Friday, July 20, 2007

Limits on Toxins

Limits for Aflatoxins
This website shows some limits on different toxins such as mycotoxins (aflatoxins) and ochratoxins and its different levels permitted in different types of foods.


Qualitative and quantitative detection of aflatoxin B1 in poultry sera by enzyme-linked immunosorbent assay

Qualitative and quantitative detection of aflatoxin B1 in poultry sera by enzyme-linked immunosorbent assay

This website introduces the principles of the qualitative and quantitative detection of aflatoxin B1 by ELISA.


Thursday, July 19, 2007

Gene Technology and Future Foods

Gene Technology and Future Foods
(This website states on principles of ELISA and othe immunoassays carried out to detect pathogens and toxins found in genetically modified foods.)

Robinson, S., PhD, Scott, N., PhD & Gackle, A.,BSc (2000). Gene technology and future foods. Retrieved June 17, 2007 from

Toxins : Aflatoxins

Aflatoxins are toxic metabolites produced by certain fungi in/on foods and feeds. They are probably the best known and most intensively researched mycotoxins in the world. Aflatoxins have been associated with various diseases, such as aflatoxicosis, in livestock, domestic animals and humans throughout the world. The occurence of aflatoxins is influenced by certain environmental factors; hence the extent of contamination will vary with geographic location, agricultural and agronomic practices, and the susceptibility of commodities to fungal invasion during preharvest, storage, and/or processing periods. Aflatoxins have received greater attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. As it is realized that absolute safety is never achieved, many countries have attempted to limit exposure to aflatoxins by imposing regulatory limits on commodities intended for use as food and feed.

In Raw Agricultural Products:
Aflatoxins often occur in crops in the field prior to harvest. Postharvest contamination can occur if crop drying is delayed and during storage of the crop if water is allowed to exceed critical values for the mold growth. Insect or rodent infestations facilitate mold invasion of some stored commodities.
Aflatoxins are detected occasionally in milk, cheese, corn, peanuts, cottonseed, nuts, almonds, figs, spices, and a variety of other foods and feeds. Milk, eggs, and meat products are sometimes contaminated because of the animal consumption of aflatoxin-contaminated feed. However, the commodities with the highest risk of aflatoxin contamination are corn, peanuts, and cottonseed.

In Processed Foods:
Corn is probably the commodity of greatest worldwide concern, because it is grown in climates that are likely to have perennial contamination with aflatoxins and corn is the staple food of many countries. However, procedures used in the processing of corn help to reduce contamination of the resulting food product. This is because although aflatoxins are stable to moderately stable in most food processes, they are unstable in processes such as those used in making tortillas that employ alkaline conditions or oxidizing steps. Aflatoxin-contaminated corn and cottonseed meal in dairy rations have resulted in aflatoxin M1 contaminated milk and milk products, including non-fat dry milk, cheese, and yogurt.
Corn can be used to produce flour and starch products and this links back to the problem statement such as aflatoxins is a likely toxin to be found in foods produced by the company in the problem statement.

Recent Methods of Analysis for Aflatoxins in Foods and Feeds
Sampling and Sample Preparation:
Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample preparation, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level. In this regard, specific plans have been developed and tested rigorously for some commodities such as corn, peanuts, and tree nuts; sampling plans for some other commodities have been modeled after them. A common feature of all sampling plans is that the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample.

Solid-Phase Extraction:
All analytical procedures include three steps: extraction, purification, and determination. The most significant recent improvement in the purification step is the use of solid-phase extraction.
Test extracts are cleaned up before instrumental analysis(thin layer or liquid chromatography) to remove coextracted materials that often interfere with the determination of target analytes.

Thin-Layer Chromatography:
Thin layer chromatography (TLC), also known as flat bed chromatography or planar chromatography is one of the most widely used separation techniques in aflatoxin analysis. Since 1990, it has been considered the AOAC official method and the method of choice to identify and quantitate aflatoxins at levels as low as 1 ng/g. The TLC method is also used to verify findings by newer, more rapid techniques.

Liquid Chromatography:
Liquid chromatography (LC) is similar to TLC in many respects, including analyte application, stationary phase, and mobile phase. Liguid chromatography and TLC complement each other. For an analyst to use TLC for preliminary work to optimize LC separation conditions is not unusual.
Liquid chromatography methods for the determination of aflatoxins in foods include normal-phase LC (NPLC), reversed-phase LC (RPLC) with pre- or before-column derivatization (BCD), RPLC followed by postcolumn derivatization (PCD), and RPLC with electrochemical detection.

Immunochemical Methods:
Thin layer chromatography and LC methods for determining aflatoxins in food are laborious and time consuming. Often, these techniques require knowledge and experience of chromatographic techniques to solve sepatation and and interference problems. Through advances in biotechnology, highly specific antibody-based tests are now commercially available that can identify and measure aflatoxins in food in less than 10 minutes. These tests are based on the affinities of the monoclonal or polyclonal antibodies for aflatoxins. The three types of immunochemical methods are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoaffinity column assay (ICA).

These are mostly chemical methods of detection but still provide an insight into the immunochemical methods such as ELISA and RIA which can used to detect aflatoxins in foods, such as flour and starch products produced by the company.
Confirmation of Identities of the Aflatoxins:
Although analytical methods might consist of different extraction, clean-up, and quantitation steps, the results of the analyses by such methods should be similar when the methods are applied properly. Since the reliability of the quantitative data is not in question, the problem still to be solved is the confirmation of identity of the aflatoxins. The confirmation techniques used involve either chemical derivatization or mass spectrometry (MS).

Monitoring Techniques for Assessing Human Exposure to Aflatoxins
In the last few years, new technologies have been developed that more accurately monitor individual exposures to aflatoxins. Particular attention has been paid to the analysis of aflatoxin DNA adducts and albumin adducts as surrogates for genotoxicity in people. Autrup et al.(1983) pioneered the use of synchronous fluorescence spectroscopy for the measurement of aflatoxin DNA adducts in urine. Urine samples collected after exposure to alfatoxins were found to contain 2,3-dihydroxy-2-(N7-guanyl)-3-hydroxyaflatoxin B1, trivially known as AFB-Gual. Wild et al.(1986) used highly sensitive immunoassays to quantitate aflatoxins in human body fluids. An enzyme linked immunosorbent assay (ELISA) was used to quantitate aflatoxin B1 over the range of 0.01 ng /ml to 10 ng/ml, and was validated in human urine samples. Using this method, aflatoxin-DNA adduct excretion into urine was found to be positively correlated with dietary intake, and the major aflatoxin B1-DNA adduct excreted in urine was shown to be an appropriate dosimeter for monitoring aflatoxin dietary exposure.


Wednesday, July 18, 2007

Possible Toxins Found in Flour and Starch Products

List of Molds That Can Be Found in Foods
Gulf Coast Mold Prevention, Inc. (2006). List of Common Molds. Retrieved July 21, 2007 from

(From the list of moulds, here are some selected ones to focus on. Research has been conducted to ensure that these are common molds which are commonly found in flour and strach products, as produced by the company in the problem statement. The research below focuses on the toxins which are produced from the possible moulds found.)

Ergot is a toxin caused by Claviceps purpurea. The disease causes ergotism in livestock if hays or grains are infected. Ergot occurs every year on cereals and grasses, and is more prevalent in rye and triticale. The most common sign of ergot is the dark purple to black sclerotia found replacing the grain in the heads of cereals and grasses just prior to harvest. The ergot disease occurs abundantly during wet seasons. The wet weather and wet soils favor germination of the ergot bodies.

(This research shows that grains which can be used to produce flour like cereals and rye can have the toxins of ergot present in them. If contaminated crops are used, this would be a toxin that is commonly found in the end product of flour.)

Ergot is toxic to animals, the most susceptible being cattle. Two well known forms of ergotism exist in animals, an acute form characterized by convulsions, and a chronic form characterized by agalactia and lack of mammary gland development, prolonged gestations, and early foal deaths in mares fed heavily contaminated feed. Symptoms of convulsive ergotism include heyperexcitability, belligerence, ataxia or staggering, lying down, convulsions and backward arches of the back. Symptoms of gangrenous ergotism involve the extremities of the animal including the nose ears tails and limbs. For humans consumption of food contaminated with Ergot can cause vomiting, diarrhea, hallucinations, and may lead to gangrene in serious cases. As recently as 1951 there was an outbreak of the disease in s small town in France. People who bought fresh bread from a local bakery started experiencing burning sensations in their limbs, began to hallucinate. Many other outbreaks were reported, and the chemical said to cause the hallucinations is actually LSD. Although Ergot can cause many problems, it has both medical and recreational uses for many.

(Freshly- produced bread could be contaminated and harmful to human health if the flour contaminated with ergot is used and this relates to the problem statement.)

Ergot is a disease of rye caused by the fungus Claviceps purpurea, which infects the flowers and produces hard mycelial masses (ergots) in place of the grains. The ergots contain numerous alkaloids, and if they are ground along with healthy grain the resulting flour and baked breads can cause a condition known as ergotism; historical records of epidemics indicate that the symptoms followed two different patterns.

(Rye can also be used to produce flour and starch products. Research shows that any baked products baked with the contaminated flour can also be still infected with this toxin despite being baked at high temperatures.)

Mycotoxins are mainly produced by fungi growing in contaminated foods; the compounds most commonly develop during storage and remain within the food after processing and cooking.

If eaten by humans or livestock, these toxins can have profound chronic and acute effects; mycotoxins are also highly carcinogenic. An example is a group of toxins called aflatoxins produced by the fungus Aspergillus flavus. Foods contaminated with this fungus have killed fowl and other animals, and humans have also died from eating contaminated corn (e.g., in 1974 in India) It is also possible that high rates of liver cancer among some groups of people in Asia and Africa are associated with consumption of aflatoxin-contaminated foods. More than 200 mycotoxins have been identified from 150 species of fungi; conditions that lead to food spoilage, such as warm temperatures, are important environmental triggers in some species for the production of toxins.

(Aflatoxins which is caused by the Aspergillus microbes can be found in crops such as corn so it is a possible microbe found in corn. Corn is a common crop used to produce flour and the above research also shows that if contaminated crops are used for production of e.g. flour, the toxins can still be present in the end product like baked breads after processing and cooking.)

Distributed in soils and plants worldwide, Fusarium can invade corn and barley and produce toxins at lower temperatures than many fungi. Fusarium has affected water-damaged carpets, and can cause infections in immunocompromised individuals. Frequently involved in eye, skin and nail infections, and is reported to be allergenic.

(I have not done much research much on Fusarium yet. But it’s a mould commonly found in grains like barley and corn. Corn again, here, can be used to produce flour and thus, it’s a microbe that can be included in the report, as it relates to the problem statement as well. The toxin produced is the “trichothecene” toxin.)

However, I have found a website that elaborates more on trichothecene mycotoxins:

Levetin & McMahon. (2003). Fungi and Human Health: Drugs, Poisons, Pathogens, Allergies. Retrieved July 21, 2007 from,%20etc/Fungi%20V.pdf

Friday, July 13, 2007

Package 2 - Task from HFLA Template

What are Genetically Modified Foods?
Genetically modified (GM) foods are produced from genetically modified organisms (GMO) and a food that comprise one or multiple ingredients that are derived from GM plants or organism, is also considered a GM food.

Genetically modified organisms (GMO) refers to an organism in which the genetic material has been changed through modern biotechnology in a way that does not occur naturally by multiplication and, or natural recombination. (WHO, 2005)

Modern biotechnology means the application of,
a) in vitro nucleic acid techniques, including recombinant deoxyribonucleic acid (DNA) and the direct injection of nucleic acid into cells or organelles, or
b) the fusion of cells beyond the taxonomic family, that overcome natural physiological, reproductive or recombination barriers and that are not techniques used in traditional breeding and selection.

Types of GM Foods
Genetically modified foods include medicines and vaccines, foods and food ingredients as well as feeds and fibers. Foods have been modified to make them resistant to insects and viruses and also to be herbicide- and insecticide-resistant. Crops that have been modified for these purposes include:
• Maize
• Soybean
• Oilseed rape (Canola)
• Cotton
• Corn
• Squash
• Potato

Others included:
· tomatoes with increased vitamin content
· foods such as peanuts with reduced or no allergenicity
· potatoes with higher starch content which absorbs less oil in cooking
· wheat with increased levels of folic acid to prevent spina bifida
· wheat with increased fibre to reduce the risk of colon cancer
· tomatoes that can ripen on the vine for better taste, but with a longer shelf life
· rice with increased pro-vitamin A content to help combat blindness in rice- dependent developing countries

Genetic Modification of Plants
Using techniques of genetic modification, it is possible to produce plants that may have the following properties:
· Disease and pest resistance
· Greater yields
· Herbicide tolerance
· Modified protein and oil content
· Improved nutritional properties
· Improved flavour due to delayed ripening
· Resistance to environmental stress e.g. drought, salinity, or cold
· Production of pharmaceuticals and other chemical substances
(Food Safety Authority of Ireland, 1999)

How Are Plants Modified?
Transgenic crops are referred to having received a foreign segment of DNA or gene(s) from another organism. Recombinant DNA techniques are often being used to alter the genetic makeup of different organisms by combining different selected individual genes from a few organisms or by inserting valuable genes into plant genomes.

This process of introducing foreign DNA into a plant genome is called transformation and is conducted, mainly using two methods:

a) The more popular method for genetic engineering of crop plants is natural gene transfer via an Agrobacterium tumefaciens vector, a bacterium normally found in soils. Many species of crop plants are susceptible to A. tumefaciens, which causes “crown gall” disease when the DNA (T-DNA) is transferred onto the plants in nature. However, removing the disease-causing gene from this bacterium now allows the T-DNA to transport foreign genes into plants.

A. tumefaciens cells, carrying the various selected foreign genes are then incubated with cultured cells of the recipient crop plant and a transgenic crop is regenerated from this process.

However, not all transgenic crops will be successfully regenerated and there is a need to identify the modified genes using marker genes which are closely linked to the genetic material being transferred. These marker genes also usually provide resistance to antibiotics such as kanamycin or herbicides.

b) Particle bombardment is also used for transformation of crops such as cereals. This process involves the coating of DNA on metal particles such as gold, and shooting the particles into plant cells using a particle gun. A small amount of plant cells that are hit with the coated particles receive the genetic material from the transferred DNA and transgenic crops can be regenerated from these cells.

Similarly to A.tumefaciens cells, marker genes are used for selection purposes and are only required in the initial stage of crop transformation.

Genetic Modification of Microorganisms
Genetically modified microorganisms contain genetic material that is artificially introduced and rearranged in an intentional and predetermined manner, which is unlikely to occur in nature.

Examples of genetically modified microorganisms include:
· bacteria that produce or enhance the amounts of novel and/or modified enzymes (e.g. rennet for cheese-making)
· bacteria that produce substances (peptides or proteins) with medical applications e.g. interferon for cancer treatment and insulin for diabetics
· viruses with medical applications e.g. disabled vaccinia virus which is used in gene therapy
· bacteria that can be used as live-vaccines
· bacteria that can clean up toxic compounds or protect plants from pests and frost
(Food Safety Authority of Ireland, 1999)

How Are Microorganisms Modified?
Selected genetic material is introduced into recipient microorganisms by recombinant DNA techniques. This genetic material may be integrated into the resident chromosomes of the cells or extrachromosomal structures like plasmids. The genetic information is then transferred through a process of replication, cell division and chromosomal segregation.

This can be achieved through the following procedures:
a) Electroporation - This method is the most popular technique used for transformation of many varieties of microorganisms because of its simplicity. It only requires a brief exposure to a high voltage electric field in order to introduce genetic material into a microorganism.

b) "Natural" transformation - Bacteria such as Bacillus subtilis, Streptococcus pneumoniae and Haemophilus influenzae have a natural capacity to take up DNA and this useful property is used to introduce recombinant DNA into the host’s genomes.

c) Transformation through artificial competence – This process includes the incubation of certain bacteria in certain salt solutions to result in pore formation, thus allowing the introduction of recombinant DNA.

Genes which are selected to undergo tranformation into a new transgenic food product usually has valuable properties or important traits beneficial to the host product, such as genes present with natural insect resistance or desred nutrients. Genetic engineering or biotechnology allows genetic material to be transferred between any organism, including between plants and animals. For example, the gene from a fish that lives in very cold seas has been inserted into a strawberry, allowing the fruit to be frost-tolerant and maintain its natural goodness. However, modified genes are used in the production of GM foods in an early stage of the food production chain and may not always be present in the end product.


Food Safety Authority of Ireland, Ireland. (1999). Food Safety and Genetically Modified Foods. Retrieved July 16, 2007 from

Food Safety Authority of Ireland, Ireland. (2004). Food Safety and Genetically Modified Foods. Retrieved July 16, 2007 from

Food Safety Department; World Health Organisation, Switzerland. (2005). Modern food biotechnology, human health and development: an evidence-based study. Retrieved July 15, 2007 from