Bio-based Sanitizer Delivery System for Improved Sanitation of

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Biological and Medical Applications of Materials and Interfaces

Bio-based Sanitizer Delivery System for Improved Sanitation of Bacterial and Fungal Biofilms Kang Huang, Fang Dou, and Nitin Nitin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02428 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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ACS Applied Materials & Interfaces

Full Title Bio-based Sanitizer Delivery System for Improved Sanitation of Bacterial and Fungal Biofilms Names of Authors Kang Huang1, Fang Dou1, Nitin Nitin1,2* Author Affiliation 1 Department of Food Science and Technology, University of California - Davis, Davis, CA 95616, USA 2 Department of Biological and Agricultural Engineering, University of California - Davis, Davis, CA 95616, USA Contact information for * Corresponding Author Nitin Nitin, Ph.D. Professor Department of Food Science and Technology And Biological and Agricultural Engineering University of California - Davis Davis, CA 95616 (530) 752-6208 (phone) (530) 752-4759 (fax) [email protected] (email) Keywords: Particle based sanitizer, N-halamine, Yeast cell wall particles, Biofilm, Antimicrobial, Antifungal 1

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Abstract: Biofilms can persist in food processing environments due to their relatively higher tolerance and resistance to antimicrobials including sanitizers. In this study, a novel bio-based sanitizer composition was developed to effectively target biofilms and deliver chlorine based sanitizers to inactivate bacterial and fungal biofilms. The bio-based composition was developed by encapsulating chlorine binding polymer in a bio-based yeast cell wall particles (YCWPs) microcarrier. This study demonstrates the high affinity of bio-based compositions to bind target bacterial and fungal cells and inactivate 5 logs of model pathogenic bacteria and fungi in wash water without and with high organic load (COD = 2000 mg/L) in 30 seconds and 5 minutes, respectively. For the sanitation of biofilm, this bio-based sanitizer can inactivate 7 logs of pathogenic bacteria and 3 logs of fungi after 1-hour treatment, while the 1-hour treatment using conventional chlorine based sanitizer can only achieve 2-3 log reduction for bacterial biofilm and 1-2 log reduction for fungal biofilm, respectively. The enhanced antimicrobial activity can be attributed to three factors: a) localized high concentration of chlorine bound on the YCWPs; b) high affinity of YCWPs to bind diverse microbes; c) improved stability in organic-rich aqueous environment. In summary, these unique attributes of bio-based carriers will significantly enhance the sanitation efficacy of biofilms, reduce persistence and transmission of antimicrobial resistance microbes, limit the use of antimicrobial chemicals, and improve the cost-effectiveness of sanitizers.

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1. Introduction Biofilms are recognized as a frequent source of healthcare-associated infections. Around 90% of infections in humans and 65% of nosocomial infections in the United States were found to be associated with biofilms.1 The total annual cost for biofilm infections in the U.S. is estimated to be 94 billion dollars with more than 50,000 deaths.2 The ability to form biofilms on both biotic and abiotic surfaces and their enhanced resistance to disinfectants3 are the leading challenges associated with biofilms across multiple sectors including food industry,4 drinking water facilities,5 health care,6 medical devices,7 and nautical shipping.8 Therefore, effective decontamination of biofilms formed by diverse microbes in different environments is expected to significantly enhance public safety and increase industrial productivity. Approaches developed and optimized for the inactivation of planktonic (free-living) microbes by physical, chemical, and biological approaches can often have limited efficacy for the removal and inactivation of microbes in the biofilms. This limited efficacy results from several factors in a biofilm including the extracellular polymeric substances (EPS) of a biofilm.9-12 EPS provide protection to the microbes in a biofilm by concentrating nutrients, preventing access of disinfectant agents, sequestering metals and toxins, and preventing desiccation. In addition, the presence of multiple layers of microbes and in many cases multiple species of microbes further enhance the resistance of biofilms to physical and chemical disinfectants. Some studies have quantified the effective resistance of biofilms and reported that resistance of a biofilm to a physical or a chemical disinfection can be up to 1000-fold higher compared with planktonic bacteria.13, 14 Furthermore, the fungal biofilms can often have higher resistance to disinfectants compared to bacterial biofilms in many instances.15 As a result of this enhanced resistance, commonly used disinfection methods such as halogens, peroxygens, 3



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acids, and quaternary ammonium often have limited effectiveness for decontamination of surfaces with extensive biofilms.16 To address these challenges, some studies have shown that intensive treatments such as irradiation, moist heat and high pressure can inactivate biofilms, although the use of these methods is limited due to high cost and lack of compatibility with diverse applications.11, 17, 18 Thus, there is an unmet need to develop effective decontamination methods for biofilms that are also cost effective and can be adapted for diversity of applications. In the last two decades, antimicrobial delivery systems have become a promising tool for inactivation of microbes in biofilms. A myriad of targeted delivery systems including liposomes, polymeric nanoparticles, dendrimers, and various inorganic nanoparticles have been developed to improve the inactivation of bacteria in a biofilm matrix using established antimicrobial agents.19 To further enhance the efficacy of antimicrobial delivery system, previous studies have evaluated the influence of surface charge of delivery systems18, 20 or modification of the surface properties of delivery systems with bioadhesive materials such as nanoparticle– hydrogel hybrid to enhance the affinity of particles to bind biofilms.19 These concepts though effective have significant limitations including the need to engineer compositions using expensive biological materials such as antibodies or develop novel materials that may require extensive approval processes prior to their application in food and biomedical sectors. Furthermore, the affinity biomolecules such as antibodies are often limited only to specific microbes and do not provide effective approach to treat both fungal and diverse bacterial targets. The overall motivation for the studies was to overcome some of these limitations and develop a cost-effective solution that can improve accessibility of antimicrobials to diversity of microbes both in planktonic state and in biofilms. Yeast cell wall particles (YCWPs) have emerged as an attractive delivery system as these 4


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particles represent a natural microcarrier that can be isolated from yeast cells.21-23 In some cases, these particles can be isolated from spent yeast cells from fermentation processes, thus can be highly cost effective carrier for delivery of diverse compounds. YCWPs are hollow and porous 2-4 µm microspheres prepared from Baker’s yeast composed primarily of β-1,3-ᴅ-glucan, chitin, and mannoproteins.24 The ability of these components to bind biological organisms such as E. coli and Salmonella spp.25, 26 indicates that the cell wall components after hydrolysis of cells still retain affinity for binding bacteria. We hypothesize that encapsulation of low lost antimicrobials such as chlorine in YCWPs will provide localized and high affinity delivery of an encapsulated antimicrobial agent to biofilms and enhance the effectiveness of the treatment. Furthermore, affinity of the YCWPs may also enhance removal of the biofilm. In this study, we describe a novel sanitizer prepared by encapsulating chlorine binding polymer (Polyethylenimine, PEI) in a bio-based YCWP microcarrier using a vacuum assisted infusion process. We investigated the characteristics of halamine polymer encapsulated YCWPs, including encapsulation yield, surface charge, total active chlorine content, and storage stability of chlorine. The antimicrobial efficacy of YCWPs@Halamine against planktonic cells and biofilms were tested for a model Gram-positive bacteria (Listeria innocua), a Gram-negative bacteria (E. coli O157:H7), and a fungal strain of Candida albicans, respectively. The key innovations and the unique aspects of this study are: a) to assess the influence of bio-based microcarriers for controlled release of chlorine based antimicrobials in aqueous conditions including preventing non-specific consumption of chlorine with suspended organic content; b) to evaluate the binding efficiency of YCWPs to bacterial and fungal biofilms; c) to demonstrate the enhanced efficacy of bio-based sanitizer for inactivation and removal of bacterial and fungal biofilms. These properties of bio-based carriers will significantly enhance 5



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the sanitation efficacy of biofilms and limit the use of antimicrobial chemicals. 2. Materials and methods 2.1 Chemicals and reagents Branched polyethylenimine (PEI) (mol wt. ~25,000 Da) and sodium hypochlorite (10%), were purchased from Sigma-Aldrich (St. Louis, MO). Acetic acid, acetone, ethanol, isopropanol, potassium phosphate dibasic (K2HPO4), potassium phosphate monobasic (KH2PO4), sodium hydroxide (NaOH), sodium thiosulfate (Na2S2O3), and hydrochloric acid (HCl) were purchased from Fisher Scientific (Waltham, MA). DPD free chlorine reagent powder was purchased from Hach (Loveland, CO). Luria Bertani (LB) broth, tryptic soy broth (TSB), LB agar, and tryptic soy agar (TSA) were from Fisher Scientific (Waltham, MA). Fleischmann's Active Dry yeast, Saccharomyces cerevisiae (Memphis, TN) was purchased from a local grocery store. Ultrapure water (18 MΩ cm) was obtained using the in-lab Milli-Q RG water ultrapurification system from EMD Millipore (Billerica, MA). 2.2 Preparation of yeast cell wall particles Yeast cell wall particles were prepared by a previously reported chemical hydrolysis method.27 Briefly, 20 g baker’s yeast cells were suspended in 200 mL of 1 M NaOH solution. The solution was heated to 80 ºC and stirred for 1 hour. After that, the suspension was allowed to cool for 10-15 min while maintaining stirring. The suspension was centrifuged at 2,500 ×g for 10 min to pellet down the cells. Once the supernatant was decanted, the cells were resuspended in 200 mL of 1 mM HCl and the pH was adjusted to 4.2. The acidic solution was stirred at 55 ºC for 1 hour. The yeast cell wall particles were centrifuged, and the supernatant was decanted. Then the yeast cell wall pellet was washed twice with water, four times with isopropanol, and twice with acetone. The resulting slurry was dried under vacuum at room 6


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temperature overnight to obtain YCWP powder. Prior to each experiment, YCWPs were rehydrated in Milli-Q water and washed three times by Milli-Q water by centrifuge at 2,500 ×g for 5 min. 2.3 Encapsulation of PEI into YCWPs via vacuum infusion For clarity in interpreting sanitizer composition preparation as well as results and discussion, a list of abbreviations is provided in Table 1. 0.5 g of wet YCWPs were suspended in a 5 mL ethanolic encapsulation solution consisting of phosphate buffer (100 mM, pH = 6.5), absolute ethanol, and PEI solution (200 mg/mL in ethanol). The encapsulation solution was prepared by adding various concentrations of PEI. Compositions of ethanolic encapsulation solution for each variant are defined in Table 2. The final concentration of yeast cell wall particles to ethanolic solution was maintained at 10% (w/v). Control samples without antimicrobial compounds were prepared by adding 3.25 mL phosphate buffer and 1.75 mL absolute ethanol. Samples were vortexed to facilitate dispersion. Encapsulation was carried out by vacuum infusion at 99% vacuum for 5 min. After encapsulation, the YCWPs@PEI were washed twice by Milli-Q water. Table 1 Abbreviations used to describe yeast cell wall particles Abbreviation

Description

YCWPs

Yeast cell wall particles

YCWPs@PEI

Polyethylenimine encapsulated yeast cell wall particles

YCWPs@Halamine Chlorine charged Polyethylenimine encapsulated yeast cell wall particles

Table 2 Components of ethanolic encapsulation solution

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No.

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PEI concentration Phosphate buffer Absolute ethanol PEI solution (% w/v)

(mL)

(mL)

(mL)

1

10

3.25

1.50

0.25

2

20

3.25

1.25

0.50

3

30

3.25

1.00

0.75

4

40

3.25

0.75

1.00

2.4 Characterization of YCWPs@PEI The morphology and structure of YCWPs were characterized by scanning electron microscopy (SEM). ζ-potential of YCWPs before and after encapsulation were measured using a dynamic light scattering (DLS, Nano-ZS, Malvern Instruments, Worcestershire, UK). The PEI content of the encapsulated YCWPs@PEI was quantified by measurement of primary amine content using the ninhydrin assay. Briefly, 1 mL of YCWPs sample was mixed in the ninhydrin reagent (2 mL of 0.2% w/v in 0.1 M phosphate buffer, pH = 9), and the mixture was heated in a boiling water bath for 15 min. After the mixture was cooled to room temperature, the absorbance of solution was measured at 570 nm. The primary amine content of diluted PEI was quantified to establish a standard curve for the amine quantification assays. Encapsulation yield on a wet basis was determined as follows: 𝑌𝑖𝑒𝑙𝑑 =

𝐶𝐸(𝑚𝑔) 𝐶𝑌(𝑔)

(1)

where CE is the mass of PEI from yeast cell wall microcarriers on a wet basis and CY is the mass of yeast cell wall microcarriers. For YCWPs microcarriers loaded with PEI, a fluorescent microscope was used. The

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modified PEI staining method28 was used to label PEI with Fluorescein (FITC). To prepare fluorescent labeled PEI, PEI was dissolved in ethanol to a PEI concentration of 2 g/mL. FITC was dissolved in DMSO at a concentration of 10 mM. The PEI solution was mixed with FITC for 2.5 h at 18 ºC to a final concentration of 0.1 mM FITC. The reaction mixture was dialyzed (MWCO 3,500) overnight against 1 liter of ice-cold PBS to remove any FITC not linked to PEI. The labeled PEI was encapsulated to YCWPs by vacuum infusion approach described above. After encapsulation, the fluorescent images of YCWPs and YCWPs@PEI were acquired by a laser scanning confocal microscope (IX71, Olympus) with a 60× objective. The excitation and emission filters were 470/15 nm and 530–590 nm, respectively. The exposure time for an individual image was 500 ms. 2.5 Chlorination of YCWPs@PEI The chlorination solution with active chlorine of 1% was prepared by diluting the Clorox bleach solution, and the pH of chlorination solution was adjusted by adding 5 M HCl. Typically, 0.5 g of YCWPs@PEI were resuspended in 20 mL of chlorination solution with shaking for a desired time, then charged particles (YCWPs@Halamine) were washed three times by Milli-Q water to remove any free chlorine in solution. Total active chlorine content was measured by titration method. 0.1 g of charged particles (YCWPs@Halamine) were added to 15 mL of 0.001 N sodium thiosulfate solution with shaking for 30 min. 1 mL of acetic acid (1% v/v) was added to promote the reaction. The residual sodium thiosulfate was subsequently titrated with a 0.001 N iodine standard solution. The active chlorine content (μmol/g) of the particles was calculated according to: Active chlorine content =

𝑉0 ― 𝑉𝑆 2 × 𝑚𝑆

(2)

where V0 and VS are the volumes (mL) of the iodine solution consumed in titration without and 9



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with charged particles, respectively. ms is the weight (g) of the charged particles. The storage stability of YCWPs@Halamine was evaluated by storing charged particles at 4 ºC for 1 month. The total active chlorine content was measured by titration method after every 7 days. The effect of chemical oxygen demand (COD) on the total active chlorine content was evaluated by incubating YCWPs@Halamine with simulated wash water at various levels of COD (0, 500, 2000, and 20000 mg/L). The total active chlorine content of YCWPs@Halamine was measured after exposure to simulated wash water for 2 min and 60 min, respectively. Passive release of free chlorine from YCWPs@Halamine to wash water was quantified to assess the contribution of released chlorine in antimicrobial properties of YCWPs. 0.1 g of charged particles (YCWPs@Halamine) were suspended in Milli-Q water with shaking for 10 min. After shaking for 10 min, the suspension was centrifuged at 2,500 ×g for 5 min. The supernatant was evaluated for the released free chlorine from YCWPs. The pellet was resuspended in 1 mL of Milli-Q water, and incubated for another 10 min. The concentration of free chlorine content of aqueous phase was measured via N,N-diethyl-p-phenylenediamine (DPD) colorimetric method.29 2.6 Bacterial strains and biofilm formation To evaluate broad applicability of this technology, this study evaluates the impact of this formulation on both bacteria and fungi. Listeria innocua (ATCC 33090) was selected as a surrogate for a gram-positive human pathogenic Listeria monocytogenes, and a shiga toxin negative strain of Escherichia coli O157:H7 (ATCC 700728) was selected as a model for a gram-negative foodborne pathogen. Candida albicans (ATCC 90028) was chosen as the model pathogenic yeast that is associated with human infection candidiasis. Each liquid nitrogen stock was streaked on to agar plates and grown overnight. Before each experiment, one colony was 10


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picked from an agar plate, cultured in the liquid medium, and incubated to achieve the stationary phase cultures. E. coli O157:H7 strain was grown in LB broth with constant shaking (250 rpm) at 37 ºC. L. innucua and C. albicans strains were grown in TSB with constant shaking (250 rpm) at 30 ºC. For plate cultures, LB agar plates were used for E. coli O157:H7, while TSA plates were used for L. innocua and C. albicans, respectively. After being centrifuged and washed in sterile PBS for twice, bacteria were resuspended in sterile PBS at a concentration of approximately 1.0 × 109 CFU/ml. The biofilms were grown in a sterile 24-well polystyrene plate (Corning, Corning, NY, USA). 0.1 mL of overnight bacterial culture and 0.9 mL of 1× M9 medium supplemented with 0.4% glucose and 0.4% tryptone were added to each well. The final concentration of bacteria was approximately 1 × 108 CFU/mL. The 24-well plate containing L. innocua and C. albicans were then incubated at room temperature for 3 days, respectively. The E. coli biofilm was incubated at room temperature for 4 days. After incubation, the medium in the plates was discarded and each well was gently washed three times with 1 mL of PBS in order to remove planktonic cells. 2.7 Efficiency of YCWPs to bind diverse microbes To demonstrate the ability of YCWPs to bind a broad spectrum of microbes, efficiencies of YCWPs to bind Gram-positive bacteria (L. innocua), Gram-negative bacteria (E. coli O157:H7) and fungi (C. albicans) were quantified using a fluorescence based binding assay. YCWPs were stained by Calcofluor White and washed three times by Milli-Q water. After labeling, 0.1 g of fluorescent labeled YCWPs were suspended in 1 mL of Milli-Q water and incubated with biofilm grown in 24-well plate at room temperature. After incubation for a certain period, YCWPs solution was gently removed by pipette, and each well was washed two 11



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times by Milli-Q water to remove the loosely attached YCWPs. The fluorescence intensity of YCWPs adhered on the biofilm was determined using a plate reader with excitation/emission at 350/440 nm. 2.8 Efficiency of microbes to bind different components of YCWPs Efficiency of microbes to bind different components of YCWPs was further determined by quantifying bacterial/fungal cells attached on specific molecular components of YCWPs. A modified approach was used for immobilizing YCWPs and different components on plastic surface.30 Briefly, the 24-well microplate was incubated with 2% PEI (pH 7.0) for 2 hours. After that, excess PEI was drained off and the wells were air dried. The PEI coated microplate was then treated with 1 ml of 0.1 g/mL YCWPs for 2 hours to achieve a uniform monolayer of YCWPs and washed twice by Milli-Q water. 0.2 mL of each component (chitin, mannan, and (1,3)-β-glucan, 0.1 g/mL) was added individually to PEI coated well and incubated at 37 ºC for drying., 1 mL of bacterial/fungal cells (~109 CFU/mL) were added to the coated wells of YCWPs, chitin, mannan and β-glucan, respectively and incubated at room temperature for 1 h. All the bacterial/fungal cells were labeled with SYBR Green I dye. After incubation, cell solution was gently removed by pipette, and each well was washed two times by Milli-Q water to remove the loosely attached microbes. The fluorescence intensity of cells adhered on each component was determined using a plate reader with an excitation/emission at 485/520 nm. 2.9 Antimicrobial activity of YCWPs@Halamine The antimicrobial activities of each treatment were evaluated (YCWPs@PEI, YCWPs@Halamine, conventional sanitizer containing 20 ppm free chlorine (which was equivalent to the free chlorine concentration released from YCWPs@Halamine), 20 ppm free chlorine + YCWPs, and 20 ppm free chlorine + YCWPs@PEI). The bacterial suspension with 12


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approximately 6 log CFU/mL was prepared by dilution. A volume of 1 mL of bacterial suspension was added in a test tube with 2 mg/mL of particles and incubated for various periods with rotating speed of 250 rpm at room temperature. Bacterial suspension without any particles was used as a negative control. After treatment, 0.1 mL of 0.1 M sodium thiosulfate was added to each sample for quenching the residual chlorine, and the bacterial suspension of each treatment was serially diluted using PBS. A volume of 100 µL of each dilution was inoculated onto agar plates and incubated until enumeration. 2.10 Biofilm inactivation assay The ability of YCWPs@Halamine to sanitize biofilm was assessed using biofilms formed on plastic surface of the selected microbes, i.e. L. innocua, E. coli, and C. albicans. The charged particles were suspended at a concentration of 10 mg/mL in sterile Milli-Q water and used for this assay. The control groups for these assays were treated with 1× PBS and 10 mg/mL uncharged particles (YCWPs@PEI), respectively. Conventional chlorine based sanitizer containing 20 ppm free chlorine was used as an additional control group, which was equivalent to the free chlorine content released from YCWPs@Halamine. 1 mL of sanitizer was added to each well where biofilms have formed and incubated at room temperature. After incubation, biofilm was gently washed twice by PBS to remove residual sanitizers. After sanitation treatments, the viability of biofilm was demonstrated by plate counting method and enzymatic activity of the cells was further measured to indicate the amount of viable but not culturable cells. Bacteria in biofilm were recovered from 24-well plate by adding 1 mL of sterile maximum recovery buffer supplemented with 0.1% v/v Tween-20 to each well, vortexing vigorously for 30 s and bath sonicating (Branson 2510 Ultrasonic Cleaner, Branson Ultrasonics, Danbury, CT, USA) for 2 min. The plates were sealed with parafilm to prevent 13



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leaking of recovery buffer. Quantification of viable bacteria recovered from biofilm was performed by serial dilution spread plating on agar plates and incubating at 37 ºC before counting. The enzymatic activity of bacterial cells after treatments was assessed using the Resazurin assay. 1 ml of Resazurin solution (50 µM in M9 medium) was added to each well and the curves of Resazurin reduction were monitored using a microplate reader (TECAN SpectraFluor Plus Microplate Reader, Tecan Group Ltd., Switzerland) for 24 h with excitation at 530 nm and emission at 580 nm. Biofilms were fixed in 4% glutaraldehyde solution in PBS (pH 7.4) at 4 ºC for 3-4 h and washed once using Milli-Q water. The biofilm samples were then mounted onto aluminum stubs with carbon conductive adhesive tape, and sputter coated with 10 nm of gold. Microscopy was performed on a Philips XL-30 electron microscope at 10 kV accelerating voltage. 2.11 Statistical analysis Statistical analysis was performed using the GraphPad Prism software V.5.04 (Graphpad Software, Inc., La Jolla, CA). All experiments were performed in triplicates. Unless otherwise stated in the corresponding section, the significant differences between treatments were determined through one-way ANOVA followed by Tukey’s pairwise comparisons with a 95% confidence interval.

3. Results 3.1 Characterization of YCWPs@PEI Particles Physicochemical properties of PEI encapsulated YCWPs were characterized based on SEM imaging, net surface charge, encapsulation yield, and fluorescence imaging. A representative SEM image of dried YCWPs (Fig. 1a) suggests a relatively uniform particle size of porous 14


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hollow microspheres with a diameter of 2.78 ± 0.21 µm. Fig. 1b describes the results of encapsulation of various concentrations of PEI into YCWPs using a vacuum infusion process for 5 seconds. Results indicate that the loading yield assessed based on the primary amine content in YCWPs increased significantly with an increase in the concentration of PEI from 10 to 30 mg/mL (p < 0.05). With further increase in extracellular PEI concentration, there was no significant increase in the encapsulation yield (p > 0.05). Results of dynamic light scattering measurements (Fig. 1c) show that as prepared YCWPs had a negative zeta potential of -15.7 ± 0.8 mV. After encapsulation with PEI, the zeta potential of YCWPs@PEI transformed to a net positive charge (+34.1 ± 0.3 mV). Fig. 1d illustrates the fluorescence images of YCWPs before and after encapsulation with PEI using vacuum infusion. In the control image, weak endogenous fluorescence signal from the residual biomolecules in YCWPs was observed. The fluorescence image was superimposed on the brightfield image of YCWPs to indicate the intracellular localization of the background fluorescence signal. In contrast, strong localized fluorescence signal corresponding to the intracellular localization of PEI-FITC in YCWPs was observed upon infusion of the fluorescently labeled PEI in YCWPs. Superimposed image of the brightfield and fluorescence scans illustrate intracellular localization of PEI in YCWP. In addition, the fluorescence signal intensity of YCWPs infused with the FITC labeled PEI was significantly higher than the control images.

15



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Figure 1 Characterization of yeast cell wall particles encapsulated with PEI. (a) SEM image of freeze dried YCWPs; (b) Encapsulation yield; (c) zeta potential of YCWPs and YCWPs@PEI (30 mg/mL); (d) Fluorescent image of PEI in YCWPs. PEI was encapsulated in YCWPs using vacuum infusion; the control YCWPs without PEI were treated in the same manner. From left to right, top row: White light image of control YCWPs; FITC (525/25 BP) image of control YCWPs; Composite image of control YCWPs; From left to right, bottom row: White image of PEI encapsulated in YCWPs; FITC (525/25 BP) image of PEI encapsulated in YCWPs; and Composite image of PEI encapsulated in YCWPs. The scale bars in the images represent the length of 20 µm. Different letters indicate statistically significant differences (p < 0.05). 3.2 Characterization of chlorine content of yeast cell wall particles In this study, the PEI encapsulated YCWPs were modified to encapsulate and store chlorine 16


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molecules based on formation of the halamine functional group (N-Cl linkage) between the primary amines and secondary imines of the PEI and the Cl. To form the halamine group, the PEI encapsulated YCWPs were chlorinated by incubating particles in a diluted household bleach (1% active sodium hypochlorite solution) at pH 5 for 15-60 minutes at room temperature. Results in Fig. 2a illustrate an increase in the total chlorine content of YCWPs@Halamine as a function of chlorination time. The results show an extremely high concentration of chlorine (ca. 136.4 μmol/g) bound to the particles after 15 min of incubation with bleach. With extended incubation, there was no significant increase in the total concentration of chlorine per unit mass of encapsulated YCWPs (p > 0.05). Chlorination of YCWPs without encapsulated PEI was also evaluated and the results show a significantly reduced amount of total chlorine bound to YCWPs using the same experimental conditions in Fig. S1. These results further validate the role of PEI polymer in binding chlorine and formation of YCWPs@Halamine. After chlorination of YCWPs@PEI, storage stability of chlorine bound to the encapsulated YCWPs was evaluated. The results in Fig. 2b show that no significant changes in the total chlorine content upon storage of YCWPs@Halamine in aqueous solution for 28 days under refrigerated conditions (p > 0.05). In addition, the effect of organic content on stability of chlorine bound YCWPs was also evaluated by exposure YCWPs@Halamine to aqueous solutions with different levels of chemical oxygen demand (COD). In conventional bleach formulations, organic content can rapidly deplete chlorine and other oxidizing sanitizers and thus limit their biocidal action. Results in Fig. 2c show that increasing COD concentration from 0 to 2000 mg/L had no significant effect on the total chlorine content of YCWPs@Halamine for 60 min of incubation. Although further increase in COD concentration to 20000 mg/L led to a slight reduction in the total chlorine content. The total active chlorine content bound to PEI 17



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encapsulated YCWPs after exposure to COD of 20000 mg/L for 2 min and 60 min were approximately 118.7 and 116.8 μmol/g, respectively. This suggests that the loss of total chlorine content of YCWPs@Halamine was less than 15%. In summary, these results demonstrate that YCWP@Halamine formulation prevents rapid depletion of chlorine upon incubation with organic content rich aqueous solutions.

Figure 2 Characterization of chlorine content of yeast cell wall particles. (a) Schematic illustration of chlorination of the primary amine moieties of PEI and the reversible reaction of the chlorinated amine moieties on PEI with microbes; (b) Chlorination of YCWPs@PEI; (c) Storage stability of YCWPs@Halamine; (d) Effect of organic load on the total chlorine content of YCWPs@Halamine. Different letters indicate statistically significant differences (p < 0.05). 3.3 Antimicrobial properties of YCWPs@Halamine To evaluate the antimicrobial activity of the YCWPs@Halamine, these particles were incubated with selected bacteria (L. innocua and E. coli O157:H7) and fungi (C. albicans) with and without the presence of organic matter. After 0.5 min of treatment, 2 mg/mL of YCWPs@Halamine was able to inactivate up to 5 log CFU/mL of both bacterial and fungal cells in water without organic matter (Fig. 3). The PEI encapsulated YCWPs without 18


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chlorination with bleach, i.e. YCWPs@PEI showed approximately 1 log CFU/mL reduction after 2 min exposure. To further characterize the antimicrobial activity of YCWPs@Halamine, its efficacy for inactivation of pathogenic bacteria and fungi was evaluated in the presence of high organic content (COD = 2000 mg/L). In the presence of organic content at 2000 mg/L, YCWPs@Halamine was able to inactivate 5 log CFU/mL of E. coli O157:H7 and C. albicans in 2 min. In the presence of organic content, the rate of inactivation of L. innocua cells was low compared to E. coli cells and inactivation of 5 log CFU/ml was achieved after 5 min of treatment with YCWPs@Halamine. Incubation of uncharged control particles YCWPs@PEI, resulted in approximately 1 log reduction in microbial plate count during 20 minutes of incubation.

Figure 3 Kinetics of sanitation of planktonic cells in (a-c) clean water (COD = 0 mg/L) and (d-f) water with organic load (COD = 2000 mg/L). (a, d) L. innocua; (b, e) E. coli O157:H7; (c, f) C. Albicans. 3.4 Control release of free chlorine The oxidizing halogens can act through direct transfer of active element to the biological target or through dissociation to hypochlorite in aqueous media. Therefore, release of free 19



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chlorine from YCWPs@Halamine was evaluated using the approach described in the materials and methods section. 0.1 g of charged YCWPs@Halamine was suspended in 1 mL of Milli-Q water and incubated at room temperature. After the first 10 min, suspended YCWP were removed by centrifugation and the free chlorine content released in water from YCWPs was 14.82 ± 1.55 ppm, which was equivalent to approximately 3% of the total chlorine content bound to YCWPs (Fig. 4). With extended incubation time, the concentration of free chlorine released from YCWPs@Halamine decreased significantly. During the fifth cycle, only approximately 0.62 ppm of free chlorine detected from water, which was equivalent to 0.1% of total chlorine content bound to YCWPs. Limited release of free chlorine from YVWPs@Halamine suggest stability of the bound chlorine in aqueous environment. There results also indicate that binding of YCWPs to target microbes and direct transfer of chlorine to target microbes has a significant role in inactivation of these microbes.

Figure 4 Measurement of control release of free chlorine in aqueous environment: (a) schematics of experimental approach; (b) results of cycle release of free chlorine. 3.5 Binding of microbes to YCWPs and their biochemical components In order to investigate binding of microbes to YCWPs and the role of various biochemical components of YCWPs in binding microbes, the selected microbes were incubated with physically adsorbed YCWPs and YCWPs coated with the key biochemical components of 20


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YCWPs including (1,3)-β-glucan, mannan and chitin on a plastic surface. The physical adsorption process for YCWPs and its individual biochemical components is described in the materials and methods section. The controls for these measurements included plastic surface without adsorbed cells or molecules, plastic surface coated with PEI, and plastic surface coated with YCWPs. The adhesion of planktonic bacterial/fungal cells to modified surfaces with different components of YCWPs were quantified based on measurement of fluorescent signal from bacterial/fungal cells upon binding to the plastic substrates with adsorbed cell wall particles or its biochemical components. The results in Fig. 5 demonstrate enhanced binding of bacteria/fungal cells to plastic substrates with adsorbed YCWPs and YCWPs coated with the selected biochemical components as compared to the control plastic surfaces. Overall, chitin exhibited a relatively higher binding affinity for both bacterial and fungal cells than the other three selected biochemical components and YCWPs. The adhesion of bacterial/fungal cells to adsorbed mannan biopolymer on a plastic substrate was similar to binding with surface adsorbed YCWPs, in which mannan is one of the key components in the external layer of YCWPs. Among all three tested strains, C. albicans showed a higher binding capacity to the modified surface than bacterial cells. The binding of C. albicans cells to YCWPs, chitin, mannan, and (1,3)-β-glucan were approximately 18.7%, 35.1%, 16.0%, and 14.6%, respectively. The L. innocua showed higher binding capacity to YCWPs and its components compared to the E. coli O157:H7.

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Figure 5 (a) Schematic illustration of measuring efficiency of microbes to bind different components of YCWPs; Efficiency of microbes to bind different components of YCWPs: (b) L. innocua; (c) E. coli O157:H7; (d) C. Albicans. Different letters in graphs indicate statistically significant differences (p < 0.05). 3.6 Efficiency of YCWPs to bind diverse biofilms To evaluate the relative efficiency of YCWPs to bind diverse biofilms, the fluorescence labeled YCWPs were incubated with biofilms at room temperature for 1 h. After incubation, attachment of YCWPs to selected biofilms was quantified based on measurement of fluorescence intensity. This measurement represents the relative concentration of bound YCWPs to a biofilm as described in the materials and method section. Results in Fig. 6 show that 1-hour incubation of fluorescently labeled YCWPs resulted in a significant binding of these particles to the three selected model biofilms. The binding efficiency of the fluorescently 22


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labeled YCWPs were approximately 19.8%, 22.6%, and 18.3% for L. innocua, E. coli O157:H7, and C. albicans biofilms, respectively. For C. albicans biofilm, the maximum fluorescence intensity was achieved after 20 min incubation, which was shorter than the time required for maximum fluorescence intensity in the case of L. innocua and E. coli O157:H7 biofilms. These results suggest relatively higher binding efficiency of YCWPs with C. albicans biofilm as compared to binding of YCWPs to L. innocua and E. coli biofilms.

Figure 6 Efficiency of YCWPs to bind diverse microbes: (a) L. innocua; (b) E. coli O157:H7; (c) C. Albicans. 3.7 Sanitation of biofilm The results for inactivation of biofilms of both model bacteria and fungi (including L. innocua, E. coli and C. albicans) indicate that these bio-based particles exhibit strong antimicrobial activity against all investigated biofilms (Fig. 7). The average initial populations of L. innocua, E. coli, and C. albicans recovered from untreated control biofilms were 7.37 ± 0.10 log CFU/cm2, 7.26 ± 0.14 log CFU/cm2, and 7.50 ± 0.14 log CFU/cm2, respectively (Fig. 7a-c). There was no significant difference in microbial cell population in selected biofilms before and after exposure to PBS for 1 h (p < 0.05). Exposure to uncharged YCWPs@PEI did not induce a significant inactivation of biofilms, as less than 1 log reduction in microbial plate count was achieved even after 1 h treatment of each of biofilms. L. innocua cell count in a biofilm was reduced by an approximately 5.0 log CFU/cm2 after 20 min of treatment with 23



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YCWPs@Halamine. After extended treatment for 1 h, L. innocua cells in biofilms were below the detection limit (detection limit of 1 log CFU/well), indicating more than 7 log inactivation of L. innocua cells. In the case of E. coli O157:H7 biofilm, 20 min treatment by YCWPs@Halamine resulted in more than 7 log reduction, indicating E. coli O157:H7 cells were less resistant to the treatment than L. innocua. C. albicans exhibited the most resistance to YCWPs@Halamine treatment. After exposure for 20 min and 1 hour, inactivation of C. albicans in biofilms were 2.1 log and 3.5 log CFU/cm2, respectively. In addition to measuring influence of YCWP@Halamine on cell culturablity, effect of the treatments on cell viability and metabolic activity were also assessed Fig. 7(d-f). For this assessment, L. innocua, E. coli O157:H7, and C. albicans biofilms after 1 h treatment were incubated with Resazurin for 24 h. In this assay, Resazurin was reduced by reactions associated with respiration of microbes. The time required to achieve maximum fluorescence intensity indicates the relative level of metabolic activity of bacterial cells under different treatments. The results demonstrate significant reductions in metabolic activity of bacterial and fungal biofilms treated with the YCWPs@Halamine compared to the control samples. When comparing biofilms treated with YCWPs@Halamine to the untreated controls, a time lag in bioconversion Resazurin was observed. e.g., for the untreated L. innocua and E. coli O157:H7 biofilms, the maximum fluorescence intensity was achieved after 4-5 hours of incubation while no peaks were observed from the kinetic curves after treatment of bacterial biofilms with YCWPs@Halamine. This indicates that bacteria in biofilms were inactivated after 60 min of treatment. This conclusion was consistent with the plate counting results. For C. albicans biofilms treated with YCWPs@Halamine, there is a 5-hour delay until the fluorescence intensity reaches maximum value as compared to the untreated control. This lag likely comes 24


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from a reduced number of viable cells present in the treated biofilm compared to the untreated. Similar to the results from plate counting assay, treatment with YCWPs@PEI did not cause significant inactivation of both bacterial and fungal cells in the biofilms.

Figure 7 Inactivation of biofilms: (a) L. innocua; (b) E. coli O157:H7; (c) C. Albicans. Metabolic activity of biofilm after 1 h treatment: (d) L. innocua; (e) E. coli O157:H7; (f) C. Albicans. The detection limit is 1 log CFU/well. Visual confirmation of the effect of bio-based sanitizer on biofilm eradication was obtained using SEM. To determine that the YCWPs@Halamine demonstrated high efficacy against performed L. innocua, E. coli O157:H7, and C. albicans biofilms, the results were compared with those obtained from untreated biofilms. As observed, the plastic surface supported extensive biofilm formation for both bacterial and fungal strains before treatment (Fig. 8). After 1-hour treatment with YCWPs@Halamine , significant removal of cells was observed from L. innocua and E. coli O157:H7 biofilms, thus confirming that the treatment with YCWPs@Halamine not only inactivated the bacteria, but also was effective in eradicating 25



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performed biofilms. A similar trend was also demonstrated for the removal of C. albicans biofilm, although C. albicans biofilm was more resistant to the treatment as compared to bacterial biofilms based on the microbial plate counting results.

Figure 8 Representative SEM images of biofilm before (a-c) and after (d-f) treated by YCWPs@Halamine for 1 h: (a, d) L. innocua; (b, e) E. coli O157:H7; (c, f) C. Albicans. The scale bars in the images represent the length of 20 µm. 4. Discussion 4.1 Enhanced Antimicrobial Activity of Chlorine Encapsulated YCWPs This study demonstrates that encapsulation of chlorine in bio-based carriers significantly improves its antimicrobial efficacy in the presence of organic rich wash water and biofilms. This bio-based sanitizer can inactivate 5 logs of model pathogenic bacteria and fungi in wash water without and with high organic load (COD = 2000 mg/L) in 30 seconds and 5 minutes, respectively (Fig. 5). In contrast, 20 ppm of free chlorine (equivalent concentration to the biobased sanitizer composition) added to wash water needs 1-2 minutes to achieve 5 log reduction in the wash water without organic load. Furthermore, 20 ppm of chlorine was not able to 26


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inactivate more than 1 log of bacteria and fungi in the presence of organic load (COD = 2000 mg/L) after 20 min treatment. In terms of sanitation of biofilm, this bio-based sanitizer can inactivate 7 logs of pathogenic bacteria and 3 logs of fungi after 1-hour treatment (Fig. 6), while the 1-hour treatment using conventional chlorine based sanitizer at equivalent concentration levels can only achieve 2-3 log reduction for bacterial biofilm and 1-2 log reduction for fungal biofilm, respectively. The enhanced antimicrobial activity can be attributed to three factors: a) localized high concentration of chlorine bound to the YCWPs; b) high affinity of YCWPs to bind diverse microbes; and c) improved stability in organic-rich aqueous environment. Based on these three features, this novel bio-based sanitizer can be used for effective sanitation of multi-species biofilms in the future work. 4.2 Localized High Concentration of Chlorine This study demonstrates the ability of YCWPs@PEI to bind 136.4 μmol/g of active chlorine per gram of YCWPs after 15 min chlorination (Fig. 2a), which enhances the localized delivery of high concentration of chlorine to the target microbes in suspension and in biofilm. The approach developed in this study is distinct from prior efforts to increase the active chlorine content on the particles. Previous studies31-33 focusing on synthesis of N-halamine coated particles (i.e., silica particles, polystyrene nanoparticles, and titanium dioxide nanoparticles) have demonstrated the feasibility of binding 5-25 μmol/g of active chlorine on the surface of microscale and nanoscale particles. In addition, the chlorination process in these studies needs 2-6 hours to achieve the maximum value of total active chlorine content.32 We hypothesize that this limitation results from the smaller amount of amine groups bound on the particle surface as compared to relatively hollow bio-based carriers, such as YCWPs. These hollow particles could encapsulate chlorine binding polymers at much higher concentration than surface coating 27



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approaches. 4.3 High Affinity of YCWPs to Bind Diverse Microbes It has been demonstrated that the bio-based carriers used in this study have high affinity to bind target bacterial and fungal cells. After 1-hour incubation, approximately 20% of the YCWPs were attached to the biofilms (Fig. 3). This result is in agreement with previous publications,25, 26, 34 which report binding yeast cell wall particles to enteropathogenic bacteria such as E. coli F4 and Salmonella Typhimurium. The results of these studies indicate binding of YCWPs ranged between 10% to 40% for diverse enteropathogenic microbes. High binding of YCWPs to microbes will lead to an efficient delivery of sanitizer and an enhanced inactivation of pathogens in a biofilm. Previous studies have suggested that there is variation in the binding capability of microbes to yeast cell wall, and the binding potentials were strainspecific and yeast sample type-specific.34, 35 This binding is mediated by range of biochemical compositional factors including chitin, mannan and galacto oligosaccharides (MOS) in the yeast cell wall.34 The results in this study suggest the affinity of chitin to bind bacterial and fungal cells was higher than that of mannan and β-glucan (Fig. 4). However, chitin is generally a minor component in the yeast cell wall, accounting for only 1-2% of the cell wall dry mass.36 It is important to note that the deposition of these selected biomolecules on a plastic surface may not fully represent the orientation and availability of these molecules on YCWPs. Furthermore, the biocomplexity of mannan oligosaccharides may be not be fully represented by the mannan used in this study. Further work is needed to improve the binding affinity of biobased carriers by using different yeast cell wall preparation methods, selection of different microbial strains for bio-based carriers, or surface modification of cell wall particles with chemicals that have high affinity to bind target microbes. 28


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4.4 Improved Stability in Organic Rich Aqueous Environment The results in this study demonstrate that the bio-based carriers decreased the release of free chlorine to the aqueous environment with high organic load, thus resulting in an enhanced storage stability of chlorine in YCWP carriers. The formulation developed in this study released less than 5% of free chlorine in the aqueous environment after 1-hour of incubation with agitation and was able to retain over 95% of the active chlorine after 4 weeks of refrigerated storage. Furthermore, exposure to wash water with extremely high organic load (20,000 COD) only reduced less than 10% of the active chlorine content in this formulation. Lack of nonspecific reactions of encapsulated chlorine in YCWPs further enhance its antimicrobial efficacy in high-organic-load (nutrient-rich) environment such as biofilms and wash water with COD = 2000 mg/L as compared to conventional chlorine based sanitizer. Lack of immediate release of high concentration of chlorine from these bio-based carriers provides advantages including i) the limited consumption of chlorine by organic content enables the extended use of this biobased sanitizer; ii) the passive release of free chlorine to aqueous environment significantly reduces the environmental impact and health effects.37 Therefore, the limited release of free chlorine enables this formation to become an attractive approach to stabilize chlorine against high organic load and reduce the amount of total chlorine used for sanitation.

5. Conclusions In summary, this study demonstrates a novel bio-based sanitizer to enhance sanitation effectiveness against bacterial and fungal biofilms. Encapsulation of chlorine bound polymer Polyethylenimine in yeast cell wall particles resulted in a bio-based system offering unique advantages in formulating material with high localized concentration of chlorine, extended 29



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stability of chlorine, high affinity of binding diverse microbes, and targeted inactivation of microbes in both planktonic state and biofilms. These advantages enable rapid inactivation of model pathogenic bacteria (L. innocua and E. coli O157:H7) and fungi (C. Albicans) in wash water without and with high organic load, respectively. In addition, this bio-based sanitizer was able to significantly inactivate both pathogenic bacteria and fungi in the biofilms. The results of this study illustrate the potential of this approach to address the unmet needs for improving sanitation of both bacterial and fungal biofilms.

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ASSOCIATION CONTENT Supporting Information Chlorination of YCWPs, inactivation kinetics of planktonic cells by conventional chlorine based sanitizer, and inactivation of biofilm by conventional chlorine based sanitizer data are available in Supporting Information section. AUTHOR INFORMATION Corresponding author *E-mail: [email protected] Notes The authors declare no competing financial interest. Acknowledgements This research was supported by funding from USDA-NIFA Program Enhancing Food Safety through Improved Processing Technologies (A4131) grant 2015-68003-23411 and the USDANIFA Foundational Food Safety Program grant 2018-67017-27879.

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(25) Ganner, A.; Stoiber C.; Uhlik, J. T.; Dohnal, I., Schatzmayr, G. Quantitative Evaluation of E. Coli F4 and Salmonella Typhimurium Binding Capacity of Yeast Derivatives. AMB Express 2013, 3, 62. (26) Ganner, A.; Stoiber, C.; Wieder, D.; Schatzmayr, G. Quantitative In Vitro Assay to Evaluate the Capability of Yeast Cell Wall Fractions from Trichosporon Mycotoxinivorans to Selectively Bind Gram Negative Pathogens. J. Microbiol. Methods 2010, 83, 151-2. (27) Soto, E. R.; Ostroff, G. R. Characterization of Multilayered Nanoparticles Encapsulated in Yeast Cell Wall Particles for DNA Delivery. Bioconjugate Chem. 2008, 19, 840-848. (28) Saito, M.; Saitoh, H. Labeling of Polyethylenimine with Fluorescent Dye to Image Nucleus, Nucleolus, and Chromosomes in Digitonin-Permeabilized HeLa Cells. Biosci. Biotechnol. Biochem. 2012, 76, 1777-1780. (29) Helbling, D. E.; VanBriesen, J. M. Free Chlorine Demand and Cell Survival of Microbial Suspensions. Water Res. 2007, 41, 4424-4434. (30) D'Souza, S. F.; Melo, J. S.; Deshpande, A.; Nadkarni, G. B. Immobilization of Yeast Cells by Adhesion to Glass Surface Using Polyethylenimine. Biotechnol. Lett. 1986, 8, 643648. (31) Sun, Y.; Sun, G. Novel Refreshable N-Halamine Polymeric Biocides: N-Chlorination of Aromatic Polyamides. Ind. Eng. Chem. Res. 2004, 43, 5015-5020. (32) Li, L.; Ma, W.; Cheng, X.; Ren, X.; Xie, Z.; Liang, J. Synthesis and Characterization of Biocompatible Antimicrobial N-Halamine-Functionalized Titanium Dioxide Core-Shell Nanoparticles. Colloids Surf. B Biointerfaces 2016, 148, 511-517. (33) Dong, A.; Xue, M.; Lan, S.; Wang, Q.; Zhao, Y.; Wang, Y.; Zhang, Y.; Gao, G.; Liu, F.; Harnoode, C. Bactericidal Evaluation of N-Halamine-Functionalized Silica Nanoparticles Based on Barbituric Acid. Colloids Surf. B Biointerfaces 2014, 113, 450-457. (34) Ganner, A.; Stoiber, C.; Uhlik, J. T.; Dohnal, I.; Schatzmayr, G. Quantitative Evaluation Of E. Coli F4 and Salmonella Typhimurium Binding Capacity of Yeast Derivatives. AMB Express 2013, 3, 62. (35) Posadas, G.A.; Broadway, P.R.; Thornton, J.A.; Carroll, J.A.; Lawrence, A.; Corley, J.R.; Thompson, A.; Donaldson, J.R. Yeast Pro- and Paraprobiotics Have the Capability to Bind Pathogenic Bacteria Associated with Animal Disease. Translational Animal Science 2017, 1, 60-68. (36) Ohno, N. 2.17 - Yeast and Fungal Polysaccharides. 2007, 559-577. (37) Lattemann, S.; Höpner, T. Environmental Impact and Impact Assessment of Seawater Desalination. Desalination 2008, 220, 1-15. 34


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Graphical Abstract (For Table of Contents Only)

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Bio-based Sanitizer Delivery System for Improved Sanitation of

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