Optimization of Enzymatic Assisted Extraction of Bioactive Compounds from Olea Europaea Leaves

1. Introduction

One of the most widely produced crops is the cultivation of olives, with 65%, 16%, and 15% of the world’s output occurring in Europe, Asia, and Africa, respectively [1]. Moreover, Proietti et al., [2] estimated that an olive tree produces approximately 11,777 leaves, most of which are thrown away as waste. However, the leaves can be used as a profitable raw material for continuous large-scale industrial production for a considerable amount of time [3]. Furthermore, since their import is probably going to include waste management services for olive oil producers, the business that processes stand a very high opportunity of increasing its profit margin.

Along with macronutrients and micronutrients, a typical diet also includes specific chemical compounds, mostly found in fruits and vegetables, which have been shown in several studies to have a significant biological impact. These substances are referred to as bioactive substances and have a variety of functions for human health. Bioactive chemicals are regarded as secondary metabolites of plants and are crucial for pest and disease resistance as well as species preservation. Each year’s action and promotion of health advantages pique their attention [4]. The olive (Olea Europe L.), one of the most significant fruit plants in the Mediterranean region, is prized for its nutritional and health benefits all over the world. Olive oil is made from the fruits of the olive by mechanical methods. The phenolic compounds’ antioxidant activity is responsible for the health-promoting properties, and their pharmacological actions have been documented in the literature [5]. Because of their potent antioxidant activity, by-products from the olive oil industry provide a prospective source of phenolic compounds [4]. An increasing number of people have been interested in giving these goods more value in the recent past, for both nutritional and environmental reasons. Numerous investigations have been conducted in this context to comprehend the function of the numerous natural chemicals found in these products. Based only on the quantity of phenol subunits present, phenols can be divided into simple phenols and polyphenols. Therefore, simple phenols, phenolic acids, coumarins, flavonoids, and stilbenes, as well as hydrolyzed and condensed tannins, lignans, and lignins, are all included in the phrase "plant phenolics." In vitro biological actions of phenols include the scavenging of free radicals, the regulation of enzyme activity, and the prevention of cell proliferation. According to Cherng et al., [6], they have anti-inflammatory, anti-ulcer, anti-allergic, and antibiotic properties. They also show antibacterial action.

The biological cycle of the olive affects significant variations in phenolic chemicals, both quantitatively and qualitatively [7]. Phenolic compounds are present in both fruits and leaves in substantial amounts, and they are transported from the fruit to the olive oil during processing. These compounds determine the taste and antioxidant activity of the olive oil, which is why they are crucial to its quality. The class of molecules known as phenolics includes over 8000 naturally occurring substances. These compounds always have an aromatic ring with at least one hydroxyl substituent, or a phenol [4]. According to Abaza et al., [8], olive leaves contain flavonoid and phenolic compounds that exhibit a range of biological activities. These chemicals may also be accountable for the pharmacological effects of olive leaves, or at the very least, for the synergistic increase of these effects.

Oleuropein and its derivatives are the principal constituents of olive leaves. hydroxytyrosol, additional polyphenols, and triterpenes, which comprise flavonoids (rutin and diosmin) and oleanolic acid. These elements give the tree, as well as its fruits and foliage, resilience against disease and insect damage [4]. Oleuropein, which was initially discovered in 1908, is thought to be the cause of many of the medicinal benefits of extracts from olive leaves and causes for the bitter flavour of olive oil and olives as well [9]. Strong antioxidant and free radical scavenging properties, as well as antimicrobial, hypoglycemic, anti-toxoplasmosis, antiviral, antifungal, anti-aggregation, and platelet hypolipidemic properties have all been reported for oleuropein [10].

Figure 1. Shows the degradation process [11].

Figure 1. Shows the degradation process [11].

The amount found in the leaf is significantly higher than in other parts of the tree, while being present in the olive fruit and oil [12]. The chemical composition of olive leaves is influenced by several elements, including olive variety, temperature, extraction methods, wood ratio, tree age, genetics, and cultivation methods [13]. Since the concentration of phenolic components varies among plant materials, it is essential to determine the best extraction conditions and characterize the extract’s antioxidant activity and composition. Tomato pastes, sauces, and other functional foods such as spreads can all include leaf extracts. Moreover, according to Baiano et al. [14] and Čukelj et al., [15], they can be added to breads or bakeries/snacks that have a good market potential and the ability to contribute key nutrients to the consumer’s daily diet. Furthermore, leaf extracts can be marketed as special medicinal supplements or added to refined oils to replace some of the bioactive chemicals lost during processing. Another potential use of the olive leaves application is the usage on the food packaging materials [16]. Thus, it is advised to use whole leaf extracts rather than only their individual components, such as oleuropein, when making nutraceuticals, functional foods, and food additives [17]. Evaluating various (new) technologies that support polyphenolic stability during extractions is crucial. At the same time should be more effective, efficient, and environmentally friendly [18].

The aims of the study were: (i) to develop a green enzymatic extraction process for the recovery of polyphenols from the olive leaves, (ii) to evaluate the synergistic effects of three (3) commercial enzymes in the enzymatic extraction process recovery of polyphenols from the olive leaves and (iii) by using the optimal enzyme mixture found in step (ii) the optimal extraction conditions for the recovery of polyphenols from the olive leaves were evaluated.

To the best of our knowledge step (ii) and step (iii) of the current study are for first time reported and are the innovative points of this study.

2. Materials and Methods

Plant Material

Olive (Olea Europaea) leaves, Koroneiki variety harvested year 2023, were obtained from a producer in the region of Agrinio, Greece. The leaves were dried at 50 °C for 5 h [32]. Dried olive leaves were stored in a metal container at room temperature until used.

Chemicals and Reagents

For analytical purposes, the following reagents were used: 2,2-Diphenyl-1-picrylhydrazyl (DPPH·) (Sigma-Aldrich, Germany). Absolute methanol (CH₃OH), sodium carbonate (Na₂CO₃), and acetate buffer (CH₃COONa × 3H₂O) (Merck Darmstadt, Germany). Folin Ciocalteu reagent from Sigma-Aldrich. Gallic acid (3,4,5-trihydrobenzoic acid) 99% isolated from Rhus chinensis Mill. (JNK Tech. Co., Republic of Korea). All the other reagents and solvents used were of analytical grade.

Enzyme Preparations

The following commercial enzyme preparations were used: pectinolytic preparation Pectinex XXL, cellulolytic preparation Celluclast and Viscozyme L. (Cellulase, Hemicellulase, Xylanase), (all from Novozymes A/S, Bagsvaerd, Denmark).

Enzyme-Assisted Extraction

Finely grounded (particle size < 700 μm) olive leaves were mixed with water (10:1, v/w), acidified (pH 4.0) with HCl, and left for 1 h for rehydration at 25 °C. After pH adjustment (pH 4.0), the suspension (100.0 g) was placed in a 50 °C water bath (Memmert Schutzart DIN 40050 – IP 20, Germany) for 20 min before enzymes were added. After incubation at 50 °C, the sample was placed in a boiling water bath for 10 min to inactivate the enzymes, then immediately cooled and finally filtered through a paper filter under vacuum and weighed to determine the extract yield [20].

Phytochemical Analyses

All measurements were performed with a SHIMADJU UV/VIS spectrophotometer (UV-1900, Kyoto, Japan) using 1 cm pathlength cuvettes. The content of total polyphenols (TPP) was determined using the method of Karabagias et al [19] with the following modifications: in a 5 mL volumetric flask, 0.20 mL of the ethanolic extracts of grape origin followed by 2.50 mL of distilled water and 0.25 mL Folin-Ciocalteu reagent were added. After 3 min, 0.50 mL of saturated sodium carbonate (Na₂CO₃, 30% w/v) was also added into the mixture. Finally, the obtained solution was brought to 5 mL with distilled water. This solution was left for 2 h in the dark at room temperature and the absorbance was measured at λ = 760 nm. The results were presented as equivalents of gallic acid (GAE). Each sample was analyzed in triplicate (n = 3).

The total antioxidant capacity was determined using the DPPH (free radical scavenging activity). DPPH assay was based on the method of Karabagias et al [19] with small modifications as follows: 1.9 mL of absolute ethanol solution of DPPH·(1.34 × 10−4 mol/L) and 1 mL of acetate buffer 100 mmol/L (100 Mm) (pH = 7.10 ± 0.01) were placed in a cuvette, and the absorbance of the DPPH· was measured at t = 0 (A₀). Subsequently, 0.1 mL of each extract studied, was added to the above medium and the absorbance was measured at regular time periods, until the absorbance value reached a plateau (steady state, At). The reaction in all cases was completed in 15 min and the absorbance was measured at λmax = 517 nm. Each sample was measured in triplicate (n = 3). For this antioxidant test ethanol and acetate buffer (2:1, v/v) were used as the blank sample.

Experimental Design

According to a recent publication [20], a simplex-centroid design for a mixture with three components was applied (Figure 2). Enzyme preparations (single or mix) were used as 1.2% (v/v) solution and the incubation time was 120 min.

This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

An optimal central composite design (OCCD) of type 2n + 2n + n0 was applied. The influence of the independent variables was determined by means of the response surface methodology [20]. Table 1 shows the levels of the two independent variables – enzyme dose (0.02–0.18%) and reaction time (30–210 min). The enzyme used was a 1:1 mixture of the Viscozyme L. and pectinolytic (Pectinex XXL) preparations. The experimental data were fitted to a second-degree regression equation:

(1)

where:

y – the dependent variable (response),

b₀ – the model intercept,

b_i, b_ii, b_ij – the linear, quadratic, and interaction regression coefficients, respectively,

xi, xj – the independent variables,

n – equal to the number of the tested factors (n = 2 in this study).

Table 1. Independent variable values and corresponding levels.

Factor	Minima	Centre point	Maxima	Axial point. a
Enzyme dose (%E/Sa) – Х1	0.02	0.1	0.18	-a = -1 +a = +1
Time (min.) – X2	30	120	210	-a + = -1 +a = +1

^a mL enzyme preparation per 100g substrate.

Table 1. Independent variable values and corresponding levels.

Factor	Minima	Centre point	Maxima	Axial point. a
Enzyme dose (%E/Sa) – Х1	0.02	0.1	0.18	-a = -1 +a = +1
Time (min.) – X2	30	120	210	-a + = -1 +a = +1

^a mL enzyme preparation per 100g substrate.

Statistical Analysis

The results reported in the present study are the mean values of at least three analytical determinations and the coefficients of variation expressed as the percentage ratios between the standard deviations and the mean values were found to be < 5% in all cases. The means were compared using one-way ANOVA and Tukey’s test at a 95% confidence level.

3. Results

Selection of the Mixture of Enzyme Preparations

Table 2 shows the results after the extractions, evaluating the synergy of the enzymes in order to find the most effective enzyme combination for polyphenol extraction from the olive leaves.

Significant increases in the recovery rates of total polyphenols and antioxidants were observed due to the enzymatic treatments (see Table 2, Figure 3a and 3b). The binary combination containing Viscozyme and pectinolytic preparations (Χ2:Χ3 = 1:1) resulted in the highest yield of total polyphenols, reaching a 5.9% higher value as compared to the control sample (without enzymatic treatment).

This value (468.19 mg GAE/L) is higher than microwave-assisted enzymatic extraction (34.53 mg GAE/g) [29] but lower than that (54.92 mg GAE/g) reported for 80% ethanolic extract [24]. However, possible differences in the polyphenolic content of the raw materials should be taken into account.

Interestingly, similar effects concerning the total polyphenols and antioxidants were observed for the Viscozyme and cellulolytic preparations mixture (Χ1:Χ3 = 1:1), and for all enzyme mixtures (X1:X2:X3 = 1:1:1) which is probably due to the secondary xylanase activity of commercial pectinase [21].

Optimization of the Process Parameters

Table 3 shows the total polyphenols and the antioxidant capacity of olive leaves after extraction with the most effective combination of enzymes as shown in Table 2, modifying the dose and the incubation time to evaluate the most optimum extraction conditions using enzymes.

Significant variations in the yields of total polyphenols and antioxidants were observed in response to the different enzymatic treatments (see Table 3).

In contrast with other researchers [20,22] an increase in the enzyme dose does not affect the recovery rate of total polyphenols, against that in some cases the increase of enzyme dose decreases the total polyphenols content. The negative effects of the higher enzyme dose indicate that the pectinase greatly decreased total phenolics due to the loss of rutin [23] one of the most abundant flavonoids in olive leaves [24] but not the antioxidant activity which remains stable.

The same results were obtained for the incubation time on the total polyphenol yield, an increasing incubation time decreases the total polyphenol yield due to thermal degradation, same results with other researchers [25,26].

The experimental data in table 3 were used to determine the coefficient of two second-order polynomial equations as follows:

Y₁ =553,87+9028,4.X₁ - 6.27.X₂ -100211,1*X₁²+0,06.X₂²-8,44.X₁.X₂ +278661,1.X₁³-0.00015.X₂³-0,075.X₁.X₂²+137,2.X₁².X₂, (mg GAE/L)

(2)

R²=0.99

Y₂ = 53,999 + 40,0077.X₁ + 0,021.X₂ – 125,25.X₁² - 0.079.X₁.X₂ - 0.00000636777.X₂², (AA%)

(3)

R² = 0.97

where:

Y₁, Y₂ – the predicted responses for TPP and DPPH, respectively,

X₁ – the enzyme dose,

X₂ – the incubation time.

After the optimization process of enzyme dose and extraction time (Table 3) the final result of the optimal conditions (605.55 mL GAE/L) are still higher than the results as mentioned above and also higher than the total polyphenols content that obtained using cyclodextrins and glycerin as co-solvents (54.33 mg GAE/g) [27] but lower than the microwave and ultrasound extractions (104.22 mg GAE/g and 80.52 mg GAE/g) respectively [28].

All of the R² (coefficient of determination) values were greater than 0.95, implying that the models accurately represent the experimental data [29].

Both the incubation time and enzyme dose produced positive linear and negative quadratic effects on total polyphenols. This means that the yield of total polyphenols (Figure 4a) increases when the incubation time or enzyme dose increases up to a certain point, after which they begin to decrease. Positive linear and negative quadratic effects of incubation time were also reported for total polyphenols in extracts from rose petals and saffron tepals [30,31].

Positive linear effects of incubation time and negative quadratic effects of enzyme dose were obtained for the total antioxidant capacity values (Figure 4b), suggesting similar changes to those observed for the total polyphenols.

A graphical optimization of the extraction conditions was carried out in order to maximize the yields of total polyphenols and antioxidants. Figure 5 shows the overlapping region, defining the intervals of variation of the enzyme mixture dose (0.06–0.15%) and treatment time (30–120 min) that satisfy the optimization criterion.

4. Discussion

This work presents a novel method for more effectively extracting polyphenols from olive leaves, leading to environmentally friendly extracts and procedures. The results show that by using green extraction techniques, it is possible to limit the usage of organic solvents by developing easy and cheap methods for extracting bioactive plant polyphenols. Table 4 summarizes the results of other studies on extracting polyphenols from olive leaves in comparison with the current method. As you can see after using the optimal combination of enzymes and incubation time, the total polyphenols obtained using enzymatic-assisted extraction are higher than all other methods except extraction with microwave-assisted extraction and ultrasound-assisted extraction which are higher.

The results of this study indicate the recovery of polyphenolic antioxidants from olive leaves is improved by enzyme-assisted extraction, particularly when a binary enzyme combination consisting of Viscozyme L. and pectinase preparations (1:1) is used. The optimum range to obtain extracts with a high concentration of total polyphenols and antioxidants is defined by the variable intervals of the enzyme mixture dose (0.06–0.15%) and incubation time (30–120 min). This novel method provides an environmentally friendly replacement for conventional extraction methods, rendering it a green technology.

5. Conclusions

The obtained results of this study clearly show that the recovery of polyphenols from olive leaves is enhanced by enzyme-assisted extraction, particularly when ternary enzyme combinations, including pectinolytic and Viscozyme preparation, are used. This enzymatic and organic solvent free extraction method has an environmentally friendly substitute. The innovative and very promising results of this work motivate us in further research which should be done based on the polyphenolic profile of the olive leaves and the olive tree varieties to achieve the maximum quantity and the highest quality of polyphenolic yields.