ENHANCING FOOD SAFETY AND QUALITY THROUGH NATURAL ANTIMICROBIALS DERIVED FROM PLANTS AND BACTERIA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1009823
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Project Director
Monu, EM.

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Poultry Science

Non Technical Summary
Food safety is an important consideration for processors and consumers. With current trends moving towards "all natural" additives and "clean eating" and away from synthetic food additives, it is essential to have safe effective products that fit into these categories, which includes plant and microbial derived compounds. Although there are existing antimicrobial interventions used in the food industry, outbreaks due to organisms such as Salmonella, Escherichia coli, Campylobacter and Listeria monocytogenes still occur in raw foods of animal origin along with cross-contamination to ready-to-eat (RTE) foods due to inadequate processing or personal hygiene. Although, these contaminants are somewhat inevitable there is a need to minimize this by implementing safety measures throughout the farm-to-table food supply. This neccesitates the development of antimicrobial interventions that can be utilized in a variety of foods including RTE deli meats, produce and bakery items as well as raw and partially cooked meat products.In addition to providing desired sensory properties to food products, plants have been used traditionally in many cultures for therapeutic purposes to treat a variety of diseases and ailments, and their antimicrobial activity is well documented. Although there has been extensive antimicrobial activity in vitro, many of these traditionally plant extracts have not been investigated for potential application in food, but primarily as alternative drugs against human pathogens.Bacteriocins are small, ribosomally-synthesized antimicrobial peptides, many of which have a narrow spectrum of activity against closely related organisms. There has been extensive research conducted on the application of bacteriocins from lactic acid bacteria (LAB) isolated from food as food antimicrobials. Application of these bacteriocins is limited as they are often produced in low amounts, have a narrow range of activity and synthesis is expensive. For these reasons, it is important to find new sources of bacteriocins and to determine ways they can be applied to food with other antimicrobial interventions in order to optimize their utilization as food antimicrobials.This project will determine the most effective way to utilize these two sources of natural antimicrobials to increase consumer safety as well as the shelf-life of selected food products.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4099	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

natural antimicrobial

bacteriocins

food safety

plant extracts

Goals / Objectives
The overall goal of the project is the development of natural antimicrobials as additives to ensure food safety and extend product shelf-life using plant and bacterial sources. This includes 1) discovery of new antimicrobial compounds and sources of compounds; 2) evaluating efficacy of these compounds in vitro and in foods to determine the effect of environment and food components on activity; and 3) developing methods to better target antimicrobial food preservatives to foodborne pathogens

Project Methods
Antimicrobial compounds will include spice essential oils and their active compounds, extracts from medicinal and culinary plants, and bacteriocin-producing organisms. Cultures of the target organisms will be grown in laboratory media for evaluation of antimicrobial activity. Screening of the antimicrobial activity of compounds against these organisms will be conducted using agar disk diffusion and spot-on-lawn assays in which zones of inhibition will be measured on agar plates seeded with target pathogens. Following screening, the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of those compounds exhibiting antimicrobial activity will be determined using microbroth dilution assays, in which antimicrobial compounds will be added to 96-well microtiter plates inoculated with target pathogens. Growth will be monitored by measuring optical density (OD) using a microplate reader. Possible synergistic antimicrobial activity of combinations of antimicrobial compounds will also be evaluated using the microdilution checkerboard method. The fractional inhibitory concentration (FIC) will be calculated by measuring ODs of target pathogens in a microtiter plate to which combinations of antimicrobial compounds will be added in a twofold dilution scheme. An FIC < 1 indicates synergy.The effect of antimicrobial compounds on the growth kinetics of target pathogens will be determined. by time-kill curve generation using either a microdilution method where OD will be measured over time in a plate reader, or a macrodilution method where samples will be plated periodically from test tubes containing combinations of growth broth, target pathogens and antimicrobial compounds.Once the antimicrobials with the greatest potential have been identified and selected, antimicrobial efficacy will be evaluated at recommended as well as abusive storage temperatures in food products. This will be accomplished through challenge studies in which antimicrobials will be added to food products that have been inoculated with the target pathogen. The effect on natural spoilage microflora of the food product and product quality aspects such as color, sensory and texture will also be evaluated in food products that have not been inoculated with pathogenic organisms.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Fellow researchers in the areas of agriculture, food science and food safety were reached through presentations at national,international and local conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A Doctoral student (Sayma Afroj) was trained under this project under joint supervision of Mark Liles in the Departmentof Biological Sciences. An additional Doctoral student (Janet Gradl) was also trained under this project under joint supervision of Michelle Worosz of in the Department of Agricultural Economics and Rural Sociology and succesfully defended her dissertation in March of 2019. An undergradiate student from the Food Science and Technology Program at Zamorano Agricultural University in Honduras was also trained in Dr. Monu's laboratory during the reporting period and succesfully defended his undergraduate research thesis based on the resulting data in August 2019. Two new MS students have begun research in this project (Zoila Chevez Tepas and Shijie Qin). An undergraduate researcher (Amanda Marinello) also began a research project in Dr. Monu's lab in the Fall semester of 2019. A visiting Fulbright researcher from Nigeria began conducting research in Dr. Monu's lab in August of 2019. How have the results been disseminated to communities of interest?The results have been disseminated through an undergraduate research thesis, PhD dissertation and presentations at international conferences, as well as one publication. What do you plan to do during the next reporting period to accomplish the goals?Based on the generated data, publications will be submitted to peer-reviewed journals, and graduate students will continue to work on experiments.Generated data will also be used to apply for external funding from the USDA. Work will continue to optimize an antimicrobial based on the plant extract P. guajava that was discussed in the 2017 -2018 reporting period. Fractions will be produced of the crude extract from leaves and the fraction with the greatest antimicrobial activity will be identified.

Impacts
What was accomplished under these goals? The project conducted by the PhD student Janet Gradl had several objectives: (1) assess the scientific basis of the agricultural water provision of the Produce Safety Rule (PSR), (2) determine the average E. coli presence in agricultural water used to grow produce during a growing season at three locations across Alabama, and (3) determine which food safety practices a sample of AAES farm managers implement. The study found that different E. coli enumeration techniques resulted in a variation in E. coli counts. The study also found that ground water sources had lower E. coli counts than surface water sources, although all sources had E. coli levels within tolerance of the PSR limits. The effectiveness of a produce wash for leafy greens using mustard essential oil was evaluated and compared to the standard chlorine wash used by the produce industry, and chlorine dioxide. The compound at 1% (v/v) was as effective as the standard chlorine wash at reducing E. coli on romaine lettuce, and both were more effective than the chlorine dioxide.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Porter, A. and Monu, E.A. 2019. Evaluating the antimicrobial efficacy of white mustard essential oil alone and in combination with thymol and carvacrol against Salmonella. Journal of Food Protection 82 (12): 2038-2043
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Monu, E., Pacheco, W., Starkey, C. and Caldas-Cuerva, J. 2019. Thermal inactivation of the Salmonella surrogate Enterococcus faecium ATCC 8459 during extended feed conditioning. PSA Annual Meeting, Montreal, Canada.
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Gradl, J. 2019. Assessing the Scientific Basis of the Food Safety Modernization Act Produce Safety Rule and Microbial Quality of Water Used to Grow Produce in Alabama

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Fellow researchers in the areas of agriculture, food science and food safety were reached through presentations at national, international and local conference. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A Masters student (Shelli Laskowitz) has completed a thesis based on the conducted research and graduated in Summer 2018. A Doctoral student (Sayma Afroj) was trained under this project under joint supervision of Mark Liles in the Department of Biological Sciences. An additional Doctoral student (Janet Gradl) was also trained under this project under joint supervision of Michelle Worosz of in the Department of Agricultural Economics and Rural Sociology. An undergradiate student from the Food Science and Technology Program at Zamorano Agricultural University in Honduras was also trained in Dr. Monu's laboratory during the reporting period. How have the results been disseminated to communities of interest?Results of the research have been presented at international and national conference on Food Science and Food Safety. Presentations have also been delivered internally at Auburn University at the AU This Is Research Symposium. What do you plan to do during the next reporting period to accomplish the goals?Based on the generated data, publications will be submitted to peer-reviewed journals, and additional graduate students will be recruited to work on experiments. Generated data will also be used to apply for external funding from the USDA.

Impacts
What was accomplished under these goals? A novel metagenomic approach was used to discover bacteriocins encoded by soil microorganisms. A number of Class III bacteriocins, Lasso peptides, lanthipeptides, sactipeptides and bottromycin-like bacteriocins were identified and five predicted gene sequences were selected PCR amplified, subcloned and successfully expressed. Bacteriocin expression was optimized in these clones. The crude extracts of Cryptolepis sanguinolenta and Psidium guajava were further investigated as they exhibited the highest antimicrobial activity of the West African extracts tested against foodborne microorganisms in the previous year. antimicrobial activity of P. guajava at 1.00% (w/v) and C. sanguinolenta at 0.50% (w/v) against E. coli and Salmonella were evaluated in milk at room temperature for 72 h. The extracts were overall less effective in reducing bacterial populations when tested in a model food system, rather than the ideal conditions in microbiological media, but were still able to have a bacteriostatic effect against the bacteria. It was also found that the antimicrobial activity of the extracts against the two bacteria was highest at a pH of 7, compared to pH 4.5 - 6.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Afroj, S. and Monu, E. 2018. Identification and heterologous expression of novel antimicrobial bacteriocins from a soil metagenome. IAFP Annual Meeting, Salt Lake City, UT.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Monu, E. and Laskowitz, S. 2018. Preliminary investigation of extracts indigenous to West Africa as alternative antimicrobial agents against common foodborne pathogens and spoilage organisms. IFT Annual Meeting, Chicago, IL.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Laskowitz, S. and Monu, E. 2018. The effect of pH on the antimicrobial activity of Cryptolepsis sanguinolenta and Psidium guajava against Salmonella and Escherichia coli. IAFP Annual Meeting, Salt Lake City, UT.
• Type: Theses/Dissertations Status: Accepted Year Published: 2018 Citation: Laskowitz, S. 2018. Investigation of West African plant extracts as alternative, natural sources of antimicrobials against foodborne pathogens and spoilage organisms.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students John Adam Porter and Shelli Laskowitz (MS) in the Depratment of Poultry Science (Food Microbiology specialization) were trained under this project. A Doctoral student (Sayma Afroj) was also trained under this project under joint supervision of Mark Liles in the Department of Biological Sciences. They performed the experiments and also presented at conferences. John Adam Porter also completed his thesis. 3 undergraduates were trained in Dr. Monu's laboratory under this project; one student from the Food Science progam in the Department of Poultry Science, and 2 interns from the Food Science and Technology Program at Zamorano Agricultural University in Honduras. How have the results been disseminated to communities of interest?Results have been presented to the scientific community at national conferences of Food Safety and Poultry Science. Presentations have also been delivered internally at Auburn University at the AU This Is Research Symposium. What do you plan to do during the next reporting period to accomplish the goals?Based on the generated data, publications will be submitted to peer-reviewed journals, and additional graduate students will be recruited to work on experiments. Using the data gathered during this reporting period, concentrations and blends of antimicrobials will be further refined to optimize use in food products.

Impacts
What was accomplished under these goals? The efficacy of white mustard essential oil as an antimicrobial against Salmonella in chicken was evaluated. It was found that an additive effect was achieved between was achieved between WMEO and carvacrol as well as thymol (antimicrobial compounds found in thyme and oregano). In addition, 0.75% WMEO and 0.1% carvacrol combination had a bacteriostatic effect against Salmonella at a temperature abuse of 10 °C, increasing shelf life. At 4 °C the application of this combination effectively controlled the growth of Salmonella, reducing levels of Salmonella by approximately 1 log CFU/g through 12 days of storage. A novel metagenomic approach was used to discover bacteriocins encoded by soil microorganisms. A number of Class III bacteriocins, Lasso peptides, lanthipeptides, sactipeptides and bottromycin-like bacteriocins were identified and five predicted gene sequences were selected PCR amplified, subcloned and successfully expressed. Bacteriocin expression was successful in three of these clones. The bacteriocins were purified and can now be tested against foodborne bacteria. This is a potential novel source of new antimicrobials. Freeze dried extracts of the plants Alchornea cordifolia, Senna alata, Psidium guajava, Cryptolepsis sanguinolenta, Solanum torvum, Piper guineense and Aframomum melegueta were obtained from the Centre for Scientific Research into Plant Medicine in Mampong, Ghana. Two extracts were made from each plant, one with 70% ethanol and one with methanol. After screening against several strains of E. coli, L. monocyogenes and Salmonella using a well diffusion assay, it was found that the ethanolic extracts of Psidium guajava, Cryptolepsis sanguinolenta and Senna alta were effective. L. monocytogenes was resistant to all of the extracts tested except the ethanol extract of Senna alta.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Porter, J.A. and Monu, E.A. 2017. Antimicrobial activity of white mustard essential oil on Salmonella spp. in vitro and in ground chicken. IAFP Annual Meeting, Tampa, Florida.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Porter, J.A. and Monu, E. 2017. Effect of white mustard essential oil and carvacrol on Salmonella in ground chicken. PSA Annual Meeting, Orlando, Florida
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Afroj, S., Zhou, J., Mead, D.A., Liles, M.R. and Monu, E. 2017. Cloning, expression and purification of bacteriocin from soil samples using metagenomic technique. ASM Annual Meeting, New Orleans, Louisiana.
• Type: Theses/Dissertations Status: Accepted Year Published: 2017 Citation: Evaluating the Antimicrobial Efficacy of White Mustard Essential Oil against Salmonella