Detection and quantification of GMO in food and raw material

Detection of food and cosmetics pathogens

Acredited laboratory

Since April 1th 2006 (CIA)

usage of highly sensitive system PCR a Real on Time PCR

based on standard normative methods

based on moleculár genetics methods

PCR – Polymerase chain reaction

DNA chips

-Salmonella sp. -Listeria monocytogenes -Campylobacter sp. -Cronobacter sakazakii -Staphylococcus auresus

based on imunochemical methods - Listeria monocytogenes - Staphylococcus auresus

Tests of microbial cosmetics contamination

member of ENGL (European Network of GMO laboratories)

member of GMO laboratories network in CR and SR

Topics for presentations

1. Mechanism of penicillin +vancomycin and bacitracine effect on bacteria and resistence.

2. Antibiotics interfering with bacterial proteosynthesis, and mechanism of resistence

3. Effect of various types of thermal treatment on food – impact on microorganisms.

4. Effect of physical (excluding thermal) on food - impact on microorganisms.

5. Impact of bacterial sporulation on food.

6. Biofilm control strategies in food industry.

7. Typical spoilage microorganisms of cereals.

8. Means of disinfection and sanitation in food industry.

9. Survey of major food-born epidemies in the last 5 years in Europe.

10. Typical spoilage microorganism of beverages

11. Principle of continuous cultivation – production of biomass

• Avetian David

• Guilbault Helene

• Le Stum Sophie

• Olshtrem Anastasiya

• Pasquelins Anais

• Ras Amandine

• von Korff Pauline

• Ilpars Emre

• Jordao Leal Cátia Daniela

• Simoes Cadete Mylene

• Ballon Aurélie 5 (6)

• García Cases Ananai 10

• Jiyoon Kang

• Yeoseung Chot

• Collet Felix

• Lozano Rios Blanka 10

• Betbeder Marie-Hélène 5 (6)

• 22.2. Microbial growth curve, thermal inactivation, primary and secondary products Pazlarová

• 1.3.Factors influencing growth of bacteria Demnerová

• 8.3. Survey of food-borne pathogens– part 1

• 15.3. Survey of food-borne pathogens – part 2

• 22.3. Classic methods of detection and quantification – Purkrtová

• 29.3. Rapid methods of detection and quantification + EU legislative– Purkrtová

• 5.4. Microbial fermentative processes, biomass, enzymes, etc. – Pazlarová

• 12.4. Micromycetes,mycotoxines – Demnerová

• 19.4. Lactic acid bacteria – probiotics Demnerová

• 26.4. Spoilage bacteria, biogenous amines - Pazlarová

• 3.5. Presentations

• 10.5. Presentations

Microorganisms on the Earth

Some numbers for beginning……

• Bacteria 3,6 mil. years

• Eucaryota 2,1 mil. years

• 99% of microorganisms (bacteria) present on the planet we are not able to cultived

• In the human body there are together 1013 cells

• In the human body there are 1014 microorganisms

• 7-10 thousands species???

• Human MICROBIOM – „second human genom“

Perception of risks from intake foreign compounds

The public Experts

Rezidua of pesticides

Contaminants of environments

Food aditives

Microbiological contamination

Natural toxins

Perception of risks from intake foreign compounds

The public Experts

Rezidua of pesticides Microbial contamination

Contaminants of environments Natural toxins

Food aditives Contaminants of environments

Microbial contamination Rezidua of pesticides

Natural toxins Food aditives

How we can define safe and healthy food?

• Eggs?????

• Vegetables????

• Meat and their products????

• Flour and their products????

• Milk and dairy products????

Microorganisms important in food

industry

atractive undesirable

Natural microflora

Pure cultures used in food industry

Pathogenic microorganisms

Microorganisms having unwanted enzymic activity

Which are sources of spoilage?

FAT TOM

explains what allows foodborne pathogens to grow. You'll also learn some important food

safety terms.

Conditions influencing the growth of microorganisms in food

• Food

• Acidity

• Time

• Temperature

• Oxygen

• Moisture

The growth of microorganisms in food

•water

•pH

•structure

•oxygen

•temperature

•time

Food

Microorganisms need nutrients to survive. Food provides these nutrients in the form of proteins and carbohydrates.

Acidity x alkalinity

• Bacteria grow best in food that contains little or no acid, measured as pH.

• On the pH scale of 0 to 14, a value of 0 is highly acidic, while a value of 14 is not acidic (alkaline) and a value of 7.0 is neutral.

• Bacteria grow best in food that is neutral to slightly acidic, represented by a pH of 6.5 -7.5

• Yeasts 4.5 – 5.5 • Molds a widerange of pH

Time and environment

• Bacteria are able to double within 20 to 30 minutes.

• Food should be removed from "the danger zone" within two-four hours, either by cooling or heating. While most guidelines state two hours, a few indicate four hours is still safe.

Temperature Food-borne pathogens grow best in temperatures between 5 to 60 °C, a range referred to as the temperature danger zone (TDZ). They thrive in temperatures that are between 21 to 40 °C.[

Atmosphera/Oxygen

Almost all foodborne pathogens are aerobic, that is requiring oxygen to grow. Some pathogens, such as Clostridium botulinum, the source of botulism, are anaerobic

Moisture - water activity

• Water is for growth and reproduction of microorganisms a limiting factor.

• Moisture is measured in %

• Water activity (aw) is measured on a scale of 0 to 1.0. Foodborne pathogens grow best in foods that have aw between 0.95 and 1.0. FDA regulations for canned foods require aw of 0.85 or below

Food safety

Commission Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs

• Foodstuffs should not contain micro-organisms or their toxins or metabolites in quantities that present an unacceptable risk for human health

• ‘micro-organisms’ means bacteria, viruses, yeasts, moulds, algae, parasitic protozoa, microscopic parasitic helminths, and their toxins and metabolites

Microbial criteria for foodstuffs • Food safety criteria

• Process hygiene criteria

a) Meat and products thereof

b) Milk and dairy products

c) Egg products

d) Fishery products

e) Vegetables, fruits and products thereof

• Rules for sampling and preparation of test samples

a) General rules for sampling and preparation of test samples

Food safety criteria Commission Regulation (EC) No 1441/2007 on

microbiological criteria for foodstuffs

• Listeria monocytogenes

• Salmonella

• Enterobacter sakazakii Cronobacter

• Escherichia coli

• Biogenic amines

• Staphylococcal enterotoxins

• Bacillus cereus

Process hygiene criteria Meat and products thereof: Aerobic colony

count, Enterobacteriaceae, Salmonella, Escherichia coli

Milk and dairy products: Enterobacteriaceae, Coagulase-positive staphylococci, E.coli, Bacillus cereus

Egg products: Enterobacteriaceae

Fishery products: E.coli, Coagulase-positive staphylococci

Vegetables, fruits and products thereof: E.coli

Staphylococcus aureus

Elektronový mikroskop Světelný mikroskop (po obarvení dle Grama) Typické kolonie na selektivním médiu

• growth in range of 7 – 48 °C • growth in range of pH 4 - 10 • limit concentration of NaCl 15 % • limit value of aw 0,85 (production of toxinu aw 0,98)

Escherichia coli

Elektronový mikroskop Světelný mikroskop (po obarvení dle Grama) Typické kolonie na selektivním médiu TBX

• Growth in range of 10 – 47 °C • Growth in range of pH 3,8 - 9,5 • limit concentration of NaCl

8,5% • limit value of aw 0,93

Bacillus cereus

Electron microskop Light microskop y Typické kolonie na selektivním médiu

• growth in range of 7 – 50 °C • growth in range of pH 4,3 - 9,3 • limit concentration of NaCl 7,5% • limit value of aw 0,95 • sporforming bacteria

Salmonella

Elektronový mikroskop Světelný mikroskop (po obarvení dle Grama) Typické kolonie na selektivním médiu

• growth in range of 5 – 47 °C • growth in range of pH 3,8 - 9,5 • limit concentration of NaCl 9 % • limit value of aw 0,92

Enterobacter sakazakii / Cronobacter spp.

Elektronový mikroskop Světelný mikroskop (po obarvení dle Grama) Typické kolonie na selektivním médiu

• growth in range of 5 – 45 °C • growth in range of pH 3,8 - 9,5 • limit value of aw 0,93

Listeria monocytogenes

Elektronový mikroskop Světelný mikroskop (po obarvení dle Grama) Typické kolonie na selektivním médiu

• growth in range of 7 – 48 °C • growth in range of pH 4 - 10 • limit concentration of NaCl 15 % • limit value of aw 0,85 (production of toxinu aw 0,98)

Campylobacte jejuni/coli

Elektronový mikroskop Světelný mikroskop (po obarvení dle Grama) Typické kolonie na selektivním médiu

• growth in range of 32- 45°C • growth in range of pH 4.9 - 9 • limit concentration of NaCl 1.5 % • limit value of aw 0.98 (microaerophilic)

What about molds??? • They spoiling food and producing mycotoxins

Aspergillus brassiliensis

Trends of kampylobacteriozis in ČR

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Trends of salmonelozis in ČR

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Occurence of salmonellas in plant products – epidemiologic cases in EU

Year Country Vehikulum Serotype Num. of cases

2000 Netherland (NL) sprounts of mungo Enteritidis 27

2002 Germany anise tea Agona 42

2006 Danmark (D) basil pesto Anatum >200

2006 Norway (N) wild rocket Thompson 21

2007 D, UK, NL basil pesto Senftenberg 51

2007 N, D, Finland sprouts of alfaalfa Weltverden 45

2008 Finland iceberg lettuce Newport/Reading 107

2009 Finland sprouts of alfaalfa Bovismorbificans 42

2010 UK sprouts of alfaalfa Bareilly 213

Occurence of salmonellas in plant products – epidemiologic cases in USA

Year Number of

states Vehikulum Serotype Number of cases

2007 44 peanat butter Tennessee

2008 43 tomatoes Saintpaul 1407

2009 14 alfa alfa sprouts Saintpaul 235

2009 ? White pepper Rissen 87

2010 26 alfa alfa sprouts 4,5,12:i.- 140

2010 11 alfa alfa sprouts Newport 44

2010 44 pepper Montevideo 272

2011 25 papaya Agona 106

2011 5 alfa alfa sprouts Enteritidis 25

2011 10 melon Panama 20

2011 5 pine nuts Enteritidis 43

2012 20 peanat butter Bredeney 42

2012 15 mango Braenderup 127

2012 24 melon Typhimurium 261

2012 ? germinating seeds Cubana ?

Trends of listeriózis in ČR

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Trends of virus hepatitidis in CR

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VHA VHE

How we can fight with microorganisms???

• HACCP

• Improvement of hygiene and sanitation

The need of modern, fast methods:

• chromogenic media

• molecular biology methods

• imunochemical methods

• mass spectrometry, MS

• nanobiosensors

HACCP Hazard Analysis and

Critical Control Point

Nowadays BIG problems

*Resistence of bacteria to antimicrobial compounds *Biofilm formation

Louis Pasteur (1822-1895)

„Sirs, they are microbes, whose have the last word“

Thank you for your attention