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The effect of microbial lipase on the lipolysis during the ripening of Tulum cheese
Journal of Food Engineering 69 (2005) 269–274 www.elsevier.com/locate/jfoodeng
The effect of microbial lipase on the lipolysis during the ripening of Tulum cheese Gu¨ldane Yilmaz, Ahmet Ayar *, Nihat Akin Selc¸uk University, Agricultural Faculty, Food Eng. Dept. Campus, Konya, Turkey Received 23 April 2004; accepted 6 August 2004
Abstract The effect of microbial lipase enzyme addition on the ripening of Tulum cheese was investigated. Commercial microbial lipase (Piccantase A) was added to milk before rennet addition at a level of 0, 0.04, 0.06 and 0.11 g L 1 of cheese milk. Free fatty acids (FFA) (C2–C18:1) were analysed in the samples during the ripening period. Total solids, fat, salt, total nitrogen, titratable acidity, acidity index, total free fatty acids of cheeses slightly increased during the ripening period. Total FFAs and volatile FFA contents in cheeses were affected by the level of microbial lipase and their contents were increased significantly during the ripening period (P < 0.01). The relative amounts of volatile FFAs varied with ripening time and lipase levels. The addition of lipase to cheese milk could be recommended for the acceleration of flavour development in Tulum cheese over a short ripening period. 2004 Elsevier Ltd. All rights reserved. Keywords: Tulum cheese; Lipolysis; Ripening; FFA
1. Introduction Processing of dairy products on a commercial basis in modern plants dates back to 35 years ago. Despite its fragmented structure, Turkey is the largest producer of milk and dairy products in the region. Cheese is an important integral part of Turkish diet. It is consumed almost three times a day. Cheese is consumed with fruit or as a sort of dessert after lunch or dinner. Generally, cheeses and olives are consumed for breakfast in Turkey. The variety of cheese depends on certain factors: cultural habits and tastes, natural conditions, the species and variety of the animals providing the milk, and the production methods employed. There are more than 100 varieties of cheese in Turkey, but if they are grouped * Corresponding author. Tel.: +90 33 222 32923; fax: +90 33 224 10108. E-mail addresses: [email protected], [email protected] (A. Ayar).
0260-8774/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2004.08.017
according to their similarities, they are limited to about 30 different cheese types. In addition to three major cheese types (White pickled cheese, Kasar cheese, Tulum cheese), there are many traditional cheeses locally produced and consumed in local regions, that are mainly Mihalic¸ cheese, Oltu cheese, Van otlu cheese, Abhazian cheese, Civil cheese, Golot cheese, Ku¨lek cheese, Urfa cheese, and Salinized Diyarbakir o¨rgu¨ cheese etc. Tulum cheese, a special cheese produced in Turkey, is not well known in other countries. It is preferred for its characteristically natural mouldy taste and flavour. During the ripening period, natural contaminating microorganisms especially moulds grow and contribute to the ripening process. Tulum is a semi-hard cheese that can be made from whole, semi skimmed or skimmed sheepÕs, goatÕs, cowÕs, and buffaloÕs milk or their mixture. It has a crumbly texture and a strong flavour. Tulum means the skin of goat or sheep. Traditionally, Tulum cheese is manufactured from skimmed milk (after butter production) for family use, and yogurt is sometimes added for taste. By removing the water of the curd, a crumbly
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texture is obtained. The crumbled curd is salted and packed firmly in goatÕs skins or in cloth sacks and aged for 3–6 months. Ripening takes place at 6–10 C, 65–85% RH. It can be used in bo¨rek or similar foods and is also a great appetizer when mixed with walnuts. FFAs are released usually by the actions of lipases (from different sources) during lipolysis. They contribute directly to cheese flavour, particularly when they are properly balanced with products of proteolysis and other reactions. However, extensive lipolysis is considered to be undesirable for some cheeses (Molimard & Spinnler, 1996; Fox, Singh, & Mcsweeney, 1995). Short-chain fatty acids themselves contribute directly to aroma in many ripened cheese varieties (Urbach, 1997). In addition to their direct impact on cheese flavour, FFAs also act as precursor molecules in a series of catabolic reactions that lead to the production of other flavour compounds such as methyl ketones, esters, thioesters (Urbach, 1993; Fox & Wallace, 1997; Mcsweeney & Sousa, 2000). Ripening time of Tulum cheese is at least four months and the shortening of ripening time without any lost of flavour characteristics could be very advantageous. It is highly desirable to improve its flavour. The studies on FFAs contents of Tulum cheese are limited. The objective of this study was to investigate the effect of a microbial lipase on the acceleration of the ripening process of Tulum cheese.
2. Material and methods 2.1. Materials Raw cowÕs milk was obtained from a farm in Lalebahc¸e in Konya (Turkey). Tulums were produced from goatskin in S ß eker Su¨t Dairy Plant in Konya. Starter culture (Frozen culture Yb 41, Streptococcus salivarus subsp. thermophilus + Lactobacillus delbrueckii subsp. bulgaricus) and microbial rennet (Chymax 15 T was produced by Aspergillus niger var. awamori with completely chymosin) were obtained from Peyma-Chr HansenÕs Inc. (Istanbul-Turkey). Lipase (Piccantase A), as a powder that was produced from Rhizomucor miehei, was obtained from Gist-brocades Inc. (Istanbul-Turkey). 2.2. Methods Experimental cheeses were made in the S ß eker Su¨t Dairy Plant in Konya. Flow diagram of production process of Tulum cheese is shown in Fig. 1. The lots of 200 L raw milk were heated to 31 C and divided into four portions, followed by addition of 0.1 g kg 1 of frozen culture. All portions were left to pre-ripening until pH reached 6.25. The first portion was used as control (A0). To the other portions, microbial lipase was added
Fresh raw milk
Heat to 310 C Starters, Rennet,
Addition
Lipase enzyme Coaguluation (within 30 min.)
Curd cutting (1 cm x 1 cm x 1 cm) and hold the curd for 10-15 min. Excess whey drainage Curd draining under pressure (25 kgm-2 until end of draining ) and pre-fermentation at 26 0C until pH 5.10
Curd breaking up by hand (Curd is break up small pieces on wooden table for 3 time)
Dry salting (30 g kg-1 NaCl w/w)
Pressure (25 kgm-2 for 16 h for 3 time)
Milling
Curd placement into goatskin (tulum)
Ripening at 8 0C for 90 days
Tulum cheese
Fig. 1. Flow diagram of manufacture of Tulum cheese.
at a rate of 0.04 g L 1 (A1), 0.06 g L 1 (A2) and 0.11 g L 1 (A3) to milk, respectively. Rennet (strength, 1:15,000) was added to coagulate the milk within 30 min. The coagulum was cut into 1 cm · 1 cm · 1 cm cubes and the curd pieces were transferred into vats lined with cheese cloth and pressed 25 kg m 2 press weight until pH 5.10 for pre-fermentation at 26 C. Subsequently, when curd reached the desired moisture content and pH value (4.90). It was broke up by hand and dry salted (30 g kg 1). The curd was put into cotton bags to remove whey. Then pressed using 25 kg m 2 press weight for 16 h. This press operation was repeated for three times. The salted fresh cheese was milled and transferred in the goatskins (specially prepared Tulums) without air holes. They ripened at 8 ± 1 C, 85 % RH for 90 days. The experiment is carried out as duplicate. Total solids, fat, total nitrogen, salt, pH and titratable acidity (SH) in cheese samples were determined according to the procedures described by Kirk and Sawyer (1991), acidity index according to Renner (1986). FFAs
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of Patir, Ates, and Dincoglu (2001); Atesß and Patir (2001) for Tulum cheese. The pH decreased slightly whereas the acidity was increased during ripening. The acidity index of the fat values increased up to 90 days of ripening. In the control cheese samples, acidity index increased in small amounts, but cheeses made from microbial lipase added milks had a higher increase of acidity index (Table 1). Statistically significant differences (P < 0.01) were found among the ages of ripening for the acidity index. The acidity index of the cheeses made with microbial lipases were significantly (P < 0.01) higher than that of control sample. The final acidity index values in lipase added Tulum cheeses are higher than those of found in traditional Tulum cheese made from raw milk by Gu¨ler and Uraz (2003) and in C ¸ imi Tulum cheese made from raw goat milk by Kılıc¸, Karagozlu, and Akbulut (2000). The FFAs composition of Tulum cheese samples made from microbial lipase added milk is given in Tables 2 and 3. In the control cheese samples, volatile FFAs (C2–C10:0) were present in small amounts, but cheese made from microbial lipase added milk had a higher content of all volatile FFAs. The higher volatile FFA was observed in A3 coded (added 11 g microbial lipase per 100 L cheese milk) cheese and the dominant volatile FFA was butyric acids for all cheese samples made with microbial lipase added milk. The results reported by Aydemir et al. (2001) for white cheese and by Omar, El-Zayat, and Ashour (1986) for Ras cheese
in cheeses were determined by gas chromatography according to the method of Deeth, Fitz-Gerald, and Snow (1983). Cheeses were analyzed at 1, 15, 30, 60 and 90 days in duplicate. The effect of addition of lipase enzyme and ripening period was evaluated by analysis of variance (Minitab, 1991). The Duncan Multiple Comparison Test was used to determine the groups significantly different (P < 0.01) from each other. All data are reported as means.
3. Results and discussion The mean chemical composition analysis data obtained from the experimental Tulum cheeses are presented in Table 1. Examination of this table revealed that the addition of lipase had no significant effect on total solids, salt, fat, total nitrogen, pH and titratable acidity of the resultant cheeses. Similarly, several researchers (Nasr, 1983; Aydemir, Akin, & Koc¸ak, 2001; Akın, Aydemir, Koc¸ak, & Yıldız, 2003) have reported that the use of lipase had no significant effect on the gross composition of cheese during the ripening period, but Bedel and Kilic (2000) have reported that the use of enzyme in Tulum cheese had significant effect on the gross composition. Because of water lose from goat skin pores. Total solids, salt, fat and total nitrogen of cheese samples were increased during ripening (P < 0.01). This finding is in accordance with the results
Table 1 Effect of the addition of microbial lipase on the chemical composition of Tulum cheese Group
Ripening period (Days)
Total solids (g 100 g 1)
Salt (g 100 g 1)
Salt in total solids
Fat (g 100 g 1)
Fat in total solids
Total nitrogen (g 100 g 1)
Total protein (Nx6.38) in total solids
pH
A0
1 15 30 60 90
52.49 53.09 53.50 53.04 53.98
a a a a a
2.13 2.19 2.21 2.21 2.23
b a a a a
4.06 4.13 4.12 4.16 4.13
a a a a
28.25 29.00 29.25 29.50 29.50
a a a a a
53.83 54.62 54.67 55.63 54.64
a a a a a
1.94 1.99 2.00 2.05 2.13
b ab ab ab a
2358 2391 2385 2466 2517
a a a a a
5.15 5.07 5.05 5.03 4.99
A1
1 15 30 60 90
53.50 54.39 54.56 54.91 54.70
b ab ab a ab
1.76 1.90 2.01 2.08 2.09
b ab a a a
3.30 3.49 3.69 3.78 3.82
b ab ab a a
27.75 28.00 28.25 29.00 29.50
a a a a a
51.88 51.48 51.78 52.81 53.93
a a a a a
1.84 2.10 2.03 2.04 2.05
b a a a a
2194 2463 2374 2370 2391
a a a a a
A2
1 15 30 60 90
53.86 53.87 54.01 54.02 54.33
b b b b a
1.84 1.99 2.01 2.06 2.50
c bc bc b a
3.42 3.69 3.72 3.81 4.59
c bc bc b a
27.25 28.75 28.75 28.75 29.25
a a a a a
50.60 53.36 53.23 53.22 53.83
a a a a a
1.85 1.90 2.03 2.10 2.11
b b ab a a
2191 2250 2398 2480 2478
A3
1 15 30 60 90
53.53 53.96 54.12 54.12 54.16
a a a a a
2.14 2.17 2.17 2.23 2.70
b b b b a
4.00 4.02 4.00 4.11 4.99
b b b b a
28.00 28.00 29.00 29.50 29.50
a a a a a
52.31 51.88 53.59 54.51 54.46
a a a a a
1.78 1.86 1.99 2.00 2.03
b ab a a a
2122 2199 2346 2358 2391
Titratable acidity (SH)
Acidity index mg KOH g 1 fat
a ab ab ab b
83.00 d 89.00 c 91.50 bc 97.00 b 113.00 a
1.53 1.70 2.47 5.01 5.54
5.07 5.04 5.01 5.01 5.01
a a a a a
86.50 d 92.50 c 96.50 c 102.50 b 112.50 a
2.68 e 4.67 d 7.21 c 14.79 b 20.95 a
a a a a a
5.06 5.04 4.94 4.97 4.94
a a a a a
89.50 c 94.50 bc 97.50 b 106.00 a 111.50 a
3.41 e 6.18 d 9.37 c 15.86 b 26.61 a
a a a a a
5.08 5.04 4.99 4.94 4.92
a ab ab b b
91.00 d 96.00 c 101.50 b 106.50 a 109.50 a
4.64 e 9.51 d 13.81 c 24.05 b 36.96 a
A0: Control; A1: 0.04 g microbial lipase L 1 milk; A2: 0.06 g microbial lipase L 1 milk; A3: 0.11 g microbial lipase L 1 milk. a–e means with different letters were significantly different among ripening periods within the rows of each cell (P < 0.01).
e d c b a
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Table 2 Effect of the addition of microbial lipase on volatile free fatty acids (C2–C10:0) of Tulum cheese (mg 100 g
1
cheese)
Cheese group
Ripening period (Days)
Acetic
Propionic
Butyric C4:0
Caproic C6:0
Caprylic C8:0
Capric C10:0
A0
1 15 30 60 90
1.58 3.02 5.92 6.98 7.75
0.30 0.30 0.41 0.74 1.10
d d c b a
1.07 2.96 3.87 4.56 8.56
1.12 1.52 2.00 2.52 3.96
1.92 c 1.70 d 1.91 c 2.935 b 4.12 a
2.96 4.01 5.86 6.01 8.01
A1
1 15 30 60 90
1.43 e 1.96 d 5.00 c 8.56 b 16.74 a
1.29 1.22 1.30 2.35 3.00
c d c b a
7.73 e 12.64 d 15.92 c 18.79 b 21.49 a
3.98 e 6.08 d 7.95 c 8.31 b 11.61 a
1.86 2.62 3.51 4.03 6.12
3.97 e 4.26 d 6.68 c 7.78 b 10.02 a
A2
1 15 30 60 90
1.19 e 1.61 d 6.81 c 13.86 b 23.96 a
1.21 1.29 1.70 2.00 3.07
e d c b a
14.36 23.31 33.23 28.11 50.09
e d c b a
8.44 e 13.35 d 17.98 c 20.25 b 23.39 a
3.43 e 5.05 d 7.01 c 9.59 b 11.31 a
4.87 e 8.59 d 11.05 c 14.08 b 20.52 a
A3
1 15 30 60 90
1.56 e 2.00 d 8.61 c 19.76 b 30.61 a
1.96 2.02 2.87 3.56 4.11
e d c b a
18.76 28.39 36.36 40.62 65.23
e d c b a
9.11 e 16.81 d 18.57 c 25.44 b 31.96 a
3.72 e 4.01 d 6.20 c 10.47 b 13.09 a
8.56 e 10.75 d 13.38 c 17.47 b 22.61 a
e d c b a
e d c b a
e d c b a
e d c b a
e d c b a
A0: Control; A1: 0.04 g microbial lipase L 1 milk; A2: 0.06 g microbial lipase L 1 milk.; A3: 0.11 g microbial lipase L 1 milk. a–e means with different letters were significantly different among ripening periods within the column of each cell (P < 0.01).
Table 3 Effect of the addition of microbial lipase on free fatty acids (C12:0–C18:1) of Tulum cheese (mg 100 g
1
cheese)
Cheese group
Ripening period (Days)
Lauric C12:0
Myristic C14:0
Palmitic C16:0
Stearic C18:0
Oleic C18:1
Total FFAs1
A0
1 15 30 60 90
4.79 e 5.76 d 7.84 c 9.95 b 10.56 a
9.04 e 13.69 d 16.35 c 20.49 b 26.15 a
30.74 37.83 41.11 47.77 50.86
e d c b a
7.14 e 10.48 d 11.64 c 15.25 b 18.15 a
24.74 36.71 39.11 46.86 49.30
e d c b a
85.39 117.78 136.02 164.06 188.52
A1
1 15 30 60 90
4.03 e 4.95 d 6.27 c 8.44 b 11.02 a
10.50 12.31 15.17 20.12 25.24
e d c b a
33.64 34.46 39.01 43.37 59.91
e d c b a
7.91 e 8.12 d 9.71 c 13.75 b 16.10 a
28.14 30.16 35.52 42.20 61.55
e d c b a
104.47 118.78 146.04 177.70 242.79
A2
1 15 30 60 90
8.74 e 9.44 d 13.14 c 16.21 b 19.61 a
14.09 15.94 17.01 25.18 31.31
e d c b a
35.61 37.01 45.08 59.23 70.58
e d c b a
9.01 e 9.76 d 10.02 c 15.38 b 19.06 a
27.91 35.01 46.13 53.97 73.47
e d c b a
128.87 160.37 209.14 257.85 346.37
A3
1 15 30 60 90
9.49 e 10.61 d 12.48 c 15.90 b 20.60 a
14.49 16.33 18.98 24.45 36.73
e d c b a
40.38 43.19 47.97 58.50 79.30
e d c b a
9.02 e 10.71 d 12.64 c 17.79 b 20.04 a
32.17 e 39.20 d 46.18c 54.00 b 82.75 a
149.22 184.01 224.25 287.97 407.03
A0: Control; A1: 0.04 g microbial lipase L 1 milk; A2: 0.06 g microbial lipase L 1 milk; A3: 0.11 g microbial lipase L 1 milk. a–e means with different letters were significantly different among ripening periods within the column of each cell (P < 0.01). 1 Total FFAs was the sum of Acetic, Propionic, Butyric, Caproic, Caprylic, Capric, Lauric, Myristic, Palmitic, Stearic, Oleic acids.
are in agreement with our findings. All volatile FFAs were found in all (A0, A1, A2 and A3) cheese samples and their distribution varied with microbial lipase levels. In the early stages of ripening, butyric acid content of control cheese (A0) was remarkably lower, while acetic
acid content was higher than that of cheeses made with microbial lipase. The results also show that volatile FFAs contents of cheese increased during the ripening period, particularly in the cheese made with microbial lipase added milk. The level of propionic, caproic,
G. Yilmaz et al. / Journal of Food Engineering 69 (2005) 269–274
caprylic and capric acid were low. Acetic and propionic acid is not a product of lipolysis. The presence of they may be due to bacterial fermentation of lactose through glycolytic pathway and decarboxylation and oxidative deamination of amino acids (Kosikowski, 1982). Acetic and propionic acids were included in this study because they are the major volatile acid extracted with FFAs and they contribute greatly to the final flavour of cheese (Lesage, Voilley, Lorient, & Bezard, 1993; Abd El-Salam, Alichanidis, & Zerfiridis, 1993). The FFAs (C12:0–C18:1) of cheese samples were increased when microbial lipase levels increased in cheese samples. The FFAs contents of the cheeses samples increased throughout the ripening time in all cheese samples, which was in agreement with the results reported by Kılıc¸ et al. (2000) for C ¸ imi Tulum cheese made from raw goat milk. The level of the FFAs and volatile FFAs of A3 cheese with the highest lipase addition was higher than that of the other cheese samples at 90 days of ripening. Similar results were observed by Aydemir et al. (2001) in white cheese and Woo, Kollodge, and Lindsay (1984) for several different cheeses. But FFAs levels in our lipase added cheeses were higher than those reported by Kılıc¸ et al. (2000) for C ¸ imi Tulum cheese and by Akin (2001) for Tulum cheeses made from different types raw milks. The differences in amounts of these fatty acids in cheese samples, made different enzyme levels, are possibly due to large reductions in numbers of bacterial spices in milk capable of liberating intracellular lipases by autolysis. The differences in the free fatty acid concentrations found by various authors may be related, either
273
to the techniques used for the distillation and extraction and type of cheese. Of the FFAs, the long-chain fatty acids dominated. The most abundant one was palmitic acid (C16:0), followed by oleic acid (C18:1) and myristic acid (C14:0). The rate of increase in the concentration of short-chain FFA (C4:0–C8:0) during ripening was higher than that of medium-chain FFA (C10:0–C14:0), which in turn was higher than that of long-chain FFA (C16:0–C18:1). This could be explained by the specificity of lipases towards the FFAs located at the positions sn-1, 3 of the triglyceryde, and generally, short-chain fatty acids are predominantly esterified at the sn-3 position (Juarez, 1986). The application of Duncan Multiple Comparison Test of all the individual FFAs showed that a significant increase (P < 0.01) occurred at each sampling point (Table 3). Short-chain FFAs at 90 days represented approximately 24–25 g 100 g 1 of the total FFA content in all cheeses. Medium and long-chain FFA represented approximately 21–22 g 100 g 1 and 52–53 g 100 g 1 respectively, of all FFA in all cheeses. Despite the quantitative importance of medium- and long-chain FFA, they are not the main contributors to cheese flavour (Freitas & Malcata, 1998; Buffa, Guamıs, Pavıa, & Trujıllo, 2001). Acetic, butyric, caproic, caprylic and capric acids were the short-chain FFA that showed the highest relative increase in cheeses made with microbial lipase during ripening period. It is known that shortchain FFA have a very important effect on the flavour of cheese (Rahmat & Richter, 1996).
Table 4 Effect of the addition of microbial lipase on sensory properties in Tulum cheeses Cheese group
Ripening period (Days)
Appearance (30)
Flavour (40)
Body and texture (20)
Odour (10)
A0
1 15 30 60 90
16.67 19.17 26.67 28.33 16.67
b b a a b
31.67 31.67 33.33 36.67 35.00
a a a a a
16.67 17.50 19.17 19.17 14.17
ab ab a a b
7.50 a 8.33 a 9.17 a 10.00 a 6.67 a
A1
1 15 30 60 90
18.33 19.17 23.33 28.33 19.17
b b ab a b
31.67 36.67 35.00 38.33 11.67
a a a a b
16.67 16.67 19.17 19.17 13.33
ab ab a a b
8.33 7.50 9.17 9.17 6.67
A2
1 15 30 60 90
18.33 28.33 26.67 28.33 26.67
b a a a a
30.00 31.67 36.67 38.33 15.83
a a a a b
16.67 18.33 19.17 20.00 14.17
ab ab a a b
8.33 a 8.33 a 9.17 a 10.00 a 6.67 a
A3
1 15 30 60 90
16.50 18.33 16.67 13.33 12.50
b a ab b b
30.00 bc 26.67 c 38.33 a 35.00 ab 5.00 d
16.67 17.50 19.17 13.33 10.83
ab a a b b
9.00 a 10.00 a 10.00 a 7.50 a 5.83 b
A0: Control; A1: 0.04 g microbial lipase L 1 milk; A2: 0.06 g microbial lipase L 1 milk; A3: 0.11 g microbial lipase L 1 milk. a–e means with different letters were significantly different among ripening periods within the column of each cell (P < 0.01).
a a a a a
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The results of organoleptic examination of different Tulum cheeses at the ages 1, 15, 30, 60 and 90 days are presented in Table 4. There is no interaction between microbial lipase levels and ripening period for all analysed sensory properties. Microbial lipase levels and ripening periods, for each of the mean properties scored by sensory test, were compared. According to statistical analysis, results can be summarised as follows: (i) when microbial lipase levels were increased, sensory scores (appearance, flavour, body and texture and odour) were not significantly increased; (ii) sensory scores in microbial lipase added cheeses were rapidly increased by the age 60 days of ripening, but in 0.11 g microbial lipase L 1 milk added cheese was increased by the age 30 days; (iii) different ripening times had effect on sensory scores; (iv) after 60 days ripening, sensory scores were significantly lower. Despite the increasing in the FFAs observed for cheeses groups, decrease in sensory scores of cheeses were observed. This supports justifies the view that other factors in addition to FFAs, such as total acidity and protein degradation as well as headspace volatiles are also important for the development of cheese flavour (Horwood, Llound, & Stark, 1981). In conclusion, the present results indicate that the use of microbial lipase in the manufacture of Tulum cheese increases the FFA content, but has only a slight effect on the pattern of the FFAs. The addition of microbial lipase to cheese milk could be recommended for accelerating the development of flavour in this cheese over a short ripening period.
References Abd El-Salam, M. H., Alichanidis, E., & Zerfiridis, G. K. (1993). Domiati and Feta type cheeses. In P. F. Fox (Ed.), Cheese: Chemistry, physics and microbiology (pp. 301–335). London, UK: Elsevier Applied Science. Akin, N. (2001). A study of free fatty acids contents of Tulum cheese ¨ Ziraat Faku¨ltesi Dergisi, 15, made from different type milks. S. U 64–73. Akın, N., Aydemir, S., Koc¸ak, C., & Yıldız, M. A. (2003). Changes of free fatty acid contents and sensory properties of white pickled cheese during ripening. Food Chemistry, 80, 77–83. Atesß, G., & Patir, B. (2001). Investigations on sensorial, chemical and microbiological features of Tulum cheese with starter culture during maturation. Firat Universitesi Saglik Bilimleri Dergisi, 15, 45–56. Aydemir, S., Akin, N., & Koc¸ak, C. (2001). Effect of lipase enzyme on the ripening of white pickled cheese. Journal of Food Lipids, 8, 205–213. Bedel, A., & Kilic, S. (2000). The effects of starter culture and commercial enzyme on ripening of Izmir Tulum cheese. Ege Universitesi Ziraat Fakultesi Dergisi, 37, 73–80. Buffa, M., Guamıs, B., Pavıa, M., & Trujıllo, A. J. (2001). Lipolysis in cheese made from raw, pasteurized or high-pressure-treated goatsÕ milk. International Dairy Journal, 11, 175–179.
Deeth, H. C., Fitz-Gerald, C. H., & Snow, A. J. (1983). Gas chromatographic method for the quantitative determination of free fatty acids in milk and milk products. New Zealand Journal of Dairy Science and Technology, 18, 13–20. Fox, P. F., Singh, T. K., & Mcsweeney, P. L. H. (1995). Biogenesis of flavour compounds in cheese. In E. L. Malin & M. H. Tunick (Eds.), In Chemistry of structure–function relationships in cheese (pp. 59–98). New York, USA: Plenum Press. Fox, P. F., & Wallace, J. M. (1997). Formation of flavour compounds in cheese. Advances in Applied Microbiology, 45, 17–85. Freitas, A. C., & Malcata, F. X. (1998). Lipolysis in Picante cheese: Influence of milk type and ripening time on free fatty acid profile. Lait, 78, 251–258. Gu¨ler, Z., & Uraz, T. (2003). Proteolytic and lipolytic composition of Tulum cheeses. Milchwissenschaft, 58, 502–505. Horwood, J. F., Llound, G. T., & Stark, W. (1981). Some flavour components of Feta cheese. The Australian Journal of Dairy Technology, 36, 34–37. Juarez, M. (1986). Physico-chemical characteristics of goatÕs milk as distinct from those of cowÕs milk. In International Dairy Federation, Bulletin 202 (pp. 54–67). Brussels: International Dairy Federation. Kılıc¸, S., Karagozlu, C., & Akbulut, N. (2000). A research on change of fatty acids in Cimi Tulum cheeses during ripening. Ege Universitesi Ziraat Fakultesi Dergisi, 37, 17–24. Kirk, R. S., & Sawyer, R. (1991). PearsonÕs composition and analyses of foods (9th ed.). London: Longman Scientific and Technical Publisher. Kosikowski, F. (1982). Cheese and fermented milk foods (2nd ed.). Brooktondale, New York: F.V. Kosikowski and Associates, p. 710. Lesage, L., Voilley, A., Lorient, D., & Bezard, J. (1993). Sodium chloride and magnesium chloride affected by ripening of Camembert cheese. Journal of Food Science, 58, 1303–1306. Mcsweeney, P. L. H., & Sousa, N. I. (2000). Biochemical pathways for the production of flavour compounds in cheeses during ripening: A review. Lait, 80, 293–324. Minitab. (1991). Minitab Reference Manual Release 7,1, Minitab Inc. State Cdl. PA 16801, USA. Molimard, P., & Spınnler, H. E. (1996). Review: Compounds involved in the flavour of surface mould-ripened cheeses: Origins and properties. Journal of Dairy Science, 79, 169–184. Nasr, M. (1983). Acceleration of Romi cheese ripening by addition of fungal esterase lipase power. Egyptian Journal of Dairy Science, 11, 309–315. Omar, M. M., El-Zayat, A. I., & Ashour, M. (1986). Flavor enhancement by lipase addition of Ras cheese made from reconstituted milk. Food Chemistry, 19, 277–286. Patir, B., Ates, G., & Dincoglu, A. H. (2001). Investigations on microbiological and chemical changes during ripening of traditionally produced Tulum cheese. Firat Universitesi Saglik Bilimleri Dergisi, 15, 1–8. Rahmat, A., & Richter, R. (1996). Formation of volatile free fatty acids during ripening of Cheddar-like goat cheese. Journal of Dairy Science, 79, 717–724. Renner, E. (1986). Milchpraktikum. Fachgebiet milcwissenschaft. Germany: Justuc–Liebig–Universitat Giessen. Urbach, G. (1993). Relations between cheese flavour and chemical composition. International Dairy Journal, 3, 389–422. Urbach, G. (1997). The flavour of milk and dairy products. II. Cheese: Contribution of volatile compounds. International Journal of Dairy Technology, 50, 79–89. Woo, A. M., Kollodge, S., & Lindsay, R. C. (1984). Quantification of free fatty acids in several cheese varieties. Journal of Dairy Science, 67, 874–878.
The effect of microbial lipase on the lipolysis during the ripening of Tulum cheese Gu¨ldane Yilmaz, Ahmet Ayar *, Nihat Akin Selc¸uk University, Agricultural Faculty, Food Eng. Dept. Campus, Konya, Turkey Received 23 April 2004; accepted 6 August 2004
Abstract The effect of microbial lipase enzyme addition on the ripening of Tulum cheese was investigated. Commercial microbial lipase (Piccantase A) was added to milk before rennet addition at a level of 0, 0.04, 0.06 and 0.11 g L 1 of cheese milk. Free fatty acids (FFA) (C2–C18:1) were analysed in the samples during the ripening period. Total solids, fat, salt, total nitrogen, titratable acidity, acidity index, total free fatty acids of cheeses slightly increased during the ripening period. Total FFAs and volatile FFA contents in cheeses were affected by the level of microbial lipase and their contents were increased significantly during the ripening period (P < 0.01). The relative amounts of volatile FFAs varied with ripening time and lipase levels. The addition of lipase to cheese milk could be recommended for the acceleration of flavour development in Tulum cheese over a short ripening period. 2004 Elsevier Ltd. All rights reserved. Keywords: Tulum cheese; Lipolysis; Ripening; FFA
1. Introduction Processing of dairy products on a commercial basis in modern plants dates back to 35 years ago. Despite its fragmented structure, Turkey is the largest producer of milk and dairy products in the region. Cheese is an important integral part of Turkish diet. It is consumed almost three times a day. Cheese is consumed with fruit or as a sort of dessert after lunch or dinner. Generally, cheeses and olives are consumed for breakfast in Turkey. The variety of cheese depends on certain factors: cultural habits and tastes, natural conditions, the species and variety of the animals providing the milk, and the production methods employed. There are more than 100 varieties of cheese in Turkey, but if they are grouped * Corresponding author. Tel.: +90 33 222 32923; fax: +90 33 224 10108. E-mail addresses: [email protected], [email protected] (A. Ayar).
0260-8774/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2004.08.017
according to their similarities, they are limited to about 30 different cheese types. In addition to three major cheese types (White pickled cheese, Kasar cheese, Tulum cheese), there are many traditional cheeses locally produced and consumed in local regions, that are mainly Mihalic¸ cheese, Oltu cheese, Van otlu cheese, Abhazian cheese, Civil cheese, Golot cheese, Ku¨lek cheese, Urfa cheese, and Salinized Diyarbakir o¨rgu¨ cheese etc. Tulum cheese, a special cheese produced in Turkey, is not well known in other countries. It is preferred for its characteristically natural mouldy taste and flavour. During the ripening period, natural contaminating microorganisms especially moulds grow and contribute to the ripening process. Tulum is a semi-hard cheese that can be made from whole, semi skimmed or skimmed sheepÕs, goatÕs, cowÕs, and buffaloÕs milk or their mixture. It has a crumbly texture and a strong flavour. Tulum means the skin of goat or sheep. Traditionally, Tulum cheese is manufactured from skimmed milk (after butter production) for family use, and yogurt is sometimes added for taste. By removing the water of the curd, a crumbly
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texture is obtained. The crumbled curd is salted and packed firmly in goatÕs skins or in cloth sacks and aged for 3–6 months. Ripening takes place at 6–10 C, 65–85% RH. It can be used in bo¨rek or similar foods and is also a great appetizer when mixed with walnuts. FFAs are released usually by the actions of lipases (from different sources) during lipolysis. They contribute directly to cheese flavour, particularly when they are properly balanced with products of proteolysis and other reactions. However, extensive lipolysis is considered to be undesirable for some cheeses (Molimard & Spinnler, 1996; Fox, Singh, & Mcsweeney, 1995). Short-chain fatty acids themselves contribute directly to aroma in many ripened cheese varieties (Urbach, 1997). In addition to their direct impact on cheese flavour, FFAs also act as precursor molecules in a series of catabolic reactions that lead to the production of other flavour compounds such as methyl ketones, esters, thioesters (Urbach, 1993; Fox & Wallace, 1997; Mcsweeney & Sousa, 2000). Ripening time of Tulum cheese is at least four months and the shortening of ripening time without any lost of flavour characteristics could be very advantageous. It is highly desirable to improve its flavour. The studies on FFAs contents of Tulum cheese are limited. The objective of this study was to investigate the effect of a microbial lipase on the acceleration of the ripening process of Tulum cheese.
2. Material and methods 2.1. Materials Raw cowÕs milk was obtained from a farm in Lalebahc¸e in Konya (Turkey). Tulums were produced from goatskin in S ß eker Su¨t Dairy Plant in Konya. Starter culture (Frozen culture Yb 41, Streptococcus salivarus subsp. thermophilus + Lactobacillus delbrueckii subsp. bulgaricus) and microbial rennet (Chymax 15 T was produced by Aspergillus niger var. awamori with completely chymosin) were obtained from Peyma-Chr HansenÕs Inc. (Istanbul-Turkey). Lipase (Piccantase A), as a powder that was produced from Rhizomucor miehei, was obtained from Gist-brocades Inc. (Istanbul-Turkey). 2.2. Methods Experimental cheeses were made in the S ß eker Su¨t Dairy Plant in Konya. Flow diagram of production process of Tulum cheese is shown in Fig. 1. The lots of 200 L raw milk were heated to 31 C and divided into four portions, followed by addition of 0.1 g kg 1 of frozen culture. All portions were left to pre-ripening until pH reached 6.25. The first portion was used as control (A0). To the other portions, microbial lipase was added
Fresh raw milk
Heat to 310 C Starters, Rennet,
Addition
Lipase enzyme Coaguluation (within 30 min.)
Curd cutting (1 cm x 1 cm x 1 cm) and hold the curd for 10-15 min. Excess whey drainage Curd draining under pressure (25 kgm-2 until end of draining ) and pre-fermentation at 26 0C until pH 5.10
Curd breaking up by hand (Curd is break up small pieces on wooden table for 3 time)
Dry salting (30 g kg-1 NaCl w/w)
Pressure (25 kgm-2 for 16 h for 3 time)
Milling
Curd placement into goatskin (tulum)
Ripening at 8 0C for 90 days
Tulum cheese
Fig. 1. Flow diagram of manufacture of Tulum cheese.
at a rate of 0.04 g L 1 (A1), 0.06 g L 1 (A2) and 0.11 g L 1 (A3) to milk, respectively. Rennet (strength, 1:15,000) was added to coagulate the milk within 30 min. The coagulum was cut into 1 cm · 1 cm · 1 cm cubes and the curd pieces were transferred into vats lined with cheese cloth and pressed 25 kg m 2 press weight until pH 5.10 for pre-fermentation at 26 C. Subsequently, when curd reached the desired moisture content and pH value (4.90). It was broke up by hand and dry salted (30 g kg 1). The curd was put into cotton bags to remove whey. Then pressed using 25 kg m 2 press weight for 16 h. This press operation was repeated for three times. The salted fresh cheese was milled and transferred in the goatskins (specially prepared Tulums) without air holes. They ripened at 8 ± 1 C, 85 % RH for 90 days. The experiment is carried out as duplicate. Total solids, fat, total nitrogen, salt, pH and titratable acidity (SH) in cheese samples were determined according to the procedures described by Kirk and Sawyer (1991), acidity index according to Renner (1986). FFAs
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271
of Patir, Ates, and Dincoglu (2001); Atesß and Patir (2001) for Tulum cheese. The pH decreased slightly whereas the acidity was increased during ripening. The acidity index of the fat values increased up to 90 days of ripening. In the control cheese samples, acidity index increased in small amounts, but cheeses made from microbial lipase added milks had a higher increase of acidity index (Table 1). Statistically significant differences (P < 0.01) were found among the ages of ripening for the acidity index. The acidity index of the cheeses made with microbial lipases were significantly (P < 0.01) higher than that of control sample. The final acidity index values in lipase added Tulum cheeses are higher than those of found in traditional Tulum cheese made from raw milk by Gu¨ler and Uraz (2003) and in C ¸ imi Tulum cheese made from raw goat milk by Kılıc¸, Karagozlu, and Akbulut (2000). The FFAs composition of Tulum cheese samples made from microbial lipase added milk is given in Tables 2 and 3. In the control cheese samples, volatile FFAs (C2–C10:0) were present in small amounts, but cheese made from microbial lipase added milk had a higher content of all volatile FFAs. The higher volatile FFA was observed in A3 coded (added 11 g microbial lipase per 100 L cheese milk) cheese and the dominant volatile FFA was butyric acids for all cheese samples made with microbial lipase added milk. The results reported by Aydemir et al. (2001) for white cheese and by Omar, El-Zayat, and Ashour (1986) for Ras cheese
in cheeses were determined by gas chromatography according to the method of Deeth, Fitz-Gerald, and Snow (1983). Cheeses were analyzed at 1, 15, 30, 60 and 90 days in duplicate. The effect of addition of lipase enzyme and ripening period was evaluated by analysis of variance (Minitab, 1991). The Duncan Multiple Comparison Test was used to determine the groups significantly different (P < 0.01) from each other. All data are reported as means.
3. Results and discussion The mean chemical composition analysis data obtained from the experimental Tulum cheeses are presented in Table 1. Examination of this table revealed that the addition of lipase had no significant effect on total solids, salt, fat, total nitrogen, pH and titratable acidity of the resultant cheeses. Similarly, several researchers (Nasr, 1983; Aydemir, Akin, & Koc¸ak, 2001; Akın, Aydemir, Koc¸ak, & Yıldız, 2003) have reported that the use of lipase had no significant effect on the gross composition of cheese during the ripening period, but Bedel and Kilic (2000) have reported that the use of enzyme in Tulum cheese had significant effect on the gross composition. Because of water lose from goat skin pores. Total solids, salt, fat and total nitrogen of cheese samples were increased during ripening (P < 0.01). This finding is in accordance with the results
Table 1 Effect of the addition of microbial lipase on the chemical composition of Tulum cheese Group
Ripening period (Days)
Total solids (g 100 g 1)
Salt (g 100 g 1)
Salt in total solids
Fat (g 100 g 1)
Fat in total solids
Total nitrogen (g 100 g 1)
Total protein (Nx6.38) in total solids
pH
A0
1 15 30 60 90
52.49 53.09 53.50 53.04 53.98
a a a a a
2.13 2.19 2.21 2.21 2.23
b a a a a
4.06 4.13 4.12 4.16 4.13
a a a a
28.25 29.00 29.25 29.50 29.50
a a a a a
53.83 54.62 54.67 55.63 54.64
a a a a a
1.94 1.99 2.00 2.05 2.13
b ab ab ab a
2358 2391 2385 2466 2517
a a a a a
5.15 5.07 5.05 5.03 4.99
A1
1 15 30 60 90
53.50 54.39 54.56 54.91 54.70
b ab ab a ab
1.76 1.90 2.01 2.08 2.09
b ab a a a
3.30 3.49 3.69 3.78 3.82
b ab ab a a
27.75 28.00 28.25 29.00 29.50
a a a a a
51.88 51.48 51.78 52.81 53.93
a a a a a
1.84 2.10 2.03 2.04 2.05
b a a a a
2194 2463 2374 2370 2391
a a a a a
A2
1 15 30 60 90
53.86 53.87 54.01 54.02 54.33
b b b b a
1.84 1.99 2.01 2.06 2.50
c bc bc b a
3.42 3.69 3.72 3.81 4.59
c bc bc b a
27.25 28.75 28.75 28.75 29.25
a a a a a
50.60 53.36 53.23 53.22 53.83
a a a a a
1.85 1.90 2.03 2.10 2.11
b b ab a a
2191 2250 2398 2480 2478
A3
1 15 30 60 90
53.53 53.96 54.12 54.12 54.16
a a a a a
2.14 2.17 2.17 2.23 2.70
b b b b a
4.00 4.02 4.00 4.11 4.99
b b b b a
28.00 28.00 29.00 29.50 29.50
a a a a a
52.31 51.88 53.59 54.51 54.46
a a a a a
1.78 1.86 1.99 2.00 2.03
b ab a a a
2122 2199 2346 2358 2391
Titratable acidity (SH)
Acidity index mg KOH g 1 fat
a ab ab ab b
83.00 d 89.00 c 91.50 bc 97.00 b 113.00 a
1.53 1.70 2.47 5.01 5.54
5.07 5.04 5.01 5.01 5.01
a a a a a
86.50 d 92.50 c 96.50 c 102.50 b 112.50 a
2.68 e 4.67 d 7.21 c 14.79 b 20.95 a
a a a a a
5.06 5.04 4.94 4.97 4.94
a a a a a
89.50 c 94.50 bc 97.50 b 106.00 a 111.50 a
3.41 e 6.18 d 9.37 c 15.86 b 26.61 a
a a a a a
5.08 5.04 4.99 4.94 4.92
a ab ab b b
91.00 d 96.00 c 101.50 b 106.50 a 109.50 a
4.64 e 9.51 d 13.81 c 24.05 b 36.96 a
A0: Control; A1: 0.04 g microbial lipase L 1 milk; A2: 0.06 g microbial lipase L 1 milk; A3: 0.11 g microbial lipase L 1 milk. a–e means with different letters were significantly different among ripening periods within the rows of each cell (P < 0.01).
e d c b a
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Table 2 Effect of the addition of microbial lipase on volatile free fatty acids (C2–C10:0) of Tulum cheese (mg 100 g
1
cheese)
Cheese group
Ripening period (Days)
Acetic
Propionic
Butyric C4:0
Caproic C6:0
Caprylic C8:0
Capric C10:0
A0
1 15 30 60 90
1.58 3.02 5.92 6.98 7.75
0.30 0.30 0.41 0.74 1.10
d d c b a
1.07 2.96 3.87 4.56 8.56
1.12 1.52 2.00 2.52 3.96
1.92 c 1.70 d 1.91 c 2.935 b 4.12 a
2.96 4.01 5.86 6.01 8.01
A1
1 15 30 60 90
1.43 e 1.96 d 5.00 c 8.56 b 16.74 a
1.29 1.22 1.30 2.35 3.00
c d c b a
7.73 e 12.64 d 15.92 c 18.79 b 21.49 a
3.98 e 6.08 d 7.95 c 8.31 b 11.61 a
1.86 2.62 3.51 4.03 6.12
3.97 e 4.26 d 6.68 c 7.78 b 10.02 a
A2
1 15 30 60 90
1.19 e 1.61 d 6.81 c 13.86 b 23.96 a
1.21 1.29 1.70 2.00 3.07
e d c b a
14.36 23.31 33.23 28.11 50.09
e d c b a
8.44 e 13.35 d 17.98 c 20.25 b 23.39 a
3.43 e 5.05 d 7.01 c 9.59 b 11.31 a
4.87 e 8.59 d 11.05 c 14.08 b 20.52 a
A3
1 15 30 60 90
1.56 e 2.00 d 8.61 c 19.76 b 30.61 a
1.96 2.02 2.87 3.56 4.11
e d c b a
18.76 28.39 36.36 40.62 65.23
e d c b a
9.11 e 16.81 d 18.57 c 25.44 b 31.96 a
3.72 e 4.01 d 6.20 c 10.47 b 13.09 a
8.56 e 10.75 d 13.38 c 17.47 b 22.61 a
e d c b a
e d c b a
e d c b a
e d c b a
e d c b a
A0: Control; A1: 0.04 g microbial lipase L 1 milk; A2: 0.06 g microbial lipase L 1 milk.; A3: 0.11 g microbial lipase L 1 milk. a–e means with different letters were significantly different among ripening periods within the column of each cell (P < 0.01).
Table 3 Effect of the addition of microbial lipase on free fatty acids (C12:0–C18:1) of Tulum cheese (mg 100 g
1
cheese)
Cheese group
Ripening period (Days)
Lauric C12:0
Myristic C14:0
Palmitic C16:0
Stearic C18:0
Oleic C18:1
Total FFAs1
A0
1 15 30 60 90
4.79 e 5.76 d 7.84 c 9.95 b 10.56 a
9.04 e 13.69 d 16.35 c 20.49 b 26.15 a
30.74 37.83 41.11 47.77 50.86
e d c b a
7.14 e 10.48 d 11.64 c 15.25 b 18.15 a
24.74 36.71 39.11 46.86 49.30
e d c b a
85.39 117.78 136.02 164.06 188.52
A1
1 15 30 60 90
4.03 e 4.95 d 6.27 c 8.44 b 11.02 a
10.50 12.31 15.17 20.12 25.24
e d c b a
33.64 34.46 39.01 43.37 59.91
e d c b a
7.91 e 8.12 d 9.71 c 13.75 b 16.10 a
28.14 30.16 35.52 42.20 61.55
e d c b a
104.47 118.78 146.04 177.70 242.79
A2
1 15 30 60 90
8.74 e 9.44 d 13.14 c 16.21 b 19.61 a
14.09 15.94 17.01 25.18 31.31
e d c b a
35.61 37.01 45.08 59.23 70.58
e d c b a
9.01 e 9.76 d 10.02 c 15.38 b 19.06 a
27.91 35.01 46.13 53.97 73.47
e d c b a
128.87 160.37 209.14 257.85 346.37
A3
1 15 30 60 90
9.49 e 10.61 d 12.48 c 15.90 b 20.60 a
14.49 16.33 18.98 24.45 36.73
e d c b a
40.38 43.19 47.97 58.50 79.30
e d c b a
9.02 e 10.71 d 12.64 c 17.79 b 20.04 a
32.17 e 39.20 d 46.18c 54.00 b 82.75 a
149.22 184.01 224.25 287.97 407.03
A0: Control; A1: 0.04 g microbial lipase L 1 milk; A2: 0.06 g microbial lipase L 1 milk; A3: 0.11 g microbial lipase L 1 milk. a–e means with different letters were significantly different among ripening periods within the column of each cell (P < 0.01). 1 Total FFAs was the sum of Acetic, Propionic, Butyric, Caproic, Caprylic, Capric, Lauric, Myristic, Palmitic, Stearic, Oleic acids.
are in agreement with our findings. All volatile FFAs were found in all (A0, A1, A2 and A3) cheese samples and their distribution varied with microbial lipase levels. In the early stages of ripening, butyric acid content of control cheese (A0) was remarkably lower, while acetic
acid content was higher than that of cheeses made with microbial lipase. The results also show that volatile FFAs contents of cheese increased during the ripening period, particularly in the cheese made with microbial lipase added milk. The level of propionic, caproic,
G. Yilmaz et al. / Journal of Food Engineering 69 (2005) 269–274
caprylic and capric acid were low. Acetic and propionic acid is not a product of lipolysis. The presence of they may be due to bacterial fermentation of lactose through glycolytic pathway and decarboxylation and oxidative deamination of amino acids (Kosikowski, 1982). Acetic and propionic acids were included in this study because they are the major volatile acid extracted with FFAs and they contribute greatly to the final flavour of cheese (Lesage, Voilley, Lorient, & Bezard, 1993; Abd El-Salam, Alichanidis, & Zerfiridis, 1993). The FFAs (C12:0–C18:1) of cheese samples were increased when microbial lipase levels increased in cheese samples. The FFAs contents of the cheeses samples increased throughout the ripening time in all cheese samples, which was in agreement with the results reported by Kılıc¸ et al. (2000) for C ¸ imi Tulum cheese made from raw goat milk. The level of the FFAs and volatile FFAs of A3 cheese with the highest lipase addition was higher than that of the other cheese samples at 90 days of ripening. Similar results were observed by Aydemir et al. (2001) in white cheese and Woo, Kollodge, and Lindsay (1984) for several different cheeses. But FFAs levels in our lipase added cheeses were higher than those reported by Kılıc¸ et al. (2000) for C ¸ imi Tulum cheese and by Akin (2001) for Tulum cheeses made from different types raw milks. The differences in amounts of these fatty acids in cheese samples, made different enzyme levels, are possibly due to large reductions in numbers of bacterial spices in milk capable of liberating intracellular lipases by autolysis. The differences in the free fatty acid concentrations found by various authors may be related, either
273
to the techniques used for the distillation and extraction and type of cheese. Of the FFAs, the long-chain fatty acids dominated. The most abundant one was palmitic acid (C16:0), followed by oleic acid (C18:1) and myristic acid (C14:0). The rate of increase in the concentration of short-chain FFA (C4:0–C8:0) during ripening was higher than that of medium-chain FFA (C10:0–C14:0), which in turn was higher than that of long-chain FFA (C16:0–C18:1). This could be explained by the specificity of lipases towards the FFAs located at the positions sn-1, 3 of the triglyceryde, and generally, short-chain fatty acids are predominantly esterified at the sn-3 position (Juarez, 1986). The application of Duncan Multiple Comparison Test of all the individual FFAs showed that a significant increase (P < 0.01) occurred at each sampling point (Table 3). Short-chain FFAs at 90 days represented approximately 24–25 g 100 g 1 of the total FFA content in all cheeses. Medium and long-chain FFA represented approximately 21–22 g 100 g 1 and 52–53 g 100 g 1 respectively, of all FFA in all cheeses. Despite the quantitative importance of medium- and long-chain FFA, they are not the main contributors to cheese flavour (Freitas & Malcata, 1998; Buffa, Guamıs, Pavıa, & Trujıllo, 2001). Acetic, butyric, caproic, caprylic and capric acids were the short-chain FFA that showed the highest relative increase in cheeses made with microbial lipase during ripening period. It is known that shortchain FFA have a very important effect on the flavour of cheese (Rahmat & Richter, 1996).
Table 4 Effect of the addition of microbial lipase on sensory properties in Tulum cheeses Cheese group
Ripening period (Days)
Appearance (30)
Flavour (40)
Body and texture (20)
Odour (10)
A0
1 15 30 60 90
16.67 19.17 26.67 28.33 16.67
b b a a b
31.67 31.67 33.33 36.67 35.00
a a a a a
16.67 17.50 19.17 19.17 14.17
ab ab a a b
7.50 a 8.33 a 9.17 a 10.00 a 6.67 a
A1
1 15 30 60 90
18.33 19.17 23.33 28.33 19.17
b b ab a b
31.67 36.67 35.00 38.33 11.67
a a a a b
16.67 16.67 19.17 19.17 13.33
ab ab a a b
8.33 7.50 9.17 9.17 6.67
A2
1 15 30 60 90
18.33 28.33 26.67 28.33 26.67
b a a a a
30.00 31.67 36.67 38.33 15.83
a a a a b
16.67 18.33 19.17 20.00 14.17
ab ab a a b
8.33 a 8.33 a 9.17 a 10.00 a 6.67 a
A3
1 15 30 60 90
16.50 18.33 16.67 13.33 12.50
b a ab b b
30.00 bc 26.67 c 38.33 a 35.00 ab 5.00 d
16.67 17.50 19.17 13.33 10.83
ab a a b b
9.00 a 10.00 a 10.00 a 7.50 a 5.83 b
A0: Control; A1: 0.04 g microbial lipase L 1 milk; A2: 0.06 g microbial lipase L 1 milk; A3: 0.11 g microbial lipase L 1 milk. a–e means with different letters were significantly different among ripening periods within the column of each cell (P < 0.01).
a a a a a
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G. Yilmaz et al. / Journal of Food Engineering 69 (2005) 269–274
The results of organoleptic examination of different Tulum cheeses at the ages 1, 15, 30, 60 and 90 days are presented in Table 4. There is no interaction between microbial lipase levels and ripening period for all analysed sensory properties. Microbial lipase levels and ripening periods, for each of the mean properties scored by sensory test, were compared. According to statistical analysis, results can be summarised as follows: (i) when microbial lipase levels were increased, sensory scores (appearance, flavour, body and texture and odour) were not significantly increased; (ii) sensory scores in microbial lipase added cheeses were rapidly increased by the age 60 days of ripening, but in 0.11 g microbial lipase L 1 milk added cheese was increased by the age 30 days; (iii) different ripening times had effect on sensory scores; (iv) after 60 days ripening, sensory scores were significantly lower. Despite the increasing in the FFAs observed for cheeses groups, decrease in sensory scores of cheeses were observed. This supports justifies the view that other factors in addition to FFAs, such as total acidity and protein degradation as well as headspace volatiles are also important for the development of cheese flavour (Horwood, Llound, & Stark, 1981). In conclusion, the present results indicate that the use of microbial lipase in the manufacture of Tulum cheese increases the FFA content, but has only a slight effect on the pattern of the FFAs. The addition of microbial lipase to cheese milk could be recommended for accelerating the development of flavour in this cheese over a short ripening period.
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