Journal Pre-proof

Preservation of strawberries using chitosan incorporated with lemongrass essential

oil: An X-ray computed tomography analysis of the internal structure and quality
parameters

Nguyen Thi Hang Phuong, Tran Thi Van, Francis Ngwane Nkede, Fumina Tanaka,
Fumihiko Tanaka
PII: S0260-8774(23)00335-7
DOI: https://doi.org/10.1016/j.jfoodeng.2023.111737
Reference: JFOE 111737

To appear in: Journal of Food Engineering

Received Date: 10 June 2023

Revised Date: 13 August 2023
Accepted Date: 6 September 2023

Please cite this article as: Thi Hang Phuong, N., Thi Van, T., Ngwane Nkede, F., Tanaka, F., Tanaka,
F., Preservation of strawberries using chitosan incorporated with lemongrass essential oil: An X-
ray computed tomography analysis of the internal structure and quality parameters, Journal of Food
Engineering (2023), doi: https://doi.org/10.1016/j.jfoodeng.2023.111737.

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 Credit Author Statement (CAS):

Nguyen Thi Hang Phuong: Conceived and designed the experiments, conducted experiments,
analyzed data, and wrote the manuscript.

Tran Thi Van: Contributed to data analysis and interpretation, provided critical feedback on the
manuscript.

Francis Ngwane Nkede: Contributed to data collection and provided critical feedback on the
manuscript.

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Fumina TANAKA: Assisted in experimental design, performed data collection, and reviewed the
manuscript.
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Fumihiko TANAKA: Supervised the overall project, secured funding, and critically reviewed the
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manuscript.
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 1 Preservation of strawberries using chitosan incorporated with lemongrass essential oil: An X-ray

2 computed tomography analysis of the internal structure and quality parameters

4 Nguyen Thi Hang Phuonga,b, Tran Thi Vana,c, Francis Ngwane Nkedea, Fumina TANAKAa and

5 Fumihiko TANAKAa*

6
a
7 Division of Bio-production Environmental Sciences, Department of Agro-environmental Sciences,

8 Faculty of Agriculture, Kyushu University, W5-873,744 Motooka, Nishi-ku Fukuoka shi 819-0395, Japan
b
9 Department of Food Technology, Faculty of Agriculture and Food Technology, Tien Giang University,

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10 My Tho city, 119 Ap Bac, Vietnam

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c
11 Department of Preservation Technology Research on Agricultural Product, Vietnam Institution of

12 -p
Agricultural Engineering and Postharvest Technology, Hanoi 10000, Vietnam
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13

14 *Corresponding Author: +81-(0)92-802-4636, Email: fumit@bpes.kyushu-u.ac.jp

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15
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16 Abstract: The effects of two different edible coatings, chitosan (CH) and chitosan-loaded lemongrass
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17 essential oil (CH+LMO), on the quality parameters of strawberries, were investigated over a 24-d storage
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18 period at temperature of 2 ℃. The coatings effectively reduced fruit weight loss, retained moisture
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19 content, total soluble solids (TSS), firmness, color, and delayed pH changes of strawberries. X-ray

20 computed tomography (CT) technique was used to monitor the changes in the internal structures of

21 strawberries during storage. Tissue degradation was slower in the coated strawberries than uncoated

22 strawberries (control). The accuracy of predicting moisture content, TSS content, pH, and firmness was

23 higher than the accuracy of predicting weight loss of strawberries. The CH coating exhibits better

24 preservation than the CH+LMO coatings in several quality parameters. The CH and CH+LMO coatings

25 effectively may extend the shelf life and controlled the quality characteristics of strawberries. The

26 combination of the X-ray CT technique and the partial least squares (PLS) model can be effectively used

27 to analyse and monitor fruit quality.

28

29 Key Words: Edible coating, storage, essential oil, strawberry, X-ray CT, PLS regression
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30

31 1. Introduction

32 Popular for their distinctive taste and appealing aroma, strawberries (Fragaria × ananassa) are

33 one of the most widely consumed fruits in the world (Velickova et al., 2013). Strawberries are rich in

34 vitamins, carotenoids, minerals, and antioxidants (Dong and Wang, 2017; Shankar et al., 2021). However,

35 strawberries have a very short postharvest life as they are susceptible to mechanical damage, water loss,

36 texture softening, and vulnerability to fungi (Shahbazi, 2018). Amaou strawberry is one of the most

37 popular strawberry varieties, and it is known as the "king of strawberries" in Japan. Despite their

38 popularity, Amaou strawberries are highly perishable and tend to degrade quickly in quality over time.

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39 This can be attributed to the high moisture content and the thin skin of the strawberries. Several

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40 preservation methods have been used to improve the shelf life of strawberries. Cooling, irradiation,

41 -p
chemical treatment, controlled atmosphere, and edible coatings are methods that have been applied to
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42 enhance the shelf life of strawberries (Dong and Wang, 2017; Kahramanoğlu, 2019; Perdones et al., 2012;
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43 Shankar et al., 2021). Edible coatings containing essential oils are widely used to preserve strawberries as

44 these coatings limit the respiration rate, help retain firmness, and inhibit microbial growth (Dong and
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45 Wang, 2017; Shahbazi, 2018; Velickova et al., 2013). The effectiveness of incorporating essential oils
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46 into chitosan (CH) to preserve strawberries, including Menthe spicata (Shahbazi, 2018), oregano (Lee et

47 al., 2022), lemon (Perdones et al., 2012), and Thymus capitatus (Martínez et al., 2018) have been
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48 previously reported. Therefore, we evaluated the effects of CH and chitosan+lemongrass (CH+LMO)

49 coatings on the quality of Amaou strawberries. The physicochemical properties and changes in the

50 internal structure of the strawberries were monitored by conducting basic analytical experiments and

51 using the X-ray computed tomography (CT) technique, respectively, during the strawberry storage

52 experiments.

53 CH is a high-molecular-weight polysaccharide extracted from yeast, fungi, crustaceans, and

54 insects (Flórez et al., 2022; Shahbazi, 2018). CH is considered a sustainable packaging material as it

55 exhibits high biocompatibility, biodegradability, and antimicrobial properties (Al-Naamani et al., 2016;

56 Martins et al., 2012). The U.S. Food and Drug Administration has classified CH into the category of

57 Generally Recognized as Safe (GRAS). Thus, it is potentially a safe food packaging material (Friedman

58 and Juneja, 2010). CH can easily form edible films that exhibit excellent mechanical and antibacterial
2
59 properties. CH contains free amino groups, allowing for cross-linking, controlled release of active

60 substances, and modulation of inter- and intramolecular hydrogen bonding interactions (Han Lyn and Nur

61 Hanani, 2020). CH can be used alone or in combination with other natural polymers or essential oils for

62 food preservation. CH contains many hydrophilic groups that limit its commercial application, and this

63 can be attributed to its high water vapor permeability (Wang and Jing, 2017). Hydrophobic essential oils

64 have been incorporated into CH to improve the moisture barrier (Amalraj et al., 2020; Atarés et al., 2010;

65 Perdones et al., 2014; Vázquez-m et al., 2014). Lemongrass essential oil (LMO) is extracted from the

66 leaves and stems of lemongrass. LMO is primarily composed of monoterpenes and several effective

67 antimicrobial compounds, such as neral, geranial, geraniol, citronellol, and limonene (Ahmad and

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68 Viljoen, 2015; Ali et al., 2015). Therefore, the addition of LMO to CH coatings is expected to improve

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69 the quality of strawberries. Ali et al. (2015) reported that CH coatings enriched with 0.5 and 1.0% (v/v)

70 -p
LMO could effectively protect bell peppers against anthracnose causal agents.
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71 X-ray CT is a non-destructive technique used for radiation density mapping. This technique can

72 be used to obtain high-quality images of the internal structure of fruits. The technique is based on X-ray
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73 attenuation contrast, and it can be used to differentiate between low- and high-density materials (Barcelon
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74 et al., 1999; Brecht et al., 2019). X-ray CT technique can be used to study the internal structure of the
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75 materials based on the differences in the degree of X-ray attenuation attained. The results reflect the
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76 structural variations in the three-dimensional (3D) structure of objects. It can be inferred that X-ray CT is
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77 an excellent technique to realize the qualitative and quantitative visualization of the porous structures of

78 products (Diels et al., 2017; Herremans et al., 2013; Magwaza and Opara, 2014). X-ray CT images were

79 analyzed to visualize the internal structure of chestnuts, tart cherries, pineapples, and pickled cucumbers

80 (Donis-González et al., 2014). Researchers have also studied the internal structure of pomegranates

81 (Arendse et al., 2016), the extent of internal browning realized (Chigwaya et al., 2021; Herremans et al.,

82 2013), and the quality of pears (Van De Looverbosch et al., 2020) using this technique. The aim was to

83 investigate and visualize the changes in the internal structures of strawberries during storage.

84 The partial least squares (PLS) regression analysis method is used to combine features from

85 multiple linear regression and principal component analysis methods. The PLS regression model was

86 used to predict a set of dependent variables from a set of independent variables or predictors. The PLS

87 regression model has been previously used to evaluate attribute’s perception and consumer liking of
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 88 apples (Mendes da Silva et al., 2021), to predict the changes in quality parameters of red bayberry juice

89 (Zheng et al., 2011), and to measure the ripeness of oil palm fresh fruit bunch (Iqbal et al., 2019). In this

90 study, the PLS regression method was used to predict the physicochemical parameters of strawberries,

91 including weight loss, moisture content, total soluble solid (TSS) content, pH, and firmness.

92 The study aimed to assess the effectiveness of a CH+LMO coating in preserving strawberry

93 quality during cold storage. The experiments were conducted at 2 °C and 70% relative humidity (RH).

94 The study also aimed to investigate changes in the internal quality of strawberries using the X-ray CT-

95 based grayscale (GS) value visualization method and predict the physicochemical parameters of

96 strawberries, including weight loss, moisture content, TSS content, pH, and firmness.

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97

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98 2. Materials and Methods

99 2.1. Coating solutions -p

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100 CH (deacetylation degree: 87%; molecular weight: 96 kDa), acetic acid, glycerol, and Tween 80

101 were obtained from FUJIFILM Wako Pure Chemical Corporation (Japan). LMO was purchased from
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102 Aubrey Organics Inc. (USA). CH (1%, w/v) was dissolved in a solution of acetic acid (0.5%, v/v), and
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103 glycerol (30%, w/w CH) was used as a plasticizer. CH-incorporated LMO solution was prepared by
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104 adding LMO (0.1%, w/w CH) and Tween 80 (50%, w/v essential oil; emulsifier) to a solution of CH. The
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105 solutions were homogenized at 18,000 rpm for 5 min using a homogenizer (Ultra-Turrax T-25, IKA Japan
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106 Cooperation). Following this, the solutions were degassed in a vacuum oven (ADP300, Yamato Scientific

107 Co., Ltd., Japan). These solutions were named CH and CH + LMO solutions, respectively.

108

109 2.2. Fruit coating

110 Fresh strawberries (Hakata amaou) were purchased from JA Itoshima (Fukuoka, Japan).

111 Strawberries were divided into three treatment groups: control, CH, and CH+LMO. The samples in the

112 control group were treated with distilled water, and the samples in the other groups were immersed in CH

113 solutions for 1 min. Following this, the samples were dried at room temperature (20 ± 2 oC) for 6 h. The

114 samples were transferred to polyethylene trays (20 × 30 cm) post drying, and the samples were stored at 2

115 °C and 70% RH in a chamber (ISUZU CAP TPAV-120-20; Japan) for 24 d. All samples were evaluated

116 for their physicochemical properties (weight loss, moisture content, firmness, pH, TSS content) and the
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117 degree of X-ray attenuation achieved. Data were collected at predetermined time intervals (0, 4, 8, 12, 16,

118 20, and 24 d).

119

120 2.3. Analysis of the physicochemical properties of strawberries

121 2.3.1. Weight loss

122 The loss in weight of the strawberries was determined using an electrical balance (AND Fx-300i;

123 A&D Co. Ltd., Tokyo, Japan). The same six strawberries from each treatment group were weighed on the

124 sampling day over the 24-d storage period. The weight loss results are presented as a percentage (relative

125 to the initial weight). The loss in weight was calculated.

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126

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127 %WL = [(Wi – Wf)/Wi] x 100, (1)

128 -p
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129 where %WL is the percentage weight loss, Wi is the initial weight of the fruit (g), and Wf is the final

130 weight of the fruit (g) at the indicated times.

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131
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132 2.3.2. Moisture content

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133 The moisture content of the strawberries was determined based on the difference in weight of the
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134 fruit pulp before and after drying. The samples were dried in an oven (EYELA WFO-520, EYELA
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135 Corporation, Tokyo, Japan), and the experiments were conducted at 105 ℃ over 24 h. The weight of the

136 sample (before and after drying) was measured using a 0.01 mg high-precision balance (BM-252, A&D

137 Corporation, Tokyo, Japan). The results represent the average values of nine replicates. The moisture

138 content was calculated as follows:

139

140 %MC = [(Wi – Wf)/Wi] x 100, (2)

141

142 where %MC is the wet basis moisture content (%), Wi is the initial weight of the sample (g), and Wf is the

143 final weight of the sample (g).

144

145 2.3.3. Total soluble solid (TSS) content and pH

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146 The TSS content and pH of strawberry juice were determined using a digital refractometer

147 (Asone IPR-101α, Atago Co. Ltd., Tokyo, Japan) and a compact pH meter (LAQUAtwin-pH-22, Horiba

148 Ltd., Kyoto, Japan), respectively. The results are the mean of nine replicates for each treatment group.

149 The results were recorded at 0, 4, 8, 12, 16, 20, and 24 d of storage.

150

151 2.3.4. Color change

152 Six strawberries from each treatment group were used to study the change in color of the samples

153 using a colorimeter (CR-20; Konica Minolta Inc., Japan). The colorimeter was calibrated with a standard

154 white plate, and the measurements for each strawberry were taken at three positions on sampling days.

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155 The experiment was conducted over a 24-d storage period. The color parameters of lightness (L*),

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156 redness to greenness (a*), and blueness to yellowness (b*) were determined. The color difference (ΔE)

157 was calculated as follows: -p

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158

∆𝐸 = √∆𝐿2 + ∆𝑎2 + ∆𝑏 2 ,
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159 (3)

160
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161 where ∆𝐿 = 𝐿∗𝑠 − 𝐿∗𝑖 ; ∆𝑎 = 𝑎𝑠∗ − 𝑎𝑖∗ ; ∆𝑏 = 𝑏𝑠∗ − 𝑏𝑖∗ . The subscripts s and i indicate storage and initial,
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162 respectively.

163
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164 2.3.5. Firmness

165 The firmness of the six strawberry samples from each treatment group was determined using a

166 creep meter (Rheoner RE-3305, YAMADEN Co. Ltd., Tokyo, Japan) which was connected to a

167 cylindrical probe (diameter: 3 mm). Strawberries were placed on the stage of the creep meter. The probe

168 penetrated to a maximum depth of 5 mm into the sample at a speed of 1 mm s-1. The maximum

169 penetration force (N) recorded during tissue breakage represented the firmness of strawberries.

170

171 2.4. Analysis of X-ray CT scan data and recorded images

172 An X-ray CT scanner (CosmoScan FX; Rigaku Corporation, Tokyo, Japan) was used to obtain X-

173 ray CT images of the samples. The operating conditions were set (voltage: 90 kV; current: 88 µA; 0.06-

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174 mm Cu filter; 0.5-mm Al filter; field of view (FOV): 72 mm for standard CT scanning), and the samples

175 were placed in holding tubes (scanning time: 2 min; pixel size: 144 μm).

176 Viewer software in the CT scanner was used for image acquisition, reconstruction, processing,

177 and 3D volume rendering. X-ray images were reconstructed at 520–1000 HU, and 16-bit bitmap images

178 were analyzed to evaluate the average CT value of each intact fruit during the storage period. The X-ray

179 data were calibrated using distilled water as the standard.

180 Fiji (NIH, Madison, USA) was used for digital image processing to determine the average GS

181 value and generate the appropriate histogram (GS threshold value: 0–255). The GS pixel values for pure

182 black and white were recorded to be 0 and 255, respectively. The Multilevel Otsu thresholding algorithm

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183 was used to identify the low-density and high-density regions. The 3D images were analyzed to

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184 understand the process of tissue breakdown. The GS profile and contrast-to-noise ratio (CNR) values

185 -p
were analyzed for the quantitative evaluation of CT images. CNR values were calculated as follows:
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186

187 CNR = (ROICore – ROIFlesh)/Flesh, (4)

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188
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189 where ROICore is the mean GS value of the core of the sample, ROIFlesh is the mean GS value for the flesh
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of the sample, and Flesh is the standard deviation recorded for the flesh region.
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190

191
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192 2.5. PLS regression model

193 The dataset was randomly split into a calibration dataset of 84 samples (28 fruits for each batch)

194 and a test dataset of 42 samples (14 fruits for each batch). The calibration dataset was used to validate the

195 PLS model. The testing data were held out during the validation process and used solely for assessing the

196 generalization capabilities of the models.

197 The PLS algorithm (Unscrambler X version 10.4, Camo Analytics, Norway) was used to

198 determine the relationship between the GS value and quality parameters of strawberries. The multivariate

199 regression model is based on multiple linear regression models, and it can be expressed as follows:

200

201 𝑌 =𝐴×𝑋+𝐵 (5)

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203 Matrix Y consisted of the weight loss, moisture content, TSS content, pH, and firmness data, and Matrix

204 X reflected the GS value. The regression coefficients are given by matrix A, and the regression residuals

205 are given by matrix B.

206 The quality of the calibration model used for the calibration set and predictions of the testing set

207 are given by the root mean square error (RMSE) as follows:

208

∑𝑛 ̅𝑖 )2
𝑖=1(𝑦𝑖 −𝑦
209 𝑅𝑀𝑆𝐸 = √ (6)
𝑛

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210

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211 where the elemental analysis concentrations are represented by yi, and the concentrations predicted via the

212 PLS regression model are represented by 𝑦̅𝑖 . The number of sample test sets in the calibration or

213 prediction set is n.

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214
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215 2.6. Statistical analysis

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216 Multiple-range tests were conducted to statistically analyze the obtained results using
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217 Statgraphics® Centurion XV (StatPoint Technologies Inc., Warrenton, Virginia, USA). The differences
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218 between the mean values were considered statistically significant at P<0.05.
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219

220 3. Results and discussion

221 3.1. Effect of coatings on the quality parameters recorded during the storage period

222 3.1.1. Weight loss and moisture content

223 Due to their thin skin, strawberries experienced significant water loss, resulting in surface

224 shrinkage and tissue weakening. The weight loss corresponding to the control and treated strawberries

225 was evaluated over 24 d (Table 1). There was an increase in the extent of weight loss realized in the

226 samples of all the treatment groups during the cold storage period. The control and treated strawberries

227 showed a statistically significant difference in weight loss on sampling days. The weight loss for the

228 control strawberries was higher than that realized for the CH- and CH+LMO-coated strawberries. These

229 observations were made during the cold storage period. The control strawberries lost 8.13% of their

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230 weight at the end of the storage period, whereas the CH- and CH+LMO-treated strawberries lost 5.78 and

231 6.36% of their weight, respectively. The CH and CH+LMO coatings could effectively reduce the extent

232 of weight loss realized in strawberries. Until the 24th day of cold storage, the CH-coated strawberries

233 experienced less weight loss compared to the CH+LMO-coated strawberries. The weight loss recorded

234 during strawberry storage could be attributed to moisture loss through thin peels. Moisture could also be

235 lost during respiration (Atress et al., 2010; Pizato et al., 2022). It was observed that CH formed a

236 semipermeable barrier on the fruit surface, delaying water loss from strawberries (Pizato et al., 2022). The

237 incorporation of essential oils into the CH film improved the moisture barrier, and this could be attributed

238 to increased hydrophobicity (Ali et al., 2015; Ojagh et al., 2010; Shankar et al., 2021). These results were

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239 consistent with the results reported by Huu Huong Nguyen and Vu Hong Nguyen, 2020. A weight loss of

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240 2.4% was recorded at 2 °C after 12 d for strawberries coated with 0.2% nanochitosan. Similar results

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were reported by Elmansy et al., (2017). They reported that strawberries coated with 1% CH-containing
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242 LMO (0.1%) or thymine (0.1%) systems exhibited weight losses of 4.36 and 4.31%, respectively. These

243 data were recorded on the 15th day of storage (temperature: 4 °C).
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244 Table 1 presents data on the moisture content of the coated and uncoated strawberries. The data
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245 were recorded at 2 ℃ over a period of 24 d. On day 0, the moisture content was approximately in the
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246 range of 90.32–90.45%. This result agreed well with the result reported by Hassan (2017), who reported
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247 that the initial moisture content of fresh strawberries varied in the range of 93.4–77%. During the storage
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248 period, there was a progressive increase in the moisture content of the samples belonging to all treatment

249 groups. However, the moisture content of the CH-coated strawberries was significantly (P<0.05) lower

250 than that for the control strawberries. The coating treatments did not result in any significant difference

251 (P<0.05) in the moisture content over the 16–20 d period of storage. The lowest moisture content was

252 recorded for control strawberries (82.34%), whereas the maximum moisture content was recorded for the

253 CH-coated strawberries (86.47%) at the end of the storage period. The moisture content of the CH+LMO-

254 CH+LMO-coated strawberries was recorded to be 85.91%. After 24 d of storage, the moisture content of

255 control strawberries reduced by 8.09%, whereas the moisture contents of the CH- and CH+LMO-coated

256 strawberries reduced by 4.09 and 4.46%, respectively. These results demonstrated that the CH and

257 CH+LMO coatings could effectively maintain the moisture content of strawberries.

258
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259 3.1.2. TSS content and pH of strawberries

260 The TSS contents of the control and coated strawberries recorded over the storage period are

261 presented in Table 2. The TSS content of fresh strawberries was in the range of 11.29–11.51% at the

262 beginning of the storage period, and similar values were reported by Kahramanoğlu (2019) and Peretto et

263 al. (2014). The TSS content of fresh strawberries varied among cultivars and was in the range of 5.3–

264 13.5% (Gol et al., 2013; Shankar et al., 2021; Velickova et al., 2013; Wang et al., 2019). The TSS content

265 gradually decreased in the coated and uncoated strawberries, and this could be attributed to the continued

266 metabolism of soluble solids during storage. There was no significant difference in the TSS content

267 (P>0.05) of the CH- and CH+LMO-coated strawberries up to 20 d of storage. A significant difference

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268 (P<0.05) in the TSS content of the CH- and CH+LMO-coated strawberries was recorded at the end of the

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269 storage period. The TSS content of the control strawberries was lower than that of the CH and CH+LMO-

270 -p
treated strawberries. The measurements were taken over a 24-d storage period. The soluble solid content
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271 in control strawberries decreased faster than the soluble solid content in the chitosan-treated samples. The

272 TSS content of the control samples was 9.41%, and the TSS contents recorded for the CH-and CH+LMO-
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273 coated strawberries were 10.32 and 10.08%, respectively. The data were collected at the end of the
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274 storage period. These results agreed well with the results reported by Velickova et al. (2013), who
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275 reported a decrease in the TSS content of strawberries at the end of the storage period and attributed the
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276 decrease in the TSS content to respiration. The CH and CH+LMO coatings helped maintain high TSS
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277 concentrations, and this could be attributed to the inhibition of respiration and metabolic activity (Dong

278 and Wang, 2017). These results are in agreement with those reported by Gol et al. (2013) and Wang et al.

279 (2019), who concluded that CH reduced the TSS content of strawberries by decelerating the process of

280 respiration and hindering the process of ripening.

281 The effects of CH and CH+LMO coatings on the pH of strawberries are presented in Table 2. The

282 pH of fresh strawberries was in the range of 3.18–3.20, as reflected in the results reported by Gol et al.

283 (2013) and Shahbazi (2018). The pH of strawberries was found to vary in the range of 3.0–3.7 depending

284 on the cultivar (Campos-Requena et al., 2017; Perdones et al., 2012). It was observed that the pH of the

285 strawberries increased gradually during the 24-d storage period. A significant difference (P<0.05) in the

286 results was recorded for the results obtained for the coated and uncoated strawberries. The minimum

287 increase in pH was recorded for the strawberries coated with CH and CH+LMO (3.47 and 3.51,
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288 respectively; recorded at the end of 24 d of storage), whereas the pH of the control samples was recorded

289 to be 3.59 at the end of this period. The increase in the pH of strawberries during storage is directly

290 related to the decrease in the contents of organic acids, which are metabolically converted to sugars that

291 function as energy sources for the process of fruit ripening (Perdones et al., 2012; Quintana et al., 2021;

292 Vargas et al., 2006). No significant difference in pH was observed over 24 d of storage for the CH- and

293 CH+LMO-treated strawberries. However, the pH of the strawberries coated with CH and combination

294 coats (CH+LMO) was significantly lower (P<0.05) than the pH of the control samples. The data were

295 recorded at the end of the 24-d storage period. These results reveal that CH and essential oils may affect

296 the metabolic activity of fruits, which in turn results in a decrease in the rate of change of pH. Similar

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297 results were reported by Quintana et al. (2021), who treated strawberries with essential oil (rosemary,

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298 thyme, and mugwort)-incorporated CH. The results indicated that the CH and CH+LMO coatings slowed

299 down the rate of change of pH in strawberries.-p

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300

301 3.1.3. Firmness and color change

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302 The data on the firmness of strawberries evaluated on the sampling days are shown in Table 3.
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303 Strawberry firmness initially ranged between 3.24 and 3.44 N, and this was consistent with the results
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304 reported by Shankar et al. (2021). They reported that the values were in the range of 3.60–4.35 N.
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305 Firmness of the control and coated strawberries decreased gradually during the storage period, and this
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306 could be attributed to the ripening of the fruits and the degradation of the middle lamella of cell walls

307 (Barikloo and Ahmadi, 2018). Although the firmness of the coated strawberries was higher than that of

308 the control samples, the difference in firmness between the samples was not significant (P>0.05). The

309 control strawberries showed a rapid decrease in firmness from 3.24±0.25N to 1.89±0.13N at the end of

310 the storage period. In contrast, strawberries coated with CH and CH+LMO retained their firmness, and

311 the firmness values were recorded to be 2.31±0.18N and 2.21±0.17N, respectively, on day 24. These

312 results demonstrate that edible coatings form a protective layer around the fruit. This slows down the

313 processes of moisture loss and softening. Fruit softening is influenced by senescence, cellular breakdown,

314 pectin hydrolysis, and depolymerisation (Elmansy et al., 2017; Paniagua et al., 2013), and similar trends

315 in strawberry firmness have been reported in strawberries coated with CH and essential oils. Khodaei et

316 al. (2021) reported that the firmness of strawberries decreased on day 16 of storage. They also reported
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317 that there were no significant differences between the results obtained for the edible substance-coated

318 strawberries and the control samples.

319 Surface color is an important parameter that determines consumer acceptance. Lightness (L*),

320 redness (a*), yellowness (b*), and total color difference (ΔE) were evaluated over 24 d of storage at 2 °C.

321 The data are presented in Table 3. The L*, a*, and b* values for fresh strawberries were approximately

322 34.46–35.00, 35.52–36.74, and 22.31–22.90, respectively. The L*, a*, and b* values decreased gradually

323 with an increase in the storage period, while the ΔE value increased significantly for all treated samples.

324 No significant differences (P>0.05) were observed in the L*, a*, b*, and ΔE values between the control

325 and treated strawberries during the first four days of storage. The L*, a*, and b* values recorded for the

of
326 control strawberries at the end of the storage period were lower than the values recorded for the treated

ro
327 strawberries. This can be potentially attributed to the event of moisture loss. The redness of the skin

328 -p
decreased as the amount of moisture lost over the storage period increased. No significant differences in
re
329 L*, a*, and b* values were observed between the treated strawberries during the storage period. The ΔE

330 value recorded during storage for the control strawberries was higher than the ΔE value recorded for the
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331 treated strawberries. The ΔE value recorded for the control was 13.15, whereas the ΔE values of the CH-
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332 and CH+LMO-coated strawberries were 8.17 and 9.49, respectively. The values were recorded after 24 d
n

333 of storage. The most significant color change during storage was observed for the control samples, and
ur

334 this could be attributed to dehydration. These results revealed that CH-based edible coatings helped
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335 maintain the color of strawberries. A similar observation was reported by, Khodaei et al. (2021), who

336 found that the surface color of strawberries could be effectively retained when the strawberries were

337 coated with edible coatings.

338

339 3.2. Effects of CH and essential oil on the internal quality of strawberries

340 3.2.1. Properties of X-ray CT images

341 Analysis of X-ray CT images revealed noticeable differences in the internal structures of the

342 control and treated strawberries. The experiments were conducted during the storage period. Black spots

343 (dark regions) were observed in all the treated samples after 12 d of storage (Fig. 1). The formation of

344 black spots can be attributed to the disintegration of tissues. The formation of the spots could be clearly

345 observed when the surface plots and the GS values corresponding to the cross-section of strawberries
12
346 were analyzed (Fig. 2). The tissue structure changed from the outer shell of the fruit to the core.

347 Differences in the pixel intensity (observed by analysing the X-ray CT images) were related to the

348 changes in the tissue structure. The pixel intensity at the core was higher than that at the flesh. Healthy

349 tissue is characterized by high density and high moisture content. Therefore, it appears as a high-intensity

350 area. Broken tissue is characterized by low density and low moisture content. Hence, it appears as a low-

351 intensity area and forms black spots in the flesh region. Analysis of surface plots revealed a decrease in

352 the connective tissue content and an increase in the extent of tissue breakdown realized. The extent of

353 tissue degradation increased with the storage period. The control strawberries degraded rapidly, while the

354 CH- and CH+LMO- treated strawberries degraded slowly. The 3D rendering images present the changes

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355 in the appearance of the samples as a function of storage time (Fig. 3). The CH+LMO-treated samples

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356 appeared fresh until the end of the storage period, while the outer skin of the CH-treated samples

357 -p
appeared slightly wrinkled after 24 d of storage. The degradation of the control samples was apparent
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358 from their appearance after 12 d of storage. These results confirmed that the quality of the strawberries

359 could be preserved using a combination of CH and LMO. These results agreed well with the results
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360 reported by Muziri et al. (2016), who studied the relationship between the distribution of water in pears
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361 and the internal differences that indicated the structural disorders in the cortex tissue with large pores in
n

362 mealy pears by the X-ray absorption properties of pears. Janssen et al. (2020) reported that the observed
ur

363 dark regions and spots (low grayscale value) corresponded to air in the pores inside apple and cell
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364 structures.

365 As shown in Fig. 4b, the GS value

(a) rapidly decreased for the core cavity and increased for the

366 flesh. The GS values recorded for the control and coated strawberries are shown in Fig. 4c. The data were

367 recorded at 2 ℃ over the 24-d storage period. The GS values corresponding to the control and coated

368 strawberries increased gradually during the 8-d storage period. Following this, the values decreased by a

369 small amount. It was observed that the GS values of the coated strawberries were higher than the values

370 recorded for the control strawberries during the period of cold storage. Moreover, the GS value of the

371 CH+LMO-treated strawberries was higher than that of the CH-treated strawberries. The results indicate

372 that the GS values were affected by the moisture content of the samples and the extent of cell destruction

373 realized. The maximum GS value (198.82±6.09) was recorded for strawberries coated with CH+LMO,

374 while the minimum GS value (179.52±5.50) was recorded for the control samples after 24 d of storage. It
13
375 can be concluded that LMO can be incorporated into CH to maintain the quality of strawberries

376 effectively. This can be attributed to the fact that the hydrophobic LMO system can help create an

377 efficient water vapor barrier to prevent moisture loss. It was also observed that the interaction between

378 LMO and charged CH groups could increase the resistance of the fruit surface to gas permeability, which

379 could eventually hinder the process of fruit respiration and reduce the extent of cell destruction realized

380 (Hasheminejad and Khodaiyan, 2020). The reduction in the GS value was also reported for cucumbers

381 (Tanaka et al., 2018) and apricot (Karmoker et al., 2018). Histogram of GS values for the control and

382 coated strawberries on 0-d and 24-d storage period are shown in Fig. 4d. The peak of the histogram

383 decreased for all treatments by the end of storage, with a noticeable shift to the left observed only in the

of
384 control strawberries. The GS values were calibrated using distilled water. The decrease in histogram peak

ro
385 and the shift in histogram profile may have a strong correlation with the physicochemical properties of

386 -p
the strawberries. This is because the GS value of each pixel could be linked to factors such as moisture
re
387 content, air, voids, and tissue structure within the fruit. Similar outcomes were documented in earlier

388 studies (Karmoker et al., 2018; Tanaka et al., 2018).

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389 The core appeared brighter than the flesh, indicating that the amount of X-rays absorbed by the
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(a)
390 core was higher than the amount of X-rays absorbed by the flesh (Fig. 5a). The image predominantly
n

391 shows the properties of the flesh of the strawberries. The GS values of the flesh and core regions were
ur

392 significantly different between treatment groups and sampling days (Fig. 5c and d). The GS value
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393 corresponding to the core region decreased gradually with time during the 24 d storage period, and the GS

394 value of the flesh region increased by a small amount till day 4 of storage. Following this, the value

395 decreased. The GS recorded for the core was higher than that recorded for the flesh of the fruit. The data

396 were collected during the storage period. CH+LMO treatment helped maintain the GS values of the flesh

397 (180.75±8.24) and core (194.14±6.24). The data were recorded at the end of the storage period. The

398 values recorded for the core and flesh of the CH-treated samples were 173.30±7.91 and 180.57±6.71,

399 respectively, while the values recorded for the control group were 159.06±5.73 and 167.25±6.31,

400 respectively. The CNR value decreased in a small amount on day 8 for all treated samples. Following

401 this, the values increased gradually until day 24 (Fig. 5b). There was no significant difference in the CNR

402 values between the control and treated strawberries until day 8 of storage. There was a significant

403 difference in the CNR values of the control and treated strawberries in the period of 12–24 d. The
14
404 maximum CNR value was recorded for the control strawberries (3.10±0.33), and the minimum CNR

405 value was recorded for the CH+LMO-treated strawberries (2.34±0.16). The CNR value of the CH-treated

406 strawberries was 2.60±0.32 at 24 d of cold storage. The results indicate that the internal structure of the

407 CH- and CH+LMO-treated strawberries consisted of fewer low-density regions compared to the control

408 strawberries. The results were recorded over 24 d of storage. It was observed that tissue degradation

409 predominantly occurred in the flesh. Kim et al. (2022) reported that the CNR values recorded for

410 mangoes, chestnuts, and tangerines increased during this time, and the CT number corresponding to the

411 flesh decreased during the storage period.

412

of
413 3.2.2. Analysis of the physicochemical properties using the PLS regression model

ro
414 The moisture content, extent of weight loss realized, degree of firmness, pH, and TSS content of

415 -p
strawberries were predicted from the GS values using the PLS model. The cross-validation method was
re
416 used to calibrate the dataset to identify the optimal PLS model. The model predicted the quality

417 parameters for the calibration and test data. The predicted values were compared with the measured
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418 values in terms of the RMSE and R-square values (Table 4). The RMSE of the test data was lower than
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419 that of the calibration data for the parameters of weight loss, pH, and firmness. The RMSE values
n

420 corresponding to the calibration data obtained for the moisture content and TSS content were lower than
ur

421 the values recorded for the test data. A good linear fit was obtained for the statistical parameters used to
Jo

422 predict the moisture content, TSS content, pH, and firmness of the control and treated strawberries

423 (R>0.8). It could be inferred that the results obtained using the predictive model for weight loss exhibited

424 a good linear fit, with an R-value exceeding 0.7.

425 Fig. 6 presents the relationship between the measured and predicted values of the weight loss, the

426 moisture content, the TSS content, the pH, and the firmness. Satisfactory results were obtained. The

427 predicted moisture content, TSS content, pH, and firmness values were in good agreement with the

428 measured values obtained based on chemical analysis for all treatment groups. The values corresponding

429 to weight loss were predicted with less accuracy. The R-square values for the test set data obtained for the

430 prediction of moisture content, TSS, pH, and firmness were greater than 0.8, whereas the R-square values

431 corresponding to the test set data used for weight loss prediction were less than 0.8. The fact that all R-

432 square values were close to 1 indicated that the PLS models could be used to obtain high-accuracy results.
15
433

434 4. Conclusion

435 The results revealed that the CH and CH+LMO coatings helped to reduce the extent of weight

436 loss realized in fruits. These coatings delayed pH changes and the changes in the ∆𝐸 values recorded for

437 strawberries. It was observed that the moisture content, total soluble solid (TSS) content, firmness, and

438 color of the fruits could effectively be retained when the fruits were coated with CH and CH+LMO. In

439 particular, the CH- and CH+LMO-treated strawberries retained 67.15 and 67.17% of the initial firmness,

440 respectively, while the control retained 58.33% of the initial firmness. The weights of the CH- and

441 CH+LMO-treated strawberries were lost by 5.78 and 6.36%, respectively, at the end of the storage period.

of
442 The weight loss recorded for the control sample was 8.13%. The X-ray CT imaging method is an

ro
443 effective non-destructive method for studying the changes in the internal structure of strawberries. The

444 -p
GS value correlated well with the internal quality of the strawberries. CH and CH+LMO coatings reduced
re
445 the extent of tissue degradation in strawberries. The weight loss, moisture content, TSS content, pH, and

446 firmness of all treated samples were predicted accurately and rapidly using the PLS model and the X-ray
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447 CT data. These results revealed that CH and CH+LMO coatings could be effectively used to preserve
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448 food items. The X-ray CT technique and PLS model can be used to readily and rapidly analyze the
n
ur

449 internal structures of food items and predict the values of different parameters that describe food

450 conditions.
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451

452 CRediT authorship contribution statement

453 Nguyen Thi Hang Phuong: Methodology, investigation, data curation, writing-original draft. Tran Thi

454 Van: Methodology, visualization. Francis Ngwane Nkede: Methodology, visualization. Fumina

455 Tanaka: Visualization, supervision. Fumihiko Tanaka: Supervision, writing-review, editing.

456

457 Declaration of competing interest

458 The authors declare that they have no known competing financial interests or personal relationships that

459 could have appeared to influence the work reported in this paper.

460

461 Data availability

16
462 Data will be made available on request.

463

464 Acknowledgments

465 We sincerely thank the Project for Human Resource Development Scholarship by Japanese

466 Grant Aid (JDS Project) and JSPS KAKENHI Grant Number 21H04748 providing the fund to

467 do this research.

468

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640 Wang, Y., Li, R., Lu, R., Xu, J., Hu, K., Liu, Y., 2019. Preparation of chitosan/corn

641 starch/cinnamaldehyde films for strawberry preservation. Foods.

642 https://doi.org/10.3390/foods8090423

643 Zheng, H., Jiang, L., Lou, H., Hu, Y., Kong, X., Lu, H., 2011. Application of Artificial

644 Neural Network (ANN) and Partial Least-Squares Regression (PLSR) to predict the

645 changes of anthocyanins, ascorbic acid, total phenols, flavonoids, and antioxidant

646 activity during storage of red bayberry juice based on fractal analysis and Red, Green,

647 and Blue (RGB) intensity values. J Agric Food Chem 59, 592–600.

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648 https://doi.org/10.1021/jf1032476

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650 Tables

651 Table 1

652 Weight loss and moisture content of control and treated strawberries during storage at 2oC.

Parameter/Storage time Treatment

(day) Control CH CH+LMO

Weight loss (%)

4 1.10a ± 0.19 1.10a ± 0.16 0.95a ± 0.09

8 2.78a ± 0.19 1.80c ± 0.08 2.15b ± 0.13

12 4.12a ± 0.14 2.62c ± 0.19 3.06b ± 0.18

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16 5.28a ± 0.25 3.69c ± 0.15 4.27b ± 0.14

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20 6.99a ± 0.26 4.93b ± 0.19 5.18b ± 0.27

24 8.13a ± 0.21 -p 5.78c ± 0.30 6.36b ± 0.20

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Moisture content (%)

90.43a ± 0.51 90.56a ± 0.62 90.37a ± 0.46

P

4 89.12b ± 0.58 89.91a ± 0.40 90.11a ± 0.41

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8 88.05b ± 0.83 89.80a ± 0.38 89.27b ± 0.45

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87.56c ± 0.43 89.32a ± 0.47 89.79b ± 0.34

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16 86.33b ± 0.29 88.76a ± 0.57 88.39a ± 0.40

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20 83.97b ± 0.28 87.55a ± 0.40 87.29a ± 0.40

24 82.34c ± 0.35 86.47a ± 0.48 85.91b ± 0.43

653 Results are expressed as mean ± standard deviation. Different letters in the same row indicate

654 statistically significant differences at P<0.05

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655 Table 2

656 Total soluble solid (TSS) and pH of control and treated strawberries during storage at 2oC.

Parameter/Storage time Treatment

(day) Control CH CH+LMO

TSS (%)

0 11.51a ± 0.32 11.41a ± 0.28 11.29a ± 0.30

4 10.60b ± 0.22 11.04a ± 0.25 11.06a ± 0.29

8 10.36b ± 0.29 10.87a ± 0.34 10.91a ± 0.26

12 10.18b ± 0.25 10.81a ± 0.24 10.63a ± 0.20

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16 9.97b ± 0.20 10.66a ± 0.30 10.39a ± 0.23

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20 9.69b ± 0.24 10.46a ± 0.28 10.28a ± 0.21

24 9.41c ± 0.21 -p 10.32a ± 0.26 10.08b ± 0.16

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pH value

3.20a ± 0.07 3.20a ± 0.05 3.18a ± 0.04

P

4 3.32a ± 0.03 3.25b ± 0.03 3.25b ± 0.03

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8 3.41a ± 0.07 3.28b ± 0.03 3.31b ± 0.03

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3.46a ± 0.02 3.35b ± 0.04 3.38b ± 0.04

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16 3.47a ± 0.06 3.41b ± 0.03 3.43ab ± 0.04

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20 3.53a ± 0.04 3.45b ± 0.04 3.47b ± 0.06

24 3.59a ± 0.03 3.47b ± 0.04 3.51b ± 0.02

657 Results are expressed as mean ± standard deviation. Different letters in the same row indicate

658 statistically significant differences at P<0.05

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659 Table 3

660 Firmness and color change of control and treated strawberries during storage at 2oC.

Parameter/Storage time Treatment

(day) Control CH CH+LMO

Firmness (N)

0 3.24b ± 0.25 3.44a ± 0.24 3.29ab ± 0.33

4 2.83b ± 0.34 3.14a ± 0.24 3.17a ± 0.22

8 2.70b ± 0.17 3.13a ± 0.27 2.90ab ± 0.37

12 2.58a ± 0.25 2.77a ± 0.29 2.77a ± 0.24

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16 2.33b ± 0.17 2.71a ± 0.23 2.60a ± 0.25

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20 2.07b ± 0.15 2.51a ± 0.16 2.42a ± 0.28

24 1.89b ± 0.13 -p 2.31a ± 0.18 2.21a ± 0.17

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Color change
P

L* value

0 34.46a ± 0.87 35.00a ± 0.77 34.78a ± 1.37

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4 33.29a ± 1.65 34.16a ± 1.45 34.11a ± 1.06

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8 31.04b ± 1.60 33.87a ± 1.01 33.97a ± 1.50

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12 30.63b ± 1.45 32.07a ± 1.26 32.58a ± 1.00

16 27.96b ± 1.21 30.68a ± 1.30 30.36a ± 1.10

20 27.23b ± 1.59 29.81a ± 0.97 29.16a ± 1.50

24 25.44b ± 1.36 28.33a ± 0.84 27.71a ± 1.43

a* value

0 36.74a ± 1.66 36.19a ± 0.96 35.52a ± 1.26

4 34.66a ± 1.75 34.67a ± 0.61 34.91a ± 1.70

8 32.74b ± 1.50 34.47a ± 1.44 34.36a ± 1.46

12 31.89b ± 1.50 33.87a ± 1.47 33.91a ± 1.17

16 31.36b ± 1.11 33.39a ± 1.63 33.32a ± 1.17

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 20 30.39b ± 1.39 32.94a ± 1.59 32.34a ± 1.20

24 28.97b ± 1.36 31.98a ± 1.50 31.32a ± 1.12

b* value

0 22.31a ± 0.85 22.43a ± 0.96 22.90a ± 1.33

4 21.02a ± 1.10 22.07a ± 0.81 21.74a ± 1.75

8 20.61a ± 0.64 21.62a ± 1.59 21.34a ± 1.63

12 19.88a ± 1.15 20.86a ± 1.08 20.60a ± 1.21

16 18.63b ± 1.57 20.44a ± 1.13 20.21a ± 1.52

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20 16.22b ± 0.90 20.19a ± 0.94 19.73a ± 1.12

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24 15.44b ± 1.55 19.22a ± 1.15 18.44a ± 1.02

ΔE value -p
4 3.33a ± 1.32 2.34b ± 0.58 2.96ab ± 0.69
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8 5.32a ± 1.09 3.03b ± 0.73 3.32b ± 0.64
P

12 6.51a ± 2.08 3.99b ± 1.00 3.98b ± 0.86

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16 9.21a ± 1.00 5.25b ± 1.66 5.92b ± 1.19

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20 11.05a ± 1.56 6.29b ± 0.89 7.42b ± 1.28

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24 13.15a ± 0.94 8.17c ± 1.26 9.49b ± 1.32

661 Results are expressed as mean ± standard deviation. Different letters in the same row indicate

662 statistically significant differences at P<0.05

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663 Table 4

664 Statistic parameters using PLS models for training set and test set to predict the internal quality

665 values of strawberries

Treatment Training set Test set

RMSE R square RMSE R square

Weight loss (%) Control 1.82 0.86 1.47 0.71

CH 1.56 0.79 0.96 0.75

CH+LMO 1.18 0.70 0.97 0.79

Moisture content (%) Control 0.79 0.91 0.72 0.92

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CH 0.58 0.83 0.46 0.88

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CH+LMO 0.48 0.91 0.76 0.70

TSS (%) Control -p 0.17 0.93 0.24 0.87

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CH 0.20 0.80 0.13 0.93
P

CH+LMO 0.19 0.84 0.15 0.86

pH Control 0.04 0.84 0.03 0.90

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CH 0.04 0.81 0.02 0.96

n
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CH+LMO 0.05 0.81 0.03 0.92

Firmness (N) Control 0.18 0.85 0.14 0.91

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CH 0.16 0.83 0.16 0.86

CH+LMO 0.18 0.82 0.13 0.88

666

29
667 Figures

0 day 12 day 24 day

Control

CH

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668 Fig. 1. X-ray CT images showing the internal section of control and chitosan treated strawberries
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669 during storage at 2 oC.

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 0 day 12 day 24 day

Control

CH

CH+LMO

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Fig. 2. Surface plots of GS value in cross section of strawberries during storage at 2 oC.
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0 day 12 day 24 day
P

Control
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CH

CH+LMO

671 Fig. 3. 3D rendering images of control and chitosan treated strawberries during storage at 2 oC.

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 (a)
Core Core
Flesh
Flesh

(b) Core cavity

672
(c)
(d)

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674
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Fig. 4. Region of interest (red line) used to acquire the GS plot profile (a). GS plot profile of strawberry
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675 X-ray CT image (b), changes in GS value (c), and changes in GS value histogram of strawberries during
P

676 storage at 2 oC (d). Vertical bars represent the standard error of the means.
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(b)
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Core
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Flesh

677

(c) (d)

678

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679 Fig. 5. Red boxes in (a) represent ROIs of 1.95 mm x 1.95 mm for CNR calculation and indicate the

680 core region and flesh region. Changes in average CNR of strawberries (b). GS value of the flesh

681 region (c) and the core region (d) during storage at 2 oC. Vertical bars represent the standard error of

682 the means.

(a) (b)

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(d)
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Training set

Test set

(e)

685

686 Fig. 6. Relationship between the measured values and the predicted values for weight loss (a), moisture

687 content (b), TSS (c), pH (d) and firmness (e) by PLS models. Each point represents mean value.

33
Highlights

• Combination of chitosan and lemongrass essential oil maintained strawberry quality.

• X-ray CT showed that tissue degradation in coated strawberries decreased.
• X-ray CT was useful for monitoring the internal structure of strawberries.
• PLS model was useful for predicting the quality parameters of strawberries.

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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:

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