Journal Pre-proofs

An on-package colorimetric sensing label based on a sol-gel matrix for fish

freshness monitoring

Xiuying Liu, Keke Chen, Jiayan Wang, Yu Wang, Yiwei Tang, Xue Gao, Lijie
Zhu, Xuepeng Li, Jianrong Li

PII: S0308-8146(19)31704-2
DOI: https://doi.org/10.1016/j.foodchem.2019.125580
Reference: FOCH 125580

To appear in: Food Chemistry

Received Date: 30 April 2019

Revised Date: 4 August 2019
Accepted Date: 23 September 2019

Please cite this article as: Liu, X., Chen, K., Wang, J., Wang, Y., Tang, Y., Gao, X., Zhu, L., Li, X., Li, J., An on-
package colorimetric sensing label based on a sol-gel matrix for fish freshness monitoring, Food Chemistry (2019),
doi: https://doi.org/10.1016/j.foodchem.2019.125580

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 1 An on-package colorimetric sensing label based on a sol-gel matrix for fish

2 freshness monitoring

3 Running title: On-package colorimetric sensing label for fish freshness

4 monitoring

5 Xiuying Liu, Keke Chen, Jiayan Wang, Yu Wang, Yiwei Tang, Xue Gao, Lijie Zhu, Xuepeng Li*

6 and Jianrong Li*

7 College of Food Science and Technology, Bohai University; Food Safety Key Lab of Liaoning Province;

8 National & Local Joint Engineering Research Center of Storage, Processing and Safety Control

9 Technology for Fresh Agricultural and Aquatic Products; Jinzhou, Liaoning, 121013, China

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21 * Corresponding author: Xuepeng Li, xuepengli8234@163.com Tel/Fax: +86-416-3719190; Jianrong

22 Li, Email: lijr6491@163.com Tel/Fax: +86-416-3400008.

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23 Abstract:

24 Fish freshness monitoring is important for consumers. This study aims to develop an colorimetric

25 sensing label based on bromocresol green (BCG) and a sol-gel matrix layer coated onto filter paper to

26 monitor fish freshness. Characterization results showed that the sol-gel layer was successfully coated,

27 and the coating yield was 14.25%. The fish freshness could be detected clearly by the naked eye as the

28 color of the sensing label changed. A Hue Saturation Value (HSV) model was used to correlate the

29 response of the sensing label to the freshness of fish samples. Hue (H) values showed a linear response

30 to the total volatile basic nitrogen (TVB-N) concentration in the range of 16.4–23.11 mg/100 g at room

31 temperature, and in the range of 9.28–24.12 mg/100 g at a chilled temperature. The sensing label was

32 applied to other types of fish, and showed an intense color change during the spoilage trial.

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34 Keywords: Fish freshness; Sol-gel matrix; Colorimetric sensing label; Bromocresol green

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36 1. Introduction

37 People consume fish as a healthy and high-value food in their daily diet because of its high

38 nutrient contents and pleasant taste (Tan, Huang, Feng, Li, & Cai, 2018). However, treatment and

39 storage conditions after capturing the fish can strongly affect fish freshness and quality (Sampels, 2015;

40 Tolstorebrov, Eikevik, & Bantle, 2016). Monitoring of fish freshness is of great importance for

41 consumers, retailers, and industries. Currently, the commonly used methods for investigation of fish

42 and seafood freshness are sensory assessment or microbial and chemical techniques, which generally

43 require long analysis time and professional operators (Olafsdottir et al., 1997; Grigorakis, Alexis,

44 Gialamas, & Nikolopoulou, 2004; Lv, Huang, Aheto, Mu, & Tian, 2018). Novel approaches such as

45 electronic noses and hyperspectral imaging techniques have also been developed in recent years (Wu,

46 Pu, & Sun, 2018; Shi et al., 2018; Shi et al., 2019). However, these techniques have high costs and

47 require complicated instruments. Hence, developing methods that are rapid, simple, low-cost, and

48 non-destructive in order to evaluate the real-time freshness of fish and seafood products is valuable.

49 Smart packaging is a small, inexpensive tag or label attached to primary or secondary packaging

50 to indicate the freshness and condition of packaged food (Ghaani, Cozzolino, Castelli, & Farris, 2016).

51 For typical smart packaging, the indicator on the package is capable of changing color via reacting with

52 the chemicals generated from the packaged food. For the development of smart packaging,

53 bromocresol green (BCG) is a highly sensitive pH indicator that belongs to the sulfone phthalein dye

54 group. A slight increase in pH can immediately trigger a visible color change of BCG. Several studies

55 have been conducted based on using BCG indicator. For example, Rijian Mo et al made an intelligent

56 label based on BCG and a porous anodic aluminum membrane to detect freshness of fish (Mo et al.,

57 2017). However, the metallic aluminum substrate they used to immobilize the indicator is expensive

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58 due to its complicated preparation process. Dermot Diamond's group entrapped the BCG molecule

59 within a polymer matrix (Byrne, Lau, & Diamond, 2002; Pacquit et al., 2007). However, leaching of

60 the dye from the polymer matrix may occur over time, which may result in inaccurate indications.

61 Hence, besides the colorimetric indicator, a solid support for immobilization of the indicator is

62 important for smart packaging.

63 Sol-gel processing is well-known as a powerful technique for designing materials and has

64 attracted considerable interest in the sensing field (Owens et al., 2016; Monton, Forsberg, & Brennan,

65 2011). It has a number of advantages, including uniform porosity, high surface areas and pore volumes,

66 optical transparency, low chemical reactivity, and mechanical rigidity (Wang, Pamidi, & Rogers, 1998).

67 Therefore, Sol-gel films have been proposed as an excellent solid matrix for the entrapping of

68 chemicals or biomolecules in chemical sensors and biosensors (Gupta, & Chaudhury, 2007; Jerónimo,

69 Araújo, & Montenegro, 2007).

70 For this work, we aimed to design a highly sensitive colorimetric label by coating filter paper with

71 a layer of sol-gel matrix entrapping the BCG indicator. Compare with the other BCG labels that have

72 been reported in the literature, filter paper was selected as the solid support in our study. Filter paper is

73 disposable, simple to use, and inexpensive. However, the filter paper prepared via the traditional

74 dip-coating method has many disadvantages, including being fragile and vulnerable to damage,

75 allowing dye to leak, and can be easily affected by humidity. To improve the above disadvantages, we

76 coated a layer of sol-gel on the surface of the filter paper. The moisture absorption properties,

77 morphology, and the colorimetric response of the designed sensing label were investigated. Finally, we

78 applied our sensing label to fish samples to assess the TVB-N levels in the fish packaging headspace

79 and monitor the fish freshness at room temperature and at a chilled temperature.

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80 2. Material and methods

81 2.1 Reagents and solutions

82 Bromocresol green (BCG), boric acid (H3BO3), and hydrochloric acid (HCl) were purchased from

83 Tianjin Fuchen Chemical Co. (Tianjin, China). Other chemicals including tetraethoxysilane (TEOS),

84 methyltriethoxysilane (MTEOS), potassium chloride (KCl), sodium borate (Na2B4O7·10H2O),

85 magnesium oxide (MgO), methyl red (C15H15N3O2), sodium hydroxide (NaOH), citric acid (C6H8O7),

86 and sodium citrate (Na3C6H5O7·2H2O) were purchased from Aladdin-Reagent Co. (Beijing, China). All

87 reagents used were of reagent grade and used as supplied, without further purification.

88 2.2 Preparation of the BCG sol-gel sensing label

89 The sol-gel preparation was based on the method of Ismail et al. with modification (Ismail, Malins,

90 & Goddard, 2002). The sol-gel solution was prepared first. A 20 mg quantity of BCG was added into a

91 mixture of 3.68 mL ethanol and 0.85 mL of a 0.1 mol/L hydrochloric acid solution. This solution was

92 stirred vigorously while a second solution of silicon alkoxide precursor mixtures (812 μL 100% TEOS,

93 394 μL 50 ％ TEOS-50%MTEOS, and 411 μL 100 ％ MTEOS) was slowly added dropwise into the

94 stirred solution. The above sol solution was combined with 9.2 mL of deionized water and stirred

95 continuously for 1 h. To prepare the BCG sol-gel sensing label, the filter paper was soaked in the sol

96 solution overnight and was then removed and dried at room temperature. Before being applied to the

97 fish samples, the sensing label was cut into a round shape with a diameter of 1 cm by using a hole

98 punch.

99 2.3 Characterization of the BCG sol-gel sensing label

100 Filter paper is a common solid support substrate for making sensor labels due to its low cost.

101 Some studies have reported designing sensing films by dip-coating filter paper with various dyes

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102 (Kuswandi, & Nurfawaidi, 2017). We hoped to overcome the disadvantages of traditional dip-coated

103 labels, therefore, we compared our sol-gel sensing label with two control samples including plain filter

104 paper (control 1) and the traditional BCG dye dip-coated filter paper (control 2) in terms of Scanning

105 Electron Microscopy (SEM) morphology, moisture absorption, and thermo-gravimetric analysis.

106 2.3.1 The moisture absorption of the BCG sol-gel sensing label

107 Detection of moisture absorption was based on the method of Pirsa et al. with modification (Pirsa,

108 Karimi Sani, & Khodayvandi, 2018). The prepared sensing label and the control labels were cut into a

109 round shape with a diameter of 2 cm by using a hole punch, and then were placed in the inner chamber

110 of a Conway diffusion cell. The outer chamber was filled with 10 mL of saturated sodium chloride

111 solution. The cell maintained a relative humidity (RH) of 76%. The Conway diffusion cell was sealed

112 with a cap and kept below a temperature of 30 °C. The weights of the labels were measured every 12 h

113 over a period of 3 days, and the moisture absorption was calculated by the following equation:

114
115 where Wt is the weight (mg) of label at certain time and Wi is the initial weight (mg).

116 Three samples were tested and the moisture adsorption was calculated as an average ± the

117 standard deviation (SD).

118 2.3.2 Scanning electron microscopy images

119 Sample surface morphologies were examined using an S-4800 scanning electron microscope

120 (Hitachi, Japan) at a voltage of 3 kV. Before SEM examination, sensing label samples were prepared

121 by cutting them into small pieces and sputtering them with gold to make them conductive. Three types

122 of samples including plain filter paper (control 1), traditional BCG dip-coated filter paper (control 2),

123 and the BCG sol-gel coated sensing label were tested. Three separate trials were run for each of the

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124 three sample types.

125 2.3.3 The thermo-gravimetric analysis (TGA) of the BCG sol-gel coated sensing label

126 The thermal stability and the coating yield of the samples was evaluated by a PYRIS DIAMOND

127 TG/DTA thermo-gravimetric analysis instrument (TGA, PerkinElmer, USA) over the temperature

128 ranged from 30 °C to 800 °C. Samples were kept under a nitrogen atmosphere and the rate of

129 temperature increase was 20 °C /min.

130 2.4 Total volatile basic nitrogen (TVB-N) measurement

131 TVB-N levels were measured according to Huang et al. with modification (Huang et al., 2019).

132 The fish was peeled and minced with a meat grinder. A 5 g sample of homogenized fish was weighed,

133 and carefully transferred into a digestion tube. Then, 1 g of magnesium oxide powder was added into

134 the digestion tube and mixed well with the fish sample. Finally, the digestion tube containing the

135 prepared fish sample was connected to a Kjeltec 8400 fully automated Kjeldahl analyzer (Foss,

136 Denmark), and was analyzed automatically. This analysis was replicated three times. The TVB-N level

137 was calculated and expressed in mg/ 100 g.

138 2.5 Determination of pH

139 The pH was measured according to Li et al. with modification (Li et al., 2012). For the pH

140 determination, 2 g of minced fish sample was homogenized in 20 ml of 0.1 mol/L potassium chloride

141 solution at room temperature. The above mixture was analyzed using a pH meter (PHS-3E, Leici,

142 China). This analysis was replicated three times.

143 2.6 Application of the sensing label to fish samples

144 Live red drum (Sciaenops ocellatus) fish were purchased from a local market and transported to

145 the lab within a period of 30 min. To ensure the freshness of the test samples, live fish were slaughtered

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146 at the lab and were processed into test samples immediately. Test samples were processed as follows:

147 After the fish were slaughtered, the skin of the fish was peeled and the bone was removed. The fish

148 meat was divided into portions of 10 g and put into containers sealed with transparent plastic wrap for

149 freshness monitoring. To ensure representative monitoring, three samples, each taken from different

150 fish, were tested.

151 Sol-gel sensing labels (1 cm in diameter) were placed in the headspace of each container. Only

152 the meat on both sides of the fish was used in our study. The experiments were conducted at room

153 temperature and also at a chilled temperature (4 o C). The TVB-N level, pH values, and the color of the

154 sensing labels were recorded every 2 hours for samples at room temperature and were recorded every

155 day for samples kept at the chilled temperature.

156 Other types of fish samples, including whole live turbot (Scophthalmus maximus) and yellow

157 croaker (Larimichthys crocea), were also purchased from the local market. The live fish were

158 slaughtered at the lab and then were processed into test samples immediately. After removing the skin

159 and bones, the fish were cut into fillets for use in additional tests.

160 2.7 Images and data collection

161 Images of the sensing labels were collected by Smartphone and then further processed with

162 Photoshop software to extract the RGB values. Every R, G, and B value was read from three spots

163 located on the upper, middle and lower part of each sensing label. If the sensing label showed a

164 different color on its edge, data had to be collected equally, both on the edge and in the center of the

165 sensing label. Data from the three independent analyses were combined into an average ± the standard

166 deviation (SD).

167 3. Results and discussion

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168 3.1 UV-vis spectra of BCG solutions at various pH values

169 The color changes of the BCG indicator in different pH solutions ranging from pH 3 to pH 8 were

170 measured. Several buffer solutions with different pH values were used. Acidic conditions, including pH

171 values of 3, 4, 5, and 6 were created using a citric acid-sodium citrate buffer solution. An alkaline

172 condition (pH=8) was created using a boric acid-borax buffer solution. A pH 7 buffer was made by

173 adjusting the boric acid-borax buffer solution with aqueous NaOH solution. As shown in Fig. 1(a), we

174 could observe the visible color changes of the BCG dissolved in the different pH buffers. The BCG

175 was yellow under strongly acidic conditions (pH 3-4) and green at pH 5. As the pH value of the buffer

176 increased from 6 to 8, the BCG became blue, and the blue color gradually intensified. Correspondingly,

177 the maximum adsorption also changed, as clearly shown in the UV-vis spectra presented in Fig. 1(b).

178 Under acidic conditions, BCG showed a maximum absorption peak at 444 nm, while this peak shifted

179 to 614 nm under alkaline conditions. The intensity of the peak increased with the increasing alkalinity

180 of the solutions. The intense color changes demonstrate that BCG is a good candidate as a sensing

181 indicator for making colorimetric sensing labels.

182 3.2 Characterization of the sensing label

183 3.2.1 Surface micromorphology analysis

184 Fig. 2 shows the SEM images of three samples (plain filter paper, BCG dip-coated paper, and the

185 BCG sol-gel sensing label) under magnifications of 500 times, 20.0 K times and 50.0 K times. We

186 could see the surfaces of these three samples (Fig. 2 (a), (b) and (c)) were similar using a magnification

187 of 500 times. They appeared as fluffy and fibrous, thus providing larger surface area to interact with the

188 gas generated from fish. This is favorable for a sensing label application. As the magnification of the

189 SEM image increased to 20.0 K times, different micro-morphology of the three samples could be

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190 detected. The plain filter paper (Fig. 2 (d)) at a magnification of 20.0K still shows the fluffy fibrous

191 structure. However, after dip-coating the plain filter paper with the BCG dye, we could see a lot of

192 spherical particles were fixed between the fibers of the filter paper (Fig. 2 (e)). These were attributed to

193 the BCG dye particles. Compared to the dip-coated filter paper (control 2), the surface of the BCG

194 sol-gel sensing label is smoother (Fig. 2 (f)). The spherical dye particles between fibers were covered

195 with a layer of sol-gel matrix, which was good for avoiding dye leaking. The differences between the

196 surface structures of these three samples were shown even more clearly using a magnification of 50.0

197 K times (Fig. 2 (g), (h) and (i)). Therefore, we concluded that the sol-gel layer was successfully coated

198 on the surface of plain filter paper and the sol-gel layer could fix BCG dye particles more tightly and

199 with greater stability.

200 3.2.2 Moisture absorption of the sensing label

201 Moisture absorption is one of the most important properties of a sensing label. When a sensing

202 label is placed in the headspace of fish packaging, the high relative humidity in the package may have a

203 negative effect on the color change of the sensing label. This can lead to an inaccurate result. Also,

204 after absorbing the moisture, the sensing label may become fragile and vulnerable to damage.

205 Therefore, lower moisture absorption by the sensing label is a desirable factor. As shown in Fig. S1 in

206 the supplementary material, the moisture absorption of all three samples increased gradually and

207 reached saturation as time progressed. At a relative humidity (RH) of 76%, the moisture absorption at

208 72 h for the filter paper and the traditional dip-coated paper were 14.1% and 9.0%, respectively.

209 However, after coating the filter paper with a layer of sol-gel, the moisture absorption value decreased

210 to 4.1%. Compared to the filter paper, the moisture absorption property of our sol-gel sensing label was

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211 approximately 3.5 times lower. This result is favorable for preventing moisture from interfering with

212 the accuracy of the results when the sensing label is applied to fish freshness monitoring.

213 3.2.3 Thermo-gravimetric analysis of the sensing label

214 In our study, filter paper was selected as the solid support substrate for coating with the sol-gel

215 sensing layer. To calculate the coating yield, the TGA analysis of the filter paper and the sol-gel

216 sensing label were recorded and the results are provided in Fig. S2 of the Supplementary material.

217 Sample labels were cut into small pieces and then heated to 800 o C at a rate of 20 o C /min. From the

218 TGA curves, we could find that the rate of weight loss of both the plain filter paper and of our sol-gel

219 sensing label slightly increased between 30 °C and 300 °C. This was mainly attributed to the

220 evaporation of water molecules. The weight loss values were 2.39% and 1.12%, respectively. When the

221 temperature was higher than 350 °C, both the plain filter paper and sol-gel sensing label decomposed

222 severely, and the weight loss values increased to 87.81% and 73.56% respectively at a temperature of

223 450 o C. The weight that remained within the temperature range of 450-800 o C could be attributed to

224 residual inorganic matter remaining after sample decomposition was complete. The subtracted weight

225 of sol-gel sensing label is higher than filter paper indicated that the sol-gel layer was grafted on the

226 surface of filter paper successfully and the coating yield was calculated as 14.25%.

227 3.3 Trials of monitoring fish freshness

228 3.3.1 TVB-N and pH analysis of fish samples

229 Generally, when fish experiences spoilage, the amount of TVB-N increases because of the

230 formation of trace amounts of volatile amines. Therefore, the TVB-N level is a widely used fish

231 spoilage indicator (Chang et al., 2017). Also, generation of amines from spoiling fish causes a change

232 in the pH values of fish meat. Fig. 3 shows the changes in TVB-N level and pH for the sample of red

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233 drum fish stored at room and at chilled temperatures. At room temperature, the amount of TVB-N

234 increased gradually from an initial TVB-N value of 7.20±0.09 mg/100 g for fresh fish (shown in Fig. 3

235 (a)). The level of TVB-N increased dramatically after 22 h of storage as the value increased from

236 23.11±2.22 mg/100 g at 22 h to 43.37±1.78 mg/100 g at 28 h of storage. Correspondingly, the pH

237 values of the fish meat were nearly stable (below 6.75±0.01) within the first 22 h of storage and then

238 increased quickly to reach a value of 7.45±0.02 at 28 h. As shown in Fig. 3 (b), for red drum sample

239 stored at the chilled temperature, the TVB-N level increased gradually during the early stages of

240 storage (24.12±1.34 mg/100 g at 6 days), and then increased rapidly. There were no significant changes

241 in the pH values during these six days. The pH values of the fish meat samples started increasing after

242 6 days of storage and reached a maximum value of 7.5±0.02 at day 10. Some studies on fish freshness

243 have reported that the limit of acceptability for TVB-N is about 25 mg/100 g (Qiong, Tuzhi, & Liju,

244 1998; Limbo, Sinelli, Torri, & Riva, 2009). In our work, samples did not exceed this acceptable limit

245 during 22 h of storage at room temperature or within 6 days of storage at the chilled temperature. This

246 implies that 22 h and 6 days can be considered as the threshold of spoilage at room and chilled

247 temperatures, respectively.

248 3.3.2 Color changes of the sensing label during fish spoilage trial

249 To evaluate the response of our sensing label during the fish spoilage trials, the color changes of

250 the sensing label during the fish storage period were recorded. The results, along with the TVB-N

251 values are given in Fig. 4 (a) and (b), respectively. As shown in Fig. 4 (a), at room temperature, the

252 color of the sensing label placed in the headspace of the fish package was yellow initially and did not

253 obviously change during the first 4 hours. The amount of TVB-N was lower than 10 mg/100 g before 4

254 hours of storage, which indicated that the fish sample was very fresh. The edge of the sensing label

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255 started changing color from yellow to green by the 4th hour, and then totally changed to green by the

256 10th hour. In the range of 10 to 22 h of storage time, the green color of the sensing label gradually

257 became darker. Correspondingly, the TVB-N level of the fish samples gradually increased to

258 23.11±2.22 mg/100 g (at 22 h) thereby reaching the threshold of spoilage. When the amount of TVB-N

259 exceeded the threshold of spoilage (25 mg/100 g), the color of the sensing label first turned somewhat

260 blue and eventually became dark blue. A similar response trend is shown in Fig. 4 (b). At the chilled

261 temperature, the sensing label changed color from yellow to green prior to 6 days, and then to blue by

262 day 7. Correspondingly, the TVB-N level increased gradually and reached the threshold of spoilage

263 when the color of the sensing label changed from green to dark blue. We were able to observe a color

264 change from green to dark blue using the naked eye, when fish freshness became unacceptable. We

265 have therefore concluded that our BCG sol-gel sensing label is a feasible design to allow real-time

266 monitoring of fish freshness by retailers and consumers.

267 3.3.3 Conversion of color changes to digital data

268 During the fish spoilage experiment, our sensing label showed a clear color change that was

269 feasible for monitoring fish freshness using the naked eye. However, to achieve quantitative evaluation

270 of fish freshness using the sensing label, we explored methods to convert colors to numerical values.

271 The RGB color model is an additive color model in which red (R), green (G), and blue (B) colors are

272 added together in various ways to produce a broad array of colors. In some reports, RGB values of

273 images are directly used for making calibration curves. However, this is not always effective. The Hue

274 Saturation Value (HSV) color model has been reported to be an alternative representation of the RGB

275 color model and has been widely used in computer vision and image analysis (Hong, & Chang, 2014).

276 In the HSV color space, the Hue (H) value represents the type of color and changes as the wavelength

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277 shifts. Therefore, we herein extracted R, G, and B values from the sensing label images and converted

278 them to Hue values according to the following equations (Sun, Li, Wang, Xiao, Wang, & Feng, 2015):

279 If R= max, H=(G-B)/(max-min) × 60

280 If G= max, H=120+(B-R)/(max-min) × 60

281 If B= max, H=240+(R-G)/(max-min) × 60

282 If H<0, H=H+360

283 The TVB-N level is a traditional indicator for quantitative evaluation of fish freshness. Hence, H

284 values were plotted along with the concentrations of TVB-N in the headspace of the fish packages and

285 the calibration curves are given in Fig. 5. At room temperature (Fig. 5 (a)), the H value slowly

286 increased with the increase of the TVB-N concentration from 7.2 to 14.23 mg/100 g, and then showed

287 a sharply rising trend over the range of 14.23 to 23.11 mg/100 g. Finally, the H value reached a plateau

288 at the TVB-N concentration from 23.11 to 43.34 mg/100 g. Correspondingly, the color of the sensing

289 label changed distinctly from yellow to green and then to dark blue. In the inset of Fig 5 (a), H values

290 showed a linear response to TVB-N concentrations over the range of 16.4 to 23.11 mg/100 g (R2 =

291 0.961, y = 9.74x - 34.03). As the linear range of the TVB-N concentration fell within the threshold of

292 spoilage, this linear calibration curve could be used to calculate the amount of TVB-N present at levels

293 below the threshold of spoilage. At the chilled temperature, as shown in Fig. 5 (b), the H value

294 displayed a similar tendency in the calibration curve. As TVB-N level increased, the H value increased

295 gradually and then remained nearly constant. A linear response was obtained in the TVB-N

296 concentration range of 9.28 to 24.12 mg/100 g (inset of Fig 7 (b), R2=0.973, y = 2.26x + 116.02).

297 These results indicate that the color change of the sensing label responded proportionally to the

298 concentration of TVB-N over a certain range. Therefore, consumers or retailers could not only judge

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299 the freshness of fish using the naked eye, but could also assess an accurate TVB-N level of the fish

300 based on RGB values extracted from sensing label images.

301 3.4 Real fish sample detection

302 Besides red drum other types of fish, including turbot and yellow croaker, were also tested using

303 our sol-gel sensing label. Fig. 6 shows the application of the sol-gel sensing label to the freshness of

304 turbot and yellow croaker in a package at room temperature. In Fig. 6 (a) when the turbot was very

305 fresh, the color of the sensing label was yellow, which indicated that the turbot in the package would

306 have its best flavor. When the turbot was slightly spoiled, the color of the sensing label turned to green.

307 Even though the turbot was still fresh, the green color conveyed a message that the turbot inside should

308 be sold or eaten as soon as possible. When the turbot spoiled, the sensing label changed to dark blue,

309 which suggested that the product must be discarded. The TVB-N value reached 26.44±1.18 mg/100 g,

310 which exceeds the maximum acceptable limit of 25 mg/100g for turbot (Scophthalmus maximus) (Lv et

311 al., 2018; Ojagh et al, 2010). In Fig. 6 (b), the sensing label showed a similar response when it was

312 used for assessing the freshness of yellow croaker. The TVB-N value was 26.34±1.22 mg/100 g when

313 the sensing label changed to dark blue, which exceeded the highest acceptable level of 25 mg/100g for

314 yellow croaker (Larimichthys crocea) (Li et al., 2012). According to the above results, it can be

315 concluded that the sensing label we designed in this study shows great potential as a freshness monitor

316 for different types of fish products.

317 4. Conclusions

318 In summary, this work developed a colorimetric sensing label based upon the indicator BCG and a

319 sol-gel matrix coated on the surface of filter paper, for the purpose of allowing real-time fish freshness

320 monitoring. Benefiting from the sol-gel layer, the moisture resistance property of the sensing label was

15
321 improved. This is favorable for preventing moisture from interfering with the accuracy of the sensing

322 label. Based on test results, the colorimetric sensing label had an accurate response to fish freshness

323 and showed intense color changes from yellow to green and then to dark blue when fish experienced

324 spoilage. These changes can be easily detected by the naked eye. Additionally, the sensing label

325 response was found to correlate with the concentration of TVB-N in red drum fish samples at room and

326 chilled temperatures. Thus, consumers or retailers can not only judge the freshness of fish by the naked

327 eye, but also assess an accurate TVB-N level in the fish based on RGB values extracted from sensing

328 label images. Finally, the sensing label was successfully applied to other types of fish including turbot

329 and yellow croaker, which indicated that the sensing label can be used as an effective tool for fish

330 freshness monitoring.

331 Acknowledgements

332 This study was supported by the Natural Science Foundation of Liaoning Province, China

333 (20170540017).

334 References

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336 from selected fish species using reflectance spectroscopic measurements of pH sensitive films.

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343 Grigorakis, K., Alexis, M., Gialamas, I., & Nikolopoulou, D. (2004). Sensory, microbiological, and

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416

417

418

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420 Figure captions

421 Fig. 1. (a) Photograph of BCG solutions at different pH values and (b) UV-vis spectra of BCG

422 solutions at different pH

423 Fig. 2. SEM images of (a, d and g) plain filter paper, (b, e and h) dip-coating paper with BCG dye and

424 (c, f and i) BCG sol-gel sensing label with magnification of 500, 20.0 K and 50.0 K times

425 Fig. 3. Changes in TVB-N and pH values of fish meat versus the storage time at (a) room and (b)

426 chilled temperature

427 Fig. 4. Color Changes of the BCG sol-gel sensing label in response to spoiling fish sample at (a) room

428 and (b) chilled temperature

429 Fig. 5. Hue values of sensing labels in response to different amount of TVBN at (a) room and (b)

430 chilled temperature

431 Fig. 6. Application of the BCG sol-gel sensing label to (a) turbot and (b) yellow croaker in package at

432 room temperature

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434

435 Fig. 1. (a) Photograph of BCG solutions at different pH values and (b) UV-vis spectra of BCG solutions at different pH

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437

438 Fig. 2. SEM images of (a, d and g) plain filter paper, (b, e and h) dip-coating paper with BCG dye and (c, f and i) BCG sol-gel

439 sensing label with magnification of 500, 20.0 K and 50.0 K times

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441

442 Fig. 3. Changes in TVB-N and pH values of fish meat versus the storage time at (a) room and (b) chilled temperature

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444

445 Fig. 4. Color Changes of the BCG sol-gel sensing label in response to spoiling fish sample at (a) room and (b) chilled

446 temperature

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448

449 Fig. 5. Hue values of sensing labels in response to different amount of TVBN at (a) room and (b) chilled temperature

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451

452 Fig. 6. Application of the BCG sol-gel sensing label to (a) turbot and (b) yellow croaker in package at room temperature

453 On-package colorimetric sensing label was developed for fish freshness.

454 Label was based on bromocresol green indicator and sol-gel matrix layer.

455 A Hue Saturation Value model was used to correlate the response of sensing label.

456 Three kinds of fish fillets showed intense color change during the spoilage trial.

457

458

459 Fig. S1. Moisture absorption properties of plain filter paper (control 1), dip-coating paper with BCG dye (control 2) and BCG

460 sol-gel sensing label

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462

463 Fig. S2. TGA analysis of (a) plain filter paper and (b) BCG sol-gel sensing label

464

465

466 Declaration of Interest Statement

467 The authors declare that they have no competing interests.

468

469

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