BIOFILMS 2: In Food, Food Processing And Food Serving Environments – Control

Photographs taken from the washrooms of three different supermarkets

How are biofilms controlled in the food sector?

Application of On-line technologies 

On-line tools for monitoring the development, adhesion, growth and removal of biofilms from surfaces in big industrial environment is helping to reduce the cost of cleaning operations and minimizes production down times to enable regular maintenance operations so that cleaning operations can be planned, targeted and better managed.

In addition to the more recent technological approaches for monitoring and detecting biofilms and ultimately leading to their control, several new and classical specific, targeted methods could be used in individually or in a multimodal design and applications. Control operations should always be preceded by a proper identification of the microbes in the complex community.  The control methods include the application of:

i. Chemical control

A range of chemical families are used in disinfection and sanitation in the food and related industry.  The challenge in the use of one chemical group is that a biofilm may compose of a complex group of pathogens, even of the same species, which may response to the chemical in different ways. While some may be killed, others may only be weakened or inhibited, only to emerge again when the chemical stress is reduced or removed.

It is important to elucidate the complex microbial community of biofilms as far as possible using effective identification methods before applying chemical controls. Identification helps to make a target use of chemicals and obtain quick and cost-effective results. The main draw-back of chemical application is the development of different levels of resistance among the microbes in the biofilm community even when a specific species is dominant in that ecosystem. The biofilms provide microbes with the necessary apparatus for resistance against chemical through various mechanisms.

Chlorine based disinfectants

Chlorine based biocides or disinfectants are widely used in the food industry. Resistance to chlorine treatments has been known in some microbes. It is also considered that multiple applications of specific chemicals ultimately enhance the microbial selection mechanism and process to leaving some microbes behind to continue the pathogenic and /or spoilage fight.

 In Staphylococcus enterica, chlorine resistance was correlated to the cellulose production phenotype, which in turn depended on the environmental stress conditions found in the food processing plants.

Among the chlorine product family, aqueous chlorine dioxide (ClO₂) is the most widely used disinfectant in the food industry. It has been shown to be more effective against Bacillus cereus endospores present in biofilms on food steel surfaces. In the case of E. coli O157:H7 biofilms, aqueous ClO2 was more effective than sodium hypochlorite (NaOCl) in particular if the application is designed so that the treated factory surface is allowed to dry after application. 

Another negative side, chlorine-based treatments are usually applied at very low ppm concentrations, they often still leave chlorine odour on treated surfaces. Additional care may be required not to contaminate food materials particular dairy or fatty food. The possibility of residues on surfaces is also important.

Biofilms formed in raw milk on stainless steel and polypropylene surfaces by Staphylococcus aureus and S. enterica were effectively eradicated by chemical treatment with sodium hypochloride (NaOCl). Similar disinfection with NaOCl of Cronobacter sakazakii biofilms in the same environment was not effective, indicating the importance of environment factors such as surfaces in the application of a specific treatment regime and the inherent variability of results.

Quaternary ammonium compounds

Quaternary ammonium compounds, often referred to as ‘quats’ are commonly used in the food industry both for general disinfection and eradication of biofilms. They are often made of a combination of different types of quaternary ammonium moieties, which is claimed to enhance the efficacy of this group of products.  Quats are soluble in water and commonly presented as water-based, non-alcohol disinfectants.

The positive charge ions in the solution disrupt the bacterial cells to exact a killing effect. However, genes responsible for antimicrobial resistance (AMR) was found in certain places and microbial species and strains due to the use of quats.  A multimodal application, combining various methods has been shown to be more effective and may well reduce the potential for the development of AMR. It was also found that a combination of NaOCl, H2O2, iodophor and benzalkonium chloride (quat) with steam heating eliminated ecosystems of biofilms formed by E. coli O157:H7, S. enterica and L. monocytogenes while achieving decreases in both the concentrations of disinfectants use and the duration of the application.

Hydrogen peroxide (H₂O₂)

H₂O₂ is a potent oxidising disinfectant commonly used to eradicate pathogens in the food industry. It is non-toxic to users and does not leave residue on surfaces or on food.  H₂O₂ generates free radicals when in contact with the microbial biofilm structures.  The free radicals destroy the biofilms structures at very low to moderate concentrations (0.08% –5%) without leaving toxic side effects.

In combination with acetic acid, H₂O₂ generates peracetic acid in situ. The resulting solution is a strong oxidant with low acidic pH (pH2.8). This is used at low concentrations with high efficacy against biofilm community of Listeria monocytogenes, known to survive with or without oxygen, and S. aureus population in water pipes.

ii. Biosurfactants

Biosurfactants, like lichenysin are added to detergent formulations used in industry to control biofilms. Biosurfactants, biological surfactants or microbial surfactants non-synthetic chemicals generally of microbial origin that have similar characteristics as the synthetic surfactants with added value of being more readily biodegradable, less or even non-toxic to human and environment and in some cases more effective. The overall characteristics and performance depend on the class and source of the biosurfactant.

iii. Bacteriophages

The use of phages as biocides or sanitisers is regarded as an environmentally sustainable approach and a good alternative to antibiotic or chemical biocide applications as phages are innocuous to humans, animal and the environment. Commercial phages such as Listex P100 are successfully used to eliminate biofilms in processed meat factory work surfaces.

Bacteriophage application already authorised in the United States by the Department of Agriculture with the status of GRAS (Generally Recognized as Safe) biological agent. S. enterica or E. coli are among other bacteriophages targeted for commercial applications at industrial scales.

There may be limitations to the use of phases in biofilm control. They possess limited ability to access and target bacterial cell inside the specific biofilm community due to the collective protective mechanisms provided by the biofilm ecosystem.

The complex biofilm structure and the extracellular materials act as physical obstacle to the ability of phage to diffusion or penetrate into the bacterial cells. To counter this obstacle, some phages have developed specific enzymes, exopolysaccharide depolymerases to help take the battle to biofilms and win. These enzymes amply facilitate the diffusion by enhancing their capability to invade and disperse within the biofilm being treated.  

iv. Bacteriocins

Bacteriocins offer a type of solution to the control of biofilms. They are antimicrobial agents of protein or peptide-based toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains. Some of the identified advantages of using bacteriocins in the food industry include:

• prevention or preservation to stop the formation of biofilms in the first place
• extend the expiry or shelf life of food materials in storage including during refrigeration by stopping changes in the food
• reduce the use of chemical preservations both in terms of number and quantity applied

The World Health Organisation (WHO) approved a peptide, Lactococcus lactis, as an antimicrobial agent since 1969.  The USA, Food and Drug Administration (FDA) approval followed in 1988 on grounds of being safe for consumption in animals and humans. Nisin is still the only FDA approved bacteriocin in the food industry. It is used as spray on surfaces used for food processing or manufacturing. It prevents adhesion and biofilm formation by L. monocytogenes.  

Other bacteriocins, which are either in use or in ongoing research include: bacteriocins produced particularly by bacteria regarded as GRAS (Generally Recognized as Safe) such as lactic acid bacteria, novel bacteriocins produced by Enterococcus spp andactive against L. monocytogenes, lactocins active against Brochothrix thermosphacta and produced by Lactococcus spp. and garvicin produced by Lactococcus garvieae, which is active against pathogenic strains of the same bacterium.

v. Quorum Sensing (QS) inhibition

This is based a complex biological systemic disruption mechanism. Bacterial biofilm formation and antimicrobial resistance development require different signalling pathways.  These include the exchange of small organic molecules or proteins and the transmission of electrical signals.  These activities will trigger the binding of inhibitors to the QS receptors, disrupting the enzymatic, biosynthetic and genomic systems.

Of the several identified signalling pathways, QS is one of the best characterised. It is a widely distributed intercellular signalling mechanism, used by bacteria to regulate gene expression in response to high environmental concentrations of small diffusible signalling molecules such as acyl homoserine lactones, peptides and the autoinducer-2. These QS regulated mechanisms include genes involved in biofilm formation, which can be inhibited.

vi. Essential oils

Plant-based essential oils have antibiofilm properties and are primarily a species-specific complex mixture of chemicals, which have been group based on type:  monoterpenoids like borneol, camphor, carvacrol, eucalyptol, limonene, pinene, thujone; sesquiterpenoids like caryophyllene, humulene and flavonoids such as cinnamaldehyde and other phenolic acids. Some essential oils are commonly available and are widely regarded as from ‘natural sources’ with good for the environmental profile. It should be noted that some plant based antibiofilm agents are also classified as toxic or irritant to humans, animal and the environment.  

vii. Enzyme disruption

Some detergents are being formulated with enzymatic components capable of disrupting the microbial cell structure. The enzymes used are mainly proteases or peptidases, glysosidases or DNase. They catalyse hydrolytic biological and genomic processes in the microbial cells and their extracellular matrices. The enzymes act on the bacterial cells and extracellular matrix of the biofilm.  

viii. Steel coating

Steel surface maybe modified by coating with silver, copper or zinc nanoparticles, or by using the novel antibiofilm polymers with lysozyme or bacteriocins. Coated surface prevents the formation of biofilms. Antimicrobial surface coating is a growing area in the food and beverage industry and more evidence of its efficacy is demanded by consumers. 

Other type of surface-based controls are repelling surfaces with monolayers, hydrogels or modified topography, inhibiting bacteria or microbes from binding onto the surfaces.

ix. High hydrostatic pressure (HHP)

HHP treatment is better used as a key part of a multimodal approach by integrating other methods such as essential oil application, chemical application or Thermal treatment between 50^oC – 100^oC. HHP at 300–900MPa, for example, can destroy vegetative bacterial cells but leave the endospores unharmed. The endospores will go on to germinate. Thermal treatment ensures the endospores are destroyed.

By carrying out the operation in two steps, a better result is obtained. A low pressure (300 – 400 MPa) pre-treatment phase, which allows the endospores to germinate before application of the HHP to eradicate the vegetative bacterial cells.

An significant advantage of using HHP is that it has no effect on the organoleptic and nutritional qualities of the food such as taste, vitamin content, colour, etc.

x. Non-thermal plasma

Non-thermal plasma treatment consists of using partially ionised gas at low temperature to treat biofilms on surfaces. The ionised has antimicrobial properties. The gas is produced at atmospheric pressure by mixing UV light with oxygen, nitrogen, ozone, and water and helium, under an electrical discharge. The resulting ionised gas is able to relatively rapidly destroy bacteria in complex biofilms ecosystems made up of Gram-negative bacteria including Pseudomonas spp. and S. enterica or Gram-positive such as Bacillus spp. species in as little as 10 min.

This is a very expensive technology to deploy. Hence, though it has proven to be highly effective, it limited to certain applications only, mainly laboratory. A key advantage is that the ionised gas can be used effectively in difficult to reach surfaces, corners, compartments and crevices.

xi. Photocatalysis

Nanotechnological applications employing photocatalytic properties of specific types of metals have been developed or are in development. Photocatalytic properties of nanoparticles enable the absorption of a specific wavelength, which generates or accelerates a chemical reaction, including destruction of microbial cells, generally due to the generation reactive oxygen species (ROS) inactivating bacterial cells on the surfaces.

Biofilm contamination in the food industry can constitute significant challenge to any affected company both economically, socially and for the wider health of the affected human population. They are difficult and expensive to treat and eradication could be a prolonged strategic battle for any affected organisation. A business may suffer reputational damage due to public announcements of the recall of biofilm contaminated products. This the business may take time to recover

The biofilms can be recurrent and corrode machineries necessitating a considerable downtime or even total closure to allow for eradication. It may be necessary to replace costly parts after the treatment. Affected products should be recalled for either treatment where possible or disposal. Some bacterial species, such as Pseudomonas spp. and Bacillus spp., secrete many different proteolytic and lipolytic enzymes that convey unpleasant odours or rancidity or bitter taste. In this latter case, affected products must be withdrawn from the market.

Some type of products may be recovered by administering appropriate treatments which may include a multimodal approach ensuring both microbial spores and vegetative stages are eradicated.

The health impact to the community may also be significant. Biofilm formation in food factories is a critical public health issue for the communities where the pathogenic microbes can interfere in the wellbeing of the community. Biofilm ecosystem that is principally of B. cereus and S. aureus are known to cause food intoxication.  Strains of E. coli (O157:H7) and S. enterica may cause gastroenteritis and other systemic diseases (L. monocytogenes).

The health impact of biofilm formation and the role in the development of antimicrobial resistance will be among our next topics of discuss.

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