GC-NPD P-pesticide

GC-NPD
Detector Type: Nitrogen Phosphorus detector

This detector is specific to Phosphorus compound, which is suitable for detection of phosphorus pesticide (same function to ECD detecting Cl-pesticide)
The detector works by electrically heating a glass bead containing an alkali metal (Rb) until electrons are emitted. These electrons are captured by stable intermediates to form hydrogen plasma. Phosphorus pesticide is ionised and directed into this plasma. An electrical field directs these resulting ions to an anode creating an increased current. The fluctuation of current will create a chromatogram.
http://www.encolabs.com/gc1.htm

Plasma: The fourth state of a matter (S,L,G,Plasma), gas becomes so hot, electron breaks away from atom.

GC-ECD Cl-Pesticide

GC-ECD

Gas Chromatograph that uses ECD (Electron Capture Detector) as detector. Compound gets separated as per normal and in column. Halogenated Compound will be detected by ECD in the ECD cell and produce a signal.

In the cell, radioactive Ni-63 Emits β-particles (Electron), which will form an electron cloud in the ECD cell. Electrical Current (electron) passes through the cell to form a close circuit. When electronegative species (Cl-pesticide) passes through the cell, electrons are being captured, resulting in a decrease in electron. Electrical current is the flow of electron. When there is a decrease in electron, there is a decrease in electron flow, i.e. decrease in electrical current. Therefore when electronegative species are present, electrical current decreases, which will translate into a chromatogram.

GC-ECD is therefore good for detecting electronegative species, such as Halogenated compound, organochloro-pesticide.

GC-Mass Spec.

GC-MS (Gas Chromatography Mass Sepectrometery)
Carrier: gas, usually N2 or He
Stationary phase: Silicon related compound
Detector: Mass Spectrometer (MS)

In the MS’ ionising chamber, compounds are being bombarded with electron, causing the compound to ionise into captions, and formation of radicals. Such radicals and compound are unstable. It will eventually break up into fragments.

At the quadripole, different electromagnetic field is applied at specific time. Compounds of the right electrical charge will pass through the quadripole.

Positively charged compound will be attracted to the High Energy Dynode (HED), which will later discharged electron. Electrons pass through the electron multiplier, which will produce more electrons when electrons hit the multiplier. This amplifies the compound’s signal. The electron will eventually be converted to electrical signal, producing a spectrum.

The spectrum is compared with a reference spectrum for identification. GC-MS may be used for further identification from other chromatographic methods. It is used as an initial screening for exceeding amount of pesticide. For further quantification and detection of minor amounts of pesticide, compound specific GC detector are used.

HPLC-DAD

HPLC-DAD ( High Performance Liquid Chromatography Diode Array Detector)

Carrier solvent: liquid, usually Methanol-Water Mixture
Stationary phase: Silicon related compound
DAD: UV ray

Analyte after separation, will be detected by DAD, which uses UV ray, DAD will detect the optical density of compound. A UV spectra of the compound will be produce, the spectra will , match with the reference spectra to determine the identity of compound. HPLC is therefore suitable for compound like
Allergens
Toxin
Antibiotics
Growth promoters (hormones)

Aflatoxin and Orchratoxin

The above toxin has a special reaction UV ray. In the presence of UV ray, toxins will fluorescence at different wavelength. E.g. Orchratoxin at 330nm. Therefore HPLC is very suitable for detection of the above captioned toxin.

Chromatographic Methods

Chromatographic Methods (HPLC-DAD, GC-MS, GC-ECD, GC-NPD)

Generally Chromatography works on a similar principle. Chromatography is a method of separation. The unit that does the separation is the column. It comprises of a mobile phase (The analyte and carrier fluid) and a stationary phase (The ‘Separator’ which will interact with the analyte which will cause separation of the mixture of compound). The separation of compounds is base on the affinity of analyte with the stationary phase. The higher the affinity, the longer the compound will interact with the stationary phase, therefore achieving separation. The time that the compound interacts with the stationary phase is termed as the retention time. The chromatogram that comes out will produce the intensity of analyte signal with reference to retention time.

Intensity of signal can be correlated with the amount of analyte present.

Specific analyte will interact differently with the different types of stationary phase. Therefore for different types of compound of interest, one may use specific types of column. All these are subjected to the chemical engineer and manufacturer’s recommendation and advisory.

With a group specific retention time of analyte on specific column, one can zoom down a group of compound. A spectroscopic method or detector needs to determine and confirm the identity of compound.

Chemical Analysis AAS Heavy Metals

Atomic Absorption Spectroscopy (AAS)

Reference:

Pockets Encyclopedia, 1997, Bob Gordon, Helen Parker, Dorling Kindersley, London, UK

Principles of Instrumental Analysis, 2004, Teaching Team of PIA Dip. Chem.Engn., Temasek Polytechnic Applied Science School, Singapore

AAS is a method to detect heavy metals, such as Mercury, Arsenic and Etc. It is also used to detect other metals.

General Principles of AAS.

Light Source (Cathode Lamp) of specific wavelength absorbed by heavy metal is emitted
Sample (heavy metals) gets vaporised and atomised using flame
Atomised Heavy Metal absorbed light wave
Monochromator Separates the light spectrum
Detector detects the intensity of light spectrum that is of interest
Electrical signal converts to chromatogram

1. Explanation on how light source emits specific wavelength that the hevy metal will absorb

Definition of light:
· A fast moving form of energy
Electromagnetic Spectrum

Occurrence of light:

Atoms have many orbital
Each level of orbital represents an energy level
The outer most shell have the highest energy level
Inner most shell have the least energy level
When electrons move from a lower energy level to a higher energy level and jumps back to the lower level of energy, it will emit photons, i.e. light

Occurrence of specific wavelength in Cathode lamp

The Lamp contains a Anode (+ terminal) and Cathode (- terminal)
When a electrical current pass through Anode will be highly + charged
Cathode will be highly – charged
Metal atoms’ electron (-) will lose it electron to the Anode (Process of ionisation)
I.E. electron move from lower energy level to higher energy level
When electrical current switched off, electrons will move from a higher energy level to a lower energy level
Therefore photons (of specific metal wavelength) is emitted
Absorption of Energy

· When metals are burnt by flame, energy is imparted, the metal will ionise or atomised
· When electrons from a higher energy level move to a lower energy level, photons is emitted
· When electrons move from a lower energy level to higher energy level, photons is absorbed
· Specific wavelength of light ( e.g Hg) is emitted
· With that wavelength of light, only Hg will absorbed it
· Therefore the amount of absorbance of light from the known amount of emitted light is correlated with the amount of metals present

Clostridias

Clostridium spp (including C. botulinun, C. perfringene) (Gram Positive)

This is a method with reference to the Thai Pharmacopoeia’s Method, according to Thai Pharmacopoeia, Vol. 1, 1987

Information retrieved from 2nd Revised Draft WHO guidelines on assessing safety and quality of herbal medicine with reference to contaminants and residue, July 2004 , 2004, WHO, Geneva Switzerland

Enrichment
· Boil 2 tubes of 100ml Cook Meat Medium for 100˚C for 10 min
· Cool to 37˚C
· Weigh 10 g of sample to tubes
· Seal up on tube immediately with parafilm
· Another heat to 65˚C for 30min
· Seal the heated tube
· Incubate both tubes for 37˚C for 4X24hrs

· At every 24hrs check for changes

Differentiation between C. perfringnes , C. botulinum , C. tentani

1. All Clostridium spp will not digest any of the meat
2. C. perfringnes will cause the meat to turn pink
3. C. botulinum will cause the tubes to have bubbles and white sediments
4. C. tentani will give off burnt organic smell

Confirmation step

1. Inoculate the broth in duplicates into 5% Defibrinated Sheep’s Blood agar
2. Incubate at 37˚C for 48hrs, one aerobically, another anaerobically.

Differentiation between C. perfringnes , C. botulinum , C. tentani colonies
Colony Morphology
Gram Stain
Spore Stain

Colony morphology
C. botulinum: Irregular
Translucent
Granular surface
Fimbrated spreading edge
Haemolysis

C. perfringnes: Round convex
Translucent
Smooth
Entire
Double zone of haemolysis


C. tentani : Transparent
Long Feathering projection
Haemolysis

Gram stain
All Gram Positive

Spore stain
C. botulinum: Oval, cocci

C. perfringnes: No exosporium observed


C. tentani : Drum Stick Shape

Rapid Microbiological Method

Enzyme Link Immnosorbent Assay ( ELISA)

After Enrichment and isolation of the target pathogen, Antigen from the isolate is being release into a well (Vessel) containing antibody against specific pathogen’s antigen. Antigen-Antibody complex forms.

An enzyme is dispense into the well and will bind specifically to Antigen-Antibody Complex.

An Enzyme substrate is then added to the vessel.

If the enzyme has bind to the Antigen-Antibody Complex, enzyme will bind to enzyme substrate, catalysing a particular reaction to the substrate, which will yield a colour.

The presence of such colour can be correlated to the presence of pathogen.

This method may be used for many pathogens like salmonella spp.

Genetic Method

After Enrichment and isolation of the target pathogen, cell undergoes cell lysis.
Cell lysis causes the microbes cell to rapture and release the DNA .
Endonuclease is added to cleave the desired set of gene on the DNA, which is unique to a particular microorganism.

PCR: Addition of DNA Polymerase will make copies of that Gene to a detectable level by electrophoresis.

After PCR reaction, Agarose Gel electrophoresis will separate the DNA fragments into bands. The bands are then compared against the known / target pathogens DNA fragment bands to check for the presence of that pathogen.

This method is also use to detect GM food.

For more information, please see Sarah’s and Farziana’s blog, also Dip. In Biotech notes Molecular Biology.

Vibrio spp.

Vibrio spp. Gram Negative

Enriched Sample in peptone water
Incubate for 37˚C for 6h
For fresh fish sample, incubate 41.5˚C for 6hrs
Culture into Thiosulphate Citrate Bile salt Sucrose (TCBS) agar
Culture into Sodium Dodecyl Sulphate polymixin Sucrose (SDS) agar
Incubate 37˚C for 24hrs


Positive Vibrio chlorae 01 colonies are Yellow (2-3mm) (TCBS)
Positive Vibrio parahaemolyticus colonies are Green (2-3mm) (TCBS)

Do a colony count

Positive Vibrio cholerae 01 colonies are Yellow (2-3mm) with halo (SDS)
· Positive Vibrio parahaemolyticus colonies are purple or green (2-3mm) (SDS)


Do a Stab on to TCBS agar
Do an oxidase test

Vibrio chlorae 01 will give yellow TCBS agar and a positive oxidase test
Vibrio parahaemolyticus will give TCBS agar green colour and oxidase test positive

Thiosulphate Citrate Bile salt Sucrose TCBS
· Bile salts suppresses G+ growth
· Sucrose differentiates Vibrio fermentor( V. cholerae) from non fermentor (V. parahaemolyticus)

Sodium Dodecyl Sulphate polymixin Sucrose (SDS) agar
Polymixin antibiotic, inhibits other microorganism’s growth
Sucrose differentiates Vibrio fermentor(V. cholerae) from non fermentor (V. parahaemolyticus)


For more information on Culture techniques, please refer to Food Microbiology: A Laboratory Manual, 2003,Ahmed E. Yousef, Carolyn Carlstron, John Wiley& Sons Inc., N.J , USA and Practical Food Microbiology 3rd Edition, 2003,D. Roberts& M. Greenwood, Blackwell Publishing Ltd, Oxford , UK

For Information on a detail Biochemical test and theories, please refer to Food Microbiology: Fundamentals and Frontiers, 2nd edition, Thomas J. Montville and Karl R. Mathews, 2001 ASM press , Washington, USA

Listeria spp.

Listeria monocytogenes (Gram Positive)

Enriched sample in UVM 1 broth
Incubate 30˚C, 24Hrs
Draw 1 ml into Fraser broth, Incubate 35˚C 24hrs
Do a streak plate on MOX and PALCAM
Incubate 35˚C for 24 Hrs.
From each plate pick 1 colony and streak onto TSAYE agar ( Half for MOX and Half for PALCAM)

Positive TSAYE agar will yield 0.2 to 1.5mm blue grey colony

From Each plate choose another colony stab into TSA blood agar

Positive TSA blood agar will yield clear zones in colony
From TSAYE agar, do a gram stain
From TSAYE agar, do a catalase test

Positive catalase test will yield oxygen bubble

With all the positive test, it can then be confirm that Listeria spp. is present. Above test can be confirm at the genus level only.

PALCAM agar medium
Contains Lithium Chloride, polymyxin, ceftazidime are antimicrobial agents that will inhibits other microorganisim, my Listeria spp. are resistant to it.
Listeria spp. hydrolyses Esculin, which will react with ferric amminium citrate. Esculin-Ferric ammonium citrate yield black ppt. Therefore Esculin differentiates Listeria spp. from other microorganisim.
Manitol and phenol red differentiates Manitol fermenting microorganism which will be in yellow colour

Trypton Soy Agar (TSA) with Blood

Listeria spp. haemolyses blood which will give a colony with clear zone.

TSA Yeast Extract

It’s a non selective enriched media. Clear media allows the examination of colony morphology that will undergo catalase reaction


University of Vermont Broth (UVM 1 broth)
· Nalidxic acid inhibits G-.
· Acrflavin suppresses other G+, but not Listeria spp.

Modified Oxford Agar (MOX)
· Contains Lithium chloride ,esculine, ferric ammonium citrate, colistin sulfate and moxalactam.
· The above are antimicrobial agents against G- and other G+, but not Listeria spp.Lithium Chloride select for osmotolerants like Listeria spp.

Escherichia coli 0157:H7

Escherichia coli 0157:H7 (E. coli) 0157:H7 (Gram Negative)

Media: Rainbow agar

Enrich sample with EC-Novobiocin broth
Incubate 35˚C 24 hrs
Do suitable serial dilution
Isolate in Rainbow agar, spread plate (3 replicates for each dilution)
Positive Escherichia coli 0157:H7 will form black colonies
From each plate choose a representative colony and inoculate into Phenol Red Sorbitol Broth (PRSB)
From each plate choose a representative colony and inoculate into Tryptose Broth
Incubate 35˚C 24 hrs


A positive result in PRSB will give yellow colour with gas produce. ( E.coli 0157:H7 don’t ferment Sorbitol, it’s PRSB negative)

From Tryptose Broth, Do an Indole test with Kovac reagent

A positive result in indole test will form a red layer in the alcohol layer

From Tryptose Broth, do a 0157 antigen test

Indole test
E. coli and Enterohaemorrhagic E. coli(EHEC) including 0157:H7 decompose tryptophan (comes from peptone) which will produce indole

Escherichia coli (EC)broth with Novobiocin (EC-Novobiocin)
Lactose enriches lactose fermentors like Coliform
Bile salts suppresses G+ and favours members of Enterobacteriaceae.
Novobiocin ( Wide spectrum antibiotics)suppresses G+ and a some G-, it generally select against non Novobiocin resistant bacteria and favours Enterobacteriaceae i.e. resistant against Novobiocin

PRSB
Sorbitol will differentiate E. coli 0157:H7 from other strains as 0157:H7 strain does not ferment Sorbitol. Sorbic acid causes phenol red to turn yellow.

Rainbow agar
Tellurite inhibits other micro organism and selects for E. coli 0157:H7
Novobiocin inhibits Tellurite reducing bacteria and Proteus spp.
E.coli 0157:H7 produces beta-galactosidase.
Indicator will turn black when there is beta-galactosidase activity

Tryptose broth
Provides a source of peptone/ tryptophan for strain 0157:H7, to facilitate Indole test

Salmonella spp.

Salmonella spp. (Gram Negative)

Enriched sample in Lactose broth, incubate 35˚C 24Hrs
Draw 1 ml of Broth to Selenite Cysteine (SC)Broth, incubate 35˚C 24Hrs
Draw 1 ml of Broth to Tetrathionate (TT) Broth, incubate 35˚C 24Hrs
Prepare BS , HE, XLD agar plates
Divide the perti dishs into half, 1 for SC and 1 for TT
2 phase streak SC and TC to all the plates with the designated halfs
Incubate 35˚C for 24hrs
From each media and section, pick 1 positive colony
Inoculate into TSI and LIA broth

Other method available is ELISA

BS (Inhibit against Coliform and Gram positives) positive will yield Black-Green colony with metallic sheen
HE agar (select against gram positives) positive will be blue colony with black centre
XLD ( Select for Salmonellae) agar positive colony will be red with black centers.
TSI Positive Red slant, yellow butt with black ppt.
LIA positive , purple slant, purple butt with black ppt.

Bismuth Sulfiteagar (BS)
Contains sulfite which will differentiate desulphydrase producer, which will yield a black ppt (H2S).
Bismuth suppresses gram Coliform
Brilliant green suppresses G+


Hektoen Enteric Agar
Bile salts, bromothymol blue, acid fuchsin suppresses G+ bacteria
bromothymol blue, acid fuchsin acts as indicator, salmonella consumes peptone which will give off alkaline pdt. pH Indicator will turn green .
Thiosulphate provides an indicator for activity of desulphydrase (salmonella), which will yield H2S ( Black ppt.)

Lysin Iron Agar (LIA)
Contains Lysine, Fe , Glucose, thiosulphate,bromocresol purple indicator
After depletion of glucose, it will utilise(‘ve lysine decarboxlyase) lysine and produce base pdt, which will cause indicator to turn purple ( purple butt)
Desulphydrase will give out H2S, which combines with Fe to give a black ppt.

Triple Sugar Iron agar ( TSI)
Contains glucose, sucrose, lactose, Fe, and thiosulphate, indicator phenol red
Salmonella ferments either or all the sugars to produce acid at the butt , i.e. yellow.
Desulphydrase will give out H2S, which combines with Fe to give a black ppt
Xylose Lysine Desoxycholate Agar (XLD)Desoxycholate inhibits non-members of Enterobacteriaceae. Thiosulphate and Fe has same fuction as the above media. Salmonella ferments xylose, therefore favours xylose fermentors. Lysine will differentiate xylose and non xylose fermentor(similar function as (LIA), phenol red indictor will turn red when pH is basic.

Coliforms

Coliform

Gram Negative Bacteria
Media VRBA

Outline:
Presumptive by VRBA
Confirmatory Test by BGLB

Procedure
Do a serial dilution of concentration 10E-2, 10E-3, 10E-4
Do a Spread Plate on VRBA
Do 3 replications fro Each Dilution
Incubate 35˚C, 24 Hrs
Do a colony count (positive’ll be purple red colony, >0.5mm)
Select 3 representative colony an inoculate into BGLB for confirmation
Incubate 35˚C 24hrs
If all the tube are positive (with gases), then above colony count is valid
If only a fraction is valid, take (fraction of positive tube X colony count)

Violet Red Bile Agar

Bile salts suppresses gram positive and non Enterobacteriaceae family members
Lactose differentiates lactose fermentors and non-lactose fermentors. Lactic acid will cause a decrease in pH, Neutral Red indicator will then turn red giving a red colony

Lauryl Sulphate Tryptose Broth (LST)
· Lactose differentiates Lactose fermentors and non ferementors
· Lauryl Sulphate suppresses non-coliform growthTryptose and NaCl provides the right osmopressure for Coliform to ferment

Staphylococcus aureus (S. aureus)

Staphylococcus aureus (S. aureus)

Gram Positive Bacteria
Media BPA

Outline:
Presumption by BPA
Confirmation by BHI, MSA, coagulase test and thermonulease test


Weigh 11g sample to 99ml of peptone water
Put into stomacher for 2 min
Do serial Dilution to achieve 10E-2 and 10E-3
Do spread plate on BPA
3 duplicate for each dilution
Incubate 35˚C for 48 hrs
Observe the colonies
Classify them into Typical, suspect, non typical
Do a colony count for typical and suspect
Pick 4 representative colony of typical colony
Divide BHI agar and MSA agar into 4 sections
Streak 4 colonies into the each of the section
Inoculate 2 colonies into 2 BHI broth (One colony for each broth)
Incubate BHI aerobically 35˚C for 24hr for BHI and MSA anaerobically
Pick 2 colony from BHI for coagulase test


Colonies in BPA will be black shinny convex colon with clear zone
Colonies in BHI will be Yellow round colony
Colonies in MSA will be yellow colonies with yellow zone


Positive coagulase test will show clotting
Thermonuclease test
From Step 13 , after incubation, put the BHI broth in water bath 100˚C for 15min
Divide Toludine Blue DNAase agar into half
Streak each tube (BHI broth)into the respective half
Positive colony will be pink colour in Thermonuclease test

Do the same for suspect colonies

If BHI, MSA , coagulase test and Thermonuclease test is positive, the colony count for typical is confirmed in BPA

If BHI, MSA , coagulase test and Thermonuclease test is positive, the colony count for suspect is confirmed in BPA.

Media Explanation

BPA (Baird-Parker Agar)
· Selective differential media
· LiCl and Potassium Tellurite suppresses non-osmophile
· Egg Yolk contains lecithin
· S.aureus produces lecithinase, therefore it will produce colony with clear zone

Blood-Brain-Infusion (BHI)
· Contains may micronutrients which will enriched S .aureus

MSA (Mannitol Salt Agar)
· Suppresses non-osmophile
· Differentiate non mannitol fermenters
· S. aureus ferments manitol carbohydrate to acid
· When pH decreases phenol red will turn yellow

Toludine Blue DNA agar
· Differentiate heat resistant DNAase producers (S.aureus) from heat resistant DNAase non producers
· Heating will isolates heat stable enzymes activity
· DNAase is produce, DNA hydrolysed into nucleotide
· Nucleotide reacts with toludine, a pdt which is blue colour

Microbiology Provision

Test for micro organism may be tested through conventional media, and also through ELISA and Genetic Methods (PCR and Electrophoresis).

Media for Microorganism

1. Staphylococcus aureus (S. aureus)
2. Escherichia coli 0157:H7 (E. coli) 0157:H7
3. Coliform
4. Salmonella spp.
5. Vibrio spp
6. Listeria Monocytogenes
7. Clostridium botulinum
8. Clostridium perfrigenes

For all test and positive and negative control should be plated

Contents for Pkg II

Areas covered:
Allergens
Toxin
Microbial
Heavy Metals
Growth promoters
Pesticides
Antibiotics
Detection for GM food

Instruments for used for
Allergens
Toxin
Antibiotics
Growth promoters (hormones)

Uses GC-MS for Identification and HPLC for quantification

Instrument used for detection and quantification of Aflatoxin and Orchratoxin:

HPLC-DAD
TLC with UV ray
Or other Instruments that uses UV ray in the instrument to cause fluoresce to the toxin

Instrument for Pesticides:
Initial Screening by GC-MS scan mode / SIM for any high amount of Pesticide that exceeds legal limit
GC-ECD for quantification of Chlorinated pesticide
GC-NPD for quantification of Phosphorus pesticide

Heavy metals:
AAS (Atomic Absorption Spectroscopy)

Detection for GM Food:
PCR (Polymerase Chain Reaction)

Reference

References:
· Food Microbiology, 2nd Edition, M.R Adams & M.O Moss, The Royal Society of Chemistry, Cambridge U.K
· Food Microbiogy: Fundemental & Frontiers, 2nd Edition, Michael P. Doyle, Larry R. Beuchat, Thomas J.Montville, ASM press, Washington D.C USA
· How to Prevent Food Poisoning a practical guide to safe cooking, safe eating and food handling, Elizabeth Scott & Paul Sockett, John Wiley & Sons Inc., USA
· Hen eggs, their basics and applied science,Takehiko Yamamoto,Leknraj Juneja,,Hajime Hatta, Mujo Kim, 1997, CRC Press Inc. Florida USA
· Hygiene for Management , 7th Edition, Richard A. Sprenger, Highfield.Co.UK Ltd., South Yorkshire,UK.

Taxonomy

Taxonomy

Family >Tribe>Genus>Species

Family: Enterobacteriaceae

Tribes:
· Escherichiae
· Klebsielleae
.Proteae
.Yersinieae
.Erwinieae

Descending Genus of Tribe Escherichiae
· Escherichia
· Salmonella
· Shigella
· Edwardsiella
· Citrobacter

Descending Genus of Tribe Klebsielleae
· Klebsiellea
· Enterobacter
· Hafinia
· Serratia

Meaning of true Coliform

· Family of Enterobacteriaceae
· Ferment Lactose

Some definite Coliform

· Escherichia
· Citrobacter
· Klebsiellea
· Enterobacter

Certain strains of Salmonella and Shigella ferment lactose


Faecal Coliform:
Coliform that survive 44-45.4 Degree

Reference microorganism, guide from Codex in Heat "Sterilizing" food.

Reference microorganism, guide from Codex in Heat "Sterilizing" food.

Food With Short shelf life: Listeria monocytogenes: z =7.5 Centigrade D-70 Centigrade: 0.33 min

Food With Medium shelflife: Enterococcus faecalis: z =10 Centigrade D-70 Centigrade : 2.95min

Food With Long shleflife: Clostridium boltulinum Type E toxin: z =10 Centigrade D-90 Centigrade 1.6min

Regulations

Regulations:

Singapore Sale of Food Act CP 283:

Regulation
35 Para. 1
Ready to eat human food cannot have E. coli or any pathogen exceeding 20 CFU/ml or CFU/g

Schedule 12
TPC 37 Degree C 48 hrs Meat Ready for Consumption : Less than 1 million

Minced Meat (European Union)

c=2
n=5

TPC:
· m: 5*10 exp+5
· M:Less than or eq. 5*10exp+6 {10exp+6 = million}{exp+ = Exponential}


E.coli:
· m: 50
· M:Less/= than 500

S.aureus:
· m:100
· M: Less than/= 5000

Salmonella:
· Absent in 25g

Roast Beef : (ICMSF)
Salmonella :
n =20
M=0

Raw Chicken (ICMSF)

TPC
n=5
c=3
m: 5*10exp+5
M: 5*10exp+7

Cooked Poultry, Frozen, Ready to Eat , or Reheated:(ICMSF)

S. aureus:
· n=5
· c=1
· m=10exp+3
· M=10exp+4

Pasteurised, Heat treated Eggs and Pdt ( United Kingdom)

· S. aureus: 0
· Salmonella : Abscent in 25 g or ml
· TPC: Less than/= 10exp+5
· Enterobacteriaceae: Less than/= 10exp+2


Guideline in Ready to Eat Food (Public Health Laboratory Services {PHLS} UK )

· TPC 30 Degree Centigrade, 2 Hrs:
m: 1000
M: 10000

· Enterobacteriaceae

m:100
M: 1000

E. coli
m: 20
M: 100

· Listeria spp.

m: 20
M: 100

· Salmonella spp., Campylobacter spp., E.coli 0157, VTEC, V. cholerae

Absent in 25g

· Listeria monocytogene, S. aureus, Clostridium perfringens
m:20
M: 100

· Bacillus spp.

m:1000
M:10000

Heat Treatments to poultry egg (US FDA)

9 log cycles of Salmonella enterica serotype Typhimurium (Salmonella typhi)

Product Recall

Product Recall

Product ( Pdt) Recall Objective
· Recall Pdt that is potentially Hazardous to consumer

Meaning of Pdt recall
Permanent removal from market
Temp. removal for correction ( Like label print wrongly)

Types of product recall
Class 1
Class 2

Class 1 pdt recall
Life Threatening to consumer
Class 2 pdt recall
Won't cause serious harm to user ( like Nutrition Label print wrongly, cooking times specified wrongly, but under cooking won't cause serious poisoning problem)

Level of Product recall
1.Wholesale
2.Retail
3.Consumer

Initiation of Pdt. Recall

· Government ( Mkt surveillance ,and discovered undersiable subtances that have adverse effect to human like N&N Poultry farm having to much Antibiotics in egg)

· Company (When there is anyforms of defect)

Responsibility of Company
· Inform Gov.
· Notify Relev. Levels of recall
· Relev. Authorities associated with hazard
· Alert General public
· Maintaining proper sale record , Min. 2 Yrs
· Est. SOP for Pdt. Recall

Guidelines to Product recall

1.Mkt survallience by AVA
2.Receive information of product defect
3.Report to AVA and relevant authorities in which the health hazard may cause within 24 hours
4.Disscussion and decission of level of product recall
5.Cease all Sale of defect Immediately
6.Quarantine of Defective Stock
7.Initiate relev. Levels of pdt. Recall Orally
8.Initiate relev. Levels of pdt. Recall With Official Letter
9.Arrangement of collection of defective stock with relv. levels
10Qurantine recalled product in warehouse
11.Submit Recall report to AVA
12.Destruction of product
13.Submit proof of Action to AVA
14.Formulate or Review corrective and preventive action

(Have not verify yet)

Health Hazards

Health Hazards:

Escherichia coli :

· Incubation: Usually 3-4 days , range 2-12 days
· 1-2nd day: Non Bloody diarrhoea
· 3-10th days: Bloody Diarrhoea (some books write 4-10days)
· Usually self limiting, about 2-3days

Health Implications:
· HUS ( Haemolytic Uraemic Syndrome){Kidney failure)
· TTP ( Thrombolic Thrombocytopenic Purpura) { Platelette count drop}
· Haemorrhagic colitis (Bloody Diarrhoea)

Infectious dose:

· 0.3-15 CFU /g

Listeria monocytogene:
Infant:
· Pneumonia
· Speticaemia (Blood Poisoning)
· Abscesses ( Widely disseminated Granulomas)

Adult:
· Speticaemia
· Meningitis (swelling of Brain Tissue)
· Meningocephalitis
· Endocarditis


Pregnant :
· Influnenza symdromes
· Fever
· Headache
· GI symptoms

Health implications for Pregnant mothers:

· Abortion
· Still birth
· Premature labour

Infectious dose :

· 0.3 CFU-100 CFU /g

Campylobacter jejuni:

· Incubation : 1-11days
· Usually: 3-5days
· Usually Self-limiting to 1 to 3 wks
· Diarrhoea
· Systemic illness
· Guillain-Barre Syndrome

Infectious Dose:
· 1000 CFU /g

Guillain Barre Syndrome:
· Acute Paralysis
· Small portion of people die
· Req. assisted ventilator

Salmonella typhi :
.Cause for typhoid
.Incubation 3-56 days
.Usually 10 -20 days
.Mild fever
.Speticaemia
.Bile infection
.Intestinal Ucle
.Fever
.Diarrhoea
.Infectious dose:
10-100 cells


Salmonella enterditis
Incubation : 6-48 Hrs
Mild fever
Nausea
Diarrhoea
Infectious dose , Less than 200 CFU/g

Clostridium botulinum :

· Incubation: Usually 12-36hr but can be up to 14 days

Health Implications:
· Nausea
· Visual Impairment
· Double vison
· Fixed/ dilated pupil
· Loss of oral function
· Lack of muscle coordination
· Respiratory impairment
· Abdominal pain
· Death

Infectious dose of toxin:

· 0.4ng/kg

Clostridium perfingnes
Incubation: 8-16 hrs
Diarrhoea
Severe abdominal cramp
Spontaneously resolved in 12 to 24 hoursInfectious dose : 10E7 CFU/g

Egg Hazard Preventive measure

Recommendations for the measures to be taken to prevent future occurrence:

Specific preventive measures to reduce Chemical Hazards

Provide withdrawal period after the last administration of antibiotics to hen, before the eggs are being use for commercial sales
Carry out sampling in eggs and chicken to check for exceeding limits of antibiotics
Practice good husbandries to the hen

Specific measures to reduce microbiological hazards:

-Heat Treat Egg Product at least 9 log cycles

-Avoid imparting stress to hens. i.e Avoid feeding interruptions and temperature fluctuation around hen’s environment. This is to reduce salmonella spp (SE). To be imparted into the egg.

-feasibility of large-scale use of an in-shell pasteurization process, a relatively new technology

-Ionisation of shelled eggs to reduce salmonella spp. and other microorganism in and on eggs

-cleaning and disinfecting hen houses between flocks

-adopting strict rodent control measures

-clean eggs properly

-putting in place biosecurity measures

-monitoring mortality of chickens

-Using SE-free chicks and pullets.

-Remove sick and injured hens

-Conduct ‘health check ups’for hen

-Sampling of hen’s feces for salmonella

-Reducing Salmonella in chickens by spraying newly hatched chickens with .The bacteria, which the chicks ingest when they peck at their wet feathers, reduce Salmonella colonization in the chicks' intestines.

-eggs packed for consumer use to be refrigerated during distribution at a temperature not to exceed 7 degrees Celsius and to include a label on packages that refrigeration is needed.

-Refrigerating eggs between transport and storage

Please refer to regulations for more details
General Measures
Sampling of hen’s serum for undesired substances

Poultry Egg Hazard

Poultry Egg Hazard

The possible hazards are as follows:
1. Chemical
2. Biological
3. Little Physical Hazards

Types of Chemical Hazards

Antibiotics:
a) Doxcycline
b) Chloramphenicol
c) Nitrofuran

The above antibiotics is to treat avian diseases like salmonella infections

Mode of entry

1. Imparted into eggs when hen is sick and be administered with the above antibiotics
2. Antibiotic enter the hen’s circulatory system and during the formation of egg, it’s being imparted into the egg
3. Site of entries are blood and ovary

Specific preventive measures

a) Provide withdrawal period after the last administration of antibiotics to hen, before the eggs are being use for commercial sales
b) Carry out sampling in eggs and chicken to check for exceeding limits of antibiotics
c) Practice good husbandries to the hen.

Biological hazards:
Microoragnisms:
a) Salmonella spp.

Poultry of Economic Importance:

Poultry of Economic Importance:

· Chicken
· Duck
· Turkey

Operations in Slaughtering Poultry:

· Transportation
· Hang Poultry on its feet
· Electrical Stunning
· Cutting at Carotid Artery
· Scalding at 50 Centigrade
· Mechnical Removal of Feather
· Chill in water

Areas of High CFU counts : Skin (hide)
: Feather
: GI Tract

Cause of Establishment of microorganism, pathogen in Poultry:

· Crowded transportation of Poultry in Lorry, causing rapid transmision of pathogen from poultry
· Poor Sanitation of lorry, bird droppinggs containing fecal coliforms causing to contaminate other birds
· Close proximity slaughtering of Poultry causes rapid transmision of microbes
· Electrical stunning causes birds to to get shock, flapping of wings causes uprise of air-borne pathogen to contaminate other birds
· Feather may also contain Salmonella spp. and Campylobacter spp.
· Mechnical defeathering using Rubber Fingers causes microbes, pathogen to pass from one bird to another
· Worn out Rubber Fingers accumulate microorganism
· Water scalding and Chilling causing Cross contamination from previous birds
· Carcass not washed
· Skin not removed
· Internal organs not removed

Important Biological Hazard Profile in

· Chicken
· Duck
· Turkey

Poultry are suscetible to
· Campylobacter jejuni
· Salmonella spp.
· Listeria Monocytogene

Relative Susceptibility to Campylobacter jejuni, base on frequency in sampling results
· Chicken 14-98%
· Duck 48%
· Turkey 3-25%

Meat of Economic importance:

Meat of Economic importance:

1. Cattle
2. Pig
3. Sheep

Operations in slaughtering Meat ( Non-Poultry)

1. Washing
2. Slaughtering
3.Rigor Mortis
4.Removal of Hide & Viscera (internal organs)
5.Wash Carcass
6.Chill
7. Deboning

Meat Data:

Areas of High CFU counts : Skin (hide)
: GI Tract

Cause of Establishment of microorganism, pathogen in meat:

· Puncturing of Viscera
· Cross contamination from GI Tract
· Cross contamination from Hide
· Cross Contamination from Deboning Equipment.

Importan Biological Hazard Profile in
1. Cattle
2. Pig
3. Sheep

Cattle:
· Escherichia coli 0157:H7 ( High Importance)
· Listeria monocytogene
· Samonella spp.
· Clostridium botulinum
· Prion {Proteinious Infectious Protein, causes bovine encephalopathy spongiform, or Mad Cow Disease (MCD) }

Pig:
· Salmonella spp.
· Trichinella spiralis (Round Worm, common in pig)
· Listeria monocytogenes
· "Pig Flu" ( Havent found out whats the Nonmenclature yet)

Sheep:
· Listeria monocytogenes
· Prion
· Clostridium botulinum

Other Microbilogical Hazrds associated generally in meat and poultry
Clostridium perfringens

Link

Links:
http://www.ava.gov.sg/javascript/module7/press/05apr05_NN_lifted.pdf
http://www.rirdc.gov.au/reports/EGGS/01-111.pdf
http://www.cfsan.fda.gov/~dms/fs-eggs3.html

Assignment: Microbiological Aspect of Egg

This page sumarises the microbioloical aspect of egg's safety

The webpage focuses on the microbiological aspect of egg's food safety, HACCP
It's informative because it covers the safety of egg by covering

• Handling of Egg
• Managing the Hens
• 'Hen's lifestyle ' relating to infections
• HACCP plans
• Hen's living condition
• Handlers

It's worth viewing this page because it provides the above points a detail inoformation, explaination of each CCP in egg 'production'

Most egg are bacteria free when laid. Eggs get contaminated through the egg shell and the environment. Cage birds have lower risk of Salmonella infection (therefore lower risk imparting to egg) than free range birds Cage birds ( Hen) have better Shell structure, therefore reduce chances of bacteria (Salmonella enteritidis ) entry via trans- shell method. To reduce poultry infection from salmonella spp. the sugesstion are as follows:
· obtaining Salmonella-free stock
· Salmonella-free feed
· Exercise pest control
· using providing rodent
· wild bird-free housing
· providing protective clothing for workers
· disinfecting footwear
· frequent cleaning of water supply systems
· water chlorination
· removing sick or dead birds
· removing droppings and litter and cleaning, with subsequent decontamination of houses after removal of flocks
Possible source of Chemical contaminations
• Free range birds on contaminated soils
• Insecticide sprays used while birds are present
• Water medication at incorrect rate
• Eggs washed in non-approved solutions
• Egg washing compound mixed at high concentrations
• Systemic pesticides used in grower shed

•Shed fumigation while birds present
• Chemical feed additives included at wrong rate
• Use of antibiotics

Information Retrieved from

http://www.rirdc.gov.au/reports/EGGS/01-111.pdf
Food Safety Risk Management in Different Egg Production Systemsby R.C. Dawson, J.M. Cox, A. Almond, and A. Moses October 2001 RIRDC Publication No 01/111 RIRDC Project No MS989-28

Miself

Hi,
This is Joseph, from Temasek Polytechnic From Applied Science School, Diploma is Applied Food Science and Nutrition, Okay this is not graded so don't have to write so detail, anyway She say cannot write birthday so must be also can disclose my age la. Any way I stay far away from TP, so Far, I hate taking the bus, but MRT is too expensive.

However, I prefer to take bus cause is cheaper.

I prefer to work Noise-less, so that I can think proberly, and in group wk if U don't think U want to cooperate, pls tell me, don't get me irritated, so that I can alcate the job to (100) some one else.

Expectations:

1. Try to keep me inform about the meetings
2. Be punctual, if not make a phone call
3. Please not make stupid excuses to go away from meetings
4. Project come first THANK YOU
5. Time slot fixed for Project is FOR Project, PLEASE do not do anything else

Ok, anything else I will add on to the list

O ya, When I mean Process Raw data, I MEAN PUT THEM INTO PROPER REPORT FROMAT dont change Font Here, change para, and not data process not done with all the SHORT FORMs and Typo error That I made.