Progress 09/01/23 to 08/31/24
Outputs
Target Audience:We presented this project to the researchers working in the coating, polymer, antimicrobial areas to bring awareness to the non-biodegradable plastics that are harmful to the environment. Specially, we presented at these meetings: Agricultural Division ofthe American ChemicalSociety at New Orleans,American Oil Chemists Society ChineseDivisionatChina, and Research Symposium in the College of Agriculture and Nature Resources at University of Delaware. The meetings were informative and productive. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This past year at the ERRC facility, the postdoctoral researcher was trained to make antimicrobial bio-polymers andto characterize the materials using various instruments. He participated in meetings with stakeholders to promote the research project. Healso presented the research at the American Chemical Society meeting at New Orleans. One PhD student at the University of Delaware was trained to conduct toxicity and biodegradability studies for the curing agents and bio-polymers. She gained valuable knowledge and analytical skills in these technical areas and presented the project at the Research Symposium in the College of Agriculture and Nature Resources at the University of Delaware. How have the results been disseminated to communities of interest?This project was disseminated to a group of high school students who visited the Eastern Regional Research Center on Career Day and to the civilians and farmers at the annual Pennsylvania Farm Show. What do you plan to do during the next reporting period to accomplish the goals?We will evaluate the bio-polymers as coatings on fabrics, papers, and stainless materials. We will seek collaborators to build a life cycle assessment model. We will continue evaluating the biodegradability of the products. We will conduct more toxicity studies for the new bio-based products generated in the project.
Impacts
What was accomplished under these goals?
Over the past year, the team has continued to make significant progress in developing antimicrobial bio-polymers. These bio-polymers are water-resistant, reusable, and have strong adhesive properties. Under these Objectives, five different antimicrobial bio-polymers were prepared with a commercial resin (bisphenol A diglycidyl ether) and five different ratios of curing agents. The curing agent ratios (ranging from 0 to 100%) were made up of arylated (phenolic) fatty amine and non-arylated (non-phenolic) fatty amine. To synthesize the bio-polymers, the curing agent/resin was solution cast in a vial and a dogbone-shaped container at room temperature, followed by thermally cured at 120oC for 2 hours. The bio-polymers prepared in the dogbone mold were manually removed to be further analyzed by thermogravimetric analysis for thermal stability, differential scanning calorimetry for glass transition temperature, and near-infrared for total hydroxyl in the phenolic group. Results showed that the bio-polymers' mechanical and antimicrobial properties strongly depend on the ratio of the curing agent mixture. Thermogravimetric analysis showed that each of the bio-polymers was thermally stable up to 350°C. Glass transition temperatures or the temperatures at which the bio-polymers move from a glassy (rigid) state to a more pliable state ranged from 21.2°C (100% non-arylated bio-polymer) to 54.6°C (100% arylated bio-polymer). This indicates that at room temperature, by increasing the amount of arylated curing agent, a more rigid bio-polymer is produced. Near-infrared spectroscopic analysis of these bio-polymers revealed a growing activity centered around 6923cm-1, mainly displaying the phenolic compounds' hydroxyl groups, increasing with the ratio of arylated curing agent. Mechanical property analysis demonstrated tensile strengths ranging from 10-20MPa, elongation at break values from 2-18%, and Young's Moduli from 465-800MPa, again depending on the arylated fatty amine/non-arylated fatty amine ratio of bio-polymers. Interestingly, the bio-polymer with a ratio of 50:50 arylated fatty amine/non-arylated fatty amine showed the highest tensile strength and elongation at break, demonstrating a desirable combination of stiffness and stress tolerance has been achieved. This ratio of bio-polymer also displayed the highest antimicrobial activity against both Listeria innocua Seelinger and Escherichia coli, two common representatives of Gram-positive and -negative foodborne pathogens, respectively. To test the products' application, we coated these bio-polymers onto cotton fabrics using various treatment processes with the aim of transforming the fabrics into antimicrobial, water-repellent, and reusable materials. Preliminary results showed that the fabrics could be coated with up to 50% of bio-polymers without affecting the fabrics' quality and at the same time enhanced the fabrics' hydrophobicity. This work is ongoing. To further improve the mechanical properties of the bio-polymers, another set of bio-polymers was prepared without the use of the commercial resin. This set of bio-polymers has superior antimicrobial activities against E. coli and is reusable. Thermogravimetric analysis results showed that the products were thermally stable to temperatures of about 300°C. That is, they did not begin to thermally degrade as measured by mass reductions until temperatures approached 300°C. This is about 50 oC lower than the bio-polymers prepared with bisphenol A diglycidyl ether. In addition, the glass transition temperatures for these samples ranged from -3°C to 19°C, under room temperature, which demonstrated the flexible material properties of each of the synthesized bio-polymers at room temperature. This work is also ongoing. The team has also advanced this development by incorporating refined phenolic fractions from the pyrolysis process into the fatty acid. Pyrolysis-derived phenols are incorporated in the synthesis, thus making the phenolic fatty acid product even more biorenewable and economical. Catalytic fast pyrolysis of biomass produced bio-oils with sufficiently high concentrations of phenol and cresols (biophenolics). These one-ring biophenolics were extracted and redistilled, yielding a fraction of greater than 75 percent phenolics. Antimicrobial tests showed activities against strains of Listeria bacteria, thus expanding the applicability of fast pyrolysis oil products and lipids. Future research will be copolymerizing these biophenolic fatty acids to bio-polymers. In another research direction, the team has explored these arylated fatty acid materials on food applications. In recent years, foodborne disease outbreaks and recalls have been linked with fresh apples due to Listeria monocytogenes contamination occurring during postharvest handling. To reduce the risk of Listeria contamination on apples. We formulated an anti-listeria coating composition made from our arylated fatty acid as a bio-based antimicrobial. When applied to apple fruit, the arylated fatty acid coating reduces the population of Listeria innocua, a surrogate of Listeria monocytogenes, by more than 99%. In addition, the arylated fatty acid coating preserves the freshness of apples during a simulated shelf-life study by reducing 36% of moisture loss. Therefore, the novel coating formulation provides the apple industry with a simple tool to minimize the risk of Listeria contamination while maintaining fruit quality.These materials are buried in closed vessels that contain prepared soil under certain conditions with pH 6-8 and moisture holding capacity 80-100%. A potassium hydroxide base solution and distilled water are placed inside the vessel. The amount of carbon dioxide produced is determined by titrating the remaining base solution with hydrochloric acid solution every week for 8 weeks.Preliminary results showed that there was a slight biodegradation with the arylated bio-polymer (6.21±1.70%).A longer biodegradation study (up to 16 weeks) is ongoing for a more comprehensive understanding of the potential biodegradation of the bio-polymer materials. Finally, the team conducted tests on the safety of these materials. In silico simulation of toxicity showed that the arylated fatty acid, its ester and amine derivatives demonstrated developmental toxicity while having minimal mutagenicity. Therefore, further experiments were conducted to verify the developmental toxicity and dose response using a chicken embryonic assay. These products showed varying toxicity at different dosages, which was due to their distinct chemical mixtures and structures. Arylated fatty acid ester exhibited the highest mortality rates among test dosages (33-1333 µg/kg egg), while the highest malformation rates were identified in both arylated fatty acid and its amine derivative treatments. Arylated fatty acid showed the highest oxidative stress with the lowest liver weight at the dosage of 300 µg/kg. In the Ames test, the mutagenic index was below 2, suggesting that all test products are non-mutagenic, which aligns with the simulation results.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Ryu, V., Uknalis, J., Lew, Ngo, Jin, J., Fan, X. (2024) Coating with Phenolic Branched-Chain Fatty Acid Reduces Listeria Innocua Populations on Apple Fruit. International Journal of Food Microbiology, 419, 110748.
- Type:
Peer Reviewed Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Helen Ngo, Karen Wagner, Steven Cermak, Xuetong Fan, Masoud Kazem-Rostami, Majher I. Sarker, Victor Ryu, Yaseen Elkasabi. Branched Chain Fatty Acids Based on Biomass Fast Pyrolysis Phenolics. European Journal of Lipid Science Technology.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Xinwen Zhang, Helen Ngo, Karen Wagner, Xuetong Fan, Changqing Wu. (2024) Developmental Toxicity and Estrogenic Activity of Antimicrobial Phenolic-Branched Fatty Acids using In Silico Simulations and In Vivo and In Vitro Bioassay. Frontiers in Toxicology.
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:We presentedthis project to the researchers working in the coating, polymer, antimicrobial areas to bring awareness to the non-biodegradable plastics that are harmful to the environment. Specially, we presented at these meetings: Biopolymer Division at the American Oil Chemists Society, Sustainable Polymer Workshop, and International Association for Food Protection. The meetingswere informative and productive, and we received many requests to collaborate. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This past year at the ERRC facility, we hiredapostdoctoral researcher to synthesize the curing agents from waste trap grease and convertthem to reusable antimicrobial bio-polymers with capability to repeatedly kill bacteria. We also hired a summer undergraduate intern to assist the project. Both employees hadgained valuable knowledge in the bio-polymer area. They also gained experience in analytical and co-polymer synthetic skills. The postdoctoral researcher presented the project at the American Oil Chemists Society meeting in the bio-based polymer division, while the intern presented a poster at her University's undergraduate program. Additionally, two graduate students at the University of Delaware were trained to conduct toxicity and performrelease profile and biodegradability studies for the curing agents and bio-polymers. They gained valuable knowledge and analytical skills in these technical areas and presented the project at the International Association of Food Protection Annual Conference. How have the results been disseminated to communities of interest? This project was disseminated to a group of high school students who visited the Eastern Regional Research Center on Career Dayand to the civilians and farmers at the annual Pennsylvania Farm Show. What do you plan to do during the next reporting period to accomplish the goals?We will continue to optimize the co-polymer synthesis to enhance the mechanical properties of the bio-polymers. We will seek collaborators with facilities that can test them against mold, mildew, and fungi. Wewill evaluate the biodegradability of the antimicrobial bio-polymers before their commercial application. We will conduct the process cost and life cycle assessment to evaluate the circular economy of the bio-polymers.
Impacts
What was accomplished under these goals?
In this past year, major progress has been made for all four objectives. Objective 1, successful production of antibacterial bio-polymers from waste trap grease has been achieved through a four-step process. First, fatty acids from waste grease were obtained by distillation. Second, the distilled fatty acids were arylated (branched) with phenolics from beechwood, thymol or phenol through arylation reaction to give the phenolic branched chain fatty acid (PBC-FA) products. Third, PBC-FAs wereconverted into an amine containing PBC-FA products through amidation reaction to form the desired PBC-FA amine curing agents. All curing agents were characterized by various chromatography and spectroscopy methods. Results showed that the curing agents can be produced at high yields and high conversions. Finally, to prepare the antibacterial bio-polymers, a mixture of the curing agent and epoxy resin (bisphenol A diglycidyl ether) was solution casted in silicon molds, air dried, and thermally cured. The silicone molds used for this purpose were designed and built in-house because the molds made of flexible and non-stick materials are not commercially available. The casted bio-polymers were cooled to room temperature and then manually extracted from the molds. The initial examination of these bio-polymers revealed that they are hard but brittle, which is a common characteristic of epoxy bio-polymers. Based on the temperature study results, they have excellent thermostability (up to 260oC) and good glass transition temperature (up to 50oC). For comparison purposes, non-arylated curing agent (with no phenolic branches) bio-polymers were prepared. Results showed that these bio-polymers are softer than the arylated bio-polymers with phenolics. These interesting properties have led us to study experiments where two types of arylated and non-arylated curing agents are being combined in various ratios and co-polymerized. The resulting bio-polymers are being tested to determinewhich ratio of the curing agents offers the most desired set of properties.Theyare also being characterized with various means and methods to determine their thermal stability, glass transition temperature, composition, and total hydroxy value. This work is ongoing. In objective 2, bio-polymers coated onto the bottom of glass vials were tested for their antimicrobial activity in repeated exposures to bacteria cultures. One milliliterculture of non-pathogenicListeria (L.) innocuaandE. colibacteria was added into the vials that had been coated with the bio-polymers as described in objective 1. The vials containing the cultures were then incubated at the desired temperature and time. The culture suspensions in the vials were then plated onto tryptic soy agar plates which were incubated to determine if there was any bacterial survival after exposure to the bio-polymers. Afterward, the bacterial cultures were removed, and fresh batches of bacteria were added to the same vials. The above procedure was repeated until the bio-polymers lost their antimicrobial activity. Results showed that bio-polymers exhibited antimicrobial efficacy for 2 consecutive exposures againstL. innocuaand for 5 consecutive exposures againstE. coli, demonstrating that the bio-polymers can be repeatedly used to inactivate bacteria. To evaluate if the bio-polymers could be regenerated after loss of antimicrobial activity due to repeated uses, the bio-polymers after exposures to the bacteriawere washed with ethanol followed by decanting and heating to evaporate residual ethanol. The bio-polymers were then tested again for the antimicrobial activity. Results showed that the bio-polymers after washing with ethanol have regained their antimicrobial activity and capacity, indicating that they can be easily regenerated. To determine the times needed for the bio-polymers to inactivate the bacteria, the bacterial populations were enumerated periodically during incubationin the presence of the bio-polymers. Results showed that bacterial populations decrease over time, and it takes about 4 hours to completely inactivate theL. innocua and 1.5 hours to completely inactivate theE. colibacteria. In objective 3, tensile and mechanical properties of the synthesized arylated bio-polymers were measured according to the American Society for Testing and Materials D638, using Type V shaped specimens that were cast into silicone molds and allowed to cool. The edges of the specimens were ground down to remove the lip and make the samples flat. Specimens were tested with a defined distance between the two grips and a constant strain rate (crosshead speed). The mechanical properties including tensile strength, elongation at break, and Young's Modulus were obtained using an Insight 5 and Testworks-4 data acquisition software. Bio-polymers were tested in a conditioning room set at room temperature and 50% relative humidity. Results showed that thearylatedbio-polymers arestiff and rigid and donot exhibit the toughness expected for coating applications. However as described in objective 1, by varying the ratio of starting materials and the co-polymerization process, it is expected that antimicrobial bio-polymers with a range of tensile properties will be developed for multiple applications. In objective 4, to explore the potential release of curing agents from the bio-polymers, the bio-polymers were immersed in ultrapure water whose absorption spectra was recorded every day for 14 days with an ultraviolet-visible spectrometer. Results showed a minor but gradually growing peak at around wavelength of 270 nm during the test period. The liquid from this study was then concentrated about 15 times with freeze-drying technique and then analyzed by a liquid chromatography instrument. Despite using higher injection volumes, the results of this preliminary study indicated the concerning amounts are so negligible that fall near or below the detection limits of the instrument, which is promising. Additionally, developmental toxicity studyof thePBC-FA amine curing agents and PBC-FA was investigatedviaa chicken embryonic assay. All productswere tested at three different concentrations in the solvent based on the antimicrobial studiesused in objective 2. A vehicle control (VC) of the solvent in phosphate-buffed saline was included in the test. Results showed that PBC-FA amine and PBC-FA treatments have a mortality rate up to 25%. For PBC-FA amine treatments, the highest mortality rate of 38% was observed at lowest test concentration of 10 µg/mL. Deformed embryos (stunting) were found in three test groups: VC, PBC-FA at 10 µg/mL and 400 µg/mL. Thiobarbituric acid reactive substances (TBARS) level as oxidative biomarkers was assessed in chicken embryonic liver samples after treatments. Most treatment groups except for PBC-FA amine withconcentration of 90 µg/mLinduced slightly differentTBARS levels when compared to the VC TBARS value. The highest TBARS value was observed in PBC-FA amine at concentration of 90 µg/mL, without a significant difference versus the VC group (p> 0.05), indicating that the oxidative stress might not be the only mechanism to induce the toxic effects.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Title: Bio-based phenolic branched-chain fatty acid emulsion achieved similar reductions of Listeria innocua population on apple fruit as chlorinator water. Submitted to Food Control.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Title: Antibacterial Agents from Waste Grease: Arylation of Brown Grease Fatty Acids with Beechwood Creosote and Derivatization. Accepted in ACS Sustainable Chemistry and Engineering.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:The target audiences are people working in the antimicrobial bio-polymer synthesis and production. The project has provided experiential learning opportunities for two undergraduate students from Drexel University and one PhD graduate student from University of Delaware. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two undergraduate students from Drexel University were trained to use various analytical instruments (total acid titrator, gas-chromatography mass spectroscopy). A PhD graduate student from University of Delaware obtained more knowledge and skills to design experiments and determine toxicities for the new phenolic monomers. With assistance from the team, the graduate student attained greater proficiency in analysis of the results and preparation of manuscript which was submitted for publication. How have the results been disseminated to communities of interest?A co-PI on the team presented a paper entitled "Bio-based antimicrobials derived from fatty acids" at The Korean Society of Food Science and Nutrition International Symposium and Annual Meeting at South Korea on October 19-21, 2022. One research paper and one critical review article were submitted for publication. What do you plan to do during the next reporting period to accomplish the goals?At the PI's facility, we will work on improving the efficiency of the phenolic monomer production, preparing bio-polymers, and evaluating the antimicrobial activity, reusability, and biodegradability of the bio-polymers. At the co-PI's facility, more estrogenic activity evaluations of newly phenolic monomers, especially focusing on those who have both good performances in antimicrobial activity and low developmental toxicity in the chicken embryo model will be evaluated. Also, various toxicities for the newly synthesized bio-polymers at the PI's facility will be examined.
Impacts
What was accomplished under these goals?
Four specific objectives have been designed to create advanced antimicrobial bio-polymers with excellent stability, reusability, and bioactive properties from non-edible oils. Objective 1 focuses on creating antimicrobial thermosetting bio-polymers derived from non-edible oils. Non-edible oils such as waste fats from a wastewater treatment plant were selected as feedstocks to make the phenolic monomers for bio-polymer production. The selected wastewater plant operates a grease receiving process that aggregates grease from the regional waste management companies. This would ensure the consistency of the wastes used. In collaboration with Drexel University researchers, three waste fats (i.e., poultry, meatball, and brown grease fats) were investigated. Since these fats were heavily contaminated with many unwanted deposits, they must be purified before processing. Two purification methods (solvent/water extraction and distillation) were explored. The extraction method provided insights of the deposits but was not a good method for large scale because it generated a lot of waste solutions. In contrast, the distillation method was more efficient, did not generate any waste materials, and the purified fats were cleaner. The total acid number (measures carboxylic acid), iodine value (measures unsaturation), and gas chromatograph-mass spectrometry (provides fatcomposition profiles)results were comparable to the soybean feedstock originally used to make the phenolic monomers. This means that these non-edible waste fats are good alternative feedstocks to build upon the ARS patented arylation catalytic branching process to produce the phenolic monomers. In a high-pressure stainless reactor, the purified fats, phenolics (i.e.,phenol, thymol), and catalysts were mixed and heated to the desired temperatures and times, about 35% to 55% of the phenolic monomers were formed. These results, however, are lower than the anticipated (about 80%). One speculation is the structure of the fats has been altered from cis to trans conformation, where trans fats are less reactive than the cis. Additionally, these new phenolic monomers have different isomeric structural variation than the ones obtained from the soybean, which may lead to another type of bio-polymer with unique properties. Objective 2 focuses on evaluating the antimicrobial properties of the synthesized phenolic monomers and their bio-polymers. Since the antimicrobial phenolic monomers are key parent compounds to produce targeted antimicrobial bio-polymers, the phenolic monomers were tested for their antimicrobial activities using the microdilution method. Minimum inhibitory concentrations and minimum bactericidal concentrations were obtained against Gram-positive and Gram-negative bacteria. Results showed that their antimicrobial activities were influenced by the alkyl chain length, saturation, and conformation of the fats. This information will help to design effective thermosetting bio-polymers with strong bactericidal properties. Objective 3 focuses on the structure property relationship between the antimicrobial activity and performance of the bio-polymers. By increasing the types of bio-polymers derived through different chemical reactions (bio-epoxies and bio-polyurethanes) and the length and saturation states of the fat starting materials, we envision the development of antimicrobial bio-polymers with mechanical properties ranging from flexible, such as might be valuable in food wrapping/preservation, to rigid, which may be more amenable to coating applications. At this point of the project, limited results have been obtained for this objective because of the lack of developmental research due to a critical research vacancy. However, results of a 2021 study by our group showed the antimicrobial properties of bio-based epoxy polymers that were derived from phenolic monomers. In that study epoxy polymers were synthesized by reacting various phenolic monomers with polyamines and then reacting the resulting products with a commercial epoxy resin to produce the antimicrobial bio-polymers. Results of that study showed that the phenol and creosote-containing monomers that had been reacted with ethylenediamine were most active in antimicrobial applications. Unfortunately, because additional phenolic monomers have not been prepared yet, bio-polymer development has been delayed thus hindering any study of structure/function relationships. In objective 4, a study was initiated to enhance our insights into the potential safety of four phenolic monomers. An in silico simulation (i.e., Toxicity Estimation Software Tool, T.E.S.T) was conducted as the first step for toxicity prediction because of its cost-effectiveness and efficiency compared to experimental studies. The T.E.S.T simulation predicted that the phenolic monomers had much higher toxicities to aquatic organisms than free phenolics did. Interestingly, the opposite was predicted for rats, where toxicity of phenolic monomers was lower than that of free phenolics. All the tested phenolic monomeric compounds were classified as non-mutagenic developmental toxins. Acute and developmental toxicities increased slightly when the fats contained a double bond. However, the location of phenolic branch (structural isomers) had no effect upon in silico simulation results. Chicken embryonic assay was also used for evaluation of developmental toxicity, as this assay is a promising alternative to traditional in vivo models with increasing applications in pharmacological and toxicological studies. In contrast to rodent animals (e.g., mice and rats), chicken embryos are isolated biological systems of the early life stage of animals. Unlike rodents, chicken embryos are abundant, cost-effective, and easily manipulated. We investigated the developmental toxicity during chicken embryogenesis for four phenolic monomers (i.e., creosote, phenol, thymol, carvacrol). A non-monotonic dose response was observed in free creosote or creosote-containing monomer treatments by in vivo chicken embryonic assay for developmental toxicity. Creosote-containing monomer had much lower mortality rates than that of free creosote at the same dosages. Although creosote-containing monomer with the highest purity had the lowest mortality rate, phenol-containing monomer showed the highest death rate, malformation number, and altered developmental index. Comparable mortality rates were observed for thymol- and carvacrol-containing monomers, while carvacrol-containing monomer significantly increased oxidative stress levels (p< 0.05) from fetal liver samples based on thiobarbituric acid-reactive substance values. Furthermore, the potential estrogenic activity of phenol-containing monomer at two concentrations (0.1 to 10 μM) was assessed by an MCF-7 cell proliferation assay. The MCF-7 cell proliferation assay findings revealed that the phenol-containing monomer showed estrogenic activity while the free phenol had little estrogenic activity at 10 µM.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
The Korean Society of Food Science and Nutrition International Symposium and Annual Meeting, Jeju Island, South Korea
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Food and Chemical Toxicology
- Type:
Other
Status:
Submitted
Year Published:
2022
Citation:
Critical Reviews in Food Science and Nutrition
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