Kanuka woodchips were obtained from the East Cape of New Zealand and used for liquid smoke production. Kanuka (Kunzea ericoides) is a native tea tree species in New Zealand and is commonly used for food smoking (Essien et al., 2019). Maltodextrin was used as the encapsulation carrier, purchased from Bulk Powders New Zealand. Chemicals and standards used in this study were purchased from Sigma-Aldrich (New Zealand), including gallic acid, catechin hydrate, Folin-Ciocalteu’s phenol reagent, DPPH (1, 1-diphenyl-2-picrylhydrazyl), Trolox (6-hydroxy-2,-5,-7,-8-tetramethylchroman-2-carboxylic acid), aluminium chloride, sodium carbonate, sodium nitrite and GC-grade dichloromethane.
Kanuka woodchips were fast-pyrolysed at 450 °C in a fluidised-bed reactor to prepare liquid smoke. The details of the reactor are illustrated in a previous study (Xin et al., 2021). The process produced liquid smoke with a yield of 30 wt% and valuable by-products, biochar and pyrolysis oil, with yields of 24 and 18 wt%. The prepared liquid smoke was an aqueous phase liquid with a brown to red colour. Maltodextrin was diluted in the fast pyrolysis liquid smoke to prepare the feed solutions for spray-drying and freeze-drying. Three feed solutions were prepared at ratios of 6:1, 5:1 and 4:1 (liquid smoke/maltodextrin, w/w).
Spray-drying of each feed solution was achieved in a Büchi B-290 spray dryer (Maia et al., 2019). Spray-drying process was maintained at an air inlet temperature of 150 °C, aspirator rate of 100%, atomising gas flowrate of 11.1 L/min (standard temperature and pressure) and feeding pump rate of 10%. The outlet temperature was in the range of 60–80 °C. Spray-dried products from the cyclone container and drying chamber were collected and weighed. The three powder products were labelled as SPD-6/1, SPD-5/1 and SPD-4/1, respectively, corresponding to the three feed solution ratios of 6:1, 5:1 and 4:1.
Freeze-drying of each feed solution was achieved using a Christ Alpha 1–2 LDplus freeze dryer (Condurache et al., 2019). The three solutions were frozen at -18 °C for 48 h before placing in the freeze-dryer chamber. The process conditions were a condenser temperature of -58.8 °C, a pressure of 6.11 mbar, a vacuum of 0.42 mbar and a drying time of 48 h. After freeze-drying, the glass-like samples were ground using a mortar and pestle following a previous study (Rezende et al., 2018). The three powder products labelled as FZD-6/1, FZD-5/1 and FZD-4/1, respectively, corresponding to the three feed solution ratios of 6:1, 5:1 and 4:1.
Six smoke powder samples were obtained from spray and freeze-drying and stored in a desiccator at around 20 °C until the analysis in the following 4 weeks. Spray-dried powders had a yellow colour like butter, while freeze-dried ones had a brown colour like chocolate.
The product yield is calculated according to Eq. (1):
$${\mathrm{Y}}_{\mathrm{product}}=\frac{{\mathrm{M}}_{\mathrm{powder}}}{{\mathrm{M}}_{\mathrm{TSS}}}\times 100$$
(1)
where \({\mathrm{Y}}_{\mathrm{product}}\) is the yield of powder product (wt.%), \({\mathrm{M}}_{\mathrm{powder}}\) is the mass of obtained powder product and \({\mathrm{M}}_{\mathrm{TSS}}\) is the mass of total soluble solid in the feed solution.
The smoke powder products and kanuka liquid smoke were dissolved and diluted in distilled water to 10 mg/mL. These solutions were analysed for total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power assay (FRAP) and DPPH scavenging activity (DPPH) using an ultraviolet–visible (UV) microplate reader (EnSpire 2300, PerkinElmer).
Total phenolic content (TPC) was determined by the Folin-Ciocalteu assay by following a previous study (Munir et al., 2018). The solutions (0.025 mL) and gallic acid standards were mixed with 0.125 mL of tenfold freshly diluted Folin-Ciocalteu reagent in 96 well plates. Then, 0.125 mL of 7.5% sodium carbonate was added to each well. The plates were incubated in the dark for 60 min at room temperature. The absorbance values were measured at 765 nm using the UV microplate reader. The results were expressed as milligrams of gallic acid equivalent per gram of smoke powder or liquid smoke (mg GAE/g).
Total flavonoid content (TFC) was determined using quercetin as the standard by an aluminium chloride method (Essien et al., 2020). The solutions (0.025 mL) and quercetin standards were mixed with 0.1 mL of distilled water and 0.01 mL of 5% sodium nitrite in 96 well plates. Then, 0.015 mL of 10% AlCl3 and 0.050 mL of 1 mol/L NaOH were added to the well plates. After adding 0.05 mL of distilled water, the plates were incubated in the dark for 60 min at room temperature. The absorbance values were measured against a reagent blank at 510 nm. The results were expressed as milligrams of quercetin equivalent per gram of smoke powder or liquid smoke (mg QE/g).
Ferric reducing antioxidant power assay (FRAP) was conducted following a previous report (Kheirkhah et al., 2019). FRAP reagent was prepared by mixing the 2,3,5-triphenyltetrazolium chloride (TPTZ) solution (10 mmol/L TPTZ in 40 mmol/L HCl), 20 mmol/L FeCl3 and 300 mmol/L sodium acetate buffer (pH 3.6) at a ratio of 1:1:10. Smoke samples (0.001 mL) and Trolox standards were mixed with 0.2 mL of FRAP reagent in 96 well plates. The absorbance values were measured at 593 nm against a reagent blank after incubation for 60 min at room temperature. The results were expressed as milligrams of Trolox equivalent per gram of smoke powder or liquid smoke (mg TE/g).
A previous study was followed to determine DPPH scavenging capacity (Essien et al., 2020). The DPPH (1,1-diphenyl-2-picrylhydrazyl) reagent was firstly dissolved in ethanol at a concentration of 40 mg/L. Then, 0.2 mL of DPPH solution was mixed with 0.01 mL of each sample, Trolox standards and blank in 96 well plates. The absorbance values were measured at 517 nm after incubation for 60 min at room temperature. The DPPH scavenging capacity was expressed as milligrams of Trolox equivalent per gram of smoke powder or liquid smoke (mg TE/g).
The retention efficiency of each bioactive value is calculated according to Eq. (2):
$$\mathrm{RE}=\frac{{V}_{\mathrm{Powder}}\times {\mathrm{Y}}_{\mathrm{Product}}}{ {V}_{\mathrm{LS}}/{\mathrm{P}}_{\mathrm{TSS}}}\times 100$$
(2)
where RE is retention efficiency (%), \({V}_{\mathrm{Powder}}\) is the bioactive value (TPC, TFC, FRAP or DPPH) of powder products, \({\mathrm{Y}}_{\mathrm{Product}}\) is the yield of powder products, \({V}_{\mathrm{LS}}\) is the bioactive value of pure liquid smoke and \({\mathrm{P}}_{\mathrm{TSS}}\) is the mass percentage of total soluble solid in the feed solution.
Gas chromatography-mass spectrometry (GC–MS) analysis was conducted to identify the volatile compounds. The smoke powder products and pure liquid smoke were dissolved and diluted in distilled water to a concentration of 250 mg/mL. Then the solutions were mixed with dichloromethane at a volume ratio of 1/2 (solution/dichloromethane), and the mixtures were agitated at 200 rpm for 6 h. The dichloromethane extracts were then filtered by 0.2 µm syringe filters. Each dichloromethane extract was prepared in triplicate for GC–MS analysis. This preparation method followed a previous study with minor modifications (María D. Guillén & Ibargoitia, 1998).
The GC–MS instrument (Shimadzu QP-5000) was equipped with a DB-5HT column (30 m × 0.25 mm × 0.1 μm). Dichloromethane extract (1 μL) was injected at an injection temperature of 280 °C. The oven temperature was increased from 50 to 250 °C by 20 °C/min and then was held for 5 min. Mass spectra were operated in electron ionisation mode at 70 eV, and the mass range was 50–300 amu for acquisition. Volatile compounds were identified by comparing the mass spectra with those in the library NIST and by comparing the data results with previous studies (Petzold et al., 2014; Taruna Syah et al., 2016; Xin et al., 2021). Their relative abundances were expressed as the peak area percentages of the total ionisation chromatogram (TIC).
Principal component analysis (PCA) as a useful chemometric tool can reduce the experimental data dimension for ease of interpretation. It can transform a large set of interrelated variables into principal components. This study used principal component analysis (PCA) to understand the difference between liquid smoke and produced powders in terms of their chemical composition. The smoke flavouring samples were set as the observations, and peak area percentages of identified compounds by GC–MS were set as the variations. A MATLAB toolbox was used with MATLAB R2020a software to conduct PCA (Ballabio, 2015). The toolbox gives visualising results, including plots of scores and loadings and numerical values of eigenvalues.
The morphology of the smoke powders was determined using a scanning electron microscope (Hitachi SU-70 Schottky field SEM). Smoke powder samples were sprinkled on the double-sided conductive carbon tabs and glued onto the SEM mounts. The samples were coated with platinum for 100 s at room temperature.
Particle sizes of the smoke powders and pure maltodextrin were measured by a particle size analyser (Malvern Mastersizer 2000, Malvern Instruments Ltd) with a dry dispersion module (Scirocco 2000). The weighted-average volume diameter was expressed as diameter D[4,3], assuming spherical particles with the same volume as the actual particles. Particle size distribution (PSD) is calculated according to Eq. (3):
$$\mathrm{PSD}=\frac{\mathrm{d}\left(0.9\right)-\mathrm{d}(0.1)}{\mathrm{d}(0.5)}$$
(3)
where d(0.9), d(0.5) and d(0.1) are respectively particle diameters at 90, 50, and 10% of the cumulative size distribution curve from Mastersizer analysis results (Tupuna et al., 2018).
Bulk density and tapped density were determined by following a reported method (Caliskan & Dirim, 2016). Briefly, 20 g of each powder sample or maltodextrin was gently loaded into a 100 mL graduated cylinder. The sample weight was divided by the measured volume to obtain bulk density (ρbulk, g/mL). The tapped density (ρtapped, g/mL) was obtained after tapping the cylinder 120 times and then measuring the sample volume.
Carr index (CI) and Hausner ratio (HR) were evaluated for the flowability and cohesiveness of the six powder samples and maltodextrin (Caliskan & Dirim, 2016). The CI and HR values are calculated according to Eqs. (4) and (5):
$$\mathrm{CI}=\frac{{\rho }_{tapped}-{\rho }_{bulk}}{{\rho }_{tapped}}\times 100$$
(4)
$$\mathrm{HR}=\frac{{\rho }_{tapped}}{{\rho }_{bulk}}$$
(5)
Water content of the powder samples and maltodextrin was measured by weight difference before and after placing the sample in a drying oven at 105 °C until no more weight loss.
All of the drying processes and analyses in this study were conducted in triplicate, and the results were presented as mean values ± standard deviation (n = 3). Analysis of variance (ANOVA) was performed, and the difference between means was analysed using Duncan’s test. Statistical significance was considered at p < 0.05. All statistical analysis was performed using SPSS 9.05 (Chicago, USA).