food safety - mycotoxins in foods

World of Food Safety 2013

Reinforcing quality and mitigating food safety risk to drive consumer confidence and profits

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

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.

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 .

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

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].

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

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.

Occurrence data (EFSA chemical occurrence database)

Distribution of total Aflatoxins by sampling year in cereals and milling products

Distribution of total Aflatoxins by sampling year in cereals and milling products

Occurrence data (EFSA chemical occurrence database)

Distribution of total Aflatoxins by sampling year in processed cereal products

Occurrence data (EFSA chemical occurrence database)

Mycotoxin occurrence

Field Crops Harvesting Drying/Silo storage

Feed mills Local farms Processing

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

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

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

Mycotoxin regulations In Europe, ~ 99% of the 39 countries have specific mycotoxin regulations.

Reference : Worldwide regulations for mycotoxins in food and feed in 2003

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

Mycotoxin regulations Regulated levels of mycotoxins differ from nation to nation.

Reference : Worldwide regulations for mycotoxins in food and feed in 2003

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

Codex Standard 193-1995

Commission Regulations (EC) No. 1881/2006

Commission Regulations (EC) No. 1881/2006

Commission Recommendation of 27 Mar 2013 (2013/65/EU)

Presence of T-2 and HT-2 toxins in cereal and cereal products

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

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

50g/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

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.

Emerging Issues February 2012 Glycoside derivatives of T-2 and HT-2 toxins in contaminated wheat and oats.

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.

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

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

Emerging Issues

Emerging Issues

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.

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

Emerging Issues (3) Emerging mycotoxins

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.

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%.

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

Analytical Strategies Direct Approach

Analytical Strategies

Full scan mass spectra of deoxynivalenol-3-glucoside in ACN (1000 g/kg)

Analytical Strategies Direct Approach

Analytical Strategies

Analytical Strategies Indirect Approach

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 glucoside HT-2 glucoside

Investigation of hidden mycotoxins

Analytical Strategies Indirect Approach

World Mycotoxin Journal, 2013

Flow Injection-Mass Spectrometry

Strategies for ASEAN National Food Testing Laboratories

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.

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

ASEAN Reference Laboratory (1) Mycotoxin Training workshops Three rounds of Mycotoxin workshop organised for ASEAN national

laboratory staff in 2005, 2007 and 2009.

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

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

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Strategies for ASEAN National Food Testing Laboratories

Challenge 3 : Development of testing methodologies suitable for determination of

multiple mycotoxins in Asian food products.

Thank You for your attention.

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