Source: RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY submitted to
FURTHER STUDIES OF PLEUROCIDIN, A NATURAL ANTIMICROBIAL PEPTIDE, FOR FOOD APPLICATIONS.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0190620
Grant No.
(N/A)
Project No.
NJ10142
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 9, 2011
Project End Date
Sep 8, 2016
Grant Year
(N/A)
Project Director
Lee, TU.
Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559
Performing Department
Food Science
Non Technical Summary
Every year, food-borne diseases are estimated to affect between 68.7 and 275 million people in the U.S. alone, and cost about $15 billion in medical care and lost productivity. Food contamination, caused by pathogens in various raw or cooked fish and other foods and food products, affect millions of people annually. This project will examine the effectiveness of a new antimicrobial substance, found in fish, against food-borne pathogens and its effectiveness for the improvement of the stability and safety of various food products. Overall, the results expected from our research will provide information as to why and how antimicrobial peptides can be used as natural food antimicrobial agents. Based on this background we can broaden the spectrum of food applications from raw to processed foods. Furthermore, we will demonstrate and verify the feasibility of using Pleurocidin and our newly developed nanotechnology-enabled biopolymer films (membranes) coated with pleurocidin as anti-microbial agent in model food systems and in real perishable food products.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7124010108025%
7125010109050%
7123799110325%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria; 3799 - Cultured aquatic animals, general/other; 5010 - Food;

Field Of Science
1090 - Immunology; 1103 - Other microbiology; 1080 - Genetics;
Goals / Objectives
Overall, we will demonstrate and verify the feasibility of using Pleurocidin and our newly developed nanotechnology-enabled biopolymer films (membranes) coated with pleurocidin as anti-microbial agent in various model food systems and other real perishable food products Our first objective as we continue this research is to obtain a large amount of purified pleurocidin as custom synthetic peptide that is dependable and affordable by commercial companies (e.g. Biomatik, Peptide 2.9, etc.) for our future experiments. Secondly, we want to verify the antimicrobial activity of the synthetic peptide against food-related pathogenic microorganisms and physical properties. Thirdly, we want to develop new nanotechnology-enabled stability and quality improvement and reusable nano-biopolymer films (membranes) specially coating with pleurocidin for foods applications Fourthly, we will conduct studies to determine the efficacy of pleurocidin in additional real food samples and various food systems by studying its effect on the safety and the shelf-life extension aspects of different types of food samples. Finally, we will conduct studies to determine the efficacy of our newly-developed nanotechnology-enabled stability and quality improvement and reusable nano-biopolymer films (membranes) that are coated with pleurocidin in real food samples and various food systems, studying its effect on the safety and the shelf-life extension aspects of different types of various food model systems and real food samples.
Project Methods
The present project will be conducted in four major stages: Stage 1 to obtain large amount of purified pleurocidin as custom synthetic peptide that is dependable and affordable by commercial companies (e.g. Biomatik, Peptide 2.9, etc.) for our future experiments. Stage 2 - We want to verify the antimicrobial activity of the synthetic peptide against food-related pathogenic microorganisms and physical properties. Stage 3 - We want to develop new nanotechnology-enabled stability and quality improvement and reusable nano-biopolymer films (membranes) specially coated with pleurocidin for food applications Stage 4- we will conduct studies to determine the efficacy of pleurocidin in actual food samples and various food systems by studying its effect on the safety and the shelf-life extension aspects of different types of food samples. Stage 5- we will conduct studies to determine the efficacy of our newly- developed nanotechnology-enabled stability and quality improvement and reusable nano-biopolymer films (membranes) specially coating with pleurocidin in real food samples and various food systems by studying its effect on the safety and the shelf-life extension aspects of different types of food samples.

Progress 09/09/11 to 09/08/16

Outputs
Target Audience:Based on what we have learned from both the aspect of basic understanding of scientific knowledge and practical application of pleurocidin in food system, through this project, the target audience has been the following: The fisherman and seafood industry. The aquacultural industry. The food scientists and the food industry. General consumers. Academic research people (e.g., microbiologist, clinical, chemist, ...etc.) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?We have published the data in professional journals and presented data in scientific meetings (e.g., Institute of Food Technologists (IFT) National Annual Meetings, etc....) and public meetings to disseminate the useful information for scientific and general public application. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Previous work by our group revealed that pleurocidin is heat stable, salt tolerant and active over a wide pH range. It is also non-toxic and non-hemolytic to human cells. Pleurocidin is produced naturally by an edible fish; thus, it has great potential application in food preservation. If large-scale production of pleurocidin is achieved it will improve the marketability and shelf-life of many food and could be used to reduce the level of infection in farmed fish. Longer shelf-life of food products will generate higher incomes for both providers and technologists and will therefore benefit the food industry both in New Jersey as well as internationally. In addition, the overall benefit of replacing conventional food preservatives with natural antimicrobial peptides such as pleurocidin, while concomitantly uses it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistant, would be an extremely important benefit to be gained from our research. Overall, we have demonstrated and verify the feasibility of using Pleurocidin and our newly developed electrospun nanofiber nanotechnology-enabled pleurocidin as anti-microbial agent in various model food systems and other real perishable food products During this investigation, we have succeeded to accomplish the following goals: 1) We have succeeded to obtain large amount of purified pleurocidin as custom synthetic peptide (GWGSFFKKAAHVGKHVGKAALTHYL) that is dependable and affordable from a commercial company (Genscript USA Inc. 860 Centennial Avenue, Piscataway, New Jersey 08854) for our future experiments. 2) We have verified the antimicrobial activity of the synthetic peptide against food-related pathogenic microorganisms and physical properties. 3) We have revealed that the efficacy of Pleurocidin in food system was reduced because pleurocidin interacted with various proteins from food. This unexpected important finding is critical for the antimicrobial peptide (e.g. pleurocidin) application. Therefore, we have investigated the mechanism of these types of interaction and possible ways of remediation (e.g. using Nano technological approach for delivery). 4) We have successfully developed a Pleurocidin- poly (vinyl alcohol) electrospun nanofiber, a novel new nanotechnology-enabled stability and quality improvement product for foods application. (See Wang, Yue and Lee Wang (2016), Development of Pleurocidin-poly (vinyl alcohol) electrospun antimicrobial nanofibers to retain antimicrobial activity in food system application. Food Control 54, 150-157) as described in the following: This study aims to preserve the bioactivity of Pleurocidin in real foodstuffs by incorporating it in poly (vinyl alcohol) electrospun nanofiber. A pattern of sustained release of Pleurocidin from fiber, following initial burst release was successfully achieved, which could be tuned by varying environmental temperature. In vitro study clearly illustrated that Pleurocidin-incorporated fiber could efficiently inhibit foodborne pathogens. More importantly, released Pleurocidin showed higher inhibition efficiency than free Pleurocidin against Escherichia coli O157:H7 in a real food system - apple cider, mainly due to the protection from negative influence of environmental components by polymeric fiber matrix. The maintenance of Pleurocidin activity suggests an extensively promising application of electrospinning technology in antimicrobial peptide immobilization towards improved food safety. In summary, our research succeeded in both aspect of basic understanding and practical applications of Pleurocidin application in food systems.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, P.. Lee, Tung-Ching, Xu, X., (2016) The contribution of gluten in macropolymer depolymerization to the deterioration of frozen steamed bread dough quality. Food Chemistry. 2016 Nov 15: 211: 27-38
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, X., Yue. T., Lee, Tung-Ching (2016) Development of Pleurocidin-poly (vinyl alcohol) electrospun antimicrobial nanofibers to retain antimicrobial activity in food system application. Food Control 54, 150-157


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Scientific communiity and general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students (all on Ph.D. Program) received training in research and professional development associated with this project. How have the results been disseminated to communities of interest?We have published the data in professional journals and presented data in scientific meetings and public meetings to disseminate the useful information for scientific and general public application. What do you plan to do during the next reporting period to accomplish the goals?We will continue our effort to fellow our objectives as cited in our proposal. Specifically, we want continue to further evaluation the efficacy of our newly developed PVA (Ple/PVA) electrospun antimicrobial fibers capable for maintain the antimicrobial activity of Ple in the application of food systems We will first to verify the antimicrobial activity of our newly developed PVA (Ple/PVA) electrospun antimicrobial fibers for food-related pathogenic microorganisms and its physical properties. We will continue to conduct studies to determine the efficacy of the newly developed PVA (Ple/PVA) electrospun antimicrobial fibers in additional real food samples and various food systems by studying its effect on the safety and the shelf-life extension aspects of different types of food samples. We will conduct studies to improve the stability and quality of our newly-developed developed PVA (Ple/PVA) electrospun antimicrobial fibers to be use in real food samples and various food systems. We plan to conduct the following experiments: 1. Release profiles of Ple from PVA fiber 2.. Minimum inhibition concentration (MIC) detection of Ple against E. coli 3. Loading efficiency of Ple/PVA fiber 4. Evaluation of antimicrobial activity of Ple/PVA fibers in vitro 5. Application of free Ple in media and apple cider against E. coli 6. Antimicrobial activity comparison of free Ple and Ple/PVA fiber against E. coli in apple cider

Impacts
What was accomplished under these goals? Pleurocidin (Ple) is a novel antimicrobial peptide with advantages of broad microbial inhibition spectrum and thermal/pH/salt-tolerance. However, direct application of Pleurocidin into food systems caused its activity loss due to it interaction with food proteins in the food systeas aswe have shown in our previous studies. In order to make a breakthrough from this problem,We focused our effort based on" Development of Pleurocidin-poly(vinyl alcohol) electrospun antimicrobial nanofibers to retain antimicrobial activity in food system applicationon". We aims to preserve the bioactivity of Pleurocidin in real foodstuffs by incorporating it in poly(vinyl alcohol) electrospun nanofiber. Electrospinning has been widely employed to fabricate ultrafine fibers based on polymers to deliver peptides, proteins, enzymes and drugs due to its low production cost and high surface area-tovolume. The entrapment of a variety of active agents in polymers via electrospinning technique might represent an alternative strategy to overcome the critical problems associated with direct application of these bioactive agents in real systems. In this period, we have firstspent a lot of time and effort to successfullyestablish an " electrospinning system.", then. wehave used this system to develop a successfully Ple incorporated PVA (Ple/PVA) electrospun antimicrobial fibers capablefor maintain the antimicrobial activity of Ple in the application of food systems.In addition, morphology of electrospun nanofibers and the release profiles of Ple from PVA fiber were determined. For the characterization of electrospun fibers, the surface images of electrospun fiber were characterized by atomic force microscopy (AFM). We also studied the Release profiles of Ple from PVA fiber and theLoading efficiency of Ple/PVA fiber. All these data are required for our further evaluation the efficacy of our developed PVA (Ple/PVA) electrospun antimicrobial fibers capable for maintain the antimicrobial activity of Ple in the application of food systems.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhou, Zhengkun., Jiang, Feihong., Lee, Tung-Ching., Yue, Tianli.,(2014) Two-step preparation of nano-scaled magnetic chitosan particles using Triton x-100 reversed phase water-in-oil microemulsion system Journal of Alloys and Compounds V 681, Dec, 2013, pp843-848 DOI:10 1016/j jallcom 2013.07 207
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhou, Zhengkun., Jun, Jin., Lee, Tung-Ching., Yue, Tianli.,(2014) Optimization of Covalent immobilization of extracellular ice nucleators from Erwinia herbicola on magnetic Fe3O4/chitosan nanoparticles for potential application in freeze concentration Food Process Technol 92014) 7:3259-3268. DOI10.1007/s11497-014-1318-6


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Scientific community and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Four graduate students (all on Ph.D. Program) received training in research and professional development associated with this project. How have the results been disseminated to communities of interest? We have published the data in professional journals and presented data in scientific meetings and public meetings to disseminate the useful information for scientific and general public application. What do you plan to do during the next reporting period to accomplish the goals? We will continue our effort to fellow our objectives as cited in our proposal. Specifically, we want to verify the antimicrobial activity of the synthetic peptide against food-related pathogenic microorganisms and physical properties and develop new nanotechnology-enabled stability and quality improvement and reusable nano-biopolymer films (membranes) specially coating with pleurocidin for foods applications. We will conduct studies to determine the efficacy of pleurocidin in additional real food samples and various food systems by studying its effect on the safety and the shelf-life extension aspects of different types of food samples.We will conduct studies to determine the efficacy of our newly-developed nanotechnology-enabled stability and quality improvement and reusable nano-biopolymer films (membranes) that are coated with pleurocidin in real food samples and various food systems.

Impacts
What was accomplished under these goals? During this period we have investigated: 1) The interaction between proteins and pleurocidin :Four chosen proteins (BSA, β-lactoglobumin, Albumin from egg white and Gelatin from bovine skin) showed different influence on the antimicrobial activity of pleurocidin. With proteins of 12 mg/ml, equally, BSA was 179 μM, β-lactoglobumin was 652 μM, Albumin from egg white was 279 μM and Gelatin from bovine skin was 240-300 μM, pleurocidin represented different minimum inhibiting concentration against E.coli, respectively for these four proteins; 5.52μM, 17.71 μM, 8.85 μM and 5.52 μM. 2) the feasibility of using Pleurocidin in apple juice system. 3.68μM, 7.36μM and 14.72μM pleurocidin was added into apple juice which was inoculated with E.coli, The results indicated that pleurocidin could inhibit the growth of E.coli, and with the increasing of its concentration, the inhibition increased. 3) The nanotechnological application of immobilizing Pleurocidin in various films for food application: A) Immobilization of pleurocidin on modified PE film, No antimicrobial activity of film against s.aureus was detected. B) Immobilization of Pleurocidin and Nisin on zein film. Based on the result of film and released solution against micrococcus, results indicatedthat Nisin solution and film showed activity. It indicated that Nisin could release from zein film. However, the Pleurocidin released from zein film kept activity, but released quickly in three days, in contrast to Nisin, which released slowly. Thus, Zein film can be used to encapsulate Pleurocidin and Nisin. Consequently, more work is needed to improve (A) and (B) and/or develop other better nanotechnological approachesfor future investigation.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Shi, K., Yu, Hailong,, Lee, Tung-Ching., and Huang, Q., 92014) Improving ice nucleation activity of zein film through layer-by-layer deposition of extracellular ice nucleators ACS Applied Materials & Interfaces, 2014, 5 (21) :pp 10456-64. Doi:10.1021/am401657
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Zhou, Zhengkun., Lin, Shiqi., Lee, Tung-Ching., Yue, Tianli.,(2014) Adsorption of food dyes from aqueous solution by glutara-aldehyde cross-linked magnetic chitosan nanoparticles Journal of Food Engineering, 2014 (126), pp133-141
  • Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Shi, K., Yu, Hailong. and Lee, Tung-Ching.,(2013) A novel approach for improving yeast viability and baking quality of frozen dough by biogenic ice nucleators from Erwinia herbicola Journal of Cereal Science 2013, 57 (2), pp 237-243; http://dx.doi.org/10.1916/jes.2012.11.010


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Scientific community and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? We have published the data in professional journals and presented data in scientific meetings and public meetings to disseminate the useful information for scientific and general public application. What do you plan to do during the next reporting period to accomplish the goals? We will continue our effort tofellowour objectives as cited in our proposl.

Impacts
What was accomplished under these goals? Previous work by our group revealed that pleurocidin is heat stable, salt tolerant and active over a wide pH range. It is also non-toxic and non-hemolytic to human cells. Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in food preservation. If large-scale production of pleurocidin is achieved it will improve the marketability and shelf-life of many foods and could be used to reduce the level of infection in farmed fish. Longer shelf-life of food products will generate higher incomes for both providers and technologists and will therefore benefit the food industry both in New Jersey as well as internationally. In addition, the overall benefit of replacing conventional food preservatives with natural antimicrobial peptides such as pleurocidin, while concomitantly uses it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistant, would be an extremely important benefit to be gained from our research. During this period we have investigated: 1) the antimicrobial activity of pleurocidin against E. Coli and Listeria and 2), the effect of various proteins on the pleurocidin activity and the minimum inhibiting concentration of pleurocidin against E. Coli with a certain proteins. The results showed that pleurocidin could inhibit the growth of E. Coli and Listeria with MIC of 1.84μM and 34.68-69.36μM respectively. More importantly, all four chosen proteins (BSA, β-lactoglobumin, Albumin from egg white and Gelatin from bovine skin) showed different influence on the reduction of antimicrobial activity of pleurocidin. With proteins of 12mg/ml, pleurocidin represented different minimum inhibiting concentration against E. Coli. Based on these result, we have revealed that the efficacy of Pleurocidin may be reduced because pleurocidin interacted with various proteins from food. This unexpected important finding is critical for the antimicrobial peptide (e.g. pleurocidin) application. Therefore, we need to investigate the mechanism of these types of interaction and possible ways of remediation immediately.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Gezgin, Z., Lee, Tung-Ching; and Huang, Q. (2013) "Engineering Functional Nano-Thin Multilayers on Food Packaging: Ice Nucleating Polyethylene Films" Journal of Agricultural and Food Chemistry (In Press)
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Shi., K., Yu, Hailong., Jun, Jin., and Lee, Tung-Ching., (2013)  Improvement to Baking Quality of Frozen Bread Dough by Novel Zein-based Ice Nucleation Films Journal of Cereal Science x.doi.org/10.1016/j.jcs. 2013.01.010
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Li, J., Li,Y.,Lee, Tung-Ching, and Huang, Q., (2013), Structure and Physical Properties of Zein/Pluronic F127 Composite Films (2013) Journal of Agric. & Food Chemistry 2013,61,1309-1318. dx.doi.org/10.1021/jf3043055
  • Type: Theses/Dissertations Status: Accepted Year Published: 2013 Citation: Development of Zein-Based Ice Nucleator Films for Frozen Food Applications Ke Shi (Dissertation Director: Dr. Tung-Ching Lee), Ph.D. Dissertation, Rutgers University October 2013.


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: For our first objective we succeeded in obtaining a large amount of purified pleurocidin as custom synthetic peptide (GWGSFFKKAAHVGKHVGKAALTHYL). It is dependable and affordable, from a commercial company (Genscript USA Inc. 860 Centennial Avenue, Piscataway, New Jersey 08854), for our future experiments. For our second objective, we succeeded in verifying the antimicrobial activity of the above synthetic peptide against food-related pathogenic microorganisms. We will continue to analyze the spectrum and mode of action of the synthetic Pleurocidin against pathogenic food borne microorganisms including 1) Antimicrobial screening: Food borne organisms, including those associated with fish spoilage will be tested against pleurocidin. Liquid growth inhibition assays will be used to determine Minimal Inhibitory Concentrations (MIC) at varying conditions of temperature, salt and pH. For our third objective, we have initiated our work to develop new nanotechnology-enabled stability and quality improvement and reusable nano-biopolymer films (membranes) specially coated with pleurocidin for food applications. We hypothesize that the antimicrobial activity of Pleuroidin will be maintained after its immobilization on natural biopolymer film surfaces. The resulting films can improve the safety of food against food- borne microorganisms and reduce quality deterioration during storage. Coating Pleuroidin to the biopolymer film surface should significantly improve the stability of Pleuroidin. In addition, the coated Pleuroidin is re-usable. This proposed research will cover both basic understanding and practical applications. Unfortunately, our preliminary results indicate a negative response of the immobilized Pleurocidin in the food system we tested. At present, we will investigate the negative mechanism and seek improvement. Furthermore, we succeeded in showing that the efficacy of using our pleurocidin in Apple juice-type product to eliminate the containment problem of E. Coli .This finding has extensive application to solve a long existing commercial problem. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Previous work by our group revealed that pleurocidin is heat-stable, salt-tolerant and active over a wide pH range. It is also non-toxic and non-hemolytic to human cells. Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in food preservation. If large-scale production of pleurocidin is achieved it will improve the marketability and shelf-life of many foods and could be used to reduce the level of infection in farmed fish. Longer shelf-life of food products will generate higher income for both providers and technologists and will, therefore, benefit the food industry in New Jersey as well as internationally. In addition, the overall benefit of replacing conventional food preservatives with natural antimicrobial peptides such as pleurocidin, while concomitantly using it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistant, will be an extremely important benefit to be gained from our research.

Publications

  • Shi, K,. Yu, H., Seema, L., and Lee, T-C. (2012) Improved Mechanical Property and Water Resistance of Zein Films by Plasticization with Tributyl Citrate. J of Agricultural and Food Chemistry 2012, 60 (23), pp.5988-5993.
  • Shi, K., Yu, H., Lee, Tung-Ching., (2012) Investigation of Adsorption Behavior of Bacterial Ice Nucleators on Zein Using Quartz Crystal Microbalance with Dissipation Monitoring. Proceeding of 2012 Annual Meeting of the Institute of Food Technologists, Las Vegas, Nevada
  • Shi, K., and Lee, Tung-Ching., (2012) Development of film with ice nucleation function and its application in frozen dough. Proceeding of 2012 Annual Meeting of the Institute of Food Technologists, Las Vegas, Nevada
  • Shi, K., Yu, H., Rao, T., Huang, Q., Lee, Tung-Ching., (2012) Improved Mechanical Property and Water Resistance of Zein Films by Plasticization with Tributyl. Proceeding of 2012 Annual Meeting of the Institute of Food Technologists, Las Vegas, Nevada


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Our project has progressed well in this period. For our first objective, we succeeded in obtaining large amounts of purified pleurocidin as custom synthetic peptide (GWGSFFKKAAHVGKHVGKAALTHYL) that is dependable and affordable from a commercial company (Genscript USA Inc. 860 Centennial Avenue, Piscataway, New Jersey 08854) for our future experiments. As for our second objective - We want to verify the antimicrobial activity of the synthetic peptide against food-related pathogenic microorganisms and physical properties. Preliminary results verify the positive antimicrobial activity of the synthetic peptide. We will continue to analyze the spectrum and mode of action of the synthetic Pleurocidin against pathogenic food borne microorganisms including 1) Antimicrobial screening: Food borne organisms, including those associated with fish spoilage will be tested against pleurocidin. Liquid growth inhibition assays will be used to determine Minimal Inhibitory Concentrations (MIC) at varying conditions of temperature, salt and pH. and 2) Additional cytotoxic evaluation of pleurocidin will be investigated in addition to our previous studies of its hemolytic activity of human red blood cells and cytotoxic effect on human intestinal epithelical cells. For our third objective - we initiated our work to develop new nanotechnology-enabled stability and quality improvement as well as reusable nano-biopolymer films (membranes) coated with pleurocidin for food applications. Our approach is based on the layer-by-layer (LbL) method to form nanoscale biopolymer multilayer films, which will then be used to immobilize Pleuroidin. This proposed research will cover both basic understanding and practical applications. We hypothesize that the antimicrobial activity of Pleuroidin will be maintained after its immobilization on natural biopolymer film surfaces. The resulting films can improve the safety of food against food- borne microorganisms and reduce quality deterioration during storage. Coating Pleuroidin to biopolymer film surfaces should significantly improve the stability of Pleuroidin. In addition, the coated Pleuroidin is re-usable. PARTICIPANTS: KeShi (0.5 Graduate) Jun Jin (0.5 Graduate) ZhengKun Zhou (0.5 Graduate) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Previous work by our group revealed that pleurocidin is heat stable, salt tolerant and active over a wide pH range. It is also non-toxic and non-hemolytic to human cells. Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in food preservation. If large-scale production of pleurocidin is achieved, it will improve the marketability and shelf-life of many foods and could be used to reduce the level of infection in farmed fish. Longer shelf-life of food products will generate higher income for both providers and technologists and will therefore benefit the food industry, both in New Jersey as well as internationally. In addition, the overall benefit of replacing conventional food preservatives with natural antimicrobial peptides such as pleurocidin, while concomitantly using it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistant, would be an extremely important benefit to be gained from our research.

Publications

  • Gezgin, Z., Huang, Q., Lee, Tung-Ching. 2011. Food Nanotechnology on the Shelf: Fabicating Nano-thin Ice Nucleating Layers on Polyethylene Films. Proceeding of 2011 Annual Meeting of the Institute of Food Technologists, New Orleans, LA.
  • Shi, K., Huang, Y., Huang, H., Lee, Tung-Ching and Huang, Q. 2011. Reducing the Brittlenes of Zein Films Through Chemical Modification. Journal of Agric. & Food Chemistry, 2011,59,56-61,DOI:10,1021/if10314r.
  • Shi, K., Yu, H., Huang, Q., Lee, Tung-Ching. 2011. Protective Effects on Freeze Thaw Stability of Frozen Bread Dough by Extracellular Ice Nucleators from Erwinia herbicola. Proceeding of 2011 Annual Meeting of the Institute of Food Technologists, New Orleans, LA.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Effect of environmental factors (i.e. concentration of salt, pH, and temperature) on the efficacy of pleurocidin was evaluated. The efficacy of untreated pleurocidin on the growth of V. parahemolyticus was conducted and its effectiveness was first confirmed. Results of the effect of environmental factors on the efficacy of pleurocidin show that pleurocidin retains full activity after incubation with 5% salt. The lag phase of the test organism was extended by 15 hours at 7.5% salt. Being salt tolerant pleurocidin is ideally suited for use as a natural preservative in foods susceptible to halophilic bacteria, such as marine shell fish (shrimp, oysters and clams). Moreover, being active against a fish pathogen such as V. parahemolyticus, pleurocidin has significant potential application in aquaculture against other Vibrio species which are pathogenic to fish such as V. anguillarum (Jia et al., 2000). The result also shows pleurocidin to be active over a wide range of pH (pH 4 to 8), which makes this peptide a potentially ideal preservative in both high and low acid foods or to be used synergistically with acidic preservatives. Pleurocidin not only retained full activity after 10 minutes of boiling, as reported by Cole et al., (1997), but also retained activity after autoclaving for 1h (15 psi and 121 degrees C). The heat resistance of pleurocidin indicates its suitability for use in heat processed foods, or foods exposed to temperature abuse. These results have significant food safety implications because they indicate that pleurocidin could potentially be used as a natural food preservative against halophilic, acidophilic and thermophilic food borne microorganisms. These findings also form the basis on which further research on the efficacy of pleurocidin in actual foods may be pursued. PARTICIPANTS: Ke Shi (0.5 Graduate) Jun Jin (0.5 Graduate) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Previous work by our group revealed that pleurocidin is heat stable, salt tolerant and active over a wide pH range. It is also non-toxic and non-hemolytic to human cells. Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in food preservation. If large-scale production of pleurocidin is achieved it will improve the marketability and shelf-life of many foods and could be used to reduce the level of infection in farmed fish. Longer shelf-life of food products will generate higher income for both providers and technologists and will therefore benefit the food industry both in New Jersey as well as internationally. In addition, the overall benefit of replacing conventional food preservatives with natural antimicrobial peptides such as pleurocidin, while concomitantly using it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistant, would be an extremely important benefit to be gained from our research.

Publications

  • Huang, Qingrong., Gezgin, Z., and Lee, Tung-Ching., 2010. Engineering novel biopolymeric functional films for frozen food application. Proceeding of 2010 Annual Meeting of the Institute of Food Technologists, Chicago Illinois
  • Shi, K., Deng, J., Huang, Q., Lee, Tung-Ching. 2010. Optimized Production and Characterization of Cell-free Ice Nucleators from Xanthomonas translucens. Proceeding of 2010 Annual Meeting of the Institute of Food Technologists, Chicago, Illinois
  • Huang, Qingrong, Gezgin, Z. and Lee, Tung-Ching. 2010. Engineering novel biopolymeric functional films for frozen food application. Proceeding of 2010 Annual Meeting of the Institute of Food Technologists, Chicago, Illinois
  • Varma, Supriya, Karwe, M.V., and Lee, Tung-Ching. 2010. Effect of High Hydrostatic Pressure Processing on Lycopene Isomers. International Food Engineering, Vol. 6, Iss. 5, article 14., Available at http:// www.bepress.com/ijfe/iss5/art14, DOI: 10.2002/1556-3758.1752
  • Ke,Shi, Huang, Y., Huang, H., Lee, Tung-Ching, and Huang, Q. 2011. Reducing the Brittleness of Zein Films through Chemical Modification. Journal of Agric. & Food Chemistry,2011, 59, 56-61, DOI:10,1021/if103164r


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Since Professor Lee, P.I. of this project was on a one year sabbatical from Rutgers University and he was awarded a National Research Council Chair Professorship in Taipei, Taiwan, the activity of the present project at Rutgers was thus minimized to some extent during this period. An experiment was designed to determine the efficacy of pleurocidin in food samples. Shrimp was chosen as the test food material for pleurocidin. The rationale for using shrimp as the test food material for our shelf life studies is due to the fact that shrimp is a commonly consumed shellfish and like other forms of shellfish, shrimp is prone to contamination from foodborne bacterial pathogens such as V. parahemolyticus and V. vulnificus (CDC Report, 1999; IFT Expert Report, 2002). The FDA guidelines for shrimp were revoked in 1996 and are currently being revised; therefore, there is no set standard treatment or guidelines for acceptable levels of microorganisms in shrimp. Pleurocidin, which naturally exists in an edible marine organism (winter flounder), would be an ideal antimicrobial agent for use on shrimp. The objective of this study was to determine the efficacy of an antimicrobial peptide on shrimp; therefore, microbiological indicators of spoilage were tested. Because pH increases during shrimp spoilage due to deamination of proteins in fish, the pH of the samples was tested to corroborate microbiological tests. V parahemo/vticus, as well as other Vibrio species, normally grow at mesophilic temperatures (18 - 37 C) and therefore may not grow on iced shrimp or will be outgrown by psychrophillic species such as Pseudomonas. Therefore, it may not be possible to isolate this organism from shrimp stored on ice. In order to ascertain whether it would be possible to isolate V parahemolyticus from iced shrimp, a pilot study was conducted in which the growth of V. parahemolyticus inoculated in broth stored in ice (4 C) was compared to growth at room temperature (25 C) and growth at 37 C. Based on the results of the pilot study, the efficacy of pleurocidin on shrimp samples stored on ice was investigated. Results show that V parahemolyticus does survive storage on ice; this formed the basis of our studies on shrimp samples. Therefore, we will follow up by conducting a shelf life study to evaluate the efficacy of pleurocidin in shrimp. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Previous work by our group revealed that pleurocidin is heat stable, salt tolerant and active over a wide pH range. It is also non-toxic and non-hemolytic to human cells. Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in food preservation. If large-scale production of pleurocidin is achieved it will improve the marketability and shelf life of many foods and could be used to reduce the level of infection in farmed fish. Longer shelf life of food products will generate higher income for both providers and technologists and will therefore benefit the food industry in New Jersey as well as internationally. In addition, the overall benefit of replacing conventional food preservatives with natural antimicrobial peptides such as pleurocidin, while concomitantly using it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistant, would be an extremely important benefit to be gained from our research.

Publications

  • Jiang, S. and Lee, Tung-Ching. 2009. Chapter 39 Freezing Seafood and Seafood Products: Principles and Application in Handbook of Food Science, Technology, and Engineering (Ed. Y.H. Hui, et al.) Vol 1, pp 39-1-19-35 Taylor & Francis Group, Boca Raton, London and New York
  • Burrowes, O., Diamond, J., Matthews, K. and Lee, Tung-Ching. 2009. The Efficacy of Pleurocidin in Food Systems in preparation for submitting to International Journal of Food Microbiology
  • Burrowes, O., Diamond, J. and Lee, Tung-Ching. 2009. Effect of Environmental Factors on the Efficacy of Pleurocidin in preparation for submitting to International Journal of Food Microbiology


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Since Professor Lee, P.I. of this project is on one year sabbatical leave (from June 1, 2008 to May 30, 2009) from Rutgers University and he was awarded as a National Research Council Chair Professorship in Taipei, Taiwan, the activity of the present project thus is minimized to some extent for this period. An experiment was conducted to determine the efficacy of pleurocidin in food samples. Apple cider was chosen as the test food material for pleurocidin because there is wide-spread concern regarding the survival of E.coli 0157:H7 in apple cider. E. coli 0157:H7 has been shown to survive for over 21 days in apple cider stored at refrigeration temperature (Fisher and Boyd, 1998; Miller and Kasper, 1994; Zhao and Doyle, 1993). We found pleurocidin to be active against E. coli 0157:H7, and Penicillium expansum isolated from rotting apple, it is tolerant to low pH (pH 4) and is heat resistant even at autoclaving temperature. It is, therefore, an ideal candidate to be used as a preservative for apple cider, which is acidic in nature and in addition to being susceptible to E. coli 0157:H7 might also be infected by yeasts and molds, both of which have been shown to be sensitive to pleurocidin. The efficacy of pleurocidin in apple cider was revealed in our investigation. A 2 log 10 reduction of E. coli 0157:H7 in apple cider (pH 3.69) was obsrved within 3 days and complete killing within 15 days. E. coli 0157:H7 cells survived in untreated cider sample up to the end of the test period (18 days). Furthermore, the stability of pleurocidin in apple cider is confirmed. Pleurocidin gradually depleted over time and was reduced from 259 ug/ml to 48 ug/ml by day 15. However, pleurocidin remained at a level that was still inhhibitory to E. coli 0157:H7. Our result shows that pleurocidin is very unique and useful for the extension of shelf-life and food safety applications of apple cider, which does not have alternative methods avialable. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Previous work by our group revealed that pleurocidin is heat stable, salt tolerant and active over a wide pH range. It is also non-toxic and non-hemolytic to human cells. Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in food preservation. If large-scale production of pleurocidin is achieved it will improve the marketability and shelf-life of many foods and could be used to reduce the level of infection in farmed fish. Longer shelf-life of food products will generate higher income for both providers and technologists and will, therefore, benefit the food industry in New Jersey as well as internationally. In addition, the overall benefit of replacing conventional food preservatives with natural antimicrobial peptides, such as pleurocidin, while concomitantly using it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistan, would be an extremely important benefit to be gained from our research.

Publications

  • Gezgin,Z.; Lin, Y.; Huang, Qingrong and Lee, Tung-Ching. 2009. "Layer by layer (LbL) deposition of oppositely charged biopolymers to produce energy saving packaging materials." Proceeding of 2008 Annual Meeting of the Institute of Food Technologists, June 6-10, Anaheim, California. Accepted.
  • Varma, S.; Karwe, Mukund and Lee, Tung-Ching. 2009. "Effect of High Hydrostatic Pressure Processing on Lycopene Isomers." Proceeding of 2008 Annual Meeting of the Institute of Food Technologists, June 6-10, Anaheim, California. Accepted.
  • Jiang, s, and Lee, Tung-Ching. 2007. Chapter 39. "Freezing Seafood and Seafood Products: Principles and Application" in Handbook of Food Science, Technology and Engineering (Ed. Y. H. Hui, et al). Vol 1, pg 39-1 - 39-35. Taylor and Francis Group, Boca Raton, London and New York.
  • Burrowes, O., Diamond, J., Matthews, K. and Lee, Tung-Ching. 2008. "The Efficacy of Pleurocidin in Food Systems" submitted to International Journal of Food Microbiology.
  • Burowes, O., Diamond, J., and Lee, Tung-Ching. 2008. "Effect of Environmental Factors on the Efficacy of Pleurocidin" submitted to International Journal of Food Microbiology.


Progress 01/01/07 to 12/31/07

Outputs
In order to standardize the potency of antimicrobial activity measurement of "Pleruocidin" for our investigation in this project, we put forward a major effort and spent a substantial time during this period, to synthesize Pleurocidin in our laboratory through a chemical method. Thus far we demonstrated our synthetic Pleurocidin (1 microgram per micro liter) effectively inhibits the growth of Bacillus subtilis, Enterobacter sakazakii, Escherichia coli and Vibrio parahaemolyticu. The inhibitory concentration exhibited by pleurocidin is much lower than the levels of traditional preservatives used in these areas. Hydroxybenzoate esters and sulfites, which are often used as preservative for food products, are usually used at levels of 200 to 1000 micro grams per milliliter (Russell and Gould, 1991). It is also well below the legal limit of nisin in foods which is 10,000 International Units per gram (2.5 milligrams per gram). In order to determine the efficacy of Pleurocidin on the shelf-life extension of different types of actual food samples we selected apple cider as a first test food. We choose apple cider for testing Pleurocidin because there is wide-spread concern regarding the survival of E. coli O157:H7 in apple cider. E. coli O157:H7 has been shown to survive for over 21 days in apple cider stored at refrigeration temperature (Fisher and Boyd, 1998; Miller and Kasper, 1994; Zhao and Doyle, 1993). Experimentation is currently on going for this investigation.

Impacts
Previous work by our group revealed that Pleurocidin is heat stable, salt tolerant and active over a wide pH range. It is also non-toxic and non-heolytic to human cells. Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in food preservation. If large-scale production of Pleurocidin is achieved it will improve the marketability and shelf-life of many foods and could be used to reduce the level of infection in farmed fish. Longer shelf-life of food products will generate higher income for both providers and technologists and will, therefore, benefit the food industry both in New Jersey as well as internationally. In addition, the overall benefit of replacing conventional food preservatives with natural antimicrobial peptides such as Pleurocidin, while concomitantly using it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistant, would be an extremely important benefit to be gained from our research.

Publications

  • Jiang, S and Lee, Tung-Ching. 2007. Chapter 39, Freezing Seafood and Seafood Products: Principles and Application in Handbook of food Science, Technology and Engineering (Editor, Y.H. Hui, et al)vol 1, pp. 39-1 to 39-35. Taylor and Francis Group. Boca Raton, London and New York.
  • Varma, S., Huang, M., and Lee, Tung-Ching. 2007. In-vitro Anti-proliferative Effect of Cis and All Trans Lycopene Isomers on Prostrate Cancer Cells. Proceeding of 2007 Annual Meeting of the Institute of Food technologists, July 28 to August 1, 2007, Chicago, Illinois.
  • Burrowes, J.J., Diamond, G. and Lee, Tung-Ching. 2006. Recombinant Expression of Pleurocidin cDNA using the Pichia pastoris Expression System. Journal of Biomedicine and Biotechnology 4:374-384.
  • Pyo, Young-Hee and Lee, Tung-Ching. 2007. The Potential Antioxidant Capacity and Angiotensin I-coverting Enzyme Inhibitory Activity of Monascus-fermented Soybean Extracts: Evaluation of Monascus-fermented Soybean Extracts as Multi-functional Food Additives. J of Food Science 72(3):S218-223.
  • Zhu, Zun-Tao and Lee, Tung-Ching. 2007. Application of Biogenic Ice Nucleator for Food Processing: Effect of Extra-cellular Ice Nucleator (ECIN) on the Reduction of Freeze-thaw (FT) Cycles Abuse of Textural Gel-forming Capacity of Frozen Fish Actomyosin. Journal of International Food Science and Technology.
  • Lee, Tung-Ching. 2007. Biogenic Ice Nucleators for Energy Saving and Quality Improvement in the Freezing and Storage of Foods in the Proceeding of the 5th International Congress on Food technology, volume, 1, Thessaloniki (Editor, E.S. Lazos). Published by Hellenic Association of Food Technologists, Thessaloniki, Greece, pp. 578-587.


Progress 01/01/06 to 12/31/06

Outputs
Efficacy of Pleurocidin in food Samples (shrimp) was conducted. Fresh Pacific White Shrimp (Litopenaeus vannamai) obtained from the Indian River Aquaculture Shrimp Farm (Vero Beach, FL, USA), were shipped, in ice packs, overnight, to our laboratory on the day of harvesting. Tests were initiated on the day of arrival to the laboratory. Shrimps were de-headed and immersed in 55 ug/ml of pleurocidin. Samples were stored in stainless steel containers placed in ice, in a large cooler box which allowed for proper draining of melting ice; ice was replenished as required. Samples were evaluated for pH, texture, odor detection (sensory panel of 21 untrained panelists) and the presense of V. parahemolyticus, coliforms and total aerobic plate count. Results show that the level of surviving V. parahemolyticus cells reduced in the presence of pleurocidin was comparable to the control sample throughout the study, however, by day 12 of the study there was a 1.5 log 10 difference between pleurocidin-treated samples and control samples, demonstrating the efficacy of pleurocidin against V. parahemolyticus. Although there was no significant difference between the odor of pleurocidin-treated and untreated shrimp (as evaluated by the sensory panel), the firmness (as a measure of texture) was retained for a longer period of time in pleurocidin-treated samples than in untreated control samples. Statistical analysis of the average force required to rupture shirmp samples revealed that treated samples retained firmness longer than untreated control samples. The pH of shrimp samples gadually increased over time for both control and pleurocidin treated samples. However, pH increased more rapidly for untreated samples. During spoilage shrimp undergo biochemical changes resulting in an increase in total votalile basic substances that causes an increase in pH (Miget, 1991; Benner et al., 2003; Goncalves et al., 2003). The increase of pH observed in the study implies a breakdown of protein by proteolysis organisms present on the shrimp; this was even more evident on un-inoculated shrimp than on shrimp inoculated with V. parahemolyticus. This result further supports our results showing inhibition of growth by pleurocidin. Our results also show that coliform populations initially increased over the first 24 h period for both test and control samples, however, there was a greater increase in coliforms on control samples, compared to the pleurocidin-treated samples. No growth was detected on pleurocidin-treated samples by day 4 while growth was detected on control samples up to day 7.

Impacts
Previous work by our group revealed that pleurocidin is heat stable, salt tolerant and active over a wide pH range. It's also non-toxic and non-hemolytic to human cells. The present work on shelf-life studies with shrimp using pleurocidin as an antimicrobial agent provides very important information about the usefullness of this antimicrobial peptide. Our results reveal that pleurocidin is not only effective against foodborne pathogens in broth culture, but also in actual food matricies and its use is not limited to seafood but extends to other types of foods/beverages. Moreover the demonstrated efficacy of pleurocidin against the marine food pathogen Vibrio parahemolyticus is of great benefit to the industry because this pathogen is the major foodborne pathogen in seafood. Presently, however, there is no set standard or treatment for Vibrio species in seafood. Our results indicate that pleurocidin could be applied in this sector. Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in seafood preservation. If large-scale production of pleurocidin is achieved it will improve the marketability and shelf-life of seafood and could reduce the level of infection in farmed fish. Longer shelf-life of seafood products will generate highter income for both providers and technologists and will, therefore, benefit the seafood industry both in New Jersey and interntaionally. This research is of major significance because it is the first comprehensive study of a novel marine antimicrobial peptide for application in food.

Publications

  • Burrows, O., Diamond, J. and Lee, Tung-Ching. 2007. Effect of Environmental Factors on the Efficacy of Pleurocidin. Submitted to the International Journal of Food Microbiology.
  • Moretti, D., Zimmermann, Muthayya, S., Thankachan, P., Lee, Tung-Ching, Kurpad, A.V. and Hurrell, R.F. 2006. Extruded Rice with Micronized Ground Ferric Pyrophosphate Reduce Iron Deficiency in Indian School Children: A Double-blind, Randomized, Controlled Trial. American Journal of Clinical Nutrition 84:822-829.
  • Yang, R-Y., Tsou, S., Lee, Tung-Ching, Wu, J. and Lai, P.Y. 2006. Distribution of 127 Edible Plant Species for Antioxidant Activities by Two Assays. Journal of the Science of Food and Agriculture 86:2395-2403.
  • Yang, R-Y., Tsou, S.C.S., Lee, Tung-Ching. 2006. Moringa, a Novel Plant Rich in Antioxidants, Bioavailable Iron, and Nutrients in Herbs: Challenges in Chemistry and Biology (Eds. Wang, et al.) pp. 224-239. American Chemical Society Symposium Series 925.
  • Jiang, S. and Lee, Tung-Ching. 2007. Chapter 84. Processing Frozen Seafoods in Handbook of Food Product Manufacturing (Ed. Y.H. Hui). John wiley & sons, Inc. (in press).
  • Burrowes, O., Diamond, J., Matthews, K. and Lee, Tung-Ching. 2007. The Efficacy of Pleurocidin in Food Systems. Submitted to the International Journal of Food Microbiology.


Progress 01/01/05 to 12/31/05

Outputs
In order to ascertain the efficacy of pleurocidin in foods, effect of environmental factors on the efficacy of pleurocidin was further investigated. We studied the efficacy of pleurocidin in varying concentrations of salt (NaCl), pH and temperature (intrinsic and extrinsic factors of food) using Vibrio parahemolyticus as the test organism. The results show that pleurocidin is highly heat resistant, retaining activity after autoclaving for 1 hour (121 degrees C/15psi), is salt tolerant up to 7.5% salt (NaCl) and is effective across pH range 4-8. These results are of microbiological significance because they show unique characteristics of pleurocidin which may be exploited by its use in both basic and acidic foods, in salted products as well as in thermally processed foods.

Impacts
Being salt tolerant, pleurocidin would be ideally suited for use as a natural preservative in foods susceptible to halophilic bacteria such as marine shell fish (shrimp, oysters and clams). Moreover, being active against a fish pathogen such as V. parahemolyticus, pleurocidin has significant potential application in aquaulture against other Vibrio species, such as V. anguillarum, which are pathogenic to fish. Results also show pleurocidin to be active over a wide range of pH (pH 4 to 8), which makes this peptide a potentially ideal preservative in both high and low acid foods or to be used synergistically with acidic preservatives. Pleurocidin not only retained full activity after 10 minutes of boiling, as reported by Cole et al., (1997) but also retained activity after autoclaving for 1h (15 psi and 121 degress C). The heat resistance of pleurocidin indicates its suitability for use in heat procesed foods, or foods exposed to temperature abuse. These results have significant food safety implications because they indicate that pleurocidin could potentially be used as a natural food preservative against halophilic, acidophilic and thermophilic foodborne microorganisms. These findings also form the basis on which further research on the efficacy of pleurocidin in actual foods may be pursued. The overall benefit of replacing conventional food preservatives and/or the use of therapeutic levels of antibiotic in aquaculture, with a natural antimicrobial peptide such as pleurocidin is an exteremely important benefit to be gained from this reaearch.

Publications

  • Burrowes, O.J., Diamond, G. and Lee, Tung-Ching. 2005. Recombinant Expression of Pleurocidin cDNA using the Pichia pastoris Expression System. Journal of Biomedicine and biotechnology 4:374-384.
  • Moretti, D., Lee, Tung-Ching, Zimmermann, M.B., Nuessli, J. and Hurrell, R.F. 2005. Development and Evaluation of Iron-fortified Extruded Rice Grains. Journal of Food Science 70(5)S330-336.
  • Moraru, C. and Lee, Tung-Ching. 2005. Kinetic Studies of Lycopene Isomerization in a Tributyrin Model System at Gastric pH. Journal of Agricultural and Food Chemistry 53:8997-9004.
  • Pyo, Y-H., Lee, Tung-Ching and Lee, Y-C. 2005. Effect of Lactic Acid Fermentation on Enrichment of Antioxidant Propeties and Bioactive Isoflavones in Soybean. Journal of Food Science 70(3)S215-220.
  • Pyo, Y-H., Lee, Tung-Ching, Rhee, Y-K. and Lee, Y-C. 2005. Hydrolysis of Isoflavone Glycosides in Soymilk Fermented with Some Bacterial Strains. Food Research International 38:551-559.
  • Jiang, S. and Lee, Tung-Ching. 2005. Part III: Freezing Seafood and Seafood Products - Chapter 22. Principles and Applications in Encyclopedia of Frozen Food. Marcel Dekker (in press).
  • Fleisher, D.H., Logendra, L., Moraru, C., Both, J-J., Lee, Tung-Ching and Janes, H.W. 2005. Effect of Temperature Perturbation on Tomato (Lycopersicon esculentum Mill.). Quality and Production Scheduling. Journal of Horticultural Science and Biotechnology (in press).
  • Yang, R-Y., Tsou, S.C.S., Lee, Tung-Ching, Chang, L.C., Kuo, G. and Lei, P.Y. 2006. Moringa, a Novel Plant Rich in Antioxidants, Bioavailable Iron, and Nutrients in: Challenges in Chemistry and Biology of Herbs (ed. Ho), American Chemical Society Symposium Series (in press).


Progress 01/01/04 to 12/31/04

Outputs
Apple cider was chosen as the test food material for pleurocidin because there is wide-spread concern regarding the survival of E. coli 0157:H7 in apple cider. E. coli 0157:H7 has been shown to survive for over 21 days in apple cider stored at refrigeration temperature (Fisher and Boyd 1998; Miller and Kasper 1994; Zhao and Doyle 1992). We found pleurocidin to be active against E. coli 0157:H7 and Penicillium expansum isolated from rotting apple, it is tolerant to low pH (pH4) and is heat resistant even at autoclaving temperature. It is therefore an ideal condidate as a preservative for apple cider which is acidic in nature and in addition to being susceptible to E. coli 0157:H7, might be also infected by yeasts and molds, both of which have been shown to be sensitive to pleurocidin.

Impacts
The work done on the shelf-life studies in apple cider using pleurocidin as an antimicrobial agent provides very important information about the usefulness of this antibmicrobial peptide. Our results reveal that pleurocidin is not only effective against floodborne pathogens in broth culture but also in actual food matricies, and its use is not limited to seafood, but also to other types of foods/beverages (apple cider). The overall benefit of replacing conventional food preservatives and/or the use of therapeutic levels of antibiotic in aquaculture, with a natural antimicrobial peptide such as pleurocidin, would be an extremely important benefit to be gained from this research.

Publications

  • Burrowes, O-J., Hadjicharlambous, C., Diamond, G. and Tung-Ching Lee (2004). Evaluation of Antimicrobial Spectrum and Cytotxic Activity of Pleurocidin, for Food Applications. Journal of Food Science 69:(3) FMS 66-71.
  • Jhoo, J-W., Sang, S-G., Wei, G-J., Lee, Tung-Ching, Rosen, R.T. and Ho, C-T. (2004). Enzmatic Synthesis of Theaflavins and Epitheaflavic Acid from Tea Catechins and Their Antioxidant Activity. Journal of Food Lipids 11:89-103.
  • Pyo, Y-H., Lee, Tung-Ching, Logendra, L., Rosen, R.T. (2004). Antioxidant Activity and Phenolic Compounds of Swiss Chard (beta vulgaris subspecies cycla) Extracts. Food Chemistry 85:19-26.
  • Hanson, P.M., Yang, R-Y., Wu, J., Chen, J-T., Ledesma, D., Tsou, S.C.S. and Lee, Tung-Ching (2004). Variation for Antioxidant Activity and Antioxidants in Tomato. Journal of American Society for Horticultural Science 129(5) 704-711.


Progress 01/01/03 to 12/31/03

Outputs
Our result revealed Pleurocidin to be active against clinically important microorganisms such as Klebsiella pneumoniae, which is often implicated in pneumonia, particularly in children, as well as in urinary tract infections. This organism is highly resistant to traditional antibiotics, such as peniclllin and has been shown to have a number of emerging resistant strains (Lim, 1989). Pleurocidin may offer a novel alternative to treat such infections. The sensitivity of Psedomonas aeruginosa is significant, both from the clinical point of view, as well as in food preservation as this organism is known to cause lower respiratory and urinary tract infections, and is implicated in burns and other skin wounds, as well as being a spoilage organism in seafood. Pleurocidin could, therefore, be important in the inibition of this organism in seafood as well as in clinical treatment.

Impacts
We have continued our effort to establish that Pleurocidin has a broad spectrum of activity and is effective against some clinically important microorganisms such as Psedomonas aeruginosa, Klebsiella pneumoniae and E. coli. In recent years, these microorganisms have posed a serious public health hazard to consumers, particularly children, the elderly and the immune-compromised, and are complicated by the fact that they are resistant to conventional anti-microbial agents. Pleurocidin could, therefore, be important in the inhibition of this organism in seafood as well as in clinical treatments.

Publications

  • Burrowes, O., Hadjicharalambous, C., Lee, Tung-Ching and Diamond, G. 2004 "Evaluation of Antimicrobial Spectrum and Cytotoxic Activity of Pleurocidin for Food Application" Journal of Food Science, in press.
  • Vandenbergh, J.G., Lee, Tung-Ching, et al. 2003 "Animal Biotechnology: Science-Based Concerns" The National Academies Press, Washington, D.C., pp 1-181.
  • Burrowes, O-J, Lee, Tung-Ching and Diamond, G. 2003 "Effect of Environmental Factors on the Efficacy of Pleurocidin, an Antimicrobial Peptide Isloated from Winter Flounder" Proceeding of the 2003 Annual Meeting of the Institute of Food Technologists, July 2003, Chicago, Illinois.
  • Boonsupthip, W. and Lee, Tung-Ching. 2003 "Applicaton of Antifreeze Protein for Food Preservation: Effect of Antifreeze Protein for Preservation of Gel-forming Capacity of frozen and Chilled Actomyosin" Journal of Food Science 68:1804-1809.


Progress 01/01/02 to 12/31/02

Outputs
We have made steady, significant and timely progress in the project up to this point. Thus far, we have tested synthetic pleurocidin against a wide spectrum of microorganisms including Gram positive and Gram negative bacteria, yeast and molds. Pleurocidin has been demonstrated to be effective against the yeast Saccharomyces cerevisiae, certain molds, and both Gram positive bacteria and Gram negative bacteria, both of clinical importance as well as for food applications. These findings are very significant as yeasts such as Saccharomyces cerevisiae, although a very useful organism in the field of biotechnology, are known to cause spoilage in alcoholic beverages as well as non-alcoholic beverages, particularly fruit juices. The level of pleurocidin required to inhibit the growth of S. cerevisiae was found to be 5.5mM (15mg/ml). This is much less than that of chemical preservatives used to preserve such juices. Citric acid and benzoic acids, which are often used for this purpose, are generally used at levels in excess of 1000mg/ml. The fact that pleurocidin has been found to be active against both Gram positive organisms such as Staphylococcus aureus and Lactobacillus alimentarius as well as Gram negative organisms such as E. coli O157: H7 and Salmonella typhimurium and also yeast and molds shows the versatility of this antimicrobial peptide as a potential food preservative when compared to nisin. E. coli O157:H7 is one of the important emerging foodborne pathogens which is resistant to conventional antibiotics, therefore its susceptibility to pleurocidin is a very significant finding. Also of major significance is the susceptibility of the well-known and important seafood pathogen Vibrio parahemolyticus to pleurocidin. This has paved the way for further research on the use of pleurocidin in the specific area of preservation of shellfish after harvesting, as well as for prophylactic treatment against common pathogens in aquaculture. Nisin is the only antimicrobial peptide presently approved by the FDA to be used as a food preservative and has been shown to be active against Gram positive organisms but not against Gram negatives, yeasts or molds unless used in combination with other antimicrobial agents (Paster et al., 1999). Perhaps our most significant finding is the high efficacy of pleurocidin (3.1mM/8.25mg/ml) against the fish spoilage organism Vibrio alginolyticus, an organism isolated from the gills of the horse mackarel and its inhibitory effect on Vibrio parahemolyticus isolated from shrimp (69mM/ 187mg/ml). The versatility of pleurocidin as an antimicrobial agent is also substantiated by the fact that, unlike other antimicrobial peptides which are active only against bacteria, pleurocidin is inhibitory to significant foodborne fungi such as Penicillium expansum (MIC=20.6mM/56mg/ml), a mold isolated from rotting apples, which is of particular significance since it produces the toxin patulin which is known to cause spoilage in fruits (Samson and Pitt, 1989). The inhibitory concentration levels exhibited by pleurocidin are much lower than the levels of traditional preservatives used in these areas.

Impacts
Pleurocidin is produced naturally by an edible fish, thus, it has great potential application in seafood preservation as well as in other perishable foods, such as meats and dairy products. Our results show that it is active against Lactobacillus species isolated from marinated fish, against Vibrio species (common fish pathogens isolated from the gills of the hourse mackarel and from shell-fish) and against Pseudomonas aeruginosa, an organism often implciated in seafood spoilage. The significance of the fact that pleurocidin is also active against E. coli 0157:H7 cannot be under estimated because this organism is an important public health microbiological hazard in a variety of foods. These results sustantiate the potential use of pleurocidin as a food preservative. The overall benefit of replacing conventional food preservatives with natural antimicrobial peptides such as pleurocidin, while concomitantly using it in clinical applications to replace traditional antimicrobial agents to which more and more bacterial species are becoming resistant, would be an extremely important benefit to be gained from our research.

Publications

  • Burrowes, O.J., Hadjicharalambous, C., Lee, T-C. and Diamond, G. 2002. Biological Activity of Pleurocidin, a Novel Antimicrobial Peptide Isolated from the Winter Flounder, Annual Conference and Food Expo of the Institute of Food Technologists, Anaheim, California, June 15-19, 2002.