1. Good Hygiene Practices along the coffee chain Fungal Overview Module 1.1

2. Slide 2 Module 1.1 – Fungal Overview Background: yeasts and moulds (fungi) in food  Eukaryotic cell structure  More complex than prokaryotic (bacteria)  Yeasts  Unicellular (3 – 5μm)  Can divide rapidly (but slower than bacteria - 2-3h)  Moulds  Tubular cells (30 - 100μm) (hyphae)  Grow by apical extension (can grow very long - filamentous fungi)  Reproduce by sexual and asexual production of spores  Adapted to lower moisture conditions than most bacteria

3. Slide 3 Module 1.1 – Fungal Overview Background: fungi in food  ‘Useful’ fungi  Edible mushrooms  Used in processing / preservation  Spoilage fungi  Can grow on foods with lower available water than most bacteria (some as low as a w = 0.65)  Typically spoil semi-moist foods – cheeses, cured meats, bread, cakes, fruit preserves etc  Cereals, grains, nuts, coffee, cocoa that are incorrectly stored (damp, moist conditions) – huge food and feed losses annually  Toxigenic fungi

4. Slide 4 Module 1.1 – Fungal Overview Toxigenic fungi: Overview of mycotoxins  Fungal metabolites  When ingested, inhaled or absorbed through skin cause lowered performance, sickness or death in man or animals, including birds.  Acute effects Headache, fever, nausea, diarrhœa, vomiting, weakness, tremors, convulsions In some cases death  Chronic or long-term effects Cancer Genetic or birth defects  Over 200 kinds of mycotoxin, produced by about 150 different fungi  Certain crops are commonly associated with certain mycotoxins  Ecological associations of mould with crop plants  Certain post-harvest conditions can favour certain moulds

5. Slide 5 Module 1.1 – Fungal Overview Mycotoxins of major significance Mould speciesMycotoxins Aspergillus parasiticusAflatoxins B 1, B 2, G 1, G 2 Aspergillus flavusAflatoxins B 1, B 2 Fusarium sporotrichiodesT-2 toxin Fusarium graminearumDeoxynivalenol, Zearalenone Fusarium moniliformeFumonisin B 1 Penicillium verrucosumOchratoxin A Aspergillus ochraceusOchratoxin A Penicillium expansumPatulin

6. Slide 6 Module 1.1 – Fungal Overview Aflatoxins  Commonly associated with maize, groundnuts, tree nuts, spices, dried fruit etc.  Carry-over from animal feed to foods of animal origin for humans: e.g. Aflatoxin M1 in milk  International guidelines exist for prevention and control CZ CBS CYA

7. Slide 7 Module 1.1 – Fungal Overview Other important mycotoxins  Trichothecenes – Fusarium spp  Associated with a variety of cereals and wet harvest conditions  Zearalenone – Fusarium spp  Associated with maize grown in temperate climates  Fumonisins – Fusarium spp  Primarily associated with maize  Patulin - Penicillium spp, Aspergillus spp  Associated with apple products  Ochratoxin – Aspergillus spp, Penicillium spp  Associated with cereals, wine, grape juice, dried fruit, coffee and cocoa

8. Slide 8 Module 1.1 – Fungal Overview OTA contamination in coffee  OTA long known as a renal toxin and carcinogen which is also teratogenic (produces birth defects)  Evidence of genotoxicity published in the early 1990’s - if true, categorizes OTA with aflatoxin  Studies in Europe on dietary exposure concluded the most significant sources are grain and grain products; beer; wine; dried fruit; coffee  Several countries have already adopted maximum levels of contamination in coffee  Some importers have rejected contaminated batches  EU harmonised limits for roasted and soluble coffees - in force from January 2005

9. Slide 9 Module 1.1 – Fungal Overview OTA producers in coffee  OTA producers in coffee:  Aspergillus ochraceus (and related)  Aspergillus carbonarius  Aspergillus niger complex  Elsewhere:  Penicillium verrucosum  Penicillium nordicum  These organisms interact with other coffee-associated organisms, and not just Coffee Berry Borer (CBB) and Colletotrichum etc. The fungi include:  Fusarium stilboides  Candida edax  Cryptococcus album  Additional context are the conditions man’s activities impose in the orchard and during processing and trading Cladosporium spp. Penicillium brevicompactum Auriobasidium pululans Eurotium repens A. Colonies of A. flavus from Aspergillus flavus group. B. & C. Typical colonies of Penicillium spp. A C B

10. Slide 10 Module 1.1 – Fungal Overview Conditions for activity of OTA producers  Not all isolates of a species that is known to produce a mycotoxin will do so:  A. niger complex  5% usually weak  A. carbonarius  80% often strong  A. ochraceus and similar  80% often strong  The range of conditions over which a mycotoxin producer can grow is broader than those over which it can produce mycotoxin:  A. niger complex: Aw and temperature limits n.a.  A. carbonarius: Aw limits  0.92 and 0.85 temperature limits  35˚C and 37˚C  A. ochraceus: Aw limits  0.82 and 0.78 temperature limits  40˚C and 42˚C  The interaction of physiological and ecological properties is too complex - thus laboratory studies are only indicative  At this stage of our understanding, only field studies can clarify the limiting conditions for OTA contamination in coffee production

11. Slide 11 Module 1.1 – Fungal Overview Effect of pH and A w on mould growth X H Xerophile pH 3.0 4.0 5.0 7.0 Aw 0.99+ 0.98 0.94 0.905 Hydrophile pH 3.0 4.0 5.0 7.0 Aw 0.99+ 0.98 0.94 0.905 Mesophile pH 3.0 4.0 5.0 7.0 Aw 0.99+ 0.98 0.94 0.905

12. Slide 12 Module 1.1 – Fungal Overview Factors controlling mould growth  Initial contamination?  Oxygen / gaseous environment?  Nutrients?  Temperature?  Water activity?  What is it?  How do we measure it? A w =

13. Slide 13 Module 1.1 – Fungal Overview Moisture content (m.c.) and A w  m.c. describes the sample; Aw predicts microbial growth potential  In commerce, m.c. is measured but the microbial stability is only predicted by Aw so we need to inter-convert  So we need to understand the precision of this inter- conversion

14. Slide 14 Module 1.1 – Fungal Overview Evaluating moisture in commodities  Chemical methods  Oven method  Temperature?  Time?  Air circulation?  Vacuum?  Electrical methods  Capacitance  Conductance  Other gravimetric methods  Empirical / traditional sensory methods Moisture content - dry or wet basis?

15. Slide 15 Module 1.1 – Fungal Overview Evaluating moisture in commodities  Internal equilibration?  Equilibration with chamber air? Water activity

16. Slide 16 Module 1.1 – Fungal Overview Precision and accuracy of measurement  Uniformity of commodity  Sampling  Calibration  Methodology  Frequency  Quality of standards  Instrument stability  Robustness  Kind of use ‘EDABO’ distillation method of moisture determination developed in Brazil SINAR moisture meter One type of low-cost moisture meter investigated under the ‘global coffee project’

17. Slide 17 Module 1.1 – Fungal Overview Moisture and A w in complex systems  The husk is more hygroscopic than the bean - it forms a barrier that slows water loss during drying and slows water ingress during re-wetting.  From the perspective of mould growth, the significance of a given moisture content of bean and cherry is quite different. BEAN LIMIT--HUSK LIMIT OTA prod limit