Identification and characterization of lipoxygenase in fresh culinary herbs

ABSTRACT

The aim of this project was to study the biochemical characterization of lipoxygenase extracted from basil (Ocimum basilicum L.), rosemary (Rosmarinus officinalis L.), and sage (Salvia officinalis L.) leaves. The lipoxygenase extracted from these culinary herbs was frozen with liquid N₂ and ground into powder before adding ice-cold phosphate buffer (pH 7). The crude extracts from the three aromatic plants showed various specific activities and the highest specific activity and the lowest protein content were observed for the basil extracts. The optimum pH values for lipoxygenase extracted from basil and sage ranged between 5 and 6, while pH 6 was the optimum value for lipoxygenase activity for rosemary. The optimum temperature was 40°C for all of the crude extracts analyzed. Considering the biochemical characteristics evaluated for lipoxygenase from each culinary herb, suitable strategies for the inactivation of the enzyme can be proposed in order to reduce its detrimental effects on fresh aromatic herbs.

KEYWORDS:

• Basil
• Sage
• Rosemary
• Lipoxygenase
• Kinetic parameters
• pH
• temperature optima

Introduction

Herbs and spices grown in various regions of the world have been used, especially for culinary purposes, since ancient times.^[1] The main commercial form of culinary herbs is the dried product, mainly because dried vegetable materials are easy to transport and market and can be stored for long periods of time.^[2,3] Nevertheless, fresh herbs are considered to be more flavorful and superior in quality than dried herbs, even if they can only be purchased from local or regional markets. Most of the economically important culinary herbs grown in the temperate zone belong to the Lamiaceae group and include basil: Ocimum basilicum L., rosemary: Rosmarinus officinalis L., and sage: Salvia officinalis L. which are traditional and popular aromatic plants which are used both fresh and dried in European and Mediterranean cuisines.^[4] The increased use of aromatic plants has led to the demand for higher quality and since the availability of fresh herbs is limited due to their seasonality, it is essential to develop better preservation and processing methods. Drying and freezing are the most common methods for preserving aromatic plants.^[2,3] Drying treatments inhibit microbial growth and retard biochemical changes; however, they can also negatively affect product appearance and lead to loss of aroma and bioactive compounds.^[5] Blanching is currently carried out prior to freezing in order to inactivate enzyme activities.^[6] With the aim of determining the effectiveness of the blanching process, the peroxydase enzyme has been proposed as indicator of sufficient heat treatment.^[7] However, lipoxygenase (LOX), which is less thermally stable than peroxidase, has been associated with the quality deterioration of many vegetables during frozen storage due to its involvement in off-flavor and odor production, the loss of pigments such as carotenes and chlorophylls and the hydrolysis of essential fatty acids.^[8–11] LOX catalyses the oxygenation of polyunsaturated fatty acids containing 1,4-cis,cis-pentadiene system, thus resulting in the production of hydroperoxides which are precursors of aldehydes and alcohols that are responsible for off-flavors or off-aromas.^[8,9,12] However, heating culinary herbs to sufficiently high temperatures to inactivate peroxydase generally destroys undesirable enzymes, since it has not yet been proven that peroxydase activity is directly responsible for quality deterioration during the frozen storage of vegetables.^[13] It is advisable to use LOX as an indicator of adequate blanching since shorter blanching times could save a large amount of energy. Moreover, shorter blanching times may enhance quality retention and provide a better appearance. Many previous studies on plant LOXs have shown a higher heterogeneity in their biochemical characteristics, such as kinetic parameters, molecular masses, optimum pH, and temperature dependence.^[14] Since to date no studies on LOX in aromatic herb leaves have been carried out, this is the first study to determine the presence of LOX in some commonly used kitchen herbs (basil, rosemary, and sage) by extracting it from the plant leaves and quantifying the amount contained. Moreover, some of the biochemical properties and the kinetic parameters of the extracted LOX forms might be used to define the optimum blanching process for aromatic culinary herbs in order to improve their storage stability and preserve their freshness.

Materials and methods

All chemicals were obtained from Sigma Aldrich (Milan, Italy) and were of analytical grade.

Preparation of crude enzyme extract

Basil (Ocimum basilicum L.), rosemary (Rosmarinus officinalis L.), and sage (Salvia officinalis L.) leaves were purchased from a local market. Before the experiments, all samples were washed and dried prior to enzyme extraction. Three replicates (one bag each), consisting in 15–20 stems with leaves, were used for each culinary plant species, all leaves were of good quality without visible marks or damage. A 25 g of leaves was frozen in liquid N₂ and then ground into a powder with a mortar and pestle and then mixed with 50 mL of extraction buffer: ice-cold (50 mm phosphate buffer, pH 7.0, 1% (w/v) polyvinylpyrrolidone, 0.1% (v/v) Triton X-100, and 0.04% (w/v) Na₂S₂O₅. The homogenate obtained was filtered through two layers of cheesecloth and the extract was subsequently subjected to centrifugation at 10,000 rpm for 20 min at 4°C and the supernatant was collected. The enzyme solution was fractionated with solid (NH₄)₂SO₄ and the precipitate was collected by centrifugation at 11,000 rpm for 20 min, re-dissolved in 5 mM phosphate buffer (pH 6.3) and then dialyzed against the same buffer. The solution obtained was used as the crude enzyme extract. The protein determinations were carried out with the dye-binding method proposed by Bradford.^[15] A standard curve was constructed using bovine serum albumin in the 50–1200 μg/mL concentration range, in which a linear response was observed.

Spectrophotometric assay of LOX activity

LOX activity was measured spectrophotometrically according to the procedure described by Gökmen et al.^[16] All substrate solutions (1.25, 2.5, 5.0, 7.0, 10.0, and 13.0 mM) were prepared by mixing different amounts of linoleic acid with Tween 20.0, 10.0 mL distilled water and 1.0 mL 1.0 N NaOH, and then diluting them to a final volume of 200 mL with 0.067 M sodium phosphate buffer (Na₂HPO₄/NaH₂PO₄) pH 6.0. The substrate solution (29.0 mL) was transferred into a 100.0 mL flask which was placed into a temperature controlled water bath set at 37.0°C. The substrate solution was aerated with a gentle stream of air for 2 min and the reaction was started by adding 1 mL of crude enzyme extract to the flask. Subsequently aliquots of 1.0 mL from the reaction medium were transferred into glass tubes containing 4.0 mL of 0.1 N NaOH solution at time intervals of 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min. Mixing with 0.1 N NaOH either stopped the enzymatic reaction or ensured optical clarity by forming Na-salt of unreacted linoleic acid prior to the absorbance reading. Enzyme assays were carried out using a double beam ultraviolet-visible (UV-Vis) spectrophotometer (SHIMADZU UV 2450, Shimadzu Corporation Tokyo, Japan). The formation of hydroperoxides was monitored spectrophotometrically by observing the increase in absorbance at 234 nm due to the presence of a conjugated hydroperoxydiene moiety. The blank solution was prepared by mixing 1 mL of substrate solution with 4.0 mL of 0.1 N NaOH solution. One unit of LOX activity was defined as an increase in absorbance of 0.001 at 234 nm per min per milligram of protein under assay conditions. All measurements were taken at least in triplicate.

Kinetic study of LOX

Kinetic parameters, V_max (maximum reaction velocity), K_M (Michaelis–Menten constant), and V_max/K_M (catalytic efficiency) were determined according to the Michaelis–Menten equation, fitting experimental data with a non-linear regression procedure (GraphPad Prism 5.0, GraphPad software, Inc.). The goodness-of-fit of each data set to its best-fit theoretical kinetic curve was assessed as the square of the correlation coefficient (R²). The K_M is equal to the substrate concentration when the initial velocity is one-half of the maximum one (V_max), and indicates catalytic efficiency. The results reported are the average of three independent determinations ± standard error of the mean.

Temperature optimum for LOX activity and stability

The effects of temperature on LOX activity were investigated in a sodium phosphate buffer at pH 6.0 under the same assay conditions described above. The enzyme extracts were placed in screw-capped test vials and incubated in a water bath at 20, 30, 40, 60, 80, and 90°C. Each vial was removed at a specific time, immediately cooled in an ice water bath and assayed for LOX activity. Relative enzymatic activity was expressed as percentage of the activity at optimum pH (RA%).

Optimum pH for LOX activity

In order to determine the optimum pH level, the activity of crude LOX extracts were measured in McIlvaine buffer at various pH values (2.2, 4.0, 5.0, 6.0, 7.0, and 9.0) at substrate concentration (linoleic acid 10 mM) as described above. Relative enzymatic activity was expressed as a percentage of the activity at optimum pH (RA%).

Results and discussion

Kinetic study of crude LOX extracts

The LOX forms isolated from plants are generally the most effective toward linoleic acid, which is the most commonly applied substrate for LOX activity determination.^[9,17] In this study, in order to establish the enzymatic reaction velocities, linoleic acid concentrations were varied in the reaction solution and the values of both Michaelis–Menten parameters, K_M, and V_max, for each type of crude enzyme extract were calculated by means of a non-linear regression procedure where the quality of the regression was evaluated by the coefficient of determination (R²). In our study, all of the extracts obtained from the Mediterranean culinary herbs oxidized the linoleic acid. The plots of the LOX kinetic assays, followed the hyperbolic behavior of the Michaelis–Menten equation (Fig. 1) and the kinetic parameters obtained are reported in Table 1. The R² values indicated that all three non-linear regression procedures properly fit the experimental data. The V_max values measured for basil were significantly higher than those obtained for the rosemary and sage samples for which the V_max values were approximately 43 and 58% of those reported for basil. Moreover, LOX extracted from rosemary and sage have considerably lower K_M values than that extracted from basil. The higher activity of LOX observed for basil would explain the increased off-flavur development observed during the frozen storage of the blanched product.^[18–20] Although all samples in this study belong to the same Lamiaceae family, they have different leaves morphology.^[21,22] Sekya et al.^[23] studied the distribution of LOX in 28 green leaves from various plants, the authors found that the amount of LOX activity varied with the plant species. That the amount of LOX activity varies with the plant species has also been reported by Hatanaka.^[24] The green leafy odors, containing mixtures of fatty acid derivates such as C6 aldheydes, alcohols, and esters that are produced by the LOX pathway can account for the “aerial bouquets” which enable phytophagous insects to detect host plants.^[25]

Table 1. Kinetic parameters (V_max and K_M) measured, at pH 6.0 and temperature of 37°C, for the oxidation of linoleic acid by LOX extracted from the three different culinary herbs. For each sample the protein content was also reported.

Sample	Protein (mgBSA/mL)	Vmax	KM	R^Citation2
Basil	0.81 ± 0.06	62.67 ± 2.85	3.09 ± 0.42	0.99
Rosemary	2.56 ± 0.0081	26.93 ± 2.52	8.90 ± 1.60	0.99
Sage	2.85 ± 0.01	36.66 ± 3.55	9.81 ± 1.76	0.99

Figure 1 Specific activity (SA, U*mg⁻¹_protein*min⁻¹), determined at pH 6.0 and temperature of 37°C, toward linoleic acid measured for the crude LOX extracted from basil (

), rosemary (

), and sage (

).

Effects of pH and temperature on LOX activity

Optimum pH values for the activity of LOX extracted from basil, rosemary and sage were determined and can be seen in Fig. 2. All of the LOX crude extracts generally showed maximum activity at pH values between 5.0 and 6.0. More specifically, the optimum pH dependent activity of LOX from basil and sage was pH 5.0, but LOX also proved to be highly active at pH 6.0 (97.18 and 93.53%, respectively). The activity then decreased sharply toward the higher pH value the alkaline side only for the crude extract from the sage sample (less than 15%), while LOX from basil preserved more than 40% of its activity at pH 4.0 and 7.0. No activity was observed either below pH 2.0 or above pH 9.0 for both of these crude extracts. Contrastingly, the optimal pH of crude LOX from rosemary was 6.0 and the enzyme retained its catalytic activity at physiological pH values (66.99% at pH 5 and 33.99% at pH 7). The optimum pH for LOX activity generally depends on the plant species: eggplant, pH 7.0; sweet corn germ, pH 6.0–7.0; lupines, pH 6.0–8.0.^[26–28] Gökmen et al.^[16] reported that the optimum pH for LOX obtained from green pea was 6.0., while a significantly lower activity was observed at pH 7.0 with all enzymes stable and within the 5.0–6.5 pH range. In many studies, the value of the pH optimum has been commonly used for classifying LOX isoenzymes.^[9,29,30] LOX isoenzymes are classified as follows: type 1 (LOX-1), with an optimum pH in the alkaline region and converting linoleic acid preferentially; type 2 (LOX-2), characterized by the largest mass and a peak of activity at pH 6.2, produces the 9-hydroperoxide compound; while LOX-3a and LOX-3b show maximal activities at pH 6 and yield a mixture of hydro peroxides as products.^[31–33] However, due to the limited amount of available data, we were not able to apply this consideration to our results. The optimum temperature for linoleic acid oxidization was 40°C for basil, rosemary and sage extracts (Fig. 3). The LOX activities for rosemary and sage also proved to be reasonably high at 20°C (over 30%) while only a trace activity was observed for the LOX of B leaves (13.95%) at this temperature. At 60°C, approximately 80% of LOX activity was retained by the sage extract, while the crude LOX extracts of basil and rosemary lost 50 and 65% of their activity, respectively. Above 60°C, all LOX activity decreased with increasing temperature and the enzyme was completely inactivated at 90°C. Kuo et al.^[34] also reported an optimum temperature of 40°C for banana leaf (Musa acuminata) LOX, while Daglia et al.^[14] reported an optimum of 38.5 for LOX extracted from chicory (Cichorium intybus cv. Silvestre). Contrastingly, Kuribayashi et al.^[35] reported a considerably lower optimum temperature (25°C) for LOX extracted from oyster mushrooms. Williams et al.^[36] reported that only 30% of residual LOX activity in green pea homogenate remained after heating to 60°C. The results for inactivation of LOX enzymes reported in other studies may not be directly comparable due to differences in various factors: plant species, maturity, enzyme heating, and assaying techniques. However, some authors reported that the space within the active site and the orientation of the substrate are both important determinants for the positional specificity of plant LOXs and are modified by additional factors such as temperature and pH.^[12,37]

Figure 2 Effect of pH on lipoxygenase activity extracted from basil (B,

), rosemary (R,

), and sage (S,

). Local optimum (100% relative activity, RA) was found at 37°C.

Figure 3 Effect of temperature on lipoxygenase extracted from basil (B,

), rosemary R,

), and sage (S,

).Local optimum (100% relative activity, RA) was found at pH 6.0.

Conclusions

In this study, the LOX from basil (Ocimum basilicum L.), rosemary (Rosmarinus officinalis L.), and sage (Salvia officinalis L.) leaves, was characterized using linoleic acid as substrate. The crude extracts examined presented several kinetic properties, with basil expressing the highest specific activity. In order to complete the catalytic characterization of crude enzyme extracts, their pH and temperature optima were examined. The optimum pH value for LOX extracted from both basil and sage ranged between 5 and 6, while the optimum pH for rosemary was 6. All crude extracts at 40°C showed the optimum of temperature. The preliminary results regarding the mechanism of LOX inhibition can serve as a starting point for future in-depth studies, with the aim of implementing effective technological strategies for the inactivation of this enzyme. By inactivating LOX, it may be possible to prolong sensory shelf life and improve aroma retention for minimally processed herbs.