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Food Safety - Mycotoxins in Foods

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From Singapore Health Sciences Authority

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Food Safety - Mycotoxins in Foods

  1. 1. World of Food Safety 2013 Reinforcing quality and mitigating food safety risk to drive consumer confidence and profits
  2. 2. Overview of Presentation Overview of mycotoxins in foods Mycotoxin challenges in 21st century Impact of mycotoxin on food industry and food regulations Emerging analytical solutions for detection of mycotoxins Strategies for ASEAN national food safety laboratories
  3. 3. Mycotoxins - Definition  The word mycotoxin stems from the Greek word "mykes" meaning mould and "toxicum" meaning poison.  Mycotoxins are secondary metabolites naturally produced by moulds (fungi) that may contaminant agriculture produce.  They can contaminate various agricultural commodities either before harvest or under post-harvest conditions.
  4. 4. Discovery of mycotoxins  Serious worldwide concern began in the early 1960s after “Turkey X disease” was discovered in UK.  More than 100,000 young turkeys on poultry farms died in the course of a few months.  Investigation of the early outbreaks showed that they were all associated with feeds, namely Brazilian peanut meal .
  5. 5. Mycotoxins Chemically and toxicologically diverse compounds Aflatoxin B1 Carcinogenic to human (Group 1) Patulin Genotoxic Fumonisin B1 Hepatotoxic, Nephrotoxic Ochratoxin A Potentially Carcinogenic (Group 2B) Nephrotoxic Citrinin Zearalenone Skin/membrane Irritant
  6. 6. Mycotoxins in Food [1] Berthiller F., Sulyok M., Krska R., Schuhmacher R., Int. J. Food Microbiol. 2007; 119:33–37. [2] Environ Health Perspect. 2010 Jun;118(6):818-24. doi: 10.1289/ehp.0901388. Epub 2010 Feb 19  Today, 300-400 mycotoxins are known[1].  Aflatoxin B1 has been classified by the IARC to be a Class 1 human carcinogen.  A 2010 study by Liu, Y. Wu in collaboration with WHO estimated that Aflatoxin causes between 5-30% of all liver cancer cases in the world[2].
  7. 7. Mycotoxins in Food  There are six agriculturally important mycotoxins : aflatoxins, trichothecenes, fumonisins, zearalenone and ochratoxin. Mycotoxins Crops Aflatoxins Groundnut, Maize, Almond, Fig, Pistachios, Hazenut, Sunflower seed, Sorghum, Cashew, Chestnut, Nutmeg, Rice, Chilli, Pepper, Turmeric, Milk Ochratoxins Coffee, Grape, Paprika, Fig, Pepper, Barley, Nutmeg, Corn Fumonisins Maize, Wheat, Rice Trichothecenes Wheat, Oats, Corn Zearalenone Corn, Wheat, Soyabean, Rice, Barley Ergot alkaloids Rye, Barley, Wheat, Oats References : RSAFF, Int. J. Mol. Sci., 2008, 9, pp2062-2090
  8. 8. Occurrence data (EFSA chemical occurrence database) 2,183 samples retrieved from EFSA chemical occurrence database on 15 Mar 13. Samples collected between 2007-2012. Analytical data on Aflatoxins (B, B1, G, G1). Samples include cereal and milling products, processed cereal products. Sampling carried out in 16 European countries.
  9. 9. Occurrence data (EFSA chemical occurrence database) Distribution of total Aflatoxins by sampling year in cereals and milling products
  10. 10. Distribution of total Aflatoxins by sampling year in cereals and milling products Occurrence data (EFSA chemical occurrence database)
  11. 11. Distribution of total Aflatoxins by sampling year in processed cereal products Occurrence data (EFSA chemical occurrence database)
  12. 12. Mycotoxin occurrence Field Crops Harvesting Drying/Silo storage Feed millsLocal farmsProcessing Field mycotoxins contamination Field mycotoxins contamination Storage mycotoxins contamination Storage mycotoxins contamination Storage mycotoxins contamination Storage mycotoxins contamination Biological factors, Agronomic practices, Environmental factors(Temperature, Humidity, Time) Mycotoxin contamination of meat/milk supply
  13. 13. Impact of Mycotoxins  Significant economic losses are associated with their impact on human health, animal productivity, and both domestic and international trade.  It is estimated that 25% of the world's food crops, including many basic foods, are affected by mycotoxin producing fungi.  According to FAO estimates global losses of foodstuffs due to mycotoxins are in the range of 1000 million tonnes per year [1].  Over $100 billion of exported commodities all over the world are susceptible to mycotoxin contamination [2]. [1] FAO wesbite (http://www.fao.org/food/food-safety-quality/a-z-index/mycotoxins/en/) [2] Cardwell K. F. (2001), Food and nutrition Bulletin, 21:488-492
  14. 14. Mycotoxin control - regulations  Legislation is established in many countries worldwide.  In Asia/Oceania, 26 countries have specific mycotoxin regulations. Reference : Worldwide regulations for mycotoxins in food and feed in 2003
  15. 15. Mycotoxin regulations In Europe, ~ 99% of the 39 countries have specific mycotoxin regulations. Reference : Worldwide regulations for mycotoxins in food and feed in 2003
  16. 16. Mycotoxin regulations The United States and Canada have five regulations on mycotoxins in 2003. Reference : Worldwide regulations for mycotoxins in food and feed in 2003
  17. 17. Mycotoxin regulations Regulated levels of mycotoxins differ from nation to nation. Reference : Worldwide regulations for mycotoxins in food and feed in 2003
  18. 18. Codex Standard 193-1995 Maximum limits exists for Total Aflatoxins, Aflatoxin M1, Ochratoxin A and Patulin. Codex standards (Maximum limits) on 14 types of foodstuffs EU Legislations on mycotoxins in foodstuffs Regulatory standards (Maximum limits) on 62 types of foodstuffs Mycotoxin regulations Reference : JRC Technical Notes, Mycotoxin Factsheet, 2011
  19. 19. Codex Standard 193-1995
  20. 20. Commission Regulations (EC) No. 1881/2006
  21. 21. Commission Regulations (EC) No. 1881/2006
  22. 22. Commission Recommendation of 27 Mar 2013 (2013/65/EU)  Presence of T-2 and HT-2 toxins in cereal and cereal products
  23. 23. 0 1 2 3 4 5 6 7 Aflatoxins B & G Aflatoxin M1 Ochratoxin A Patulin Fumonisins Deoxynivalenol Zearalenone  Legislation established on regulatory limits of mycotoxins in ASEAN countries. Mycotoxin regulations
  24. 24. Mycotoxin regulations  Regulated levels of mycotoxins differ from nation to nation. Country Limits for Patulin Indonesia Fresh apple, canned apple, apple extract, nectar, alcoholic drinks 50g/kg Apple puree 25 g/kg Apple puree for infants and children 10 g/kg Malaysia Apple juice (includes apple juice as ingredients in other beverages) 50 g/kg Singapore Juices 10 g/kg Vietnam Fruit and fruit juices 50 g/kg Concentrated fruit juices and all product derived from them5 50 g/kg
  25. 25. Emerging Issues (1) Masked Mycotoxins In mycotoxin contaminated commodities, many structurally related compounds generated by plant metabolism or by food processing can co-exists with the native mycotoxin. These derivatives may have very different chemical behaviour, easily escaping routine detection. These derivatives can be hydrolysed to its precursors in the digestive tract & can exert toxic effects comparable to the free form.
  26. 26. Emerging Issues February 2012 Glycoside derivatives of T-2 and HT-2 toxins in contaminated wheat and oats.
  27. 27. Emerging Issues “Occurrence of deoxynivalenol and deoxynivalenol-3-glucoside in durum wheat”, C. Dall'Asta, A. Dall'Erta, P. Mantovani, A. Massi, G. Galaverna, World Mycotoxin Journal, Dec 2012, pp. 83-91. Abstract The occurrence of deoxynivalenol and deoxynivalenol-3-glucoside in durum wheat samples (n=150; 25 lines × 2 reps × 3 environments) collected in 2010 from 3 areas located in north-central Italy was evaluated. In addition, the co-occurrence of other trichothecenes was considered. An optimised extraction method based on the use of salts followed by ultra-high performance liquid chromatography-mass spectrometry analysis was used for the quantification of the mycotoxins. All samples were found positive for deoxynivalenol at concentrations ranging between 47 and 3,715 μg/kg. A ubiquitous occurrence of deoxynivalenol-3-glucoside was found; 85% of the analysed samples contained this masked mycotoxin at concentrations varying between 46 and 842 μg/kg. In addition to glycosylated deoxynivalenol, acetylated forms of deoxynivalenol (3- and 15- acetyldeoxynivalenol) were also found in most of the durum wheat samples. The deoxynivalenol-3- glucoside/deoxynivalenol ratio, reaching up to 30% in many samples, was similar to that already found in other cereals such as soft wheat and barley. These data open the way for further investigations on the role of glycosylating activity as a possible Fusarium head blight-resistance mechanism in durum wheat, as already proved in the case of soft wheat.
  28. 28.  According to an article in Chemistry Research in Toxicology, current government limits on mold toxins in grain crops fail to account for so-called “masked mycotoxins,” which transform from harmless when outside the body to potentially harmful when inside.  Chiara Dall’Asta and colleagues say that plants protect themselves by conjugating glucose, sulfur or other substances to the mycotoxin, producing conjugated mycotoxins that are not harmful to the grain.  However, Dall’Asta’s research has shown for the first time that bacteria present in the human large intestine deconjugates deoxynivalenol (DON) and zearalenone (ZEN), the two most widespread mycotoxin contaminants. Once deconjugated, DON and Zen revert to their toxic forms. http://www.foodengineeringmag.com/articles/90405-food-safety-regulations-miss-masked-mycotoxins Emerging Issues
  29. 29. Ochratoxin A moieties T-2 /HT-2 toxins moieties Open-lactone-ochratoxin A 4-deoxy T-2 toxin Ochratoxin-A-hydrochinon 3-acetyl T-2 toxin 4R/4S-Hydroxy-ochratoxin A T-2 toxin 3-glucoside Ochratoxin alpha T-2 triol Ochratoxin alpha glucoronide T-2 tetraol Ochratoxin A glucuronide Glycoside derivative of T-2 11-Hydroxy-ochratoxin A Glycoside derivative of HT-2 Fumonisins moieties Deoxynivalenol moieties Hydrolysed B1 3 acetyl DON Hydrolysed B2 15 acetyl DON Emerging Issues
  30. 30. Emerging Issues
  31. 31. Emerging Issues
  32. 32. Emerging Issues (2) Effect of climate change on mycotoxin geographic distribution pattern EFSA’s Emerging Risks Unit identified changing patterns in mycotoxin contamination in cereals such as wheat, maize and rice, due to climate change as a potential emerging hazard. In particular, aflatoxins (AFs) which are frequent in tropical and sub-tropical areas may become a concern in the EU. With climate change and expected increasing temperature and decreasing rain, the fungi, Aspergillus flavus and A. parasiticus may find conditions that are more suitable for their development. Reference : “Modelling, predicting and mapping the emergence of aflatoxins in cereals in the EU due to climate change”, 18 Jan 2012, http://www.efsa.europa.eu/en/supporting/doc/223e.pdf.
  33. 33. Emerging Issues + 2oC climate change + 5oC climate change Predictive modelling : Map of Aflatoxin B1 contamination risk (Maize) in the EU in different climate change scenarios
  34. 34. Emerging Issues (3) Emerging mycotoxins
  35. 35. Emerging Issues (3) Co-occurrence of multiple mycotoxins Published 8 March 2013 139 mycotoxins & metabolites were detected in 83 feed samples.  All samples were co- contaminated with 7–69 different mycotoxins.
  36. 36. Emerging Issues (3) Co-occurrence of multiple mycotoxins Beauvericin was found most often, in 98% of sample. Enniatins were second most, in 96% sample followed by DON and emodin, each 89%.
  37. 37. Major approaches for masked mycotoxin detection includes : (a) Direct method using LC/MS/MS - Pros : Specific, Quantitative to target mycotoxin - Cons : Expensive, Lack of reference standards (b) Indirect method requiring acidic/enzymatic cleavage, LC/MS/MS strategies, etc. - Pros : Cheaper (?), Detection of uncharacterised conjugates - Cons : Not specific, not quantitative, Efficiency of hydrolysis Analytical Strategies
  38. 38. Analytical Strategies Direct Approach
  39. 39. Analytical Strategies Full scan mass spectra of deoxynivalenol-3-glucoside in ACN (1000 g/kg)
  40. 40. Analytical Strategies Direct Approach
  41. 41. Analytical Strategies
  42. 42. Analytical Strategies Indirect Approach
  43. 43. Analytical Strategies Probing extracts for masked mycotoxin through investigation of parent ions chromatogram of product ions (m/z 185, 215) Parent : 442 Parent : 604 Parent : 484 Parent : 646 Parent : 400 Flow injection ESI Product Ion Scan T-2 glucosideHT-2 glucoside Investigation of hidden mycotoxins
  44. 44. Analytical Strategies Indirect Approach World Mycotoxin Journal, 2013 Flow Injection-Mass Spectrometry
  45. 45. Strategies for ASEAN National Food Testing Laboratories
  46. 46. Strategies for ASEAN National Food Testing Laboratories Challenge 1 : Strengthen the national food testing laboratory capacity to ensure safety of food supply particularly those of significance to ASEAN.
  47. 47. ASEAN Reference Laboratory Field of Expertise Designated ARL Mycotoxin Health Sciences Authority (HSA), Singapore Pesticide Residues Agri-food and Veterinary Authority (AVA), Singapore Heavy Metals & Trace Elements Department of Medical Sciences, Ministry of Public Health, Thailand Veterinary Drug Residues in foodstuffs of animal origin Department of Livestock Development, Ministry of Agriculture and Cooperatives, Thailand Microbiology Quatest 3, MOST, Vietnam Genetically Modified Organisms Department of Chemistry, Malaysia
  48. 48. ASEAN Reference Laboratory (1) Mycotoxin Training workshops Three rounds of Mycotoxin workshop organised for ASEAN national laboratory staff in 2005, 2007 and 2009.
  49. 49. ASEAN Reference Laboratory (2) Mycotoxin Proficiency Testing Programme Annual PT was organised for all ASEAN national laboratories since 2010. 2010 Ochratoxin A in Cereals 2011 Aflatoxins B & G in Peanuts 2012 Aflatoxins B & G in Nutmeg Powder 2013 Aflatoxins B & G and Ochratoxin A in Chilli Powder
  50. 50. Strategies for ASEAN National Food Testing Laboratories Challenge 2 : Development of testing methodologies suitable for determination of mycotoxins in Asian food products. Nutmeg Powder B1 – 1.8 g/kg B2 – 42 g/kg Peanut B1 – 8.5 g/kg B2 – 1.8 g/kg √ √
  51. 51. Strategies for ASEAN National Food Testing Laboratories Challenge 3 : Development of testing methodologies suitable for determination of multiple mycotoxins in Asian food products.
  52. 52. Thank You for your attention.
  53. 53. AFRIS. AsianFoodRegulationInformationService. We have the largest database of Asian food regulations in the world and it’s FREE to use. We publish a range of communication services, list a very large number of food events and online educational webinars and continue to grow our Digital Library. We look forward to hearing from you soon! www.asianfoodreg.com adrienna@asianfoodreg.com