biofilm : a recurring issue in food industry€¦ · biofilm expert • research partners :...
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Biofilm : a recurring issue in food industry
Our DNA
• Reinvent hygiene that ensures the well-being of human and its environment
OUR MISSION
• Leader in enzymatic detergents and biofilm treatment and prevention
OUR AMBITION
• Loyalty
• Reliablity
• Transparency
• Enthusiasm
OUR VALUES
ISO 9001Certification
54 employees
13 patents
T.O. 10,5 M12 – 15 %
Reinvested in R&D46 % Exportation
Key facts
• The International Biofilm Summit is a worldwide conference with high-quality presentations focused on biofilm’s issues in the Food & Beverage industry
• Info and registration : www.biofilmsummit.com
To give you
• an introduction about biofilms
• tools for detection and cleaning
• industrial case studies
To make you aware of the presence of biofilms in food industries and their consequences on the quality and the safety of the final product.
Objectives
Introduction
What is a biofilm ?
Source of constant contamination created by an accumulation of microorganisms who develop a protective matrix made of organic polymers (EPS) polysaccharides, proteins, DNA, lipids, etc.
• Some slime producers who build biofilm matrix▪ Listeria monocytogenes
▪ Bacillus cereus & mycoïdes
▪ Salmonella spp.
▪ Stenotrophomonas maltophilia
▪ Pseudomonas spp.
▪ Leuconostoc spp.
▪ Burkholderia spp.
▪ Etc.
• Other microorganisms
▪ Viruses
▪ Phages
▪ molds & yeast
Which microorganism can be found into a biofilm
Some facts about biofilms
• 80% of the microbial biomass on our planet
• Microorganisms are 10 to 1000 times more resistant to antimicrobial agents or biocids
• 60% of foodborne illness is causedby biofilms
• If a microorganim is found in the environment, there is a 70% Riskof it being present in the food
• Biofilm is a constant source of contamination due to the unpredictable release of microorganisms and organic polymers.
Biofilm growth cycle
FIXING GROWING MATURING SPREADING
Sources of contamination in industries
Source of microbial
contaminationPersonnel/staff
Facility
Flow of personal,
material, waste
Equipment Process
Utilities
Water/gas
Materials
Microbes attach easily on the manufacturing surfaces (e.g. processing pipes, feeding lines), forming biofilms
which are difficult to remove(Van Houdt & Michiels, 2010)
Adverse effects of biofilms in a foodprocess
• Product contamination leads to economic losses (recall of products) or strategic risks (loss of public confidence)
ECONOMIC LOSSES
• Reduces the quality and shelf life of products
QUALITY
• Reduces productivity (e.g. membrane filters) and performance (e.g. heat exchangers)
PRODUCTIVITY
• Bacterial resistance to disinfection (neutralizing biocides or changing their physiological status)
RESISTANCE
• Surface corrosion (biocorrosion)
CORROSION
Industrial case : Listeria monocytogenes in Polony
203deaths
1.034sick people
$
3.667 tonsdestroyed
+ 525 millions Cost US $ 60
weeks
Products contaminated by
Listeriamonocytogenes
The current situation of biofilm cleaning and detection
Resistance of micro organisms against cleaning and disinfection
Tolerance of microorganisms to a high concentration of chlorine when there are inside a biofilm
A total of 58 samples were collected from four breweries in Belgium between August and December2014, before and after cleaning in place (CIP).
Total bacterial load (CFU/cm2) varied between 102
and 108 CFU/cm2 before CIP and between 101 and 109 CFU/cm2 after CIP.
Genetic diversity enhances the tolerance of microbialcommunities against antimicrobial treatment.
Resistance of micro organisms against cleaning and disinfection
0% Tolerance : all the microorganims are detroyed100 % Tolerance : NO microorganims are detroyed
Resistance of micro organisms against cleaning and disinfection
Tolerance of microorganism to the CIP process
Resistance of micro organisms to cleaning and disinfection treatments
Sampling and detection methods
• Estimated recovery for contact plates: 26 to 56% depending of the microorganism and the method (Behling et al., 2010)
• The effectiveness of swabbing is poor; previous studies have reported recovery rates ranging from 25% to only 0-1% of the initial inoculum (Niskanen and Pohja 1977; Roelofsen et al. 1999; Moore and Griffith 2002a; Taku et al. 2002; Obee et al. 2004).
• Bacteria become increasingly difficult to extract once they have adhered to a surface, especially if they are associated with a biofilm (Bredholt et al. 1999). The efficiency of sampling is reduced by 40% if the bacteria are in biofilms (Keeratipibul et al., 2017).
=> UNDER ESTIMATION OF THE RISK
Sampling and detection methods
Food process
Microbiological test
Viable, cultirable / VNC
Classical microbiology , Contacts methods
Molecular biology
Metagenetics
qPCR
Biochemical test
Proteins, carbohydrates, lipids
ATP, NADPH, catalase
EPS Biofilm detection kit (Realco)
Classcial microbiologyContact plates, (RODAC)PetrifilmTM (3M)Contact Agar Slides (dipslides)
Protein detection : CleanTrace(3M), Clean Test (LiofilChem), PRO-Clean (Hygiena) Carbohydrates: SpotCheck Plus [Hygiena]
Sampling and detection methods
Microbiological methods
• Very long (18 – 72 h)
• Accessibility required
• Unable to detect food residues
• Only able to detect culturablemicroorganisms
• Possible identification of the microorganisms----------
• Not suited to check the effectiveness of cleaningprocedures
• Suited to check the effectiveness of disinfectionprocedures OR to validateroutine methods
Molecular biology
• Very long (18 – 72 h)
• Accessibility required
• Unable to detect food residues
• Able to detect cultivable microorganisms an non cultivable
• Possible identification of the microorganisms----------
• Not suited to check the effectiveness of cleaningprocedures
• Suited to check the effectiveness of disinfectionprocedures OR to validateroutine methods
Biochemical tests
• Rapid
• More or less accessible areas
• Minimal training required
• Not affected by the physiological state of the bacteria (except ATP, NAD)
• +/- sensitive, +/- reproducible----------
• Not suited to check the effectiveness of disinfectionprocedures
• Suited to check the effectiveness of cleaningprocedures
Sampling and detection methods
Biofilm detection and cleaning
• Detection and visualisation of Biofilm contamination
• Detection of resistant organicresidues
DETECTION / LocalisationBiofilm Detection Kit
DETECTION > LocalisationBiofilm Detection Kit : blue coloration
Blue coloration stick specifically to organic residues EPS (Extracellular Polymeric Substances) which forms the biofilm matrix.
Diagnosis 1: Cleaning Quality
Good cleaning Satisfactory cleaningCleaning quality
problem
Diagnosis 2: Biofilm Presence
No biofilm Biofilm presence
An in-depth cleaning for a total removal
!!!!! Poor hygiene !!!!!!!!
High risks of contamination
Optimal Hygiene
No risks of contamination
Biofilm before
treatment
Superficial
action
Bacteria’s remain
trapped in biofilm
structure
Biofilm before
treatment
IN DEPTH CLEANING
Bacteria’s are free and accessible to disinfectants
BIO
REM
Cle
anin
g
Ch
em
ical
Cle
anin
g
✓✗
Enzymatic solution breaks down biofilm matrix
(Xiaobo Liu and al. 2014)
Why setting up preventive treatment?
TIME
3 weeks min Biofilm-Free Program
BIO
FILM
MA
TUR
ATI
ON
TIME
Tolerance area
Biofilm indicator
CONTAMINATION RISKS
TREATMENT
Hard cleaning & disinfection
Preventative treatment & disinfection
Curative treatment & disinfection
High potential contamination area
BIO
FILM
MA
TUR
ATI
ON
Case studies
Industrial Case 1
ISSU
E
Massive product recallPresence of macroscopic residues observed after few days of production
SOLU
TIO
N Biofilm Free Program• Detection• Curative treatment• Preventive treatment
RES
ULT
S • Back to microbiological conformity
• No more product recall
Beverage production line (mixing tank, heat exchanger, aseptic tank, fillers)
1.0.E+00
1.0.E+01
1.0.E+02
1.0.E+03
1.0.E+04
1.0.E+05
1.0.E+06
1.0.E+07
1.0.E+08
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Process Water
(reference)
BA
CT
/ M
L
T0
T1 = T0 + 60’
Treatment performance indicator :living germs extraction from equipment during enzymatic treatments
0
20
40
60
80
100
120
140
160
180
Customer complaints
ISSU
E
Customer complaintsOrganoleptic issues (off-flavours)Presence of Pediococcus, Lactobacillus, Zymomonas & wild yeast
SOLU
TIO
N Biofilm Free Program• Detection & Characterisation• Curative treatment• Preventive treatment
RES
ULT
S • Back to microbiological conformity
• No more customercomplaints
Beer production process – 150 K HL/year (keg & can fillers, BBT’s, pasteurizers, bottle rinsing system )
Biofilm free program
Industrial Case 2
Baby food : contamination of water equiments
ISSU
E Ponctual product rejects due to high contamination
SOLU
TIO
N Biofilm Free Program• Detection• Curative treatment• Preventive treatment
RES
ULT
S • No more batch production destruction
• Improved quality control
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Eau1 Eau2 Eau3
bacteria others* Xanthomonadaceae (family)
Thermus sp. Thermus scotoductus
Streptococcus (genus) Streptococcus thermophilus
Stenotrophomonas maltophilia Staphylococcus sp.
Sphingobacterium sp. Sphingobacterium multivorum
Pseudomonas sp. Propionibacterium acnes
Micrococcales (order) Microbacterium sp.
Microbacterium laevaniformans Microbacteriaceae (family)
Lactobacillales (order) Klebsiella sp.
Klebsiella pneumoniae Klebsiella oxytoca
Gordonia sputi Gordonia otitidis
Geobacillus stearothermophilus Gammaproteobacteria (class)
Enterobacteriaceae (family) Enterobacter sp.
Dermacoccus sp. Bacteria (kingdom)
Bacillaceae (family) Actinobacteria (class)
Industrial Case 3
Biofilm EXPERT
Biofilm Expert
• Research partners : University of Liege, Quality Partner and Realco
• Objectives : Evaluation of enzymatic cleaning on the microbial flora in the installations and the final products from several food industries
• Timing : 2 years
• Food industries: Ready to eat food, collective kitchens, meat cutting plant, cheese factory, butchery
• Communications : scientific publications, talks and posters in scientific congress
• Financed by the Walloon Region (DGO6)
Materials and methods
• Monitoring of a food company producing Italian dishes for 3 months with different cleaning treatments: ▪ conventional cleaning▪ reinforced enzymatic cleaning▪ routine enzymatic cleaning
• All factors remained constant and only the cleaning solutions were replaced with the sanitation treatments
• A total of 189 samples were analyzed, including surface samples pipes and finished products (Italian Meat dishes) (at day 0, at the end of shelf life) by classical microbiology and 16S rDNA metagenetic
• Statistical analysis was carried out with the R project software with different packages (non parametric tests)
Results
• Decrease of the total flora during the enzymatic treatments
• Increase of the total flora when the enzymatic treatment is stopped
• Conventional cleaning vs enzymaticcleaning Mann & Withney test : p value < 0.05
Total aerobic flora for the final products analyzed at day 0 in relation with the cleaning process (log cfu/g) (Plate Count Agar incubated at 22°C during 72h)
Results
Conventional cleaning vs enzymatic cleaning -Mann & Withney test :Day 0 - p value < 0.05End of the shelf life – p value < 0.05End of the shelf life +7 - p value < 0.05
16S rDNA metagenetic analysis on the finished products at day 0, end of the shelf life and end of the shelf life +7 days in relation with the cleaning process
Discussion
• A significant reduction of the total flora concentration and spoiling bacteria after implementation of enzymatic cleaning
• A minimum frequency of enzymatic cleaning is necessary to maintain a low proportion of spoiling bacteria in food products
• The microbiological quality of the finished products troughout the shelf life is improved thanks to the implementation of enzymatic cleaning of the installations
Conclusions
Take home message
Biofilms • are everywhere in the process• cause quality and safety issues• are not detected/cleaned/destroyed with
conventional methods
But you can deal with it using:• specific monitoring plan• specific detection methods• periodic enzymatic cleaning
Laurent Paul - Key account managerBenoit Duculot - Expert and Technical Services
Laurent Delhalle – R&D Senior scientist
THANK YOU
FOR YOUR
ATTENTION