Six fungal genera including thirteen species were isolated and identified in Agric. Microbiology Dept., Fac. of Agric., Damietta University, Damietta, Egypt as following: Geotrichum candidum; Aspergillus ochraceus; A. alliaceus; A. oryzae; A. niger; A. nidulans; Emericella nidulans; A. flavus; A. glaucus; Penicillium sp.; Mucor sp.; Rhizopus stolonifer and A. flavipes [13] [14] .

The fungal strains were maintained on potato dextrose agar (PDA) medium slants [16] at 5˚C till use. Before use, the fungal strains were subcultured on new slants of PDA and incubated at 25˚C for 5 days.

Using a microscope (Bio-4o2-B Microtech, made in Japan) and a digital microscope eye piece (640 × 480) (10× WF made in China). The fungal spores were photographed and the concentrations were calculated.

Spores suspensions were prepared as described by Osman [17] . Fungi were grown on PDA slant at 25˚C for 10 days. The spores were harvested in sterilized tap water. 10 ml of sterilized saline solution (0.09% NaCl) was added to the slants and the spores were loosened by gently brushing with a sterile inoculating loop. A vortex mixer (VM-300 power: 220 VAC, 50 Hz, 0.16 A/made in Taiwan-associated with Cannic, Inc., USA) was used for one min. to remove all spores from slant [18] . Spores count was performed in a Hematocytometer slide and the following equation was used for fungal spores count:

Spores count/ml = mean of spores count in 10 squares × slid factor × dilution ratio.

where slid factor = 2.5 × 10⁶ Hematocytometer slide (model Buerker MOM BUDA pest).

Lactose fermentation was done on a medium containing the following composition (g/L): peptone, 5.0; beef extract, 3.0; lactose, 5.0; bromothymol blue, 0.03 ml; and the pH was adjusted to 7.0. Each test tube was supplemented with Durham’s fermentation tube. Sterilization was done in Arnold sterilizer at 100˚C for 60 min for three successive days. All tubes were incubated with approximately 1 × 10⁶ spores per tubes. The tubes were incubated in a digital incubator (Switc, MPM Instruments s.r.l., Bernareggio/made in Italy) at 25˚C. The developed color was observed during the incubation period (10 days), the production of acid was recognized by the change in color from blue to yellow and the production of gas was noted in Durham’s tubes [19] .

Casein and milk fat hydrolysis were done on two different cultivation media. The first one was modified potato dextrose agar (MPDA) medium and the second was modified Czapek agar (MCA) medium [13] [14] . The measurement was carried out using modified potato dextrose agar (MPDA) medium, one ml of sterilized skim milk or milk fat was added to PDA medium. All plates were spot inoculation by tested fungal strains. After incubation at 25˚C, the plates of casein and milk fat were flooded with hydrochloric acid (10%) or cupper sulfate (10%), respectively [20] . The results were recorded by measuring the diameter of growth and clear zone using a digital vernier caliper, Made in Jiangsu China [14] . In case of modified Czapek agar (MCA) medium, the chemical composition (g/L) was K2HPO4, 1; Czapek concentrate, 10 ml and agar agar, 15. The pH was adjusted to 5.2, sterilized by autoclaving at 121˚C for 15 min [21] . A modification was occurred in this medium to be suitable for casein and fat hydrolysis. One ml of sterilized skim milk or fat of milk was added to every Petri dish. Skim milk and fat of milk were separately sterilized in Arnold sterilizer at 100˚C for 60 min for three successive days and this medium was used for testing the isolated fungi for proteolysis and lipolysis, respectively [14] .

The chemical composition of Czapek concentrate was as follow (g/100ml): NaNO₃, 30; KCl, 5; MgSO₄・7H₂O, 5 and FeSO₄・7H₂O, 0.1 Czapek concentrate will keep indefinitely without sterilization. The precipitate of Fe(OH)₃ which forms in time can be resuspended by shaking before use [21] .

Ras Cheese was manufactured in El-Ghazy laboratory, Elsawalem, Damietta , Egypt . The procedure suggested by Yossef [22] and Abou-Donia [23] for making Ras cheese was adopted. After completely salting process (dry salting for forty-five day in salting rooms), the Ras cheese wheels were stored at about 15˚C for one month in a commercial ripening room, then the Ras cheese wheels were transferred in a digital incubator at 60˚F (15.5˚C) for another two months, thereafter samples were taken after one, two and three months. Ras Cheese was analyzed for fat, soluble nitrogen (SN) and non-protein nitrogen contents (NPN) according to Ling [24] .

Spores of all the tested fungal strains were counted by a Hemocytometer slide (Figure 1). It was observed that, Aspergillus nidulans was the highest value of spores count being 60 × 10⁶ spore/ml, but Mucor sp. was the lowest number being 7 × 10⁶ spore/ml (Table 1). Spores suspension were prepared to give approximately 1 × 10⁶ spores per ml using the data presented in Table 1, by adding the volume of solution A (spore suspension) to the volume of solution B (saline solution) for adjustment the fungal spores count to give a final concentration of 1 × 10⁶ spores/ml using a micropipette (Micro Volume pipettor-Accumax A Made in China), thus the count of spores became fixed in the following experiments.

Most lactose in milk is lost in whey as lactose or lactate during cheese manufacture. However, low levels of lactose remain in the curd at the end of manufacturing (0.8% - 1.0%). Residual lactose is metabolized quickly to lactate (Glycolysis) during the early stages of ripening at a rate largely determined by temperature and the salt- in-moisture (S/M) levels of the curd [25] [26] . Figure 2 showed that, all fungal strains gave surface growth, but negative results in lactose assimilation except A. nidulans (F6) which produced acid without gas after 24 hr. This observation was explained by two theories, the fungal deacidification and the formation of alcohols. P. camemberti metabolises the lactate to CO₂ and H₂O, which results in deacidification of the cheese surface within three weeks.

The outer part of Camembert undergoes considerable modification of texture and the curd, which is firm and brittle at the beginning of ripening, later becomes soft. The surface flora establishes a pH gradient from the surface (basic) to the interior (acidic) due to consumption of lactic acid and NH₃ production. The increase in pH and breakdown of α_s1-casein by rennet are responsible for the softening of the curd, which gradually extends towards the center, and is visible in a cross-section of the cheese. The production of methyl ketones by P. roqueforti are inhibitory to further mould growth, and may be a factor in preventing excessive mould development in blue veined cheese. The strong reducing conditions in hard cheeses may favour the formation of alcohols from aldehydes. The findings of [1] [4] [7] [14] [27] may explain why the acid formation decreased.

The volume of solution A (spore suspension) was added to the volume of solution B (saline solution) for adjustment the fungal spores count to give a final 1 × 10⁶ spores/ml using a micropipette (Micro Volume pipettor-Accumax A made in China).

All fungal strains did not give gas in the Durham’s fermentation tube, but small air pebbles were observed under the fungal growth in case of Geotrichum candidum, Aspergillus ochraceus, Emericella nidulans, Penicillium sp., Mucor sp. and Aspergillus flavipes (Figure 2). It was also observed that, A. nidulans could produce a soluble red pigment which dissolved in the medium after 10 days of incubation. The finding which reported by Kure et al. [28] could explain the formation of gas where G. candidum is able to grow in environments with high levels of CO₂.

As Jarlsberg cheese releases higher amounts of carbon dioxide than Norvegia cheese, Jarlsberg cheese may provide an environment that selects for G. candidum. Similar results were obtained by Kure and Skaar [15] who reported that, P. roqueforti was capable of growth in an atmosphere with high levels of carbon dioxide. The growth was unaffected, or slightly stimulated, by high levels of CO₂, especially when levels of O₂ were low. This species is also resistant to weak acid preservatives. This may explain why P. roqueforti var. roqueforti is the dominant species with 39.5% of the total strains.

All fungal strains were tested for proteolysis on tow cultivation media. The first one was done on plates of modified PDA medium and incubated at 25˚C for 4 days. It was found that, fungal growth was very strong and caused a very rapid clear zone and was not suitable for enzymes activity evaluation. Thus the further experiments were controlled and examined every day on the second medium of modified Czapek agar (MCA). Also, all tested strains were inoculated on plates containing Czapek agar (CA) medium without skim milk addition as a control. Results of proteolysis were presented in Table 2. All tested fungal strains showed positive results. The fungal growth diameters were corresponding to clear zone diameter and it was varied between 16.40 to 94.53 mm in the cultures of Aspergillus nidulans and Rhizopus stolonifer, respectively. Also, Geotrichum candidum, A. alliaceus, A. oryzae and E. nidulans achieved the following values 21.76, 35.97, 26.71 and 31.87, respectively (Figure 3).

The presence of casein in the cultural medium caused a strong growth comparing with the growth on control. Changing of growth resulting from casein milk addition was in the highest values in the cultures of Rhizopus stolonifer, A. flavus and A. niger being 87.81, 58.32 and 41.58 mm, respectively. On the other hand, the lowest values were achieved by A. nidulans and Geotrichum candidum being 7.09 and 13.71 mm. Florez et al. [29] reported that, Geotrichum candidum is commonly found in mould-ripened cheese varieties. The strains consume lactate and produce several enzymes that break down proteins and fats. These processes involved in the softening of the cheese matrix and in the formation of aroma components (e.g., alcohols, methyl ketones, esters and various sulphides) in mature cheeses. P. roqueforti was also found in similar numbers of G. candidum suggesting that they also play a significant role in maturation of Cabrales cheeses.

Proteolysis is the most important event and the most complex for cheese texture by hydrolyzing the para-ca- sein matrix which gives cheese its structure and by increasing the water binding capacity of the curd (i.e. to the new α-carboxylic and α-amino groups produced on cleavage of peptide bonds) [2] [5] [30] . However, the major role of proteolysis in cheese flavour is in the production of amino acids which act as precursors for a range of catabolic reactions which produce many important volatile flavour compounds. Small peptides produced from

^*Changing of growth (mm) = growth diameter on MCA medium (mm) − growth diameter on CA medium as a control (mm).

hydrolysis of casein, can be broken down into short chain amino acids, peptides, amines, alcohols and sulphur containing compounds, which contribute to flavour formation, by proteinases and peptidases [3] [26] [27] [31] [32] . This observation is in agreement with the results of those [13] [14] [33] who reported that, during the aging of Kefalograviera, Cheddar, Domiati and Feta cheeses, the protein matrix was converted to a smoother structure and softening occurs. These changes are probably due to proteolysis of α_s1-casein, mainly by residual coagulant.

Lipids in foods may undergo hydrolytic or oxidative degradation. However, in cheese, oxidative changes are very limited due to the low oxidation/reduction potential (about −250 mV). However, triglycerides in all cheese varieties undergo hydrolysis by the action of lipases, which result in the liberation of fatty acids in cheese during ripening [25] [26] .

After Ras cheese manufactured and completely salting process, the Ras cheese wheels were stored at about 15˚C for one month in a commercial ripening room, then the Ras cheese wheels were transferred in a digital incubator at 60˚F (15.5˚C) for another two months, thereafter samples were taken after one, two and three months. The storage of Ras cheese at this temperature accelerates lipolysis. Similar results were obtained by Mahony et al. [34] who studied the effect of temperature from 4˚C to 12˚C for ripened of commercial Cheddar cheeses for a total of 270 days. They found that, the levels of total and individual free fatty acids increased with increasing ripening temperature and progressive ripening time.

Increasing ripening temperature and time resulted in increases in the levels of short-(C4:0-C8:0), medium- (C10:0-C14:0) and long-(C16:0-C18:3) chain of free fatty acids. The results also suggested that the use of higher temperatures during the early stages of ripening (1 to 60 day) was most effective at accelerating lipolysis. Cheddar cheese ripened at low temperature (4˚C) did not attain the flavour and aroma characteristics typical of mature Cheddar cheese.

All the tested fungal strains were inoculated on plates containing Czapek agar (MCA) medium with milk fat addition as a control and incubated at 25˚C. Also, all tested species were inoculated on plates containing Czapek agar (CA) medium without milk fat addition as a control. Data in Table 3 and Figure 4 showed that, all strains gave positive results for lipolysis except Aspergillus nidulans. The fungal growth diameter was varied between 9.17 to 30.67 mm in the case of A. niger and A. alliaceus, respectively. The presence of fat milk in the cultural medium caused a changing of growth in the case of A. glaucus; A. alliaceus and A. flavus being 28.11; 27.42 and 25.19 mm, respectively. On the other hand A. niger, Rhizopus stolonifer and A. flavipes achieved lowest values being 5.62, 10.80 and 11.82 mm, respectively.

Results showed also that fungus A. nidulans gave negative reaction, but the changing of growth resulting from milk fat addition was 17.70 mm when compared with the value of control (3.58 mm). It may be due to the nutrients of milk fat addition such as fat and protein. Similar results were obtained by Hayaloglu and Kirbag [9] who reported that, G. candidum affects the cheese biochemistry during ripening. It releases lipases and proteases into the cheese matrix.

Lipolysis is particularly extensive in hard Italian cheese and blue mould chesses and is essential to correct flavour development in these cheeses. Extensive lipolysis is considered undesirable in other types of cheese varieties such as Cheddar, Gouda and Swiss cheeses; high levels of fatty acids in these cheeses lead to rancidity. However, low concentrations of free fatty acids contribute to the flavour of these cheeses, particularly when they are correctly balanced with the products of proteolysis or other reactions [27] . However, the most lipolytic organisms associated with cheese are Penicillium spp., which grow on or in mould-ripened varieties. Penicillium roqueforti, which causes the extensive lipolysis in blue cheese, possesses two lipases with pH optima of 7.5 - 8 and 9 - 9.5, respectively. Penicillium camemberti produces an extracellular lipase that is optimally active on tributyrin at pH 9 and 35˚C [14] [25] [26] .

Malek et al. [35] isolated 35 strains of Enterococcus faecium isolated from two Egyptian cheeses (“Ras” and “Domiati”) in order to determine their ability for acidification, proteolysis and lipolysis activities, autolysis, texture, flavour development, antibiotic resistance and β-haemolytic activity. They demonstrate that, some indigenous strains of E. faecium displayed interesting technological properties for cheese manufacture, together with good safety characteristics. They could be useful for the manufacture of typical products in Egypt and Middle East .

^*Changing of growth (mm) = growth diameter on MCA medium (mm) − growth diameter on CA medium as a control (mm).

The moisture percentage (M%) was 38.86% in fresh Ras cheese wheels (Table 4), this value decreased to 34.67% after salting (after 45 day). This value gradually decreased during storage period (3 months at 15.5 C) from 31.29% in the first month to 30.25% in the third month. Dry matter percentage (DM%) was corresponded to moisture and took a reserve pattern of moisture. The total solids gradually increased towards the end of storage period. These findings are in agreement with those Kheadr et al. [2] ; Tejada et al. [36] ; Abdalla et al. [37] ; Hamid and Abdelrahman [38] ; Habib [14] and El-Fadaly, et al. [39] who reported that, the total solids contents of cheese increased during storage period. This increase was due to continuous loss of moisture from the crud a result of lactic acid development which caused crud contraction. However, the decrease in dry matter in the third month was possibly due to proteolytic effect of microorganisms on the protein and composed volatile components such as ammonia and volatile fatty acids into the ripening rooms.

Fat percentage was 24.05% in fresh Ras cheese wheels, this value increased to 25.89% after salting (after 45 day), this increase was due the lost of water and the increasing of dry matter. During ripening period the values of fat in Ras cheese wheels were 30.53%, 35.98% and 32.38% after the 1^st, 2^nd and 3^rd month. The increasing of fat percentage was due to the fungal enzymes activities specially lipase (Table 2). These results are in accordance with the findings of Kheadr et al. [2] ; Habib [14] and El-Fadaly, et al. [39] and in disagreement with those Hamid and Abdelrahman [38] and Abdalla et al. [37] who reported the decrease in fat content during storage period.

During cheese ripening the lost of weight and the decrease in dry matter was occurred. This decrease was due to the complete oxidation of lactate to CO₂ and H₂O and deamination of amino acids to the corresponding ketoacid and NH₃ [14] . In addition, volatile compounds which composed during ripening period also caused the decrease in dry matter. This compounds such as; hydrogen sulphide and methanethiol (resulting from decomposition of sulphur amino acids); ketones; methyl ketones; aldehydes; primary alcohols (e.g. ethanol); other aliphatic primary alcohols (e.g. 1-butanol, 1-pentanol and 1-hexanol); branched-chain primary alcohols (e.g. 2- methyl-1-butanol, 2-methyl-1-propanol 3-methyl-1-butanol and 3-Methyl-1-butanol) secondary alcohols (e.g. 2-propanol and 2-butanol); butanone, diacetyl, phenol (a major flavour compound in surface-ripened cheeses) and free fatty acids containing four carbons or more can originate from the lipolysis of milk fat or from breakdown of amino acids [1] .

The highest value of F/DM% was 46.42% after 3 months of storage. The highest values of total nitrogen (TN%), soluble nitrogen (SN%) and non-protein nitrogen (NPN%) percentages of Ras cheese wheels were 4.18%; 43.97% and 18.98%, respectively. These values tended to increase with advanced storage. This increase was due to the fungal enzymes activities specially casinease and protease (Table 3). Awad et al. [40] reported the chemical composition of Ras cheese and they found that, moisture was % 47.46, fat was % 36.00 and protein was % 26.78. Dabiza and El-Deib [11] reported the chemical analysis of traditional Ras cheese and they found that, total nitrogen, soluble nitrogen and SN/TN were 16.01, 7.31 and 7.4, respectively.

Similar trends were obtained by El-Soda et al. [41] ; Hassan et al. [42] ; Mohedano et al. [43] ; Osman and Abbas [44] ; Abou-Donia [23] ; Awad et al. [45] ; Osman [46] ; Habib [14] and El-Fadaly, et al. [13] . The proteolysis indices expressed as SN/TN and NPN/TN. The values of both were gradually increased during ripening period. The role of lipolysis and proteolysis in improving quality of Ras cheese was demonstrated by Hattem et

Where: M% = Moisture percentage; DM% = Dry Matter percentage; F% = Fat percentage; F/DM% = Fat on Dry Matter percentage; TN% = Total Nitrogen percentage; SN% = Soluble Nitrogen percentage and NPN% = Non-Protein Nitrogen percentage.

al. [12] . This role could be considered from the values of TVFA, SN/TN and NPN/TN which were greatly correlated with the sensorial properties of the mature cheese.