Lecture 16 - Mycotoxins

Question 1

what are mycotoxins?

Answer 1

the toxins produced when molds (filamentous fungi) develops on food commodities)

Question 2

what are hte main producers of mycotoxins?

Answer 2

fungal species belonging to the genera Asperigillus, Fusarium and Penicillin

Question 3

what are the major classes of mycotoxins?

Answer 3

1. aflatoxins
2. ochratoxins
3. trichothecens
4. fumoisins and patulin
5. zearalenone

Question 4

what are examples of aflatoxins?

Answer 4

B1; B2; M1; M2; G1 and G2

Question 5

what are examples of ochratoxins?

Answer 5

ochratoxin A

OTA

Question 6

what are examples of trichothecens

Answer 6

DON (deoxynivalenol)
T2
HT2

Question 7

what are examples of fumonisins

Answer 7

FBs: FB1, FB2, FB3

Question 8

where are patulins found?

Answer 8

apples and apple products

Question 9

what is the order of the food chain “farm to plate”

Answer 9

1. preharvest farm
2. harvest farm
3. post harvest storage
4. processing factory
5. distribution transportation
6. marketing wholesalers and retailers
7. consumption

Question 10

what are 2 categories of mold?

Answer 10

1. field fungi

2. storage fungi

Question 11

where are aflatoxins found?

Answer 11

sorghum
soybeans
corn
wheat
barley

Question 12

where are trichothecens found?

Answer 12

barley
oats
sorghum
soybeans
corn
wheat

Question 13

where are zearalenone found?

Answer 13

wheat
sorghum
corn
barley
silage (forage grass, legume crops and corn)

Question 14

what are the 3 methods of control and detoxification?

Answer 14

1. physical methods: grinding and rinsing, heat treatment, irradiation, degradation, inorganic absorption
2. chemical methods: alkalization, ozone degradation
3. biological methods: microbial absorption, microbial degradation, biological degradation

Question 15

examples of AFB1?

describe the properties of AFB1

Answer 15

• eg. aspergillus flavus and aspergillus parasiticus

solubility:
- soluble in polar organic solvents
- poorly soluble in water
- insoluble in petroleum ether, ethyl acetate and hexane

pH:

• stable in neutral solutions
• resistant to strong acids
• rapid decomposition in alkaline

UV:

• produces fluoresence (group B&G)
• destructive for low conc of AFB1

heat:

• stable in 200deg C
• decomposed until 268degC
• hard to destroy under normal temp

Question 16

what are examples of Zearalenone ZEN

describe properties

Answer 16

eg. fusarium graminearum and fusarium culmorum

solubility:

• insoluble in water
• slightly soluble in hexane
• more soluble in benzene, acetonitrile, methylene, chloride, methanol, ethanol, acetone
• soluble in aqueous alkali

pH:

• stable in neutral and acidic
• ester bond will be open in alkaline environment

UV:
- exhibits blue-green

heat:

• melting point is 161degC
• hard to destroy under normal cooking

Question 17

what are examples of deoxynivalenol?

describe properties

Answer 17

eg. fusarium and gibberella

solubility:

• soluble in water and polar solvents
• insoluble in hexane and diethyl ether

pH

• sensitive in alkaline environment
• stable in neutral and acid

UV:

• absorption peak under short wave UV
• decomposes under high tense UV

heat

• resistant to heat
• stable in 120degC for 1 hr
• decomposes at 150degC for 15 min

Question 18

what are the types of pre-treatment/extraction in mycotoxin analysis

Answer 18

1. liquid-liquid extraction (LLE)
2. supercritical fluid extraction (SFE)
3. solid phase extraction (SPE)
4. solid phase micro-extraction (SIME)

Question 19

what are 3 types of clean up detection in mycotoxin analysis?

Answer 19

1. thin layer chromatography
2. gas chromatography
3. high performance liquid chromatography

Question 20

how is a mycotoxin sampling plan defined?

Answer 20

by a mycotoxin test procedure and a defined accept/reject limit

Question 21

how much can the number of containers sampled vary?

Answer 21

can vary from 1/4th (for small containers less than 20 metric tons) to the square root of the total number of containers (for large containers greater than 20 metric tons)

Question 22

in sampling, how do you ensure that the analytical test portion has the same conc of toxins as the original sample?

Answer 22

the whole primary sample must be ground and mixed

Question 23

describe the liquid-liquid extraction

Answer 23

• two immiscible phases shaken together

- compounds partition to their preferred phase (into aqueous or organic solvent)

Question 24

describe the solid phase extraction/clean up and concentration

what are advantages?

Answer 24

• SPE is based on molecularly imprinted polymers that are syntehtic receptors for the mycotoxins
• used for sample extraction, clean up and concentration
• C18 (octadecylsilane), silica gel, anionic and cationic exchange materials, immunosorbets and molecular imprinted polymers

adv
- faster and most popular method

Question 25

describe the solid phase concentration what are advantages?

Answer 25

- uses immunoaffinity columns (IACs) - used for purification of samples which are contaminated with different mycotoxins - the mycotoxins (analytes) are bound selectively to the antibodies on the column - rinsing removes most possible interferences. toxins can be eluted by antibody denaturation adv: - offers high selectivity

Question 26

describe thin layer chromatography (TLC) what gel is used? what are advantages and disadv?

Answer 26

- used for detection step (for quantitative and semi-quantitative measurements of mycotoxins) - detects by fluorodensitometry or visual procedures - uses silica gel, F254 fluorescent silica gel, or silica gel impregnated with organic acid (for detection of aflatoxins, citrinin, fumonisin) adv: - costs less - simple - rapid screening dis - lack of automation has let to TLC being replaced by other techniques

Question 27

describe gas chromatography for detection

Answer 27

- detection is achieved by linking the system to mass-spectrometry (MS), flame ionization or fourier transform infrared spectroscopy

Question 28

why is GC not a technique suitable for commercial purposes?

Answer 28

not suitable for commercial purposes because of its limitations to volatile and thermostable compounds

Question 29

when detecting mycotoxins with GC, why do mycotoxins need to be derivatised first? what are examples of chemical reactions used for derivatisation?

Answer 29

since mycotoxins are not volatile thus, derivitisation by chemical reactions (eg. silylation or polyfluoroacylation) are needed in order to be quantified

Question 30

what is the most widely accepted official method for determination of mycotoxins?

Answer 30

HPLC | high performance liquid chormatography

Question 31

describe detection by HPLC

Answer 31

HPLC = high performance liquid chromatography

Question 32

what kind of detection is used in HPLC?

Answer 32

UV fluorescence amperometric spectrofluorimetric

Question 33

how can mycotoxins be detected directly by HPLC-fluorensence (HPLC-FD)

Answer 33

when mycotoxins have natural fluorescence (ochratoxin or citrinin)

Question 34

what has increased detection sensitivity in comparison to HPLC-FD?

Answer 34

Mass spectroscopy with electrospray or pressure chemical ionization

Question 35

what is thel imit of detection by HPLC-MS?

Answer 35

0.1 and 1 microg/Kg

Question 36

what are advantages of HPLC? disadv?

Answer 36

adv - high resolution - improved limit of detection - possibility to be coupled to multiple detection automated systems disadv: - most chromatographic assays are expensive, time consuming and require expensive equipment and clean up

Question 37

what is ELISA?

Answer 37

enzyme linked immuno sorbent assay - based on the ability of a specific antibody to distinguish the 3D structure of a specific mycotoxin - the direct competitive ELISA is commonly used in mycotoxin analysis