Further Studies of Pleurocidin, a Natural Antimicrobial Peptide, for Food Applications.

<p>NON-TECHNICAL SUMMARY:<br/> 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.
<p>APPROACH:<br/> 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.
<p>PROGRESS: 2012/10 TO 2013/09<br/>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.
<p>PROGRESS: 2011/10/01 TO 2012/09/30<br/>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.
<p>PROGRESS: 2011/01/01 TO 2011/12/31<br/>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.
<p>PROGRESS: 2010/01/01 TO 2010/12/31<br/>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.
<p>PROGRESS: 2009/01/01 TO 2009/12/31<br/>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.
<p>PROGRESS: 2008/01/01 TO 2008/12/31<br/>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.
PROGRESS: 2007/01/01 TO 2007/12/31In 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.
<p>PROGRESS: 2006/01/01 TO 2006/12/31<br/>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.
<p>PROGRESS: 2005/01/01 TO 2005/12/31<br/>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.
<p>PROGRESS: 2004/01/01 TO 2004/12/31<br/>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.
<p>PROGRESS: 2003/01/01 TO 2003/12/31<br/>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.
<p>PROGRESS: 2002/01/01 TO 2002/12/31<br/>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.