Oxidative stability of spray dried matcha-tuna oil powders

Journal Pre-proofs Oxidative stability of spray dried matcha-tuna oil powders Meng Shi, DanYang Ying, Mya Myintzu Hlaing, JianHui Ye, Luz Sanguansri, Mary Ann Augustin PII: DOI: Reference:

S0963-9969(20)30075-2 https://doi.org/10.1016/j.foodres.2020.109050 FRIN 109050

To appear in:

Food Research International

Received Date: Revised Date: Accepted Date:

23 September 2019 10 January 2020 31 January 2020

Please cite this article as: Shi, M., Ying, D., Myintzu Hlaing, M., Ye, J., Sanguansri, L., Ann Augustin, M., Oxidative stability of spray dried matcha-tuna oil powders, Food Research International (2020), doi: https:// doi.org/10.1016/j.foodres.2020.109050

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Oxidative stability of spray dried matcha-tuna oil powders Meng Shia, DanYang Yingb, Mya Myintzu Hlaingb, JianHui Yea, Luz Sanguansrib, Mary Ann Augustinb* aZhejiang bCSIRO

University Tea Research Institute, Hangzhou 310058, China

Agriculture and Food, 671 Sneydes Road, Werribee, Victoria 3030, Australia

*Corresponding author. E-mail: [email protected]

ABSTRACT Matcha-tuna oil and matcha-maltodextrin-tuna oil emulsions (25% oil, dry basis), formulated to have protein: carbohydrate ratios of 1:1.1, 1:2, 1:3 and 1:4, were spray dried. Confocal laser scanning microscopy showed effective emulsification of oil in all emulsions. All powders had low surface fat (2.9-4.2%). The addition of maltodextrin enhanced the bulk density and flowability of powders. Water sorption isotherms indicated that addition of maltodextrin increased water uptake of powders. The oxidative stability of the powders under accelerated conditions in an Oxipres was highest for the matcha-tuna oil powder. Increasing amounts of added maltodextrin decreased oxidative stability. A comparison of levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in neat oil and tuna oil powders over 12 weeks at 40°C, demonstrated that % remaining EPA and DHA were higher for all spray dried powders compared to neat oil. There was a significant correlation (p<0.01) between the amount of the loss of tea catechins and % remaining EPA and DHA after 12 weeks at 40°C, suggesting that the catechins had a major role in protecting the tuna oil against oxidation. This study has demonstrated the potential of using a whole biomass (matcha) as the single encapsulant for protection and delivery of omega-3 oils.

Keywords: matcha, tuna oil, microencapsulation, omega-3, catechins

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1. Introduction The consumption of long chain omega-3 fatty acids, eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6), have been associated with decreasing incidence of cardiovascular disease, cancer and neurodegenerative diseases (Barber, Ross, Voss, Tisdale, & Fearon, 1999; Kris-Etherton, Harris, & Appel, 2002; Palacios-Pelaez, Lukiw, & Bazan, 2010). There has been increasing interest in the development of functional foods with enhanced levels of long chain polyunsaturated fatty acids. Omega-3 fatty acids are susceptible to oxidation, which results in loss of sensory appeal and nutritional value (Encina, Vergara, Giménez, Oyarzún-Ampuero, & Robert, 2016). Encapsulation of omega-3 fatty acids protects the oils against degradation and allows omega-3 oils to be delivered in a convenient powder format for incorporation into manufactured foods (Sanguansri & Augustin, 2016). Only food grade encapsulating matrices are permitted for delivery of bioactives into functional foods. For the stabilisation of oils, proteins (notably milk proteins) and carbohydrates have commonly been used for formulation of encapsulated bioactives (Roos & Livney, 2017). The use of whole vegetable and plant biomass, in place of isolated combinations of isolated proteins and carbohydrates which have traditionally been used as the encapsulating matrix, has recently been explored as delivery systems for fish oils (Augustin & Sanguansri, 2019). To increase the stability of oils within encapsulated systems, antioxidants may be added, with the preference being for natural plant antioxidants and extracts. For example, the oxidative stability of microencapsulated fish oil was improved by the addition of various antioxidants (vitamin E, rosmarinic acid, ethylenediaminetetraacetic acid and citric acid, plus carnosic acid and ascorbyl palmitate), antioxidant blends (tocopherols, ascorbyl palmitate and lecithin with rosemary extract), quercetin, rosemary and laurel extracts, while microencapsulated linseed oil was protected by a polyphenolic rich extract from murta leaves (Azizi, Li, Kaul, &

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Abbaspourrad, 2019; Barrett, Porter, Marando, & Chinachoti, 2011; Rubilar et al., 2012; Serfert, Drusch, & Schwarz, 2009; Yeşilsu & Özyurt, 2019). Powdered green tea leaves (matcha) were chosen because they contain proteins and carbohydrates, which are necessary for stabilising the structure of the microcapsule. Yamamoto et al. (1997) reported protein and carbohydrate contents of green tea powder are around 30% and 39% respectively. Although tea leaf has a low amount (1.8-3.7%, dry basis) of hot-water soluble protein (Selvendran, Perera, & Selvendran, 1972), there are other components in the tea that bind and encapsulate oils. For example, the water-insoluble proteins in green tea can stabilise emulsions (Ren, Chen, Zhang, Lin, & Li, 2019), and therefore have a contributory role in the encapsulation of oils. Green tea is rich in polyphenols and other phytonutrients which can serve as natural antioxidants for protecting the oils against oxidation. Leaves of whole green tea (Camellia sinensis) contain polyphenols, which are known to act as radical scavengers and reactive oxygen quenchers (Zokti, Sham Baharin, Mohammed, & Abas, 2016). Green tea catechins have been shown to retard oxidation in different lipid systems (Frankel, Huang, & Aeschbach, 1997; Huang & Frankel, 1997; Rababah, Hettiarachchy, & Horax, 2004). Green tea extract and -tocopherol improve the oxidative stability of fish oil and high linoleic sunflower oil (Dwyer, O’Beirne, Ní Eidhin, & O’Kennedy, 2012; Yin, Becker, Andersen, & Skibsted, 2012). Tea polyphenols also significantly prevent the oxidation of tree peony seed oil at the concentration of 0.04% (Bai et al., 2018). Maltodextrin was chosen to be used in combination with matcha in some formulations as it is commonly used in combination with proteins for encapsulation of tuna oil (Hogan, O'riordan, & O'sullivan, 2003). Maltodextrin has also been reported to protect tea catechins through the encapsulation process (Tengse, Priya, & Kumar, 2017). Apart from its potential for improving the oxidative stability of oil, the combination of tuna oil and tea catechins has synergetic effects on nutritional functionalities such as anti-amyloidogenic properties and prevents mitochondrial dysfunctions (Dwyer,

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O’Beirne, Ní Eidhin, & O’Kennedy, 2012; Vacca & Valenti, 2015). This makes the combination of matcha and fish oil an attractive functional food ingredient. It was hypothesised that matcha would serve as an effective delivery vehicle for omega-3 oils in powdered formats due to its inherent gross composition and presence of natural antioxidants in green tea. In this study, matcha alone or in combination with maltodextrin was used for the formulation of oil-in-water emulsions, which were subsequently spray dried. The physical and chemical characteristics of tuna oil emulsions and powders formulated with matcha alone or matcha in combination with maltodextrin were examined. The oxidative stability of the oil and the powders was assessed under accelerated conditions using an Oxipres. In addition, the % remaining EPA and DHA, and the loss of major catechins over 12 weeks storage at 40C were determined.

2. Materials and methods 2.1 Material and chemicals Tuna oil (TO) was purchased from Nu-Mega Ingredients Pty Ltd. (Australia). Matcha powder was obtained from Zhejiang Tea Group Co., Ltd. (China), and tapioca maltodextrin (MD, DE10) was obtained from Melbourne food ingredients depot (Australia). The reference fatty acid methyl esters standards (GLC-569, GLC-411; Nu-Chek Prep) and saturated triglyceride heptadecanoic acid (C17:0 TAG; Nu-Chek Prep) were purchased from Adelab Scientific (Australia). Hexane, HCl, dichloromethane, toluene, 3 N methanolic HCl, Nile red, Fluorescein

5(6)-isothiocyanate

(FITC),

(-)-epigallocatechin

gallate

(EGCG),

(-)-

epigallocatechin (EGC), (-)-gallocatechingallate (GCG), (-)-epicatechin gallate (ECG), (-)epicatechin (EC), (-)-gallocatechin (GC), (-)-catechin gallate (CG), and (+)-catechin (C) were purchased from Sigma-Aldrich (New South Wales, Australia). Methanol, formic acid, ethanol, diethyl ether, and petroleum spirit were obtained from VWR chemicals (Queensland, Australia).

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2.2. Chemical analyses of raw ingredients 2.2.1. Chemical analysis of matcha The moisture content was measured by drying at 105 °C until constant weight was obtained (In & Horwitz, 1990). The nitrogen content was determined using the Leco (Leco Corp., St Joseph, MI, USA), and calculated with conversion factor of 6.25. Total fat was determined according to Australian Standard 2300.1.30. The ash content was measured by incineration of samples in a muffle furnace at 760 °C. Insoluble fibre and soluble fibre were outsourced to an analytical laboratory (DTS Food Assurance, Melbourne, Australia) using an ANKOM dietary fibre analyser. For the determination of eight individual catechins (total catechins) and caffeine, matcha (0.15 g) was extracted with 25 mL 50% ethanol for 30 min at 70 °C and cooled down to room temperature. Then the supernatant was filtrated through a 0.22 μm membrane and sampled for HPLC analysis (Bae, Ham, Jeong, Kim, & Kim, 2015; Liang et al., 2007). An Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm) was used at temperature of 32 °C. Injection volume was 10 μL and flow rate was set at 1 mL min−1. Peaks were detected at 280 nm. The mobile phase A = acetonitrile/acetic acid/ water (6:1:193, v), mobile phase B = acetonitrile/acetic acid/ water (60:1:139, v), linear gradient elution: from 75% (v) A and 25% (v) B to 35% (v) A and 65% (v) B during the first 35 min and then 75% (v) A and 25% (v) B until 40 min. The standards of epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG) and (-)-epicatechin (EC), (+)-gallocatechin (GC), (+)-catechin (C), (-)-gallocatechin gallate (GCG), (-)-catechin gallate (CG) and caffeine were used for quantification. The glass transition (Tg) temperature of matcha was measured using differential scanning calorimetry (DSC). Briefly, samples (10 mg) were placed in 40 L aluminium crucibles (ME27331, Mettler-Toledo limited). The sample was cooled to −35 °C, heated to 90 °C, then cooled to −35 °C and heated to 150 °C, with a constant rate of 5 °C /min. The midpoints of step change

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in slope was taken as Tg.

2.2.2. Chemical analysis of tuna oil and lipid fraction of matcha The fatty acid methyl ester (FAME) composition of tuna oil, matcha powder and the matchatuna oil powders was analysed by gas chromatography (GC). The 75 L and 50 L of C17:0 TAG internal standard solution (10 mg/ml in toluene) was added to the powder sample (10 ± 0.01 mg) and oil sample (5.0 ± 0.01 mg) weighed in argon-flushed 2 mL GC vial respectively. The mixture of the powder/oil with the internal standard solution were resuspended in 1N methanolic HCl (0.9 mL) and dichloromethane (0.1 mL). Then the vial was blanketed under argon and tightly sealed. This mixture was incubated in a water bath shaker at 100 rpm and 80 °C for 2 h. After incubation, 0.3 mL of 0.9% NaCl was added to the vial, and FAME were extracted with 0.3 mL hexane (centrifuged at 1700 ×g, room temperature for 5 min). The upper phase (transesterified fatty acids, FAME) was transferred to a new GC vial followed by adding with 0.3 ml hexane for GC analysis. The GC analysis conditions were according to published paper (Shen, Augustin, Sanguansri, & Cheng, 2010). FAME solution (1 µL) was injected at a split ratio of 1:50 into the GC column (BPX 70 fused silica column, 30 m, 0.25 mm i.d. and 0.25 μm films, SGE, Australia), installed in a 7890A GC system (Agilent Technologies Australia Pty Ltd., Mulgrave, Victoria, Australia) equipped with an Agilent Technologies 7693 auto-sampler and a flame ionization detector (FID). The GC column was programmed from 150 to 210 °C at a rate of 3 °C/min, then increasing at a rate of 50 °C/min to a final temperature of 240 °C. The injector and detector (FID) temperatures were held at 240 and 250 °C, respectively. Agilent Chemstation software [B.04.02 SP2 (256)] was used to integrate the GC peak areas Fatty acids in samples which were identified on the basis of retention times of the reference fatty acid methyl ester standards. Individual polyunsaturated fatty acids contents (eicosapentaenoic acid, EPA, C20:5ω3 and docosahexaenoic acid, DHA, C22:6ω3) in the sample (mg fatty acid/g dry weight) were

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calculated as described in the AOCS (2009) official method (Let, Jacobsen, & Meyer, 2007) and the % fatty acids profile was calculated from the fatty acid composition of the samples. As there is a contribution to the fatty acid profile from the fat inherent in the matcha and therefore influenced the amount of matcha in the powders, the EPA and DHA remaining were expressed as a % of the original content of these fatty acids in the powder formulations instead of the absolute amounts in various powders.

2.3. Preparation of powders Four tuna oil emulsions (15% total solids (TS)) were formulated with matcha alone or matcha-maltodextrin mixtures to obtain tuna oil in powder (25% w/w, dry basis) upon drying. Formulations were prepared with different protein: carbohydrate (CHO) ratio. The TS (15% w/w) and tuna oil content (3.75% w/w) were kept constant in all oil-in-water formulations. For the preparation of the tuna oil powders, the required amount of matcha or a mixture of mactha and maltodextrin was dispersed in water (45 °C) and stirred for 1 h. The mixture was heated to 60°C, followed by an addition of preheated tuna oil. The mixture was homogenised by a Silverson emulsifier-mixer (Silverson L4R, Silverson Machines Ltd., Chesham, Buckinghamshire, UK) for 3 min at maximum speed and further homogenized by a two-stage homogeniser at 250/100 bar (Avestin Emulsiflex C5, Avestin Inc., Ottawa, Ontario, Canada). The emulsions were spray dried using a lab-scale spray dryer (Armfield, Armfield Ltd, Ringwood, England), fitted with twin fluid nozzle operated at 1.5 bar pressure. The inlet air temperature was kept at 185 °C and outlet temperature was kept at 80 °C. The flow rate was 12 g emulsion/min. The spray dried tuna oil powders were stored at 20 °C. Two independent trials were carried out to produce the tuna oil powders. The calculated formulation of the powders and the ratio of protein to carbohydrate (CHO) are as follows: 

Matcha-TO powder 1: 75% matcha powder (protein:CHO=1:1.1 ) with 25% tuna oil on a dry basis

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

Matcha-MD-TO powder 2: 54% matcha and 21% maltodextrin (protein:CHO=1:2) with 25% tuna oil on a dry basis



Matcha-MD-TO powder 3: 43% matcha and 32% maltodextrin (protein:CHO=1:3) with 25% tuna oil on a dry basis



Matcha-MD-TO powder 4: 35% matcha and 40% maltodextrin (protein:CHO=1:4) with 25% tuna oil on a dry basis

2.4. Microscopy of emulsions One drop of the oil-in-water emulsion, either emulsions prior to drying or emulsions reconstituted by dispersion of the powder in water, was placed onto a slide and covered with a cover slip. The droplet was observed by a Leica DM6000B light microscope with Leica DFC450C camera (Leica Microsystems GmbH, Wetzlar, Germany) or a confocal laser scanning microscopy (Leica TCS SP5, Leica microsystems GmHB, Mannheim, Germany). Auto-fluorescence of unstained emulsions were observed (excitation: 488 nm, emission: 600680 nm). Emulsions, fluorescently labelled with FITC (excitation: 488 nm, emission: 510-530 nm, 0.1% in ethanol) and Nile red (excitation: 543 nm, emission: 610-630 nm, 0.1% in ethanol) were also visualised.

2.5. Viscosity of emulsions The viscosity of the emulsion was measured by a Brookfield Viscometer (Model DVII, Brookfield Engineering Laboratories Inc., Middleboro, Massachusetts, USA) with RV Spindle 4 Attachment at different shear rate (0.1 – 1000 s-1) at 60 °C. The temperature of 60 °C was chosen for measurement as this was the temperature of the feed into the dryer.

2.6. Particle size distribution measurement The particle size distribution of matcha dispersed in water, emulsions prior to spray drying and spray dried emulsions re-constituted in water were measured by a MasterSizer 3000 laser

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diffraction particle size analyser with wet system (Malvern Instruments Ltd., Malvern, Worcestershire, UK).

2.7. Characteristics of powders 2.7.1. Composition The moisture content of the tuna oil encapsulated powders (1 g) was measured by a Sartorius MA 30 Moisture Analyser (Sartorious AG, Goettingen, Germany), where the sample was heated at temperature of 105 °C until constant weight (Kristensen, Felby, & Jørgensen, 2009). The total fat was measured according to Australian Standard 2300.1.30. The surface free fat content of powders was analysed according to a published method with slight modification (Kim, Chen, & Pearce, 2005). Briefly, powder (1 g) was placed on a filter paper (No. 4, Whatman, Maidstone, Kent, UK), and washed with 5 mL petroleum ether through a funnel and into a flask. The solvent was evaporated under vacuum (Buchi Rotavapor R-124, Essen, Germany), and the remaining fat dried at 102 °C for 1 h and expressed as % surface free fat in powder. The encapsulation efficiency (%) was calculated as [100(Total fat - surface fat)] / total fat].

2.7.2. Bulk density and Hausner ratio The poured powder bulk density was measured by placing 5 g of powder into a 25 mL graduated cylinder. Poured bulk density was determined by dividing weight of the sample by its volume (g/ml) (Binsi et al., 2017). The cylinder was then tapped for 180 times using a Stampfvolumeter (J. Engelsmann AG, Germany) to obtain the tapped density. After tapping, the ratio of sample weight and volume (g/ml) was calculated as tapped bulk density. The flowability (Hausner ratio) was measured by the ratio of tapped bulk density to poured bulk density (Abdullah & Geldart, 1999).

2.7.3. Water activity and dynamic vapour sorption isotherms 10

The water activity of the powders was measured by an Aqualab water activity meter 4TE (METER). Water vapour sorption properties of matcha, maltodextrin and tuna oil powders were determined at 25 °C by a dynamic vapour sorption system (DVS Series 2000, Surface Measurement System Ltd., London, UK) according to Ying et al. (Ying, Sun, Sanguansri, Weerakkody, & Augustin, 2012). The sorption isotherms were also expressed as a function of the whole powder as well as non-fat solids (encapsulant only) of the powders.

2.8. Assessment of oxidative stability 2.8.1. Oxygen uptake The oxidative stability of neat tuna oil and microencapsulated tuna oil powders (containing an equivalent of 3 g tuna oil) was analysed by an ML Oxipres apparatus installed with Paralog software (DK-8270, Mikrolab Aarhus A/S, Højbjerg, Denmark) at 80 °C and 5 bar oxygen pressure. The decrease in oxygen pressure was recorded against time. The induction period (IP) is a point when oxygen pressure drops significantly which marks the starting point of accelerated oxidation of the sample. As controls, matcha alone (9 g – equivalent to the matcha amount of Matcha-TO powder 1) and a physical mix of matcha and tuna oil (9 g matcha plus 3 g tuna oil equivalent to Matcha-TO powder 1) were also tested.

2.8.2. Changes in composition of lipids during storage One gram of powder or 0.25 g of tuna oil was placed into 22 mL headspace vial. The vials were tightly sealed and stored at 40 °C for 12 weeks. Two sample vials including neat oil and powders from two spray drying trial batches were stored for at T=0, 4, 8 and 12 weeks. The stored samples were used for FAME analysis. The % remaining EPA and DHA during the storage time was determined.

2.8.3. Changes of four major catechins at 12 weeks The storage vials from Section 2.8.2 were used for tea catechins extraction. For the determination of catechins in matcha (0.05 g), samples were extracted with 10 mL 50% ethanol 11

for 30 min at 70 °C and cooled down to room temperature. Then the supernatant was filtrated through a 0.22 μm membrane and sampled for Waters Acquity UHPLC analysis. An BEH C18 column (1.7µm, 2.1×50 mm) was used at temperature of 40 °C. Injection volume was 3 μL and flow rate was set at 0.3 mL min−1. Peaks were detected at 280 nm. The mobile phase A = 0.1% formic acid in water, mobile phase B = 0.1% formic acid in acetonitrile, gradient elution from 85%A/15%B to 70%A/30% B during the first 35 min and then to 85%A /15%B till 40 min. The standards of the four major catechins (EGC, EC, EGCG and ECG) were used for quantification.

2.9. Statistical analysis All measurements were conducted in triplicate. Statistical significance of EPA and DHA percentage remaining (%) and catechins amount losses were analysed by one-way ANOVA on Origin 8.0 (Origin Lab Inc., MA, USA). The Pearson’s correlation coefficients were determined using Origin 8.0 software.

3. Results and discussion 3.1. Characteristics of raw ingredients 3.1.1 Composition of matcha The moisture content of matcha was 2.2%. On a dry weight basis, matcha had 35.5% crude protein (Nx6.25), 5.9% fat, 40% dietary fibre (33.2% insoluble and 6.8% soluble), 6% fat, 3.5% sugars, 6.0% ash, 3.2% caffeine and 13.0% total catechins. Crude protein was likely overestimated when the conversion factor of 6.25 is used. There is no published conversion factor for tea leaves. However, from studies of leaves of 90 plant species, lower conversion factors of 3.28-5.15 have been found to be more appropriate (Yeoh & Wee, 1994). If these conversion factors were used the calculated protein content would be 18.6-29.2%. The chemical composition of matcha is close to or within the range of reported values of 15.0-30.7% protein, 5.3-8.0% fat, 2.4-3.0% sugar, 5-7.4% ash, 3.2-3.9% caffeine, 9.4-17.9% 12

tea catechins (Graham, 1992; Lvova, Legin, Vlasov, Cha, & Nam, 2003; Yamamoto, Juneja, & Kim, 1997; Goto, Nagashima, Yoshida, & Kiso, 1996 ). EGCG, EGC, ECG and EC were found to be the major catechins with 8.7, 2.4, 1.3 and 0.3% respectively while C, GCG, GC and CG were found in found at trace levels (in total less than 0.2%). The measured value for EGCG is within that reported in the literature, where EGCG in matcha ranges from 5.9-10.1% (Weiss & Anderton, 2003; Goto, Nagashima, Yoshida, & Kiso, 1996). The content of EGC, ECG and EC in most variety of matcha were 1.6-4.3, 1.1-2.0 and 0.5-1.1, while the total C, GCG, GC and CG content less than 0.5% (Goto, Nagashima, Yoshida, & Kiso, 1996). Our results were close to the reported values. Apart from the water soluble phytonutrients, matcha has been reported to contain lipid soluble phytonutrients such as quercetin (0.18%, dry basis), beta-carotene (0.03%, dry basis), kaempferol (0.13-0.26%, dry basis), myricetin (0.08-0.1%, dry basis) and proanthocyanidins. (1.2-3.0%, dry basis) (Perva-Uzunalić, Škerget, Knez, Weinreich, Otto, & Grüner, 2006; Peterson et al., 2005; Turkmen, Sarı, & Velioglu, 2009).

3.1.2 Fatty acids composition of tuna oil and oil extracted from tea leaves The tuna oil composition was 20.7% palmitic acid (C16:0), 5.3% C18:0, 13.3% oleic acid (C18:1), 7.7% EPA, 30.6% DHA and 22.4% of other fatty acids. The fatty acids of the lipid extracted from matcha contained 17.5% palmitic acid (C16:0), 2.3% C18:0, 6.2% oleic acid (C18:1), 18.4% -linoleic acid (C18:2), 50.6% linoleic acid (C18:3) and 5.0% of other fatty acids. Palmitic, oleic, linoleic and -linoleic acids were reported to be present in higher amounts than other fatty acids in green tea (Bhuyan, Tamuly, & Mahanta, 1991).

3.2 Characterization of matcha dispersions and oil-in-water emulsions 3.2.1 Viscosity of emulsions prior to drying The initial viscosity of all emulsions (15% TS) were 0.8 - 10 Pa.s at a shear rate of 0.1 1/s. Although the initial viscosity of the emulsions was high, it was possible to spray dry the emulsions as they exhibited shear thinning characteristics (data not shown). The viscosity 13

significantly decreased to < 200 mPa.s at a shear rate of 100 1/s. This viscosity is equivalent to the shear rate at the nozzle during atomisation at velocity of 150 m/s (Rozali, Paterson, Hindmarsh, & Huffman, 2019). A low viscosity of the pre-spray drying emulsion decreases the air inclusion in the powder resulted from the droplet ballooning during drying (Drusch, 2007).

3.2.2 Microscopy and particle size analysis Fig. 1 (A1 and A2) shows the light micrographs of the matcha dispersed in water and emulsions prior to drying and after reconstitution of tuna oil powders in water. There were signs of flocculated particles (>400µm) in all samples at 15% TS. Flocculation decreased with the addition of maltodextrin in the formulation. Depletion flocculation has been observed previously in caseinate-stabilised fish oil emulsions beyond a critical level of polymer (caseinate) concentration (Day, Xu, Hoobin, Burgar, & Augustin, 2007). Depletion flocculation can also be increased with the increasing of polymer concentration, and the dispersion could be stabilizing at higher polymer concentration due to the depletion stabilization (Feigin & Napper, 1980; McClements, 2000). Fig. 1 (B1, B2, C1 and C2) shows the confocal micrographs of matcha, the emulsions (15% TS) prior to drying and after reconstitution of tuna oil powders in water. There was evidence of autofluorescence in all samples. The data suggests that most of the auto fluorescent compounds present in matcha were partitioned into the oil phase when tuna oil was added to make tuna oil – matcha stabilised emulsions. The emulsions prior to drying contained small oil droplets (Fig. 1, B1 and C1) which auto fluoresced and there appeared to be some coalescence of oil droplets upon drying, as evidenced by the larger oil droplets in reconstituted emulsions (Fig. 1, B2 and C2). This is probably partly due to the coalescence of oil droplets during reconstitution (Turchiuli, Munguia, Sanchez, Ferre, & Dumoulin, 2014). In diluted emulsions used for analysis of particle size using light scattering, the particle size distribution of emulsions prior to drying did not show the presence of aggregates > ~100 μm,

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suggesting that particles were dispersed upon dilution (Fig. 2a). The D[4,3] of the emulsions prior to drying used for formulations Matcha-TO powder 1, Matcha-MD-TO powder 2, Matcha-MD-TO powder 3, and Matcha-MD-TO powder 4 were 35.1, 31.1, 29.8 and 29.0 μm respectively, showing that the addition of maltodextrin slightly reduced particle size. The D[4,3] of Matcha-TO powder 1, Matcha-MD-TO powder 2, Matcha-MD-TO powder 3, Matcha-MDTO powder 4 reconstituted in water were 41.0, 37.0, 32.3 and 31.0 μm respectively (Fig. 2b), which were bigger than the emulsions before drying, possibly due to flocculation of particles and inefficient reconstitution. In all emulsions, there were spherical particles (i.e. oil droplets) between 0.1 to 1 μm in diluted emulsions in light scattering data (Fig. 2a) in addition to larger oil droplets seen in the micrographs (Fig. 1, B1 and C1). The presence of small size of the droplets suggested that there was efficient homogenisation of the tuna oil with matcha alone or with matcha-maltodextrin. The proteins components in the matcha are expected to stabilise the interface of the fat globules. Proteins are known to have good emulsifying properties and have been commonly used for stabilisation of emulsions (Lam & Nickerson, 2013; McClements, 2004). Proteins have also been used in combination with carbohydrates such as maltodextrins. These non-surface active carbohydrate form part of the matrix for delivery (Bae & Lee, 2008; de Barros Fernandes, Borges, & Botrel, 2014).

3.3 Powder properties 3.3.1 Characteristics of powders The visual appearance of different formulations and selected powder characteristics are shown in Table 1. Powders had an olive green colour. Matcha had a moisture content of 2.71%, a water activity of 0.28 and a glass transition temperature (Tg) of 43 ºC. The Tg of the matcha used was lower than the reported value 45.8 ºC at the water activity of 0.43 (Li, Taylor, & Mauer, 2011). This could be explained by the different composition and varieties

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of tea. The moisture contents of tuna oil powders were 2.77 to 3.61 % with water activity of 0.10 to 0.16 (Table 1). The total fat content ranged from 27.1 to 28.4%, with the calculated tuna oil after accounting for fat from matcha component being 24-26%. The surface free fat and encapsulation efficiency of powders ranged from 2.9-4.3% and 84.9- 89.3% respectively (Table 1). The poured bulk density of the samples ranged from 0.25 to 0.36 g/ml. The tapped bulk density ranged from 0.33 to 0.43 g/mL. The poured bulk density and tapped bulk density were higher with an increasing maltodextrin content. This trend is similar to that for the bulk density of spray dried mountain tea powder, which was higher with an increase in the maltodextrin concentration of the feed (Nadeem, Torun, & Özdemir, 2011). A comparison of bulk density between various powders is difficult as it is affected by many factors, such as chemical composition, particle size, moisture content and processing conditions (Beristain, Garcıa, & Vernon-Carter, 2001). The Hausner ratio is an indicator of the flowability of powders in a wide range of industries (Grey & Beddow, 1969). The Hausner ratio of the tuna oil powders ranged from 1.31 to 1.19 (Table 1). A Hausner ratio greater than 1.22 is an indication of poor flowability, and a Hausner ratio below 1.18 is regarded as good flowability (Hao, 2015). Adding maltodextrin resulted in improved flowability, which was similar to that previously reported for sumac powder formulations (Caliskan & Dirim, 2016). 3.3.2 Water sorption The water sorption isotherm for matcha, maltodextrin and tuna oil powders formulated with matcha or matcha-maltodextrin mixtures are shown in Fig. 3. All the isotherms showed a nonlinear and sigmoidal shape (type II), typical for food materials. Maltodextrin had higher water uptake capacity compared with matcha. Each material will have a different water sorption property that is an inherent characteristic of the material, which is dependent on its structure. In the case of matcha, there is a mixture of components in a matrix that has the

16

ability to adsorb water, they will have contributions to the observed uptake which will also be impacted by processing. After encapsulation, the water uptake for tuna oil powders decreased due to the oil content (25%, w/w) (Fig. 3a). The moisture uptake was also plotted as a function of the non-fat solids (Fig. 3b) as the contribution of fat to water uptake is negligible (Maher, Roos, & Fenelon, 2014). The expected trend of water uptake as a function of non-fat solids was Matcha-MD-TO powder 4> Matcha-MD-TO powder 3> Matcha-MD-TO powder 2> Matcha-TO powder 1. Generally, the water uptake of tuna oil powders formulated with matcha and maltodextrin (on a non-fat solid basis) was between that for maltodextrin alone and matcha alone as might be expected. However, there were differences between water uptake on a non-fat encapsulant matrix basis for matcha alone or matcha-tuna oil powder. This suggested that there were process-induced changes during the processing of the matchatuna oil powders. A possible explanation is that the process increased the solubility of matcha component, which contributed to the higher water uptake of matcha-tuna oil powders. Knowledge about water sorption properties is useful it has an influence on the flowability and storage stability of powders when exposed to different humidity environments during transport and storage.

3.4. Assessment of oxidative stability of microcapsules 3.4.1 Oxygen uptake The oxidative stability of matcha, tuna oil, a physical mixture of matcha and tuna oil and spray dried emulsions using Oxipres® System is shown in Fig. 5. The decrease in oxygen pressure is directly related to rate of sample oxidation. The order in oxidative stability of samples were: Matcha > Matcha-TO powder 1 ~ Physical Mix of Matcha and TO > MatchaMD-TO powder 2 > Matcha-MD-TO powder 3 > Matcha-MD-TO powder 4 > neat TO. The same trend in oxygen uptake for the different oil powders was obtained for both production batches of powders produced on independent occasions.

17

There is no clear IP (h) for Matcha, Matcha-TO powder 1 and Matcha-TO powder 1 (physical mix). When IP is not clear, a slow decline means that the sample is more stable. When IP is obvious, a longer IP (h) indicates a more oxidatively stable sample. IP were only obvious for Matcha-MD-TO powder 3 (66.3h), Matcha-MD-TO powder 4 (21.9h) and neat TO (9h). The absence of an obvious IP in other powders was possibly due to the fact that the samples had not reached the auto-oxidation state for the fat. The results indicated that matcha had potential for stabilising tuna oil against oxidation, with the protection afforded to the oils being greater with increasing amount of matcha in the formulation. The physical mixture of matcha and tuna oil and the equivalent spray dried Matcha-TO powder had similar rate of decline in oxygen pressure. This suggested the antioxidants in matcha were the most significant contributor to the oxidative stability of tuna oil. Although it has been suggested that surface free fat oxidised at a faster rate than encapsulated oil, resulting in rancidity and off-flavours (Chang et al., 2018), this trend did not hold for matcha-tuna oil powders (Compare Table 1 and Fig. 4). 3.4.2 Fatty acid composition of microencapsulated tuna oil The EPA and DHA contents of the original tuna oil were 60.5 mg/g and 245.5 mg/g respectively. The tuna oil powders (~24-26% tuna oil, dry basis) contained the following levels of EPA and DHA after powder manufacture on a mg/g sample basis: Matcha-TO powder 1 (15.0% EPA, 60.7% DHA), Matcha-MD-TO powder 2 (14.7% EPA, 59.6% DHA), MatchaMD-TO powder 3 (15.1% EPA, 60.9%) and Matcha-MD-TO powder 4 (16.1% EPA, 65.2% DHA). EPA and DHA deceased over the 12-week storage at 40 C, with the oxidation of DHA being faster than that of EPA as expected (Fig. 5). The protection afforded to EPA and DHA increased with the increasing level of matcha in the formulation and the stability of EPA and DHA was enhanced in all formulations compared to the neat oil. The stability of the tuna oil in the powders was Matcha-TO powder 1 > Matcha-MD-TO powder 2 > Matcha-MD-TO powder

18

3 > Matcha-MD-TO powder 4. The EPA and DHA remaining in Matcha-TO powder 1 were 65.5% and 58.6% respectively at 12 weeks, which was significantly higher compared with the neat oil with the value of 14.7% and 6.0% respectively (p<0.05) (Fig. 5). The same trend in oxidative stability of EPA and DHA for the different oil powders was obtained for both production batches of powders produced on independent occasions. Tea constituents are effective antioxidants for lipid system and are responsible for the protecting oil against oxidation (Gramza & Korczak, 2005). Matcha is particularly rich in catechins. While catechins are water soluble, they are also partitioned into the lipid phase (Moen, Stoknes, & Breivik, 2017). In addition, there are also lipid soluble phytonutrients such as quercetin and carotene present in matcha which also have anti-oxidative properties. It is suggested that the mix of the anti-oxidative phytonutrients, which are partitioned between the aqueous and oil phase, work synergistically to protect oils against oxidation in emulsion systems. 3.4.3 Losses of catechins Table 2 shows the contents of EGC, EC, EGCG and ECG before and after 12 weeks storage at 40C. The catechin compounds significantly (p<0.05) declined after 12 weeks storage. The % of total four catechins lost were similar among the formulations, with loss being 19.6-20.8%. The amount of catechin lost (mg/g powder, dry basis) after 12 weeks were: 8.2 mg/g EGC, 7.6 mg/g EGCG, 3.0 mg/g ECG and 0.9 mg/g EC for Matcha-TO powder 1, 7.4 mg/g EGC, 4.7 mg/g EGCG, 1.7 mg/g ECG and 0.4 mg/g EC for Matcha-MD-TO powder 2, 5.8 mg/g EGC, 4.1 mg/g EGCG, 1.0 mg/g ECG and 0.4 mg/g EC for Matcha-MD-TO powder 3, as well as 3.9 mg/g EGC, 3.4 mg/g EGCG, 1.6 mg/g ECG and 0.3 mg/g EC for Matcha-MD-TO powder 4. The losses in EGC and EGCG were higher than the other catechins measured, which might be associated with their excellent protective effects against lipid oxidation. Chen and Chan (1996) reported the potency of catechins against the canola oil oxidation was in the order of

19

EGC>EGCG>EC>ECG. The postive correlation (r=0.948, p<0.01) was observed between the total amount of four catechins lost and the %remaining of (EPA+DHA), suggesting that the major catechins protected the oil aganist oxidation.

4. Conclusion Matcha, which has 35.5% crude protein, 40.0% dietary fibre and a high level of natural antioxidants (including 13.0% tea catechins), can be used in the formulation of tuna oil emulsions which are subsequently spray dried. The protein and carbohydrate components in matcha provide the structural components for effective encapsulation. The high level of catechins in matcha protected the omega-3 fatty acids in matcha-tuna oil and matchamaltodextrin-tuna oil powders against oxidation. These results indicate that matcha have the potential to be used as the matrix for delivering fish oil and other polyunsaturated oils in a convenient powder format, whilst also affording protection of sensitive lipids against oxidation. For future work, studies on the sensory properties of matcha-tuna oil powders will be required to determine the sensory appeal of these powders. It would be also desirable to examine the stability of the catechins and bio-accessibility of the matcha components during in vitro digestion. It would be important to assess the bioavailability of the oil and especially the omega-3 fatty acids, when the matcha-tuna oil powder is consumed as a supplement or incorporated into a food matrix. Conflict of interest The authors declare no competing financial interest. Acknowledgements Meng Shi gratefully acknowledges the support of China Scholarship Council. References Abdullah, E. C., & Geldart, D. (1999). The use of bulk density measurements as flowability indicators. Powder Technology, 102(2), 151-165.

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Captions of Figures Fig. 1. Light microscopy and Confocal Laser Scanning Microscopy (CLSM) of emulsions (15% TS, 3.8% tuna oil) and spray dried powder re-constituted in water (15% TS). A1: light microscopy of emulsion; A2 light microscopy of powders; B1 CLSM - autofluorescence of emulsions; B2 CLSM-autofluorescence of powders; C1 CLSM - FITC (green = protein) & Nile red (red = fat) stained emulsions; C2 CLSM – FITC & Nile red stained powders. (see formulation details in section 2.3). Fig. 2. Particle size distribution of emulsions (15% TS, 3.8% tuna oil) and matcha dispersion in water (a); and particle size distribution of spray dried powders reconstituted in water (b). (See formulation details in section 2.3). Fig. 3. Dynamic vapour sorption isotherms of 25% tuna oil powders (a); and corresponding encapsulant matrix (EM) without oil (b). (See formulation details in section 2.3). Fig. 4. Oxipres test result showing the induction period (h) at 80 C of tuna oil, 25% tuna oil encapsulated powders, matcha powder and physical mix of matcha powder and oil. (Oxygen uptake for Matcha-TO powder and Physical Mix of Matcha and Tuna oil are the same; See formulation details in section 2.3). Fig. 5. Percentage remaining (%) of EPA (a) and DHA (b) in spray dried powders (25% w/w tuna oil, dry basis) after 0, 4, 8 and 12 weeks storage at 40 ºC. (See formulation details in section 2.3) All measurements were carried out in triplicate and expressed as mean ± SD; Different capital letters (A-E) among samples indicate a significant difference at a specific storage time (p<0.05); Different letters (a-d) of each sample indicate a significant difference during storage (p<0.05). 26

Table 1 The characterisation of powders (spray dried emulsions) a. Formula

Matcha-

Matcha-

Matcha-

Matcha-

TO

MD-TO

MD-TO

MD-TO

powder 1

powder 2

powder 3

powder 4

L*

50.8±0.9

52.3±0.3

55.0±0.5

57.1±0.8

a*

-10.1±0.3

-9.9±0.2

-9.7±0.2

-8.5±0.2

b*

33.9±0.5

35.2±0.8

36.1±0.6

35.6±0.8

Moisture (%)

3.61±0.12

2.79±0.14

2.77±0.08

3.24±0.07

aw

0.16±0.00

0.12±0.00

0.10±0.00

0.14±0.00

0.25±0.01

0.29±0.00

0.33±0.01

0.36±0.01

Tapped bulk density V180 0.33±0.01

0.38±0.00

0.42±0.01

0.43±0.01

1.30±0.04

1.26±0.00

1.19±0.02

Visual Appearance Colour

Bulk density (g/ml) Poured bulk density V0 Hausner ratio (V180/V0)

1.31±0.05

Total fat (%)

28.39±0.09 27.01±0.35 28.27±0.28 27.20±0.24

Matcha lipidb

4.5%

3.2%

2.6%

2.1%

Tuna oil (By difference)

23.9%

23.8%

25.7%

25.1%

4.28±0.11

2.90±0.08

3.61±0.01

3.43±0.09

84.9±0.4

89.3±0.3

87.2±0.0

87.4±0.3

Surface free fat (% in powder) Encapsulation efficiency (%) c a

All measurements are the mean ± standard deviation of each sample group from two batches of

experiments. b

Calculated using determined level of lipid in matcha powder (5.9%, dry basis) and formulated amount

of matcha in powders. c

Encapsulation efficiency (%)= [100 x (total fat – surface fat)/total fat]

(See formulation details in section 2.3)

27

Table 2 Content of selected catechins and total catechins (mg/g powder, dry basis) in tuna oils powders after manufacture (T=0 week) and after 12 weeks storage at 40°C (T=12 weeks). (See section 2.3 for formulation of powders)a Sample

EGC

EC

EGCG

ECG

Total four catechins

Matcha-TO

T=0 week

17.0±0.0 a

2.4±0.0 a

powder 1 T=12 weeks

8.8±0.1 b

1.5±0.1 b

64.0±0.9

11.5±0.2

95.0±0.7 a

a

a

57.0±1.6

8.5±0.5 b

75.9±2.2 b

b

Matcha-MD-

Amount lost

8.2±0.2

0.9±0.1

7.6±1.0

3.0±0.7

19.7±1.7

% Lost

48.0±0.8

37.3±5.0

11.9±1.6

26.2±5.3

20.8±1.8

T=0 week

14.7±0.3 a

1.2±0.1 a

47.5±0.3

8.5±0.2 a

71.9±0.6 a

6.8±0.0 b

57.3±0.1 b

TO powder 2

a

T=12 weeks

7.3±0.5 b

0.8±0.0 b

42.4±0.4 b

Matcha-MD-

Amount lost

7.4±0.1

0.4±0.1

4.7±0.4

1.7±0.2

14.5±0.6

% Lost

50.0±3.2

32.8±3.3

10.7±3.3

20.3±1.4

20.2±0.5

T=0 week

11.0±0.0 a

1.1±0.1 a

38.0±0.2

6.9±0.0 a

57.0±0.3 a

5.9±0.1 b

45.9±1.2 b

TO powder 3

a

T=12 weeks

5.3±0.1 b

0.4±0.1 b

34.0±1.2 b

Matcha-MD-

Amount lost

5.8±0.1

0.4±0.0

4.1±0.2

1.0±0.0

11.2±1.3

% Lost

52.0±1.3

38.2±2.6

10.6±3.3

14.2±0.4

19.6±2.2

T=0 week

8.6±0.0 a

0.8±0.0 a

30.6±0.0

5.6±0.0 a

45.6±0.1 a

4.0±0.1 b

36.4±0.2 b

TO powder 4

a

T=12 weeks

4.7±0.0 b

0.5±0.0 b

27.2±0.2 b

a Amount

Amount lost

3.9±0.1

0.3±0.1

3.4±0.3

1.6±0.1

9.2±0.3

% Lost

45.2±0.5

35.8±11.7

11.1±0.8

29.1±1.2

20.2±0.6

lost = Amount lost after 12 weeks = Amount at T=0 week – Amount at 12 weeks, % Lost =

(100 x Amount lost after 12 weeks/ Amount at T=0 week; All measurements are the mean ± standard deviation of each sample group from two batches of experiments; Different letters (a-b) within a column of the sample represent significant differences (p<0.05) in the selected catechin after 12 weeks storage.

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Graphical abstract

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Highlights



Green tea (matcha)–tuna oil emulsions were used for producing 25% tuna oil powders



Matcha was superior to matcha–maltodextrin for reducing oxidation in oil powders



The catechins in matcha may have protected omega-3 fatty acids against oxidation

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

The authors Meng Shi, DanYang Ying, Mya Myintzu Hlaing, JianHui Ye, Luz Sanguansri, Mary Ann Augustin do not have any conflict of interest

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Author Credits M Shi performed the experiments and analysed the data. MA Augustin and L Sanguansri developed the concept and the experimental design. M Hlaing performed fatty acid analysis and interpreted the data. DY Ying carried out powder production trials and interpreted data on dynamic vapor sorption. J-H Ye contributed to cosupervision of the work.

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