Food Sci. Biotechnol. 22(3): 613-620 (2013) DOI 10.1007/s10068-013-0122-9
RESEARCH ARTICLE
Effect of Processing Techniques on Antioxidative Enzyme Activities, Antioxidant Capacity, Phenolic Compounds, and Fatty Acids of Table Olives Yasemin Sahan, Asuman Cansev, and Hatice Gulen
Received: 9 August 2012 / Revised: 7 January 2013 / Accepted: 8 January 2013 / Published Online: 30 June 2013 © KoSFoST and Springer 2013
Abstract In this study, olive fruits (Olea europeae cv. Gemlik) of the most common sources of table olives in Turkey were used. Total polyphenol content (TPC), antioxidant capacity (AC), and antioxidant enzymes (catalase, CAT; ascorbate peroxidase, APX; and glutathione reductase, GR) of table olives were compared by 4 different methods of ripe table olive processing. Results revealed that TPC of the processed olives ranged from 117.44 to 418.69 mg gallic acid equivalents/g fresh weight (f.w.). The highest AC as mg Trolox equivalents of 189.58/ g f.w. was obtained from unprocessed black olives. CAT, APX, and GR activities of unprocessed olives were higher than those obtained in all processed olives. In conclusion, TPC, AC, and antioxidant enzyme activities are strongly affected by fruit ripening and processing in table olives of ‘Gemlik’ cultivar. In addition, the best processing technique is untreated black olives in brine for antioxidant properties. Keywords: table olive, processing technique, antioxidant property, antioxidant enzyme, fatty acid composition
Introduction Olive (Olea europea L.) is the most widespread and important plant in the Mediterranean countries. Indeed, Yasemin Sahan () Food Engineering Department, Faculty of Agriculture, Uludag University, 16059, Bursa, Turkey Tel: +90 224 29 41 502; Fax: +90 224 29 41 402 E-mail:
[email protected] Asuman Cansev, and Hatice Gulen Horticulture Department, Faculty of Agriculture, Uludag University, 16059, Bursa, Turkey
98% of olive production worldwide is concentrated in the Mediterranean basin (1). The olive fruits are rarely consumed as a natural fruit due to its extreme bitterness; it undergoes various processes for direct human consumption. Table olives comprise the most popular fermented food in Turkey with an estimated annual worldwide production of 2,565,000 tons (2). Turkey contributes about 11.9% of this amount. Olea europaea L. cv. ‘Gemlik’ is the most abundant olive variety for table olives in Turkey. Table olives are rich sources of a wide range of essential micronutrients, essential fatty acids, and biologically-active phytochemicals containing antioxidant compounds and phenolics which promote health benefits (2-4). Although the phenolic compounds show variations in both quality and quantity, oleuropein, hydroxytyrosol (β(3-4-dihydroxyphenyl) ethanol), tyrosol (β(4-hydroxyphenyl) ethanol), and verbascoside comprise the main phenols in olive (5). Antioxidant enzymes are one of the most active and efficient protective mechanisms against oxidative stress. Antioxidant enzymes such as superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; 1.11.1.11), and glutathione reductase (GR; 1.6.4.2) are involved in scavenging of reactive oxygen species (6,7). Activities of these enzymes change with stress conditions in fruits as well as they display differences depending on the physiological state of the fruit (8). Although changes in some enzymes have been investigated with regard to maturation stages and processing techniques of olive fruits, to the best of our knowledge, no study has investigated antioxidant enzymes. The phenolic fraction, antioxidant properties, and fatty acid compositions of table olives are very complicated and variable, as they depend on upon cultivars, degree of maturation, agricultural practice, growing conditions, and processing methods (9). Olive fruits processing methods
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are diverse and related to the chosen maturation stage of the green or black olive. The bitterness of olives may be removed by alkaline treatment, by immersion in a liquid to dilute the bitter compound oleuropein, or by biological processes (5,10). In fact, the processing technology greatly influences the chemical composition and physical properties of the final product of olive fruits. Lye-treatment and fermentation, both are commonly used in most table olive preparations, cause chemical and physical changes affecting the lipid constituents, the phenols and antioxidant properties (5). Several studies have focused on the phenolic content of olive fruits (11-13). However, little work was conducted regarding the differences on antioxidant properties and fatty acid composition of table olive processing techniques (14,15). The purpose of this study was (i) to determine the total polyphenol content (TPC), antioxidant capacity (AC), and antioxidant enzymes (catalase, CAT; ascorbate peroxidase, APX; and glutathione reductase, GR), (ii) to identify and quantify the main phenolic compounds [oleuropein, hydroxytyrosol (β(3-4-dihydroxyphenyl) ethanol], tyrosol [β(4-hydroxyphenyl) ethanol), and verbascoside] and (iii) to determine the fatty acid concentration with regard to maturation stages and processing methods of table olives. Hence it is possible to determine the best processing method to yield the optimum nutritional value of olive.
Materials and Methods Chemical reagents, standards, and solvents All reagents used were analytical-grade purity. Oleuropein, verbascoside, tyrosol, and hydroxytyrosol were obtained from Extrasynthese (Genay, France). Folin-Ciocalteau phenol reagent DPPH and gallic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA); Trolox was purchased from Aldrich Chemicals Co. (Steinheim, Germany). All standard solutions were prepared in methanol (Merck, Darmstadt, Germany). All chemicals used for antioxidant enzyme activities were purchased from Sigma-Aldrich. High-quality water, obtained using a Milli-Q system (Millipore, Bedford, MA, USA), was used exclusively. Samples Olives cv. Gemlik were handpicked in a grove located near Iznik, Bursa, Turkey (40o22'58.60''N; 29o38'23.15''E) in 2008 at 2 different stages of ripening. Twelve olive trees older than 10 years were selected and tagged. Fruit samples were randomly obtained from the same tagged trees. About 10 kg green olives and 20 kg black olives were collected. From each tree, only healthy fruits were picked. The olive maturation index (MI) was determined on a representative sample according to the method proposed by Esti et al. (11), based on the
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evaluation of the color of the skin. The MI values were 1 and 6 for the olives picked in October and December, respectively. The olives were placed into 10 kg plastic boxes and immediately transferred at 4±1oC to the laboratory and properly processed. Black olives were divided into 3 of 5 kg batchs and processed with 3 different techniques. Total polyphenols, antioxidant capacity, phenolic characterization, and fatty acid composition of both fresh and processed olives were determined. For control, approximate 1.5 kg raw olives each MI (raw green olives, R-Green; raw black olives, R-Black), were stored at −80oC until analyzed. Processing of olive fruits Although numerous processing methods are used around the world only some of them are economically important from a global standpoint (10). The most common sources of table olives in Turkey are the Spanish style for green olives, the Californian style for oxidized black olives and naturally black olives (untreated black olives in brine and dry salt). Spanish style green olives (S-Style): Olives were treated by NaOH (1.5%) in which they remained until the lye has penetrated 2/3 of the way through the flesh. The lye was then replaced by tap water, which removes any remaining residue and the process was repeated. After washing, the olives were placed in glass bottles containing 8% brine. The brine level was adjusted, when necessary, to avoid air penetration which causes the growth of oxidative yeasts and moulds on the surface (2). Californian style black olives (C-Style): Olives were treated by 0.5% NaOH solution on 3 consecutive days, until the lye penetrated the cuticle layers on the 1st day, 12 mm of pulp on the 2nd day, and pit on the 3rd day. In the intervals between alkali treatments, water was added to cover the olives and air was bubbled for 24 h. Water addition and aeration continued until the pH reached 8; then a 5 g/L ferrous gluconate solution was added and air was bubbled for another 24 h. Finally, the olives were put into glass bottles in brine containing 8% NaCl (16). Natural black olives in brine (B-Black): Olives were washed and immersed in 8% NaCl brine. In the beginning of fermentation, glass bottles were tightly sealed in order to avoid the olives to be exposed to air. The brine stimulates the microbial activity for fermentation and reduces the bitterness of the oleuropein (2). Natural black olives in dry salt (DS-Black): Olives were washed and placed in baskets with alternating layers of dry salt equivalent to 15% of the weight of the olives. When the olives bitterness resolved, salt was removed, and the olives were transferred to glass bottles. Antioxidant enzyme activities Enzymes were extracted at 0-4oC from 0.5 g of olives, by grinding with mortar and pestle in 1.0% polyvinyl pyrrolidone (PVPP) and 2 mL of
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the following extraction solution, for CAT: 100 mM Kphosphate, pH 7.0, 0.1 mM EDTA, 0.1% Triton; for APX: 50 mM K-phosphate buffer, pH 7.8, 50 mM ascorbate; for GR: 50 mM K-phosphate, pH 7.6, 0.1 mM EDTA. Homogenates were centrifuged at 760×g for 10 min for CAT, at 10,000×g for 15 min for APX and at 15,000×g for 20 min for GR at 4oC. Supernatants were used for enzymatic assays. CAT was assayed by monitoring the consumption of H2O2 at 240 nm (17). The activity was calculated using the extinction coefficient of 39.4/mM·cm for H2O2. APX activity was determined by measuring the decrease in absorbance of the oxidized ascorbate at 290 nm, as described by Nakano and Asada (18). The concentration of oxidized ascorbate was calculated using extinction coefficient (£=2.8/mM·cm), 1 unit of APX was defined as 1 µmol/mL ascorbate oxidized/min. GR activity was determined by following the oxidation of NADPH at 340 nm (extinction coefficient 6.2/mM·cm) (19). Extraction of phenolic content and antioxidant capacity One-hundred g of olives and table olives from each type were processed. The flesh was separated from the seed and homogenized in a blender. A quantity (1.5 g) of sample was extracted 5 times with 5 mL methanol, the extracts were combined, methanol was evaporated under nitrogen and the residue was dissolved in 5 mL methanol/water (8/ 2) (15). All olive extracts filtered through a 0.45-µm filters (Hydropinilic PVDF Millex-HV; Millipore, Bedford, MA, USA) and stored in −18oC until use. All obtained extracts were used for determination of total phenolic, DPPH scavenging activity, and phenolic profiles. Total polyphenol content (TPC) The amount of total phenols in the extracts was determined with Folin-Ciocalteu reagent using a method described by Apak et al. (20) with slight modifications. Diluted plant extracts in 1 mL were reacted with 5 mL H2O and 0.5 mL of Folin-Ciocalteu reagent. The tubes were vortexed and the mixture was allowed to stand for 3 min. At the end of this period, 1 mL 7.5% Na2CO3 was added to the mixture which was then shaken intermittently for 1 h at room temperature under dark conditions. The absorbance was measured at 750 nm against blank with UV2-100 UV/Vis spectrophotometer (ATI Unicam, Cambridge, UK). TPC was calculated from a standard curve of gallic acid and results were expressed as mg of gallic acid equivalents (GAE)/g fresh weight (f.w.). Antioxidant capacity (AC) The DPPH free radical scavenging activity of each sample was determined using the UV2-100 UV/Vis spectrophotometer (ATI Unicam) according to the slightly modified method described by Boskou et al. (15). The initial absorbance of the DPPH in
methanol was measured at 515 nm and did not change throughout the period of assay. Briefly, all diluted extracts (0.1 mL) were mixed with 3.9 mL of methanolic solution of DPPH radical (6×10−5 M). The reaction then was allowed to take place in dark for 60 min, and absorbance was measured. Methanol was used as blank to determine the concentration of remaining DPPH. Standard curve was prepared using different concentrations of Trolox. Results were expressed as µmol Trolox equivalent (TE)/g f.w. Percent inhibition of the DPPH radical by the samples was calculated according to the following formula where A means absorbance at 515 nm. DPPH scavenging effect (%) =[(Acontrol −Asample)/Acontrol]×100 Chromatographic analysis of phenolic compounds About 1.5 g of each sample was subjected to extraction as previously described. The methanolic extracts were then passed through an Isolute C18 (EC) (Sorbent AB, Sollentuna, Sweden) column, previously conditioned with 6 mL of methanol and 14 mL of acidified water (pH 2 with HCl). The loaded cartridge was washed with 6 mL of nhexane to eliminate the lipid fraction and the retained phenolic compounds were then eluted with methanol. The injection volume for HPLC analysis was 20 µL (12). Chromatographic separation was carried out using an analytical HPLC unit (LC 10 A; Shimadzu, Kyoto, Japan), containing a Hypersil ODS column (250×4.6 mm, 5 µm particle size, Thermo Electron Corp., Waltham, MA, USA). The mobile phase consisted of A, methanol and B, formic acid (0.05%). The flow rate was 1 mL/min, and the injection volume was 10 µL. The following elution program was employed; 0-50 min linear to 5% A; 50-55 min linear gradient to 50% A; 55-60 min linear gradient to 100% A; 60-65 min linear gradient to 5 and 0% A at 65 min. Detection was achieved with a diode array detector, and chromatograms were recorded at 280 and 320 nm. Fatty acid composition Five-hundred g of olives were separated from the seeds. The flesh was homogenized in a blender. A quantity (25 g) of homogenate was centrifuged 3 times at 2,750×g for 10 min (NF 400; Nuve, Ankara, Turkey). After centrifugation, the oil was collected. The analytical methods for determining fatty acid composition were described in regulation standard method (21). Fatty acids were converted to fatty acid methyl esters before analysis by shaking a solution of 0.6 g of oil and 4 mL of 2,2,4-trimethylpentane with 0.2 mL of 2 N methanolic KOH. The converted fatty acid methyl esters were analyzed using a GC (GC-17 A; Shimadzu), equipped with a capillary column (DB wax; 30 m×0.25 mm; 0.25 µm, Agilent Technologies, Santa Clara, CA, USA), a split-
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splitless injector, and a flame ionization detector (FID). Flow rate of the carrier gas (nitrogen) was 1 mL/min. The temperatures of the injector, detector, and oven were held at 250, 250, and 210oC, respectively. Statistical analysis All analyses were carried out in triplicate. Results were given as mean±standard deviation (SD) of 5 independent determinations. Statistical analyses were performed using SPSS software. One-way analysis of variance (ANOVA) was used to compare the means, and differences were considered significant at p Californian style black olives > untreated black olives in dry salt > Spanish style green olives. Compared to Spanish style green olives, the black olives exhibited higher antioxidant capacity at all processed
Phenolic compounds composition Phenolic composition of fresh olive and olives by different processing techniques is given in Table 1. Several factors are known to affect the quantitative phenolic profiles of table olives. Phenolic content of table olive is closely related to cultivar, agronomic traits, and ripening level of the fruit, including the processing phases of production methods (3,15). There are some notable differences in phenolic composition among
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the green and black stages and processed table olives that are recognized to a series of chemical and enzymatic alterations of some phenolics during the maturation phase and processing techniques (5). An increase in phenolic compounds was determined with ripening of olive fruits, except for oleuropein, in fresh olives studied (Table 1). Similarly, Ziogas et al. (13) reported that the amount of phenols in olives cv. Chondrolia and cv. Amfissis increased at the black stage. In addition, Bouaziz et al. (3) reported an increase in phenolic content of olives cv. Chemlali at the last maturation period. Oleuropein is mainly responsible for the bitterness of fresh green olives. In early growth of fruit, oleuropein content of olive is most abundant, although in some varieties its levels may drop down to zero in black stage (4,5). Elenolic acid glucoside and demethyloleuropein are glucosylated derivatives of oleuropein which accumulate during olive maturation. Levels of these compounds increase simultaneously with a fall in oleuropein content (28). In this study, oleuropein was found in only fresh green maturation stage (42.06 mg/kg), while oleuropein could not be detected in samples in the black stage. The results of the present study are in good agreement with the findings of Bouaziz et al. (3) who observed a decline in oleuropein content with fruit maturity. They reported that the change in oleuropein content may be related to the increased activity of hydrolytic enzymes during maturation. Oleuropein, assessed by HPLC, was under quantification limits in processed olives. This state may be due to break down of oleuropein in process of olives. On the other hand, Zoidou et al. (29) reported that oleuropein levels were very low (45 and 60 µg/fruit, respectively) in 2 different commercial type Greek style olives (naturally black olives in brine) and very high (1.2 mg/fruit) in 1 commercial type naturally black olives in dry salt, while oleuropein could not be detected in the 6 commercial type olive (Spanish style green olives, green olives in brine, olives cv. Kalamata, naturally black olives in dry salt, and Greek style olives). These significant differences could be explained by the cultivated variety and the specific processes applied on the fruits, especially the use of brine, lye, or dry salt. Oleuropein content of the olives was found to be too low after application of different processing methods in the present study, while hydroxytyrosol content was very high, suggesting the breakdown of oleuropein to hydroxytyrosol under hydrolytic conditions during olive fermentation. Several studies reported a decrease in the oleuropein content of olive with maturation of the fruit and an increase of its derivatives (3,27). Many chemical and enzymatic reactions cause the decrease of oleuropein concentration and the increase of hydroxytyrosol concentration. Hydroxytyrosol is one of the principal degradation products of oleuropein. The 1st observed major phenolic compound was hydroxy-
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tyrosol in all samples tested. Its concentration was 132.35 mg/kg at the green maturation stage which increased to 294.62 mg/kg in the fully mature black fruits. The present results with regard to hydroxytyrosol content are in good accord with cv. Thrubes Crete (2 mg/100 g), whereas cv. Tsakistes (114 mg/100 g) was shown to have a much greater concentration of hydroxytyrosol (15). In addition, Jemai et al. (27) reported that hydroxytyrosol concentration in olives cv. Chemlali at the beginning of maturation attains a value of 300 mg/kg and reaches its maximum in January (760 mg/kg). Among processed olives, untreated black olives in brine had the highest hydroxytyrosol level (532.01 mg/kg) which was followed by Californian style black olives (521.33 mg/kg). In the present study, verbascoside was the 2nd most abundant phenolic compound after hydroxytyrosol, except R-Black and S-Style. Previous studies reported that the rise of verbascoside in fully developed fruits coincides with a decline in oleuropein in mature fruits, although not to the same extent and that a part of oleuropein degradation during maturation might contribute to a rise in verbascoside (5). In other studies, it was noted that degradation of oleuropein could produce hydroxytyrosol, which then could be converted into verbascoside (27). The verbascoside concentration increased from green maturation (98.05 mg/ kg) to black maturation stage (220.50 mg/kg) in cv. Gemlik. The highest verbascoside concentration was detected in untreated black olives in brine (410.33 mg/kg), whereas the lowest concentration was in Spanish style green olives (43.01 mg/kg) among the processed olives. Tyrosol is a phenolic alcohol, usually present in olives, although in lower amounts than hydroxytyrosol. Tyrosol concentration increased from 13.66 to 57.33 mg/kg green maturation to black maturation in the unprocessed samples. This increase may arise from ligistroside transformation or the hydrolysis of other compounds containing tyrosol (3). Unexpectedly, in the present study, tyrosol concentration was the highest (79.01 mg/kg) in the processed olives. This behavior may be due to slow hydrolysis of tyrosol glucoside in green olives. Correlation analysis was used to explore the relationships between contents of AC, TPC, and phenolic compounds in olives. Significant correlation (r= 0.86) was found between TPC and AC. In addition, the results of this study demonstrate a positive correlation between AC and oleuropein (r=0.43). However, a negative correlation was detected between AC and hydroxytyrosol (r= −0.40), tyrosol (r= −0.52) in olives. When the changes in phenolic content in olives during ripening processes were compared, considerable differences were seen between raw material and ripe olives after process (Table 1). The richest qualitative and quantitative phenolic content of different processed olives was found in untreated black olives in brine. This situation might be
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Antioxidative Properties of Different Table Olives Table 1. Phenolic composition of the unprocessed olive and processed olive from different processing techniques Phenolic compound (mg/kg) Hydroxytyrosol Tyrosol Verbascoside Oleuropein
RT(min)1) 08.62 12.67 35.22 42.32
R-Green
R-Black
S-Style
C-Style
B-Black
DS-Black
132.35±5.9e2) 194.62±9.8d 264.56±11.6c 521.33±17.2a 532.01±16.8a 317.66±11.5b 0 13.66±1.5d 0057.33±4.1bc 79.01±6.3a 066.33±4.7ab 74.01±5.3a 48.60±4.1c 0 98.05±5.4d 220.50±9.7c 43.01±2.9e 367.40±14.9b 410.33±15.4a 208.33±8.8c0 42.06±3.5 ND ND ND ND ND
1)
Retention time; R-Green, fresh green olives; R-Black, fresh black olives; S-Style, Spanish style green olives; C-Style, Californian style black olives; B-Black, untreated black olives in brine; DS-Black, untreated black olives in dry salt Each value is represented as the mean±SD (n=4); ND, not dedected
2)
Table 2. Fatty acid composition of the fresh olive and olive fruit from different processing techniques Fatty acid (%) Palmitic acid(C16:0) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) Linoleinic acid (C18:3) Arachidic acid(C20:0) Eikosanoic acid(C20:1)
R-Green1)
R-Black 2)
13.30±0.05c 01.04±0.01d 02.98±0.01ab 073.9±0.06a 07.61±0.01e 00.53±0.01b 00.40±0.01ab 00.24±0.01a
13.44±0.07b 01.43±0.02b 02.88±0.01abc 72.74±0.09a 10.24±0.03ab 00.65±0.01a 00.37±0.01c 00.22±0.02a
S-Style
C-Style
B-Black
DS-Black
13.84±0.10a 01.02±0.04d 03.13±0.01a 72.79±0.09a 07.95±0.02d 00.53±0.02b 00.41±0.04a 00.24±0.03a
11.80±0.01e 01.58±0.05a 02.52±0.03cd 71.41±0.03a 10.21±0.02b 00.68±0.09a 00.38±0.01bc 00.21±0.01a
11.72±0.12e 01.35±0.04c 02.71±0.01bc 72.99±0.13a 09.96±0.02c 00.65±0.01b 00.37±0.02c 00.24±0.02a
12.20±0.13d 01.48±0.09b 02.32±0.60d 72.51±0.52a 10.27±0.07a 00.63±0.01b 00.36±0.01c 00.22±0.02a
1)
R-Green, fresh green olives; R-Black, fresh black olives; S-Style, Spanish style green olives; C-Style, Californian style black olives; B-Black, untreated black olives in brine; DS-Black, untreated black olives in dry salt Each value is represented as the mean±SD (n=4).
2)
explained by unused chemical, low salt value, and natural fermentation process. Especially, alkaline aerobic oxidation during ripe olive processing induced significant changes in the total phenolic concentration (16). Fatty acid composition The effect of the degree of ripeness and processing techniques on the main fatty acid composition in the fruits of cv. Gemlik is shown in Table 2. It is well known that, in addition to the maturity stage and environmental factors such as rainfall and geographical origin, fatty acid composition can be affected by processing techniques (1). Oleic acid (C18:1) is the major mono unsaturated fatty acid in olive oil and its content ranged between 71.41 and 73.90% according to ripeness and processing techniques. The content of oleic acid in cv. Gemlik samples was similar to that of cv. Ayvalik (70.073.3%) (30), but was higher than in Chétoui (56.9565.15%) (4). The highest percentage was observed in unprocessed green olives (73.90%), whereas the lowest was in Californian style black olives (71.41%). These differences are related to maturation and the differences in the processes. Thus, the use of NaOH in the Californian style may decrease the oleic acid concentration in these olives. The contents of other fatty acids, including palmitoleic (C16:1), linoleic (C18:2), and linolenic (C18:3) acid were increased from green maturation to black maturation stage. On the other hand stearic (C18:0) and arachidic (C20:0) showed an opposite trend. Statistical evaluation showed
significant variation among the results (p
