Presumptive Bacillus cereus testing
Food Safety versus Food Quality
Dr. ir. T Wijtzes
WFCWFC is one of the foremost microbiological laboratories in the Netherlands. We combine expert knowledge in Food Science with excellence in Microbiology to assure the safety and quality of our customers’ products.
WFC-AnalyticsQuality control
100 % microbiological laboratory
ISO 17025 accredited laboratory for quality control
Microbiological lab (Arkel)
Chemical analyses (Outsourced)
Food Quality and Food Safety
Food safety and food quality are determined by
Chemical composition
Physical composition
(Micro-) biological composition
Chemical food composition
Food quality Carbohydrates,
Proteins
Fat
Salt
Food Safety Lead
Cadmium
Aflatoxins
Fumonisins
Physical food composition
Food quality Emulsion stability
Firmness of a product
Food Safety Presence of external physical hazards
eg. glass, needles
Presence of product specific hazards
eg. bone splinters, fish bone, nut shell.
Microbiological food composition
Inert Presence of enterobacteriaceae in soil
Positive Fermentation
Alcohol formation
Gas formation (beer)
Degradation of waste materials (bio fuels)
Negative Spoilage
Illness
Microbiological food composition
Spoilage Degradation of food quality parameters
Pathogenic (human health) Toxin formation (Intoxication)
Presence of the pathogen (Infection)
Bacillus cereus
Human pathogen transmitted by food products
Both outside human host and inside host
Vomit toxin
Enterotoxin
ISO 7932 “Presumptive B. cereus”
MYP Agar – Mannitol Yolk Polymyxin Agar Mannitol: Carbohydrate Yolk: Nutrients and lecithin source Polymyxin: Inhibits other microbes, but not Bc species
Inoculate, incubate at 30oC for 18-24 hrs, count
Confirm “presumptive Bc” using Blood Agar
However
Other Bacillus species grow on MYP
B. cereus
B. subtilis
B. mycoides
B. anthracis
B. pseudomycoides
B. weihenstephanensis
B. megatarium
B. amyloliquifaciens
B. thuringiensis
and others…
Straining to Observe Crystal Inclusions
Isolate 5-15 colonies
Incubate 24 – 96 hrs
ISO 7932 Confirmation
Blood Agar
Observe Crystals
• Non-standardize method
• Few colonies counted
• Variability between technicians
Alternative crystal method(under validation)
Modified method
• Standard procedure ISO 7932
• Followed by confirmation
• Twice on Trypton Soy Agar
• HCT agar
• Phase contrast microscopy
Much more reliable
ISO/TC34/SC9 WG20Use of the parasporal crystal differentiation of cereus and thuringiensis
Isolate
DNA
Shared marker
Bc and BT
Specific markers
BT strains
Simultaneous amplification real-time qPCR
Quantification of
BT Strains using qPCRBacillus cereus
Bt-based product
Standard procedure
(ISO 7932)
Comparative Benefits of
qPCR method
ISO 7932 Crystal Method qPCR
Colonies counted Total in sample Percentage Total in sample
Accuracy 1.5 Log Adequate 1 Log
Limit of Detection 2 Log Percentage of MYP counts
3 Log
Time to results 18-24 h Additional 5 days 36 to 96 h
Confirm strain identity
No Partly Yes
Some food safety issues
Maximum allowed levels of BC differ per EU member state
• Lowest levels < 1000 cfu/gram
• Highest levels < 100.000 cfu/gram
Some food safety issues
How to find 1.000 BC
in a pool of 1.000.000 presumtive BC
Specific qPCR for BC Are they harmful ?
Toxin production (qPCR ?) Endotoxin(s)Vomitotin
What about BT and BS ?
Toxin production BC
ISO 18465:2017(en)
Quantitative determination of emetic toxin (cereulide) using LC-MS/MS
After harm has been done
Thank You for Your Attention!
M. Wuite or T. WijtzesAddress: Kolk 27, 4241 TH Arkel, NL
+31 183 629 749
http://www.foodconsult.nl/