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biofilm

    Handbook of hygiene control in the food
    industry
    Biofilm risks

    Introduction: biofilm formation and detection

    Biofilm formation, sampling and detection methods, pathogens in biofilms, persistent and non-persistent microbes, prevention of biofilm formation and biofilm removal as well as future trends in biofilm control in the food industry.

    Microbes that inhabit contact and environmental sites in food processing are mostly harmful because microbial communities in the wrong places lead to contamination of surfaces and of the product produced in the process.

    This study showed that the slow-growing strains covered tested surfaces with 2±4% biofilm in 10 days; fast biofilm producers had already covered the whole surface in 2 days.

    In addition to the problems in food industry, biofilm formation also causes problems in food-related systems, e.g. industrial water systems as well as the paper and packaging industry.

    On the positive side, however, biofilms have also been applied successively in food-related processes, e.g. in brewing and in water treatment .

    Factors affecting biofilm formation

    In order to be able to survive hostile environmental factors such as heat and chemicals, microbes in micro colonies have a tendency to form protective extracellular matrices, which mainly consist of polysaccharides and glyco- proteins, and are called biofilms.

    The microcolony formation is the first stage in biofilm formation, which occurs under suitable conditions on any surface ± both inert and living.

    Microbes can start up this formation when there is water or moisture available.

    Physical parameters such as fluid flow rate, charge, hydrophobicity and micro-topography of the surface material affect the attachment of cells to the surface.

    Cells must overcome the energy-intensive repulsion barrier, which affects the particle surfaces.

     Bacteria with pili could conceivably overcome this barrier to achieve micro-colonisation and biofilm formation.

    It has been found that temperatures below 50°C promote biofilm formation.

    In the food industry, equipment design plays the most important role in combating(نبرد) biofilm formations.

    The choice of materials and their surface treatments as well as roughness, e.g. grinding and polishing, are important factors for inhibiting the formation of biofilm and making surfaces easier to clean.

    Treating surface materials so that they reject biofilms can be performed actively to remove or passively to retard biofilm reoccurrence.

    The cleanliness of surfaces, training of personnel and good manufacturing and design practices are the most important tools in combating biofilm problems in the food industry.

    Biofilm formation on food processing surfaces

    It is also important to remember that about 85±96% of a biofilm consists of water, which means that only 2±5% of the total biofilm volume is detectable on dry surfaces.

    Biofilm can generally be produced by any microbes under suitable conditions, although some microbes naturally have a higher tendency to produce biofilm than others.

    A biofilm consists of microbial cell clusters with a network of internal channels or voids in the extracellular polysaccharide and glycoprotein matrix.

     This allows nutrients and oxygen to be transported from the bulk liquid to the cells.

    It has been suggested that the mechanisms of microbial attachment and biofilm build-up occur in 2, 3,5 and eight-step processes.

    The two-step process is divided into reversible and irreversible biofilm formation.

    The reversible phase involves the association of cells near to but not in contact with the surface.

    Biofilm formation on food processing surfaces

    Cells associated with the surface synthesise exopolymers, which irreversibly bind the cells to the surface.

    Characklis described biofilm build-up using the following five steps:

    Biofilm formation on food processing surfaces

    1- transportation of cells to a wetted surface, absorption of the cells into a conditioning film, adhesion of microbial cells to the wetted surface, reaction of the cells in the biofilm and detachment of biofilm from the surface.

    Bryers and Weightman (1995) divided the biofilm build-up into the following eight steps:

    1-preconditioning of the surface by macromolecules, 2- transport of cells to the surface,3 and 4- reversible and irreversible adsorption to the surface, 5- cell replication, 6- transport of nutrients and metabolism, 7- production of extracellular polymers and, finally,8- detachment.

     

    Sampling and detection of biofilm formation in food processing sites

    Methods for studying biofilm formation include microbiological, chemical, microscopical and molecular biological methods.

    Practical methods for assessing microbes and organic soil on processing surfaces are needed to establish the optimal cleaning frequency of the equipment. 

    Hygiene monitoring is currently based on conventional cultivation using swabbing, rinsing or contact plates.

    Surface sampling can be improved by wetting the surface in advance.

     In methods that use swabs, sponges or something similar, the detachment of surface-bound microbes is a limiting factor.

    Sampling and detection of biofilm formation in food processing sites

    In the cultivation of biofilm microbes, it is important for the sample to be detached and mixed properly.

    Sampling and detection of biofilm formation in food processing sites

    Agitation used too forcefully in the detachment of the biofilm from the surface may harm the cells, making them unable to grow on the agar plates, whereas insufficient mixing may result in clumps and inaccurate results.

    Ultrasonics detaches about ten times the number of cells from the surface compared with swabbing.

    Sampling and detection of biofilm formation in food processing sites

    In biofilm detection the planktonic cell counts of processing fluids should be interpreted with caution because they are not always representative of the sessile(چسبیده) organisms found on surfaces, especially in badly designed equipment and process lines.

    Sampling and detection of biofilm formation in food processing sites

    Organisms from extreme environments are difficult to culture and therefore standard plate counts do not give accurate estimates.

    The choice of agar and incubation conditions during the cultivation is governed by the characteristics of the microbes that are considered to be the most important. 

    Sampling and detection of biofilm formation in food processing sites

    Conventional culturing techniques are used to measure the number of viable cells able to grow on the chosen agar at given circumstances.

    The plates and slides are usually incubated at 25±30 °C for 2±3 days.

    The agars are either nutrient agars, which may contain tryptose, yeast, glucose and agar-agar, or selective agars based on growth inhibitors, e.g. nutritional, antibiotic or acidic compounds.

    Sampling and detection of biofilm formation in food processing sites

    The international standard methods for the detection and enumeration of spoilage and pathogenic microbes are based on culturing techniques.

    Impedance techniques can be used to enumerate microorganisms directly on surfaces as the increase in conductance and capacitance due to the metabolic activity of the microbes in the sample leads to a decrease in the impedance.

    The measurement of the change in impedance value at suitable time intervals provides an impedance curve and thus the detection time of microbial growth in the sample.

    The detection time depends on the number of microbes in the sample.

     Results are achieved more rapidly with impedance measurements than with cultivation.

    Impedance measurement is used in the food industry to control product quality and to assess the effect of cleaning agents and disinfectants.

    The chemical methods used in the assessment of biofilm formation are indirect methods based on the utilisation or production of specific compounds, e.g. organic carbon, oxygen, polysaccharides and proteins, or on the biofilm microbial activity, e.g. living cells and ATP (adenosine 5' -triphosphate) content.  

    ATP measurement is a luminescence method based on the luciferine ± luciferase reaction.

     The ATP content of the biofilm is proportional to the number of living cells in the biofilm and provides information about their metabolic activity.

    Kinetic data obtained for freely suspended cells should not be used to assess immobilised biomass growth, e.g. biofilm.

    The ATP method is insensitive and therefore not suitable for hygiene measurements in equipment where absolute sterility is needed, because with most of the reagents used today a count of at least 103 bacterial cells is needed to obtain a reliable ATP value.

    Important tools in modern biotechnology-related research are based on microscopical techniques.

     One advantage of microscopical analysis is that it can measure surface-adhered cells, rather than cells that have been detached from the surface.

    Various microscopical techniques for studying cell adhesion and biofilm formation on surface materials are available including:

     epifluorescence, scanning and transmission electron microscopy, Fourier transformation infrared spectrometry, quartz-crystal microbalance and infrared spectroscopy as well as confocal laser scanning and atomic force microscopying techniques.

    Fluorescence is a type of luminescence in which light is emitted from molecules for a short period of time following the absorption of light.

     Fluorescence occurs when an excited electron returns to a lower-energy orbit and emits a photon of light.

    Many different fluorochromes have been used for the staining of microbes in food samples, biofilms and environmental samples.

    Flow cytometry using fluorescent probes is a direct optical technique for the measurement of functional and structural properties of individual cells in a cell population.

    The cells are forced to flow in single file along a rapidly moving fluid stream through a powerful light source.

    This technique has been used to determine the viability of protozoa, fungi and bacteria.

    It measures the viability of a statistically significant number of organisms (5000±25 000 cells per sample).

    The advantages of flow cytometry are accuracy, speed, sensitivity and reproducibility.

    In the food industry, the first step is to identify the biofilm problems in a particular process or site. 

    Subsequently, it is important to use the best possible methods for isolation and detection of the biofilm for further characterisation in the laboratory using molecular biology and biochemical methods. 

    These methods can be utilized in the detection and identification of microbes in two ways by performing identification either directly from sample material or indirectly from pure cultures obtained from the samples.

    The two major techniques applied in the molecular detection and identification of bacteria are the polymerase chain reaction and the hybridisation technique.

    Pathogens in biofilms

    It is somewhat alarming to know that pathogens such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, Campylobacter jejuni and Yersinia enterocolitica can easily produce biofilms or be part of biofilm communities that cause severe disinfection and cleaning problems on surfaces in the food industry.

    According to a study by Peters et al. (1999) pathogens were isolated from biofilm communities.

    In this study Listeria spp. were found in 35% of food contact sites and 42% of environmental sites, with Staphylococcus aureus being
    present in a total of 7% and 8%, respectively.

    Joseph et al. (2001) have reported pathogenic bacteria such as Klebsiella spp., Campylobacter spp. and entero- haemorrhagic E. coli in biofilms.

    In laboratory studies, specific properties of pathogens in biofilms have been studied, and it has been found that biofilm cells of Listeria were more resistant than planktonic cells to disinfectants containing, e.g., chlorine, iodine, quaternary ammonium and anionic acid compounds.

    L. monocytogenes strains in biofilm studies on glass surfaces at static conditions of 37 °C for up to 4 days.

    After 3 h incubation bacterial cells from all 13 strains had attached themselves to the glass slides and they formed biofilms within 24 hours.

    Two poultry isolates of Salmonella were used to study biofilm formation on three commonly used food contact surfaces viz. plastic, concrete and stainless steel.

     Both isolates, i.e. Salmonella Weltevreden and Salmonella FCM 40, showed similar patterns in the biofilm formation with the greatest growth on plastic followed by concrete and stainless steel.

    In the following chapters there are more examples of the biofilm formation capability of some Gram-negative and Gram-positive pathogenic bacteria.

    Salmonella biofilms

    Salmonella is a genus within the family Enterobacteriaceae in which approxi- mately 2200 serotypes are recognised.

    Some of these strains are specifically adapted to hosts and largely restricted to them, e.g. S. Typhi in man and S.Dublin in cattle.

    Salmonella is a non-spore-forming rod-shaped, motile Gram- negative bacterium with non-motile exceptions such as S. Gallinarum and S. Pullorum.

    Salmonella serotypes are traditionally named as if they were separate species but, because of their genetic similarity, a single species, S. enterica, has been proposed, with food-poisoning serotypes mostly classified subspecies, also named enterica.

    The growth range for salmonellae is 5±47 ëC at pH 4.0±9.0, with optimum growth at 35±37 ëC and pH 6.5±7.5. Salmonellae are not particularly salt-tolerant, although growth can occur in the presence of 4% The lower limit of water activity (aw) permitting growth is 0.93.

    Foods commonly associated with the disease include raw meats, poultry, eggs, milk and dairy products.

    Milk-borne salmonellosisis common in parts of the world where milk is neither boiled nor pasteurised.

    It occurs, but much less frequently, in developed countries where the main products implicated are pasteurised milk, powdered milk and certain cheeses.

    Formation of a biofilm by Salmonella on various types of surfaces used in the food processing industry has been reported by several groups.

    These studies have shown that Salmonella spp. can form biofilms on food contact surfaces and that the cells in biofilms are much more resistant to sanitisers compared to planktonic cells.

    Mokgatla and co-workers (1998) studied the resistance of Salmonella sp. isolated from a poultry abattoir and found out that it will grow in the presence of in-use concentrations of hypochlorous acid.

     The presence of Pseudomonas fluorescens in the biofilm resulted in the increased resistance of S. Typhimurium to chlorine.

    Escherichia coli biofilms

    Escherichia coli is a Gram-negative, rod-shaped bacterium.

    Because many microbes from faeces are pathogenic in animals and humans, the presence of the intestinal bacterium E. coli in water and foods indicates a potential hygiene hazard.

    Most strains of E. coli are harmless. However, a few strains with well- characterised traits are known to be associated with pathogenicity.

    Those of greatest concern in water and foods are the intestinal pathogens, which are classified into five major groups:

    the enterohaemorrhagic E. coli (EHEC), the enterotoxigenic E. coli (ETEC), the enteroinvasive E. coli (EIEC), the enteropathogenic E. coli (EPEC) and the enteroaggregative E. coli (EAEC).

    Growth can occur at 7±46 ëC with the maximal growth rate at 35±37 ëC. The minimum aw for growth ranges from 0.94 and 0.97.

    Escherichia coli biofilms

    The optimum pH for growth is approximately 7.0, with a minimum and maximum pH for growth of 4.5 and 9.0. EHEC has been shown to grow poorly at temperatures of 44 °C.

    Escherichia coli has been isolated from a large number of foods and drinks, e.g. fermented meat sausage, dairy products, vegetables, meat, poultry and fish products, water and apple cider.

    These agents can cause diarrhoeal outbreaks.

    Unpasteurised milk is a common vehicle of E. coli O157:H7 transmission to humans .

    E. coli can also survive for extended periods of time in several acidic foods, e.g. cheese and yogurt.

    Acid-adapted E. coli O157:H7 has shown enhanced survival and prevalence in biofilms on stainless steel surfaces.

    In a hygiene survey performed in the food industry by Holah et al. (2002), microbial strains, e.g. E. coli and L. monocytogenes, were found either on surfaces or in productsor in both, and some of these strains were persistent.

    Faille et al. (2002, 2003) found out that E. coli cells were poorly adhered to surfaces.

     The cells were embedded in the organic matrix of the biofilm, which shows that the structure ofthe biofilm formed affects the way in which the surfaces should be cleaned.

    Oulahal-Lagsir et al. (2003) showed in their studies that proteolytic and glycolytic enzyme treatment together with ultrasonics enhance the removal of E. coli biofilm from stainless steel soiled with milk.

     These findings correspond with results obtained in the food industry.

     

    Campylobacter biofilms

    Listeria monocytogenes biofilms

    Staphylococcus aureus biofilms

    Bacillus cereus biofilms

    Clostridium perfringens biofilms

    Mycobacterium biofilms

     

    Biofilms and microbial contamination in food processing

    Prolonged or persistent contamination of some Listeria monocytogenes strains, which means that they have caused food plant contamination for long periods of up to several years, has been reported in several food industry areas, e.g. meat, poultry, fish, dairy and fresh sauces.

    Biofilms and microbial contamination in food processing

    Escherichia coli and Salmonella isolates are also known to be persistent in food and fish feed factories.

    Persistent L. monocytogenes plant contamination appears to be the result of the interaction of several different factors.

    Properties that influence survival, including enhanced adherence to food contact surfaces and adaptation to disinfectants, in addition to such predisposing factors in the processing line as complex processing machines and poor zoning may lead to persistent plant contamination.

    Biofilms and microbial contamination in food processing

    The eradication of persistent contamination of L. monocytogenes has been shown to be difficult but not impossible.

    Targeted and improved sanitation has led to successful eradication.

    In studies performed by LundeÂn (2004), persistent L. monocytogenes strains were observed to adhere to stainless steel surfaces in higher cell numbers than non-persistent strains after short contact times.

    Such enhanced adherence increases the likelihood of the survival of the persistent strains due to increased resistance against prevention methods and may have an effect on the initiation of persistent plant contamination.

    If the adherence period of strains was prolonged then the adherence level of non-persistent strains was close to the adherence level of persistent strains.

     The initial resistance of persistent and non-persistent L. monocytogenes strains to disinfectants varied, and the increase in resistance was similar for persistent and non-persistent strains.

    Prevention of biofilm formation and biofilm removal

    Harmful microbes may enter the manufacturing process and reach the end-product in several ways, e.g. through raw materials, air in the manufacturing area, chemicals employed, process surfaces or factory personnel.

    Once a biofilm is formed, either on food contact or environmental surfaces, it can be a source of contamination for foods passing through the same processing line.

    For example, Listeria monocytogenes is difficult to remove from the factory environment once it has become a part of the house microbiota.

     Therefore, it is especially important for the persistent growth of pathogenic and harmful microbes to be prevented in the food processing line using all available means.

     

    In the food industry, equipment design and the choice of surface materials are important in fighting microbial biofilm formation.

     Attention should also be paid to the quality of additives and raw materials as well as the processing water, steam and other additives, because using poor quality materials leads to the easy spoiling of the process.

    Prevention of biofilm formation and biofilm removal

    The aim of microbial control in a process line is two-fold: to reduce or limit the number of microbes in liquids and products and to reduce or limit their activity and to prevent and control the formation of biofilms on surfaces.

    At present the most efficient means for limiting the growth of microbes are good production hygiene, the rational running of the process line, and the well-designed use of cleaning and decontamination processes.

     The cleanliness of surfaces, the training of personnel and good manufacturing and design practices are important in combating biofilm problems in the food industry.

    Hygienic equipment design

    Several conferences and literature reviews have shown that the design of the equipment and process line in the food processing and packaging industry are important in preventing biofilm formation to improve the process and production hygiene.

    The most significant laws regarding the food industry are the EU directive 98/37/EU and machine standard EN 1672-2:1997.

     EN 1672 draws particular attention to dead spaces, corners, crevices, cracks, gaskets, seals, valves, fasteners and joints owing to their ability to harbour microorganisms that can subsequently endure adverse/harmful process conditions.

    Equipment that causes problems in food processing and packaging includes slicing and cutting equipment, filling and packing machines, conveyors, plate heat exchangers and tanks with piping.

    These types of equipment can cause contamination through spoilage microbes and pathogens as they are difficult to clean, e.g. the pathogen Listeria monocytogenes is often associated with harbourage in poorly designed equipment.

    Biofilm removal

    The elimination of biofilms is a very difficult and demanding task, because many factors affect the detachment, such as temperature, time, mechanical forces and chemical forces.

    Sanitation, i.e. cleaning and disinfection, is carried out in food processing plants in order to produce safe products with an acceptable shelf-life and quality.

     The key to the effective cleaning of a food plant is the understanding of the type and nature of the soil and of the microbial growth on the surfaces to be removed.

    The intelligent integration of decontamination programmes in the manufacturing are essential to achieve both successful cleaning and business profit.

     

    Lelieveld as early as 1985 wrote that there is a trend towards longer production runs with short intervals for sanitation, because the sanitation should be performed as cost- effectively and safely as possible.

    The mechanical and chemical power, temperature and contact time in the cleaning regime should be carefully chosen to achieve an adequate cleaning effect.

    An efficient cleaning procedure consists of a sequence of rinses and detergent and disinfectant applications in various combinations of temperature and concentration, finally letting the equipment and process lines dry in well-ventilated areas.

    The basic task of detergents is to reduce the interfacial tensions of soils so that the soil attached to surfaces, for example biofilm, becomes miscible(مخلوط شدنی) in water.

    The effect of the surfactants is increased by the mechanical effect of turbulent flow or water pressure, or by abrasives, for example salt crystals.

    Prolonged exposure of the surfaces to the detergent makes removal more efficient.

     Detergents to be used in the cleaning of open systems are formulated to be effective at temperatures in the range 35±50 C.

    In closed systems the detergents are formulated to be used at temperatures in the range of 55±80 C.

    Elimination of biofilms in open systems is performed as follows: gross soil should be removed by dry methods, e.g. brushing, scraping or vacuuming, and, if the process is wet, the visible soil can be rinsed off with low-pressure water.

    The effective elimination of biofilms from open systems is achieved by dismantling the equipment in the process line and cleaning is then carried out using either foam or gel.

     

    Foams are most effective in situations where contact with the soil for an extended contact time is necessary.

    The surfactants, which suspend the adhered particles and microbes from the surfaces in the water, are added to increase the cleaning effect, which is also increased by using water of sufficient volume at the correct temperature and pressure.

    The dismantled equipment and utensils should thereafter be stored on racks and tables, not on the floor.

    The cleaning is mostly carried out in combination with a final disinfection, because viable microbes on the surfaces are likely to harm production.

    In the cleaning regime for closed processes, pre-rinsing with cold water is carried out to remove loose soil.

     Cleaning-in-place (CIP) treatment is normally performed using hot cleaning solutions, but cold solutions can also be used in the processing of fat-free products.

    The warm alkaline cleaning solution, normally 1% sodium hydroxide, is heated to 75±80 ëC and the cleaning time is 15±20 min.

    The equipment is rinsed with cold water before the acid treatment is performed at approximately 60±70 ëC for 5 min.

    The effect of chlorine-based agents can be divided into three phases: loosening of the biofilm from the surface, breakage of the biofilm and the disinfective effect of the active chlorine.

     

    The cleaning solutions should not be re-used in processes in order to achieve total sterility because the reused cleaning solution can contaminate the equipment.

    Single-phase CIP is more commonly used nowadays because the processing industry wants to save time.

    In single-phase cleaning procedures the time it takes to carry out one cleaning process, normally the acid treatment, and a rinsing step can be saved .

    The photobacterial test can be used to test that rinsing has been performed properly.

     

     

    Bacterial biofilm, bacillus subtilis (scale bar is 10 μm), which grows in big colonies, all packed together in parallel chains.

    Biofilm on a stainless steel surface

    Scanning electron microscopy photomicrograph of a 6 old B. cereus biofilm formed on a stainless steel surface. x 6330 magnification; bar = 5 micron

    Biofilm Formation

    Scanning electron micrograph of P. fragi on stainless steel surface grown in tryptic soy broth for 24 h at 23°C. Thin, hair-like projectiles can be seen going from the cell wall to the stainless steel surface. These frimbrils physically attach the cell to the surface and are the first phase in the development of a biofilm. The other material visible on the slide is debris from the culture medium[10].

     

    Five stages of biofilm development: (1) Initial attachment, (2) Irreversible attachment, (3) Maturation I, (4) Maturation II, and (5) Dispersion. Each stage of development in the diagram is paired with a photomicrograph of a developing P. aeruginosa biofilm. All photomicrographs are shown to same scale.

    An image from an electron scanning microscope of a Staphylococcus aureus biofilm on a vascular prosthesis.

    Thermophilic bacteria in the outflow of Mickey Hot Springs, Oregon, approximately 20 mm thick


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