EFSTATHIA KOGKAKI Supervisor: Prof. NARESH MAGAN 2 nd Supervisor: - - PowerPoint PPT Presentation

“THE EFFECT OF OZONE ON INHIBITION OF GERMINATION, GROWTH & MYCOTOXIN PRODUCTION BY SPOILAGE FUNGI”

EFSTATHIA KOGKAKI

Supervisor: Prof. NARESH MAGAN 2nd Supervisor: Ms. KALLIOPI MYLONA

Fungi and mycotoxins

• Fungi exist in a wide range of ecosystems
• Mycotoxins are secondary metabolites
• Produced by filamentous fungi
• Negative impact
• Major groups:
• a. Aflatoxins
• b. Ochratoxins
• c. Trichothecenes, zearalenone, fumonisins

Control strategies

• Gaseous modification for control of mycotoxigenic

fungi in cereals

• Sulphur dioxide (SO2) fumigation of cereals
• Ozone (O3) fumigation of grain
• CO2/O2 modified atmospheres
• Chemical additives

Aims of project

• Effect of O3 (0-200 ppm) on germination of:

Penicillium verrucosum, Fusarium graminearum and Aspergillus flavus

• Effects mycelial growth: three mycotoxigenic species
• Impact of O3 treatment on mycotoxin production by P.

verrucosum (ochratoxin) and A. flavus (aflatoxin B1)

• Fumigation with O3 on natural wheat grain mycobiota and

that inoculated with F. graminearum or P. verrucosum

Materials and methodology

• Fungal strains:

i. Aspergillus flavus ii. Fusarium graminearum

• iii. Penicillium verrucosum

Ozone exposure treatment

Exposure for 30 mins at 6 L/min

Mycotoxins extraction and HPLC system used for toxin analyses

Results

• The effect of O3 on spore germination in vitro
• a. Penicillium verrucosum 0.95 aw

20 40 60 80 100 120 24 48 72 96 168 spore germination (%) Τime (Hours) 20 40 60 80 100 120 24 48 72 96 168 spore germination (%) Τime (Hours) control 50 ppm 100 ppm 200 ppm

• b. P. verrucosum 0.90 aw

The effect of O3 on spore germination in vitro

• a. Fusarium graminearum 0.98 aw

20 40 60 80 100 24 48 72 96 168 spore germination (%) 10 20 30 40 50 60 70 80 90 100 24 48 72 96 168 spore germination (%) control 50 ppm 100 ppm 200 ppm

• b. F. graminearum 0.94 aw

The effect of O3 on spore germination in vitro

• a. Aspergillus flavus 0.95 aw

20 40 60 80 100 120 24 48 72 96 168 Hours spore germination (%) 20 40 60 80 100 120 24 48 72 96 168 spore germination (%) control 50 ppm 100 ppm 200 ppm

• b. A. flavus 0.90 aw

The effect of O3 exposure on in vitro mycelial growth

• a. Penicillium verrucosum 0.95 aw

5 10 15 20 25 30 35 40 con 50 100 200 Colony diameter (mm) Concentrations (ppm) 2 4 6 8 10 12 14 16 con 50 100 200 Colony diameter (mm) Concentrations (ppm)

• b. P. verrucosum 0.90 aw

The effect of O3 exposure on in vitro mycelial growth

• a. Fusarium graminearum 0.98 aw

10 20 30 40 50 60 70 80 con 50 100 200 Colony diameter (mm) Concentrations (ppm) 2 4 6 8 10 12 con 50 100 200 Colony diameter (mm) Concentrations (ppm)

• b. F. graminearum 0.94 aw

The effect of O3 exposure on in vitro mycelial growth

• a. Aspergillus flavus 0.95 aw

5 10 15 20 25 30 35 40 con 50 100 200 Colony diameter (mm) Concentrations (ppm) 5 10 15 20 25 con 50 100 200 Colony diameter (mm) Concentrations (ppm)

• b. A. flavus 0.90 aw

Mycotoxin analyses

• a. Aflatoxin B1 production by A. flavus

5 10 15 20 25 30 35 40 45 20 40 60 80 100 120 Aflatoxin average µg/g O3 Concentrations (ppm) 0.9 0.95

Mycotoxin analyses

• b. Ochratoxin A production by P. verrucosum

0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 50 100 150 200 250 Ochratoxin average µg/g O3 Concentrations (ppm) 0.9 0.95

Effects of O3 treatment

• f wheat grain

inoculated with F. graminearum

• Effect of ozone on wheat grain inoculated with F.

graminearum at 0.94 and 0.98 aw MEA after 20 days storage

0,5 1 1,5 2 con 100 200 Log CFUs population Concentrations (ppm) 0.94 0.98

Effects of O3 treatment of wheat grain inoculated with P. verrucosum

• Effect of ozone on wheat grain inoculated with P.

verrucosum at 0.90 and 0.95 aw MEA after 20 days storage

2 4 6 8 10 12 con 100 200 Log CFUs population Concentrations (ppm) 0.95 0.9

Discussion

• Effects of gemination and mycelial growth:
• P. verrucosum : few spores germinated after 72 hrs exposure
• F. graminearum : only a few germinated 48 hrs after treatment;

some spores germinated at 0.94 aw after 168 hrs in high O3 (200 ppm)

• A. flavus : complete inhibition at > 100 ppm O3 after 48 hrs

regardless of the aw level For all three species there was no effect up to 200 ppm O3 on mycelial growth regardless of aw level used

Mycotoxin analyses

• The amount of aflatoxin was significantly reduced at 50 and

100 ppm after O3 for 30 mins at 0.90 aw However, at 0.95 aw aflatoxin production was stimulated

• At 0.95 aw, ochratoxin A was stimulated by O3 exposure for 30

mins No toxin was produced at 0.90 aw

Effect of O3 treatment

• f natural wheat on

fungal populations isolated

• F. graminearum at 0.94 and 0.98 aw MEA: 0.05 log reduction
• f total populations observed in the O3 treatments regardless
• f the water activity
• P. verrucosum at 0.90 and 0.95 aw MEA : 2 log reduction at

0.95 aw but only 1 log reduction obtained at 0.90 aw at 200 ppm

Conclusions

• P. verrucosum spore germination was inhibited, yet at >96 hrs

even at low aw levels germination recovered completely (≈100%)

• However, the macroconidial germination of F. graminearum was

inhibited, especially at 0.94 aw

• A. flavus conidial germination was more sensitive to O3 since

complete inhibition occurred even after 48 hrs

• No effect was found against mycelial growth of the three species
• No significant reduction in total fungal populations by P.

verrucosum or F. graminearum occurred due to O3 at 200 ppm for 30 mins

Future Work

• More studies are required of dose x time of exposure
• The effect of 200-500 ppm for 30 and perhaps 60 mins
• The potential effects of exposure with O3 on grain

quality

Thank you for your attention! Any questions