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/