Supercritical fluid extraction of torch ginger: Encapsulation, metabolite profiling, and antioxidant activity

2.1 Torch ginger sample and chemicals

Torch ginger (Etlingera elatior) inflorescences were procured from a farm in Kuala Pilah, Negeri Sembilan, Malaysia. The sample was harvested and delivered to the authors’ laboratory within the same day. Subsequently, the sample was stored in the laboratory's refrigerator for storage under refrigerated conditions (±4 °C). Analytical grade organic solvents and chemicals were used in the experiments for an optimum precision. The N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) used for the analysis was bought from Thermo Fisher Scientific (Waltham, MA, United States of America). Denatured ethanol (99% purity) and acetic acid (glacial, ≥99% assay) were obtained from HmbG Chemicals (Hamburg, Germany). Additionally, hydrochloric acid (fuming 37%), gallic acid, Folin-Ciocateu phenol reagent, and ferric chloride hexahydrate were purchased from Merck (Darmstadt, Germany). Meanwhile, anhydrous pyridine (99.8%), methoxyamine hydrochloride (98%), sodium acetate (anhydrous), sodium carbonate (anhydrous), gallic acid (TraceCERT®), 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH), and (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were procured from Sigma-Aldrich (St. Louis, MO, United States of America).

2.2 Preparation

2.2.3

2.2.3 Spray drying encapsulation of torch ginger extract

Prior to the process, a mixture made up of torch ginger extract (10%), water (80%), maltodextrin as an encapsulating agent (7%), and emulsifiers (glyceryl monostearate and sodium stearoyl lactylate) each at 1.5% was prepared. Subsequently, the prepared mixture was evenly mixed and homogenised for 15 min at 6000 rpm by using a Silverson L5M-A laboratory mixer (Silverson Machines, Inc., East Longmeadow, Massachusetts, United States of America). The aforementioned machine was operated with a short stop at every consecutive 5-minute to allow the cool down of the rotor blades. The mixture was further homogenised for 30 min by applying speed at 5800 rpm using a GEA Lab Homogeniser PandaPLUS 2000 (GEA Group Aktiengesellschaft, Düsseldorf, Germany). The formed emulsion was dried using a Büchi B-290 spray dryer (Büchi Labortechnik AG, Flawil, Switzerland) which equipped with an atomiser nozzle (0.5 mm in diameter) at 15 MPa. The spray drying process was conducted under the following conditions: inlet air temperature at 130 °C, outlet air temperature at 50 ± 1 °C, flow rate fixed at 150 L/h, and feed suspension rate set at 180 mL/h. Upon completion, the obtained powder was transferred into an amber glass bottle and tightly sealed. The bottle was stored in a refrigerator for storage at a refrigerated condition (±4 °C). Schematic diagram of the supercritical fluid extraction (SFE) process and the encapsulation of torch ginger extract by spray drying is showed in Fig. 1.

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Fig. 1

A schematic representation illustrating the SFE and the encapsulation process of TGEP.

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Yield of the encapsulated torch ginger extract powder (TGEP) obtained from the process was determined according to the equation used by Navarro-Flores et al. (2020) which given as follows:

(1)

$$\mathit{Yiel}{d}_{\mathit{TGEP}}\left(\%\right)=Powderobtained\left(g\right)/Solidsinthefeedsolution\left(g\right)x100\%$$

2.3 Analysis of powder characteristics

2.4 Metabolites profiling by GC–MS

Prior to Gas Chromatography-Mass Spectrometry (GC–MS) analysis, TGEP was derivatized according to the method used by Robinson et al. (2005). Briefly, 25.0 mg of TGEP was placed inside a 2.0 mL centrifuge tube, and about 50.0 μL of anhydrous pyridine was then added into it. The mixture was subjected to sonication for a duration of 10 min at 30 °C by using an Elmasonic S 30 (H) ultrasonic device (Elma Schmidbauer GmbH, Singen, Germany). Subsequently, about 100 μL of methoxyamine HCl (20 mg/mL in pyridine) was pipetted into the solution and it was vortexed for one minute. Two consecutive incubation was conducted by which the solution was initially incubated for 2 h at 60 °C and it was again incubated for 30 min at 60 °C upon the addition of 300 μL of MSTFA. Lastly, the solution was filtered using a 0.22 μm nylon syringe filter, and the filtered liquid was then transferred to an amber vial to be left at room temperature overnight.

The GC–MS method used in our previous study was followed for the identification of metabolites in TGEP and some modifications were made to improve the detection (Naziruddin et al., 2022). The derivatized fraction of TGEP was analysed by using a TSQ Quantum XLS GC–MS system (Thermo Scientific, United States of America). TGEP’s aliquot was injected (1 μL injection volume) into an Agilent J&W DB-5MS column (length: 30 m, inner diameter: 0.25 mm, and film thickness: 0.25 mm) (Agilent Technologies, California, United States of America) in split-less mode and the carrier gas used was helium at 1.0 mL/min. The column was first held at 80 °C for 5 min and afterwards increased at 8 °C/min to 200 °C. Subsequently, the temperature of the oven was gradually raised to 280 °C at a ramp rate of 4 °C/min, and maintained at that level for 15 min. The temperature of the ion source and interface were regulated at 280 °C and 250 °C, respectively. The GC–MS analysis was performed in a total ion chromatography (TIC) mode and the full scan data was collected within a mass scan range of 40 to 600 m/z. To identify the compounds present, the acquired mass spectra for each chromatographic peak were compared with a retention time index and mass spectral libraries for GC–MS that were created by the National Institute of Standards and Technology (NIST). The data version used for this analysis was NIST17 (NIST, 2017).

2.5 Analysis of antioxidant activity

3.1 Yield of the encapsulated torch ginger-extract powder

In the process, about 400 g of the prepared emulsion with a total solid mass of 80.0 g (50:50 torch ginger’s extract and encapsulating agent) was fed into the spray dryer, which produced 47.8 g of TGEP. Hence, based on the determination by Eq. (1), the process managed to yield around 59.8% of TGEP. The obtained yield in this study was found to be substantially higher than the yields reported by Anuar et al. (2021) which were in the range of 15 % – 36 % based on various formulations tested.

3.2 Particle size analysis and BET analysis

Fig. 2 displays the distribution of particle size for encapsulated torch ginger-extract powder. The median diameter, D_0.5 for TGEP was measured at 6.188 ± 0.771 μm. Meanwhile, the sample revealed two distinct particle sizes concentrating at 2.2 μm and 17.4 μm, respectively. This non-uniform size distribution was likely due to the formation of large agglomerates as a result of spray drying at low inlet air temperature (≤140 °C) (Both et al., 2020). Additionally, Siccama et al. (2021) also mentioned that the presence of high residual moisture content and low glass transition temperature of the spray dried powder might increase its stickiness which leads to agglomeration. Nonetheless, the produced TGEP showed relatively small size of particles which was less than 50 μm.

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Fig. 2

Particle size distribution of TGEP.

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In BET analysis (Table 1), TGEP was examined with the presence of considerably high surface area, pore volume, and pore size features. Surface area purportedly affected the functionality of powders: solubility, flowability, rehydration, and wetting characteristics. Furthermore, high surface area of functional powder also contributed to a high degree of solvation which is paramount for the absorption of retained compounds (Burgain et al., 2017; Koç & Kaymak-Ertekin, 2014). The outcomes’ trend of BET analysis were in agreement with results reported by Zhang et al. (2018), as the spray-dried powder obtained from their research exhibited higher surface area (ranging from 1.54 to 2.18 m²/g) with greater porosity. The authors also reported that the physical characteristics of spray-dried powder often affected by the spray drying inlet air temperature and type of atomiser. Zhang et al. (2018) also reported that the inlet air temperature in between 120 °C and 160 °C can induced the increased of powder’s surface area due to formation of particles with dryer and harder coating. The aforementioned inlet air temperature reflected to this study as the spray drying process was conducted at 130 °C. Hence, similar powder characteristics reported by Zhang et al. (2018) were expected.

3.3 FTIR analysis

The FTIR spectrum of TGEP is displayed in Fig. 3, and the peaks in the fingerprint region were assigned based on their corresponding functional groups in Table 2. A wide band was observed extending from 3000 cm⁻¹ to 3600 cm⁻¹, which corresponded to the O–H stretching vibration of alcohols and carboxylic acids in the sample. Several researchers have been reported the present of alcohols, such as 1-Dodecanol, Tetradecanol, and 1-Undecanol in the torch ginger extract. Meanwhile, carboxylic acid that predominantly fatty acids such as Hexacosanoic acid, Decanoic acid, and Hexadecanoic acid also has been reported to present in torch ginger extract (Al-Mansoub et al., 2021; Anzian et al., 2020; Marzlan et al., 2020). The spectra showed two intense sharp peaks at 2853 cm⁻¹ and 2925 cm⁻¹ which were in relation to the C–H stretching vibration of alkyl group. Additionally, the presence of alkene in TGEP can be identified by a medium peak formed at 1463 cm⁻¹ which corresponded to C–H bending of methylene group and a 1652 cm⁻¹ shoulder band which representing the double bonds (C = C) vibrational stretching of alkenyl group. Alkane and alkene which belong to the class of hydrocarbons also has been reported by Al-Mansoub et al. (2021), as the author managed to identify the presence of Cyclododecane, Heptadecane, 1-Decene, and 1-Tetradecene in the ethanolic torch ginger extract. The sample also exhibited a sharp peak at 1727 cm⁻¹ which indicating the C = O stretching of carbonyl group such aldehyde, ester, and carboxylic acid. This finding was in agreement with results reported by Marzlan et al. (2020), as the authors identified esters such as Lauryl acetate, Myristyl myristate, and (E)-9-Tetradecen-1-ol acetate in torch ginger oil extracted by supercritical fluid extraction. Anhydride group also potentially present in TGEP as indicated by the intense peak formed at 1025 cm⁻¹ which attributing to the CO-O-CO stretching of the functional group. Subsequently, a sharp peak present at 1200 cm⁻¹ signalled the C-N stretching of amine, while a broad peak formed at 1250 cm⁻¹ to 1310 cm⁻¹ might be corresponded to the C-O stretching of aromatic ester. Lastly, a noticeable peak formed at 750 cm⁻¹ could be associated to C–H bending of 1,2-disubstituted and monosubstituted compounds that present in TGEP. Hence, the results proved that the presence of chemically-diverse compounds in TGEP was valid.

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Fig. 3

FTIR spectrum of TGEP.

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3.4 Metabolite profiling and identification by GC–MS

GC–MS chromatogram of the detected metabolites in TGEP was showed in Fig. 4. Broad array of masses was acquired (scan range 40 – 600 m/z) within the scan time of 5.0 to 55.0 min and presence of metabolites were monitored at various retention times (RTs). The metabolites’ identities were confirmed by comparing the generated spectral pattern with those of established spectral library developed by National Institute of Standards and Technology (NIST), data version NIST17 (NIST, 2017). Additionally, the detected metabolites were classified by referring to the comprehensive database of FooDB which was developed by the renounce research teams specialising in metabolomics (TMIC, 2021).

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Fig. 4

The chromatogram obtained by GC–MS (TIC) displays the metabolites that have been identified in TGEP.

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Altogether, about 59 metabolites belong to the 19 different classes of compounds were identified in TGEP as listed in Table 3. The number of metabolites able to be detected in this study is substantially higher and more diversified than other reported torch ginger’s metabolite studies. In literature, studies conducted by Marzlan et al. (2020) and Anzian et al. (2020) reported the numbers of metabolite found in torch ginger’s essential oil at 20 and 33 respectively. In brief, most of the detected metabolites were belong to four major classes which were fatty acids (30.5%), terpenes and derivatives (20.3%), fatty acid esters (16.9%), and alcohols (8.47%).

Among these major classes of metabolites, alcohols particularly 1-Dodecanol was reported to render a potent antibacterial activity against several bacterial species (Marzlan et al., 2022; Marzlan et al., 2020). Additionally, the pharmacological activities of terpenoids were also well-known and heavily studied in the literature. Uvaol, a triterpene, was reported by Agra et al. (2016) to be effective as an active ingredient for the treatment of inflammation caused by allergic reaction.

Torch ginger notoriously known for its distinctive aroma which is a vital characteristic of Southeast Asian’s cuisine. Expert described that torch ginger embodies a sweet, tangy, and lemongrass-like aroma profile (Khor et al., 2017). Although the key aromatic compounds that responsible for torch ginger’s aroma have never been reported in literature, such study was already being conducted for ginger (Zingiber officinale Roscoe) and galangal (Kaempferia galanga L.) which also belong in the Zingiberaceae family. The researchers identified the presence of metabolite composition that consist of aldehyde, alcohol, hydrocarbon, ketone, terpene, and ester to be accountable for the distinctive aroma of both herbs (Hasegawa et al., 2016; Pang et al., 2017). In spite of the differences in species, similar composition as previously mentioned was also found in TGEP and the aroma profiles exude by the compounds could be assumed similar to torch ginger. Pang et al. (2017) mentioned that the presence of primary odorants in ginger namely, monoterpenes and sesquiterpenes could be associated to the woody, minty, citrusy, and herbal-like aroma. The researchers also attributed the sweet notes (balsamic and floral) of ginger to the presence of metabolites in the class of alcohol, aldehyde, terpene and terpene derivative. Additionally, humulene, a sesquiterpene found in TGEP was also reportedly presence in galangal and it was presumed to emits the unique galangal-like aroma (Hasegawa et al., 2016). The formerly defined aroma profile of ginger and galangal are noticeably similar to the general description of torch ginger’s aroma. Therefore, it is presumed that the key aromatic compounds of torch ginger were retained in the TGEP, which would be beneficial for the application in food.

3.5 Antioxidant activity

The antioxidant capacity of TGEP was evaluated based on the performance of TPC, DPPH, and FRAP as showed in Table 4. The quantified value of TPC for TGEP was found to be higher than other reported values in literature which were in the range of 2.12 – 19.4 mg GAE/g (Anzian et al., 2017; Yan & Asmah, 2010). The TPC values of the aforementioned studies were based on the quantification in the fresh and dried forms of torch ginger. Therefore, it indicates that the phenolic compounds present in torch ginger retained at an exceptional level in TGEP. Additionally, this claimed also supported by the presence of Hexadecyl-(E)-p-coumarate which is a phenolic acid identified in TGEP by the GC–MS.

The free radical scavenging capacity of TGEP was predicted based on its antioxidants ability to reduce DPPH radical. The activity of scavenging DPPH radicals was measured by the EC₅₀ value in which it indicates the effective TGEP’s concentration needed to reduce the DPPH radical’s absorbance by 50%. The value of EC₅₀ for TGEP was determined at 1.31 ± 0.002 mg/mL, which was substantially lower than such value reported by Nurain et al. (2013) at 3.47 ± 0.420 mg/mL based on the determination in ethanolic torch ginger’s extract. This low value of EC₅₀ for TGEP signified its high antioxidant activity which could potentially linked to its high TPC value. Additionally, the quantified FRAP value of TGEP also was found to be substantially higher as compared to such values reported by Bunleu and Buavaroon (2018) and Wijekoon et al. (2011) which were in the range of 9.0 – 130 μM Fe(II)/g. The excellent performance of TGEP’s antioxidants in the assay demonstrated its high reactivity against the Fe³⁺ - TPTZ and effectively reduced it to Fe²⁺ - TPTZ. Hydroxyl and carbonyl-rich compounds in plants have been associated with excellent reducing capabilities and stabilisers (Mohamad et al., 2014; Pradeep et al., 2022). Based on Table 3, TGEP possessed abundant compounds with sufficient hydroxyl and carbonyl groups present, such as dodecanal (aldehyde), xanthophyll, and β-Sitosterol (terpene) (Mahavy et al., 2022; Tovey, 2019). The oxidation–reduction abilities of these compounds allow the binding of metals and inactivate them via chelation (Azri et al., 2019).

The outstanding performance of TGEP in the aforementioned antioxidant assay could be linked to the existence of various metabolites in the class of terpenoids as profiled in Table 3. Terpenoid has been vastly studied in literature and the evidences for its potency as an antioxidant were well recorded. Terpenoids, namely humulene and uvaol found in TGEP have been reported in multiple studies able to induce the reduction of oxidative stress by effectively control the autoxidation reaction (Allouche et al., 2010; Gunawan et al., 2016). Additionally, the abundance of TGEP’s metabolites with a hydroxyl, methoxy, and carboxylic acid groups could potentially contribute to the high antioxidant capacity as these functional group were reported to render a vital effect on the antioxidant ability (Chen et al., 2020).

3.6 Potential future as a plant-based functional food additive

The development of functional food additives derived from plants has aroused the interest of consumers for a much healthier alternative and reduced the dependency on its artificial counterparts (Domínguez et al., 2021). However, various shortcomings in applying plant extract at the industrial scale might induce complexity in the manufacturing process and not be economically sound. Plant extract must be handled with the utmost care as it is highly volatile, hydrophobic, and prone to stability issues when exposed to environmental stressors (e.g., extreme temperature, light) (Kfoury et al., 2016; Rezaei & Nasirpour, 2019). The encapsulation technique has been known to offset these problems as it can trap the plant’s bioactive compounds in an encapsulating agent and makes it more stable (Muñoz-Shugulí et al., 2021). Based on the evaluations, TGEP has demonstrated the retention of various metabolites with different bioactive functions and maintained its antioxidant capability. This technique also captures the key aromatic compounds of torch ginger, which would be vital for its application as a food additive.