الفهرس 
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Abstract
The present work was an extensive survey of filamentous fungi and yeasts contaminating dry milk and dried milk products commonly consumed by infants and adults. Moreover, the proteolytic and lipolytic activity of some of the isolated filamentous fungi was examined and detection of AFM1 contaminating some of the examined products. Finally, an experimental work was carried to study the effect of some additives like natamycin, potassium sorbate and cinnamon oil on controlling the growth of A. flavus and A. parasiticus which are toxigenic and commonly contaminating the examined products.
A total of 180 samples of milk products as milk powder (packaged and unpackaged), ice cream powder (packaged and unpackaged), milk based infant formula, and milk based wheat and rice baby food, (30 samples each) were collected from different pharmacies and supermarkets in Assiut governorate during the period from March 2019 to March 2020.
I- Mycological evaluation of dry milk and dried milk based products :
The mycological analysis revealed that a total of 87 species related to 26 genera of filamentous fungi were recovered from all of the six investigated dried milk products collected randomly during the period from March 2019 to March 2020 on dichloran rose bengal chloramphenicol agar (DRBC).
The broadest spectrum of fungal genera and species was recovered from unpackaged milk powder (17 genera 46 species) followed by infant formula (12 & 39), unpackaged ice cream (13 & 38) and baby food (16 & 37). Whereas, the lowest was from packaged ice cream samples (9 genera & 24 species) followed by packaged milk powder (10 & 28).
The unpackaged milk powder was heavily contaminanted with fungi accounting for 618 colony forming units/30 g followed by baby food and infant formula (447 and 371 CFUs/30 g respectively), while the lowest contaminanted product was the packaged ice cream comprising 99 CFUs/30 g then packaged milk powder and unpackaged ice cream (232 and 274 CFUs/ 30 g respectively). Filamentous fungi were represented 100 % of the total fungal counts of the packaged milk powder while ranging from 95.96 % to 98.11 % of all remaining products except for baby food which gathered 61.44 % of the total fungal count. The yeast fungi were comprised minor percentages from all products ranging from 0 - 4.04 % of total fungal counts whereas only from baby food, they was constituted 38.56 %.
II- Enzymatic activity of some of the isolated filamentous fungi:
Eighty-two strains out of the hundred filamentous fungi (82%) were showed lipolytic activity, fifty-six strains (56 %) were possessed proteolytic activity, while forty-seven strains were showed both lipolytic and proteolytic activities together.
1- Lipolytic activity:
Eighty-two strains were possessed lipolytic activity. from these, thirty-four strains were highly lipase producers: Alternaria dianthicola (1 strain), Aspergillus brasiliensis (6), A. flavus (1), A. niger (16), A. pseudoglaucus (2), A. tubingensis (4), Cladosporium cladosporioides (1), Cochliobolus oryzae (1), Penicillium aurantiogriseum (1), and Talaromyces duclauxi (1).
Fourteen strains were moderately lipase producers: Acremonium rutilum (1), Alternaria tenuissima (1), Aspergillus flavus (1), A. fumigatus (1), A. heteromorphus (1), A. niger (1), A. parasiticus (1), Cladosporium cladosporioides (1), C. herbarum (2), Mucor hiemalis f. hiemalis (1), Penicillium aurantiogriseum (1), P. chrysogenum (1), and Sarocladium strictum, while thirty-four strains were showed low lipolytic activities.
2- Proteolytic activity:
Fifty-six strains were possessed proteolytic activity. Twenty-one strains were highly protease producers: Alternaria dianthicola (one strain), Aspergillus brasiliensis (3), A. flavoforcatis (1), A. flavus (2), A. flavus var. columnaris (1), A. fumigatus (1), A. heteromorphus (1), A. niger (1), A. parasiticus (1), A. tamari (1), A. terreus (1), A. tubingensis (1), Cladosporium sphaerospermum (1), Penicillium chrysogenum (1), Penicillium citrinum (1), Rhizopus microspores (1), R. stolonifer (1), Talaromyces purpureogenus (1).
Eighteen strains were showed moderate proteolytic activities: Aspergillus brasiliensis (3), A. flavus (2), A. niger (5), A. pseudoglaucus (1), A. tubingensis (2), Curvularia australiensis (1), Penicillium aurantiogriseum (1), P. chrysogenum (2), P. crustosum (1), but seventeen strains have low proteolytic activities.
Six fungal strains were high producers of lipase and protease enzymes which were Alternaria dianthicola (one strain), Aspergillus brasiliensis (3), A. niger (1), and A. tubingensis (1), while thirteen fungal strains were possessed high lipolytic and moderate proteolytic activities; Aspergillus brasiliensis (3 strains), A. flavus (1), A. niger (5), A. pseudoglaucus (1), A. tubingensis (2), and Penicillium aurantiogriseum (1).
III- Aflatoxigenic fungal strains isolated from the examined dry milk and dried milk based products on CAM: IV-
A total of forty-eight strains of aflatoxigenic Aspergillus flavus group isolated from examined dry milk and dried milk products; A. flavus (39 strains) and A. parasiticus (9) were examined for their aflatoxin production ability, that expressing as fluorescence intensity (at 365 mm) on coconut agar medium (CAM).
Sixteen strains out of the examined strains showed intense blue color under UV light indicating very high aflatoxin production ability; A. flavus (13 strains) and A. parasiticus (3), nineteen strains showed blue color indicating high aflatoxin production ability; A. flavus (13) and A. parasiticus (6) and only 13 A. flavus strains showed faint blue color indicating weak aflatoxin production ability.
IV- Incidence of AFM1 in the examined dry milk and dried milk based products:
Twenty samples of dry milk and dried milk products (unpackaged milk powder, unpackaged ice cream powder, infant formula and baby food, 5 samples each) which were previously mycologically evaluated, were examined for the presence of AFM1.
Aflatoxin M1 (AFM1) was detected in 100% (5 samples) of unpackaged milk powder samples ranging from 0.85 to 5.65 µg/kg. All the positive milk powder exceed the Egyptian regulation, 1999 and the European Commission EC regulation, 2006.
Aflatoxin M1 (AFM1) was detected in 40% (2 samples) of baby food samples with concentrations of 1.00 and 1.20 µg/kg, while 20 % (one sample each) of both unpackaged ice cream and infant formula samples were contaminated with AFM1 at concentrations of 0.250 and 1.17 µg/kg respectively. All the positive baby food, unpackaged ice cream and infant formula samples exceed the Egyptian regulation, 1999 and the European Commission EC regulation, 2006.
V- Effect of added antifungal treatments on growth and multiplication of some A. flavus group strains in vitro:
In vitro, preliminary screening of antifungal activities of some preservatives (natamycin and potassium sorbate) and essential cinnamon oil were carried out. The effect of natamycin, cinnamon oil separately and mixtures of both natamycin with potassium sorbate, potassium sorbate with cinnamon oil, and natamycin with potassium sorbate and cinnamon oil, were tested against the most common strains of Aspergillus flavus and A. parasiticus from the investigated samples.
Based on the preliminary screening; the minimum inhibitory concentrations (MICs) of the most powerful treatments; natamycin, natamycin with potassium sorbate, natamycin and potassium sorbate with cinnamon oil on the tested species were determined using the agar well diffusion method to evaluate their effectiveness in controlling them. The MIC was considered as the lowest concentration which inhibited the growth of the respective filamentous fungi species.
The MICs of the antifungal natamycin on the tested strains was ranging from 63 μg/ml when used separately, and 0.312 μg/ml when used in a mixture with potassium sorbate that its MIC value was 31.3 μg/ml. While the MICs of natamycin, potassium sorbate with cinnamon oil when used together was 19 μg/ml, 210 μg/ml and 20.63 μl/ml, respectively. The addition of cinnamon oil to the mixture of natamycin and potassium sorbate increased their antimicrobial efficiency and reduced their MICs.
The three antifungal treatments; natamycin, natamycin with potassium sorbate, and natamycin, potassium sorbate with cinnamon oil have the same MICs on the tested strains.
Thus, the danger of the presence of these toxins in the various dairy products under study on the health of the consumer and the need to avoid their presence in these products in order to preserve his health was discussed.