Biofilm formation and its elimination from food processing equipment: Dissertation

Microbes adhered to surfaces have a tendency to form protective extracellular matrices called biofilms. Biofilm and biofouling refer to biological deposits on any surface. European Union legislation on food hygiene and the hygienic design of machinery together with increasing public awareness of product quality make sound scientific testing of equipment cleanability and surface cleanliness an important facet of the food industry. However, there is at present a lack of reliable, rapid methods for hygiene assessment. Determination of the spectra of disinfectants and antimicrobial agents is usually performed in suspensions, which do not mimic the growth conditions on surfaces were the agents are expected to inactivate the microbes. A comparison of surface and suspension tests of cleaning agents and disinfectants was carried out to study the antibacterial effects on different food spoilage and opportunistic pathogenic bacteria in biofilms and suspensions. The results of this work showed that none of the cleaning agents or disinfectants tested in the recommended concentrations were efficient enough to eradicate all the bacteria growing in biofilms on the surface. In practice the suspension tests do not indicate the efficiency of the agents on surfaces. The tested agents were efficient against vegetative cells in suspensions, reducing the amount of living cells by as much as 5 log units. However, they were not efficient against spores of Bacillus subtilis. If a reduction of 3 log units was considered acceptable in the surface test, the chlorine- and iodophore-based compounds were effective against biofilms of Enterococcus hirae, Listeria monocytogenes and Pseudomonas fragi. The quaternary ammonium compound was effective against biofilms of B. subtilis and P. fragi and the anionic tenside was effective against biofilms of B. subtilis, L. monocytogenes and P. fragi. Methods generally used in studying biofilm build-ups include microbiological methods, physical, chemical and microscopy methods. In this work conventional cultivation using the swabbing technique, glycocalyx measurement and epifluorescence microscopy combined with image analysis were compared for studying the build-up of biofilm on surfaces. The results showed that the age of the biofilm is very important in choosing the best method for hygiene assessment. The traditionally used swab method could only remove some layers of the biofilm, resulting in unreliable data concerning the hygienic status of the surfaces. Furthermore, the results of the swab method did not provide information about the thickness and age of the biofilm. The image analysis turned out to be a helpful tool for studying biofilm build-up. The elimination of biofilm is a very difficult and demanding task because many different factors affect the detachment. In complex cases where several factors have an important role, statistical experimental design enables studies of these interactions. In this work factors affecting the cleaning of surfaces were evaluated using the Taguchi method. The chosen parameters were surface material, soil, cleaning procedure, use of detergent, storage of samples, bacterium and growth conditions for the bacterium. The surface structure turned out to be a very important factor when cleaning surfaces. The results showed that the species of bacteria did not affect the results when the comparison was carried out using the same type of washing procedures. The detergent had a profound effect on the cleaning when the surfaces were presoiled. Different cleaning procedures were also evaluated. Simple rinsing and cleaning-in-place (CIP) procedures could not remove all of the three Bacillus sp. biofilms tested. The addition of a chelating agent in the CIP procedure improved the cleaning efficiency remarkably. These results showed that the cleaning procedures can be tested using standard organisms. In case of problems with some particular microbe, final conclusions should however be drawn only after tests performed with the microbes concerned. There are relatively few, predominantly dairy based, international standards for cleanability testing of equipment. In the present work a simple microbial cleanability test was developed. The method was based on measuring the luminescence of Photobacterium leiognathi. According to the results the luminescent bacterium P. leiognathi was promising for use in this type of assays. However, the cleanability method based on bioluminescence must be developed further. Microbes adhered to surfaces have a tendency to form protective extracellular matrices called biofilms. Biofilm and biofouling refer to biological deposits on surfaces. The biofilm can be a hygienic risk in food processing. Legislation on food hygiene and the hygienic design together with increasing public awareness of product quality make sound testing of equipment cleanability and surface cleanliness an important facet of the food industry. A comparison of surface and suspension tests of cleaning agents and disinfectants was carried out to study the antibacterial effects on different foodborne bacteria in biofilms and suspensions. The results showed that none of the cleaning agents or disinfectants tested in the recommended concentrations were efficient enough to eradicate all the bacteria growing in biofilms on the surface. In practice the suspension tests do not indicate the efficiency of the agents on surfaces. Methods generally used in studying biofilm build-ups include micro-biological methods, physical, chemical and microscopy methods. In this work conventional cultivation technique, glycocalyx measurement and epifluore-scence microscopy combined with image analysis were compared for studying biofilm build-up on surfaces. The results showed that the age of the biofilm is very important in choosing the best method for hygiene assess-ment. The swabbing used in cultivation removed only some layers of the biofilm. The image analysis turned out to be a helpful tool in biofilm studies. The elimination of biofilm is a very difficult and demanding task because many different factors affect the detachment. In complex cases where several factors have an important role, statistical experimental design e.g. the Taguchi method enables studies of these interactions. Different cleaning procedures were also evaluated. Simple rinsing and cleaning-in-place (CIP) procedures could not remove all the biofilm from the surfaces. The results showed that the cleaning procedures can be tested using standard organisms. In case of problems with some particular microbes, final conclusions should however be drawn only after tests performed with the microbes concerned. There are relatively few international standards for cleanability testing of equipment. In the present work a simple microbial cleanability test was developed. The method was based on measuring the luminescence of Photo-bacterium leiognathi ATCC 33469. According to the results the luminescent bacterium was promising for use in this type of assays, which must be developed further.