Variations of peroxidase activity among Salvia species

Journal of Food Engineering 79 (2007) 375–382 www.elsevier.com/locate/jfoodeng

Variations of peroxidase activity among Salvia species Serap Dog˘an

a,*

, Pınar Turan b, Mehmet Dog˘an b, Oktay Arslan b, Mahir Alkan

b

a b

Department of Biology, Faculty of Science and Literature, Balikesir University, 10100 Balikesir, Turkey Department of Chemistry, Faculty of Science and Literature, Balikesir University, 10100 Balikesir, Turkey Received 11 November 2005; accepted 1 February 2006 Available online 23 March 2006

Abstract Peroxidase was partially purified from Salvia species such as Salvia tomentosa Miller, Salvia virgata Jacq and Salvia viridis L. using solid (NH4)2SO4 precipitation and dialysis methods. We investigated the effect of some kinetic parameters such as buffer concentration, pH, temperature and substrate specificity on peroxidase activity. To clarify the role of peroxidase (POD) in enzymatic browning, oxidation of substrates such as 2,2 0 -azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), o-dianisidine, o-phenylenediamine, catechol and guaiacol catalyzed by partially purified peroxidase was followed spectrophotometrically. POD was observed to oxidize some phenolic compounds in the presence of H2O2, leading to enzymatic browning. From the experimental results, we found that (i) POD activities varied with the buffer concentration as depending on the substrate studied, (ii) optimum pH values were 4.5, 2.5, 5.0, 7.0 and 6.0 for S. tomentosa Miller POD; 4.0, 2.5, 6.0, 6.0 and 7.0 for S. virgata Jacq POD; and 3.5, 2.5, 6.0, 7.0 and 7.0 for S. viridis L. POD using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates, respectively, (iii) optimum temperatures were 40, 30, 50, 50 and 50 C for S. tomentosa Miller; those for S. virgata Jacq 60, 50, 60, 80 and 60 C; and those for S. viridis L. 50, 50, 60, 20 and 50 C using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates, respectively, and (iv) the substrate specificity of Salvia species was different from specie to specie and the best substrate for Salvia PODs was ABTS. Again, S. tomentosa Miller was the species with the highest POD activity, followed by S. virgata Jacq and S. viridis L. S. tomentosa Miller can be the most suitable Salvia species for dark-tea preparations because of the highest Vmax/Km values.  2006 Elsevier Ltd. All rights reserved. Keywords: Salvia species; Peroxidase; Substrate specificity; pH; Temperature

1. Introduction Post-harvest changes account for over 50% of losses of fruits and vegetables worldwide (Martinez & Whitaker, 1995). Browning of damaged tissues of fruits and vegetables during post-harvest handling and processing is one of the main causes of quality loss (Mathew & Parpia, 1971). Fruits and vegetables are very susceptible to undesirable alterations as a consequence of injuries suffered during storage, handling, and processing (Watada, Abe, & Yamuchi, 1990). Among such alterations, changes in texture, flavor, and color, which decrease the market value of the product,

*

Corresponding author. E-mail address: [email protected] (S. Dog˘an).

0260-8774/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2006.02.001

can lead to substantial economic loss. From among the organoleptic properties that determine a food’s acceptance by consumers, the appearance is the most important, and the color is the main characteristic of this property (Almeida & Nogueira, 1995). One of the most important causes of color alterations is due to either formation or degradation of pigmented compounds usually present in the produce. This phenomenon is mediated by endogenous enzymatic activities such as polyphenol oxidase (PPO) and peroxidase (POD). This process ultimately leads to the formation of dark brown polymers of a quinoidal nature (Lee, 1992). These reactions, known as enzymatic browning are not generally desirable for the food industry, but in some plants used for preparation of dark tea. Peroxidases (donor: H2O2 oxido-reductase; EC 1.11.1.7) constitute a group of glycoproteins the main function of

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which is the oxidation of different substrates at the expense of H2O2. Peroxidases are ubiquitous, iron-containing enzymes, which oxidize phenolic compounds and related substances, using activated oxygen released from H2O2 or organic peroxidase (Robinson, 1991). Peroxidases are oxido-reductive enzymes that participate in the wall-building processes such as oxidation of phenols, suberization, and lignification of host plant cells during the defense reaction against pathogenic agents (Chittoor, Leach, & White, 1999; Kolattukudy, Mohan, Bajar, & Sherf, 1992). Peroxidase catalyzes the oxidation of phenols (guaiacol, p-cresol), aromatic amines (aniline, o-dianisidine), and some other organic compounds in the presence of hydrogen peroxide (Vamos-Vigyazo, 1981). POD is one of the most heatstable and widely distributed enzymes in the plant kingdom. Active peroxidases are known to alter food flavor, color, texture and nutritional qualities of raw and processed foods (Svensson, 1977). The observed post-harvest changes are hard to relate to specific reaction mechanisms of peroxidase activity. Much of the difficulty in understanding the effects of peroxidase activity in foods is due to the very broad type of action of this enzyme, catalysing a large number of bio-conversions. Involvement of PODs in enzymatic browning has been assumed by numerous authors (Burnette, 1977; Nicolas, Richard-Forget, Goupy, Amiot, & Aubert, 1994; Williams, Lim, Chen, Pangborn, & Whitaker, 1985) and has also been reported in slow processes such as pineapple internal browning (Teisson, 1972). This involvement remained however questionable for two main reasons, i.e. the high affinity of POD for its natural substrate and the low H2O2 level in fruits. Salvia, with over 900 species from both the Old and New World, is the largest genus in the Lamiaceae (Walker, Sytsma, Treutlein, & Wink, 2004), and is found in both subtropical and temperate parts of the world (Polunin & Huxley, 1967). In Turkey, it is represented by 86 species (Davis, 1982/1988). Many species of the Lamiaceae are aromatic and are often used as herbs, herbal tea, spices, folk medicines, antioxidant, and fragrances in Turkey and World (Ravid & Putievsky, 1985). All of these properties make the Salvia very important in the food industry. Furthermore, Salvia genus are known as garden sage (or island-tea) in Turkey (Gu¨ndog˘maz, Dog˘an, & Arslan, 2003). In addition, Salvia species are grown in parks and gardens as ornamental plants (Nakipoglu, 1993). There are many works related to Salvia genus such as antioxidant activities (Ollanketo, Peltoketo, Hartonen, Hiltunen, & Riekkola, 2002; Stashenko, Puertas, & Martinez, 2002) antifungal activities (Sokovic, Tzakou, Pitarokili, & Couladis, 2002), essential oil composition (Mirza & Sefidkon, 1999; Sefidkon & Khajavi, 1999) and polyphenol oxidase (Gu¨ndog˘maz et al., 2003). However, there are many works related to POD from different plants, fruits and vegetables such as black tea (Subramanian, Venkatesh, Ganguli, & Sinkar, 1999), Thymus vulgaris L. (Zheng & Shetty, 2000), Cynara scolymus L. (Lopez-Molina et al., 2003), banana (Cano, Marin, & Fuster, 1990), pear (Richard-For-

get & Gauillard, 1997), marula (Mdluli, 2005), blackberry (Gonzalez, de Ancos, & Cano, 2000). Salvia species are of growing commercial importance. Malencic et al. (2002) and Malencic et al. (2000) investigated the antioxidant properties of two wild growing sage species from the Serbian flora, Salvia nemorosa L. and Salvia glutinosa L.; and the wild growing sage species, Salvia reflexa Hornem, and (i) found that investigated species exhibited high superoxide dismutase and peroxidase activities as well as the content of total flavonoids, and (ii) the dominant naturally occurring compound in both species was rosmarinic acid. Furthermore, Rounkova and Gaspar (1976) studied peroxidase and isoperoxidase pattern of roots and apices of light-grown Salvia and matthiola treated by GA and CCC. We previously investigated the effects of pH, temperature and inhibitors on polyphenol oxidase activity obtained from different organs such as stem, leaf and flowers of three different Salvia species such as Salvia tomentosa Miller, Salvia virgata Jacq and Salvia viridis L. using catechol as a substrate (Gu¨ndog˘maz et al., 2003). But, as seen in above, a few published data has been found in the literature, which discusses the kinetic properties of POD obtained from these Salvia species. Therefore, this study was made to determine the pH, buffer concentration and temperature dependence, and substrate specificity of partially purified peroxidase contributing to the quality loss in Salvia species. 2. Material and methods 2.1. Materials Salvia species such as S. tomentosa Miller, S. virgata Jacq and S. viridis L. used in this study were freshly collected in spring from field near Balikesir in Turkey, and kept for two days in the refrigerator at 4 C before extracting POD. H2O2, catechol and (NH4)2SO4 were obtained from Merck and guaiacol, o-phenylenediamine, 2,2 0 azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and o-dianisidine from Sigma Chemical Co. All other chemicals used in this study were of analytical grade and were used without further purification. 2.2. Preparation of Salvia extracts The extraction and assay procedures for peroxidase were adapted from the methods described by Sakharov, Blanco, and Sakharova (2002). Varying time of extraction, composition and pH of extracting buffer the extraction of peroxidase was carried out with the proportion of sample of Salvia plants to solution being 1:10 (w/v) at ambient temperature with constant agitation. The sample obtained (10 g) was homogenized in a blender with 100 ml of buffer whose composition was as a follows: 0.1 M phosphate buffer; 28 mM ascorbic acid; 5 mM EDTA; 5% NaCl; pH 7.0. The homogenate was incubated for 1 h at ambient temperature and then centrifuged at 20,000 · g for 15 min at

S. Dog˘an et al. / Journal of Food Engineering 79 (2007) 375–382

20 C. The pellet was discarded. 12 g of (NH4)2SO4 was added in 20 ml of the supernatant, and held at 4 C for 1 h. The precipitate formed was collected by centrifugation (30,000 · g; 20 min; 4 C), dissolved in 2 ml of 10 mM phosphate buffer (pH 6.0) and dialyzed against 10 mM phosphate buffer (pH 7.0) for two days with three time changes of buffer. The dialyzed sample was used as the POD enzyme in the following experiments.

377

tested by heating the standard reaction solutions (buffer and substrates) to the appropriate temperatures before introduction of the enzyme. The desired temperatures were provided by using a temperature controller attached to the

14000 Guaiacol ABTS o-dianisidine o-phenylenediamine

12000

2.4. pH stability profile The partially purified enzymes were originally isolated in 100 mM phosphate buffer (pH 7.0). The pH ranged from 2.5 to 8.0 obtained by a number of different buffering systems all 0.1 M: sodium acetate (pH 2.5–5.5), potassium phosphate (pH 5.8–7.0) and Tris–HCl (pH 7.0–8.0). All assays were made with hydrogen peroxide and various reducing substrate concentrations. All assay was made at least in triplicate and the average of triplicate readings was taken.

Catechol

8000 6000

2000 0 2.5

3.5

4.5

(a)

5.5 pH

6.5

7.5

14000 Guaiacol ABTS o-dianisidine o-phenylenediamine Catechol

Activity (EU min-1)

12000 10000 8000 6000 4000 2000 0 2.5

3.5

4.5

(b)

5.5 pH

6.5

7.5

7000 Guaiacol ABTS o-dianisidine o-phenylenediamine Catechol

6000 5000 4000 3000 2000

2.5. Effect of temperature For determining the optimum temperature values of the enzyme, POD activity was measured at different temperatures in the range from 10 to 60 C for S. tomentosa Miller, 10 to 90 C for S. virgata Jacq and 10 to 70 C for S. viridis L. under optimal pH and buffer concentration for each substrate. The effect of temperature on the activity of POD was

10000

4000

Activity (EU min-1)

The POD activity in the sample was measured using various substrates such as ABTS, o-dianisidine, o-phenylenediamine, catechol and guaiacol. All analyses were conducted within the linear range and made at least in triplicate. When studying substrate specificity of Salvia PODs, the activity was measured under optimal conditions determined for each substrate. Temperature was controlled using a circulating water bath with a heater/cooler. Initial rates of free radical formation for substrates were monitored at maximum wavelength of each substrate. The changes in absorbance were read for 3 min using a double beam UV–visible spectrophotometer (a Carry j1Ej g UV– visible, Varian, Australia). The following wavelengths were used in the assays at 414 nm for ABTS, 420 nm for o-dianisidine, 445 nm for o-phenylenediamine, 295 nm for catechol and 470 nm for guaiacol which are substrates of POD. At these wavelengths, maximum absorbance was obtained due to the disappearance of the substrate or due to product appearance. One unit of the activity was defined as the amount of enzyme that caused an absorbance change of 0.001 per min under standard conditions (Dogan & Dogan, 2004). Kinetic parameters were determined using the Lineweaver–Burk double reciprocal plot. From the plots of 1/V versus 1/[S], the kinetic parameters such as Km and Vmax were determined for all substrates.

Activity (EU min-1)

2.3. Peroxidase activity

1000 0 2.5

(c)

3.5

4.5

5.5 pH

6.5

7.5

Fig. 1. The changing of POD activity with pH: (a) Salvia tomentosa Miller, (b) Salvia virgata Jacq and (c) Salvia viridis L.

S. Dog˘an et al. / Journal of Food Engineering 79 (2007) 375–382

378

cell-holder of the spectrophotometer. Once temperature equilibrium was reached, enzyme was added and the reaction was followed spectrophotometrically at constant temperature at given time intervals. As mentioned, each assay was repeated in triplicate using the same stock of enzyme extract (Dog˘an, Turan, Ertu¨rk, & Arslan, 2005). 3. Results and discussion It is well known that uncontrolled oxidative reactions mediated by PPO and POD are responsible for quality deterioration in several food products derived from plant sources (Whitaker, 1994). We previously investigated the effects of pH, temperature and inhibitors on PPO activity obtained from different organs such as stem, leaf and flowers of three different Salvia species such as S. tomentosa Miller, S. virgata Jacq and S. viridis L. using catechol as a substrate. The major biocatalytic properties, which influence the activity of Salvia PODs were the buffer concentration, pH, temperature and substrate specificity. Firstly, in this study, the effect of pH is discussed, followed by buffer concentration, temperature and substrate specificity on POD activity. 3.1. Effect of pH The effect of acid H+ ions or basic OH ions on the activity of an enzyme is probably caused by a change in stereo configuration at or in the neighbourhood of the active sites (Fersht, 1984; Whitaker, 1994). As in almost all protonation reactions, these reactions will occur very fast. The different configurations are instantaneously in equilibrium. The protonated and hydroxylated enzymes are assumed to be completely inactive or at least less active (Seyhan, Tijskens, & Evranuz, 2002). Optimum pH values for Salvia peroxidases were determined in the pH ranges of 2.5–8.0 using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates. Fig. 1 has shown the changing of POD activity with pH for S. tomentosa Miller, S. virgata Jacq and S. viridis L. peroxidases using ABTS, ophenylenediamine, o-dianisidine, catechol and guaiacol as substrates. As seen in Fig. 1, optimum pH values were 4.5, 2.5, 5.0, 7.0 and 6.0 for S. tomentosa Miller POD; 4.0, 2.5, 6.0, 6.0 and 7.0 for S. virgata Jacq POD; and 3.5, 2.5, 6.0, 7.0 and 7.0 for S. viridis L. POD using ABTS,

o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates, respectively. From Fig. 1, for ABTS, o-dianisidine, catechol and guaiacol substrates, there is a sharp pH optimum. POD activity has increased with increasing pH of the buffer solution and has declined at pH values above the optimal level. On the other hand, optimum pH value for three Salvia PODs was the same as and 2.5 using o-phenylenediamine as a substrate. From the experimental results, it can be said that Salvia PODs with o-phenylenediamine substrate have optimum pH value at a broader pH range. The results above have shown that optimum pH values are substrate- and specie-dependent. Table 1 has shown optimum pH values of peroxidases obtained from various sources using different substrates. As seen in Table 1, it can be said that optimum pH values obtained for ABTS substrate are approximately the same as. But the optimum pH values obtained for Salvia PODs in this study are different from those of other sources. We found that Salvia PODs with o-phenylenediamine substrate were more active at lower pH values. Optimum pH values of POD enzyme obtained from various sources in the literature (Table 1) and in this study have changed in the range of 4.5–6.0 for o-dianisidine, 4.5–7.0 for catechol and 3.5–7.0 for guaiacol substrates, respectively. 3.2. Buffer concentration The enzyme activity depends upon buffer concentration (Sakharov, 2001). As seen in Fig. 2, the experimental data obtained have showed that the POD activity of Salvia species varied with the buffer concentration. Maximum activities for S. tomentosa Miller POD were observed at 20, 80, 10, 20 and 20 mM; those for S. virgata Jacq POD 10, 80, 100, 20 and 20 mM; and those for S. viridis L. POD 10, 80, 60, 40 and 40 mM buffer concentrations using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates, respectively. From the results, it can be said that POD activities of Salvia species varied with the buffer concentration as depending on the substrate studied. These results suggest that the studied substrates having different chemical structures react with different regions of the active site of Salvia PODs. Therefore, in the latter studies, enzyme activity was measured at maximum buffer concentrations for both Salvia species and substrates.

Table 1 The comparison of optimum pH values of PODs obtained from various sources POD sources

Corn steep water Sweet potato tubers Royal palm tree African oil palm tree Salvia tomentosa Miller Salvia virgata Jacq Salvia viridis L.

Optimum pHs ABTS

o-Phenylenediamine

o-Dianisidine

Catechol

Guaiacol

References

3.4 4.5 3.0 3.0 4.5 4.0 3.5

5.2 5.0 5.0 5.5 2.5 2.5 2.5

5.2 4.5 5.2 5.5 5.0 6.0 6.0

– 5.5 5.0 4.5 7.0 6.0 7.0

3.7–5.2 3.5 and 5.5 5.5 5.5 6.0 7.0 7.0

Gray and Montgomery (2003) Leon et al. (2002) Sakharov (2001) Sakharov et al. (2002) In this study In this study In this study

S. Dog˘an et al. / Journal of Food Engineering 79 (2007) 375–382

3.3. Effect of temperature Enzyme activity is generally measured as the amount of some specific substrate converted per unit time. This activ-

379

ity, as observed by activity measurements, is a combination of a true concentration of the enzyme, multiplied by its specific reaction rate constant. The specific rate constant increases with increasing temperature according to the Arrhenius law. At higher temperatures the enzyme starts

8000 18000

7000

Guaiacol

Activity (EU min-1)

6000 5000 4000 3000 2000

16000

o-phenylenediamine o-dianisidine ABTS

12000 10000 8000 6000 4000

1000 0 0.01

Catechol

14000

Activity (EU min-1)

Guaiacol ABTS o-phenylenediamine o-dianisidine Catechol

2000

0.03

0.05

0.07

0.09

0

Concentration (mol L-1)

(a)

10

20

30

(a)

40

50

60

t (°C)

16000 35000 Guaiacol ABTS o-phenylenediamine o-dianisidine Catechol

Activity (EU min-1)

12000 10000 8000 6000 4000

Guaiacol o-phenylenediamine

2000 0 0.01

0.03

0.05 0.07 Concentration (mol L-1)

20000 15000 10000

10

30

50

(b)

70

90

t (°C) 7000

5000 4000 3000 2000

Guaiacol Catechol o-phenylenediamine

6000

Activity (EU min-1)

Guaiacol ABTS o-phenylenediamine o-dianisidine Catechol

6000

Activity (EU min-1)

ABTS

0

0.09

7000

1000

(c)

o-dianisidine

25000

5000

(b)

0 0.01

Catechol

30000 Activity (EU min-1)

14000

o-dianisidine

5000

ABTS

4000 3000 2000 1000 0

0.03

0.05

0.07

10

0.09

Concentration (mol L-1)

Fig. 2. The changing of POD activity with buffer concentration: (a) Salvia tomentosa Miller, (b) Salvia virgata Jacq and (c) Salvia viridis L.

(c)

20

30

40

50

60

70

t (°C)

Fig. 3. The changing of POD activity with temperature: (a) Salvia tomentosa Miller, (b) Salvia virgata Jacq and (c) Salvia viridis L.

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Table 2 Experimental conditions for the determination of substrate specificity of Salvia species and kinetic data Salvia species

Substrates

k (nm)

[H2O2] (mM)

pH

[Buffer] (mM)

t (C)

Km (mM)

Vmax (EU min1)

Vmax/Km (EU min1 mM1)

Salvia tomentosa Miller

ABTS o-Phenylenediamine o-Dianisidine Catechol Guaiacol

414 445 420 295 470

8 16 4 8 16

4.5 2.5 5.0 7.0 6.0

20 80 10 40 20

40 30 50 50 50

0.027 3.333 0.081 2.220 2.030

27,900 610 5060 3260 22,400

1,040,000 183 62,700 1460 11,000

Salvia virgata Jacq

ABTS o-Phenylenediamine o-Dianisidine Catechol Guaiacol

414 445 420 295 470

16 16 4 8 16

4.0 2.5 6.0 6.0 7.0

10 80 80 20 20

60 50 60 80 60

0.161 0.169 1.310 1.650 3.190

38,800 526 22,600 2750 7750

241,000 3110 17,200 1670 2430

Salvia viridis L.

ABTS o-Phenylenediamine o-Dianisidine Catechol Guaiacol

414 445 420 295 470

8 16 4 8 16

3.5 2.5 6.0 7.0 7.0

10 80 60 40 40

50 50 60 20 50

0.189 0.331 0.460 8.180 4.620

10,800 481 11,700 2980 6180

57,100 1450 25,400 364 1340

to denature, thereby effectively decreasing the amount or concentration of the active enzyme configuration. Eventually, the enzyme loses its activity entirely (Seyhan et al., 2002). The effect of temperatures on POD activities of different Salvia species such as S. tomentosa Miller, S. virgata Jacq and S. viridis L. was assayed using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates, and the results are shown in Fig. 3. It was found that optimum temperatures for S. tomentosa Miller POD were 40, 30, 50, 50 and 50 C; those for S. virgata Jacq POD 60, 50, 60, 80 and 60 C; and those for S. viridis L. POD 50, 50, 60, 20 and 50 C using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates, respectively. After optimum temperatures, POD activity decreased with increasing temperature and showed very little activity at higher temperatures. Similar result was also found for soybean seed hull peroxidase. The highest enzymatic activity for soybean seed hull peroxidase was observed at the temperatures of 80 C and is about three times higher than the activity obtained at room temperature. Furthermore, more than 85% of the highest enzyme activity was retained between 75 and 95 C, and >80% between 68 and 85 C. The rate of enzyme reaction is favored at higher temperatures (Geng, Rao, Bassi, Gijzen, & Krishnamoorthy, 2001). These observations indicate that the Salvia PODs are quite resistant to heat. 3.4. Substrate specificity Phenolic compounds make up 25–35% of the dry matter content of plants. Flavanol compounds were 80% of the phenols while the remainder was proanthocyanidins, phenolic acids, flavonols and flavones. During tea fermentation the flavanols are oxidized enzymatically to compounds which are responsible for the color and flavor of tea. Flavor intensity of tea is correlated with the total content of the phenolic compounds (Gu¨ndog˘maz et al.,

2003). The kinetic characterization of POD from S. tomentosa Miller, S. virgata Jacq and S. viridis L. using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates led to the determination of the Vmax and Km values for all substrates. The effect of substrate concentration on the rate of POD-catalyzed reaction was tested. Increasing the concentration led to a greater enzyme activity (data not shown). The enzyme kinetic was measured for a period of 3 min. The slope of the straight line was used to compute the enzyme reaction rate throughout the experiment. The Km and Vmax values for POD were determined from Lineweaver–Burk plots for S. tomentosa Miller, S. virgata Jacq and S. viridis L. using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates. From the Lineweaver–Burk double reciprocal plots, the values of Km and Vmax were obtained (Table 2). As seen in Table 2, we found that (i) Km values for S. tomentosa Miller POD were 0.027, 3.333, 0.081, 2.220 and 2.030 mM; those for S. virgata Jacq POD 0.161, 0.169, 1.310, 1.650 and 3.190 mM; and those for S. viridis L. POD 0.189, 0.331, 0.460, 8.180 and 4.620 mM using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates, respectively; and (ii) Vmax values for S. tomentosa Miller POD were 27,900, 610, 5060, 3260 and 22,400 EU min1; those for S. virgata Jacq POD 38,800, 526, 22,600, 2750 and 7750 EU min1; and those for S. viridis L. POD 10,800, 481, 11,700, 2980 and 6180 EU min1 using ABTS, o-phenylenediamine, o-dianisidine, catechol and guaiacol as substrates, respectively. In Table 2, Vmax/Km ratio is called ‘catalytic power’ and is a good parameter for finding the most effective substrate (Dog˘an, Arslan, & Dogan, 2002). As seen from Km and Vmax/Km values in Table 2, S. tomentosa Miller,S. virgata Jacq and S. viridis L. PODs have a relatively high affinity for ABTS, which was the best substrate of those tested because both the lowest Km and the highest Vmax/Km ratio were obtained with ABTS. As a result, it can be said that S. tomentosa Miller was the most suitable Salvia species

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for dark-tea preparations because of the highest Vmax/Km. On the contrary, for green-tea, then S. viridis L. was the most appropriate Salvia species. Similar result was found for African oil palm tree POD (Sakharov et al., 2002). Again, it was found that the substrate specificity of Salvia species also was different from specie to specie. The best substrate for S. tomentosa Miller POD was ABTS, followed by o-dianisidine, guaiacol, catechol and o-phenylenediamine; that for S. virgata Jacq POD ABTS, followed by o-dianisidine, o-phenylenediamine, guaiacol and catechol; and that for S. viridis L. POD ABTS followed by o-dianisidine, o-phenylenediamine, guaiacol and catechol, respectively. Again, Km and Vmax values for POD activity have varied with the type of substrate and buffer. 4. Conclusions In this study, we found that (i) peroxidase activity varied with buffer concentration, and was quite resistant to heat, (ii) optimum pH and temperature depended on enzyme source and substrate used, (iii) the best substrate for Salvia species was ABTS. Moreover, we previously found that the type with the highest polyphenol oxidase activity was S. tomentosa Miller (Gu¨ndog˘maz et al., 2003). Similarly, in this study, we determined that S. tomentosa Miller had the highest POD activity. Therefore, it can be said that S. tomentosa Miller has more phenolic compound content than other species. References Almeida, M. E. M., & Nogueira, J. N. (1995). The control of polyphenol oxidase activity in fruits and vegetables. A study of the interactions between the chemical compounds used and heat treatment. Plant Foods for Humon Nutrition, 47, 245–256. Burnette, F. (1977). Peroxidase and its relationships to food flavor and quality: A review. Journal of Food Science, 42, 1–5. Cano, M. P., Marin, M. A., & Fuster, C. (1990). Effects of some thermal treatments on polyphenoloxidase and peroxidase activities of banana (Musa cavendishii, cv. Enana). Journal of the Science of Food and Agriculture, 51, 223–231. Chittoor, J. M., Leach, J. E., & White, F. F. (1999). Induction of peroxidase during defense against pathogens. In S. K. Datta & S. Muthukrishnan (Eds.), Pathogenesis: Related proteins in plants (pp. 291). Boca Raton, FL: CRC Press. Davis, P. H. (1982/1988). Flora of Turkey and the East Aegean Islands (Vols. 7, 10). Edinburgh University Press. Dog˘an, M., Arslan, O., & Dog˘an, S. (2002). Substrate specificity, heat inactivation and inhibition of polyphenol oxidase from different aubergine cultivars. International Journal of Food Science and Technology, 37, 415–423. Dogan, S., & Dogan, M. (2004). Determination of kinetic properties of polyphenol oxidase from Thymus (Thymus longicaulis subsp. chaubardii var. chaubardii). Food Chemistry, 88, 69–77. Dog˘an, S., Turan, Y., Ertu¨rk, H., & Arslan, O. (2005). Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.). Journal of Agricultural and Food Chemistry, 53, 776–785. Fersht, A. W. H. (1984). Enzyme structure and mechanism. New York: Freeman. Geng, Z., Rao, K. J., Bassi, A. S., Gijzen, M., & Krishnamoorthy, N. (2001). Investigation of biocatalytic properties of soybean seed hull peroxidase. Catalysis Today, 64, 233–238.

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