Determination of mycotoxins in samples

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					               MOULDS, MEALS , MAN AND MYCOTOXINS

                              S O FAPOHUNDA, Ph.D.

                                    President, NMASN

                                 Telephone 234-8033709492
                                 oystak@yahoo.co.uk

                                             or

                               info@mycotoxin -ng.org

Abstract

The health and economic impacts of mycotoxins are discussed. Global challenges

,expressed as varied or lack of permissible limits, arising from differences in sampling

techniques, analytical procedures and poverty are highlighted. The need to adopt a

holistic management approach from ‗the farm to the fork‘ is emphasized.



Introduction

Mycotoxins ,an important class of xenobiotics (in terms of morbidity), are biologically

active ,non volatile secondary metabolites that are chemically poisonous to man and

animals in low concentrations. They are produced by toxigenic strains of Aspergillus,

Fusarium, Penicillium, Alternaria Claviceps and a few atypical ones that grow on grains,

seeds, fruit crops and even beer when kept in favourable conditions of temperature and

humidity. Factors that enhance mould growth and mycotoxin production include moisture

content above 15% for wheat and maize, and above 8% for peanuts ,temperature regime

of 25-30 0c, presence of broken grains, mites and insects and prolonged storage time.

Increased consumer cost , less nutritious foodstuff, greater susceptibility to disease , and
reduced export value are some of the economic implications. Unlike other natural plant

poisons, or microorganisms, mycotoxins ,which affect about 25% of global crops, are

extremely stable and may not be destroyed by cooking. Direct consumption can result in

the deposit of their residues in organs like the kidney, liver, heart and products such as

milk and egg inciting metabolic disorders in them .They are present in foods and feeds

even when the moulds that produced them are no longer present.. The modes of entry to

man and animal include ingestion, skin contact and inhalation of spore-borne toxin. Once

exposed to it, the human body runs a triple risk to its effect. These are direct toxic action,

acquisition of mutated RNA from the toxin parent fungus and creation of an internal

biofilm that can harbour a ‗soup‘ of disease.

Classification It is a challenge to classify this group of metabolites because although

mycotoxins are of fungal origin, not all compounds made by fungi are mycotoxins e g

fungal products that attack bacteria are called antibiotics, those that attack plants are

phytotoxins (just as those secreted by higher plants). Ethanol and mushroom poisons are

most times excluded .More than 300 are known but the common ones are:



--Aflatoxins—These include B1 , B2, from Aspergillus flavus and G1, G2 from

Aspergillus parasiticus and other atypical ones, M1 and M2 are the hydroxylated

metabolites of B1 and B2 and are excreted in milk ..B2 and G2 are the dihydro-

derivatives of the parent compounds. In decreasing order of toxicity it is B1,G1, G2, and

B2.

--Trichothecenes—e g deoxynivalenone DON or vomitoxin from Fusarium spp

--Zearalenone---from Fusarium spp
--Ochratoxins from Aspergillus spp and Penicillium spp

--Fumonisins from Fusarium spp



Health Implications

Sporadic cases of human mycotoxicosis, particularly aflatoxicosis, expressing as liver

cancer,chronic hepatitis,jaundice hepatomegaly and cirrhosis due to repeated

consumption of low levels of the toxin have reported in parts of Africa and Asia. They

also incite reproductive and neuro-disorders as well as immunosuppression. The

Europeam Mycotoxin Awareness Network reported that autopsy of brain specimens

from kwarshiokor children and other children who had died from a variety of other

diseases showed high levels of aflatoxin presence . Cases like this have been noticed in

Kenya, India and Nigeria.

The global health and trade concern gave rise to the launching of the International

Society for Mycotoxicology(ISM) in 2005. The Nigeria Mycotoxin Awareness and Study

Network(NMASN) was formed in 2006.



Determination of mycotoxins in samples

This process involves stages like sampling, sample preparation, analysis (Fig 1)Because

of the critical nature of the sample preparation , about 60% of the total analysis time and

effort is devoted to it. Mistakes or carelessness at this stage accounts for the curious

sources of errors and variations in the results obtained by different laboratories.

The mycotoxin concentration in a bulk lot of human foods and animal feeds is estimated

by analyzing small samples taken from the lot(Whittaker 2006)although due to some
uncertainties, the exact concentration in the lot cannot be measured with 100% certainty.

Because contaminated particles may not be evenly distributed throughout the lot, random

sampling is strongly advised so that analytical sample is fully representative of the

consignment. The standard recommendation is about 200g as an increment; and one

incremental portion is taken for every 200kg of product. The gathered incremental

portions is called the bulk sample from which the final comminuted test sample is taken.

In static lot( e g bags, cans , drums etc)the standard number of containers sampled may

vary from 25% of total(if less than 20 tonnes) to the square root of the total number of

containers (if more than 20 tonnes)(FAO/WHO 2001) in dealing with a moving stream of

sample e g from a production line , in a factory, small increments can be taken at regular

intervals. Whether static or streaming, sample variance generally decrease with an

increase in sampling size ( Johansson et al 2000). For peanuts, and similar crops,

subsampling by dry milling is accepted because it is easy to apply and fast in

comminuting samples into analytical portions,if the sample are less than 4kg, beyond

which there can the incidence of cloggering and buttering. In recent times slurry mixing

is recommended by Spanjer et al ,2006 because it can handle larger samples, up to 10kg

and there is a drastic reduction in subsampling errors as well as chances as false positive

or negative values. A statistical model is now available to estimate uncertainty associated

with results of measurement of mycotoxins based on small numbers of

increments(MacArthur et al ,2006)



EXTRACTION
Extraction is performed from a sample when 2 immiscible liquid phases or a solid and a

liquid phase are present with the solvent selected to extract the analyte(desired

mycotoxin) quantitatively. The factors that can affect efficiency of the process include

volatility,stability, recoverability, water immiscibility and transparency to UV light(Kos

and Krska 2001). The following are some common extraction solvents :

Aflatoxin—acetonitrile/water; methanol/water

Type A Trichothecenes----acetonitrile / water ; methanol/ water

Type B Trichothecene-----acetonitrile/water ;water/PEG ;chloroform/methanol

Zearalenone--- ethyl acetate, methanol,acetonitrile,chloroform, and mixtures therof

Ochratoxin A----chloroform, acetonitrile/water;

Fumonisins----- methanol /water(3:1); acetonitrile/water

Patulin-----ethyl acetate, acetone(from Krska et al 2001)




DETECTION

Methods of analysis and detection include the Thin Layer Chromatography TLC ,Gas

chromatography GC,Liquid Chromatography with Mass Spectrometric detection(LC-

MS) and Biosensors ,ELISA and the HPLC (with the Fluorescence detector). Detection

by TLC and HPLC is based on their fluorescence under UV radiation and both can

achieve quantification. The HPLC technique , although sensitive is more expensive than

TLC. .The efficiency of TLC analysis, which is popular due to the relative ease of use

and less cost , can depend on factors like
a . nature and quality of the coat ,silica gel, which may reflect on the ability of the

aflatoxins to separate out from each other and from interference and

b. reference standards the level of purity of which will determine the reliability of result

.When not in use, the standards are to be stored in the dark and at low temperatures

c. development, it is advised that only one chromatogram should be developed at a time

and as soon as plates are spotted. Development time varies with the solvent,silica gel

particle size and moisture content




In a solid sample , the summary of procedure is presented in the following chart

Fig 1 Determination of mycotoxins in solid samples

Sampling

     ↓

Grinding and Homogenisation

     ↓

Extraction 10-200g sample

     ↓

Filtration
     ↓

Clean up, Separation of matrix from analyte

          ↓

Preparation for analysis, pre concentration with defined volume of solvent

      ↓

Analysis

(Source: EMAN Analytical methodology-sampling preparation techniques for the

determination of mycotoxins)




Setting Permissible Limits

To reduce the risk associated with contamination, regulations and law3s are made and

endorsed by countries and international authorities. Regulatory limits for Food additives,

residues of veterinary drugs and pesticides, naturally occurring toxicants and human

derived contaminants in commodities involved in world trade are set by the Codex

Alimentarius Commission. This body, currently consisting about 165member coutries

was formed after the World War 11,to safeguard international public health and enhance
trade. The scientific advisory body for Codex is the joint FAO/WHO expert committee

on food additives—JECFA. By the end of 2003, the number of countries that had set

regulatory limits on foods and feeds had reached 100, particularly for total aflatoxins and

aflatoxin B1. regularly, the European countries meet to set those standards which are

maximum permissible, measured in parts per million ppm or as parts per billion ppb, or

as microgram per kilogram. As stated in 2004 at the EU special workshop held in

Brussels permissible limits are not perfect as errors arising from diverse sampling

models, for bulk commodities ,sample homogenization and measurement uncertainties

during analytical procedures, can account for varied limits among countries.. For example

in the case of edible groundnuts, the US and Australia set 20ppb for total aflatoxins,

while the EU countries have a low limit of 4ppb for same and 2ppb for aflatoxin B1.

Setting permissible limits has to strike a balance between the risk associated with eating

contaminated foodstuff with that of not having enough to eat, particularly in a rising

economically-challenged population as found in Africa .




Factors Determining Safe Limits

    Chemical class of the toxin. Aspergillic acid, vomitoxin and Ergot alkaloid will

       not act same way.
 Presence of other mycotoxins. Particularly in field situations when they exhibit

    synergy and potentiation.

 Species of consumer involved. For example, ducklings are about 10 times more

    sensitive to aflatoxin than laying hens.

 Health status of man or animal involved. Stress, nutritional and disease status can

    determine the response to a given mycotoxin.

 Parameters measured.—eg disease resistance and weight gain respond differently

    to same concentration.

 Sampling and assay procedures ---Safe limits are dictated by the accuracy of

    methods.



   SOME PERMISSIBLE LIMITS

 <20ppb( USFDA )aflatoxin in human foods and animal feeds.

 10μg/kg for total aflatoxin for grains to be subjected to further sorting (Codex)

 0.5μg/L for aflatoxin in milk(FDA)

 2ppb for aflatoxin B1 ;4ppb for total aflatoxin for edible groundnut (EU)

 0.5μg/kg for OTA for raw cereals 0.3µg/kg for malt(EU)

 Fumonisin B1—5ppm for horses, 50ppm for cattle and poultry(AAVLD).

 0.05ug/kg of body weight of zearalenone per day .As at 2001,zearalenone levels

    in foodstuff are not regulated anywhere

 0.4mg/kg of body weight per day for patulin(FAO-WHO)

 1ppm for DON(vomitoxin) for human consumption.(FDA)

 For beef cattle,DON is 50ppb, zearalenone is 250ppb
    Nigeria, has not set her own specific mycotoxin limits. This however, does not

       mean the problem is ignored




MANAGEMENT OF MYCOTOXIN CONTAMINATION

No one control method has been adjudged satisfactory till date. Strategies like

microbial,chemical and thermal inactivation of toxins, physical separation of

contaminated feedstuff, irradiation, ammoniation and ozone degradation have their

drawbacks in time consumed and cost(Dawson 2001)

Binders like bentonites, zeolites,aluminosilicates and a yeast-derived glucomannan are

sold commerciallty. Of all these , a glucomannan derived from yeast cell

walls(Mycosorb TM) proved fairly effective against a wide range of toxins(Swamy et al

2002). This organic polymer presents a wide surface area and an enough specificity to

allow effective mycotoxin binding at a low level of dietary inclusion. A biological

approach involving competition of other fungal species led to a reduction in the aflatoxin-

producing ability of Aspergillus flavus(Horn and Dorner 2002). The use of Lactic acid

bacteria, LAB, is also an attraction to an extent. However,a holistic concept of

intervention from the farm through processing to the dining table , which takes in to

consideration the salient features of Good Agricultural Practices (GAP) and Good
Manufacturing Practices (GMP)with emphasis on the HACCP guidelines ,is most

attractive.




SAFETY PRECAUTIONS

While working on mycotoxin analysis in the laboratory, the following precautions are

advised

-a whole lab or a part should be exclusively reserved for analysis.

-the bench top should be of nonabsorbent material

-fume cup board or extraction hood should be used always

-avoid use of Bunsen burners, or sparking apparatus because the solvents can be

flammable e g acetone

-avoid eating ,smoking, or drinking in the lab

-while grinding to achieve fine particles during sample preparation , there is the risk of

absorbing toxin into the skin or inhalation of dust. It is advisable to wear protective

clothing, masks and spectacles

-glassware and TLC plates can be decontaminated by soaking for 2 hours in a 15 sodium

hypochlorite . after this time 5% acetone should be added and the glassware soaked for

another30 mins(Anon, 1980)

-
SOME AGENCIES CONCERNED WITH APPROVAL OF ANALYSES AND

LIMITS

   AOAC

   USDA

   FDA

   IUPAC

   AACC

   EU

   ISO( International Organization for Standardisation)

   FAO-WHO Expert Committee on Food Additives and Contaminants

   FAO-IAEA

   Codex Alimentarius Commission (an organ of the WHO)

   National bodies e.g. NAFDAC established by Decree 15 of 1993 as amended,

     was set up to ―control and regulate the manufacture, importation, exportation,

     distribution, advertisement, sale and use of drugs, food, cosmetics, chemicals,

     detergents, medical devices and all kind of drinks‖.

   SON

   Institute of Public Health Japan
    The IPAN is the only professional body in Nigeria empowered to carry out

       scientific analysis of consumable items for mycotoxin levels.




NOTE

One part per million ppm is equivalent to

-one penny in USD 10,000

-one minute in 2 years

-a single corn in 2 tons of corn

One part per billion ppb is equivalent to

-one penny in USD 10,000,000

-One second in 22 years

- a single kernel in 2000 tons of corn




References

-Anon 1980 Laboratory decontamination and destruction of aflatoxins B1, B2, G1 G2 in

Laboratory wastes (ed Castegnaro et al ) International Agency for Research on Cancer

Publ 37-41

-Dawson K A 2001 the application of yeast and yeast derivatives in the poultry industry.

Proc Australian Poultry Science Symposium 13;100-105
-FAO/WHO 2001. Proposed draft revised sampling plan for total aflatoxin in peanuts

intended for further processing. Joint FAO/WHO Food Standard program. CODEX

Alimentarius commission 24th session, Geneva, Switzerland, 2-7 July 2001 Rome

FAO/WHO pp 276-280

-Horn B W and Dorner J W 2002 Effect of competition and adverse culture conditions

on aflatoxin –production by Aspergillus flavus through successive generations Mycologia

94 (5) 741-751

-Krska R 1998 Performance of modern sample preparation techniques in the analysis of

Fusarium mycotoxins in cereals J. Chrom A 815: 49-57

-Krska, R; Baumgastner , S and Josephe, R 2001 The state of the art analysis of type A

and type B Trichothecene mycotoxins in cereals. Feesenius Journal of Analytical

Chemistry 371; 285-299

-Macarthur R; Macdonald S; Breeton P and Murray A 2006 Statistical modeling as an aid

to the design of retail sampling plans for mycotoxins in food. Food Additives and

Contaminants 23 (1) 84-92

-Spanjer M C , Scholen, J M ; Karstup S; Jansen U; Schazka T F and Toyonoko N J 2006

Sample comminution for mycotoxin analysis; dry milling or slurry mixing. Food

Additives and Contaminants 23 (1) 73-83

-Swamy, H V; Smith T K ;Scotter P F ; Boerman H J and Sefton A E 2002 Effect of

feeding blend of grains naturally contaminated with Fusarium mycotoxins in production

and metabolism in broilers Poultry Science 81: 966-975

-Whitaker T B 2006 sampling foods for mycotoxin Food Additives and Contaminants 23

(1) 50-61
-

Acknowledgements: Documents from EU, FDA, Engormix, EMAN, were consulted in

writing this paper and are hereby duly acknowledged .

				
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