Fungi isolated from Ras cheese samples
In this study, Ras cheese was prepared with different three treatments by using different probiotic bacterial strains as adjunct culture plus the control and it was stored for 90 days. A total of 15 species and one species variety belonging to 6 fungal genera were recovered from Ras cheese samples at 0 and 90 days using two isolation media at 28°C (Table, 1). On CzDA medium, 14 species and 1 variety belong to 6 genera. On the PDA medium, 12 species and 1 variety belong to 5 genera (Table 1). Aspergillus (represented by 6 species plus one variety on CzDA medium and 5 species plus one variety on PDA medium) and Penicillium (represented by 4 species on each of the two used media) were the most common fungal genera being isolated. Only one species of each Cladosporium, Mucor and Rhizopus were isolated on the two media used. On the other hand, one species of Paecilomyces was recorded on Czapek agar only.
Milk and its products provide a favorable environment for the growth of various microorganisms, whereas Penicillium and Aspergillus are the common fungal contaminants of cheese (Gandomi et al. 2009). Our results are in harmony with several other studies. El-Fadaly et al. (2015) isolated 66 fungal isolates from Ras cheese and classified them into13 species belonging to 6 genera of which, Aspergillus was the most predominant. These results are also confirmed by Elramly et al. (2019) who isolated Aspergilus niger, A. flavus, A. fumigatus, A. ochraceus, A. candidus, A. terreus, Cladosporium spp., Mucor spp., Paeciliomyces spp., Penicillium spp. and Rhizopus spp. from Egyptian Ras cheese.
On Czapek’s medium
The results recorded in Table 2 clearly show that the total number of fungi in fresh Ras cheese in all three treatments (ranged from 277 to 348 CFU/g cheese) were less than those recorded in control samples (525 CFU/g cheese). Also, the number of total fungal colonies in storage control samples (334 CFU/g) was highly decreased than those recorded in the fresh control (525 CFU/g). At the end of the ripening period (90 days), the highest count of fungi was isolated from the sample of T1 which was 344 CFU/g, while the least count was isolated from the sample of T2 which was 302 CFU/g.
Aspergillus was represented by 6 species and one variety. Penicillium was represented by 4 species. The rest of the genera represent by one species. The percentages of Aspergillus species from the recorded total fungal counts in the fresh and stored samples were the highest compared to the other species (Table, 2). Aspergillus flavus and Aspergillus niger were the most common and have the highest percentages. However, the treated cheese samples showed lower percentages of these species compared to the control, which could reflect the positive effect of adding the Lactobacillus starters to cheese.
The number of species per sample ranged from 7-10 species/sample. The highest number was isolated from T2 sample after 90 days of ripening (10 species).
On PDA medium
With reference to Table (3), the total number of fungal colonies of Ras cheese samples on PDA medium ranged from 243-363 CFU/g. The total number of fungi in control, T1, T2 and T3 fresh cheese samples were 287, 363, 265 and 296 CFU/g, while were 301, 271, 267and 243 CFU/g after 90 days, respectively. The highest count was isolated from the sample of T1 at fresh time (363 CFU/g), while the least count was isolated from the fresh sample of T2 (265 CFU/g). At the end of ripening, the highest count was isolated from the control sample (301 CFU/g), while the least count was isolated from T3 sample (243 CFU/g).
Aspergillus and Penicillium also were the most common isolated genera. Aspergillus percentage was higher in the fresh control cheese (87.8%) compared to the treated cheese (ranged from 78.49% to 84.45%), however, it ranged from 49.16% to 79.83% after 90 storage days (Table, 3).
The number of species ranged from 5 to 9 species/sample. The highest number was isolated from the fresh control sample (9 species). Aspergillus was represented by 5 species and one variety, while Penicillium was represented by 4 species. The other isolated fungal genera were represented by only one species for each (Table, 3). Also, it is noted that each of A. flavus, A. fumigatus, A. niger, P. chrysogenum, P. corylophium, P. funiculosum and Rhizopus stolonifer were isolated from control plus all the three treatments cheese, however each of A. versicolor and P. duclauxii appeared on control cheese samples at zero time only (Table, 3).
These results in Table 2 and 3 suggest that probiotic bacteria incorporated in the manufacture of Ras cheese prevented and reduced the fungal growth in cheese. This inhibition of growth could be due to the facultative anaerobic condition created by bacteria in the cheese (Batish et al. 1997), or due to their lactic and organic acids production, which decreases the pH of the growth environment (Bonestroo et al. 1993; Caplice and Fitzgerald 1999). Also, these bacteria have the potential to produce other components, e.g., reuterin, hydrogen peroxide, proteinaceous compounds, hydroxyl fatty acids and phenolic compounds which have a negative effect on fungal growth (Dalié et al. 2010).
Occurrence of mycotoxins in cheese samples
The presence of mycotoxins were confirmed in different types of cheese by several studies (Siemens and Zawistowski 1993; Gandomi et al. 2009). Lieu and Bullerman (1977) showed that aflatoxins B1 and G1 were stable in cheese during storage at 5 °C. The high presence of Aspergillus species, especially A. flavus, in our studied cheese supported the hypothesis of the presence of mycotoxins in these cheeses. Therefore, Ras cheese samples were examined for the natural occurrence of mycotoxins. The results showed that the examined Ras cheese samples were completely free from the presence of mycotoxins. This result gives an assumption that the raw milk used in the preparation of Ras cheese was free from mycotoxins. Also, the use of probiotic bacteria as an adjunct starter as well as the production and storage conditions could be the reason to prevent the mycotoxins formation by the contaminated fungi.
In this respect, several bacterial species of Bacillus, Lactobacilli, Pseudomonas and Ralstonia have shown the ability to inhibit fungal growth and production of mycotoxins (Nesci et al. 2005; Palumbo et al. 2006; Zohri et al. 2018). El-Nezami et al. (1998); Wiseman and Marth (1981) found that the fungal growth and mycotoxins production are inhibited by antifungal metabolites produced by lactic acid bacteria which have the potential to bind aflatoxins. Gomah and Zohri (2015) found that Lactobacillus rhamnosus completely inhibited fungal growth as well as deoxynivalenol, zearalenone and fumonisin B1 production by Fusarium graminearum, F. culmorum and F. proliferation, respectively.
On the other hand, Aiad and Abo El-Makarem (2013) found that 56 % of examined Ras cheese samples were contaminated with aflatoxins with levels ranging from 7.40 to 111.50 ng/kg. Seddek et al. (2016) found that two out of three Ras cheese samples contaminated by aflatoxins B1, B2 and G2. Also, Elramly et al. (2019) recorded the contamination of Ras cheese samples by aflatoxins and Ochratoxin A.
Mycotoxins production by fungi isolated from Ras cheese
A total of 29 fungal isolates from different Ras cheese samples were examined for their ability to mycotoxin production (Table 4). These isolates belong to Aspergillus (18 of A. flavus and 11 of A. niger). Seven out of the 18 tested A. flavus isolates had the ability to produce aflatoxin B1 and two isolates had the ability to produce aflatoxin B1 and G1. Three isolates out of the 10 tested A. niger had the ability to produce Ochratoxin A (Table 5). This observation raises the possibility of contamination of cheese with the aflatoxins and ochratoxin, especially if it is stored for periods longer than 90 days.
In a study by Mohamed (2020), several isolates of A. flavus and A. niger groups showed positive production of aflatoxins B1, B2, G1, G2, and ochratoxin A from different dairy sources. Also, Varma and Verma (1987) found that three out of seven A. flavus isolates have the ability to produce aflatoxin B1. Sánchez-Hervás et al. (2008) found that 49.2% of A. niger strains were able to produce ochratoxin A.
Extracellular lipases and proteases secreted by filamentous fungal isolates
Lipases and proteases enzymes are considered very important factors that can affect the sensory and the characteristics of Ras cheese during the ripening period (Seddek et al. 2016). Twenty-eight fungal isolates belonging to the species of Aspergillus (18 of A. flavus and 10 of A. niger) were collected from Ras cheese samples and were examined for their abilities to produce lipases and proteases (Table 5). All the tested fungal isolates showed lipase positive results. Seventeen possessed high lipase activities (activity depth more than 20 mm), while 3 and 8 had moderate and low ability to produce the enzyme, respectively. Two isolates of Aspergillus flavus showed the highest producers of lipase (24 mm as clear zone). This result is in agreement with Refaie (2013), who found that Aspergillus flavus was the highest producer of lipase enzyme with 30 mm clear zone as a depth of activity. Also, Seddek et al. (2016) recorded that A. niger was the highest lipase producer, followed by A. ustus and A. flavus.
Also, all the 28 fungal isolates tested in this study showed high and moderate positive results for their ability to proteases production. Nine of them possessed high production levels (more than 20 mm), where one of Aspergillus flavus isolates presented the highest producer with 31 mm clear zone. Nineteen isolates had moderate production (10-20 mm). The lowest producer of proteases (16 mm) was Aspergillus niger (T1a / PDA / fresh) which was isolated from Ras cheese samples made with adding Lb. acidophilus as adjunct culture (Table 5).
The obtained results are in agreement with that reported by Seddek et al. (2016) who found that A. flavus and A. niger were the highest producers of casienase. In addition, El-Fadaly et al. (2015) found that the presence of casein in the fungal media caused a strong growth of the fungal strains compared with the growth on control medium without casein. Our results, in addition to the results found in previous studies, confirm the negative impact of the growth of filamentous fungi on Ras cheese and its expected defects on the sensory properties as a result of the secretion of lipolytic and protein degrading enzymes.