Scholarly article on topic 'Effect of Wild Strains Used as Starter Cultures on Free Fatty Acid Profile of Urfa Cheese'

Effect of Wild Strains Used as Starter Cultures on Free Fatty Acid Profile of Urfa Cheese Academic research paper on "Animal and dairy science"

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Academic research paper on topic "Effect of Wild Strains Used as Starter Cultures on Free Fatty Acid Profile of Urfa Cheese"

DE GRUYTER

Original article Section: Food Technology

Pol. J. Food Nutr. Sci., 2016, Vol. 66, No. 4, pp. 303-310 DOI: 10.1515/pjfns-2015-0034 http://journal.pan.olsztyn.pl

Effect of Wild Strains Used as Starter Cultures on Free Fatty Acid Profile of Urfa Cheese

H. Avni Kirmaci*

Department of Nutrition and Dietetic, Karabük University, 78050 Karabük, Turkey

Key words: Urfa cheese, wild type strains, free fatty acids, lipolysis, sensory analysis

In the present study, the influences of wild-type lactic acid bacteria including Lactococcus lactis subsp. lactis 1B4, Lactococcus garvieae IMAU 50157, Enterococcus faecium ATCC 19434, Enterococcus durans IMAU 60200 and Enterococcus faecalis KLDSO.034 on the composition and free fatty acid contents of Urfa cheeses were evaluated throughout the ripening period. Three different combinations of the strains were employed in the manufacture of cheeses from pasteurised milk. These are: cheese A (strains 1B4+ATCC 19434+IMAU 50157), cheese B (strains 1B4+IMAU 60200+ATCC 1934) and cheese C (strains ATCC 19434+1B4+IMAU 50157+IMAU 60200+KLDS0.0341). The control cheese (cheese D) was produced from raw ewe's milk without starter culture. The basic composition of ripened cheese samples was not significantly affected by wild type strains. C cheese had a higher level of lipolysis than the other cheeses at all stages of ripening (p<0.05). Sensory evaluation of the cheese samples revealed that control cheese had significantly higher aroma and flavour scores than the other cheeses.

INTRODUCTION

Urfa cheese which is a brined type cheese native to Southeastern Anatolia region of Turkey, is an artisanal cheese made from either raw milk of Awassi sheep breed or mixture of caprine and ovine milks at appropriate ratios. In traditional Urfa cheese-making practices, the milk is not pasteurised and no starter cultures are used. The milk is coagulated with animal rennet at 30-32°C for about 90 min, cut into small cubes and drained by gravity drainage in special moulds of triangular shape called "parzin". Afterwards, the cheese blocks are dry salted and/or kept in brine (~16 g/100 g NaCl, w/v) for 5-6 months [Ozer et al., 2002].

Lipolysis in cheese is a result of action of biolytic enzymes called hydrolases (lipases and esterases) that split the ester linkage between a fatty acid and the glycerol moiety of the tri-acylglycerol [Lopez et al, 2006; McSweeney, 2004]. Among the short-chain free fatty acids propionic, butyric, isobutyric and iso-valeric acids play active role in aroma formation; the medium-chain free fatty acids, such as hexanoic, octanoic and decanoic acids, are visibly arisen from the lipolytic breakdown of milk fat [Randazzo et al, 2008].

Owing to increasing consumers' demand towards healthier and safer foods, some modifications in the manufacturing practices of Urfa cheese have been made [Ozer et al, 2004]. The major modification is the replacement of raw milk by pasteurised milk. The brine concentration has also been reduced from 16-18% (w/v) to 10-12% (w/v). The use of pasteurised

milk in cheese manufacturing has made the incorporation of lactic starters including lactococcal strains necessary. This eventually has resulted in lacks of characteristic aroma and flavour attributes of raw milk Urfa cheeses in its industrial counterparts. Therefore new strains that are isolated and identified from traditional Urfa cheeses should be employed in industrial cheese-making practices. At this point, characterisation of traditional cheese flora and preparation of proper strain combinations are of critical importance for obtaining industrial cheese with sensory and physicochemical properties close to its traditional counterpart. Such an attempt was made by Kirmaci [2010] who characterised the predominant microflora of traditional Urfa cheese made from raw sheep's milk. The author demonstrated that the natural lactic flora of Urfa cheese contained the salt-resistant lactococci (mainly L. lactis, L. garvieae) and enterococci (mainly E. durans, E. faecalis and E. faecium). Although, enterococcal strains are generally recognised as a sign of unsafe production, some of these strains have been used successfully in starter combinations of different European cheeses such as Mozzarella [Parente et al, 1989], Feta [Lit-opoulu-Tzanetaki et al, 1993], Venaco [Casalta & Zennaro, 1997] and Cebreiro [Centeno et al, 1999]. The present study aimed to determine the FFA profiles of cheeses made from selected starter culture (wild type) combination that was isolated from traditional raw milk Urfa cheese.

MATERIAL AND METHODS

* Corresponding Author: Tel: +903704330202, Fax: +903704330262; E-mail: avnikirmaci@karabuk.edu.tr;

Materials

Raw sheep's milk supplied from Harran University Agricultural Faculty Dairy Plant (Sanliurfa, Turkey) was used

© Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences

© 2016 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerUnau5heni'cated (http://creativecommons.org/licenses/by-nc-nd/3.0/). Downlead Date | 3/23/17 8:53 AM

in the manufacture of Urfa cheese. Calf rennet was used to coagulate milk in liquid form (declared coagulating power of 1:16,000 IMCU, Mayasan A.S., Istanbul, Turkey). Starter strains were selected among the isolates obtained from traditional Urfa cheese produced from raw sheep's milk. The strains used were previously characterised by phenotypic and genotypic 16S rDNA sequence analysis by Kirmaci [2010]. The strains were chosen according to their contributions to the milk microflora and rate of acidification. The strains used with percentage of identity in brackets were: Lactococcus lac-tis subsp lactis B14 (99%), Lactococcusgarvieae IMAU 50157 (96%), Enterococcus faecium ATCC 19434 (97%), Entero-coccus durans IMAU 60200 (96%) and Enterococcus faecalis KLDS0.0341 (96%). All chemicals were derived from Sig-ma-Aldrich Co. (Interlab A.S., Istanbul, Turkey) and Merck (Merck Pharmaceutical and Chemical Trading. Inc., Istanbul, Turkey), and were of analytical grade.

Cheese-making protocol

A batch of sheep's milk (100 liters) was divided into four equal parts. Three parts of milk (samples A to C) were pasteurised at 72°C for 2 min (vat system) and cooled to 32°C. The last part was not subjected to heat treatment and converted to cheese without starter culture (sample D, control). Bacterial isolates were enriched in M17 broth at 37°C for 24 h. Then, the isolates were harvested by centrifugation and washed with peptone water. The isolates were then inoculated into sterile skim milk (10 mL) for pre-enrichment for bulk culture and kept at 37°C for 24 h. This culture was used to prepare working culture for cheese making in the same way. The cheeses A, B and C were inoculated with the following starter combinations at a level of 1 g/100 g: Cheese A: E. faecium ATCC 19434 + L. lactic subsp. lactis 1B4 + L. garvieae IMAU 50157 (with ratio of 1:1:0.5), Cheese B: E. durans IMAU 60200 + E. faecium ATCC 19434 + L. lactic subsp. lactis 1B4 (with ratio of 1:1:1), and Cheese C: E. faecium ATCC 19434 + E. durans IMAU 60200 + L. garvieae IMAU 50157 + L. lactic subsp. lactis 1B4 + E. faecalis KLDS0.0341 (with ratio of 1:1:1:1:1). Food grade calcium chloride was added to the pasteurised milk at a level of 20 g/100 L to restore ionic calcium balance after heat treatment. After inoculation, cheese milk was left at 32°C for 30 min to allow acidity development prior to renneting. The milk was coagulated by means of calf rennet within 90 min. Following coagulation, the curd was cut into cubes (1 cm3) and put into special cheese cloths of triangular shape, locally called "parzin". The parzins were hung up on a horizontal bar and whey-ing off was achieved by gravity drainage for about 18 h at room temperature. Each parzin contained approximately 500 g of curd yielding about 100 g of fresh cheese after wheying-off. The cheese blocks were dry salted overnight and then put into pasteurised brine solution (12 g/100 g NaCl, w/v). The cheeses were ripened at 8°C for 180 days.

Chemical analysis

Total solids [ISO, 2004], titratable acidity [Anonymous, 2006] and salt [IDF 1988] were determined. The pH was determined by means of a combined electrode pH-meter (Mettler Toledo GmbH, Giessen, Germany). The fat was determined by the Gerber method [Anonymous, 2006].

Free fatty acid analyses

Fatty acids were extracted as outlined by Garcia-Lopez etal. [1994]. A cheese sample (10 g) was grinded and extracted using a mixture of methanol:methylene chloride (1:9). Nonaic acid was used as internal standard. The extract was vacuum extracted (40°C) and methylated with regard to the method of Sukhija & Palmquist [1988]. Fatty acid methyl esters were analysed by GC (Shimadzu GC-17 AAF, V3, 230 V series; Shimadzu Corpora tion, Kyoto, Japan) fitted with flame ionisation detector (FID).. FAME was passed through fused silica capillary column (SP-2380, 100 m - 0.25 mm; Supelco Inc., Bellefonte, PA). Helium was used as a carrier gas at the rate of 2 mL/mon. Injection of 1 ¿L sample was applied with a split ratio of 1:30 into the injector. Injector and detector temperature were adjusted as 250°C. The initial oven temperature was 40°C for 1.0 min, and then increased to 240°C at the rate of 5°C/min. The final temperature was maintained for 10 min. Fatty acids were identified by contrasting their retention times with a standard fatty acid mixture containing 37 fatty acids (Sigma-Aldrich Chemicals 189-19).

Sensory evaluation

Samples were organoleptically utilised according to the scheme proposed by Bodyfelt et al. [1988]. The panel group comprised of ten trained panelist who were familiar with Urfa cheese. A 10 point hedonic scale was used to appraise over perception, aroma and flavour, appearance and colour, saltiness, body and texture scores (1: strongly unacceptable; 10: strongly acceptable).

Statistical analyses

Data obtained were analysed with one-way analysis of variance using SPSS package statistical program (SPSS for Windows release 5.0.1., SPSS Inc., Chicago, IL, USA). Duncan's multiple range test was applied to identify the differences among the cheese groups. The study including cheese-making was done in triplicate.

RESULT AND DISCUSSION

Compositional analyses

The gross compositions of cheese samples are presented in Table 1. A continuous decline in total solids levels of the cheeses throughout ripening period was observed. This may be attributed to the formation of new peptides with high water absorbing capacity as a result of advanced proteolysis [Atasoy et al, 2008]. In fact, a decrease in moisture content of the cheeses was expected along with the increase in salt-in-dry matter since salt and water diffusions occur in opposite ways [Luo et al, 2013; Papademas, 2006; Madadlou et al, 2007]. Salt penetration was almost complete within the first 30 days of ripening and then the salt-in-moisture levels of the cheeses changed within a very narrow margin. Regarding the salt-in-moisture levels of the cheeses in the same ripening day, no significant variations were noted between the samples (p>0.05). The pHs of the experimental cheeses increased until 120 d and then declined gradually towards the end of ripening. The titratable acidity level of the control cheese (sample D) declined continuously throughout ripening

TABLE 1. Gross composition of Urfa cheese made using different starter culture systems.

Storage days Cheeses Total solids (g/100 g) Fat-in-dry matter (g/100 g) pH Titrable acidity (g/100 g lactic acid) Salt-in-moisture (g/100 g)

A 54.92a1 26.75a1 4.69a1 0.829a2 3.95a1

1 B 55.26a1 26.88a1 4.69a1 0.809a2 4.09a1

C 53.97a1 26.38a1 4.89a1 1.088a3 4.20a1

D 54.41a1 26.50a1 4.72a1 0.668a1 3.86a1

A 51.69b1 25.13b1 4.88b1 0.674ab2 6.21b1

30 B C 51.66b1 50.87b2 25.63b1 25.25b1 4.69a1 4.79a1 0.688b2 0.837b3 6.37b1 6.25b1

D 50.47b2 25.50b1 4.77ab1 0.621b1 6.42b1

A 48.73c2 25.25b1 4.91b1 0.679ab2 7.58bc1

60 B C 49.50c2 48.65c1 25.13c1 25.00b1 4.96b1 4.91a1 0.638b12 0.719c2 6.83b1 7.11bc1

D 49.05c2 25.13b1 4.74a1 0.567c1 6.99bc1

A 48.86c1 24.75b2 5.09c12 0.744ab2 7.70bc1

90 B C 48.70d1 48.25c1 24.38d12 24.25c12 5.14b2 5.08b1 0.727ab2 0.699c2 7.98c1 7.87cde1

D 47.33d1 23.38c1 5.05b1 0.509d1 8.00bc1

A 48.40c1 24.00c1 5.10c2 0.735ab2 7.95c12

120 B 48.35d1 24.13d1 5.13b3 0.638b12 8.34c12

C 47.49d1 24.00cd1 5.12b23 0.699c12 8.86e2

D 47.10de2 23.75c1 4.96b1 0.509d1 8.36d1

A 46.04d1 23.50c1 5.07c1 0.572b12 8.03bc1

150 B C 46.51e1 47.07d2 23.50e1 23.88cd2 5.07b1 5.06b1 0.672b12 0.716c2 8.52bc1 8.75de1

D 45.48f1 23.38c1 4.95ab1 0.457e1 8.77cd1

A 46.73d1 23.50c1 4.90b1 0.667ab2 8.00bc1

180 B C 46.08e1 46.93d2 23.25e1 23.50d1 4.98b1 4.78a1 0.618b2 0.719c2 7.88b1 8.07cd1

D 46.61e1 23.30c1 4.93ab1 0.426e1 7.85 bc1

Cheese A: Enterococcus faecium ATCC19434+ Lactococcus lactis subsp. lactis 1B4+ Lactococcus garvieae IMAU50157 (1:1:0.5), Cheese B: Enterococcus durans IMAU60200+ Enterococcus faecium ATCC19434+ Lactococcus lactis subsp. lactis 1B4 (1:1:1), Cheese C: Enterococcus faecium ATCC19434+ Enterococcus durans IMAU60200+ Enterococcus faecalis KLDS0.0341 + Lactococcus garvieae IMAU50157+ Lactococcus lactis subsp. lactis 1B4 (0.6:0.4) and Cheese D: control. *Samples showing common superscripts numbers (during storage days) and superscripts letters (between cheeses at the same storage day) do not differ significantly (p>0.05).

period, indicating the catabolism of lactic acid and the production of ammonia by deamination of free amino acids [Grappin & Beuvier, 1997; Prieto et al, 2000]. On the other hand, the titratable acidity levels of the cheeses A, B and C fluctuated after 90 days of ripening. This may be attributed to almost complete salt penetration to cheese after 30 days of ripening as increasing salt level in cheese results in decreasing total acidity in brined-type cheeses [Upreti & Metzger, 2007]. The combined effects of starter strains and ripening period on the development of acidity were significant at p<0.05. The fat-in-dry matter content of the cheeses decreased significantly (p<0.05) during ripening proportionally to decreasing total solids contents of the samples. The effect of starter combinations on the fat-in-dry matter level was insignificant (p>0.05). The total nitrogen (TN) contents of the experimental cheeses declined continuously throughout ripening; however, no considerable differences between the cheeses were noted regarding TN concentrations (p>0.05). The reduction in the TN levels of the cheeses could be due to hydrolysis of proteins to water-soluble nitrogen (WSN) compounds and their migration into the brine. This may also explain why

total solids levels of the cheese declined in the present case during the ripening period.

FFA analysis

Lipolysis in cheese is mainly affected by the type of milk used, stage of lactation, form of milking and milk collection, cooling and agitation in farm tanks. All these factors activate the membrane-bound lipoprotein lipase and thus trigger enzymatic hydrolysis of milk fat [Hassan et al, 2013; Lopez et al, 2006].

The variations in concentrations of individual short chain FFAs [(EC4.0-C8.0)], in the cheese samples during ripening stage are shown in Table 2. Level of lipolysis in the cheeses increased significantly throughout ripening period (P<0.05). Butyric acid concentrations of all samples increased during ripening period. This is most probably due to the high specificity of bacterial lipolytic enzymes towards FFA located in the position sn-1,3 of the triglyceride, where short chain free fatty acids SCFFA are predominantly esterified [Avila et al, 2007]. These findings are in good agreement with previous studies for different brined cheeses [Haya-

TABLE 2. Short chain free fatty acids (mg/100 g cheese) of cheese samples at during ripening.

Samples Storage days Butyric acid (C4:û) Caproic acid (CJ Caprylic acid (CJ Total FFA (2C4:0-C8:0)

1 1.41±0.05a1 1.31±0.13a1 0.93±0.25b1 3.65±0.33a1

30 1.93±0.10b2 1.83±0.04a2 1.39±0.13b2 5.15±0.27b2

60 2.87±0.03a3 2.54±0.18a3 2.33±0.09b3 7.74±0.30ab3

A 90 4.12±0.03b4 3.21±0.01a4 3.46±0.19b4 10.79±0.23b4

120 4.53±0.05a45 5.23±0.22a5 4.67±0.22b5 14.43±0.49ab5

150 5.07±0.18a5 5.65±0.13a56 5.08±0.23b56 15.80±0.54ab6

180 5.99±0.33ab6 6.25±0.31a6 5.60±0.28c6 17.84±0.92a7

1 30 1.42±0.14a1 1.77±0.13a12 1.34±0.11b1 1.89±0.12a2 0.98±0.07a1 1.43±0.05a2 3.74±0.32ab1 5.09±0.30b2

60 3.05±0.14b2 2.81±0.23b3 2.47±0.32ab3 8.33±0.69b3

B 90 3.45±0.34a2 3.42±0.21ab4 3.49±0.39a4 10.36±0.94a4

120 4.79±0.34b23 5.27±0.45ab5 4.77±0.31a5 14.83±1.10b5

150 5.30±0.32b4 5.98±0.57b6 5.26±0.66a56 5.73±0.49bc6 16.54±1.55bc6

180 6.26±0.47b5 6.31±0.52a7 18.30±1.48ab7

1 1.56±0.12b1 1.42±0.03b1 1.08±0.06a1 4.06±0.21b1

30 1.89±0.09b12 1.97±0.21a12 1.62±0.12a2 5.48±0.42c2

60 3.18±0.38b2 3.06±0.23b3 2.64±0.25a3 8.88±0.86c3

C 90 4.21±0.29b3 3.89±0.35b4 3.62±0.15a4 11.72±0.79c4

120 5.22±0.35c4 5.31±0.48b5 5.11±0.58a5 15.64±1.41c5

150 5.41±0.22b45 6.12±0.31b6 5.46±0.41ab56 16.99±0.94c6

180 6.47±0.32b5 6.50±0.41b6 5.92±0.396 18.89±1.12b7

1 1.36±0.14a1 1.24±0.02a1 0.85±0.11a1 3.45±0.27a1

30 1.71±0.25a12 1.89±0.22a2 1.31±0.07a2 4.45±0.44a2

60 2.77±0.12a2 2.41±0.12a3 2.23±0.27a3 7.41±0.51a3

D 90 3.79±0.12ab3 3.30±0.25a4 3.24±0.34a4 10.33±0.61a4

120 4.62±0.36a4 5.11±0.32a5 4.28±0.32a5 13.91±0.86a5

150 4.95±0.25a45 5.45±0.19a5 4.86±0.30a56 15.26±0.74a6

180 5.43±0.25a5 6.16±0.21a6 5.37±0.19a6 17.76±0.65a7

Cheese type description as in Table 1. *Samples showing common superscripts numbers (during storage days) and superscripts letters (between cheeses at the same storage day) do not differ significantly (p>0.05).

loglu et al. 2013; Atasoy & Turkoglu, 2009; Bohdziewicz, 2006, Perotti et al, 2005; Georgala et al, 2005; Buffa et al, 2001]. At the beginning and end of ripening, the C cheeses had the highest butyric acid content than the other cheeses. The concentrations of caproic acid and caprylic acid showed similar trend to butyric acid in all samples. The total concentration of SCFFA [(EC4.0-C8.0)] increased significantly (p<0.01) in all cheese samples during ripening period (Table 2). The increase in FFAs in cheese during ripening has been reported previously in matured cheese [Delgado et al, 2009; Mallatou et al, 2003]. At the end of storage, the volatile FFA content of the C cheese was significantly (p<0.01) higher compared to the other cheeses.

The addition of wild type bacterial strains to the pasteurised milk affected the volatile FFAs levels significantly during ripening period (p<0.01). In contrast, Atasoy & Turk-oglu, [2009] showed that Urfa cheese made from raw milk had higher total FFAs levels than those made from pasteurised milk. Short chain fatty acids (C4 to C8) were at much higher concentrations in the C cheeses. Low and high levels of short chain fatty acids indicate low and high lipolytic action of the natural microflora of the corresponding cheeses [Collins et al, 2003]. Generally, the levels of butyric, caproic

and caprylic acids were low in all experimental cheeses, indicating low lypolitic activity of lipases/esterases from the wild type strains. As a result, rancid flavour was not noticed in the cheese samples during ripening.

The concentrations of capric (C10:0) and lauric (C12:0) acids in the cheese C were significantly higher than the cheeses B, C and D (control) at all stages of ripening (p<0.001). Among medium-chain FFA, lauric acid had the lowest concentration in all cheese samples (Table 3). However, myristic acid was the predominant medium-chain FFA at the beginning of ripening period. After 120 days of ripening, lauric and myristic acid concentrations were close to each other in all cheese groups. Similar results were reported by Atasoy &Turkoglu [2008, 2009]. Despite the quantitative importance of medium and long chain FFA, they are not the main factor for the cheese flavour [Freitas & Malcata, 1998; Attaie & Richert, 1996].

The total long chain FFA contents of the cheeses were higher than those of the medium chain FFAs (Table 4). At the beginning of ripening, palmitic acid (C16:0) concentration of the cheese C was significantly (p<0.001) higher than the other cheeses and this remained unchanged at the later stages of the ripening. Among the FFA, the palmitic (C16)

TABLE 3. Middle chain free fatty acids (mg/100 g cheese) of cheese samples at during ripening.

Ç Q m nlpç Storage Capric acid Lauric acid Myristic acids Total FFA

kj dill Ult ¡5 days (C10:0) (C12:0) (C14:û)

1 4.36±0.18b1 2.22±0.24a1 9.67±0.31a1 16.25±0.73a1

30 4.90±0.19a2 3.04±0.22a2 10.30±0.44a2 18.54±0.85a23

60 7.24±0.22b3 4.93±0.38ab3 12.13±0.51a3 24.30±1.11b34

A 90 10.62±0.13a34 5.80±0.19a4 14.29±0.29b4 30.71±0.61b4

120 14.35±0.31a5 7.86±0.36a5 15.49±0.38b5 37.70±1.05a5

150 16.41±0.33ab6 8.12±0.27a6 17.20±0.19b6 41.73±0.79a6

180 17.67±0.21a7 9.20±0.44a7 18.76±0.72a7 45.73±1.37b7

1 4.40±0.33b1 2.28±0.22a1 10.00±0.38b1 16.78 ±0.93b1

30 5.25±0.19b12 3.08±0.29a2 10.43±0.21a1 19.56±0.69b2

60 6.92±0.25ab3 5.19±0.13b3 12.23±0.26b2 24.34±0.64b3

B 90 10.58±0.09a 5.89±0.32a4 13.74±0.19a3 30.21±0.60a4

120 14.44±0.34a5 7.77±0.19a5 15.39±0.43b4 37.70±0.96a5

150 16.96±0.41b6 8.58±0.36b6 17.26±0.61b5 42.80±1.38b6

180 18.11±0.39b7 9.44±0.26b7 18.56±0.57a6 46.11±1.22b7

1 4.72±0.18c1 2.58±0.22b1 10.69±0.19c1 17.99±0.59c

30 5.33±0.24b2 3.35±0.32b2 11.30±0.22b2 19.78±0.78b

60 7.41±0.31b3 5.57±0.41c3 12.54±0.43b3 25.52±1.15c3

C 90 11.57±0.64 b4 6.53±0.21b4 14.95±0.35c4 33.05±1.20c4

120 15.64±0.24 b5 7.89±0.61a5 16.08±0.61c5 39.31±1.46b5

150 17.89±0.36c6 8.95±0.32c6 17.81±0.34c6 44 44.65±1.02c6

180 18.90±0.21c7 9.96±0.42c7 19.66±0.45b7 48.52±1.08c6

1 4.08±0.21a1 2.10±0.29a1 9.83±0.22a1 15.91±0.72a1

30 4.72±0.29a2 3.31±0.18b2 10.23±0.18a1 18.16±0.65a2

60 6.65±0.32a3 4.78±0.41ab3 11.90±0.25a2 23.23±0.98a3

D 90 10.44±0.33a4 5.64±0.22a4 13.72±0.33a3 29.60±0.88ac4

120 14.55±0.41a5 7.66±0.47a5 15.01±0.41a4 37.67±1.29a5

150 16.06±0.23a6 8.34±0.55b6 16.74±0.55a5 41.14±1.33a6

180 17.44±0.52a7 9.12±0.51a7 18.51±0.38a6 44.97±1.41a6

Cheese type description as in Table 1. *Samples showing common superscripts numbers (during storage days) and superscripts letters (between cheeses at the same storage day) do not differ significantly (p>0.05).

and oleic (C18:1) acids were the principal fatty acids in all cheeses at all ripening stages. The highest volatile FFAs contents were determined in C cheeses. These results are consistent with other long-time maturated cheeses [Delgado et al., 2011; Talpur et al, 2008].

Generally, the total FFAs contents found in the cheeses were significantly higher in the cheese C. This may be related to higher lipolytic activity of enterococcal strains in the cheese C. Compared to many cheese varieties, the total FFAs content of Urfa cheese was markedly lower. Storing in high salt brine (>12%, w/v) may be the main reason of low lipoysis in Urfa cheese. The inhibitory effect of NaCl on FFAs was emphasised by other researchers [Katsiari et al, 2000; Pavia et al, 2000].

Sensory evaluations

The cheese samples were evaluated organoleptically based on appearance and colour, aroma and flavour, body and texture, saltiness and overall perception. The results for 180 day old cheeses are presented in Table 5. With respect to the appearance and colour, no differences were found between cheese samples. Control cheese (D) received significantly lower body and texture scores at 180 d (p<0.05).

This could be due to the lower titration acid value than other cheeses at the end of ripening, which has a significant impact on the development of the textural properties of cheese. However, the body and texture scores of the other cheeses showed similarities at 180 d. Conversely, the cheese D had significantly higher aroma and flavour scores than the other cheeses (p<0.01). All cheeses had similar saltiness scores at 180 d. The cheese C had the lowest overall perception scores at 180 d. The cheese C was criticized for the slight bitterness at day 180. All the samples were found to be consumable by the panelists with less pronounced in the sample C. Atasoy & Turkoglu, [2009] used termophillic and mesophillic cultures in the production of Ufa cheese. The cheeses made by thermophillic culture showed similar sensory evaluations with cheeses made from raw milk, on the other hand panelists gave lower flavour scores to cheeses made by the mesophilic culture.

CONCLUSION

Based on the results obtained from the present study, the use of various combinations of L. lactis subsp. lactis B14, L. garvieae IMAU 50157, E. faecalis KLDS0.0341,

TABLE 4. Long chain free fatty acids (mg/100 g cheese) of cheese samples at during ripening.

Samples Storage days Palmitic acid (CiJ Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) Total FFA (^C16:0-C18:2) Total FFA (^C4:<TC18:2)

1 7.85±0.65a1 19.99±0.18b1 16.01±0.32a1 1.22±0.12b1 44.57±1.27a1 64.47±2.19a1

30 12.52±0.54a2 27.50±0.43b2 20.29±0.18a2 1.82±0.09a2 62.01±1.24b2 85.70±3.36b2

60 16.88±0.28a3 37.55±0.33b3 26.34±0.76a3 2.83±0.31a3 83.40±1.68a3 115.44±3.09ab3

A 90 23.67±0.19b4 48.03±1.21b4 32.78±0.99b4 3.33±0.11a4 107.81±1.50ab4 149.31±2.34b4

120 29.20±0.81a5 58.35±0.64a5 38.81±0.43b5 4.74±0.38a5 131.10±2.26a5 183.23±3.80a5

150 35.01±0.54a6 64.35±0.86a6 42.36±1.15a6 5.20±0.19a6 146.92±1.74a6 204.45±3.07d6

180 38.37±0.42a7 69.22±0.92ab7 46.99±1.32a7 6.42±0.61a7 161.34±2.27ab7 224.91±4.16a7

1 20.17±0.29b1 8.20±0.09ab1 16.14±0.31a1 1.02±0.12a1 45.53±0.81b1 66.05±1.93b1

30 27.99±0.17b2 12.31±0.14a2 20.34±0.17a2 1.88±0.07a2 65.52±0.55c2 90.17±1.34c2

60 37.10±0.44a3 17.71±0.32b3 26.63±1.03a3 2.79±0.07a3 84.23±1.86b3 116.90±2.79b3

B 90 49.84±0.62c4 23.78±0.11b4 32.17±0.31a4 3.29±0.21a4 109.08±1.25b4 149.65±1.99b4

120 59.31±0.39b5 30.14±0.41b5 38.15±0.67a5 4.69±0.05a5 132.39±1.52b5 184.92±2.88b5

150 64.95±0.21ab6 35.74±0.62b6 42.45±0.89a6 5.84±0.13b6 148.98±1.85b6 208.32±3.35b6

180 69.42±0.53b7 39.75±0.33b7 46.80±1.14a7 6.39±0.41a7 162.34±2.38b7 226.75±4.08a7

1 20.63±0.55c1 8.55±0.32b1 17.01±0.65b1 1.28±0.09b1 47.47±1.61c1 69.52±2.41c1

30 28.50±0.43c2 13.91±0.27b2 21.14±0.31b2 2.23±0.06b2 65.78±1.07c2 91.04±2.07d2

60 38.08±0.39c23 18.72±0.44c3 27.44±0.98b3 3.03±0.12b3 87.27±1.93c3 121.67±3.44c3

C 90 50.50±0.71d4 24.38±0.49c4 33.90±1.01c4 3.87±0.26b4 112.65±2.47c4 157.42±3.86c4

120 60.38±0.64c5 31.61±0.17c5 40.36±0.43c5 4.93±0.11b5 137.28±1.35c5 192.23±3.22c5

150 65.62±1.32b6 36.53±0.87c6 44.91±0.21c6 5.99±0.08b6 153.05±2.48c6 214.69±3.84c6

180 70.87±0.42c7 40.64±0.21c7 48.43±0.86b7 7.02±0.41b7 166.96±1.89c7 234.37±3.09b7

1 19.18±0.75a1 7.96±0.07a1 16.12±0.51a1 1.14±0.03ab1 44.40±1.36a1 63.76±2.35a1

30 26.24±0.64a2 12.45±0.31a2 20.26±1.05c2 1.78±0.10a12 60.73±2.10a2 83.34±2.89a2

60 36.90±0.38a3 17.20±0.43ab3 26.54±0.75a3 2.86±0.07a3 83.60±1.63a3 114.24±3.06a3

D 90 47.36±1.12a4 22.83±0.18a4 32.13±0.21a4 3.44±0.25a4 105.76±1.76a4 145.69±2.95a4

120 58.23±0.98a5 29.86±0.56b5 38.31±0.38 5 4.76±0.32a5 131.16±2.24a5 182.74±4.09a5

150 64.52±1.38a6 34.89±0.87a6 42.97±1.05 b6 5.12±0.19a6 147.30±3.49a6 203.70±5.16c6

180 69.13±0.87a7 38.07±0.21a7 46.77±0.49 a7 6.37±0.41a7 159.74±1.98a7 221.67±3.64a7

Cheese type description as in Table 1. *Samples showing common superscripts numbers (during storage days) and superscripts letters (between cheeses at the same storage day) do not differ significantly (p>0.05).

TABLE 5. Sensory evaluation of 180 day-old cheese samples.

Cheese samples Appearance and colour Aroma and flavour

Body and texture

Saltiness

Overall perception

A B C D

4.54±0.21a 4.75±0.36b 4.35±0.15a 4.80±0.49b

8.18±0.58b 8.32±0.84b 7.24±0.54a 9.06±0.66c

4.22±0.24b 4.13±0.19b 4.63±0.42c 3.35±0.33a

3.32±0.11a 3.42±0.08b 3.28±0.10a 3.60±0.18a

20.26±0.88a 20.62±0.81b 19.50±0.69a 20.81±0.95b

Cheese type description as in Table 1. Samples showing common superscripts letters (between cheeses at the same storage day) do not differ significantly (p>0.05).

E. faecium ATCC 19434 and E. durans IMAU 60200 offers certain advantages compared to raw ewe's milk cheeses in regard to the development of lipolysis. Especially, the starter combination including all above strains in equal proportions (cheese C) was found to be more efficient for faster development of lipolysis in Urfa cheese made from pasteurised ewe's milk. Whereas panelists gave the lowest overall perception degree to the C cheeses. Surely, there are a number of parameters that need to be properly addressed before offering a starter or starter combination to cheese

industry. Although the lipolytic performances of the strains employed in the present case were superior to that of raw milk microflora, the safety of enterococcal strains for cheese applications has to be assured.

ACKNOWLEDGEMENT

This study was supported financially by Harran University Scientific Research Projects Authority (HUBAK Project No: 13004).

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Submitted: 3 November 2015. Revised: 9 March 2015. Accepted: 16 April 2015. Published on-line: 27 October 2015.