Biofilms in the food industry: problems and potential solutions

Contamination of food by spoilage and pathogenic micro‐organisms costs the food industry millions of dollars annually. Much of this contamination may be attributed to the presence of biofilms in the processing plant. In this review, we examine the properties of micro‐organisms and the surfaces of processing equipment that influence the formation of biofilms. Of particular concern is the increased resistance of biofilms to cleaning and disinfection processes. Alternative means of controlling biofilm development are described and some aspects where more information is required are identified.     

Biofilms in the Spotlight: Detection,Quantification, and Removal Methods

Microorganisms can colonize and subsequently form biofilms on surfaces, which protect them from adverse conditions and make them more resistant than their planktonic free‐living counterparts. This is a major concern in the food industry because the presence of biofilms has significant implications for microbial food contamination and, therefore, for the transmission of foodborne diseases. Adequate hygienic conditions and various preventive and control strategies have consequently been developed to ensure the provision of safe, good‐quality food with an acceptable shelf‐life. This review focuses on the significance of biofilms in the food industry by describing the factors that favor their formation. The interconnected process among bacteria known as “quorum sensing,” which plays a significant role in biofilm development, is also described. Furthermore, we discuss recent strategic methods to detect, quantify, and remove biofilms formed by pathogenic bacteria associated with food processing environments, focusing on the complexity of these microbial communities.     

Removal of Salmonella enterica serovar Typhimurium and Cronobacter sakazakii biofilms from food contact surfaces through enzymatic catalysis

Bacterial biofilms are highly difficult to control, hence significant economic resources have been allocated to develop strategies to eradicate them. This study evaluated the effect of an enzymatic treatment to be used as a cleaning product to control the presence of biofilms. Two different materials used in the food industry, polystyrene and stainless steel, were tested using Salmonella Typhimuirum and Cronobacter sakazakii. Biofilm formation was carried out by inoculating the surfaces with a standardized concentration of 4 log (CFU cm⁻²) and incubated for 48 hr with renewal of nutrients. The biofilm formation and subsequent enzymatic treatment were quantified using fluorescence microscopy and the conventional culture method. The enzymatic treatment showed significant reductions of 2–3 log (CFU cm⁻²) in biofilm cells, which was attributed to the degradation of the extracellular matrix and the further detachment of both microorganisms. The maximum biofilm detachment obtained with the preventive formula was 46.67%; however, this percentage could be increased by applying an aggressive treatment or by adding a subsequent disinfection step that would eliminate adhered microbial cells. Further, the enzymatic cleaning treatment could be exploited as a potent technology to control bacterial adherence and biofilm formation in the food industry.     

Promising strategies to control persistent enemies: Some new technologies to combat biofilm in the food industry—A review

Biofilm is an advanced form of protection that allows bacterial cells to withstand adverse environmental conditions. The complex structure of biofilm results from genetic-related mechanisms besides other factors such as bacterial morphology or substratum properties. Inhibition of biofilm formation of harmful bacteria (spoilage and pathogenic bacteria) is a critical task in the food industry because of the enhanced resistance of biofilm bacteria to stress, such as cleaning and disinfection methods traditionally used in food processing plants, and the increased food safety risks threatening consumer health caused by recurrent contamination and rapid deterioration of food by biofilm cells. Therefore, it is urgent to find methods and strategies for effectively combating bacterial biofilm formation and eradicating mature biofilms. Innovative and promising approaches to control bacteria and their biofilms are emerging. These new approaches range from methods based on natural ingredients to the use of nanoparticles. This literature review aims to describe the efficacy of these strategies and provide an overview of recent promising biofilm control technologies in the food processing sector.     

Biofilm Formation in Milk Production and Processing Environments; Influence on Milk Quality and Safety

Abstract: Bacteria in milk have the ability to adhere and aggregate on stainless steel surfaces, resulting in biofilm formation in milk storage tanks and milk process lines. Growth of biofilms in milk processing environments leads to increased opportunity for microbial contamination of the processed dairy products. These biofilms may contain spoilage and pathogenic microorganisms. Bacteria within biofilms are protected from sanitizers due to multispecies cooperation and the presence of extracellular polymeric substances, by which their survival and subsequent contamination of processed milk products is promoted. This paper reviews the most critical factors in biofilm formation, with special attention to pseudomonads, the predominant spoilage bacteria originating from raw milk. Biofilm interactions between pseudomonads and milk pathogens are also addressed, as emerging risks and future research perspectives, specifically related to the milk processing environment.     

An Ecological Perspective of Listeria monocytogenes Biofilms in Food Processing Facilities

Listeria monocytogenes can enter the food chain at virtually any point. However, food processing environments seem to be of particular importance. From an ecological point of view, food processing facilities are microbial habitats that are constantly disturbed by cleaning and sanitizing procedures. Although L. monocytogenes is considered ubiquitous in nature, it is important to recognize that not all L. monocytogenes strains appear to be equally distributed; the distribution of the organism seems to be related to certain habitats. Currently, no direct evidence exists that L. monocytogenes-associated biofilms have played a role in food contamination or foodborne outbreaks, likely because biofilm isolation and identification are not part of an outbreak investigation, or the definition of biofilm is unclear. Because L. monocytogenes is known to colonize surfaces, we suggest that contamination patterns may be studied in the context of how biofilm formation is influenced by the environment within food processing facilities. In this review, direct and indirect epidemiological and phenotypic evidence of lineage-related biofilm formation capacity to specific ecological niches will be discussed. A critical view on the development of the biofilm concept, focused on the practical implications, strengths, and weaknesses of the current definitions also is discussed. The idea that biofilm formation may be an alternative surrogate for microbial fitness is proposed. Furthermore, current research on the influence of environmental factors on biofilm formation is discussed.     

A review of current and emergent biofilm control strategies

Microbial adhesion to surfaces and the consequent biofilm formation has been documented in many different environments. Biofilms constitute a protected mode of growth that allows microorganisms to survival in hostile environments, being their physiology and behavior significantly different from their planktonic counterparts. In dairy industry, biofilms may be a source of recalcitrant contaminations, causing food spoilage and are possible sources of public health problems such as outbreaks of foodborne pathogens. Biofilms are difficult to eradicate due to their resistant phenotype. However, conventional cleaning and disinfection regimens may also contribute to inefficient biofilm control and to the dissemination of resistance. Consequently, new control strategies are constantly emerging with main incidence in the use of biosolutions (enzymes, phages, interspecies interactions and antimicrobial molecules from microbial origin).The present review will focus on describing the mechanisms involved in biofilm formation and behavior, deleterious effects associated with their presence, and some of the current and emergent control strategies, providing new insight of concern for food industry.     

食品加工过程中细菌生物被膜的危害及控制

生物被膜中的微生物生活在一个由胞外聚合物（EPS）形成的环境中,它的形成是微生物生长过程中的一个保护模式,允许细胞在恶劣的环境中生存并分散到新的环境中。食品加工过程中有害菌形成的生物被膜对食品工业的危害极大,可使微生物残存增加,加工设备无法严格清洗、消毒,导致产品受到污染。该文在收集、研究现有文献的基础上归纳介绍了生物被膜的特点及其形成过程和形成机制,概述了生物被膜的危害、控制及检测方法,旨在提高人们对生物被膜的认识,推动该领域的研究发展。     

Assessment of a regulatory sanitization process in Egyptian dairy plants in regard to the adherence of some food-borne pathogens and their biofilms

Food-borne pathogens may develop certain strategies that enable them to defy harsh conditions such as chemical sanitization. Biofilm formation represents a prominent one among those adopted strategies, by which food-borne pathogens protect themselves against external threats. Thus, bacterial biofilm is considered as a major hazard for safe food production. This study was designed to investigate the adherence and the biofilm formation ability of some food-borne pathogens on stainless steel and polypropylene surfaces using chip assay, and to validate regular sanitizing process (sodium hypochlorite 250mg/L) for effective elimination of those pathogens. Sixteen pathogenic bacterial strains, previously isolated from raw milk and dairy products at Zagazig city, Egypt (9 Staphylococcus aureus, 4 Cronobacter sakazakii and 3 Salmonella enterica serovar Typhimurium), were chosen for this study. Strains showed different patterns of adherence and biofilm formation on tested surfaces with minor significance between surfaces. The ability of sodium hypochlorite to completely eradicate either adhered or biofilm-embedded pathogens varied significantly depending on the strain and type of surface used. Whilst, sodium hypochlorite reduced tested pathogens counts per cm(2) of produced biofilms, but it was not able to entirely eliminate neither them nor adherent Cronobacter sakazakii to stainless steel surface. This study revealed that biofilm is considered as a sustainable source of contamination of dairy products with these pathogens, and also emphasized the need of paying more attention to the cleaning and sanitizing processes of food contact surfaces.     

Development and Control of Bacterial Biofilms on Dairy Processing Membranes

Membrane fouling is a major operational problem that leads to reduced membrane performance and premature replacement of membranes. Bacterial biofilms developed on reverse osmosis membranes can cause severe flux declines during whey processing. Various types of biological, physical, and chemical factors regulate the formation of biofilms. Extracellular polymeric substances produced by constitutive microflora provide an effective barrier for the embedded cells. Cultural and microscopic techniques also revealed the presence of biofilms with attached bacterial cells on membrane surfaces. Presence of biofilms, despite regular cleaning processes, reflects ineffectiveness of cleaning agents. Cleaning efficiency depends upon factors such as pH of the cleaning agent, temperature, pressure, cleaning agent dose, optimum cleaning time, and cross‐flow velocity during cleaning. Among different cleaning agents, surfactants help to prevent bacterial attachment to surfaces by reducing the surface tension of water and interfacial tension between the layers. Enzymes mixed with surfactants and chelating agents can be used to penetrate the biofilm matrix formed by microbes. Recent studies have shown the role of quorum‐sensing‐based cell‐to‐cell signaling, which provides communication within bacterial cells to form a mature biofilm, and also the role of applying quorum inhibitors to prevent biofilm formation. Major cleaning applications are also summarized in Table 1 .     

Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and Non-O157 Strains and Their Tolerance to Sanitizers Commonly Used in the Food Processing Environment

Shiga toxin-producing Escherichia coli (STEC) strains are important foodborne pathogens. Among these, E. coli O157:H7 is the most frequently isolated STEC serotype responsible for foodborne diseases. However, the non-O157 serotypes have been associated with serious outbreaks and sporadic diseases as well. It has been shown that various STEC serotypes are capable of forming biofilms on different food or food contact surfaces that, when detached, may lead to cross-contamination. Bacterial cells at biofilm stage also are more tolerant to sanitizers compared with their planktonic counterparts, which makes STEC biofilms a serious food safety concern. In the present study, we evaluated the potency of biofilm formation by a variety of STEC strains from serotypes O157:H7, O26:H11, and O111:H8; we also compared biofilm tolerance with two types of common sanitizers, a quaternary ammonium chloride-based sanitizer and chlorine. Our results demonstrated that biofilm formation by various STEC serotypes on a polystyrene surface was highly strain-dependent, whereas the two non-O157 serotypes showed a higher potency of pellicle formation at air-liquid interfaces on a glass surface compared with serotype O157:H7. Significant reductions of viable biofilm cells were achieved with sanitizer treatments. STEC biofilm tolerance to sanitization was strain-dependent regardless of the serotypes. Curli expression appeared to play a critical role in STEC biofilm formation and tolerance to sanitizers. Our data indicated that multiple factors, including bacterial serotype and strain, surface materials, and other environmental conditions, could significantly affect STEC biofilm formation. The high potential for biofilm formation by various STEC serotypes, especially the strong potency of pellicle formation by the curli-positive non-O157 strains with high sanitization tolerance, might contribute to bacterial colonization on food contact surfaces, which may result in downstream product contamination.     

Biofilm formation and contamination of cheese by nonstarter lactic acid bacteria in the dairy environment

Defects in cheese, such as undesirable flavors, gas formation, or white surface haze from calcium lactate crystals, can result from growth of nonstarter lactic acid bacteria (NSLAB). The potential for biofilm formation by NSLAB during cheese manufacturing, the effect of cleaning and sanitizing on the biofilm, and bacterial growth and formation of defects during ripening of the contaminated cheese were studied. Stirred-curd Cheddar cheese was made in the presence of stainless steel chips containing biofilms of either of two strains of erythromycin-resistant NSLAB (Lactobacillus curvatus strain JBL2126 or Lactobacillus fermentum strain AWL4001). During ripening, the cheese was assayed for total lactic acid bacteria, numbers of NSLAB, and percentage of lactic acid isomers. Biofilms of L. curvatus formed during cheese making survived the cleaning process and persisted in a subsequent batch of cheese. The starter culture also survived the cleaning process. Additionally, L. curvatus biofilms present in the vat dislodged, grew to high numbers, and caused a calcium lactate white haze defect in cheese during ripening. On the other hand, biofilms of L. fermentum sloughed off during cheese making but could not compete with other NSLAB present in cheese during ripening. Pulsed-field gel electrophoresis results verified the presence of the two biofilm strains during cheese making and in the ripening cheese. Probable contamination sites in the plant for other NSLAB isolated in the cheese were identified, thus supporting the hypothesis that resident NSLAB biofilms are a viable source of contamination in the dairy environment.     

Salmonella enterica Enteritidis biofilm formation and viability on regular and triclosan-impregnated bench cover materials

Contamination of food contact surfaces by microbes such as Salmonella is directly associated with substantial industry costs and severe foodborne disease outbreaks. Several approaches have been developed to control microbial attachment; one approach is the development of food contact materials incorporating antimicrobial compounds. In the present study, Salmonella enterica Enteritidis adhesion and biofilm formation on regular and triclosan-impregnated kitchen bench stones (silestones) were assessed, as was cellular viability within biofilms. Enumeration of adhered cells on granite, marble, stainless steel, and silestones revealed that all materials were prone to bacterial colonization (4 to 5 log CFU/cm(2)), and no significant effect of triclosan was found. Conversely, results concerning biofilm formation highlighted a possible bacteriostatic activity of triclosan; smaller amounts of Salmonella Enteritidis biofilms were formed on impregnated silestones, and significantly lower numbers of viable cells (1 × 10(5) to 1 × 10(6) CFU/cm(2)) were found in these biofilms than in those on the other materials (1 × 10(7) CFU/cm(2)). All surfaces tested failed to promote food safety, and careful utilization with appropriate sanitation of these surfaces is critical in food processing environments. Nevertheless, because of its bacteriostatic activity, triclosan incorporated into silestones confers some advantage for controlling microbial contamination.     

食源性混合菌生物被膜的形成和相互作用机制

在食品加工中,食源性病原菌生物被膜的存在会引发大量食品污染等安全问题。目前,国内外学者深入研究了单一菌生物被膜,然而对混合菌生物被膜的研究较少。本文在现有的研究基础上,归纳总结了食源性混合菌生物被膜的形成规律及混合菌生物膜中菌种间的相互作用,初步探讨了群体感应在混合菌生物膜中的作用,以期为食品领域应对和破解食源性混合菌生物被膜污染问题提供思路。     

群体感应抑制剂调控食源性微生物生物膜形成的研究进展

食源性微生物的生物膜是附着在固体表面上形成的具有空间组织的群落,因其能够附着在食品工业环境中的生物或非生物表面,且对消毒剂和抗菌剂能产生抗药性,通常难以控制,被认为是造成设备损坏、能源成本增加、食物变质和引起食源性疾病的原因之一,给食品工业带来了巨大的挑战。研究发现群体感应在生物膜的形成中起至关重要的作用,可以通过阻断群体感应系统来控制生物膜的形成。因此,群体感应抑制剂可以作为控制生物膜形成的新策略,在食品工业中对控制生物膜的形成具有巨大潜力。本文综述了生物膜的形成、群体感应系统及其对生物膜的调控作用,介绍了群体感应抑制剂的调控机制及其分类,为通过群体感应抑制剂调控生物被膜的形成提供研究思路。     

Candida krusei development on turbulent flow regimes: Biofilm formation and efficiency of cleaning and disinfection program

In food processing lines or in complex equipment such as pumps or valves, microorganisms are exposed to varying hydrodynamic conditions caused by the flow of liquid food, and biofilms are thus grown under a wide distribution of local hydrodynamic strengths. Using an industrially relevant strain of Candida krusei, we demonstrated that biofilms formed on stainless steel for 4 days at Reynolds (Re) numbers ranging from 294,000 to 1.2 × 10⁶ proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in an extracellular matrix and biofilm formation increases when increasing Reynolds number and time. In all growth phases, the morphology of C. krusei biofilm revealed the influence of hydrodynamic drag. Indeed, we study the effect of cleaning and sanitation procedure in the control of turbulent flow-generated biofilm. This procedure involves alkali (NaOH 0.5%) and sodium hypochlorite (500 ppm). In terms of total biofilm mass, removal decreases with increasing biofilm age. The largest reduction post-treatment (between 57% and 62%) was observed, to all Reynolds numbers, on 24 and 48 h-old biofilms. Removal was between 39% and 46% on 72 h-old biofilms and was close to 30% for all Reynolds numbers on 96 h-old biofilm.     

Combating biofilms of foodborne pathogens with bacteriocins by lactic acid bacteria in the food industry

Most foodborne pathogens have biofilm-forming capacity and prefer to grow in the form of biofilms. Presence of biofilms on food contact surfaces can lead to persistence of pathogens and the recurrent cross-contamination of food products, resulting in serious problems associated with food safety and economic losses. Resistance of biofilm cells to conventional sanitizers urges the development of natural alternatives to effectively inhibit biofilm formation and eradicate preformed biofilms. Lactic acid bacteria (LAB) produce bacteriocins which are ribosomally synthesized antimicrobial peptides, providing a great source of nature antimicrobials with the advantages of green and safe properties. Studies on biofilm control by newly identified bacteriocins are increasing, targeting primarily onListeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli. This review systematically complies and assesses the antibiofilm property of LAB bacteriocins in controlling foodborne bacterial-biofilms on food contact surfaces. The bacteriocin-producing LAB genera/species, test method (inhibition and eradication), activity spectrum and surfaces are discussed, and the antibiofilm mechanisms are also argued. The findings indicate that bacteriocins can effectively inhibit biofilm formation in a dose-dependent manner, but are difficult to disrupt preformed biofilms. Synergistic combination with other antimicrobials, incorporation in nanoconjugates and implementation of bioengineering can help to strengthen their antibiofilm activity. This review provides an overview of the potential and application of LAB bacteriocins in combating bacterial biofilms in food processing environments, assisting in the development and widespread use of bacteriocin as a promising antibiofilm-agent in food industries.     

Mixed-species biofilms in the food industry: Current knowledge and novel control strategies

Abstract

Attachment of microorganisms to food contact surfaces and the subsequent formation of biofilms may cause equipment damage, food spoilage and even diseases. Mixed-species biofilms are ubiquitous in the food industry and they generally exhibit higher resistance to disinfectants and antimicrobials compared to single-species biofilms. The physiology and metabolic activity of microorganisms in mixed-species biofilms are however rather complicated to study, and despite targeted research efforts, the potential role of mixed-species biofilms in food industry is still rather unexplored. In this review, we summarize recent studies in the context of bacterial social interactions in mixed-species biofilms, resistance to disinfectants, detection methods, and potential novel strategies to control the formation of mixed-species biofilms for enhanced food safety and food quality.     

Prevalence of microbial biofilms on selected fresh produce and household surfaces

Investigations of biofilms in domestic environments are sparsely represented in the literature. In this study, samples of various household surfaces, including food, laundry and kitchen items, were analyzed for evidence of biofilm presence. Visualization of the surfaces was carried out using cryostage scanning electron microscopy (CSEM) and light microscopy. Qualitative evidence of the presence of biofilm formation was obtained from all of the sample groups analyzed, suggesting the widespread existence of microorganisms in biofilms on domestic surfaces. This suggests that biofilms may be important in household hygiene, and highlights the need for standardized, approved biofilm methods suitable for consumer products testing.     

单核细胞增生李斯特菌生物被膜交叉污染评估进展

单核细胞增生李斯特菌（Listeria monocytogenes）（以下简称单增李斯特菌）是一种引发李斯特菌病罹患者高住院率和高死亡率的食源性致病菌,其可在冷、热、干燥和消毒剂处理等不利条件下黏附于食品接触表面并进一步形成难以清除的生物被膜。交叉污染是单增李斯特菌传播的主要途径,生物被膜的形成提高了单增李斯特菌在工厂和厨房环境持续传播和污染的可能性,可引发相关食源性疾病暴发和食品召回等,从而造成健康和经济损失。本文首先介绍了单增李斯特菌生物被膜的胞外聚合物组分,并从外部生存环境和内部微生物自身因素两方面总结了影响单增李斯特菌生物被膜交叉污染转移的因素;进一步重点从研究类型和细菌收集两方面阐述了生物被膜交叉污染的相关研究进展;最后,归纳总结了针对单增李斯特菌生物被膜形成早期的防控策略,展望该领域的研究前景,以期为科学评估和早期精准防控单增李斯特菌生物被膜交叉污染的潜在风险提供理论依据。