Journal of Food Engineering 79 (2007) 13911396 www.elsevier.

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Antioxidant activities of Satureja cilicica essential oil in butter and in vitro

Gulcan Ozkan *, Bedia Simsek, Hakan Kuleasan
Suleyman Demirel University, Faculty of Agriculture, Department of Food Engineering, 32260 Isparta, Turkey Received 14 November 2005; accepted 12 April 2006 Available online 4 May 2006

Abstract Satureja (Labiatae) species are a well-known aromatic plant which is used to produce essential oil and aromatic water in the mountain regions of the Mediterranean part of Turkey. In our study, it was aimed to determine antioxidant activities of Satureja cilicica essential oil in butter and in vitro. Antioxidant activities of the oils at dierent concentrations were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and phosphomolybdenum methods. Also the essential oil with 0.5%, 1.0% and 2.0% were added in butter as antioxidants and were assayed during 60 days storage of butter at +4 and +20 C. For this reason, it was analyzed peroxide value, pH, titratable acidity and total lactic acid bacteria as a criterion to assess the antioxidant activity of essential oil at 20th, 40th and 60th days of storage. Antiradical activity was found as IC50 = 32.02 0.58 lg/ml and in vitro antioxidant capacity was 101.16 3.32 lg/ml by phosphomolybdenum methods. On the other hand, the essential oil of S. cilicica exhibited a strong antioxidant activity in butter. Antioxidant activities of oils were higher when the essential oil concentration was increased. In addition to that peroxide value pH, titratable acidity and number of viable lactic acid bacteria were compared to the control. In addition, titratable acidity and total number of lactic acid bacteria of samples stored at +20 C were determined higher than the other storage temperature during the storage time. According to our results, essential oil of S. cilicica could be used as both natural antioxidant and aroma agent in butter. 2006 Elsevier Ltd. All rights reserved.
Keywords: Satureja cilicica; Essential oil; Butter; Starter culture

1. Introduction Satureja cilicica L. is an aromatic and endemic medicinal plant belonging to the Lamiaceae family. The aerial material has a distinctive taste and can be added to stung, meat pies, sausages and some milk products as a seasoning. Fresh sprigs can be boiled with pulses, such as peas, beans or lentils, for avouring or, alternatively, they can be used instead of parsley and chervil as a garnish. The leaves, owers and stems of Satureja species are used as herbal tea, in production of traditional medicine, to treat various ailments, such as cramps, muscle pains, nausea, indigestion, diarrhea and infectious diseases (Baydar, Sagdic,

Corresponding author. Tel.: +90 246 2111666; fax: +90 246 2370437. E-mail address: gozkan@ziraat.sdu.edu.tr (G. Ozkan).

Ozkan, & Karadogan, 2004; Gulluce et al., 2003; Hajhashemi, Sadraei, Ghannadi, & Mohseni, 2000). Herbs and derivatives have been used as culinary and medicinal purposes for a long time and added in the food to prevent the formation of undesirable oxidation products. Not only are many of the avours and aromas distinctively pleasant, but they can also be used to conceal o-avours and odors. The researches conducted on the opportunity of antioxidant and antimicrobial agent of herbs in vitro an in food generally focused on their essential oils in the last 1520 years (Tainter & Grenis, 1993). The oxidation of lipids is very important with respect to the shelf life of a product. In the rst stage of oxidation, peroxides are formed by the reaction between unsaturated fatty acids and oxygen molecule. Butter is also a milk product with relatively longer shelf life. In butter making, the fat content of the milk is concentrated approximately

0260-8774/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2006.04.020

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20 times. By contrast, the other constituents in milk, i.e. protein, lactose, salts and water are reduced. Butter is produced from milk, cream and yoghurt in dierent areas of Turkey (Konar & Hayaloglu, 1999). The rate of consumption of butter is about 2.6% for each person in Turkey (Atamer, 1993). In addition, lactic acid bacteria are commonly used as starter cultures to provide diacetyl which is the major aroma compound in the production of various dairy products. They cause rapid acidication of the raw material with the production of organic acids, mainly lactic acid. Their production of acetic acid, ethanol, aroma compounds, and several enzymes is also important. In this way they enhance shelf life, microbial safety, improve texture, and contribute to the pleasant sensory prole of the end product. The characteristics and or fatty acid composition of butter and milk fat have been investigated by many researchers (Bilgin, 1996; Collomb, Butikofer, Sieber, Jeangros, & Bosset, 2002; Glew, Okolo, Chuang, Huang, & Vanderjagt, 1999; Sagdic, 2000; Sagdic, Arici, & Simsek, 2002). But according to our knowledge there were a little literature on opportunity of herb essential oil as both natural antioxidant and aroma agent in butter. The aim of this study was to determine the antioxidant activities and aromatic properties of S. cilicica essential oil when it is used in butter production. In addition, the eect of essential oil on oxidation, chemical properties and stability of butter was determined. Since the presence of starter culture is crucial for the aroma development of butter, the eect of essential oil on viability of Lactococci used as starter culture was also investigated. 2. Materials and methods 2.1. Essential oil Essential oil of S. cilicica was obtained from the bi-national project of Deutscher Akademischer Austauschdienst (DAAD) in Bonn, Germany (reference number: A/04/17627). Schulz, Ozkan, Baranska, Kruger, and Ozcan (2005) were reported in their article from this project that S. cilicica main components were the thymol (22.76%), carvacrol (18.90%), p-cymene (19.52%) and c-terpinene (13.40%). 2.2. Butter production For butter production, cream and butter were produced in Suleyman Demirel University, Un-Sut Dairy Plant. The rst, cream was analyzed, after cream was standardized 35% fat and pasteurized at 90 C for 15 min and cooled to 1011 C, cream churned. After churning cream was inoculated with butter culture (2%) (Wiesby, Probat 505, Germany). After fermentation, the butters were divided into two parts (A: +4 C stored and B: +20 C stored) and each part was divided into four parts again. essential oil was added into each part (1UA, 0.5%; 2UA, 1.0%; 3UA, 2%; KA, control: non-essential oil; 1UB, 0.5%;

2UB, 1.0%; 3UB, 2%; KB, control: non-essential oil). Then the samples were packaged as 100 g and stored at 4 and 20 C for 60 days until analysis. All determinations were done in duplicate. 2.3. Determination of antiradical activity The free radical scavenging activity of essential oil was examined by comparing to those of known antioxidant such as BHT by 1,1-diphenyl-2-picrylhydrazyl (DPPH) using the method of Lee et al. (1998). Briey, a 1.0 ml solution of the essential oil in methanol (10 ppm) was mixed with 2.0 ml of methanolic solution of DPPH (10 mg/l). The mixture was shaken vigorously and allowed to stand at room temperature for 5 min. Then the absorbance was measured at 517 nm against methanol as the blank in a spectrophotometer. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The percent of DPPH discoloration of the samples was calculated according to the formula: Antiradical activity (%) = 100 (absorbance of control absorbance of sample/absorbance of control). Extract concentration providing 50% inhibition (IC50) was calculated from the plot of inhibition percentage against extract concentration. Tests were carried out in triplicate. 2.4. Evaluation of antioxidant activity The antioxidant activity of essential oil was evaluated by the formation of phosphomolybdenum complex method according to Prieto, Pineda, and Aguilar (1999). Briey, an aliquot of 0.4 ml of sample solution (10 ppm in methanol) was combined in a vial with 4 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The blank solution contained 4 ml of reagent solution and the 1 ml of methanol. The vials were capped and incubated in a water bath at 95 C for 90 min. After the samples had cooled to room temperature, the absorbance of the mixture was measured at 695 nm against a blank. The antioxidant activity was expressed relative to that of ascorbic acid. All determinations were done in triplicate. 2.5. Peroxide value analysis The antioxidant activity of the essential oil was tested in butter and expressed as the decrease in the rate of peroxide formation. And its peroxide value was also found as 0.35 mequiv/kg. Oil (2 g) was accurately weighted and the essential oil was added directly to the oil at a concentration of 0.5%, 1% and 2% (v/wt). Control samples of butter without antioxidant were also placed under same conditions. All samples were incubated in 100 ml capped plastic beakers at +4 and +20 C in the dark. The peroxide values of the samples were determined at 20th, 40th and 60th days of storage in terms of the method of the American Oil

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Chemists Society (AOAC, 1990). All determinations were done in triplicate. 2.6. Physicochemical analyses of butter and cream The pH value of butter and cream were determined by pH meter (WTW instruments, pH 330-Germany). Titratable acidity (as lactic acid %) of cream and butter was determined as suggested by Anonymous (1975, 1989). Moisture was found by heating in an over at 102 C, until a constant weight was obtained. The contents of fat (%) and non-fat solid of cream and butter were measured according to the standard methods (Anonymous, 1975, 1989). All determinations were done in triplicate. 2.7. Lactic acid bacteria counts Commercial butter starter which contains Lactococcus and Leuconostoc species were added in butter to develop desired avour. Lactic acid bacteria counts were done in every 20 days of storage period. For this purpose 10 ml butter sample was taken into sterile tubes and melted in a 45 C water bath. Then proper dilutions were done and each dilution was plated on MRS agar plates in triplicates. After incubation at 30 C for 24 h, colony counts were recorded. The same experimental procedures were applied to both samples stored at 4 and 20 C. The results were presented as average of three counts. 2.8. Statistical analysis Results of the research were tested for statistical signicance by one-way ANOVA. Dierences were considered statistically signicant at the P < 0.05 level. 3. Results and discussion In our study, antiradical activity and antioxidant capacity of the essential oil obtained from S. cilicica were studied as in vitro and in food. Antiradical activity of essential oil from S. cilicica tested by the DPPH model system and the antiradical activity of essential oil was found as IC50 = 32.02 0.58 lg/ml. BHT used as a synthetic antioxidant in food industry showed lower antiradical activity (IC50 = 96,13 0.01 lg/ml) when it was compared to the same concentration of essential oil. The model of scavenging the stable DPPH radical is a widely used method to evaluate antioxidant activities in a relatively short time compared with other methods (Gulc in, Sat, Beydemir, Elmastas, & Kufrevioglu, 2004). The addition of the essential oil to the DPPH solution caused a rapid decrease in the optical density at 517 nm. The degrees of discoloration indicate the scavenging capacity of the essential oil. Free radicals cause autoxidation of unsaturated lipids in food (Kaur & Perkins, 1991). The eect of antioxidant on DPPH radical scavenging was thought to be due to their hydrogen donating ability or radical scavenging activity (Baumann,

Wurn, & Bruchlausen, 1979). Antioxidants cease the free radical chain of oxidation and to donate hydrogen from the phenolic hydroxyl groups. Therefore formed stable end-product does not permit further oxidation of the lipid (Sherwin, 1978). The antioxidant capacity of the essential oil (equivalent to ascorbic acid) was found as 101.16 3.32 lg/ml by phosphomolybdenum method. The phosphomolybdenum method is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compounds and the formation of a green Mo(V) complex with a maximal absorption at 695 nm (Prieto et al., 1999). The antioxidants break the free radical chain by donating a hydrogen atom (Gordon, 1990). Jayaprakasha, Selvi, and Sakariah (2003) were reported that the antioxidant activity of the essential oil depends on the presence of polyphenols which may act as reductons. According to our knowledge, there was no available literature on antiradical activities and antioxidant properties of essential oil from S. cilicica for a further discussion. Peroxide value is a widely used measure of the primary lipid oxidation indicating the amount of peroxides formed in fats and oils during oxidation. Antioxidant activity of the essential oil, compared with control samples, was tested. The results given in Figs. 1 and 2 showed that all concentrations of essential oil reduced the oxidation rate of butter at +4 and +20 C in terms of formation peroxides. After 60 days all essential oil showed antioxidant eect in varying degrees in butter compared with the control. Peroxide value of control sample increased from 0.35 mequiv/kg to 1.00 and 1.14 mequiv/kg at +4 and +20 C, respectively. In terms of retarding the formation of oxidation products, the eectiveness of the essential oil was determined at 2% concentration. The essential oil at all concentration was found more eective than the control and the peroxide value of butter stored at +4 C had lower value than at +20C. Eectiveness of essential oil concentration (%) at days of 60th can be put into the following order: 2 > 1 > 0.5% and with the values of 0.50, 0.65 and 0.71 mequiv/kg at +4 C and 0.69, 0.94 and 1.04 mequiv/ kg at +20 C, respectively. Some researchers are also reported that herb and propolis extract had high antioxidant activity in butter (Ayar, Ozcan, Akgul, & Akin, 2001; Ozcan & Ayar, 2003). Before the production of butters, the physicochemical characteristic of cream was analyzed. The fat, solidnon- fat, pH, titratable acidity and moisture values of cream were determined as 72.666 0.2880%, 8.9766 0.3534%, 4.713 0.0115, 0.2566 0.0011% and 18.3566 0.0702%, respectively. And then, the fat, solid-nonfat, pH, titratable acidity and moisture values of butters were analyzed and found as 84.166 0.2880%, 1.5366 0.2663%, 6.373 0.0115, 0.028 0.0005% and 14.2976 0.0321%, respectively (rst days). The titratable acidity value of butters added essential oil decreased according to control during the storage period. This value increased at control samples during the storage period. The lactic acid (%) value of samples stored at

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Peroxide value, per meq kg

1.20 1.00 0.80 0.60 0.40 0.20 0.00 0 20 40 60 KA 1 UA 2 UA 3 UA

Storage time, days

Fig. 1. Peroxide values of essential oil from S. cilicica at 2%, 1% and 0.5% and control during storage at 4C for up to 60 days in the dark.

Peroxide value, per meq kg

1.20 1.00 0.80 0.60 0.40 0.20 0.00 0 20 40 60 KB 1 UB 2 UB 3 UB

Storage time, days

Fig. 2. Peroxide values of essential oil from S. cilicica at 2%, 1% and 0.5% and control during storage at 20 C for up to 60 days in the dark.

+20 C was higher than other storage temperature during the storage time. The pH value of samples added essential oil at +20 C was lower than other storage temperature. This value was degreased at control samples during the storage period at both +4 and +20 C. There was an insignicant dierence between titratable acidities and pH of the butters (Table 1). Butter samples stored at +4 C were more stable than those kept at +20 C. The results of physicochemical characteristics were similar to those given by Sagdic et al. (2002) and Sagdic et al. (2003). As a result, the antiradical and antioxidant activities of the herb extracts and essential oils are generally ascribed to the diterpenes and polyphenols (Couladis, Tzakou, Verykokidou, & Harvala, 2003; Lu & Foo, 2001; Miliauskas, Venskutonis, & van Beek, 2004; Yildirim et al., 2004).

The main components of essential oil sample used in our study were the thymol (22.76%), carvacrol (18.90%), pcymene (19.52%) and c-terpinene (13.40%) (Schulz et al., 2005). The antioxidant properties could be attributed to these diterpenes and phenolics, especially thymol and carvacrol. The results showed that antiradical and antioxidant activity of essential oil may depend on not only the essential oil content but also dierent concentrations. Because 2% concentration of essential oil was found the most eective to reduce the oxidation of butter. In addition to its antioxidant activity, the essential oil did not show any undesired eect on the viability of starter cultures which are necessary for development of aroma in butter and chemical properties analyzed. As it is shown in the Figs. 3 and 4 there was a slight decrease in the number of lactic

Table 1 Titratable acidities and pH values of the butter samples (mean standard deviation) Butter Lactic acid 20th day KA 1UA 2UA 3UA KB 1UB 2UB 3UB 0.095 0.002 0.067 0.002 1.067 0.002 0.058 0.002 0.101 0.002 0.086 0.002 0.083 0.002 0.080 0.002 40th day 0.0988 0.00 0.0665 0.00 0.0639 0.00 0.1113 0.00 0.1113 0.00 0.0874 0.00 0.0832 0.00 0.0821 0.00 60th day 0.103 0.00 0.075 0.00 0.069 0.00 0.114 0.00 0.114 0.00 0.093 0.00 0.088 0.00 0.087 0.00 pH 20th day 4.99 0.02 5.09 0.03 5.16 0.03 4.52 0.02 4.50 0.05 4.97 0.05 4.86 0.07 5.13 0.05 40th day 4.62 0.02 4.93 0.03 5.07 0.05 5.19 0.01 4.36 0.02 4.55 0.05 4.53 0.01 4.78 0.01 60th day 4.91 0.56 4.83 0.04 4.90 0.04 4.98 0.05 4.28 0.04 4.49 0.05 4.51 0.02 4.72 0.02

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Lactic acid bacteria counts (log cfu/ml)

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7.5 7 6.5 6 5.5 5 4.5 0 20 40 60

KB 1 UB 2 UB 3 UB

Storage time, days (4 C)

Fig. 3. Viable number of lactic acid bacteria during 60 days of storage at 4 C with control and essential oil from S. cilicica.
Lactic acid bacteria counts (log cfu/ml)

7.5 7 6.5 6 5.5 5 4.5 0 20 40 60 KA 1 UA 2 UA 3 UA

Storage time, days (20 C)

Fig. 4. Viable number of lactic acid bacteria during 60 days of storage at 20 C with control and essential oil from S. cilicica.

acid bacteria in the rst 20 days of storage. After 20 days, the numbers increased and the viable numbers of lactic acid bacteria reached from 105 to 106 cfu/ml. The results also showed that there was an insignicant dierence between the samples stored in refrigeration temperature and those which stored in high temperature regarding the viable numbers of lactic acid bacteria. When dierent concentrations of essential oils added in butter samples were compared, similar results were obtained which indicates that essential oil even at high concentrations did not aect viability of starter thus aroma development of the product. The results of the present study indicate that S. cilicica essential oil could be used as easily accessible source of natural antioxidant and aroma agent for butters. References
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