Non Technical Summary
Microbial food spoilage represents a significant economic and environmental issue: it is reported that 40% of food goes to waste, two thirds due to spoilage. Natural, food grade antimicrobials can be used to prevent such waste, and synthetic metal chelators like EDTA are commonly added in foods to enhance their antimicrobial activity. However, consumers are increasingly demanding removal of such label-unfriendly synthetic additives from foods. Advanced packaging technologies are a potential means to improve the quality, shelf-life, and therefore economic and environmental sustainability, of packaged foods. Current active packaging approaches require migration of the active agents to be active, have unacceptable effects on material mechanical properties, and typically exert low activity. We propose a non-migratory active packaging technology, in which hydroxamate chelating agents are grafted from a packaging materials surface by covalent linkages and enhance the activity of natural antimicrobials against spoilage organisms without migration to the food . The proposed materials will prevent growth of spoilage organisms, thus improving shelf life and reducing waste of packaged foods. The long term impact of the proposed research is in support of the USDA NIFA's goals to improve packaging technologies to enhance the quality and shelf life of foods and enhance the economic and environmental sustainability of agricultural and food systems.
Animal Health Component
Research Effort Categories
Goals / Objectives
The overall goal of this hypothesis-driven research proposal is to maintain food quality and reduce the loss of food due to spoilage using a novel non-migratory active packaging material. We hypothesize that design of packaging materials with non-migratory, chelating activity will result in extended food shelf life, improve food quality and reduced loss of beverages and semi-viscous foods due to microbial spoilage. We further hypothesize that such materials would enable removal of synthetic chelators from product formulations while retaining enhanced activity of natural antimicrobials due to chelator/antimicrobial synergies. Chelating moieties will be grafted from the surface of a common, affordable packaging material to enhance the activity of natural antimicrobials against spoilage organisms.
Material Synthesis - Ultraviolet Light Initiated Graft Polymerization: Clean PET films will be submerged in a nitrogen flushed solution of monomer and exposed to ultraviolet irradiation. Grafts are then converted to chelating polyhydroxamate grafts by exposure to hydroxylamine . Films will be rinsed in copious deionized water to extract unreacted monomer and non-covalently linked oligomers. The degree of grafting will be quantified gravimetrically and by cross-sectional SEM (below). We will utilize an iterative approach in designing our materials and testing for antimicrobial enhancement (objectives 1 and 2). The amount and strength of chelating capacity will be optimized by varying monomer concentration, irradiation dose, inclusion of buffering agents, time, and temperature.Surface Analysis: Changes in surface chemistry, topography, and morphology will be characterized to guide material design and ensure uniform grafting of hydroxamate chelating moieties on the surface of the films. ATR-FTIR spectra will be collected on an IRPrestige 21 spectrometer (Shimadzu Corporation) with a diamond ATR crystal. The resultant spectra will be analyzed using Know-It-All software (BioRAD Laboratories). Advancing and receding water contact angles will be measured on a DSA100 (Kruss) to determine the effect of chelator grafting on surface hydrophilicity, an important indicator of changes in surface chemistry. Atomic Force Microscopy (AFM) will be used to quantify the effect of chelator grafting on nanoscale surface topography. For roughness calculations, the arithmetic average of absolute values (Ra) will be calculated from a 3D roughness profile. Cross-sectional SEM images of control and modified film samples will be imaged on a JOEL 6320FXV Field Emission SEM (Peabody, MA, USA). Films will be immersed in liquid nitrogen for 60 minutes, followed by freeze fracturing while submerged. Fractured samples will be sputter coated with gold and imaged at a 90-degree angle to acquire cross-sectional images in order to quantify the degree of grafting in thickness.Iron Chelating Capacity: Two methods will be utilized to quantify iron chelation by the films: ferrozine assay, a colorimetric technique to facilitate rapid screening during material design/synthesis, and ICP-MS (inductively coupled plasma mass spectrometry) for direct, sensitive, multi-ion quantification. Dissociation behavior, film mechanical properties, and relative binding constants will also be quantitatively determined.Microorganisms: Because of their prevalence in food spoilage, Alicyclobacillus acidoterrestris, Bacillus cereus, Lactobacillus plantarum, Pseudomonas fragi, and Pseudomonas fluorescens will be used as challenge organisms in testing antimicrobial enhancement by the films. At least two different strains of each organism will be tested, including isolates from a food processing environment and/or food source when possible. We will obtain isolates from the American Type Culture Collection as well as environmental isolates from the collection of Dr. McLandsborough.Antimicrobial Enhancement Assays: Antimicrobial enhancement will be assessed in terms of change in bacterial populations after periods of defined contact with the antimicrobials and chelating packaging film; such methodology enables comparisons to be made between controls and test surfaces at a single time point and as a growth over time. Antimicrobial enhancement will be further characterized in terms of change in minimum inhibitory concentration (MIC) of the antimicrobials due to use of chelating film. In this objective, we will test the antibacterial activity of the materials in conjunction with lysozyme and nisin in pure cultures in growth media; additional challenge studies against organisms in the presence of complex food matrices will be performed as part of Objective 3. We will test each organism at a treatment temperature similar to their optimal growth temperature to assess antimicrobial activity of the test surfaces under optimal conditions. Defined incubation intervals will be 2 to 48 hours; shorter intervals (every 15 minutes up to 2 hrs) will also be tested to elucidate short term inactivation kinetics. Unmodified PET and unmodified PET with EDTA in solution will be used as controls. By including a range of controls in our experimental design, we will be able to isolate effects of chelator, antimicrobial agent, and the synergy.Preparation of food systems: Organic mayonnaise (containing oil, pasteurized eggs, seasoning, and not containing EDTA), homogenized, pasteurized fluid whole milk and pasteurized apple juice will be purchased from a local grocery store. In all of these foods, endogenous iron concentrations are adequate to enable microbial growth. However, in order to accurately interpret the results, iron concentration in all the products will be determined using ICP-MS.Migration of active agent into food: The benefit of covalently linking the active agents to the surface of the packaging films is that migration from the package into the food products is unlikely, which offers benefits in terms of both regulatory approval and consumer perception (clean labeling). Demonstration of the stability of the biomimetic chelating moieties on our active packaging films is therefore a key metric of our success. Total immersion migration tests, adapted from the European Union regulations, will be performed.