To prevent the spread and development of altering or pathogenic micro-organisms. The food industry is required to comply with high hygiene standards. One of the most feared events is biofilm formation. Research is focussed on the development of technologies to prevent it or mitigate its consequences.
A clean working environment is critical to preventing prevent food-borne diseases. Processing residues contain the nutrients that microorganisms use to grow. The most classic among the sanitization procedures involves a first step with alkaline product to attack organic compounds such as fat and protein residues, a second step with acid to remove inorganic residues, and, finally, the use of a sanitizer. By doing so, and respecting time and temperatures, good results are obtained in terms of cleaning, descaling, sanitization and prevention of biofilm formation, a layer of organic matter, i.e. organic polymer agglomerates (proteins, polysaccharides, glycoproteins and nucleic acids) and micro-organisms adhering to surfaces that come into contact with food.
When the biofilm is in its initial state (a few microns thick), it is easily removed with a sanitizer. Far more difficult is its treatment when it is well rooted and of considerable thickness. In this case, the thickness hinders the diffusion of the sanitizer, which loses its effectiveness because its active substance is neutralized by the organic matrix surrounding the micro-organisms. Cleaning and sanitization make hygienically safe the environments and processing plants, hence also the food produced there.
There are many variables to consider when setting up these tasks. In the food industry, plants are large and complex, and production is usually long and significant in quantity; many of the raw materials used have not undergone previous processing and may therefore have high microbial loads or be contaminated by micro-organisms that are potentially pathogenic to the consumer. The parts of the plant that need to be monitored with particular attention are those that are most difficult to reach in the usual cleaning and sanitization procedures.
The formation of biofilm
Biofilm is formed in several stages. Some microbial cells, found in free form in raw materials, adhere with weak bonds to the surface of the processing plant. Most come off and are dragged away from the product flow, the rest sticks to the surface with weak bonds. Above this first layer, new microbial cells are deposited, in addition to other organic substances derived partly from the foodstuff and partly from the cells that split themselves and integrate with the new micro-organisms, also from other species. This forms a small ecosystem protected by a matrix of polymeric compounds (polysaccharides, exogenous proteins or DNA). Its structure facilitates communication between cells, in particular the diffusion of nutrients and signal molecules, and conveys waste substances and any microbial exotoxins to the outside.
The Plant Configuration
The possibility of biofilm formation and the effectiveness of sanitization depend on a number of factors, such as plant layout and material, more or less rough food-contact surfaces, and the absence of dead spots. Stainless steel is the most widely used material in food processing industry because it is chemically inert, easy to clean, corrosion resistant at different processing temperatures. However, it is subject to wear, especially when the processed product contains hard or abrasive particles that flow or are forced along the pipe walls. Wear leads to the formation of surface irregularities that facilitate the adhesion of biofilm.
The nature of the processed food may promote or hinder its formation, and residues of foods rich in fat, proteins and carbohydrates, facilitate microbial development. Equally important are the morphological characteristics of microbial cells, the components of their cell membranes, their appendages (pili, flagella, fimbriae), the biopolymers produced by bacterial secretion. In addition, different biofilm-forming abilities between species of strains of different genotypes and serotypes have been identified.
Lipases and proteases released by micro-organisms in the biofilm may promote the alteration of the organoleptic characteristics of the products. Biofilm can be an indirect source of food-borne diseases when its constituent micro-organisms produce toxins that pass easily into the food. This complexity makes sanitization difficult, and facilitates the development of microbial resistance to sanitizers, rarely attributable to genetic mutations of micro-organisms, but due to multicellular strategies and/or the ability of individual cells – residing in the biofilm – to differentiate into a phenotypic state tolerant to the action of disinfectants. Other mechanisms responsible for resistance may be the delayed penetration of the antimicrobial agent into the biofilm matrix, a slower growth rate of the constituent micro-organisms, or physiological changes of those micro-organisms in response to environmental conditions.
Sanitization
Sanitizing principles are divided into two broad categories: Highly rinseable products ( e.g. hydrogen peroxide) and products with residual effect (e.g. quaternary ammonium salts). Non-foaming, completely rinseable detergents and disinfectants are to be preferred, suitable for water with different hardness, effective at low concentration. The food industry tends to use specially formulated products. According to the environments and equipment to be treated, classical sanitization involves the use of hypochlorites, quaternary ammonium compounds, amphoteric compounds, peroxides (peracetic acid and hydrogen peroxide), aldehydes (formaldehyde, glutaraldehyde, paraformaldehyde), alkyl amines, chlorine dioxide, alcohols, phenolic compounds.
The lack of product rotation facilitates the development of bacterial resistances. Sodium hypochlorite (NaOCI) is still used. Its efficacy derives from the production of hypochlorous acid (HOCl) and hypochlorite ion (Ocl), strong oxidising agents that kill cells by crossing the cell membrane and oxidising the cell membrane and oxidizing the sulfhydryl groups of certain enzymes. Under physiological pH conditions, the sanitizer reacts with proteins, amino acids, lipids, peptides and DNA. On the other hand, its effectiveness is mitigated by the presence of organic matter that compose food residues. Free chlorine reacts with these molecules, transforms into inorganic chloramines that generate trihalomethanes. Chlorine dioxide in aqueous solution is sometimes more effective than sodium hypochlorite, especially when the plant is dried immediately after sanitization.
Hydrogen peroxide is oxidizing. In contact with biofilm it generates free radicals that destroy it without toxic side effects. Peracetic acid is a powerful oxidizer and is often used against biofilms, because it does not interact with organic matter residues and acts at low temperatures. Quaternary ammonium salts (benzalkonium chloride, cetrimide, didecyldimethylammonium chloride, cetylpyridinium chloride) are cationic surfactants. They reduce the surface tension between biofilm and metal and form micellae that disperse in a liquid. They interact with the cytoplasmic membrane of bacteria and yeasts and are also effective against viruses containing lipids. Ozone is a toxic gas with a powerful oxidizing activity; it destroys different types of microorganisms, biofilms, viruses and protozoa, by intervening on cell membranes.
Enzymes
Enzymes are chosen because of their low environmental impact; in fact, they are biodegradable and not toxic. As mentioned above, the biofilm matrix consists of organic macromolecules (mainly proteins and polysaccharides), which can be attacked by protease (e.g. serine protease, proteinase K, pepsin, and trypsin) and glycosidase (e.g. amylase, dextranase, and pectinase), which facilitate the removal of the biofilm. Pectin methylesterase is an enzyme used for preventive purposes as pretreatment in bioreactors, pipes and other plant parts. Cellulase, lyase, glycosidase (such as dispersin B) and DNase are often components of industrial detergents and can be used both as routine detergents and for more drastic biofilm removal.
Alpha-amylase is effective in the degradation of biofilms containing S.aureus, whereas proteases are less specific and are therefore useful in the treatment of organic-based biofilms. When the matrix is partially degraded, the biofilm is removed mechanically and its micro-organisms become more sensitive to the action of disinfectants. Subtilisins counteract biofilm formation from P. Aeruginosa or L. monocytogenes. The widespread implementation of these enzymatic alternatives is slowed down by the high treatment costs, mainly due to patent protections.
Pre-treatments of steel surfaces
Among the alternative approaches, nanotechnologies are the most promising. Nanomaterials have two possible functions in sanitization: They are bactericides or they improve the efficiency of other sanitization treatments. Many nanoparticles are effective by direct contact with microbial cell walls. Their action is therefore not reduced by the protection mechanisms described above, and specific microbial resistances are unlikely to be established. Among the most studied nanoparticles there are carbon-based nanoparticles (fullerenes and carbon nanotubes).
They act as dendrimeres with cavities hosting other organic nanocompounds or metals (silver, gold, copper oxides, zinc iron). They are used to reduce bacterial adhesiveness by intervening in factors that promote it, such as tendency to repel water, presence of electric charges, functional groups. Antibacterial surfaces are usually composed of inert materials where the repelling property is provided by hydrogel coatings, coatings with intrinsically antibacterial materials and antibiofilms. Thus, functionalized surfaces are obtained, for example by using lysozyme-functionalized polymers.
Biosurfactants are natural compounds, usually of microbial origin, that modify the hydrophobic characteristics of the bacterial surface. One of the most studied molecules is lichenisin, a cyclic nonribosomal lipopeptide produced by B. licheniformis. Fengycin, iturin and surfactin are lipopeptides similar to lichenisine but are produced by B. amyloliquefaciens and B.subtilis in the case of surfactin. These compounds act on the surface of the corresponding target micro-organism, altering its binding capacity surface. These molecules insert themselves into microbial cell membranes, altering their permeability, disrupting it and causing cell death.
