1. Introduction
Oxidative stress is a condition of oxidative damage when the critical balance between free radical generation and antioxidant defenses is disrupted [
1]. Short-term oxidative damage may cause human health conditions, such as trauma, infection and heat injury [
2]. Hence, antioxidants play an important role in stabilising free radicals by donating an electron to neutralise them. Plants contain a promising amount of antioxidants to protect and preserve their physical and metabolic integrity [
3].
Murraya koenigii, which is commonly known as curry leaf, is commonly used as a natural flavouring agent because of its aromatic scent and medicinal value [
4,
5].
M. koenigii belongs to the Rutaceae family, which is native in Malaysia, India and other South Asian countries [
6].
M. koenigii leaves contain several bioactive compounds that possess anti-tumour, antioxidant, anti-inflammatory and hypoglycemic properties [
7]. Essential oils, which can be found in the leaves of
M. koenigii, are significant contributors to antioxidant activity [
8,
9,
10]. However, the proper preservation of these pharmacological properties and their corresponding bioactive compounds through the processing of herbal plants remains challenging.
Drying is a challenging process to retain pharmacological properties. It is usually performed to preserve herbal plants and their bioactive compounds in post-harvesting. This process involves the removal of water from herbal plants to reduce their microbiological activity and preserve their bioactive compounds [
11,
12]. Traditional drying methods, such as sun drying and air drying, have been used for a long time for the dehydration of food due to their simplicity and low operating cost. However, open-air drying and long drying duration cause food to be contaminated with dust and microbes [
13]. Another type of drying method that is widely used in dehydrating agricultural products is convective hot-air drying (CD). This drying method involves two types of moisture diffusions during heat transfer. The first type is external diffusion, where the surface moisture content diffuses to the drying medium; the second type is internal diffusion, where internal moisture content diffuses out to the drying surface [
14]. One of the disadvantages of CD is long drying duration due to the internal diffusion process. The drying process requires higher energy compared with other production processes due to the extremely low energy efficiency of dryers and high latent heat of water evaporation [
15]. Drying consumes 10% to 15% of the total national industrial energy demand in the USA and 20% to 25% in Europe [
16]. Thus, the drying industry is faced with challenges to resolve this problem by optimising drying conditions or developing an alternative drying method, such as hybrid drying.
Hybrid drying combines two or more drying methods into one drying system. Hybrid drying systems occur in two types, namely, single-stage hybrid drying and two-stage hybrid drying. Single-stage hybrid drying combines two drying methods, such as microwave drying and vacuum drying, into a one-unit system called microwave vacuum drying (MVD). However, one of the disadvantages of MVD is that the intensive water evaporation from the leaves may exceed the capacity of the vacuum pump. Thus, this technique requires the reduction of raw materials or an increase in the capacity of the vacuum pump [
17]. This problem can be overcome using CD as a pre-drying to efficiently remove excess moisture and decrease the load of the vacuum system in MVD. Convective hot-air pre-drying before MVD (CPD-MVFD) allows a reduction in the total cost of the drying process and improves the quality of products [
18]. To overcome the energy consumption issue of the drying process, two-stage hybrid drying methods, such as MVD and CPD-MVFD, were used in this study to compare its specific energy consumption with commercial drying method (CD) and reduce energy consumption during the drying process.
In this present study, four drying methods, namely, freeze-drying (FD), CD, MVD, and two-stage hybrid CPD-MVFD were used to estimate and reduce energy consumption during the drying of M. koenigii leaves without compromising the quality of the dried leaves. Five quality parameters, including antioxidant capacity (ABTS and FRAP), total polyphenolic content (TPC) analysis, profiling of volatile compounds, colour analysis and water analysis, were analysed. The Pearson correlation analysis was performed to study the correlation between ABTS, FRAP, TPC, volatile compounds and colour parameters and to identify the main contributor to the antioxidant capacity.
3. Material and Methods
3.1. Chemical Reagents
Methanol, hydrochloric acid, Folin–Ciocalteu reagent, sodium carbonate, 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), potassium persulphate, ferric reducing antioxidant power (FRAP), 2,4,6-tripyridyl-1,3,5-triazine (TPTZ), iron (III) chloride, gallic acid and sodium sulphate were purchased from Sigma-Aldrich (Steinheim, Germany). Cyclohexane and 2-undecanone were provided by United Quantum Factory (Wrocław, Poland)
3.2. Sample Preparation
Approximately 5 kg of fresh
M. koenigii leaves were purchased from Ukay Nursery (Kuala Lumpur, Malaysia) and identified at the Forest Research Institute Malaysia (FRIM) with the voucher number of 031/18. The bone drying weight of
M. koenigii leaves were obtained using an oven dryer at 105 °C for 24 h, in accordance with the ASTM standard (D1348-94) [
15].
3.3. Drying of Fresh M. koenigii Leaves
Fresh
M. koenigii leaves were dried using the four drying methods, namely, CD, VMD, CPD-MVFD, and FD. Freeze-dried samples were used as the control samples [
17,
19,
29].
3.3.1. Convective Hot-Air Drying (CD)
Approximately 40 g of fresh M. koenigii leaves were dried using a convective hot-air dryer designed and constructed in the Institute of Agriculture Engineering (Wrocław, Poland) at 40, 50 and 60 °C. The leaves were placed on a wire mesh tray and spread evenly. The wire mesh tray was placed on top of the drying chambers. Weight loss was determined at 5 min interval for the first 1 h, at 30 min interval for the next 5 h and an hour interval until the mass loss difference was 0.05 g or less using an analytical balance (PS600/C/2, Radwag , Wrocław, Poland).
3.3.2. Microwave Vacuum Drying (MVD)
Approximately 40 g of fresh M. koenigii leaves were dried using a vacuum microwave dryer (Plazmatronika, Wrocław, Poland) with 6, 9 and 12 W/g microwave power. The leaves were first placed in an organic glass container connected to a vacuum system and then rotated at a constant speed of 6 rpm throughout the drying process to prevent the local overheating of leaves. The temperature of the leaves was measured immediately after the samples were taken out of the dryer. The weight loss of the leaves was recorded for every 4, 3, and 2 min intervals for 6, 9 and 12 W/g, respectively, until the difference in the mass loss was 0.05 g or less using an analytical balance (PS600/C/2, Radwag, Wrocław, Poland).
3.3.3. Convective Hot-Air Pre-Drying and Microwave Vacuum Finishing-Drying (CPD-MVFD)
M. koenigii leaves were first subjected to CPD by using a convective hot-air dryer at 50 °C. The partially dried samples were then subjected to a vacuum microwave dryer (Plazmatronika, Wrocław, Poland) at 9 W/g to thoroughly dry the leaves. A convective hot-air temperature of 50 °C and a microwave wattage of 9 W/g were used in CPD-MVFD to ensure the good quality of the dried product [
18,
30].
3.3.4. Freeze Drying (FD)
M. koenigii leaves were dehydrated using a freeze dryer (OE-950, Hungary) at a vacuum pressure of 65 Pa. The freezing temperature was −60 °C. The heating plate was set to 30 °C for sublimation.
3.4. Energy Consumption and Specific Energy Consumption of CD, MVD, and CPD-MVFD
Energy consumption (
E) is the energy used expressed in kilojoules. Specific energy consumption is an energy performance indicator to evaluate or measure the performance of energy efficiency. In this study, the specific energy consumption of dried
M. koenigii leaves was expressed in kilojoule per gram of fw (
) and kilojoule per gram of water (
). The energy consumption and specific energy consumption of CD, MVD, and CPD-MVFD at all drying conditions were determined using the equations shown in
Section 3.4.1,
Section 3.4.2 and
Section 3.4.3 [
17,
18].
3.4.1. Energy Consumption in Convective Hot-Air Drying Method
Energy consumed (
, during CD (kJ)) was calculated using Equation (1):
where
is the power consumption by fans blowing air to six pipes (kW),
is the power consumption of the electric heater (kW) and
is the drying time (s).
3.4.2. Energy Consumption in Microwave Vacuum Method
The energy consumed during MVD (kJ) was calculated using Equation (2).
where
is the output power (kW), and
is the efficiency of magnetrons.
is the power consumption by the vacuum pump (kW),
is the power consumption by the electric engine rotating the container (kW), and
is the time (s).
3.4.3. Specific energy Consumption
Specific energy consumption was expressed in (a) the ratio of energy consumption to the initial mass,
, and (b) the ratio of energy consumption to the mass of water removed from the material,
). Equations (3) and (4) show the specific energy consumption for CD, and Equations (5) and (6) show the specific energy consumption for MVD.
3.5. Modelling of Drying Kinetics
The drying kinetics was modelled in this study to understand the transport mechanism, simulate or scale up the entire optimisation process and control the operating conditions [
31]. Therefore, the drying kinetics of
M. koenigii leaves dried with CD, MVD, and CPD-MVFD at all drying conditions was determined based on the mass loss throughout the drying process. Drying curves were plotted as a function of moisture ratio (
) against time.
was determined using Equation (7).
where
is the moisture content at the respective time (g water/g dw),
is the equilibrium moisture content (g water/g dw) representing the lowest moisture content obtainable at equilibrium under the drying conditions used and
is the initial moisture content (g water/g dw)
The drying rate (
) was calculated using Equation (8):
where
is the moisture content at the respective time (
),
is the moisture content at respective time + 1 (
) and
is the time difference.
Lewis Newton (Equation (9)), Modified Page (Equation (10)) and Midilli–Kucuk (Equation (11)) were adopted in this study to describe the drying kinetics of dried
M. koenigii leaves. These three thin-layer models have been frequently used to model the drying kinetics of leaves. They have shown good fitting in describing the drying kinetics of leaves and have been used to simulate or scale up the entire drying process for further optimisation [
32,
33,
34].
where
denotes the moisture ratio,
denotes the model constants,
denotes the drying constant,
is the dimensionless empirical constant and
is the drying time.
Goodness of fitting was evaluated and compared using the statistical measures, such as , RMSE and . Goodness of fit was identified based on the highest value and the lowest RMSE and .
3.6. Total Polyphenolic Content (TPC) and Antioxidant Capacity Analysis of Dried M. koenigii Leaves
3.6.1. Extraction of TPC from M. koenigii Leaves
The extraction of TPC from dried leaves of
M. koenigii was conducted using the procedure described by Chua et al. [
23] with some modifications. Approximately 0.5 g of powdered sample was extracted with 9 mL of 80% methanol acidified with 1% hydrochloric acid. The extraction was performed in an ultrasonic bath (Sonic 6D; Polsonic, Warsaw, Poland) for 15 min with a frequency of 50 Hz under room temperature. The extract solution was stored at 4 °C overnight and sonicated again at the same extraction conditions. The extracts were centrifuged at 10,000 rpm for 5 min (MPW-350R; Warsaw, Poland) and subjected to TPC and antioxidant capacity analysis.
3.6.2. TPC Analysis
TPC was determined using the Folin–Ciocalteu method described by Hamrouni et al. [
35] and Wojdyło et al. [
22] with some modifications. Approximately 0.1 mL of sample extract was mixed with 2 mL of distilled water and 0.2 mL of Folin–Ciocalteu reagent. The mixture was then incubated for 3 min at room temperature and added to 1 mL of 20% sodium carbonate. TPC was determined after 1 h of incubation at room temperature in the dark. Absorbance values were determined using UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan) at 765 nm. The results were generated in triplicates and expressed in gallic acid equivalence per 100 g of dry weight (mg GAE/100 g dw).
3.6.3. Antioxidant Capacity Analysis
The in vivo mechanisms of action of the antioxidant-protecting effect of
M. koenigii leaves are complex. Therefore, at least two or more
in vitro antioxidant assays must be conducted to evaluate different aspects of the reactivity of compound (s) toward reactive oxygen species and reactive nitrogen species [
36,
37]. Two antioxidant assays, namely, ABTS radical scavenging method and FRAP, were conducted in this study to quantify the antioxidant capacity of
M. koenigii leaves.
ABTS Radical Scavenging Assay
ABTS radical scavenging assay was determined following the procedure described by Chua et al. [
20]. ABTS was dissolved in distilled water at a final concentration of 7 mM and then mixed with 2.45 mM potassium persulphate to obtain ABTS radical cation (ABTS). The mixture was incubated in the dark at room temperature for 12–16 h. Thereafter, the mixture was diluted with distilled water until an absorbance value of 0.700 ± 0.02 at 734 nm was reached using a UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). Subsequently, 3 mL of the diluted radical solution was added to 30 µL of the extracted supernatant. Absorbance values were determined at 734 nm after 6 min. All determined values were expressed in µM Trolox per 100 g of dry weight and measured in duplicate.
FRAP Assay
FRAP assay was performed following the procedure described by Benzie et al. [
38]. FRAP reagent was prepared by mixing acetate buffer (300 µM at a pH 3.6) with 10 µM of TPTZ solution, which was subsequently added with 40 µM of HCl and 20 µM of FeCl
3 at a ratio of 10:1:1 (v/v/v). Approximately 3 mL of FRAP reagent was then added to 1 mL of the sample solution and mixed well. Absorbance values were read at 593 nm after 10 min. A standard curve was plotted using different Trolox concentrations. All readings were expressed in µM Trolox per 100 g of dry weight, and the results were obtained in duplicate.
3.7. Hydrodistillation of Volatile Compounds/Essential Oils
The essential oil was extracted using the hydrodistillation method via Deryng type apparatus, as described by Wróblewska et al. [
39]. Approximately 1 g of
M. koenigii leaves was transferred into a 150 mL round bottom flask with 50 mL of distilled water. The bottom flask was connected to the apparatus and heated using a heating mantle. Distillation was finished after 30 min. The essential oils were collected from the three-way tap. Approximately 1 mL of a mixture of cyclohexane and 2-undecanone (concentration 5 mg/mL) (UQF, Wrocław, Poland) was added into the samples as an internal standard for the identification and quantification of volatile compounds using GC-MS. The collected essential oils were dried using anhydrous Na
2SO
4 and stored in the fridge for the profiling of volatile compounds.
Identification and Quantification of Volatile Compounds Using GC-MS
The detailed analysis of the chemical composition of
M. koenigii leaves was carried out using a gas chromatograph coupled to a mass spectrometer (Shimadzu 2020 Kyoto Japan). The separation was performed on ZB-5 (Phenomenex, Torrance, CA, USA) fused silica capillary tubes column (30 m × 0.25 mm ID × 0.25 μm film thickness). The stationary phase consisted of 5% phenyl and 95% dimethylpolysiloxane. The mass spectrometer was equipped with a quadrupole analyser set at 1508 for all analyses at an electron multiplier voltage of 1350 V. Scanning (1 scan/s) was performed in the range of 39–450
m/
z by using electron impact (
EI) ionisation at 70 eV. The components were identified by comparison of: (a) the obtained
EI mass spectra with NIST17 (NIST MS Search 2.0d software, Gaithersburg, MD, USA); (b) the retention times of the authentic standards and analysed signals; (c) the retention Kovats indices (RI exp.) and theoretical value (RI lit. from Adams [
40], MassFinder and NIST17 entries). For the calculation of retention indices, homologous series of C7–C20
n-alkanes (UQF Wrocław, Poland) were used. The carrier gas was helium with a flow rate of 1 mL/min. The starting temperature of the isolation was 50 °C (2 min), which was raised to 130 °C at 4 °C/min and then reached a final temperature of 270 °C in 10 °C/min. This temperature was retained for 5 min. Injector and detector temperatures were 220 and 300 °C, respectively. The volume of the injected solution was 1 μL with a split ratio 1:40. The identified compounds were quantified by comparing the peak area of individual compounds with the peak area of the standard used with a concentration of 5 mg/mL.
3.8. Quality Analysis of M. koenigii Leaves
3.8.1. Colour Analysis
The loss of the colours in the leaves may cause a loss in the bioactive compounds, given that the colour of plant leaves is correlated to the bioactive compounds contributed to the pharmacological properties [
41]. Therefore, the colour analysis was conducted in the current study to evaluate the correlation between the colour parameters of the dried leaves and the antioxidant activity. The colour of
M. koenigii leaves dried via CD, MVD, CPD-MVFD, and FD were determined following the procedure described by Chua et al. [
23]. The
M. koenigii leaves after the drying process were grounded into powder form. The colours were determined with a Minolta a Chroma Meter CR-200 (Minolta Co. Ltd., Osaka, Japan). Colour data were expressed in
,
and
. Each measurement was carried out five times, and the mean value was obtained.
3.8.2. Water Activity Analysis
Water activity corresponds to the amount of water available for the degradation reactions of antioxidant activity caused by the microorganisms [
42]. Hence, the water analysis of dried
M. koenigii leaves for CD, MVD, CPD-MVFD, and FD was conducted following the procedure described by Chua et al. [
20] to evaluate the stability of dried
M. koenigii leaves. The water content of dried leaves was determined using a water activity meter (Aqualab 4TE, Pullman, WA, USA). Powdered leaves were placed and spread up across the sample cup and inserted into the measuring chamber. The average temperature of the measuring chamber was 24.9 ± 0.05 °C.
3.9. Statistical Analysis
Results were expressed as mean ± standard deviation. The error bars in the figures indicated the standard deviation. Differences between means were analysed using one-way ANOVA through SPSS 23 (IBM, New York, NY, USA). Significant differences (
p ≤ 0.05) between means were determined using Tukey’s test. The thin-layer modelling of CD, MVD and CPD-MVFD was performed using Table Curve 2D windows v2.03 (Jandel Scientific Software, San Jose, CA, USA), and goodness of fit was evaluated using
, RMSE and
. The correlation analysis was determined using the Pearson correlation test through SPSS 23 (IBM, New York, NY, USA) [
17].
4. Conclusions
The energy usage in the current drying process of herbal plants is a major concern as the commercial drying method consumes up to 60% of the total energy. Hence, the hybrid drying methods were proposed to study the energy consumption in comparison to the energy usage of the convective hot-air drying (CD). Both hybrid drying methods (MVD and CPD-MVFD) effectively reduced the specific energy consumption. Notably, MVD as the finishing drying method reduced the specific energy consumption by 67.3% (kJ/g fw) and 48.9% (kJ/g water) in comparison to CD at 50 °C. In the drying modelling of CD, MVD and CPD-MVFD, the modified Page model demonstrated good fit to the empirical data obtained. FD showed a promising antioxidant capacity result and MVD at 6 W/g retained a promising amount of TPC. Retained TPC was contributed to the antioxidant capacity (ABTS and FRAP) in M. koenigii leaves. β-phellandrene (2.647 mg/g dw), α-pinene (1.711 mg/g dw) and sabinene (1.374 mg/g dw) were identified as the major volatile compounds in dried M. koenigii leaves. Colour analysis showed MVD’s high performance in preserving the colour parameters of M. koenigii leaves under all conditions. In the case of water activity, all drying methods showed that the sample conditions were shelf-stable. Furthermore, the brightness of the dried M. koenigii leaves was correlated to the TPC with a Pearson coefficient of 0.880 (p ≤ 0.01). However, further investigation on the correlations of the single pigments to the antioxidant capacity and TPC are required. In summary, MVD showed a promising reduction in energy consumption as well as high recovery in TPC and volatile compounds.