SURFACE MICROBIOLOGY Biofilm Formation & Its Elimination

Transcription

1 SURFACE MICROBIOLOGY Biofilm Formation & Its Elimination The Annual Veterinary Congress 2015 Helsinki Fair Center, Helsinki, Finland December 4, 2015 Gun Wirtanen DTU National Food Institute Lyngby, Denmark Example: Poor hygienic design 1 (21)

2 Example: Poor hygienic design Surface - profile in m Surface Profiles of Various Stainless Surfaces # 2D # 2B # 7 Size of a bacterial cell Surface profile Profile height Cold rolled, annealed and pickled surface, The centre of the surface 2 D plus, cold rolled surface 2 B plus, 400 grit polished surface 2 (21)

3 Size of Microbes Bacterial strain Length [µm] Thickness - Ø [µm] Clostridium botulinum Clostridium perfringens Mould Aspergillus niger 2-10 Spores Ø [µm] Typical representation of a stainless steel surface profile of R a =0.6 µm roughness achieved by 240 grit mechanical polish. No surface defects or damage. Source: Fraunhofer AVV, Leibniz Institut für Polymerforschung Dresden SIZE OF MICROBIAL CELLS The average diameter of a microbial cell are: bacterial cell 1 m mould mycelia 2 10 m yeast cell 2 12 m virus nm Electron microscope Light microscope 3 (21)

4 Microscopying of 5 d Pseudomonas fragi biofilm stained with acridine orange on various stainless steel surfaces (AISI 304): (a) glass blasted, b) lapped and c) mechanically polished (according to Wirtanen, Saarela, Mattila-Sandholm Biofilms Impact on hygiene in food industries. In: Bryers (Ed.) Biofilms II: Process analysis and applications. Wiley-Liss Inc. Pp ) Microbes Biofilm consists of: Extracellular polymers (EPS; polysaccarides, glycoproteins etc. from microbes) Water (85-98%) Captured particles and other dissolved materials from the process stream a) 6 d biofilm of Lactobacillus brevis b) the same biofilm after swabbing 4 (21)

5 Phases in the formation of biofilms according to Busscher & van der Mei (2000) in "Initial microbial adhesion events: mechanisms and implications". In Proc. 59th SGM Symposium, p , Cambridge University Press, ISBN Temperature FACTORS AFFECTING MICROBIAL GROWTH Atmosphere (oxygen) Humidity, water PALM BEACH ph Nutrients 5 (21)

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7 Biofilm Problems on Food Contact Surfaces: on food contact surfaces of stainless-steel on conveyors on gaskets on blancher extractor surfaces in air handling systems on floors and in drains in manufacturing of paper-based packaging material in packaging machines in milk transfer lines cont. Biofilm Problems on Food Contact Surfaces: in heat exchangers, pasteurizers and cooling systems in ultrafiltration and reverse osmosis membranes on mixers and slicers on rubber fingered pluckers in poultry processing equipment and on vegetable processing lines etc. 7 (21)

8 Contamination Routes - Surfaces Source: Roland Cocker, Cocker Consulting Source: Roland Cocker, Cocker Consulting Contamination Routes - Waterborne condensate / wet films stagnant water or liquid, drops poor installations allowing backflow non potable water Drain opened Source: Soro, Ainia Source: Soro, Ainia 8 (21)

9 Problematic equipment in various food industries based on a Finnish survey 1. Packaging machines (20/44) 2. Conveyors (17/44) 3. Slicing machines (15/44) 4. Dispensers (15/44) 5. Cooling equipment (9/44) 6. Proofing drawers (6/44) 7. Brining equipment (5/44) Aarnisalo et al., The hygienic working practices of maintenance personnel and equipment hygiene in the Finnish food industry. Food Control 17, Biofilm Effects in Food Processing energy losses in processing e.g. prolonged pasteurisation deterioration on spesific chemical reactions e.g. contamination of immobilised cell systems deterioration of products e.g. the product can be of lowered quality already after production limited shelf-life of the products occurrence of pathogenic microbes which increase the risks of foodborne poisoning 9 (21)

10 Biofilm Effects in Food Processing cleaning and disinfection processes are affected and the choice of chemicals, flow, time and temperature must be altered increased need of maintenance in process lines and equipment e.g. prolonged down-time or laborious demounting of equipment e.g. heat exchangers in the process lines, in water supply systems, in granular activated carbon columns, in reverse osmosis membranes, in ion exchange systems, in degasifiers, in water storage tanks and in microporous membrane filters due to cleaning and hygiene, energy losses & blockages. Biofilm Problems in Food Products: RTE-products of meat and poultry fermented meat sausages fish cakes unpasteurized milk dairy products e.g. ice-cream & cheese spices & herbs iceberg lettuce, minimally processed vegetable salads & various deli 10 (21)

11 Biofilm Problems in Food Products: vegetables caramel apples canned products pastries, biscuits & pizza fruit juices, apple cider & beer etc. Contamination routes Airborne - Problem high pressure cleaning Source: Trainer Toolbox DVD (21)

12 Phases in an open, wet cleaning & disinfection procedure in a food factory Dry cleaning e.g. brushing or vacuum cleaning Removal of gross soil from process equipment and facilities through rinsing Removal of visible soil in process facilities through cleaning and rinsing Phases in an open, wet cleaning & disinfection procedure in a food factory Removal of visible soil from production equipment (i.e. cleaning of equipment ) foam/gel in open processes Rinsing of equipment - Removal of remaining soil and cleaning agents from process surfaces through multipressure in open system 12 (21)

13 Phases in an open, wet cleaning & disinfection procedure in a food factory Disinfection to kill living microbial residues left on surfaces and inhibition of microbial growth in periods when no processing is on Fogging i.e. disinfection of process facilities Drying of equipment and processing facilities using efficient air conditioning Hygiene Assessment - Physical Observation Use mirrors (also endoscopic e.g. inside pipelines) and lamps to get a view of hardto-see places Use special light e.g. UV-light to see residues of organic soil Feel if the surfaces are greasy or encrusted Smell inside containers and tanks 13 (21)

14 Hygiene Assessment Visual Observation Look for product residues or raw material Look for scale Feel if the surfaces are greasy or encrusted Use camera to take pictures for documentation Smell in premises Problems in Hygiene Monitoring Attached microorganisms and biofilms are difficult to detach from surfaces Improved sampling methods are needed Conventional hygiene control often underestimates the number of microorganisms or amount of soil Methods based on culturing are slow Improved detection methods are needed 14 (21)

15 Microbiological Assessment What to sample in the process? Surfaces (also air, water, raw material & semi-ready products) Where to sample? Avoid easy-to-sample areas, worst case areas are important When to sample? Before, during and after production How to sample? Consistent sampling, use contact agar applications, final rinse, swabs, sponges or gauzes How to interpret the results? Internal standards Basic Idea of On-site Microbial Testing Sample transported in liquid Sample transported on contact agar e.g. dipslide n hours x bacteria n hours Factor 2 3n 2 0 =1 2 3 =8 2 6 = = =4096 x * 2 3n bacteria Example: x=5 5*1=5 5*8=40 5*64=320 5*512=2560 5*4096=20480 n hours x colonies x colonies Colony number unchanged 15 (21)

16 Aerosols are efficient transport vehicle for airborne contaminants to surfaces, where the biofilm growth starts Source: Diversey Inc Sizes of Airborne Particles (Ø in μm) Viruses Bacterial spores Fungal spores Bacteria Brown, (21)

17 Movements of Particles in Air Sedimentation of particles in still air / 1 meter Diameter of particle m Sedimentation time min Removal of Microorganisms from Surfaces Technique Suitability Limitations Swab, sponge & gauze Rinse Direct contact Open surfaces, large areas or irregular surfaces Enclosed areas or small items Critical spot areas with low contamination Need suspension which will lower the sensitivity Dilution of the contamination is inevitable decreasing the sensitivity Only small areas can be examined 17 (21)

18 Enumeration of Relevant Microbes SWAB Suspension Spread on agar Incubation Counting RINSE Spread on agar Incubation Counting CONTACT (Spread on agar) Incubation Counting Usually sufficient to know the total amount of microbes Sometimes important to know what kind of microbes Choice of agar Choice of incubation conditions (time, temperature & atmosphere) How to Sample? Microbial Sampling of Surfaces Quantification of the actual number of microbes from surfaces is difficult due to strong microbial adherence of biofilms - traditional swabbing method is often improper The process hygiene may not be spoiled during sampling of surface Rapid results are needed to be able to act upon the results and prevent hazards 18 (21)

19 Visual observation of cleaning results is an important practical method for: 1) open food contact surfaces, 2) difficult-to-see areas & 3) surfaces in closed equipment Soil can also be visualised with UV-light. This type of assessment is a practical choice especially in large equipment such as fermentation tanks. Surface hygiene can be carried out using ATP and protein based kits Microbiological Assessments in Cleaning Validation How to Interpret the Results? Establish internal standards Conduct a series of min 10 correct cleaning programmes Determine the level of microbes after each programme Mean result provides a standard Trend analysis Repeated sampling and testing over a period of time Trend analysis provides a baseline These concepts apply to both equipment and environment 19 (21)

20 We can see that biofilm can generate problems anywhere in the food process if the design and maintenance is improper! European Hygienic Engineering & Design Group Microbial growth after pasteurisation Hygienic filling line 2010 EHEDG Advanced Course in Hygienic Design held at DTU on 24 th of November 2015 Hazards in Hygienic Food Processing 20 (21)

21 European Hygienic Engineering & Design Group Microbial growth after sterilisation without recontamination after heat treatment Aseptic filling line 2010 EHEDG Advanced Course in Hygienic Design held at DTU on 24 th of November 2015 Hazards in Hygienic Food Processing European Hygienic Engineering & Design Group Microbial growth after sterilisation, when recontamination has occurred after heat treatment Recontamination due to process air, process surfaces or packaging material Filling line 2010 EHEDG Advanced Course in Hygienic Design held at DTU on 24 th of November 2015 Hazards in Hygienic Food Processing 21 (21)