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Recovery of Phenolic Antioxidants from Green Kiwifruit peel using

Subcritical Water Extraction

Francesca Guthrie Yiting Wang Natasha Neeve Siew Young Quek

Khosrow Mohammadi Saeid Baroutian

PII: S0960-3085(20)30423-5
DOI: https://doi.org/doi:10.1016/j.fbp.2020.05.002
Reference: FBP 1254

To appear in: Food and Bioproducts Processing

Received Date: 8 April 2020

Revised Date: 2 May 2020
Accepted Date: 6 May 2020

Please cite this article as: Guthrie, F., Wang, Y., Neeve, N., Quek, S.Y., Mohammadi, K.,
Baroutian, S.,Recovery of Phenolic Antioxidants from Green Kiwifruit peel using Subcritical
Water Extraction, Food and Bioproducts Processing (2020),
doi: https://doi.org/10.1016/j.fbp.2020.05.002

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Highlights

Highlights

 Subcritical water effectively extracted phenolic bioactives from kiwifruit peel.

 The highest yield was attained at 160°C and pH 2 for 20 min with 2% solid content.

 Significant correlation existed between antioxidant content and activity.

 Subcritical water showed a better extraction ability compared to ethanol.

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Graphical Abstract

Graphical Abstract

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Manuscript File Click here to view linked References

1 Recovery of Phenolic Antioxidants from Green Kiwifruit peel

2 using Subcritical Water Extraction
3 Francesca Guthrie 1, Yiting Wang 2, Natasha Neeve 1, Siew Young Quek 2, Khosrow
4 Mohammadi 3, Saeid Baroutian 1,*
1
5 Department of Chemical & Materials Engineering, Faculty of Engineering, The University of
6 Auckland, Auckland, New Zealand
2
7 School of Chemical Sciences, Faculty of Science, The University of Auckland, Auckland, New
8 Zealand
3
9 Department of Food Hygiene, Tabriz Branch, Islamic Azad University, Tabriz, Iran
10

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11 Abstract

12 This study investigated the recovery of phenolic antioxidants from kiwifruit peels using subcritical

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13 water extraction (SWE) method. The use of water is particularly advantageous when extracting

14 medically and commercially important phenolic compounds from food and food-processing by-
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15 products. The effects of extraction parameters (pH, temperature, solid:solvent ratio and time) on
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16 the recovery of polyphenols, flavonoids and the antioxidant activity of the extracts were studied.

17 The optimum antioxidant extraction was found to be 160 °C, pH 2, and 2% solid:solvent ratio
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18 using 20 minute extraction. Under the optimized condition, the total phenolic content (TPC) and
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19 total flavonoid content (TFC) were 51.2 (mg GAE/g DW) and 22.5 (mg CE/g DW), respectively.

20 Comparing the subcritical water with conventional ethanol extraction revealed that the method is
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21 technically and economically feasible for extracting antioxidants from kiwifruit peels.
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22 Keywords: Antioxidant; bioactive compounds; kiwifruit peel; polyphenols, subcritical water

23 extraction

24

25

26 Corresponding author: Saeid Baroutian (s.baroutian@auckland.ac.nz)

27

Page 3 of 26
28 List of abbreviations

SWE subcritical water extraction

TPC total phenolic content

TFC total flavonoid content

mg GAE/g DW milligrams of gallic acid equivalents per gram dry weight of kiwifruit peel

mg CE/g DW milligrams catechin equivalents per gram dry weight of kiwifruit peel

ROS reactive oxygen species

UV ultra violet

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CCD central-composite design

RSM response surface methodology

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DPPH 1, 1-diphenyl-2-picrylhydrazyl

FRAP ferric-reducing antioxidant power

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ABTS azino-bis 3-ethylbenzothiazoline-6-Sulphonic acid
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µL microlitre

UV/Vis ultraviolet–visible

M molar
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NaOH sodium hydroxide

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mL milliliter

nm nanometer
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mM TE/g DW millimolar Trolox equivalent per gram dry weight of kiwifruit peel
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TPTZ 2,4,6-tripyridyl-s-triazine

HCl hydrochloric acid

FeCl3 Ferric chloride

H2O water

v/v volume per volume

K2S2O8 potassium persulfate

Page 4 of 26
29 1. Introduction

30 Kiwifruit is notorious for its green-flesh, rich nutritious value and unique flavour (Lim et al., 2014).

31 Global kiwifruit production was 4.3 million tonnes in 2018. Both kiwifruit farming and processing

32 industry are growing worldwide which produce considerable amount of by-products and waste

33 such as kiwifruit peels and pomace (Reynolds et al., 2016). Since waste products are generally

34 transported to landfills, disposal of these by-products has escalated markedly in recent years due

35 to its negative impacts on the environment (Lam et al., 2014).

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36 Feeding, anaerobic fermentation, combustion, composting and charring are examples of

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37 established schema in phytowaste processing (Maroušek et al., 2020). From the environmental

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38 protection point of view, handling the large amount of phytowaste by traditional methods have a

39 series of disadvantages and often lead to environmental pollution. A key for phytowaste
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40 management which would be environmentally-friendly and technologically-promising is yet to be
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41 found. As regards biogas production, only about 5-10% of the energy is accessible for anaerobic

42 bacteria to transfer to methane, compared to 50% for the equivalent aerobic reaction (Feilden,
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43 1983). As for compost, it maintains nutrients in the soil by ion exchange mechanism and leads to

44 low quality (Maroušek et al., 2016). large amounts of nitrogen and chlorine were detected in
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45 phytomass which forms chloric acid and various carcinogenic components during combustion
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46 (Mardoyan and Braun, 2015).

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47 Kiwifruit industry has a high focus on sustainable production and utilisation of environmental

48 performance including efforts in waste minimisation, clean production, carbon footprint reduction,

49 use of water resources and reducing packaging and transportation. Whereas a large amount of

50 effort has been expended in finding alternative uses for kiwifruit pomace (Zhu et al., 2019), less

51 work has been done on utilisation of kiwifruit peels.

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52 Peels of fruits contain generally higher amounts of nutrients than the flesh, especially the

53 antioxidants (Remorini et al., 2008). Antioxidants perform as sacrificing substances in prevention

54 or inhibition of reactive oxygen species (ROS) development in countless biological systems, thus

55 they are proven to show a favourable reaction on inhibiting oxidation processes and improving

56 tissue damage caused by ROS (Gonçalves et al., 2013). Compressed propane and n-hexane

57 extraction revealed that kiwifruit seed oil is a superior source of α-linolenic acid around 60% of

58 the total fatty acids (Coelho et al., 2016). High content of unsaturated fatty acids and antioxidant

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59 capacity protected lymphocytes DNA from oxidative damage (Deng et al., 2018). Ethanol extract

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60 of kiwifruit peel potentiated pentobarbital-induced sleep in mice as dose-dependently decreased

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61 sleep latency and increased sleep duration (Yang et al., 2013).

62 The most commonly used method for recovery of phenolic antioxidants from fruit peels and plant
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63 materials is organic solvent extraction. Whilst this process is efficient, it is not ideal when the
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64 product is more likely ending up in functional food or pharmaceutical products. Therefore,

65 stringent solvent removal procedures are required whenever products or extracts are prepared for
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66 food or medicinal uses. Furthermore, the solvents themselves can be costly to obtain a high purity

67 and often cannot be easily disposed of, taking both time and money. Consequently, there is a need
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68 for extraction techniques and/or solvents that are non-toxic.

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69 Subcritical water (SWE) has been utilised to extract phenolic compounds from plants and algae
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70 (Zakaria and Kamal, 2016), potato peels (Singh and Saldaña, 2011a), green tea (Ko et al., 2014a),

71 grape marc (Todd and Baroutian, 2017), onion skin (Munir et al., 2018), carrot leaves (Song et al.,

72 2018), pomegranate seed (He et al., 2012), and kiwifruit pomace (Kheirkhah et al., 2019).

73 Subcritical water extraction (SWE) is a green and techno-economically viable alternative to

74 current solvent extraction techniques. a techno-economic study on the extraction of bioactive

75 phenolic compounds from New Zealand grape marc revealed that manufacturing cost by SWE

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76 (NZ$89.60/kg product) was equivalent to existing solvent extraction techniques (NZ$87.0/kg)

77 (Todd and Baroutian, 2017). Accordingly, SWE remains an applicable and encouraging

78 technology in food and pharmaceutical industries which is employed by commercial companies,

79 e. g. NZ Extracts Ltd. (New Zealand) and Mazza Innovation Ltd. (Canada). SWE utilizes water as

80 an extractant which is readily available and cheap, non-flammable, renewable and while

81 extraction, it does not form toxic by-products. Moreover, in comparison to traditional methods,

82 extraction time and steps are low with higher yield and purity of the extract (Liang et al., 2013).

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83 During SWE, the dielectric constant of water decreases significantly (Abdelmoez et al., 2014). The

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84 dielectric constant of water is a measure of its polarity and under elevated temperature of SWE,

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85 the reduced dielectric constant of water enhances its dissolution properties (Speight, 2005).

86 Therefore non-polar substances being more water-soluble in subcritical water (Kronholm et al.,
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87 2007). Furthermore, the mass transfer rate also increases due to the reduced viscosity and surface
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88 tension of the water as well as its increased diffusivity (Asl et al., 2013).

89 This study aims to investigate the effect of SWE on the quality and quantity of phenolic
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90 antioxidants extracted from kiwifruit peels. The results from SWE were compared with those

91 obtained from commonly used ethanol extraction. The extraction process was optimised by taking
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92 into consideration the highest yield of total phenolic and flavonoid compounds as well as the
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93 radical scavenging and antioxidant activity.

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94

95 2. Materials and Methods

96 2.1. Sample Preparation

97 Peels of ripe green kiwifruit (Actinidia deliciosa) cultivar ‘Hayward’ was obtained from a local

98 kiwifruit processing company (Kiwifruitz Ltd.). The peels were washed in a stainless steel sieve

99 with low pressure distilled water for 5 minute to remove impurities and were subjected to freeze

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100 drying (Laconic, FreeZone-12, USA & Canada) at −81 °C, for 2 days to preserve bioactive species

101 and ensure complete extraction. Dried samples were manually shredded and pulverised in a

102 commercial food grade high-speed multifunction blade grinder and then sieved to obtain an

103 average particle size of 200-500 µm. Size reduction of the substrates before extraction maximises

104 the surface area, which in turn enhances the mass transfer of bioactive compounds from the

105 kiwifruit peel to the solvent. Finally, powdered samples were mixed with deionised water using a

106 magnetic stirrer to make aqueous mixtures with 2-6 wt % total solids.

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107

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108 2.2. Extraction

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109 2.2.1. Subcritical Water Extraction (SWE)

110 SWE of phenolic compounds from kiwifruit peels was carried out using a high-pressure autoclave
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111 reactor (Amar Equipments Pvt. Ltd., Mumbai, India). The extraction vessel was filled with 500
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112 mL of the aqueous mixture of kiwifruit peels following the vessel was purged with N2 gas to

113 remove air from the extractor and was then pressurised to 30 bar with N2 gas to keep the aqueous

mixture in the liquid state during the extraction process. Extracts of 15 mL were taken at time
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114

115 intervals of 0, 5, 10, 20 and 30 minute and vacuum filtered using filter paper (Whatman No.1).
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116 Collected samples were kept in UV-safe tubes, flushed with nitrogen gas and stored at −8 °C until
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117 further analyses.

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118 For SWE, a rotatable central-composite design (CCD) of response surface methodology (RSM)

119 was used to design the extraction experiment. The process input variables were extraction

120 temperature (120, 140 and 160 °C), pH (2, 3.75 and 5.5), and solid:solvent ratio (2, 4 and 6%); and

121 the responses were total phenolic contents (TPC), total flavonoid content (TFC), 1, 1-diphenyl-2-

122 picrylhydrazyl (DPPH) radical scavenging activity, ferric-reducing antioxidant power (FRAP),

Page 8 of 26
123 and azino-bis 3-ethylbenzothiazoline-6-Sulphonic acid (ABTS). The experiments at the centre

124 point (160 °C, pH 3.75 and solid:solvent ratio 4%) were replicated five times.

125 Pressure was not included in the study variables as the increase of the pressure has no significant

126 effect on extraction efficiency (Ozel et al., 2003). The main role of pressure in SWE is to keep

127 water above the boiling point, in its liquid form. Practically, SWE is weakly dependent on pressure,

128 so any additional pressure has no significant influence on extraction efficiency. Under pressure,

129 the hot water penetrates matrixes which would not be contacted under normal atmospheric pressure

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130 (Crescenzi et al., 2000).

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131

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132 2.2.2. Ethanol Extraction

133 Duplicate ethanol extractions were carried out using 50% ethanol concentration, pH (2, 4.5 and 7)
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134 and time (2, 13 and 24 h). These experimental conditions were selected based on the previous
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135 studies (Lee et al., 2014). The extracts were concentrated on a rotary evaporator before analyses.

136

137 2.3. Analysis

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138 2.3.1. Determination of Total Phenolic Content (TPC)

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139 TPC was determined according to an improved version of the procedure explained by Singleton et

140 al. (1965) and the method suggested by Tang et al. (2015). Briefly, 25 µL of each diluted gallic
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141 acid standard and diluted sample were transferred into a 96-well plate in triplicate, mixed with 125
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142 µL of 10-fold freshly diluted Folin-Ciocalteu reagent and allowed to react for 10 minute. Then 125

143 µL of sodium carbonate solution (7.5%) was added to each and the plate was incubated for 60

144 minute in a dark place. Absorbance was measured at 765 nm using a 96-well ultraviolet-visible

145 (UV/Vis) microplate reader (PerkinElmer 2300 EnSpire Multimode Reader, USA) against a

146 reagent blank. The total phenolic content in each sample was determined using a standard curve

Page 9 of 26
147 prepared by gallic acid and the results were expressed as mg of gallic acid equivalent per gram dry

148 weight of kiwifruit peel (mg GAE/g DW).

149

150 2.3.2. Determination of Total Flavonoid Content (TFC)

151 Flavonoid content was measured by the aluminium chloride colorimetric assay as described by

152 Albishi et al. (2013). Briefly, 200 µL of each standard and sample were transferred to 12 mL test

153 tubes, and then diluted with 800 µL of Milli-Q water. 60µl of aqueous solution of sodium nitrite

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154 (5%) was added to each tube. After 5 minute of incubation, 60 µL of aqueous aluminium chloride

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155 (10%) was added to each standard and sample. After 6 minute of incubation 400 µL of 1M NaOH

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156 solution was added. Finally, the volume of each standard and sample solution was added up to 2

157 mL by 480 µL of Milli-Q water, and 200 µL of each sample was transferred into a 96-well plate.
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158 The absorbance was measured in a 96-plate reader at 510 nm against a prepared reagent blank
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159 using the same microplate reader as above. The total flavonoid content was expressed as mg of

160 catechin equivalent to per gram dry weight of kiwifruit peel (mg CE/g DW).
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161

162 2.3.3. Radical Scavenging Activity Assay (DPPH)

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163 The radical scavenging activity (DPPH) of the extracts was assessed using the modified DPPH
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164 methods of (Ozgen et al., 2006) and (Tang et al., 2015). In brief, the Trolox stock solution (2.5 mg
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165 Trolox in 10 mL ethanol) was divided into concentrations of 0.5, 0.4, 0.3, 0.21, 0.105 and 0.053

166 mg/mL. Ethanol and subcritical water extraction samples were diluted 20 and 10 times,

167 respectively. Each standard and sample (10 µL) were transferred into a 96-well plate. DPPH

168 solution (200 µL) was added to each well and the plates were stored in darkness for 60 minute

169 until used. The absorbance was measured twice in a 96-well microplate reader at 517 nm. Results

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170 of DPPH are given in millimolar Trolox equivalent per gram dry weight of kiwifruit peel (mM

171 TE/g DW).

172

173 2.3.4. Ferric-Reducing Antioxidant Power (FRAP) assay

174 Ferric-reducing antioxidant power (FRAP) was assessed according to (Ozgen et al., 2006), with a

175 few modifications described by (Kheirkhah et al., 2019). Briefly, the FRAP reagent was prepared

176 by mixing 300 mM acetate buffer (pH 3.6) with TPTZ solution (10 mM TPTZ in 40 mM HCl) and

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177 20 mM FeCl3·6H2O, at a ratio of 10:1:1 (v/v/v) and kept in a water bath at 35 °C for 60 minute.

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178 Trolox solution (10 µL), and a freshly prepared FRAP reagent (200 µL) were added and the

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179 absorbance was measured at 593 nm against the reagent blank. Results of FRAP are given in

180 Trolox equivalent per gram dry weight of kiwifruit peel (mM TE/g DW).
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181
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182 2.3.5. Azino-bis 3-Ethylbenzothiazoline-6-Sulphonic Acid (ABTS) Assay

183 ABTS assay was performed according to the method described by (Ozgen et al., 2006), with
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184 moderate changes. ABTS was prepared by mixing an ABTS stock solution (7 mM in water) with

185 2.45 mM potassium persulfate (K2S2O8) (1:1) and stored in dark at room temperature for 12-16
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186 hours. ABTS was dissolved in 20 mM acetate (pH 4.5) and prepared with potassium persulfate as
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187 described above and then diluted in the acidic medium of 20 mM sodium acetate buffer (pH 4.5)
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188 to an absorbance of 0.7 ± 0.01, at 734 nm. The antioxidant activity was determined after 1 h

189 incubation and expressed as millimolar Trolox equivalent per gram dry weight of kiwifruit peel

190 (mM TE/g DW).

191

192 3. Results and Discussion

193 3.1. Subcritical Water Extraction

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194 3.1.1. Total Phenolic and Flavonoid Contents of kiwifruit peel wastes

195 The effects of temperature, pH, solid:solvent ratio and time on the TPC and TFC of extracts are

196 presented in Figures 1 and 2, respectively. As can be seen in Fig. 1 (a-c), the temperature had the

197 most significant influence on TPC and TFC, followed by solid:solvent ratio and pH.

198 The total phenolic contents (TPC) of the kiwifruit peel extract were increased significantly with

199 an increase in temperature from 120 °C to 140 °C, at pH 3.75 and using 2% solid:solvent ratio,

200 and tended to increase slightly once the extraction time extended to 30 minute. By increasing the

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201 temperature up to 160 °C, the extraction yield was decreased (Fig. 1c), due to the degradation of

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202 TPC at higher temperatures (Tunchaiyaphum et al., 2013). An increase in temperature results in

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203 higher mass transfer due to the added energy, a lower dielectric constant and decreased viscosity

204 of the water. This effect of temperature on the extraction of phenolic compounds is in agreement
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205 with the previous studies (Spigno and De Faveri, 2007). Singh and Saldaña (2011b) reported that
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206 the optimal temperature for extracting phenolic compounds from potato peel was 180 °C out of a

207 range of 100-240 °C.

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208 The correlation between temperature and solid:solvent ratio was statistically significant (p < 0.05).

209 At the higher solid:solvent ratio, more phenolics were extracted when the extraction temperature
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210 was around 145°C. However, at lower solid:solvent ratio (2%), the TPC was increased
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211 significantly as the temperature increased. Moreover, at lower temperatures, the impact of
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212 solid:solvent ratio was not significant on the total phenolic content.

213

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Page 12 of 26
 60

50
(a)
TPC (mg GAE / g DW)
40

30

20

10

0
0 5 10 15 20 25 30
Time (min)
(T: 120 C; pH 2; Solid 2%) (T: 120 C; pH 2; Solid 4%) (T: 120 C; pH 2; Solid 6%)
(T: 120 C; pH 3.75; Solid 2%) (T: 120 C; pH 3.75; Solid 4%) (T: 120 C; pH 3.75; Solid 6%)

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(T: 120 C; pH 5.5; Solid 2%) (T: 120 C; pH 5.5; Solid 4%) (T: 120 C; pH 5.5; Solid 6%)

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60

50 (b)
TPC (mg GAE / g DW)

40

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30

20
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10

0
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0 5 10 15 20 25 30
Time (min)
(T: 140 C; pH 2; Solid 2%) (T: 140 C; pH 2; Solid 4%) (T: 140 C; pH 2; Solid 6%)
(T: 140 C; pH 3.75; Solid 2%) (T: 140 C; pH 3.75; Solid 4%) (T: 140 C; pH 3.75; Solid 6%)
(T: 140 C; pH 5.5; Solid 2%) (T: 140 C; pH 5.5; Solid 4%) (T: 140 C; pH 5.5; Solid 6%)
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60
(c)
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50
TPC (mg GAE / g DW)

40
u

30

20
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10

0
0 5 10
(T: 160 C; pH 2; Solid 2%)
(T: 160 C; pH 3.75; Solid 2%)
(T: 160 C; pH 5.5; Solid 2%)
214
15 20
Time (min)
(T: 160 C; pH 2; Solid 4%)
(T: 160 C; pH 3.75; Solid 4%)
(T: 160 C; pH 5.5; Solid 4%)
25 30
(T: 160 C; pH 2; Solid 6%)
(T: 160 C; pH 3.75; Solid 6%)
(T: 160 C; pH 5.5; Solid 6%)

215 Figure 1. Effects of extraction temperature, pH and solid:solvent ratio on total phenolic content
216 during 30 minute subcritical water extraction. (a) 120 °C; (b) 140 °C; (c) 160 °C

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Page 13 of 26
 25
(a)
TFC (mg CE/ g DW) 20

15

10

0
0 5 10 15 20 25 30
Time (min)
(T: 120 C; pH 2; Solid 2%) (T: 120 C; pH 2; Solid 4%) (T: 120 C; pH 2; Solid 6%)
(T: 120 C; pH 3.75; Solid 2%) (T: 120 C; pH 3.75; Solid 4%) (T: 120 C; pH 3.75; Solid 6%)

f
(T: 120 C; pH 5.5; Solid 2%) (T: 120 C; pH 5.5; Solid 4%) (T: 120 C; pH 5.5; Solid 6%)

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25
(b)
20
TFC (mg CE/ g DW)

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15

10
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5

0
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0 5 10 15 20 25 30
Time (min)
(T: 140 C; pH 2; Solid 2%) (T: 140 C; pH 2; Solid 4%) (T: 140 C; pH 2; Solid 6%)
(T: 140 C; pH 3.75; Solid 2%) (T: 140 C; pH 3.75; Solid 4%) (T: 140 C; pH 3.75; Solid 6%)
(T: 140 C; pH 5.5; Solid 2%) (T: 140 C; pH 5.5; Solid 4%) (T: 140 C; pH 5.5; Solid 6%)
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25
(c)
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20
TFC (mg CE/ g DW)

15
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10
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0
0 5 10
(T: 160 C; pH 2; Solid 2%)
(T: 160 C; pH 3.75; Solid 2%)
217 (T: 160 C; pH 5.5; Solid 2%)
15 20
Time (min)
(T: 160 C; pH 2; Solid 4%)
(T: 160 C; pH 3.75; Solid 4%)
(T: 160 C; pH 5.5; Solid 4%)
25 30
(T: 160 C; pH 2; Solid 6%)
(T: 160 C; pH 3.75; Solid 6%)
(T: 160 C; pH 5.5; Solid 6%)

218 Figure 2. Effects of extraction temperature, pH and solid:solvent ratio on total flavonoid content
219 during 30 minute subcritical water extraction. (a) 120 °C; (b) 140 °C; (c) 160 °C

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220 Another important factor for TPC extraction was pH which increased by an increase in pH values

221 from 2 to 3.75 but decreased as the pH value increased to 5.5. The maximum amount of TPC was

222 achieved at pH 3.75 and this result is in line with Ruenroengklin et al. (2008) report on the

223 extraction of phenolics from litchi fruit pericarp tissue under various pH. They reported that at pH

224 4 the extraction of total phenolics was at the highest level (50.2 mg GAE / g DW). The increased

225 extraction efficiency under the acidic conditions could be as a result of change in the ionic strength

226 of solvent and hydrodynamic revolutions due to the reduction in interfacial tensions (Saien and

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227 Ojaghi, 2010). Moreover, acidic solvent prevents enzymatic oxidation by inactivating oxidative

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228 enzymes such as peroxidases, glycosidases, polyphenol oxidases and esterases, which are inherent

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229 in most of plant tissues, are released during the extraction process and could affect the stability of

230 phenolic compounds (Li and Jiang, 2007).

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231 Other studies indicated high polyphenolic content of kiwifruit peel compared to flesh (Kim et al.,
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232 2009). According to our results, the optimal conditions for the maximum extraction (57.8 mg

233 GAE/g DW) of the TPC was temperature 140 °C, pH 3.75, and 2% solid:solvent ratio. this measure
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234 was higher than 15.58 mg GAE/g FDW which was previously reported in Hayward Kiwifruit peels

235 (Jiao et al., 2018). According to their results, TPC in peels of three cultivars including SunGold,
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236 Sweet Green and Hayward peels were 3.44 times much more than the flesh.
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237 Based on the results shown in Figures 1 and 2, the highest yields of TPC and TFC were achieved
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238 at 30 minute of extraction time but the difference of means between 20 minute and 30 minute

239 extractions were not statistically significant.

240 As shown in table 1 of appendix 1, the R2 values for the regression model of TPC and TFC were

241 95.68% and 95.07%, respectively, which indicate that the model reasonably fits to the

242 experimental data. The lack of fit was tested to assess the adequacy of the model's fit. The lack of

243 fit of 0.0001 And 0.5322 indicates the adequacy of the model with both TPC and TFC.

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Page 15 of 26
244 The extraction of TFC increased constantly by an increase in temperature from 120 °C to 140 °C

245 (Fig. 2 a-b) and continued to increase up to 160 °C (Fig. 2c). Increasing temperature moderately

246 increases the total flavonoid content within the extracts which is in agreement with other studies.

247 Ko et al. (2014b) used a range of fruit and vegetable peels to find optimum conditions for different

248 classes of flavonoids and found the optimal temperature for most classes was 190°C. Plaza et al.

249 (2013) extracted flavonoids from apple by-products and found that although TPC increased with

250 temperature the TFC began to reduce after 170°C.

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251 As for the TFC results, the more acidic samples possessed the highest flavonoid content of 23 mg

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252 CE/g DW, possibly demonstrating that flavonoids are more stable in acidic conditions. This is in

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253 agreement with the results from the study conducted by Firlbeck et al. (2013) on olive waste.

254 The effects of solid:solvent ratio for SWE of TFC of kiwifruit peels were mostly similar to TPC;
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255 the maximum concentration of TFC (23.11 mg CE/g DW) was achieved using 2% solid:solvent
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256 ratio at 160 °C and pH 2. This is much higher than the value reported by (Jiao et al., 2018) who

257 extracted flavonoids from Hayward kiwifruit peel (2.47 mg CE/g FDW).
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258

259 3.1.2. Antioxidant Activity

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260 The effects of extraction parameters on antioxidant activities of kiwifruit peel extracts were
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261 evaluated by DPPH, FRAP and ABTS assays and are shown in Figure 3 (a-i).
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262 The results from statistical analysis showed that the antioxidant activities (DPPH=195.1,

263 FRAP=202.8, ABTS=269.4 mM TE/g DW) obtained after 20 minute extraction was significantly

264 (p < 0.05) different from those obtained after 10 minute and 30 minute. For antioxidant activity,

265 there was a significant difference of means between 20 minute and 10 minute extraction in each

266 experiment, but the difference of means between 20 minute and 30 minute extractions was not

267 statistically significant.

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 DPPH (mM TE/ g DW)

DPPH (mM TE/ g DW)

DPPH (mM TE/ g DW)

200 200 200

(a) (b) (c)
160 160 160

f
120 120

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120

80 80 80

40 40 40

0 0

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0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
0 5 10 15 20 25 30 Time (min)
Time (min) Time (min)

FRAP (mM TE/ g DW)

FRAP (mM TE/ g DW)

200 200
200
(e) (f)

e-
(d)

FRAP (mM TE/ g DW)

160 160 160

120 120
120

Pr
80 80
80
40 40
40
0 0
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
0 5 10 15 20 25 30 Time (min)
Time (min) l Time (min) 300

ABTS (mM TE/ g DW)

300
ABTS (mM TE/ g DW)

300
na
ABTS (mM TE/ g DW)

240 (g) 240 (h) 240 (i)

180 180 180

120 120 120

ur

60 60 60

0 0 0
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0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (min) Time (min) Time (min)

268

269 Figure 3. Effects of extraction temperature, pH and solid:solvent ratio on antioxidant activity (DPPH, FRAP and ABTS) of subcritical
270 water extracts. (a, d, g) 120 °C; (b, e, h) 140 °C; (c, f, i) 160 °C

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271 With regards to extraction time, no significant (p>0.05) correlation was obtained among all

272 antioxidant assays. All antioxidant assays were either weakly or moderately correlated with each

273 other except for TPC and DPPH. It was found that TPC was correlated with DPPH (R2= 0.860)

274 which suggested that phenolic compounds might be the major contributors for DPPH radicals

275 scavenging capacities of crude extract under the effect of extraction time.

276 This result confirms that the antioxidant potential of the extracts correlates with the phenolic

277 content of the extract (Kheirkhah et al., 2019). However, as the extraction time kept increasing,

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278 there were only slight changes in antioxidant activity, which indicated that an excess extraction

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279 time would lead to the degradation or oxidation of phenolic compounds. This result was consistent

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280 with a previous report from Yoswathana and Eshtiaghi (2013) where the extraction was also

281 optimised with a 20 minute-extraction. results by Jiao et al. (2018) showed that antioxidant activity
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282 in the peel of three different kiwifruit cultivars (SunGold, SweetGreen, and Hayward) was
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283 significantly (p < 0.05) higher than that of flesh extract.

284 Acidic conditions are known to increase the stability of polyphenolic compounds, the main
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285 compounds which exhibit antioxidizing effects in kiwifruit peels, allowing them to be extracted

286 into the solvent. It is, therefore, sound that lowering the pH of the extraction fluid increases the
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287 levels of antioxidizing activity in the extracts. The effect of lowering pH was consistent across all
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288 three indicators, with conditions of pH equal to 2 resulting in the highest yields.
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289 According to the results, antioxidant activity of the extracts was significantly (p < 0.05) affected

290 by extraction temperature. These results are in accordance with previous study on extraction of

291 phenolic compounds from parsley and pomegranate seed oil indicating that the effect of

292 temperature is more significant compared to solid:solvent ratio and time (Abbasi et al., 2008).

293

294

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295 3.2. Ethanol Extraction

296 3.2.1. Total Phenolic and Total Flavonoid Content

297 Figure 4 shows the effect of time, pH and solid:solvent ratio on the yield of TPC and TFC during

298 the extraction with 50% ethanol solvent. The amount of TPC and TFC increased by extending the

299 extraction time from 2 to 24 h within the limit of 22-26.5 mg GAE/g DW for TPC and 15.5-19 mg

300 CE/ g DW for TFC.

301 In terms of TPC, pH strongly affected TPC of the extract in the combination with both time and

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302 solid:solvent ratio. While the effect of extraction time did not show a significant change in TPC

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303 but resulted in a slight increase in TPC with longer extraction time. Extraction with lower

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304 solid:solvent ratio resulted in a higher TPC, but the effect was not as significant as pH. As can be

305 seen in Fig. 4c, the highest amount of TPC was obtained under pH 2, 2 % solid:solvent ratio, and
e-
306 24 h extraction time.
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307 The highest TFC was yielded under the same extraction conditions as of TPC.

308
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309 3.2.2. Antioxidant Activity

310 Figures 5 (a-c) show the effects of extraction parameters on antioxidant activity in terms of DPPH,
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311 FRAP, and ABTS. A strong correlation was found between antioxidant activity and total phenolic
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312 contents. At pH 7, extraction time was found to be insignificant, while the antioxidant activity was
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313 increased with longer extraction time under lower pH.

314 Higher antioxidant activities were observed for the extracts obtained using lower solid:solvent

315 ratio. Based on all trends, it can be concluded that pH and solid:solvent ratio were the main

316 influencing factor, and the antioxidant activity could both be achieved under lower acidic pH,

317 lower solid:solvent ratio and longer extraction time.

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318

319

f
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30 30 30
(a) (b) (C)
Solid Content (%)

TPC (mg GAE/ g DW)

TPC (mg GAE/ g DW)

TPC (mg GAE/ g DW)

20 20 20

pr
10 10 10

e-
0 0 0

Pr
2 4 6 2 4 6 2 4 6
Solid Content (%) Solid Content (%)
(t=2 h; ph 2) (t=2 h; pH 4.5) (t=13 h; ph 2) (t=13 h; pH 4.5) (t=24 h; ph 2) (t=24 h; pH 4.5)

30 30
30 l (e) (f)
na
(d)
TFC (mg CE/ g DW)

TFC (mg CE/ g DW)

TFC (mg CE/ g DW)

20 20
20
ur

10 10 10
Jo

0 0 0
2 4 2 4 6 2 4 6
Solid Content (%) 6 Solid Content (%) Solid Content (%)
(t=2 h; ph 2) (t=2 h; pH 4.5) (t=2 h; pH 7) (t=13 h; ph 2) (t=13 h; pH 4.5) (t=24 h; ph 2) (t=24 h; pH 4.5)

320
321 Figure 4. Effects of extraction time, pH and solid:solvent ratio on total phenolic content (a-c) total flavonoid content (d-f) of ethanol
322 extracts

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 300 300

Antioxidant Activity (mM TE/ g DW)

Antioxidant Activity (mM TE/ g DW) (a) (b)
250 250

200 200

f
150 150

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100 100

50 50

pr
0 0
2 4 6 2 4 6
Solid Content (%) Solid Content (%)

e-
DPPH (t=2 h; ph 2) DPPH (t=2 h; pH 4.5) DPPH (t=2 h; pH 7) DPPH (t=13 h; ph 2) DPPH (t=13 h; pH 4.5) DPPH (t=13 h; pH 7)
FRAP (t=2 h; ph 2) FRAP (t=2 h; pH 4.5) FRAP (t=2 h; pH 7) FRAP (t=13 h; ph 2) FRAP (t=13 h; pH 4.5) FRAP (t=13 h; pH 7)
ABTS (t=2 h; ph 2) ABTS (t=2 h; pH 4.5) ABTS (t=2 h; pH 7) ABTS (t=13 h; ph 2) ABTS (t=13 h; pH 4.5) ABTS (t=13 h; pH 7)

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300
Antioxidant Activity (mM TE/ g DW)

323 (C)
250
324
l 200
na
325
150
326
100
ur

327
50
328
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0
2 4 6
329 Solid Content (%)
DPPH (t=24 h; ph 2) DPPH (t=24 h; pH 4.5) DPPH (t=243 h; pH 7)
330 FRAP (t=24 h; ph 2) FRAP (t=24 h; pH 4.5) FRAP (t=243 h; pH 7)
ABTS (t=24 h; ph 2) ABTS (t=24 h; pH 4.5) ABTS (t=243 h; pH 7)
331

332 Figure 5. Effects of extraction time, pH and solid:solvent ratio on antioxidant activity (DPPH, FRAP and ABTS) of ethanol
333 extracts. (a) 2 h extraction; (b) 13 h extraction; (c) 24 h extraction

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334 3.3. Comparison of Subcritical Water with Ethanol Extraction under Optimum Conditions

335 Numerical hill-climbing algorithms were employed to search out the most desirable outcomes

336 from subcritical water and ethanol extractions. The optimal SWE conditions for the maximum

337 extraction yield of the phenolic antioxidants with the highest antioxidant activity was predicted to

338 be at 160 °C and pH 2 using 2% solid:solvent ratio. For ethanol extraction, the optimised extraction

339 was found under pH 2 using 2% solid:solvent ratio and 24 h extraction time.

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340 Analysis of subcritical water extracted samples showed significantly higher results than those of

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341 solvent extracted samples. Paired t-test analysis between two methods shows that the recovery of

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342 total phenolics by subcritical water was nearly two times of that by solvent extraction (Table 1).

343 Table 1. Experimental and Predicted results for subcritical water and ethanol extractions at the
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344 optimum conditions
Pr
Subcritical Water Ethanol

0 min 5 min 10 min 20 min 30 min 24 h

Experimental 10.17 39.89 48.23 51.24 48.23 26.15

al

TPC
(mg GAE/g DW) Predicted 10.11 41.32 46.82 49.96 48.35 25.79
rn

TFC Experimental 4.78 21.08 21.67 22.49 23.11 18.93

(mg CE/g DW) Predicted 4.74 21.46 22.76 22.95 23.12 18.98
u

Experimental 30.84 142.98 184.38 203.89 200.12 178.47

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DPPH
(mM TE/g DW) Predicted 33.48 148.57 187.42 204.45 197.52 174.33

FRAP Experimental 41.48 142.47 172.15 212.52 208.66 94.19

(mM TE/g DW) Predicted 40.06 143.91 170.65 202.82 194.82 93.01

ABTS Experimental 84.13 210.78 264.37 282.2 274.27 233.29

(mM TE/g DW) Predicted 85.93 219.17 246.5 269.43 264.27 228.17
345

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346 Moreover, the antioxidant activities of subcritical water extracts were significantly higher than

347 those obtained by ethanol extraction. This result is in agreement with the studies conducted by

348 other researchers on cocoa beans (Othman et al., 2007) and mushroom (Cheung et al., 2003).

349

350 4. Conclusion

351 Subcritical water and ethanol extraction techniques were used to recover phenolic compounds

352 from green kiwifruit peel. Different extraction conditions were evaluated and found to have a

f
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353 significant effect on extracts. The results showed that the efficiency of SWE was significantly

354 dependent on the extraction variables. Under the optimum SWE (160 ˚C, pH 2, 2% solid:solvent

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355 ratio and 20 minute extraction time), 51.24 mg GAE/g DW of phenolic compounds was recovered
e-
356 from kiwifruit peel. The kiwifruit peel extracts obtained by subcritical water showed high

357 antioxidant activities. In comparison with traditional ethanol extraction, subcritical water showed
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358 significantly higher extraction yield and antioxidant activities than that of ethanol extraction. This

359 research satisfies the desire of using environmentally-friendly solvents to separate compounds with
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360 health-promoting ability from food processing residues and by-products. The results also indicate
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361 that there is a clear niche for this process to be developed in commercial scale and to be flourish

362 in future years. Future studies will look into identifying unique compounds present in this extract.
u

363 In addition, the effects of other extraction parameters such as particle size and pH need to be
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364 studied.

365

366 Acknowledgement

367 The authors acknowledge Kiwifruitz Ltd. for providing the peel samples and Hamid Kheirkhah

368 for his technical assistance.

369

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