Source: UNIVERSITY OF ARKANSAS submitted to
ELECTROSTATIC SPRAY TREATMENTS WITH ORGANIC ACIDS AND PLANT EXTRACTS TO DISINTEGRATE BIOFILMS AND DECONTAMINATE FRESH PRODUCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0213362
Grant No.
2008-35201-18855
Project No.
ARK02182
Proposal No.
2007-01919
Multistate No.
(N/A)
Program Code
32.0
Project Start Date
Apr 1, 2008
Project End Date
Mar 31, 2013
Grant Year
2013
Project Director
Hettiarachchy, N.
Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703
Performing Department
FOOD SCIENCE
Non Technical Summary
In light of recent contaminated produce outbreaks, reduction of incidence of food-borne outbreaks related to fresh produce is made a national priority by FDA. Several approaches are in practice to overcome pathogen contamination on fresh produce. However, the commercial methods available do not completely eliminate pathogens attached to the surface and internal tissue. Non-uniform attachment of antimicrobials on the surface leaves blank spots where microorganism can grow and attach to the surface. We are proposing an electrostatic antimicrobial spray to effectively kill/inhibit the growth of pathogens and disrupt bacterial attachments. This research is focused on electrostatic spray for efficient delivery of organic acids, plant extracts, or combinations to kill/inhibit human pathogens Salmonella spp / E. coli O157:H7 on the surface of spinach, lettuce, sprouts, cantaloupe cubes, and tomatoes. These produce will be inoculated with Salmonella / E. coli O157:H7 and sprayed with organic acids/ grape seed/ green tea extracts, or combinations. Log number of survivors will be evaluated during storage. Inhibition of bacterial attachments, disruption of attached bacteria, and killing/inhibiting of internalized pathogens will also be investigated. Treatments will be optimized by statistical method to identify maximum antimicrobial activity. Color, texture, and sensory attributes of the non-inoculated produce will also be evaluated for the treatments that show maximum antimicrobial activity. Success of this study will provide an alternative efficient technology to overcome the outbreaks of Salmonella/E.coli 0157:H7 to the fresh produce industry.
Animal Health Component
30%
Research Effort Categories
Basic
25%
Applied
30%
Developmental
45%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7121199110010%
7121430110080%
7121460110010%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1199 - Deciduous and small fruits, general/other; 1430 - Greens and leafy vegetables; 1460 - Tomato;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The overall objective of this proposal is to decontaminate/kill Salmonella and E. coli O157:H7 and extend shelf life of spinach, lettuce, alfalfa sprouts, cantaloupe cubes, and tomatoes using electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination. The specific objectives are 1. Investigate the effect of electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination on the inhibition/killing of Salmonella spp. and E. coli O157:H7 inoculated on the surface and in wounded/torn spinach, lettuce, alfalfa sprouts, cantaloupe cubes, and tomatoes stored at 4 and 8 degree C and optimize the levels of malic acid, grape seed, green tea extracts alone and in combination by response surface method (RSM). Evaluate the number of survivors at 0, 3, 6, 9, 12, and 15 days of storage (Year 1). 2. Investigate the effect of electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination on the biofilms formed by Salmonella spp./ E. coli O157:H7 and Enterobacter agglomerans (latter will help in biofilm formation) on the surface and in wounded/torn spinach, lettuce, alfalfa sprouts, cantaloupe cubes, and tomatoes stored at 4 and 8 degree C. Evaluate the number of survivors in the biofilm for 15 days as above. Evaluate the thickness of the biofilm and distribution of Salmonella/E. coli O157:H7 and Enterobacter agglomerans under confocal microscope at 0, 5, 9, and 15 days of storage at 4 and 8 degree C (Year 2). 3. Investigate the effect of electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination on the color and texture of the spinach, lettuce, alfalfa sprouts, cantaloupe cubes, and tomatoes stored at 4 and 8 degree C. Evaluate color and texture at 0, 5, 9, and 15 days of storage (Year 3). 4. Investigate the effect of electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination on the sensory attributes of the spinach, lettuce, alfalfa sprouts, cantaloupe cubes, and tomatoes stored at 4 degree C. Evaluate sensory attributes at 0, 5, 9, and 15 days of storage (Year 3).
Project Methods
Four major activities are covered in this proposal. The first activity will be investigating the effect of electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination on the inhibition/killing of Salmonella spp. and E. coli O157:H7 inoculated on the surface and in wounded/torn spinach, lettuce, alfalfa sprouts, cantaloupe cubes, and tomatoes stored at 4 and 8 degree C and optimizing the levels of these antimicrobials by response surface method (RSM). The second activity will be focused on the effect of electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination in the levels optimized by response surface method (RSM) from objective 1 on the number of survivors of Salmonella spp./ E. coli O157:H7 in the biofilm formed on the surface and in wounded/torn produce stored at 4 and 8 degree C. In this activity we will also evaluate the thickness of the biofilm and distribution of Salmonella/E. coli O157:H7 and Enterobacter agglomerans under confocal microscope. The third activity will be evaluating the effect of electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination in the levels optimized by response surface method (RSM) from objective 1 on the color and texture of the produce. The fourth activity will be investigating the effect of electrostatic spray containing malic acid, grape seed, green tea extracts alone and in combination in the levels optimized by response surface method (RSM) from objective 1 on the sensory attributes of the produce stored at 4 degree C. All values will be collected and reported as means of three determinations. Analysis of variance (ANOVA) will be conducted using the Statistical Analysis System (SAS 8.1, SAS Institute Inc., Cary, NC, 2000). Duncans multiple-range test will be performed using the SAS program for pair comparison. Significance of difference will be defined at p < 0.05. Successful completion of this project should provide a useful intervention strategy utilizing food acid, natural grape seed and green tea extracts alone or in combination to effectively control Salmonella spp. and E. coli O157:H7 on spinach, lettuce, alfalfa sprouts, cantaloupe cubes, and tomatoes. The cost benefit ratio to adopt new technology will be calculated based on the log reduction of Salmonella spp. and E. coli O157:H7. Major fresh producers will be contacted to discuss the collaboration and application of the electrostatic spray containing malic acid, grape seed and green tea extracts alone or in combination that shows maximum killing/inhibition of Salmonella spp. and E. coli O157:H7. It is anticipated that the initial collaboration will be limited to a pilot plant scale operation to scale up the method without inoculation at the Department of Food Science, University of Arkansas, to serve as a measure of scale-up of the technology.

Progress 04/01/08 to 03/31/13

Outputs
Target Audience: Food scientists, food microbiologists, food safety personnel, food companies, Wal-Mart, Ozark natural foods. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? NavamHettiarachchy (PI), Vijayalakshmi Ganesh, Leighanna Massey, Satchithanandam Eswaranandam. Provided hands on experience for graduate students and training for their professional career development. How have the results been disseminated to communities of interest? Following ways used to disseminate the positive outcome of this project to nonscientific audience 1. Food productiondaily.com New Electrostatic salad spraying technique shows &lsquo;efficacy&rsquo; against E.coli &ndash; study A new US study claims to show that electrostatic spraying using organic acids could offer the fresh produce industry more effective protection against E.coli than current sanitation methods. July 09, 2012 http://www.foodproductiondaily.com/Quality-Safety/New-Electrostatic-salad-spraying-technique-shows-efficacy-against-E.coli-study 2. Research Frontiers, University of Arkansas.Pathogen Protection &ndash; University of Arkansas, Research Frontiers.Spring 2010. http://researchfrontiers.uark.edu/10254.php 3. Food processors can go natural, researchers say. Experiments show green tea and grape seed are effective alternatives to chemicals. Mark Huffman. ConsumerAffairs.com. November 12, 2010. Ref: http://www.consumeraffairs.com/news04/2010/11/food-processors-can-go-natural-researchers-say.html#ixzz16Epj2Wci 4. Green tea, grape seed can protect food against contamination: Scientists &ndash; India Vision, NANOVIP.com, Oneindia news, Asian News International and MeD Citizen (Mariam). November 14, 2010. Ref: http://news.oneindia.in/2010/11/14/greentea-grape-seed-can-protect-food-againstcontamination.html Ref: http://www.nanovip.com/green-tea-grape-seed-can-protect-food-against-contamination-scientists.html Ref: http://www.highbeam.com/doc/1G1-242715524.html? key=01-42160D517E1A106B140B071A046A4B2E224E324D3417295C30420B61651B617F137019731B7B1D6B39 Ref: http://www.medcitizen.com/news/4309.html 5. Prevent Food Contamination With Green Tea and Grape Seed. Tanya Thomas, November 18, 2010. General Health News. Ref: http://www.medindia.net/news/Prevent-Food-Contamination-With-Green-Tea-and-Grape-Seed-76849-1.htm#ixzz16EtDnAP9\ 6. Natural Alternatives to Chemical Preservatives. Claire Mitchell. Food Safety News &ndash; Science &amp; Research. November 20, 2010. Ref: http://www.foodsafetynews.com/2010/11/a-natural-alternative-to-chemicals-in-food-processing/ 7. Natural Extracts Can Cut Chemical Additives in Food Processing. Nov 29, 2010. Environment Protection &ndash; Articles. Ref: http://eponline.com/articles/2010/11/29/ natural-extracts-can-cut-chemical-additives-in-food-processing.aspx 8. Researchers Discover Natural Alternatives to Protect Against Food Pathogens. December 2010. Holly Case, Natural Food Network Newsletter Editor. Ref: http://www.naturalfoodnet.com/ME2/Audiences/dirmod.asp?sid=&amp;nm=&amp; type=MultiPublishing&amp;mod=PublishingTitles&amp; mid=8F3A7027421841978F18BE895F87F791&amp;tier=4&amp; id=C7E7815EB7F94BEFBAF45215D707E558&amp;AudId=51E6700A094C4B86B050DBCEF699BED2 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The electrostatic spray of phosphoric (PA), malic (MA), and lactic (LA) acids and grape seed extract (GSE) as an intervention technology to control E. coli 0157:H7 (E.c.) and SalmonellaTyphimurium (S.T.) in spinach and comparing the effectiveness of electrostatic spray with conventional spray and dipping were investigated. Malic acid (2%) in combination with GSE (3%) sprayed electrostatically showed reductions of 2.6-3.3 log CFU/g of S.T. compared to lower log reductions (0.0-0.3 log CFU/g) on day 14 when sprayed conventionally, on spinach. The same combination inhibited E.c. on spinach by 1.5-3.8 log CFU/g between the days 1 to 14 of incubation. Lactic acid (2%) alone caused 1.6-1.8 and 0.1-1.4 log CFU/g reductions of S.T. and E.c. on spinach, respectively. Phosphoric acid (2%) and GSE (3%) showed reductions of 2.0 and 2.7 logs CFU/g of S.T. on days 1 and 14 of storage while PA (1.5%) and PA (1.5%)&ndash;GSE (2%) exhibited 1.1-2.1 log CFU/g inhibition of E.c. on spinach during the 14-day storage. In the response surface method, MA and LA (3% each) combined inhibited S.T. by 2.4-4.3 log CFU/g and E.c. by 2.0-4.0 log CFU/g on spinach. The same combination showed 1.1-2.5 log CFU/g reduction of E.c. on lettuce. On the cantaloupe cubes, electrostatic spray of malic acid (4%) alone and combined with LA (2%) showed 3.3 and 3.6 log CFU/g reduction of S. T. and 4.6 log CFU/g reduction of E.c.On cherry tomatoes, electrostatic spray of lactic acid (4%) alone and combination of MA (2%) and LA (3%) reduced S.T.by 3.6 log CFU/g. Reduction of E. c. was seen when combination of MA (3%) and LA (2%) was sprayed (2.3 log CFU/g). Sensory evaluation was performed using consumer panelist (60 people) showed no changes in color, flavor, and texture of tomatoes untreated and treated with antimicrobials. As storage period increased, panelists were able to detect differences in color, texture, and flavor of treated and untreated cantaloupe cubes. The flavor of cantaloupe cubes treated with MA 4% to was reported to be slightly better than untreated cubes and cubes treated with MA 4% + LA 2% on day 0. Yet, as storage increased, MA 4% treated cubes did not show any significant difference from the other cantaloupe samples for flavor aspect. Panelist liked the color of untreated and cubes treated with MA 4% + LA 2% better than cubes treated with MA 4%. Flavor differences were detected by consumer panelist for spinach treated with and without antimicrobials. Consumers found that there was a significant difference between MA 3%+LA 3% treated spinach and control spinach, while the consumer liked the control spinach better. Attached S.T. and E.c. on spinach was killed by MA 3% + LA 3% treatment when observed under the epifluorescent microscope with the aid of the LIVE/DEAD Baclight kit. Green Fluorescent Protein and dsRed labeled E.c. and S.T. formed biofilm on spinach leaf when observed under confocal laser scanning microscope. Confocal images showed the disruption of biofilm formation by electrospray of MA 2%. Crystal violet biofilm assay of spinach extract also showed MA 3% + LA 3% had the lowest absorbance (0.13 &ndash; 0.27) at 595 nm compared to control non treated (0.8-1.31).

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Massey, LM, Hettiarachchy, NS, Martin, EM, and Ricke, SC. 2013. Electrostatic Spray of Food-Grade Organic Acids and Plant Extract to Reduce Escherichia coli O157:H7 on Fresh-cut Cantaloupe Cubes. J. Food Safety 33(1): 71-78.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Ganesh, V., Hettiarachchy, N.S. Griffis, C. L. Martin, E. M. and Ricke S. C. 2012. Electrostatic spraying of food-grade organic and inorganic acids and plant extracts to decontaminate Escherichia coli O157:H7 on spinach and iceberg lettuce. J Food Sci. 77(7): M391-M396
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: 5. Ganesh, V., Hettiarachchy, N.S., Ravichandran, M., Johnson, M. G., Griffis, C.L. Martin, E.M., Meullenet, J.F. Ricke. S.C., 2010 Electrostatic sprays of food-grade acids and plant extracts are more effective than conventional sprays to decontaminate Salmonella Typhimurium on Spinach. J Food Sci. 75(9): M574-M579.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Massey, L. M. and Hettiarachchy N.S. 2013. Reduction of SalmonellaTyphimurium on Tomatoes by the Application of Natural Antimicrobials Using Electrostatic SprayerIFT annual meeting, Chicago, Ill. Food Microbiology Division: Food Microbiology Posters 204-26
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Massey, L. M. and Hettiarachchy N.S. Martin, E. M. Ricke, S. C. 2013. Electrostatic Spray of Food-grade Organic Acids and Plant Extract to Reduce Escherichia coli O157:H7 on Fresh-cut Cantaloupe Cubes. Food Microbiology Division: Food Microbiology Posters 204-05
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: 16. Massey, L.M., Hettiarachchy, N.S., Martin, E.M., and Ricke, S.C. 2012. The Application of Electrostatic Spray with Organic Acids and Plant Extracts to Reduce Escherichia coli O157:H7 on cantaloupe cubes. IFT Annual Meeting and Food Expo. Las Vegas, NV. June 25  28, 2012. Book of Abstracts #035-122.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Massey L. and Hettiarachchy N. 2012. The application of electrostatic spray with organic acids and plant extracts to reduce SalmonellaTyphimurium on cantaloupe cubes. Ozark Food Processors Association  Annual meeting Fayetteville AR
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Massey L. and Hettiarachchy N. 2012. The application of electrostatic spray with organic acids and plant extracts to reduce SalmonellaTyphimurium on cantaloupe cubes. Arkansas Association for Food Protection (AAFP) meeting, Fayetteville AR
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Ganesh, V. Hettiarachchy, N.S. Griffis, C.L. Martin, E.M. Ricke, S.C. Huff G.R. 2012. A comparative study of electrostatic spraying of food grade organic and inorganic acids with natural grape seed extract for decontamination of Escherichia coli O157:H7 on Spinach. Ozark Food Processors Association  Annual meeting, Fayetteville AR
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Ganesh, V., Hettiarachchy, N.S., Griffis, C.L., Martin, E.M., Ricke, S.C., Huff, G.R. 2012. Electrostatic spraying of food-grade organic and inorganic acids and natural grape seed extract for decontamination of Escherichia coli O157:H7 on Spinach and Iceberg lettuce. AAFP meeting, Fayetteville, AR.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Ganesh, V., Hettiarachchy, N.S., Johnson, M.G., Griffis, C.L., Martin, E.M., Meullenet, J-F., Ricke, S.C. 2010. Electrostatic sprays of food-grade acids and plant extracts are more effective than conventional sprays to decontaminate SalmonellaTyphimurium and Escherichia coli O157:H7 on spinach. Institute of Food Technologists Annual Meeting and Food Expo. Chicago, Illinois. July 17- 20. Book of Abstracts # 038-17.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2009 Citation: Ganesh, V., Hettiarachchy, N.S., Johnson, M.G., Meullenet, J-F., Ricke, S.C. 2009. Electrostatic spray treatments with organic acids and plant extracts to decontaminate SalmonellaTyphimurium in spinach. Institute of Food Technologists Annual Meeting and Food Expo. Anaheim, California. June 6- 9. Book of Abstracts # 123-02.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2008 Citation: Eswaranandam, S., Hettiarachchy, N.S., Johnson, M.G. 2008. Electrostatic spray of organic acids and grape seed extract to decontaminate E. coli O157:H7 on Spinach. Institute of Food Technologists Annual Meeting and Food Expo. New Orleans, Louisiana. June 28-July 1. Book of Abstracts # 052-03.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2008 Citation: Eswaranandam, S., Hettiarachchy, N.S., Hanning, I., Johnson, M.G., Slavik, M.F. 2008. Inhibition of biofilm formation by organic acids in model system. Institute of Food Technologists Annual Meeting and Food Expo. New Orleans, Louisiana. June 28-July 1. Book of Abstracts # 052-27.
  • Type: Theses/Dissertations Status: Other Year Published: 2013 Citation: Massey, LM. 2013. Electrostatic Spray Application of Food-grade organic Acids and Plant Extract to Decrease Escherichia coli O157:H7 and Salmonella Typhimurium on Select Produce. M.S. Thesis, Dept. of Food Science, Uni. of Arkansas, Fayetteville, AR, 160 pp. (Expected to complete in June 2013)
  • Type: Theses/Dissertations Status: Other Year Published: 2013 Citation: Ganesh, V. 2013. Effect of Electrostatic spraying of food-grade organic and inorganic acids and grape seed extract on Salmonella Typhimurium and Escherichia coli O157:H7 on spinach and iceberg lettuce M.S. Thesis, Dept. of Food Science, Uni. of Arkansas, Fayetteville, AR, 180 pp. (Expected to complete in June 2013)
  • Type: Journal Articles Status: Other Year Published: 2013 Citation: Massey, L.M., Hettiarachchy, N.S. 2013. The Effects of Organic Acids and Plant Extract Electrostatically Sprayed on Cantaloupe Cubes Inoculated with Salmonella Typhimurium. Journal of Food Science, Manuscripts in preparation
  • Type: Journal Articles Status: Other Year Published: 2013 Citation: Massey, L.M., Hettiarachchy, N.S. 2013. Application of Organic acids and Plants Extracts by Electrostatic Spray to Reduce Salmonella Typhimurium and E. coli O157:H7 on Tomatoes. Journal of Food Science, Manuscripts in preparation
  • Type: Journal Articles Status: Other Year Published: 2013 Citation: Massey, L.M., Hettiarachchy, N.S., 2013. The Effects of Organic Acids and Plant Extract Electrostatically Sprayed on Cantaloupe Cubes Inoculated with Salmonella Typhimurium Journal of Food science, Manuscripts in preparation
  • Type: Journal Articles Status: Other Year Published: 2013 Citation: Almasoud, A. Eswaranandam S. Hettiarachchy, N.S. 2013. Effect of organic acid electrostatic spray on disruption of biofilm formation by Escherichia coli O157:H7. Journal of Food Science. Manuscripts in preparation.
  • Type: Journal Articles Status: Other Year Published: 2013 Citation: Massey, L.M., Hettiarachchy, N.S., Seo, H. Meullenet, J-F. 2013. Quality and Sensory Evaluation of Fresh Produce Electrostatically Sprayed with Organic Acids. Journal of Food Science. Manuscripts in preparation
  • Type: Journal Articles Status: Other Year Published: 2013 Citation: Massey, L.M., Hettiarachchy, N.S. 2013. Application of Natural Antimicrobials by Electrostatic Spraying to Reduce Foodborne Pathogens on Alfalfa Sprouts. Journal of Food Protection. Manuscripts in preparation
  • Type: Journal Articles Status: Other Year Published: 2013 Citation: Massey, L.M., Hettiarachchy, N.S., Martin, E.M. 2013. The Effects of Organic Acids and Plant Extract Electrostatically Sprayed on Cantaloupe Cubes Inoculated with Salmonella Typhimurium. Journal of Food Science, Manuscript in preparation


Progress 04/01/11 to 03/31/12

Outputs
OUTPUTS: Effects of organic acids and plant extract alone and in combination were applied by electrostatic spraying on E. coli O157:H7 and Salmonella Typhimurium inoculated cantaloupe cubes. Surface inoculated cantaloupe cubes were sprayed with malic (M) and lactic (L) acid and grape seed extract (G) at different concentrations then stored at 4 degrees C. Under optimized concentrations, it was observed that ML 2% was able to reduce E. coli O157:H7 by 1.9 logs after 12 days of storage. This reduction was significantly different from the controls (P < 0.05). Increasing the concentration of M (4%) alone and in combination with L (2%) caused an increase in log reduction (4.6 logs). The initial Salmonella load inoculated on cantaloupe cubes was reduced after spraying (Day 0) by all antimicrobial treatments. However, ML (3%) and MLGSE (3%) were able to reduce Salmonella Typhimurium by approximately 2 logs CFU/g after 12 days of storage. Texture analysis showed no difference in firmness between treated cantaloupe cubes and untreated cantaloupe cubes. Color analysis indicated that cantaloupe cubes treated with MLGSE and ML (3%) showed changes in lightness (L* value) over 12 day observation period, but change in lightness was not different from controls. These treatments can be used as an alternative to chemicals in order to improve the safety of fresh-cut cantaloupe cubes. Multiple hurdle technology of electrostatic sprayer combined with natural antimicrobials can be effective in improving the safety of food products. In another study, Green Fluorescent Protein (GFP) and dsRed labeled E.coli O157:H7 and Salmonella Typhimurium and plant pathogen Pantoea agglomerans formed biofilm on spinach (observed under confocal laser scanning microscope). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Malic acid (4%) alone and in combination with lactic acid (2%) was able to reduce E. coli O157:H7 on fresh cut cantaloupe cubes when sprayed with the electrostatic sprayer. Over the storage period, antimicrobial activity increased by reducing pathogen each day. Applying the organic acid with the electrostatic sprayer is multiple hurdle technology that can be adopted by the produce industry to improve foodborne safety.

Publications

  • Ganesh, V., Hettiarachchy, N.S. Griffis, C. L. Martin, E. M. and Ricke S. C. 2012. Electrostatic spraying of food-grade organic and inorganic acids and plant extracts to decontaminate Escherichia coli O157:H7 on spinach and iceberg lettuce. J Food Sci. 77(7): M391-M396
  • Ganesh, V., Hettiarachchy, N.S., Ravichandran, M., Johnson, M. G., Griffis, C.L. Martin, E.M., Meullenet, J.F. Ricke. S.C., 2010 Electrostatic Sprays of Food-grade Acids and Plant Extracts are More Effective than Conventional Sprays to Decontaminate Salmonella Typhimurium on Spinach. J Food Sci. 75(9): M574-M579.
  • Perumalla, A.V.S. and Hettiarachchy, N.S. 2011. Green tea and grape seed extracts - Potential applications in food safety and quality. A review. Food Res Intl. 44(4): 827-839
  • Massey, L.M., Hettiarachchy, N., Elizabeth, M., Ricke, S. 2012. Electrostatic Spray of Food-grade Organic Acids and Plant Extract to Reduce Escherichia coli O157:H7 on Fresh-cut Cantaloupe Cubes. J Food Science, Manuscript ID JFS-2012-1022. Submitted 25-Jul-2012


Progress 04/01/10 to 03/31/11

Outputs
OUTPUTS: Organic acids (malic and lactic acids) along with grape seed extract were tested for their effectiveness as antimicrobials at varying concentrations on cantaloupe cubes. Cantaloupes were washed with water, the rind and seeds discarded and the melon cut into cubes of ~9 cu.cm (weighing ~4-6 g) using a sterile knife. A second day culture of Salmonella Typhimurium/ Escherichia coli O157:H7 was added to 1 liter of sterile deionized water to make up the inoculum (7.0 log CFU/g). The cantaloupe cubes were immersed in the inoculum for 1 min with constant shaking, dried under the laminar hood for 1 hour and used for the antimicrobial studies. Test solutions consisted of malic and lactic acids, and grape seed extract (2% and 3% concentrations) alone and in combinations, filtered through a Whatman No. 1 filter paper before application onto the cantaloupe cubes by electrostatic spraying. Control samples were cubes sprayed with deionized water adjusted to a pH of 2.3 (similar to test solutions) and samples without any treatment. Treated samples were allowed to dry under the laminar hood for 30 min, packaged in Ziploc bags and stored at 4 degree Celsius. Sampling was conducted on the days 0, 1, 3, 7 and 12 and following serial dilutions with phosphate buffer saline (pH 7.4), were plated onto XLT4 agar (for S. Typhimurium)/ MacConkey sorbitol agar with cefixime tellurite supplement (for E. coli O157:H7). A combination of malic and lactic acids (at 3%) and malic-lactic acids-grape seed extract (at 3%) showed a 2 log CFU/g reduction of S. Typhimurium and E. coli O157:H7 on the cantaloupe cubes. Biofilm assays were carried out using crystal violet solution (0.2%) and dilutions from cantaloupe samples onto 96-well plate and measuring the absorbance at 595 nm. Results showed a decreased biofilm formation of S. Typhimurium and E. coli O157:H7 on samples treated with combination of malic-lactic acids-grape seed extract. Color and texture determination studies on cantaloupe cubes sprayed electrostatically with the above combinations showed no significant difference from that of the controls. PARTICIPANTS: Navam Hettiarachchy, P.I., Vijayalakshmi Ganesh, and Leighanna Massey. Provided hands on experience for graduate students and training for their professional career development. TARGET AUDIENCES: Food scientists, food microbiologists, food safety personnel, food companies, Walmart, Ozark natural foods PROJECT MODIFICATIONS: Green tea extract was not included in the trials with cantaloupe cubes owing to the poor antimicrobial efficacy in comparison to grape seed extract.

Impacts
Electrostatic spraying of malic and lactic acids in combination with grape seed extract can be used as post-harvest multiple hurdle technologies to control Salmonella Typhimurium and Escherichia coli O157:H7 on cantaloupe cubes. The treatments effectively reduce the biofilms formed by the pathogens on the cantaloupe samples without compromising the color and textural qualities of the fruit. The application of organic acids and natural plant extracts using electrostatic spraying can be used as a safe and promising alternative to chemical disinfectants to enhance commercial food safety.

Publications

  • Ganesh, V., Hettiarachchy, N.S., Johnson, M.G., Griffis, C.L., Martin, E.M., Meullenet, J-F., Ricke, S.C. 2010. Electrostatic sprays of food-grade acids and plant extracts are more effective than conventional sprays to decontaminate Salmonella Typhimurium and Escherichia coli O157:H7 on spinach. Institute of Food Technologists Annual Meeting and Food Expo. Chicago, Illinois. July 17-July 20. Book of Abstracts # 038-17.
  • Ganesh, V., Hettiarachchy, N.S., Massey, L.M., Griffis, C.L., Martin, E.M., Ricke, S.C. 2011. Effective decontamination of Escherichia coli O157:H7 using electrostatic spraying of food-grade organic and inorganic acids and plant extracts on spinach and iceberg lettuce. (Manuscript in preparation to J Food Science)
  • Massey, L.M., Hettiarachchy, N.S., Griffis, C.L., Martin, E.M., Ricke, S.C. 2011. The application of electrostatic spray with organic acids and plant extracts to reduce Salmonella Typhimurium and Escherichia coli O157:H7 on cantaloupe cubes. (Manuscript in preparation to J Food Science)


Progress 04/01/09 to 03/31/10

Outputs
OUTPUTS: The inhibitory properties of malic, tartaric, lactic and phosphoric acids, and grape seed extract (GSE) alone and in combinations when applied by electrostatic spraying or conventional spraying and compare their effectiveness against Salmonella Typhimurium and Escherichia coli O157:H7 inoculated spinach were evaluated. Fresh spinach leaves were cleaned of dirt, disinfected by dipping in sodium hypochlorite solution (0.04% v/v) for 30 minutes and rewashed with sterile deionized water. Inoculation was carried out by soaking them in 6 litres of sterile water containing 10 ml of a second day culture of Salmonella Typhimurium/ E. coli O157:H7 (7.0 log CFU/ml). After 24 h incubation at room temperature, the spinach leaves were washed again with sterile deionized water, to remove the non-adhered bacteria. These were treated with individual and combinations of organic acids/phosphoric acid with GSE or deionized water (control) adjusted to the same pH as that of the test solutions either electrostatically or conventionally and the leaves were refrigerated at 4 oC. S. Typhimurium and E. coli O157:H7 counts were taken on the 1st, 7th and 14th days of storage in triplicates by plating on xylose lysine tergitol 4 (XLT4) agar and MacConkey sorbitol agar supplemented with cefixime tellurite respectively. Malic acid (2%) in combination with GSE (3%) sprayed electrostatically showed reductions of 2.6-3.3 log CFU/g of S. Typhimurium compared to lower log reductions (0.0-0.3 log CFU/g) on the 14th day when sprayed conventionally. The same combination inhibited E. coli O157:H7 by 1.5-3.8 log CFU/g between the 1st and 14th days of incubation. Lactic acid (2%) alone caused 1.6-1.8 log CFU/g reduction of S. Typhimurium and E. coli O157:H7 on spinach. Phosphoric acid (2%) and GSE (3%) showed reductions of 2.0 log CFU/g and 2.7 log CFU/g on the 1st and 14th days of storage of S. Typhimurium while only 0.2-0.4 log CFU/g reduction of E. coli O157:H7. Of the food grade acids tested, malic and lactic acids offered better antimicrobial activities against the pathogens and hence response surface method was designed to optimize their concentrations for maximum potency. Malic and lactic acids (3% each) combined inhibited S. Typhimurium by 2.4-4.3 log CFU/g and E. coli O157:H7 by 2.0-4.0 log CFU/g. PARTICIPANTS: Navam Hettiarachchy, P.I., Vijayalakshmi Ganesh, and Madhuram Ravichandran. Provided hands on experience for graduate students and training for their professional career development. TARGET AUDIENCES: Food scientists, food microbiologists, food safety personnel, food companies, Walmart, Ozark natural foods PROJECT MODIFICATIONS: We tried grape seed and green tea extracts in our study but found grape seed extract to have a higher potency to inhibit pathogens than green tea extract. Thus in our experiments grape seed extract was alone used in combination with organic acids and the concentration optimized using response surface method.

Impacts
Electrostatic spraying of MA, LA and GSE can be efficiently used to inhibit S. Typhimurium and E. coli O157:H7 on spinach in comparison to conventional spraying due to uniform deposition of antimicrobials on the surface and retention of antimicrobials. Malic acid in combination with GSE/ lactic acid solutions applied by electrostatic spraying can be used for commercial applications to enhance food safety. The combination of these antimicrobials applied by electrostatic spraying can serve as effective multiple hurdle technologies to disrupt the biofilms formed by the food borne pathogens and decontaminate the produce surfaces.

Publications

  • Ganesh, V., Hettiarachchy, N.S., Johnson, M.G., Meullenet, J-F., Ricke, S.C. 2009. Electrostatic spray treatments with organic acids and plant extracts to decontaminate Salmonella Typhimurium in spinach. Institute of Food Technologists Annual Meeting and Food Expo. Anaheim, California. June 6-June 9. Book of Abstracts # 123-02.
  • Ganesh, V., Hettiarachchy, N. S., Ravichandran, M., Johnson, M. G., Griffis, C. L., Martin, E. M., Meullenet, J-F., Ricke, S. C. 2009. Electrostatic Sprays of Food-grade Acids and Plant Extracts are more Effective than Conventional Sprays to Decontaminate Salmonella Typhimurium on Spinach. (Manuscript in preparation to JFS: Food microbiology and safety)


Progress 04/01/08 to 03/31/09

Outputs
OUTPUTS: The electrostatic spray of tartaric acid (TA), and malic (MA) acids and grape seed extract (GSE) as an intervention technology to control E.coli 0157:H7 in spinach and compare the effectiveness of electrostatic spray with conventional spray and dipping were investigated. Spinach samples were washed and dipped in sodium hypochlorite solution (6.25 ml/L) and washed with sterile de-ionized water. These were inoculated with E.coli 0157:H7, washed to remove unattached bacteria and sprayed with/dipped in antimicrobial test solution. Excess solution was allowed to drip in a beaker, air dried and stored at 4 degrees Celsius. On the day of testing spinach samples were transferred into Whirlpak bag, ground, serially diluted and plated in MacConkey sorbitol agar supplemented with Cefixime Tellurite. Results showed that electrostatic spray of TA (2%), + GSE (2%), and MA (2%) + GSE 92%) had more reductions in log numbers (3.3-3.6 and 1.5-3.8) log CFU/g, respectively) in comparison to control. Conventional spraying resulted in the growth of surviving E. coli 0157:H7, while dipping showed less inhibition/killing of E.c. during storage. In another study, organic acids on the inhibition of biofilm formation by Salmonella Typhimurium in 96 well plate assays. Diluted culture was inoculated into 96-well plates. Test solutions containing citric, malic, lactic, and tartaric acids alone and in combination (1:1) at concentrations of 0.2-1.6% w/v was also added to the wells and inoculated at 37 degrees Celsius for 24 hours to form biofilm. Then the plates were rinsed, stained with crystal violet, and allowed to dry. Stain was eluted with ethanol and absorbance at 595 nm was determined on a microplate reader. Bacteria attached and formed biofilms on the wells will intake crystal-violet stain and give higher absorbance. Results showed that citric, malic, and tartaric acids alone and in combination inhibited biofilm formation at 0.4% w.v concentration (OD595 0.01-0.06) compared to control at the same pH (OD595 1.07). Lactic acid inhibited the biofilm formation at 0.6% w/v concentration (OD595 0.01). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Electrostatic spray of TA, MA, and GSE can be efficiently used to inhibit E. coli 0157:H7 on spinach in comparison to conventional spray and dipping due to uniform deposition of antimicrobials on the surface. Electrostatic spray of of TA, MA, and GSE has the potential to enhance microbial safety of spinach in comparison to other methods of application. Citric, malic, and tartaric acids has the potential to inhibit the biofilm formation and improve the efficiency of antimicrobials on food surfaces including fresh produce and meat products.

Publications

  • Eswaranandam, S., Hettiarachchy, N.S., Johnson, M.G. 2008. Electrostatic spray of organic acids and grape seed extract to decontaminate E. coli O157:H7 on Spinach. Institute of Food Technologists Annual Meeting and Food Expo. New Orleans, Louisiana. June 28-July 1. Book of Abstracts # 052-03.
  • Eswaranandam, S., Hettiarachchy, N.S., Hanning, I., Johnson, M.G., Slavik, M.F. 2008. Inhibition of biofilm formation by organic acids in model system. Institute of Food Technologists Annual Meeting and Food Expo. New Orleans, Louisiana. June 28-July 1. Book of Abstracts # 052-27.
  • Over, K., Hettiarachchy, N.S., Johnson, M.G. 2008. Effect of organic acids and plant extracts on Salmonella typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 in broth culture and chicken meat model. Institute of Food Technologists Annual Meeting and Food Expo. New Orleans, Louisiana. June 28-July 1. Book of Abstracts # 052-09.