Understanding the threat from mycotoxins


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It has been known for some years that cereal commodities can contain mycotoxins. With the gradual increase of human life expectancy, it has now been proven that these natural toxins contribute to certain forms of cancers in both humans and animals.

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Understanding the threat from mycotoxins

  1. 1. Digital Re-print - March | April 2014 Understanding the threat from mycotoxins www.gfmt.co.uk Grain & Feed MillingTechnology is published six times a year by Perendale Publishers Ltd of the United Kingdom. All data is published in good faith, based on information received, and while every care is taken to prevent inaccuracies, the publishers accept no liability for any errors or omissions or for the consequences of action taken on the basis of information published. ©Copyright 2014 Perendale Publishers Ltd.All rights reserved.No part of this publication may be reproduced in any form or by any means without prior permission of the copyright owner. Printed by Perendale Publishers Ltd. ISSN: 1466-3872
  2. 2. MILL MAINTENANCE Understanding the threat from mycotoxins - A comparison of mycotoxin testing techniques, regulations and best practices for flour and feed milling by Clare Milligan, Product Manager, R-Biopharm Rhône Ltd I t has been known for some years that cereal commodities can contain mycotoxins. With the gradual increase of human life expectancy, it has now been proven that these natural toxins contribute to certain forms of cancers in both humans and animals. Invasion of foods and feed by moulds under certain storage or environmental con- ditions, such as high moisture and/or specific temperatures, can lead to the development of these highly toxic components. There are many different types of myco- toxins, the most common and dangerous of which are aflatoxins (B1, B2, G1 and G2). However, legislation also exists for other mycotoxins such as ochratoxin A, deoxyni- valenol, fumonisins, zearalenone and patulin. Aflatoxins are produced by the fungi Aspergillus flavus and Aspergillus parasiticus and are often associated with commodities grown in tropical regions including various nuts, maize, dried fruits and spices. Mycotoxins such as ochratoxin A are found in cold to temperate climates, but can often be found in similar commodities to aflatoxins including dried fruits, coffee, cocoa, cereals, herbs and animal feed ingredients. Fumonisins, deoxynivalenol, zearalenone and T-2 and HT-2 toxins are produced by vari- ous Fusarium species and are mainly found in maize and maize-based commodities while patulin is commonly found in apple juice and purees. Surveillance plans for cereals Due to the prevalence of mycotoxins in cereals, cereal products and animal feed there has been growing concern for human and animal safety resulting in increased sur- veillance plans for cereals and retail products to generate data on consumer exposure to the range of Fusarium mycotoxins in the human diet. Legislation is currently in place (1126/2007/EC) for most of the Fusarium mycotoxins including DON, fumonisin, zea- ralenone and in March 2013 recommenda- tions were introduced for T-2 and HT-2 (2013/165/EU). See Figure 1 for further information on current EU Legislation in Food. Currently there are a wide variety of tests available for the detection of mycotoxins. These include ‘traditional’ methods such as thin layer chromatography and more rapid tests such as enzyme immunoassays or lateral flow devices commonly known as dipstick tests. However, for more sensitive analysis of mycotoxins, immunoaffinity columns are commonly used to allow legislative levels to be easily and accurately quantified by HPLC or LC-MS/MS. With the increase in mycotoxin legisla- tion there is a growing need for fast, simple and accurate methods for both quantifying or screening mycotoxins, which are, not only capable of detecting the legislative levels but also of meeting the analytical criteria laid down by the European Commission. R-Biopharm have developed a complete range of test formats including ELISAs, cards, lateral flow tests and immunoaffinity columns to allow users to comply with both European and International Mycotoxin regulations. The method of choice Immunoaffinity columns are the standard method of choice for regulatory mycotoxin analysis, however, there is also the need for columns, which can offer multi-mycotoxin analysis in conjunction with either HPLC or LC-MS/MS using a single extraction method. The company has therefore developed a number of new multi-mycotoxin immu- noaffinity columns including AFLAOCHRA PREP® , EASI-EXTRACT® T-2 & HT-2 and AO ZON PREP® for use in conjunction with HPLC and the most recent additions to it’s product line are DZT MS-PREP® and AOF MS-PREP® for simultaneous detection of mycotoxins in conjunction with LC-MS/MS (see Figure 2). The multi-mycotoxin columns are pro- duced using more than one monoclonal antibody bound to a solid support within Figure 1: Overview of current EU Legislation in Food Toxin Commodity Max Level (ppb) Deoxynivalenol Unprocessed cereals (excluding durum wheat, oats and maize) 1,250 Unprocessed durum wheat and oats 1,750 Unprocessed maize 1,750 Cereals intended for direct human consumption, cereal flour (including maize flour, maize meal, and maize grits), bran as end product intended for direct consumption and germ 750 Bread, biscuits, pastries, cereal snacks and breakfast cereal 500 Dry pasta 750 Processed cereal based baby and infant food 200 Toxin Commodity Max Level (ppb) Zearalenone Unprocessed cereals other than maize 100 Unprocessed maize 350 Cereals intended for direct human consumption, cereal flour, bran as end product for direct human consumption and germ 75 Maize intended for direct human consumption, maize based snacks and maize based breakfast cereals 100 Refined maize oil 400 Bread, pastries, biscuits, cereal snacks and breakfast cereals 50 Processed cereal based foods and baby foods for infants and young children 20 Processed maize based foods and baby foods for infants and young children 20
  3. 3. a column. This enables the isolation and concentration of target mycotoxins and the use of monoclonal antibodies makes the test highly specific and offers improved sensitivity. In all cases the toxins are extracted by blending the food or feed sample with a solvent, the extract is then filtered, diluted and a small volume passed through the immunoaffinity column by the analyst. If the target mycotoxins are present in the test sample, they are selectively bound to the antibodies in the column; the toxins are then released from the column using a solvent. The eluate is then injected onto the HPLC or LC-MS/MS system ready for quan- tification (see Figure 3). The advantage of these new multi-myco- toxin immunoaffinity columns is that only one sample preparation method is required for quantifying all mycotoxins in a single run therefore having greater sample throughput and a reduction in the use of solvents and consumables. The development of faster, accurate tests enables easier and more thor- ough monitoring of foods by the supplier. If such testing is used in conjunction with preventative measures at all stages in the production process, contamination risk to the consumer will be significantly reduced. With the new recommendations in March 2013 for T-2 and HT-2 (2013/165/ EU) Member States, should with the active involvement of food and feed business operators, perform monitoring for the pres- ence of the toxins in food and feed samples. Member States are encouraged to test samples simultaneously for the presence of T-2 and HT-2 and other Fusarium toxins such as deoxynivalenol, zearalenone and fumonisin B1 and B2 to allow the extent of co-occurence to be assessed. T-2 is rapidly metabolised in a large Toxin Commodity Indicative Max Level of T-2 & HT-2 (ppb) T-2 & HT-2 Unprocessed barley and maize 200 Unprocessed oats (with husk) 1,000 Unprocessed wheat, rye and other cereals 100 Oats for direct human consumption 200 Maize for direct human consumption 100 Other cereals for direct human consumption 50 Oat, bran and flaked oat products for human consumption 200 Cereal bran except oat bran, oat milling products other than oat bran and flaked oats, and maize milling products 100 Other cereal milling products 50 Breakfast cereals including formed cereal flakes 75 Bread (including small bakery wares), pastries, biscuits, cereal snacks, pasta 25 Cereal-based foods for infants and young childern 15 Toxin Commodity Max Level of B1 & B2 (ppb) Fumonisins Unprocessed maize 4,000 Maize and maize based foods intended for direct human consumption 1,000 Maize based breakfast cereals and maize based snacks 800 Processed maize based foods and baby foods for infants and young children 200 Figure 2: Example Immunoaffinity Column Precision Sensorsfor inline production Analog & Digital RF-Solutions •• Suitable for solid, granular and powdered materials •• High speed measurement •• Non-nuclear technology www.work-microwave.de Premium Quality FoodsSensor M
  4. 4. number of cereal products, HT-2 being a major metabolite. It is also recommended that if the method employed by the opera- tor allows, that the presence of masked mycotoxins in particular the mono and di-glycosylated conjugates of T-2 and HT-2 should also be reported by Member States. Masked mycotoxins just as toxic A masked mycotoxin is a compound who's 'parent' mycotoxin is chemically altered into a conjugated or structurally related form of the original mycotoxin. Masked mycotoxins are thought to be just as toxic as the original toxin but may behave very different chemically and can be more complex to analyse. R-Biopharm Rhone's immunoaffinity columns have been tested and have been found to cross react with the many of the masked mycotoxins and are suitable for use with LC-MS/MS allowing the analysis of all toxins in a single run. With more commodities being covered by legislation comes the need for large and small food companies and importers to test for mycotoxins. R-Biopharm Rhone have developed a range of test formats to meet the growing demands of the market. For those customers who require quantitative analysis of mycotoxins, but who do not have an HPLC or LC-MS/MS, a wide range of ELISAs have been developed for a range of mycotoxins. For port health authorities and small laboratories who need a fast, qualitative test, there are a range of test kits that can be used to screen cereals and cereal products. The AFLACARD, OCHRACARD and RIDA® QUICK tests allow for visual screening of the samples. These kits allow users to analyse samples quickly and easily yet be assured that they are keeping in line with current legislation. Lateral flow dipsticks RIDA® QUICK lateral flow dipsticks are available for screening for deoxynivalenol, aflatoxins, zearalenone and fumonisins. Interpretation of results is easy; a visible test band appears for positive samples and no line is visible for negative samples. RIDA® QUICK SCAN is available to read the test bands in order to give customers a quantita- tive result (see Figure 4). Enzyme linked immunosorbent assay (ELISA) can also be used for screening various mycotoxins. The RIDASCREEN® ELISAs use the high specificity of antigen and antibody interaction to detect and quantify the mycotoxin of interest by pho- tometric detection. RIDASCREEN® T-2 / HT-2 R3805) has been recently added to the range and is a competitive enzyme immunoassay for the quantitative analysis of T-2 and HT-2 toxins in oat, corn, barley and wheat. The mycotoxins are extracted from the cereal samples with methanol / water and the concentration of mycotoxin present is determined by means of the standard curve (range: 1 - 36 ppb (μg/kg)). The limit of detection is approximately 30 ppb (30 μg/ kg) and the recovery in natural contaminated cereal samples (Trilogy® reference mate- rial, TR-MT100) was determined to be 105 percent ± 15 percent and in spiked samples was found to be 95 percent ± 15 percent. The performance, stability and quality of RIDASCREEN® ELISA kits are validated both internally and externally before being officially released to the market for use. Accreditations from recognised bodies such as AOAC, UKAS and AFNOR are also sought. It is important to stress that the research and development of a product does not end with its launch into the market. Work continues and is carried out on a regular basis at laboratories in Germany and Scotland to ensure products continue to meet ever changing legislation and the differ- ent types of matrices that these regulations cover. Customer feedback is very much an integral part in ensuring that the company continues to develop successful products to be a leading supplier in this sector. Drastically reducing contamination The development of faster, accurate tests enables easier and more thorough monitor- ing of foods by the supplier. If such testing is used in conjunction with preventative meas- ures at all stages in the production process; this will drastically reduce contamination and lower the risk to the consumer. Since the formation of the company in the late 1980s R-Biopharm Rhone has established itself as a leading developer of test solutions for food and feed analysis, manufacturing test kits that offer high pre- cision and accuracy. With over 30 years experience in the development of various test formats, methods and services for the detection of mycotoxins, the company plans to continue to develop and offer exciting prospects in the field of mycotoxins ahead of legislation and to provide complete myco- toxin solutions for both large and small food companies. The company is well positioned to advance the development of innovative and versatile fast tests necessary to obtain accurate results from a wide range of new challenges. More inforMation: Claire Milligan Product Manager, R-Biopharm Rhône Ltd Tel: +44 141 945 2924 Fax: +44 141 945 2925 Email: info@r-biopharmrhone.com. Website: www.r-biopharm.com Figure 3: Overview of Immunoaffinity Column Principle  Mycotoxins  Other materials Figure 4: Overview of RIDA® QUICK DON Method GRAIN&FEED MILLING TECHNOLOGY24 | March - April 2014 M
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  7. 7. www.gfmt.co.uk LINKS • See the full issue • Visit the GFMT website • Contact the GFMT Team • Subscribe to GFMT A subscription magazine for the global flour & feed milling industries - first published in 1891 INCORPORATING PORTS, DISTRIBUTION AND FORMULATION In this issue: • Researching and reporting: the roller flour milling revolution • What is Fumigation? A technique of pest control using a toxic gas • VIV Europe preview Our pull out centre section March-April2014 • Bread is the foundation of civilization • Conserving grains: through drying • Flour miller values weighbridge technology on the island of Zanzibar first published in 1891 This digital Re-print is part of the March | April 2014 edition of Grain & Feed Milling Technology magazine. Content from the magazine is available to view free-of-charge, both as a full online magazine on our website, and as an archive of individual features on the docstoc website. Please click here to view our other publications on www.docstoc.com. To purchase a paper copy of the magazine, or to subscribe to the paper edi- tion please contact our Circulation and Subscriptions Manager on the link adove. INFORMATION FOR ADVERTISERS - CLICK HERE Article reprints All Grain & Feed Milling Tecchnology feature articles can be re-printed as a 4 or 8 page booklets (these have been used as point of sale materials, promotional materials for shows and exhibitions etc). If you are interested in getting this article re-printed please contact the GFMT team for more informa- tion on - Tel: +44 1242 267707 - Email: jamest@gfmt.co.uk or visit www.gfmt.co.uk/reprints