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 2 nd 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 (SO 2 ) fumigation of cereals  Ozone (O 3 ) fumigation of grain  CO 2 /O 2 modified atmospheres  Chemical additives

Aims of project  Effect of O 3 (0-200 ppm) on germination of: Penicillium verrucosum , Fusarium graminearum and Aspergillus flavus  Effects mycelial growth: three mycotoxigenic species  Impact of O 3 treatment on mycotoxin production by P. verrucosum (ochratoxin) and A. flavus (aflatoxin B 1 )  Fumigation with O 3 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 O 3 on spore germination in vitro a. Penicillium verrucosum 0.95 a w b. P. verrucosum 0.90 a w 120 120 100 100 spore germination (%) spore germination (%) 80 80 control 60 60 50 ppm 100 ppm 40 40 200 ppm 20 20 0 0 24 48 72 96 168 24 48 72 96 168 Τime (Hours) Τime (Hours)

The effect of O 3 on spore germination in vitro b. F. graminearum 0.94 a w a. Fusarium graminearum 0.98 a w 100 100 90 spore germination (%) spore germination (%) 80 80 70 60 60 control 50 50 ppm 40 40 100 ppm 30 200 ppm 20 20 10 0 0 24 24 48 48 72 72 96 96 168 168

The effect of O 3 on spore germination in vitro a. Aspergillus flavus 0.95 a w b. A. flavus 0.90 a w 120 120 100 100 spore germination (%) spore germination (%) 80 80 control 60 60 50 ppm 40 100 ppm 40 200 ppm 20 20 0 0 24 48 72 96 168 24 48 72 96 168 Hours

The effect of O 3 exposure on in vitro mycelial growth a. Penicillium verrucosum 0.95 a w b. P. verrucosum 0.90 a w 16 40 14 35 Colony diameter (mm) Colony diameter (mm) 12 30 10 25 8 20 6 15 4 10 2 5 0 0 con 50 100 200 con 50 100 200 Concentrations (ppm) Concentrations (ppm)

The effect of O 3 exposure on in vitro mycelial growth b. F. graminearum 0.94 a w a. Fusarium graminearum 0.98 a w 80 12 70 10 Colony diameter (mm) Colony diameter (mm) 60 8 50 40 6 30 4 20 2 10 0 0 con 50 100 200 con 50 100 200 Concentrations (ppm) Concentrations (ppm)

The effect of O 3 exposure on in vitro mycelial growth b. A. flavus 0.90 a w a. Aspergillus flavus 0.95 a w 40 25 35 Colony diameter (mm) Colony diameter (mm) 20 30 25 15 20 10 15 10 5 5 0 0 con 50 100 200 con 50 100 200 Concentrations (ppm) Concentrations (ppm)

Mycotoxin analyses a. Aflatoxin B 1 production by A. flavus 45 40 Aflatoxin average µg/g 35 30 25 20 0.9 15 0.95 10 5 0 0 20 40 60 80 100 120 O 3 Concentrations (ppm)

Mycotoxin analyses b. Ochratoxin A production by P. verrucosum 0,08 Ochratoxin average µg/g 0,07 0,06 0,05 0,04 0.9 0,03 0.95 0,02 0,01 0 0 50 100 150 200 250 O 3 Concentrations (ppm)

Effects of O 3 treatment of wheat grain inoculated with F. graminearum  Effect of ozone on wheat grain inoculated with F. graminearum at 0.94 and 0.98 a w MEA after 20 days storage 2 Log CFUs population 1,5 1 0.94 0.98 0,5 0 con 100 200 Concentrations (ppm)

Effects of O 3 treatment of wheat grain inoculated with P. verrucosum  Effect of ozone on wheat grain inoculated with P. verrucosum at 0.90 and 0.95 a w MEA after 20 days storage 12 10 Log CFUs population 8 6 0.95 0.9 4 2 0 con 100 200 Concentrations (ppm)

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 a w after 168 hrs in high O 3 (200 ppm) A. flavus : complete inhibition at > 100 ppm O 3 after 48 hrs regardless of the a w level For all three species there was no effect up to 200 ppm O 3 on mycelial growth regardless of a w level used

Mycotoxin analyses  The amount of aflatoxin was significantly reduced at 50 and 100 ppm after O 3 for 30 mins at 0.90 a w However, at 0.95 a w aflatoxin production was stimulated  At 0.95 a w , ochratoxin A was stimulated by O 3 exposure for 30 mins No toxin was produced at 0.90 a w

Effect of O 3 treatment of natural wheat on fungal populations isolated  F. graminearum at 0.94 and 0.98 a w MEA: 0.05 log reduction of total populations observed in the O 3 treatments regardless of the water activity  P. verrucosum at 0.90 and 0.95 a w MEA : 2 log reduction at 0.95 a w but only 1 log reduction obtained at 0.90 a w at 200 ppm

Conclusions  P. verrucosum spore germination was inhibited, yet at >96 hrs even at low a w levels germination recovered completely (≈100%)  However, the macroconidial germination of F. graminearum was inhibited, especially at 0.94 a w  A. flavus conidial germination was more sensitive to O 3 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 O 3 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 O 3 on grain quality

Thank you for your attention! Any questions