Articles Service
Research article
Removal of Salmonella Typhimurium Biofilm from Food Contact Surfaces Using Quercus infectoria Gall Extract in Combination with a Surfactant
1Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok 10140, Thailand
2Food Safety Center, Institute for Scientific and Technological Research and Services (ISTRS), KMUTT, Bangkok 10140, Thailand
3Maintenance Technology Center, ISTRS, KMUTT, Bangkok 10140, Thailand
J. Microbiol. Biotechnol. 2021; 31(3): 439-446
Published March 28, 2021 https://doi.org/10.4014/jmb.2101.01014
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Foodborne diseases are a significant public health concern and also an impediment to social development globally [1].
In the food industry, some of the commonly used sanitizers and disinfectants (
Materials and Methods
Preparation of Q. infectoria Gall Crude Extract
Nutgalls were washed with distilled water and then physically crushed using a mortar. The nutgall powder (100 g) was submerged in 95% ethanol (500 ml) at room temperature for seven days. After filtration, the excess solvent was removed using a rotary evaporator (Model R-205, Canada) at 60°C until completely dry. [22].
Preparation of Surfactant and Nutgall Extract Stock Solutions
The stock solutions of CTAB (Ajax Finechem, Australia) and SDS (Ajax Finechem) were individually prepared by dissolving the surfactants in sterile distilled water to obtain the initial concentrations of 40 and 200 mg/ml, respectively. To prepare the stock solution of nutgall extract, the crude extract was dissolved in 20%dimethylsulfoxide (DMSO; Fisher Scientific, UK) to achieve the initial concentration of 512 mg/ml. The stock solutions were stirred using a magnetic stirrer until completely dissolved prior to use in a microbroth dilution assay.
Determination of Total Phenolic Content
The total phenolic content of the crude extract of nutgall was determined using a modified Folin-Ciocalteu colorimetric method as described by Dewanto
Preparation of S . Typhimurium Culture for Antimicrobial Assay
MIC and MBC Assays
MIC values of the nutgall extract or surfactants were determined using the broth microdilution assay in a 96-well plate. A volume of 100 μl of the extract and surfactants were serially 10-fold diluted in a 96-well plate to obtain desired concentrations. Then, 100 μl of
Biofilm Formation on Polypropylene and Stainless Steel Coupons
The PP (C.A.P. Intertrade Co., Ltd., Thailand) and SS Type 304 (AEC Industrial Services Co., Ltd., Thailand) coupons (10 × 20 mm) were sanitized in 70% ethanol for 15 min. Then, the coupons were autoclaved at 121°C for 15 min and were dried at 60°C. For biofilm formation, the coupons were transferred to conical tubes containing TSB with approximately 6 log CFU/ml of
Biofilm Removal Effect
After a 72-h incubation, the coupons were removed from the conical tubes using sterile forceps and then washed three times in phosphate-buffered saline (PBS; HiMedia) to remove loosely attached cells. The coupons were submerged in conical tubes individually containing 4 ml of 2x MBC of nutgall extract (256 mg/ml) + 2x MBC of CTAB (2.5 mg/ml), nutgall extract alone (256 mg/ml), CTAB alone (2.5 mg/ml), distilled water, and 100 ppm sodium hypochlorite (NaOCl; adjusted to pH 7 using 0.1N HCL) for 5, 15, and 30 min. CTAB and NaOCl solutions were prepared by dissolving CTAB and NaOCl individually in sterile distilled water to obtain the target concentrations. The solution of nutgall extract in combination with CTAB (nutgall extract + CTAB) was prepared by dissolving nutgall crude extract in CTAB solution to achieve the concentration of 256 mg/ml of nutgall extract+ 2.5 mg/ml of CTAB. All sanitizing agents were stirred using a magnetic stirrer. The untreated control samples were included to determine sessile cell numbers of
Following sanitizing treatments, the coupons were removed from the conical tubes using sterile forceps and then washed three times in PBS to remove loosely attached cells or antimicrobial residues. Then, cotton swabs moistened with 0.1% peptone water (PW; HiMedia) were used to scrape the submerged parts (1 cm2 × 2 sides) of the coupons. The swabs were transferred to test tubes containing 10 ml of 0.1% PW, followed by vortex agitation for 1 min. The resulting samples were serially diluted in 9 ml of 0.1% PW and then spread on TSA plates. After a 24-h incubation at 37°C,
Sample Preparation for SEM Analysis
The PP and SS coupons containing biofilms were treated with sanitizing solutions for 30 min. After that, the coupons were washed with PBS, followed by sterile distilled water. For sample fixation, the coupons were submerged in 4% glutaraldehyde for 2 h at 4°C. After fixation, the coupons were washed with sterile distilled water and then subjected to a 15-min gradual dehydration in 25%, 50%, 75%, 95%, and 100% ethanol, respectively. After drying, the coupon samples were sputter-coated with PdAu and then subjected to SEM (Quanta 450, FEI, USA) observation.
Statistical Analysis
Populations of
Results
Total Phenolic Content
The total phenolic contents of ethanolic extract of
MICs and MBCs of Nutgall Extract and Surfactants
The MIC and MBC of nutgall ethanolic extract against
Several studies have evaluated the antimicrobial effect of
In this study, cationic and anionic surfactants were utilized. The cationic surfactant CTAB is a quaternary ammonium compound widely used as a sanitizer and disinfectant for manual processing lines and surfaces in the food industry [38]. SDS is an anionic surfactant generally employed for many cleaning applications and is also highly effective in removing oily stains and residues [39]. CTAB [38, 40] and SDS [35, 39] have been reported to exhibit antimicrobial activities. CTAB could form an electrostatic bond with negatively charged sites on microbial cell walls, leading to stress in the cell wall, cell lysis, and death [40]. It has also been reported that CTAB could induce superoxide stress in microbial cells [38]. SDS has been shown to denature membrane-located proteins and damage microbial cell membranes, resulting in leakage of the cytoplasmic constituents and potentially depolarization of the membrane [35]. In the present study, CTAB was more effective in inhibiting and inactivating planktonic cells of
Biofilm Removal Effect of Sanitizers against Preformed Biofilm on Polypropylene and Stainless Steel Coupons
Tables 1 and 2 present the mean survivors (log CFU/cm2) of
-
Table 1 . Mean survivors (log CFU/cm2) of
S . Typhimurium sessile cells on polypropylene coupons after treatment with sanitizing agents.Treatment Mean survivors (log CFU/cm2) Sanitizer exposure time (min) 5 min 15 min 30 min Nutgall extract (256 mg/ml) 3.54 ± 0.02d 3.14 ± 0.03f 2.51 ± 0.07h Nutgall extract (256 mg/ml) + CTAB (2.5 mg/ml) 3.29 ± 0.03e 2.84 ± 0.06g 2.14 ± 0.12j CTAB (2.5 mg/ml) 3.46 ± 0.06d 3.09 ± 0.09f 2.35 ± 0.12i Water 5.20 ± 0.03b 5.16 ± 0.03bc 5.03 ± 0.04c NaOCl (100 ppm) 3.31 ± 0.03e 2.94 ± 0.09g 2.43 ± 0.10hi Untreated control 5.66 ± 0.02a 5.66 ± 0.02a 5.66 ± 0.02a Numbers across rows and columns not sharing the same letter are significantly different (
p < 0.05).The untreated control represents sessile cells on polypropylene coupons without sanitizing treatment.
-
Table 2 . Mean survivors (log CFU/cm2) of
S . Typhimurium sessile cells on stainless steel coupons after treatment with sanitizing agents.Treatment Mean survivors (log CFU/cm2) Sanitizer exposure time (min) 5 min 15 min 30 min Nutgall extract (256 mg/ml) 3.53 ± 0.02d 2.92 ± 0.08g 2.61 ± 0.06h Nutgall extract (256 mg/ml) + CTAB (2.5 mg/ml) 3.18 ± 0.05f 2.84 ± 0.10g 2.20 ± 0.17j CTAB (2.5 mg/ml) 3.57 ± 0.02d 3.17 ± 0.04f 2.47 ± 0.11i Water 5.24 ± 0.02b 5.20 ± 0.02bc 5.10 ± 0.02c NaOCl (100 ppm) 3.38 ± 0.04e 3.10 ± 0.07f 2.60 ± 0.07hi Untreated control 5.97 ± 0.01a 5.97 ± 0.01a 5.97 ± 0.01a Numbers across rows and columns not sharing the same letter are significantly different (
p < 0.05).The untreated control represents sessile cells on stainless steel coupons without sanitizing treatment.
PP and SS are food contact surface materials commonly used in the food industry and household [41]. In this study, the effects of nutgall extract + CTAB for removing
SEM Analysis
The morphology of
-
Fig. 1. SEM images of S. Typhimurium on polypropylene coupons after treatment with sanitizing agents for 30 min. (A) Nutgall extract; (B) nutgall extract + CTAB; (C) CTAB; (D) water; (E) NaOCl; (F) untreated control. The untreated control sample did not receive sanitizing treatment.
-
Fig. 2. SEM images of
S . Typhimurium on stainless steel coupons after treatment with sanitizing agents for 30 min. (A) Nutgall extract; (B) nutgall extract + CTAB; (C) CTAB; (D) water; (E) NaOCl; (F) untreated control. The untreated control sample did not receive sanitizing treatment.
Conclusions
In this study, overall, the biofilm removal efficacy of the tested sanitizing agents against
Acknowledgments
This research was supported by the Faculty of Science, King Monkut’s University of Technology Thonburi Grant No. SCI60-001 and the KMUTT Research Fund. The authors would like to acknowledge Mr.Picha Panmongkol from AEC Industrial Services Co., Ltd. for the kind provision of stainless steel coupons. Also, the authors would like to thank Dr. Sirirat Wachiralurpan from the ISTRS, KMUTT, for assistance with sample preparation for SEM analysis.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- World Health Organization (WHO). WHO estimates of the global burden of foodborne diseases, 2015. Available from https://apps.who.int/iris/bitstream/handle/10665/199350/9789241565165_eng.pdf?sequence=1. Accessed Oct. 8, 2020.
- World Health Organization (WHO).
Salmonella (non-typhoidal), 2018. Available from https://www.who.int/news-room/factsheets/detail/salmonella-(non-typhoidal). Accessed July 21, 2020. - Center for Disease Control and Prevention (CDC).
Salmonella , 2020. Available from https://www.cdc.gov/salmonella/. Accessed July 21, 2020. - European Food Safety Authority (EFSA). 2018. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017.
EFSA J. 16 : e05500-e05500. - Department of Disease Control (DDC). Food Poisoning, 2019. Available from https://www.boe.moph.go.th/boedb/surdata/506wk/y62/d03_5262.pdf. Accessed. July 21, 2020.
- Scanlan CM. 2004. Genus
Salmonella , pp. 116-119.In: Bacterial diseases of domestic animals , 2nd Ed. Brown Paw Educational Media, College Station, Texas. USA. - Chmielewski RAN, Frank JF. 2003. Biofilm formation and control in food processing facilities.
Compr. Rev. Food Sci. Food Saf. 2 : 22-32. - Jun W, Kim MS, Cho BK, Millner PD, Chao KL, Chan DE. 2010. Microbial biofilm detection on food contact surfaces by macro-scale fluorescence imaging.
J. Food Eng. 99 : 314-322. - Srey S, Jahid IK, Ha S-D. 2013. Biofilm formation in food industries: A food safety concern.
Food Control 31 : 572-585. - LeChevallier MW, Cawthon CD, Lee RG. 1988. Inactivation of biofilm bacteria.
Appl. Environ. Microbiol. 54 : 2492-2499. - Kumar CG, Anand SK. 1998. Significance of microbial biofilms in food industry: a review.
Int. J. Food Microbiol. 42 : 9-27. - Simões M, Simões LC, Vieira MJ. 2010. A review of current and emergent biofilm control strategies.
LWT-Food Sci. Technol. 43 : 573-583. - Van Houdt R, Michiels CW. 2010. Biofilm formation and the food industry, a focus on the bacterial outer surface.
J. Appl. Microbiol. 109 : 1117-1131. - Corcoran M, Morris D, De Lappe N, O'Connor J, Lalor P, Dockery P,
et al . 2014. Commonly used disinfectants fail to eradicateSalmonella enterica biofilms from food contact surface materials.Appl. Environ. Microbiol. 80 : 1507-1514. - Cogan TA, Bloomfield SF, Humphrey TJ. 1999. The effectiveness of hygiene procedures for prevention of cross-contamination from chicken carcasses in the domestic kitchen.
Lett. Appl. Microbiol. 29 : 354-358. - Schlegelova J, Babak V, Holasova M, Konstantinova L, Necidova L, Sisak F,
et al . 2010. Microbial contamination after sanitation of food contact surfaces in dairy and meat processing plants.Czech J. Food Sci. 28 : 450-461. - Djordjevic D, Wiedmann M, McLandsborough LA. 2002. Microtiter plate assay for assessment of
Listeria monocytogenes biofilm formation.Appl. Environ. Microbiol. 68 : 2950-2958. - Hapidin H, Rozelan D, Abdullah H, Wan Hanaffi WN, Soelaiman IN. 2015.
Quercus infectoria gall extract enhanced the proliferation and activity of human fetal osteoblast cell line (hFOB 1.19).Malays. J. Med. Sci. 22 : 12-22. - Baharuddin NS, Abdullah H, Abdul Wahab WNAW. 2015. Anti-Candida activity of
Quercus infectoria gall extracts againstCandida species.J. Pharm. Bioallied Sci. 7 : 15-20. - Voravuthikunchai S, Chusri S, Suwalak S. 2008.
Quercus infectoria .Oliv. Pharm. Biol. 46 : 367-372. - Satirapathkul C, Leela T. 2011. Growth inhibition of pathogenic bacteria by extract of
Quercus Infectoria galls.Int. J. Biosci. Biochem. Bioinformatics 1 : 26-31. - Chusri S, Voravuthikunchai SP. 2009. Detailed studies on
Quercus infectoria Olivier (nutgalls) as an alternative treatment for methicillin-resistantStaphylococcus aureus infections.J. Appl. Microbiol. 106 : 89-96. - Chusri S, Voravuthikunchai SP. 2011. Damage of staphylococcal cytoplasmic membrane by
Quercus infectoria G. Olivier and its components.Lett. Appl. Microbiol. 52 : 565-572. - Mohammadi-Sichani M, Karbasizadeh V, Dokhaharani SC. 2016. Evaluation of biofilm removal activity of
Quercus infectoria galls againstStreptococcus mutans .Dent. Res. J. 13 : 46-51. - Voravuthikunchai S, Limsuwan S, Mitchell H. 2006. Effects of
Punica granatum pericarps andQuercus infectoria nutgalls on cell surface hydrophobicity and cell survival ofHelicobacter pylori .J. Health Sci. 52 : 154-159. - Voravuthikunchai S, Suwalak S. 2009. Changes in cell surface properties of shiga toxigenic
Escherichia coli byQuercus infectoria G. Olivier.J. Food Prot. 72 : 1699-1704. - Chusri S, Phatthalung PN, Voravuthikunchai SP. 2012. Anti-biofilm activity of
Quercus infectoria G. Olivier against methicillinresistantStaphylococcus aureus .Lett. Appl. Microbiol. 54 : 511-517. - Wan Nor Amilah WA, Masrah M, Hasmah A, Noor Izani NJ. 2014. In vitro antibacterial activity of
Quercus infectoria gall extracts against multidrug resistant bacteria.Trop. Biomed. 31 : 680-688. - Falcó I, Verdeguer M, Aznar R, Sánchez G, Randazzo W. 2018. Sanitizing food contact surfaces by the use of essential oils.
Innov. Food Sci. Emerg. 51 : 220-228. - Halden RU. 2014. On the need and speed of regulating triclosan and triclocarban in the United States.
Environ. Sci. Technol. 48 : 3603-3611. - Xue R, Shi H, Ma Y, Yang J, Hua B, Inniss EC,
et al . 2017. Evaluation of thirteen haloacetic acids and ten trihalomethanes formation by peracetic acid and chlorine drinking water disinfection.Chemosphere 189 : 349-356. - Soni KA, Oladunjoye A, Nannapaneni R, Schilling MW, Silva JL, Mikel B,
et al . 2013. Inhibition and inactivation ofSalmonella Typhimurium biofilms from polystyrene and stainless steel surfaces by essential oils and phenolic constituent carvacrol.J. Food Prot. 76 : 205-212. - Dewanto V, Wu X, Adom KK, Liu RH. 2002. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity.
J. Agric. Food Chem. 50 : 3010-3014. - Bazargani MM, Rohloff J. 2016. Antibiofilm activity of essential oils and plant extracts against
Staphylococcus aureus andEscherichia coli biofilms.Food Control 61 : 156-164. - Ruengvisesh S, Loquercio A, Castell-Perez E, Taylor TM. 2015. Inhibition of bacterial pathogens in medium and on spinach leaf surfaces using plant-derived antimicrobials loaded in surfactant micelles.
J. Food Sci. 80 : M2522-2529. - Nanasombat S, Kuncharoen N, Ritcharoon B, Sukcharoen P. 2018. Antibacterial activity of thai medicinal plant extracts against oral and gastrointestinal pathogenic bacteria and prebiotic effect on the growth of
lactobacillus acidophilus .Chiang Mai J. Sci. 45 : 33-44. - Haque ASA, Ahmad W, Khan RM, Hasan A. 2016. Ethnopharmacology of
Quercus infectoria galls: a review.Hippocratic J. Unani Med. 11 : 105-118. - Nakata K, Tsuchido T, Matsumura Y. 2011. Antimicrobial cationic surfactant, cetyltrimethylammonium bromide, induces superoxide stress in
Escherichia coli cells.J. Appl. Microbiol. 110 : 568-579. - Bhattarai A, Niraula T, Chatterjee S. 2014. Sodium dodecyl sulphate: A very useful surfactant for scientific investigations.
J. Knowledge Innov. 2 : 111-113. - Simões M, Pereira MO, Vieira MJ. 2005. Action of a cationic surfactant on the activity and removal of bacterial biofilms formed under different flow regimes.
Water Res. 39 : 478-486. - Vidacs A, Kerekes E, Rajko R, Petkovits T, Alharbi NS, Khaled JM,
et al . 2018. Optimization of essential oil-based natural disinfectants againstListeria monocytogenes andEscherichia coli biofilms formed on polypropylene surfaces.J. Mol. Liq. 255 : 257-262. - de Souza EL, Meira QGS, de Medeiros Barbosa I, Athayde AJAA, da Conceição ML, de Siqueira Júnior JP. 2014. Biofilm formation by
Staphylococcus aureus from food contact surfaces in a meat-based broth and sensitivity to sanitizers.Braz. J. Microbiol. 45 : 67-75. - da Silva Meira QG, de Medeiros Barbosa I, Alves Aguiar Athayde AJ, de Siqueira-Júnior JP, de Souza EL. 2012. Influence of temperature and surface kind on biofilm formation by
Staphylococcus aureus from food-contact surfaces and sensitivity to sanitizers.Food Control 25 : 469-475. - Wang H, Wang H, Xing T, Wu N, Xu X, Zhou G. 2016. Removal of
Salmonella biofilm formed under meat processing environment by surfactant in combination with bio-enzyme.LWT - Food Sci. Technol. 66 : 298-304. - Amaral VCS, Santos PR, da Silva AF, dos Santos AR, Machinski Jr M, Mikcha JMG. 2015. Effect of carvacrol and thymol on
Salmonella spp. biofilms on polypropylene.Int. J. Food Sci. Technol. 50 : 2639-2643. - Guo J, Gao Z, Li G, Fu F, Liang Z, Zhu H,
et al . 2019. Antimicrobial and antibiofilm efficacy and mechanism of essential oil from Citrus Changshan-huyou Y. B. chang againstListeria monocytogenes .Food Control 105 : 256-264. - Rodrigues JBD, de Souza NT, Scarano JOA, de Sousa JM, Lira MC, de Figueiredo R,
et al . 2018. Efficacy of using oregano essential oil and carvacrol to remove young and matureStaphylococcus aureus biofilms on food-contact surfaces of stainless steel.Lwt-Food Sci. Technol. 93 : 293-299.
Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2021; 31(3): 439-446
Published online March 28, 2021 https://doi.org/10.4014/jmb.2101.01014
Copyright © The Korean Society for Microbiology and Biotechnology.
Removal of Salmonella Typhimurium Biofilm from Food Contact Surfaces Using Quercus infectoria Gall Extract in Combination with a Surfactant
Peetitas Damrongsaktrakul1, Songsirin Ruengvisesh1*, Arewan Rahothan1, Nuttamon Sukhumrat1, Pravate Tuitemwong2, and Isaratat Phung-on3
1Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi (KMUTT), Bangkok 10140, Thailand
2Food Safety Center, Institute for Scientific and Technological Research and Services (ISTRS), KMUTT, Bangkok 10140, Thailand
3Maintenance Technology Center, ISTRS, KMUTT, Bangkok 10140, Thailand
Correspondence to:Songsirin Ruengvisesh,
songsirin@gmail.com
Abstract
Quercus infectoria (nutgall) has been reported to possess antimicrobial activities against a wide range of pathogens. Nevertheless, the biofilm removal effect of nutgall extract has not been widely investigated. In this study, we therefore evaluated the effect of nutgall extract in combination with cetrimonium bromide (CTAB) against preformed biofilm of Salmonella Typhimurium on polypropylene (PP) and stainless steel (SS) coupons in comparison with other sanitizers. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of nutgall extract and surfactants (CTAB and sodium dodecyl sulfate; SDS) were assessed. CTAB showed a more efficient antimicrobial activity than SDS and was selected to use in combination with nutgall extract for removing biofilm. To determine the biofilm removal efficacy, the PP and SS coupons were individually submerged in 2x MBC of nutgall extract (256 mg/ml) + 2x MBC of CTAB (2.5 mg/ml), nutgall extract alone (256 mg/ml), CTAB alone (2.5 mg/ml), distilled water, and 100 ppm sodium hypochlorite for 5, 15, and 30 min. The remaining sessile cells in biofilm were determined. Overall, the greatest biofilm removal efficacy was observed with nutgall extract + CTAB; the biofilm removal efficacy of sanitizers tended to increase with the exposure time. The SEM analysis demonstrated that S. Typhimurium biofilm on PP and SS coupons after exposure to nutgall extract + CTAB for 30 min displayed morphological alterations with wrinkles. This study suggests nutgall extract + CTAB may be an alternative to commonly used sanitizers to remove biofilm from food contact surfaces in the food industry and household.
Keywords: Quercus infectoria gall extract, Salmonella Typhimurium, biofilm removal, food contact surfaces, surfactants
Introduction
Foodborne diseases are a significant public health concern and also an impediment to social development globally [1].
In the food industry, some of the commonly used sanitizers and disinfectants (
Materials and Methods
Preparation of Q. infectoria Gall Crude Extract
Nutgalls were washed with distilled water and then physically crushed using a mortar. The nutgall powder (100 g) was submerged in 95% ethanol (500 ml) at room temperature for seven days. After filtration, the excess solvent was removed using a rotary evaporator (Model R-205, Canada) at 60°C until completely dry. [22].
Preparation of Surfactant and Nutgall Extract Stock Solutions
The stock solutions of CTAB (Ajax Finechem, Australia) and SDS (Ajax Finechem) were individually prepared by dissolving the surfactants in sterile distilled water to obtain the initial concentrations of 40 and 200 mg/ml, respectively. To prepare the stock solution of nutgall extract, the crude extract was dissolved in 20%dimethylsulfoxide (DMSO; Fisher Scientific, UK) to achieve the initial concentration of 512 mg/ml. The stock solutions were stirred using a magnetic stirrer until completely dissolved prior to use in a microbroth dilution assay.
Determination of Total Phenolic Content
The total phenolic content of the crude extract of nutgall was determined using a modified Folin-Ciocalteu colorimetric method as described by Dewanto
Preparation of S . Typhimurium Culture for Antimicrobial Assay
MIC and MBC Assays
MIC values of the nutgall extract or surfactants were determined using the broth microdilution assay in a 96-well plate. A volume of 100 μl of the extract and surfactants were serially 10-fold diluted in a 96-well plate to obtain desired concentrations. Then, 100 μl of
Biofilm Formation on Polypropylene and Stainless Steel Coupons
The PP (C.A.P. Intertrade Co., Ltd., Thailand) and SS Type 304 (AEC Industrial Services Co., Ltd., Thailand) coupons (10 × 20 mm) were sanitized in 70% ethanol for 15 min. Then, the coupons were autoclaved at 121°C for 15 min and were dried at 60°C. For biofilm formation, the coupons were transferred to conical tubes containing TSB with approximately 6 log CFU/ml of
Biofilm Removal Effect
After a 72-h incubation, the coupons were removed from the conical tubes using sterile forceps and then washed three times in phosphate-buffered saline (PBS; HiMedia) to remove loosely attached cells. The coupons were submerged in conical tubes individually containing 4 ml of 2x MBC of nutgall extract (256 mg/ml) + 2x MBC of CTAB (2.5 mg/ml), nutgall extract alone (256 mg/ml), CTAB alone (2.5 mg/ml), distilled water, and 100 ppm sodium hypochlorite (NaOCl; adjusted to pH 7 using 0.1N HCL) for 5, 15, and 30 min. CTAB and NaOCl solutions were prepared by dissolving CTAB and NaOCl individually in sterile distilled water to obtain the target concentrations. The solution of nutgall extract in combination with CTAB (nutgall extract + CTAB) was prepared by dissolving nutgall crude extract in CTAB solution to achieve the concentration of 256 mg/ml of nutgall extract+ 2.5 mg/ml of CTAB. All sanitizing agents were stirred using a magnetic stirrer. The untreated control samples were included to determine sessile cell numbers of
Following sanitizing treatments, the coupons were removed from the conical tubes using sterile forceps and then washed three times in PBS to remove loosely attached cells or antimicrobial residues. Then, cotton swabs moistened with 0.1% peptone water (PW; HiMedia) were used to scrape the submerged parts (1 cm2 × 2 sides) of the coupons. The swabs were transferred to test tubes containing 10 ml of 0.1% PW, followed by vortex agitation for 1 min. The resulting samples were serially diluted in 9 ml of 0.1% PW and then spread on TSA plates. After a 24-h incubation at 37°C,
Sample Preparation for SEM Analysis
The PP and SS coupons containing biofilms were treated with sanitizing solutions for 30 min. After that, the coupons were washed with PBS, followed by sterile distilled water. For sample fixation, the coupons were submerged in 4% glutaraldehyde for 2 h at 4°C. After fixation, the coupons were washed with sterile distilled water and then subjected to a 15-min gradual dehydration in 25%, 50%, 75%, 95%, and 100% ethanol, respectively. After drying, the coupon samples were sputter-coated with PdAu and then subjected to SEM (Quanta 450, FEI, USA) observation.
Statistical Analysis
Populations of
Results
Total Phenolic Content
The total phenolic contents of ethanolic extract of
MICs and MBCs of Nutgall Extract and Surfactants
The MIC and MBC of nutgall ethanolic extract against
Several studies have evaluated the antimicrobial effect of
In this study, cationic and anionic surfactants were utilized. The cationic surfactant CTAB is a quaternary ammonium compound widely used as a sanitizer and disinfectant for manual processing lines and surfaces in the food industry [38]. SDS is an anionic surfactant generally employed for many cleaning applications and is also highly effective in removing oily stains and residues [39]. CTAB [38, 40] and SDS [35, 39] have been reported to exhibit antimicrobial activities. CTAB could form an electrostatic bond with negatively charged sites on microbial cell walls, leading to stress in the cell wall, cell lysis, and death [40]. It has also been reported that CTAB could induce superoxide stress in microbial cells [38]. SDS has been shown to denature membrane-located proteins and damage microbial cell membranes, resulting in leakage of the cytoplasmic constituents and potentially depolarization of the membrane [35]. In the present study, CTAB was more effective in inhibiting and inactivating planktonic cells of
Biofilm Removal Effect of Sanitizers against Preformed Biofilm on Polypropylene and Stainless Steel Coupons
Tables 1 and 2 present the mean survivors (log CFU/cm2) of
-
Table 1 . Mean survivors (log CFU/cm2) of
S . Typhimurium sessile cells on polypropylene coupons after treatment with sanitizing agents..Treatment Mean survivors (log CFU/cm2) Sanitizer exposure time (min) 5 min 15 min 30 min Nutgall extract (256 mg/ml) 3.54 ± 0.02d 3.14 ± 0.03f 2.51 ± 0.07h Nutgall extract (256 mg/ml) + CTAB (2.5 mg/ml) 3.29 ± 0.03e 2.84 ± 0.06g 2.14 ± 0.12j CTAB (2.5 mg/ml) 3.46 ± 0.06d 3.09 ± 0.09f 2.35 ± 0.12i Water 5.20 ± 0.03b 5.16 ± 0.03bc 5.03 ± 0.04c NaOCl (100 ppm) 3.31 ± 0.03e 2.94 ± 0.09g 2.43 ± 0.10hi Untreated control 5.66 ± 0.02a 5.66 ± 0.02a 5.66 ± 0.02a Numbers across rows and columns not sharing the same letter are significantly different (
p < 0.05)..The untreated control represents sessile cells on polypropylene coupons without sanitizing treatment..
-
Table 2 . Mean survivors (log CFU/cm2) of
S . Typhimurium sessile cells on stainless steel coupons after treatment with sanitizing agents..Treatment Mean survivors (log CFU/cm2) Sanitizer exposure time (min) 5 min 15 min 30 min Nutgall extract (256 mg/ml) 3.53 ± 0.02d 2.92 ± 0.08g 2.61 ± 0.06h Nutgall extract (256 mg/ml) + CTAB (2.5 mg/ml) 3.18 ± 0.05f 2.84 ± 0.10g 2.20 ± 0.17j CTAB (2.5 mg/ml) 3.57 ± 0.02d 3.17 ± 0.04f 2.47 ± 0.11i Water 5.24 ± 0.02b 5.20 ± 0.02bc 5.10 ± 0.02c NaOCl (100 ppm) 3.38 ± 0.04e 3.10 ± 0.07f 2.60 ± 0.07hi Untreated control 5.97 ± 0.01a 5.97 ± 0.01a 5.97 ± 0.01a Numbers across rows and columns not sharing the same letter are significantly different (
p < 0.05)..The untreated control represents sessile cells on stainless steel coupons without sanitizing treatment..
PP and SS are food contact surface materials commonly used in the food industry and household [41]. In this study, the effects of nutgall extract + CTAB for removing
SEM Analysis
The morphology of
-
Figure 1. SEM images of S. Typhimurium on polypropylene coupons after treatment with sanitizing agents for 30 min. (A) Nutgall extract; (B) nutgall extract + CTAB; (C) CTAB; (D) water; (E) NaOCl; (F) untreated control. The untreated control sample did not receive sanitizing treatment.
-
Figure 2. SEM images of
S . Typhimurium on stainless steel coupons after treatment with sanitizing agents for 30 min. (A) Nutgall extract; (B) nutgall extract + CTAB; (C) CTAB; (D) water; (E) NaOCl; (F) untreated control. The untreated control sample did not receive sanitizing treatment.
Conclusions
In this study, overall, the biofilm removal efficacy of the tested sanitizing agents against
Acknowledgments
This research was supported by the Faculty of Science, King Monkut’s University of Technology Thonburi Grant No. SCI60-001 and the KMUTT Research Fund. The authors would like to acknowledge Mr.Picha Panmongkol from AEC Industrial Services Co., Ltd. for the kind provision of stainless steel coupons. Also, the authors would like to thank Dr. Sirirat Wachiralurpan from the ISTRS, KMUTT, for assistance with sample preparation for SEM analysis.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

-
Table 1 . Mean survivors (log CFU/cm2) of
S . Typhimurium sessile cells on polypropylene coupons after treatment with sanitizing agents..Treatment Mean survivors (log CFU/cm2) Sanitizer exposure time (min) 5 min 15 min 30 min Nutgall extract (256 mg/ml) 3.54 ± 0.02d 3.14 ± 0.03f 2.51 ± 0.07h Nutgall extract (256 mg/ml) + CTAB (2.5 mg/ml) 3.29 ± 0.03e 2.84 ± 0.06g 2.14 ± 0.12j CTAB (2.5 mg/ml) 3.46 ± 0.06d 3.09 ± 0.09f 2.35 ± 0.12i Water 5.20 ± 0.03b 5.16 ± 0.03bc 5.03 ± 0.04c NaOCl (100 ppm) 3.31 ± 0.03e 2.94 ± 0.09g 2.43 ± 0.10hi Untreated control 5.66 ± 0.02a 5.66 ± 0.02a 5.66 ± 0.02a Numbers across rows and columns not sharing the same letter are significantly different (
p < 0.05)..The untreated control represents sessile cells on polypropylene coupons without sanitizing treatment..
-
Table 2 . Mean survivors (log CFU/cm2) of
S . Typhimurium sessile cells on stainless steel coupons after treatment with sanitizing agents..Treatment Mean survivors (log CFU/cm2) Sanitizer exposure time (min) 5 min 15 min 30 min Nutgall extract (256 mg/ml) 3.53 ± 0.02d 2.92 ± 0.08g 2.61 ± 0.06h Nutgall extract (256 mg/ml) + CTAB (2.5 mg/ml) 3.18 ± 0.05f 2.84 ± 0.10g 2.20 ± 0.17j CTAB (2.5 mg/ml) 3.57 ± 0.02d 3.17 ± 0.04f 2.47 ± 0.11i Water 5.24 ± 0.02b 5.20 ± 0.02bc 5.10 ± 0.02c NaOCl (100 ppm) 3.38 ± 0.04e 3.10 ± 0.07f 2.60 ± 0.07hi Untreated control 5.97 ± 0.01a 5.97 ± 0.01a 5.97 ± 0.01a Numbers across rows and columns not sharing the same letter are significantly different (
p < 0.05)..The untreated control represents sessile cells on stainless steel coupons without sanitizing treatment..
References
- World Health Organization (WHO). WHO estimates of the global burden of foodborne diseases, 2015. Available from https://apps.who.int/iris/bitstream/handle/10665/199350/9789241565165_eng.pdf?sequence=1. Accessed Oct. 8, 2020.
- World Health Organization (WHO).
Salmonella (non-typhoidal), 2018. Available from https://www.who.int/news-room/factsheets/detail/salmonella-(non-typhoidal). Accessed July 21, 2020. - Center for Disease Control and Prevention (CDC).
Salmonella , 2020. Available from https://www.cdc.gov/salmonella/. Accessed July 21, 2020. - European Food Safety Authority (EFSA). 2018. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017.
EFSA J. 16 : e05500-e05500. - Department of Disease Control (DDC). Food Poisoning, 2019. Available from https://www.boe.moph.go.th/boedb/surdata/506wk/y62/d03_5262.pdf. Accessed. July 21, 2020.
- Scanlan CM. 2004. Genus
Salmonella , pp. 116-119.In: Bacterial diseases of domestic animals , 2nd Ed. Brown Paw Educational Media, College Station, Texas. USA. - Chmielewski RAN, Frank JF. 2003. Biofilm formation and control in food processing facilities.
Compr. Rev. Food Sci. Food Saf. 2 : 22-32. - Jun W, Kim MS, Cho BK, Millner PD, Chao KL, Chan DE. 2010. Microbial biofilm detection on food contact surfaces by macro-scale fluorescence imaging.
J. Food Eng. 99 : 314-322. - Srey S, Jahid IK, Ha S-D. 2013. Biofilm formation in food industries: A food safety concern.
Food Control 31 : 572-585. - LeChevallier MW, Cawthon CD, Lee RG. 1988. Inactivation of biofilm bacteria.
Appl. Environ. Microbiol. 54 : 2492-2499. - Kumar CG, Anand SK. 1998. Significance of microbial biofilms in food industry: a review.
Int. J. Food Microbiol. 42 : 9-27. - Simões M, Simões LC, Vieira MJ. 2010. A review of current and emergent biofilm control strategies.
LWT-Food Sci. Technol. 43 : 573-583. - Van Houdt R, Michiels CW. 2010. Biofilm formation and the food industry, a focus on the bacterial outer surface.
J. Appl. Microbiol. 109 : 1117-1131. - Corcoran M, Morris D, De Lappe N, O'Connor J, Lalor P, Dockery P,
et al . 2014. Commonly used disinfectants fail to eradicateSalmonella enterica biofilms from food contact surface materials.Appl. Environ. Microbiol. 80 : 1507-1514. - Cogan TA, Bloomfield SF, Humphrey TJ. 1999. The effectiveness of hygiene procedures for prevention of cross-contamination from chicken carcasses in the domestic kitchen.
Lett. Appl. Microbiol. 29 : 354-358. - Schlegelova J, Babak V, Holasova M, Konstantinova L, Necidova L, Sisak F,
et al . 2010. Microbial contamination after sanitation of food contact surfaces in dairy and meat processing plants.Czech J. Food Sci. 28 : 450-461. - Djordjevic D, Wiedmann M, McLandsborough LA. 2002. Microtiter plate assay for assessment of
Listeria monocytogenes biofilm formation.Appl. Environ. Microbiol. 68 : 2950-2958. - Hapidin H, Rozelan D, Abdullah H, Wan Hanaffi WN, Soelaiman IN. 2015.
Quercus infectoria gall extract enhanced the proliferation and activity of human fetal osteoblast cell line (hFOB 1.19).Malays. J. Med. Sci. 22 : 12-22. - Baharuddin NS, Abdullah H, Abdul Wahab WNAW. 2015. Anti-Candida activity of
Quercus infectoria gall extracts againstCandida species.J. Pharm. Bioallied Sci. 7 : 15-20. - Voravuthikunchai S, Chusri S, Suwalak S. 2008.
Quercus infectoria .Oliv. Pharm. Biol. 46 : 367-372. - Satirapathkul C, Leela T. 2011. Growth inhibition of pathogenic bacteria by extract of
Quercus Infectoria galls.Int. J. Biosci. Biochem. Bioinformatics 1 : 26-31. - Chusri S, Voravuthikunchai SP. 2009. Detailed studies on
Quercus infectoria Olivier (nutgalls) as an alternative treatment for methicillin-resistantStaphylococcus aureus infections.J. Appl. Microbiol. 106 : 89-96. - Chusri S, Voravuthikunchai SP. 2011. Damage of staphylococcal cytoplasmic membrane by
Quercus infectoria G. Olivier and its components.Lett. Appl. Microbiol. 52 : 565-572. - Mohammadi-Sichani M, Karbasizadeh V, Dokhaharani SC. 2016. Evaluation of biofilm removal activity of
Quercus infectoria galls againstStreptococcus mutans .Dent. Res. J. 13 : 46-51. - Voravuthikunchai S, Limsuwan S, Mitchell H. 2006. Effects of
Punica granatum pericarps andQuercus infectoria nutgalls on cell surface hydrophobicity and cell survival ofHelicobacter pylori .J. Health Sci. 52 : 154-159. - Voravuthikunchai S, Suwalak S. 2009. Changes in cell surface properties of shiga toxigenic
Escherichia coli byQuercus infectoria G. Olivier.J. Food Prot. 72 : 1699-1704. - Chusri S, Phatthalung PN, Voravuthikunchai SP. 2012. Anti-biofilm activity of
Quercus infectoria G. Olivier against methicillinresistantStaphylococcus aureus .Lett. Appl. Microbiol. 54 : 511-517. - Wan Nor Amilah WA, Masrah M, Hasmah A, Noor Izani NJ. 2014. In vitro antibacterial activity of
Quercus infectoria gall extracts against multidrug resistant bacteria.Trop. Biomed. 31 : 680-688. - Falcó I, Verdeguer M, Aznar R, Sánchez G, Randazzo W. 2018. Sanitizing food contact surfaces by the use of essential oils.
Innov. Food Sci. Emerg. 51 : 220-228. - Halden RU. 2014. On the need and speed of regulating triclosan and triclocarban in the United States.
Environ. Sci. Technol. 48 : 3603-3611. - Xue R, Shi H, Ma Y, Yang J, Hua B, Inniss EC,
et al . 2017. Evaluation of thirteen haloacetic acids and ten trihalomethanes formation by peracetic acid and chlorine drinking water disinfection.Chemosphere 189 : 349-356. - Soni KA, Oladunjoye A, Nannapaneni R, Schilling MW, Silva JL, Mikel B,
et al . 2013. Inhibition and inactivation ofSalmonella Typhimurium biofilms from polystyrene and stainless steel surfaces by essential oils and phenolic constituent carvacrol.J. Food Prot. 76 : 205-212. - Dewanto V, Wu X, Adom KK, Liu RH. 2002. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity.
J. Agric. Food Chem. 50 : 3010-3014. - Bazargani MM, Rohloff J. 2016. Antibiofilm activity of essential oils and plant extracts against
Staphylococcus aureus andEscherichia coli biofilms.Food Control 61 : 156-164. - Ruengvisesh S, Loquercio A, Castell-Perez E, Taylor TM. 2015. Inhibition of bacterial pathogens in medium and on spinach leaf surfaces using plant-derived antimicrobials loaded in surfactant micelles.
J. Food Sci. 80 : M2522-2529. - Nanasombat S, Kuncharoen N, Ritcharoon B, Sukcharoen P. 2018. Antibacterial activity of thai medicinal plant extracts against oral and gastrointestinal pathogenic bacteria and prebiotic effect on the growth of
lactobacillus acidophilus .Chiang Mai J. Sci. 45 : 33-44. - Haque ASA, Ahmad W, Khan RM, Hasan A. 2016. Ethnopharmacology of
Quercus infectoria galls: a review.Hippocratic J. Unani Med. 11 : 105-118. - Nakata K, Tsuchido T, Matsumura Y. 2011. Antimicrobial cationic surfactant, cetyltrimethylammonium bromide, induces superoxide stress in
Escherichia coli cells.J. Appl. Microbiol. 110 : 568-579. - Bhattarai A, Niraula T, Chatterjee S. 2014. Sodium dodecyl sulphate: A very useful surfactant for scientific investigations.
J. Knowledge Innov. 2 : 111-113. - Simões M, Pereira MO, Vieira MJ. 2005. Action of a cationic surfactant on the activity and removal of bacterial biofilms formed under different flow regimes.
Water Res. 39 : 478-486. - Vidacs A, Kerekes E, Rajko R, Petkovits T, Alharbi NS, Khaled JM,
et al . 2018. Optimization of essential oil-based natural disinfectants againstListeria monocytogenes andEscherichia coli biofilms formed on polypropylene surfaces.J. Mol. Liq. 255 : 257-262. - de Souza EL, Meira QGS, de Medeiros Barbosa I, Athayde AJAA, da Conceição ML, de Siqueira Júnior JP. 2014. Biofilm formation by
Staphylococcus aureus from food contact surfaces in a meat-based broth and sensitivity to sanitizers.Braz. J. Microbiol. 45 : 67-75. - da Silva Meira QG, de Medeiros Barbosa I, Alves Aguiar Athayde AJ, de Siqueira-Júnior JP, de Souza EL. 2012. Influence of temperature and surface kind on biofilm formation by
Staphylococcus aureus from food-contact surfaces and sensitivity to sanitizers.Food Control 25 : 469-475. - Wang H, Wang H, Xing T, Wu N, Xu X, Zhou G. 2016. Removal of
Salmonella biofilm formed under meat processing environment by surfactant in combination with bio-enzyme.LWT - Food Sci. Technol. 66 : 298-304. - Amaral VCS, Santos PR, da Silva AF, dos Santos AR, Machinski Jr M, Mikcha JMG. 2015. Effect of carvacrol and thymol on
Salmonella spp. biofilms on polypropylene.Int. J. Food Sci. Technol. 50 : 2639-2643. - Guo J, Gao Z, Li G, Fu F, Liang Z, Zhu H,
et al . 2019. Antimicrobial and antibiofilm efficacy and mechanism of essential oil from Citrus Changshan-huyou Y. B. chang againstListeria monocytogenes .Food Control 105 : 256-264. - Rodrigues JBD, de Souza NT, Scarano JOA, de Sousa JM, Lira MC, de Figueiredo R,
et al . 2018. Efficacy of using oregano essential oil and carvacrol to remove young and matureStaphylococcus aureus biofilms on food-contact surfaces of stainless steel.Lwt-Food Sci. Technol. 93 : 293-299.