O potencial uso do ozônio como agente antifúngico e antiaflatoxigênico em castanhas e seu efeito na qualidade nutricional

Ali, E. M.; Abdallah, B. M.

E. M. Ali

King Faisal University, College of Science, Department of Biological Sciences, Al-Ahsa, Saudi Arabia

Cairo University, Faculty of Science, Department of Botany and Microbiology, Cairo, Egypt

http://orcid.org/0000-0002-9794-1557

B. M. Abdallah ^**e-mail: babdallallah@kfu.edu.sa

King Faisal University, College of Science, Department of Biological Sciences, Al-Ahsa, Saudi Arabia

http://orcid.org/0000-0003-0324-8055

*e-mail: babdallallah@kfu.edu.sa

Figuras (5)
Tabelas (2)

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Figure 1
Effect of ozone gas on selected fungal species isolated from nut samples. (A) Effect of ozone gas on the sporulation of some selected fungal species isolated from nut samples. The sporicidal activity of ozone was carried out by spore germination assay. The selected fungal species were exposed to 4 ppm ozone for 0 (control), 30, 60, 90, and 180 min. Spores were allowed to grow on CDA medium, then were harvested when the cultures were fully sporulated. Columns labeled with the same letters are not significantly different at p < 0.05 by Duncan’s test. All values presented as the average of three replicates. Error bars represent the standard deviation. (B) Effect of ozone gas on radial growth of A. flavus. A. flavus was exposed to 4 ppm ozone for 0 (control), 30, 60, 90, and 180 min. Mycelial disks (5 mm in diameter) were transferred to plates filled with PDA media. Radial mycelial growth was determined after 5 days by the calculation of the average of two perpendicular colony diameters for each replicate.

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Figure 2
Effect of ozone on cell membrane leakage and components of cell wall. (A) Effect of ozone gas on Leakage of DNA. A. flavus was exposed to 4 ppm ozone for 0 (control), 30, 60, 90, and 180 min. Spore suspension in each case was centrifuged for 15 min at 4000 rpm at 4 ºC. The optical density (OD) of the supernatant was read at 260. (B) Leakage of proteins. A. flavus was exposed to 4 ppm ozone for 0 (control), 30, 60, 90, and 180 min. Spore suspension in each case was centrifuged for 15 min at 4000 rpm at 4 ºC. The optical density (OD) of the supernatant was read at 280. (C) The content of β-1,3-glucan. A. flavus was exposed to 4 ppm ozone for 0 (control), 30, 60, 90, and 180 min. The fluorescence dye-binding microassay for β-1,3-glucan was applied. The fluorescence intensity was measured with at an excitation wavelength of 398 nm and an emission wavelength of 502 nm. (D) The content of chitin. A. flavus was exposed to 4 ppm ozone for 0 (control), 30, 60, 90, and 180 min. Lyophilized mycelia were dehydrated with 95% ethanol. Columns labeled with the same letters are not significantly different at p < 0.05 by Duncan’s test. All values are presented as the average of three replicates. Error bars represent the standard deviation.

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Figure 3
Ultrastructure modifications of A. flavus following ozone treatment (4ppm, 180 min.). (A) Scanning electron micrographs showing untreated control (a, b) where development of mycelium and conidiophore was normal with abundant conidia and ozonated conidiophores (c, d) where conidiophore development was clearly abnormal and mycelia were shriveled compared to untreated controls (The arrows represent hyphae or conidiophore). (B) Transmission electron micrographs of mycelial morphology of untreated control (a, b) and ozonated (c, d) A. flavus. Amorphous layer on the surface of the control group however, this layer was injured on the ozonated mycelia. Quantification analysis displayed that the cell wall thickness of the control was 170.12 ± 22.18 nm and that of the ozonated group was 121.58 ± 17.33 nm.

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Figure 4
Effect of ozone on the aflatoxins production. (A) Effect of ozone gas on production of aflatoxins (µg/g dry mass). A. flavus was exposed to 4 ppm ozone for 0 (control), 30, 60, 90, and 180 min. A. flavus was inoculated on yeast extract sucrose and incubated for seven days. Aflatoxins were extracted and then analyzed quantitatively using HPLC. (B) Effect of ozone gas on total aflatoxins concentration recovered from the investigated nut samples determined as µg/kg dry mass. 100 g of ozonated and non-ozonated nut samples homogenized with sodium chloride and methanol–water (2:3) mixture and extracted. Aflatoxins were quantified using an HPLC system. Columns labeled with the same letters are not significantly different at p < 0.05 by Duncan’s test. All values are presented as the average of three replicates. Error bars represent the standard deviation.

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Figure 5
Effect of ozone gas on nuts components. Effect of 4 ppm of ozone on the total contents of carbohydrate, protein and lipid in pistachio (A), Almond (B) and peanut (C). 500 g of each ground samples were exposed to ozone concentration of 4 ppm applied for 0 (control), 90, and 180 minutes. Values are mean ± SD of three independent experiments. Columns labeled with the same letters are not significantly different at p < 0.05 by Duncan’s test. All values are presented as the average of three replicates. Error bars represent the standard deviation.

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Table 1
Count of fungal species isolated from three selected nut samples, after the treatment with 4 ppm ozone gas for 90 and 180 minutes.

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Table 2
Effect of 4 ppm of ozone gas applied for 90 and 180 minutes on the fatty acids composition of some selected nut samples

Fungal species	Exposure time (minutes)
	0 (control)				90				180
	Pistachio	Peanut	Almond	Total count	Pistachio	Peanut	Almond	Total count	Pistachio	Peanut	Almond	Total count
Aspergillus flavus	1900ⁱ	1122^h	913^f	3935	920^g	610^e	430^d	1960	22^b	18^a	28^c	68
A. fumigatus	633^g	313^f	200^e	1146	312^f	156^d	95^c	563	27^b	11^a	11^a	49
A. niger	1122ⁱ	855^h	716^g	2693	611^f	410^e	333^d	1354	18^b	11^a	27^c	56
A. oryzae	0.0a	180^f	122^e	302	0.0^a	93^d	65^c	158	0.0^a	19^b	0.0^a	19
A. parasiticus	0.0^a	744^f	544^e	1288	0.0	367^d	280^c	647	0.0	22^b	23^b	45
A. tamarii	100^e	0.0^a	5^c	105	48^d	0.0^a	2^b	50	0.0^a	0.0^a	0.0^a	0.0
A. terreus	15^d	0.0^a	7^c	22	6^c	0.0^a	4^b	10	0.0^a	0.0^a	0.0^a	0.0
A. japonicas	610^h	412^g	333^f	1355	305^e	205^d	156^c	666	22^b	17^a	18^a	57
A. nomius	21^f	15^e	0.0^a	36	11^d	9^c	0.0^a	20	0.0^a	5^b	0.0^a	5
A. wentii	0.0	22^c	0.0^a	22	0.0	11^b	0.0^a	11	0.0^a	0.0^a	0.0^a	0.0
A. ochraceous	0.0	0.0^a	10^c	10^e	0.0	0.0^a	4^b	4^d	0.0^a	0.0^a	0.0^a	0.0
A. flavipes	55^g	40^f	33^e	128	22^d	18^c	18^c	58	0.0^a	8^b	0.0^a	8
Penicillium puberulum	90^g	77^f	0.0^a	167	42^e	32^d	0.0	74	7^b	16^c	0.0^a	24
P. aurantiogriseum	0.0	184^f	155^e	339	0.0	0.0	78^d	78	0.0^a	22^c	11^b	33
P. oxalicum	0.0^a	0.0^a	49^d	49	0.0	32^c	22^b	54	0.0^a	0.0^a	0.0^a	0.0
P. chrysogenum	166ⁱ	133^h	100^g	399	72^f	66^e	53^d	191	11^b	9^a	13^c	33
P. citrinum	110^g	90^f	75^e	284	53^d	41^c	41^c	135	9^a	18^b	18^b	45
P. comembeti	0.0^a	0.0^a	16^c	16	0.0^a	0.0^a	9^b	9	0.0^a	0.0^a	0.0^a	0.0
P. funiculosum	0.0a	0.0^a	19^c	19	0.0a	0.0^a	11^b	11	0.0a	0.0^a	0.0^a	0.0
P.. verrucosum	0.0a	88^e	71^d	159	0.0a	0.0^a	33^c	33	0.0a	0.0^a	16^b	16
P. crustosum	18^e	10^d	10^d	38	8^c	4^b	4^b	16	0.0^a	0.0^a	0.0^a	0.0
P. fellutanum	0.0^a	13^b	^0.0a	13	0.0^a	0.0^a	0.0^a	0.0	0.0^a	0.0^a	0.0^a	0.0
P. italicum	60^g	32^f	29^e	121	28^d	13^c	14^c	55	2^b	0.0^a	0.0^a	2
Alternaria alternate	45^g	32^f	28^e	105	20^d	16^c	13^b	49	0.0^a	0.^0a	0.0^a	0.0
A. tenuissima	63^h	45^g	35^f	143	32^e	21^d	19^c	72	0.0^a	0.0^a	14^b	14
Cladosporium cladosporoides	66^f	55^e	0.0^a	121	35^d	24^c	0.0^a	59	0.0^a	11^b	0.0^a	11
Cochliobolus lunatus	77^d	33^c	0.0^e	110	32^c	15^b	0.0	47	0.0^a	0.0^a	0.0^a	0.0
Botryotrichum piluliferum	22^e	0.0^a	15^d	37	10^b	0.0^a	12^c	22	0.0^a	0.0^a	0.0^a	0.0
Fusarium oxysporum	120^g	66^f	59^e	245	58^d	30^c	32^c	120	12^b	13^b	0.0^a	25
F. moniliforme	166^g	98^f	0.0^e	264	75^e	50^d	0.0^a	125	11^b	22^c	0.0^a	33
Rhizopus oryzae	156^e	133^h	122^g	411	80^f	74^e	63^d	217	13^b	19^c	15^a	47
R. stolonifer	33^e	21^d	0.0^a	54	15^c	10^b	0.0^a	25	0.0^a	0.0^a	0.0^a	0.0
Trichoderma hamatum	71^e	0.0^a	55^d	126	35^c	0.0	2^b	37	0.0^a	0.0^a	0.0^a	0.0
T. harzianm	133^f	0.0^a	75^e	208	65^d	0.0	33^c	98	11^b	0.0a	11^b	22
Bispora antennata	47^d	33^c	0.0^a	80	24^b	23^b	0.0^a	47	0.0^a	0.0^a	0.0^a	0.0
Total count	5899ⁱ	4846^h	3796^g	14541	2919^f	2330^e	1812^d	7061	165^a	233^c	205^b	603

fatty acids (g/100g)	Exposure time (minutes)
	0 (control)			90						180
	Pistachio	Peanut	Almond	Pistachio	% I	Peanut	% I	Almond	% I	Pistachio	% I	Peanut	% I	Almond	% I
C_14:0	0.4	0.0	0.0	0.3	8.8	0.0	0.0	0.0	0.0	0.2	33.33	0.0	0.0	0.0	0.0
C_16:0	11.9	12.4	7.7	9.3	21.8	8.9	28.22	5.2	32.46	8.11	31.84	7.2	41.9	3.9	41.02
C_18:0	2.5	4.6	2.3	1.4	44	2.8	39.13	1.8	21.73	0.98	60.8	2.1	54.34	1.1	52.17
C_20:0	0.4	2.6	0.4	0.2	50	2.7	3.7	0.2	50	0.18	55	2.0	23.07	0.16	60
C_22:0	0.4	3.5	0.0	0.3	25	3.1	11.42	0.0	0.0	0.25	37.5	2.5	28.57	0.0	0.0
C_24:0	0.0	3.2	0.0	0.0	0.0	1.9	40.62	0.0	0.0	0.0	0.0	1.1	65.62	0.0	0.0
C_16:1	2	0.4	0.7	1.3	35	0.2	50	0.45	35.71	0.98	51	0.17	57.5	0.32	54.28
C_18:1	59.7	43.8	69.3	55.2	7.5	39.8	9.1	66.33	4.28	51.23	14.18	34.22	21.87	61.23	11.64
C_18:2	29.7	37.8	25.3	24.8	16.4	34.2	9.52	22.2	12.25	21.3	28.28	31.5	16.66	17.85	29.44
C_18:3	0.8	0.5	0.0	1.77	3.7	0.33	34	0.0	0.0	0.4	50	0.22	56	0.0	0.0

[1] *e-mail: babdallallah@kfu.edu.sa

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O potencial uso do ozônio como agente antifúngico e antiaflatoxigênico em castanhas e seu efeito na qualidade nutricional

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