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Photodynamic Treatment: a Novel Method for Sanitation of Food Handling and Food Processing Surfaces

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Photodynamic Treatment: a Novel Method for Sanitation of Food Handling and Food Processing Surfaces 1020 Journal of Food Protection, Vol. 72, No. 5, 2009, Pages 1020–1024 Copyright ᮊ, International Association for Food Protection Photodynamic Treatment: A Novel Method for Sanitation of Food Handling and Food Processing Surfaces LUBOV Y. BROVKO,1* ANN MEYER,1 ARVINDER S. TIWANA,1 WEI CHEN,2 HAN LIU,2 CARLOS D. M. FILIPE,2 AND MANSEL W. GRIFFITHS1 1Canadian Research Institute for Food Safety, University of Guelph, Guelph, Ontario, Canada N1G 2W1; and 2Department of Chemical Engineering, McMaster University, Hamilton, Ontario, Canada L8S 4L7 MS 08-507: Received 6 October 2008/Accepted 14 December 2008 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/5/1020/1677894/0362-028x-72_5_1020.pdf by guest on 01 October 2021 ABSTRACT The photodynamic bactericidal effect of the photoactive dyes acriflavine neutral, rose bengal, phloxine B, and malachite green (oxalate salt) at concentrations of 5 to 5,000 ␮g/ml against two gram-negative strains (Escherichia coli LJH 128 and Salmonella Typhimurium C1058), two gram-positive strains (Bacillus sp. C578 and Listeria monocytogenes LJH 375), and yeast (Saccharomyces cerevisiae C1172) was investigated. Incubation of the investigated bacteria with acriflavine neutral under illumination resulted in a significant reduction in cell numbers compared with dark incubation. Rose bengal caused a significant killing effect for bacteria incubated both in the dark and under illumination. Malachite green was active against gram-positive bacteria under illumination and did not affect gram-negative bacteria or yeasts. Incubation with phloxine B resulted in a significant decline in cell numbers for gram-positive bacteria, both in the dark and under illumination; gram-negative bacteria and yeasts were unaffected. Conjugation of rose bengal and phloxine B with poly(vinyl amine) resulted in an enhanced bactericidal effect during both dark and light incubation. This was explained by electrostatic interaction of the polymer with the cell surface, which resulted in closer contact of the photoactive dye and cell. No killing effect was observed for yeasts incubated with dye conjugates. Filter paper treated with dye–poly(vinyl amine) conjugates showed high photodynamic bac- tericidal activity against the bacterial strains, but not against the yeasts. The extent of bacterial killing depended on the nature and concentration of the dye conjugate and the type of microorganism. The presented data suggest that a photodynamic approach for constructing ‘‘self-decontaminating’’ materials has potential. Numerous outbreaks of foodborne illness have been the need for novel cleaning and sanitation strategies has attributed to postprocess contamination of product due to been recognized, especially to combat biofilms. inadequate sanitation of food contact surfaces (8). Recent The bactericidal effect of visible light illumination on research has indicated that pathogens can acquire resistance bacteria treated with nontoxic photosensitizers has been to commonly used sanitizers and, as a result of such ad- shown (13, 17, 18). Their efficacy depended on both cell aptation, cross-resistance to antibiotics has been observed type and the nature of the photosensitizer used. The method (4, 12). Emergence of multiantibiotic resistant pathogens is relies on illumination of microorganisms treated with nontoxic a risk to animal health and to the safety of food products. photosensitizers by low-power visible (red, blue, or white) The problem of removing bacteria from food processing light. Interaction of light with photosensitizers produces surfaces is compounded by the fact that microorganisms highly active, short-lived free radicals that are able to de- growing in a biofilm secrete extracellular polymeric sub- stroy cell components in close vicinity of the dye. This stances, which can remain attached to the cell in a capsular results, in some cases, in a 5- to 7-log cycle reduction in form or, alternatively, be released as a slime in which the bacterial counts, indicating the promise of the approach. cells form a complex multicellular structure (6, 12, 20). Development of resistance to antimicrobial photodynamic Bacteria in biofilms are more resistant to sanitizers. Deter- treatment has not been reported, which makes this approach gents can be formulated to remove particular types of soils, more attractive for investigation. As visible light can pen- for example, proteinaceous, fatty, carbohydrate or mineral etrate thick layers, antimicrobial photodynamic treatment soils, rather than to remove microorganisms. Gibson et al. may also provide a method for eracidating biofilms in situ (9) reported that detergents did not significantly improve (18). the removal of attached gram-positive and gram-negative Clinical applications of antimicrobial photodynamic organisms from food contact surfaces. Many commonly treatment are being investigated, including treatment of used enzymatic cleaners also fail to reduce the viable bac- wound infections and burns, infections in body cavities, terial load or remove the bacterial extracellular polymeric such as mouth, ear, sinus, stomach, and surface infections substances from surfaces (2, 16). In view of these findings, of cornea and skin (7, 10, 19). Currently, photoantimicro- bials are used for the disinfection of blood products, e.g., * Author for correspondence. Tel: 519-824-4120, Ext 58301; Fax: 519- the Marco Blueflex system (19, 21). 763-0952; E-mail: [email protected]. In a preliminary investigation (3), we had shown that J. Food Prot., Vol. 72, No. 5 PHOTODYNAMIC SURFACE SANITATION 1021 TABLE 1. Physicochemical and photochemical properties of the photoactive dyes Molecular Absorption/emission Molar extinction Dye mass (Da) wavelength (nm) (1) coefficient (MϪ1 cmϪ1) Current applications Acriflavin neutral 665.8 436/520 3.6 ϫ 104 Biological stain, topical antiseptic, and treatment of bacte- (euflavine) rial, fungal, and parasitic infection in fish Rose bengal 1,017.6 525,540/550–600 7.28 ϫ 104 Biological stain, eye drops to assess the damage of con- junctiva and corneal cells, and treatment of certain can- cers Malachite green 927.02 629/NAa 15.0 ϫ 104 Dye for silk, leather and paper, biological stain, topical antiseptic, and treatment for parasitic, fungal, and bac- terial infections in fish Phloxine B 829.64 524/600 10.1 ϫ 104 Colorant for food, cosmetics and drugs, biological stain, disinfection and detoxication of waste water, toxicant Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/5/1020/1677894/0362-028x-72_5_1020.pdf by guest on 01 October 2021 for fruit fly, bacteriocidal agent in plants a NA, not available. photoactive dyes such as toluedine blue, and tetramethyl The PVAm (degree of hydrolysis of 97% and viscosity average rosamine and its derivatives possess high photodynamic molecular mass of 150 kDa; BASF, Mississauga, Ontario, Canada) killing activity against both gram-positive and gram-nega- was further hydrolyzed with 5% NaOH at 75ЊC for 48 h, under tive pathogens present as planktonic cells and in biofilms, nitrogen purge, which was followed by thorough dialysis in water as well as against bacterial spores and viruses. The objec- and freeze-drying (5). One hundred milligrams of hydrolyzed PVAm was dissolved in 4 ml of water (pH 5.5), and 150 mg of tive of this study was to assess the degree of inactivation the dye was added to the solution. Conjugation was started by of microorganisms as a result of photodynamic treatment addition of 100 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbo- with common photoactive dyes that are compatable with diimide hydrochloride (EDC; Sigma-Aldrich, Canada). The mix- food industry requirements. The second objective was to ture was stirred for 24 h in the dark and at room temperature. The explore the possibility of designing self-decontaminating sample was then dialyzed (molecular weight cutoff of 3,500; materials on the basis of the dyes that would show a strong Spectra/Pro 3 Dialysis Membrane, Spectra Laboratories, Rancho bacteriocidal effect on illumination with incident light. Dominguez, CA) against deionized water for 1 week to separate uncoupled dye and excess EDC from the PVAm. The concentra- MATERIALS AND METHODS tion of labeled PVAm in these samples was determined from the Bacterial cultures and cultivation. The microorganisms absorbance at 540 and 550 nm for PVAm-PhB and PVAm-RB, used in the study were obtained from the culture collection of the respectively. The final concentration of PVAm-PhB was 0.46 mg/ Canadian Research Institute for Food Safety (University of ml, and 0.48 mg/ml for PVAm-RB, with dye contents of 9.2 and Guelph, Ontario) and comprised two gram-negative strains (Esch- 9.6%, respectively. This corresponded to dye concentrations of 42 ␮ ␮ erichia coli LJH 128 and Salmonella Typhimurium C1058), two g/ml for PhB and 46 g/ml for RB. gram-positive strains (Bacillus sp. C578 and Listeria monocyto- Photoinactivation in broth. Photoinactivation was per- genes LJH 375), and yeast (Saccharomyces cerevisiae C1172). All formed in a 96-well microtiter plate by adding 150 ␮l of bacterial microorganisms were cultivated at 30 or 37ЊC, with shaking (120 suspension (105 to 106 CFU/ml) in PBS and 150 ␮l of the dye rpm; Incubator Shaker, New Brunswick, Scientific, Edison, NY) solution to each well. The plate was then illuminated for 30 min for 24 h by using Luria-Bertani broth (Difco, Becton Dickinson, by white light under the Illumatool illuminator (Montreal Biotech, Sparks, MD) for E. coli and Salmonella, brain heart infusion broth Inc., Montreal, Quebec, Canada). The light source (halogen lamp) (Difco, Becton Dickinson) for L. monocytogenes and Bacillus sp., was placed at a distance of 6.5 cm from the surface and provided and yeast broth (Difco, Becton Dickinson) for S. cerevisiae. Enu- uniform illumination with an average intensity of 0.45 Ϯ 0.04 meration of bacterial cells was performed by using the spread- mW/cm2. An identical plate was kept in the dark and used as a plate method in which serial dilutions of bacterial suspension were control. After 30 min of incubation, the bacterial population in inoculated onto the plate with the respective agar-containing me- each well was enumerated by the spread-plate method as described dia.
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