Recipient Organization
ANTIMICROBIAL MATERIALS, INC.
5201 S SWEETBRIAR CT
SIOUX FALLS,SD 57108
Performing Department
(N/A)
Non Technical Summary
We propose to develop effective, robust, rechargeable and economic antimicrobial coatings to fight cross-contamination in food manufacturing and improve food safety. The severe consequences of microbiological contamination (e.g., foodborne illnesses, food recalls, brand damage, and economic losses) demand food industry to re-examine the current food safety control strategies and introduce novel technologies for controlling the microbial crosscontamination. Persistent pathogens have become a big threat to food industry, especially on non-food-contact surfaces. Even the bacteria-killing procedures cannot fully eliminate the contamination from these surfaces with the ready-to-eat foods. One potential solution is to modify non-food-contact surfaces with anti-microbial coatings to control cross-contamination. We will focus on achieving the durability and scalability of the antimicrobial coating systems through the combination of advanced coating chemistry and formulation. There are four supporting objectives: 1) Obtain coating thickness and adhesion that can survive 3 months of sanitation scrubbing. 2) Prove that the coating is compatible and non-reactive to normal sanitation chemicals. 3) Validate that the coating is effective against multiple listeria persistent strains. 4) Validate that the coating prevents biofilms forming for up to 3 months in-service. If successful, the new coating system will offer a practical and sustainable solution to control foodborne contaminants and greatly enhance the food safety.
Animal Health Component
50%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
Goals / Objectives
Our goal is to further understand the properties of our newly developed DOHA antimicrobial coating and develop optimized coating formulation that can provide desired performance for supporting commercial applications. After completing the National Science Foundation (NSF) ICorps Team program with over 90 in-person interviews with sanitation/food safety/operational managers in the food industry, we validated a need and built a model for application in ready-toeat food manufacturing (figure 4). Ideally, our coating needs to retain ample chlorine to be effective for up to 1 week without bleach rinse recharge, and also remain in-tact and effective for 3 months before coating reapplication during quarterly planned maintenance. This use case is derived from plant protocols to sanitize with chlorine-based cleaners at least weekly and perform planned maintenance every quarter. After communicating with industry experts, we also understand that Lm is currently the environmental pathogen of most concerned, and thus we will use multiple Lm resistance strains as model environmental pathogens. Based on customer feedback, key criteria for the proposed work are:1) Obtain coating thickness and adhesion that can survive 3 months of sanitation scrubbing. 2) Prove that the coating is compatible and non-reactive to normal sanitation chemicals. 3) Validate that the coating is effective against multiple listeria persistent strains. 4) Validate that the coating prevents biofilms form forming for up to 3 months in-service
Project Methods
Task 1. Determine the optimized formulation for achieving highest coating thickness in one spray-coating cycle. A standard stainless steel 304 sheet (#8 surface finish) will be placed vertically and be spraying coated with designed formulas using a sprayer gun. The dried samples will be sent out for determining coating thickness using a Spectroscopic Ellipsometer (Woollam). Based on the result, we will define the major factors that contribute to the coating thickness and then establish a mathematical relationship of coating thickness vs polymer concentration and cross-linker ratio. With the instruction of this established mathematic model, we will be able to obtain the maximize coating thickness (>200 nm) for the following study.Task 2.Determine optimum chlorination condition which gives highest chlorine content without damaging the coating. Chlorination solution will be prepared from Clorox chlorine bleach to desired concentration and pH will be adjusted to 7.0 with acetic acid. Then the samples with optimized formulation will be used for chlorination with different treatment conditionsunder room temperature and dried overnight.Surface chlorine content will be quantified by a iodometric/thiosulfate titration method and the active chlorine content can be calculated. Surface characteristics of coating will also be observed with Scanning Electron Microscopy (SEM). The chlorine conditions which gives highest chlorine content without damaging the coating structure will be used for the chlorination condition in the following study.Task 3. Determine wet-scrub resistance of coating and failure cycle. The scrubbability of coating will be determined using ISO11998 with modifications and a wet-scrub tester will be used. The ability of a coating film to sustain less than a specified loss in film thickness, averaged over a defined area, when exposed a defined wet-scrub cycles, is defined as we-scrub resistance ability. After each of 10 wet-scrub cycles, the coating thickness will be determined by Ellipsometer. Then, the coating will be recharged and applied for a second cycle. The cycle which causes coating thickness <100 nm (or chlorine content < 1017 atoms/cm2) will be defined as the wet-scrub failure cycle of coating and that number of wet-scrub cycles will be recorded. A mathematical model can also be established for coating thickness vs wet-scrub cycles for different soil agents.Task 4. Correlate standard scrubbing test cycle with commercial sanitation scrubbing cycles. Correlate 3-month of daily cleaning/scrubbing procedures in a commercial ready-to-eat food plant with the standard scrubbing test cycles. We will visit one of our industry collaborator Rich's food plants and observe their sanitation practices to obtain real-world data about the typical number of scrub cycles per day and sanitation tools they used on food equipment. We will select 10 points of different non-food contact areas and scrub will be averaged in three different days. Next, multiply each-day average cycles by 90 days to calculate a 3-month expected cycles. Then, do this scrubbing manually using the sanitation tool from their plant on our stainless-steel samples. After every 30-day equivalent scrubbing, the samples will be sent out for determining coating thickness using Ellipsometry. Record the number of sanitation cycles until fail (<100 nm) and compare results with standard scrubbability test cycles that have same degree loss of coating. Based on these data, a correlation between real industrial scrubbing and the standard tester scrubbing can be established. The scrub cycles (SN) from standard tester that are equivalent to 1-month, 2-month, 3-month in-service sanitation scrubbing in commercial plant will be recorded and used for the following studies.Task 5. Determine the stability of surface N-halamine in different environment. Surface chlorine will be determined through iodometric/thiosulfate titration as described in task 2 over 4-week storage time and under the following storage conditions. A mathematic model can be established for chlorine content vs temperature and relative humidity (RH%). This result can be used to validate that if the designed one-week recharging cycle will be enough to maintain potent antimicrobial function against Lm (5 log reduction in less than 2 hours).Task 6. Determine the compatibility of coating with cleaning sanitation chemicals. Several chemical agents that are used in the food industry will be used to treat the coating for fixed time (the exact time for each chemical treatment will be determined after communicating with our food industry partners). After each treatment, the surface will be washed with DI water, dried, and subjected to a second treatment. Then, the surface will be recharged every 6 cycles and titrated for N-halamine content to measure the chlorine withholding capacity. Coating thickness will also be determined by Ellipsometer every 6 cycles. Repeat this process for 60 cycles to simulate the 3-month targeted service for one newly applied coating.Task 7. Validate the antimicrobial control effects against multiple Lm persistent strains at different timepoint of the 3-month service cycle. Prepare four groups of coating samples on stainless steel that are equivalent to 0, 1, 2, 3-month of service after 30-day equivalent scrubbing and 6-day treatment with multiple chemical (task 6) recorded in task 6 and validate for antimicrobial function against Lm. A cocktail of five L. monocytogenes strains will be obtained used for this study. The bacterial cells will be adapted to grow in tryptic soy broth plus 0.6% yeast extract supplemented with nalidixic acid (50 g/ml) (TSBYE-N). Equal volumes of individual cultures will be mixed to form a cocktail of L. monocytogenes. The N-halamine-coated and control surfaces will be artificially contaminated with the bacterial cocktail (e.g. 106 CFU/cm2) and stored in a container for 30 min for bacterial attachment. Both coated and untreated surfaces will be swabbed with foam at different time points (e.g., 10 min, 30 min, 1 h, 6 h and 12 h) and immersed in a washing solution. For microbiological analysis, treated and untreated (control for the initial counts determination) inoculated surface swabbing foams will be mixed with sterile 0.1% peptone water. The mixture will then be stomached for 2 min at 260 rpm and serially diluted in sterile 0.1% peptone and surface-plated on tryptic soy agar with 0.6% yeast extract supplemented with 50 g/ml nalidixic acid (TSAYE-N). TSAYE-N plates will be incubated at 35ºC for 3 days before enumeration.Task 8. Verify biofilm prevention effect against Lm at different timepoint of the 3-month service cycle. A biofilm control test will be used to verify the biofilm prevention effect following a previously reported method.29 Same samples and Lm cocktails cells will be prepared following the method in task 5. The samples will be inoculated with bacteria for 4 hours at 25 °C to induce biofilm formation. Then the samples will be taken out and further incubated under controlled environment. After incubation, biofilm samples will be removed, gently rinsed in phosphate-buffered saline (PBS) at pH 7.2, and the adherent bacteria will be fixed by 2.5% glutaraldehyde (GDA) in PBS solution for at least 1 h, then rinsed 3 times with PBS to remove unreacted GDA. After fixation, samples will be treated with Tween 20 (0.05% in PBS buffer) for 10 min, rinsed with sterile PBS, and stained in the dark with SYTOX green (Life Technologies) for 30 min. Samples will imaged by fluorescence microscopy (Olympus BX63) using a 20X air objective, and biofilm fluorescence will be observed through a GFP filter. The fluorescence intensity of biofilms will be quantified by image analysis. At same time bacteria will be counted. For these samples with less than 4 log reduction compared with control will be defined as failure point.