SURFACE MICROBIOLOGY Biofilm Formation & Its Elimination · Source: Trainer Toolbox DVD 10. 12 (21)...

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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015

Gun Wirtanen , DTU National Food Institute

SURFACE MICROBIOLOGYBiofilm Formation & Its Elimination

The Annual Veterinary Congress 2015

Helsinki Fair Center, Helsinki, Finland

December 4, 2015

Gun WirtanenDTU National Food InstituteLyngby, Denmark

National Food Institute, Technical University of Denmark

Example: Poor hygienic design

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Example: Poor hygienic design


Surface Profiles of Various Stainless Surfaces

2 B plus, 400 grit polished surface

2 D plus, cold rolled surface

Cold rolled, annealed and pickled surface

Surface profile

Profile height

The centre of the surface

Size of a bacterial cell

,

# 2D # 2B # 7

Surface profile

-

in m

200

100

0 0

100

200

1

1

2

2

3

3

4

5

4

5

4

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Size of Microbes

Mould Spores – Ø [µm]

Aspergillus niger 2-10

Bacterial strain Length [µm] Thickness - Ø [µm]

Clostridium botulinum 3-8 0.5 – 0.8

Clostridium perfringens 4-8 1.0 – 1.5

Typical representation of a stainless steel surface profile of Ra =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 10 – 100 nm

Electron microscope Light microscope

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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. 2000. Biofilms – Impact on hygiene in food industries. In: Bryers (Ed.) Biofilms II: Process analysis

and applications. Wiley-Liss Inc. Pp. 327-372.)


Biofilm consists of:

Microbes

Extracellular polymers (EPS; polysaccarides, glycoproteins etc. from microbes)

Water (85 - 98%)

Captured particles and other dissolved materials from the process stream

b) the same biofilm after swabbing

a) 6 d biofilm of Lactobacillus brevis

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Phases in theformation of biofilms

according to Busscher & van der Mei (2000) in "Initial microbial adhesion events: mechanisms and implications".In Proc. 59th SGM Symposium,p. 25-36, Cambridge University Press, ISBN 0 521 79302 5.


PALM BEACH

pH

Atmosphere (oxygen)Temperature

Nutrients

Humidity, water

FACTORS AFFECTING MICROBIAL GROWTH

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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.

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Contamination Routes - Surfaces

Source: Roland Cocker, Cocker Consulting

Source: Roland Cocker, Cocker Consulting


– condensate / wet films

– stagnant water or liquid, drops

• poor installations allowing backflow

• non potable water

Source: Soro, AiniaSource: Soro, Ainia

Drain opened

Contamination Routes - Waterborne

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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)

Problematic equipment in various food industries based on a Finnish survey

Aarnisalo et al., 2006. The hygienic working practices of maintenance personnel and equipment hygiene in the Finnish food industry. Food Control 17, 1001-1011


Biofilm Effects in Food Processing

energy losses in processing e.g. prolongedpasteurisation

deterioration on spesific chemical reactionse.g. contamination of immobilised cell systems

deterioration of products e.g. the product can beof lowered quality already after production

limited shelf-life of the products

occurrence of pathogenic microbes whichincrease the risks of foodborne poisoning

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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 Effects in Food Processing


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

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Biofilm Problems in Food Products:

vegetables

caramel apples

canned products

pastries, biscuits & pizza

fruit juices, apple cider & beer etc.


Airborne - Problem high pressure cleaning

Contamination routes

Sou

rce:

Tra

iner

Too

lbox

DVD

10

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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


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


in a food factory

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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


in a food factory


Hygiene Assessment -Physical Observation

•Use mirrors (also endoscopic e.g. inside pipelines) and lamps to get a view of hard-to-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

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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

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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

n hours

Sample transported in liquid Sample transported on contact agar e.g. dipslide

x bacteria x * 23n bacteria

x colonies x colonies

Colony number unchanged

n hours01234

Factor 23n

20=123=826=6429=512212=4096

n hoursExample: x=55*1=55*8=405*64=3205*512=25605*4096=20480

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Source: Diversey Inc

Aerosols are efficient transport vehicle for airborne contaminants to surfaces, where the biofilm growth starts


Sizes of Airborne Particles (Ø in μm)

Viruses

Brown, 1996

Bacterial spores Fungal spores

Bacteria

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Sedimentation of particles in still air / 1 meter

Diameter of particle Sedimentation timem min

2 1304 338 8

16 2

Movements of Particles in Air


Removal of Microorganisms from Surfaces

Technique Suitability Limitations

Swab, sponge & gauze

Open surfaces, large areas or

irregular surfaces

Need suspension which will lower the sensitivity

RinseEnclosed areas or

small items

Dilution of the contamination is inevitable decreasing

the sensitivity

Direct contactCritical spot areas

with low contamination

Only small areascan be examined

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Enumeration of Relevant Microbes

SWAB

RINSE

CONTACT

Suspension Spread on agar Incubation Counting

Spread on agar Incubation Counting

Incubation Counting(Spread on agar)

• 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)


• 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

How to Sample? –Microbial Sampling of Surfaces

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• 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


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

Microbiological Assessments in Cleaning Validation –

How to Interpret the Results?

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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

Advanced Course in Hygienic Designheld at DTU on 24th of November 2015Hazards in Hygienic Food Processing

© 2010 EHEDG

Microbial growth after pasteurisation

Hygienic filling line

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© 2010 EHEDG

Microbial growth after sterilisation without recontamination after heat treatment

Aseptic filling line



© 2010 EHEDG

Microbial growth after sterilisation, when recontamination has occurred

after heat treatment

Filling line

Recontamination due to process air, process surfaces or packaging material

SURFACE MICROBIOLOGY Biofilm Formation & Its Elimination · Source: Trainer Toolbox DVD 10. 12 (21)...

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Transcript of SURFACE MICROBIOLOGY Biofilm Formation & Its Elimination · Source: Trainer Toolbox DVD 10. 12 (21)...