1. Filamentous fungi on meat products,
their ability to produce mycotoxins
and a proteome approach to study
mycotoxin production
by Marie Sørensen

2. Outline
• Background
• Problems in the meat industry
• Objectives
• Results and conclusions of the experimental work
– Part 1: A survey of the mycobiota in Danish meat processing environments
– Part 2: Determination of mycophenolic acid in inoculated meat products
– Part 3: A proteome analysis used to study fumonisin production by
Aspergillus niger
• Perspectives
2

3. Background
1.00
Bacteria
Filamentous fungi
Yeasts
0.90
Water
activity
0.80
0.70
3

4. Filamentous fungi on meat products
Frequently isolated genera
Penicillium
Aspergillus
Eurotium
Cladosporium
Alternaria
Mucor
Rhizopus
Scopulariopsis
0 5 10 15 20 25 30 35 40 45
Number of studies
Fermented sausages Dry-cured hams Other meat products
4

5. Meat-associated toxinogenic species
Penicillium species:
P. brevicompactum Mycophenolic acid
P. chrysogenum Roquefortine C, secalonic acid, PR toxin
P. citrinum Citrinin
P. commune Cyclopiazonic acid
P. cyclopium Penicillic acid, xanthoemegnins
P. expansum Roquefortine C, patulin, citrinin and others
P. nordicum Ochratoxin A
P. palitans Cyclopiazonic acid
P. roqueforti Roquefortine C, mycophenolic acid
P. verrucosum Ochratoxin A, citrinin
P. viridicatum Penicillic acid, xanthoemegnins, viridic acid
Aspergillus species:
A. flavus Aflatoxin B1, cyclopiazonic acid, 3-nitropropionic acid
A. ochraceus Ochratoxin A, penicillic acid, xanthoemegnin and others
A. niger Ochratoxin A, fumonisin B2
A. versicolor Sterigmatocystin
5

6. Factors influencing mycotoxin production
Time / development
Primary metabolism
• Biosynthetic origin
• Availability of cofactors
Species Environment
• Physiology • Nutrients, pH, water activity
Secondary metabolism • Temperature, relative humidity
• Gene induction and regulation
• Atmosphere
• Chromosomal location • Antimicrobial compounds
• Other microorganisms
Mycotoxin production
6

7. Traditional meat products
”Salame di Felino” ”Ålerøget spegepølse”
7

8. Problems with moulds – and mycotoxins ?
8

9. Initiation of the project
9

10. Primary objectives
• To determine if filamentous fungi from meat processing environments
can produce toxic secondary metabolites during growth on meat products
• To enhance the understanding of conditions that can influence the
production of toxic secondary metabolites on meat products
10

11. Part 1:
A survey of Danish meat processing plants
Plant A Plant B Plant C Plant D
Fermented Fermented Liver pâté Liver pâté
sausage sausage
Spring Autumn Spring Autumn
Air 30 30 28 38
Surfaces 39 51 39 45
Raw materials 2 10 - -
Mouldy products 18 2 4 -
Total 89 93 71 83
11

12. Plant A Plant B Plant C Plant D
Main isolated genera
Fermented Fermented Liver pâté Liver pâté
-
sausage sausage
% positive samples
Spring Autumn Spring Autumn
Aspergillus 5% 2%
Botrytis 5% 1%
Cladosporium 4% 60 % 6% 57 %
Epicoccum 3% 8% 1%
Eurotium 1% 9%
Penicillium 38 % 22 % 7% 13 %
Phaeoacremonium 8%
Phoma 3% 11 %
≤ 5 % : Alternaria, Acremonium, Aureobasidium, cf. Clonostachys,
Fusarium, Paecilomyces, cf. Pseudohansfordia, cf. Sporotrix, Trichoderma
12

13. Plant A Plant B Plant C Plant D
Main isolated
Penicillium species Fermented Fermented Liver pâté Liver pâté
- sausage sausage
% positive samples
Spring Autumn Spring Autumn
P. brevicompactum /
7% 4% 3% 4%
P. bialowiezense
P. fagi 13 %
P. milanense (ined.) 9% 4%
P. palitans 10 %
P. solitum 21 % 4% 1% 8%
Other ++ + + ++ + +
≤ 5 % : P. chrysogenum, P. commune, P. corylophilum / cf. P.
corylophilum, P. cyclopium, P. decaturense, P. glabrum, P. hordei, P.
implicatum, P. purpurogenum, P. roqueforti, P. spinulosum
13

14. Laboratory-scale inoculation study
P. brevicompactum (1) Smoked
Salted beef Cooked ham Smoked fillet saddle Sausage
P. bialowiezense (3) of pork
P. commune (2) Smoked
Smokedbeef
Salted Cooked ham Smoked fillet saddle Sausage Liver pâté
P. palitans (5)
Salted beef Cooked ham Smoked fillet saddle Sausage Liver pâté of pork
of pork
Smoked
Cooked ham Smoked fillet saddle Sausage Liver pâté
of pork
Mycophenolic acid
Asperphenamate
Quinolactacin
Xanthoepocin
Cyclopiazonic acid
Viridicatol
Cyclopeptin
Viridicatin
14

15. Part 2
Determination of mycophenolic acid in
inoculated meat products
15

16. OH CH3
O
O
O
CH3 OH
pKa = 4.7
Analytical method
O
CH3
16

17. MPA in dry-cured hams
MPA concentration (μg/kg)
Centre Middle Surface
Inoculated hams
A1 230 1100 4200
A2 190 2900 6000
A3 220 2100 4400
A4 200 3200 11000
Control hams
E2 nd nd nd
E4 nd nd nd
Method performance in dry-cured ham:
RSD = 20 4 %, LOD = 6 μg/kg, LOQ = 100 μg/kg
17

18. Fermented sausages
MPA concentration (μg/kg)
Centre Middle Surface
Inoculated sausages
A1 nd nd nd
A2 nd nd nd
A3 nd nd nd
Control sausages
E1 nd nd nd
E2 nd nd nd
E3 nd nd nd
Method performance in fermented sausages:
RSD = 7 4 %, LOD = 4 μg/kg, LOQ = 100 μg/kg
18

19. MPA in liver pâté
MPA concentration (μg/kg)
Bottom Middle Surface
Inoculated patés
A1 nd3 4100 9400
A2 < LOQ 4500 11000
A3 150 6100 14000
A4 < LOQ 5200 8600
A5 < LOQ 3800 12000
Control patés
B1 - - nd
B2 - - nd
B3 - - nd
19 Method performance in liver pâté: RSD = 4 2 %, LOD = 6 μg/kg, LOQ = 100 μg/kg.

20. Conclusions part 1 and 2
• Important fungi in terms of spoilage:
– Penicillium and Eurotium on fermented sausages
– Penicillium and Cladosporium on liver pâtés
• Prevalent toxinogenic species:
– P. brevicompactum and P. palitans
• P. brevicompactum was able to produce MPA on ham and liver pâté at
realistic processing conditions
– MPA was detectable in centre/bottom fractions
• Meat products that are or has been covered with uncontrolled, unknown
mould growth may contain fungal metabolites and mycotoxins
20

21. Part 3
A proteome analysis used to study fumonisin
production by Aspergillus niger
Change in conditions Cellular response Physiological response
Transcriptome
Condition 1 Survival
…………..2 Growth
…………..3 Reproduction
Proteome Metabolome
Protein Mycotoxin Physiological
f ingerprint production measures
Combination of data in a holistic analysis
21

22. Model organism
• Aspergillus niger
– Genome sequenced
– Present on meat products
– Produces two important mycotoxins: Fumonisin B2 and ochratoxin A
Conditions to study
• Nutrients
– Glucose
– Lactate
– Fat
– Starch
22

23. Starch and lactate increases FB2 production
20
Fumonisin B2 (ug/cm2) 3 % Starch
+ 3 % Lactate
15
3 % Starch
+ 1.5 % Lactate
10
3 % Starch
5
0 3 % Lactate
40 60 80 100 120
Time (hours)
23

24. Fumonisin B2 Fumonisin B4 Ochratoxin A Ochratoxin alpha
% of maximum
% of maximum
% of maximum
% of maximum
100 100 100 100
0 0 0 0
40 60 80 100 120 40 60 80 100 120 40 60 80 100 120 40 60 80 100 120
Time (hours) Time (hours) Time (hours) Time (hours)
Malformin A Malformin C Orlandin Desmethylkotanin
% of maximum
% of maximum
% of maximum
% of maximum
100 100 100 100
0 0 0 0
40 60 80 100 120 40 60 80 100 120 40 60 80 100 120 40 60 80 100 120
Time (hours) Time (hours) Fumonisin B2 Time (hours) Time (hours)
% of maximum
Kotanin
100 Aurasperone B Pyranonigrin A Tensidol B
% of maximum
% of maximum
% of maximum
% of maximum
100 0 100 100 100
40 60 80 100 120
Time (hours)
0 0 0 0
40 60 80 100 120 40 60 80 100 120 40 60 80 100 120 40 60 80 100 120
Time (hours) Time (hours) Time (hours) Time (hours)
3 % Starch 3 % Starch + 3 % Lactate 3 % Lactate
24

25. Supplemented carbon source n Fumonisin B2 (µg/cm2) after 66 h
3 % Starch 18 2.89 ± 0.63 a
3 % Starch + 3 % maltose 3 2.61 ± 0.74 a
3 % Starch + 3 % xylose 3 2.06 ± 0.28 a
3 % Starch + 3 % lactate 14 7.49 ± 2.10 b
3 % Starch + 3 % pyruvate 3 5.06 ± 0.60 b
3 % Lactate 15 0.86 ± 0.34 c
25

26. 4000
C:Documents and Settingsjajor06Desktophold. BMaldi STRBSA int kalib BSA.massml (16:37 11/24/06)
3815.7
<3592.7>
3307.8
2984.2
2868.4
2666.3
2626.1
2612.1
2529.2 2543.1
2512.1
2498.2
m/z
2301.1
2045.0
1880.9 1894.9
1864.5
1724.8 1740.8
1738.8
Description: BSA int kalib BSA
1639.9
1567.7 1537.8
1479.8
1439.8 1423.4
1399.6
1292.6
1283.7
BSA kalib BSA
1000
1001.5
927.48
804.29
50
0
Intensity
SL
S
L
26

27. 27

28. 28

29. No. of spots
Cluster groups
Total Identified
Higher levels on SL* 62 27
Lower levels on SL* 68 20
Higher levels if starch is present 45 3
Lower levels if starch is present 52 0
Higher levels if lactate is present 21 4
Lower levels if lactate is present 35 0
Possibly an effect, instability 58 3
No effect, instability and noise 308 1
Total 649 58
29
* Or tendency for it

30. Uncharacteris
ed, 7 Polysaccharide
degradation, 5
Central
Miscellaneous carbon/energy
, 10 metabolism, 1
8
Redox balance
and oxidative
Nitrogen and
stress, 6
amino acid
Protein metabolism, 5
synthesis, pro
cessing and
turnover, 8
30

31. SL
SL (tendency)
SL (tendency, noisy)
SL
SL & S
SL & L
31

32. SL
SL (tendency)
SL (tendency, noisy)
SL
SL & S
SL & L
32

33. Conclusions part 3
• Lactate added in a rich substrate containing starch increased FB2
production by A. niger
• Cellular mechanisms that may affect FB2 production:
– The levels of carbon passing through acetyl-CoA
– The capacity to regenerate NADPH
• A new hypothesis: Regulation of FB2 production according to the nutrient/
energy state with acetyl-CoA as a relevant candidate
33

34. Perspectives
• Continued focus on prevalent species
• Procedures for discharge of products with (unknown) mould growth
• Development of methods for determination of mycotoxins
• Evaluation of potential mycotoxin production on the given products
• Establishment of maximum mycotoxin levels
34

35. Acknowledgements
• Involved companies
• Technical staff
– CMB (Hanne, Jesper, Lisette, Kir)
– Danish Meat Research Institute
– Meat Research Group at LIFE, Copenhagen University
– Protein Research Group at University of Southern Denmark
• Supervisors • Colleagues at CMB
– Per Væggemose Nielsen – Kristian Fog Nielsen
– Jens Christian Frisvad – Mikael Rørdam Andersen
– Anette Granly Koch – Fellow ph.d. students
– Tomas Jacobsen
– Rene Lametsch • Family and friends
35