Sanitization of food-grade materials: Combined effect of an enzymatic product and two chemical sanitizers on Listeria monocytogenes biofilms

i

This article has been updated. A Correction to this article was published on .

i

The Original Article was published on

i

The Original Article was published on

i

This article has been updated

i

This article was retracted on

i

This article has been updated

Abstract

In industrial practice, different food-grade materials could be subjected to contamination by microorganisms that are responsible for the spoilage of foods. The chemical sanitizers used to inactivate microbes on the food-grade surfaces have proved effective on vegetative cells, but may not be successful on biofilms, thus encouraging food companies to search alternative ways to eradicate these microbes. For this reason, in this work, two commercially available chemicals based on quaternary ammonium salts (QAC) and alcohols were tested alone or in combination with an enzymatic product against seven-day-old biofilms of three Listeria monocytogenes strains on stainless steel (SS) or poly-tetra-fluoro-ethylene (PTFE). When the sanitizers were applied without any pre-treatment, with the QAC, no survivors were detected after 10 minutes at a concentration of 0.75–1.0% on SS; on PTFE, it was necessary to increase the concentration to 1.0% to obtain a total inactivation of the sessile cells for all the tested strains. The use of the alcoholic product enabled us to obtain total inactivation of the sessile cells at a concentration of 50% for contact times greater than 2.5–5 minutes on SS; on the contrary, on PTFE, it was necessary to use the undiluted sanitizer for 10 minutes for all the tested strains. When the enzymatic solution was used as a pre-treatment, the achieved logarithmic reductions were significantly higher than the ones obtained when using the sanitizing products alone: In half of the cases, this combination enabled us to obtain a total inactivation on the biofilm-forming cells.

Keywords

biofilm / sanitization / ammonium quaternary salts / QAC / ethanol / enzymes / Listeria monocytogenes

CHANGE HISTORY

A Correction to this paper has been published:
https://doi.org

The Retraction Note was updated to reflect reception of statements of disagreement from all authors.

This article has been retracted.Please see the Retraction Notice for more detail:
https://doi.org

References

[1] Czechowski MH (1990) Bacterial attachment to Buna-N gaskets in milk processing equipment. Aust J Dairy Technol 45: 113-114.

[2] Mafu AA, Roy D, Goulet J, et al. (1990) Attachment of Listeria monocytogenes to stainless steel, glass, polypropylene, and rubber surfaces after short contact times. J Food Prot 53: 742-746. https://doi.org/10.4315/0362-028X-53.9.742

[3] Bonsaglia ECR, Silva NCC, Fernades Jr A, et al. (2014) Production of biofilm by Listeria monocytogenes in different materials and temperatures. Food Contr 35: 386-391. https://doi.org/10.1016/j.foodcont.2013.07.023

[4] Zara G, Budroni M, Mannazzu I, et al. (2020) Yeast biofilm in food realms: occurrence and control. World J Microbiol Biotechnol 36: 134-144. https://doi.org/10.1007/s11274-020-02911-5

[5] Kostakioti M, Hadjifrangiskou M, Hultgren SJ (2013) Bacterial biofilms: Development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. Cold Spring Harb Perspect Med 3: a010306. https://doi.org/10.1101/cshperspect.a010306

[6] Blasi F, Scaramuzza N, Chierici S, et al. (2025) Effect of chemical sanitizers alone or combined with an enzymatic product against Pseudomonas fluorescens biofilms on food-grade materials. Curr Microbiol 82: 512. https://doi.org/10.1007/s00284-025-04485-7

[7] Shree P, Kant Singh C, Sodhi KK, et al. (2023) Biofilms: Understanding the structure and contribution towards bacterial resistance in antibiotics. Med Microecol 16: e100084. https://doi.org/10.1016/j.medmic.2023.100084

[8] Parrilli E, Tutino ML, Marino G (2022) Biofilm as an adaptation strategy to extreme conditions. Rend Fis Acc Lincei 33: 527-536. https://doi.org/10.1007/s12210-022-01083-8

[9] Stoica M (2018) Sustainable sanitation in the food industry, In: Sustainable Food Systems from Agriculture to Industry, New York: Academic Press, 309-339. https://doi.org/10.1016/B978-0-12-811935-8.00009-3

[10] Verma N, Agarwal V (2022) Proceedings of the conference BioSangam 2022: Emerging trends in Biotechnology (BIOSANGAM 2022), Prayagraj, India. https://doi.org/10.2991/978-94-6463-020-6_13

[11] Hua Z, Zhu MJ (2024) Comprehensive strategies for controlling Listeria monocytogenes biofilms on food-contact surfaces. Compr Rev Food Sci Food Saf 23: e13348. https://doi.org/10.1111/1541-4337.13348

[12] Wang S, Zhao Y, Breslawec AP, et al. (2023) Strategy to combat biofilms: A focus on biofilm dispersal enzymes. npj Biofilms Microbiomes 9: 63. https://doi.org/10.1038/s41522-023-00427-y

[13] Lewis T, Stone WL (2025) Biochemistry, Proteins Enzymes, Treasure Island (FL): StatPearls Publishing. Available from: https://www.ncbi.nlm.nih.gov/books/NBK554481/.

[14] Molobela IP, Cloete TE, Beukes M (2010) Protease and amylase enzymes for biofilm removal and degradation of extracellular polymeric substances (EPS) produced by Pseudomonas fluorescens bacteria. Afr J Microbiol Res 4: 1515-1524. Available from: http://www.academicjournals.org/ajmr.

[15] Orgaz B, Neufeld RJ, SanJose C (2007) Single-step biofilm removal with delayed release encapsulated Pronase mixed with soluble enzymes. Enzyme Microb Technol 40: 1045-1051. https://doi.org/10.1016/j.enzmictec.2006.08.003

[16] Thomas VC, Thurlow LR, Boyle D, et al. (2008) Regulation of autolysis-dependent extracellular DNA release by Enterococcus faecalis extracellular proteases influences biofilm development. J Bacteriol 190: 5690-5698. https://doi.org/10.1128/JB.00314-08

[17] Brindle ER, Miller DA, Stewart PS (2011) Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or Dispersin B. Biotechnol Bioeng 108: 2968-2977. https://doi.org/10.1002/bit.23245

[18] Iñiguez-Moreno M, Gutiérrez-Lomelí M, Avila-Novoa MG (2021) Removal of mixed-species biofilms developed on food contact surfaces with a mixture of enzymes and chemical agents. Antibiotics 10: 931. https://doi.org/10.3390/antibiotics10080931

[19] Johansen C, Falholt P, Gram L (1997). Enzymatic removal and disinfection of bacterial biofilms. Appl Environ Microbiol 63: 3724-3728. https://doi.org/10.1128/aem.63.9.3724-3728.1997

[20] Pei R, Lamas-Samanamud GR (2014) Inhibition of biofilm formation by T7 bacteriophages producing quorum-quenching enzymes. Appl Environ Microbiol 80: 5340-5348. https://doi.org/10.1128/AEM.01434-14

[21] Wiktorczyk-Kapischke N, Skowron K, Grudlewska-Buda K, et al. (2021) Adaptive response of Listeria monocytogenes to the stress factors in the food processing environment. Front Microbiol 12: e710085. https://doi.org/10.3389/fmicb.2021.710085

[22] Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs (Text with EEA relevance). Available from: http://data.europa.eu/eli/reg/2005/2073/oj.

[23] Silva S, Teixeira P, Oliveira R, et al. (2008) Adhesion to and viability of Listeria monocytogenes on food contact surfaces. J Food Prot 71: 1379-1385. https://doi.org/10.4315/0362-028x-71.7.1379

[24] Mazaheri T, Cervantes-Huamán BRH, Turitich L, et al. (2022) Removal of Listeria monocytogenes biofilms on stainless steel surfaces through conventional and alternative cleaning solutions. Int J Food Microbiol 381: e109888. https://doi.org/10.1016/j.ijfoodmicro.2022.109888

[25] Nguyen UT, Burrows LL (2014) DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms. Int J Food Microbiol 187: 26-32. https://doi.org/10.1016/j.ijfoodmicro.2014.06.025

[26] Mazaheri T, Ripolles-Avila C, Rodríguez-Jerez JJ (2023) Elimination of mature Listeria monocytogenes biofilms formed on preconditioned and non-preconditioned surfaces after the application of cleaning treatments and their cell regeneration. LWT 173: e114316. https://doi.org/10.1016/j.lwt.2022.114316

[27] Rodríguez-López P, Carballo-Justo A, Draper LA, et al. (2017) Removal of Listeria monocytogenes dual-species biofilms using combined enzyme-benzalkonium chloride treatments. Biofouling 33: 45-58. https://doi.org/10.1080/08927014.2016.1261847

[28] Cho HY, Lee JE, Lee JH, et al. (2022) Anti-biofilm effect of egg white ovotransferrin and its hydrolysates against Listeria monocytogenes. LWT 165: e113759. https://doi.org/10.1016/j.lwt.2022.113759

[29] Yushina YK, Nasyrov NA, Zaiko EV, et al. (2023) Evaluating the effect of various types of disinfectants on bacterial biofilms. Theory Pract Meat Process 8: 162-167. https://doi.org/10.21323/2414-438X-2023-8-2-162-167

[30] Haase JK, Didelot X, Lecuit M, et al. (2014) The ubiquitous nature of Listeria monocytogenes clones: A large-scale Multilocus Sequence Typing study. Environ Microbiol 16: 405-416. https://doi.org/10.1111/1462-2920.12342

[31] George SM, Lund BM, Brocklehurst TF (1988) The effect of pH and temperature on initiation of growth of Listeria monocytogenes. Lett Appl Microbiol 6: 153-156. https://doi.org/10.1111/j.1472-765X.1988.tb01237.x

[32] UNI EN 13697:2023. (2023) Chemical disinfectants and antiseptics-Quantitative non-porous surface test for the evaluation of bactericidal and yeasticidal and/or fungicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas without mechanical action-Test method and requirements without mechanical action (phase 2, step 2). Available from: https://webstore.uni.com/en/uni-en-13697-2023.

[33] Kostaki M, Chorianopoulos N, Braxou E, et al. (2012) Differential biofilm formation and chemical disinfection resistance of sessile cells of Listeria monocytogenes strains under monospecies and dual-species (with Salmonella enterica) conditions. Appl Environ Microbiol 78: 2586-2595. https://doi.org/10.1128/AEM.07099-11

[34] Geesey GG, Wigglesworth-Cooksey B, Cooksey KE (2000) Influence of calcium and other cations on surface adhesion of bacteria and diatoms: A review. Biofouling 15: 195-205. https://doi.org/10.1080/08927010009386310

[35] Di Bonaventura G, Piccolomini R, Paludi D, et al. (2008). Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: Relationship with motility and cell surface hydrophobicity. J Appl Microbiol 104: 1552-1561. https://doi.org/10.1111/j.1365-2672.2007.03688.x

[36] Purkrtová S, Turoňová H, Pilchová T, et al. (2010) Resistance of Listeria monocytogenes biofilms to disinfectants. Czech J Food Sci 28: 326-332. https://doi.org/10.17221/153/2010-CJFS

[37] Mandakhalikar KD, Rahmat JN, Chiong E, et al. (2018) Extraction and quantification of biofilm bacteria: Method optimized for urinary catheters. Sci Rep 8: 8069. https://doi.org/10.1038/s41598-018-26342-3

[38] Webber B, Canova R, Esper LM, et al. (2015) The use of vortex and ultrasound techniques for the in vitro removal of Salmonellaspp. biofilms. Acta Sci Vet 43: 1332.

[39] Kobayashi H, Oethinger M, Tuohy MJ, et al. (2009) Improved detection of biofilm-formative bacteria by vortexing and sonication: A pilot study. Clin Orthop Relat Res 467: 1360-1364. https://doi.org/10.1007/s11999-008-0609-9

[40] Harada AMM, Nascimento MS (2021) Efficacy of dry sanitizing methods on Listeria monocytogenes biofilms. Food Contr 124: e107897. https://doi.org/10.1016/j.foodcont.2021.107897

[41] Fagerlund A, Heir E, Møretrø T, et al. (2020) Listeria monocytogenes biofilm removal using different commercial cleaning agents. Molecules 25: 792. https://doi.org/10.3390/molecules25040792

[42] Mendez EA, Tande B, Vipham J, et al. (2022) Preliminary investigation of the effect of chemical sanitizers and UV-C light on Listeria monocytogenes biofilm survivability. Food Prot Trends 42: 278-283. https://doi.org/10.4315/FPT-21-025

[43] Korany AM, Hua Z, Green T, et al. (2018) Efficacy of ozonated water, chlorine, chlorine dioxide, quaternary ammonium compounds and peroxyacetic acid against Listeria monocytogenes biofilm on polystyrene surfaces. Front Microbiol 9: 2296. https://doi.org/10.3389/fmicb.2018.02296

[44] Mertz AW, O'Bryan CA, Crandall PG, et al. (2015) The elimination of Listeria monocytogenes attached to stainless steel or aluminum using multiple hurdles. J Food Sci 80: 1557-1562. https://doi.org/10.1111/1750-3841.12926

[45] Hua Z, Korany AM, El-Shinawy SH, et al. (2019) Comparative evaluation of different sanitizers against Listeria monocytogenes biofilms on major food-contact surfaces. Front Microbiol 10: 2462. https://doi.org/10.3389/fmicb.2019.02462

[46] Iñiguez-Moreno M, Avila-Novoa MG, Gutiérrez-Lomelí M (2018) Resistance of pathogenic and spoilage microorganisms to disinfectants in the presence of organic matter and their residual effect on stainless steel and polypropylene. J Glob Antimicrob Resist 14: 197-201. https://doi.org/10.1016/j.jgar.2018.04.010

[47] Chaves RD, Kumazawa SH, Khaneghah AM, et al. (2024) Comparing the susceptibility to sanitizers, biofilm-forming ability, and biofilm resistance to quaternary ammonium and chlorine dioxide of 43 Salmonella enterica and Listeria monocytogenes strains. Food Microbiol 117: e104380. https://doi.org/10.1016/j.fm.2023.104380

[48] Nahar S, Mizan MFR, Ha AJ, et al. (2018) Advances and future prospects of enzyme-based biofilm prevention approaches in the food industry. Compr Rev Food Sci Food Saf 17: 1484-1502. https://doi.org/10.1111/1541-4337.12382