Biofilm Formation in Dairy: A Food Safety Concern—Biofilms in the milking machine, from laboratory scale to on-farm results.

Fresh milk contains a complex microbial community, including micro-organisms of nutritional and technological importance, such as lactic acid bacteria (LAB). The micro-organisms can be altered, which could influence milk composition, processing, spoilage, and consumer health. They can also form biofilms, which are structured bacterial communities embedded in a self-produced polymeric matrix, which provides specific advantages to the component species and makes them resistant to cleaning and disinfection methods. Biofilms can contribute to pitting and corrosion of equipment and pipes in the dairy industry. However, the main concern is that dairy biofilms, through both inter- and intra-species interactions, including LAB, pathogenic bacteria, and spore-forming bacteria, serve as sources of milk and product contamination, and can contain human pathogens such as Bacillus cereus, Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus. The sections below provide more detail.

The microbial environment in milk:

Milk is an ideal medium for hosting a diverse microbial community. The community includes bacteria and yeasts of technological interest for dairy products, such as LAB, Corynebacteriaceae, Geotrichum candidum, and Kluyveromyces lactis. Contamination during the milk production process and inadequate hygiene practices can lead to the presence of spoilage and pathogenic microorganisms, including psychrotrophic bacteria (Pseudomonas spp.), spore-forming and thermoduric bacteria (Clostridium spp. and Bacillus spp.), and pathogenic species (Listeria monocytogenes, Salmonella spp., Escherichia coli, Campylobacter spp. and mycotoxin-producing fungi). In addition, cows with bacterial infections, such as mastitis, can contribute to milk contamination by pathogenic species such as Staphylococcus spp., Pseudomonas spp. Streptococcus spp., and Klebsiella spp.

Spoilage bacteria in fresh milk are often psychrotrophic and are of particular concern to the dairy industry due to their ability to produce heat-stable enzymes. Additionally, the microbiota of raw milk and its associated biofilms are influenced by storage temperature. For example, Pseudomonas predominates at 4°C, whereas Lactobacillus is more prevalent at 25°C. Among these, Pseudomonas spp., particularly Pseudomonas fluorescens, is one of the most significant bacterial species responsible for dairy product spoilage. The impact of spoilage is further exacerbated when strains possess biofilm-forming ability, which is frequently the case.

The sporulated form of micro-organisms makes them resistant to heat, pressure, and cleaning and disinfection methods. Sporulated bacteria, such as Bacillus and Geobacillus, pose significant challenges to the dairy industry due to their ability to withstand heat treatment and form both mono- and multispecies biofilms. Because of their heat resistance, spores can survive milking machine cleaning, pasteurization, and milk sterilization. Later, they can germinate into their vegetative form and cause milk spoilage. They can also form biofilms, serving as a continuous source of contamination from the milking machine to the final product. During growth, many spore-forming bacteria produce proteolytic enzymes that cause spoilage.

The biofilm challenge:

Biofilms are complex microbial communities which produce an extracellular matrix, primarily composed of extracellular polymeric substances (EPS), along with proteins, carbohydrates, extracellular DNA, lipids, and signaling molecules. This extracellular matrix acts as a protective physical barrier, restricting the diffusion of antimicrobial agents and facilitating nutrient and waste exchange. The development of biofilm results from the successful attachment and subsequent growth of micro-organisms on a surface. The transition from the planktonic to biofilm mode is a dynamic, multistage process influenced by environmental and physiological factors, including nutrient availability, cellular stress, and intercellular communication mediated by quorum sensing (a process by which bacteria communicate and synchronize gene expression in response to variations in cell density and species composition). These environmental factors can influence microbial interactions, leading to changes in quorum sensing, thereby optimizing biofilm development. Additionally, microbial metabolic activity and gene expression are crucial, as they regulate the production of EPS that form the biofilm matrix and enhance microbial adhesion to surfaces.

Biofilms may consist of a single species or multiple species and can form either single layers or complex 3-dimensional structures. These structures may contain water channels, which facilitate nutrient distribution, metabolite exchange, and waste removal, contributing to biofilm adaptability. Interactions among species can be cooperative, where bacteria enhance biofilm formation by improving adhesion, promoting growth, and increasing resistance to biocides. Time wise, biofilms can form within several hours or over several weeks, depending on environmental conditions. Dynamic biofilm structures adapt to their environment, with their formation significantly influenced by hydrodynamics. Fluid shear, in particular, plays a critical role in shaping the physical characteristics of biofilms. Shear stress caused by fluid flow impacts both the structural integrity and density of biofilms, with higher shear conditions often leading to denser biofilms. Additionally, biofilms formed in zones exposed to shear forces exhibit substantially greater resistance to mechanical stress.

Biofilms in the milking machine environment:

The milking machine is a complex system composed of numerous pipelines and components with varying geometries and materials depending on their function and therefore provides an environment conducive to biofilm formation, with microbial colonization affecting both equipment functionality and milk quality. This is evidenced by the frequent detection of Pseudomonas spp., Lactococcus lactis, Klebsiella, Staphylococcus spp., and Enterococcus faecalis. The presence of Listeria monocytogenes, the causative agent of listeriosis, has also been documented on stainless steel, rubber, and plastic components of milking machines. This pathogen has been isolated from critical areas such as milk meters, teat cup liners, and bulk tank outlets. Pseudomonas spp. are particularly adept at forming robust biofilms with high metabolic activity, even at low temperatures, making them resistant to disinfection and persistent within milking machine pipes. Milking equipment biofilms have also been identified as a source of Staph. aureus contamination during milking, further compromising the microbiological quality of the milk.

Milk composition plays a critical role, as residues form conditioning films composed of proteins, lipids, minerals, and carbohydrates that promote bacterial adhesion and biofilm development. These films are often incompletely removed during cleaning, resulting in persistent contamination. In addition, the circulation of milk at 38°C in milking machines raises the system’s temperature, promoting the growth of mesophilic bacteria such as LAB and pathogens such as Escherichia coli. Moist areas that remain after milk passage or cleaning further encourage bacterial and mold growth. Additionally, the continuous flow of milk provides nutrients to bacteria, facilitating their distribution throughout the system.

Different components of milking machines, such as teat cup liners, pipelines, and bulk milk storage chambers, are prone to biofilm formation due to undrained liquid. Stagnant liquid (water or milk) in these areas create conditions that promote microbial growth. Poor structural design of milking machines, particularly inadequate hygienic aspects, creates areas susceptible to bacterial adhesion and biofilm persistence and complicates cleaning efforts. Mechanical action and pressure within milking machines can cause abrasions and surface imperfections, creating prime sites for bacterial adhesion. These micro-environments protect bacteria from cleaning agents and physical removal. Regular maintenance is crucial to limiting biofilm development. Replacing worn parts, such as teat cup liners and milk hoses, helps prevent fouling and microbial adhesion.

Efficacy of cleaning and preventative actions:

Experimental results demonstrated that even stringent cleaning and disinfection procedures cannot eliminate all microorganisms. This is due to the complex structural design of milking machines, which hinders thorough cleaning, along with intrinsic biofilm factors such as multiple resistance mechanisms to disinfectants.

Numerous studies have examined the effects of various chemical compounds on biofilm elimination. One study compared the effect of cationic surfactants, specifically quaternary ammonium compounds (QAC), on planktonic cells and mature biofilms of Pseudomonas aeruginosa and Staph. aureus. The results showed that QAC exhibit significant bactericidal activity against planktonic cells but are much less effective to biofilms. Strong oxidizing agents, such as chlorine and peracetic acid, are the most commonly used disinfectants for biofilm control. However, chlorination can lead to the formation of by-products, such as trihalomethanes and haloacetic acids, which pose risks to consumer health. Additionally, biofilms exhibit significant resistance to common chemical compounds such as QAC and chlorine, primarily due to the protective EPS matrix and phenotypic adaptations of bacterial cells. One promising alternative is chlorine dioxide; however, biofilm thickness remains a limiting factor in its effectiveness. Another promising procedure is by using electrolyzed water, provided that the sequential order of the procedure is followed. Nevertheless, control and elimination of biofilms through treatment are mostly only partly effective.

Biofilm control on farm:

Effective biofilm management requires a comprehensive approach that integrates machine design, optimized cleaning and disinfection protocols, and proper farm management practices. The interaction between machine components, milk residues, and microbial communities complicates biofilm control, reinforcing the need for precise and effective cleaning procedures. Cleaning the milking machine remains fundamental in biofilm management to prevent the establishment of spoilage and pathogenic flora. To achieve this, hygienic equipment design and adherence to cleaning recommendations from dairy organizations and product suppliers are essential.