Characterization and Comparison of Free Fatty Acid Profiles of Eleven Varieties of Turkish Cheeses

Abstract

Free fatty acids profiles of 11 different cheese varieties sold in Turkey were determined to assess the development of lipolysis. Results obtained showed that the concentrations of short chain fatty acids (C₄ and C₆ free fatty acids) were close in all cheeses (P > 0.05), except for Canak cheese. However, significant differences were noted among the samples for other free fatty acids including C₈ to C_18:2 (P < 0.05). Palmitic (C₁₆) and oleic (C_18:1) acids were the most abundant free fatty acids in all cheese samples. Principal component analysis was applied to simplify interpretation of the data and distinguish the variety of the cheeses on the plot. Canak cheese gave a dramatically different free fatty acid profile from the other cheeses, probably because of the fact that ripening of this variety is achieved in the earthenware pots for about 1 year. Van Otlu (ripened with special herbs) and Civil (ripened by spontaneously molding on its surface) cheeses differed from the others by ANOVA and principal component analysis techniques. In conclusion, the degree of lipolysis in the cheeses could be classified into extreme (Canak), high (Civil, Divle Tulum, Mihalic, and Van Otlu), mild (Ezine, Orgu, and Urfa), and low (Dil, Hellim, and Malatya) based on their FFA profiles.

Keywords:

• Cheeses
• Turkey
• Free fatty acids
• Gas chromatography
• Ripening

INTRODUCTION

Cheese ripening is a complex and dynamic biochemical process that includes protein breakdown, fat hydrolysis, and lactose metabolism. The relative importance of these events depends on cheese variety, and these reactions have powerful impact on formation of sensorial characteristics in the cheeses.[1– 3] In general, the rate of protein hydrolysis is measured by conventional or modern analytical methods and the levels of hydrolyzed products are used as the ripening indices in many of the matured cheeses. Hydrolysis of lipids is also used as a quality parameter for most types of matured/ripened cheeses, since changes triggered by lipid hydrolysis affect the physical, chemical, and sensory properties of the cheese. Despite the marginal effects of lipid hydrolysis on cheese quality, the degree of contribution of free fatty acids to the cheese quality have been less investigated compared to the proteolysis.[4] Free fatty acids (FFAs) are important precursors of catabolic reactions, which produce volatile compounds, including esters, alcohols, aldehydes, and ketones, and these compounds contribute to cheese flavor.[2, 4] The level of lipolysis depends on the types of cheese; that is, extensive lipolysis takes place in the surface ripened and some Italian-type cheeses but it is low in brined varieties.[5] Pasteurization of cheese milk inactivates the majority of indigenous milk lipase and, therefore, there are considerable differences between raw or pasteurized milk cheeses with regards to the level of lipolysis.[6] However, lipolytic agents are not only originated from raw milk, but also are originated from rennet paste, starter, adjunct starter, non-starter bacteria, and/or exogenous lipases.[2, 4]

There are more than 50 varieties of cheese in Turkey, including brine ripened (e.g., white cheese), scalded at very high temperature (>90°C) (e.g., Halloumi), pasta-filata type (e.g., Kashar or Kashkaval), herby or spicy cheeses (e.g., Van Otlu cheese). Canak cheese, for example, is ripened in earthenware materials for at least 6 months underground and strong lipolytic taste is noted by consumers. Also, Civil cheese is ripened by molds grown spontaneously on the surface of the cheese and it is characterized by a rancid taste although it has a low level of fat. Some varieties of cheeses (Dil, Hellim, Malatya, Orgu, and Urfa) are scalded by dipping cheese blocks into hot (90°C or more) water or whey and then ripened under dense brine. Limited lipolysis is expected for these types of cheeses. However, information regarding the individual FFA profiles of these cheeses is limited. Another cheese variety is Divle Tulum, which is ripened in a natural cave (4–5°C and 80% ± 5 relative humidity) for a long period. The production practices of Van Otlu cheese present some differences from the other local cheese varieties as some herbs, including Allium, Thymus, Silene, and Ferula species, are added into the cheese curd individually or in appropriate mixtures in the manufacture of this variety.[7– 9] In the past, the cheeses were manufactured in villages for home consumption by traditional methods using raw milk. Recently, a majority of the cheeses are produced in mechanized dairy plants and the pasteurization process is used widely in their manufacture. However, it is still possible to find artisanally-produced cheeses in local retails and they are preferred by consumers due to their unique taste. To understand the intervarietal differences in the cheeses with increasing popularity, 75 cheese samples belonging to 11 different types were collected and characterized based on their individual FFA profiles. Although proteolysis is the main index for ripening in most bacterially ripened cheeses, the FFA profile has been suggested to be considered as a ripening index for cheese as well.[10] The aims of this study were (i) to determine the FFA profiles of different artisanal Turkish cheese varieties and (ii) to understand whether or not their FFA profiles were related with their manufacturing practices. In the previous works, only the acid degree values or free fatty acid compositions (as of % total) were taken into account to determine the level of lipolysis in Turkish cheese varieties.[11 ⁻ 16] FFA profiles of a few Turkish cheese varieties have been laid open should they be investigated.[17 ⁻ 20] These parameters yield limited data to characterize the cheeses based on their lipolytic potentials and to understand intervariation among the varieties.

MATERIALS AND METHODS

Materials

Seventy-five samples of 11 different types of cheese, namely Civil (moldy/mildewy), Canak, Dil, Divle Tulum, Ezine, Hellim, Malatya, Mihalic, Orgu, Urfa, and Van Otlu cheeses were collected from different regions of Turkey in 2009. The following criteria were taken into account during sampling: the samples should be as old as 2–8 months or longer, and the fully ripened ones and ready-for-sale samples were selected as far as possible. The production dates on the label were noted for some samples; however, some samples are still produced by artisanal methods without packaging and labeling. The production dates were estimated based upon the manufacturers' declaration. The samples were kept in sterile plastic bags and transported to the laboratory. The samples were stored at −20°C until analysis.

Determination of Moisture, Fat, and Titratable Acidity

Cheeses were analyzed in duplicate for moisture by IDF,[21] fat by the Van Gulik method,[22] and titratable acidity as described in AOAC.[23]

Determination of FFAs

FFAs were extracted according to De Jong and Badings[24] and analyzed using a gas chromatography-flame ionization detector (GC-FID) system. For this purpose, 1.0 g of cheese was ground with 3 g of anhydrous Na₂SO₄ (Merck KGaA, Darmstadt, Germany) and then added with 0.3 mL of 2.5 м H₂SO₄ (Merck KGaA) and 1 mL internal standard solution (it contains C₅, C₁₃, C₁₇ fatty acids at 0.5 mg/mL each). Then 3 mL of diethyl ether:heptane mixture (1:1, v/v) was added to the extract and shaken for 3 min using a vortex mixer and then centrifuged (at 500 × g) for 2 min at room temperature. Extraction was repeated three times and the supernatant was transferred to a screw-capped tube containing 1.0 g of anhydrous Na₂SO₄. After the column, an AccuBond II 3 mL 500 mg SPE cartridge (Agilent Technologies, Santa-Clara, CA, USA) was conditioned with 10 mL of heptane; the pooled diethyl ether:heptane extract was introduced into the column to separate neutral lipids from FFA. The neutral lipids and FFA were eluted with 10 mL of chloroform:2-propanol (2:1) and 10 mL diethyl ether containing 2% formic acid, respectively. A 500 μg nonanoic acid (C₉) was added to the solution to calculate the recovery of the internal standards. One microliter of sample was injected to the GC-FID system (GC-2010 model, Shimadzu Corporation, Kyoto, Japan) equipped with a capillary column (DB-FFAP, 60 m, 0.25 um, 0.25 mm, J&W Scientific, Folsom, CA, USA). Column oven temperature was programmed to 140°C and held at this temperature for 3 min, then raised to 245°C at a rate of 10°C/min and held for 45 min. Total running time was arranged to 58.5 min. The detector temperature was 260°C. The carrier gas was helium at a flow rate of 30 mL/min. Retention time of individual fatty acids in the samples were determined by using known individual fatty acid standards (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) including butyric (C₄), valeric (C₅), caproic (C₆), caprylic (C₈), nonanoic (C₉), capric (C₁₀), lauric (C₁₂), tridecanoic (C₁₃), myristic (C₁₄), palmitic (C₁₆), margaric (C₁₇), stearic (C₁₈), oleic (C_18:1), and linoleic (C_18:2) acids. Gas chromatography of these fatty acid standards were the same conditions as in cheese samples. The FFA of the cheese samples were quantified using internal standards C₅ (for C₄–C₈), C₁₃ (for C₁₀–C₁₄) and C₁₇ (for C₁₆–C_18:2) due to different responses of the FFAs in FID and their extraction yields in cheese matrix. Correction factors were used for calculation of peak areas of the low (C₄–C₈), medium (C₁₀–C₁₄), and long (C₁₆–C_18:2) chain FAAs separately. Results were expressed as mg FFA/100 g cheese and all samples were analyzed in duplicate.

Statistical Analysis

Data from GC chromatograms were analyzed by ANOVA to determine the difference among 11 different types of cheese in terms of FFAs using SPSS package program version 9.0 for Windows (SPSS Inc., Chicago, IL, USA). Duncan's multiple comparisons test was used as a guide for pair comparisons of the means. Differences among means were determined at P < 0.05. To simplify interpretation of the results, principal component analysis (PCA) was performed using the varimax rotation between the concentrations of the FFA in cheeses on the ANOVA results. Hierarchical cluster analysis (HCA) was performed using Euclidean distance and average linkage without standardizing the variables. HCA was performed without the data for Canak cheese samples due to extremely higher levels of FFA than the other varieties analyzed. To perform the multivariate analyses of the GC-FID results, the same package program (SPSS) was also used.

RESULTS AND DISCUSSION

Levels of Moisture, Fat, and Titratable Acidity

The moisture levels (as mean values) of the cheeses varied from 39.9 (Divle Tulum) to 56.3% (Civil) and wide variations were noted among the samples due to lack of standardization in manufacturing. The lowest and highest titratable acidity values were determined in Hellim (0.18%) and Van Otlu (1.54%) cheeses, respectively. It was thought that the large differences of titratable acidity among cheese varieties may have been linked to the levels of individual FFA. The values for fat varied from 1.79 (Civil) to 25.92% (Divle Tulum). Civil cheese that contained the lowest fat level is traditionally produced from reduced fat milk.

Concentrations of FFA

Results of GC analysis of FFA are shown in Fig. 1. Fatty acids from C₄ to C_18:2 were determined in the cheeses and average values are also given in Table 1. Due to the limited information on FFA profile of Turkish cheeses, the results obtained in this study were discussed with different varieties of cheeses from other countries.

Table 1 Mean concentrations (mg/100 g cheese) and standard deviations (SD) of free fatty acid (FFA) profiles of 11 Turkish cheeses

		Free fatty acids (mg/100 g of cheese)
Cheese varieties		C4	C6	C8	C10	C12	C14	C16	C18:0	C18:1	C18:2	Total
Civil (n = 8)	Mean	14.7^a	8.6^a	4.0^ab	3.2^a	5.9^abc	22.3^b	64.5^d	20.2^de	83.1^d	5.3^b	231.8^c
	SD	4.1	1.3	2.9	4.5	6.6	22.6	55.9	17.5	97.4	5.5
Canak (n = 6)	Mean	87.2^b	44.2^b	37.4^e	35.6^d	38.9^d	139.7^c	356.7^e	56.6^e	386.3^e	20.7^c	1203.3^d
	SD	48.7	8.2	6.5	9.2	11.2	36.3	78.7	7.8	67.8	6.9
Dil (n = 6)	Mean	8.6^a	9.1^a	3.1^a	2.4^a	3.3^a	8.8^a	28.2^ab	6.6^a	23.4^abc	2.4^a	95.9^a
	SD	2.0	0.4	1.5	0.3	0.4	0.8	0.9	1.8	4.5	0.7
Divle Tulum (n = 8)	Mean	13.9^a	10.7^a	14.9^cd	19.5^c	12.5^c	32.8^b	75.8^bcd	22.5^bcd	87.2^cd	7.9^b	297.7^bc
	SD	2.2	0.8	6.1	7.7	3.0	9.9	2.8	7.2	57.7	2.4
Ezine (n = 8)	Mean	17.6^a	11.4^a	9.0^b	9.2^b	7.3^abc	19.5^ab	54.9^abcd	17.8^abc	57.4^abc	7.2^ab	211.3^abc
	SD	4.3	0.5	2.5	3.7	2.7	9.4	26.6	7.1	34.8	3.3
Hellim (n = 6)	Mean	10.0^a	8.5^a	2.5^a	1.8^a	2.7^a	6.2^a	19.9^a	6.1^a	17.6^a	2.6^a	77.9^a
	SD	1.9	1.0	0.9	0.3	0.4	0.9	3.7	1.4	3.5	0.6
Malatya (n = 8)	Mean	11.0^a	8.5^a	2.0^a	2.2^a	2.8^a	7.4^a	24.9^ab	8.1^a	21.6^ab	2.6^a	91.1^a
	SD	1.5	0.1	1.0	0.7	1.1	2.3	10.0	1.9	7.8	0.9
Mihalic (n = 8)	Mean	9.2^a	8.7^a	10.0^bc	9.0^b	10.2^bc	31.0^b	91.2^cd	25.1^cd	82.1^bcd	9.2^b	285.7^bc
	SD	1.7	0.9	3.8	5.7	4.6	14.8	46.3	12.5	40.9	3.9
Orgu (n = 5)	Mean	13.1^a	11.0^a	5.3^ab	3.9^a	5.6^ab	16.0^ab	49.1^abc	11.4^ab	43.3^abc	5.0^ab	163.7^bc
	SD	2.0	0.6	2.2	1.8	2.5	8.7	26.7	6.5	25.9	3.6
Urfa (n = 6)	Mean	11.3^a	10.5^a	6.5^ab	5.1^b	5.3^ab	16.2^ab	42.6^abc	9.3^a	32.7^abc	4.1^ab	143.6^ab
	SD	1.5	0.5	2.5	3.7	2.5	9.1	20.7	4.5	19.5	2.5
Van Otlu (n = 6)	Mean	19.1^a	11.2^a	14.2^d	17.2^c	11.1^c	30.4^b	74.6^abcd	22.9^cd	54.1^abc	5.4^ab	260.2^bc
	SD	3.9	0.4	9.2	14.3	8.3	7.9	16.5	16.7	39.5	2.8
Means of pairs in the same column with different superscript are significantly different (P < 0.05).
SD: standard deviation.

Figure 1 A representative gas chromatogram of free fatty acid extracted from Canak cheese and internal standards (C₅, C₉, C₁₃, and C₁₇) used.

The most abundant FFAs found in the samples were palmitic (C₁₆) and oleic acids (C_18:1) with the levels of 19.9–356.7 and 17.6–386.3 mg/100 g, respectively. These free fatty acids were also present at large quantities in other cheeses, such as Beyaz peynir, Tulum, and Kashar cheeses,[25] Urfa,[20] Teleme,[26] Manchego,[27] and Cheddar.[4] In general, the highest concentration of FFA was determined in Canak cheese, which is a raw milk cheese and ripened underground for around a year and the total FFA level was 1203 mg/100 g cheese. The lowest level of total FFAs was found in Hellim cheese (77.8 mg/100 g), due probably to the curd scalding at high temperature.

Concentration of butyric acid (C₄) in Canak cheese samples were considerably higher (87.2 mg/100 g) than the other types of cheeses (varied between 8.6 and 19.1 mg/100 g) (P < 0.05). The high level of butyric acid in this variety may be attributed to the growth of fungi on the surface of this cheese, as the ripening conditions of this variety are suitable for the growth of molds. The levels of the butyric acid in the cheeses except for Canak were similar to the results of 180 day-old Kefalogreviera cheese[28] but higher than those for 180 day-old Teleme cheeses.[26] Similarly, caproic acid (C₆) values exhibited the same trend with butyric acid. C₆ level was extremely high (44.2 mg/100 g) in Canak cheese, varying from 8.5 to 11.4 mg/100 g.

The concentrations of caprylic (C₈) and capric (C₁₀) acids presented similar behavior in all samples. The mean values for caprylic acid in the cheeses were significantly different. The highest (37.4 mg/100 g) and lowest values (2.0 mg/100 g) were found in Canak and Malatya cheeses, respectively. Hellim cheese, which has similar manufacturing and ripening protocols to Malatya cheese, also had low levels of caprylic acid (2.5 mg/100 g). Interestingly, higher values for caprylic acid values were determined in Divle Tulum (14.9 mg/100 g) and Van Otlu (14.2 mg/100 g) cheeses than the other cheeses. The last two cheeses are separated from the others due to their production practices (Van Otlu cheese is added with herbs) and ripening conditions (Divle Tulum is ripened in a buried earthenware container for a long period). Similar trends were noted for capric acids in all samples and the lowest levels of capric acids were determined in Civil, Dil, Hellim, and Malatya cheeses, while the highest levels were in Canak, Divle Tulum, and Van Otlu cheeses (P < 0.05). The capric acid values for Civil, Dil, Ezine, Hellim, Malatya, Mihalic, Orgu, and Urfa cheeses were similar to the values reported for Teleme cheese.[26] When compared to the short chain fatty acids (C₄ to C₁₀), these fatty acids were at a much higher concentration in Canak, Divle Tulum, and Van Otlu cheeses. Low and high levels of these short chain fatty acids indicate the low and high lipolytic action of the natural microflora of the corresponding cheeses.[2, 4] Differences were observed between the samples in terms of lauric (C₁₂) and myristic (C₁₄) acids (P < 0.05) and high levels of these two fatty acids were noted in Canak, Divle Tulum, Mihalic, and Van Otlu cheeses; however, they were at lower concentrations in Dil, Hellim, and Malatya cheeses. The last three cheeses are manufactured by curd-scalding and this process inhibits the enzymes from milk and microorganisms. Therefore, lipolysis developed fairly slowly in these types of cheese. According to the Duncan's multiple comparison test, the cheeses were separated into three groups based on their lauric and myristic acid levels. One group included Dil, Hellim, and Malatya cheeses (high temperature curd-scalding and short time ripening); the second group included Orgu and Urfa (curd-scalding but long ripening period); and the last group included the other cheeses (raw milk in production and very long ripening period and growth of complex microflora). These findings indicate that the manufacturing and ripening conditions were considerably significant for the formation of medium chain length FFA in cheese. Use of raw milk[20] and artisanal rennet[4, 29] in the manufacture of cheese can improve lipolysis in cheese during ripening.

One of the major FFAs was palmitic (C₁₆) acid in the cheeses investigated and significant differences were found for palmitic acid among the cheeses (P < 0.05). The highest (356.7 mg/100 g) and lowest (19.9 mg/100 g) concentrations of palmitic acid were found in the samples of Canak and Hellim cheeses, respectively. Palmitic acid levels in other cheeses varied from 24.9 mg/100 g (Malatya) to 75.8 mg/100 g (Divle Tulum) and that fatty acid in each group of cheeses were significantly different. Palmitic and oleic acids were reported to be the major fatty acids in Feta,[29] Teleme,[26] and Kefalograviera[28] cheeses, whereas stearic and oleic acids were the most abundant acids in white-brined cheese.[17] Atasoy and Turkoglu[20] identified the palmitic acids as the most abundant fatty acids in Urfa cheeses, but they found the stearic and oleic acids at close levels. The long chain fatty acids (C₁₄ to C_18:2) were dominant in many cheeses; however, they do not contribute to the cheese flavor at a considerable level as much as short chain fatty acids.[4, 26] Stearic acid (C_18:0) concentrations of the samples differed in majority of the cheese samples (P < 0.05); however, Dil, Hellim, Malatya, and Urfa cheeses contained similar levels of this fatty acids (P > 0.05). Stearic acid level was the highest in Canak cheese followed by Mihalic, Van Otlu, and Divle Tulum cheeses in decreasing order and, the differences between the samples were significant (P < 0.05). Oleic (C_18:1) acid showed the same profile as palmitic acid in the cheeses and it was the second most abundant fatty acid in these cheeses. Its concentrations were higher in Canak (386.3 mg/100 g), Divle Tulum (87.2 mg/100 g), Civil (83.1 mg/100 g), and Mihalic (82.1 mg/100 g) cheeses than the other varieties varying from 17.6 mg/100 g (Hellim) to 57.4 mg/100 g (Ezine). Dil, Hellim, and Malatya cheeses contained similar levels of oleic acid, indicating that heating of milk or scalding of cheese reduced the formation of FFA in the cheese as emphasized by many workers.[4, 6, 29] Linoleic (C_18:2) acid concentration in Canak cheese samples (as mean value of 20.7 mg/100 g) was much higher than other cheeses varying from 2.6 to 7.9 mg/100 g (P < 0.05). According to the linoleic acid contents, the cheeses were grouped into three groups: (1) Civil, Divle Tulum, and Mihalic cheeses; (2) Ezine, Orgu, Urfa, and Van Otlu cheeses; and (3) Dil, Hellim and Malatya cheeses.

Principal Component Analysis of FFAs

Principal component (PC) analysis was performed on the concentrations of free fatty acids as variables to clarify separation of the cheese samples and interpretation of the results. PC1 and PC2 accounted for 92.5 and 4.9% of the variance, respectively, as shown in Fig. 2. Figure 3 shows the hierarchical cluster analysis (HCA) of the data and the cheeses separated into three groups. Due to extremely high concentrations of the FFAs, Canak cheese was not included in the data sheet. Except for Canak cheese, all separations were in accordance in both PCA and HCA.

Figure 3 A dendrogram from HCA of data obtained from free fatty acid profiles of the cheeses.

Figure 2 Principal component analysis of the chromatographic data from free fatty acid profiles of the cheeses. (Color figure available online.)

PCA showed that Canak, Civil, and Van Otlu cheeses differed from the other cheeses, and Canak and Van Otlu cheeses were characterized with higher levels of medium and long chain fatty acids. As shown in Fig. 2, the cheeses were distinguished into four main groups. The cheeses with more than 300 mg FFA/100 g have a characteristic lipolytic flavor note and lipolysis plays a major role in the ripening of these types of cheese.[30] Therefore, the samples could be classified based on the concentrations of FFA by using the multivariate analyses. First, extreme lipolysis was observed in Canak cheese and the cheese located on the plot individually. The second main group contained cheeses with a high degree of lipolysis including Civil, Divle Tulum, Mihalic, and Van Otlu cheese. Civil and Van Otlu cheeses are separated individually due to their different FFA profiles, but closely located and positioned under the same group both by PCA and HCA. The third group was mid-lipolysis degree cheeses including Ezine, Orgu, and Urfa cheeses. Finally, the last main group contained cheeses with a degree of lipolysis including Dil, Hellim, and Malatya varieties, which had close concentrations of FFA and they clustered on the same position on the HCA dendrogram (Fig. 3).

CONCLUSIONS

The results obtained showed that 11 varieties of cheeses had different FFA profiles. The free fatty acid concentrations of the cheeses were largely affected by the manufacturing practices and ripening conditions. The higher concentrations of FFA were identified in the raw milk cheeses ripened for a long period, whereas they were at lower concentrations in the curd-scalded cheeses ripened for a shorter period. Limited lipolysis was determined in Dil, Hellim, and Malatya cheeses, while mild- or high-lipolysis was observed in Civil, Canak, Divle Tulum, Mihalic, and Van Otlu cheese. PCA of the results of free fatty acids analyses revealed that Canak cheese was grouped individually and it was completely different from the other cheeses. PCA of the results proved to be useful information about FFA profiles of the cheeses. Among the FFA, the palmitic (C₁₆) and oleic (C_18:1) acids were the principal fatty acids in all cheeses, as with other Turkish cheeses including Beyaz peynir, Tulum, and Kashar. Long-chain free fatty acids (C₁₄ to C_18:2) were present in greater proportion (about 70 to 80% of total fatty acids). It is known that long chain free fatty acids do not contribute to cheese flavor due to higher perception thresholds. The short (C₄–C₈) and medium (C₁₀–C₁₂) chain fatty acids have considerably lower perception thresholds and give the characteristic flavor to cheese. Therefore, Canak, Divle Tulum, Ezine, and Van Otlu cheeses were expected to have a strong characteristic flavor note since the levels of short chain free fatty acids were high (>40 mg/100 g) in these cheeses.

ACKNOWLEDGMENTS

This study was funded in part by the Scientific Research Projects Unit of Inonu University, Turkey (Project No. 2007/31). The authors would like to thank H. Tanaci-Kaptanoglu (Antteknik Co., Inc., Ankara, Turkey) for her helpful technical assistance during the evaluation of FFA and Professor B. Ozer for critical reading of the manuscript.