Microbial contamination on surfaces is a problematic and potentially deadly issue, particularly in high-risk settings such as healthcare, aged care, and food and beverage. In this research, we present an antimicrobial waterborne polyurethane additive that will be of interest to the coatings community due to its high compatibility with a variety of coating systems, which results in a range of highly diverse and useful applications.

This next-generation antimicrobial uses a nano-composite approach to create a silver-based active ingredient that is non-toxic and truly non-leaching. The presented technology produces strong binding between silver and the polymer backbone. This prevents leaching while retaining high activity with proven results against a range of pathogens.

Introduction

Each day, approximately 1 in 31 U.S. hospital patients and 1 in 43 nursing home residents contract at least one infection associated with their healthcare.¹ Healthcare-associated infections accrue direct costs to U.S. hospitals of at least $28.4 billion each year.² Healthcare settings are just one example of a high-risk area where the transmission of disease is dangerous and expensive.

Silver is well known for its excellent antimicrobial properties, and many products have been produced that exploit this.^3-5 These products typically work via the controlled release of silver ions or nanoparticles, resulting in concerns over the release of silver into the environment and the interactions it subsequently undergoes.^6-8

Countering this risk, a novel process has been developed that binds the silver active ingredient directly to a polymer backbone, creating a nanocomposite from which the silver does not leach.^9,10 This produces an antimicrobial material that is not only sustainable and environmentally friendly, but also displays incredible longevity as the active ingredient is not depleted.

In this research, a water-based silver-polyurethane nanocomposite material has been developed that displays antimicrobial activity against bacteria, viruses, and fungi. Its high potency means it can be utilized as an additive in other coating systems, and its compatibility with a range of materials has been shown, including acrylates, polyurethanes, and other waterborne systems.

As a result, this antimicrobial additive can be used effectively in a range of highly diverse applications (textiles, walls and floors, furniture, high-touch surfaces, filters, etc.).

Experimental

The silver-polyurethane composite was produced using Inhibit Coatings’ proprietary functionalization process.

Characterization

X-ray diffraction (XRD) was used to confirm the crystalline structure and composition of the silver particles within the polymer. A PANalytical X’Pert PRO diffractometer was utilized with a copper K-alpha X-ray source at a wavelength of 1.5405 Å, operating at 45 kV and 40 mA.

A JEOL 2010 transmission electron microscope (TEM), operated at 200 kV, was used to visualize the silver particles within the polymer. The silver-polyurethane composite was diluted and then drop cast onto 200 mesh copper grids and plasma treated with a JEOL EC-52000IC ion cleaner prior to TEM analysis.

Independent Testing

Antimicrobial Testing

Antibacterial testing of the silver-polyurethane composite applied to a polyester textile was conducted according with the AATCC 100-2019 standard¹¹ by Microbe Investigations Switzerland (MIS). The sample was tested against Escherichia coli (E. coli) (ATCC 8739) for a contact time of 24 hours.

Antiviral testing of the silver-polyurethane composite in a polyurethane coating on acrylic was conducted according to the Japanese Industrial Standard JIS Z 2801 standard (modified for viruses)¹² by Microchem Laboratory. The sample was tested against Human coronavirus (ATCC VR-740, Strain: 229E) and Influenza A virus (H1N1) (ATCC VR-1469, Strain: A/PR/8/34), for a contact time of 2 hours.

Antifungal testing of the silver-polyurethane composite in a polyurethane coating on acrylic was conducted according to the ASTM G21 standard¹³ by Microbe Investigations Switzerland (MIS). The test details utilised are provided in Table 1.

Leach Testing

Leaching of silver from the silver-polyurethane composite was conducted by Nanosafe, Inc. Testing of the composite applied to fabric samples followed U.S. EPA Method 1311 “Toxicity Characteristic Leaching Procedure,”¹⁴ adapted for the assessment of silver-leaching potential from textiles.¹⁵ Testing of the composite applied to wood samples was conducted under guidance of OECD 313 “Estimation of Emissions from Preservative-Treated Wood to the Environment.”¹⁶

Toxicology Testing

Toxicology testing, as outlined in Table 2, was conducted by STILLMEADOW, Inc., on the silver-polyurethane composite.

Preservative Testing

Preservative testing was run using a Difco Paddle Tester over three weeks, using three concentrations of the silver-polyurethane composite in a polyurethane dispersion, and testing weekly for bacterial growth.

Results and Discussion

Silver Particle Characterization

Characterization techniques have verified that metallic silver nanoparticles have formed.

The XRD pattern of the silver-polyurethane composite has shown peaks with 2θ values of 38.2°, 44.3°, 64.5°, and 77.6° (Figure 1), which can be attributed to the (111), (200), (220), and (311) crystallographic planes of face-centered cubic (fcc) silver crystals, respectively.¹⁷

Activated carbon (AC) has been utilized in several biomedical applications such as wound care and hemoperfusion, and in this work, experimental procedures were designed and carried out to better understand the effect of AC on bacteria, viruses, and parasites.

Antimicrobial properties of AC are relevant to the coatings industry because they can be incorporated into gel, liquid, or other coatings to impart antipathogenic properties. Possible biomedical applications include gels used for catheter tubing, drug capsule shells, and medical implants.

These experiments analyzed the ability of various sources of AC to exhibit antimicrobial properties by prohibiting the growth and propagation of pathogens. Wood, coconut, and coal-based AC were evaluated for activity against a series of bacterial dilutions with Escherichia coli (E. coli) and Staph aureus (S. aureus), a series of viral dilutions with T1 and Φ11 bacteriophages, and a series of parasitic dilutions with Euglena gracilis. Results were compared and analyzed using microscopy and other standard plating techniques.

Overall, the results collected from various trials displayed a significant reduction in the activity of the pathogens. Now that the concentration requirements and responsivity for the different pathogens are known, the potential mechanisms of interaction can be investigated.

In the presence of AC, the Euglena gracilis were observed to become immobile upon contact with AC. Unable to remain mobile, the Euglena expired. The immobilization of the parasite suggests that adsorption may play a key role in antipathogenic activity, although more studies are needed to be conclusive. Further research will be conducted to use AC in new biomedical applications, which will serve not only the biomedical industry but other research areas as well.

INTRODUCTION

Activated carbon has been used in biomedical research for decades due to both its adsorptive qualities and the ease of its production. AC is most commonly generated through heterogeneous reactions that increase surface area and create highly porous structures. Many claim that the increased porosity is key to providing antimicrobial properties as their capacity for adsorption increases.¹ Accordingly, the chemical production process determines the chemical and physical characteristics, which ultimately provides the adsorptive properties of activated carbon.

AC can also be chemically tailored, as demonstrated in a study where chemical activation of olive-stone derived AC with phosphoric acid was achieved during the production process.² The phosphoric acid functionalized AC is significant because the activation of AC through chemical reactions can alter the compound’s surface chemistry and thus its performance. In addition, through this activation, the micropore, mesopore, and the narrow micropore volumes were determined as well as the apparent surface area and the pore size distribution.² The extent to which the chemical activation affects these physical properties remains unknown.

The identification and utilization of the antimicrobial properties of AC is relevant to several commercial applications, as its adsorption capacity and cost-effectiveness could provide an alternative to more expensive antimicrobial products. Activated carbon is designated as GRAS (generally recognized as safe) by the FDA in some forms such as medical-grade, coconut-shell-based AC, and as such it has the potential to be quickly implemented as an antimicrobial alternative in biomedical applications.

The use of AC as an antimicrobial agent has long been of interest to researchers, as several peer-reviewed studies explore its ability to inhibit the propagation of bacteria. For example, a study performed in 2001 examined the adsorption of enterohemorrhagic E. coli through its exposure to AC.³ The results for the study were promising, as the propagation of the bacteria and its toxins were limited or inhibited by the presence of the AC (with better results shown at higher concentrations of AC exposure⁴).

REFERENCES

1. Lozano-Castello, D.; Cazorla-Amoros, D.; Linares-Solano, A.; Quinn, D. F. Activated Carbon Monoliths for Methane Storage: Influence of Binder. Elsevier 2002, 40 (15), 2817–2825.
2. Ruiz-Rosas, R.; García-Mateos, F. J.; Gutiérrez, M. del; Rodríguez-Mirasol, J.; Cordero, T. About the Role of Porosity and Surface Chemistry of Phosphorus-Containing Activated Carbons in the Removal of Micropollutants. Frontiers in Materials 2019, 6.
3. Naka, K.; Watarai, S.; Tana; Inoue, K.; Kodama, Y.; Oguma, K.; Yasuda, T.; Kodama, H. Adsorption Effect of Activated Charcoal on Enterohemorrhagic Escherichia Coli. Journal of Veterinary Medical Science 2001, 63 (3), 281–285.
4. Thambiliyagodage, C.; Mirihana, S.; Gunathilaka ,
   H. Porous Carbon Materials in Biomedical Applications. Biomedical Journal of Scientific & Technical Research 2019, 22 (4), 16905–16907.
5. Thomas, B. N.; George, S. C. Production of Activated Carbon from Natural Sources. iMedPub Journals 2015,
   1 (1), 1–5.

CoatingsTech | Vol. 18, No. 9 | September 2021

The COVID-19 pandemic has highlighted the need to prevent microbial contamination of many different types of surfaces. In turn, that has shined a spotlight on antimicrobial coatings and the benefits they can provide.

Several market research reports on the global value and growth rate for the antimicrobial coating market were issued in 2020. All the research firms (Meticulous Research, Expert Market Research, MarketsandMarkets, Technavio, Grand View Research, and Allied Market Research) forecast strong growth for these coatings, as compound annual growth rate (CAGR) estimates fall between 8% and 13.5%.

Even so, projected values for the market vary significantly: $5.6 billion and $7.36 billion by 2025 (MarketsandMarkets and Meticulous Research, respectively) to $11.6 billion and $18.8 billion by 2027 (Grand View Research and Allied Market Research, respectively). Technavio predicts the global antimicrobial coatings market will expand by $682.06 million during 2020-2024, while Expert Market Research anticipates the market will reach a volume of 922 million kilotons by 2026.

The wide variations in estimates may be attributed to the numerous types of antimicrobial coatings and end-use applications. Among the major end-uses include:

• Building and construction, including HVAC systems and mold remediation
• Medical and life sciences/healthcare and pharmaceutical
• Food and beverages
• Packaging
• Machinery and equipment
• Automotive OEM and components
• Consumer electronics and appliances
• Protective clothing
• Miscellaneous materials

In addition to being classified by the type of microorganisms they protect against, these coatings can be categorized by the type of antimicrobial agent, such as metal-based actives, including silver, copper, titanium dioxide, and zinc-based compounds, and organic or polymeric materials, such as graphene, certain polymeric substances, and quaternary ammonium salts. Silver is expected to the most prominent due to its high efficacy and durability.

These various types of antimicrobial coating can be formulated as solventborne or water-based liquids, powders, and aerosols. Liquid coatings comprise the largest share of the market, with water-based system predominating, but powder coatings are expected to witness the fastest CAGR.

While the dollar estimates differ widely, there is a consensus on the key drivers for the growing demand projected for antimicrobial coatings. The COVID-19 pandemic has been a primary factor. Demand for antimicrobial coatings has been increasing drastically in the medical and healthcare sectors, as well as for packaging products, protective clothing, consumer electronics, appliances, and other goods. During peak infection waves, temporary facilities were erected to treat COVID-19 patients. In most cases, high-touch surfaces (beds, handles, medical devices, instruments, and protective gear) were protected with antimicrobial coatings to prevent hospital-acquired infections (HAIs).

Antimicrobial coatings are also widely used in established healthcare facilities and elder-care facilities to prevent healthcare-associated infections in general. Growing concerns about air quality are also driving demand for high-quality air-purification systems, from consumer-based products to complete HVAC (heating, ventilation and air conditioning) solutions—all protected with antimicrobial coatings. As a result, the indoor-air-quality sector has become another significant contributor to market growth.

Aside from the pandemic, there is also growing demand for antimicrobial coatings on implantable devices used in treatments for cardiovascular and other diseases. There is also demand for mold-remediation solutions, which include antimicrobial coatings. Food recalls are another driver; the use of antimicrobial coating in food packaging has increased as companies look to reduce the likelihood of microbial contamination in packaged food products, further spurring demand. Finally, increased awareness of the benefits of antimicrobial coatings will provide additional opportunities for growth in emerging economies such as Latin America, South East Asia, and Africa.

Respiratory viruses can be spread through contact transmission (directly with a person or with a contaminated surface), droplet transmission (breathing in large and small respiratory droplets when near and infected person), and airborne transmission (breathing of smaller droplets and particles suspended for an extended period in a larger area).

New questions are also being raised about transmission via surface contact. Early in the pandemic, one study found that SARS-CoV-2 can survive on smooth surfaces (plastics, stainless steel) for three days and one day on paper and cardboard. The quantity of virus placed on these surfaces, however, was much greater than would occur naturally unless an infected person coughed or sneezed directly onto a surface.

It has therefore been thought for some time that surface transmission would only occur if someone touched a contaminated surface on which a large amount of virus was recently deposited by an infected individual through coughing or sneezing in close proximity. The person who touched the surface would shortly thereafter have to touch his/her eyes, nose, or mouth without any handwashing.

New research by Australia’s national science agency (CSIRO), however, suggests that in the dark SARS-Cov-2 can survive on smooth surfaces including paper money and cell phone screens for nearly one month and porous surfaces such as cloth for two weeks. It has also been found that in areas where COVID-19 outbreaks have occurred or infected people have quarantined, viral DNA is detected.

However, in the Australian study (and many others), the virus was not applied to surfaces in fresh human mucus, which contains white blood cells and antibodies that actively attack viruses, and clearly the conditions (cool, stable temperature, and humidity in the dark) do not mimic real-world scenarios (sunlight is known to inactivate the virus).

In addition, recent investigations have found that while virus particles recovered from infected surfaces left for days in laboratories could be cultured and thus remained infections, real-world samples collected in COVID-19 isolation wards at a hospital and from a quarantine hotel were not infectious.

The overall consensus is that for many common surfaces there isn’t a need to worry about disinfecting them. There are some exceptions—such as high-touch objects, particularly for people at higher risk from COVID-19. Hospitals and other health-care facilities also need to ensure cleanliness.

In addition, for many people, knowing that surfaces in public spaces—railway cars and buses, community centers, churches and libraries, government building, grocery and retail stores, etc.—are clean and protected gives them assurance during an ongoing time of real uncertainty.

Researchers at the University of Birmingham, in partnership with the University of Cambridge, Dupont Teijin Film (DTF), Innospec, and FiberLean, hope to help in that regard. They are developing materials that can be added to detergents and cleaners or integrated with current packaging processes to form sub-micron-thick films on surfaces that are designed to capture aerosol droplets and inactivate SARS-CoV-2 by disrupting the protective environment that exists in those droplets.

The initial phase of the 18-month project involves gaining an understanding of the interactions of the virus with different surface materials with varying characteristics, such as porosity, rigidity, and roughness, by leveraging the team’s expertise in soft matter, surface chemistry, formulation engineering, and microbiology.

The commercial partners will contribute their product development expertise in polyester film, performance chemicals, and micro-fibrillated cellulose, with the goal of facilitating rapid commercialization. All three companies have collaborated with the School of Chemical Engineering at Birmingham for a decade via the Centre for Formulation Engineering.

For additional reading:

• “COVID-19 transmission—up in the air,” The Lancet Respiratory Medicine, October 9, 2020. https://www.thelancet.com/journals/lanres/article/PIIS2213-2600(20)30514-2/fulltext
• Watson, Stephanie. “Coronavirus on Surfaces: What’s the Real Risk?” September 3, 2020. https://www.webmd.com/lung/news/20200903/coronavirus-on-surfaces-whats-the-real-risk
• “Covid Virus ‘Survives for 28 days’ in Lab Conditions,” October 11, 2020. https://www.bbc.com/news/health-54500673
• “It’s Time to Talk About Covid-19 and Surfaces Again,” October 20, 2020. https://www.wired.com/story/its-time-to-talk-about-covid-19-and-surfaces-again/
• ��“Researchers Tackle the Surface Transmission of COVID-19 in New Partnership,” October 23, 2020. https://www.birmingham.ac.uk/news/latest/2020/10/researchers-tackle-the-surface-transmission-of-covid-19-in-new-partnership.aspx

Viruses such as SARS-CoV-2, the virus that causes COVID-19, generally range in size from 100–300 nm and are much smaller than bacteria. They comprise nucleic materials (DNA or RNA) surrounded by proteins that protect them from the environment and enable them to bind to specific sites on host cells. Enveloped viruses (including SARS-CoV-2) also contain an outer layer composed of lipids, polysaccharides, and proteins that allow the virus to more easily fuse with and enter host cells. To survive and reproduce, viruses must eventually infect a living host. Coronaviruses such as SARS-CoV-2, however, have been shown to survive on metal, glass, wood, fabric, and plastic surfaces for several hours to days. Therefore, destroying or damaging viruses while they exist on surfaces can prevent infection and the spread of disease. Many different types of nanoparticles have been shown to be effective at inactivating viruses, including coronaviruses such as SARS-CoV-2. For example, nanoscale zinc oxide, cuprous oxide, silver, copper iodide, gold on silica, and quaternary ammonium cations (quats) have all shown promise.¹

Coronaviruses such as SARS-CoV-2, however, have been shown to survive on metal, glass, wood, fabric, and plastic surfaces for several hours to days. Therefore, destroying or damaging viruses while they exist on surfaces can prevent infection and the spread of disease.

Due to the COVID-19 pandemic, much research is being focused on the development of antiviral coatings. Antiviral coatings are a subset of antimicrobial coatings. Some antimicrobial coatings can kill viruses as well as bacteria and other microorganisms (i.e., mold and fungi), while others are not designed to destroy viruses. One example of a coating approved by the U.S. Environmental Protection Agency that targets viruses is Caliwel™ BNA Coating from Allistagen Corporation. This coating is based on calcium hydroxide and has been proven effective against more than 20 life-threatening bacteria, viruses, and fungi, according to the company.²

Nova Surface-Care Centre Pvt. Ltd. believes its NANOVA HYGIENE+™ product, a low-surface-
energy coating (repels both oil and water) containing a combination of nano-actives (quats, positively charged silver nanoparticles) that can be applied to fabrics, plastics, metals, and concretes has the potential to inactivate SARS-CoV-2.¹ The coating has been shown to be effective against bacteria, fungi, and algae. An initial test against the small nonenveloped RNA virus MS2 Bacteriophage (Poliovirus) showed 99.9% antiviral efficacy in just two hours according to global standard AATCC 100-2012. A test is currently underway to establish the coating’s performance against SARS-CoV-2.

Researchers at the Hong Kong University of Science and Technology report the development of an antiviral that, when sprayed onto frequently touched surfaces, could provide 90 days of “significant” protection against bacteria and viruses, including SARS-CoV-2.³ The coating is based on what the scientists refer to as a multilevel antimicrobial polymer (MAP-1) and contains millions of nano-capsules formed with heat-sensitive polymers that contain disinfectants. The polymers release the disinfectants when warmed by human contact. Clinical tests were conducted at a Hong Kong hospital and a home for the elderly, and now the coating is commercially available through the university’s industrial partner Chiaphua Industries Ltd. A local charity stepped in to help, spraying the non-toxic coating around the homes of more than a thousand low-income families in Hong Kong.

Nippon Paint and Corning, Inc. began work to develop an antiviral coating in June 2019.⁴ The COVID-19 pandemic accelerated work on the project. The resulting coating, Nippon Paint’s Antivirus Kids Paint, contains Corning’s Corning Guardiant™ Antimicrobial Particles and was tested by Microchem Laboratory, an accredited, independent laboratory in the United States, in January. The paint was shown to inactivate > 99.9% of the Feline Calicivirus (an EPA-approved representative for human norovirus) and to kill > 99.99% of harmful bacteria including Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa under the latest test protocols that mimic contamination in everyday indoor environments. Nippon Paint kids paint by itself is environmentally friendly and TÜV and GREENGUARD certified. In March, Nippon Paint China and Corning Inc. donated 5 million RMB worth of the antiviral coating, which was specially produced for frontline hospital use and is not yet available on the market, to four Hubei coronavirus-designated hospitals.

Researchers at the Hong Kong University of Science and Technology report the development of an antiviral that, when sprayed onto frequently touched surfaces, could provide 90 days of “significant” protection against bacteria and viruses, including SARS-CoV-2.3 The coating is based on what the scientists refer to as a multilevel antimicrobial polymer (MAP-1) and contains millions of nano-capsules formed with heat-sensitive polymers that contain disinfectants.

In one other example, materials company Nobio, Ltd. received a $205,000 grant in April 2020 from the Israel Innovation Authority to support the development and manufacture of a long-acting surface coating containing its patented antimicrobial technology.⁵ Nobio received FDA clearance in 2019 for its Infinix antimicrobial dental restorations (fillings) and expects to launch them in the second half of 2020. With the new grant money, the company will apply similar nanoparticle-based antimicrobial technology to develop antiviral facemasks and long-acting surface coatings. Nobio is currently working with contract manufacturers to enable scale-up and production of the face masks and coatings.

References

1. Swapan Kumar Ghosh, “Anti-Viral Surface Coating to Prevent Spread of Novel Coronavirus (COVID-19) Through Touch,” Coatings World, April 15, 2020. https://www.coatingsworld.com/content-microsite/cw_covid-19/2020-04-15/anti-viral-surface-coating-to-prevent-spread-of-novel-coronavirus-covid-19-through-touch.
2. Allistagen, “EPA-Approved Antimicrobial Surface Coating Represents Breakthrough in the Control and Spread Of Infectious Diseases,” Press Release, March 19, 2020. https://www.prnewswire.com/news-releases/
   epa-approved-antimicrobial-surface-coating-represents-breakthrough-
   in-the-control-and-spread-of-infectious-diseases-301027074.html.
3. Reuters, “HK scientists say new antiviral coating can protect surfaces for 90 days,” Health News, April 27, 2020. https://www.reuters.com/article/us-health-coronavirus-hongkong-coating/hk-scientists-say-new-antiviral-coating-can-protect-surfaces-for-90-days-idUSKCN2290S5.
4. Nippon Paint, “Nippon Paint and Corning Incorporated donated lab-tested antivirus coatings to hospitals in Hubei,” Press Release, March 16, 2020. https://prnmedia.prnewswire.com/news-releases/
   nippon-paint-and-corning-incorporated-donated-lab-tested-antivirus-
   coatings-to-hospitals-in-hubei-301024778.html.
5. Nobio Ltd., “Nobio Secures Grant from the Israel Innovation Authority for Protective Measures Against Covid-19,” Press Release, April 9, 2020. https://prnmedia.prnewswire.com/news-releases/nobio-secures-
   grant-from-the-israel-innovation-authority-for-protective-measures-
   against-covid-19-301038123.html.

CoatingsTech | Vol. 17, No. 7 | July 2020

Consumer and regulatory pressure to replace bisphenol-A (BPA)-based materials in food contact metal packaging coatings has increased in recent years. Regardless of the controversy around BPA, consumers expect canned foods to be free of substances perceived to have negative health impacts while maintaining current shelf life and flavor characteristics. To address the market needs, formulators must innovate to deliver BPA-non-intent (BPA-NI) solutions that can meet or exceed the performance of BPA-based materials. This presents a challenge with regard to improving the resistance to food sterilization and stability during pack testing, and simultaneously balancing mechanical performance that allows the BPA-NI coating to withstand the aggressive canning process.

One response to these technical challenges has been the development of BPA-NI polyester resin technology through innovation on a monomer basis. This monomer innovation provides protective performance attributes such as resistance to corrosion and chemical attack, while enabling flexibility and adhesion through innovative resin and formulation design. Fundamental techniques such as electrochemical impedance spectroscopy (EIS) and cathodic disbonding were employed in combination with industrial fitness-for-use evaluations to demonstrate the improved protective barrier properties of novel non-BPA resins in formulated coatings. In addition, hydrophobicity and interfacial properties were studied to understand the impact of resin structure on coating performance from both experimental and computational perspectives. Applying this suite of methods and analysis builds strong structure-property correlations as part of a resin development strategy for novel non-BPA resins in metal packaging coating applications.

INTRODUCTION

Bisphenol-A (BPA) is a chemical commonly used in food contact plastics and coatings applications such as the BPA-epoxy-based linings of metal cans containing food or beverages. In recent years, the use of BPA in food contact applications has come under scrutiny. The 2003–2004 National Health and Nutrition Examination Survey (NHANES III) conducted by the Centers for Disease Control and Prevention (CDC) found detectable levels of BPA in 93% of 2517 urine samples from people six years and older.¹ In 2008, the National Toxicology Program of National Institute of Health (NIH) determined that BPA may pose risks to human development, raising concerns for early puberty, prostate effects, breast cancer, and behavioral impacts from early-life exposures.² Due to the potential health concerns, France has banned the use of BPA in all packaging, containers, and utensils intended to come into direct contact with food since 2015.³

With increasing pressure from food brands, formulators and can makers are actively looking for alternative solutions that can meet or exceed the performance of BPA-based coatings. From a technical standpoint, it is challenging to find the right alternatives due to the rigorous performance requirements for the coatings as well as the low price of BPA-epoxy resins. For example, the coating must be able to endure high temperatures, high pressure food sterilization, and long-time direct contact while exposed to the food materials, which include hydrolytic and corrosive environments such as low pH, acids, sulfur, and salt. Adhesion of the coating to the metal can is also crucial for both preventing corrosion and withstanding the can forming process. To respond to the technical challenges, coating scientists and chemists must innovate to develop new resin technologies.

Among all the new resin technologies today, polyesters with a balance of key performance attributes have emerged as one of the most promising alternative solutions. For polyester resins in this application, it should be noted that enabling high glass transition temperature (T_g) in combination with good mechanical properties, such as flexibility and toughness, is critical to the final film performance. These considerations are often applied when selecting monomers for resin design. Therefore, several qualified specialty glycol monomers such as 1, 4–cyclohexanedimethanol (CHDM), isosorbide, tricyclodecane dimethanol (TCDDM), and 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD) have received broad attention as building blocks. TMCD also demonstrated superior hydrolytic stability to other specialty monomers in a degradation kinetics study based on model compounds (Figure 1). Through previous work, polyesters containing TMCD are also known to demonstrate a variety of excellent properties such as good temperature resistance, toughness, chemical resistance, and hydrolytic stability. They have been successfully used in BPA-free specialty plastics applications such as durable water bottles.^4-6 Applying the monomer innovation to resin development, TMCD-based resin systems have shown attractive performance attributes such as resistance to corrosion and chemical attack, while enabling flexibility and adhesion through innovative resin and formulation design.

To better understand the structure–property relationships of polyester resins, a series of fundamental methodologies including electrochemical impedance spectroscopies (EIS) and cathodic disbonding tests were developed to study barrier properties, interfaces, and adhesion of metal packaging coatings. EIS is a widely used corrosion evaluation tool based on an electrical analogue of corrosion processes. It uses simple electrical circuits typically comprising of resistive and capacitive elements. For polymer coated metal systems, the EIS test is sensitive to the electrochemical changes at metallic interfaces as well as the resistive properties of organic coatings in a variety of aggressive or corrosive environments. In the past decades, both academia and industry have been using this technique to characterize coating barrier properties including diffusivity and polarity,⁷ to characterize water uptake,⁸ to detect formation of blisters and pinholes, and to recognize the loss of adhesion.⁹ The cathodic disbonding test is also an effective electrochemical technique to evaluate adhesion performance. Cathodic disbonding is an important delamination mechanism associated with interfacial corrosion of organic coatings on metal substrates that lead to an exposure of bare metal to the aqueous food environment.¹⁰

In this study, the electrochemical techniques have been employed in combination with industrial fitness-for-use (FFU) evaluations to better understand the barrier properties, interface, and adhesion of metal packaging coatings. This work provides fundamental insights on the relative performance of polyester resins in a variety of formulations where improved barrier properties and enhanced coating-
metal interfacial strength is observed with the TMCD-based resins.

Experimental Procedures

Materials and Sample Preparation

In this study, BPA-NI Resin A and Resin B were TMCD-based polyester resins developed and produced by Eastman Chemical Company, while Control A and Control B are the benchmark resins based on commercial BPA-NI polyesters. All of the polyester resins were classified by molecular weight: both Resin A and Control A (Category A) have absolute-number average molecular (Mn) greater than 10,000 g/mol while Resin B and Control B (Category B) have absolute-Mn in the range of 4,000–7,000 g/mol. Formulation components in this study were chosen for the purpose of representing BPA-NI interior lacquers. All coatings based on these polyester resins were formulated and applied in the Eastman Chemical coating development laboratories. The details of resins and formulation components are given in Table 1 and Table 2.

Prior to formulating, Resin A and Control A were reduced with Aromatic 100 to achieve 50% solids; Resin B was reduced with Aromatic 100 to 55% solids; Fascat 9102 catalyst was diluted to 10% of the original supplied concentration with Aromatic 100; likewise, Nacure 5925 catalyst was also diluted to 20% of the original supplied concentration with Aromatic 100. The formulation details of gold benzoguanamine phenolic formulation, clear PU formulation, and white PU formulation are provided in Table 3 and Table 4, respectively.

Electro tin plate (ETP) substrate panels were described by the vendor, Lakeside Metals Inc. as 0.25 # Bright T-1 0.009–0.010 x 4.0 x 12.0 in. All formulated paints were drawn down onto 4.0 x 12.0 in. (10.16 cm x 30.48 cm) tinplates (provided by Lakeside Materials Inc.) using an appropriate wire wound rod targeting a dry film thickness (DFT) of 0.4 mils (10.2 μm) for clear and gold formulations, or 0.6 mils (15.2 μm) for white formulations. Following a 15 min room temperature flash-off at constant temperature and humidity conditions (73°F ± 2, 50% RH ± 5), panels were baked at 200°C for 12 min in an air oven.

Testing and Evaluations

Electrochemical Impedance Spectroscopy (EIS)

A Gamry Instrument “Reference 600” Potentiostat, equipped with Gamry framework and Echem Analyst, was used in electrochemical impedance measurements. The flat coated panels (3.0 x 4.0 in.) were masked by a Gamry designated masking tape with a hollow circle at the center. A cylindrical glass cell with a rubber O-ring attached to the grooved bottom of the cylindrical cell and a clamp fixture was used to hold the samples. A nickel electrode and a graphite electrode were used as reference and auxiliary electrodes, respectively. To simulate food, several food simulants were used as the electrolyte and corrosive environments. The potential was applied in a range of ± 5 mV from open circuit potential and the frequency was varied from 10⁵ to 10^-1 Hz.

Cathodic Disbonding Test

The cathodic disbonding test is an internally developed coating disbonding test modified from a number of standard test methods.¹¹ It helps to differentiate coatings on the basis of their susceptibility to adhesion failure in the presence of a defect. The scheme of the cathodic disbonding experimental setup and the lab setup are shown in Figure 2a and 2b. The flat coated panels (3.0 x 4.0 in.) were masked by a Gamry designated masking tape with a hollow circle at the center. In the unmasked area at the center, the coatings were scribed with an “X” mark (as shown in Figure 2b using a knife. A cylindrical glass cell with a rubber O-ring attached to the grooved bottom of it and a clamp fixture was used to hold the samples. A coated panel with X scribe (cathode) was connected with the negative electrode of the voltage supply while a graphite (anode) was connected with the positive electrode of a direct current (DC) voltage supplier. Between cathode and anode, the cylindrical glass cell was filled with electrolyte made by 3.5 wt% NaCl solution with 150 ppm Manoxol OT solution to ensure both parallel electrodes were submerged in the electrolyte solution. The DC voltage applied between the parallel cathode and anode was 5 volts (V) for 60 sec. After this electrochemical process was completed, the sample was rinsed with DI water and followed by air drying.

Under the applied DC electric field, the hydrogen bubbles were initially generated at the X scribe and then propagated to the coating-tinplate interface following the cathode reaction as shown in equation (1). In addition to the hydrogen bubbles generated at the metal surface lifting up the coating from the tinplate, the generation of ����¯ also weakens the coating–tinplate adhesion, resulting in coating delamination.

(1)

Food Simulants and Retort

The recipes of food simulants used in this study are shown below:

(i) Lactic acid–Acetic acid-Salt (LAS) food simulant: 1 wt% lactic acid, 1 wt% acetic acid, and 1 wt% NaCl in 97 wt% DI water;

(ii) 3% Acetic acid food simulant: 3% acetic acid in 97% DI water; and

(iii)�� 2% Lactic acid food simulant: 2% lactic acid in 98% DI water.

A coupon measuring 2.5 x 4.0 in. was cut from the coated panel for retort testing. The coupons were scribed by a knife with an X mark on the bottom-half of the panel and then placed in 250 mL closed-cap glass jars half filled with 3% acetic acid food simulant where half the coupon is out of the food simulant and the other half is submerged in food simulant. The retort test was conducted on a coated panel with an X scribe at 131°C for 60 min in an autoclave.

Computational Modeling

Calculated LogP values which estimate the value of the octanol-water partitioning coefficient were determined by using ACD/Labs Chemsketch software. The calculated logP values were also validated by ChemBioDraw Ultra 13.0, Molinspiration and Accelrys Materials Studio 5.5. Molecular structures of trimer model compounds represented as glycol1-terephthalate-glycol2 (G1-T-G2) with hydroxyl end groups were used in the experiments and calculations.¹²

Solubility parameters were calculated for 30x30x30 ų amorphous cells with 1 g/cm³ cell density for 3 terephthalate and glycol units that were constructed by using Accelrys Materials Studio 5.5 software. PCFF forcefield was used to build and minimize cells and calculate cohesive energies, which led to the calculation of cohesive energy densities followed by calculation of solubility parameters.¹³ Average solubility parameters were calculated for 500 different amorphous cells followed by 5000 steps of molecular mechanics geometry optimization with Ewald Summation method¹⁴ and 12.5 Å vdW cut-off distance for each composition.

Results and Discussion

EIS and Corrosion Mechanisms

EIS is a powerful technique to understand corrosion mechanisms and barrier properties of coatings by providing an accurate in-situ measurement for characterizing polymer-coated metals and changes in coating performance during exposure in corrosive environments. The term “impedance” refers to the frequency-dependent resistance to current flow of circuit elements such as resistors, capacitors, inductors, etc. In practice, the corrosion resistance is integrated from all types of resistance involved and can be approximately estimated by using |Z(w®0)|, the impedance value at low frequencies. In this study, corrosion resistance is identified by the impedance value at 0.1 Hz. However, to describe the corrosion process quantitatively, Bode plots are fitted using electrical equivalent circuits corresponding to the appropriate stage of corrosion.

Stage Zero: Dry Film

Like a dielectric layer, before immersion in an electrolyte solution a dry polymer film often plays a role like a pure capacitor as shown in Figure 3.�� When the film responds to the frequency, the overall impedance can be presented as shown in equation (2):

(2) therein ω = 2πf

where j is the complex number (  j ²=-1); R_sis the solution resistance which is the resistance of the food simulant; C_cis the coating capacitance; w is the angular frequency; and_�� f _����is the frequency. The Bode plot for Stage zero is simply demonstrated in Figure 3.

��

Stage I: Food Simulant Absorption

Before the corrosion process starts, the dry coating must absorb the electrolyte until the polymer film gets fully saturated by the electrolyte as shown in Figure 4. The electrolyte is the food simulant in this case. In Stage I, the coating is no longer a pure capacitance due to the presence of water and ions. Instead, the combination of the capacitance component and the resistance component of the coating contribute to the overall impedance. With higher water and ion uptake, the value of the capacitance component increases while the value of the resistance component decreases. They change independently as a function of time before the film gets fully saturated by the food simulant. In general, the diffusion kinetics are highly dependent on the physical properties of the polymer film such as crosslinking density. When the film responds to the frequency, the overall impedance can be presented as shown in equation (3):

(3) where j is the complex number (  j ²=-1); R_sis the solution resistance which is the resistance of the food simulant; C_c�� is the coating capacitance; w is the frequency; and R_c��is the coating resistance. As shown in Figure 4, a single time-constant can be indicated by a frequency–independent impedance plateau at low frequency followed by a frequency–dependent impedance plot in the medium frequency region. The increase of C_cas a function of exposure can be used to determine the diffusion coefficient as well as the food simulant uptake.

Stage II: Corrosion Initiation

After the film gets fully saturated, water and ions in the food simulant start to be delivered to the coating-tinplate interface and initiate the corrosion process. In this process, the redox reactions between the metal and food simulant (H⁺��either from food simulant or from hydrolyzed water) lead to corrosion. At this stage, the newly formed oxidized layer with semi-dielectric character exists under the polymer film, playing a role as the combination of a double layer capacitance and a charge transfer resistance, as presented in equation (4):

(4)
where R_s��is the solution resistance which is the resistance of the food simulant; C_c�� is the coating capacitance; w is the angular frequency; C_dl is the double layer capacitance; R_ct�� is the charge transfer resistance; and R_c�� is the coating resistance.

Stage III ~ IV: Pore/breakthrough Formation and Delamination

A process that depends on diffusion of reactants toward or away from the surface has a particular low-frequency character. The impedance with this characteristic is usually described as “Warburg” impedance (as shown in Figure 7), which indicates the breakthrough of a barrier and localized disbonding. At this stage, the overall impedance can be presented as shown in equation (4) (pore/breakthrough formation) and equation (5), (delamination), respectively:

(5)

(6) where R_s�� is the solution resistance which is the resistance of the food simulant; C_c�� is the coating capacitance;�� w is the frequency; C_dl�� is the double layer capacitance; R_ct�� is the charge transfer resistance; R_po�� is the pore resistance; k is the redox reaction rate; and D is the diffusion coefficient at this stage. At Stage III ~ IV, the barrier has been damaged locally, even though the defects might not be able to be detected by the naked eye. In this case, R_ct�� representing pore resistance is used to describe the concept of “coating resistance” because this value now is highly dependent on the number of pores in the film or capillary channels resulting from the formation of ionically conducting paths through the coating, instead of the intrinsic physical properties of the coating barrier.

To demonstrate the progression of coating failure as a function of LAS food simulant exposure time, Control B was chosen to formulate a clear coating, then subjected to the EIS test in LAS food simulant. As shown in Figure 7, Control B in a clear PU formulation starts to show Stage I corrosion (one time-constant) after 4 min of exposure, indicating that the food simulant absorption has begun. After 15 min, the characteristics of Stage II corrosion (e.g., two time-constants) have been observed, and this is followed by the Bode plot with Warburg character (Stage III ~ IV) after 5 h of exposure in LAS food simulant.

To compare BPA-NI polyester resins in clear PU formulations, Resin A and commercial Control A were selected to formulate the paints followed by appropriate baking. A comparison of the EIS spectrum of Resin A and Control A in these two coatings in Figure 8a shows similar corrosion resistance and barrier properties after 5 h of exposure. Resin A-based clear PU shows a corrosion resistance of 53.7 mega Ohms, which is slightly higher than that of the Control A-based coating (39.8 mega Ohms). Both coatings demonstrate excellent barrier properties and almost two orders of magnitude improvement on corrosion resistance after 5 h of exposure as compared to the low T_g polyester control in the same formulation (0.69 mega Ohms, as shown in Figure 7).

In Figure 8, Resin A was formulated in both clear PU and gold benzoguanamine phenolic formulations. Through the comparison of EIS data after 5 h of exposure (Stage I corrosion), it has been found that the gold benzoguanamine phenolic formulation exhibits significantly better corrosion resistance (170 mega Ohms) as compared to the corrosion resistance of clear PU formulation (53.7 mega Ohms) at the same dry film thickness. The authors believe that the presence of triazine and aromatic structures in benzoguanamine-formaldehyde and phenolic-formaldehyde crosslinkers may provide better hydrophobicity and barrier properties as compared to IPDI trimer-based PU structures.

Time-based Corrosion Resistance

In many cases, corrosion is an electrochemical process that requires multiple steps, and each step is associated with a different mechanism and kinetics. The estimation or prediction of long-term corrosion performance, such as the corrosion observed in a pack test, often relies on continuous time-based corrosion observations over a relatively long interval of testing instead of a single data point at a short exposure time. As part of a resin design strategy, a continuous in-situ EIS test has been conducted on white PU coatings based on four BPA-NI polyester resins including Control A (high-T_g high Mn polyester), Control B (low-T_g low Mn polyester), Resin A (high-T_g high Mn polymer), and Resin B (med-T_g low Mn polyester) in 2% lactic acid food simulant. The EIS test in one testing period was set up to continuously run for 48 h. Ten hours of relaxation time was given prior to the next testing period. Figure 9a and 9b demonstrate a decay of corrosion resistance as a function of exposure time for each sample during the 1st and 2nd 48-h test intervals. After a total of 106 h of exposure, all the white PU samples still remain in the Stage I corrosion process. During the 2nd 48-h test period, the decay of corrosion resistance for each sample becomes significantly slower, followed by a plateau of impedance at longer times. The two high Mn polyesters seem to be separated from the other two low Mn polyesters after the 2nd 48-h interval, where the high Mn polyester-based coatings show higher values of corrosion resistance. By the end of the test, the comparison of all four white PU coatings shows a ranking on corrosion resistance: Resin A > Control A > Resin B > Control B in white PU formulations (Figure 10). Since all of the samples are still in Stage I corrosion, the decay kinetics indicate the diffusion coefficient while the films absorb the food simulant, whereas the plateau level reflects the solubility of the electrolyte solution in the coating film. Therefore, higher corrosion resistance correlates with higher hydrophobicity or lower solubility in 2% lactic acid food simulant in this case. In Figure 11, LogP and Hildebrand solubility parameters were calculated for glycol (G1)-terephthalic acid (T)-glycol (G2) trimer model compounds through computational modeling. In general, a higher LogP value or lower Hildebrand solubility parameter indicates better hydrophobicity of a polymer. With the same molecular weight (trimers) and acid composition (terephthalic acid) in the model compounds, it has been hypothesized that the glycols with higher LogP values or lower Hildebrand solubility parameter lead to a lower concentration of the aqueous food simulant in the bulk of the film, thus reducing the rate of corrosion.¹² Considering the molecular weight contributions in the resins, the comparison between a TMCD-based resin and a control polyester resin at a similar molecular weight and T_g range (Resin A vs Control A and Resin B vs Control B) indicates that the improvement of corrosion resistance in coatings formulated with Resin A and Resin B is primarily due to the hydrophobicity contributions from TMCD (Figure 11).

Interface and Adhesion

To better understand what happened during late stage corrosion, the EIS comparison between Resin A- and Control A-based clear PU coatings (shown in Figure 8a) was extended to longer exposure times. After 12 days in LAS food simulant, it has been observed that both Resin A- and Control A-based clear PU coatings are in Stage III ~ IV corrosion with clear Warburg impedance in the Bode plots (Figure 12). Although the pores and capillary channels that provide conducting paths (e.g., Warburg impedance) through a coating have already formed in both Control A- and Resin A-based clear PU coatings, the interface, with its excellent double-layer capacitance and charge transfer resistance, can still provide excellent corrosion prevention.

In Figure 12, Resin A shows a higher plateau in the middle—frequency range, which indicates the value of charge transfer resistance corresponding to equation (6). When a redox reaction occurs, electrons enter the metal and metal ions diffuse into the electrolyte. Thus, charge is being transferred. The current density of the charge transfer process at the applied potential follows Faradays Law [equation (7)]:

(7) where i_o��is exchange current density;�� C_o is the concentration of oxidant at the electrode surface; C_o*����is the concentration of oxidant in the bulk; C_R�� is the concentration of reductant at the electrode surface; C_R*������is the concentration of reductant in the bulk; h is the overpotential (difference between applied potential and open circuits potential, OCP); F is Faradays constant; T is absolute temperature; R is the ideal gas constant; a is the reaction order; and n is the number of electrons involved. When the overpotential is very small (± 5 mV vs OCP in this experiment) and the electrochemical system is at equilibrium (C_o�� =�� C_o*���� and�� C_{R����}=  C_R*����), charge transfer resistance can be represented, as shown in equation (8):

(8) With the same experimental conditions, the difference between the two clear PU coatings on charge transfer resistance is believed to be due to the number of electrons involved, which correlates to the percentage of area without polymeric barrier due to the loss of adhesion. Several studies^15-17 for different applications have found that charge transfer resistance is correlated to adhesion experimentally. For the comparison shown in Figure 12, the authors believe that the Resin A-based clear PU with significantly higher charge transfer resistance indicates that Resin A provides a clear PU coating with a stronger coating-tinplate interface and better adhesion as compared to Control A.

Besides the EIS tests, gloss loss after a 3% acetic acid retort test was measured as a way to evaluate the barrier performance of the coatings. As the retort test was conducted on a coated panel at 131°C for 60 min in an autoclave, the presence of high temperature and pressure has significantly accelerated the corrosion formation at the coating-tinplate interface, causing the development of “blisters” as the result of under-film corrosion. This process often leads to gloss loss due to (i) the changes in surface and interface smoothness caused by under-film corrosion; (ii) rust stains the coating surface caused by broken “blisters”; and (iii) a change in coating refractive index caused by water retention. Similar to what occurs during the late stage corrosion process, the corrosion development and coating delamination at X scribes could be much faster than that in other areas because the barrier layer has been broken through manually. In this scenario, adhesion performance can be evaluated based on a visual observation. As shown in Figure 13a, no significant difference can be observed between Resin A- and Control A-based clear PU coatings from gloss reduction, indicating that Resin A provides similar barrier properties as Control A in the clear PU formulation. This retort result is consistent with the insights obtained from EIS data (Figure 8a).

In Figure 13b, when the aggressive cathodic disbonding test was applied under 5V on X scribed panels, the Control A-based clear PU coating with a large disbonded area (less desirable) is differentiated from the Resin A-based coating with very little disbonded area (more desirable). The disbonded area was identified by a high-resolution camera and the areas of bare tinplate were quantified by pixel-counting image analysis software (Figure 14). In this case, the disbonded area, in percentage of the total area, of Control A-based clear PU coating was determined as approximately 52%, while only a minor disbonded area (< 5% disbonded area) was found for Resin A-based clear PU coating. In this case, a smaller disbonded area indicates better adhesion or interfacial strength. The cathodic disbonding result in Figure 13b is consistent with the EIS results (Figure 12) regarding adhesion performance. The combination of EIS and cathodic disbonding results indicates that Resin A provides a stronger interface to tinplate as compared to Control A. Considering the molecular weight and T_g effects, the comparison between TMCD-based Resin A and Control A polyester resin at a similar molecular weight and T_g range indicates that the improvement of adhesion in coatings containing Resin A could be due to the hydrophobicity contributions from TMCD.

CONCLUSIONS

In this study, fundamental methodologies based on EIS and cathodic disbonding tests were successfully developed and applied to understand the barrier properties, interfaces, and adhesion of BPA-NI metal packaging coatings. Corresponding to EIS Bode plot characteristics, a series of equivalent circuit models indicating the stages of corrosion process were developed to demonstrate the degradation mechanisms including (i) food simulant absorption, (ii) corrosion initiation, and (iii) pore/breakthrough formation. These circuit models also enable quantitative analysis of coating performance to design resins that are in tune with coating properties in metal packaging applications.

A combination of electrochemical techniques and industrially relevant FFU evaluations were conducted to differentiate the corrosion resistance of TMCD containing vs non-TMCD containing clear and white PU coatings. In the early stage corrosion process, Resin A exhibited a slightly improved barrier performance in the coatings as compared to Control A due to the low permeability after being fully saturated by the food simulants. This is believed to be due to the hydrophobicity contribution from TMCD in polyester Resin A. A similar conclusion has been obtained when comparing the TMCD-containing Resin B to the Control B resin in both white and clear PU coatings.

In addition to PU formulations, TMCD containing Resin A and Control A resin were also formulated with benzoguanamines and phenolic crosslinkers to evaluate these resins in gold lacquer applications. A comparable barrier performance was observed during the early stage corrosion processes in a clear PU coating with Resin A in and Control A. However, the comparison between Resin A in the clear PU formulation and gold benzoguanamine phenolic formulations indicates that the gold formulation is significantly better on barrier performance with the same resin and film thickness.

EIS results for a late-stage corrosion process based on Resin A- and Control A-based clear PU coatings are consistent with the results obtained from cathodic disbonding tests. Analysis of these results demonstrates that the coating made with TMCD-containing Resin A provides a significantly improved coating-tinplate interface that leads to superior adhesion performance as compared to Control A resin in a clear PU formulation.

ACKNOWLEDGMENT

Technical expertise provided by Damiano Beccaria, Sandra Case, Peter Chapman, Alain Cagnard, Samuel Puaud, John Maddox and Carlos Carvajal from Eastman Chemical Company is gratefully acknowledged. The Computational Modeling results presented in this paper were generated in collaboration with Erol Yildirim, Harold Freeman, and Melissa Pasquinelli, Fiber and Polymer Science Program, North Carolina State University. The contribution made by Dr. Yildirim for this paper is highly appreciated. The authors also appreciate the technical support from Dr. Li-piin Sung through the Polymer Surface Interface Consortium, Engineering Laboratory, National Institute of Standards and Technology.

References

1. �� https://www.niehs.nih.gov/health/topics/agents/sya-bpa/index.cfm.

2.���� https://www.ewg.org/research/timeline-bpa-invention-phase-out#.W7YW9FRKi9I.

3. �� https://www.foodpackagingforum.org/news/france-bans-bpa.

4������ Kelsey, D.R., Scardino B.M., Grebowicz, J.S., and Chuah, H.H., “High Impact, Amorphous Terephthalate Copolyesters of Rigid 2,2,4,4-Tetramethyl-1,3-cyclobutanediol with Flexible Diols, Macromolecules, 33, 5810-5818 (2000).

5.���� Zhang, M., Moore, R.B., and Long, T.E., “Melt transesterification and characterization of segmented block copolyesters containing 2,2,4,4-tetramethyl-1,3-cyclobutanediol,” J. Polym. Sci. Part A: Polym. Chem., 50, 3710-3718 (2012).

6.���� Clark, R.D., Treatment of 2, 2, 4, 4-tetraalkyl-1, 3-cyclobutanediols, US3236899A, in: Eastman Kodak Co., 1966.

7. �� Feng, L. and Iroh, J.O., “Corrosion resistance and lifetime of polyimide-b-polyurea novel copolymer coatings,” Prog. Org. Coat., 77, 590-599 (2014).

8. �� Kefallinou, Z., Lyon, S.B., and Gibbon, S.R., “A bulk and localised electrochemical assessment of epoxy-phenolic coating degradation,” Prog. Org. Coat., 102, 88-98 (2017).

9. �� Mansfeld, F., “Use of electrochemical impedance spectroscopy for the study of corrosion protection by polymer coatings,” J. Appl. Electrochem., 25, 187-202 (1995).

10. Bi, H. and Sykes, J., “Cathodic disbonding of an unpigmented epoxy coating on mild steel under semi- and full-immersion conditions,” Corros. Sci., 53, 3416-3425 (2011).
11. Holub, J., Wong, D.T., and Tan, M., “Analysis of CDT methods and factors affecting cathodic disbondment,” from NACE International Corrosion Conference and Expo, Nashville, TN, pp. 5199, 2007.
12. Yildirim, E., Freeman, H., Pasquinelli, M., and El-Shafei, A., “Role of chemical structure on the alkaline hydrolysis of industrially-relevant copolyester model compounds,” from 252th ACS National Meetings, Philadelphia, PA, 2016.
13. Dechant, J., Polymer Handbook, 3rd Edition. Brandrup, J. and Immergut, E.H. (Eds.), ISBN 0-471-81244-7. New York/Chichester/Brisbane/Toronto/Singapore: John Wiley & Sons, 1989. ca. 1850 pages; Acta Polymerica, 41, 361-362 (1990).
14. Tosi, M.P., “Cohesion of Ionic Solids in the Born Model,” Based on work performed under the auspices of the U.S. Atomic Energy Commission, in: Solid State Physics, Seitz, F. and Turnbull, D. (Eds.), Academic Press, pp. 1-120, 1964.
15. Wang, M. and Sakamoto, J., “Correlating the interface resistance and surface adhesion of the Li metal-solid electrolyte interface,” J. Power Sources, 377, 7-11 (2018).
16. Lu, F., Song, B., He, P., Wang, Z., and Wang, J., “Electrochemical impedance spectroscopy (EIS) study on the degradation of acrylic polyurethane coatings,” RSC Advances, 7, 13742-13748 (2017).
17. Qian, Y., “Electrochemical Impedance Spectroscopy Investigation of a Polyimide Coating on Q345 Steel,” Int. J. Electrochem. Sci., 12, 3063–3071, 2017.

*This paper received the Best Paper Award at the 2019 CoatingsTech Conference, sponsored by the American Coatings Association, April 8–10, in Cleveland, OH.

CoatingsTech | Vol. 16, No. 6 | June 2019

Antimicrobial powder coatings are new warriors in this battle. The value of the global antimicrobial coatings market is expected to reach $4.19 billion by 2021, rising at a compound annual growth rate of 12.1% from 2016 to 2021, according to market research firm MarketsandMarkets. The main applications for these materials are in air conditioning and ventilation systems, medical implants/devices, mold remediation, and various uses in the building and construction (manufacturing, public venues, office building, schools, restaurants, and residential housing), food and beverage (food processing and production), and textile industries. Medical applications account for the largest market share, followed by protection of indoor air/HVAC systems. North America is the largest and fastest growing market for antimicrobial coatings.

Looking to meet the growing need for antimicrobial coatings in the construction industry, AkzoNobel has developed the antimicrobial Interpon AM range of hygienic coatings that provide a combination of outstanding decorative characteristics and the ability to inhibit the growth of microbes such as bacteria and mold. The coatings are used in hygiene conscious environments��such as hospitals, clinics, changing rooms, schools and public transport areas, according to the company.�� For example, an Interpon AM coating was recently specified for 2,350 door handles in a clinic in Abu Dhabi.

The antimicrobial protection is provided by BioCote^® antimicrobial technology, which reduces up to 99.99% of bacteria and mold on a protected surface, makes it easier to keep hygienically clean, and reduces the negative effects such as odors and staining. BioCote has been developing antimicrobial solutions for over 20 years. “We work successfully with industry-leading, global companies such as AkzoNobel to help them create more hygienic, premium antimicrobial products,” says Jennifer Collier, partner development at BioCote Ltd. “We are extremely proud of our partnership with AkzoNobel and to be part of such a high-quality, reliable product,” she adds.

Demand for these types of antimicrobial coatings is increasing due to growing consumer concern for hygiene, according to Mark Reekie, global segment manager for AkzoNobel’s industrial powder coatings business. As a leader in powder coatings, he also notes that AkzoNobel is committed to setting standards for quality, service, and innovation. “InterponAM is a prime example of how we continue to develop world-class products for our customers while always looking to grow and improve,” he asserts.

The antimicrobial properties of two disparate bio-based coating additives were evaluated in a polyvinyl alcohol (PVA) food packaging coating for antimicrobial activity. Chitosan, a shrimp and crustacean shell derived polysaccharide, and an antimicrobial peptide were evaluated in a dissolvable food package coating for reductions in microbial growth after contacting agar patties serving as food simulants. Where the antimicrobial components of such packaging coatings are chosen to be generally recognized as safe by worldwide regulatory agencies, migration from the packaging into headspaces and food-contact surfaces can provide enhanced efficacy against foodborne pathogens, including viruses. The techniques and coatings presented in this article suggest that dramatic improvements in food safety can be achieved using coatings containing non-toxic bio-based biocides.

Introduction

Food packaging is an asset that can preserve foods going to market and extend shelf-life, but it can also become a liability when the packaged food itself is contaminated or becomes so during the packaging process. The economic damage to the food industry of reduced shelf-life for packaged foods is great, and contamination of packaged food is a major public health concern. There are several contamination points during food preparation and packaging and numerous examples of outbreaks of foodborne illness leading to the recall of goods.^1-3 Major incidences have occurred around the world over the decades (e.g., E. coli O157:H7 contamination from the United States Jack in the Box restaurant,⁴ E. coli O104:H4 spread from German sprouts,⁵ listeriosis throughout Europe in frozen corn,⁶ E. coli O157:H7 on Canadian pork,⁷ listeriosis from processed meat in South Africa⁸) and continue today. Table 1 highlights the diversity of products and pathogens associated with some of the most recent outbreaks in the United States.⁹ Even as recently as July 2018, there was a recall of packaged vegetable trays due to a multistate outbreak of parasitic cyclosporiasis in the United States.¹⁰

Such outbreaks have led not only to changes in guidelines and adoption of new regulations,^11-13 but also spurred interest in new technologies such as “smart” packaging.^14,15 Packaging coatings that can reduce such contamination while the food is traveling to its point of sale are long-sought goals of the food industry. Research for improved food packaging has generally focused on two areas: edible films that can be applied directly to food and are safe to eat,^16-19 and chemical or physical modifications to plastic packaging materials that are permanent and specifically do not leach onto or into the packaged food product.^20-22 In this work, we combined the best attributes of both research areas into one product by retaining the physical barrier properties afforded by plastic packaging film and gaining the effectiveness of dissolvable (but safe to eat) bio-based antimicrobial additives. Bio-based additives have the advantage of often having low toxicity to humans, and many have notifications with the Food and Drug Administration that they have been determined to be Generally Recognized as Safe (GRAS). Though not a prerequisite for development of an antimicrobial food contact coating in the present study, the selection of materials, whether polymeric coating components or bio-based additives, with GRAS notices previously filed was a consideration. Both polyvinyl alcohol and chitosan fall into this category and were chosen for this study.^23-26 Other considerations were the likely efficacy of the additive against microorganisms commonly associated with foodborne illness (i.e., bacteria like Escherichia coli), known general lack of toxicity, and biodegradability. Finally, as the numbers and types of foodborne pathogens are varied, consideration was given to bio-based biocides that were potentially capable of controlling not only bacteria, but also spores of Gram-positive species, fungi, algae, and viruses (ergo, the inclusion of antimicrobial peptides).

Two quantitative assays were developed to analyze effectiveness of the coatings using modern microbiological methods and statistical software. The first used clear-coated plastic disks through which bacterial colonies could be enumerated on the agar surface beneath. The second used agar “patties” serving as a food simulant in a vacuum-sealed food packaging system. The contaminated agar can be contacted on one or both sides with an antimicrobial coating and the microbial growth inhibition evaluated. Statistical evaluation was undertaken to detect antagonistic, additive, and/or synergistic relationships between chitosan and an antimicrobial peptide, AMP7, in the food contact coating.^27-29 The assays were successful in detecting antimicrobial activity against E. coli for both the bio-based additives analyzed, and the statistical methods used detected an antagonistic effect at most of the concentrations evaluated and only slightly antagonistic or additive effects at higher concentrations. These methods proved useful in screening the candidate bio-based additives presented here and are currently being used to evaluate other promising bio-based additives for incorporation into food packaging.

Materials and Methods

Reagents and Bacterial Strains

Polyvinyl alcohol (PVA) was obtained from Sigma-Aldrich (Cat# 348406, reported Mw 13,000-23,000, 98% hydrolyzed). Chitosan was obtained from bulksupplements.com. E. coli K12 was obtained from Presque Isle Cultures (Erie, PA), and the peptide AMP7 was obtained from Reactive Surfaces, Ltd. (Austin, TX). Isopropanol (91%) was purchased locally. All growth media used Difco Tryptic Soy Agar (TSA) or tryptic soy broth from Becton, Dickinson, and Co. (Sparks, MD). MacConkey, Eosin Methylene Blue (EMB), and Luria-Bertani (LB) agar were obtained from Carolina Biological Supply Co. (Burlington, NC). 4-Methylumbelliferyl-β-D-glucuronide dehydrate (MUG) was obtained from Fisher Scientific (Waltham, MA).

Preparation of Coated Films

Disks of 0.5-in. diameter were cut using a 40W CO2 laser (Glowforge®, Seattle, WA) from a 3-mil thick sheet of clear Dura-Lar polyester (Grafix, Maple Heights, OH) substrate. For the vacuum-sealed food simulant experiment, 8.5 cm diameter disks were hand-cut from Dura-Lar sheets, or from commercial substrates, including Opalen™ (Bemis, Parc de L’Alliance Braine L’Alleud, Belgium), a clear, PET film and Trayforma™ (Stora Enso, Stockholm, Sweden), a PET-coated paperboard. A 5% (w/w) PVA solution, 1% (w/w) chitosan in 2% (v/v) acetic acid solution, and 10% (w/w) AMP7 in 5% (w/w) PVA solution were prepared and mixed before application to create the final concentrations indicated in the following experiments. In each case, the bio-based additive levels reported are based on the percentage by weight in the final, dry coating. All Dura-Lar films were coated by applying a specific volume of the liquid coating directly to the film, so that the final film thickness was approximately 1 mil. The coated films were left to dry at room temperature overnight before being used in any of the antimicrobial tests.

Antimicrobial Coated Disk Treatment of E. coli Contaminated Agar Plates

Traditional zone of inhibition testing uses paper disks infused with the target active. The paper disks are incubated with microbes, and the zone of clearing seen around the disk (i.e., lack of microbial colony growth due to leaching of the active from the disk) is measured to indicate the effectiveness of the active. In our experiments, using the transparent disks with a clear, dissolvable coating allowed inspection of cell growth directly beneath the disk as well as any observable zone of inhibition (Figure 1). A cotton swab was dipped into a suspension of E. coli K12 (~5×106 CFUs/mL) and was spread over 15 cm diameter TSA plates (done in triplicate). After the plates had dried for approximately 15 min, the disks were placed, coated side down, on top of the E. coli layer. The plates were incubated at 30°C to 36°C overnight. Each disk was photographed using a dissecting microscope for magnification, and colonies were counted using ImageJ software from the National Institutes of Health (Bethesda, MD). The dose response of individual additives was evaluated using log-logistic regression model with the R package drc.³⁰ For experiments involving the combination of bio-based additives in coatings, the interaction between the two bioadditives (i.e., synergistic, additive, antagonistic) was evaluated using the zero interaction potency (ZIP) model with the R package synergyfinder.³¹

Preparation of Vacuum-Sealed Food Simulants

To mimic packaged food, agar patties were cast into petri dishes and then gently removed from the dishes once firm to serve as food-patty simulants. These patties were vacuum sealed with plastic film inserts containing mixtures of PVA, chitosan, and AMP7 as studied in the small disk assays (Figure 2). Several selective and differential agar media were evaluated for visualization of E. coli colonies. These included MacConkey agar, LB agar with MUG, and EMB agar. It was determined that MacConkey agar consistently produced clearly visible and easily observable E. coli colonies, and thus was primarily used. Each agar patty was placed in a vacuum bag, and an aliquot of diluted E. coli was spread over one surface so that approximately 200 CFUs were added (each test done in triplicate). Coated films sized to match the agar patties were placed, coating side facing the bacteria, on the agar patties, and uncoated films were used as controls. The vacuum seal bags were sealed using the “Low” vacuum setting and the default seal setting on a Harvest Keepers Commercial vacuum sealer. The vacuumed samples were incubated for 24 h at 30°C, and colonies were counted using ImageJ software. For experiments measuring the effect of AMP7 and chitosan combinations, the interaction between the two bioadditives (i.e., synergistic, additive, antagonistic) was evaluated using the R package synergyfinder, with the Bliss model being used to calculate predicted response because the number of combinations was low.

Experiments and Results

Clear Disk Antimicrobial Assay

The efficacy of the bio-based antimicrobials in food-safe coatings was assessed by placing the coated 0.5-in. diameter Dura-Lar disks, coating-side down, onto prepared lawns of E. coli (Figure 3). The disks were coated with PVA-based coatings dosed with AMP7 at concentrations from 5,000 [0.5% (w/w)] to 20,000 ppm [2% (w/w)], and with chitosan at concentrations from 10,400 [1.04% (w/w)] to 90,800 ppm [9.08% (w/w)], or combinations of these two additives.

The dose response, as determined by percent reduction in colony numbers compared to the PVA negative control, was determined for AMP7 and chitosan. The effective dose to kill 50% of the bacterial population (ED50) of AMP7 was around 4,500 ppm and 30,000 ppm for chitosan (Figures 4A and 4B). The responses of the bacteria to various combinations are displayed in the heatmap (Figure 4C), which shows the relative response as a color from red (highest percent growth inhibition) to green (lowest percent growth inhibition). To test whether antagonism or synergy exists between these two compounds, the responses from AMP7 and chitosan combinations were used to determine the synergy score using the ZIP method, which returns a score based on the deviation of the actual response from the expected response.³² These scores are visualized for each combination in a contour plot (Figure 4D), which suggests that the two antimicrobials generally interact in an antagonistic manner, with the highest regions of antagonism existing for concentrations of AMP7 between 5,000 ppm [0.5% (w/w)] and 10,000 ppm [1.0% (w/w)] and for concentrations of chitosan ranging from 10,400 ppm [1.04% (w/w)] to 47,600 ppm [4.76% (w/w)]. However, beyond this region of antagonism, all other combinations of AMP7 and chitosan appear to interact with reduced levels of antagonistic behavior. This suggests that the AMP7 peptide and chitosan are interfering with each other’s function, but that the negative interaction can be overcome as the concentrations of both additives increase. The antagonistic response could be happening because both additives target the cellular membrane of microbes to produce a biocidal effect.

Moist-Food Simulant Packaging Study

To test the scalability of the results from the clear disk assay, we simulated the common vacuum-sealed storage of moist foods (meats, fruits, vegetables, etc.) using a nutrient agar patty contacting a bio-based antimicrobial coated plastic disk while stored inside a vacuum sealed bag. Single additives were tested as well as combinations at corresponding concentrations. These results are summarized in the table in Figure 5A. Addition of 1% (w/w) AMP7 to 2.17% (w/w) chitosan-PVA coatings markedly worsened the efficacy of chitosan at this concentration, suggesting stronger antagonistic behavior in this concentration regime, which is consistent with the original model from the small disk study. Interaction between the two additives was evaluated by calculating synergy scores, using the Bliss model to calculate predicted responses. The contour plot of these scores is shown in Figure 5B. Although the combination of 0.5% (w/w) AMP7 with 2.17% (w/w) chitosan-PVA coating exhibited improved antimicrobial activity compared with the 0.5% AMP7 or 2.17% chitosan PVA coatings alone, the percent growth inhibition was less than predicted for an additive combination response, resulting in a negative synergy score (Figure 5B). Representative plates for this combination, as well as for the single additives and control, are shown in Figure 6.

Commercial Packaging Study at Elevated Concentrations and Reduced Coating Thickness

The vacuum-sealed patty studies had good agreement with the results seen in the disk assay, which confirmed that the disk assay is a good method for screening the effectiveness of these bio-based additives alone and in combination. To complete our study with this combination of materials, we wanted to overcome the antagonistic effects of these two components by increasing the final concentration in the PVA coating to 16% (w/w) chitosan and 1.5% (w/w) AMP7, and evaluating if cost-effective materials could be made by reducing the overall coating thickness on the test films. Because the bioactive coatings used in these studies are soluble, dosage of the active ingredients can be varied both by their concentrations in the coating mixture and by the amount of the coating added to the films (higher volumes applied to the films resulted in thicker coatings and a higher dose of actives). This test was conducted using disks of commercial packaging products. Disks of both types of commercial packaging were tested with either a 0.2-mil thick coating or a 0.6-mil thick coating (compared to the 1-mil thick coatings used in the earlier studies). As seen in Figure 7, it was confirmed that the bio-based additives could be used with commercial packaging materials to get efficient kill of E. coli contamination by controlling coating thickness to achieve enough of the chitosan and AMP7 to overcome their antagonistic effects (see Table 2).

Conclusions

Individually, and in combination, AMP7 and chitosan in a simple PVA coating demonstrated effective antimicrobial activity in reducing bacterial growth in a food simulant contacting the food packaging coating. It was determined that combinations of AMP7 and chitosan had an antagonistic interaction, rather than additive or synergistic activity. This was not unexpected, as these additives, though biochemically dissimilar with one being a polysaccharide and the other an amino acid oligomer, act upon the same cellular target (the external cell membrane of bacteria and other microorganisms). When combined, they may compete for physical interaction with the cellular membrane to disrupt the membrane and produce a biocidal effect; so it is possible that one interfered with the other’s effectiveness. The antagonistic effects were shown to be overcome at high concentrations of both additives, and they are both still good candidates for the development of antimicrobial food packaging systems because they have demonstrated antimicrobial activity and are known to possess low toxicity; for example, chitosan has previously been used in antimicrobial edible coatings^33,34 and the antimicrobial peptide used has previously been demonstrated to exhibit no discernable toxicity in rodent oral administration evaluations.³⁵

The analytical methods used here offer a powerful tool for screening potential bio-based actives for additive, antagonistic, or synergistic activity. Other bio-based antimicrobials can be selected to act on different cellular targets, and combinations that target different cellular components would likely produce additive or synergistic antimicrobial effects. Selection of antimicrobials that interact synergistically in combination is ideal, because this increases the antimicrobial activity of both additives while decreasing the concentration needed, thereby reducing overall production costs in a commercial application. Enzymatic additives may be selected to catalyze destructive reactions on lipids, proteins, sugars, and cellular wall components that sustain microbial life. Enzymes can be selected to be specific to the biochemistry of target microorganisms, such as selecting an enzyme that preferentially degrades bacterial cell walls vs the cell walls of fungi. Alternatively, some enzymatic additives could be selected to exert nonspecific antimicrobial effects, such as certain oxidases that produce reactive oxygen species that attack most microbial biomolecules, including DNA. Other non-enzymatic peptide bio-additives, such as nisin and AMP7, have varying modes of action, typically through disrupting microbial membranes and cell walls, but due to their small molecular sizes, may be more suitable for applications where diffusion from a food preservative coating may aid in getting better protective coverage of the food item during storage. Future studies using different combinations of these types of bio-additives may produce coatings to safely enhance the shelf-life of food, and in some cases, be tailored to protection of specific food items from microbes, particularly pathogens, that preferentially contaminate those products.

References

1. Carney, E., O’Brien, S.B., Sheridan, J.J., McDowell, D.A., Blair, I.S., and Duffy, G., “Prevalence and Level of Escherichia coli O157 on Beef Trimmings, Carcasses and Boned Head Meat at a Beef Slaughter Plant,” Food Microbiol., 23 (1), 52-59 (2006).
2. Møretrø, T. and Langsrud, S., “Residential Bacteria on Surfaces in the Food Industry and Their Implications for Food Safety and Quality,” Comprehensive Reviews in Food Science and Food Safety, 16 (5), 1022-1041 (2017).
3. Jay, M.T., Cooley, M., Carychao, D., Wiscomb, G.W., Sweitzer, R.A., Crawford-Miksza, L., Farrar, J.A., Lau, D.K., O’Connell, J., Millington, A., and Asmundson, R.V., “Escherichia coli O157: H7 in Feral Swine Near Spinach Fields and Cattle, Central California Coast,” Emerging Infectious Diseases, 13 (12), 1908 (2007).
4. Pennington, H., Escherichia coli O157. The Lancet, 376 (9750), 1428-1435 (2010).
5. Frank, C., Werber, D., Cramer, J.P., Askar, M., Faber, M., an der Heiden, M., Bernard, H., Fruth, A., Prager, R., Spode, A., and Wadl, M., “Epidemic Profile of Shiga-toxin–producing Escherichia coli O104: H4 Outbreak in Germany,” New Engl. J. Med., 365 (19), 1771-1780 (2011).
6. Listeria monocytogenes: Update on Foodborne Outbreak. .
7. Honish, L., Punja, N., Nunn, S., Nelson, D., Hislop, N., Gosselin, G., Stashko, N., and Dittrich, D., “Escherichia coli O157: H7 Infections Associated with Contaminated Pork Products—Alberta, Canada, July–October 2014,” Canada Communicable Disease Report= Releve Des Maladies Transmissibles Au Canada, 43 (1), 21-24 (2017).
8. World Health Organization. Listeriosis–South Africa
9. Centers for Disease Control and Prevention. List of Selected Multistate Foodborne Outbreak Investigations. .
10. Del Monte Fresh Produce N.A., Inc. Voluntarily Recalls Limited Quantity of Vegetable Trays in a Multistate Outbreak of Cyclospora Illnesses in Select Retailers in Illinois, Indiana, Iowa, Michigan, Minnesota,
    and Wisconsin, Because of Possible Health Risk. https://www.fda.gov/Safety/Recalls/ucm610992.htm.
11. Bennett, S.D., Sodha, S.V., Ayers, T.L., Lynch, M.F., Gould, L.H. and Tauxe, R.V., “Produce-associated Foodborne Disease Outbreaks, USA, 1998–2013,” Epidemiol. Infect., 1-10 (2018).
12. Littlefield, R.S., “Jack in the Box: Lessons Learned by Accepting Responsibility,” Lessons Learned About Protecting America’s Food Supply, 35 (2005).
13. Swanger, N. and Rutherford, D.G., “Foodborne Illness: The Risk Environment for Chain Restaurants in the United States,” Int. J. Hosp. Manage., 23 (1), 71-85 (2004).
14. Malhotra, B., Keshwani, A. and Kharkwal, H., “Antimicrobial Food Packaging: Potential and Pitfalls,” Front. Microbiol., 6, 611 (2015).
15. Vanderroost, M., Ragaert, P., Devlieghere, F. and De Meulenaer, B., “Intelligent Food Packaging: The Next Generation,” Trends in Food Sci. & Technol., 39 (1), 47-62 (2014).
16. Embuscado, M. and Huber, K.C. (Eds.), Edible Films and Coatings for Food Applications. New York: Springer, 2009.
17. Sapper, M. and Chiralt, A., “Starch-Based Coatings for Preservation of Fruits and Vegetables,” Coatings, 8 (152); DOI:10.3390/coatings8050152 (2018).
18. Yu, D., Regenstein, J.M., and Xia, W., “Bio-based Edible Coatings for the Preservation of Fishery Products: A Review,” Crit. Rev. Food Sci. Nutrition, DOI: 10.1080/10408398.2018.1457623 (2018).
19. Zimoch-Korzycka, A. and Andrzej Jarmoluk, A., “Polysaccharide-Based Edible Coatings Containing Cellulase for Improved Preservation of Meat Quality during Storage,” Molecules, 22 (390); DOI:10.3390/
    molecules22030390 (2017).
20. Appendinia, P. and Hotchkiss, J.H., “Review of Antimicrobial Food Packaging,” Innov. Food Sci. Emerg. Technol., 3 (2), 113-126 (2002).
21. Ahmed, I., Lin, H., Zou, L., Brody, A.L., Li, Z., Qazi, I.M., Pavase, T.R., and Liangtao Lv, L., “A Comprehensive Review on the Application of Active Packaging Technologies to Muscle Foods,” Food Control, 82, 163-178 (2017).
22. Wang, H., Wei, D., Ziaee, Z., Xiao, H., Zheng, A., and Zhao, Y. “Preparation and Properties of Nonleaching Antimicrobial Linear Low-Density Polyethylene Films,” Ind. & Eng. Chem. Res., 54 (6), 1824-1831; DOI: 10.1021/ie504393t (2015).
23. Agency Response Letter GRAS Notice No. GRN 000141 from George H. Pauli, Acting Director, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, FDA, to David R. Schoneker (on behalf of Colorcon) (April 28, 2004),
24. GRAS Notice No. GRN 000397 from Dennis M. Keefe, Director, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, FDA, to Véronique Maquet (on behalf of KitoZyme S.A.) (Dec. 19, 2011)
25. GRAS Notice No. GRN 000170 from Lee B. Dexter, Technical Consultant (on behalf of Primex ehf.), to Robert Martin, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, FDA (April 25, 2005)
26. Goy, R.C., Britto, D.D. and Assis, O.B., “A Review of the Antimicrobial Activity of Chitosan,” �ʴDZ�í������Dz�, 19 (3), 241-247 (2009).
27. Wales, M.E., McDaniel, C.S., Everett, A.L., Rawlins, J.W., Blanton, M.D., Busquets, A., Wild, J.R., and Gonzolez, C.F., “Next Generation Antimicrobial Additives for Reactive Surface Coatings,” Paint & Coat. Ind., 22 (7) 62-64, 66, 68-70 (2006).
28. European Patent no. EP1644452B.
29. United States Patent Application no. 10/884,355.
30. Ritz, C., Baty, F., Streibig, J.C., Gerhard, D., “Dose-Response Analysis Using R,” PLoS One, 10 (12), e0146021 (2015).
31. He, L., Kulesskiy, E., Saarela, J., Turunen, L., Wennerberg, K., Aittokallio T., et al., “Methods for High-throughput Drug Combination Screening and Synergy Scoring,” In: Cancer Systems Biology: Methods and Protocols. von Stechow L., (Ed.), New York, NY: Springer New York, 351-98, 2018.
32. Yadav, B., Wennerberg K., Aittokallio T., and Tang, J., “Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model,” Computational and Structural Biotechnology J., 13: 504-513 (2015).
33. Elsabee, M.Z., Abdou, E.S, “Chitosan Based Edible Films and Coatings: A Review,” Materials Sci. and Eng.: C., 33 (4): 1819–41 (2013).
34. Cagri, A., Ustunol, Z., and Ryser, E.T., “Antimicrobial Edible Films and Coatings,” J. Food Protect., 67 (4) 833–848 (2004).
35. Kuhn, J.O., ProteCoat Final Report Acute Oral Toxicity Study (UDP) in Rats OPPTS No. 870.1100 StillMeadow, Inc., Sept. 1, 2010 (Unpublished Study).

*Tyler W. Hodges is also affiliated with William Carey University, Hattiesburg, MS.
**Aayushma Kunwar is also affiliated with Reactive Surfaces.

CoatingsTech | Vol. 15, No. 9 | September 2018

“The benefits of microbial control technologies cannot be overstated. For instance, these technologies are needed to prevent paints from breaking down during storage and losing all viscosity, texture, and adhesion ability. Despite this critical role, people know very little about the benefits these technologies bring. That is why MCEC was founded. It is dedicated to educating different stakeholders about the benefits and safe use of microbial control technologies to ensure their continued availability,” says Michael Schäfer, chair of the MCEC.

The management of the MCEC works on a rolling basis, with LANXESS’s Michael Schäfer currently installed as chair of the organization, and Troy’s W. Brian Smith as vice-chair. The members frequently liaise and hold meetings in Brussels (and elsewhere) to discuss recent developments, future activities, and upcoming opportunities.

The MCEC’s primary concern is communicating with its target audience of industry leaders, academics, and downstream users. “Our goal is to highlight the broad and varied impact microbial control technologies have on our lives and how, unfortunately, they often go unnoticed. We focus largely on our commitment to the betterment of public health through the advancement of sustainable technologies in a variety of applications. It is the aim of MCEC members to inform key stakeholders about the sustainable use of these technologies and the critical role they play in our society,” Schäfer adds.

To communicate this message effectively, MCEC has been active in engaging with trade media, attending events and speaking opportunities, and promoting anti-microbial technologies via its online platforms, according to Schäfer. The primary tool for communicating with its target audience is through online engagement and attending relevant industry events. Following its formation, the organization created an online information center (https://www.microbial-control.com/) that gives visitors access to up-to-date and relevant information on how biocides help contribute to a healthier and safer environment. The information center provides an overview on how anti-microbial technology improves the world through a wide range of applications such as paints and coatings, marine shipping, wastewater management, and oil and gas recovery. Additionally, the MCEC has an active LinkedIn page where its activities are shared.

Recently, the MCEC has engaged with industry and downstream users by attending large industry events such as the Conference on Biocides in Kassel, Germany, where the group presented on the benefits of microbial control technologies in the construction sector. At these events, MCEC literature is distributed to raise awareness around the importance of anti-microbial technologies and the various solutions offered by biocides. The MCEC also distributes a bi-annual newsletter to subscribers with brief updates on recent activities and future actions.

With respect to paints and coatings, MCEC members have attended both the 2017 European Coatings Show in Nuremburg, Germany and the 2018 American Coatings Show in Indianapolis, Indiana. Its members, including Schäfer, were present to discuss the organization’s activities and plans for the coming year with visitors and industry representatives. “MCEC’s activities at these events are centered on promoting the benefits of microbial control technologies and the sustainable and safe use of biocides in the context of a variety of different applications, coatings included,” observes Schäfer.

For the remainder of 2018 and 2019, MCEC members will be attending a number of events and speaking opportunities to update industry stakeholders on the MCEC’s goals and core messages, according to the group’s chair. Members will be present at the European Coatings Show in Nuremburg from March 19–21, 2019. “The MCEC will use this as an opportunity to highlight the benefits of microbial control technologies in the context of coatings and promote the importance of biocides in that particular application, such as lengthening product shelf life and protecting coatings from microbial growth on the dry film,” he explains. “Members will also use the European Coatings Show 2019 as a platform to inform the group’s target audience that without microbial control products, the performance of products would broadly decline. For example, water-based paints would very quickly deteriorate in the can without the use of biocides,” Schäfer adds.

Looking to the future, Schäfer emphasized that the MCEC aims to promote the safe and sustainable use of biocides in a whole host of applications and focus on market needs and trends, as well as how innovation has contributed to the improvement of the safety and sustainability of biocides.

The utility of a high-throughput, spectrophotometric assay in screening over 23 enzyme and peptide-based additives against bacterial coating spoilage agents was evaluated. Candidate additives were then evaluated using ASTM D2574 for in-can coating spoilage challenges, and additives that may be used to substitute for traditional biocides were identified that eliminated recoverable bacterial growth.��

Introduction

Traditional biocides approved for in-can and in-film preservation, as well as antifouling activity, (e.g., alkylating agents and crosslinkers) can have direct impact on humans, while others such as the isothiazolinones may cause sensitization following continued exposure.^1-3 Such health risks and potential environmental impacts have prompted many countries to restrict the levels of use and/or require special labeling of various chemical preservatives.^1-5 Combined with the trend towards the use of lower levels of volatile organic compounds (VOCs) that have preservative qualities, there is a rapidly increasing demand for development of novel preservatives that are highly effective, economically reasonable, and present minimal regulatory hurdles.^6-8

Enzymes, peptides, and natural product small molecules have the potential to overcome some of the challenges associated with traditional biocides, and have been the subject of increasing interest in food and materials preservation.^9-18 An extensive technology portfolio involving antimicrobial peptides (AMPs) and enzymes in coatings and materials, chiefly those exhibiting in-film efficacy, has been developed.^12-17 AMPs may be able to provide a novel approach to in-can coating preservation based on principles used by organisms to protect themselves against microbial invasion. Various bio-based molecules that target different components of the microbial cell, including enzymes that degrade the cell wall (lysozyme), glycocalyx or biofilm (alginate lyase), or that generate reactive oxygen (glucose oxidase) can then be used in combination with AMPs to disrupt the cellular function synergistically. Recently, Reactive Surfaces was asked to use its bio-based biocides to completely replace traditional in-can preservatives in a well-known commercial internal architectural coating, the proprietary study, which is extended and discussed here.

The time and crude nature of techniques that have been used historically can make developing and screening of novel agents for coating preservation difficult and not data-intensive enough to detect useful trends in biocidal effects. One goal of the current study is to use modern microbiological screening methods for selection of candidates. We chose a rapid cell viability assay that quantitatively measures the metabolic ability of living cells to reduce the tetrazolium dye XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) to a colored product that is spectrophotometrically measured at 492 nm.¹⁹ This technique allows screening of candidate biocides in-can and in-film using very large arrays (of concentrations, combinations, microbial strains, coatings formulations, etc.). This preliminary screening allows quick down selection of biomolecules that inhibit metabolic activity for subsequent correlation with the traditional ASTM D2574 “Standard Test Method for Resistance of Emulsion Coatings in the Container to Attack by Microorganisms” microbiological assay when tested alone or combined with traditional chemical biocides. Such a “bookend” approach can then be used to evaluate the ultimate efficacy of antimicrobial formulations, providing the formulation chemist and microbiologist with the largest and most complete database from which to control microbes from raw materials introduction, through production, into the marketed container, and ultimately into dry films.

Materials and Methods

Reagents and Bacterial Strains

Glucose oxidase, alginate lyase, nisin (2.5%), α-amylase, β-glucosidase, β-mannosidase, β-glucanase, amyloglucosidase, cellulose, trypsin, pectinase, cinnamaldehyde, citral, and protease were obtained from Sigma-Aldrich (St. Louis, MO). Lysozyme was obtained from Bio-Cat (Troy, VA). Peroxidase was obtained from TCI America (Portland, OR). Chymotrypsin was obtained from MP Biomedicals (Santa Anna, CA). OPDtox™ and the peptides AMP-6, AMP-7, and AMP-LKLK were obtained from Reactive Surfaces, Ltd. (Austin, TX). Monolaurin was obtained by grinding Lauricidin^® pellets from Med-Chem Labs, Inc. (Goodyear, AZ) into a fine powder. XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)) was obtained from Biotium (Fremont, CA). Bacterial broth cultures used either SelenoMet^TM (SM) minimal media from Molecular Dimensions (Altamonte Springs, FL) or Bacto Tryptic Soy Broth (TSB) from Becton, Dickinson, and Co. (Sparks, MD). All growth media used Difco Tryptic Soy Agar (TSA) from Becton, Dickinson, and Co. (Sparks, MD). Pseudomonas aeruginosa (#155250A), Pseudomonas putida (#155265), Pseudomonas fluorescens (#155255), Alcaligenes faecalis (#154835A), Bacillus cereus (#154870) and Enterobacter aerogenes (#155030) cultures were obtained from Carolina Biological Supply (Burlington, NC).

Measuring Cell Viability Using XTT

Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Alcaligenes faecalis, Bacillus cereus, and Enterobacter aerogenes cultures were grown in 5 mL SM or TSB broth overnight at 30°C with agitation, then diluted 1:10 in SM or TSB for assays measuring the effect of single additives of the growth of individual strains. In experiments looking at the effects of additives on mixed cultures, 1 mL from each overnight culture was first combined in a sterile tube, and the microbial mixture was diluted 1:10 with SM media. A stock of menadione was prepared at a concentration of 1.7 mg/mL in acetone, and was diluted 1:120 into a solution of 1 mg/mL XTT in PBS (filter-sterilized with 0.45 mm nylon filter (Fisher Scientific)) immediately before setting up the assay. Stock solutions were prepared in dimethyl sulfoxide (DMSO) for monolaurin, cinnamaldehyde, and citral, or sterile water for all other additives so that the stock concentration was 20X the final test concentration. Each well in a 96-well microplate, in triplicate, received 10 mL of the additive to be tested, 20 mL of the XTT/menadione solution, and 100 mL of diluted cells, with the remainder consisting of growth media so that the final volume for each well was 200 mL. Wells containing diluted cells without additives (or with 10 mL DMSO for additives requiring DMSO for solubility) were included as negative controls. The absorbance at 492 nm was measured before and after incubation at 30°C for 20 h. The percent increase in absorbance and percent reduction in metabolism was calculated for each treatment as described.²³ Testing was performed in triplicate using three different concentrations of the bio-based additives.

Coating Challenges

Coating challenges were conducted as described in the ASTM International Standard procedure D2574-16.²⁴ Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Alcaligenes faecalis, Bacillus cereus, and Enterobacter aerogenes cultures were grown in 5 mL TSB broth for 24 h at 30°C with agitation. Sterile inoculation loops were used to pass a loop-full of each culture into new 5 mL TSB broths, which were incubated for 24 h at 30°C with agitation. The cultures were passed and incubated again for 24 h. Coating samples were prepared by hand mixing additives into 25 mL of acrylic latex coating using sterile glass rods. Sterile swabs were used to sample each coating and streak TSA plates to ensure that the coatings were sterile prior to inoculation. One mL from each broth culture was combined into a sterile tube and mixed well immediately prior to inoculation of the coating. The coating samples were inoculated with either 25 mL or 250 mL of the microbial mixture, and the coatings were incubated at 30°C for the duration of the test. Sterile swabs were used to sample each coating and streak duplicate TSA plates on days 1, 3, 5, and 7 following inoculation. The TSA plates were incubated at 30°C for one week, after which the amount of bacterial recovery from the coating was scored as described in the ASTM standard procedure:

0—no bacterial recovery

1—trace contamination (1 to 9 colonies)

2—light contamination (10 to 99 colonies)

3—moderate contamination (> 100 distinct colonies)

4—heavy contamination (continuous smear of growth, colonies have grown together and are indistinguishable)

Results

From an initial panel of 30 enzymes, peptides, and small molecule natural products, 23 listed in Table 1 were selected for evaluation. These were screened against individual members of a microbial contamination panel listed in Table 2 using the XTT assay. Nine of the 23 bio-based additives were found to reduce cellular metabolism in the XTT assay by ≥ 50% (bold in Table 1).

Following the screening of individual strains, the XTT assay was used to evaluate effectiveness of individual additives or additive combinations against a mixed inoculum of all six test strains. This was similar to the inoculation procedure used in ASTM D2574 where all strains are grown separately and then inoculated as a mixture into the coating. Several complex mixtures, particularly with those bio-based additives that target different molecular components of the cell, were analyzed for additive or synergistic effects. Figure 1 shows the results of the mixed bacterial inoculum XTT testing.

FIGURE 1—Effects of various bio-based additives, alone and in combination, on the growth of a mixture in equal parts of A. faecalis, B. cereus, E. aerogenes, P. aeruginosa, P. fluorescens, and P. putida. The blue bars represent the highest concentration tested 0.5 mg/mL, the orange bars represent the middle concentration tested of 0.05 mg/mL, and the yellow bars represent the lowest concentration tested, 0.005 mg/mL, for each compound.

Several additives displayed similar trends in activity against the mixed inoculum as was seen with individual strains—with cinnamaldehyde, monolaurin, and AMP-7 showing the greatest reduction in cellular metabolism of 55%, 61%, and 54%, respectively. In addition, a combination containing AMP-7/glucose oxidase had a reduction in cellular metabolism of 49%. AMP-6, AMP-LKLK, and a complex combination of lysozyme/AMP-7/glucose oxidase/alginate lyase showed approximately 40% reduction in cellular metabolism. All other bio-based additives tested were below 25% reduction in metabolism.

Based on the XTT results, select bio-based additives and combinations were tested against the microbial test panel using direct coating challenges as described in ASTM D2574 followed by sampling for recoverable growth over seven days. A biocide-free acrylic latex negative control coating and biocide positive control acrylic latex coating containing Kathon™ LX 1.5% (final concentration 0.15 wt%) were prepared and used in this study (Table 3).

ASTM D2574 paint challenges were performed using increasing bio-based additive concentration levels from those showing greater than 50% reduction in metabolism in the XTT assay. The coating containing Kathon LX 1.5% showed efficient control of the mixed microbial test panel with a score of 0 on day 1. Cinnamaldehyde at 1 mg/mL showed gradual decline in recoverable growth, achieving a 0 rating by day 7. This rate of decline was accelerated in a combination containing 0.05 mg/mL glucose oxidase to a 0 rating by day 5. A more rapid rate of decline was also seen at higher test concentrations of cinnamaldehyde comparable to those resulting in over 80% reduction in metabolism in the mixed panel XTT assay (determined to be 89% at 5 mg/mL). In a separate experiment at the higher test concentrations, a combination containing cinnamaldehyde and glucose oxidase showed a slightly greater impact on day 1 with a 2.5 (one plate scoring a 2 and the other scoring a 3) on day 1 vs a 3 for the cinnamaldehyde coating alone, and both samples reached a 0 by day 3. At the tested concentrations, neither AMP-7, AMP-6, monolaurin, glucose oxidase alone or in combination with other bio-based additives targeting different molecular targets, decreased the score from 4 by day 7. However, in the analogous proprietary studies using different coatings formulations with or without traditional biocides and a different panel of microbial strains, these same bio-based additives demonstrated efficacy. These differences suggest to the authors that bio-based additives, much like traditional biocides, should be selected to protect a given type of coating formulation or category of raw material against the microbial challenges encountered in a particular application.

Following these encouraging results, the XTT assay was utilized to evaluate the potential for synergistic effects between bio-based additives and traditional biocides. MIT (2-methyl-4-isothiazolin-3-one) and DMDM (1,3-dihydroxymethyl-5,5-dimethylhydantoin) were tested as described above for the bio-based additives. The concentration of MIT and DMDM was kept constant at 15 ppm, either alone or in combinations with varying concentrations of bio-based additives (Figure 2).

FIGURE 2—Effects of various bio-based additives, alone and in combination with traditional chemical-based biocides, on the growth of a mixture in equal parts of A. faecalis, B. cereus, E. aerogenes, P. aeruginosa, P. fluorescens, and P. putida. The blue bars represent no chemical biocide present, the orange bars represent 15 ppm DMDM present, and the yellow bars represent 15 ppm MIT present, with each bio-based additive tested.

The most dramatic impact on activity of the traditional biocides was seen with glucose oxidase. DMDM went from approximately 15% reduction in metabolism at 15 ppm to nearly 70% reduction in metabolism when combined with the two highest test concentrations of glucose oxidase (which had under 30% reduction alone). MIT increased from less than 15% reduction in metabolism alone, to greater than 60% reduction in metabolism when combined with glucose oxidase at any of the test concentrations.

Discussion

The XTT assay of single bacterial challenge microbes successfully identified bio-based enzymes, peptides, and small-molecule natural product additives that could reduce cellular metabolism by 50%, with those having highest activity defined as reducing metabolism to over 80% being comparable to heat-killed experiments where no viable cells are detectable. For the single additives, high activity against all the individual strains correlated to high activity in the mixed inoculum test results. In some cases, all members of a mixed inoculum were eliminated similarly to a traditional biocide in the same coating. In other cases, all but a single up to only a few microbial contaminants were eliminated from a mixed inoculum by use of a bio-based biocidal formulation—which results were distinguished by a lower kill-rate exhibited by the metabolic assay, and a single species lawn or isolated colonies in the standard ASTM plating assay. Species-specific differences were detected in the current study, for instance, P. putida being susceptible to more classes and concentrations of bio-based additives tested as compared to P. aeruginosa. In certain instances (not reported here), in-film antimicrobial efficacy was observed against bacterial, mold, and algal challenges of fixed films of the in-can preserved coatings. These results indicate the potential to substitute bio-based biocides for traditional biocides.

Several in-can challenge methodologies routinely used by the industry can vary in their degree of specificity, and it can be difficult to draw conclusions between them.²⁵ For instance, a score of 2 or 3 in the ASTM could be a single colony type or multiple colony types, and does not discern if each strain is remaining viable in the coating. Even for a score of 4, visual differences in colony types present could be observed in some cases (Figure 3).

FIGURE 3—Comparison of colony types present on plates from paint samples that scored 4 on day 7. Comparison of day 7 plates from the control acrylic latex sample (A) and acrylic latex + 0.05 mg/mL glucose oxidase + 2 mg/mL dextrose (B) shows that the paint with bio-based additive, though scoring 4 according to the ASTM guidelines, had at least one fewer colony type compared to the control.

Having more data-intensive results is critical where the contamination, as it almost always is, is the result of a microbial community, not a single microbe. In a subsequent Part II, we will demonstrate and discuss this finding. In this study, comparing activities against single bacterial isolates to a mixed inoculum containing all six test bacterial strains can be used to quickly focus the preservative formulation to those contaminants that exhibit initial recalcitrance. For example, glucose oxidase and alginate lyase showed activity against one or more test strains alone, but had < 20% reduction in metabolism against the mixed inoculum. This indicates that less than complete kill was achieved for at least one of the mixed strains, allowing for continued survival and growth in the culture media. One rationale to overcome a resistant contaminant is to select disruptive properties impacting distinct cellular components to achieve synergistic effects, with a lysozyme, AMP-7, glucose oxidase, and alginate lyase combination as an example. Alginate lyase targets the extracellular polysaccharide layer, AMP-7 disrupts cellular membranes, lysozyme cleaves bacterial cell wall peptidoglycans, and glucose oxidase produces hydrogen peroxide that can induce cellular damage.²⁶

The current lead candidates based on the XTT and ASTM data include AMPs, glucose oxidase, and lysozyme. AMPs play an important role in host defenses against microorganisms, and have demonstrated antimicrobial activity in solution and in dry-film formulations against various microorganisms, including bacteria (including Pseudomonas), fungi, algae, and viruses.^12-17 Reactive Surfaces is currently undertaking EPA registration of AMP-7. Glucose oxidase and lysozyme are both listed as Generally Regarded as Safe for various intended uses and have been used in the food industry.^21-22 They are produced on commercial scale and available in bulk quantities. Lysozyme is also notable for being particularly effective against Gram-positive bacteria.¹³

These results indicate that an initial metabolic or other rapid-throughput assay can be used to predict likelihood of success of bio-based biocides as in-can preservatives. For example, such testing shown here resulted in an 80% reduction in metabolism using the mixed inoculum translated into a complete kill in the ASTM challenge method. In addition, molecular methods are being developed to conduct direct analysis of microbial community structure in coating samples, such as rapid identification of each microbial species using DNA amplification and sequencing techniques. This will allow for real-time monitoring of individual bacterial growth patterns in the coating sample and impacts on each strain of the consortium following bio-based additive treatment.

References

1.�� Chan, P.K., Baldwin, R.C., Parsons, R.D., Moss, J.N., Stiratelli, R., Smith, J.M., and Hayes, A.W., “Kathon Biocide: Manifestation of Delayed Contact Dermatitis in Guinea Pigs Is Dependent on the Concentration for Induction and Challenge,” J. Invest. Dermatol., 81, No. 5, pp. 409–411 (1983).

2. Schwensen, J.F., Menné, T., Andersen, K.E., Sommerlund, M., and Johansen, J.D.,���� “Occupations at Risk of Developing Contact Allergy to Isothiazolinones in Danish Contact Dermatitis Patients: Results from a Danish Multicentre Study,” (2009-2012), Contact Dermatitis, Nov. 771, No. 5, pp. 295-302 (2014).

3. Schwensen, J.F., Lundov, M.D., Bossi, R., Banerjee P., Giménez-Arnau, E.,�� Lepoittevin, J.P.,�� Lidén, C.,�� Uter, W.,�� Yazar, K.,�� White, I.R., and Johansen, J.D., “Methylisothiazolinone and Benzisothiazolinone Are Widely Used in Coating: A Multicentre Study of Coatings from Five European Countries,” Contact Dermatitis, 72, No. 3, pp. 127–138 (2015).

4. Bollmann, U.E., Fernández-Calviño, D., Brandt, K.K., Storgaard, M.S., Sanderson, H., Bester, K., “Biocide Runoff from Building Facades: Degradation Kinetics in Soil,” Environ. Sci. Technol., 51, No. 7, pp. 3694–3702 (2017), Apr. 4, 51 (7), pp. 3694–3702.

5. Lundov, M.D., Kolarik, B. Bossi, R., Gunnarsen, L., and Johansen, J.D., “Emission of Isothiazolinones from Water-based Coatings,” Environ. Sci. Technol., 48, No. 12, pp. 6989–6994 (2014).

6. Cresswell, T., Richards, J.P., Glegg, G.A., and Readman, J.W., “The Impact of Legislation on the Usage and Environmental Concentrations of Irgarol 1051 in UK Coastal Waters,” Mar. Pollut. Bull., 52, No. 10, pp. 1169–1175 (2006).

7.�� Brinch, A., Hansen, S.F., Hartmann, N.B., and Baun, A., “EU Regulation of Nanobiocides: Challenges in Implementing the Biocidal Product Regulation (BPR),” Nanomaterials (Basel), 6, No. 2, pp. E33 (2016).

8. “White Paper: Antimicrobial Building Products Should Be Avoided Whenever Possible,”

9. Bhatia, S. and Bharti, A., “Evaluating the Antimicrobial Activity of Nisin, Lysozyme and Ethylenediaminetetraacetate Incorporated in Starch-based Active Food Packaging Film,” J. Food Sci. Technol., 52, No. 6, pp. 3504–3512 (2015).

10. Yuan, S., Wan, D., Liang, B., Pehkonen, S.O., Ting, Y.P., Neoh, K.G., Kang, E.T., “Lysozyme-coupled Poly(poly(ethylene glycol) methacrylate) – Stainless Steel Hybrid and their Antifouling and Antibacterial Surfaces,” Langmuir, 27, No. 6, pp. 2761–2774, (2011).

11. Zodrow, K.R., Schiffman, J.D., and Elimelech, M., “Biodegradable Polymer (PLGA) Coatings Featuring Cinnamaldehyde and Carvacrol Mitigate Biofilm Formation,” Langmuir, 28, No. 39, pp. 13993–13999 (2012).

12. Williams, E., Carvajal, J.C., Schwausch, M., McDaniel, C.S., Wales, M., Rawlins, J., “Nature Versus Natural: Engineered Oligopeptides that Prevent Microbial Degradation of Coatings.” 37th International Waterborne, High-Solids and Powder Coatings Symp., New Orleans, LA, 2011.

13. McDaniel, C.S., Anti-fouling Coatings and Coatings, U.S. patent application 14/097,128, July 9, 2015.

14. McDaniel, C.S., Antifungal Coatings and Coatings, U.S. patent 8,497,248, July 30, 2013.

15. McDaniel, C.S., Coating Compositions having Peptidic Antimicrobial Additives and Additives of Other Configurations, U.S. patent 8,618,066, December 31, 2013.

16. McDaniel, C.S., Antifungal Coatings and Coatings, U.S. patent 7,932,230, April 26, 2011.

17. McDaniel, C.S., Anti-fouling Coatings and Coatings, U.S. patent 7,939,500, May 10, 2011.

18. Cloutier, M., Mantovani, D., and Rosei, F., “Antibacterial Coatings: Challenges, Perspectives, and Opportunities,” Trends in Biotechnology, 33, No. 11, pp. 637–652 (2015).

19. XTT Cell Proliferation Assay Kit Instruction Manual, American Type Culture Collection, Manassas, VA, 2011.

20. Tiina, M. and Sandholm, M., “Antibacterial Effect of the Glucose Oxidase-Glucose System on Food-Poisoning Organisms,” Internat. J. Food Microbiol., 8, No.2, 165–174 (1989).

21. Lysozyme Safety Toxicology and Safety Statement, https://www.fordras.com/lysozyme-safety/ (accessed December 2017).

22. Bankar, S.B., Bule, M.V., Singhal, R.S., and Ananthanarayan, L., “Glucose Oxidase—an Overview,” Biotechnology Advances, 27, No. 4, pp. 489–501 (2009).

23. Khurram, M., Lawton, L.A., Edwards, C., Iriti, M., Hameed, A., Khan, M.A., Khan, F.A., and Rahman, S.U., “Rapid Bioassay-Guided Isolation of Antibacterial Clerodane Type Diterpenoid from Dodonaea viscosa (L.) Jaeq.” Int J Mol Sci., 16, No. 9, pp. 20290-20307 (2015).

24. “ASTM Standard D2574-16, Standard Test Method for Resistance of Emulsion Coatings in the Container to Attack by Microorganisms,” ASTM International, West Conshohocken, PA, (2016), DOI: 10.1520/D2574-16.

25. Winkowski, K., “Efficacy of In Can Preservatives,” European Coatings Journal, 1-2, pp. 87-91 (2001).

26. Hall-Stoodley, L., Costerton, J.W., Stoodley, P., “Bacterial Biofilms: From the Natural Environment to Infectious Diseases,” Nat. Rev. Microbiol., 2, No. 2, pp. 95-108 (2004).

This article was presented at the Waterborne Symposium, February 4–9, 2018, in New Orleans, LA.

*Certain of the coauthors from Reactive Surfaces and the University of Southern Mississippi received the inaugural American Coatings Award in 2008 for work leading to the research reported here. An expansion of this study will be reported in Part II discussing the in-film efficacy of proteins and peptides in packaging for enhanced food preservation.

^†Tyler W. Hodges and Kyle L. Wilhelm are also affiliated with William Carey University, Hattiesburg, MS.