Antioxidant, Antibacterial, and Antibiofilm Properties of Polyphenols

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Antioxidant, Antibacterial and Antibiofilm Properties of Polyphenols from Muscadine Grape (Vitis rotundifolia Michx.) Pomace Against Selected Foodborne Pathogens Changmou Xu, Yavuz Yagiz, Wei-Yea Hsu, Amarat Simonne, Jiang Lu, and Maurice R. Marshall J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf501073q • Publication Date (Web): 27 May 2014 Downloaded from http://pubs.acs.org on June 16, 2014

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Journal of Agricultural and Food Chemistry

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Antioxidant, Antibacterial and Antibiofilm Properties of Polyphenols from

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Muscadine Grape (Vitis rotundifolia Michx.) Pomace Against

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Selected Foodborne Pathogens

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Changmou Xu†, Yavuz Yagiz†, Wei-Yea Hsu‡, Amarat Simonne‡, Jiang Lu§∥, Maurice R. Marshall†*

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†Department

of Food Science and Human Nutrition, University of Florida, Gainesville 32611, USA

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‡Department

of Family, Youth and Community Sciences, University of Florida, Gainesville 32611, USA

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§College

of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083,

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People's Republic of China

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∥Center

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Tallahassee 32317, USA

for Viticulture and Small Fruit Research, Florida Agricultural & Mechanical University,

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*Corresponding

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Maurice R. Marshall – University of Florida, Food & Environmental Toxicology Lab, 1642 SW

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23rd Dr., Bldg. 685, PO Box 110720, Gainesville, FL 32611-0720, USA

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Tel.: +1-352-392-1978 Ext. 405; Fax: +1-352-392-1988; E-mail: [email protected]

author:

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ACS Paragon Plus Environment


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Abstract: Polyphenols are predominantly secondary metabolites in muscadine grapes

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playing an important role in the species’ strong resistance to pests and diseases. This

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study examined the above property by evaluating the antioxidant, antibacterial and

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antibiofilm activities of muscadine polyphenols against selected foodborne pathogens.

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Results showed that antioxidant activity for different polyphenols varied greatly ranging

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from 5 – 11.1 mmol Trolox/g. Antioxidant and antibacterial activity for polyphenols

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showed a positive correlation. Muscadine polyphenols exhibited a broad spectrum of

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antibacterial activity against tested foodborne pathogens, especially Staphylococcus

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aureus (MIC 67 – 152 mg/L). Muscadine polyphenols at 4 × MIC caused nearly a 5 log10

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CFU/mL drop in cell viability for S. aureus in 6 h with lysis, while at 0.5 x MIC, they

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inhibited its biofilm formation, and at 16 × MIC, they eradicated biofilms. Muscadine

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polyphenols showed synergy with antibiotics and maximally caused a 6.2 log10 CFU/mL

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drop in cell viability at sub-inhibitory concentration.

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Keywords: Polyphenols, Staphylococcus aureus, MIC, biofilm

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INTRODUCTION

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Foodborne disease is a common, costly—yet preventable—public health problem.

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The United States Centers for Disease Control and Prevention (CDC) estimates that one

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in six Americans (or 48 million people) get sick, 128,000 are hospitalized, and 3,000 die

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from foodborne diseases each year.1 The most commonly identified foodborne pathogens

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include Staphylococcus aureus, Salmonella typhimurium, Shigella sonnei, and


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Escherichia coli O157: H7.1 The increasing incidence of methicillin-resistant

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Staphylococcus aureus (MRSA),2 multiantibiotic-resistant Salmonella typhimurium,3

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multidrug-resistant Shigella sonnei4 and Escherichia coli5 are worldwide problems in

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both food, and clinical or medical settings. For example, although Staphylococcus aureus

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is not always pathogenic, it is a common cause of food poisoning, skin infections (e.g.

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boils), and respiratory disease (e.g. sinusitis).6 Furthermore, the National Institutes of

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Health estimates that 80% of all bacterial infections occurred when bacteria are at the

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biofilm mode of growth.7 Treatment of these infections is complicated by intrinsic

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resistance to conventional antibiotics. Therefore there is an urgent need for new

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compounds or innovative treatment strategies.

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Muscadine grape (Vitis rotundifolia Michx.) is indigenous to the southeastern

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United States and contains a large variety of antioxidant phenolic compounds. Muscadine

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phenolic compounds are well known for their nutraceutical benefit, such as anticancer

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activities8, 9 and improving cardiovascular health.10 Recent studies also confirmed that

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muscadine grape polyphenols and wine have antibacterial activity against foodborne

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pathogenic bacteria E. coli O157:H7,11,

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monocytogenes14, and Streptococcus15. However, few studies have been published about

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the antibacterial activity of specific muscadine polyphenols against these bacteria, the

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correlation between antioxidant and antibacterial activity of polyphenols, the treatment

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use of muscadine polyphenols with/without antibiotics, and their inhibition to biofilm

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formation and eradicating biofilm capability.

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Salmonella typhimurium,13 Listeria

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With increasing demands or requirements for natural food preservatives and

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antibiotics, this work was a comprehensive study to examine the antioxidant, antibacterial



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and antibiofilm activities of muscadine grape pomace polyphenols against foodborne

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pathogens. Polyphenols time–kill curves and synergism with antibiotics also were

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investigated. Ultimately, it was hoped that muscadine polyphenols might have potential

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as antimicrobial and/or antibiofilm agents for use in the food and healthcare industries.

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MATERIALS AND METHODS

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Bacterial Strains. Three Staphylococcus aureus strains (ATCC 35548, ATCC

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12600-U, and ATCC 29247), one Salmonella strain (S. typhimurium), one Shigella strain

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(S. sonnei ATCC 25931), and one Escherichia coli O157:H7 strain (204P) were used for

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the antimicrobial activity study. All the ATCC bacterial strains tested were purchased

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from American Type Culture Collection (ATCC, Manassas, VA, USA). S. typhimurium

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and E. coli O157:H7 (204P) strains were obtained from Dr. Tung-shi Huang’s laboratory

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at Auburn University, Auburn, AL, USA.

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Antibiotics and Chemicals. Ampicillin and streptomycin were purchased from

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Fisher Scientific (Fair Lawn, NJ, USA). Nalidixic acid was purchased from Sigma–

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Aldrich (St Louis, MO, USA). Plant-derived phenolic standards, including gallic acid,

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caffeic acid, catechin, ellagic acid, and quercetin were purchased from Sigma–Aldrich (St

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Louis, MO, USA). All the above commercially purchased chemicals were dissolved in

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sterile distilled water to a concentration of 5000 mg/L. Folin & Ciocalteu’s phenol

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reagent (2N), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 6-hydroxy-2, 5, 7, 8-

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tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma–Aldrich (St.


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Louis, MO, USA). Sodium carbonate and HPLC grade organic solvents were purchased

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from Fisher Scientific Co. (Pittsburgh, PA, USA).

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Grape Materials, and Extraction, Separation, and Identification of Grape

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Phenolic Compounds. Fully ripened Muscadine grape (Vitis rotundifolia Michx.) cv.

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Noble (red) and cv. Carlos (bronze) were harvested from the Center for Viticulture and

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Small Fruit Research (latitude 30.65 N, longitude 84.60 W) at Florida A&M University

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on August 24, 2012. Harvested samples were shipped to the University of Florida on the

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same day and stored at refrigeration (4 °C). Grape skin and seeds were separated

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manually from berries and freeze-dried in a freeze drier (Advantage, The Virtis

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Company, NY, USA) within the following three days. The freeze-dried samples were

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stored in vacuum-packaged polyethylene pouches at -20 °C until analyzed.

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Plant-extracted phenolic compounds (Figure 1) were prepared in-house at the

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Food and Environmental Toxicology Laboratory from Muscadine grape skin and seeds.

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Phenolic compounds from grape were extracted and isolated by a method adapted from

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previous study16. Freeze-dried grape skin and seeds (defatted) were ground sufficiently

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with a stainless-steel grinder (Omni-Mixer 17105, OCI Instruments, CT, USA) to a fine

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powder (≤ 0.25 mm). Powder (5 g) from each sample was extracted with 30 mL of 70%

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methanol. The extraction flasks were vortexed for 30 s, sonicated for 10 min, kept at

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room temperature (22 °C) for 60 min, and sonicated for an additional 5 min. The extracts

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were transferred to tubes and centrifuged at 2820 x g, 0 °C for 10 min (J-LITE®JLA-

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16.250, Beckman Coulter Inc., CA, USA), and the supernatant was collected in separate

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glass tubes. Residue was re-extracted with the same procedure. The collected supernatant

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(60 mL) was evaporated in a rotary evaporator (Büchi, Labortechnik AG, Flawil,



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Switzerland) under reduced pressure at 40 °C to remove solvent. The concentrates

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obtained after evaporation were re-dissolved in 5 mL of distilled water and sonicated for

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5 min.

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Solid-phase extraction technique adapted from previous study17 was used to

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separate fractions of phenolic compounds. Briefly, two C18 Sep-Pak cartridges

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(Whatman® Chromatography, ODS-5) were connected and preconditioned by

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sequentially passing 12 mL ethyl acetate, 12 mL absolute methanol, and 12 mL of 0.01 N

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aqueous hydrogen chloride (HCl) through the cartridges. The prepared re-dissolved

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extract (0.5 mL) was loaded onto cartridges. Each cartridge was rinsed separately with 12

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mL of 0.01N aqueous HCl (Fraction 1, F1), 12 mL of 10% methanol (F2), and 12 mL of

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30% methanol (F3) for Carlos skin extract; for Noble skin extract, the cartridges were

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rinsed with 4 mL of 0.01N aqueous HCl (F1), 15 mL of 10% methanol (with 0.1% HCl)

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(F2), and 12 mL of methanol (with 0.1% HCl) (F3); and finally for Carlos and Noble

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seed extracts, the cartridges were rinsed with 12 mL of 0.01N aqueous HCl (F1), 12 mL

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of 30% methanol (F2), and 12 mL of 50% methanol (F3). To obtain a sufficient volume

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for subsequent study, four individual separations of each extract was performed and then

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combined. The collected fractions were evaporated in a rotary evaporator under reduced

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pressure at 40 °C to remove the solvent. The concentrates obtained after evaporation were

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dissolved in distilled water and stored at 4 °C until analyzed.

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The above described extracts and their fractions were subjected to

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chromatographic analyses by HPLC (Hitachi, L-7000 series, Japan) with an analytical

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SB-C18 column (Zorbax Stablebond, 4.6 mm x 250 mm, 5 µm, Agilent Technologies).

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Mobile phase was 0.5% formic acid in water (solvent A) and 0.5% formic acid in 60%


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methanol (solvent B) under the following gradient: 0–3 min: 5% B, 3–8 min: 30% B, 8–

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25 min: 50% B, 25–30 min: 70% B, 30–35 min: 80% B, 35–47 min: 100% B, 47-51min:

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5% B. The flow rate was 0.9 mL/min. Injection volume was 20 µl. Detection was

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accomplished with a UV detector (Hitachi, L-7400, Japan) set at an absorbance

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wavelength of 280 nm. The retention times for each eluted components in the analyzed

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samples were compared to those of the following standards: gallic acid, caffeic acid,

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catechin, epicatechin, epicatechin gallate, trans-resveratrol, ellagic acid, quercetin, and

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cyanidin-3,5-diglucoside.

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Standards for caffeic acid hexoside, mono- and digalloyl glucose, pentagalloyl

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glucose, ellagic acid hexoside, xyloside and rhamnoside, delphinidin-3,5-diglucoside, and

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peonidin-3,5-diglucoside were not available. Thus, a second HPLC system was employed

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to confirm the identification of these by ESI–MS detection in the Selective reaction

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monitoring (SRM) mode. The LC–MS analyses were performed on a Thermo-Finnigan

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Surveyor HPLC system (Thermo-Finnigan, St. Jose, CA, USA) equipped with a

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TSQuantum controlled by XCalibur data analysis software (version 1.3, Thermo-

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Finnigan). The column and mobile phase used were the same as described above in

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HPLC analysis. The MS acquisition was with ESI interface in negative ionization mode

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at the following conditions: Sheath gas (N2) pressure, 42 arb; auxiliary gas (N2) pressure,

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20 arb; spray voltage 3.96 kV; capillary temperature, 414 °C; collision gas pressure, 1.5

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mTorr; collision energy, 22 V. These compounds were identified on the basis of their

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mass fragmentation data compared with and identical to those reported by the literature18.

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Phenolic Content and Antioxidant Activity Assay. Total phenolic content of

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each extract or fraction was determined by the Folin-Ciocalteu colorimetric method19

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with an ultraviolet–visible Beckman Coulter DU-640 spectrophotometer (Beckman

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Instruments, Fullerton, CA, USA) and expressed as mg gallic acid equivalents per gram

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dry matter. The unitary antioxidant activity of each extract or fraction was evaluated

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using the DPPH radical scavenging capacity assay20 and expressed as mol Trolox

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equivalents per gram polyphenols.

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Antibacterial Activity Assay. Antibacterial activities of the phenolics

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(experimental compounds) were determined by agar disc diffusion method21, and

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pathogenic bacteria for the test were listed above. Isolated colonies of test bacterial

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strains grown overnight on tryptic soy agar plates (TSA; Difco, Becton Dickson, Sparks,

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MD, USA) were cultured in Mueller-Hinton broth (MHB; Difco, Becton Dickinson) at 37

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°C for 18 h. The turbidity of bacterial suspensions was adjusted to approximately 2.0 ×

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108 CFU/mL (Optical Density at 600 nm = 0.2). Bacterial suspensions were evenly swab-

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inoculated on surfaces of Mueller-Hinton agar (MHA; Difco, Becton Dickinson). Then

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each sterile blank disc (6-mm in diameter, Difco, Becton Dickinson) was placed on the

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surface of the test MHA plates, impregnated with 20 µL of experimental compounds

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(Table 2, in triplicate per compound), and incubated at 37 °C for 24 h. Commercial

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antibiotics (ampicillin, nalidixic acid, and streptomycin at 10 µg/disc) and distilled water

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prepared in the same manner as the experimental compounds were used as positive and

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negative controls, respectively. The size of the zones of inhibition was measured and the

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antibacterial activity was expressed in terms of the average diameter of the zone of


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inhibition in millimeters. The absence of a zone of inhibition (≤6 mm) was interpreted as

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the absence of antibacterial activity.

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The MIC was determined using an Alamar blue assay adapted from previous

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study21. Briefly, serial dilution of experimental compounds in MHB (100 µL) with test

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bacteria at a density of 5 x 106 CFU/mL were incubated in flat-bottom, polystyrene, non-

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tissue-culture-treated 96-well microtiter plates at 37 °C without shaking for 20 h. After

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20 h, 10 µL Alamar blue (AB; Invitrogen, Life Technologies, Carlsbad, CA, USA) was

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added to the wells and the plates were shaken gently and incubated for 1 h at 37 °C.

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Plates were gently shaken again, and absorbance at 570 nm and 600 nm were obtained in

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a Microplate Reader (ELX808 Universal, Bio-Tek Instruments, USA). Controls included

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media alone (blank), diluted media plus experimental compounds plus AB (negative),

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and media plus cells plus AB (positive). Percent reduction of AB was calculated using

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the manufacturer’s formula (absorbance difference between 570 nm and 600 nm for each

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experimental well divided by the difference in positive control). Assays were performed

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in triplicate, and the average % reduction was used to determine MIC. The AB MIC was

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defined as the lowest concentration of the experimental compound resulting in ≤50%

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reduction of AB and a purplish well 60 min after the addition of AB22.

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To investigate the correlation of Alamar blue reduction to CFU/mL, CFU/mL was

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obtained from the same wells used to obtain AB absorbances. Before adding AB to all

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treated wells, 10 µl of the well culture was removed and serially diluted (10-fold) in 0.1%

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peptone water followed by plating (20 µl of the dilution) on TSA. Colonies were counted

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after incubation at 37 °C for 24 h and recorded as CFU/mL. Pearson’s two tailed

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correlations were calculated between Alamar blue reduction and CFU/mL.



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Time–kill and Synergy Assay. Time–kill assays were performed using a method

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adapted from previous study23. Briefly, bacteria were cultured to exponential phase (1.0 ×

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108 CFU/mL, OD600 = 0.15) in MHB and challenged with antibiotics or experimental

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compounds at 4 × MIC or 500 mg/L. Synergy between antibiotics and phenolic

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compounds was tested at 0.9 x MIC. Bacterial viability was monitored every hour (0 – 6

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h) by plating cultures onto TSA, and counting colonies after incubation at 37 °C for 24 h

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as described previously. To detect bacterial lysis following the challenge with

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antibacterial agents, the culture turbidity of early exponential phase cultures was

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monitored every hour (0 – 6 h) and finally 24 h by absorbance measurements at 600 nm

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at 37 °C. Synergy between antibiotics and phenolic compounds was defined as follows,

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using log10 CFU/mL at 24 h: synergy was >2-log10 kill, additivity was >1- to 2-log10 kill,

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indifference was 1-log10 kill to 2-log10 growth, and antagonism was >2-log10 growth

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compared to the most active drug alone. Bactericidal activity was defined as a ≥3-log10

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CFU/mL kill at 24 h from baseline24.

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Antibiofilm Activity Assay. The planktonic cell susceptibility to polyphenols and

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polyphenols inhibiting biofilm formation were determined using the Alamar blue assay

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adapted from previous study21. Briefly, serial dilution of experimental compounds in

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MHB (100 µL, with diluted 2,500 to 5 mg/L) with bacteria at a density of 5 x 106

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CFU/mL were incubated in round-bottom, polystyrene, non-tissue-culture-treated 96-well

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microtiter plates at 37 °C without shaking for 20 h. After 20 h, visual MICs were

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identified by the dot at the bottom of the well (the well before dots appear is defined as

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MIC point); then, the content of each well was aspirated and transferred to new flat-

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bottom plates; 10 µL Alamar blue was subsequently added to the new plate wells and


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analyzed as the previous procedure to determine the MICs of planktonic cells. After this,

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the original round-bottom plate wells (after transferring) were gently washed twice with

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250 µL sterile physiological saline to remove non-adherent bacteria; 100 µL sterile

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distilled water was added to these wells followed by 10 µL Alamar blue, and analyzed as

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the previous procedure to determine the Minimum Biofilm Inhibitory Concentration

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(MBIC).

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To determine the reduction capability of polyphenols on preformed biofilm,

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assays were performed in flat-bottom plates with bacteria incubated at a density of 5 x

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106 CFU/mL in 100 µL MHB. First, plates were incubated at 37°C without shaking for

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24 h to allow biofilm formation. Then, two-fold dilutions (diluted 2,500 to 5 mg/L) of

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experimental compounds in MHB were prepared external to the plates. After 24 h

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incubation, well contents were aspirated from all experimental and control wells; and

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wells were gently washed twice with 250 µL sterile physiological saline and 100 µL of

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the appropriate diluted compounds was added. Preformed biofilms were exposed to

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compounds for 20 h at 37 °C without shaking. After 20 h, 10 µL Alamar blue was added

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to the wells and analyzed previously.

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Statistical Analysis. Experimental results were expressed as means ± standard

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error (SE) of three independent replicates. Data were subjected to ANOVA and

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significant differences were tested by post hoc comparison test (Student Newman Keuls)

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at P < 0.05. Microsoft Excel 2008 and SPSS 20.0 for Windows were used for this

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analysis and for calculating Pearson’s correlation coefficients.

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RESULTS



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Muscadine Grape Phenolic Compounds and Antioxidant Activity. Phenolic

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compounds from Muscadine grape (Noble and Carlos) skin and seeds were separated into

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three fractions, respectively, and chromatograms of identified phenolics from Noble skin

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and seed fractions were presented in Figure 1. Caffeic acid hexoside, hydrolyzable

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tannins, and gallic acid were the major phenolics in fraction 1 of Noble skin polyphenols

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(Figure 1a). Anthocyanins were the major phenolics in the corresponding fraction 2

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(Figure 1b), and ellagic acid and its conjugates were in fraction 3 (Figure 1c). Polyphenol

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fractions from Carlos skin were different from Noble skin, with richer ellagic acid and

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conjugates in fraction 3, but almost no anthocyanins in fraction 2 (data not shown).

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However, polyphenol fractions from Noble and Carlos seed showed similar profiles, with

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hydroxybenzoic acid, hydrolyzable tannins, and little flavan-3-ols distributed in fraction 1

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(Figure 1d), major flavan-3-ols and condensed tannins distributed in fraction 2 (Figure

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1e), and ellagic acid and its conjugates distributed in fraction 3 (Figure 1f). Overall,

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anthocyanins (fraction 2, 22.6 mg/g) were the major phenolics for Noble skin, while it

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was ellagic acid and its conjugates (fraction 3, 20.5 mg/g) for Carlos skin; flavan-3-ols

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and condensed tannins (fraction 2, 38.8 and 40.8 mg/g, respectively) were the major

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phenolics for Noble and Carlos seeds (Table 1).

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Antioxidant activity of phenolic compounds varied greatly among tested samples

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(Table 1). For the polyphenol standards, gallic acid showed the strongest antioxidant

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activity per unit, and ranked in order as: Gallic acid > Ellagic acid > Quercetin >

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Catechin > Caffeic acid. For the polyphenol extracts, fraction 3 showed the strongest

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antioxidant activity per unit, and ranked in order as: fraction 3 (ellagic acid and


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conjugates) > fraction 1 (hydroxybenzoic acid and hydrolyzable tannins) > fraction 2

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(anthocyanins or flavan-3-ols and condensed tannins). As expected, antioxidant activity

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for the non-separated grape skin or seed extracts varied among their corresponding

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activities of the fractions.

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Antibacterial Activity of Phenolic Compounds. Antibacterial activity of

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phenolic compounds was initially screened by the standard agar disc diffusion method,

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and then subjected to the MIC test. Table 2 showed that the inhibition of bacterial growth

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was quite drug and species dependent. Muscadine grape skin and seed polyphenol

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extracts exhibited a broad spectrum of antibacterial activity against Gram-positive

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bacteria (S. aureus), but exhibited little to no antibacterial efficacy against Gram-negative

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bacteria (Salmonella typhimurium, S. sonnei ATCC 25931, E. coli O157:H7 204P).

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Inhibition by seed polyphenols (Carlos Seed vs. Noble Seed) against S. aureus was

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positively dependent on concentration while skin polyphenols (Carlos Skin vs. Noble

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Skin) was negatively dependent. Although Carlos Skin was used at higher polyphenol

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concentration than Carlos Seed, its inhibition was significantly weaker than the later.

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These indicated that the antibacterial activity of muscadine grape phenolic compounds

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were not only concentration dependent, but also phenolic specific dependent. Gallic acid

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exhibited good antibacterial activity on tested S. aureus strains while ellagic acid showed

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slightly less inhibition at 100 µg/disc. Caffeic acid, catechin, and quercetin exhibited little

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to no antibacterial efficacy against S. aureus at this concentration. None of these

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polyphenol standards was found to show inhibition on Gram-negative bacteria in this

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study. The susceptibility of all tested bacteria to the three antibiotics (positive control, 10

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µg/disc) varied significantly with zones of inhibition ranging from 0 to 43.8 mm. In



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general, ampicillin and streptomycin showed strongest inhibition than nalidixic acid in

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this study.

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Muscadine grape polyphenols were further demonstrated to have good

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antibacterial activity against Gram-positive bacteria (S. aureus) as shown by an MIC of

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74 – 367 mg/L for skin polyphenols and 67 – 173 mg/L for seed polyphenols (Table 3).

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Inhibition for different phenolic fractions was significantly different. In general, the non-

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separated skin and seed polyphenols exhibited comparable or even better inhibition than

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some of their corresponding fractions. This indicated there might be a synergistic effect

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between different phenolics in inhibiting bacteria growth. Muscadine grape polyphenols

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also showed good antibacterial activity against Gram-negative bacteria (S. sonnei ATCC

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25931) with an MIC of 112 – 735 mg/L, while exhibiting fair antibacterial efficacy

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against S. typhimurium and E. coli O157:H7 204P with an MIC of 224 – 1,976 mg/L and

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304 – 1,540 mg/L, respectively. The phenolic concentration required to inhibit Gram-

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negative bacteria ranged from 1 to 16 times higher than that required for Gram-positive

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bacteria. Unexpectedly, almost no inhibition (≥2,500 mg/L) was found for the

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commercial polyphenol standards on all tested bacterial strains. The inhibition of

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antibiotic controls on all study strains, even though within the same species, varied

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greatly and were either very strong (≤20 mg/L) or almost none (≥1,250 mg/L). By

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contrast, muscadine grape polyphenols displayed consistent antibacterial activity within

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one species, such as S. aureus (Table 3) and Salmonella typhimurium (data not

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completely shown). This indicated that muscadine grape polyphenols had a broad

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spectrum of antibacterial activity and possibly could be used to treat these antibiotic-

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resistant strains.


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Evaluation of Bacterial Killing and Synergistic Effect by Phenolic

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Compounds. In order to further evaluate the bactericidal activity of phenolic compounds,

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the killing kinetics of these compounds were assessed against S. aureus ATCC 35548

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alongside established antibiotics. At 4 x MIC, Noble skin and seed polyphenols caused a

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reduction in cell viability of 4.7 and 4.9 Log10 CFU/mL after 6 h in MHB, respectively,

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while Streptomycin caused a reduction of 6.1 Log10 CFU/mL (Figure 2a). Gallic acid and

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Ellagic acid caused only a slight bacteriostatic effect at 500 mg/L. Significant synergism

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between antibiotics and polyphenols at sub-MIC were observed. Ampicillin (150 mg/L),

329

with a high MIC of 2,500 mg/L, when added with Gallic acid (500 mg/L), Ellagic acid

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(500 mg/L), Noble skin (0.9 x MIC), and Noble seed (0.9 x MIC) caused a reduction in

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cell viability of 0.5, 3.9, 5.1 and 4.8 Log10 CFU/mL, respectively (Figure 2b). While

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Streptomycin added with Gallic acid, Ellagic acid, Noble skin, and Noble seed caused a

333

reduction in cell viability of 1.6, 4.0, 5.0 and 6.2 Log10 CFU/mL, respectively (Figure

334

2c). Although both Gallic acid and Ellagic acid had little effect on bacterial viability

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individually, when combined with antibiotics, Ellagic acid showed considerably stronger

336

bactericidal activity than Gallic acid, especially after 2 h. Synergy of Streptomycin and

337

Noble Seed (at 0.9 x MIC) was better than Streptomycin or Noble seed (at 4 x MIC)

338

alone, and was the strongest bactericidal activity among all the treatments. These

339

indicated that combining grape polyphenols with antibiotics could maximize the

340

bactericidal activity while reducing the usage of antibiotics.

341

Bacterial lysis showed that, at 4 x MIC, Noble skin and seed polyphenols caused a

342

reduction in culture turbidity over a 6 h period while Streptomycin exhibited an increase

343

(Figure 2d). This indicated Noble skin and seed polyphenols were bactericidal and killed



344

bacterial with concomitant cell lysis while Streptomycin was nonlysis but bactericidal.

345

Antibiotics combined with Noble seed polyphenols exhibited stronger bacterial lysis

346

ability than skin polyphenols (Figure 2e, 2f). Overall, there was a positive correlation (r =

347

0.84) between bacterial number (Log10 CFU/mL) and culture turbidity (OD600) at 6 h.

348

After 24 h, as the turbidity of the control continued to increase, all treatments except

349

Gallic acid caused a reduction in culture turbidity. This suggests that with plenty of time,

350

phenolic compounds promoted a loss of culture turbidity as well as a loss of bacterial

351

viability.

352

Antibiofilm Activity of Phenolic Compounds. To investigate the resistance of

353

biofilm versus planktonic-grown strains, Alamar blue MICs of Nalidixic acid against a

354

variety of pathogenic bacteria and MBICs against their preformed biofilm were studied

355

(Table 4). Alamar blue MBICs increased at least four-fold relative to planktonic MICs.

356

These data are consistent with previous reports of many-fold increases in drug resistance

357

of biofilm versus planktonic-grown strains22, 25.

358

A variety of antibiotics and phenolic compounds with different mechanisms were

359

further studied on planktonically grown S. aureus susceptibility, inhibition of biofilm

360

formation and resistance of preformed biofilm (Table 5). Visual and Alamar blue MICs

361

for planktonically grown strains were identical with Ellagic acid as an exception. Thus,

362

MICs can be generally determined visually or spectrophotometrically. MBICs for

363

inhibiting biofilm formation were within two 2-fold dilutions lower than MICs, while

364

MBICs of preformed biofilm were at least 16-fold higher than MICs. For example, the

365

MIC of Carlos seed polyphenols against planktonic-grown S. aureus ATCC 35548 was

366

40 mg/L, against biofilm formation was 20 mg/L, and against preformed biofilm was as


Page 16 of 38

Page 17 of 38


367

high as 641 mg/L. These findings indicated that grape polyphenols were able to inhibit

368

bacteria biofilm formation at a sub-MIC concentration without killing the bacteria;

369

however, to eradicate the preformed biofilm a high concentration of phenolic compounds

370

is needed.

371 372

DISCUSSION

373 374

This study demonstrated that muscadine grape skin and seed polyphenols had

375

potent bactericidal activity against planktonic cells and biofilm formation of Gram-

376

positive bacteria, S. aureus. The fact that polyphenols acted to cause cell lysis and

377

therefore compromise the integrity of the bacterial membrane may explain why these

378

compounds have the ability to eradicate S. aureus biofilms; potent antibiofilm activity is

379

a property that is often associated with membrane-perturbing agents26. The limited

380

activity of grape polyphenols against Gram-negative bacteria was probably because of

381

the limited ingress across the outer membrane27. However, another intrinsic mechanism

382

that has been insufficiently studied is efflux, and multidrug efflux pumps have been

383

shown to be associated with the insusceptibility of E. coli to various biocidal agents28.

384

A positive correlation (r = 0.72) between antioxidant (Table 1) and antibacterial

385

(Table 2) activities of polyphenol standards against S. aureus on agar medium was found.

386

Also a weak positive correlation (r = 0.53) between antioxidant (Table 1) and

387

antibacterial (Table 3) activities of muscadine polyphenols against S. aureus was

388

observed. These findings suggested that the stronger the antioxidant activity per unit

389

polyphenols, the better the antibacterial activity against S. aureus. However, strong



390

antibacterial activity against S. aureus doesn’t necessarily mean strong antioxidant

391

activity since no antioxidant activity was demonstrated for antibiotics (ampicillin, nalidix

392

acid, and streptomycin) (data not shown). Similar to a previous finding29, there was a

393

positive correlation (r = 0.77) between total polyphenol content and total antioxidant

394

activity for these grape extracts and fractions. Nonetheless, higher phenolic levels do not

395

necessarily correlate with increased inhibition against S. aureus but rather the type and

396

concentration of compounds present in these extracts.

397

Interestingly, compared to grape phenolic extracts, commercial phenolic standards

398

Gallic acid and Ellagic acid exhibited stronger antioxidant activity per unit, 11.1 and 9.8

399

mmol/g, respectively (Table 1); and also showed good antibacterial activity on tested S.

400

aureus strains using the disc diffusion method (Table 2). However, further Alamar blue

401

MIC test (Table 3) and time-killing study (Figure 2) suggested that Gallic acid and

402

Ellagic acid possess negligible antibacterial efficacy against S. aureus. This contradictory

403

finding indicated the very different antibacterial mechanism of Gallic acid and Ellagic

404

acid on a solid surface versus in a liquid. Other phenolic standards Caffeic acid, Catechin,

405

and Quercetin exhibited little to no inhibition on the tested strains in all assays.

406

Therefore, it seems that antibacterial activities of commercial phenolic standards were

407

weaker compared to the muscadine grape phenolic extracts. One possible reason was that

408

the antibacterial activities of these phenolics were lost during their processing or these

409

phenolics existed as ineffective isomer forms. Another possible reason was the synergism

410

between multiple phenolics increased their antibacterial activities. These findings can be

411

further supported by other authors’ reports that pure catechin and epicatechin possess

412

negligible antibacterial activity against Gram-positive and Gram-negative bacteria30;


Page 18 of 38

Page 19 of 38


413

while plant extracted whole polyphenols exhibited good antibacterial activity against

414

several food-borne pathogenic bacteria31, for instance, the mean MICs against S. aureus

415

ranged from 98 to 389 mg/L and against E. coli ranged from 450 to 1519 mg/L. Also, in

416

this study, whole extracted skin and seed polyphenols exhibited comparable or even

417

better inhibition than their corresponding fractions. Therefore, whole extracted

418

polyphenols were used in the following studies.

419

This study also investigated the correlation between Alamar blue (AB) percent

420

reduction by bacteria metabolic activity22, and the existing bacteria number per well on

421

Carlos seed polyphenols and Ellagic acid at various concentrations. Results showed that

422

percent reduction of AB had a significant correlation (r = 0.94 and 0.92, respectively)

423

with existing bacteria number (Figure 3). Carlos seed polyphenols at 40 mg/L inhibited

424

the bacteria number at initial level (6 Log10 CFU/mL) with a 24.9% reduction of AB after

425

20h incubation, while at 160 mg/L (4 x MIC) killed the bacteria with no reduction of AB.

426

Ellagic acid at 2,500 mg/L reduced bacteria number by 1.4 Log10 CFU/mL with a 39.2%

427

reduction of AB after 20h incubation. These confirmed that the colorimetric AB assay is

428

a reliable, reproducible means of determining bacteria antibiotic susceptibility. A

429

previous study also showed that the AB assay was a good means of determining biofilm

430

antibiotic susceptibility and had good to excellent correlation with two other biofilm

431

susceptibility methods, XTT reduction and viable counts22.

432

The antibacterial mechanisms of phenolics are attracting more and more research

433

in recent years. The membrane-interaction of phenolics is a prevailing theory30. Phenolics

434

are partially hydrophobic; this character may allow them to interact with the lipid bilayer

435

of bacterial cytoplasmic membrane and lipopolysaccharide interfaces more effectively by



436

decreasing membrane stability32. For example, Epicatechin gallate could rapidly enter the

437

cytoplasmic membrane, causing an immediate reduction in bilayer fluidity33. The

438

cytoplasmic membrane constitutes the selective barrier controlling cellular ingress and

439

egress of materials and houses many of the enzymes involved in bioenergetic functions,

440

cell wall synthesis and macromolecule secretion. Penetration of phenolics into this

441

phospholipid bilayer will have a profound outcome on a wide range of essential cell

442

functions. Since intercalation of these chemical structures could induce membrane

443

disruption that permits solute equilibration across the bilayer, resulting in cell death, but

444

more subtle interactions at sub-inhibitory concentrations induce re-organization of

445

membrane architecture with implications for the capacity of the target bacteria to cause

446

disease in the susceptible host32. Phenolics also were found to prevent the formation of

447

biofilms in this study (Table 5) as well as others’ report34, 35. This may further support the

448

membrane-interactive mechanism since potent antibiofilm activity is a property that is

449

often associated with membrane-perturbing agents26. Thus, phenolics exhibit at least two

450

biological activities: inhibition of bacterial growth at high concentrations and/or induce

451

bacteria membrane disruption and prevention of biofilm formation at lower

452

concentrations. In view of these activities, phenolics may be useful in a synergistic

453

approach with conventional antibiotics against antibiotic-resistant forms of pathogens

454

(e.g. multidrug-resistant clinical isolates MRSA) in clinical medicine. Our time-killing

455

study (Figure 2) indicated that muscadine Noble seed phenolics combined with

456

Ampicillin or Streptomycin at subinhibitory concentrations greatly reduced S. aureus

457

ATCC 35548 (MRSA) cell viability by 4.8 or 6.2 Log10 CFU/mL, respectively.


Page 20 of 38

Page 21 of 38


458

Naturally occurring molecules, predominantly secondary metabolites, are the

459

source of the majority of drugs in clinical use today and they continue to be an important

460

basis of new therapeutics for bacterial infectious diseases36. Phenolics are secondary

461

metabolites in muscadine grapes and play important roles in the strong resistant character

462

of this species to pests and diseases, including Pierce’s disease37. Evidence is emerging

463

that phenolics may be useful in the control of common oral infections, such as dental

464

caries and periodontal disease38, 39. In this case, phenolics can be incorporated into rinse

465

and toothpaste formulations. Muscadine phenolics also have been proven to possess

466

strong anticancer activities, such as inhibiting proliferation of colon8 and prostate9 cancer

467

cells by inducing apoptosis. Importantly, phenolics are widely distributed in edible

468

grapes40; they have minimal toxicity41. Therefore, even though outstanding bactericidal

469

properties cannot be achieved because of a relatively high concentration required in this

470

study and the partial metabolism of these by intestinal bacteria42, phenolic agents still

471

have promise as supplements for nutraceutical value and conventional antibacterial

472

chemotherapies by inhibiting the growth of foodborne pathogens in the body. The broad

473

ability of muscadine phenolics to have strong antibacterial activity, to inhibit biofilm

474

formation, and to synergistically work with antibiotics may stimulate a muscadine by-

475

product market as natural food preservatives, potential antibiotic replacements and/or as

476

natural sanitizers for processing equipment where foodborne pathogens reside.

477 478 479 480



481

ACKNOWLEDGEMENTS

482 483

The authors are grateful to the Center for Viticulture and Small Fruit Research at Florida

484

A&M University for providing muscadine grape materials and to USDA/IR4 Lab at

485

University of Florida for assisting in HPLC-MS/MS analysis.

486 487

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619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636


Page 28 of 38

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637


Figure captions

638 639

Figure 1. Phenolic compounds and their structures found in muscadine grape skin and

640

seeds.

641

(a), (b), and (c) are Noble skin fractions 1, 2, and 3 respectively; (d), (e), and (f) are

642

Noble seed fractions 1, 2, and 3 respectively.

643 644

Figure 2. Evaluation of killing (a, b, c) and lytic (d, e, f) action of experimental

645

compounds and their synergism with antibiotics against S. aureus ATCC 35548 in MHB.

646

(Means of three independent replicates; MICs of Noble Skin, Noble Seeds, and

647

Streptomycin were 113, 88, and 20 mg/L, respectively).

648 649

Figure 3. Percent reduction of Alamar blue (bars) and Log10 CFU/mL () for

650

phenolic compounds on S. aureus ATCC 35548.

651

(Means of three independent replicates; Test strains were inoculated at 5 x 106 CFU/mL

652

in each well).

653

* Values differ significantly (P < 0.05) from values without phenolic compounds

654



Page 30 of 38

Table 1. Phenolic content and antioxidant activity of experimental compounds Experimental compounds Phenolic content1 Antioxidant activity per unit2 (mg/g) (mmol/g) Polyphenol Standards Gallic Acid 11.1 ± 0.2a Caffeic Acid 5.1 ± 0.2e Catechin 5.3 ± 0.3e Ellagic Acid 9.8 ± 0.6ab Quercetin 9.4 ± 0.4b 3 Polyphenol Extracts Carlos Skin (CK) Polyphenols 39.0 ± 2.1c 6.3 ± 0.5d f CK-F1 9.1 ± 0.8 7.4 ± 0.3cd f CK-F2 8.7 ± 0.7 5.9 ± 0.2de e CK-F3 20.5 ± 1.4 8.7 ± 0.6bc d Noble Skin (NK) Polyphenols 35.9 ± 2.5 5.3 ± 0.3e h NK-F1 5.5 ± 0.3 7.9 ± 0.5c e NK-F2 22.6 ± 1.8 5.0 ± 0.4e g NK-F3 7.5 ± 0.3 8.9 ± 0.8bc b Carlos Seed (CS) Polyphenols 50.8 ± 3.4 10.1 ± 0.4ab h CS-F1 5.1 ± 0.2 8.4 ± 0.2bc c CS-F2 38.8 ± 1.2 7.4 ± 0.3cd g CS-F3 7.1 ± 0.4 10.8 ± 0.5a a Noble Seed (NS) Polyphenols 58.2 ± 3.5 9.2 ± 0.3b g NS-F1 7.6 ± 0.6 8.6 ± 0.2bc c NS-F2 40.8 ± 3.1 6.9 ± 0.1d f NS-F3 8.9 ± 0.6 11.1 ± 0.5a 1 2 3

Phenolic content of extracts was expressed as mg gallic acid equivalents per gram dry matter. Antioxidant activity was determined by DPPH assay and expressed as mmol Trolox equivalents per gram polyphenols. Fractions 1-3 for Carlos and Nobel skins and seeds were eluted from tandem C18 Sep-Pak cartridges with different solvents: Skins, F10.01 N HCl; F2-10% MeOH; F3-30% MeOH and Seeds, F1-0.01 N HCl; F2-30% MeOH; F3-50% MeOH. Results were expressed as means of triplicate determinations ± SE; different superscript letters within columns were significantly different (P < 0.05).


Page 31 of 38


Table 2. Antibacterial activity of experimental compounds against selected foodborne pathogens using the disc diffusion method Zone of inhibition (mm) on MHA Experimental compounds Gram-positive bacteria Gram-negative bacteria S. aureus S. aureus S. aureus S. typhimurium S. sonnei E. coli Antibiotic Controls Ampicillin (10 µg/disc) Nalidixic Acid (10 µg/disc) Streptomycin (10 µg/disc) Polyphenol Standards Gallic Acid (100 µg/disc) Caffeic Acid (100 µg/disc) Catechin (100 µg/disc) Ellagic Acid (100 µg/disc) Quercetin (100 µg/disc) Polyphenol Extracts Carlos Skin (390 µg/disc) Noble Skin (290 µg/disc) Carlos Seed (340 µg/disc) Noble Seed (450 µg/disc)

ATCC 35548

ATCC 12600-U

ATCC 29247

ATCC 25931

O157:H7 204P

0 0 15.2 ± 0.3a,C

43.8 ± 1.2a,A 0 14.0 ± 0.2b,D

13.5 ± 0.5b,D 0 0

24.2 ± 0.8a,B 0 19.8 ± 0.6b,A

19.0 ± 0.5a,C 19.7 ± 0.3a 14.0 ± 0.2b,D

9.2 ± 0.1b,E 0 17.6 ± 0.4a,B

14.7 ± 0.5a,B 0 0 12.7 ± 0.3* 0

11.3 ± 0.3c,C 0 0 12.3 ± 0.5* 0

17.7 ± 0.6a,A 8.2 ± 0.3ef 0 14.0 ± 0.2* 8.8 ± 0.2e

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

9.0 ± 0.3e,A 10.6 ± 0.2d,A 12.4 ± 0.2c,A 14.1 ± 0.1b,A

8.2 ± 0.1e,B 10.1 ± 0.2d,A 11.4 ± 0.3c,AB 13.5 ± 0.2b,AB

7.8 ± 0.3f,B 10.7 ± 0.2d,A 12.0 ± 0.1c,A 14.0 ± 0.5b,A

0 0 0 0

0 0 7.2 ± 0.3c,C 7.7 ± 0.2c,C

0 0 0 0

Results were expressed as means of triplicate determinations ± SE; Results with different superscript lowercase letters in columns and different superscript uppercase letters in rows were significantly different (P < 0.05); Results > 6 mm were considered effective against bacteria, ≤ 6 mm were considered non-effective and recorded as 0. * Partial inhibition within a zone and not included in the statistical analysis.



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Table 3. Minimum inhibitory concentration (MIC) of experimental compounds against human pathogenic bacteria Mean MIC (mg/L) value1 Experimental compounds Gram-positive bacteria Gram-negative bacteria S. aureus S. aureus S. aureus S. typhimurium S. sonnei E. coli Antibiotic Controls Ampicillin Nalidixic Acid Streptomycin Polyphenol Standards Gallic Acid Caffeic Acid Catechin Ellagic Acid Quercetin Polyphenol Extracts2 Carlos Skin (CK) Polyphenols CK-F1 CK-F2 CK-F3 Noble Skin (NK) Polyphenols NK-F1 NK-F2 NK-F3 Carlos Seed (CS) Polyphenols CS-F1 CS-F2 CS-F3 Noble Seed (NS) Polyphenols NS-F1 NS-F2 NS-F3

ATCC 35548

ATCC 12600-U

ATCC 29247

ATCC 25931

O157:H7 204P

2,500a,A 20g,C 20g,B

2,500A

2,500

2,500a 2,500a 2,500a >2,500 >2,500

>2,500 2,500a >2,500 >2,500 >2,500

2,500a >2,500 >2,500 >2,500 >2,500

2,500a >2,500 >2,500 >2,500 >2,500

>2,500 2,500a >2,500 >2,500 >2,500

152c,C 119d,C 183b,D 149c,D 113d,C 112d,C 145c,C 74f,D 67f,C 87ef,C 148c,B 78e,C 88ef,C 123d,D 69f,D 97e,C

304c,B 238d,B 367b,C 149e,D 226d,B 112ef,C 290c,C 148e,C 67g,C 173e,C 74g,C 156e,B 88fg,C 123ef,D 138e,C 97f,C

152e,C 119f,C 367c,C 298d,C 113f,C 112f,C 145e,C 74gh,D 67h,C 173e,C 74gh,C 78gh,C 88g,C 123f,D 69h,D 97g,C

608c,A 476d,A 1,470b,A 1,191b,A 903c,A 224e,B 1,159b,A 297e,B 1,069b,A 1,386b,A 1,181b,A 624c,A 1,403b,A 1,976a,A 1,107b,A 1,540b,A

304d,B 119f,B 735b,B 595b,B 226e,B 112f,C 579b,B 148f,C 268e,B 348d,B 148f,B 156f,B 352d,B 492c,C 276e,B 193ef,B

304d,B 476c,A 1,470b,A 1,191b,A 903bc,A 448c,A 1.159b,A 593c,A 1,069b,A 1,386b,A 1,181b,A 624c,A 1,403b,A 988bc,B 1,107b,A 1,540b,A

1

MICs were determined by the Alamar blue assay. MIC defined as the lowest experimental compound concentration resulting in ≤ 50% reduction of AB (average of three experiments) and a purplish well 60 min after addition of AB. Results with different superscript lowercase letters in columns and different superscript uppercase letters in rows were significantly different (P < 0.05). MIC values > 2,500 mg/L were not included in the statistical analysis. Test strains were inoculated at 5 x 106 CFU/mL in each well. 2 Fractions 1-3 for Carlos and Nobel skins and seeds were eluted from tandem C18 Sep-Pak cartridges with different solvents: Skins, F1-0.01 N HCl; F2-10% MeOH; F3-30% MeOH and Seeds, F1-0.01 N HCl; F2-30% MeOH; F3-50% MeOH.


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Table 4. Minimum inhibitory concentration (MIC) of Nalidixic acid against pathogenic bacteria and Minimum Biofilm Inhibitory Concentration (MBIC) against their preformed biofilm reduction Pathogenic bacteria MIC1 MBIC2 (mg/L) (mg/L) Gram-positive bacteria S. aureus ATCC 35548 20 1,250* S. aureus ATCC 12600-U 156 625*,# S. aureus ATCC 29247 20 1,250* Gram-negative bacteria Salmonella typhimurium 2,500 >2,500 Shigella sonnei ATCC 25931 2,500* 1, 2

MIC or MBIC defined as the lowest nalidixic acid concentration resulting in ≤50% reduction of AB (average of three experiments) and a purplish well 60 min after addition of AB. * Significant differences between MICs of nalidixic acid versus MBICs (P < 0.05). # Significant differences between MBICs of nalidixic acid against biofilms within the same S. aureus species (P < 0.05).



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Table 5. Minimum inhibitory concentration (MIC) of experimental compounds against S. aureus ATCC 35548, and Minimum Biofilm Inhibitory Concentration (MBIC) against its biofilm formation and preformed biofilm reduction Experimental MIC1 (mg/L) MIC2 (mg/L) MBIC3 (mg/L) MBIC4 (mg/L) compounds (visual) (planktonic cell) (biofilm formation) (biofilm reduction) * Ampicillin 2,500 2,500 1,250 >2,500# * Nalidixic Acid 20 20 5 1,250*,# * Streptomycin 20 20 5 625*,# Gallic Acid >2,500 >2,500 >2,500 >2,500 Ellagic Acid 312 >2,500 2,500 >2,500 Carlos Skin Phenolics 122 122 61* 1,950*,# * Noble Skin Phenolics 113 113 57 1,808*,# * Carlos Seed Phenolics 40 40 20 641*,# * Noble Seed Phenolics 51 51 25 801*,# 1

Visual MIC was identified by the dot at the bottom of the well. The well before dots appear defined as MIC point. Planktonic cell MIC or MBIC defined as the lowest experimental compound concentration resulting in ≤50% reduction of AB (average of three experiments) and a purplish well 60 min after addition of AB. * Significant differences between MICs (visual and planktonic cell) of experimental compounds versus MBICs (biofilm formation and reduction) (P < 0.05). # Significant differences between MBICs of experimental compounds against biofilm formation versus preformed biofilm reduction (P < 0.05). 2, 3, 4


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Figure 1. Phenolic compounds and their structures found in muscadine grape skin and seed. ACS Environment (a), (b), and (c) are Noble skin fractions 1, 2, and 3 respectively; (d), (e), and (f)Paragon are Noble Plus seed fractions 1, 2, and 3 respectively.

Journal of Agricultural and Food Chemistry 10

0.5

Log10 CFU/mL

Control 8

Gallic Acid (500 mg/L) Ellagic Acid (500 mg/L)

6

Noble Skin (4 x MIC) Noble Seed (4 x MIC) Streptomycin (4 x MIC)

4

Absorbance OD600

(a)

Page 36 of 38

(d)

0.4 0.3 0.2 0.1 0

2 0

1

2

3

4

5

0

6

1

2

3

Ampicillin (150 mg/L)

Log10 CFU/mL

0.5

Control

(b)

8 Ampicillin (150 mg/L) + Gallic Acid (500 mg/L) Ampicillin (150 mg/L) + Ellagic Acid (500 mg/L) Ampicillin (150 mg/mL) + Noble Skin (0.9 x MIC) Ampicillin (150 mg/L) + Noble Seed (0.9 x MIC)

6

4

2

Absorbance OD600

10

5

6

24

5

6

24

5

6

24

(e)

0.4 0.3 0.2 0.1 0

0

1

2

3

4

5

6

0

1

2

3

Time (h) 10

0.5

8

Streptomycin (0.9 x MIC) Streptomycin (0.9 x MIC) + Gallic Acid (500 mg/L) Streptomycin (0.9 x MIC) + Ellagic Acid (500 mg/L) Streptomycin (0.9 x MIC) + Noble Skin (0.9 x MIC) Streptomycin (0.9 x MIC) + Noble Seed (0.9 x MIC)

6

4

2 1

2

3

4

5

6

Absorbance OD600

(c)

0

4

Time (h)

Control

Log10 CFU/mL

4

Time (h)

Time (h)

(f)

0.4 0.3 0.2 0.1 0 0

Time (h)

1

2

3

4

Time (h) Figure 2. Evaluation of killing (a, b, c) and lytic (d, e, f) action of experimental compounds and their synergism with antibiotics against S. aureus ATCC 35548 in MHB. (Means of three independent replicates; MICs of Noble Skin, Noble Seed, and Streptomycin were 113, 88, and 20 mg/L, respectively.


Page 37 of 38


100

(a) r = 0.94

% Reduction AB

10

CFU/mL

* 8

* 60

6

40

4

Log10 CFU/mL

% Reduction of Alamar blue

80

* 2

20

* * 0

0 160

80

40

20

10

5

0

Phenolic Compound Concentration (Carlos Seeds, mg/L) % Reduction AB

CFU/mL 10

(b) r = 0.92

80

9

*

60

40

8

*

Log10 CFU/mL

% Reduction Alamar blue

100

7

6

20 2500

1250

625

312.5

156.2

78.1

0

Phenolic Compound Concentration (Ellagic Acid, mg/L) Figure 3. Percent reduction of Alamar blue (bars) and Log10 CFU/mL () for phenolic compounds on S. aureus ATCC 35548. (Means of three independent replicates; Test strains were inoculated at 5 x 106 CFU/mL in each well). * Values differ significantly (P < 0.05) from values without phenolic compounds.



Table of Content Graphic


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Antioxidant, Antibacterial, and Antibiofilm Properties of Polyphenols

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