Mycotoxins: occurrence, legislation and analysis

Transcription

1 5 th CEFSER Training Course Analysis of chemical contaminants in food and the environment Faculty of Technology, University of Novi Sad, Novi Sad, Republic of Serbia 7-11 May 2012 Mycotoxins: occurrence, legislation and analysis Prof. Dr. Biljana Škrbić University of Novi Sad, Faculty of Technology, Centre of Excellence in Food Safety and Emerging Risks (CEFSER)

2 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Mycotoxins are secondary metabolites produced by a wide variety of fungal species that cause nutritional losses and represent a significant hazard to the food chain. In fact, mycotoxins have been ranked as the most important chronic dietary risk factor, higher than synthetic contaminants, plant toxins, food additives or pesticide residues.

3 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Several environmental factors such as temperature, humidity and soil or storage conditions influence the occurrence of these toxic substances on agricultural commodities. Prolonged periods of high humidity and warm temperatures (25-30 o C) are the most favorable conditions for mold growth, while several other factors, such as mechanical injury, insect damage, chemical treatment, rapidity of drying, leakage in storage and hot spots can also affect the mold growth.

4 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Approximately 300 to 400 substances are recognized as mycotoxins, comprising a broad variety of chemical structures, but the most important groups of mycotoxins that occur quite often in food are:

5 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) aflatoxins (AFs): AFB1, AFG1, AFB2, AFG2, AFM1 produced by Aspergillus species, ochratoxin A (OTA) produced by both Aspergillus and Penicillium species, patulin produced by both Aspergillus and Penicillium species,

6 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) trichothecenes - type A: HT-2 and T-2 toxin, and - type B: deoxynivalenol (DON) zearalenone (ZON), and fumonisins B1 and B2 (FBs: FB1 and FB2) produced by Fusarium species.

7 Examples of high mycotoxin occurrence in cereals in Europe Hungaria: 1998: DON was found in 88% (n=99) wheat samples, with the highest concentration of 4300 μg/kg, being 3.4 times higher than the EC limit (Fazekas et al., 2000) Croatia: 2004: maximum level of DON in cereal grains was 2.7 times higher than the EC limit (Sokolović and Šimpraga, 2006)

8 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Northern Italy: aflatoxin contamination exceeding the EU limit in corn (Giorni et al. 2007) Romania: 1997: in all analyzed wheat samples (n=25) DON was presented 100%, while 32% of samples had the concentration above the EC limit with the maximum concentration of 5600 μg/kg, being about 4.5 times higher than the limit (Curtui et al. 1998) : 30% of the examined corn samples had of AFs, 20% of them above the EU limit (Tabuc et al. 2009)

9 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Germany: 1999 frequency of DON occurrence in wheat flour samples (n=60) was very high, with almost 100%, with the maximum concentration of 1379 μg/kg, being about 2 times higher than the EC limit (Schollenberger et al. 2002). Czech Republic: 1999 in all analyzed wheat samples (n=48) DON was presented 100%, with the maximum concentration of μg/kg, being about 2 times higher than the EC limit (Hajslova et al. 2007).

10 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) When present in food in sufficiently high amounts, these fungal metabolites can have toxic effects that range from acute to chronic symptoms. The chemical and biological properties of the mycotoxins, as well as their toxic effects are extremly variable. Some mycotoxins were shown to be mutagenic, teratogenic, or/and carcinogenic.

11 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) The International Agency for Research in Cancer (IARC) has classified aflatoxins (AFs) as carcinogenic to humans, while ochratoxin A (OTA) and fumonisins B (FBs) were classified as possibly carcinogenic. Trichothecenes (TRs) and zearalenone (ZON) were classified as noncarcinogenic but cause other adverse effects.

12 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Generally, they are stable chemical compounds and can neither be completely removed from the food supply nor destroyed during processing and heat treatment.

13 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Therefore, to protect consumers and to control the mycotoxin occurrence in products intended for human or animal consumption, regulatory authorities have set limits for maximum residue levels of several toxins.

14 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) The major factors affecting maximum levels are the toxicity of the respective mycotoxin, its occurrence in food products and the intake of the concerned food products by the population.

15 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) The latest Official Bulletin of the Republic of Serbia, No 28/11 (26 April 2011) established the maximum level for mycotoxins (AFs, OTA, DON, ZON, PAT, FB1 and FB2) in different foodstuffs in line with the relevant EU regulations.

16 COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs Ochratoxin A Maximum levels (μg/kg) Unprocessed cereals and All products derived from unprocessed cereals, including processed cereal products and cereals intended for direct human consumption with the exception of foodstuffs listed in 3.0 Dried vine fruit (currants, raisins and sultanas) 10.0 Roasted coffee beans and ground roasted coffee, excluding soluble coffee 5.0 Soluble coffee (instant coffee) 10.0 Wine (including sparkling wine, excluding liqueur wine and wine with an alcoholic strenght of not less than 15% vol) and fruit wine Automatised wine, aromatised wine-based drinks and aromatised wine-product cocktails Grape juice, concentrated grape juice as reconstituted, grape nectar, grape must and concentrated grape must as reconstituted, intended for direct human consumption 2.0 Processed cereal-based foods and baby foods for infants and young children 0.50 Dietary foods for special medical purposes intended specifically for infants 0.50 Green coffee, dried fruit other than dried vine fruit, beer, cocoa and cocoa products, liqueur wines, meat products, spices and liquorice

17 Patulin Maximum levels (μg/kg) Fruit juices, concentrated fruit juices as reconstituted and fruit nectars 50 Spirit drinks, cider and other fermented drinks derived from apples or containing apple juice Solid apple products, including apple compote, apple puree intended for direct consumption with the exception of food-stuffs Apple juice and solid apple products, including apple compote and apple puree, for infants and young children and labelled and solid as such Baby foods other than processed cereal-based foods for infants and young children T2 and HT-2 toxin Sum of T-2 and HT-2 toxin Unprocessed cereals and cereal products -

18 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) It should be noted that regulations regarding HT-2 and T-2 toxins in food have not yet been established due to scarce data on the occurrence of these toxins; nevertheless, they are in the preparation.

19 However, according to some literature data (Erikson and Alexander, 1998), limit for T2 in food and feed is proposed to be 100 µg/kg. Canada also recomends the maximum of 100 µg/kg of HT-2 in cattle and poultry feed, Israel has the 100 µg/kg as a tolerance limit for T-2, as well as Russia uses the same values as a limit for cereals, wheat flour and bran. * Eriksen GS, Alexander J (eds.), Fusarium toxins in cereals a risk assessment. Nordic Council of Ministers; TemaNord 1998: 502, pp and 45-58; Copenhagen.

20 COMMISSION REGULATION (EC) No 1126/2007 of 28 Septembar 2007 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards Fusarium toxins in maize and maize products Deoxynivalenol ( 17 ) μg/kg Unprocessed cereals ( 18 ) ( 19 ) other than durum wheat, oats and maize Unprocessed durum wheat and oats ( 18 ) ( 19 ) Unprocessed maize ( 18 ), with the exception of unprocessed maize intended to be processed by wet milling (*) Cereals intended for direct human consumption, cereal flour, bran and germ as end product marketed for human consumption, with the exception of foodstuffs listed in 2.4.7, and ( 20 ) Pasta (dry) ( 22 ) 750 Bread (including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals Processed cereal-based foods and baby foods for infants and young children ( 3 ) ( 7 ) 200 Milling fractions of maize with particle size > 500 micron falling within CN code or and other maize products with particle size > 500 micron not used for direct human consumption falling within CN code Milling fractions of maize with particle size 500 micron falling within CN code and other maize milling products with particle size 500 micron not used for direct human consumption falling within CN code ( 20 ) ( 20 )

21 Zearalenone ( 17 ) Unprocessed cereals ( 18 ) ( 19 ) other than maize 100 Unprocessed maize ( 18 ) with the exception of unprocessed maize intended to be processed by wet milling (*) Cereals intended for direct human consumption, cereal flour, bran and germ as end product marketed for direct human consumption, with the exception of foodstuffs listed in 2.5.6, 2.5.7, 2.5.8, and μg/kg 350 ( 20 ) Refined maize oil 400 ( 20 ) Bread (including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals, excluding maize-snacks and maize-based breakfast cereals Maize intended for direct human consumption, maize based snacks and maize-based breakfast cereals Processed cereal-based foods (excluding processed maize-based foods) and baby foods for infants and young children ( 3 ) ( 7 ) ( 20 ) Processed maize-based foods for infants and young children ( 3 ) ( 7 ) 20 ( 20 ) Milling fractions of maize with particle size > 500 micron falling within CN code or and other maize milling products with particle size > 500 micron not used for direct human consumption falling within CN code Milling fractions of maize with particle size 500 micron falling within CN code and other maize milling products with particle size 500 micron not used for direct human consumption falling within CN code ( 20 ) 300 ( 20 )

22 Fumonisins Sum of B1 and B2 Unprocessed maize (18), with the exception of unprocessed maize intended to be processed by wet milling (*) ( 23 ) Maize intended for direct human consumption, maize-based foods for direct human consumption, with the exception of foodstuffs listed in and ( 23 ) Maize-based breakfast cereals and maize-based snacks 8 00 ( 23 ) Processed maize-based foods and baby foods for infants and young children ( 3 ) ( 7 ) 200 ( 23 ) Milling fractions of maize with particle size > 500 micron falling within CN code or and other maize milling products with particle size > 500 micron not used for direct human consumption falling within CN code Milling fractions of maize with particle size 500 micron falling within CN code and other maize milling products with particle size 500 micron not used for direct human consumption falling within CN code ( 23 ) ( 23 ) (*) The exemption applied only for maize for which it is evident e.g. through labelling, destination, that is intended for use in a wet milling process only (starch production).

23 COMMISSION REGULATION (EC) No 165/2010 of 26 February 2010 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins Foodstuffs Maximum levels (μg/kg) Aflatoxins B 1 Sum of B 1, B 2, G 1 and G 2 M 1 Groundnuts (peanuts) and other oilseeds, to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs, with the exception of: groundnuts (peanuts) and other oilseeds for crushing for refined vegetable oil production Almonds, pistachios and apricot kernels to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs Hazelnuts and Brasil nuts, to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs Tree nuts, other than the tree nuts listed in and 2.1.3, to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs Groundnuts (peanuts) and other oilseeds and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs, With the exception of: crude vegetables oils destined for refining refined vegetable oils

24 Foodstuffs Maximum levels (μg/kg) Aflatoxins B 1 Sum of B 1, B 2, G 1 and G 2 M 1 Almonds, pistachios and apricot kernels, intended for direct human consumption or use as an ingredient in foodstuffs Hazelnuts and Brasil nuts, intended for direct human consumption or use as an ingredient in foodstuffs Tree nuts, other than the tree nuts listed in and 2.1.7, and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs Dried fruit to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs Dried fruit and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs All cereals and all products derived from cereals, including processed cereal products, Maize and rice to be subjected to sorting or other physical treatment before human consumption or use as an ingredient in foodstuffs Raw milk, heat treated milk and milk for the manufacture of milkbased products

25 Foodstuffs Maximum levels (μg/kg) Aflatoxins B 1 Sum of B 1, B 2, G 1 and G 2 M 1 Following species of spices: Capsicum spp. (dried fruits thereof, whole or ground, including chillies, chilli powder, cayenne and paprika) Piper spp. (fruits thereof, including white and black pepper) Myristica fragrans (nutmeg) Zingiber officinale (ginger) Curcuma longa (turmeric) Mixtures of spices containing one or more of the above mentioned spices Processed cereal-based foods and baby foods for infants and young children 0.10 Infant formulae and follow-on formulae, including infant milk and follow-on milk Dietary foods for special medical purposes intended specifically for infants

26 COMMISSION REGULATION (EC) No 105/2010 of 5 February 2010 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A Ochratoxin A Capsicum spp. (dried fruits thereof, whole or ground, including chillies, chilli powder, cayenne and paprika) Piper spp. (fruits thereof. Including white and black pepper) 30 μg/kg as from until Myristica fragrans (nutmeg) Zingiber officinale (ginger) Curcuma longa (turmeric) 15 μg/kg as from Mixtures of spices containing one or more of the above mentioned spices Liquorice (Glycyrrhiza glabra, Glycyrrhiza inflate snd other species) Liquorice root, ingredient for herbal infusion Liquorice extract, for use in food in particular beverages and confectionary 20 μg/kg 80 μg/kg

27 COMMISSION REGULATION (EC) No 683/2004 of 13 April 2004 amending Regulation (EC) No 466/2001 as regards aflatoxins and ochratoxin A in food for infants and young children Products Maximum levels aflatoxins (μ/kg or ppb) B 1 B 1 +B 2 +G 1 +G 2 M 1 Sampling method Reference analysis method Baby foods and processed cereal-based foods for infants and young children 0.10 Directive 1998/53/EC Directive 1998/53/EC Infant formulae and followon formulae, including infant milk and follow-on-milk Directive 1998/53/EC Directive 1998/53/EC Dietary foods for special medical purposes intended spacifically for infants Directive 1998/53/EC Directive 1998/53/EC

28 Product Maximum levels Ochratoxin A (μg/kg or ppb) Sampling method Reference analysis method Baby foods and processed cereal-based foods for infants and young children 0.50 Directive 2002/26/EC Directive 2002/26/EC Dietary foods for special medical purposes intended spacifically for infants 0.50 Directive 2002/26/EC Directive 2002/26/EC

29 COMMISSION REGULATION EC No 401/2006 defines the quality control parameters in order to assure the reliability of methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs...

30 COMMISSION REGULATION (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs Performance criteria for aflatoxins Criterion Concentration range Recommended Value Maximum permitted Value Blanks All Negligible Recovery-Aflatoxin M μg/kg 60 to 120 % > 0.05 μg/kg 70 to 110 % < 1.0 μg/kg 50 to 120 % Recovery-Aflatoxin B 1, B 2, G 1, G μg/kg 70 to 110 % > 10 μg/kg 80 to 110% Precision RSD R All As derived from Horwitz Equation 2 x value derived from Horwitz Equation Precision RSD r may be calculated as 0.66 times Precision RSD R at the concentration of interest.

31 Performance criteria for ochratoxin A Level μg/kg Ochratoxin A RSD r % RSD R % Recovery % < to to 110 Performance criteria for patulin Level μg/kg Patulin RSD r % RSD R % Recovery % < to to 105 > to 105

32 Performance criteria for deoxynivalenol Level μg/kg Deoxynivalenol RSD r % RSD R % Recovery % > to 110 > to 120 Performance criteria for zearalenone Level μg/kg Zearalenone RSD r % RSD R % Recovery % to 120 > to 120 Performance criteria for fumonisin B 1 and B 2 Level μg/kg Fumonisin B 1 and B 2 RSD r % RSD R % Recovery % to 120 > to 110

33 Performance criteria for T-2 and HT-2 toxin Level μg/kg T-2 toxin RSD r % RSD R % Recovery % to 130 > to 130 Level μg/kg HT-2 toxin RSD r % RSD R % Recovery % to 130 > to 130

34 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) In order to ensure compliance with the current legislation it is necessary to have reliable and accurate mycotoxin analytical methods, which allow their unambiguous identification and confirmation, as well as an accurate quantification at very low concentration levels.

35 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Generally, they consist of preparatory step based on extraction and clean-up followed by the analysis.

36 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) The purpose of the preparatory step is to remove co-extracting compounds that can interfere with the mycotoxin analysis; these substances requires multiple extraction and clean-up steps before mycotoxins can be quantified.

37 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Thus, different strategies have been performed, including solid phase extraction (SPE), liquid liquid extraction, supercritical fluid extraction, etc.

38 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Also, multifunctional columns (MycoSep) and selective or specific antibodies (immunoaffinity columns: IAC) for isolation and purification from the matrix compounds have been extensively used.

39 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Mycotoxins are usually extracted with (combinations of) organic solvents (methanol, acetonitrile or acetone) and water.

40 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Modern analysis of mycotoxins relies mostly on high-performance liquid chromatography (HPLC) or gas chromatography (GC) in combination with a variety of detectors:

41 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) fluorescence detection (FLD) UV detection flame ionization detection (FID) electron-capture detection (ECD) and more recently, mass spectrometry (MS).

42 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Different types of mycotoxins produced by a single or several fungal species may be present simultaneously in a commodity, which is of importance since exposure to mixtures of mycotoxins may have unexpected synergistic or additive toxic effects differing from those of an individual compound.

43 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Thus, determination of all mycotoxins subjected to the EU regulations in particular matrix by routine analysis in one single extract, and, if possible, in a single analytical run is of great relevance.

44 However, several difficulties are found to reach this objective: Very different maximum levels are admitted for mycotoxins as a function of their toxicity and type of food (e.g. babyfood has more restricted regulation). The matrix food composition is also highly variable, and, finally, Mycotoxins present a great diversity in their physicochemical properties.

45 Considering the wide range of polarities of the analytes, it would be advantageous to keep sample preparation to a minimum and to inject the crude extract. While lower detection levels are achievable in more purified extracts using a clean-up step, excluding this step provides a faster analysis.

46 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Recently, several authors have validated the crude matrix extracts analysis in contrast to usual preparatory methods widely used by many routine laboratories for analysis of single toxin or multiple toxins belonging to the same group in one particular sample matrix.

47 Sulyok et al. firstly validated a method for the determination of 39 mycotoxins in wheat and maize using a single extraction step followed by liquid chromatography coupled to triple qudrupole mass spectrometer equipped with electrospray ionization source (LC-ESI-MS/MS) without the need for any clean-up.

48 Later, the authors extended this method to perform semi-quantitative analysis of 87 fungal metabolites.

49 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Several more simple multi-mycotoxin analysis have been developed recently and they referred primarily to Fusarium toxins in (crude extracts of) cereals and/or cerealbased products:

50 References Matrix No. of Mycotoxins analyzed Sample preparation Separation/Detection Herebian et al. J. Sep. Sci. 32 (2009) wheat, maize mg of sample µl ACN/H2O/HAc (79:20:1) Vortex, Shaking, Centrifugation Dilution 5:1 with the same extraction solvent HPLC-ESI-MS/MS Spanjer et al. Food Addit. Contam. 25 (4) (2008) peanut, pistacio, wheat, maize, cornflake, raisin, fig g of sample ml ACN/H 2 O (80:20), 25 g of sample + 70% MeOH/H 2 O - figs and raisisns Shaking Dilution 1:3 with H 2 O HPLC-ESI-MS/MS Sulyok et al. Rapid Commun. Mass Spectrom 20 (2006) wheat, maize g of sample + 2 ml ACN/H 2 O/HAc ( ) Shaking, Centrifugation Dilution 1:1 with ACN/H 2 O/HAc ( ) HPLC-ESI-MS/MS Beltran et al. Rapid Commun. Mass Spectrom 23 (12) (2009) maize, dry pasta, multicerea l baby food g of sample + 10 ml ACN/H 2 O (80:20) + 0.1% HCOOH Shaking, Centrifugation Dilution 1:1 with HPLC grade water UHPLC-ESI-MS/MS

51 Ferrer Amate et al. Anal Bioanal Chem 397 (2010) spices (e.g. paprika, curry, black and white pepper, chilli, curcuma, numteg, ginger) 4 1 g of sample +10 ml ACN Shaking, Sonication, Centrifugation Dilution 1:1 with high-purity water HPLC-ESI-MS/MS Frenich et al. Food Chem, 117 (2009) maize, walnuts, biscuits, breakfast cereals 12 5 g of sample +10 ml ACN/ H 2 O (80:20) (for biscuits- 20ml) Vortex, Rotary agitator, Centrifugation UHPLC-ESI-MS/MS Elbert et al. AB Sciex Pte. Ltd. (2010) wheat, rye, barley, oat g of sample + 40 ml ACN/H 2 O (84:16) Mixing, Filtrating Dilution 1:10 with water and 5 mm NH 4 Ac HPLC-ESI-MS/MS Škrbić et al. Food Cont 22 (2011) wheat g of sample + 50 ml ACN/H 2 O (84:16) Mixing, Filtrating Dilution 1:1 with water-methanol mixture (1:1, v/v) HPLC-APCI-MS/MS Škrbić et al. Food Control 25 (2012) Wheat flour 11 5 g of sample + 20 ml ACN/H 2 O (84:16) Mixing, Filtrating Dilution 1:3 with UHPLC mobile phase of the initial content UHPLC-HESI-MS/MS

52 In these multimycotoxin analysis, ultra high pressure liquid chromatography (UHPLC) coupled to tandem mass (triple quadrupol) spectrometry (MS/MS or QqQ) has been preferred analytical methods for selective identification/quantification of target toxins.

53 Centre of Excellence in Food Safety and Emerging Risks - (CEFSER) Some of the first studies on the multitoxin analysis in wheat, wheat flour, maize and spices collected in Serbia have been conducted by the CEFSER researchers and the presentation of these results follows.

54 5 th CEFSER Training Course Analysis of chemical contaminants in food and the environment Faculty of Technology, University of Novi Sad, Novi Sad, Republic of Serbia 7-11 May 2012