Disinfection in Food Processing – Efficacy Testing of Disinfectants

Reviews in Environmental Science and Bio/Technology 2: 293–306, 2003.
2004 Kluwer Academic Publishers. Printed in the Netherlands. 293 Disinfection in food processing – efficacy testing of disinfectants G. Wirtanen* & S. Salo VTT Biotechnology, Microbiology, Tietotie 2, P.O. Box 1500, FIN 02044, VTT, Finland (*author for correspondence: e-mail: gun.wirtanen@vtt.fi) Key words: biofilm, microbes, chemical residues, cleaning, disinfection, food processing Abstract The key to effective cleaning and disinfection of food plants is the understanding of the type of the soil to be removed from the surfaces. An efficient cleaning and disinfection procedure consists of a sequence of rinses using good quality water with application of detergents and disinfectants. Disinfection is required in food plant operations, where wet surfaces provide favourable conditions for the growth of microbes. The efficacy of disinfectants is usually determined in suspensions, which do not mimic the growth conditions on surfaces where the agents are required to inactivate the microbes. Therefore, the suspension tests do not give adequate information and reliable carrier tests, which mimic surface growth, are needed. In developing a proposal for the testing of disinfectants on surfaces to an analytical standard, it is important to identify the major sources of variation in the procedure. In response to the need for a relatively realistic, simple and reliable test for disinfectant efficacy a method for culturing laboratory model biofilms has developed. The use of artificial biofilms i.e. biofilm-constructs inoculated with process contaminants in disinfectant testing can also be used for screening the activity of various disinfectants on biofilm cells. Both biofilm carrier tests showed clearly that the biofilm protects the microbes against the disinfectants. The chemical cleanliness is also essential in food plants. The total cleanliness of the process lines is mainly based on measuring the microbial load using culturing techniques. These results can give an incorrect picture of the total cleanliness, because the viable microbes do not grow when disinfectants are left on the surface. The luminescent bacteria light inhibition method offers a useful alternative for testing chemical residue left on surfaces after cleaning and disinfection operations. 1. Introduction The general aims for microbial control including biofilm removal are to prevent spoilage of products and to ensure that the quality specifications of the product are met. The most important means for maintaining efficient microbial control include: (1) minimizing the microbial load from outside sources to the process, (2) efficient control of growth at microbiologically vulnerable sites and (3) adequate cleaning and disinfection of the process lines (Wirtanen et al. 2000). Physical, chemical and microbiological cleanliness is essential in food processing plants. Physical cleanliness means that there is no visible waste, foreign matter or slime on the equipment surfaces. Chemically clean surfaces are surfaces from which undesirable chemical residues have been removed, whereas microbiologically clean surfaces imply freedom from spoilage microbes and pathogens (Gould 1994). Attached microbes or microbes in biofilms can be a problem in food processing, because they adhere to the surfaces and if the cleaning is insufficient the remaining cells start to grow and contaminate the product (Hood & Zottola 1995). The selection of detergents and disinfectants in the food industry depends on the efficacy, safety and rinsability of the agent as well as where it is corrosive or affects the sensory values of the products manufactured. An independent quality control system to monitor the cleaning results for a food plant can be integrated in the Hazard Analysis 294 Critical Control Points (HACCP) program. The key to effective cleaning and disinfection of food plants is the understanding of the type and nature of the soiling agent (sugar, fat, protein, mineral salts etc.) and the microbial growth to be removed from the surfaces (Czechowski & Banner 1990; Troller 1993). 2. Cleaning and disinfection Cleaning and disinfection is carried out in order to produce safe products with acceptable shelf life and quality. In the food industry there is a trend towards longer production runs with short intervals for sanitation. The cleaning programmes should be performed as cost-effectively and safely as possible, which means as infrequently as possible, in the shortest possible time, with low chemical, energy and labour costs, producing as little waste as possible and with no damage to the equipment (Lelieveld 1985; Holah 1992). The mechanical and chemical power, temperature and contact time in the cleaning regime should be carefully chosen to achieve an adequate cleaning effect (Czechowski & Banner 1990; Mosteller & Bishop 1993). The use of effective cleaning agents and disinfectants on surface-attached microbes minimises contamination of the product, enhances shelf life and reduces the risks of foodborne illness. A prolonged exposure of the surfaces to cleaning agents and disinfectants enhances the removal (Troller 1993). Attention should also be paid to the quality of the processing water, steam and other additives. Using additives of poor quality easily spoils the process. Furthermore, the tools and methods used must also suit the process and the personnel must be properly trained and responsible to maintain a good level of plant hygiene (Lelieveld 1985; Mattila-Sandholm & Wirtanen 1992). An efficient cleaning and disinfection procedure consists of a sequence of rinses and detergent and disinfectant applications in various combinations of temperature and concentration (Frank & Koffi 1990; Holah 1992; Troller 1993; Gould 1994; Wirtanen 1995). In a wet open process the gross soil should be removed by dry methods, e.g. brushing, scraping or vacuuming and visible soil rinsed off with low-pressure water. Using water of sufficient volume and temperature increases the cleaning effect. However, a pure water washing system is not practical due to ineffectiveness and cost limitation. Surfactants, which suspend the adhered particles and microbes from the surfaces in the water, are added to increase the washing effect. After a production run the equipment should be dismantled and cleaned. The cleaned utensils should be stored on racks and tables, not on the floor (Mattila-Sandholm & Wirtanen 1992; Holah & Timperley 1999). The cleaning of open process surfaces and surfaces in the processing environment is carried out using either foam or gel cleaning. The foam-units are constructed to form foam of varying wetness and durability depending on the cleaning to be performed. The application of gels extends the contact time with a soiled surface and can be used with low-pressure system. The cleaning is mostly carried out in combination with a final disinfection, because there are likely to be viable microbes on the surfaces that could harm continued production. Furthermore good ventilation in the process facilities is needed to enable drying of the process equipment and process lines (Holah 1992; Wirtanen et al. 2000). In the cleaning of closed processes, prerinsing with cold water is carried out to remove loose soil. The 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 of 1–2% sodium hydroxide, is heated to 75–80 C and the cleaning time is 15–20 min. The equipment is rinsed with cold water before the acid treatment is performed at approximately 60 C for 5 min. The cleaning solutions should not be reused in processes aiming at total sterility because the reused cleaning solution can contaminate the equipment. The design of the tank should ensure that also parts directly above the spray ball is also cleaned. Drainage, minimisation of internal probes, crevices and stagnant areas, arrangement of valves, couplings and instrument ports and instrumentation should be planned carefully so that the equipment is easily cleanable. Problems caused by equipment constructions and materials cannot be eliminated with CIP, because the CIP treatment was not designed to eliminate biofilms (Czechowski & Banner 1990; Brackett 1992; Chisti & MooYoung 1994; Zottola & Sasahara 1994; Wirtanen et al. 1997). 295 3. Common disinfectants Disinfectants have been developed to destroy microbes. Microbes have nevertheless, been found in disinfectant solutions, which is due to their ability to form resistant strains and build-up of protective biofilms (Gilbert & Allison 1999; McBain et al. 2000; Wirtanen et al. 2002). This means that microbial contaminants can be spread on the surface to be cleaned instead of being cleansed. As early as 1967, it was reported that chlorhexidine mixtures were contaminated with Pseudomonas sp. Pseudomonas sp. have also been found in concentrated iodine solutions (Marrie & Costerton 1981). Serratia marcescens was found to be viable even after 27 months in a disinfectant containing 2% chlorhexidine. A concentration of 0.1% chlorhexidine is sufficient to kill the cells of S. marcescens if they are freely suspended in liquid (Marrie & Costerton 1981; Costerton & Lashen 1983). Microbial contamination of e.g. Alcaligenes faecalis, Enterobacter cloacae, Eschericia coli, Flavobacterium meningosepticum and Pantoea agglomerans has also been found in solutions of Table 1. Advantages and disadvantages of some disinfectants used in the food processes (according to Flemming 1991; Troller 1993 and Wirtanen 1995) Disinfectant type Advantages Disadvantages Alcohols Effective against vegetative cells, non-toxic, easy-to-use, colourless, harmless on skin, soluble in water, volatile Effective in low concentration, broad microbial spectrum, kills spores, penetrates biofilms, non-toxic (fi acetic acid and water) Decomposes to water and oxygen, relatively non-toxic, easy to use; weakens biofilms and supports detachment Microbistatic, ineffective against spores Peracetic acid Hydrogen peroxide Corrosive, unstable High concentrations needed, corrosive Chlorine Effective in low concentration, broad microbial spectrum, Toxic by-products, resistance easy to use, supports microbial detachment, cheap development, residues, corrosive, reacts with EPS, discolouration, explosive gas Hypochlorite Cheap, effective in a broad microbial spectrum, easy to use, supports detachment Unstable, toxic, oxidative, corrosive, rapid regrowth, no prevention of adhesion, discolouration of products Chlorine dioxide Effective in low concentration, can be produced on-site, low dependency in pH Effective, non-toxic, prevents regrowth, supports microbial detachment, non-irritating, non-corrosive, odourless, flavourless Toxic by-products, explosive gas Quaternary ammonium agents Inactivated in low pH and by salts (Ca2+ and Mg2+), resistance development, ineffective against Gram-negative bacteria Iodophor Non-corrosive, easy to use, non-irritating, broad activity spectrum Expensive, flavour, odour, forms purple compounds with starch Ozone Similar effect as chlorine, decomposes to oxygen, no residues, decomposes biofilm Corrosive, inactivated easily, reacts with organics (fi epoxides) Glutaraldehyde Effective in low concentrations, cheap, non-corrosive Low penetration in biofilms, degrades to formic acid, increased DOC 296 quaternary ammonium compounds, aldehydes and amfotensides (Heinzel 1988). Disinfection is required in food plant operations where wet surfaces provide favourable conditions for the growth of microbes. The aim of disinfection is to reduce the surface population of viable microbes after cleaning and to prevent microbial growth on surfaces before restart of production. Disinfective agents do not penetrate the biofilm matrix left on the surfaces after an ineffective cleaning procedure very well, and thus do not destroy all the living cells in biofilms (Bloomfield 1988; Pontefract 1991; Brackett 1992; Holah 1992; Carpentier & Cerf 1993). Disinfectants are most effective in the absence of organic material, e.g. fat-, sugar- and protein-based materials. Interfering organic substances, pH, temperature, concentration and contact time generally control the efficiency of disinfectants (Czechowski & Banner 1990; Mosteller & Bishop 1993; Gould 1994). The disinfectants must be effective, safe and easy to use, and easily rinsed off surfaces, leaving no toxic residues or residues that affect the sensory values of the product. The use of disinfectants in food plants depends on the material used and the adhering microbes. Disinfectants (Table 1) approved for use in the food industry are alcohols, chlorine-based compounds, quaternary ammonium compounds, oxidants (peracetic acid, hydrogen peroxide and ozone), persulphates, surfactants and iodophors. They should be chosen based on the process (Sequeira et al. 1989; Larson & Morton 1991; Troller 1993; Wirtanen 1995): • Is the agent effective in the pH range used? • Is the agent stable when diluted? Does it vaporize? • Is the agent toxic, safe or irritating? • What is the spectrum of the agent? • How does temperature affect the activity of the agent? • Is the agent corrosive on the surface? • Is the agent surface active? • Is the agent stable when reacting with organic material? • Is the agent effective, and what are the costs? There are several chlorine or chlorine-based compounds, which are approved for use in food plants, e.g. gaseous chlorine, chlorine dioxide, sodium and calcium hypochlorites. The antibacterial active moiety is formed when the chlorine com- pound is added to water. Hypochlorous acid is formed and it dissociates further into protons and hypochlorite anions. Stabilised hypochlorites are used when a long duration is required. The range of microbes killed or inhibited by chlorine-based compounds is probably broader than by any other approved sanitizer (Troller 1993; Stewart et al. 1994; Sanderson & Stewart 1997). Hydrogen peroxide has been found to be effective in removing biofilms from equipment used in hospitals. The effect of hydrogen peroxide is based on the production of free radicals, which affect the biofilm matrix. The microbicidal effect of peracetic acid on microbes in biofilms was shown to vary (Exner et al. 1987; Christensen 1989; Kramer 1997). Aldehydes did not break the biofilm, but rather seemed to improve its stability. The biofilm must be disrupted in some way before chemical agents such as peracetic acid and aldehydes can be used effectively (Exner et al. 1987). The effect of ozone treatment has been found to vary depending on the processing circumstances and the microbes tested, e.g. ozonation has proved very effective in treatment of cooling water systems (Lin & Yeh 1993). Also, iodophors are used in the food industry. In the disinfection the iodine compound takes part in the oxidation of essential parts of the microbial cells. Like chlorine-containing products, iodophors are active against Gram-positive and Gramnegative bacteria, yeasts and molds (Holah et al. 1990; Holah 1992; Troller 1993). Bacterial spores, however, are highly resistant to iodophors. Iodophors cannot be used in food plants where starchcontaining products are produced because iodine forms a purple complex with starch. Quaternary ammonium compounds are used as sanitizers in dairies and in the food industry, because they have good wetting properties and are nonspecifically described as cationic surface active agents in which the cationic part is hydrophobic. The greatest effect of quaternary ammonium compounds is observed against Gram-positive bacteria, whereas Gram-negative organisms, many of them significant in the contamination of food, may not be affected (Troller 1993). Fogging can be defined as chemical disinfection using automatic spraying of disinfectant in a closed room. The aim of disinfection testing on an industrial scale using fogging is to study the efficiency of the disinfection on surfaces at different 297 places in the room. Controlled experiments have been carried out at two cheese-producing dairies. Neither of the fogging trials showed clear reduction of the microbial load. Critical control points in fogging are the amount of fog used, the disinfectant concentration used for the fog, thorough rinsing of equipment and drying of facilities (Wirtanen et al. 1997, 2002). 4. Efficacy and residue testing methods Efficacy of disinfectants and antimicrobial agents are usually determined in free cell suspensions, which do not mimic the growth conditions on surfaces where the agents are required to inactivate the microbes (Frank & Koffi 1990; Wirtanen 1995). The agent must reduce the microbial populations by 5 log units in suspensions in order to be considered effective. The goal for reduction of surface-attached bacteria with disinfectants is 3 log units (Mosteller & Bishop 1993). The standard suspension tests have proved sufficiently reliable because the variations of results are within acceptable limits when replication is adequate. There can, however, be problems with the repeatability and reproducibility of suspension tests performed with an organic load. It is obvious that the surface tests are even more difficult to perform than suspension tests because of the carrier material used and the viability of dried cells on the surfaces (Bloomfield et al. 1994). In developing a proposal for the testing of disinfectants on surfaces to an analytical standard, it is important to identify the major sources of variation in the procedure. Microbes growing or dried on surfaces are not susceptible to disinfectants from all sides as they are in suspensions. Due to the requirement for penetration, disinfectants are thus used in higher concentrations on surfaces than in suspensions (Mattila-Sandholm & Wirtanen 1992). 4.1. Suspension tests Determination of disinfectant efficiency is often performed in suspension tests with ready-to-use dilutions. The European Committee for Standardization (CEN) has launched many standards. The microbes used are standard test organisms as well as spoilage bacteria, pathogens and spores of concern in hygiene. All disinfectants passing the efficacy test should reduce the number of vegetative cells by ‡5 log units and the number of bacterial spores by ‡ 1 log unit (Wirtanen 1995). The suspension tests in European Standards are: • in EN 1040:1997 the basic bactericidal activity of a disinfectant is tested against both Gram-negative (Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus), • in EN 1275:1997 the basic fungicidal activity of a disinfectant is tested against both yeast (Candida albicans) and mould (Aspergillus niger), • in the quantitative suspension test EN 1276:1997 the bactericidal activity of a disinfectant for use in food, industrial, domestic and institutional areas is tested against both Gram-negative (P. aeruginosa, and Escherichia coli) and Gram-positive (S. aureus and Enterococcus hirae) bacteria (additionally the following bacteria Salmonella typhimurium, Lactobacillus brevis and Enterobacter cloacae can be used if needed) in hard water, • in the quantitative suspension test EN 1650:1997 the fungicidal activity of a disinfectant for use in food, industrial, domestic and institutional areas is tested against both yeast (C. albicans and if needed Saccharomyces cerevisiae can additionally be used) and mould (A. niger) in hard water, • in the quantitative suspension test EN 1656:2000 the bactericidal activity of a disinfectant for use in veterinary field is tested against both Gram-negative (P. aeruginosa, and Proteus vulgaris) and Gram-positive (S. aureus and Enterococcus hirae) bacteria in hard water with an organic load of bovine albumin or a mixture of bovine albumin and yeast extract or skimmed milk, • in the quantitative suspension test EN 1657:2000 the fungicidal activity of a disinfectant for use in veterinary field is tested against both yeast (C. albicans) and mould (A. niger) in hard water with organic load of bovine albumin or a mixture of bovine albumin and yeast extract or skimmed milk, • in the quantitative suspension test prEN 13704:1999 the sporicidal activity of a disinfectant for use in food, industrial, domestic and institutional areas is tested against 298 bacterial spores of Bacillus subtilis (if needed spores of B. cereus and Clostridium sporogenes can additionally be used) in hard water, • in EN 1499:1997 the basic activity of hygienic handwash products is tested against E. coli on test persons’ hands, • in EN 1500:1997 the basic activity of hygienic handrub products is tested against E. coli on test persons’ hands. The activity of disinfectants is at VTT Biotechnology tested using a Dutch 555-suspension test protocol. The 555-suspension test is performed to find out the bactericidal, fungicidal and sporicidal activity of the disinfectant. The activity is measured after a challenging time of 5 min. The product has both bactericidal and fungicidal activity if the microbial reduction is at least 5 logunits for vegetative cells and sporicidal activity if the reduction of spores is at least 1 log-unit. The microbes used are Salmonella Choleraesuis, P. aeruginosa, S. aureus, B. cereus (spores) and S. cerevisiae and the test is carried out using bovine albumin as the organic load. In a modified 555-suspension test the disinfectant is tested against a chosen panel of process contaminants (consisting of bacteria, yeasts and/or moulds) in bovine albumin solution (Wirtanen 1995; Wirtanen & Juvonen 2002). 4.2. Tests using biofilms Various surface tests have shown that surface-attached cells are more resistant to disinfectant treatment than are cells in suspension (Wirtanen 1995; Wirtanen et al. 1997). Results obtained using only one assessment method in testing can be inaccurate. For example, cultivation and CTCDAPI staining in a comparison based on biofilms showed underestimation of viable bacteria in the cultivation (Wirtanen et al. 1997). Microscopy techniques have often been used as a reference method for cultivation based on swabbing. It has been reported that counting cells by direct microscopy consistently give a result at least one log unit higher than the cultivation method (Holah et al. 1988). This may be because the bacteria do not grow under the conditions provided in the plate count method or that large numbers of bacteria remain on the surfaces after swabbing (Holah 1992). In our experiments, epifluorescence micros- Figure 1. Diagram of the biofilm-based disinfectant test. Biofilm is grown on test coupons using an inoculated filter paper placed on top of a suitable nutrient agar (Charaf et al. 1999). copy clearly revealed that even vigorous swabbing detached only a small portion of the actual biofilm and the cells within it (Wirtanen 1995). Therefore methods used in assessment and in detachment should both be chosen carefully. One testing procedure, based on cultivation, image analysis, impedance and metabolic indicators e.g. CTCDAPI staining, seems to give a good estimation of both removal of biofilm from surfaces and killing of bacteria on surfaces (Wirtanen et al. 1997). Microbial cells dried on test surfaces have also been used in disinfectant carrier tests e.g. Draft prEN 13697. The model biofilms have many of the characteristics of ‘‘wild’’ biofilms. The microbial cells are adhered to test surfaces, they produce slime and they show increased resistance to disinfectants. In response to the need for a relatively realistic, simple and reliable test for disinfectant efficacy, Charaf et al. (1999) have developed a method for culturing laboratory model biofilms. The method involves growing biofilm on test coupons using inoculated filter papers placed on top of a suitable nutrient agar (Figure 1). After the removal of the filter paper, the coupons are subjected to disinfectant testing as per suspension tests. The above mentioned methods for disinfectant testing, however, require further validation. 4.3. Tests with biofilm constructs The poloxamer hydrogels demonstrate thermoreversible gelation properties, being liquid and fully miscible with water at temperatures <15 C, but firm gels at temperatures >15 C. This means high cell densities can be cultured within the gels at 30 C and subsequently exposed to a disinfectant. After treatment, a full recovery of the individual cells can be achieved simply by moving the hydrogels into neutraliser solutions/diluents at <15 C (Wirtanen 299 Figure 2. Test protocol in efficacy testing of disinfectants using biofilm constructs (Wirtanen et al. 1998, 2001, 2003). et al. 1998, 2001, 2003). Poloxamer F127 is a diblock co-polymer of polyoxyethylene and polyoxypropylene. It has been investigated earlier for its potential as an agar substitute in microbiology. Solutions are unaffected by autoclaving and appear to be non-toxic to all the bacterial species so far tested (Gilbert et al. 1998; Wirtanen et al. 1998, 2001). The poloxamer matrices we have studied not only reproduce the reaction-diffusion resistance properties of the biofilms but also simulate other aspects of the biofilm mode of growth (Figure 2). The use of artificial biofilms i.e. biofilm-constructs inoculated with process contaminants in disinfectant testing is suitable for screening the activity of various disinfectants. 4.4. Microbial based residue test As mentioned above, chemical cleanliness is also essential in food plants. Chemically clean surfaces are surfaces from which undesirable chemical residues have been removed. The total cleanliness of the process facilities prior to initiating production is mainly based on measuring the microbial load using culturing techniques. It is possible that these results do not indicate the total cleanliness, they only show that there are no viable microbes on the surface. It is possible that there are chemical residues of the cleaning agents and disinfectants left on the surface. This is not allowed but usually no tests are run to avoid this. The luminescent bacteria light inhibition method can be used to measure low amounts of residues both in liquids and on surfaces. The luminescence inhibition method with the photobacterium Vibrio fischeri is a useful tool to estimate the toxicity of different samples, which is based on the reduction in light output due to interactions between bacteria and toxic compounds (Lappalainen 2001; Lappalainen et al. 2003). Specified volumes of the test sample or diluted sample is mixed with the suspension of luminescent bacteria in a cuvette (Figure 3). The decrease in light output is measured in a luminometer after a contact time of 5 min. The values measured are compared to a control sample and the changes in intensity are taken into account by using a correction factor when calculating the results (Lappalainen et al. 2003). 5. Results of disinfectant efficacy and residues 5.1. Suspensions Figure 3. Test protocol for testing of chemical residues (Lappalainen 1991; Lappalainen et al. 2003). Tests carried out at VTT Biotechnology have shown that microbes in suspensions are easily 300 Table 2. The microbicidal effect of four commercial disinfectants (alcohol-based, hydrogen peroxide-based, hypochlorite-based and persulphate-based products) against Listeria innocua, L. monocytogenes, E. coli, Salmonella Infantis, S. Choleraesuis and Bacillus cereus grown in suspension (reduction unit is log cfu/ml) Disinfectant/Concentration Escherichia coli Listeria innocua Listeria monocytogenes Salmonella Choleraesuis Salmonella Infantis Bacillus cereus (spores) Alcoholbased Hydrogen peroxide-based Hypochlorite-based Persulphate based 100% 75% 1% 0.50% 0.25% 1.40% 0.70% 0.35% 1/1.251 >6.7 >6.4 >6.3 >6.5 >6.8 ND >6.7 >6.4 >6.3 >6.5 >6.8 0.14 >6.7 >6.4 >6.3 >6.5 >6.8 0.20 >6.7 >6.4 >6.3 >6.5 >6.8 0.26 >6.7 >6.4 >6.3 >6.5 >6.8 ND >6.7 >6.4 >6.3 >6.5 >6.8 ND >6.7 >6.4 4.9 >6.5 >6.8 ND >6.7 >6.4 >6.3 >6.5 >6.8 ND >6.7 >6.4 >6.3 >6.5 >6.8 ND Control 7.46 7.11 7.04 7.29 7.67 6.04 ND = no microbicidal effect observed. destroyed, which is in agreement with several studies from other laboratories (Table 2). LeChevallier et al. (1988) showed that unattached Gram-negative bacteria in drinking water were susceptible to chlorine disinfectants whereas attached cells were not. Eagar et al. (1988) reported that planktonic cells of Pseudomonas fluorescens were more sensitive towards glutaraldehyde than sessile cells. Best et al. (1990) stressed that the selection of appropriate disinfectants for use on contaminated surfaces should be carried out carefully. They tested the effects of disinfectants on Listeria monocytogenes in carrier and suspension tests and found that sodium dichloroisocyanurate was the only effective solution in the presence of milk containing 2% fat. Available reports about the susceptibility of foodborne yeasts to various chemicals used in the food industry are sporadic and covers only a few disinfectants against some spoilage yeasts (Juvonen et al. 2001). McGrath et al. (1991) showed that ready-to-use concentrations of a hypochlorite disinfectant killed most of the S. cerevisiae tested in suspension by 15–20 min. The ascospore-containing cultures of Pichia and Saccharomyces species are more resistant to disinfectants than the vegetative populations (McGrath et al. 1991). Hypochlorite, peracetic and phosphoric acid as well as anionic compound in ready-to-use concentrations were effective against suspended yeasts isolated from orange juice (Winniczuk & Parish, 1997). Chlorine dioxide appeared to be effective against yeast and mould contaminants (Han et al. 1999). The efficacy of various types of disinfectants against food-spoilage was thoroughly studied using a modified 5-5-5 suspension tests against yeast contaminants isolated from various food processing environments. The results of the suspension tests (Table 3) showed that the alcohol, peroxide and tenside based disinfectants were efficient. The disinfectants containing chlorine and persulphate did not destroy suspended yeast cells (Wirtanen & Juvonen 2002). 5.2. Biofilms The microbicidal effect of the four commercial disinfectants (hydrogen peroxide-based, alcoholbased, persulphate-based and hypochlorite-based products) were also tested using biofilms of six different bacteria (L. innocua, L. monocytogenes, E. coli, S. Infantis, S. Choleraesuis and B. cereus). The bacteria were allowed to form biofilms on the surface of steel coupons (Figure 1) according to the method described by Charaf et al. (1999). When the results of the suspension test and the biofilm test were compared, the protection of the biofilm against the disinfectants was shown clearly. In the suspension tests all disinfectants had a sufficient microbicidal effect towards all vegetative bacterial types, but in the biofilm tests the microbicidal effects of the same disinfectants was lower. Only the alcohol-based and the hydrogen peroxide-based agents were efficient enough giving a logreduction greater than three for most vegetative bacteria tested. Spore forming bacteria were also used in the biofilm test. The test showed (Table 4) that the biofilm protects also spores against the disinfectants. Table 3. The in-use concentrations of alcohol-based (A1-A3), hydrogen peroxide-based (H1-H3), chlorine-based (C1-C2), tenside-based (T1-T2) and persulphate-based (S1) disinfectants needed to kill various yeast isolates obtained from different food processes. The disinfectant efficacy was tested using a modified 5-5-5 suspension test using Saccharomyces servazzii, S. cerevisiae, Zygosaccharomyces rouxii, Candida krusei, C. lambica, C. lipolytica, C. boidinii, C. intermedia, C. parapsilosis, Cryptococcus albidus, Debaromyces hansenii, Dekkera anomala, Rhodotorula glutinis, R. rubescens and R. mucilaginosa (Wirtanen & Juvonen 2002) Yeast S. servazzii C-00362 S. cerevisiae C-00370 S. cerevisiae C-96203 Z. rouxii C-00363 Z. rouxii C-00367 Z. rouxii C-95218 C. lambica C-00365 C. lipolytica C-00365 C. lipolytica C-00380 C. boidinii C-00366 C. intermedia C-00372 C. parapsilosis C-00373 C. parapsilosis C-00381 C. krusei C-00371 C. albidus C-00392 C. albidus C-00397 D. hansenii C-00382 R. glutinis C-00391 R. rubescens C-00393 R. mucilaginosa C-00396 D. anomala C-91183 R. mucilaginosa Disinfectant treatmentsa Control 4.7–5.1 5.8–6.4 5.0–5.6 5.9–6.1 4.6–4.7; 4.6–5.1 5.6–6.0 5.0–5.7; 4.9–5.1; 5.7–6.1; 6.3–6.5 6.0–6.7 5.9–6.4 5.9–6.1 4.9–5.4 4.6–5.2 5.3–6.1 5.3-6.0 5.7–6.0 5.8–6.1 6.3–7.0 5.6–6.1 6.1–6.3 6.2–6.5 6.7 6.5–6.6 A1b A2 A3 H1 H2 H3 C1 C2 Tl T2 S1 100% 100% 100% 100% 100% 100% ME 2.0 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% ME 2.7 100% 100% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 75% 0.5% 0.5% 0.25% 0.25% 0.25% 0.25% ME 1.4 ME 1.5 1.0% 0.25% 1.0% 1.0% 1.0% 0.5% 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% 0.5% 1.0% 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% ME 3.6 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% 0.25% 0.25 % 0.25% 0.25% 0.25% 0.3% 1.3% 0.3% 0.3% 0.3% 0.3% 0.3% ME 2.2 1.3% 0.3% ME 2.8 1.3% ME 3.3 0.3% 0.3% 0.3% 0.3% 0.3% 0.3% 1.3% 0.3% 1.3% 0.3% 1.0% 0.7% ME 3.7 0.3% 0.3% 0.7% ME 2.1 1.0% 0.7% 1.0% ME 1.3 1.0% ME 3.7 0.7% 0.3% 0.3% 0.3% ME 3.0 – 0.3% – 1.0% ME 4.3 1.0% 0.1 % 1.0% 1.0% ME 4.0 ME 2.8 ME 3.1 ME 4.3 1.0% ME 3.5 ME 1.7 ME 3.0 ME 2.3 ME 2.7 ME 3.9 1.0% ME 3.0 ME 2.5 1.0% ME 4.4 0.2% 2% 0.2% 0.2% 0.2% 0.2% 2% 0.2% 0.2% 0.2% 2% 2% 2% ME 4.4 0.2% 0.2% 0.2% 0.2% 2% 0.2% 0.2% 0.2% 2% 2% 2% 2% 2% 4% 2% 2% 2% 2% 2% 2% 2% ME 3.7 2% 2% 2% 2% 2% 2% 2% 2% – – – – – – – – ME 1.1 – – – ME 0.9 – – – 1 tab/1.25 l – – – ME 3.5 – a The percentage given is the lowest in-use concentration of the agent tested,which kills the yeast tested; if a microbial effect (ME) value is given in a shadowed cell the highest concentration of the agent tested is given (ME unit is log cfu/ml). b The agent A1 was tested only as undiluted (recommended concentration). 301 302 <-20 3% 100.0 -20 - 0 10 % 50.0 0 - 20 21 % Inh/ % 50 - 100 38 % 0.0 0 100 200 300 400 500 -50.0 -100.0 20 - 50 28 % -150.0 Figure 4. Residue test results of 501 dairy samples. The results are divided in five groups according to the inhibition result. The distribution of all results shows clearly that there are residues left on the surfaces in about 40% of the samples, in which the inhibition was greater than 50% (Lappalainen et al. 2003). tunistic pathogens (Wirtanen & Juvonen 2002). The yeast also readily form biofilms on process surfaces at both low and elevated temperatures. Therefore, the spoilage yeast can be a hygiene risk, when failures occur in the cleaning and disinfection procedure. The aim of our study was to assess the efficacy of various types of disinfectants against yeast isolated from various food processes growing on surfaces (Charaf et al. 1999). The results of the surface test showed that the alcoholbased agent was the most efficient disinfectant against the yeasts tested (Table 5). After 5 min Abundant growth of unwanted yeast during production can lead to defects in the final products and, furthermore, problems in process hygiene (McGrath et al. 1991; Winniczuk & Parish 1997; Han et al. 1999; Juvonen et al. 2001). Low pH, high sugar or salt content favours the growth of yeast. Spoiling yeast takes part in the deterioration of syrups, pralines, jams, berries and fruits, fruit juices, pickled vegetables and dairy products. In many cases the risk caused by growth of spoilage yeast in products has been underestimated because many of these yeast are not known to be oppor- Table 4. The microbicidal effect of four commercial disinfectants (alcohol-based, hydrogen peroxide-based, hypochlorite-based and persulphate-based products) against biofilms of Listeria innocua, L. monocytogenes, E. coli, Salmonella Infantis, S. Choleraesuis and Bacillus cereus (reduction unit is log cfu/cm2) Disinfectant/Concentration Escherichia coli Listeria innocua Listeria monocytogenes Salmonella Choleraesuis Salmonella Infantis Bacillus cereus (spores) Alcohol-based Hydrogen peroxide-based 100% 75% 1% 0.50% 0.25% 1.40% 0.70% >5.07 [0.00] >2.99 [0.00] >5.30 [0.00] >5.07 [0.00] >2.99 [0.00] >5.30 [0.00] 429 [0.80] 2.81 [2.10] >2.99 >2.99 [0.00] [0.00] 4.72 >5.30 [1.01] [0.00] 1.44 [3.18] >2.99 [0.00] 4.20 [1.91] 2.11 [0.84] >299 [0.00] –0.47 [0.99] >3.14 [0.00] >3.14 [0.00] 1.81 [1.20] 2.47 [1.17] 1.80 [1.31] >6.06 [0.00] ND >6.06 [0.00] ND 5.09 [1.68] ND 4.30 [1.95] ND 5.48 [1.01] ND ND = no microbicidal effect observed. Hypochloritebased Control 0.35% Persulphate based 1/1.251 0.44 [0.77] 1.76 [0.63] –0.91 [0.24] 0.29 [0.65] 2.68 [0.55] –1.19 [0.08] 1.54 [0.29] 2.35 [1.11] 0.00 [0.00] 5.39 [0.51] 3.32 [0.37] 5.62 [0.00] 1.98 [2.02] 1.81 [1.26] 2.22 [0.67] 0.58 [1.15] 3.47 [0.32] 1.55 [0.52] ND 0.34 [0.15] ND 0.43 [0.06] ND 1.61 [0.13] 0.36 [0.23] 6.38 [0.25] 5.21 [0.59] 303 likely effects of a formulation against microbial contamination in situ, they enable discrimination between the disinfectant formulations at normal use levels and the choice of the most effective one. The survival levels obtained made the test able to distinguish the performances of many different disinfectant formulations against a variety of test strains where this had been impossible with conventional testing methods (Wirtanen et al. 1998, 2001, 2003). Conventional suspension tests fail to discriminate between the agents in terms of their efficacy and are therefore, not of assistance in the final selection of agents (Gilbert et al. 1998; Wirtanen et al. 1998). The results showed that Gramnegative bacteria are more resistant to disinfectant treatment the hydrogen peroxide-based disinfectant was also effective in some cases and especially when the duration of the disinfectant treatment was extended to 15 min the peroxide-based and quaternary ammonium containing disinfectants were also efficient (Table 5). The disinfectants containing chlorine and persulphate were ineffective in destroying yeast biofilms. 5.3. Biofilm constructs The biofilm construct test is a severe measure of disinfection efficiency giving reproducible results. Whilst the results do not necessarily reflect the Table 5. Efficacy of disinfectant treatments on yeast biofilms grown on stainless steel after 5 min (upper table) and 15 min (lower table): The yeast isolates used were Saccharomyces cerevisiae, Candida lipolytica, C. intermedia, C. parapsilosis, C. krusei, Cryptococcus albidus, Debaromyces hansenii, Rhodotorula glutinis, R. rubescens; R. Mucilaginosa, Dekkera anomala and Trichosporon asahii Disinfectant treatments Yeasts Alcohol-based Concentration 100% 75% Hydrogen peroxide-based 50% S. cerevisiae C-00370 S. cerevisiae C-96203 C. lipolytica C-00380 C. intermedia C-00372 C. parapsilosis C-00373 C. parapsilosis C-00381 C. krusei C-00371 C. albidus C-00392 D. hansenii C-00382 R. glutinis C-00391 R. rubescens C-00393 R. mucilaginosa C-00396 D. anomala C-91183 T. asahii G-10 S. cerevisiae C-00370 C. lipolytica C-00380 C. C. C. C. R. intermedia C-00372 parapsilosis C-00373 parapsilosis C-00381 albidus C-00392 mucilaginosa C-00396 – – – – – – – – – – – – – – – – – Persulphatebased 0.5% 0.25% 1.4% 0.7% 4% 2% 0.2% 1 tab/ 1.25 l ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND – – Quaternary ammonium 1.0% ND – – Hypochlorite-based ND ND ND ND ND ND ND ND – – ND ND ND – – ND ND ND NO – ND ND ND ND ND ND ND ND ND ND ¼ Reduction greater than 3 log-units; h ¼ Reduction below 3 log-units but exceeding 1.5 log-units; ND ¼ reduction lower than 1.5 log-units; – ¼ Not tested. 304 treatments than Gram-positive bacteria. This is in agreement with the general observations (Nikaido & Vaara 1985; McKane & Kandel 1996) as well as studies using surfaces with dried bacterial cells (Grönholm et al. 1999) and biofilms (Wirtanen 1995; Wirtanen et al. 1997, 2002). 5.4. Microbial based residue test The method based on photobacteria is rapid to perform, because the incubation time is only 5 min. The results show that residues do exist on production surfaces prior to production start-up (Figure 4). Most of the samples taken in dairies were swab samples because of ease of sampling. There were in total 501 samples from different countries. The samples from factories can be divided into three groups: samples with very clear inhibition (inhibition percentage >50), samples with moderate inhibition (inhibition percentage between 20 and 50%) and samples that induced light production (inhibition value negative). About 40% of the samples showed a clear inhibition. In other words, this means that there are residues on surfaces before starting the production if no preventive rinsing is performed. This method offers a useful alternative for testing chemical residue left on surfaces after cleaning and disinfection. 6. Conclusions Disinfection is required in food plant operations where wet surfaces provide favourable conditions for the growth of microbes. The use of effective disinfectants minimizes contamination of the product, enhances shelf life, and reduces the risks of foodborne illness. A prolonged exposure of the surfaces to disinfectants enhances the microbicidal effect. Disinfectants approved for use in the food industry are alcohols, oxidants, iodophor- and chlorine-based compounds, persulphates, surfactants and quaternary ammonium compounds. The efficacy testing based on suspensions gives an answer to whether the disinfectant is effective against the microbe tested. According to the standard tests available a reduction of 5 log-units is needed for the agent to be effective against vegetative microbial cells and 1 log-unit for bacterial spores. Various laboratory studies have shown that surface-attached cells are more resistant to disinfectant treatment than suspended cells. This has lead to development of various types of carrier tests, e.g. tests using microbial cells dried on surfaces, biofilm-constructs, and biofilms grown on test coupons. The above mentioned methods require further validation and improved repeatability. Research has shown that there can be chemical residues on the surfaces prior to production. The results from using a photobacterial method can be categorized into the following levels: very clear, moderate and no inhibition. This method offers a useful alternative for testing chemical residue on surfaces and is worthy of further study. 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