Part I RisksRisks
4.2 Factors influencing the effectiveness of cleaning and disinfectiondisinfection
irradiation) or chemical methods. In general, physical methods are preferred as they are very reliable and leave no residues behind. However, physical methods cannot always be applied owing to restrictions such as temperature, safety of personnel and design of the equipment. In those cases chemical disinfectants are used (Krop, 1990).
In this chapter the mode of action of the main disinfectants, the behaviour/
response of pathogenic bacteria towards chemical disinfectants and some future developments are discussed. The effect of physical methods is not discussed.
4.2 Factors influencing the effectiveness of cleaning and disinfection
A wide range of disinfectants is available that can be divided in the following groups (see also Table 4.1):
· halogen-releasing agents (HRA);
· quaternary ammonium compounds (QAC);
· peroxygens;
· alcohols;
· aldehydes;
· (bis)phenols;
· biguanides.
Each of the different groups has its own applications within the food industry and its own restrictions in use. It is important to realise what the proposed effect of a disinfectant is on a target-organism and what possible protection mecha-nisms are present within the organism. In the following sections, the different compounds, their mode of action and their applications are discussed.
Table 4.1 Disinfectants and their mode of action
Biocide Mode of action Target
Halogen-releasing agents Halogenation/oxidation Nucleic acids, proteins Quaternary ammonium Electrostatic (ionic) Cell surface, enzymes,
compounds (QACs) interaction proteins
Peroxygens Oxidation Lipids, proteins, DNA
Alcohols (ethanol) Protein denaturation Plasma membrane
Aldehydes Alkylation reaction Cell wall
(bis)Phenols Penetration/partition Phospholipid bilayer phospholipids bilayer
Biguanides Electrostatic (ionic) Cytoplasmic membrane interaction (bacteria)/plasma
membrane (yeasts) 70 Handbook of hygiene control in the food industry
4.2.1 Halogen-releasing agents (HRA)
Chlorine-based compounds are the most frequently applied HRAs. They include sodium hypochlorite, chlorine dioxide, and the N-chloro compounds such as sodium dichloroisocyanurate (NaDCC). A very cheap and frequently applied formulation is an aqueous solution of sodium hypochlorite producing hypo-chlorous acid (HClO) (Krop, 1990; McDonnell and Russell, 1999) (Table 4.3 on page 77). HClO is the active component and results in the inactivation of all types of microorganisms such as bacteria, viruses and spores (Sofos and Busta, 1999). Another applied form of chlorine is chlorine dioxide (ClO2). It is synthesised by the reaction of chlorine and sodium hypochlorite. However, chlorine dioxide is much more unstable than a standard hypochlorous solution and decomposes chlorine into gas at temperatures higher than 30 ëC when exposed to light (Beuchat, 1998). This can lead to dangerous situations as high concentrations of chlorine gas are explosive (Speek, 2002; Codex, 2003).
However, when the solution is kept cool and protected from light the disinfectant can be kept stable at concentrations up to 10 g lÿ1(Erco Worldwide, 2004).
Mode of action of hypochlorous acid
Although the exact mode of action is not known, the main disinfecting effect of chlorine is caused by oxidative activity. In particular, nucleic acids and proteins are destroyed, resulting in irreversible changes and disruption of DNA-protein synthesis (Krop, 1990). The mechanism of killing of spores differs owing to their thick proteinaceous coat. Therefore higher concentrations are needed than for inactivation of vegetative cells. Young and Setlow (2003) concluded that hypochlorite affects spore germination possibly because of the severe damage to the spore's inner membrane. For spore suspensions, Young and Setlow (2003) showed that a concentration of 50 mg lÿ1during 10 min at room temperature is sufficient to achieve 4 decimal reductions of Bacillus subtilis spores. A concentration of 50 mg lÿ1 resulted in 1 decimal reduction of B. cereus spores after 1.5 min (Wang et al., 1973). These results show that the minimal inhibitory concentration can vary per species.
Mode of action of chlorine dioxide
Chlorine dioxide (ClO2), if applied properly, appears to be 2.5 times more oxidative than sodium hypochlorite (Speek, 2002; Rodgers et al., 2004), and is effective against bacteria, viruses and spores (Hoxey and Thomas, 1999). The action of chlorine dioxide involves disruption of the cell's protein synthesis and membrane permeability control mechanism. It produces no harmful by-products as trihalomethans, nor does it react with ammonia. After treatment with chlorine dioxide, spores of Bacillus subtilis can undergo the initial steps in spore germination but the process stops because of membrane damage (Young and Setlow, 2003). An aqueous chlorine dioxide treatment of alfalfa seeds inoculated with E. coli for 10 min at a concentration of 25 mg lÿ1resulted in approximately 1 log reduction of the microorganism (Singh et al., 2003). Compared with Pathogen resistance to sanitisers 71
standard chlorine solutions (sodium hypochlorite) a concentration of 3 mg lÿ1 chlorine dioxide has the same inactivating effect on E. coli O157:H7 and L.
monocytogenes as 200 mg lÿ1 of chlorine when applied for decontamination of fruit surfaces (Rodgers et al., 2004).
Iodine
Iodine is widely used for sanitising food processing equipment and surfaces.
Iodine is less reactive than chlorine and less affected by the presence of organic matter but also has disadvantages such as staining human skin, plastic parts of equipment, and also has a relatively high price as compared with chlorine (Krop, 1990; Hugo and Russell, 1999). Solutions of 15% active chlorine are commercially available for ¨0.20±0.30 per kg whereas a 6% solution of iodine in 70% ethanol costs approximately ¨400 per kg (Boom Chemicals). Iodine is applied in three possible formulations: ethanol-iodine, aqueous iodine solutions and iodophores. The iodophores are most frequently applied and have high solubility in water, produce no vapour (below 50 ëC), are less corrosive to stainless steel than chlorine-containing solutions, and are generally effective against Gram-negative and Gram-positive vegetative cells, yeasts, moulds and viruses (Bernstein, 1990; Beuchat, 1998). Bacterial spores (B. cereus, B. subtilis and C. botulinum type) are more resistant to iodophors (D-values are 10±100 times higher) and higher concentrations are necessary to achieve inactivation.
Mode of action of iodine
Similar to chlorine, the exact mode of action of iodine is not known. Iodine penetrates into microorganisms and attacks specific groups of proteins, nucleotides and fatty acids in a way comparable to chlorine (McDonnell and Russell, 1999). The effective concentration of iodine is approximately 100 mg lÿ1which is as effective as 300 mg lÿ1of chlorine (Krop, 1990).
4.2.2 Quaternary ammonium compounds (QACs)
QACs can be divided in two main subgroups (Mohr and Duggal, 1997; Reuter, 1998):
· tri-alkylbenzyl-ammonium compounds (e.g. benzalkonium chloride);
· tetra-alkyl-ammonium compounds (e.g. didecyldimethyl-ammonium chloride).
QACs combine antimicrobial properties with surface-active properties and are therefore useful for hard surface cleaning and deodorisation (McDonnell and Russell, 1999). Compared with chlorine they are more expensive but have the advantage of having residual action. QACs remain active on surfaces for approximately 1 day (e.g. fish industry) and therefore discourage further bacterial growth (Tatterson and Windsor, 2001). This adherence to the surface also has disadvantages. Removing the disinfectant from the surface by flushing with water becomes difficult, resulting in possible residues in the product (Kraemer, 1998).
72 Handbook of hygiene control in the food industry
In general QACs are effective against vegetative bacteria but have greatest effectiveness against Gram-positive bacteria. Yeast and moulds can be inactivated to some extent but higher concentrations are necessary (Krop, 1990; Bernstein, 1990) (see Table 4.2). QACs are most effective in the range of pH 6 and 10 (Beuchat, 1998), which limits their applicability in acid environments.
Mode of action
The principal actions of QACs are lowering of surface tension, inactivation of enzymes and denaturation of cell proteins. As a result of adsorption of QACs onto the microorganism's surface, the cell's permeability is changed dramatic-ally. This results in leakage of intracellular low-molecular compounds, degrada-tion of proteins and nucleic acids, and cell wall lysis by autolytic enzymes (McDonnell and Russell, 1999). The concentration applied depends on the type of microorganisms present in the product, the processing system and the environment. Concentrations typically used are in the range between 150 and 250 mg lÿ1of active Quaternary Ammonium (QA) (Bernstein, 1990; Beuchat, 1998). Allerberger and Dierich (1988) showed a bactericidal effect on E. coli at a concentration of 100 mg lÿ1. Low concentrations (0.0005% w/v = 5 mg lÿ1) of benzalkonium chloride are sporostatic, inhibiting outgrowth but not germination. QACs are not sporicidal (Russell, 1990).
4.2.3 Peroxygens
Hydrogen peroxide and peracetic acid are the main representatives of the group of peroxygens. Hydrogen peroxide is widely applied within the food industry and is commercially available in concentrations varying between 3% and 90%
w/v, with 35% routinely used in the food industry (McDonnell et al., 2002). It is Table 4.2 Efficacy of quaternary ammonium compounds on different infectious agents
Infectious agent Efficacy Comments Source
Bacteria Russell (1995)
Gram-positive
Gram-negative MIC higher than Gram +
Spores ÿ Sporostatic Russell (1990)
Viruses Quinn and Markey
Lipid (1999)
Small non-lipid ÿ
Non-lipid =ÿ
Mycobacteria ÿ Russell (1996)
Yeast/moulds Moulds more resistant Russell (1999c)
, effective, ÿ ineffective, =ÿ, limited efficacy
Pathogen resistance to sanitisers 73
applied for sterilising packaging material prior to filling (Mohr and Duggal, 1997), sterilising contact lenses and sterilising the surface of fruit and vegetables. Hydrogen peroxide is both bactericidal and sporicidal (Hugo and Russell, 1999), in general a concentration of 6% is bactericidal. Peroxygens are generally more active against Gram-positive bacteria than Gram-negative bacteria (Russell, 1990; McDonnell and Russell, 1999). To achieve a sporicidal effect, concentrations between 10 and 30% are necessary. Peracetic acid is commercially available in 15% solutions as a mixture of water, hydrogen peroxide and acetic acid and acts faster than hydrogen peroxide. It has a broad spectrum of efficacy against viruses, bacteria, yeast and spores (Bernstein, 1990). Compared with hydrogen peroxide, the activity of peracetic acid is hardly influenced by organic matter (Russell, 1990; McDonnell and Russell, 1999).
Disadvantages are that peroxygens corrode on tools and equipment and are aggressive to, e.g., human tissues (Reuter, 1998). However the development and use of anticorrosives has reduced this concern (Marquis et al., 1995).
Mode of action
The mode of action of peroxygens is based on free-radical oxidation (e.g.
hydroxyl radicals) of essential cell components such as lipids, proteins and DNA (McDonnell and Russell, 1999). Peracetic acid not only attacks the proteins in the cell wall but also migrates into the cell and disrupts inner cell components as well (Donhauser et al., 1991).
4.2.4 Alcohols
The most widely used alcohols for disinfection are: ethyl-alcohol (ethanol, alcohol), isopropyl alcohol (isopropanol, propane-2-ol) and n-propanol, the latter especially in Europe (Mohr and Duggal, 1997; McDonnell and Russell, 1999). In food production areas, alcohols are particularly used for the decontamination of hard surfaces of equipment (e.g. filling machines). The most effective concentration is between 60 and 70% v/v (Mohr and Duggal, 1997). The concentrations to achieve reduction of growth or complete inactivation are higher than for chorine solutions or organic acids. Alcohols are quick reacting, have a broad spectrum of antimicrobial activity and inhibit growth of vegetative bacteria, viruses and fungi. Spores are rather resistant against the effects of alcohol; however, a combination of 70% v/v concentration with temperatures up to 65 ëC results in inactivation of spores, for example Bacillus subtilis spores (Setlow et al., 2002). Compared with other disinfectants the concentrations applied are much higher (50±100 times) and in fact alcohols are only effective if used as the substance itself, instead of a low-concentration solution. This property makes alcohol more expensive in use compared with chlorine and QACs, and therefore is not frequently applied on a large, industrial scale but is used mostly for applications such as small, difficult to reach spots in equipment, temperature probes and quick wipe-downs of working surfaces and scales.
74 Handbook of hygiene control in the food industry
Mode of action
The general mode of action for inactivation of microorganisms by alcohols is by denaturation of proteins (Schlegel, 1993), with the primary site of action being the cell (plasma) membrane. As a result of deterioration of the plasma membrane, the cell wall starts to leak essential cell components such as ions (Ca2+) and low molecular weight solutes such as peptides and amino acids. Therefore, the mode of action and its effect on the metabolism of the microorganism depends very much on the concentration. Moulds and actinomycetes are most susceptible to alcohols and are inhibited at 4% (v/v) whereas most bacteria can still grow at these concentrations (Kalathenos and Russell, 2003). Application of 5.5% (v/v) shows a bacteriostatic effect on E. coli, but in order to kill this microorganism concentrations of 22.2% or higher are necessary (Allerberger and Dierich, 1988).
Yeasts are able to grow at higher alcohol concentrations (8±12% v/v), which is not surprising since they are responsible for the production of beer and wine (Saccharomyces cerevisiae). Spores are affected by ethanol. Setlow and co-workers (2002) showed that the spore coat can be permeabilized. Consequently, ethanol in combination with other components or with high temperature (> 65 ëC) is more effective than ethanol itself in activating spores.
4.2.5 Aldehydes
Two aldehyde compounds are mainly used for disinfecting, glutaraldehyde and formaldehyde. Aldehydes are active against a wide range of bacteria, viruses, moulds and spores, are easily removed from surfaces and are (bio) degradable (Mohr and Duggal, 1997). However, the activity of aldehydes is very easily influenced by remaining (protein) fouling, which necessitates sufficient cleaning prior to disinfecting. From a toxicological point of view, aldehydes do not cause problems for humans when used within the prescribed concentrations (Mohr and Duggal, 1997). On the other hand, it is possible that formaldehyde can have mutagenic effects (McDonnell and Russell, 1999).
Mode of action
The mode of action of glutaraldehyde involves a strong association with the outer layers of bacterial cells (Denyer and Stewart, 1998; McDonnell and Russell, 1999). The cell's chemical reaction with glutaraldehyde results in metabolic and replicative inhibition (Denyer and Stewart, 1998). The way formaldehyde reacts is most probably the same. Concerning processing conditions, an alkali environment is more favourable than an acid environment as more reactive sites will be formed on the cell surface. Applied concentrations vary between 0.08 and 1.6% (w/w) for inactivating E. coli. For a sporicidal effect, a solution of 2% is normally sufficient.
4.2.6 Bisphenols
Bisphenols are hydroxy halogenated derivatives of diphenyl methane, diphenyl ether and diphenyl sulphide, and are active against bacteria, fungi and algae.
Pathogen resistance to sanitisers 75
Triclosan and hexachlorophene are the most widely used (McDonnell and Russell, 1999). Triclosan, a derivative of diphenyl ether, is known as an ingredient in some medicated soaps and hand-cleansing gels and toothpastes, and is effective against staphylococci (Hugo and Russell, 1999). It is currently applied as antimicrobial layer in packaging material (Vermeiren et al., 2002; Chung et al., 2003) and conveyer belts (Quantex Laboratories, 2001; Stekelenburg and Hartog, 2002).
Unfortunately, depending on the impurity of the starting material, Triclosan can contain concentrations of dioxin and dibenzofurans, both substances highly toxic to humans (Quantex Laboratories, 2001). Therefore, it is of great importance that the origin and way of production are known prior to application in food production areas. Hexachlorophene has been used in soaps as well; in 1972 it was restricted in use by the US Food and Drug Administration (FDA) to levels less than 0.1%.
Nowadays, application as a surgical scrubber in case of certain infections is permitted (Spectrum Laboratories).
Mode of action
The exact mode of action is unknown so far but it is suggested that Triclosan affects the cytoplasmic membrane. However, current research shows that Triclosan inhibits one specific enzyme of the fatty acid synthesis of E. coli. This increases the risk of resistance against Triclosan as one mutation of a gene can result in a decreased efficacy of the disinfectant (Sixma, 2001). Hexachloro-phene affects bacteria by inducing leakage, causing protoplast lysis and inhibiting respiration.
4.2.7 Biguanides
The group of biguanides is represented by chlorhexidine, alexidine and poly-meric biguanides (McDonnell and Russell, 1999; Hugo and Russell, 1999).
Chlorhexidine is probably the most widely applied biocide in hand-washing and oral products such as mouthwash, mouth spray and throat-lozenges (Sixma, 2001) and is bacteriostatic at concentrations of 0.0001 mg lÿ1 as well as bactericidal at concentrations of 0.002 mg lÿ1(Russell, 1991). Chlorhexidine has a broad spectrum of activity and is pH-dependent (higher efficacy at alkaline rather than acid pH); its efficacy is greatly reduced by the presence of organic matter. High concentrations of chlorhexidine cause coagulation of intracellular constituents (Russell, 1990; McDonnell and Russell, 1999). Chlorhexidine is only sporicidal at elevated temperatures (>0.005 mg lÿ1 at 70 ëC) and is in general more sporostatic; it has little effect on the germination of the spore but does not prevent the outgrowth of the spore (Russell, 1991; Gorman et al., 1987). Alexidine and the polymeric biguanides are used only on a small scale.
The polymeric biguanides are used in particular by the food industry and also for the disinfection of swimming pools. An example is poly(hexamethylene biguanide) hydrochloride (PHMB) which is the main active ingredient of Vantocil, which is widely used in the food industry, hospitals, nursing homes and consumer households (Avecia, 2004).
76 Handbook of hygiene control in the food industry
Table 4.3 Summary of disinfecting agents
Biocide Application Bactericidal Sporicidal Comments
Halogen-releasing agents 50±250 mg lÿ1 >10 mg lÿ1 >50 mg lÿ1 Chlorine cheap Iodine expensive
Influenced by organic substances Quaternary ammonium compounds 150±250 mg lÿ1 >100 mg lÿ1 No Residual action (approx 1 day),
neutral, non-aggressive
Peroxygens 3±90% >6% 10±30% More effective as mixture with
acetic acid
Alcohols (ethanol) 20±70% (w/v) >22% (w/v) 60±70% (w/v) Not for large industrial application
Aldehydes 0.8±16 mg lÿ1 <10 mg lÿ1 20 mg lÿ1
Bisphenols 2±20 mg kgÿ1 >10 mg lÿ1 No
Biguanides (chlorhexidine) >150 mg lÿ1 1±60 mg lÿ1 Ð Applied in hand-washing and oral
products
Mode of action
In principle chlorhexidine attacks the outer cell layer but not sufficiently to induce lysis or cell death. However, after crossing the cell wall it damages the cytoplasmic membrane (bacteria) or plasma membrane (yeast) (McDonnell and Russell, 1999). Polymeric biguanide appears to have a non-specific mode of attack against cell membranes resulting in quick cell death.
Summary
The different effective concentrations for the biocides are summarised in Table 4.3. It is obvious that, depending on the type of application or type and metabolic state of the microorganism, the proper disinfectant must be chosen.