MATERIALS AND METHODS

The study was conducted during the month of September until December 2016.

Materials: Cow’s milk was supplied by a private farm in Fayoum governorate, Egypt. Rennet powder (CHY-MAX, 2280 IMCU mL^–1, Ch. Hansen, Denmark), yoghurt starter (DVS YC-X11) was obtained from Chr. Hansen's Laboratory (Copenhagen, Denmark). Calcium chloride and sodium chloride were obtained from a local market in Egypt. Microbial lipase (with activity of 40.000 U g^–1) from Rhizomucor miehei strain was obtained from Chr. Hansen's Laboratory (Copenhagen, Denmark). Experimental RCFC samples were produced in our pilot dairy plant of Faculty of Agriculture, at Fayoum University Egypt.

Methods
Preparation of lipolyzed cream: Lipolyzed cream was prepared by incubating cream 35% fat with the microbial lipase 12000 U kg^–1 fat at 27°C for 48 h as described by Tomasini et al.³.

Preparation of Ras cheese flavour concentrate: Ras cheese curd was prepared from skim cow’s milk according to Hofi et al.¹². The drained curd was used for the production of Ras cheese flavor concentrate with added lipolyzed cream at the rate of 0, 10, 20 and 30%. The prepared samples were incubated at 37°C for 7 days and analyzed periodically at 1, 3, 5 and 7 days of storage. The RCFC were sampled in triplicate and analyzed for some chemical composition, ripening indices and sensory evaluated at the 7th day of storage for the total flavor score.

For the determination of flavor/volatile compounds, 4 samples were analyzed using GC-MS spectroscopy. The four samples were, T1 (sample with 20% lipolyzed cream at 3rd day of storage), T2 (sample with 30% lipolyzed cream at 3rd day of storage), T3 (sample with 20% lypolized cream at seventh day of storage), T4 (sample with 30% lipolyzed cream at 7th day of storage).

Isolation of headspace volatiles: The volatiles in the headspace of each sample under investigation were isolated by using a dynamic headspace system. The samples were purged for 1 h with nitrogen gas (grade of N2 <99.99) at a flow rate 100 mL min^–1. The headspace volatiles were swept into cold traps containing diethyl ether and pentane (1:1, v/v) and held at -10°C. The solvents containing the volatiles were dried over anhydrous sodium sulfate for 1 h the volatiles were obtained by evaporation of the solvents under reduced pressure.

Gas Chromatographic Analysis (GC): The GC analysis was performed by using HP 5890 equipped with a Flame Ionization Detector (FID) (Hewlett Packard Corporation, Palo Alto, California, USA). A fused silica capillary column DB-5 (60 m×0.32 mm id,) was used. The oven temperature was maintained initially at 50°C for 5 min and then programmed from 50-250°C at a rate of 4°C min^–1. Helium was used as the carrier gas at flow rate of 1.1 mL min^–1. The injector and detector temperatures were 220 and 250°C, respectively. The retention indices (Kovats index) of the separated volatile components were calculated using hydrocarbons (C8-C22, Aldrich Co.) as references.

Gas Chromatographic-Mass Spectrometric (GC-MS) analysis: The analysis was carried out by using a coupled gas chromatography Hewlett-Packard model (5890)/mass spectrometery Hewlett-Packard MS (5970). The ionization voltage was 70 eV, mass range m/z 39-400 a.m.u. The GC condition was carried out as mentioned above. The isolated peaks were identified by matching with data from the library of mass spectra (National Institute of Standard and Technology) and compared with those of authentic compounds and previous published data¹³. The quantitative determination was carried out based on peak area integration.

Chemical analysis: The RCFC samples were analyzed for their total acidity (%), pH, moisture (%), fat/DM (%), WSN/TN (%) according to Nateghi et al.¹⁴. The Total Volatile Fatty Acids (TVFA) as described by Novikova and Ciprovica¹⁵. Analysis of the total free amino acids method was conducted according to Folkertsma and Fox¹⁶. All analysis were performed in triplicate and results reported as means±standard deviations.

Sensory analysis: Prepared RCFC samples were evaluated for flavor (100 points) according to El-Hofi and Ismail¹⁷ by 10 panelists of the staff members at Dairy Department, Agricultural Faculty, Fayoum University.

Statistical analysis: Statistical analyses were performed using one way ANOVA test. Statistical analysis was carried out using SPSS software version 16. The varying degree of the result is expressed as Mean±Standard Deviation (Mean±SD). The differences between the samples were determined using t-tests (α = 0.05)¹⁸.

RESULTS AND DISCUSSION

Addition of lipolyzed cream in different concentration to Ras cheese curd prepared from skim milk powder, in order to have RCFC showed a significant effect on the sensory, chemical and volatiles analysis based on the storage period. Based on the sensory evaluation, the volatile compounds produced in four samples, 20% cream stored for 3 days (T₁), 30% cream stored for 3 days (T₂), 20% cream stored for 7 days (T₃) and 30% cream stored for 7 days (T₄) were identified using GC-MS.

Seventeen compounds were detected included 7 esters, 5 alcohols, 4 ketones and 1 fatty acid compounds (Table 1). Samples had almost the same volatile constituents; with exception, 2-pentanol was not detected in sample (T₃). Quantitatively, esters e.g., ethyl hexanoate, ethyl octanoate and ethyl butanoate were the dominants among the identified aroma compounds in all samples, followed by alcohols especially 3-methyl butanol and 2-butanol. Ketones as 2-pentanone, diacetyl and 2-nonanone were also detected in a higher concentrations (Table 1). Butyric acid was the only short-chain fatty acid detected in all samples with concentrations ranged from 1.36-1.88%. The above results were found to be in agreement with Ayad et al.¹¹. According to Olson¹⁹ and McSweeney²⁰ the formation of aroma depend on the enzymes present, so different flavors can be obtained. Therefore, the flavor of samples under investigation in the present work results from the interaction of enzymes from the microorganisms. Alcohols detected in samples result from catabolism of amino acids and lactose, reduction of methyl ketones as well as the as degradation of linoleic and linolenic acids^21,22. However, the concentrations of the different ketones varied between the different cheese samples and are formed by enzymic oxidative decarboxylation of fatty acids, through the action of mould^23,18. Methyl-ketones are well known for their contribution to the flavor of blue mould-ripened cheeses^11,24. Among the identified ketones, 2-heptanone is an important flavor compound of Emmental, natural/creamy Gorgonzola and Ras cheeses, while 2-nonanone is the predominant methyl ketone which is present in natural Gorgonzola, ripened Ragusano and matured Ras cheese ^25,26,11.

Diacetyl found in high level in all samples (1.34-1.68%) and described as the most important diketones derived from pyruvate stemming by lactose and citrate metabolism that is produced principally due to the activity of lactic acid bacteria, especially Lactococcus lactis ssp. lactis biovar diacetylactis and Leuconostoc spp. and considered as a creamy aroma component of Camembert, Cheddar, Emmental and Ras cheese^27,10,11. Meanwhile, aldehydes were not identified in RCFC samples as they are characterized as intermediate and unstable compounds, which can undergo in the reduction into alcohols or the oxidation into acids and appear at low concentrations in the aroma profile of most cheeses¹⁵.

Significant differences (α = 0.05) in the levels of ethylesters were also encountered among samples (Table 1).

Table 1:	GC-MS analysis of volatile compounds in Ras Cheese Flavour Concentrate (RCFC)
T1: Sample with 20% lipolyzed cream at 3rd day of storage, T2: Sample with 30% lipolyzed cream at 3rd day of storage, T3: Sample with 20% lipolyzed cream at 7th day of storage, T4: Sample with 30% lipolyzed cream at 7th day of storage

These compounds are formed by chemical or enzymatic reactions of fatty acids with primary alcohols as reported by Le Bars and Yvon²⁸. Ethyl butanoate, ethyl hexanoate, ethyl pentanoate, ethyl heptanoate, ethyl octanoate, ethyl decanoate have been identified as the most common odorants and reported to play an important role in the aroma profile of aged Cheddar, Emmental, creamy Gorgonzola, Grana Padano, Pecorino, natural Gorgonzola, Ragusano, Flor de Guia and aged Ras cheeses^11,24. Fatty acids were considered as important and predominant constituents of the flavor of various cheese types and serve as precursors of methyl ketones, alcohols, lactones and esters formation. The short chain free fatty acids originate from lipolysis of milk fat by the indigenous milk lipases, moulds and exogenous lipases or the breakdown of lactose and amino acids²⁹. They could be also produced by the oxidation of ketones, esters and aldehydes²².

The chemical composition of the prepared lipolyzed cream that used for producing Ras cheese Flavor Concentrate (RCFC) was: 35% fat, 2.23% protein, 60.29% moistures, pH 5.29 and 1.50% as the total acidity. The acidity of the RCFC samples was obviously changed during the storage period due to the concentration of lipolyzed cream used in the preparation of RCFC. The acidity value (%) of the control sample was 0.42% at the 1st day of the storage compared to the corresponding values in the treated samples which were 0.69, 1.30 and 1.62% for T1, T2 and T3, respectively.

The increase of RCFC acidity was associated with a decrease of the pH values. This is might be associated with the percentage of used lipolyzed cream, whereas an accumulation of free fatty acids produced as a result of lipase reaction during the incubation of cream with the microbial lipase as reviewed by Ozcan and Kurdal³⁰. Abd El-Hamid et al.³¹ found that, the incubation temperature had a significant effect on the development of acidity as well as the protein degradation, fat hydrolysis and production of the flavor compounds during the preparation of Ras cheese slurry. The higher of the incubation temperature, the higher rate of acidity and flavor development was reported. Also, starter bacteria was reported to have a vital impact on the ripening process and the rate of developed acidification⁸.

The moisture values were higher in treated samples over the control ones during the whole storage period and decreased in all samples by the end of storage as shown in Table 2. The added cream increased the moisture content of the prepared RCFC samples, while the reduction of moisture might be due to the evaporation of water as a result of the effect of the incubation temperature.

The level of fat/DM (%) increased in the treated RCFC samples depending on the amount of added lipolyzed creamand the change of the dry matter contents as shown in Table 2 which is in agreement with Hofi et al.¹².

The rate of proteolysis in RCFC samples treated and untreated with lipolyzed cream throughout storage period as conducted with water soluble nitrogen (WSN/TN) and the accumulation of total free amino acids TFAA are shown in Fig. 1 and 2. Cheese curd treated with lipolyzed cream exhibited noticeable differences in the proteolysis rate during storage compared to the control curd.

Fig. 1:	Changes in water soluble nitrogen (WSN/TN) of RCFC during the storage period

Fig. 2:	Changes in TFAA contents of RCFC during the storage period

Fig. 3:	Changes in TVFA contents of RCFC during the storage period

Fig. 4:	Flavour scores of RCFC at the end of the storage period

Table 2:	Changes in pH values, T.A (%), moisture (%) and fat/DM (%) of RCFC during the storage period (days)
C: Ras cheese curd without lipolyzed cream, T1: Ras cheese curd treated with 10% lipolyzed cream, T2: Ras cheese curd treated with 20% lipolyzed cream, T3: Ras cheese curd treated with 30% lipolyzed cream, All values represented as Means±SD, Means in the same column with different small letter superscripts are significantly different (α = 0.05)

Proteolysis in RCFC control sample varied from 6.21% (WSN/TN) and 0.04 mg leucine/g at the 1st day of storage to 10.26% (WSN/TN) and 0.10 mg leucine/g at the 7th day of the storage. The same trend could be observed on the other samples, e.g., proteolysis of T1 ranged from 6.35-11.20% (WSN/TN) and from 0.06-0.27 mg leucine/g during the storage period, 7.20-12.38% (WSN/TN) and from 0.08 to 0.33 mg leucine/g for T2 and finally 8.87-14.33% (WSN/TN) and from 0.15-1.15 mg leucine/g for T3. This increase in the extent of proteolysis might be due to the action of rennin enzyme used in the curd preparation and the enzymes from the starter cultures. Moreover, the incubation temperature might have a notable effect on the rate of proteolysis as well. The results are in agreement with Abd El-Hamid et al.³¹, who found higher levels of WSN/TN produced in Ras cheese slurry depending on the incubation temperature used and with El Neenay et al.⁸, who produced the enzyme modified Ras cheese and found the same trend in the proteolysis development and the higher production of water soluble nitrogenous compounds.

The extent of lipolysis as assessed by the TVFA was determined for all RCFC samples during the storage periods (Fig. 3). The TVFA contents of RCFC samples increased during the progress of curd storage in comparison to the control one. This reflects continuous hydrolysis of lipid fraction in curd during storage. The obtained results suggested that lipolyzed cream affected the production of Total Volatile Fatty Acids (TVFA) which is in accordance with Abd El-Hamid et al.³¹, Degheidi et al.³², Hassan et al.²⁴ and El Neenay et al.⁸.

The sensory evaluation of the RCFC samples are presented in Fig. 4, which indicate that the level of added lipolyzed cream affected for the flavor intensity at the end of storage time. This might be due to protein and fat hydrolysis among other enzymatic and chemical reactions during the storage time by the action of lipase enzyme used in the preparation of lipolyzed cream lead to the formation of aroma constituents responsible for flavor perception and subsequent formation of the aroma profile of resultant RCFC. It is well-known that, the higher concentrations of many aroma compounds e.g., diacetyl, 2-butanol, ethylbutanoate and hexylethanoate, may affect negatively the overall flavor profile of the cheese and so, they are reduced during cheese ripening¹¹. This is in agreement to our findings and could be noted throughout the concentrations of the detected volatile constituents (Table 1) which found to be inversely proportional to the sensory grades scored for the samples under investigation.

CONCLUSION

The addition of 30% lipolyzed cream into skim milk curd which stored for 7 days at 37°C enhanced both chemical composition, organoleptic evaluation and the flavor profile of prepared RCFC samples compare with cheese aroma compounds generated during classical ri`pening processes. Fat/DM (%) increased in the treated RCFC samples depending on the amount of added lipolyzed cream and the change of the dry matter contents. Also, cheese curd treated with lipolyzed cream exhibited noticeable differences in the proteolysis rate during storage compared to the control curd.

SIGNIFICANCE STATEMENTS

This study discovers the effect of lipolyzed enzymes on acceleration of ripening of ras cheese and produce ras cheese flavor concentrate. Thus, Ras cheese curd was used to produce Ras Cheese Flavour Concentrate (RCFC). The effect of different levels from lipolyzed cream on the sensory evaluation of Ras cheese flavor was studied. This study will help the industry to increase the turnover of the production of ras cheese as well as the quality of the final product.

RECOMMENDATION

ACKNOWLEDGMENT

We are grateful to Professor Nabil Samyfrom, Dairy Science Department at National Research Centre, for his valuable discussions and opinions.