a selective broth in the NMR vessel. 2.Epidemiological study for Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella spp. in dairy farms in Maine, U.S and development of optimized enrichment broths. 3.Development of rapid protocol for the detection of E. coli O157:H7, L. monocytogenes, and Salmonella spp. by optimizing an immuno-capture system (Pathatrix) in combination with Colortrix, and VIP. 4.Study of the resistance and injury of foodborne pathogens after traditional and novel processing using innovative recovery methods developed by the P.I, namely, modified Thin Agar Layer method and Four-compartment Thin Agar Layer method . The second approach for the control and prevention of foodborne pathogens is using naturally occurring food ingredients. We will utilize natural ingredients extracted from Maine blueberry, cranberry, small fruit or other herbs which contain phenolic antioxidants to study suppressing and killing effect to control foodborne pathogens. The successful natural ingredients can be used as natural additives in foods to control food safety and may also improve food quality. This section includes: 1.Study of antimicrobial properties (killing and suppressing effects) of natural fruit ingredients (blueberries, cranberries, blackberries, raspberries, strawberries, and grapes) and herbs (Cornus fruit, Cornus officinalis Sieb. et Zucc.) for controlling E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus . 2.Study of the role of natural ingredients on injury, survival and death of foodborne pathogens (E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus) using innovative recovery methods and to study the mechanism of antimicrobial action of natural ingredients in BHI medium using Transmission Electron Microscopy (TEM). 3.Functionalities of natural ingredients in foods: the successful antimicrobial natural ingredients will be added in food to study the improvement of flavor and texture in solid and liquid foods.

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PROGRESS: 2007/10 TO 2008/09<BR>
OUTPUTS: 1.We investigated the survival of E. coli O157:H7 in apple juice and neutralized apple juice with 3% Cornus fruit extract added. 2.We studied 1) antimicrobial effects of cranberry concentrate on E. coli O157:H7 in ground beef, 2) sensory evaluation of burgers combined with cranberry concentrate. 3.We developed a dielectrophoretic system for filtering foodborne pathogens such as Escherichia coli O157:H7 from liquid samples. 4.We developed a real-time selective system for monitoring the growth of E. coli O157:H7 using a quartz crystal microbalance immunosensor. 5.We developed a circulating-flow piezoelectric biosensor, based on an Au nanoparticle amplification and verification method for real-time detection of a foodborne pathogen, Escherichia coli O157:H7. The results of projects conducted in this period are shared with the public through extension and outreach activities, which benefit local, regional, and state agriculture and the food industry. The results of the projects have also been presented in national and international meetings, shared in international workshops, and published in scientific refereed journals. <BR>PARTICIPANTS: In this period, the project provides research opportunities for training graduate students and international collaborations with professionals in the U.S. and Taiwan. P.I.: Dr. V. C.H. Wu, University of Maine. Collaborators: Dr. C-S Lin, National Chiao Tung University, Taiwan Dr. Y-H. P Hsieh, Florida State University Dr. A. Bushway, University of Maine Professional development: X. Qiu, Ph.D. student S.-H. Chen, Ph.D. student. L. Harper, undergraduate student <BR>TARGET AUDIENCES: Target audiences include professionals and scientists in the area of microbiology, food science, biotechnology, and food safety; the food industry; the general public and consumers. The results of projects are shared with the public and the food industry through extension and outreach activities, which benefit local, regional, and state agriculture and the food industry. The results of the projects are presented in national and international meetings, conferences, workshops, and published in scientific refereed journals. <BR><BR>
IMPACT: 2007/10 TO 2008/09<BR>
1.The most dramatic reduction of E. coli O157:H7 was observed in apple juice with Cornus fruit extract at room temperature. Results also showed that Thin Agar Layer (TAL) media were more efficient than selective media, MacConkey Sorbitol agar (MSA). These results may be used as a basis for an intelligent hurdle design to preserve food samples such as apple juice. TAL may be considered for use in recovering cells injured by various treatments. 2.Compared to the control beef samples, cranberry concentrate significantly reduced both total aerobic bacteria and E. coli O157:H7 after 3 days (P < 0.05). Time and cranberry concentrate concentrations in ground beef had a synergistic effect on inhibition of both total aerobic bacteria and E. coli O157:H7 (P < 0.05). Sensory evaluation results showed that no differences were found among burgers with 0%, 2.5%, and 5% cranberry concentrate for appearance, flavor, and taste. Considering the antimicrobial effects and other health benefits, burgers with cranberry concentrate may be a potential safe, healthy, and popular product preferred by consumers. 3.The dielectrophoretic filtration system has potential applications by spring water companies which still use ultra filtration systems to remove bacteria from water. 4.The QCM immunosensor selectively monitored the growth of E. coli O157:H7 during enrichment. It has potential application in real food samples for detecting viable cells and reduces entire detection time. 5.The DNA piezoelectric biosensor was able to detect targets from real food samples. The sensor with specifically oligonucleotide functionalized Au nanoparticles as amplifiers has potential for additional applications in detecting E. coli O157:H7 or other microorganisms in food, water and clinical samples. The targets for 2008 have been met.

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PROGRESS: 2006/10/01 TO 2007/09/30<BR>
OUTPUTS: 1.We developed and investigated bactericidal effectiveness of a simple and cheap ClO2 method on four foodborne pathogens, Salmonella Typhimurium, Listeria monocytogenes, Yersinia enterocolitica, and Pseudomonas aeruginosa. 2.We developed an aqueous chlorine dioxide (ClO2) method and studied its effects on controlling foodborne pathogens, yeasts, and molds on blueberries. 3.We studied and investigated the antimicrobial effect of cranberry concentrate on Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus in raw ground beef. 4.We accessed the effectiveness of the cytokinin N6-benzyladenine (6-BA), alone or in combination with the biocontrol yeast Cryptococcus laurentii, in controlling blue mold on pear fruit. 5.We developed a DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating flow system. Specific probes for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets and PCR amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. 6.We developed an innovative dielectrophoresis system for separation and concentration of the foodborne pathogens Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes and Staphylococcus aureus. The results of projects conducted in this period are shared with the public through extension and outreach activities, which benefit local, regional, and state agriculture and the food industry. The results of the projects have also been presented in national and international meetings, shared in international workshops, and published in scientific refereed journals. <BR>PARTICIPANTS: In this period, the project provides research opportunities for training graduate students and international collaborations with professionals from China and Taiwan. P.I.: Dr. Vivian C.H. Wu, University of Maine. Collaborators: Dr. Chich-Sheng Lin, National Chiao Tung University, Taiwan Dr. Xiao Dong Zheng, Zhejiang University, China Professional development: Xujian Qiu, Ph.D. student B. Kim, Ph.D. student Ting Yu, Ph.D. student Bin Huang, Ph.D. student. Sz-Hau Chen, Ph.D. student. <BR>TARGET AUDIENCES: Target audiences include professionals and scientists in the area of microbiology, food science, biotechnology, and food safety; the food industry; the general public and consumers. The results of projects are shared with the public and the food industry through extension and outreach activities, which benefit local, regional, and state agriculture and the food industry. The results of the projects are presented in national and international meetings, conferences, workshops, and published in scientific refereed journals.
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IMPACT: 2006/10/01 TO 2007/09/30<BR>
1. ClO2 showed promise as a sanitizer for killing foodborne pathogens. The high bactericidal effectiveness of aqueous ClO2 with efficient treatment time provides an advantage to producers who do not want any changes in the appearance of their produce after sanitization. Our results suggest that aqueous ClO2 is a highly effective microbial decontamination method for different foodborne pathogens.<BR> 2. The simple aqueous chlorine dioxide (ClO2) approached we investigated can be used as a sanitizer for controlling foodborne pathogens as well as yeasts and molds on blueberries. We expect that brief treatment intervals (20 or 30 min) and degradation of ClO2 over time and by organic materials (blueberries) provide advantages to food processors who seek to preserve the appearance of their product and reduce ClO2 residues while enhancing food safety with novel sanitization procedures. <BR>3. Results showed that compared to the control, cranberry concentrate significantly inhibited (P<0.05) foodborne pathogens in ground beef at both 7C and 21C. With various potential health benefits and antimicrobial effects, cranberry concentrate is very promising for use as an effective hurdle to control foodborne pathogens in the food industry.<BR> 4. Our data suggested that a combination of 6-BA and C. laurentii could integrate the dual biological activities from 6-BA and C. laurentii and might be developed into a novel protection strategy for reduction of the blue mold rot of pear fruit. 5.The results indicate that the spacer enhanced the H% when the probes were hybridized with 30-mer and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis. 6.All four pathogens were captured with our DEP system using AC (20 Vpp and 1 MHz) signals. This new DEP system can capture all four pathogens regardless of cell wall types (Gram-positive or negative) and shapes (bacilli or cocci). Since the separation depends on not only the size but also the electric properties of each bacterium, this DEP system has potential for selective separation of foodborne pathogens from food. The targets for 2007 have been met. These include: number of new natural antimicrobials developed from fruits and vegetables (2), percentage of Maine food processors learning about principles of food safety programs (30), percentage of Maine food processors establishing their own HACCP plans (40), percentage of Maine manufactures adopting new technologies to reduce microbial contamination (10).

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PROGRESS: 2005/10/01 TO 2006/09/30<BR>
1. We developed a new enrichment protocol that improved traditional enrichment procedures (primary and secondary enrichments) by the use of a Pathatrix system. Rapid, sensitive, and reliable detection of Listeria monocytogenes was achieved following with polymerase chain reaction (PCR) detection. UPE was the most effective among three broths and showed a significant recovery of injured cells and low inocula compared to LEB. With the addition of Oxyrase, Universal Pre-enrichment Oxyrase Broth (UPEO) was better than UPE (1 to 2 log difference). The UPEO was processed in the Pathatrix System and then followed by molecular detection. A same day protocol for detecting of L. monocytogenes was developed. <BR>2. We evaluate the efficacy of 3% Cornus fruit extract for controlling Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus in uncooked ground beef. Results from the 21oC incubation showed no significant difference between the counts achieved with 3% Cornus fruit extract and the control for total aerobic count, E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus on day 0, however, there was a significant suppression (2 to 6 log) of four pathogens on day 1, 3, and 5 compared to the control. Results from the 7oC incubation indicated the suppression effect (1 to 3 log) of extract started from day 0 to 5. <BR>3. We investigated the antimicrobial effects of cranberry concentrate on four foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium , and Staphylococcus aureus in both water and growth media. Results from the DW experiments showed that while no reduction of pathogens was observed in pure DW (0% treatment) at 7oC or 21oC, killing effects on four pathogens started at 1 hr in 10% and at 7 hr in 2.5, 5.0, and 7.5% concentrate. At 24 hr, no pathogens were recovered from the 10 % treatments. Both S. Typhimurium and L. monocytogenes, were reduced to non-detectable level at 5 hrs in 10% cranberry concentrate at both 7oC and 21oC. BHI data indicated that the growth of all pathogens tested was reduced compared to the negative control at both temperatures. No colonies were observed for S. Typhimurium at 10 % after 5 days at both 7oC and 21oC. <BR>4. We investigated the synergistic antimicrobial effects of cranberries and blueberries for foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus, in both water and growth media. Results from the DW experiments showed that while no reduction of pathogens was observed in pure DW at 7oC or 21oC, bactericidal effects on four pathogens started at 1 hr in berry concentrate mixture and berry powder blend. At 24 hr, no L. monocytogenes, S. Typhimurium and S. aureus were recovered from the berry concentrate mixture treatments. The berry powder blend which was developed for enhancing gastro-intestinal health showed 2 log CFU/ml reduction of E. coli O157:H7. BHI data indicated that the growth of all pathogens tested was reduced (4 to 9 log CFU/ml difference) compared to the negative control at both temperatures.
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IMPACT: 2005/10/01 TO 2006/09/30<BR>
1. The development offers an efficient enrichment protocol which provides a significant benefit for the industry to detect L. monocytogenes (low numbers or injured cells) in food products and environment samples. <BR>2. The addition of 3% Cornus fruit extract is an effective way to suppress the growth of microorganisms in ground beef both at 7oC and 21oC. The method should be considered to be applied in further food product development and used as natural food preservatives. <BR>3. The cranberry concentrate has significant killing and suppression effects on the foodborne pathogens tested. Based on various heath benefits and antimicrobial effects, cranberries may be considered for potential food applications, such as food preservatives. <BR>4. The synergistic effects of cranberries and blueberries have not only heath benefits but significant antimicrobial effects. It may be considered for food applications.

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PROGRESS: 2004/10/01 TO 2005/09/30<BR>
1. We were interested in determining if E. coli O157:H7, Listeria monocytogenes Salmonella species could be found in various locations on operating dairy farms, if there was a relationship of where positive samples were found, and what the sensitivities of any isolates to several antibiotics were. Environmental samples were taken from eight different areas on a total of fifteen commercial dairy farms. Common areas sampled on dairy farms were the calving area, the fresh cow pen, milking parlor, milk house floor, calf pens, manure handling equipment, silo floor and total mixed ration. Only farms that had clinically sick animals were positive for Salmonella. Salmonella serovars detected on farms were Typhimurium, Typhimurium var. copenhagen, and Newport. <BR>2. We investigated the anti-microbial effects of Cornus fruit extracts on four foodborne pathogens, E. coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes, in both water and growth media. Results from the DW experiments showed that while no reduction of pathogens was observed in pure DW (0% treatment) at 7C and 21C, killing effects on E. coli O157:H7 and S. Typhimurirum started at 7 hr. At 24 hr, no gram negative-organisms were recovered from the 2 and 3 % treatments. Both S. aureus and L. monocytogenes, were reduced to non-detectable levels at 7 hrs in all water extracts at both 7C and 21C. BHI data indicated that all pathogens tested had reduced growth comparing with the negative control (0% treatment) at both temperatures. No colonies were observed for S. aureus and L. monocytogenes in BHI extracts at 1, 2, or 3 % after day 1. <BR>3. A four-compartment Thin Agar Layer (TAL) system was developed to improve operation efficiency and recover four heat-injured foodborne pathogens simultaneously from ground beef and milk. No significant difference (p>0.05) occurred between the single recovery unit (non-selective agar overlaid on one selective agar in a standard petri dish) and the four-compartment unit for detection of four heat-injured pathogens in ground beef and milk samples. Both TAL methods showed greater recovery of all four heat-injured pathogens than the pathogen-specific selective media (p<0.05). 4).Using the traditional recipe, pickle fermentation was studied. The pH decreased fastest in the presence of 10 degree salometer. The salinity increased in 2 degree salometer, but decreased in 20 degree salometer by the end of fermentation. No significant change of salinity in 10 degree salometer was observed. The viable cell counts on EMB dropped to zero after two days. The counts on MRS and Kang-Fung medium increased at least 4 logs by the end of fermentation. Kang-Fung medium selected more lactic acid bacteria than MRS in a mixture of bacterial environment. The 10 degree salometer of brine was concluded as the best for Chinese pickle.
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IMPACT: 2004/10/01 TO 2005/09/30<BR>
1). Once clinically sick cows were identified on a farm, environmental samples were positive in a variety of locations reflecting movement of organisms to several areas on the farm by animate and inanimate vectors. The information provides a significant benefit for the industry. These results can also form the basis for continuing education of veterinarians in Maine. The use of this multiplier puts U Maine data at the farm level, delivered by the attending veterinarians. <BR>2). The Cornus fruit extract had significant killing and suppression effects on the foodborne pathogens tested, with greater efficacy for gram-positive than gram-negative pathogens at 7C and 21C, and may be considered for food applications.<BR> 3). This four-compartment TAL system is more efficient than the single TAL system for recovery and detection of pathogens in food in terms of operation, material and labor costs, and space of incubation. <BR>4). According to the investigations and product desirability in terms of taste, Chinese pickle fermented in 10 degree salometer brine provided the best results. Chinese pickle has special flavor and could be introduced to western as another appetizer. Kang-Fung medium selected more lactic acid bacteria than MRS in a mixture of bacterial environment. It can be used to selectively enumerate and monitor growth of LAB in mixed culture studies such as effects of starter cultures on foodborne pathogen in sausage and other fermentations.