WO2025184738A1

WO2025184738A1 - Antimicrobial microgel and method of making the same

Info

Publication number: WO2025184738A1
Application number: PCT/CA2025/050305
Authority: WO
Inventors: Mina Mekhail; Francisco Rafael CASTIELLO FLORES
Original assignee: Freshr Sustainable Technologies Inc
Current assignee: Freshr Sustainable Technologies Inc
Priority date: 2024-03-06
Filing date: 2025-03-05
Publication date: 2025-09-12
Anticipated expiration: 2026-09-06
Also published as: WO2025184738A8

Abstract

The present disclosure relates to a composition and a method of making microgel particles with antimicrobial properties, for example, for packaging, food, textiles, and medical applications. In an implementation, an antimicrobial microgel comprises: a first peptide comprising arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL; an organic acid in a concentration of about 0.6mg/mL to about 20 mg/mL, and a second peptide comprising at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL, and a method of making the same. These particles may be used as a stand-alone antimicrobial in liquid or as a coating on surfaces. The antimicrobial microgel may be used to provide antimicrobial food packaging including a non-migrating antimicrobial surface for controlled and sustained protection against spoilage or pathogenic organisms without compromising the integrity or flavor of the packaged food.

Description

ANTIMICROBIAL MICROGEL AND METHOD OF MAKING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority of U.S. Patent Application Serial No. 63/561,904 filed March 6, 2024, the entirety of which is hereby incorporated by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to antimicrobial gels, and more particularly to antimicrobial microgels and methods of making the same.

BACKGROUND

[0003] Microgel particles are composed of a network of microscopic polymer chains that form a gel-like structure, exhibiting properties such as high-water content or high-water absorption capacity, tunable porosity, and high stability [1],

[0004] Microgels have been utilized in the medical field either for cell culture, injectable scaffolds [3], sustained release of nutrients [4], or sustained drug release [5],

SUMMARY

[0005] In accordance with an embodiment is provided, an antimicrobial microgel includes: a first peptide includes arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL, an organic acid in a concentration of about 0.6 mg/mL to about 20 mg/mL , and a second peptide includes at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL.

[0006] In accordance with another embodiment is provided, a method of making an antimicrobial microgel includes: providing a first peptide includes arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL, providing an organic acid in a concentration of about 0.6 mg/mL to about 20 mg/mL, and providing a second peptide includes at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL, combining the first peptide, organic acid, and second peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] To easily identify the discussion of any particular element or act, the most significant digit or digits in a reference number refer to the figure number in which that element is first introduced.

[0008] FIG. 1 illustrates a schematic representation of the fabrication process of an antimicrobial microgel film in accordance with one embodiment.

[0009] FIG. 2 shows light microscopy and profilometry images of a microgel-coated and uncoated LDPE film. A) 3D image of the surface of a clean uncoated low-density polyethylene (LDPE) film surface. B) 2D image of a clean uncoated LDPE film surface. C) 3D image of the surface of a microgel-coated LDPE film surface. D) 2D image of the surface of a microgel- coated LDPE film surface.

[0010] FIG. 3 shows surface Fourier-transform infrared spectroscopy (FTIR) spectra of clean and microgel-coated film samples. A) poly (butylene adipate-co-terephthalate) (PBAT) film samples, the red line continuous line shows the spectra for a coated film and the blue dotted line shows the spectra for a clean PBAT film. B) LDPE film samples, the red line continuous line shows the spectra for a coated film and the blue dotted line shows the spectra for a clean LDPE film.

[0011] FIG. 4 illustrates A) Charge density of microgel-coated LDPE film samples with increased amounts of components. B) Weight increase of different microgel-coated film formulations. Each mg increase is equivalent to 1 pL of water absorbed by the microgel coating. From left to right in both figures it can be observed that by doubling and tripling the amount of Protamine sulfate (Ptm) in the formulation the charge density and the water absorption increase proportionally.

[0012] FIG. 5 illustrates antimicrobial testing results of microgel particles in a liquid culture of bacteria extracted from Atlantic Salmon. Sample size N=3.

[0013] FIG. 6 illustrates antimicrobial testing results of microgel-coated and clean LDPE films using a modified ISO 22196 protocol. A) Antimicrobial testing of the coated film using Pseudomonas species extracted from Atlantic salmon with bacteria Log reduction of 99.99 % bacteria (N=5). B) Antimicrobial testing of the coated film using Enterobacteria species extracted from Atlantic salmon with a bacteria Log reduction of 99.999 % bacteria (N=5). [0014] FIG. 7 illustrates antimicrobial testing showing the extension of shelf-life of Atlantic salmon. The fish was packed with a control and coated film and sampled daily after purchase from a local retailer and the graph shows the mesophilic total viable count (TVC) of bacteria in the fish. The dotted line represents the generally accepted spoilage threshold of Log 7 CFU/g of fish.

[0015] FIG. 8 illustrates antimicrobial testing showing the effect on shelf-life on different proteins according to the base composition of the micro gel particles at a temperature of 8 °C. A) Effect of the microgel-coated film on the shelf-life of Atlantic salmon at a composition of 5 mg/mL Ptm-1.2 mg/mL Ethylenediaminetetraacetic acid (EDTA)-0.4 mg/mL s-Poly(lysine) (s- PL). B) Effect of the microgel-coated film on the shelf-life of beef at a composition of 5 mg/mL Ptm-1.2 mg/mL EDTA-0.4 mg/mL s-PL. C) Effect of the microgel-coated film on the shelf-life of beef at a composition of 15 mg/mL Ptm-3.6 mg/mL EDTA-1.2 mg/mL s-PL. All samples were packed with a control clean film and a coated film and sampled daily after purchase from a local retailer and the graph shows the mesophilic total viable count (TVC) of bacteria in the sample. The red dotted line represents the generally accepted spoilage threshold of Log 7 CFU/g for fresh proteins.

DETAILED DESCRIPTION

[0016] Generally, the present disclosure relates to a composition and a method of making microgel particles with antimicrobial properties, for example, for packaging, food, textiles, and medical applications. These particles may be used as a stand-alone antimicrobial in liquid or as a coating on surfaces, for example, films, textiles, medical equipment, among others.

[0017] In one aspect, an antimicrobial microgel is provided including: a first peptide includes arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL, an organic acid in a concentration of about 0.6 mg/mL to about 20 mg/mL, and a second peptide includes at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL.

[0018] In another aspect is provided, a method of making an antimicrobial microgel includes: providing a first peptide includes arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL, providing an organic acid in a concentration of about 0.6 mg/mLto about 20 mg/mL, and providing a second peptide includes at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL, combining the first peptide, organic acid, and second peptide.

[0019] For the purpose of promoting an understanding of the principles of the disclosure, reference will be made to the features illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Any alterations and further modifications, and any further applications of the principles of the disclosure as described herein are contemplated as would normally occur to one skilled in the art to which the disclosure relates. It will be apparent to those skilled in the relevant art that some features that are not relevant to the present disclosure may not be shown in the drawings for the sake of clarity.

[0020] Certain terms used in this application and their meaning as used in this context are set forth in the description below. To the extent a term used herein is not defined, it should be given the broadest definition persons in the pertinent art have given that term as reflected in at least one printed publication or issued patent. Further, the present processes are not limited by the usage of the terms shown below, as all equivalents, synonyms, new developments and terms or processes that serve the same or a similar purpose are considered to be within the scope of the present disclosure.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the meaning as commonly understood in the art.

[0022] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context dictates otherwise.

[0023] Microgels Having Antimicrobial Properties

[0024] Known antimicrobial microgel compositions may result in migration or release of an active substance from the microgels for bacterial control. Such technologies are based on the migration of substances and may present several disadvantages for commercialization on the regulatory side and on the consumers perception. For instance, consumers may ingest small amounts of these substances, which may potentially lead to adverse health effects, such as antibiotic resistance or allergic reactions. Regulatory bodies often impose strict guidelines and regulations regarding the use of antimicrobial agents in food packaging. Antimicrobial agents may interact with food components in unexpected ways, altering taste, texture, or appearance. Finally, released antimicrobials agents will have to go on the food label, and consumers may be wary of food packaged with antimicrobial materials due to concerns about chemical additives and their potential health effects. Such negative perceptions regarding the safety and quality of such products could impact consumer trust and purchasing decisions.

[0025] In view of the shortcomings in existing antimicrobial packaging technologies, embodiments of the present disclosure seek to produce an antimicrobial microgel to provide antimicrobial food packaging including a non-migrating antimicrobial surface for controlled and sustained protection against spoilage or pathogenic organisms without compromising the integrity or flavor of the packaged food. Compositions disclosed herein may incorporate generally recognized as safe (GRAS) components in the making of microgels to contribute to improved safety, regulatory compliance, and consumer acceptance of the antimicrobial microgels. Moreover, including value-add components sourced from food waste streams may contribute to a circular economy.

[0026] Applications of Antimicrobial Microgels

[0027] Antimicrobial microgel compositions disclosed herein may be used in a variety of applications, to reduce food spoilage, reduce the risk of food pathogens, for other food packaging applications, or for other suitable purposes.

[0028] Antimicrobial microgel formulations disclosed herein may comprise formulations based on GRAS natural ingredients with intrinsic and highly potent antimicrobial properties, with potential use as, for example, a coating on different materials to create contact-killing surfaces, as a free-standing food additive for food preservation, or other microbial control applications where a broad-spectrum antimicrobial is required.

[0029] Example Components Used in Making and Testing an Antimicrobial Microgel and Antimicrobial Microgel Film

[0030] In an example implementation, deionized (DI) water, protamine sulfate (Ptm) amorphous powder, and cetyltrimethylammonium chloride (CTAC) 25 wt. % in H2O was purchased from manufacturer Sigma Aldrich. Hydrochloric acid (HC1) Certified ACS Plus, 36.5 to 38.0%, sodium chloride (NaCl) crystalline/certified ACS, ethylenediaminetetraacetic acid (EDTA) 99% pure, l-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) pure, N-hydroxysuccinimide (NHS) pure, acid orange 7 (AO7) 97.0+% TCI America, nutrient broth (NB) dehydrated, were purchased from the manufacturer Fisher Scientific, and s-poly lysine (s-PL) was purchased from the manufacturer MarkNature as a food grade dried powder.

[0031] Extruded Poly (butylene adipate-co-terephthalate) (PBAT) film or low-density polyethylene (LDPE) film were purchased from Farnell Packaging Limited. [0032] All reagents used were food grade and GRAS.

[0033] Example Fabrication and Coating Preparation of an Antimicrobial Microgel

[0034] First, a piece of film was cut to the desired size. For example, a sheet of dimensions 30 x 20 cm.

[0035] The surface of the film was then activated using known techniques in the field such as acid treatment, UV light, plasma, or corona discharge. In one example, an industrial corona treater equipped with a vacuum table and an adjustable treatment speed was used with a power of about 1.2 KW at a feed speed of about 10 ft/min.

[0036] Treatment conditions may be adjusted depending on the type of polymer to be coated. For instance, the power can be adjusted from about 0.2 KW to about 2 KW and/or the feed speed can be adjusted from Ift/min to 20ft/min according to the required treatment time and production speed.

[0037] Immediately after corona treatment, the film having an activated surface was laid flat on a surface and the microgel solution was prepared by combining in a separate container, about 5 mg/mL of Ptm (though the range may span from about O.lmg/mL to about 20 mg/mL), about 1.2 mg/mL EDTA (though the range may span from about 0.6 mg/L to about 20 mg/mL, and about 0.4 mg/mL s-PL (though the range may span from about 0.4 to about 8 mg/mL) are mixed, and the pH adjusted to about 5.5 (though the adjusted pH may range from about 5 to about 6).

[0038] To initiate a reaction, NHS was added to the mix at a concentration of about 2 mg/ml (though the range may span from about 0.5 mg/mL to about 16 mg/mL) and then EDC at a concentration of about 12mg/mL (though the range may span from about 3 mg/mL to about 104 mg/mL) to create crosslinking between the amine group from Ptm and s-Poly and the carboxyl groups from the EDTA, initiating the formation of the microgel particles, and later coupling the particles to the activated surface of the film.

[0039] The reaction was allowed to continue until the solution became opaque due to the formation of the first microgel particles. Then the solution was transferred onto the surface of the prepared film at a dose of about 0.16 mL/cm² (though the range may span from about 0.04 to about 0.2 mL/cm² depending on the desired coating thickness). For example, for an approximately 30 cm x 20 cm film, 96 mL of solution may be transferred. [0040] The solution may also be applied by other techniques in the field of coating such as, but not limited to, roll coating, spraying, dip coating, brushing, Mayer rod, gravure, and doctor blade.

[0041] After the solution was applied to the film, the reaction was allowed to continue at room temperature for about 40 minutes to precipitate and create a chemical covalent bond with the microgel particles to the surface of the corona activated film [8], The reaction may be performed at different temperatures in the range of about 15°C to about 80°C to control the time of deposition.

[0042] The solution was removed from the film and the film was placed in a bath with water for quenching the reaction and removing any unreacted components. After this, the coated film was subsequently rinsed with an acidic solution containing aboutlOO mM HC1 and about IM NaCl and a final rinse with DI water. Finally, the coated film was dried in an oven at about 70°C for 1 hour.

[0043] The antimicrobial microgel particles may also be collected and used independently if the reaction is allowed to proceed for about 40 min at room temperature in the reaction vessel. Once the reaction is completed, the particles may be centrifuged and rinsed similarly as described for the film coating example. These particles may be used as a free-standing antimicrobial or to coat different surfaces or materials. For example, the microgel particles could be applied to textiles, biomedical equipment, or other inorganic surfaces.

[0044] In the foregoing methodology, EDTA may be directly replaced with citric acid or other organic acids such as, but not limited to, adipic acid, oxalic acid, or malic acid. Additionally, 8- PL may be directly replaced with other small peptides containing amine groups such as Poly- L/D-Lysine. Ptm may similarly be replaced with a small arginine based synthetic peptide.

[0045] Concentrations noted above refer to concentration of the precursors or components that form the base of the microgel particles. During the fabrication process, excess or weakly bound particles may be washed from the final product.

[0046] By applying the same composition precursor composition, the same final product ratios may be obtained. By varying the composition, the microgel properties may be modified or adapted for different applications.

[0047] The proportion of organic acid may be in at least 2 times molar excess to the arginine containing peptide. Additionally, the second peptide containing an amine may be at least 1/20 molar ratio to the organic acid. [0048] FIG. 1 illustrates a visual summary of a microgel coating process for fabricating an antimicrobial film for packaging applications, in accordance with an embodiment.

[0049] Characterization of Microgel-Coated Films

[0050] Surface modification. The surface modification of the polymeric material was characterized by Light Microscopy and Profilometry. Clean film samples and coated samples were analyzed with a light microscope under a bright field using a 400X magnification factor (40X lens and 10X objective). Two-dimensional (2D) images were recorded with a top camera mounted on a microscope eyepiece with the software ToupCam 2.0. For three-dimensional (3D) images, clean and coated films were analyzed using a Profilm3D with a 100X objective.

[0051] Chemical characterization. The surface chemical analysis of the samples was carried out using a Nicolet Summit lite FTIR equipped with a diamond crystal ATR attachment module. The spectra were taken in transmission mode with 32 scans per sample from wave number range between about 4000 cm'¹ to about 550 cm'¹.

[0052] Charge density characterization. Charge density of the coated film was characterized using a negatively charged dye AO7. First, a solution of 1% (w/v) AO7 was prepared in pH 3 adjusted DI water. Then the coated and uncoated sample films were immersed in the stain and incubated at room temperature with shaking for about 1 hour. After the incubation, the films were rinsed with copious amounts of DI water to remove all the nonspecific absorbed dye. Next, the films were immersed in a 1% CTAC for about 1 hour at about 60 °C to ensure full desorption of the dye. Finally, the destained CTAC solutions were placed in a UV-vis cuvette and their absorbance was measured at 483 nm. This dye has a stoichiometric ionic interaction 1 : 1 with positive charges allowing for quantification of cationic charges in a material.

[0053] Water absorption characterization. The water absorption capacity of the microgel- coated films was assessed using a gravimetric method. The films were weighted using a microbalance before and after exposure to water. Coated pieces of film were weighed and then placed in a beaker with water at room temperature and allowed to equilibrate for about 5 min. Then, they were removed from the beaker and any excess water was carefully removed using a paper towel. Then the weight of the wet-coated film was recorded and compared to the dry film to determine the amount of water absorbed by the film. Assuming a water density of about 1 g/mL, about 1 mg of weight increase is equivalent to about 1 pL of water retained by the coated film. [0054] Antimicrobial property assessment. Antimicrobial properties of microgel particles with a composition of 5 mg/mL Ptm-1.2 mg/mL EDTA-0.4 mg/mL s-PL were tested by adding them to a microbial culture of bacteria extracted from fish. Enterobacter species were inoculated to 1.5 mL of NB and 400 pL of DI water or 400 pL microgel particles at a solution (concentration 1 mg/mL) were added to the culture. The bacteria were placed in a 24 well microplate and grown at 25 °C for 24 h with double orbital shaking and continuous monitoring the culture absorbance at 600 nm. Final quantification of the Log CFU reduction was then performed using a calibration curve based on the bacterial growth curves by assessing the time at which they reach and exponential curve with an absorbance of 0.25 as previously reported in other references [10],

[0055] Antimicrobial properties of the films were characterized by two methods. The first method follows ISO 22196⁶ to assess antimicrobial plastics with minor modifications to increase the challenge for the antimicrobial coating. First, sample bacteria extracted directly from fish samples, specificity Pseudomonas species, and Enterobacteria species were inoculated to the coated and uncoated surface of various films (N=5) using full NB as growing media instead of the typical 1/500 NB dilution specified on the ISO protocol. Following inoculation, the samples were incubated for about 24 h at about 25 °C to allow bacterial growth. After incubation, the bacteria were taken from the surface and analyzed to quantify the remaining bacterial population. This analysis was performed by counting the colony-forming units (CFUs) present before and after contact with the surface. The antibacterial activity is then calculated based on the reduction in bacterial population compared to control samples, expressed as a percentage reduction or log reduction.

[0056] A second method to characterize the antimicrobial activity and an application of the present invention for the extension of the shelf-life of fresh protein involved the application of coated and uncoated films on top of fresh salmon samples. Clean films and microgel-coated film pieces of about 7.5 x 7.5 cm were used for this test. A whole salmon filet was purchased from a local retailer and sectioned into 5 x 5 cm pieces. Then the salmon sections were randomized, and control and coated films were placed on top of the fish pieces. The pieces were placed in the fridge at about 5 °C ± 1°C and the bacteria growth was tracked over the next few days. For the quantification of bacteria, each section of the fish was cut into 4 equal pieces of about 2.5 X 2.5 cm, then processed using a stomacher for bacterial extraction. Finally, serial dilution of the bacteria extract was prepared and plated on count agar plates, and the CFU/g of fish was recorded over time. It is generally accepted that spoilage occurs once the CFU/g on the fish reaches Log 7 CFU/g.

[0057] Antimicrobial properties of the microgels disclosed herein may reside in their high cationic charge. Cationic materials may be broad-spectrum antimicrobial agents⁷ exerting a contact-killing antimicrobial mechanism. Accordingly, cationic (+) charge density may be used to measure antimicrobial activity. Increased charge density may positively correlate to antimicrobial activity [9],

[0058] Examples

[0059] Example 1

[0060] A composition of 5 mg/mL of Ptm, 1.2 mg/mL EDTA, and 0.4 mg/mL s-PL microgel particles comprising naturally derived GRAS ingredients possessing highly potent antimicrobial properties was made according to the fabrication methodology described herein.

[0061] Surface Characterization of Microgel-Coated Films

[0062] The microgel particles disclosed herein may be applied to a range of polymeric materials with slight modifications to the treatment and deposition condition of the microgel particles, for instance PBAT film material will require a lower corona treatment of 1 kW while an LDPE based material will required a 1.2 kW corona treatment for similar level of particle deposition at a constant composition. In this example, an LDPE film substrate was coated with the microgel particles. FIG. 2 shows the topography of the film surface before and after the particle deposition.

[0063] FIG. 2A shows a profilometer 3D image of a clean film before deposition. It may be seen that the surface is substantially flat without significant peaks or valleys. On the color scale and the Z-axis of the image, most of the area on the surface does not exceed a height of 1 pm. The flatness and lack of surface features may be corroborated by the 2D microscopy image in FIG. 2B

[0064] By contrast, after the microgel particle deposition, the 3D image in FIG. 2C shows change in surface topography. In particular, high areas of the image go up to about 4 pm and it may possible to observe the arrangement of peaks across the entire surface.

[0065] The deposition of the microgel particles causes this arrangement and it can be corroborated in FIG. 2D with the light microscopy image of the surface where the particulate nature of the material is shown across the image. [0066] An additional polymeric film was prepared comprising a PBAT film substrate.

[0067] Physicochemical Characterization of Microgel-Coated Films

[0068] ATR-FTIR (Attenuated Total Reflection-Infrared spectroscopy) was performed for chemical characterization of the coating on two different polymeric film substrates, namely Low Density Polyethylene (LDPE) and Poly(butylene adipate-co-terephthalate) (PBAT). The coating was performed as described herein.

[0069] In particular, infrared spectroscopy (FTIR) was used to measure the absorption of energy by chemical bonds when a light source of varying wavelength interacts with the material of interest. The energy absorption may be measured and is very specific to the chemical nature of the bond, allowing for the identification of chemical species present in the material or substance being analyzed. Additionally, with the aid of complementary modules is it possible to collect the FTIR spectra of only the outer surface of a material. In the present example, an Attenuated Total Reflection module (ATR) was used to enable collection FTIR spectra of about 1 pm to about 2 pm from the surface.

[0070] For both polymeric films, the characteristic peaks for the clean material may be observed as a dotted line. Some of the characteristic peaks of PBAT (FIG. 3A) include ester groups from both terephthalate and adipate components with a strong carbonyl stretching vibration peak around 1700 cm'¹. A C-H stretching vibration peak can be observed between 2800 cm'¹ to about 3000 cm'¹. Ether linkages in its structure, resulting from the C-O-C stretching vibrations can be observed in the range of about 1100 cm'¹ to about 1300 cm'¹. The bending vibrations of methylene (CEL) groups can be observed in the region of about 1300 cm'¹ to about 1470 cm'¹. And the stretching vibrations associated with the adipate ester groups may be seen in the range of about 1100 cm'¹ to about 1300 cm'¹. Characteristic FTIR peaks of LDPE (FIG. 3B), primarily composed of repeating ethylene (CHICLE) units are the stretching vibrations of CEL groups seen as two large peaks in the region of about 2800 cm'¹ to about 3000 cm'¹, the bending vibrations of CEE groups observed as peaks in the region of about 1350 cm'¹ to about 1470 cm'¹, and the tending vibrations of hydrogen attached to carbon atoms in the polymer backbone seen in the region of about 700 cm'¹ to about 900 cm'¹.

[0071] After both materials were coated, it was possible to observe the change in the FTIR spectra as a solid line in FIG. 3A and FIG. 3B. Some of the characteristic peaks arising from the microgel particles include an Amide A band corresponding to the N-H stretching vibration of the amide groups shown as a broad peak in the range of about 3200 cm'¹ to about 3500 cm'¹ The Amide I band arising from the C=O stretching vibration coupled with N-H bending and C- N stretching shown as a strong peak in the range of about 1600 cm'¹ to about 1700 cm'¹, the Amide II band corresponding to a combination of N-H bending and C-N stretching vibrations is seen as a medium intensity peak in the range of about 1500 cm'¹ to about 1600 cm'¹, and the stretching vibration of the C-N bond in the peptide backbone of Ptm and s-PL appears as a peak around about 1250cm'¹ to about 1350 cm'¹.

[0072] From the FTIR spectra of FIG. 3, the chemical composition on the surface of the material changes are illustrated, thus showing the successful coating of the surface. Additionally, little of the characteristic peaks from the clean materials remains after the coating, particularly for the LDPE film, demonstrating a thickness of the coating of 2 pm due to the maximum penetration of the light source in the ATR module.

[0073] Adjusting physicochemical characteristics may be facilitated by altering the amount or ratio of its basic components, particularly their charge density and the water uptake of the deposited material. These may be properties that may tailor the antimicrobial microgel particles for a desired application. For instance, in the field of packaging a food item more prone to water loss could be matched with a formulation with a larger water absorption capacity. Also, items with a larger bacteria load or with more resistant types of bacteria could benefit from a higher charge density material.

[0074] In this regard, LDPE film was coated with microgel particles formed from varying amounts of components. Charge density of the coated films was then characterized together with water absorption properties to demonstrate the degree of control possible by altering the initial composition of the same components (FIG. 4). FIG. 4A shows that as the content of Ptm doubles and triples, so does the charge density. This is largely due to Ptm being the main contributor to charge density in the formulation. Also, it is possible to observe the increase in charge density from a clean film to a coated film demonstrating the successful coating of the material.

[0075] FIG. 4B shows that increasing the content of Ptm in the formulation increases water absorption capacity of the coating. In this example, Ptm may act as a capping agent creating a larger number of particles deposited on the surface of the films.

[0076] The antimicrobial properties of the microgel particles with a composition of 5 mg/mL Ptm- 1.2 mg/mL EDTA-0.4 mg/mL s-PL were tested by adding them to a microbial culture of bacteria extracted from fish. Enterobacter species were inoculated to 1.5 mL of Nutrient broth and 400 pL of DI water or 400 pL microgel particles at a solution (concentration 1 mg/mL) were added to the culture. The bacteria were placed in a 24 well microplate and grown at 25 °C for 24 h with double orbital shaking and continuous monitoring the culture absorbance at 600 nm. Then final quantification of the Log CFU reduction was performed using a calibration curve based on the bacterial growth curves by assessing the time at which they reach and exponential curve with an absorbance of 0.25 as previously reported in other references [10], [0077] Example 2

[0078] Antimicrobial Characterization of Stand-Alone Microgel Particles

[0079] To test the antimicrobial characteristics of the microgel particles, microgel particles were prepared with an initial composition of 5 mg/mL of Ptm, 1.2 mg/mL EDTA, and 0.4 mg/mL s-PL and washed to remove any unreacted reagents and precursors. The prepared microgel particles were then added to a culture of Enterob acter species extracted from fish with an initial inoculation of approximate IxlO⁵ CFU/mL and placed in a microplate. For this assay, the bacteria log reduction was calculated in retrospective comparing the growth curves to standard bacteria cultures under similar conditions at different initial levels of inoculation.

With such standards, a calibration curve was built, and the bacterial Log reduction calculated without the need for plating.

[0080] This example yielded a 99.9% bacteria reduction produced from the microgel particles under high growth bacterial conditions (full broth, shaking and 25 °C). This assay demonstrates, for example, the antimicrobial properties of the microgel particles when applied as a stand-alone system. Such antimicrobial properties can be easily transferable to all kinds of materials and surface coatings such an antimicrobial packaging system as we will demonstrate in subsequent sections.

[0081] FIG. 5 presents the Log reduction on bacterial growth when free floating (stand-alone) microgel particles are placed on a liquid culture of Enterob acter family bacteria.

[0082] Example 3

[0083] Antimicrobial Characterization of Microgel-Coated Films

[0084] LDPE films were coated according to the methodology disclosed herein, and then exposed to two spoilage species of bacteria extracted from Atlantic salmon. The antimicrobial test was performed following protocol 22196 from the International Organization for Standardization (ISO-22196)⁶ designed to measure the antibacterial activity on plastics and other non-porous surfaces with some minor modifications. FIG. 4 shows the Log reduction in CFU/mL that the film achieved when exposed to Pseudomonas species (FIG. 4A) and Enterobacteria species (FIG. 4B) for about 24 hours at about 25 °C.

[0085] FIG. 6 shows that an initial inoculation dose for both bacteria was about 4.5 CFU/mL and each bacteria species had a slightly different growth over the about 24-hour incubation period when incubated at about 25 °C. This growth rate difference may be explained by the preferred growing temperature of each bacteria species.

[0086] A modification was made to the ISO protocol for increasing the challenge level for antimicrobial material to include high growth conditions. In particular, full NB broth was used instead of a 1/500 broth dilution as used in the ISO protocol. Under these conditions, there was still a significant Log reduction of 99.99 % for Pseudomonas bacteria (FIG. 6A) and a 99.999 % Log reduction for Enterobacter bacteria species (FIG. 6B). The large standard deviation in the coated films for both bacteria species arise from the wipeout of the bacteria in some of the replicates, for which case a zero value was used during the average calculations.

[0087] In the application of the microgel-coated film for a food packaging application, Atlantic salmon was bought from a local retailer, and packed with LDPE-coated film. The fish was refrigerated, and samples were taken daily to track the spoilage level over the next few days. Shown in FIG. 7 is the change in Log CFU per gram of fish over time. The total mesophilic bacteria were tracked and the dotted line in the graphs shows the typical spoilage level threshold of Log 7 CFU/g.

[0088] FIG. 7 shows packing the fish with the microgel-coated film delayed the bacterial growth on the salmon, keeping the fish under spoilage level for two additional days compared to the control fish packed with clean uncoated film. This demonstrates the effectiveness of microgel particle formulations disclosed herein as an antimicrobial packaging material capable of extending the shelf life of fresh products.

[0089] Example 4

[0090] To illustrate the versatility of the antimicrobial microgel system, and how its composition can be easily adjusted to adapt for different applications we performed an accelerated spoilage experiment (8 °C) were we packed two different types of proteins, namely beef and salmon. Each protein presents a unique challenge in terms of chemical composition, required packaging conditions and free water content. The beef was tested for a vacuum-packed application and the salmon was packed similarly to our previous example where a liner film was placed on the surface of piece of salmon fillet.

[0091] FIG. 8 shows a summary of the results obtained from the tracking of the total viable count of mesophilic bacteria from the different proteins. First, the extension of shelf life of the coating composition of 5 mg/mL of Ptm, 1.2 mg/mL EDTA, and 0.4 mg/mL s-PL, was compared between fish packed with a liner (FIG. 8A) and vacuum pack beef (FIG. 8B). It can be observed that there was an extension of shelf life for the fish samples pack with microgel coated film of about 3 days compared to a control packed with clean uncoated film. However, when the same coating composition was applied to the vacuum-packed beef, no extension of shelf life was observed. This could be explained by the fact that the beef samples had a larger amount of drip or water loss compared to the fish samples. After observing this fact, we prepared a film coated with a composition of 15 mg/mL of Ptm, 3.6 mg/mL EDTA, and 1.2 mg/mL s-PL, in this case, it can be observed in FIG. 8C that an extension on shelf life was achieved of about 2 days. By adjusting the formulation, it is possible to adapt the coating for different scenarios, in this case as shown earlier in FIG. 4 by adjusting the components we can prepared a coating with an increased water uptake capacity and a larger charge density which will benefit its applications for beef packing.

[0092] Example 5

[0093] Use of antimicrobial particles as disclosed herein is not limited to surface coating on polymeric film materials with a final application of food packaging. Application of such microgel may extend to use as an antimicrobial agent in liquid, in a spray format on diverse foods and substances, in combination with textiles, as a coating for biomedical applications, for example. The microgel particles may also be adapted for additional applications. By modifying the ratio of its the components, particularly that of Ptm, it is possible to adjust their physicochemical properties depending on the specific field application.

[0094] By the evidence disclosed herein, coating of multiple polymeric material films and the potent antimicrobial properties of the coated films was demonstrated in the application of a packaging material able to extend the shelf life of fresh proteins. Due to the cationic nature of the microgel particles, they may additionally be applied as a broad-spectrum antimicrobial in many fields such as food preservation, food safety, biomedical, veterinarian, and even biological warfare defense.

[0095] Embodiments [0096] In one aspect, an antimicrobial microgel includes: a first peptide includes arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL, an organic acid in a concentration of about 0.6 mg/mL to about 20 mg/mL, and a second peptide includes at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL. The antimicrobial microgel may also include where the first peptide includes arginine is protamine sulfate. The antimicrobial microgel may also include where the second peptide includes at least one amine group is selected from the group consisting of s-poly lysine, poly(L-lysine), and poly(D-lysine). The antimicrobial microgel may also include where the organic acid is selected from the group consisting of EDTA, citric acid, adipic acid, oxalic acid, and malic acid. Use of the antimicrobial microgel may also include for forming an antimicrobial microgel coating. Use of the antimicrobial microgel may also include for preserving a perishable foodstuff. Use of the antimicrobial microgel may also include for coating a surface.

[0097] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0098] In one aspect, a method of making an antimicrobial microgel includes: providing a first peptide includes arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL, providing an organic acid in a concentration of about 0.6 mg/mLM to about 20 mg/mL, and providing a second peptide includes at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL, combining the first peptide, organic acid, and second peptide. The method may also include further includes combining N- hydroxysuccinimide (NHS) with the first peptide, organic acid, and second peptide, the NHS in a concentration of about 0.5 mg/mL and 16 mg/mL. The method may also include further includes combining l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) with the first peptide, organic acid, and second peptide, the EDC in a concentration of about 3 mg/mL and 104 mg/mL. The method may also include further includes combining the first peptide, organic acid, and second peptide for about 40 minutes. The method may also include further combining washing the antimicrobial particle with water, an acid and a salt for substantially removing the NHS and the EDC. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims. The antimicrobial microgel may also include where the protamine sulfate is in a concentration of about 5 mg/mL. The antimicrobial microgel may also include where the second peptide includes at least one amine group is s-poly lysine. The antimicrobial microgel may also include where the s-poly lysine is in a concentration of about 0.4 mg/mL. The antimicrobial microgel may also include where the organic acid is EDTA. The antimicrobial microgel may also include where the EDTA is in a concentration of about 1.2 mg/mL. The method may also include where the acid is HC1 in a concentration of 100 mM. The method may also include where the salt is NaCl in concentration of 1 M. The method may also include further includes rinsing the antimicrobial particle with deionized water. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

REFERENCES

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Claims

1. An antimicrobial microgel comprising: a first peptide comprising arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL; an organic acid in a concentration of about 0.6 mg/mL to about 20 mg/mL; and a second peptide comprising at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL.

2. The antimicrobial microgel of claim 1, wherein the first peptide comprising arginine is protamine sulfate.

3. The antimicrobial microgel of claim 2, wherein the protamine sulfate is in a concentration of about 5 mg/mL.

4. The antimicrobial microgel of claim 1, wherein the second peptide comprising at least one amine group is selected from the group consisting of s-poly lysine, poly(L-lysine), and poly(D-lysine).

5. The antimicrobial microgel of claim 4, wherein the second peptide comprising at least one amine group is s-poly lysine.

6. The antimicrobial microgel of claim 5, wherein the s-poly lysine is in a concentration of about 0.4 mg/mL.

7. The antimicrobial microgel of claim 1, wherein the organic acid is selected from the group consisting of EDTA, citric acid, adipic acid, oxalic acid, and malic acid.

8. The antimicrobial microgel of claim 7, wherein the organic acid is EDTA.

9. The antimicrobial microgel of claim 8, wherein the EDTA is in a concentration of about 1.2 mg/mL.

10. Use of the antimicrobial microgel of claim 1 for forming an antimicrobial microgel coating.

11. Use of the antimicrobial microgel of claim 1, for preserving a perishable foodstuff.

12. Use of the antimicrobial microgel of claim 1, for coating a surface.

13. A method of making an antimicrobial microgel comprising:

(a) providing a first peptide comprising arginine, the first peptide in a concentration of about 0.1 mg/mL to about 20 mg/mL;

(b) providing an organic acid in a concentration of about 0.6mg/mL to about 20 mg/mL 64 mM; and

(c) providing a second peptide comprising at least one amine group, the second peptide in a concentration of about 0.4 mg/mL to about 8 mg/mL;

(d) combining the first peptide, organic acid, and second peptide.

14. The method of claim 13, further comprising combining N-hydroxysuccinimide (NHS) with the first peptide, organic acid, and second peptide, the NHS in a concentration of about 0.5 mg/mL and 16 mg/mL.

15. The method of claim 13, further comprising combining l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) with the first peptide, organic acid, and second peptide, the EDC in a concentration of about 3 mg/mL and 104 mg/mL.

16. The method of claim 13, further comprising combining the first peptide, organic acid, and second peptide for 1 - 40 minutes.

17. The method of claim 13, further combining washing the antimicrobial particle with water, an acid and a salt for substantially removing the NHS and the EDC.

18. The method of claim 17, wherein the acid is HC1 in a concentration of 100 mM.

19. The method of claim 17, wherein the salt is NaCl in concentration of 1 M.

20. The method of claim 17, further comprising rinsing the antimicrobial particle with deionized water.