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Fungi in foodProblems, methods and detection

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Overview presentation

• Fungal problems and associated mycobiota

• Detection methods

• Conclusion

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es Current fungal problems in food and feed

mycotoxigenic molds

xerophilic molds

heat-resistant molds

preservative-resistant mold

anaerobic molds

mycotoxigenic molds

xerophilic molds

heat-resistant molds

preservative-resistant mold

anaerobic molds

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Mycotoxigenic Fungi

More than 400 mycotoxins are known

Important are the aflatoxins, Fusarium-toxins, ochratoxin, patulin and sterigmatocystin

Mycotoxins are often imported in (sub)tropical foodproducts and feedstuffs

More that 125 people died in Kenya in 2004

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es Example: Fumonisinsfumonisins in relation with NTD and corn

Marasas WF et al., 2004, J Nutr. Apr;134(4):711-6

Missmer, S. et al, 2006, Environ. Health Perspect. February; 114(2): 237–241

FOOD FEED

Primary Primary

Secondary

Mycotoxicoses

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1. Aspergillus spp.

2. Penicillium spp.

3. Fusarium spp.

4. Alternaria spp.

5. Stachybotrys spp

Which fungi produce mycotoxins

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Fusarium graminearum

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Mycotoxins in daily life

Aflatoxins in nuts

Fusarium toxins in cereals, corn etc

Ochratoxin in wine, coffee, raisins, wheat

Patulin in apple juice

No toxins in fermented cheeses

Toxins in the laboratory and during sampling !

Aflatoxins in nuts

Fusarium toxins in cereals, corn etc

Ochratoxin in wine, coffee, raisins, wheat

Patulin in apple juice

No toxins in fermented cheeses

Toxins in the laboratory and during sampling !

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es Current fungal problems in food and feed

mycotoxigenic molds

xerophilic molds

heat-resistant molds

preservative-resistant mold

anaerobic molds

mycotoxigenic molds

xerophilic molds

heat-resistant molds

preservative-resistant mold

anaerobic molds

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Common Xerophilic Fungi

Eurotium species

Aspergillus penicillioides

Wallemia sebi

Chrysosporium species

Xeromyces bisporus

These species are found on dry products or in dry environments with wateractivities < 0.70 and > 0.85

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Overview projects

Eurotium herbariorum on cookiesCen

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Xeromyces bisporus

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Xerophilic fungi

Eurotium herbariorum on tablets

Eurotium herbariorum on DG18Cen

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Heat-resistant fungi

Spoilage problems: heat-resistant molds in pasteurized products like drinkyoghurts, canned food products (peas, strawberries), iced tea and fruit-juices (pectin)

• Contaminated products often associated with soil (raw ingredients!)

• Common, ascomycetes:Byssochlamys, Talaromyces and Neosartorya species

• Less common:Eupenicillium, Monascus, Hamigera species

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Dormant (asco)spore

Germinating sporeActivation

Extreme trigger/heat/high-pressure/?

Ascospores are the heat resistant vehicle. These spores are dormant and can only be waken up after a rigorous physical trigger as, for example 10 min heating at 85 °C.

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Heat-resistant fungi Byssochlamys species:

B. nivea, B. fulva, B. spectabilis(=Paecilomyces variotii)

Talaromyces species:

T. macrosporus, T. trachyspermus, T. bacillisporus

Neosartorya species:

N. fischeri, N. glabra, N. spinosa

Eupenicillium sp.

Eurotium sp.

D-values of some heat-resistant molds

Byssochlamys fulva D90= 1.3-15 min

Byssochlamys nivea D88= 0.75-0.80 min

Byssochlamys spectabilis (=Paecilomyces variotii)

D85= 45-75 min

Talaromyces macrosporus D88= 7.8 min

Neosartorya fischeriD80= 66.7 minD88= 1.4 min

Eurotium herbariorum D70= 5.5 min

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NOTE: Degree of heat resistance depends on age of ascospores and heating solution!

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es Examples of heat tolerant structures Chlamydospores

Thick-walled chlamydospores of Paecilomyces variotii

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es Examples of heat tolerant structures Sclerotia

Aspergillus flavus Aspergillus cretensis

Dectection method for heat-resistant molds

Sample:1. Pectin: 12.5 g + 230 ml H2O2. Fruit (concentrate): 100 g + 150 ml H2O3. Solid samples (eg powdery ingredients, soil): 25 g + 225 ml H2O

H2O

Stomacherbag

Homogenize

and sealAdjust pH >3.5

Heat treatment, 30 min 75-80°C

1. Cool the sample rapidly2. Mix the sample with 250 ml

handwarm double strength MEA agar + chloramphenicol

Mix thoroughly and disperse agar/product mass into approx. 7-8 Petri-dishes (diam. 14.5 cm)

1. Incubate for 14 -21 d. at 28-30°C2. Check every 7 days

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es Important step during procedure:Effect of heat treatment

1,0E+00

1,0E+01

1,0E+021,0E+03

1,0E+04

1,0E+05

1,0E+061,0E+07

1,0E+08

1,0E+09

0 20 40 60 80 100 120

Time (min)

CFU

/m

Thermal death curve of Byssochlamys nivea at 75°C

Elimination of non-heat resistant propagules and activation of heat resistant ascospores!

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Preservative-resistant fungi

Penicillium, Aspergillus and many other moulds are resistant against sorbate, benzoate and propionate

Sorbate-resistant moulds form pentadiene by decarboxylation

Typical preservative resistant species are Trichoderma, Paecilomyces variotii and Penicillium roqueforti

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Trichoderma on margarine

Penicillium roqueforti on rye bread

Moulds found in food and feed containing preservativesAcremonium kiliense

Penicillium aurantiogriseum

Penicillium chrysogenum

Eurotium herbariorum

Paecilomyces variotii

Trichoderma spp.

Penicillium brevicompactum

Penicillium roqueforti

Contactlens solution

Contactlens solution

Contactlens solution

Bakery products, cake, nuts, almonds

Margarine

Margarine

Fruit sauce

Rye bread, salads, silage

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es Modified atmosphere package and mould contamination

Modified atmosphere packaging can be a good alternative for preserving (mostly dry) food products

Low or no oxygen, however, still permits fungal growth

Mostly spoilage occur with gas packed pre-baked bread by Saccharomycopsis fibuliger and Hyphopichia burtonii

Modified atmosphere packaging is used for many products, particularly those which expected to have an extended shelf life, much attention should be given to the moulds which can grow at low oxygen

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Saccharomycopsis fibuliger

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Saccharomycopsis fibuligera

The associated mycobiota of foods and beverages

=Each product has its own mycobiota

(fungal flora)

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es Examples of associated mycobiota in processed products

Low water activity products

Heat-treated products

Products containing preservatives

Modified atmosphere packaging

….OR a combination of methods….

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Associated mycobiota

Relation between spoilage organism and food Normally less than 10 species; and often only

1-3 species are responsible for spoilage Combination of a-biotic factors play a role:

combination of e.g. temperature, pH, water activity, and atmosphere (especially for processed products)

BUT….also chemical communications via extrolites (mycotoxins!) and extracellular enzymes (especially for non-processed foods)

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es Associated mycobiota (non-processed foods)

Citrus fruits: Penicillium digitatum and P. italicum; however when treated with fungicides P. ulaienseemerges!

Apples: P. expansum (treated with fungiciden P. solitum occurs); but also P. crustosum and Alternaria species can occur

Garlic: P. alliiStored cereal grains: P. verrucosum, P. hordei, and all

members of series Viridicata of the genus Penicillium, Aspergillus flavus, A. niger, A. candidus, A. ochraceus, Eurotium sp., Alternaria sp.

Potato tubers: Fusarium sambucinum and F. coeruleum

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Overview presentation (part II)

• Fungal problems and associated mycobiota

• Methods

• Modern detection methods

• Conclusion

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es Detection methods for food-borne fungi

• Direct observations

• Direct plating and visual inspection of the sample mostly preferred to suspension and plate counts

• Quantitative estimation of fungi is often problematic

• Identification up to species level

• Mycological media for food-borne fungi

• Incubation at >5 days at 25°C

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Direct microscopical observation

Direct microscopical observation

Direct plating

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es Mycological media for food-borne fungi

Dichloran 18% Glycerol Agar (DG18 Agar) recommended as general purpose medium

Dichloran Rose Bengal ChloramphenicolAgar (DRBC) suitable for products with high aw

Maltextract Agar (MEA) with antibiotics is a good alternative medium

Sabouraud Agar or Plate Count Agar are NOT suitable!

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es Dichloran Rose Bengal Chloramphenicol agar (DRBC)

Peptone 5 gr

Glucose 10 gr

KH2PO4 1 gr

MgSO4.7H2O 0.5 gr

Dichloran (0.2% in ethanol) 1.0 ml

Rose Bengal (5% soln w/v) 0.5 ml

Chloramphenicol 0.1 gr

Agar 15 gr

Distilled water 1000 ml

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es Dichloran 18% Glycerol agarDG18

Peptone 5 gr

Glucose 10 gr

KH2PO4 1 gr

MgSO4.7H2O 0.5 gr

Dichloran (0.2 % in ethanol) 1.0 ml

Glycerol 220 gr

Chloramphenicol 0.1 gr

Agar 15 gr

Distilled water 1000 ml

Final aw= 0.955; pH 5.6

DG 18 agar 2% Malt extract agar

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Selective media

There are only a few selective mycological media

AFPA agar is useful for detection of aflatoxinogenic species

MYA50G for extreme xerophiles (eg. Xeromyces bisporus, Chrysosporium spp.)

MEA supplemented with 0.5% acetic acid for the detection of acidophilic fungi

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es MEA with 0.5% acetic acidP.roqueforti in environmental samples

Air sample Swab sample

Used media: DG18, MEA/PS (upper row) and MEA supplemented with 0.5% acetic acid (lower row)

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Modern detection methods

Molecular detection methods for certain group of fungi (ochratoxin producing Penicillia; Fusarium species) Many developed; often for detection of specific fungi;

poorly validated

No fast general detection methods Selective media are not always fast ATP, DEFT and electrical methods Physiological and biochemical methods

(API, BIOLOG)

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Identification

The identity of the species will reveal important characters of the spoilage organism

Identification of food-borne fungi is based on morphological characters and/or molecular identification methods (sequencing)

For morphological identification of Penicillium, Aspergillus and Fusarium, special media are required

Identification of yeasts is based molecular methods and/or on physiological and biochemical criteria

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Summary

• There are still many problems with spoilage and mycotoxins in food and feed• Samples have an associated mycobiota• It is important to use the appropriate detection methods • Qualitative analysis is often more important than quantitative estimations • No “fast” detection methods are present for the detection of general mycobiota• Molecular methods are being developed