BACKGROUND

[0001] Maintaining food safety and security for the increasing global population is one of the most important challenges of the 21st century. Food packaging plays a key role in maintaining food safety and quality and can serve to reduce food waste across the “farm to the fork” continuum. Films made of synthetic, petroleum-based polymers are widely used in the market as food packaging materials due to their low cost and excellent gas barrier and mechanical properties. These films can protect the contents from the external oxygen that triggers the oxidative reactions compromising food quality and shelf life. However, films with incorporated antimicrobial active ingredients (Als) have not been used widely due to their poor performance as a result of the low-surface-to-volume ratio and potential negative sensory effects.

BRIEF DESCRIPTION OF THE FIGURES

[0002] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein.

[0003] FIGS. 1 A-1 C are schematic representations enzyme- and relative humidity (RH)-triggered strategy; preparation of the solution composed of zein, starch, cellulose nanocrystals (CNCs), nature-derived free active ingredients (Als), and cyclodextrin-inclusion complexes (CD-ICs) of Als; and the synthesis of multi-stimuli responsive fibers by electrospinning, respectively.

[0004] FIG. 2 is a schematic representation of the synthesis of cyclodextrin- inclusion complexes (CD-ICs) of active ingredients (Als) by a co-precipitation method.

[0005] FIGS. 3A-3B are X-ray diffraction (XRD) patterns of y-CD, active ingredients (Als) and cyclodextrin-inclusion complexes (CD-ICs) of Als and attenuated total reflectance-Fourier transform infrared spectrometry (ATR-FTIR) spectra of y-CD, Als, and CD-IC of Ais, respectively.

[0006] FIGS. 4A-4D are scanning electron microscopy (SEM) images with average fiber diameter (AFD) distribution of a. pristine fibers; b. enzyme responsive fibers with only free active ingredients (Als), c. relative humidity (RH) responsive fibers with only cyclodextrin-inclusion complexes (CD-ICs) of Als, and d. multi stimuli responsive fibers.

[0007] FIGS. 5A-5B are X-ray diffraction (XRD) patterns and attenuated total reflectance-Fourier transform infrared spectrometry (ATR-FTIR) spectra of pristine fibers, enzyme responsive fibers with only free active ingredients (Als): thyme oil (TO), citric acid (CA), nisin, relative humidity (RH) responsive fibers with only cyclodextrin-inclusion complexes (CD-ICs) of Als: TO, sorbic acid (SA), nisin, and multi stimuli responsive fibers with both free Als (TO, CA, nisin) and CD-IC of Als (TO, SA, nisin).

[0008] FIGS. 6A-6B are plots of cumulative release (%) of thymol from enzyme responsive fibers with only free active ingredients (Als) into enzyme solution and PBS without enzymes and remaining thymol concentration (ppm) from relative humidity (RH) responsive fibers with only cyclodextrin-inclusion complexes (CD- ICs) of Als at 50% RH and 95% RH as a function of time. The error bars in the figure represent the standard deviation (SD). The significant difference among data in the same contact time was labeled with non-significant (ns): p>0.05, ¹ P < 0.05, P < 0.01 , * P < 0.001 , or **** P < 0.0001 .

[0009] FIGS. 7A-7C are plots of the antimicrobial activity of multi-responsive fiber against E. coli, L. innocua, and A. fumigatus, respectively. Aluminum foil and pristine fibers were used as controls. 2.50 and 1 .25 mg/cm2 represents the mass per surface area of fibers. Data in the same material labeled with different uppercase letters are significantly different (p < 0.05). Data in the same treatment time group labeled with different lowercase letters are significantly different (p < 0.05).

[0010] FIG. 8 is plots of the antimicrobial activity of relative humidity (RH) responsive fibers (2.50 mg/cm ² ) against E. coli at 50% RH and 90% RH. At the same RH level, data in the same material labeled with different uppercase letters are significantly different (P < 0.05). Data in the same contact time labeled with different lowercase letters are significantly different (P < 0.05). At different RH level, significant difference among data in the same contact time of RH responsive fibers was labeled with *: “* P < 0.001 , * P < 0.05. The error bars in the figure represent the standard deviation (SD).

[0011] FIGS. 9A-9B are schematic representations of experiment design for direct contact assay and relative humidity (RH) triggered antimicrobial activity test of relative humidity (RH) responsive fibers, respectively.

[0012] FIGS. 10A-10C are scanning electron microscopy (SEM) images of the degradation of enzyme responsive fibers with only free active ingredients (Als) in a. 0.1 U/mL, b. 1 U/mL, and c. 3 U/mL enzyme concentrations at the end of 12 hours.

[0013] FIGS. 11A-11 B are scanning electron microcopy (SEM) images of relative humidity (RH) responsive fibers with only cyclodextrin-inclusion complexes (CD- ICs) of Als at a. 50% RH and b. 95% RH at the end of 4 hours. [0014] FIG. 12 is a plot of cumulative release (%) of thymol from multi stimuli responsive fibers into food simulant (water) as a function of time.

SUMMARY

[0015] The disclosure provided herein relates to the development of biodegradable, biopolymer based antimicrobial fibers (also referred to herein simply as “fibers” and “multi-stimuli responsive fibers”) that can be used as, among other things, coatings on surfaces (e.g., inner surfaces) of food package materials to enhance food safety and quality. Such fibers can sense changes in relative humidity (RH) and can sense the presence of enzymes released by microorganisms that come in contact with the fibers. In response to changes in RH or enzymes released from microorganisms, the fibers described herein release antimicrobial active ingredients that help control the proliferation of microorganisms on or near food products that come in contact with them. RH responsiveness can be achieved by including cyclodextrin inclusion complexes (CD-ICs) of hydrophopic, nature-derived antimicrobials in or on the fibers, whereas the enzymatic responsiveness can be achieved via the degradation (e.g., enzymatic degradation) of the biopolymer that makes up the fibers, thereby releasing the antimicrobials described herein.

[0016] The disclosure therefore relates to a fiber, responsive to at least one of a biotic and an abiotic trigger, the fiber comprising: at least one antimicrobial active ingredients in the fiber that is released from the fiber in response to the at least one of a biotic and an abiotic trigger.

DESCRIPTION

[0017] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

Introduction

[0018] Advanced fibrous materials with high surface-to-volume ratio are better suited to incorporate minimal quantities of Als for the development of active, antimicrobial food packaging materials. For instance, researchers have developed antimicrobial nanofibers using electrospinning of zein and a cocktail of nature- derived antimicrobial agents. More recently, emphasis has shifted toward the development of “smart” or stimuli responsive packaging materials in order to provide precision in the delivery of antimicrobial Als and minimize the use of chemicals, thereby minimizing sensory and public health concerns. Such advanced materials are designed to exhibit changes in their properties in response to a desired and specific chemical, physical, and biological stimuli such as pH, relative humidity (RH), and enzymes present in the biological exudates secreted by microorganisms. If Als are incorporated into these responsive materials, such biotic and abiotic stimuli can trigger their release at the right time and at the right dose, bringing precision to the delivery.

[0019] The disclosure describes both biotic (enzymatic) and abiotic (RH) triggers that are considered in the design of a fiber. More specifically, food-associated microorganisms across various food categories including fresh produce, meat, and bread exude a range of enzymes, including cellulase, protease, and amylase. These enzymes can selectively breakdown polymers (e.g., into subunit monomers). Therefore, cellulolytic, proteolytic, and amylolytic enzymatic reactions from the presence of food related microorganisms can break down polymer-based fibers to trigger the release of antimicrobial Als to control microbial growth on an as needed basis. In addition, the sustainable use of biopolymers for food packaging materials will also reduce the plastic waste and micro-Znanoplastics environmental crisis caused by synthetic polymers.

[0020] Furthermore, RH and temperature are key environmental variables related to food safety and quality. The optimum RH for the storage of food categories such as meat and fresh produce is typically above 90%. It is also known that food-borne microorganism growth increases as the RH increases above 90%. Therefore, as described herein, high RH can be used as an abiotic trigger for the release of Als to prevent microbial growth. In addition, native cyclodextrins (CDs) are FDA GRAS compounds and have the ability to make inclusion complexes (ICs) with various types of hydrophobic compounds owing to their relatively hydrophobic cavity; this approach can be used to effectively improve the aqueous solubility and thermal stability of a range of analytes. CD-ICs are widely used for biomedical applications such as drug delivery, wound dressing, and tissue engineering. As described herein, CD-ICs are disassociated when the RH exceeds 85% as the hydrogen bonds are weakened between the hydrophobic molecules and CD, making this platform an ideal candidate for the RH triggered release of antimicrobial Als.

Fibers

[0021] The disclosure generally relates to a fiber, responsive to at least one of a biotic and an abiotic trigger, the fiber comprising: at least one antimicrobial active ingredients in the fiber that is released from the fiber in response to the at least one of a biotic and an abiotic trigger.

[0022] The fibers described herein can be made of any suitable material so long as the at least one antimicrobial active ingredients in the fiber is released from the fiber in response to the at least one of a biotic and an abiotic trigger. The fibers described herein, for example, can comprise any suitable material that is at least partially broken down by at least one enzyme (e.g., enzymatically degradable) or broken down sufficiently by the at least one enzyme to release the antimicrobial active ingredients described herein. Thus, for example, the fibers described herein can comprise any suitable polymer (e.g., biopolymer) that is at least partially broken down by at least one enzyme (e.g., enzymatically degradable) or broken down sufficiently by the at least one enzyme to release the antimicrobial active ingredients described herein.

[0023] Such suitable polymers include, but are not limited to biopolymers, such as cellulose and derivatives thereof (e.g., cellulose acetate, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and ethyl-cyanoethyl cellulose), zein, and starch alone or in combination (e.g., mixtures of biopolymers). For example, the fibers described herein can comprise a combination of two, three or more (bio)polymers in any suitable ratio. Thus, if the fibers described herein comprise a combination of two (bio)polymers, the ratio of a first (bio)polymer to a second (bio)polymer can be about 85:15, about 80:20, about 70:30 or about 50:50. An example of a suitable (bio)polymer combination in the fibers described herein includes fibers having a ratio of zei starch of about 85:15.

[0024] But these are non-limiting examples, since the fibers contemplated herein need not be made entirely of a biopolymer. That is, the fibers contemplated herein, can be made of copolymers of biopolymers and synthetic polymers, so long as at least one of the biopolymers and the synthetic polymers are at least partially broken down by at least one enzyme (e.g., enzymatically degradable) or broken down sufficiently by the at least one enzyme to release the antimicrobial active ingredients described herein. For example, the fibers described herein, can comprise naturally- or non-naturally- occurring (co)polymers that are at least partially broken down by at least one enzyme (e.g., enzymatically degradable) or broken down sufficiently by the at least one enzyme to release the antimicrobial active ingredients described herein. An example of such (co)polymers include those described in U.S. Patent No. 6,316,585, which is incorporated by reference as if fully set forth herein, having the formula (I): wherein X — is an amino acid residue selected from the group consisting of tyrosine, serine, threonine and cysteine,

Y — is a pendant group selected from drug molecules containing carboxyl groups, R — is selected from the group consisting of hydrogen, methyl and a mixture of hydrogen and methyl on the individual molecule, n — is an integer from 0 to about 100, and m — is an integer from 0 to 10.

[0025] Also contemplated herein are mixtures of the fibers described herein with naturally- or non-naturally- occurring (co)polymers, regardless of whether the naturally- or non-naturally- occurring (co)polymers are at least partially broken down by at least one enzyme (e.g., enzymatically degradable) or broken down sufficiently by the at least one enzyme to release the antimicrobial active ingredients described herein. In such circumstances, a mixture would include the fibers described herein in any suitable percent by weight. For example, mixtures comprising about 0.5 wt%, about 1 wt%, about 2 wt%, about 4 wt%, about 5 wt%, about 10 wt%, from about 0.5 wt% to about 10 wt%, or about 1 wt% to about 5 wt% of the fibers described herein with naturally- or non-naturally- occurring (co)polymers are contemplated herein.

[0026] The fibers of the disclosure can be made by any suitable method, including electrospinning, rotary jet spinning, extrusion or chemical synthesis. Electrospinning methods for making the fibers described herein are well known in the art. See, e.g., Polymer Reviews 48: 378-391 (2008).

[0027] The fibers of the disclosure can have a multipoint Brunauer-Emmett-Teller (BET) surface area of from at least about 10 m ² /g, at least about 15 m ² /g, at least about 20 m ² /g, at least about 30 m ² /g, at least about 40 m ² /g, at least about 50 m ² /g, at least about 60 m ² /g, at least about 70 m ² /g, at least about 80 m ² /g, at least about 90 m ² /g, at least about 100 m ² /g, at least about 110 m ² /g, at least about 120 m ² /g, at least about 150 m ² /g, at least about 180 m ² /g, from about 10 m ² /g to about 200 m ² /g, about 10 m ² /g to about 100 m ² /g, about 10 m ² /g to about 50 m ² /g, about 30 m ² /g to about 90 m ² /g, about 40 m ² /g to about 150 m ² /g or about 30 m ² /g to about 60 m ² /g. Alternatively, or in addition to the multipoint BET surface area, the fibers of the disclosure can have at least one of an average pore radius of at least about 1 .5 nm; and a total pore volume of at least about 0.5 x 10 ^-2 mL/g.

Antimicrobial Active Ingredients

[0028] The fibers described herein comprise at least one antimicrobial active ingredients in the fibers. The antimicrobial active ingredients in the fibers are released from the fiber in response to the at least one of a biotic and an abiotic trigger. As used herein, the term “in the fibers” generally refers to antimicrobial active ingredients that are located on (e.g., on a surface) or inside (e.g., as part of the fiber or dissolved in the fiber) the fibers described herein. Thus, for example, the antimicrobial active ingredients can form a coating on at least a portion of the fibers described herein or, in some instances, can be seen (e.g., via a microscope, such as a scanning electron microscope) as crystals on at least a portion of the fibers (e.g., on a surface of the fibers).

[0029] The types of antimicrobial active ingredients contemplated herein include naturally occurring antimicrobials, such as those described in Naturally Occurring Antimicrobials in Food, Task Force Report No. 132, April 1998, Council for Agricultural Science and Technology, which is incorporated by reference as if fully set forth herein.

[0030] Antimicrobial active ingredients contemplated herein include, but are not limited to: bacteriocins (e.g., naturally produced, small peptides with bactericidal activity usually against closely related bacteria, such as nisin, lacticins, lactococcins, dricin, dipolcoccin, lactostrepcins, mesenterocin 5, leuconosin S, leuconosin A-UAL187, leuconosin Lcm1 , pediocin A, pediocin AcH, pediocin PA- 1 , lactocins B and F, lactocins 27 and S, plantaricin F, and SAG brevecins, caseicin 80, acidocin A, helveticin J, plantacin B, sakacin, propionicin PLG-1 ), defensins (e.g., a family of small molecular weight peptides of 29 to 24 amino acids, such as cationic, cysteine-rich peptides, including magainin 2 and magainin 2 amide); antibacterial peptides found in insects (e.g., abaecin, apidaecin, attacin, cecropin, coleoptericing, diptericin, and royalisin); organic acid antimicrobials (e.g., acetic acid, benzoic acid, lactic acid, propionic acid, sorbic acid, citric acid, malic acid, caprylic acid, fumaric acid, succinic acid, and tartaric acid and salts thereof); lipid antimicrobials (e.g., fatty acids, such as short-chain fatty acids such as those with about six carbon atoms, monoacylglycerols, such as monolauryl (e.g., Ci _2: o and Ci8:3) glycerol and lipopeptides, such as polymyxin and those that are derived from the condensation of peptides or amino acids and fatty acids, such as the condensation products of D-tryptophan, D-alanine, D-methionine, D-valine, and D- aspartic acid with succiminidyl sorbate, myristate, palmitate, or caproate, including sorboyl-tryptophan, sorboyl-D-alanine, myristoyl-D-aspartic acid, and glycyl-D- alanine); antimicrobial plant substances (e.g., caffeic acid, chlorogenic acid, catechol, catechin, allicin and precursors thereof, cinnamic aldehyde, eugenol, and thymol); polypeptide antimicrobials (e.g., lytic enzymes, peroxidases, oxidases, transferrins, and antimicrobial peptides); phenolic antimicrobials (e.g., p-coumaric acid, p-3,4-dihydroxyphenylethanol, 3,4-dihydroxyphenyl acetic acid, ferulic acid, gallic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-hydroxyphenyl acetic acid, p-hydroxyphenyl propionic acid, hydroxytytrosol, kinic acid, oleuropein, protocatechu ic acid, protocatechuic aldehyde, autin, syringic acid, syringaldehyde, 3,4,5-trimethoxybenzoic acid, tyrosol, vanillic acid, p-vanillin, and veratric acid), and combinations thereof. The at least one antimicrobial active ingredient can be at least one of an essential oil, an organic acid antimicrobial or a bacteriocin, such as thyme oil, citric acid, and nisin, respectively. Other examples of essential oils include lavender oil, thyme oil, peppermint oil, cajuput oil, cinnamon oil, eucalyptus oil, clove oil, sage oil, and tea tree oil.

[0031] Antimicrobial active ingredients contemplated herein also include, but are not limited to cyclodextrin-inclusion complexes (CD-ICs) of: bacteriocins (e.g., naturally produced, small peptides with bactericidal activity usually against closely related bacteria, such as nisin, lacticins, lactococcins, dricin, dipolcoccin, lactostrepcins, mesenterocin 5, leuconosin S, leuconosin A-UAL187, leuconosin Lcm1 , pediocin A, pediocin AcH, pediocin PA-1 , lactocins B and F, lactocins 27 and S, plantaricin F, and SAG brevecins, caseicin 80, acidocin A, helveticin J, plantacin B, sakacin, propionicin PLG-1 ), defensins (e.g., a family of small molecular weight peptides of 29 to 24 amino acids, such as cationic, cysteine-rich peptides, including magainin 2 and magainin 2 amide); antibacterial peptides found in insects (e.g., abaecin, apidaecin, attacin, cecropin, coleoptericing, diptericin, and royalisin); organic acid antimicrobials (e.g., acetic acid, benzoic acid, lactic acid, propionic acid, sorbic acid, citric acid, malic acid, caprylic acid, fumaric acid, succinic acid, and tartaric acid and salts thereof); lipid antimicrobials (e.g., fatty acids, such as short-chain fatty acids such as those with about six carbon atoms, monoacylglycerols, such as monolauryl (e.g., C12:0 and C18:3) glycerol and lipopeptides, such as polymyxin and those that are derived from the condensation of peptides or amino acids and fatty acids, such as the condensation products of D-tryptophan, D-alanine, D-methionine, D-valine, and D-aspartic acid with succiminidyl sorbate, myristate, palmitate, or caproate, including sorboyl- tryptophan, sorboyl-D-alanine, myristoyl-D-aspartic acid, and glycyl-D-alanine); antimicrobial plant substances (e.g., caffeic acid, chlorogenic acid, catechol, catechin, allicin and precursors thereof, cinnamic aldehyde, eugenol, and thymol); polypeptide antimicrobials (e.g., lytic enzymes, peroxidases, oxidases, transferrins, and antimicrobial peptides); phenolic antimicrobials (e.g., p-coumaric acid, p-3,4-dihydroxyphenylethanol, 3,4-dihydroxyphenyl acetic acid, ferulic acid, gallic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-hydroxyphenyl acetic acid, p-hydroxyphenyl propionic acid, hydroxytytrosol, kinic acid, oleuropein, protocatechu ic acid, protocatechuic aldehyde, autin, syringic acid, syringaldehyde, 3,4,5-trimethoxybenzoic acid, tyrosol, vanillic acid, p-vanillin, and veratric acid), and combinations thereof. The at least one antimicrobial active ingredient can be at least one of an essential oil, an organic acid antimicrobial or a bacteriocin, such as thyme oil, citric acid, and nisin, respectively. Other examples of essential oils include lavender oil, thyme oil, peppermint oil, cajuput oil, cinnamon oil, eucalyptus oil, clove oil, sage oil, and tea tree oil. Contemplated herein are, therefore, a CD- IC, wherein the at least one antimicrobial active ingredient is an essential oil, an organic acid antimicrobial or a bacteriocin, such as thyme oil, citric acid, and nisin, respectively.

[0032] The fibers described herein can comprise any suitable content of the at least one antimicrobial active ingredients. Examples of suitable content of the at least one antimicrobial active ingredients include at least about 0.05% w/v, at least about 0.1% w/v, at least about 0.2% w/v at least about 0.5% w/v, at least about 1% w/v, at least about 2% w/v, at least about 3% w/v, at least about 4% w/v, at least about 5% w/v, from about 0.2% w/v to about 5% w/v, about 0.05% w/v to about 5% w/v, about 0.1% w/v to about 3% w/v, about 0.5% w/v to about 3% w/v, about 1 % w/v to about 3% w/v, about 0.9% w/v to about 2.5% w/v or about 1 % w/v to about 5% w/v.

Triggers

[0033] The fibers described herein are responsive to at least one of a biotic and an abiotic trigger. The biotic and the abiotic trigger each cause the release of the at least one antimicrobial active ingredient from the fiber.

[0034] An example of a biotic trigger is at least one enzyme secreted by at least one microorganism, such as bacteria. Examples of enzymes secreted by the at least one microorganism include but are not limited at least one of a cellulase, a protease, and an amylase, including microbial cellulases, microbial proteases (e.g., pepsin-like enzymes produced by Aspergillus, Penicillium, Rhizopus, and Neurospora and rennin-like enzymes produced by Endothia and Mucorspp.), and microbial amylases. See, e.g., DOI: 10.5772/intechopen.84531 (2019); Microbiol Mol Biol Rev. 1998 Sep; 62(3): 597-635; and Process Biochemistry 38: 1599-1616 (2013).

[0035] As used herein, the term “bacteria” generally refers to gram-positive and gram-negative bacteria. Gram-positive bacteria include, but are not limited to, mycobacteria. Mycobacteria, in turn, include, but are not limited to, M. africanum, M. avium, M. bovis, M. chelonei, M. farcinogenes, M. flavum, M. fortuitum, M. haemophilum, M. intracellulare, M. kansasii, M. leprae, M. lepraemurium, M. marinum, M. microti, M. parafortuitum, M. paratuberculosis, M. phlei, M. scrofulaceum, M. senegalense, M. simiae, M. smegmatis, M. thermoresistibile, M. tuberculosis, M. ulcerans, and M. xenopi. Other gram-positive bacteria include, but are not limited to, gram-positive cocci including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Group A Streptococci, Group B Streptococci, Group C Streptococci, Group G Streptococci, and vancomycin resistant Enterococci (VRE). Other gram-positive bacteria include Listeria spp. e.g., Listeria monocytogenes, Listeria innocua), Clostridium spp. (e.g., Clostridium perfringens and Clostridium botulinum), and Bacillus cereus.

[0036] Gram-negative bacteria include, but are not limited to, Pseudomonas aeruginosa, Pseudomonas spp., Serratia marcescens, E. coli, Salmonella spp., Campylobacter jejuni, Shigella, and Vibrio spp.

[0037] The bacteria can be at least one of Escherichia coli, Listeria innocua, Listeria spp. Salmonella enterica, Salmonella spp., Mycobacterium parafortuitum, Saccharomyces cerevisiae, Pseudomonas aeruginosa, Pseudomonas spp., Serratia marcescens, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermitis, methicillin-resistant Staphylococcus epidermidis (MRSE), Propionibacterium acnes, Group A Streptococci, Group B, Streptococci, Group C Streptococci, Group G Streptococci, vancomycin resistant Enterococci (VRE), and Acinetobacter baumannii.

[0038] An example of an abiotic trigger is an increase in relative humidity beyond a threshold percentage (e.g., a RH sufficient to cause Al dissociation from CD-ICs as the hydrogen bonds are weakened between the hydrophobic Al molecules and CD). For example, the abiotic trigger is a relative humidity of 80% or greater, 85% or greater, 88% or greater, 90% or greater, 95% or greater, from about 80% to about 95%, about 85% to about 95%, about 80% to about 90% or from about 82% to about 89%.

[0039] The at least one of a biotic and an abiotic trigger can cause the release of the at least one antimicrobial active ingredients from the fiber and a concomitant reduction of colony forming units of at least about 1 log, at least about 1 .5 logs, at least about 2 logs, at least about 3 logs, at least about 4 logs, at least about 5 logs, from about 1 log to about 5 logs, about 1 .5 logs to about 2.5 logs, about 2 logs to about 4 logs, about 1 .5 logs to about 3 logs, about 2 logs to about 5 logs or about 1 .5 logs to about 5 logs over after about 24 hour contact time of the at least one antimicrobial active ingredients released from the fibers described herein with at least one microorganism (e.g., bacteria). [0040] The at least one of a biotic and an abiotic trigger can cause a cumulative release (%) of the at least one antimicrobial active ingredients from the fibers described herein over a period of time. The cumulative release can be from about 1% to about 90%, about 1% to about 10%, about 5% to about 15%, about 10% to about 30%, about 15% to about 20%, about 12% to about 25%, about 13% to about 18%, about 20% to about 40%, about 20% to about 60%, about 25% to about 75%, about 25% to about 60%, about 30% to about 90% or about 40% to about 80% per unit of time, such as a time period of from about 1 hour to about 5 hours, about 1 hour to about 24 hours, about 2 hours to about 48 hours or about 3 hours to about 10 hours. As used herein, the term “cumulative release (%)” generally refers to the overall amount of the at least one antimicrobial active ingredients released in from the fibers described herein, e.g., in a four hour period, based on the total content of the at least one antimicrobial active ingredients that could be released from the fibers. Thus, for example, the cumulative release (%) of thymol from the fibers described herein as a function of time is shown in FIG. 6A. The release of thymol reached a plateau at 4 hours and remained relatively constant at 13% and 23% for PBS and enzymatic conditions, respectively.

Articles

[0041] The disclosure relates to articles comprising a plurality of the fibers described herein, whether coated onto or otherwise incorporated into (e.g., into a polymer that makes up the article). The articles can be beverage and consumer goods packaging (e.g., packaging used for produce and meats) made of any suitable material including cellulose, nanocellulose, metal, plastic, glass, paperboard, composite, or of single-layer or multilayer materials. The articles can also be barrier plastics that provide increased resistance to water vapor transmission and the permeation of various gases. Among such materials, which have been made commercially available as single layer (monolayer) or multiple layers for food beverage packaging are polymeric coextruded or laminated sheet and film material such as commercially available under the designations "EVAL" (or "EVOH") (ethylene vinyl alcohol copolymer). High barrier materials of utility are PET film (MYLAR), polyvinylidene chloride (PVDC), and a new class of barrier resins formed of amorphous nylon known as "SELAR PA". Other materials having certain barrier capabilities are high density polyethylene (HDPE), ultra high molecular weight polyethylene (UHMWPE), polypropylene (PP) and oriented polypropylene (OPP), not to mention high impact and other low to medium barrier materials such as polystyrenes (PS) as well as various copolymers such as, for example, acrylonitrile butadiene-styrene (ABS), and high nitrile barrier resins including styrene acrylonitrile (SAN), "BAREX" polymer and polyethylene terephthalate (PET and PETG). Moreover, there are also commercially available a combination of materials with barrier monolayers or employing relatively low barrier materials, such as low density polyethylene (LDPE), HDPE, PP, and HIPS.

[0042] The article can be a film, such as a food-grade film or a food-grade container. The article can also be a food-grade container that is subsequently covered with a food-grade film.

[0043] The disclosure also relates to medical/healthcare applications of the fibers described herein including, but not limited to, applications of the fibers described herein in films or disposable textiles that can be applied to wounds (e.g., first aid applications on fresh wounds, open wounds or wounds that are healing, for example, post-surgery).

[0044] As used herein, the term “produce” includes, but is not limited to, fruit and vegetables including: avocado and pome fruits such as apples and pears; nectarines and peaches; vegetables from the Solcanaceae family, for example, potatoes, peppers, eggplants and tomatoes; vegetables from the Alliaceae family, such as onions; vegetables from the Brassiaceae family also referred to as the Cruciferae family, for example cabbage; vegetables from the Cucurbitaceae family, for example, cucumbers; vegetables from the Apiaceae family also referred to as the Umbelliferae family, for example celery; the Compositae family, also referred to as the Asteraceae family, for example, lettuce; and edible fungi of the Ascomycetes/Basidiomycetes classes.

[0045] As used herein, the term “meat” includes fish, poultry (e.g., chicken, turkey, and the like), beef, deer, and the like.

[0046] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1 % to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0047] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading can occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0048] In the methods described herein, the steps can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing X and a claimed step of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0049] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

[0050] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0051] The term “substantially no” as used herein refers to less than about 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1 %, 0.5%, 0.1%, 0.05%, 0.001 %, or at less than about 0.0005% or less or about 0% or 0%.

[0052] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Thus, the description is not intended and should not be construed to be limited to the examples given but should be granted the full breadth of protection afforded by the appended claims and equivalents thereto. In addition, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Accordingly, the foregoing description of or illustrative embodiments is provided for the purpose of illustrating the principles of the present disclosure and not in limitation thereof and can include modification thereto and permutations thereof.

Examples

[0053] The disclosure can be better understood by reference to the following examples which are offered by way of illustration. The disclosure is not limited to the examples given herein.

Materials and Methods

[0054] The fibers were developed from cellulose nanocrystals (CNCs), zein, and starch incorporated with nature derived antimicrobials and their associated CD- ICs. All compounds and solvents used for the fiber “green” synthesis process are FDA approved GRAS materials to ensure no toxicity, sustainability and scalability. The morphological and physicochemical properties of CD-ICs and fibers, and dissolution kinetics of Als from fibers in the presence of the RH and enzymatic triggers were assessed as a function of time using a range of advanced analytical methods. The antimicrobial efficacy of multi-stimuli fibers was also assessed by standard microbiological methods. The developed responsive biopolymer-based antimicrobial fibers offer a novel and effective means to promote food shelf life and quality and to serve as an important tool in maintaining food security and safety.

[0055] Synthesis of cyclodextrin-inclusion complexes (CD-ICs): CD-ICs were synthesized to provide relative humidity (RH) functionality to the responsive fibers. For this purpose, y-CD, which is an FDA GRAS approved compound, and nature derived GRAS antimicrobials (thyme oil, sorbic acid and nisin) were used to synthesize the RH responsive CD-ICs. Such antimicrobials have a broad antimicrobial efficacy against a range of food related microorganisms, including Escherichia coll, Staphylococcus, Listeria monocytogenes, Candida albicans, Bacillus, and lactic acid bacteria. ^32-35 It is also worth noting that among native CDs, y-CD is known for its use in food related applications and its minimal toxicity. ³⁶ In addition, while deciding the molar ratio between the y-CD and antimicrobial molecules, the size of each Al was considered so as to maximize the loading efficacy. As such, the molar ratios of y-CD/thyme oil-IC and y-CD/sorbic acid-IC were 1 :2, whereas the molar ratio of y-CD/nisin-IC was 4:1 .

[0056] Physicochemical characterization of CD-ICs: XRD analysis was used to characterize the crystallinity of y-CD, y-CD/thyme oil-IC (1 :2), sorbic acid, y- CD/sorbic acid-IC (1 :2), nisin, and y-CD/nisin-IC (4:1 ) and more importantly, to confirm the formation of CD-ICs. (FIG. 3A) Sorbic acid has its characteristic 29 peaks at 11.8°, 13.4°, and 23.2°, whereas nisin has at 29 32.0°. The absence of characteristic of sorbic acid and nisin peaks in y-CD/sorbic acid-IC (1 :2) and y- CD/nisin-IC (4:1 ) indicates successful CD-IC formation. Furthermore, the major cage type crystalline characteristic peaks of y-CD are observed at 29 5.5°, 6.5°, 9.6°, 10.4°, 11.5°, 12.6°, 14.1 °, 15.6°, 16.7°, and 19.0°. Pristine CDs have cage type crystalline structure, but when CD-ICs are formed, a channel type crystalline structure form with characteristic peaks. ²⁸ In the CD-ICs, characteristic channel type peaks of y-CD-IC were observed at 29 7.8°, 14°-17°, and 22° for y-CD/thyme oil-IC, y-CD/sorbic acid-IC, and y-CD/nisin-IC, respectively. Collectively, these findings confirm that CD-ICs of thyme oil, sorbic acid, and nisin were successfully synthesized.

[0057] FIG. 3B shows the ATR-FTIR spectra of the CD-ICs and further confirms CD ICs synthesis. First, the peak at 1696 cm ^-1 confirms the presence of C=O stretching of sorbic acid ³⁷ in the sorbic acid-CD-IC. Second, the intensity increments of the peak at 1250 cm' ¹ corresponds to C-0 stretching of thyme oil in the spectra of the thyme oil-CD-IC ³⁸ and the peak at 1643 cm ^-1 in the spectra of nisin-CD-IC ³⁹ confirms the presence of thyme oil ³⁸ and nisin. Third, the characteristic peaks of CDs were evident at 3000-3630 cm ^-1 (OH stretching), 2929cm ^-1 (C-H stretching), 1643cm ^-1 (H-OH bending), 1150 cm ^-1 (C-O-C glycosidic antisymmetric stretching), 1078 cm ^-1 (C-0 stretching), 1020 cm ^-1 (C-C stretching). It is worth noting that the CD associated peaks shifted from 3281 cm ^-1 to 3310 cm ^-1 and 1150 cm ^-1 to 1154 cm ^-1 for thyme oil-CD-IC, 1018 cm ^-1 to 1022 cm ^-1 for sorbic acid-CD-IC, and 1018 cm ^-1 to 1021 cm ^-1 for nisin-CD-IC. These peak shifts observed in each CD-IC highlight the interactions between CD and the guest molecules and further confirms successful CD-IC formation. ⁴⁰

[0058] “Green” synthesis of stimuli responsive fibers: Table 1 summarizes the Al concentrations (%), polymer composition (%), and other operational parameters used for the synthesis of multi-stimuli responsive fibers by electrospinning, as well as for the control pristine fibers (no Als), enzyme responsive fibers (no CD-ICs, only free Als) and RH responsive fibers (no free Als, only CD-ICs). Table 1

[0059] Morphological characterization of fibers: SEM images of pristine, enzyme responsive, RH responsive, multi-stimuli responsive fibers are given in FIGS. 4A-4D, along with the average fiber diameter (AFDs) and diameter distribution graphs. All fibers exhibited bead-free and uniform morphology by SEM. The AFDs of pristine, enzyme responsive, RH responsive, and multi-stimuli responsive fibers were calculated from SEM images as 285 ± 60 nm, 205 ± 35 nm, 290 ± 50 nm, and 225 ± 50 nm. It is worth noting that AFDs were constant across various fiber types at around 200-300 nm by adjusting the operational electrospinning parameters. Fiber diameter distributions ranged from 100-500 nm, 100-350 nm, 150-450 nm, and 100-450 nm pristine, enzyme responsive, RH responsive, multi-stimuli responsive fibers, respectively.

[0060] Physicochemical characterization of the fibers: Specific surface area (SSA, m ² /g), average pore radius (nm), and total pore volume cc/g) of the fibers were determined by BET (Table 2).

> [0061] The multipoint BET surface area of pristine, enzyme responsive, RH responsive, multi-stimuli responsive fibers were 12.1 m ² /g, 19.3 m ² /g, 10.4 m ² /g, and 10.9 m ² /g, respectively. The SSA of the enzyme responsive fibers are greater than those of the others due to the lower AFD (205 nm), whereas the SSA of other fibers are all quite similar. The slight changes in AFDs resulted in the SSA differences in the fibers. The average pore radius for pristine, enzyme responsive, RH responsive, multi-stimuli responsive fibers were 2.18, 1 .64, 1 .56 and 2.16 nm, respectively. Last, the total pore volumes were 1 .32 x 10 ^-2 cc/g, 1 .58 x 10 ^-2 , 0.81 x 10 ^-2 , and 1.18 x 10 ^-2 cc/g for pristine, enzyme responsive, RH responsive, multistimuli responsive fibers, respectively. These total pore volume values are consistent with the literature as pore volume tends to increase with the SSA.

[0062] FIG. 5A shows the crystallinity of pristine, enzyme responsive, RH responsive, multi-stimuli responsive fibers using XRD. All fibers exhibited broad amorphous peaks for zein and starch at 29 5°-12.5° and 16.5°-25.0° respectively. In electrospinning, crystalline formation for small molecules is significantly hindered due to the rapid solvent evaporation during the process. ⁴² This is the case for the enzyme responsive fibers, where crystalline peak of nisin at 2930° was not evident. However, this may also be related to the miniscule quantity of nisin and the dominance of the amorphous polymers in the fibers. In addition, RH responsive and multi-stimuli responsive fibers show peaks at 29 7.8° and 14°-17° due to the channel type crystalline nature of CD-ICs. However, these peaks are more prominent in the case of multi-stimuli responsive fibers due to the lower concentration of polymers (41%, w/v) present related to the enzyme responsive fibers (47%, w/v). In summary, the XRD patterns reveal the successful incorporation of CD-ICs in the electrospun fibers and aligns well with the literature for y-CD-ICs. ⁴³

[0063] The chemical composition of the pristine, enzyme responsive, RH responsive, multi-stimuli responsive fibers was investigated by ATR-FTIR (FIG. 5B). Thyme oil has peaks at 2960-2850 cm ^-1 belonging to C-H stretching vibration of CH ₃ . ³⁸ The enzyme responsive, RH responsive, and multi-stimuli responsive fibers exhibited this broad peak which confirms the presence of thyme oil in the fibers. Nisin has two peaks at 1650 cm ^-1 and 1522 cm ^-1 which are attributed to the amide I and II bonds in the structure. ³⁹ These peaks are also evident in the enzyme responsive, RH responsive, and multi-stimuli responsive fibers, confirming the presence of nisin in the fibers. It is also worth noting that these same peaks in the spectra of pristine fibers are attributed to zein, which also has amide I and II bonds in its structure. Finally, the C=O stretching band of citric acid ⁴⁴ is observed at the spectra in 1721 cm ^-1 and as expected, this peak was only observed in enzyme responsive and multi-stimuli responsive fibers. The trans alkene peaks of sorbic acid are observed at 1000 cm ^-1 in the RH responsive and multi-stimuli responsive fibers, which have sorbic acid in the CD-IC. In summary, the FTIR spectra further confirms the successful incorporation of all Als in the various fibers.

Triggered release kinetics of antimicrobial Als:

Example 1

[0064] Bacterial enzyme triggered Al release: The cumulative release (%) of thymol from enzyme responsive fibers as a function of time is shown in FIG. 6A. The release of thymol reached a plateau at 4 hours and remained relatively constant at 13% and 23% for PBS and enzymatic conditions, respectively. Importantly, a significantly higher guantity of thymol was released in the presence of enzymes as compared to PBS, highlighting the responsiveness of the fibers and demonstrating the different mechanisms involved in cargo. In PBS, the fibers released thymol by diffusion, but in the enzyme solution, the thymol is released both by diffusion and by biopolymer degradation by enzymes. Of interest is FIGS. 10A-10C which shows the enzymatic degradation of the fibers at 0.1 , 1 , and 3 U/mL. The degradation of nanofibers at 1 U/mL increases significantly as compared to low level of enzyme (0.1 U/mL), in which degradation was minimal. However, when the concentration further increases to 3 U/mL complete degradation of nanofibers is observed. Therefore, 1 U/mL was chosen to confirm the enzymatic release of Als from the fibers.

Example 2

[0065] Relative humidity (RH) triggered release: The remaining concentration of thymol in RH responsive fibers at 50% and 95% RH levels are shown in FIG 6B. The thymol concentration reached a plateau at ~7.5 ppm after 4 hours for the 95% RH condition, significantly lower than the -11.5 ppm level for the 50% RH condition. This confirms the material behaves as designed, releasing more thymol at higher humidity. In addition, SEM images (FIGS. 11 A and 11 B) also demonstrate that RH responsive fibers are intact and not wetted at the end of the 4 hours at 50% RH, whereas the fibers at 95% RH maintain their fibrous morphology but are swollen and merged. Statistical analysis between the groups in each responsive release experiment was performed using unpaired t-test.

[0066] Al release kinetics from fibers in agueous food simulant (water): The cumulative release (%) of thymol from the multi-stimuli responsive fibers into water, a non-acidic agueous food simulant, as a function of time is shown in FIG. 12. There is a rapid Al release in 6 hours (21%) and then a plateau is reached. This is attributed to the high specific surface area of fibers as shown by BET, as well as the wettability of the fibers owing to the nature of polymers used in the synthesis.

Example 3

[0067] Antimicrobial efficacy of multi-stimuli responsive fibers: FIGS. 7A-7C summarizes the antimicrobial efficacy of multi-stimuli responsive fibers against E. coli, L. innocua, and A. fumigatus. Aluminum foil and pristine fibers were included as controls. Two levels of fiber mass per surface area were tested: 2.5 mg/cm ² and 1 .25 mg/cm ² .

[0068] E. coli and L. innocua: In general, aluminum foil and pristine fibers did not support or inhibit the growth of E. coli', the population fluctuation within 24 hours and was less than 1 log. The responsive fibers reduced E. coli and L. innocua growth by 5 logs (under the detection limit) at 1 hour and 24 hours contact time. Notably, when the mass per surface area of multi-stimuli responsive fibers decreased from 2.50 mg/cm ² to 1 .25 mg/cm ² , the antimicrobial efficacy against E. coli reduced from ~5 log to ~1 log at a contact time of 1 hour. However, for L.innocua, the treatment with a lower mass per surface area did not show reduced antimicrobial efficacy at both 1 hour and 24 hours and exhibited 5 logs reduction for both contact time. Similar fiber mass per surface dependency was found in our previous publication using antimicrobial zein fibers. ⁹

[0069] In summary, the multi-stimuli responsive fibers developed in this study showed an excellent inactivation efficacy against both Gram-negative bacteria and Gram-positive bacteria with 24 hours of contact time. The antimicrobial activity of multi-stimuli responsive fibers seem to be fiber mass per surface area and time dependent for E.coli but not L. innocua although that is dependent on the species of bacteria. However, 24 hours contact time is sufficient to release enough Als for bacterial inactivation even at the miniscule amounts of Als released. In addition, these results are also in consistent with the results reported in the literature for nature derived antimicrobials used in this study .

[0070] Antifungal efficacy. The antifungal efficacy of the multi-stimuli responsive fibers was also assessed. Since fungal spores are generally more resistant to antimicrobial agents, the higher fiber mass per surface area (2.50 mg/cm ² ) was used for the antifungal efficacy assessment. Aluminum foil and pristine fibers were used as controls and had minimal influence of the fungal growth (with less than 0.2 log population change after 24 hours contact time). As shown in FIG. 7C, a significant population reduction (1.4 log) of A. fumigatus was evident after 24 h contact time. The antifungal efficacy of the multi-stimuli responsive fibers may be attributed to the presence of thyme oil in the Al cocktail, since thymol has proven antifungal efficacy. Previous studies have reported thymol to have a minimum inhibitory concentration (MIC) of 150-190 pg/mL and a minimum fungicidal concentration (MFC) of 175-384 pg/mL against A. fumigatus.

Example 4

[0071] Relative Humidity (RH) triggered antimicrobial activity:

[0072] The antimicrobial efficacy of relative humidity (RH) responsive fibers with the change of RH conditions is shown in FIG. 8. In general, aluminum foil and pristine fibers did not support or inhibit the growth of E.coli at low (50% RH) or high (95% RH) levels. With 1 hour contact time, RH responsive fibers was able to achieve 5 logs (under the detection limit) reduction of E. coli at 95% RH, while only 1.9 log reduction at 50 % RH. Based on the statistical analysis, there was a significant (P < 0.05) difference in the population reduction of E. co//between 50 % RH and 95 % RH condition at both contact times (15 min and 1 hour) (labeled with *). At the same RH level, the population reduction of E. coli increased significantly (P < 0.05) with the increase of contact time, indicating that the antimicrobial agents incorporated in RH responsive fibers released in a time-dependent manner. However, the release rate of Al in the RH responsive fibers was much higher at 95% RH than that at 50% RH. At 95% RH, RH responsive fibers can eliminate E. coli from the aluminum foil within 1 hour contact time, indicating a sufficient release of antimicrobial agents incorporated in fibers.

[0073] RH responsive fibers showed significant lower antimicrobial activity against E. coli at 50% (~2 log reduction) compared with that at 95% (> 5 log reduction). Most of the antimicrobial agents incorporated in CD-IC, especially thymol, did not release from the core fiber at a low RH level. Based on our findings from FIG. 6B, the remaining thymol concentration at 95% RH was lower than that at 50%, indicating that more thymol was released from the fibers to provide antimicrobial activity. Previous studies also showed that the release of thymol was faster at high RH (97%) and high temperature (75°C) . ⁸⁵² Our antimicrobial results further prove the evidence that this type of fiber is RH responsive, and a strong antimicrobial activity can be achieved in a fast manner when the environmental RH is high.

[0074] FIGS. 1A-1 C outline the representation of the enzyme- and RH-triggered strategy, preparation of the polymer solution composed of zein, starch, cellulose nanocrystals (CNCs), nature-derived free active ingredients (Als), and cyclodextrin-inclusion complexes (CD-ICs) of Als; and synthesis of multi-stimuli responsive fibers by electrospinning. Example 5

[0075] Synthesis of CD-ICs of antimicrobial Als: CD-ICs of nature derived antimicrobial Als (thyme oil, sorbic acid, nisin) were synthesized using the coprecipitation method. The Als were selected because of their FDA GRAS status and their ability to inactivate a broad range of food related pathogenic and spoilage microorganisms. ^25-27 A schematic representation of the synthesis is given in FIG. 2. For y-CD/thyme oil-IC (1 :2) and y-CD/sorbic acid-IC (1 :2), y-CD (Wacker, Cavamax W8 Food) was first dissolved in water for 10 minutes, and thyme oil (Sigma Aldrich, W306509) or sorbic acid (TCI, S0053) was then added into the solution at a 1 :2 molar ratio (CD:thyme oil or CD:sorbic acid). For y-CD/nisin-IC (4:1 ), y-CD dissolved in water for 10 minutes and was then added into nisin (Alfa Aesar, J66370) that had previously been mixed with a small quantity of thyme oil. It is worth noting that nisin was initially mixed with a small volume of more hydrophobic compound (thyme oil) prior to mixing with y-CD to further facilitate IC formation. The molar ratio of CDmisin was 4:1 due to large molecular weight of nisin compared to CD. After stirring the three solutions overnight at room temperature, they were incubated at 4°C for 24 hours. CD-ICs precipitated at the bottom of the bottle and were collected by vacuum-filtration, followed by drying in hood for 48 hours. The resulting solids were ground into fine powders with an agate mortar.

Example 6

[0076] Physicochemical characterization of CD-ICs of Als: The crystallinity of y-CD, y-CD/thyme oil-IC (1 :2), sorbic acid, y-CD/sorbic acid-IC (1 :2), nisin, and y- CD/nisin-IC (4:1 ) were investigated by X-ray diffraction (XRD, Bruker D2 Phaser) in the 26 range of 5°-40° using Cu Ka radiation. XRD analysis of thyme oil could not be performed because of its liquid nature. The cage type crystalline structure of CDs converts to a channel type crystalline structure when CD-IC is formed. These characteristic peaks of cage and channel type crystalline structure allows determination of successful CD-ICs synthesis. ²⁸ The chemical composition of y- CD, thyme oil, y-CD/thyme oil-IC (1 :2), sorbic acid, y-CD/sorbic acid-IC (1 :2), nisin, and y-CD/nisin-IC (4:1 ) was analyzed using attenuated total reflectance-Fourier transform infrared spectrometry (ATR-FTIR, Thermo Scientific Nicolet IS50). The spectra were recorded between 4000 cm' ¹ and 400 cm' ¹ at a resolution of 4 cm ^-1 and 64 scans/sample were taken.

Example 7

[0077] “Green” synthesis of electrospun fibers [0078] Synthesis of pristine fibers: Polymers such as zein, which is a protein, and starch were chosen to form the backbone of the fiber because enzymes released from bacteria such as protease, amylase, and cellulase will degrade these materials. Zein which was used in a previous study and starch with a lower amylose content (approximately 55%) was selected. Acetic acid was used as a solvent for preparing the cellulose nanocrystals (CNCs) incorporated zeimstarch solutions. Notably, cellulose is not soluble in most organic solvents used in electrospinning, ²⁹ thus, CNCs were incorporated into the zeimstarch fibers to yield responsiveness to cellulose degrading enzymes. Last, in contrast to toxic organic solvents usually preferred in electrospinning, acetic acid is a GRAS solvent and can dissolve both the polymers and the selected Als.

[0079] CNCs (140 nm x 20 nm, length x diameter, 3.5%, w/w content) were synthesized as previously described. ³⁰ After vortexing for 10 seconds, 1 mL of suspension was mixed into 4 mL of acetic acid in a vial and the solution was stirred for another 20 minutes. Subsequently, zein (zein from maize, Sigma-Aldrich, Z3625) and starch (Ingredion, Hylon V, corn starch, 55% amylose content) were added at 85:15 ratio (zeimstarch) into the solution. Pristine fibers were synthesized using electrospinning by loading the solutions in a 10 mL plastic syringe (BD Luer- Lock tip) and were supplied by a syringe pump through a stainless-steel singleneedle injector (diameter: 0.6 mm, 90° blunt end) towards the collector. High voltage was applied to both the needle injector tip and collector from the power supply. The electrospinning process parameters such as flow rate, needlecollectordistance, total polymer concentrations, and applied voltage were modified to obtain bead-free fibers. The fibers were randomly deposited on aluminum foil (20 x 20 cm ² ). The mass of the fibers per surface area was adjusted to 2.5 mg/cm ² by adjusting electrospinning time.

[0080] Synthesis of multi-stimuli responsive fibers: Since the goal was to produce both enzyme and RH response functionalities to the fibers, we incorporated a cocktail of both free Als and CD-ICs of Als. The cocktail comprised of the nature- derived free Als thyme oil, citric acid, and nisin, as described in our previous study ⁹ . Separately, CD-ICs of thyme oil, sorbic acid, and nisin were incorporated into fibers to provide RH response due to the unique structure of CDs.

[0081] These multi-stimuli responsive fibers were synthesized by incorporating both the cocktail of free Als and CD-ICs of Als into the solution of CNCs, zein, and starch by direct solution integration. ⁹ First, a cocktail of Als (1%, w/v, thyme oil; 5%, w/v, citric acid (VWR, citric acid 10% w/v aqueous solution); 0.2%, w/v, nisin) was dissolved in acetic acid. The composition of the Al cocktail and its optimization in terms of food related antimicrobial efficacy was determined in our previous study. ⁹ The CNC solution was vortexed for 10 seconds and was then mixed into this solution, followed by stirring for 20 minutes. Then y-CD/thyme oil-IC (1 :2), y- CD/sorbic acid-IC (1 :2), and y-CD/nisin-IC (4:1 ) corresponding to 1% (w/v) thyme oil, 0.5% (w/v) sorbic acid, 0.2% (w/v) nisin were added into the solution while stirring. Finally, zeimstarch (85:15) was added into the solution and after a complete dissolution of all components, electrospinning was performed. The solution was loaded into a 10 mL plastic syringe (BD Luer-Lock tip) and process parameters including flow rate, needle-collector distance, and applied voltage were adjusted to yield bead-free multi-stimuli responsive fibers that were randomly deposited them on aluminum foil by electrospinning. Fibers with free Als cocktail only or with CD-ICs of Als only were also synthesized as control samples and were denoted as enzyme responsive fibers or RH responsive fibers, respectively. Two separate mass per surface area fibers were synthesized by varying the deposition time:1 .25 mg/cm ² and 2.50 mg/cm ² .

[0082] Morphological/size characterization of fibers: Morphological characterization of the fibers was performed by scanning electron microscopy (SEM, Zeiss FESEM Ultra Plus). Prior to imaging, the fiber samples were cut into small pieces and were mounted on a stub using double-sided carbon tape. The average diameter of the fibers was calculated using Imaged Software (n = 100) and the results are given as average ± standard deviation. The specific surface area (m ² /g), average pore radius (nm), and total pore volume (cc/g) of the fibers were measured by a Brunauer-Emmett-Teller (BET, Quantachrome NOVA touch LX4) surface area analyzer. Prior to BET measurement, fiber samples in 9 mm cell were degassed at 313.15° K for 12 hours. Last, low temperature (77.35° K) nitrogen adsorption isotherms were measured at relative pressures from 0.005 to 1.00.

Chemical and crystallinity characterization of fibers

[0083] The crystallinity of the fibers was investigated by XRD (Bruker D2 Phaser) in the 29 range of 5°-40° using Cu Ka radiation. The chemical composition of fibers was investigated using ATR-FTIR (Thermo Scientific Nicolet IS50) and the spectra were recorded between 4000 cm ^-1 and 400 cm' ¹ at the resolution of 4 cm ^-1 and 64 scans/sample were taken.

Example 8

Al Release Kinetics

[0084] Determination of Al content in the fibers: The Al content of fibers was determined by dissolving 15 mg of fiber samples in 10 mL of ethanol. The solutions were then filtered using cellulose acetate filter (0.45 gm), and analysis was performed by liquid chromatography with high resolution mass spectrometry (LC/HRMS) as described below. The Al concentration is calculated and compared to the theoretical values from the concentrations in solution used during electrospinning and is then used in the calculation of released thymol in the enzyme and multi-stimuli responsive fibers.

[0085] Liquid chromatography with high resolution mass spectrometry (LC/HRMS) analysis: The Al release kinetics under various stimuli were quantified in controlled dissolution experiments. Thymol, the major compound of thyme oil (60%), was selected as a surrogate of Al quantitation for chemical analysis by LC/HRMS (Dionex Ultimate 3000 Liquid Chromatograph interfaced to a Thermo Q-Exactive HRMS). The LC was run was on an Agilent SB-C18 2.1 x 150 column at 0.2 mL/min. Mobile phase A was water with 1% formic acid; mobile phase B was acetonitrile with 1% formic acid. After a 1 min hold at 50% B, there was a linear gradient for 9 minutes to 95% B where it was held for 3 minutes followed by a 3- minute re-equilibration at 50% B. The MS was operated using positive electrospray at 3.5 kV with the capillary and auxiliary gas temperatures set to 300°C, and the gas flows were as follows: sheath 50, auxiliary 15, and sweep 10. For quantitation of thymol, we isolated a 3 m/z window around the (M+H)+ ion, fragmented it at a collision energy of 20, collected a full scan of the fragments between m/z = 50 to m/z = 175 at a resolution of 17500 and monitored the sum of the m/z = 91 .0547 and m/z =109.09656 fragments. The area of each peak was converted to concentration (ppm) using standard calibration curves. The release experiments were carried out in triplicate, and the results were reported as average ± standard deviation. In more detail:

[0086] Bacterial enzyme triggered Al release kinetics from fibers: First, enzyme responsive fibers that were incorporated with only free Als (no CD-ICs) were placed in enzyme solution in phosphate buffered saline (PBS, VWR) at 0.1 U/mL, 1 U/mL, and 3 U/mL concentration for 12 hours. Enzyme solutions prepared in PBS consisted of protease (Sigma, P5147, protease from Streptomyces griceus), a-amylase (Sigma, 10065, amylase from Aspergillus oryzae) and cellulase (Sigma, C1184, cellulase from Aspergillus niger) at 1 :1 :1 ratio. Fibers were immersed in enzyme solutions in an incubator at 37°C and shaken for 12 hours. After incubation, the morphology of the fibers was observed by SEM (Zeiss FESEM Ultra Plus). Separately, 15 mg of enzyme responsive fibers were immersed in 10 mL of 1 U/mL enzyme solution (protease:amylase:cellulase, 1 :1 :1 ). The samples were kept in an incubator at 37° C for 12 hours. As a control, enzyme responsive fibers were also immersed in 10 mL of PBS without enzymes. A 1 mL of aliquot sample was withdrawn at 1 hour, 4 hour, and 12 hour and was replaced with the same volume of either enzyme or PBS solution. The solutions were passed through a cellulose acetate filter (0.45 pm), and were analyzed by LC/HRMS as described above. The measured concentration of thymol in the fibers was found to be 96% of the expected theoretical value, demonstrating highly efficient loading, and were used to convert concentration of released thymol into percent values. [0087] RH triggered Al release kinetics from fibers: 15 mg of RH responsive fibers incorporated with only CD-ICs of Als (no free Als) were added into two chambers with different RH values. The RH in the chambers were adjusted to 50% and 95% using saturated solutions of Mg (NO ₃ )2 and KNO ₃ (VWR chemicals, BDH Prolabo, Australia) to achieve a stable low and high RH conditions at room temperature, respectively. At three time points over 4 hours, samples were taken out of the chamber and placed in 10 mL of ethanol to extract the thymol left in the fibers. The concentration of thymol in the fibers was also measured by LC/HRMS and was found to be at 91 % of the expected theoretical calculation. The release experiments were carried out in triplicates, and the results are given as remaining concentration of thymol (ppm) in the fibers for the two RH conditions. Additionally, SEM (Zeiss FESEM Ultra Plus) images were also taken after fiber removal from the chambers for 4 hours to observe any changes in their morphology as a function of RH.

[0088] Al release kinetics from fibers into water: The release kinetics experiments were also performed by immersing the multi-stimuli responsive fibers (15 mg) in 10 mL of water to simulate an aqueous based food environment. A 1 mL of aliquot samples was withdrawn at 6 hour, 12 hour, and 24 hour and was then replaced with the same volume of water. The sample solutions were then filtered using cellulose acetate filter (0.45 pm) and analyzed by LC/HRMS. The initial Al concentration of fibers were determined to be 89% and was used to calculate percentage of thymol released.

[0089] Assessment of Antimicrobial efficacy of multi-stimuli responsive fibers: [0090] Strain information: Escherichia coli ATCC 25922 (E.coli) and Listeria innocua ATCC 33090 (L. innocua), Aspergillus Fumigatus ATCC 96918 (A. fumigatus) were used in this study as representative Gram-negative, Grampositive, and fungal food pathogens, respectively. E. coli ATCC 25922 has been regarded as a non-pathogenic surrogate organism for the pathogenic E. coli O157:H7 and L. innocua surrogate for the pathogenic L. monocytogenes. A. fumigatus is a widely spread fungus which causes food spoilage, as well as produces mycotoxins that cause human illness. ³¹ E. coli and L. innocua were resuscitated and streak plated from stock solutions and maintained on tryptic soy agar (TSA; Hardy Diagnostic, Santa Maria, CA) at 4°C. A single colony from TSA was transferred into 10 mL of tryptic soy broth (TSB; Hardy Diagnostic, Santa Maria, CA). After incubation at 37°C for 24 hours, the bacterial broth was centrifuged at 3000 rpm for 15 minutes (Allegra 6R, Beckman Coulter, Indianapolis, IN). After discarding the supernatant, 2 mL sterile 0.1 % (w/v) peptone water was used to resuspend the bacterial pellet. The cell density was adjusted to ~10 ⁸ colony forming unit (CFU)/mL with peptone water with a final optical density (O.D.) 600 value as 0.2. Freeze-dried A. fumigatus was rehydrated in sterile deionized water and further translated onto a Malt Extract Agar (MEA) and incubated at 30°C for 3 days. To produce mature conidia, a single colony was further incubated on MEA at 30°C for 7 days until the colony became dark green. Mature spores were harvested from the lawn and then diluted with deionized water. The final concentration of spores was adjusted to ~10 ⁷ CFU/mL using a manual hemocytometer (Diagnocine, Hackensack, NJ).

[0091] Direct contact assay: The direct contact assay to assess the antimicrobial efficacy test of fibers was conducted as described previously with minor modifications (FIG. 9A). ⁹ Briefly, 100 pL of the microbial culture concentration was diluted in 10 mL of agar slurry (0.85% NaCI, 0.3% agar). Three hundred pL of the inoculated agar slurry was then inoculated onto a 2 x 2 cm ² fibers deposited in aluminum foil and then placed into a 6-well plate (Thermo Scientific™, 145380). After 5 minutes contact time, the agar slurry formed a gel layer on the top of the fibers with a thickness less than 1 mm. The plates were then transferred into an incubator at 37°C for 1 hour and 24 hours. To avoid drying out of the gel during exposure, a reservoir full of water was used to maintain the RH at approximately 70%.

[0092] Enumeration: After 1 hour and 24 hour, each test sample was transferred into a sterile Whirl-Pak bag with 2.7 mL of PBS to reach a first 10-fold dilution. The sample bag was homogenized with a stomacher for 2 minutes at a normal speed. The homogenate and its serial dilutions were pour plated and incubated. Specifically, for E. coli and L. innocua, were cultured on TSA 100 pL of proper dilution and incubated at 37°C for 24 hours, whereas for A. fumigatus 100 pL of dilution was grown on MEA and incubated at 30°C for 48 hours.

[0093] Statistical analysis: Three independent replicates were conducted for each condition. The number counts of each microorganism were converted into log CFU/sample. Statistical analysis to illustrate difference within the same fibers (upper case letter) and within the same contact time (lower case letter) was performed by one-way ANOVA within the confidence interval of 95% (P < 0.05) (IBM SPSS Statistics for Windows, version 19.0, IBM Corp., Armonk, NY).

Example 9

[0094] Relative humidity triggered antimicrobial efficacy of fibers: For relative humidity (RH) triggered release study, only E. coll was used as an example to validate the antimicrobial efficacy of RH responsive fibers. Dry inoculation method was used to avoid the introduction of extra moisture into the chambers. The detailed experimental steps are illustrated in FIG. 9B. 10 pL of bacteria culture (~10 ⁸ CFU/mL) was applied by 10 small aliquots on an aluminium foil substrate to reach a final inoculation level of 10 ⁶ CFU/sample. The inoculated aluminium foil was further dried in a biosafety cabinet for 10 min. RH responsive fiber (2.50 mg/cm ² ) was placed on top and cover the dried bacterial cells and a binder clip was used to let the fiber and bacterial cells contact tightly. In this experiment, RH responsive fibers were peeled off from the aluminium foil after the fiber production to allow sufficient moisture transfer. The two-layer system was transported into a chamber at room temperature (22°C) with a maintained at 50% RH or 95% RH, separately. The two-layer system was kept in the desiccator for 15 min and 1 hour, respectively. Afterwards, the aluminium foil with bacteria was disassembled with the fiber and transported into a centrifuge tube with 1 mL of peptone water. The tube was then vortexed sufficiently for at least 2 min to make sure the bacteria were detached from the aluminium foil. Serial dilution was performed and followed by plating on TSA and incubation at 37°C for 24 hours. As a control, inoculated aluminum foil without fibers and attached with the pristine fibers were kept at 50% RH and 95% RH conditions for 15 min and 1 hour, the number of E. coli was also tested in the same manner. To detect if there were any bacterial cells left on the fibers, tested fibers were transported into a centrifuge tube with 1 mL of peptone and 100 pL of solution was pour plated on TSA for enumeration at 37°C for 24 hours.

[0095] Statistical analysis: Three independent replicates were conducted for each condition. The number counts of E. coli were converted into log CFU/sample. Oneway ANOVA within the confidence interval of 95% (P < 0.05) was performed for significant analysis (IBM SPSS Statistics for Windows, version 19.0, IBM Corp., Armonk, NY). Significant difference within the same fibers was labeled with upper case letters (“A” or “B” was used for 50%RH, “C” or “D” were used for 95% RH). Significant difference within the same contact time was labeled with lower case letters (“a” or “b” were used for 50%RH, “c” or “d” was used for 95% RH). To illustrate the difference of RH responsive fibers against E. coli at different RH levels, significant difference among data in the same contact time was labeled with * (*** P < 0.001 , * P < 0.05).

[0096] Developing sustainable and biodegradable active food packaging is of great importance to the food industry due to the significant negative environmental impact of petroleum-based polymers that are currently widely used. In addition, unnecessary release of antimicrobial active ingredients (Als) from film-based packaging materials can exacerbate negative sensory and health effects. Here, biopolymer based, biodegradable, enzyme- and relative humidity (RH) responsive antimicrobial fibers were developed using electrospun cellulose nanocrystals (CNCs), zein (protein), and starch and a cocktail of both free nature derived antimicrobials including thyme oil, sorbic acid, nisin and also cyclodextrin-inclusion complexes (CD-ICs) of thyme oil, sorbic acid, and nisin. The fibers were designed to release their free Als and CD-ICs of Als in response to enzyme and RH triggers, respectively, bringing precision in the Al delivery while achieving superior antimicrobial functionality (5 log for bacteria and 1 log for fungi) for a broad range of food related pathogenic bacterial and spoilage microorganisms. More importantly, RH triggered antimicrobial activity was also shown by 5 logs of reduction at 95% RH in contrast to 1 .9 log reduction at 50% RH. The use of FDA GRAS approved materials and green synthesis processes makes these nontoxic, biodegradable fibers ideal for sustainable food package materials.